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

This work describes the development and characterization of two antibody fragments that specifically target the α(v)β(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 α(v)β(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [(18)F]-FB-α(v)β(6) diabody, [(18)F]-FB-α(v)β(6) cys-diabody, and the [(64)Cu]-NOTA-α(v)β(6) cys-diabody. Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-α(v)β(6) intact antibody. The anti-α(v(β(6) 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 α(v)β(6). The diabodies were radiolabeled with [(18)F]-SFB and in addition, the anti-α(v)β(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [(64)Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ(6) (α(v)β(6)+) and DX3Puro (α(v)β(6)-)cell lines. The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC(50)=0.8 nM, cys-diabody IC(50)=0.6 nM) and flow cytometry revealed high specificity only to the DX3Puroβ(6) cell line for both diabodies. RCYs were 22.6%±3.6% for the [(18)F]-FB-α(v)β(6) diabody, 8.3%±1.7% for the [(18)F]-FB-α(v)β(6) cys-diabody and 43.5%±5.5% for the [(64)Cu]-NOTA-α(v)β(6) cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([(18)F]-FB-α(v)β(6) diabody=58.7%±6.7%, [(18)F]-FB-α(v)β(6) cys-diabody=80.4%±4.4%, [(64)Cu]-NOTA-α(v)β(6) cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. Two novel diabodies with excellent binding affinity and specificity for the α(v)β(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([(18)F]) and [(64)Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.