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

Introduction: This work describes the development and characterization of two antibody fragments that specifically target the alpha v beta(6) 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 alpha v beta 6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[F-18]fluorobenzoate ([F-18]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([Cu-64]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [F-18]-FB-alpha v beta(6) diabody, [F-18]-FB-alpha v beta(6) cys-diabody, and the [Cu-64]-NOTA-alpha v beta(6) cys-diabody. Methods: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-alpha v beta(6) intact antibody. The anti-a,136 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 alpha v beta(6). The diabodies were radiolabeled with [F-18]-SFB and in addition, the anti-alpha v beta(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [Cu-64]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puro beta(6) (alpha v beta(6)+) and DX3Puro (alpha v beta(6)-) cell lines. Results: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards alpha v beta(6) was confirmed by ELISA (diabody IC50 = 0.8 nM, cys-diabody IC50 = 0.6 nM) and flow cytometry revealed high specificity only to the DX3Puro beta(6) cell line for both diabodies. RCYs were 22.6% +/- 3.6% for the [F-18]-FB-alpha v beta(6) diabody, 83% +/- 1.7% for the [F-18]-FB-alpha v beta(6) cys-diabody and 43.5% +/- 55% for the [Cu-64]-NOTA-alpha v beta(6) cysdiabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([F-18]-FB-alpha v beta(6) diabody = 58.7% +/- 6.7%, [F-18]-FB-alpha v beta(6) cys-diabody = 80.4% +/- 4.4%, [Cu-64]-NOTA-alpha v beta(6) cys-diabody = 59.4% +/- 0.6%) regardless of the radiolabeling method used. Conclusions: Two novel diabodies with excellent binding affinity and specificity for the alpha v beta(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([F-18]) and [Cu-64] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach. (C) 2015 Elsevier Inc. All rights reserved.