Influence of Peptide-Based Chelator Sequences on HER2-Targeting Radiopeptide

In our pursuit of developing HER2-targeting radiolabeled peptides for detection and therapy of HER2-positive breast cancer, the previously reported rL-A9 peptide had shown promising potential. In the present work, a different variant of rL-A9 was designed by incorporation of peptide-based chelators for ^99mTc-labeling. Cysteine-based amino acids (CGGG, CSSG, CEEG) were conjugated at the N-terminus of the peptide and were radiolabeled with [^99mTc]NaTcO₄. Of the three radiopeptides, [^99mTc]Tc-CGGG-rL-A9 exhibited high stability, whereas [^99mTc]Tc-CSSG-rL-A9 and [^99mTc]Tc-CEEG-rL-A9 decomposed rapidly. Hence, only CGGG-incorporated radiopeptide was further investigated in vitro in human breast carcinoma SKBR3 cells and in vivo in tumor xenografted mice. Cell-binding studies demonstrated high binding affinity [K_d: 5.82 ± 0.92 nM] and specificity (63% reduction in blocking study) of [^99mTc]Tc-CGGG-rL-A9 toward HER2-expressing cells. In vivo studies showed moderate tumor uptake (3.56 ± 0.81% ID/g, 1 h) with 82.8% retention at 3 h (3.16 ± 0.22% ID/g). Moderately high tumor-to-background ratios, along with washout from the other non-target organs, were observed. Inhibition studies revealed 52.6% reduction in the tumor activity, signifying high HER2-receptor specificity of the presently studied radiopeptide, [^99mTc]Tc-CGGG-rL-A9.