p-Isothiocyanatobenzyl-desferrioxamine: a new bifunctional chelate for facile radiolabeling of monoclonal antibodies with zirconium-89 for immuno-PET imaging

Abstract

Purpose: Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mAbs) for imaging and therapy. The positron emitter zirconium-89 ((89)Zr) has attractive characteristics for immuno-PET with intact mAbs. Previously, we have described a multi-step procedure for stable coupling of (89)Zr to mAbs via the bifunctional chelate (BFC) tetrafluorophenol-N-succinyldesferal (TFP-N-sucDf). To enable widespread use of (89)Zr-immuno-PET, we now introduce the novel BFC p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) and compare its performance in (89)Zr-immuno-PET with the reference BFC TFP-N-sucDf.

Methods: Three mAbs were premodified with Df-Bz-NCS and labeled with (89)Zr at different pHs to assess the reaction kinetics and robustness of the radiolabeling. Stability of both (89)Zr-Df-Bz-NCS- and (89)Zr-N-sucDf-conjugates was evaluated in different buffers and human serum. Comparative biodistribution and PET studies in tumor-bearing mice were undertaken.

Results: The selected conjugation conditions resulted in a chelate:mAb substitution ratio of about 1.5:1. Under optimal radiolabeling conditions (pH between 6.8-7.2), the radiochemical yield was >85% after 60 min incubation at room temperature, resulting in radioimmunoconjugates with preserved integrity and immunoreactivity. The new radioimmunoconjugate was very stable in serum for up to 7 days at 37 degrees C, with <5% (89)Zr release, and was equally stable compared to the reference conjugate when stored in the appropriate buffer at 4 degrees C. In biodistribution and imaging experiments, the novel and the reference radioimmunoconjugates showed high and similar accumulation in tumors in nude mice.

Conclusions: The novel Df-Bz-NCS BFC allows efficient and easy preparation of optimally performing (89)Zr-labeled mAbs, facilitating further exploration of (89)Zr-immuno-PET as an imaging tool.

Fig. 1

Schematic representation of mAb modification with the new bifunctional chelate Df-Bz-NCS (1) and subsequent labeling with ⁸⁹Zr (2) (a). The multi-step reference procedure using desferrioxamine B as starting ligand (b)

Fig. 2

Time course of ⁸⁹Zr complexation of mAb cU36 conjugated with Df-Bz-NCS at different pH and at room temperature

Fig. 3

Biodistribution of ⁸⁹Zr-Df-Bz-NCS-cU36 (black bars) and ⁸⁹Zr-N-sucDf-cU36 (white bars) in FaDu tumor-bearing nude mice at 72 h (a) and 144 h (b) after injection. Total administered mAb dose: 100 μg. Mean (%ID/g) ± SD at each time point after injection (n = 4 animals per time point for each conjugate)

Fig. 4

Biodistribution of ⁸⁹Zr-Df-Bz-NCS-cetuximab (black bars) and ⁸⁹Zr-N-sucDf-cetuximab (white bars) in A431 tumor-bearing nude mice at 24 h (a), 48 h (b), 72 h (c), and 120 h (d) after injection. Total administered mAb dose: 500 μg. Mean (%ID/g) ± SD at each time point after injection (n = 4 animals per time point for each conjugate). Significant differences (P < 0.01) in biodistribution between both radioimmunoconjugates are marked with an asterisk

Fig. 5

HRRT PET images (coronal slices) of two different FaDu xenograft-bearing nude mice at 72 h after injection with ⁸⁹Zr-Df-Bz-NCS-cU36 (a) or with ⁸⁹Zr-N-sucDf-cU36 (b). Slices from ventral (left) to dorsal (right). Images demonstrate high level of radiolabeled antibody accumulating in the tumor (arrows point to flank tumors) and low levels of tracer uptake in nontarget tissues