Preparation Of Antibodies Against Human Neuropilin 1b Domain And Its Inhibitory Effect On Angiogenesis

As a transmembrane glycoprotein,Neuropilin 1(NRP1)plays an important role in axonal guidance,angiogenesis and tumor immunity.The study found that NRP1 is a non-tyrosine kinase receptor of vascular endothelial growth factor(VEGF),which promotes the binding of VEGF to its receptor(VEGFR),and has a significant role in promoting angiogenesis.The blb2 domain of NRP1 is the main binding site with VEGF165.Studies have found that NRP1 is highly expressed on the surface of many types of tumor cells,especially epithelial cell tumors.Blocking NRP1 can not only directly inhibit the migration of tumor cells and the occurrence and development of tumors,but also block its binding with VEGF165,indirectly inhibit the formation and development of tumor local blood vessels,and then affect the growth and development of tumors.Therefore,NRP1 is expected to become a new breakthrough point for anti-tumor therapy.In this study,human NRP1 b domain(HuNRP1b)cDNA was obtained by PCR and cloned into a prokaryotic expression vector.After expression and purification,recombinant HuNRP1b protein was obtained;mice were immunized with recombinant HuNRP1b protein to prepare anti-HuNRP1b monoclonal antibody(McAb).Subsequently,the in vivo and in vitro experiments were used to analyze the ability of McAb in binding tumor cells and inhibit the activity of angiogenesis to provide the basis for subsequent anti-tumor studies.1.Acquisition of recombinant HuNRP1b domain proteinThe coding gene of HuNRPlb was obtained by PCR and the recombinant plasmid pET30a-HuNRPlb was constructed.After verified by restriction enzyme digestion and sequencing,the plasmid was transformed into E.coli BL21(DE3)to construct the recombinant expression strain BL21(pET30a-HuNRP1b).After low temperature induction with 0.6 mM/L IPTG,the expression of the fusion protein His-HuNRP16 was analyzed by SDS-PAGE.Soluble recombinant proteins were prepared through denaturation,refolding and purification steps.The result of SDS-PAGE analysis showed that His-HuNRP1b was successfully expressed and purified at a concentration of approximately 3.0 mg/ml.2.Preparation of anti-HuNRP1b domain monoclonal antibody and analysis of its biological characteristics8 wk Balb/c mice were given abdominal subcutaneous injectionwith the purified protein His-HuNRP1b obtained above,100 ?g/each,toal three times injection with a 2 wk interval.For the first immunization,His-HuNRPlb was mixed with an equal volume of complete Freund's adjuvant.After emulsification,multiple injections were performed subcutaneously on the abdomen of the mice.The adjuvant was replaced with incomplete Freund's adjuvant for the second immunization with the same antigen at the same dosage.1 week later,blood was collected from the eyes of the subaxillary veins of the immunized mice and the serum titer was measured.The mice with high ELISA titer were chosen and immunized.For booster immunization,tail intravenous administration was performed with His-HuNRPlb without any adjuvant.3 days later,the spleens of immunized mice were removed to prepare single cell suspensions and fused with sp2/0-Ag-14 myeloma cells.The positive hybridoma cell lines were screened by ELISA and subcloned 2-3 times to obtain the stable monoclonal cell lines,and ascitic fluid contain McAbs were prepared by intraperitoneal injection in mice.The biological characteristics of McAbs were studied using Western-Blot,indirect immune-fluorescence(IFA),cell immunochemistry(ICC),and immunohistochemistry(IHC)assays.In this study,4 McAbs:1H4,2G7,3H4,and 8G9 were obtained Their ELISA titers were 1:256 000,1:64 000,1:256 000,1:128 000,and the subclasses were IgG2b,IgG1,IgG2b,IgG2b,respectively.The result of Western-Blot identification showed that McAbs:1H4,2G7,3H4,8G9 could only boundthe fusion protein HuNRP16 expressed in BL21(pET30a-HuNRP16)at band of 36 kDa but not protein expressed in BL21(pET30a),showing specific bands.The McAbs can bind to breast cancer cell lines:MCF-7,BT474,SK-BR-3,MDA-MB-231 that highly express native HuNRP1 and have specific bands at a position of about 120 kDa.It indicates that McAbs generized here not only recognizes recombinant HuNRP1 b domain protein but also the full-length HuNRP1 protein.IFA results indicate that McAb:1H4,2G7,3H4 and 8G9 can bind HuNRP1 expressed in human umbilical vein endothelial cells(HUVEC),human breast cancer cell lines(MDA-MB-231,MCF-7)and human leukemia cell line(k 562)and bright green fluorescence was observed.ICC results showed that when McAbs(1H4,2G7,3H4 and 8G9)acted on HUVEC cells and MDA-MB-231 cells,strongbrown signals were observed in the cell membrane and cytoplasm indicating that McAbs obtained here could recongnized native HuNRP1 and may had the obitity to bind and inhibit cancer cells proliferation and tumor development.IHC assay revealed that McAbs(1H4,2G7,3H4,and 8G9)could distinguish the HuNRP1 expression level on breast cancer(6/9),lung cancer(3/7),esophageal cancer(3/3)and kidney cancer(3/3)which were revealed by commercial antibody against recombinant HuNRPl at the same degree,showing that McAbs obtained can specifically recognize HuNRP1 in tumor cells and tissues.3.Anti-angiogenic activity of monoclonal antibodies against HuNRPl b domainTranswell chamber migration experiments were used to analyze the effect of McAbs in inhibiting the migration of HU VEC cells.10 ng/ml VEGF165 was added to the lower chamber and McAb:1H4,2G7,3H4 and 8G9 was added to the upper chamber respectively at a final concentration of 50 ng/well to prevent the cells from migrating downwards,the PBS group served as the control,each group had three replicates.The experimental results showed that McAbs(1H4,2G7 and 8G9)could inhibit HUVECs' migration at 15%inhibiting rate approximately compared with PBS group,McAb 3H4 showed no inhibitory effect.Tube formation of HUVEC cells on Matrigel in vitro assay was used to analyze if McAbs had the ability of inhibiting tube formation.after seeded,10 ng/ml VEGF165 and McAbs(1H4,2G7 and 8G9)with 50 ng was added to each well ifor inhibiting tubes' formation on Matrigel,PBS were used as control.The results showed that HUVEC cells in the PBS group could form a distinct tubular structure,while most cells in the McAbs group still adhered to the Matrigel surface and the inhibitory rate was about 10%.The effect of McAbs on inhibiting the development of necvascularization in tumors was analyzed using chicken chorioallantoic membrane(CAM)angiogenesis assay and Matrigel angiogenesis assay.Using the silica gel ring as a carrier,McAbs(1H4,2G7,3H4 and 8G9)were placed at a relatively low density of vascular development in CAM with the final concentration of 100 ng/each,dexamethasone group with a final concentration of 100 ?g/each was used as a positive control,and PBS with 100 ?l/each was used as a negative control.The results showed that the vascular development in McAbs 1H4,8G9 and dexamethasone groups was significantly inhibited,respectively.McAbs 2G7,3H4 had no significant inhibitory effect.Finally,Matrigel angiogenesis assay was used in SCID mice to study whether McAbs could inhibit the formation of blood vessels in animals.100 ?g of McAb 1H4,5×106 MCF-7 cells and 500 ?l of Matrigel were mixed and injected subcutaneously in the abdomen of SCID mice to form small bulges.After 6 days,the Matrigel bodies implanted in mice were harvested and pathological sections were made.Through HE staining and IHC experiments,it was found that McAb 1H4 could interfere with the attraction of vascular growth to tumor cells in the matrigel in vivo,thereby affecting the production of blood vessels in the implanted body.The above experimental results show that McAbs 1H4 obtained in this study has an inhibitory effect on the generation of tumor blood vessels.In summary,Four McAbs agianst recombinant HuNRP1b protein were successfully prepared by hybridoma technique,all McAbs were able to recognize the recombinant and natural HuNRP1.McAbs 1H4,2G7,and 8G9 have the ability to inhibit HUVEC migration and tube formation in vitro,McAbs 1H4 and 8G9 was picked out in inhibit chicken embryonic neovascularization and McAb 1H4 showed the strongest ability in inhibiting tumor angiogenesis among all the McAbs.The specific McAbs obtained in this study will provid important biological material for the study of the tumor angiogenesis and a potental drug for tumor therapy.