부산대학교 도서관

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [.sup.99m Tc(CO).sub.3 (OH.sub.2).sub.3 ].sup.+). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d.sup.+ red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d.sup.+, in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K.sub.D [almost equal to]500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [.sup.99m Tc(CO).sub.3 (OH.sub.2).sub.3 ].sup.+ at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d.sup.+ RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d.sup.+ RBCs. We conclude that rCR2-Tc.sup.99m has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.
Author(s): Adam Badar 1 , 2 , Sarah DeFreitas 1 , James M. McDonnell 3 , Norhakim Yahya 3 , David Thakor 2 , Reza Razavi 2 , Richard Smith [...]