Zirconium-89 (t1/2 = 3.27 days) is a positron emitting radionuclide which displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In these studies we report the preparation of 89Zr-DFO-J591, a novel 89Zr -labeled monoclonal antibody (mAb) construct for targeted immunoPET imaging and quantification of prostate-specific membrane antigen (PSMA) expression in vivo.
The in vivo behavior of [89Zr]Zr-chloride, [89Zr]Zr-oxalate and [89Zr]Zr-DFO was investigated by using PET imaging. High level computational studies using density functional theory (DFT) calculations have been used to investigate the electronic structure of [89Zr]Zr-DFO and probe the nature of the complex in aqueous conditions. J591 was functionalized with the hexadentate, tris-hydroxamate ligand desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature. ImmunoPET imaging experiments in male, athymic nu/nu mice bearing sub-cutaneous LNCaP (PSMA positive) or PC-3 (PSMA negative) tumors were conducted. The change in 89Zr-DFO-J591 tissue uptake in response to high- and low-specific-activity formulations in the two tumor models was measured by using acute biodistribution studies and immunoPET.
Basic characterization of three important reagents, [89Zr]Zr-chloride and [89Zr]Zr-oxalate, as well as the complex, [89Zr]Zr-DFO, demonstrated that the nature of the 89Zr species has a dramatic effect on the biodistribution and pharmacokinetics. DFT calculations provide a rationale for the observed high in vivo stability of 89Zr-DFO-labeled mAbs and suggest that in aqueous conditions, [89Zr]Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of two water molecules. 89Zr-DFO-J591 was produced in high radiochemical yield (>77%) with radiochemical purity >99% and a specific-activity of 181.7±1.1 MBq/mg (4.91±0.03 mCi/mg). In vitro assays demonstrated that 89Zr-DFO-J591 had an initial immunoreactive fraction of 0.95±0.03 and remains active for up to 7 days. In vivo biodistribution experiments revealed high uptake of 89Zr-DFO-J591 in LNCaP tumors after 24, 48, 96 and 144 h (34.4±3.2 %ID/g; 38.0±6.2 %ID/g; 40.4±4.8 %ID/g; and 45.8±3.2 %ID/g, respectively). Specificity for PSMA expression was confirmed by biodistribution studies in PC-3 (PSMA negative) tumor models and by using low specific activity competitive inhibition studies. ImmunoPET studies also demonstrated that 89Zr-DFO-J591 provides excellent image contrast with tumor-to-muscle ratios >20 for delineation of LNCaP tumors between 48 – 144 h post-administration.
These experimental and computational studies demonstrate that 89Zr-DFO-labeled mAbs show exceptional promise as radiotracers for immunoPET imaging of human cancers. 89Zr-DFO-J591 displays tumor-to-background tissue contrast in immunoPET and can be used to delineate and quantify PSMA-positive prostate tumors in vivo.
Keywords: ImmunoPET, zirconium-89, prostate-specific membrane antigen (PSMA), J591, monoclonal antibodies, density functional theory