Translational Development of a Zr-89-Labeled Inhibitor of Prostate-specific Membrane Antigen for PET Imaging in Prostate Cancer

Purpose We present here a Zr-89-labeled inhibitor of prostate-specific membrane antigen (PSMA) as a complement to the already established F-18- or Ga-68-ligands. Procedures The precursor PSMA-DFO (ABX) was used for Zr-89-labeling. This is not an antibody, but a peptide analogue of the precursor for the production of [177Lu]Lu-PSMA-617. The ligand [89Zr]Zr-PSMA-DFO was compared with [68Ga]Ga-PSMA-11 and [18F]F-JK-PSMA-7 in vitro by determination of the Kd value, cellular uptake, internalization in LNCaP cells, biodistribution studies with LNCaP prostate tumor xenografts in mice, and in vivo by small-animal PET imaging in LNCaP tumor mouse models. A first-in-human PET was performed with [89Zr]Zr-PSMA-DFO on a patient presenting with a biochemical recurrence after brachytherapy and an ambiguous intraprostatic finding with [18F]F-JK-PSMA-7 but histologically benign cells in a prostate biopsy 7 months previously. Results [89Zr]Zr-PSMA-DFO was prepared with a radiochemical purity ≥ 99.9% and a very high in vitro stability for up to 7 days at 37 °C. All radiotracers showed similar specific cellular binding and internalization, in vitro and comparable tumor uptake in biodistribution experiments during the first 5 h. The [89Zr]Zr-PSMA-DFO achieved significantly higher tumor/background ratios in LNCaP tumor xenografts (tumor/blood: 309 ± 89, tumor/muscle: 450 ± 38) after 24 h than [68Ga]Ga-PSMA-11 (tumor/blood: 112 ± 57, tumor/muscle: 58 ± 36) or [18F]F-JK-PSMA-7 (tumor/blood: 175 ± 30, tumor/muscle: 114 ± 14) after 4 h (p < 0.01). Small-animal PET imaging demonstrated in vivo that tumor visualization with [89Zr]Zr-PSMA-DFO is comparable to [68Ga]Ga-PSMA-11 or [18F]F-JK-PSMA-7 at early time points (1 h p.i.) and that PET scans up to 48 h p.i. clearly visualized the tumor at late time points. A late [89Zr]Zr-PSMA-DFO PET scan on a patient with biochemical recurrence (BCR) had demonstrated intensive tracer accumulation in the right (SUVmax 13.25, 48 h p.i.) and in the left prostate lobe (SUV max 9.47), a repeat biopsy revealed cancer cells on both sides. Conclusion [89Zr]Zr-PSMA-DFO is a promising PSMA PET tracer for detection of tumor areas with lower PSMA expression and thus warrants further clinical evaluation. Supplementary Information The online version contains supplementary material available at 10.1007/s11307-021-01632-x.


Introduction
In current clinical practice, tumor localization in patients with biochemical recurrence (BCR) of prostate cancer is the most accepted and validated field of application of PET/CT with Ga-68 or F-18-prostate-specific membrane antigen (PSMA) ligands [1][2][3]. However, in an estimated 20% of patients with biochemical recurrence the tumor will remain undetected with conventional PSMA PET imaging approaches [4][5][6].
There are two main reasons for this: First, about 5-10% of primary prostate cancers express no PSMA [7,8]. Second, different tumors can exhibit a marked heterogeneity in the proportion of PSMA-positive cells they contain [9][10][11].
It cannot be ruled out that weakly PSMA-expressing prostate carcinoma foci are overlooked when using short-lived radionuclides for a PET scan [12]. A negative finding after PSMA PET/CT examinations using Ga-68-or F-18-ligands therefore represents a selection of prostate carcinoma foci with absent or weak PSMA expression. Our aim was to develop a PSMA ligand that can localize BCRs with weak PSMA expression on the basis of late acquisition windows (2 days after injection or later) and hence achieve an improved lesion-to-background ratio. The intention of this study was not to develop an alternative to short-lived radiotracers, but to expand the range of available PET tracers if, despite rising prostate-specific antigen (PSA) values, no convincing tumor detection with Ga-68 or F-18 PSMA tracers is possible. We designed [ 89 Zr]Zr-PSMA-DFO for the rare constellation of a BCR, a preceding PSMA-negative scan with Ga-68-or F-18-PSMA ligands, and the preference for metastasis-directed therapy over androgen deprivation therapy.
To this end, we investigated a new PSMA-binding compound that exploits the longer physical half-life (78.41 h) of Zr-89: [ 89 Zr]Zr-N-sucDf-AMCHA-2Nal-EuK (N-sucDf: N-succineimidedesferrioxamine, AMCHA: tranexamic acid, 2Nal: 2-naphthyl-alanine, E: glutamic acid, u: urea, K: lysine; [ 89 Zr]Zr-PSMA-DFO). The ligand itself is an analogue of the precursor used to prepare [ 177 Lu]Lu-PSMA-617. The only difference lies in the chelating agent for zirconium binding. While in DFO-PSMA, the chelating agent is N-sucDf (blocked temporarily with Fe(III)), in PSMA-617 DOTA is used for this purpose. So in this study, it is not an antibody that was used for Zr-89 labeling but a small molecule.
Previous studies with Zr-89 have focused primarily on antibody-based PET imaging [13,14], as the physical halflife of this radionuclide fits well with the biological half-life of the commonly used antibody constructs. The radionuclide Zr-89 decays into the stable isotope Y-89 by positron emission (23%) and electron capture (77%). The E max of 897 keV and the E ave of 396.9 keV of the emitted positrons are low enough to produce PET images of good spatial resolution. The possibly distorting, spontaneous gamma decay of Zr-89 with 908.97 keV photons (99% abundance) can be masked by adjusting the energy window of the PET scanner [15]. The radionuclide has not previously been used to target PSMAexpressing prostate cancer lesions. produced by changing the reaction parameters, such as pH, amount of precursor, and incubation time, were evaluated to determine the highest radiochemical yield. The unbound Zr-89 was then efficiently removed by solid-phase extraction using a Sep-Pak™ C 18 plus light cartridge.
A complete description of the Zr-89 labeling procedure can be found in the Supplementary information as Supplementary Fig. 1.
To test the release of low molecular weight zirconium species from the PSMA-targeting radiotracer, 100 μl of [ 89 Zr]Zr-PSMA-DFO were added to either 1 ml PBS (PAA Laboratories, Pasching Austria) or 1 ml human serum. The solutions were kept at a constant temperature of 37 °C by means of a heating block (Dry Block Heater 1, IKA, Staufen, Germany) for different periods of time after thorough mixing. The samples were checked for radiochemical purity by radio-thin-layer chromatography immediately after reaching a temperature of 37 °C and again after periods of 1 h, 2 h, 24 h, 48 h, and 72 h. The [ 89 Zr]Zr-PSMA-DFO remained at the origin of the salicylic acid impregnated instant thin-layer chromatography (ITLC) strip (Agilent, CA, USA), while free Zr-89 migrated with the mobile phase (citrate buffer of 0.5 M pH 5.0). The stability of the complexes was calculated as the percentage of complexes remaining at the origin. The radiochemical purity was additionally determined by HPLC (Column: Macherey-Nagel, NUCLEODUR C 18 gravity 5 µm, 110 Å, 250 × 4 mm) at a flow rate of 1.2 ml/min. Elution with 2 min 5% B was followed by the beginning of the gradient of 5-95% B in 15 min, followed by a hold of 5 min. The A solvent was 0.1% trifluoroacetic acid in water, while 0.1% trifluoroacetic acid in acetonitrile was the B solvent. The unreacted Zr-89 was eluted at retention time R t = 2.01 min, while the [ 89 Zr]Zr-PSMA-DFO had a R t = 9.36 min.

Equilibrium Dissociation Constant K d
The equilibrium dissociation constant describing the interaction of the radiolabeled ligands with the PSMA binding sites was determined in PSMA-positive LNCaP cell lines (Cell Line Service, Eppelheim, Germany). LNCaP cells were seeded at a density of 10 6 cells/well on a 6-well plate (Corning, ME, USA) and incubated under 5% CO 2 at 37 °C for 48 h with medium (minimum essential medium Eagle supplemented with 2 mM l-glutamine, 0.1 mM non-essential amino acids (NEAA), 1.0 mM sodium pyruvate, 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin) (Lonza, Verviers, Belgium) in a humidified incubator (Thermo Heracell 150 CO 2 -Incubator, MA, USA). To determine the nonspecific binding, the medium was removed from selected wells, and 1 ml of a solution of 0.1 mM of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) in fresh medium was added. 2-PMPA reliably blocks the PSMA binding sites [20,21]. The plates were then incubated under 5% CO 2 , at 37 °C for 1 h. After that, the medium was removed and the cells incubated under the same incubation conditions for 3 h with different concentrations (0.25 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, and 75 nM) of the radiotracer under investigation in 1 ml fresh medium.
After 3 h, the cells were washed three times with 1 ml PBS, and the cells were finally lysed by adding 1 ml of 1 M NaOH and incubating them for 10 min at room temperature. All samples were measured with a gamma counter (Nuklear Medizintechnik Dresden Isomed 100, Dresden, Germany) and were decay-corrected. The equilibrium dissociation constant (K d ) and the maximum density of receptors (B max ) were calculated by non-linear regression using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, USA).

Cellular Uptake and Internalization
The cell binding determined in these experiments is the sum of specific and nonspecific cell binding. To determine the values for nonspecific cell binding and internalization, all experiments were additionally performed in the presence of 2-PMPA. The plates were incubated for 1 h; after which, 0.75 pmol of the radiotracer to be examined was added to 1 ml of fresh medium. The cells were incubated for 30 min, 1, 2, 3, and 5 h. At each time point, the supernatant was removed and the cells washed with 1 ml of PBS. To dissociate the receptorbound radioligand, the cells were washed twice with 1 ml of a 0.1 M glycine buffer solution at pH 2.8 for 5 min. The 0.1 M glycine-HCl buffer dissociated all the surface-bound complexes. The cells were then washed with 1 ml of PBS, and the internalized fraction was determined by solubilizing the cells with 1 ml of 1 M NaOH and incubating them for 10 min at room temperature. The radioactivity collected from the culture medium, 0.1 M glycine (surface-bound), and 1 M NaOH (internalized fraction) was measured in a gamma counter and decay-corrected. Cell binding was calculated from the surface-bound (0.1 M glycine) and the internalized fraction (1 M NaOH). The internalized fraction was expressed as a percentage of cell binding (internalization to cell-bound radioactivity ratio). All cell uptake experiments were run in triplicate.

Biodistribution in Animal Tissue
After application of the various radiotracers, these experiments were used to determine the radioactivity accumulation in various tissues (percent of the applied radioactivity per gram of organ) and then to calculate tumor/blood, tumor/ muscle, tumor/liver, and tumor/kidney ratios.
Animal experiments were performed in strict accordance with the European Union directive 2010/60/EU for animal experiments, and with the approval of the regional authorities (Ministry for Environment, North Rhine-Westphalia).
A total of 35 mice were used for biodistribution experiments. Male CB17-SCID mice (age: 6 weeks, weight: 17-20 g) were purchased from Charles River Laboratories (Wilmington, USA). Mice were kept in groups of 3-5 with free access to water and food in individually ventilated cages (NexGen EcoFlo, cages Mouse500; Allentown Inc., Allentown, NJ, USA) under controlled conditions (22 ± 1 °C and 55 ± 5% rh) and a 12-h light/dark schedule. The day before implantation of the LNCaP cells, 20 µl of Anti-Asialo GM1 Rabbit (1 mg ml −1 0.9% NaCl) (FUJIFILM Wako Chemicals GmbH, Neuss, Germany) was injected into each mouse. This was done in order to transiently suppress natural killer cell activity, which is preserved in SCID mice [22]. Tumor cells for implantation were harvested by trypsination (TrypLE™ Express, Life Technologies, Paisley, UK) and 8.7 × 10 6 cells in 150 µl PBS with Ca 2+ /Mg 2+ 1:1 with Corning ® Matrigel ® matrix (Corning, NY, USA) were inoculated subcutaneously into the right flank of each mouse. After inoculation, the mice were monitored periodically until the cells had formed a tumor of 300 to 600 mg (approximately 6 weeks). The mice were then used either for biodistribution studies or for PET imaging.
On The organs to be studied (blood, liver, spleen, kidneys, muscle, bone, thyroid, lungs, intestines, tumor, heart, and prostate) were dissected out and weighed. The radioactivity in samples was counted in a gamma counter and decaycorrected. The results for each labeled urea-based inhibitor are expressed as a percent of the injected dose per gram of tissue (% ID/g) and presented as means ± standard deviations (SD) (n = 5). Hence, only one mouse per radiotracer was used for small-animal PET comparisons. The binding specificity of [ 89 Zr]Zr-PSMA-DFO was tested using the PSMA blocker 2-PMPA (23 mg/kg, n = 3 with and n = 3 without 2-PMPA). Scans were conducted under anesthesia on a Focus 220 micro-PET scanner (CTI-Siemens, Germany). Prior to PET imaging, the animals were anesthetized by inhalation of 5% isoflurane/gas mixture (O 2 /air 3:7). Thereafter, the anesthesia was reduced and maintained at a concentration of 2% isoflurane/gas mixture.

Small-Animal PET in Mice Bearing an LNCaP Tumor Xenograft
Emission scans were performed for 60 min, starting 60 min after tracer injection. Additional scans of 60 min duration were performed 4 h, 21 h, and 48 h after injection of [ 89 Zr]Zr-PSMA-DFO. All emission scans were followed by a 10-min transmission scan with a Co-57 point source for attenuation correction. Summed images were reconstructed using an iterative OSEM3D/MAP procedure resulting in voxel sizes of 0.47 × 0.47 × 0.80 mm. Post-processing and image analysis was performed with VINCI 4.72 (Max-Planck-Institute for Metabolism Research, Cologne, Germany). Images were Gauss-Filtered (1 mm FWHM) and intensity-normalized to injected dose, corrected for body weight (SUV bw ). For this, every frame was divided by injected dose and multiplied by 100 * body weight. The patient had given his written informed consent for PET imaging and the scientific evaluation of his data. All procedures were performed in accordance with the Institutional Review Board and the regulations of the regional authorities in Cologne. The kidney dose was estimated on the basis of two PET scans. The following assumptions were made for the estimation: Between time 0 (injection) and the first measuring point, the time-activity-curve follows a constant progression. All measuring points were integrated numerically using trapezoidal approximation. From the last measuring point to infinity, a mono-exponential function was fitted and integrated. As the effective half-life could not be accurately determined from two measurement points, the physical half-life of Zr-89 was i., a mixed-effects analysis was used for organ uptake, and 1-way ANOVAs for the tumor-to-blood, -kidney-and -muscle ratios. For the PET experiments with the tumor xenograft-bearing mice, three separate 2-way mixed design ANOVA tests (for tumor, liver, and kidneys, respectively) were used with the factors blocking ([ 89 Zr]Zr-PSMA-DFO with or without the blocking agent 2-PMPA) and time point (repeated measures). All ANOVA tests were followed by Sidak's or Tukey's multiple comparison procedures. The significance level was always p < 0.05.

Radiochemistry and Stability
After the removal of free Zr-89 by solid-phase extraction using a Sep-Pak™ C 18 Table 1.
The Zr-89-radioligand was found to be stable over a period of 7 days at 37 °C in PBS and human serum. The stability test was performed in thin-layer chromatography (TLC) solely to identify free Zr-89 at 1 h, 2 h, 24 h, 48 h, 72 h, and 7 days. However, the stability in PBS was measured in parallel with HPLC at same time intervals, and one single peak was identified at the retention time of [ 89 Zr]Zr-PSMA-DFO (R t = 9.35 min).

Affinity, Cell Binding, and Internalization of [ 89 Zr]Zr-PSMA-DFO in Comparison with [ 18 F] F-JK-PSMA-7 and [ 68 Ga]Ga-PSMA-11
The radioligands showed no important differences with regard to their in vitro behavior when interacting with the LNCaP cells. The binding curves including Scatchard Plots are shown in Supplementary Fig. 2. Table 2 gives an overview of the results of these investigations.
Checking the relationship between B max and molar activity by linear regression reveals a clear linear relationship: B max = 25.37 A m -82.63 (R 2 = 0.9986, p = 0.002).
The specific binding of radioactively labeled ligands to LNCaP cells, as a percentage of the total activity, ranged between 45.8 and 49.4% after 5 h. The specific internalized activity in LNCaP cells, expressed as a percentage of the cell activity relative to the specific cell-bound activity, was between 58 and 62% after 5 h. More detailed information on the respective values and their statistics can be found in the Supplementary information (Supplementary Tables 1  and 2).

Small-Animal PET in Mice Bearing an LNCaP Tumor Xenograft
The LNCaP tumors were clearly visible with all the tracers tested, when measured for 60 min starting at 1 h p.i. (Fig. 2).
Owing to the longer physical half-life of Zr-89, it was also possible to obtain PET images up to 48 h after injection (N = 3, Fig. 3).

[ 89 Zr]Zr-PSMA-DFO in a Patient with PCA Recurrence
A first-in-human study was conducted with [ 89 Zr]Zr-PSMA-DFO in a 60-year-old patient with BCR (Fig. 4) Signal-to-noise ratios (SNR) of [ 89 Zr]Zr-PSMA-DFO were 1.9 and 2.0 in the first and second PET scans, respectively. These ratios were lower than those obtained for [ 18

Discussion
The following results can be derived from the first radiopharmaceutical, biochemical, and biological data obtained with [ 89 Zr]-Zr-PSMA-DFO, a PSMA-targeting agent with a longer half-life than Ga-68-or F-18-PSMA ligands, with which it was compared: 1. [ 89 Zr]Zr-PSMA-DFO has been produced with high radiochemical purity of > 99%, stability lasting at least In the search for a suitable radionuclide for the production of PSMA-targeting ligands, allowing a PET scan to be performed at least 24 h p.i., zirconium-89, which, was considered a promising candidate. The zirconium isotope has become established in immuno-positron emission tomography (PET) imaging in recent years and, with a half-life of 3.27 days, meets the requirements better than Cu-64 (0.53 day), Tb-152 (0.73 day), or Sc-44 (0.167 day)-PET radionuclides whose suitability for the production of radioactive PSMA ligands was demonstrated not so long ago [25][26][27][28][29]. The proven high long-term stability of the Zr-89-labeled, PSMA-affine PET tracer in combination with its long half-life also has practical advantages. There is no need to produce the radiopharmaceutical several times a week. Of course, one could argue that nowadays F-18 tracers can also be easily obtained via existing radiopharmaceutical networks in most areas with PET scanners. Nevertheless, the fact that the [ 89 Zr]Zr-PSMA-DFO can be stored for a longer period of time and can be applied 7 days after synthesis with a one-off tracer production run speaks for itself. Apart from that, [ 89 Zr]Zr-PSMA-DFO can also be transported over longer distances without any major loss of quality.
The labeling procedure, including cleaning and quality control, was feasible within a time frame of about 120 min. The yield based on the Zr-89 activity employed was 75% in optimum cases. After final purification, the radiochemical purity was excellent and remained > 99% even on storage in aqueous solution for 7 days.
The linear relationship between the molar activities of the radioactive PSMA vectors examined in this study and their maximum achievable concentration on 10 6 tumor cells (B max ) can be summarized as follows: The higher the molar activity of the radioligand, the more radioligand molecules can be bound to a given number of tumor cells. The assumption derived from this that with higher molar activity, there are also higher radioactivity concentrations in the tumor tissue and higher tumor-background ratios have not been confirmed (see the "Limitations" section).
Nevertheless, the key question as set out in the introduction, of whether the results obtained with [ 89 Zr]Zr-PSMA-DFO correspond to those obtained with [ 68 Ga]Ga-PSMA-11 and [ 18 F]F-JK-PSMA-7 or are better, can be answered as follows: [ 89 Zr]Zr-PSMA-DFO showed no inferiority compared to the other PSMA tracers regarding in vitro experiments for affinity, binding to prostate cancer tumor cells, and biokinetics.
PSMA-DFO, which serves as an in vivo vehicle for Zr-89, is itself a relatively small molecule with faster clearance than, for example, an antibody. This is reflected in the low levels of activity, for example in blood and muscles even at 2 h p.i. The question therefore arises whether it makes sense to combine a relatively long-lived nuclide with a ligand with a short biological half-life. Our cell biological studies showed that [ 89 Zr]Zr-PSMA-DFO showed significant internalization. In the context of this work, it can only be assumed that this leads to a re-complexation of the zirconium within the cell and a binding to intracellular molecules. This assumption is supported by a number of studies. Current publications show that Zr-89 is bound intracellularly after it has been able to penetrate the cell wall as a lipophilic complex [30,31]. Fung et al. [32] observed differences in the clearance rates of radioactivity from the tumor for two forms of the  the radioimmunoconjugates was the type of radiolabeling. The authors attribute this difference to typical zirconium trapping mechanisms within the tumor cells which do not operate in the case of non-residualizing of iodine. Similar results were found by Cheal et al. [33] when comparing an antibody against the clear cell renal carcinoma labeled with Zr-89 or I-124. These and other authors conclude that Zr-89 is a residualizing isotope and remains in cells after internalization, allowing activity to accumulate and concentrate in tumor cells while removing non-localized activity from the body, ultimately resulting in high-contrast images.
Our blocking experiment with 2-PMPA showed that in the mouse approximately two-thirds of renal radioactivity is due to specific binding of the PSMA tracer. Around one-third is not blockable, and therefore reflects renal excretion of the radiotracer and its metabolites. Forty-eight hours after injection of [ 89 Zr]Zr-PSMA-DFO, there was no detectable radioactivity in the bladder in mice. Thus, Zr-89 is well-suited to detect lesions in the genitourinary tract at late time points, when radioactivity has already cleared from the kidneys and bladder. The scheduling of tracer injection and PET scans on different days was chosen as the optimal arrangement for a few patients with a rare constellation of findings and on account of patient preference.
On the basis of the excellent preclinical imaging properties of [ 89 Zr]Zr-PSMA-DFO, we carried out the first observational study on [ 89 Zr]Zr-PSMA-DFO PET/CT in a patient with BCR. The activity used (93 MBq) was not derived from a phase-1 trial, but was analogous to the labeling of antibodies with Zr-89 [34]. The first use in humans resulted in a promising performance with regard to clear localization of PSMA-positive tumor tissue when the preceding PET with [ 18 F]F-JK-PSMA-7 had been interpreted as PSMA-negative or equivocal. The radiation exposure should be weighed against the potential benefit of metastasis-directed therapy or salvage radiotherapy. Additional clinical data in a series of patients will be published in due course and will evaluate whether [ 89 Zr]Zr-PSMA-DFO PET can improve the contrastto-noise ratio in patients with weakly PSMA-positive lesions.

Limitations
Some of the tumor/background ratios determined by measuring the radioactivity of organ samples are unexpected. The highest tumor-muscle ratio was determined just 2 h after the injection of [ 89 Zr]Zr-PSMA-DFO. The tumor-to-background ratios for [ 68 Ga]Ga-PSMA-11 were significantly lower compared to those of the other radioactive PSMA vectors. It is noticeable that, for example, the tumor/blood ratios given in the literature for [ 68 Ga]Ga-PSMA-11 at 2 h p.i. differ widely between individual studies [34][35][36][37]. In animal experiments on mice, it should be taken into account that owing to the low blood volume, and variations in the molar activities of the radioactive PSMA ligand used by different authors, different tumor models, or mouse strains can produce different results.
Our work should be seen as a first feasibility study to investigate the suitability of [ 89 Zr]Zr-PSMA-DFO for PET examinations of prostate carcinoma lesions with weak PSMA expression. The data collected are not yet sufficient to make generalizable statements about radiation exposure from the radiotracer. This is the subject of a study involving several BCR patients that will be published soon.

Conclusion
This preclinical study demonstrates that the tumor uptake and biodistribution of Funding Open Access funding enabled and organized by Projekt DEAL.

Conflict of Interest
The authors declare that they have no conflict of interest.
Ethics Approval All applicable institutional and/or national guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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