Development and Characterization of Annexin V Mutants with Endogenous Chelation Sites for <SUP>99m</SUP>Tc

[^99mTc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for ^99mTc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl<INF>2</INF> as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with ^99mTc to specific activities of at least 50−100 μCi/μg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (K<INF>d</INF>) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25−100 μCi/μg were injected intravenously in mice at a dose of 100 μg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [^99mTc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for ^99mTc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.