title: Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement.
abstract: 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 [(99m)Tc(CO)(3)(OH(2))(3)](+)). 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(+) 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(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) 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(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.
authors.affiliation: Medical Research Council Centre for Transplantation, King's College London, Guy's Hospital, London, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Badar
authors.forename: Sarah
authors.initials: S
authors.lastname: DeFreitas
authors.forename: James M
authors.initials: JM
authors.lastname: McDonnell
authors.forename: Norhakim
authors.initials: N
authors.lastname: Yahya
authors.forename: David
authors.initials: D
authors.lastname: Thakor
authors.forename: Reza
authors.initials: R
authors.lastname: Razavi
authors.forename: Richard
authors.initials: R
authors.lastname: Smith
authors.forename: Steven
authors.initials: S
authors.lastname: Sacks
authors.forename: Gregory E D
authors.initials: GE
authors.lastname: Mullen
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003167:Complement Activation / Q000276:immunology*; D015933:Complement C3d / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D055767:Immobilized Proteins; D007553:Isotope Labeling / Q000379:methods*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D055550:Protein Stability; D011877:Radionuclide Imaging / Q000379:methods*; D019275:Radiopharmaceuticals; D051381:Rats; D017464:Receptors, Complement 3d / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*; D012756:Sheep; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D013667:Technetium; D017556:Technetium Compounds
chemical_list: D055767:Immobilized Proteins; D019275:Radiopharmaceuticals; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins; D017556:Technetium Compounds; C543202:triaquatricarbonyltechnetium(I); D013667:Technetium; D015933:Complement C3d