Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody

Abstract

IntroductionThis work describes the development and characterization of two antibody fragments that specifically target the αvβ6 integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound αvβ6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([64Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [18F]-FB-αvβ6 diabody, [18F]-FB-αvβ6 cys-diabody, and the [64Cu]-NOTA-αvβ6 cys-diabody. MethodsDiabodies were constructed from the variable domains of the humanized 6.3G9 anti-αvβ6 intact antibody. The anti-αvβ6 cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to αvβ6. The diabodies were radiolabeled with [18F]-SFB and in addition, the anti-αvβ6 cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [64Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ6 (αvβ6+) and DX3Puro (αvβ6−) cell lines. ResultsThe diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC50=0.8nM, cys-diabody IC50=0.6nM) and flow cytometry revealed high specificity only to the DX3Puroβ6 cell line for both diabodies. RCYs were 22.6%±3.6% for the [18F]-FB-αvβ6 diabody, 8.3%±1.7% for the [18F]-FB-αvβ6 cys-diabody and 43.5%±5.5% for the [64Cu]-NOTA-αvβ6 cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([18F]-FB-αvβ6 diabody=58.7%±6.7%, [18F]-FB-αvβ6 cys-diabody=80.4%±4.4%, [64Cu]-NOTA-αvβ6 cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. ConclusionsTwo novel diabodies with excellent binding affinity and specificity for the αvβ6 integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([18F]) and [64Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.