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

Jyoti Roy, Elaine M Jagoda, Falguni Basuli, Olga Vasalatiy, Tim E Phelps, Karen Wong, Anita T Ton, Urs B Hagemann, Alan S Cuthbertson, Patricia E Cole, Raffit Hassan, Peter L Choyke, Frank I Lin, Jyoti Roy, Elaine M Jagoda, Falguni Basuli, Olga Vasalatiy, Tim E Phelps, Karen Wong, Anita T Ton, Urs B Hagemann, Alan S Cuthbertson, Patricia E Cole, Raffit Hassan, Peter L Choyke, Frank I Lin

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.

Conflict of interest statement

A.S.C. holds a patent on 3,2-HOPO-MSLN-mAb conjugate. Other authors declare that he/she has no potential conflict of interest.

Figures

FIG. 1.
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.
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.
FIG. 3.
Mesothelin density per cell and binding affinity. Number of mesothelin per cell (A) and binding affinities (B; Kd) 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.
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.
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.
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.

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