Development of New Peptide-based Chelating Agents for Site-specific Radiolabeling with Cu-64

Radiolabeled monoclonal antibodies (mAbs) are very useful molecular probes for nuclear imaging technique due to their high-target specificity and high-stability in the bloodstream. MAbs are generally labeled by indirect method using the combination of relatively long-lived metallic radionuclides (including Cu-64, Zr-89, and In-111) and bifunctional chelating agents which are capable of binding both antibodies and radio metals. The indirect radiolabeling method has some advantages such as high labeling efficiency and long-term retention ability within their target cells. However, this conventional labeling method can potentially lead to low-target affinity of the mAb probes, because of the non-site-specific introduction of the bifunctional chelators into the active site of the mAbs. To overcome the shortcoming, we proposed a new direct labeling method utilizing fusion proteins comprising mAbs linked to metal binding peptides at the N- or C-terminus. In this study, we synthesized new peptide derivatives possessing an N-terminal tripeptide sequence (Xaa-Yaa-His) called the amino terminal Cu2+- and Ni2+-binding (ATCUN) motif as Cu-64 binding peptides for the proposed labeling method. Moreover, we studied the stability constants of Cu2+-ATCUN peptide complexes by pH titration. From these studies, we found that a low basicity of the N-terminal amine in the peptide resulted in a high stability constant of the complex. This finding may provide valuable guidelines in designing the ATCUN peptide with high-binding affinity toward Cu-64.