Screening Of Alpaca Nanobodies Targeting PDGFR-β

PDGFR-β is mainly expressed in interstitial cells,which promotes cell growth,differentiation and migration under normal physiological conditions.However,overactivated PDGFR-β usually induces tumor growth and metastasis,as well as organ fibrosis.Antibody have high affinity and good specificity,so antibody-based therapy represents an attractive way to treat tumors.Compared with traditional antibodies,nanobodies are smaller in size,so they can penetrate into tumor tissues and cross the blood-brain barrier more easily.Additionally,nanobodies are more stable,and are easier for modification.Therefore,development of nanobodies targeting human PDGFR-β for treatment of associated carcinomas is necessary.In this study,a truncated human PDGFR-β protein was expressed using mammalian cells and purified through affinity chromatography.After 4 immunization rounds,the peripheral blood of the alpaca was collected and the peripheral blood mononuclear cells(PBMCs)inside were isolated.The total RNA was extracted and reverse-transcribed into cDNA,which was used as template to amplify the variable region of heavy-chain antibody(VHH).The amplified VHH was ligated into phagemid vector using Gibson assembly method and then electroporated into E.coli TG1 strain.Finally,phage particles displaying VHH were obtained from the library using KM 13 helper phage.The final diversity of the constructed library reached 7.25×10~6 pfu.After two rounds of panning using the purified PDGFR-β protein to specifically enrich binding phage,followed by monoclonal phage ELISA to identify positive single clones and sequencing,we obtained seven different anti-PDGFR-β nanobodies,named as 6A,6C,9H,11C,E4,G6 and E2,respectively.Nanobody and nanobody-human IgG1 Fc fusion protein were expressed using mammalian cells and purified using affinity chromatography.Next,the binding properties of the selected nanobodies were characterized.First,size-exclusion chromatography(SEC)showed that all nanobodies but not 6A could form complexes with PDGFR-β in solution.ELISA showed 6C-Fc,9H-Fc,11C-Fc,E4-Fc,G6-Fc and E2-Fc tightly bound to PDGFR-β with EC50 values of 0.06891 nM,0.05787 nM,0.1008 nM,0.04438 nM,0.4098 nM and 0.2444 nM,respectively.The binding affinity of the six nanobodies to PDGFR-β was also measured using Surface Plasmon Resonance(SPR),and the results showed that five of them including 6C,9H,11C,E4 and G6 tightly bound to PDGFR-β with equilibrium dissociation constants(KD)of 0.2295,32.52,8.349,14.19 and 26.18 nM,respectively,while E2 weakly bound to PDGFR-β with KD of 130.4 nM.Next,we tested the binding ability of the five high-affinity nanobodies to the full-length PDGFR-β using flow cytometry,and showed that all the five nanobodies could bind to the full-length PDGFR-β protein expressed on the surface of HEK 293T cells,indicating their potential to bind to native PDGFR-β,SPR was further used to detect the competition relationship between the five high-affinity nanobodies for binding to PDGFR-β.The results showed that only 6C and E4 competed with each other.Finally,competitive ELISA showed that 11C,G6 and E4 could inhibit the binding between PDGF-BB-Fc and PDGFR-β.In summary,in this study 5 alpaca nanobodies that bind PDGFR-β with relatively high affinity were produced,and all of them can interact with the full-length PDGFRβ expressed on the cell surface.6C and E4 can compete with each other when binding to PDGFR-β.11C,G6 and E4 can inhibit the binding of PDGF-BB-Fc to PDGFR-β.This study lays foundation for the development of nanobody-based therapy against PDGFR-β-associated carcinomas.