Preparation And Identification Of Anti-CD47 Nanobodies

CD47, also known as integrin-associated protein(IAP), is a broadly expressed membrane protein. As a ligand of signal regulatory protein alpha(SIRPα) which expressed on the surface of macrophages and myeloid cells, engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis. In recently years, CD47 has been found to be overexpressed on multiple human tumor types including leukemia, lymphoma and almost all solid tumors. The critical role of CD47 on the surface of resistant cancer cells has been proposed in their evasion of immunosurveillance by suppressing the phagocytosis of macrophages on tumor cells. Blocking of CD47- SIRPα signaling pathway by antibody or antagonist might provide an antitumor effect by restoring of immune cell function.Esophageal cancer(ECa) is one of the most frequent cancers with high incidence in Xinjiang. ECa is resistant to conventional chemotherapy and has a poor prognosis. The expression of CD47 on ECa was not determined. Moreover, the efficacy of anti-CD47 Ab against ECa was not investigated.Nanobodies(Nbs) are camel derived single domain heavy chain antibody fragments which naturally devoid of light chain. It is the available smallest antibody fragment with intact activity of antigen binding. Owing to single domain property of Nbs, there are several advantages over heterotetrameric mAb. Such as relatively non-immunogenic, soluble, stable, and highly tissue penetrable and binding inaccessible epitopes. These features make Nbs a promising candidates of next generation therapeutic molecules.One of the objective of this work is evaluating the value of CD47 as the therapeutic target of ECa by determining the efficacy of anti-CD47 mAb-mediated phagocytosis with macrophages against ECa. The other is selection and generation of anti-CD47 nanobodies via phage display and biopanning techniques. It will provide a novel therapy strategy for treatment of ECa by anti-CD47 Nbs.There are two main part research contents in the thesis: 1. The effects of monoclonal antibody B6H12 on macrophage mediated phagocytosis against esophageal cancer in vitro.Firstly, the expression levels of CD47 on ECa cells were determined by RT-PCR, western-blot, Flow cytometry and cell immunofluorescence microscopy. Then, the anti-tumor effect of mAb B6H12 on ECa was detected by in vitro mice macrophages phagocytosis assay. The ECa cells were fluorescently labelled with a carboxyfluorescein succinimidyl ester(CFSE). The proliferation and apoptosis of ECa cells were assayed by CCK-8 and Annexin V-FITC in FCM respectively. The results of RT-PCR, western-blot, Flow cytometry and cell immunofluorescence microscopy showed all tested ECa cells expressed CD47 on cell surface, in which, CD47 was high expressed on EC9706 and KYSE150 cells, and low expressed on Eca-109 cells. MTT assay showed that the proliferation of ECa cells were significance inhibited by anti-CD47 Ab compared with the IgG1 isotype control(p < 0.05). After incubation of murine macrophages with CFSE-labelled EC9706 and KYSE150 cells in the presence of IgG1 isotype control or CD47 mAb B6H12, the B6H12 mAb showed increased the phagocytosis of ECa cells by mouse macrophages significantly compared with treatment with an IgG1 isotype control(p< 0.01). The Index of phagocytosis reaches 0.2. These results suggest CD47 overexpressed on the surface of ECa cells. Anti-CD47 Ab might have anti- tumor effect by promoting phagocytosis of ECa cells with macrophage. 2. Identification and generation of anti-CD47 nanobodies by phage display techniquesFirstly, the CD47 extracellular domain(ECD) recombinant protein was prepared by DNA recombinant techniques. The ECD coding sequence of CD47 was recovered by PCR cloning from a plasmid containing CD47 full cDNA. After codon optimization, the gene fragment in pET30 a was expressed and purified with immobilized metal affinity chromatography(IMAC). The expressed recombinant CD47 extracellular domain proteins(rCD47-ECD) were identified by anti-CD47 mAb in western-blot and further were used to immunize a bactrian camels for constructing immunized library in future. Subsequently, the nanobodies were generated by phage display techniques. The PBLs collected from 5 camels were used as starting materials for construction VHH library. The VHH gene fragments were PCR amplified and ligated into pMECS vector by VHH-specific primers and PstI/NotI digestion. The na?ve VHH library was generated by electrotransformation of compentent E.coli TGI with pMECS-VHH ligated product. The functional size and quality of library were estimated by independently transformed clones, insert ratio(%), and diversity and quality of the VHH sequences. For screening,the phage display of VHH library was rescued by super-infection of transformed TG1 with M13KO7 helper phage and subsequent proliferation and purification with PEG8000. The CD47-specific binders were recovered by three round consecutive biopanning on CD47 coated 96 well microtiter plates. The progress of selection against immobilized CD47 was monitored by performing an ELISA using polyclonal phage from each round of panning as well as with the unselected library phage. The output of enriched phage clones containing correct VHH inserts were detected by colony PCR and confirmed with DNA sequencing. The expected VHH coding sequences were subcloned into the pET30 a expression vector. Recombinant nanobodies were expressed in BL21(DE3) strain by IPTG induction and purified with IMAC. The expressed nanobodies were identified by anti-myc mAb in ELISA assay for detection of their binding with CD47 antigen.The recombinant CD47-ECD gene fragments with 351 bp expected size in pET30 a vector were was induced with 0.2mM IPTG and expressed in BL21(DE3) cells. The purification with Ni-ion affinity yields about 90% purified recombinant CD47, which was bound with anti-CD47 mAb(B6H12). It was raised a high titer anti-CD47 humoral response(2.0×105) in CD47 antigen immunized camel, and provided the materials for constructing immune VHH library in future.The 1.4×109 cfu sized naive VHH phage display library was constructed from 5 camels. The randomly selected 91 library TG1 clones represent 85 individual different VHH clones indicated a high quality of library. By three round consecutive biopanning, 6.0×104cfu output phage were recovered. The enrithment of CD47 specific phages was 10-fold in detection with polyconal phage ELISA. The 38 clones randomly selected from 6.0×104cfu output phage showed all were positive clones in colony PCR and DNA sequencing. The three positive clones, VHH-CD47-201、VHH-CD47-204 and VHH-CD47-802 were selected and expressed in E.coli BL21 cells and showed 25 kDa、28 kDa and 20 kDa bands in SDS-PAGE respectively. The Nbs with about 90% purity were purified with IMAC. The results of ELISA with the three Nbs showed VHH-CD47-201 have CD47-binding activity, in contrast, the other two Nbs have no CD47-binding activity.The current research evidenced that CD47 was over-expressed on the surface of ECa cells, and anti-CD47 Ab may promote the phagocytosis of ECa cells(EC9706 and KYSE150) by macrophage in vitro. Moreover, taking advantage of phage display technique, the CD47 binding nanobodies were generated from a na?ve VHH library. The anti-tumor effects of the Nbs on ECa will be further identified in the future.