Generation Of Nanobody Recognizing Human CD147 Antigen And Its Functional Study

Extracellular matrix metalloproteinase inducer（CD147）is a 50⁶0 KD glycosylated transmembrane protein in the immunoglobulin superfamily.It is overexpressed on the surfaces of various tumor cells and promotes the proliferation,invasion,and metastasis of cancer cells,and regarded as a wide variety of tumor biomarkers.Therefore,it has become a hot topic that antibodies and corresponding inhibitors of CD147 are studied.With the development of antibody technology,polyclonal antibodies,monoclonal antibodies and recombinant antibodies have been developed to be used for clinic treatment and the therapeutic effect was significantly improved.The commercialized monoclonal antibody-Licartin had a significant effect on the treatment of liver cancer recurrence.Nanobodies,characterized by their small size,high affinity,high specificity,and low immunogenicity,are a diagnostic and therapeutic tool with great promise.In this study,phage display technology was used to screen anti-CD147 Nanobodies and its specificity and targeting were identified in vitro and in vivo,which can be used for the targeted diagnosis of tumor and follow-up functional studies.The main research contents are as follows:1.Screening of anti-CD147 Nanobody and prokaryotic expressionThe VHH phage display library was successfully constructed through the immunizationand construction of the VHH library with ectodomain protein of CD147(CD147^ect)as the target.The size of the phage-displayed VHH library was approximately 5×10⁸ CFU/mL,which met the requirements of subsequent screening.The phage of CD147-specific VHH was efficiently enriched after three rounds of panning.Random selection of 25 clones for PCR identification from each round showed that the library insertion rate was approximately both 100%with second and third round.The phage-ELISA identification results showed that about 40%of the specific phage clones could be combined with CD147 antigen.And then these genes were sequenced and identified.The correct genes of sequencing were inserted into the expression vector（pet-15b and pet-28a）to be used forpurifying Nanobodies by the prokaryotic expression,and the expression quantity of different sequences of Nanobody in 1 L bacteria was analyzed.2.Characterization and targeting of anti-CD147 NanobodiesTwo Nanobodies（11-1 and 12-3）were selected for a series of specific and targeted verification.The particle size of Nanobodies was determined by dynamic light scattering（DLS）.Their particle size was below 100 nm,and Nanobody 11-1 morphology was observed by transmission electron microscope.It was elliptical and the size was uniform.Nanobodies were conjugated with HRP,and the binding activity of Nanobodies 11-1 and 12-3 was verified by Western bloting.The equilibrium dissociation constant（KD,which represents the affinity of the interaction between 2 molecules）values for the 11-1 Nanobody with the CD147 and C78 proteins were 6.36×10^-10 M and 1.78×10^-9 M and the 12-3 Nanobody with CD147 was9.46×10^-9 M.The specificity of the Nanobodies in the cell level was verified by Western bloting and cell fluorescence assays.Balb/c mice bearing 4T1 tumor modelswere established to successfully confirm the targeting of Nanobodies in vivoimaging system.3.Functional study of CD147 Nanobody and its anticancer activities conjugating doxorubicin（DOX）in vitroNanobody 11-1（30μM,100μL）was injected into Balb/c mice bearing 4T1 tumor models（n=3）.The expression of RANTES,MIP-3α,MCP-1,MIG and IP-10 were detected using fluorescence activated cell sorter（FACS）.It shows that 11-1 could significantly induce the increase of chemotactic factor MIG in blood.After Nanobody 11-1 conjugating DOX,we found that DOX-11-1 was distributed in the cell membrane and cytoplasm ofHeLa,U87 and4T1 by real-time observation,whereasred fluorescence signal was not detected in 293T cells.MTT assay demonstrated that DOX-11-1 had a certain targeted therapeutic effect on tumor cells in vitro.Annexin V-FITC/PI assaypreliminarily elucidated that DOX-11-1 could cause apoptosis or necrosis.In this study,we used phage display technology to select specific phage clones and successfully prepared highly specific Nanobody.The tumor targeting of Nanobodywas verifiedin vivo imaging system.The Nanobody 11-1 couldinduce the level of chemotactic factor MIG in blood,which may indirectly exert anti-tumor effect.A preliminary study on the anti-tumor effect of Nanobody 11-1 in vitro was performed,and it elucidated that the anti-tumor mechanism was caused by cell apoptosis or necrosis.This study provides data support and reference for the further application of anti-CD147 Nanobodies.