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.

Colorectal cancer is one of the most common malignancies in the United States and has the potential to metastasize locoregionally within the peritoneum as well as extraperitoneally. Approximately 10%–20% of cases present with intraperitoneal metastasis, and many more develop future recurrence within the peritoneal cavity (1, 2). The presence of intraperitoneal dissemination, or peritoneal carcinomatosis, is a condition marked by the accumulation of mucinous ascites, and portends a poor clinical outcome with high symptom burden, recurrent bowel obstructions, and frequent hospitalizations (3–5). Median survival (MS) with metastatic intraperitoneal colorectal cancer is approximately 5–12 months with 5-year overall survival of about 20% (1, 4, 5).

Pretargeted radioimmunotherapy (RIT) offers parenteral administration of therapeutic radiation with highly specific antitumor antibodies, making it well suited for treatment of peritoneal carcinomatosis (6). However, for treatment of solid tumors, RIT often suffers from poor therapeutic index (TI) due to prolonged circulation and slow clearance of non–tumor-bound radioantibody, leading to delivery of insufficient absorbed radiation dose to tumor within tolerable administered activities, especially to blood (marrow; ref. 6). To overcome this RIT limitation, a common strategy is to “pretarget” the tumor with non-radioactive antibody, followed with separate administration of the radioactive payload (7, 8). Various approaches to PRIT have been reported for treatment of peritoneal carcinomatosis, going back to early PRIT with streptavidin/radiobiotin (9–13) and bispecific antibody/radiohapten (14), and more recently with biorthogonal click chemistry (15). Notably, PRIT has also has been studied for decades clinically, with Schoffelen and colleagues showing highly promising improvements in TI with TF2 bispecific antibody and 111In/177Lu-IMP288 for theranostic treatment of carcinoembryonic antigen (CEA)-expressing tumor (16).

We have recently described a PRIT innovation for improved TI based on pretargeting the tumor with bispecific antitumor antigen/anti-DOTA antibody (BsAb), followed by injection of a clearing agent (CA), and finally, a small molecule DOTA-radiohapten (DOTA-pretargeted RIT; DOTA-PRIT). For targeting cell-surface glycoprotein A33 antigen [GPA33 antigen, expressed on >95% of colorectal cancer (17, 18)], a BsAb was produced using the sequences for the humanized anti-GPA33 mAb huA33 and murine anti-DOTA single-chain (scFv) antibody C825 in the tetravalent IgG-scFv format [molecular weight (MW): 210 kDa] (19). C825 shows metal-DOTA specificity, with ultrahigh affinity to complexes of S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn; 655.4 Da) to isotopes of lutetium (10.8 ± 2.5 pmol/L affinity) and yttrium (15.4 ± 2.0 pmol/L affinity; ref. 20). The CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units and is designed to rapidly complex circulating non–tumor-bound BsAb and direct it to hepatic Ashwell receptors for enhanced clearance, leading to significant reductions of BsAb in blood within 1 hour post injection of CA (21). The radiohapten is captured by tumor localized BsAb or is otherwise quickly and efficiently cleared renally (22), forming the basis for high-TI therapy. With intravenous injection of GPA33 DOTA-PRIT + [177Lu]LuDOTA-Bn [i.e., DOTA-Bn radiolabeled with the theranostic beta (β)- and gamma (γ)-emitting radioisotope Lutetium-177 (177Lu)], we were able to deliver >100 Gy to established subcutaneous flank SW1222 human colorectal cancer xenografts in nude mice without detectable radiotoxicity (23).

The primary objective of our study is to demonstrate that intraperitoneal DOTA-PRIT [intraperitoneal anti-GPA33/anti-DOTA BsAb, followed by intravenous CA (given consideration of biological mechanism of CA), and intraperitoneal lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten] can be administered with sufficient TI to safely achieve complete responses and even cures. Herein we employ an aggressive SW1222-luciferase reporter peritoneal carcinomatosis nude mouse model to test the hypothesis that intraperitoneal GPA33 DOTA-PRIT using [177Lu]LuDOTA-Bn is a promising treatment modality for colorectal peritoneal carcinomatosis. Furthermore, we examine the feasibility of quantitative PET imaging of peritoneal carcinomatosis with intraperitoneal GPA33 DOTA-PRIT using DOTA-Bn radiolabeled with the positron (β+)-emitting radioisotope yttrium-86 (86Y) ([86Y]DOTA-Bn) as a diagnostic surrogate for staging and treatment monitoring.

DOTA-PRIT reagents

The BsAb and CA were prepared according to reported methods (19, 21). DOTA-Bn was obtained commercially (Macrocyclics) and used without any additional purification.

Peritoneal carcinomatosis mouse models

The GPA33-expressing human colorectal cancer cell line SW1222 was obtained from the Ludwig Institute for Cancer Immunotherapy (New York, NY). Upon receipt of the cell line, cultures were established with two passages and cryopreserved in small aliquots. No cell line authentication was performed. The luciferase-labeled tumor cell line SW1222-luc was generated by retroviral infection of the SW1222 cell line with a SFG-GFLuc vector (24), confirmed to be negative for Mycoplasma using a commercial kit in April 2018 (Lonza), and cryopreserved in small aliquots. For all experiments with SW1222-luc cells, passages were limited to ≤4. Female athymic nude mice (strain: Hsd:Athymic Nude-Foxn1nu, Envigo; ages 6–8 weeks; average weight for 6-week-old and 8-week-old animals: 18.6 and 21.0 g, respectively) were used for all experiments. To establish a peritoneal carcinomatosis model of colorectal cancer, mice were intraperitoneally inoculated with 5 × 106 SW1222-luc cells suspended in 200 μL medium, using a sterile syringe with a 28-gauge needle. Experiments were initiated 19–27 days post injection (d.p.i.) as noted. Peritoneal carcinomatosis tumor progression and survival were monitored by in vivo bioluminescence imaging (BLI) using the IVIS Spectrum In Vivo Imaging System (see Supplementary Materials and Methods for additional details). All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (MSKCC; New York, NY) following NIH guidelines for animal welfare.

IHC

For histology of SW1222 intraperitoneal tumor, excised tumor tissue was formalin fixed and embedded in paraffin. Sections of 5-μm thickness were cut and stained with hematoxylin and eosin (H&E) by the Molecular Cytology Core or Laboratory of Comparative Pathology at MSKCC (New York, NY). IHC staining for the GPA33 antigen was conducted using an anti-A33 mouse mAb (25), an anti-mouse secondary antibody, and the Leica Novolink Polymer Detection Kit. IHC staining for the nuclear antigen Ki67 was conducted by the MSKCC Molecular Cytology Core Facility or Laboratory of Comparative Pathology at MSKCC (New York, NY), and QuPath 0.2.1 was used for digital pathologic analysis (ref. 26; see Supplementary Materials and Methods for additional details).

Radiolabeling of BsAb and DOTA-bn

131I in the form of carrier-free Na131I was purchased from Nordion, 177Lu in the form of carrier-added 177LuCl3 was received from MU Research Reactor, and 86Y in the form of no-carrier-added 86YCl3 was received from the MD Anderson Cancer Center Cyclotron Radiochemistry Facility. Radioiodination of BsAb was performed as described previously (19) using the IODOGEN method to a final molar activity of approximately 22 MBq/nmol and radiochemical purity (RCP) >98% determined using radio-size-exclusion high-pressure liquid chromatography (HPLC). [177Lu]LuDOTA-Bn or [86Y]DOTA-Bn were prepared from DOTA-Bn (27) with RCP ≥ 99% determined using reverse-phase C18 radio-HPLC. Because the BsAb C825 scFv does not recognize metal-free DOTA-Bn (20), the molar activities of the corresponding radiohaptens [177Lu]LuDOTA-Bn and [86Y]DOTA-Bn (∼5.45E-3 pmol/kBq or ∼0.2 pmol/μCi) are described herein based on the specific activities of the as-received radionuclides (∼1110 GBq/mg or 30 Ci/mg for both isotopes).

Radiopharmacology of intraperitoneally administered 131I-BsAb and optimization of [177lu]LuDOTA-Bn dosing

The injection timing intervals and doses of BsAb (250 μg/1.19 nmol) and CA (25 μg/2.76 nmol) used in the current study were selected on the basis of previous anti-GPA33 DOTA-PRIT reports, showing high TI targeting of [177Lu]LuDOTA-Bn to subcutaneous flank SW1222 xenografts (19). BsAb and [177Lu]LuDOTA-Bn were administered intraperitoneally, and CA was administered intravenously [in consideration of the biological mechanism of action of the CA (i.e., active hepatocyte targeting)]. Preliminary studies to validate in vivo targeting of BsAb and [177Lu]LuDOTA-Bn to intraperitoneal SW1222 xenografts, assay the kinetics of BsAb blood clearance following injection of CA, and evaluate the impact of administration route of [177Lu]LuDOTA-Bn on tumor uptake during pretargeting were performed (see Supplementary Materials and Methods for additional details).

Dosimetry

An intraperitoneal anti-GPA33 DOTA-PRIT treatment cycle consisted of the following: on cycle day 1, the mice were injected intraperitoneally BsAb. Approximately 22 hours later, on cycle day 2, CA was given intravenously, and after an additional 4 hours, injected intraperitoneally with [177Lu]LuDOTA-Bn (Fig. 1A). Time–activity curves were generated from serial biodistribution data from groups administered either 18.5 MBq (100 pmol, n = 18) or 37 MBq (200 pmol, n = 18) of pretargeted [177Lu]LuDOTA-Bn. At each timepoint (1, 2, 4, 24, 48, and 120 hours post injection), 3 mice from each group underwent biodistribution assay. All tumors within the intraperitoneal cavity was collected from the mouse and measured in aggregate. A total of 7/36 mice (19%) presumably had failed intraperitoneal injections based on high large intestine activity (>2%IA/g) or low blood and tumor uptake (both <0.2%IA/g) as seen during preliminary studies. These mice were excluded from analysis. For each tissue, the non–decay-corrected time–activity concentration data were fit using Excel to a one-component, two-component, or more complex exponential function as appropriate, and analytically integrated to yield the cumulated activity concentration per unit administered activity (MBq-hour/g per MBq). The 177Lu equilibrium dose constant for non-penetrating radiations (8.49 g-cGy/MBq-hour) was used to estimate the tumor-to-tumor and select organ-to-organ self-absorbed doses, assuming complete local absorption of the 177Lu beta rays only, and ignoring the gamma ray and non–self-dose contributions.

Figure 1.

Intraperitoneal DOTA-pretargeting of radiohapten [177Lu]LuDOTA-Bn for theranostic β-therapy of PC of colorectal origin. Schematic of pretargeting radioimmunotherapy approach to treat peritoneal carcinomatosis (A). Peritoneal SW1222 tumor nodules (B) ex vivo analysis day 20 post injection; tumor weight: 0.519 g. H&E stain (C) and IHC analysis of GPA33-expression and Ki67 of a peritoneal carcinomatosis tumor nodule collected at 58 d.p.i.. Scale bars on the left denote 2,000 μm, and scale bars on the right denote 100 μm.

Figure 1.

Intraperitoneal DOTA-pretargeting of radiohapten [177Lu]LuDOTA-Bn for theranostic β-therapy of PC of colorectal origin. Schematic of pretargeting radioimmunotherapy approach to treat peritoneal carcinomatosis (A). Peritoneal SW1222 tumor nodules (B) ex vivo analysis day 20 post injection; tumor weight: 0.519 g. H&E stain (C) and IHC analysis of GPA33-expression and Ki67 of a peritoneal carcinomatosis tumor nodule collected at 58 d.p.i.. Scale bars on the left denote 2,000 μm, and scale bars on the right denote 100 μm.

Close modal

Radiopharmaceutical therapy of peritoneal carcinomatosis

Peritoneal carcinomatosis tumor-bearing mice with BLI average radiance of >1 × 105 p/second/cm2/sr on d.p.i. 19–25 were assigned to groups such that the baseline BLI was not statistically different between the groups (vide infra). Mice in the survival arms reached therapeutic endpoints if they were noted to have >20% weight drop from pretreatment baseline, developed severe abdominal distension from palpable tumor or ascites, or appeared moribund by investigators or on daily monitoring by MSKCC Research Animal Resource Center staff. Weekly weights, hematology, and BLI were measured. Experimenters were not blinded to group assignment or outcome assessment.

For single-cycle treatment, injections were initiated on d.p.i. 26. Groups included: no treatment control (n = 12), BsAb only (n = 9), 111 MBq (600 pmol) [177Lu]LuDOTA-Bn only (n = 4), and anti-GPA33 DOTA-PRIT + 111 MBq (600 pmol) [177Lu]LuDOTA-Bn (n = 13). For multi-cycle treatment, a total of 40 tumor-bearing mice that met inclusion criteria on d.p.i. 19 underwent three treatment cycles with 1-week delay between cycles, starting on d.p.i. 27 (cycle 2 and cycle 3 start on d.p.i. 35 and 42, respectively), with follow-up until 183 d.p.i. (study endpoint). The groups were: no treatment control (n = 6), BsAb only (n = 6), 111 MBq (600 pmol) [177Lu]LuDOTA-Bn only (n = 8), and anti-GPA33 DOTA-PRIT + 111 MBq (600 pmol) [177Lu]LuDOTA-Bn (n = 14). To verify tumor targeting for each treatment cycle, 2 animals undergoing anti-GPA33 DOTA-PRIT were sacrificed for biodistribution assay four hours post injection of [177Lu]LuDOTA-Bn; therefore, n = 8 for longitudinal assessment.

PET-CT imaging of multi-cycle treated long-term survivors

Prior to the final necropsy at 183 d.p.i., a total of 7 animals from the multi-cycle therapy study (6 mice intraperitoneal anti-GPA33 DOTA-PRIT and 1 mouse in the intraperitoneal BsAb only group) underwent PET-CT imaging with intraperitoneal anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn (see Supplementary Materials and Methods for details). For comparison, two treatment-naïve tumor-bearing mice (15 d.p.i.) were administered either with intraperitoneal anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn or intraperitoneal [86Y]DOTA-Bn only and imaged with PET/CT.

Data reporting and statistical analysis

Quantitative data were expressed as mean ± SD unless otherwise noted. The sample sizes were selected taking into account both statistical consideration and the exigencies of funding. Statistical analysis was performed using GraphPad Prism 8.1.0. A P value of <0.05 was considered significant. For t tests, an unpaired two-tailed test was used unless otherwise noted.

Establishment of peritoneal carcinomatosis model of colorectal origin

Peritoneal carcinomatosis tumors (take rate from 5 × 106 cells injected on day 0 was approximately 83% based on BLI threshold of 1 × 105 p/second/cm2/r when measured at 24 d.p.i.) were found to be well vascularized and engrained into the connective tissues and nearby organs, and were mainly located around the stomach, spleen, pancreas, and liver, but they were also noted on occasion to be along the peritoneum at the implantation site, small and large intestine mesentery, diaphragm, ovaries, and intraperitoneal fat around the bladder. Untreated mice were noted to begin having abdominal distension, cachexia, and lethargy starting 27–38 d.p.i. Preliminary necropsy of tumor-bearing mice revealed the aggregate ex vivo tumor burden mass on 10, 15, and 20 d.p.i. to be: 0.251, 0.246, and 0.519 g, respectively (Fig. 1B). In a larger study (n = 17), the aggregate ex vivo tumor 29 d.p.i. was 0.369 ± 0.246 g and was found to be significantly correlated to BLI (Pearson r = 0.55, r2 = 0.31, n = 17, P = 0.021; Supplementary Fig. S1). As the peritoneal carcinomatosis progressed, abdominal ascites (mostly hemorrhagic in nature) were observed, resulting in euthanasia after 41–108 days, with final tumor burden ranging from 1.63–5.53 g (3.19 ± 1.41 g, n = 6). Assay of histopathology of harvested tumor at 58 d.p.i. showed glandular tumor formations, homogenous GPA33 expression, and high Ki67 staining (Fig. 1C).

Effect of CA on radiopharmacology of 131I-BsAb and optimization of [177Lu]LuDOTA-bn dosing

As shown in Supplementary Fig. S2A, intravenous administration of CA was effective at rapidly reducing circulating 131I-BsAb levels following diffusion from the peritoneal cavity. After injection of CA at 24 hours post injection of 131I-BsAb, the blood radioactivity rapidly dropped from 5.82 ± 0.81%IA/g to 1.39 ± 0.77%IA/g at 0.5 hour post injection of CA, corresponding to an average percent decrease of approximately 76% (P < 0.001; t test). A significant difference in blood radioactivity was observed between groups at 48 hours post injection (3.45 ± 0.87%IA/g vs. 0.92 ± 0.71%IA/g for no CA and with CA, respectively; P < 0.005; t test). Tumor uptake of 131I-BsAb ranged from 3.23–8.61%IA/g at 72 hours post injection, confirming efficient intraperitoneal tumor targeting (Supplementary Fig. S2B). In addition, low 131I-BsAb uptake was observed in other normal tissues, including blood (average 2.57%IA/g), liver, and spleen (both average <1%IA/g).

We also documented successful pretargeting of [177Lu]LuDOTA-Bn to peritoneal carcinomatosis, with biodistribution assay showing tumor uptake of 3.70 ± 1.56%IA/g, and low uptake in all other tissues, including blood (0.563 ± 0.297%IA/g) and kidney (0.438 ± 0.230%IA/g) at 72 hours post injection of [177Lu]LuDOTA-Bn (Supplementary Fig. S2C). When comparing route of injection of [177Lu]LuDOTA-Bn, slightly higher tumor uptake was observed with intraperitoneal than with intravenous dosing of [177Lu]LuDOTA-Bn; however, this was not statistically significant (P = 0.175; t test) (Supplementary Fig. S3). Finally, there were no significant differences in [177Lu]LuDOTA-Bn uptake among any another tissues (P = 0.202–0.870; t test), suggesting that similar tumor-to-normal tissue ratios could be achieved with either injection route.

Serial biodistribution of intraperitoneal anti-GPA33-DOTA-PRIT+ [177Lu]LuDOTA-Bn and dosimetry calculations

As shown in Fig. 2A, peak tumor uptake of 177Lu-radioactivity (16.26 ± 15.01%IA/g) was rapidly achieved within 1 hour post injection [177Lu]LuDOTA-Bn (37 MBq/200 pmol), and remained constant up to 4 hours post injection 14.25 ± 10.80%IA/g. However, at 24 hours post injection, the tumor 177Lu-radioactivity fell to approximately a third of peak value (5.77 ± 0.52%IA/g), and the activity further dropped off at 48 hours (3.33 ± 0.08%IA/g) and 120 hours (0.69 ± 0.65%IA/g). Except for large intestine uptake at 4 hours post injection (3.43 ± 4.04%IA/g), the highest 177Lu-radioactivity in non-tumor tissues was found at 1 hour post injection in kidney (1.12 ± 0.78%IA/g). Efficient clearance was noted, with 0.14 ± 0.07%IA/g in kidney and 0.01 ± 0.01%IA/g in large intestine at 120 hours post injection Similar biodistribution profiles were observed during pretargeting with a lower administered [177Lu]LuDOTA-Bn dose (18.5 MBq/100 pmol; Supplementary Fig. S4).

Figure 2.

Serial biodistribution and radiopharmaceutical therapy of peritoneal carcinomatosis with single-cycle intraperitoneal anti-GPA33 DOTA-PRIT. A, Biodistribution data in groups of mice bearing intraperitoneal SW1222 xenografts (n = 3/timepoint) undergoing GPA33 DOTA-PRIT + [177Lu]LuDOTA-Bn (37 MBq; 1 mCi; 200 pmol). B, Kaplan–Meier survival curves of single-cycle regimens (treatment was initiated at 26 days after tumor inoculation, vertical dotted line). Animals were euthanized if weight loss was >20% baseline or if moribund with severe abdominal distension. Survival data reflect the progression of intraperitoneal peritoneal carcinomatosis tumor (n = 4–13 per cohort). A significant difference in median survival was observed (all data; P = 0.0071; Gehan–Breslow). Log-rank P values for MS: no treatment (all data) versus BsAb only 0.9663; no treatment (all data) versus [177Lu]LuDOTA-Bn only 0.9340; no treatment (all data) versus single-cycle anti-GPA33 DOTA-PRIT 0.0562; no treatment (with exclusion of tumor-free survivor) versus single-cycle intraperitoneal anti-GPA33 DOTA-PRIT 0.0032. C, Growth of intraperitoenal SW1222 tumor in treatment group and control groups as assessed by bioluminescence. Data are presented as mean ± SEM. #Groups reached n = 1.

Figure 2.

Serial biodistribution and radiopharmaceutical therapy of peritoneal carcinomatosis with single-cycle intraperitoneal anti-GPA33 DOTA-PRIT. A, Biodistribution data in groups of mice bearing intraperitoneal SW1222 xenografts (n = 3/timepoint) undergoing GPA33 DOTA-PRIT + [177Lu]LuDOTA-Bn (37 MBq; 1 mCi; 200 pmol). B, Kaplan–Meier survival curves of single-cycle regimens (treatment was initiated at 26 days after tumor inoculation, vertical dotted line). Animals were euthanized if weight loss was >20% baseline or if moribund with severe abdominal distension. Survival data reflect the progression of intraperitoneal peritoneal carcinomatosis tumor (n = 4–13 per cohort). A significant difference in median survival was observed (all data; P = 0.0071; Gehan–Breslow). Log-rank P values for MS: no treatment (all data) versus BsAb only 0.9663; no treatment (all data) versus [177Lu]LuDOTA-Bn only 0.9340; no treatment (all data) versus single-cycle anti-GPA33 DOTA-PRIT 0.0562; no treatment (with exclusion of tumor-free survivor) versus single-cycle intraperitoneal anti-GPA33 DOTA-PRIT 0.0032. C, Growth of intraperitoenal SW1222 tumor in treatment group and control groups as assessed by bioluminescence. Data are presented as mean ± SEM. #Groups reached n = 1.

Close modal

Time activity curves for tumor and select tissues are shown in Supplementary Fig. S5. The estimated tumor absorbed dose for an intraperitoenal anti-GPA33 DOTA-PRIT treatment cycle was 34.81 cGy/MBq, with TIs for normal tissues ranging from 12 (kidney) to 118 (stomach). Mouse dosimetry estimates for each organ are presented in Supplementary Table S1. These dosimetry estimates differed from our previous experience with intravenous targeting of subcutaneous flank SW1222 xenografts, wherein the estimated tumor absorbed dose was approximately 7-fold higher (229 cGy/MBq) with TIs for normal tissues ranging from 35 (kidney) to 545 (stomach; ref. 19). A comparison of the time–activity curves for tumor, blood, and kidney for the two approaches are provided in Supplementary Fig. S6; the largest difference was seen for tumor. For intravenous targeting, peak tumor 177Lu-radioactivity was not reached until 4 hours post injection with a mean value of 26.87 ± 3.5%IA/g, and was extrapolated by curve fitting to fall to half the peak value by 120 hours post injection (19). The ratio of absorbed dose in tissue to absorbed dose in blood is similar for intraperitoneal and intravenous route across all tissues with the exception of tumor, and to a lesser extent, lung and large intestine, as seen in Supplementary Fig. S7. The intravenous route resulted in a nearly 3-fold higher ratio of absorbed dose in tumor to absorbed dose in blood than the intraperitoneal route.

Therapy studies

A summary of the therapy study outcomes is provided in Table 1. For single-cycle treatment (Fig. 2), BLI was comparable at baseline in all groups (P = 0.738; ANOVA). Peritoneal carcinomatosis was not eradicated as evidenced by stable and/or increasing BLI (for day 51 d.p.i. P = 0.210; ANOVA), and eventually the treated mice succumbed to progressive disease, but with prolonged survival compared with controls. The MS was 49.5 d.p.i. for no treatment control, 51 d.p.i. for intraperitoneal BsAb only, 51 d.p.i. for intraperitoneal [177Lu]LuDOTA-Bn only, and 84.5 d.p.i. for the single-cycle intraperitoneally anti-GPA33 DOTA-PRIT (P = 0.007; Gehan–Breslow). Notably, there were two survivors at 110 d.p.i. that were euthanized and grossly examined for peritoneal carcinomatosis: a single treated mouse with observable tumor, and unexpectedly, a control (from no treatment group) mouse with no observable tumor.

Table 1.

Summary of outcome of radiopharmaceutical treatment studies.

Treatment/Survival
Study endpoint in days d.p.i.MS in days d.p.i.DosimetryLong-term survivors
1 × anti-GPA33 DOTA-PRIT (total [177Lu]LuDOTA-Bn: 111 MBq/mouse)/110 days No treatment: 49.5 days Tumor: 50 Gy No treatment: 1/12 
 BsAb only: 51 days Blood: 310 cGy BsAb only: 0/9 
 [177Lu]LuDOTA-Bn only: 51 days Kidney: 327 cGy [177Lu]LuDOTA-Bn only: 0/4 
 anti-GPA33 DOTA-PRIT: 84.5 days  anti-GPA33 DOTA-PRIT: 1/13 
3 × anti-GPA33 DOTA-PRIT No treatment: 44 days Tumor: 150 Gy No treatment: 0/6 
 BsAb only: 52.5 days Blood: 929 cGy BsAb only: 1/6 
(total [177Lu]LuDOTA-Bn: 333 MBq/mouse)/183 days [177Lu]LuDOTA-Bn only: 42 days Kidney: 982 cGy [177Lu]LuDOTA-Bn only: 0/8 
 anti-GPA33 DOTA-PRIT: >183 days  anti-GPA33 DOTA-PRIT: 6/8 
Treatment/Survival
Study endpoint in days d.p.i.MS in days d.p.i.DosimetryLong-term survivors
1 × anti-GPA33 DOTA-PRIT (total [177Lu]LuDOTA-Bn: 111 MBq/mouse)/110 days No treatment: 49.5 days Tumor: 50 Gy No treatment: 1/12 
 BsAb only: 51 days Blood: 310 cGy BsAb only: 0/9 
 [177Lu]LuDOTA-Bn only: 51 days Kidney: 327 cGy [177Lu]LuDOTA-Bn only: 0/4 
 anti-GPA33 DOTA-PRIT: 84.5 days  anti-GPA33 DOTA-PRIT: 1/13 
3 × anti-GPA33 DOTA-PRIT No treatment: 44 days Tumor: 150 Gy No treatment: 0/6 
 BsAb only: 52.5 days Blood: 929 cGy BsAb only: 1/6 
(total [177Lu]LuDOTA-Bn: 333 MBq/mouse)/183 days [177Lu]LuDOTA-Bn only: 42 days Kidney: 982 cGy [177Lu]LuDOTA-Bn only: 0/8 
 anti-GPA33 DOTA-PRIT: >183 days  anti-GPA33 DOTA-PRIT: 6/8 

For multi-cycle treatment (Fig. 3), BLI was comparable at baseline in all groups (P = 0.984; ANOVA). Increasing BLI signal was observed in all groups until 40 d.p.i. (i.e., 2 days prior to cycle 3) at which point anti-GPA33 DOTA-PRIT signal began to decline. On 68 d.p.i., the anti-GPA33 DOTA-PRIT BLI was below baseline (68 d.p.i. mean BLI: 3.89 × 106 vs. corresponding baseline mean BLI: 4.42 × 106), while the two remaining controls continued to be elevated. Furthermore, for anti-GPA33 DOTA-PRIT treated animals, the BLI continued to decrease in all but 1 mouse, which began increasing on 68 d.p.i., suggesting tumor recurrence. A significant change from baseline BLI was observed starting on 117 d.p.i. (P = 0.046; two-tailed paired t test) anti-GPA33 DOTA-PRIT BLI after the single recurring mouse was removed from the study due to progressive disease. Final BLI following anti-GPA33 DOTA-PRIT was reduced by 4.38 × 106 from matched baseline in surviving mice (final mean BLI: 6.30×105; matched baseline BLI: 5.01 × 106; P = 0.015; two-tailed paired t test), corresponding to a percent decrease of 87%. At 183 days, 6/8 (75%) anti-GPA33 DOTA-PRIT were alive and with no apparent clinical disease compared with 1/20 (5%) cumulative control mice. The lone surviving control mouse (BsAb only group) was noted to have a large progressing abdominal wall tumor at the original tumor implantation site. The MS was 44 d.p.i. for no treatment, 52.5 d.p.i. for BsAb only, 42 d.p.i. for [177Lu]LuDOTA-Bn only, and not reached (MS >183 days follow-up) for the multi-cycle anti-GPA33 DOTA-PRIT (P < 0.001; Gehan–Breslow). The difference in MS was statistically significant for single- versus multi-cycle anti-GPA33 DOTA-PRIT (P = 0.016; log-rank Mantel Cox).

Figure 3.

Radiopharmaceutical therapy of peritoneal carcinomatosis with multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT. C1, C2, and C3 indicate cycle 1, cycle 2, and cycle 3, respectively. A, Kaplan–Meier survival curves of multi-cycle regimens with prior single-cycle intraperitoneal GPA33 DOTA-PRIT survival curve for comparison (green dotted line). Animals were euthanized if weight loss was >20% baseline or if moribund with severe abdominal distension. Survival data reflect the progression of intraperitoneal peritoneal carcinomatosis tumor (n = 6–13 per cohort). B, Bioluminescent images of abdominal SW1222 tumors from long-term survivors. Note: At day 166, the mouse treated with intraperitoneal BsAb only had a BLI of 1.20 × 108, and mice treated with multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT had a BLI of 6.30 × 105 ± 7.11 × 105. C, Growth of intraperitoneal SW1222 tumor in treatment group and control groups as assessed by bioluminescence. Data are presented as mean ± SEM. #Groups reached n = 1; +Recurrence in single treated mouse; −Euthanasia of treated mouse with recurrence. D, Individual BLI curves for multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT.

Figure 3.

Radiopharmaceutical therapy of peritoneal carcinomatosis with multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT. C1, C2, and C3 indicate cycle 1, cycle 2, and cycle 3, respectively. A, Kaplan–Meier survival curves of multi-cycle regimens with prior single-cycle intraperitoneal GPA33 DOTA-PRIT survival curve for comparison (green dotted line). Animals were euthanized if weight loss was >20% baseline or if moribund with severe abdominal distension. Survival data reflect the progression of intraperitoneal peritoneal carcinomatosis tumor (n = 6–13 per cohort). B, Bioluminescent images of abdominal SW1222 tumors from long-term survivors. Note: At day 166, the mouse treated with intraperitoneal BsAb only had a BLI of 1.20 × 108, and mice treated with multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT had a BLI of 6.30 × 105 ± 7.11 × 105. C, Growth of intraperitoneal SW1222 tumor in treatment group and control groups as assessed by bioluminescence. Data are presented as mean ± SEM. #Groups reached n = 1; +Recurrence in single treated mouse; −Euthanasia of treated mouse with recurrence. D, Individual BLI curves for multi-cycle intraperitoneal anti-GPA33 DOTA-PRIT.

Close modal

During multi-cycle treatment, efficient tumor targeting was verified for each cycle via biodistribution assay (Supplementary Fig. S8). Furthermore, the amount of [177Lu]LuDOTA-Bn administered did not significantly impact the %IA/g loaded onto the tumors or normal tissues (37 MBq/200 pmol vs. 111 MBq/600 pmol comparison of each individual tissue; P value range = 0.207–0.861; t test).

A summary of the gross and histologic descriptions of tumor observed at necropsy is presented in Supplementary Table S2. Representative histology and IHC is shown in Fig. 4. Residual tumors were limited to intra-abdominal nodules on peritoneal surface and there was no evidence of tumors within any organs. The BsAb only treated mouse had a large viable carcinoma in the abdominal wall at the site of original tumor implantation, but no viable carcinoma inside the peritoneal cavity, consistent with a misinjection of the initial tumor implantation, explaining the later than expected demise when compared with its experimental cohort. All mice treated with anti-GPA33 DOTA-PRIT except one (mouse 3) had small intra-abdominal peritoneal tumors at various sites that were composed of viable carcinoma cells in relatively small amounts as well as necrosis, fibrosis, calcifications, and chronic inflammation. For mouse 3, the small intra-abdominal peritoneal nodules were composed of these components, but without evidence of viable carcinoma cells. Ki67 IHC staining was used as a marker of proliferation in all viable carcinoma cells. The percentage of Ki67-positive cells in the single surviving BsAb only treated mouse was 47%, which was similar to that of the untreated control mice (range, 43%–51%), and much greater than that of treated mice numbers 4 (0.7%) and 7 (1%). GPA33 IHC staining also demonstrated persistent GPA33 expression in all mice except mouse 3.

Figure 4.

H&E and Ki67 and GPA33 staining of representative intraperitoneal tumors in no treatment control, BsAb only, and multi-cycle anti-GPA33 DOTA-PRIT (residual tumor and “microscopic cure” mice). Ki67 staining demonstrated less proliferation in mice in treated mice when compared with control or BsAb only mice. Viable tumor cells demonstrated persistent membranous GPA33 antigen expression available for continued treatment. Please note that there is significant nonspecific background staining of GPA33 due to the use of an anti-mouse secondary antibody, but membranous staining is specific to GPA33. Scale bars on left denote 2,000 μm and scale bars on right denote 50 μm.

Figure 4.

H&E and Ki67 and GPA33 staining of representative intraperitoneal tumors in no treatment control, BsAb only, and multi-cycle anti-GPA33 DOTA-PRIT (residual tumor and “microscopic cure” mice). Ki67 staining demonstrated less proliferation in mice in treated mice when compared with control or BsAb only mice. Viable tumor cells demonstrated persistent membranous GPA33 antigen expression available for continued treatment. Please note that there is significant nonspecific background staining of GPA33 due to the use of an anti-mouse secondary antibody, but membranous staining is specific to GPA33. Scale bars on left denote 2,000 μm and scale bars on right denote 50 μm.

Close modal

Toxicity

For single-cycle treatment, all treatments were well tolerated, with no significant weight loss requiring euthanasia (Supplementary Fig. S9). However, during hematologic monitoring of the mice (Supplementary Fig. S10), the mice that received anti-GPA33 DOTA-PRIT were found to have mild neutropenia with nadir of post-treatment day (p.t.d.) 21 (2.35 ± 0.71 K/μL vs. 5.47 ± 2.41 K/μL pretreatment baseline on day −2; P = 0.028, t test). By p.t.d. 28, the white blood cell (WBC) count had recovered to pretreatment baseline (3.49 ± 0.94 K/μL vs. 5.47 ± 2.41 K/μL pretreatment baseline on day −2; P = 0.144; t test). Other hematologic parameters were within normal limits and did not differ from controls. For multi-cycle treatment, all treatments were well tolerated, and no significant weight loss requiring euthanasia was documented in all but 3 animals [a single death in the anti-GPA33 DOTA-PRIT (44 d.p.i.) and two deaths in the [177Lu]LuDOTA-Bn only group (both at 42 d.p.i.)]. These 3 animals were noted to have abdominal distension, and tumor was found at necropsy. Animal weight data are provided in Supplementary Fig. S9B. The most significant change in weight loss from pretreatment baseline at 24 d.p.i. was observed at 40 d.p.i. (i.e., just prior to the third treatment cycle) in the [177Lu]LuDOTA-Bn only group (83.04 ± 5.91% vs. 91.34 ± 6.98% for no treatment; P = 0.033; t test). However, this was likely not treatment related as the anti-GPA33 DOTA-PRIT did not show any significant weight loss in comparison with no treatment (e.g., at 40 d.p.i.: 88.65 ± 6.51% vs. 91.34 ± 6.98% for no treatment; P = 0.471; t test). As shown in Supplementary Fig. S11A–S11D, hematologic monitoring of the mice revealed neutropenia seen in all therapy groups that received [177Lu]LuDOTA-Bn, including [177Lu]LuDOTA-Bn alone and anti-GPA33 DOTA-PRIT (nadir: p.t.d. 23; [177Lu]LuDOTA-Bn alone n = 2: 1.18 ± 0.40 K/μL; anti-GPA33 DOTA-PRIT n = 7: 0.54 ± 0.30 K/μL). WBC counts recovered to low normal by p.t.d. 30 (anti-GPA33 DOTA-PRIT n = 7: 1.55 ± 1.05 K/μL; reference range for age/strain: 1.42–10.25 K/μL). However, they remained lower than corresponding remaining controls not receiving 177Lu treatments (3 animals total; range: 9.02–11.16 K/μL). All other hematologic parameters in groups receiving 177Lu-therapy (Hct, RBC, Plt) showed minimal deviation from no treatment and BsAb only controls.

All long-term survivors (six treated with anti-GPA33 DOTA-PRIT and one treated only with BsAb) were submitted for final necropsy (Supplementary Table S2), complete blood count with automated differential (Supplementary Table S3), and complete metabolic panel (Supplementary Table S4) at 183 d.p.i. In summary, no treatment-related toxicity was observed except for one organ: all mice that received anti-GPA33 DOTA-PRIT showed marked ovarian atrophy (6/6, 100%; Supplementary Fig. S11E and S11F). Such ovarian atrophy is not expected in mice of this strain and age, and we have seen the same finding in some other DOTA-PRIT studies in mice (28). The ovarian atrophy is compatible with radiation injury (it is identical to the ovarian change we see in mice that received whole-body irradiation), and the mechanism of ovarian injury in these studies may have an off-target effect of the treatment (e.g., cross-fire effect from adjacent organs such as the kidneys or bladder). Notably, the kidney absorbed dose was relatively high (982 cGy). Other prominent findings in anti-GPA33 DOTA-PRIT treated mice included glomerulonephritis (4/6, 66.7%) and adrenocortical hyperplastic and hypertrophic changes (6/6, 100%). Glomerulonephritis is a well-known spontaneous lesion in nude mice of this strain and age (29, 30), and therefore it is not considered treatment related. Furthermore, biomarkers of renal function of multi-cycle treated mice at 183 d.p.i., including Cr (0.220 ± 0.017 mg/dL) and BUN (26.5 ± 3.39 mg/dL) were within normal limits (Supplementary Table S4). Adrenocortical hyperplastic and hypertrophic changes are not well-defined spontaneous lesions of nude mice, but can occur in aged mice of several strains. As such, we cannot definitely rule out that this was an effect of treatment. Finally, there was no evidence of significant changes or treatment-related effects on hematology and serum chemistry panel (Supplementary Tables S3 and S4).

PET-CT imaging of multi-cycle treated survivors

As shown in Fig. 5, PET-CT using anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn can be used to image peritoneal carcinomatosis at 15 d.p.i. with high contrast (Fig. 5A) observed at 18 hours post injection of [86Y]DOTA-Bn. Furthermore, in the corresponding negative control (PET/CT imaging at 18 hours post injection of [86Y]DOTA-Bn without pre-injection of BsAb), no tumor uptake and very little residual activity is seen in the peritoneal cavity due to the low normal tissue retention and favorable renal clearance of [86Y]DOTA-Bn (Fig. 5B). Notably, residual tumor nodules were detected in multi-cycle anti-GPA33 DOTA-PRIT treated survivors (average image-based tumor region of interest: 2.45 ± 1.77%IA/g), with a tumor-to-blood ratio of 1.9 ± 0.5 and a tumor-to-liver ratio of 2.0 ± 0.5 (Fig. 5C and D).

Figure 5.

PET/CT staging and monitoring of peritoneal carcinomatosis with anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn. To demonstrate feasibility of the approach, peritoneal carcinomatosis tumor-bearing mice (15 d.p.i.) were administered either anti-GPA33 DOTA- PRIT + [86Y]DOTA-Bn (3.7 MBq; 100 μCi; 20 pmol; A) or [86Y]DOTA-Bn only (3.7 MBq; 100 μCi; 20 pmol; B) and imaged with PET/CT at approximately 8 hours post injection of [86Y]DOTA-Bn. Multi-cycle anti-GPA33 DOTA-PRIT survivors were administered anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn and imaged at 175 d.p.i.; representative image shown in C. Maximum intensity projection images are shown. D, Image-based ROI analysis for heart, tumor, and liver. Peritoneal carcinomatosis tumor can be visualized in treatment-naïve mice with anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn, while no tumor is detected and little nonspecific uptake is observed with [86Y]DOTA-Bn only. Furthermore, minimal residual disease can be detected in long-term multi-cycle anti-GPA33 DOTA-PRIT survivors.

Figure 5.

PET/CT staging and monitoring of peritoneal carcinomatosis with anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn. To demonstrate feasibility of the approach, peritoneal carcinomatosis tumor-bearing mice (15 d.p.i.) were administered either anti-GPA33 DOTA- PRIT + [86Y]DOTA-Bn (3.7 MBq; 100 μCi; 20 pmol; A) or [86Y]DOTA-Bn only (3.7 MBq; 100 μCi; 20 pmol; B) and imaged with PET/CT at approximately 8 hours post injection of [86Y]DOTA-Bn. Multi-cycle anti-GPA33 DOTA-PRIT survivors were administered anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn and imaged at 175 d.p.i.; representative image shown in C. Maximum intensity projection images are shown. D, Image-based ROI analysis for heart, tumor, and liver. Peritoneal carcinomatosis tumor can be visualized in treatment-naïve mice with anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn, while no tumor is detected and little nonspecific uptake is observed with [86Y]DOTA-Bn only. Furthermore, minimal residual disease can be detected in long-term multi-cycle anti-GPA33 DOTA-PRIT survivors.

Close modal

Despite the best available treatment, the presence of peritoneal carcinomatosis from colorectal origin is generally considered incurable (5, 31). For patients with advanced colorectal cancer with less severe disease progression, primary treatment involves a combination of cytoreductive surgery and intraperitoneal chemotherapy, and is associated with major morbidity (31, 32). Progression or recurrence of the disease presents a treatment challenge, of which there is no treatment consensus (33). Often these patients are placed on palliative chemotherapy or enrolled in clinical trials (31).

The potential of RIT in the treatment of advanced colorectal cancer has been recognized since the late 1970s (34). Highly promising results with 111In/90Y-DOTA-M5A anti-CEA humanized mAb were recently reported by Akhavan and colleagues (35). However, hematologic toxicity was dose limiting and a relatively low MTD of 10 mCi/m2 (0.37 GBq/m2) was noted, likely due to the pretreated patients having relatively low marrow reserve (35). While tumor doses achievable by RIT alone may be suitable for peritoneal carcinomatosis, marrow doses can be reduced using intracavity administration (6). For example, Modak and colleagues recently reported (36) a phase I study of intraperitoneal RIT with 124/131I-omburtumab in patients with B7H3-expressing tumors involving the peritoneum with favorable results. Also, the relatively modest whole-peritoneal radiation dose (<500 cGy) enabled initiation of a phase II trial with addition of 131I-omburtumab to standard-of-care multimodality therapy NCT04022213 (36). Taken together, while conventional RIT approaches are promising, dose limiting toxicity to normal tissues, particularly the bone marrow and liver, remains a critical barrier to RIT in solid tumors (6), presenting the need for improved TIs via intracavity administration and alternative targeting approaches such as DOTA-PRIT.

We were able to successfully establish an aggressive peritoneal carcinomatosis mouse model with SW1222, a well-differentiated GPA33-expressing human colorectal cancer cell line, and treat with intraperitoneal DOTA-PRIT while maintaining high TI with minimal toxicities. The untreated mice developed enlarging tumors that ultimately led to death in 50% of the mice by 45–50 d.p.i., which was consistent across multiple experiments. Furthermore, untreated SW1222 peritoneal carcinomatosis was highly lethal, with a majority of the animals (19/20, 95%) attaining humane endpoints as discussed above requiring euthanasia by 69 d.p.i. These tumors were well engrained into organs and spread throughout the abdominal cavity, like peritoneal carcinomatosis in the clinical setting. This intraperitoneal SW1222 xenograft model was reproducible across multiple implantations, and with high uptake rates, making it a suitable method for studying peritoneal carcinomatosis.

Our findings are consistent with previous preclinical PRIT studies aimed at managing colorectal peritoneal carcinomatosis. Rondon and colleagues recently reported (15) significantly reduced tumor growth using a biorthogonal click chemistry PRIT approach to target intraperitoneal CEA-expressing peritoneal carcinomatosis with a 177Lu-tetrazine radioligand (A431-CEA-Luc is derived from vulvar squamous epithelial carcinoma transfected with constructs encoding for both CEA and luciferase; shown to mimic colorectal peritoneal carcinomatosis). Notably, the authors compared the effect of the injection route of PRIT antibody-trans-cyclooctene 35A7-TCO (i.e., intravenous or intraperitoenal) on the in vivo performance of the system and showed no significant differences in 177Lu-tetrazine radioligand targeting to tumor. For PRIT, peritoneal tumors were first injected intravenously with 50 μg of PRIT antibody-trans-cyclooctene (35A7-TCO), followed 24 hours later by the intraperitoneal injection of 177Lu-tetrazine (40 MBq; i.e., no CA step). They reported a slightly higher tumor dose of 59 cGy/MBq (this study: 35 cGy/MBq), with 0.219 and 2.59 cGy/MBq to heart (TI: 269) and kidney (TI: 23), respectively (note: absorbed dose to blood not reported; ref. 15). In the current study, we observed less favorable dosimetry and lower TIs of 0.91 cGy/MBq to heart (TI: 38) and 2.95 cGy/MBq to kidney (TI: 12). However, as Rondon and colleagues were proof-of-concept studies, a maximum dose of 40 MBq was selected for PRIT and dose escalation was not performed (estimated tumor dose 23.692 ± 20.87 Gy/40 MBq). As another comparison, Schoffelen and colleagues reported preclinical PRIT with intravenous administration of anti-CEA TF2 bispecific antibody and 111In/177Lu-IMP288 in mice bearing intraperitoneal CEA-expressing LS174T human colorectal cancer xenografts (14). The authors did not use a CA step and no PRIT dosimetry was reported. In this study, treatment at MTD (60 MBq of 177Lu-IMP288) was initiated 14 days after mice were intraperitoneally xenografted with 1.0 × 106 LS174T, with improved overall survival (MS of 50 days for PRIT vs. 19–22 days for controls), and 3/10 animals surviving long term (study endpoint: 120 days).

Comparing our previous subcutaneous flank SW1222 xenograft targeting to this current study, we discovered both positive and negative features in terms of TIs during DOTA-PRIT therapy. Perhaps somewhat expected for intraperitoneal RIT versus systemic (intravenous targeting; ref. 6), we observed an approximately 50% reduction in estimated absorbed dose to blood (2.79 vs. 5.74 cGy/MBq for intraperitoneal PRIT vs. intravenous PRIT, respectively) and kidney (2.95 vs. 6.52 cGy/MBq for intraperitoneal PRIT vs. intravenous PRIT, respectively). However, while the initial tumor targeting was rapid and comparable (%IA/g was approximately 20%–25% within 1 hour of [177Lu]LuDOTA-Bn injection), the estimated absorbed dose to tumor was 34.81 cGy/MBq, which is approximately 7-fold less than the estimated absorbed dose to tumor observed during our prior subcutaneous flank SW1222 targeting (229 cGy/MBq).

We recognize that any sort of rationale for the differences is speculative. We note, however, that tumors were controlled effectively by radiation at these levels; we intend to explore these differences more in depth in the future.

It is unclear why pretargeted [177Lu]LuDOTA-Bn washed out faster from SW1222 peritoneal carcinomatosis than subcutaneous flank SW1222. Consequently, although the blood and kidney doses were also lower, the TIs were reduced because of the lower tumor dose. To improve our understanding of BsAb targeting of peritoneal carcinomatosis, and hence possibly improve the in vivo performance of DOTA-PRIT, experiments are currently underway to examine the impact of route of administration and dose on the kinetics of tumor uptake and clearance of excess BsAb from the peritoneal cavity. Possible approaches to optimize TIs with the current reagents include administration of BsAb both intravenous and intraperitoneal to improve peritoneal carcinomatosis tumor targeting (including well-vascularized peritoneal carcinomatosis tumor nodules that may not be readily targeted after intraperitoneal injection), variation in dose and timing of CA (including potentially shortening the time interval between CA and [177Lu]LuDOTA-Bn administration or elimination of CA step if both BsAb and [177Lu]LuDOTA-Bn are injected intraperitoneally), and taking into account the ratio between BsAb present in the peritoneal cavity and the dose of [177Lu]LuDOTA-Bn. Also, we envision possible use of an indwelling catheter for monitoring of BsAb infusion, BsAb binding kinetics to tumor and clearance from peritoneal cavity, and monitoring BsAb-radioligand binding kinetics to peritoneal carcinomatosis tumor.

Otherwise, BsAb and/or hapten innovations can be implemented; TIs can be further improved with the application of the next-generation BsAb platform Self-Assembling and DisAssembling (SADA; ref. 28), which we have recently adapted for GPA33 targeting (37). An additional benefit to the SADA platform is the elimination of the CA step, which could theoretically present safety concerns through its mechanism of action of forming high-MW BsAb-CA complexes. However, during our PRIT studies with hundreds of animals, we observed no objective toxicity to the cellular components of the fixed macrophage system, (liver, spleen, bone marrow, lung, and lymph nodes), which remove particles from the blood. Furthermore, the use of bivalent haptens to significantly improve tumor uptake and retention during PRIT has also been reported previously (38–44), including clinically [e.g., 11In/177Lu-IMP288 (16), 111In-bivalent DTPA hapten (45), or 131I-di-DTPA-indium (46)], and trivalent haptens are in development for TF2 bispecific antibody (47). However, while these multivalent DOTA constructs may lead to improved TIs, they may also have different safety and immunogenicity profiles that must be considered; fortunately, no related issues have been reported so far clinically.

Herein we establish efficacy of intraperitoneal GPA33-DOTA-PRIT + [177Lu]LuDOTA-Bn, including complete peritoneal carcinomatosis disease eradication. Single- and multi-cycle treatment regimens were well tolerated, leading to minimal weight loss and reversible myelotoxicity. After one cycle of the therapy (total 177Lu-radioactivity: 111 MBq/mouse), the mice were noted to have doubling of their MS with minimal neutropenia (MS = 84.5 vs. 49.5–51 days for controls). With a multi-cycle approach (total 177Lu-radioactivity: 333 MBq/mouse), most of the mice (6/8, 75%) had durable treatment response without clinical signs of disease recurrence. Furthermore, MS was never met in this multi-cycle administration (MS > 183 days). The difference in MS was statistically significant for single- versus multi-cycle anti-GPA33 DOTA-PRIT (P = 0.016). This apparent dose effect was also previously observed in the subcutaneous flank SW1222 model (19). We observed increasing BLI in mice without treatment, and decreasing BLI signals following multi-cycle therapy, even dropping below initial screening baselines which showed strong treatment response. The BLI imaging was also able to detect when one of the treated mice with minimal residual BLI signal started to have proliferation of disease with exponentially increasing BLI signal prior to any outward physical signs of disease recurrence. IHC assay of recurrent tumor at necropsy of a multi-cycle treated mouse was GPA33-antigen positive, thus ruling out the possibility of antigen escape. While only one mouse was found to have no microscopic disease (cure rate 12.5%), the other surviving mice had no change in BLI signals for months after treatment and a decrease of Ki67, a marker of proliferation, from >40% in control or antibody-only mice to about 1% in residual tumors, indicating a senescent phenotype of the residual carcinoma; these mice had minimal residual active disease on final necropsy. We determined that these tumor nodules could be imaged with anti-GPA33 DOTA-PRIT + [86Y]DOTA-Bn PET-CT, indicating GPA33 antigen expression. Hence, consolidative or salvage therapy may be considered should these tumored mice show signs of disease recurrence.

Furthermore, although three cycles of therapy induced cure in this model, dose escalation and/or treatment with additional cycles would be feasible. Like single-cycle therapy, multi-cycle therapy had toxicities of transient neutropenia and did not lead to death in any of the mice. As potential mouse toxicity benchmarks, a non-lethal but highly nephrotoxic dose has been reported to be 42 ± 2.3 Gy (48), and a maximum tolerated bone marrow absorbed dose has been reported to be 15–16 Gy (i.e., treated animals required euthanasia at 10–12 d.p.i. due to poor general condition; ref. 49). On final pathology, no treatment-related organ toxicity other than ovarian atrophy was detected, a finding that mirrors results in select studies of nude mice undergoing 177Lu-therapy (28, 50). The etiology of this atrophy is unclear and may be related to age or radiation dose (50). Repetto-Llamazares and colleagues (50) hypothesized that even if the observed ovarian atrophy can be in large part attributed to the crossfire effect from the nearby kidneys or bladder, this effect is unlikely to result in significant changes in humans. Indeed, the FDA label for LUTATHERA (177Lu dotatate) does note the risk of infertility, as is expected with all therapies involving radioactivity, citing estimated absorbed dose per unit activity of 0.031 Gy/GBq with a 2.2 mm maximum penetration of 177Lu (51). Finally, given that the average age of diagnosis with colorectal peritoneal carcinomatosis is 59.9 years (52), the risk of infertility associated with potentially curative therapy may not be clinically relevant in many patients.

Looking forward, it is possible that using an alpha-emitting radioisotope such as actinium-225 (Ac-225), which is compatible with our system (53), in place of the beta-emitter Lu-177 may be effective for microscopic disease encountered following surgical cytoreduction. We have studies currently underway investigating intraperitoneal DOTA-PRIT using Ac-225 radiohapten (225Ac-proteus-DOTA) in the peritoneal carcinomatosis model utilized herein, as well as low-volume-disease peritoneal carcinomatosis burden model (i.e., to simulate malignant ascites) to evaluate the efficacy of this approach (54).

In addition to examining other therapeutic radiation and BsAb platforms, another primary preclinical development focus will be to address the critical limitation of using mouse models to study and develop GPA33 targeting as the normal gut expression is absent. While non-tumored colonocyte GPA33 expression may present as a limitation of this targeting approach in humans, no gastrointestinal toxicity was observed despite uptake of 131I-huA33 in the bowel, and in fact hematologic toxicity was dose limiting (55). To demonstrate safety and efficacy in an animal model that more accurately reflects behavior in humans, studies of kinetics and differential uptake between normal tissues and tumor nodules in cynomolgus monkeys are ongoing by our group (56).

Finally, in anticipation of clinical translation, we prioritized development of a theranostic approach to peritoneal carcinomatosis treatment for “precision oncology” (57). Quantitative SPECT imaging can be utilized to show effective delivery of the therapy and provide dosimetry estimates for tumor and normal tissues during 177Lu-radiotherapy (58), including theranostic PRIT with 111In/177Lu (16); however, a PET imaging agent may be preferable (59). Owing to ultrahigh affinity of BsAb C825 scFv for both Lutetium- and Yttrium-haptens, PET-CT was also successfully utilized to detect peritoneal carcinomatosis tumor using DOTA-PRIT. Using DOTA-Bn radiolabeled with the positron-emitting isotope Y-86 (33% with a 14.7-hour half-life), we could detect treatment-naïve bulk tumor, as well as residual tumor nodules in long-term survivors. Additional PET probes for DOTA-PRIT (F-18, Zr-89, Ga-68) that may be more suitable clinically are in development, although the theranostic pairing may be complicated by the requirement of modified haptens [“proteus-DOTA” (53)] to account for differences in C825-binding selectivity.

In sum, the anti-GPA33 DOTA-PRIT system utilizing 177Lu as the radiohapten [177Lu]LuDOTA-Bn is a promising theranostic treatment approach for otherwise incurable colorectal peritoneal carcinomatosis. We therefore propose that anti-GPA33 DOTA-PRIT + [177Lu]LuDOTA-Bn can be used as the sole treatment for an advanced unresectable peritoneal carcinomatosis as a result of colorectal cancer or as an adjunct to surgery. It can either be used as neoadjuvant treatment to reduce tumor nodules prior to cytoreduction to allow for safe and complete resection or as a possible adjuvant therapy to treat residual disease following surgical cytoreduction.

E.K. Fung reports personal fees from Y-mAbs Therapeutics Inc. outside the submitted work. H. Xu reports a patent for GPA33-related patents pending. B.H. Santich reports grants from Y-mAbs Therapeutics Inc. during the conduct of the study; in addition, B.H. Santich has a patent for SADA platform pending, licensed, and with royalties paid from Y-mAbs Therapeutics Inc., a patent for GPA33 sequences pending, and a patent for DOTA haptens pending; and B.H. Santich reports grants from Jeff Gordon Children's Foundation outside the submitted work and is listed as a coinventor on multiple patents on therapeutics for the treatment of cancer all of which are owned by MSKCC. B.H. Santich is now employed by Y-mAbs Therapeutics Inc. P.B. Zanzonico reports PCT/US16/33217; and PRIT Intellectual Property Licensed to Y-mAbs Therapeutics Inc. and is a paid consultant to Novartis. N. Pandit-Taskar reports personal fees from Actinium Pharmaceuticals, Illumina, and Imaginab outside the submitted work. N.K.V. Cheung reports grants and other support from Y-mAbs Therapeutics Inc., Abpro lab, and other support from Eureka Therapeutics outside the submitted work; in addition, N.K.V. Cheung has a patent for GPA33 antibodies and radioligands pending, issued, and licensed to Y-mAbs Therapeutics Inc. S.M. Cheal reports grants from Y-mAbs Therapeutics Inc. outside the submitted work; in addition, S.M. Cheal is listed as a coinventor on multiple patents related to this work owned by MSKCC, some of which are licensed to Y-mAbs Therapeutics. S.M. Larson reports receiving commercial research grants from Y-mAbs Therapeutics, Inc., Genentech, Inc., WILEX AG, Telix Pharmaceuticals Limited, and Regeneron Pharmaceuticals, Inc.; holding ownership interest/equity in. Elucida Oncology Inc., and holding stock in Y-mAbs Therapeutics, Inc. S.M. Larson is the inventor and owner of issued patents both currently unlicensed and licensed by MSKCC to Samus Therapeutics, Inc., Y-mAbs Therapeutics Inc., and Elucida Oncology, Inc.; is or has served as a consultant to Cynvec LLC, Eli Lilly & Co., Prescient Therapeutics Limited, Advanced Innovative Partners, LLC, Gerson Lehrman Group, Progenics Pharmaceuticals, Inc., Bristol Myers Squib, and Janssen Pharmaceuticals, Inc. No disclosures were reported by the other authors.

C.S. Chandler: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. M.M. Bell: Data curation, formal analysis, validation, investigation, visualization, methodology. S.K. Chung: Data curation, formal analysis, validation, investigation, visualization, writing–original draft, writing–review and editing. D.R. Veach: Data curation, investigation, writing–review and editing. E.K. Fung: Software, formal analysis, writing–review and editing. B. Punzalan: Data curation, investigation, writing–review and editing. D. Burnes Vargas: Data curation, validation, investigation, visualization. M. Patel: Data curation, formal analysis, validation, investigation. H. Xu: Resources, writing–review and editing. H.-f. Guo: Resources, writing–review and editing. B.H. Santich: Data curation, writing–review and editing. P.B. Zanzonico: Resources, software, formal analysis, writing–review and editing. S. Monette: Resources, data curation, formal analysis, validation, investigation, writing–review and editing. G.M. Nash: Funding acquisition, writing–review and editing. A. Cercek: Funding acquisition, writing–review and editing. A. Jungbluth: Resources, data curation, validation, investigation, writing–review and editing. N. Pandit-Taskar: Supervision, writing–review and editing. N.K.V. Cheung: Funding acquisition, methodology, writing–review and editing. S.M. Larson: Conceptualization, supervision, funding acquisition, methodology, project administration, writing–review and editing. S.M. Cheal: Conceptualization, supervision, funding acquisition, investigation, methodology, writing–original draft, project administration, writing–review and editing.

We would like to thank Valerie Longo for assistance with animal imaging and Alyssa Duck for editorial support. This research was funded in part by the Donna & Benjamin M. Rosen Chair (to S.M. Larson), Enid A. Haupt Chair (to N.K.V. Cheung), The Center for Targeted Radioimmunotherapy and Theranostics, Ludwig Center for Cancer Immunotherapy of Memorial Sloan Kettering Cancer Center (to S.M. Larson), and William H. Goodwin and Alice Goodwin and the Commonwealth Foundation for Cancer Research and The Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center (to S.M. Larson). S. M. Larson was also supported in part by NIH grant P50 CA86438. This study also received support from NIH grant R01 CA233896 (to S.M. Cheal). We also acknowledge the NIH/NCI Cancer Center Support Grant (P30 CA08748) for use of the Tri-Institutional Laboratory of Comparative Pathology, Memorial Sloan Kettering Cancer Center, Weill Cornell Medicine, and The Rockefeller University, New York, NY. Technical services provided by the MSK Small-Animal Imaging Core Facility and Laboratory of Comparative Pathology were also supported by P30 CA08748.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Jayne
DG
,
Fook
S
,
Loi
C
,
Seow-Choen
F
. 
Peritoneal carcinomatosis from colorectal cancer
.
Br J Surg
2002
;
89
:
1545
50
.
2.
Lemmens
VE
,
Klaver
YL
,
Verwaal
VJ
,
Rutten
HJ
,
Coebergh
JW
,
de Hingh
IH
. 
Predictors and survival of synchronous peritoneal carcinomatosis of colorectal origin: a population-based study
.
Int J Cancer
2011
;
128
:
2717
25
.
3.
Annest
LS
,
Jolly
PC
. 
The results of surgical treatment of bowel obstruction caused by peritoneal carcinomatosis
.
Am Surg
1979
;
45
:
718
21
.
4.
Chu
DZ
,
Lang
NP
,
Thompson
C
,
Osteen
PK
,
Westbrook
KC
. 
Peritoneal carcinomatosis in nongynecologic malignancy. A prospective study of prognostic factors
.
Cancer
1989
;
63
:
364
7
.
5.
Franko
J
,
Shi
Q
,
Goldman
CD
,
Pockaj
BA
,
Nelson
GD
,
Goldberg
RM
, et al
Treatment of colorectal peritoneal carcinomatosis with systemic chemotherapy: a pooled analysis of north central cancer treatment group phase III trials N9741 and N9841
.
J Clin Oncol
2012
;
30
:
263
7
.
6.
Larson
SM
,
Carrasquillo
JA
,
Cheung
NK
,
Press
OW
. 
Radioimmunotherapy of human tumours
.
Nat Rev Cancer
2015
;
15
:
347
60
.
7.
Goldenberg
DM
,
Chang
CH
,
Rossi
EA
,
J
W
,
McBride
SRM
. 
Pretargeted molecular imaging and radioimmunotherapy
.
Theranostics
2012
;
2
:
523
40
.
8.
Verhoeven
M
,
Seimbille
Y
,
Dalm
SU
. 
Therapeutic applications of pretargeting
.
Pharmaceutics
2019
;
11
:
434
.
9.
Buchsbaum
DJ
,
Khazaeli
MB
,
Axworthy
DB
,
Schultz
J
,
Chaudhuri
TR
,
Zinn
KR
, et al
Intraperitoneal pretarget radioimmunotherapy with CC49 fusion protein
.
Clin Cancer Res
2005
;
11
:
8180
5
.
10.
Lindegren
S
,
Karlsson
B
,
Jacobsson
L
,
Andersson
H
,
Hultborn
R
,
Skarnemark
G
. 
(211)At-labeled and biotinylated effector molecules for pretargeted radioimmunotherapy using poly-L- and poly-D-Lysine as multicarriers
.
Clin Cancer Res
2003
;
9
:
3873S
9S
.
11.
Zhang
M
,
Yao
Z
,
Sakahara
H
,
Saga
T
,
Nakamoto
Y
,
Sato
N
, et al
Effect of administration route and dose of streptavidin or biotin on the tumor uptake of radioactivity in intraperitoneal tumor with multistep targeting
.
Nucl Med Biol
1998
;
25
:
101
5
.
12.
Zhang
M
,
Yao
Z
,
Saga
T
,
Sakahara
H
,
Nakamoto
Y
,
Sato
N
, et al
Improved intratumoral penetration of radiolabeled streptavidin in intraperitoneal tumors pretargeted with biotinylated antibody
.
J Nucl Med
1998
;
39
:
30
3
.
13.
Yao
Z
,
Zhang
M
,
Sakahara
H
,
Saga
T
,
Arano
Y
,
Konishi
J
. 
Avidin targeting of intraperitoneal tumor xenografts
.
J Natl Cancer Inst
1998
;
90
:
25
9
.
14.
Schoffelen
R
,
van der Graaf
WT
,
Sharkey
RM
,
Franssen
GM
,
McBride
WJ
,
Chang
CH
, et al
Quantitative immuno-SPECT monitoring of pretargeted radioimmunotherapy with a bispecific antibody in an intraperitoneal nude mouse model of human colon cancer
.
J Nucl Med
2012
;
53
:
1926
32
.
15.
Rondon
A
,
Schmitt
S
,
Briat
A
,
Ty
N
,
Maigne
L
,
Quintana
M
, et al
Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-of-concept
.
Theranostics
2019
;
9
:
6706
18
.
16.
Schoffelen
R
,
Boerman
OC
,
Goldenberg
DM
,
Sharkey
RM
,
van Herpen
CM
,
Franssen
GM
, et al
Development of an imaging-guided CEA-pretargeted radionuclide treatment of advanced colorectal cancer: first clinical results
.
Br J Cancer
2013
;
109
:
934
42
.
17.
Garinchesa
P
,
Sakamoto
J
,
Welt
S
,
Real
F
,
Rettig
W
,
Old
L
. 
Organ-specific expression of the colon cancer antigen A33, a cell surface target for antibody-based therapy
.
Int J Oncol
1996
;
9
:
465
71
.
18.
Baptistella
AR
,
Salles Dias
MV
,
Aguiar
S
 Jr
,
Begnami
MD
,
Martins
VR
. 
Heterogeneous expression of A33 in colorectal cancer: possible explanation for A33 antibody treatment failure
.
Anticancer Drugs
2016
;
27
:
734
7
.
19.
Cheal
SM
,
Xu
H
,
Guo
HF
,
Lee
SG
,
Punzalan
B
,
Chalasani
S
, et al
Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity (8)(6)Y- or (1)(7)(7)Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates
.
Eur J Nucl Med Mol Imaging
2016
;
43
:
925
37
.
20.
Orcutt
KD
,
Slusarczyk
AL
,
Cieslewicz
M
,
Ruiz-Yi
B
,
Bhushan
KR
,
Frangioni
JV
, et al
Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging
.
Nucl Med Biol
2011
;
38
:
223
33
.
21.
Cheal
SM
,
Patel
M
,
Yang
G
,
Veach
D
,
Xu
H
,
Guo
HF
, et al
An N-acetylgalactosamino dendron-clearing agent for high-therapeutic-index DOTA-hapten pretargeted radioimmunotherapy
.
Bioconjug Chem
2020
;
31
:
501
6
.
22.
Orcutt
KD
,
Nasr
KA
,
Whitehead
DG
,
Frangioni
JV
,
Wittrup
KD
. 
Biodistribution and clearance of small molecule hapten chelates for pretargeted radioimmunotherapy
.
Mol Imaging Biol
2011
;
13
:
215
21
.
23.
Cheal
SM
,
Fung
EK
,
Patel
M
,
Xu
H
,
Guo
HF
,
Zanzonico
PB
, et al
Curative multicycle radioimmunotherapy monitored by quantitative SPECT/CT-based theranostics, using bispecific antibody pretargeting strategy in colorectal cancer
.
J Nucl Med
2017
;
58
:
1735
42
.
24.
Ponomarev
V
,
Doubrovin
M
,
Serganova
I
,
Vider
J
,
Shavrin
A
,
Beresten
T
, et al
A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging
.
Eur J Nucl Med Mol Imaging
2004
;
31
:
740
51
.
25.
O'Donoghue
JA
,
Smith-Jones
PM
,
Humm
JL
,
Ruan
S
,
Pryma
DA
,
Jungbluth
AA
, et al
124I-huA33 antibody uptake is driven by A33 antigen concentration in tissues from colorectal cancer patients imaged by immuno-PET
.
J Nucl Med
2011
;
52
:
1878
85
.
26.
Bankhead
P
,
Loughrey
MB
,
Fernandez
JA
,
Dombrowski
Y
,
McArt
DG
,
Dunne
PD
, et al
QuPath: Open source software for digital pathology image analysis
.
Sci Rep
2017
;
7
:
16878
.
27.
Dacek
MM
,
Veach
DR
,
Cheal
SM
,
Carter
LM
,
McDevitt
MR
,
Punzalan
B
, et al
Engineered cells as a test platform for radiohaptens in pretargeted imaging and radioimmunotherapy applications
.
Bioconjug Chem
2021
;
32
:
649
54
.
28.
Santich
BH
,
Cheal
SM
,
Ahmed
M
,
McDevitt
MR
,
Ouerfelli
O
,
Yang
G
, et al
A Self-Assembling and DisAssembling (SADA) bispecific antibody (BsAb) platform for curative 2-step pre-targeted radioimmunotherapy
.
Clin Cancer Res
2021
;
27
:
532
41
.
29.
Pelletier
M
,
Hinglais
N
,
Bach
JF
. 
Characteristic immunohistochemical and ultrastructural glomerular lesions in Nude mice
.
Lab Invest
1975
;
32
:
388
96
.
30.
Viguera
C
,
Ward
JM
,
Nims
RM
,
Wenk
ML
,
Strandberg
JD
. 
Clinical and pathologic conditions of female nude (athymic) mice in two conventional maintained colonies
.
J Am Vet Med Assoc
1978
;
173
:
1198
201
.
31.
Verwaal
VJ
,
van Ruth
S
,
de Bree
E
,
van Sloothen
GW
,
van Tinteren
H
,
Boot
H
, et al
Randomized trial of cytoreduction and hyperthermic intraperitoneal chemotherapy versus systemic chemotherapy and palliative surgery in patients with peritoneal carcinomatosis of colorectal cancer
.
J Clin Oncol
2003
;
21
:
3737
43
.
32.
Sugarbaker
PH
. 
Intraperitoneal chemotherapy and cytoreductive surgery for the prevention and treatment of peritoneal carcinomatosis and sarcomatosis
.
Semin Surg Oncol
1998
;
14
:
254
61
.
33.
Verwaal
VJ
,
Zoetmulder
FA
. 
Follow-up of patients treated by cytoreduction and chemotherapy for peritoneal carcinomatosis of colorectal origin
.
Eur J Surg Oncol
2004
;
30
:
280
5
.
34.
Goldenberg
DM
,
DeLand
F
,
Kim
E
,
Bennett
S
,
Primus
FJ
,
van Nagell
JR
 Jr
, et al
Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning
.
N Engl J Med
1978
;
298
:
1384
6
.
35.
Akhavan
D
,
Yazaki
P
,
Yamauchi
D
,
Simpson
J
,
Frankel
PH
,
Bading
J
, et al
Phase I study of Yttrium-90 radiolabeled M5A anti-carcinoembryonic antigen humanized antibody in patients with advanced carcinoembryonic antigen producing malignancies
.
Cancer Biother Radiopharm
2020
;
35
:
10
5
.
36.
Modak
S
,
Zanzonico
P
,
Grkovski
M
,
Slotkin
EK
,
Carrasquillo
JA
,
Lyashchenko
SK
, et al
B7H3-directed intraperitoneal radioimmunotherapy with radioiodinated omburtamab for desmoplastic small round cell tumor and other peritoneal tumors: results of a phase I study
.
J Clin Oncol
2020
;
38
:
4283
91
.
37.
Chung
SK
;
Wang
M
,
Veach
D
,
Cheal
SM
,
Burnes Vargas
D
,
Seo
S
, et al
Self-sssembling 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
.
38.
He
J
,
Liu
X
,
Zhang
S
,
Liu
G
,
Hnatowich
DJ
. 
Affinity enhancement bivalent morpholinos for pretargeting: surface plasmon resonance studies of molecular dimensions
.
Bioconjug Chem
2005
;
16
:
1098
104
.
39.
Gruaz-Guyon
A
,
Janevik-Ivanovska
E
,
Raguin
O
,
De Labriolle-Vaylet
C
,
Barbet
J
. 
Radiolabeled bivalent haptens for tumor immunodetection and radioimmunotherapy
.
Q J Nucl Med
2001
;
45
:
201
6
.
40.
Gautherot
E
,
Rouvier
E
,
Daniel
L
,
Loucif
E
,
Bouhou
J
,
Manetti
C
, et al
Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and 131I-labeled bivalent hapten
.
J Nucl Med
2000
;
41
:
480
7
.
41.
Boerman
OC
,
Kranenborg
MH
,
Oosterwijk
E
,
Griffiths
GL
,
McBride
WJ
,
Oyen
WJ
, et al
Pretargeting of renal cell carcinoma: improved tumor targeting with a bivalent chelate
.
Cancer Res
1999
;
59
:
4400
5
.
42.
Gautherot
E
,
Le Doussal
JM
,
Bouhou
J
,
Manetti
C
,
Martin
M
,
Rouvier
E
, et al
Delivery of therapeutic doses of radioiodine using bispecific antibody-targeted bivalent haptens
.
J Nucl Med
1998
;
39
:
1937
43
.
43.
Goodwin
DA
,
Meares
CF
,
Watanabe
N
,
McTigue
M
,
Chaovapong
W
,
Ransone
CM
, et al
Pharmacokinetics of pretargeted monoclonal antibody 2D12.5 and 88Y-Janus-2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) in BALB/c mice with KHJJ mouse adenocarcinoma: a model for 90Y radioimmunotherapy
.
Cancer Res
1994
;
54
:
5937
46
.
44.
Le Doussal
JM
,
Barbet
J
,
Delaage
M
. 
Bispecific-antibody-mediated targeting of radiolabeled bivalent haptens: theoretical, experimental and clinical results
.
Int J Cancer Suppl
1992
;
7
:
58
62
.
45.
Barbet
J
,
Peltier
P
,
Bardet
S
,
Vuillez
JP
,
Bachelot
I
,
Denet
S
, et al
Radioimmunodetection of medullary thyroid carcinoma using indium-111 bivalent hapten and anti-CEA x anti-DTPA-indium bispecific antibody
.
J Nucl Med
1998
;
39
:
1172
8
.
46.
Kraeber-Bodere
F
,
Rousseau
C
,
Bodet-Milin
C
,
Ferrer
L
,
Faivre-Chauvet
A
,
Campion
L
, et al
Targeting, toxicity, and efficacy of 2-step, pretargeted radioimmunotherapy using a chimeric bispecific antibody and 131I-labeled bivalent hapten in a phase I optimization clinical trial
.
J Nucl Med
2006
;
47
:
247
55
.
47.
Debnath
S
,
Guan
B
,
Hsieh
JT
,
Raj
G
,
Sun
X
. 
Design and synthesis of a bifunctional theranostic scaffold for bispecific antibody pretargeting
.
J Nucl Med
2020
;
61
:
1108
.
48.
Kristiansson
A
,
Ahlstedt
J
,
Holmqvist
B
,
Brinte
A
,
Tran
TA
,
Forssell-Aronsson
E
, et al
Protection of kidney function with human antioxidation protein alpha1-microglobulin in a mouse (177)Lu-DOTATATE radiation therapy model
.
Antioxid Redox Signal
2019
;
30
:
1746
59
.
49.
Timmermand
OV
,
Elgqvist
J
,
Beattie
KA
,
Orbom
A
,
Larsson
E
,
Eriksson
SE
, et al
Preclinical efficacy of hK2 targeted [(177)Lu]hu11B6 for prostate cancer theranostics
.
Theranostics
2019
;
9
:
2129
42
.
50.
Repetto-Llamazares
AH
,
Larsen
RH
,
Giusti
AM
,
Riccardi
E
,
Bruland
OS
,
Selbo
PK
, et al
177Lu-DOTA-HH1, a novel anti-CD37 radio-immunoconjugate: a study of toxicity in nude mice
.
PLoS One
2014
;
9
:
e103070
.
51.
Strosberg
J
,
El-Haddad
G
,
Wolin
E
,
Hendifar
A
,
Yao
J
,
Chasen
B
, et al
Phase 3 trial of (177)Lu-dotatate for midgut neuroendocrine tumors
.
N Engl J Med
2017
;
376
:
125
35
.
52.
Mayanagi
S
,
Kashiwabara
K
,
Honda
M
,
Oba
K
,
Aoyama
T
,
Kanda
M
, et al
Risk factors for peritoneal recurrence in stage II to III colon cancer
.
Dis Colon Rectum
2018
;
61
:
803
8
.
53.
Cheal
SM
,
McDevitt
MR
,
Santich
BH
,
Patel
M
,
Yang
G
,
Fung
EK
, et al
Alpha radioimmunotherapy using (225)Ac-proteus-DOTA for solid tumors - safety at curative doses
.
Theranostics
2020
;
10
:
11359
75
.
54.
Chandler
C
,
Cheal
SM
,
Nash
G
,
Cercek
A
,
Punzalan
B
,
Veach
D
, et al
Multicycle pretargeted radioimmunotherapy using 177Lu for intraperitoneal GPA33-expressing colorectal xenografts
.
J Nucl Med
2020
;
61
.
55.
Chong
G
,
Lee
FT
,
Hopkins
W
,
Tebbutt
N
,
Cebon
JS
,
Mountain
AJ
, et al
Phase I trial of 131I-huA33 in patients with advanced colorectal carcinoma
.
Clin Cancer Res
2005
;
11
:
4818
26
.
56.
Veach
DRFE
,
Cheal
SM
,
Beattie
BJ
,
Cheung
NV
,
Carrasquillo
JA
,
Larson
SM
.
A non-human primate (NHP) model for pre-clinical testing of DOTA-radiohaptens a key component of "curative" pretargeted radioimmunotherapy (PRIT)
; 
2020
;
Tampa, FL
.
57.
Langbein
T
,
Weber
WA
,
Eiber
M
. 
Future of theranostics: an outlook on precision oncology in nuclear medicine
.
J Nucl Med
2019
;
60
:
13S
9S
.
58.
Frezza
A
,
Desport
C
,
Uribe
C
,
Zhao
W
,
Celler
A
,
Despres
P
, et al
Comprehensive SPECT/CT system characterization and calibration for (177)Lu quantitative SPECT (QSPECT) with dead-time correction
.
EJNMMI Phys
2020
;
7
:
10
.
59.
Konik
A
,
O'Donoghue
JA
,
Wahl
RL
,
Graham
MM
,
Van den Abbeele
AD
. 
Theranostics: the role of quantitative nuclear medicine imaging
.
Semin Radiat Oncol
2021
;
31
:
28
36
.

Supplementary data