Screening Of An Anti-FGF-2 Nanobody And Its Antineoplastic Activity On Melanoma In Vivo And In Vitro

Objective:Antibodies are important targeted drugs for tumor antigens.It has been a new trend for antibody development to investigate therapeutic antibodies with new epitopes by using nanobody strategy.As a hotspot for anti-tumor therapy at present,the anti-FGF-2 monoclonal antibodies are still unavailable on the market.It is suggested that the development of anti-FGF-2 antibody is of far-reaching significance.This research,focusing on HUVECs,CAM assay,melanoma eells and tumor-bearing mice,intends to exploit anti-FGF-2 nanobody candidates with anti-angiogenesis and anti-tumor activity in vivo and in vitro.Methods and Results:A phage library of anti-FGF-2 nanobody with a storage capacity of 1×10¹⁴ pfu/m L was constructed by immuning llama.After three rounds of panning and screening with phage-ELISA,six anti-FGF-2 nanobodie were acquired,which were mainly different on the amino sequence.The sequence of Nb _FGF-2 were inserted into the expression vector of p NCS.After expressed in E.coli and purified by Ni-NTA chromatography,the purity of Nb _FGF-2 were confirmed to be 90%?95%by RP-HPLC.Based on the bioinformatics analysis,it was found that the molecular weights of Nb _FGF-2 were between 17.5 k Da and 18.6 k Da;Their theoretical isoelectric points were below 7,suggesting that they were negatively charged in the physiological environment;As the GRAVY of Nb _FGF-2 were negative,all of them were hydrophilic protein.Besides,it was proved that Nb _FGF-2 had high target specificity,high affinity?n M?and high thermal stability by ELISA method.Furthmore,the binding epitope of Nb1,Nb2,Nb3,Nb5 and Nb6 with FGF-2 were close or similar to FGF-2 m Ab's;and the binding epitope of Nb1,Nb3,Nb5 and Nb6 with FGF-2 were close or similar to FGFR2's.In the study of anti-angiogenesis,Nb1?Nb2?Nb3?Nb5 and Nb6 could inhibit the proliferation of HUVECs in a dose-dependent manner,and the highest inhibition rates were between 29%and 43%.In the CAM assay,Nb _FGF-2 could significantly reduce the vessel area of chicken chofioallantoie membrane?**p<0.01?with a inhibition rate of 52.7%when the concentration of Nb6 was 4 nmol/L.In the melanoma cells,it was verified by CCK-8 method that Nb1,Nb2,Nb3,Nb5 and Nb6 could hinder the growth of B16 cells?25%?46%?,B16-F10 cells?31%?43%?and A375 cells?24%?36%?,respectively.The nanobody of Nb6,which was the best candidate,could obviously curb the growth and migration of melanoma cells in a dose-dependent manner?**p<0.01?.Furthermore,it was found that Nb6 could lower the phosphorylation level of Erk1/2 and Akt at 0.5 h in a dose-dependent manner by Western Blotting assay,which resulted in the inhibition of cell proliferation and migration.In the melanoma model of intermediate stage cancer,the inhibition rate of the wet weight of tumor was 40.52%when mice were administrated with Nb6 of 20 mg/kg.It was certified by HE staining that the nuclear were fusioned in tumor after treated with Nb6.And the immunohistochemical assay confirmed that Nb6 could significantly reduce the expression of FGF-2 and decrease the microvessel density in tumor?**p<0.01?.It was proved by Western Blotting method that Nb6 could lower the expression of p-Erk1/2 and Bcl-2 and increase the expression of caspase-9.In the melanoma model of early-stage cancer,the inhibition rate was 72.24%?**p<0.01?,which was higher than the results in the intermediate stage cancer with the same dose of Nb6.However,the tumor were reduced83.17%?**p<0.01?when they were treated with Nb6 and cisplatin.And there was no obviously difference between the results came from the mice treated with Nb6 alone.Conclusion:In this study,a phage library of anti-FGF-2 nanobody was constructed by immuning llama with FGF-2.Six different nanobodies,which had low molecular weight,high target specificity,high affinity?n M?and high thermal stability,were screened from the phage library.In vivo and vitro assay,Nb _FGF-2 not only could inhibite the cell proliferation in HUVECs and angiogenesis in chicken chofioallantoie membrane,but also could hinder the growth and migration of melanoma cells.Compared the results of early-stage cancer with intermediate stage cancer in mice with melanoma cells,it was found that Nb6 were more suitable for administration in early-stage cancer.Besides,there was no synergistic anti-tumor activity when Nb6 was administrated with cisplatin.