Development and characterization of annexin V mutants with endogenous chelation sites for Tc-99m

[Tc-99m]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the prepara tion and labeling of annexin V far nuclear-medicine studies, me have invest igated the addition of peptide sequences that will directly form endogenous chelation sites for Tc-99m. Three mutant molecules of annexin V, called an nexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These mo lecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis i n a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl2 as reducing agent and glucoheptonate as exchange agent, all th ree proteins could be labeled with Tc-99m to specific activities of at leas t 50-100 mu Ci/mug. The proteins retained membrane binding activity after t he radiolabeling procedure, and quantitative analysis indicated a dissociat ion constant (K-d) Of 7 nmol/L for the annexin V-117 mutant. The labeling r eaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered s aline or serum in vitro. Proteins labeled to a specific activity of 25-100 mu Ci/mug were injected intravenously in mice at a dose of 100 mug/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with pr evious results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [Tc-99m]annexin V-117. In conclusion , annexin V can be modified near its N-terminus to incorporate sequences th at form specific chelation sites for Tc-99m without altering its high affin ity for cell membranes. These annexin V derivatives may be useful for in vi vo imaging of cell death.