Pretargeted Radioimmunotherapy using ²²⁵Ac for Intraperitoneal GPA33-Expressing Colorectal Xenografts

Objectives: Peritoneal carcinomatosis (PC) occurs when tumor spreads to the peritoneal lining, as seen in end stage GI malignancies, such as colon cancer. PC is considered incurable and development of curative therapy is an ongoing need. We previously reported Pretargeted Radioimmunothearpy (PRIT) in mouse xenografts from GPA33-expressing human CRC SW1222, using IV ¹⁷⁷Lu radiohaptens targeting GPA33, a cell-surface glycoprotein on >95% of CRC^1,2, via a bispecific antibody (BsAb). In this work, we achieved cure in a multicycle flank xenograft model³ and prolonged survival in a single cycle IP model of PC.⁴ We attempt to improve outcomes in our IP PC model using a PRIT regimen using PrDOTA-PRIT labeled with the alpha emitter ²²⁵Ac and assess acute and chronic toxicity in a pilot study on low and high-volume disease.

Methods: 6 wk old female athymic nude mice were inoculated IP with 5E6 cells of a stably luciferase/GFP-transfected SW1222 in 200µL. Mice were screened with BLI on day 5 (low-volume, LV) and 19 (high-volume, HV) and included if BLI >1E5p/s/cm²/sr. LV Groups: 1 (n=4) and 2 (n=3) cycles [²²⁵Ac]PrDOTA (Non-Targeted), 1 (n=3) and 2 (n=4) cycles of HuA33-C825 BsAb,+ [²²⁵Ac]PrDOTA (A33-Targeted). HV Groups: 1 and 2 cycles Non-Targeted (n=4 each), 1 and 2 cycles A33-Targeted (n=4 each). Previous data for No Treatment and A33-BsAb was used for controls in both. Cycles were given 1 wk apart and started on days 6/13 (LV C1/2), 20/27 (HV C1/2). On cycle day 1, the mice were injected IP with 0.25 mg (1.19 nmol) BsAb. On cycle day 2, 25 µg (2.76 nmol) CCA16-DOTAY clearing agent (CA) was given IV 22 hrs from BsAb. Mice in therapy groups were injected IP with 1 µCi (0.74-.79 nmol) [²²⁵Ac]PrDOTA 4 hrs after CA. Weekly hematology, weights, and BLIs with ROIs of the IP cavity were performed. End points: weight loss was >20% baseline, moribund, or severe abdominal distension. A33-Targeted Median Survival was compared within LV and HV to all controls.

Results: In HV disease, prolonged survival was seen with 2 cycle regimen A33-targeted (MS 87 days, Log-Rank, Mantel Cox test p=0.044), but not for 1 cycle A33-targeted (MS 60 days, Log-Rank Mantel Cox Test, p=0.307) with no treatment MS of 44 days. By 103 days, all mice with had died from disease progression with no toxicity/weight loss resulting in death. In LV disease, prolonged survival was seen with 1 cycle A33-Targeted (MS 75 days, Log-Rank Mantel Cox test, p=0.004), but was not significant for 2 cycle A33-Targeted (MS 85.5 days A33-Targeted, Log-Rank Mantel Cox test, p=0.068) due to MS of Non-Targeted control of 75 days. At 113 days with LV disease, 2 C2 Non-Targeted, 2 C1 A33-Targeted, and 2 C2 A33-Targeted remain alive. 1 death in C2 Non-Targeted was due to weight loss (>20% baseline), all other group deaths were from disease progression. Mild Neutropenia occurred in C2 A33-Targeted L/HV groups on ptd 15, which resolved by next sampling. LV C2 A33-Targeted had mild neutropenia again on ptd 29, which also resolved on next sampling. All other hematological values were within normal limits. Acute blood loss anemia was noted from disease progression with tumor rupture. BLI was comparable at baseline in all groups (ANOVA, p=0.782 in LV; 0.621 in HV) with increasing signal in all groups till day 26 in early and 33 in late when HV C2 A33-Targeted, LV C1/2 A33-Targeted, and LV C2 Non-Targeted began to decline. BLI began increasing in HV C2 A33-Targeted, after day 61, and mice showed clinical signs of disease progression with euthanasia 30 days later.

Conclusions: [²²⁵Ac]PrDOTA-PRIT against GPA33 for IP SW1222 resulted in significantly prolonged survival after 2 cycles in high volume and 1 cycle in low volume disease, suggesting greater efficacy of alpha therapy in low volume disease. The anti-GPA33 PrDOTA-PRIT system using ²²⁵Ac is a promising theranostic treatment approach for otherwise incurable colorectal peritoneal carcinomatosis, particularly for early stage disease.

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