GPA33-pretargeted radioimmunotherapy with mono- and bivalent DOTA-based Lu-177-labeled radiohaptens in a mouse orthotopic liver xenograft model of metastatic human colorectal cancer

<b>Rationale:</b> Pretargeted radioimmunotherapy (PRIT), which combines systemic antibody-based targeting with ionizing radiation, is promising for treating liver metastases in patients with colorectal cancer (CRC). Previously, we established a three-step DOTA-PRIT regimen to deliver DOTA radiometal payloads to CRC using an anti-tumor/anti-DOTA bispecific antibody (BsAb) targeting cell surface glycoprotein A33 (GPA33), a tumor antigen target expressed on over 95% of primary and metastatic CRC; a clearing agent; and a monovalent ¹⁷⁷Lu radiohapten called [¹⁷⁷Lu]Lu-ABD. More recently, we developed a bivalent ¹⁷⁷Lu radiohapten called [¹⁷⁷Lu]Lu-Gemini to enhance tumor uptake and radiohapten retention. Here, we aimed to compare the efficacy and safety of bivalent vs. monovalent three-step DOTA-PRIT regimens in orthotopic CRC liver metastasis models, to mimic a clinical path forward. <b>Methods:</b> We established two orthotopic CRC liver metastasis models by inoculating either SW1222-luc (GPA33^high) or LoVo (GPA33^low) human CRC cells in athymic nude mice under ultrasonographic guidance. Tumor targeting efficacy and dosimetry of the radiohaptens were compared using <i>ex vivo</i> biodistribution studies, SPECT/CT, and quantitative autoradiography. We also performed a DOTA-PRIT experiment to compare the efficacy and safety profiles of bivalent (single-cycle [¹⁷⁷Lu]Lu-Gemini, 48 h pretargeting interval) vs. monovalent (multicycle [¹⁷⁷Lu]Lu-ABD, 24 h pretargeting interval) three-step DOTA-PRIT regimens, each designed to deliver comparable total radiation doses to tumors (around 50 Gy). <b>Results:</b> Both radiohaptens demonstrated efficient SW1222-luc tumor targeting, with [¹⁷⁷Lu]Lu-Gemini showing superior targeting and tumor activity retention compared with [¹⁷⁷Lu]Lu-ABD. In LoVo tumors, [¹⁷⁷Lu]Lu-Gemini showed superior targeting, while [¹⁷⁷Lu]Lu-ABD showed negligible targeting. Dosimetry estimates revealed higher SW1222-luc tumor mean absorbed doses for [¹⁷⁷Lu]Lu-Gemini (119.88 cGy/MBq, 48 h pretargeting interval) compared with [¹⁷⁷Lu]Lu-ABD (32.88 cGy/MBq, 24 h pretargeting interval), with more favorable blood and kidney therapeutic indices (50 and 9 for [¹⁷⁷Lu]Lu-Gemini, and 15 and 5 for [¹⁷⁷Lu]Lu-ABD, respectively). In the DOTA-PRIT experiment, both monovalent (injected activity: 3 × 44.4 MBq, 133.2 MBq total) and bivalent (injected activity: 44.4 MBq total) radiohapten regimens increased the median survival of treated mice compared with controls: 71 days for [¹⁷⁷Lu]Lu-ABD-treated mice, 81 days for [¹⁷⁷Lu]Lu-Gemini-treated mice, and 18 days for controls, without a statistical difference between treatment groups. Treatments were well tolerated, without significant weight loss or hematologic changes. Radiation-induced injuries were not identified histologically in the kidneys or bone marrow of mice submitted for necropsy. <b>Conclusions:</b> Our study demonstrates the exceptional benefit of a multivalent radiohapten strategy when treating an advanced model of CRC liver metastasis. Three-step GPA33 DOTA-PRIT with ¹⁷⁷Lu-Gemini demonstrated that multivalency 1) improves PRIT therapeutic indices for blood and kidney and 2) has the potential to greatly reduce the administered activity without compromising the efficiency of the PRIT platform in clinically relevant models of target-rich and target-poor metastatic CRC.