Breaking Barriers: Tight Junction-Directed Radiotheranostic Probes for Metastatic Colorectal Cancer
Ha Tran1, Xiaoxia Wen1, Ryan Coll1, Aiko Yamaguchi2, Nicholas Hornstein3, Tomoyuki Mashimo1, F. William Schuler1, Seok-Yong Lee1, John Paul Shen4, Scott Kopetz4, Charles Manning1,2.
1Department of Nuclear Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX
2Radiopharmaceuticals for Advanced Diagnostic Imaging and Therapy R&D Platform, Therapeutics Discovery Division, The University of Texas MD Anderson Cancer Center, Houston, TX
3Department of Hematology and Oncology, Northwell Health Cancer Institute, New York, NY
4Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX
Background: Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death1. Despite systemic therapy advances, 5-year survival for metastatic CRC (mCRC) remains below 15%2. Radiotheranostics offers a compelling strategy to address this unmet need. Tight junction proteins represent an emerging class of cancer targets3. While normally confined to cell-cell junctions in healthy epithelium, malignant transformation disrupts junctional architecture and epithelial polarity, leading to aberrant surface exposure and creating a unique opportunity for selective targeting4. Claudin-4 (CLDN4), a tight junction protein frequently overexpressed in CRC and enriched in mCRC, is particularly well suited for this approach. Here, we focus on the preclinical evaluation of CLDN4-directed radiotheranostic probes for mCRC to advance a clinically translatable tight junction–targeted radiotheranostic platform.
Methods: CLDN4 was identified as a candidate radiotheranostic target through analysis of clinical datasets from primary and metastatic CRC. A commercially available anti–CLDN4 monoclonal antibody (MAB4219) was conjugated with DFO or DOTA and radiolabeled with 89Zr, 177Lu, or 161Tb at high radiochemical purity and specific activity. For PET/CT imaging, athymic nude mice bearing subcutaneous HT29 (CLDN4+) tumors were injected with [89Zr]Zr-DFO-MAB4219 or an isotype control, followed by serial PET/CT imaging and ex vivo biodistribution. Tumor autoradiography was performed to assess intratumoral distribution. In vitro internalization was evaluated using fractionated uptake assays, and therapeutic potential was assessed using clonogenic survival assays following treatment with 161Tb- and 177Lu-labeled antibodies.
Results: PET imaging with [89Zr]Zr-DFO-MAB4219 demonstrated clear and specific tumor visualization, with sustained tumor retention and improved tumor-to-background contrast. Biodistribution analysis at 48 and 144 h post-injection (p.i.) showed that tumor uptake was higher at 48 h p.i., while liver, blood, lung, and heart exhibited moderate accumulation at this time point. By 144 h, tumor uptake remained high, whereas non-target tissues largely cleared, except for the spleen. Two-way ANOVA revealed a significant conjugate × organ interaction (F₁₃,₅₆ = 3.24, p = 0.0011), and Šidák-corrected post hoc comparisons confirmed significantly higher tumor uptake of [89Zr]Zr-DFO-MAB4219 versus isotype control (mean difference = 8.10 %ID/g, 95% CI = 5.31–10.89, p < 0.0001), demonstrating specific tumor targeting. Autoradiography showed heterogeneous intratumoral localization consistent with antibody penetration. In vitro studies confirmed efficient uptake and internalization of CLDN4-targeted radioconjugates. Both [161Tb]Tb- and [177Lu]Lu-DOTA-MAB4219 induced dose-dependent clonogenic cell kill in CLDN4+ CRC cells, with [161Tb]Tb showing enhanced cytotoxicity at lower activities, consistent with high-linear energy transfer (LET) β⁻ emission and short-range Auger electron effects, maximizing DNA double-strand break induction within targeted cells.
Conclusion: In a disease characterized by marked molecular heterogeneity and limited durable treatment options, CLDN4 represents a biologically accessible and consistently expressed target for radiotheranostic development in mCRC. CLDN4-directed antibodies can be successfully radiolabeled to enable tumor-specific imaging and targeted radiotherapy, demonstrating favorable PET performance, efficient internalization, and potent radiotoxicity in preclinical models. These findings support tight junction-directed radiotheranostics as a promising strategy to overcome target variability in CRC and provide a strong foundation for advancing CLDN4-targeted agents toward therapeutic evaluation and clinical translation.
References
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