Construction of Bispecific T-Cell Engager Radiotracer and Its Micro-PET Evaluation in Pancreatic Cancer.

Abstract

Mucin 17 (MUC17), a transmembrane mucin, is overexpressed in pancreatic cancer and is associated with tumor proliferation and metastasis. CD3 is an indispensable molecule on the surface of T lymphocytes, which is associated with T cell activation and participates in immune responses. Here, we developed a bispecific T-cell engager radiotracer, ⁸⁹Zr-M17C3, targeting MUC17 and CD3, to enable noninvasive PET imaging of both tumor cells and T-cell infiltration in pancreatic cancer. ⁸⁹Zr-M17C3 was synthesized by conjugating AMG199 with zirconium-89 and verified for its radiochemical purity and in vitro stability. The ⁸⁹Zr-M17C3 probe demonstrated excellent radiochemical purity (>99%) and stability (maintained ≥99% over 120 h). Cellular uptake assays and binding affinity studies were conducted to evaluate the probe's specificity for MUC17 and CD3. Micro-PET/CT imaging and biodistribution studies were performed in MUC17-expressing nude mice and CD3 humanized mice to assess probe uptake in tumors and T-cell-infiltrated tissues. In MUC17-expressing AsPC-1 tumors, probe uptake was significantly higher than in MUC17-negative PANC-1 tumors (SUVmax: 2.26 ± 0.18 vs 1.13 ± 0.14, P < 0.001) and was confirmed to be MUC17-dependent through blocking studies. In CD3 humanized mice, the probe was able to visualize both T-cell infiltration and MUC17-positive tumors, with peak uptake in AsPC-1 tumors (SUVmax: 2.35 ± 0.46) and spleen (SUVmax: 2.19 ± 0.40) at 216 h. Immunohistochemical analysis confirmed the spatial correlation between MUC17 expression and CD3-positive T-cell infiltration in AsPC-1 tumors but not in PANC-1 tumors. In summary, the ⁸⁹Zr-M17C3 radiotracer exhibited high affinity for MUC17 and CD3 and successfully differentiated MUC17-positive tumors from MUC17-negative tumors while simultaneously providing insight into the T-cell distribution. This study highlights the potential of ⁸⁹Zr-M17C3 as a versatile imaging tool to support patient stratification and therapeutic monitoring in tumor-targeted immunotherapy, particularly for bispecific T-cell engager-based approaches such as AMG199.