In Vitro and In Vivo Comparison of 3,2-HOPO Versus Deferoxamine-Based Chelation of Zirconium-89 to the Antimesothelin Antibody Anetumab

Abstract

Introduction: [²²⁷Th]Th-3,2-HOPO-MSLN-mAb, a mesothelin (MSLN)-targeted thorium-227 therapeutic conjugate, is currently in phase I clinical trial; however, direct PET imaging using this conjugate is technically challenging. Thus, using the same MSLN antibody, we synthesized 3,2-HOPO and deferoxamine (DFO)-based zirconium-89 antibody conjugates, [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb and [⁸⁹Zr]Zr-DFO-MSLN-mAb, respectively, and compared them in vitro and in vivo. Methods: [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb and [⁸⁹Zr]Zr-DFO-MSLN-mAb were evaluated in vitro to determine binding affinity and immunoreactivity in HT29-MSLN and PDX (NCI-Meso16, NCI-Meso21) cells. For both the zirconium-89 conjugates, in vivo studies (biodistribution/imaging) were performed at days 1, 3, and 6, from which tissue uptake was determined. Results: Both the conjugates demonstrated a low nanomolar binding affinity for MSLN and >95% immunoreactivity. In all the three tumor types, biodistribution of [⁸⁹Zr]Zr-DFO-MSLN-mAb resulted in higher tumor uptake(15.88-28-33%ID/g) at all time points compared with [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb(7-13.07%ID/g). [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb femur uptake was always higher than [⁸⁹Zr]Zr-DFO-MSLN-mAb, and imaging results concurred with the biodistribution studies. Conclusions: Even though the conjugates exhibited a high binding affinity for MSLN, [⁸⁹Zr]Zr-DFO-MSLN-mAb showed a higher tumor and lower femur uptake than [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb. Nevertheless, [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb could be used to study organ distribution and lesion uptake with the caveat of detecting MSLN-positive bone lesions. Clinical trial (NCT03507452).

Keywords: 32-HOPO; DFO; PET imaging; antibody conjugate; mesothelin; zirconium-89.

FIG. 1.

Structure of antibody conjugate and HPLC result. Structure of mesothelin antibody–chelator conjugate, 3,2-HOPO-MSLN-mAb (A) and DFO-MSLN-mAb. (B) Representative HPLC of zirconium-89 labeled conjugates, [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (C), and [⁸⁹Zr]Zr-DFO-MSLN-mAb. (D) HPLC condition: eluent, 0.1 M sodium phosphate, 0.1 M sodium sulfate, 0.05% sodium azide, 10% isopropyl alcohol (pH 6.8), flow rate 0.3 mL/min; black line UV detector, red line radiodetector.

FIG. 2.

In vitro saturation assay graphs. Representative in vitro saturation graphs of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb and [⁸⁹Zr]Zr-DFO-MSLN-mAb in HT29-MSLN, NCI-Meso16, and NCI-Meso21. For each plot Bt, bound total; Bnsp, bound nonspecific; Bsp, bound specific (Bt = Bnsp-Bsp).

FIG. 3.

Mesothelin density per cell and binding affinity. Number of mesothelin per cell (A) and binding affinities (B; K_d) of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb and [⁸⁹Zr]Zr-DFO-MSLN-mAb for mesothelin were derived from the in vitro saturation assays in HT29-MSLN, NCI-Meso16, and NCI-Meso21 cells. The assay was performed on an average two to three times for each cell type.

FIG. 4.

Competition binding assay and immunoreactivity plots. Representative plots from an in vitro [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (A) and [⁸⁹Zr]Zr-DFO-MSLN-mAb, (B) competition-binding assays using unlabeled MSLN targeted monoclonal antibody (self-displacement, Morris method) with HT29-MSLN cells. Each point (average of duplicates) represents cell-bound CPM. Representative plots for determination of the % immunoreactivity (immunoreactive fraction) of ⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (C) and [⁸⁹Zr]Zr-DFO-MSLN-mAb (D) from the same batch by the Morris method: representative plot (linear regression curve fit), immunoreactivity for ⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb = 96% and [⁸⁹Zr]Zr-DFO-MSLN-mAb = 95%. CPM, count per minute.

FIG. 5.

Biodistribution and PET imaging. Biodistribution (A) of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (HOPO) and [⁸⁹Zr]Zr-DFO-MSLN-mAb (DFO) in HT29-MSLN xenografts from 1 to 6 d. Each point represents the mean %ID/g of tissue normalized to 20 g mice ± SE (n = 9, 10 for each point). Tissue:Blood ratios (B) and Tissue:Muscle ratios (C) of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (HOPO) and [⁸⁹Zr]Zr-DFO-MSLN-mAb (DFO) in tumor and femur over a period of 6 d. Each point represents the mean ratio ± SE (n = 9, 10 for each point). Representative coronal PET/CT images (D) of HT29-MSLN mouse xenografts at days 1, 3, and 6 postinjection of (50 μCi, 1.85 MBq) of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb and [⁸⁹Zr]Zr-DFO-MSLN-mAb. Green arrow indicates tumors. Tissue:Blood of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (HOPO, 4.5 μg, control) in HT29-MSLN xenografts 3 d after receiving coinjections of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (180 μg, blocking); (E) each bar represents the mean Tissue:Blood ratios ± SD (n = 5–6 for each group).

FIG. 6.

Biodistribution and PET imaging. Biodistribution (A), Tissue:Muscle ratios (B), and imaging (C); representative coronal PET/CT images of [⁸⁹Zr]Zr-3,2-HOPO-MSLN-mAb (HOPO) and [⁸⁹Zr]Zr-DFO-MSLN-mAb (DFO) in HT29-MSLN, NCI-Meso16, and NCI-Meso21 mouse xenografts at day 3 postinjection of (50 μCi, 1.85 MBq; i.v) of either of the tracers. Each bar represents mean %ID/g of tissue normalized to 20 g mice ± SE (n = 9–10 for each point). Green arrow indicates tumors.