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Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26–27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic “cure”) at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42–52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

Supplementary Data

Summary of immunogenicity experiment.

2-step SADA-PRIT imaging by PET/CT

2-step IgG-PRIT imaging by PET/CT (no CA)

summary of toxicity data from 225Ac SADA-PRIT experiment against a neuroblastoma PDX

Supplementary Data (Tables and Figure legends)

Purpose:Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index.Experimental Design:The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses.Results:SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver.Conclusions:The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377

3-step IgG-PRIT imaging by PET/CT

summary of toxicity data from 177Lu SADA-PRIT experiment against a neuroblastoma cell line

Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26–27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic “cure”) at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42–52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

Pretargeted imaging and radioimmunotherapy approaches are designed to have superior targeting properties over directly targeted antibodies but impose more complex pharmacology, which hinders efforts to optimize the ligands prior to human applications. Human embryonic kidney 293T cells expressing the humanized single-chain variable fragment (scFv) C825 (huC825) with high-affinity for DOTA-haptens (293T-huC825) in a transmembrane-anchored format eliminated the requirement to use other pretargeting reagents and provided a simplified, accelerated assay of radiohapten capture while offering normalized cell surface expression of the molecular target of interest. Using binding assays, ex vivo biodistribution, and in vivo imaging, we demonstrated that radiohaptens based on benzyl-DOTA and a second generation “Proteus” DOTA-platform effectively and specifically engaged membrane-bound huC825, achieving favorable tumor-to-normal tissue uptake ratios in mice. Furthermore, [(86)Y]Y-DOTA-Bn predicted absorbed dose to critical organs with reasonable accuracy for both [(177)Lu]Lu-DOTA-Bn and [(225)Ac]Ac-Pr, which highlights the benefit of a dosimetry-based treatment approach.

Purpose: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. Experimental Design: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. Results: SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. Conclusions: The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose. See related commentary by Capala and Kunos, p. 377

Objectives Epithelial ovarian carcinoma (EOC) is a common and lethal gynecologic malignancy that frequently presents as advanced staged disease, such as peritoneal carcinomatosis (PC). We previously reported cures in BT-474 murine xenografts with Pretargeted Radioimmunotherapy (PRIT) using 177Lu radiohaptens targeting Her2 via a bispecific antibody (BsAb). Here-in we report the use of PRIT with alpha emitter 225Ac labelled PrDOTA to treat a PC model of SKOV3, a Her2+ cell line of EOC. Methods 6 wk old female athymic nude mice inoculated IP with 1E5 luciferase/GFP-transfected SKOV3 cells were separated into 5 groups (n=10). Treatment mice received 1 or 2 cycles of Anti-Her2-C825 BsAb + [225Ac]PrDOTA (Her2-Targeted), at 14 and 21 days after inoculation, respectively. Control groups received Anti-Her2 BsAb only, Anti-GPA33 BsAb + [225Ac]PrDOTA (Off-Targeted) or no treatment. On cycle day 1, the mice were injected IP with 0.25mg (1.19nmol) BsAb. On cycle day 2, 25µg (2.76nmol) CCA16-DOTAY clearing agent (CA) was given IV 22h from BsAb. Mice in therapy groups were injected IP with 1µCi (0.74-0.79nmol) [225Ac]PrDOTA-Bn 4h after CA. Weekly weights and BLIs with IP cavity ROIs were obtained and normalized to the respective values for each mouse at week 0 pre-treatment. End points: weight loss >20% baseline, moribund, or severe abdominal distension. At 154 days, 15 surviving treatment and 1 untreated control mice were submitted for hematology and histopathology. Results Histologic cures and prolonged survival were demonstrated in treatment mice (17/20 at 133 days) as compared to control mice (12/27 at 133 d, Logrank p0.05; all weeks). BLI of mice treated with 1 and 2 cycle of Her2 PRIT decreased 47% when compared to baseline within 1 week (T test p=0.04), suggesting treatment effects as early as 1 week. There were no differences in weights when compared to baseline (2-way ANOVA p>0.05). While the untreated mouse had high peritoneal adenocarcinoma tumor burden, there was no histologic evidence of viable neoplasia in 15/15 submitted treatment mice. Treatment mice had moderate renal tubular degenerative lesions on histology, but this did not affect renal function based on serum BUN or Cr. All hematologic parameters were within normal limits for treated mice. Conclusions 1 and 2 cycles of [225Ac]PrDOTA-PRIT against Her2 resulted in histologic cures and prolonged survival in IP SKOV3 xenografts with minimal toxicity. The anti-Her2 PrDOTA-PRIT system is a promising theranostic approach for otherwise incurable PC. Citation Format: Sebastian K. Chung, Christopher S. Chandler, Daniela Burnes Vargas, Shin H. Seo, Michael R. McDevitt, Darren Veach, Blesida Punzalan, Xu Hong, Hong-fen Guo, Garrett M. Nash, Andrea Cercek, Nai-Kong V. Cheung, Steven M. Larson, Sarah M. Cheal. Pretargeted radioimmunotherapy using 225Ac for intraperitoneal Her2-expressing epithelial ovarian carcinoma xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P168.

Objectives: There is increasing interest in the potential role of radioimmunotherapy in the treatment of peritoneal carcinomatosis (PC), locoregional metastasis throughout the peritoneal cavity. We recently described a SADA platform when applied to GD2-specific PRIT using beta and alpha emitting radioisotopes achieved exceptional therapeutic indices (TI) achieving tumor cures without myelo-, renal-, neuro-, or hepatic toxicities (1). We applied SADA technology to a tumor associated antigen GPA33 expressed by colorectal cancer in a PC model, and compared that to a 3 step PRIT which requires a clearing agent (CA), previously reported to produce durable responses including histologic cures of GPA33-expressing SW1222 subcutaneous (2) and intraperitoneal (ip) colorectal xenograft models (3). Methods: SADA BsAb built from a humanized anti-GPA33 single chain Fv (scFv) and a humanized anti-DOTA scFv were expressed using the Expi293 Expression System and purified by affinity chromatography (1). Biochemical purity of >90% was confirmed via size exclusion HPLC and antigen binding confirmed by surface plasmon resonance and by flow cytometry on GPA33(+) cell lines. 8 wk old female athymic nude mice were inoculated ip with 5E6 cells of stably luciferase/GFP-transfected SW1222 in 200 µL media. PC tumor progression was monitored by in vivo BLI. GPA33-SADA and control GD2-SADA were injected intravenously (iv) on day 26, followed 48 hr later with 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip. In the 3 step PRIT group, mice were treated with 250µg (1.19nmol) GPA33-BsAb (n=4) followed 24 hr later by 20 µg (2.21 nmol) of CA iv, before the injection of 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip 4 hr later. In an additional control group, no SADA was given before 1 mCi (37 MBq,200 pmol) [177Lu]Lu-DOTA-Bn ip. Biodistribution was performed 24 hr after injection of ip 177Lu radiohapten. Results: Radioactivity absorbed in harvested ip SW1222 xenograft tumors treated with SADA-GPA33 (4.92 + 1.59 [mean + SD] percentage injected dose per gram [%ID/g]) was greater than that of harvested tumors treated with the traditional 3 step PRIT to GPA33 (2.68 + 0.179 %ID/g, p=0.043) or with negative control SADA-GD2 (0.513 + 0.293 %ID/g, p Conclusions: SADA-PRIT targeting GPA33 has favorable TI for colorectal PC. When compared to 3 step PRIT, SADA showed more selectivity and TI. {{References}}: 1. Santich BH, Cheal SM, Ahmed M, et al. Clin Cancer Res. 2020; 2. Cheal SM, Xu H, Guo HF, et al. Eur J Nucl Med Mol Imaging. 2016;43(5):925-937.3. Chandler C, Cheal SM, Nash G, et al. Journal of Nuclear Medicine. 2020;61. Citation Format: Sebastian Chung, Mao Wang, Darren Veach, Sarah Cheal, Daniela Burnes Vargas, Shin Hyuk Seo, Blesida Punzalan, Brian Santich, Hong-fen Guo, Hong Xu, Garrett Nash, Andrea Cercek, Christopher Chandler, Steven Larson, Nai-Kong Cheung. Self-assembling and disassembling bispecific antibody platform for pretargeted radioimmunotherapy against GPA33 in a xenograft model of colorectal peritoneal carcinomatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1391.