225Ac-alpha pretargeted radioimmunotherapy (DOTA-PRIT) of human colorectal cancer: achieving 100% complete responses with single-cycle targeting of 2 distinct antigens, HER2 and GPA33.

291 Objectives: We recently reported a novel anti-tumor/anti-DOTA hapten bispecific pretargeted radioimmunotherapy (DOTA-PRIT) of the theranostic isotope pair 111In/225Ac, documenting selective targeting of 111In- or 225Ac-radiolabeled proteus-DOTA hapten ([111In]PrDOTA or [225Ac]PrDOTA; to SW1222 colon cancer xenografts in mice, J Nucl Med 2018 59:123). Herein we report the therapeutic effectiveness and safety of targeting of 2 distinct anti-tumor antigens GPA33 and HER2 expressed on SW1222 tumor cells. Methods: We conducted studies in nude mice bearing subcutaneous (s.c.) SW1222 human colon carcinoma xenografts (5 x 106 cells/mouse; treatment initiated on day 11 post-inoculation) with appropriate controls (n = 10/group). The GPA33 and HER2 antigen levels determined by FACS for SW1222 were MFI = 673 and MFI = 125, respectively (negative control MFI = 5). The DOTA-PRIT targeting regimens were administered intravenously (i.v.): bifunctional antibody 0.25 mg (1.19 nmol) of anti-huA33-C825 or anti-HER2-C825 at t = -28 hours (h), 25 µg of CCA16-DOTAY clearing agent (2.76 nmol) at t = -4 h, and [225Ac]PrDOTA (0.74-0.79 nmol, 1 µCi (37 kBq)) at t = 0 h. Efficacy of single-cycle treatment was then compared to two-cycle treatment (total administered [225Ac]PrDOTA: 2 µCi (74 kBq)), separated by 1 week, of anti-GPA33-DOTA-PRIT only. Median survival (MS; in days (d) after treatment initiation), was defined as time to tumor progression to a diameter of 15 mm or death for any reason. Statistical comparison by log-rank (Mantel-Cox) test was used to test for significance (P Results: All treatments were well tolerated, with noweight loss >10% observed during treatment. When groups of SW1222-tumor bearing mice were treated with single- or two-cycle anti-GPA33-DOTA-PRIT (0.74 nmol for cycle 1, 0.79 nmol for cycle 2; 1 µCi (37 kBq)), MS (evaluated at 66 d post-treatment initiation) was not yet reached for either treatment groups compared with MS of 20.5 d for no treatment. The MS for the group treated with one cycle of anti-HER2-DOTA-PRIT was 64 d (P = 0.0010 versus no treatment). Furthermore, all mice treated with anti-GPA33 or anti-HER2-DOTA-PRIT showed complete response (CR; tumor volume ≤10 mm3), with tumor escape observed for a majority of the animals treated with one cycle only. For single-cycle anti-HER2-DOTA-PRIT, 5/10 were still alive at 66 d, with tumor volumes of 0 mm3 (i.e., no palpable tumor), 100 mm3, 123 mm3, 197 mm3, and 501 mm3. In comparison for single-cycle anti-GPA33-DOTA-PRIT, 7/10 were still alive at 66 d, with tumor volumes of 12 mm3, 29 mm3, 40 mm3, 42 mm3, 207 mm3, 221 mm3, and 798 mm3. All mice (10/10) treated with two cycles of anti-GPA33-DOTA-PRIT were still alive at 66 d, with 9/10 CRs and a single animal with a tumor volume of ~25 mm3. Conclusions: Alpha-DOTA-PRIT targeting the GPA33 or HER2 antigens is well tolerated and effective for treatment of SW1222 colorectal cancer model. Two cycles of anti-GPA33-DOTA-PRIT was potentially curative in most animals. For single-cycle treatment with either anti-HER2- or anti-GPA33-DOTA-PRIT, treatment effect as determined by MS was significantly correlated with antigen-receptor expression. Although ~5-fold difference in GPA33 and HER2 antigen levels for SW1222 observed by FACS, treatment with anti-HER2-DOTA-PRIT had substantial efficacy when compared to treatment with anti-GPA33-DOTA-PRIT, suggesting that factors other than receptor expression may play a role in tumor absorbed radiation dose. Additional studies are underway to extend cycle number for HER2 targeting regimens, as well. Analysis of survivors for tumor status and chronic toxicity (immunohistopathology of kidney and bone marrow) at 20 weeks, is pending.