Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry

Abstract

The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel ¹⁷⁷Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1-a human, anti-CA19.9 mAb-in preclinical murine models of pancreatic cancer. The exemplary ligand, ¹⁷⁷Lu-DOTA-PEG₇-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 hours) and persistent (16.8 ± 3.9% ID/g at 120 hours) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of ¹⁷⁷Lu-DOTA-PEG₇-Tz (400, 800, and 1,200 μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation. Mol Cancer Ther; 16(1); 124-33. ©2016 AACR.

Figure 1

A cartoon depicting the four basic steps of the pretargeted radioimmunotherapy approach used in these studies is shown (A). Also depicted is the reaction between the two components (TCO and Tz) on which the system is based (B) and the two ¹⁷⁷Lu-labeled ligands used in these studies (C).

Figure 2

Biodistribution data obtained from an in vivo pretargeting experiment in which ¹⁷⁷Lu-DOTA-PEG₇-Tz (2 nmol) was injected 72 h after the administration of 5B1-TCO (1.3 nmol). The activity concentrations in selected tissues at time points ranging from 4 to 120 h after the injection of ¹⁷⁷Lu-DOTA-PEG₇-Tz were determined along with selected tumor-to-tissue activity concentration ratios (inset).

Figure 3

Plots of the average tumor volume (A) and relative tumor volume (B) for each cohort of mice during the first 31 days of the PRIT study are shown with error bars denoting standard deviation. Line graphs mapping the response of each individual mouse over entire course of the PRIT study are shown for the control groups (C) as well as each therapy cohort (D–F).

Figure 4

Tomographic slices (A) and maximum intensity projections (B) of PET images (120 h post-injection) of a mouse from the 800 µCi therapy cohort that was injected with ⁸⁹Zr-5B1 at the conclusion of the therapy study. Masson’s trichrome (C, left), autoradiography (C, middle), and anti-CA19.9 immunofluorescence (C, right) on the excised tumor of the same mouse at the conclusion of the imaging study.