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

Abstract

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

Keywords: Lu-177; colorectal cancer hepatic metastasis; multivalent radiohapten; pretargeted radioimmunotherapy; theranostics.

Figure 1

DOTA-based pretargeted radioimmunotherapy (DOTA PRIT) using monovalent and bivalent ¹⁷⁷Lu-labeled radiohaptens. A. Concept of avidity enhancement. [¹⁷⁷Lu]Lu-Gemini is a symmetrical dimer of ABD and forms highly stable complexes with a variety of radiometals (β and α-emitters). Gemini potentially allows cooperative binding, with both DOTA binding with the BsAb scFv C825 anti-(M) ABD site. The Gemini radiohapten precursor is initially radiometallated with ¹⁷⁷Lu, and the remaining empty DOTA chelators then filled with nonradioactive ¹⁷⁵Lu. Non-radioactive lutetium backfilling is necessary, as both DOTA must be metal bound (M-DOTA) to be recognized by the antibody on either side, allowing for cooperative binding and enhanced retention at the cell surface. B. Three-step DOTA PRIT protocol.

Figure 2

Monovalent [¹⁷⁷Lu]Lu-ABD and bivalent [¹⁷⁷Lu]Lu-Gemini three-step DOTA-PRIT ex vivo biodistribution comparison. A. [¹⁷⁷Lu]Lu-ABD (3.7 MBq, 0.4 nmol) and B. [¹⁷⁷Lu]Lu-Gemini (3.7 MBq, 0.2 nmol) three-step DOTA-PRIT ex vivo biodistribution. Values are represented as means, and error bars represent standard error of the mean (SEM). C. Area under the curve (AUC) analysis of ex vivo biodistribution for the tumor, kidneys, and blood. The longer biological half-life of [¹⁷⁷Lu]Lu-Gemini on the tumor (T_1/2 = 25.4 h) and higher plateau (4.5 %ID/g) compared with [¹⁷⁷Lu]Lu-ABD (T_1/2 = 13.4 h, plateau: 1.3 %ID/g) better aligns with the longer decay period of the therapeutic radioisotope (lutetium-177 half-life: 6.7 days), increasing tumor exposure time with a higher tumor activity retention (3.5-fold higher [¹⁷⁷Lu]Lu-Gemini plateau value compared with [¹⁷⁷Lu]Lu-ABD). The dashed yellow lines on the tumor graph of subfigure C represent non-linear fit one-phase decay curves for [¹⁷⁷Lu]Lu-ABD (R² = 0.91) and [¹⁷⁷Lu]Lu-Gemini (R² = 0.86) tumor biodistribution data.

Figure 3

DOTA-PRIT SPECT/CT studies with high GPA33-positive (SW1222-luc) and low GPA33-positive (LoVo) CRC liver metastasis tumor-bearing mice. Coronal multiplanar reconstruction (MPR) and maximum intensity projection (MIP) SPECT images acquired 24 h and 120 h after 44.4 MBq injection of [¹⁷⁷Lu]Lu-ABD (0.4 nmol) and [¹⁷⁷Lu]Lu-Gemini (0.2 nmol). High SW1222-luc tumor uptake is noted on the 24 h SPECT images of both [¹⁷⁷Lu]Lu-ABD and [¹⁷⁷Lu]Lu-Gemini, with higher tumor retention of [¹⁷⁷Lu]Lu-Gemini at 120 h. In LoVo tumors, superior tumor targeting and retention are noted with [¹⁷⁷Lu]Lu-Gemini, compared with [¹⁷⁷Lu]Lu-ABD which shows negligible tumor uptake. T.: liver tumor; GB: gallbladder; Spl.: spleen.

Figure 4

Quantitative autoradiography (QAR). A. Hematoxylin and eosin (H&E) micrograph of optimal cutting temperature (OCT)-frozen SW1222-luc hepatic tumors (circled in yellow), and B. concurrent QAR image 5 days after 44.4 MBq injection of [¹⁷⁷Lu]Lu-ABD (0.4 nmol) and [¹⁷⁷Lu]Lu-Gemini (0.2 nmol). Higher uptake is noted in the [¹⁷⁷Lu]Lu-Gemini tumor (mean %ID/g ± SD throughout the tumor: 2.1 ± 0.4 %ID/g) compared with [¹⁷⁷Lu]Lu-ABD (mean %ID/g ± SD throughout the tumor: 1.1 ± 0.2 %ID/g).

Figure 5

Three-step DOTA-PRIT experiment. A. Experimental three-step DOTA-PRIT protocol (with estimated tumor mean absorbed doses of 44 Gy and 53 Gy for 3 therapy cycles of [¹⁷⁷Lu]Lu-ABD (group 1) and a single cycle of [¹⁷⁷Lu]Lu-Gemini (group 2), respectively). B. Serial in vivo bioluminescence imaging (BLI). C. Serial coronal multiplanar reconstruction (MPR) CT images of a representative mouse treated with [¹⁷⁷Lu]Lu-ABD DOTA-PRIT (same mouse as in Figure 3). The initial hepatic tumor is circled in red, and the recurrent tumor in yellow. The 2 coronal images at D₁₁₁ have been acquired at different coronal levels. T.: tumor; GB: gallbladder; spl.: spleen. D. Kaplan-Meier survival curves and median survival histograms. Median survival times were compared using the log-rank (Mantel-Cox) test. ns: not statistically significant; *** p ≤ 0.001.

Figure 6

GPA33 differential expression in recurrent liver tumors post-three-step DOTA-PRIT (experimental three-step DOTA-PRIT protocol in Figure 5A). A. Representative hematoxylin and eosin (H&E) micrograph (left) and GPA33 immunostaining (right) of a recurrent GPA33(-) liver tumor 64 days post [¹⁷⁷Lu]Lu-ABD three-step DOTA-PRIT (G1M2 in Table 2). High magnification on viable cellular areas of the tumor shows negligible membranous GPA33 expression (5% immunohistochemistry (IHC) signal positivity). B. Representative H&E micrograph (left) and GPA33 immunostaining (right) of a recurrent GPA33(+) hepatic tumor 113 days post-[¹⁷⁷Lu]Lu-Gemini three-step DOTA-PRIT (G2M3 in Table 2). High magnification on viable cellular areas of the tumor shows diffuse and strong, membranous GPA33 expression (90% IHC signal positivity). Large necrotic areas, which show non-specific staining on IHC, are marked with an asterisk.