| G protein-coupled receptors (GPCRs) represent the largest family of cell-surface proteins mediating signal transmission. These receptors play a pivotal role in many physiological functions and their dysfunctions are involved in multiple human diseases. Although receptor activities of GPCRs are traditionally modulated by various small molecules, which represent 30% of all marketed drugs. In fact, only a small fraction of the GPCR members are targeted, and many more GPCRs of interest are intractable targets of small molecules, like orphan GPCRs and GPCRs with large ligand-binding sites. Besides, small molecule drugs rarely trigger GPCR-mediated apoptosis or induce direct cell killing, which is crucial for cancer therapies. Therefore, for these GPCRs, antibody-based drug would be a good alternative.;Despite being eagerly sought for, the production of monoclonal antibody (mAb) targeting GPCR has been hindered by the low expression level, relatively short exposed regions and conformational heterogeneity of GPCRs. Therefore, well established conventional methods for the generation of mAbs targeting soluble proteins usually showed frustrating results in the case of GPCRs. To bypass the production and purification of recombinant GPCR proteins, in this study, we developed a novel technology by transplanting epitopes of GPCR into an antibody scaffold to make a water soluble surrogate antigen of GPCR, named as GPCRantigenized antibody which can be easily and abundantly produced in bacteria for animal immunization. In couple with phage display technology, which allow the retrieval of specific binders from a huge number of candidates, we successfully isolated scFv antibodies for two human GPCRs, MAS receptor and chemokine receptor CXCR4. These antibodies showed good specificity in immunofluorescent staining, western immunoblot and immunoprecipitation assay. Moreover, after conversion into Fab antibodies, clone F1 and E1 showed improved binding capability on MAS and CXCR4, respectively. Clone Fab-E1 also displayed antagonistic activity against CXCR4. The results demonstrated antigenized antibody design could be used as a novel platform technology for developing anti-GPCR antibodies. |
