Covalent versus Dimeric Optimization of Nectin-4-Targeted Peptide Radiotracers: A Head-to-Head Comparison in Triple-Negative Breast Cancer Mice Models

Nectin-4 is highly expressed in several malignancies, including triple-negative breast cancer, and it represents an attractive target for molecular imaging and therapy. In this study, we report the first head-to-head comparison of two structural optimization strategies for Nectin-4-targeted peptide radiotracers: peptide dimerization (for the development of ⁶⁸Ga-DOTA-HTA-DM) and sulfonyl fluoride modification (for the development of ⁶⁸Ga-DOTA-HTA-SF). The dimeric peptide DOTA-HTA-DM achieved a pronounced affinity gain (SPR apparent <i>K</i>_D ≈ 0.37 nM), consistent with the bivalent binding mode predicted by docking and molecular dynamics simulations. In contrast to the lead compound N188, the sulfonyl fluoride-modified peptide DOTA-HTA-SF retained low-nanomolar affinity, and the ⁶⁸Ga radiolabeled probe exhibited markedly higher tumor uptake at early time postinjection (∼5% ID/g at 30 min) and sustained tumor retention (>4% ID/g at 2 h), resulting in superior tumor-to-background contrast. Both radiotracers were predominantly cleared through the renal system, and blocking studies confirmed their Nectin-4-mediated tumor accumulation. Taken together, these findings demonstrate that dimerization enhances molecular recognition through multivalency, while sulfonyl fluoride modification prolongs tumor residence and improves imaging contrast. The complementary advantages of these two strategies establish a rational framework for the design of next-generation Nectin-4-targeted radiotracers.