Theranostic GPA33-Pretargeted Radioimmunotherapy of Human Colorectal Carcinoma with a Bivalent 177Lu-Labeled Radiohapten

Abstract

Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and β-emitting isotope ¹⁷⁷Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S-2-(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [¹⁷⁷Lu]Lu-Gemini was prepared with no-carrier-added ¹⁷⁷LuCl₃ to a molar-specific activity of 123 GBq/μmol and radiochemical purity of more than 99%. The specificity of BsAb-¹⁷⁷Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [¹⁷⁷Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [¹⁷⁷Lu]Lu-S-2-(4-aminobenzyl)-DOTA ([¹⁷⁷Lu]Lu-DOTA-Bn) in naïve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/anti-DOTA IgG-scFv BsAb, a clearing agent, and [¹⁷⁷Lu]Lu-Gemini) in mouse models. Results: Initial in vivo studies showed that [¹⁷⁷Lu]Lu-Gemini behaved similarly to [¹⁷⁷Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [¹⁷⁷Lu]Lu-Gemini to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [¹⁷⁷Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor-to-normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [¹⁷⁷Lu]Lu-Gemini in DOTA-PRIT leads to improved TIs and augmented [¹⁷⁷Lu]Lu-Gemini tumor uptake and retention in comparison to monovalent [¹⁷⁷Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor-bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [¹⁷⁷Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [¹⁷⁷Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [¹⁷⁷Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [¹⁷⁷Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered ¹⁷⁷Lu activity while still achieving high TIs for both the blood (>100) and the kidneys (>30).

Keywords: 177Lu; GPA33; colorectal cancer; multivalent; pretargeted radioimmunotherapy.

Graphical abstract

FIGURE 1.

Chemical structures of bivalent [¹⁷⁷Lu]Lu-Gemini or monovalent [¹⁷⁷Lu]Lu-S-2-(4-aminobenzyl)-DOTA ([¹⁷⁷Lu]Lu-DOTA-Bn). Radiochemical synthesis of [¹⁷⁷Lu]Lu-Gemini (left) and [¹⁷⁷Lu]Lu-DOTA-Bn (right) is shown.

FIGURE 2.

In vitro anti-GPA33 DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini or [¹⁷⁷Lu]Lu-DOTA-Bn in GPA33-positive SW1222 cells up to 24 h after addition of ¹⁷⁷Lu activity. (A) Percentage of cell-associated activity. (B) Percentage of internalized activity. AUC was determined from 10 min to 24 h after addition of ¹⁷⁷Lu activity. Data shown are n = 3.

FIGURE 3.

Anti-GPA33 DOTA-PRIT serial biodistribution data in mouse model of human colorectal cancer. (A–C) Ex vivo biodistribution assay at 2 h (A), 24 h (B), or 120 h (C) after administration of GPA33-pretargeted [¹⁷⁷Lu]Lu-Gemini (∼1,850 kBq [50 µCi]/200 pmol) or [¹⁷⁷Lu]Lu-DOTA-Bn (∼1,850 kBq [50 µCi]/400 pmol) into groups of nude mice bearing subcutaneous SW1222 xenografts (n = 4–5/group). *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. Lg. = large; ns = no significant difference; Sm = small.

FIGURE 4.

Efficacy and toxicity of DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini in SW1222 tumor-bearing mice. Tumor-bearing mice were given single injection of anti-GPA33 DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini (44.4 MBq [1.2 mCi], 200 pmol) or anti-GD2 (nonspecific) DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini (44.4 MBq [1.2 mCi], 200 pmol). (A) Tumor response. (B) Kaplan–Meier survival.

FIGURE 5.

SPECT/CT images obtained from randomly selected mice undergoing DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini (44.4 MBq [1.2 mCi], 200 pmol) 24 h after administration of [¹⁷⁷Lu]Lu-Gemini. Shown is n = 3 from anti-GPA33 DOTA-PRIT + 44.4 MBq of [¹⁷⁷Lu]Lu-Gemini (mouse 1 to mouse 3) and n = 2 from anti-GD2 (nonspecific) DOTA-PRIT + 44.4 MBq of [¹⁷⁷Lu]Lu-Gemini (mouse 4 and mouse 5).

FIGURE 6.

Necropsy and histopathology were performed in surviving animals at 80 d. Representative mice treated with either anti-GPA33 DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini or anti-GD2 (nonspecific) DOTA-PRIT + [¹⁷⁷Lu]Lu-Gemini are shown. Representative sections of kidney, bone marrow (femur), and abnormal ovary are shown. Ovarian cortices in both mice were diffusely atrophied with complete absence of follicular structures and corpus luteus.