Abstract
89Zr (half-life, 78.41 h) is a positron-emitting radionuclide that displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In the current study, we report the preparation of 89Zr-desferrioxamine B (DFO)-J591, a novel 89Zr-labeled monoclonal antibody (mAb) construct for targeted immunoPET and quantification of prostate-specific membrane antigen (PSMA) expression in vivo. Methods: The in vivo behavior of 89Zr-chloride, 89Zr-oxalate, and 89Zr-DFO was studied using PET. High-level computational studies using density functional theory calculations have been used to investigate the electronic structure of 89Zr-DFO and probe the nature of the complex in aqueous conditions. 89Zr-DFO-J591 was characterized both in vitro and in vivo. ImmunoPET in male athymic nu/nu mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors was conducted. The change in 89Zr-DFO-J591 tissue uptake in response to high- and low-specific-activity formulations in the 2 tumor models was measured using acute biodistribution studies and immunoPET. Results: The basic characterization of 3 important reagents—89Zr-chloride, 89Zr-oxalate, and the complex 89Zr-DFO—demonstrated that the nature of the 89Zr species dramatically affects the biodistribution and pharmacokinetics. Density functional theory calculations provide a rationale for the observed high in vivo stability of 89Zr-DFO–labeled mAbs and suggest that in aqueous conditions, 89Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of 2 water molecules. 89Zr-DFO-J591 was produced in high radiochemical yield (>77%) and purity (>99%), with 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 remained active for up to 7 d. In vivo biodistribution experiments revealed high, target-specific uptake of 89Zr-DFO-J591 in LNCaP tumors after 24, 48, 96, and 144 h (34.4 ± 3.2 percentage injected dose per gram [%ID/g], 38.0 ± 6.2 %ID/g, 40.4 ± 4.8 %ID/g, and 45.8 ± 3.2 %ID/g, respectively). ImmunoPET studies also showed that 89Zr-DFO-J591 provides excellent image contrast, with tumor-to-muscle ratios greater than 20, for the delineation of LNCaP xenografts between 48 and 144 h after administration. Conclusion: These studies demonstrate that 89Zr-DFO–labeled mAbs show exceptional promise as radiotracers for immunoPET of human cancers. 89Zr-DFO-J591 displays high tumor–to–background tissue contrast in immunoPET and can be used to delineate and quantify PSMA-positive prostate tumors in vivo.
- immunoPET
- 89Zr
- prostate-specific membrane antigen (PSMA)
- J591
- monoclonal antibodies
- density functional theory
The National Cancer Institute estimated that in the United States during 2009, approximately 192,000 cases of prostate cancer (PC) would be diagnosed, with a projected mortality rate of over 27,000 men (>14%). Despite the high incidence of PC, standard diagnostic imaging techniques used for the detection and monitoring of therapy remain inadequate. For example, early-stage, hormonally sensitive tumors on treatment and noncastrate PCs often appear negative on PET scans using either the metabolic radiotracer 18F-FDG or the hormone-based radiopharmaceutical 16β-18F-dihydrotestosterone (18F-FDHT) for imaging the overexpression of androgen receptors (1). Therefore, there is an urgent requirement to develop new tools for the noninvasive delineation and staging of PC in vivo.
Prostate-specific membrane antigen (PSMA) is a 100-kDa, type II transmembrane glycoprotein and is one of the best characterized oncogenic markers or targets (2,3). PSMA expression has been detected in a limited range of normal tissues including benign prostatic epithelium, renal proximal tubule, small bowel, and brain (a subset of astrocytes). However, these normal sites express PSMA at levels 2–3 orders of magnitude lower than that observed in more than 95% of clinical PC specimens (4,5). In addition, these normal-tissue PSMA sites are highly polarized to the apical or luminal aspect of the benign prostatic glands, renal tubules and small bowel, basement membrane, and epithelial tight junctions, which form substantial barriers to circulating monoclonal antibodies (mAbs). PSMA expression by astrocytes is similarly sequestered behind the blood–brain barrier. As a result, antibodies to PSMA are functionally tumor-specific, whereas small-molecule PSMA ligands excreted via the renal tubular lumen are not.
PSMA expression levels have been shown to exhibit a positive correlation with increased tumor aggression, metastatic spread, and the development of castrate resistance, or resistance to hormone-based therapies. PSMA expression has also been reported in the neovasculature of most solid tumors (6). The failure of 18F-FDG PET for detecting early and treated PC and the acquired resistance of many advanced PCs to androgen-based agents have been the driving force behind recent efforts toward developing novel chemo- and radioimmunoconjugate-based drugs and imaging agents. In particular, in 1996 the U.S. Food and Drug Administration approved the use of 111In-capromab pendetide or 111In-7E11 (111In-ProstaScint; Cytogen Corp.), a murine–mAb specific for an intracellular epitope of PSMA, for SPECT of PC soft-tissue metastases. However, 111In-capromab pendetide for clinical diagnosis is suboptimal because of low sensitivity for viable tumor sites (62% for lymph node metastases, 50% for prostate bed recurrence), which is probably because the number of available targets (presented in dead or dying tissue) is limited. Furthermore, 111In-capromab pendetide does not bind to viable PC sites in bone (the most common site of metastatic disease), and in contrast to PET, SPECT remains only semiquantitative in the clinical setting. Despite these limitations, the National Comprehensive Cancer Network Clinical Practice Guidelines (5)—which propose using 111In-capromab pendetide before salvage therapy after radiotherapy or prostatectomy—still recommend the mAb as being useful for specific clinical situations. This fact is testament to the relative lack of better imaging methods for the detection of metastatic prostate cancer, especially in soft tissue.
In 1997, Liu et al. produced 4 mAbs (IgG1: J415, J533, and J591; and IgG3: E99) specific for binding to 2 distinct epitopes on the extracellular domain of PSMA (7,8). Subsequent in vitro and in vivo studies identified J591 as the most promising candidate for developing diagnostic and therapeutic immunoconjugates for the targeting of extracellular PSMA in viable tissue (9–11). Since these initial studies, J591 has been humanized, and a range of preclinical and clinical studies using J591 radiolabeled with 90Y, 177Lu, or 131I for β-therapy; 213Bi and 225Ac for α-therapy; and 111In for SPECT have been reported (9–20).
The work presented here describes the production and preclinical evaluation of 89Zr-radiolabeled humanized-J591 for targeted immunoPET of PSMA-positive tumors in vivo.
MATERIALS AND METHODS
Full details of all methods and equipment used are presented in the supplemental materials (available online only at http://jnm.snmjournals.org).
Density Functional Theory (DFT) Calculations
All calculations were conducted using DFT as implemented in the Gaussian03 suite of ab initio quantum chemistry programs (21). Full computational details and Cartesian coordinates of the optimized structures are presented in the supplemental materials. Energetic values are reported in units of kJ mol−1.
Antibody Conjugation and Radiolabeling
The humanized IgG1 mAb J591 was conjugated to the tris-hydroxamate hexadentate chelate, desferrioxamine B (DFO) (Calbiochem), using a 6-step procedure modified (22) from the approach described by Verel et al. (23) (supplemental material).
89Zr was produced via the 89Y(p,n)89Zr transmutation reaction on a TR19/9 variable-beam-energy cyclotron (Ebco Industries Inc.) in accordance with previously reported methods (23,24). The 89Zr-oxalate was isolated in high radionuclidic and radiochemical purity (RCP) greater than 99.9%, with an effective specific activity of 195–497 MBq/μg, (5.28–13.43 mCi/μg) (24).
Stability Studies
The stability of 89Zr-DFO-J591 with respect to change in RCP, loss of radioactivity from the mAb, or change in immunoreactivity was investigated in vitro by incubation in solutions of 0.9% saline and 1% bovine serum albumin for 7 d at 37°C. The RCP was determined by radio–instant thin-layer chromatography and γ-counting, and the immunoreactive fraction was measured using the LNCaP cellular binding assay.
Xenograft Models
All animal experiments were conducted in compliance with Institutional Animal Care and Use Committee guidelines. Male athymic nu/nu mice (NCRNU-M, 20–22 g, 6–8 wk old) were obtained from Taconic Farms Inc. and were allowed to acclimatize at the Memorial Sloan-Kettering Cancer Center vivarium for 1 wk before tumors were implanted. Mice were provided with food and water ad libitum. In separate animals, LNCaP (PSMA-positive) and PC-3 (PSMA-negative) tumors were induced on the left and right shoulders, respectively. Full details are provided in the supplemental material.
Acute Biodistribution Studies
LNCaP and PC-3 tumor–bearing mice were randomized before the study and were warmed gently with a heat lamp 5 min before administration of 89Zr-DFO-J591 (0.55–0.74 MBq [15–20 μCi], 3–4 μg of mAb, in 200 μL of sterile saline for injection) via injection into the tail vein (0 h). Animals (n = 3–5, per group) were euthanized by CO2 gas asphyxiation at 24, 48, 96, and 144 h after injection, and 12 organs (including the tumor) were removed, rinsed in water, dried in air for 5 min, weighed, and counted on a γ-counter for accumulation of 89Zr radioactivity. Full details are presented in the supplemental material.
Small-Animal immunoPET
PET experiments were conducted on a microPET Focus 120 scanner (Concorde Microsystems) (25). Mice were administered 89Zr-DFO-J591 formulations (10.9–11.3 MBq [295–305 μCi], 60–62 μg of mAb, in 200 μL of 0.9% sterile saline for injection) via injection into the tail vein. Approximately 5 min before PET images were recorded, mice were anesthetized by inhalation of a 1% isoflurane (Baxter Healthcare)/oxygen gas mixture and placed on the scanner bed. PET images were recorded at various times between 3 and 144 h after injection (supplemental material).
Statistical Analysis
Data were analyzed using the unpaired, 2-tailed Student t test. Differences at the 95% confidence level (P < 0.05) were considered to be statistically significant.
RESULTS
DFT Calculations
The complexation reaction between 89Zr-chloride or 89Zr-oxalate and the hexadentate, tris-hydroxamate chelate DFO is shown in Figure 1A. Structures of the octahedral complex [89Zr(HDFO)]2+ (1), the 7-coordinate complexes with mono-H2O coordination in the axial and equatorial sites [89Zr(HDFO)-ax-(H2O)]2+ (2-ax) and [89Zr(HDFO)-eq-(H2O)]2+ (2-eq), and the 8-coordinate complex [89Zr(HDFO)-cis-(H2O)2]2+ (3-cis) were fully optimized using DFT. The optimized structure of the 8-coordinate complex 3-cis is shown in Figure 1B (Supplemental Tables 1–7; Supplemental Figs. 1 and 2).
(A) Complexation reaction between [89Zr(C2O4)4]4− and DFO. (B) DFT-optimized structure of 8-coordinate complex [89Zr(HDFO)-cis-(H2O)2]2+ (3-cis).
The calculations revealed that expansion of the coordination sphere to either 7 or 8 coordinates by the addition of 1 or 2 water molecules is thermodynamically favorable. Interestingly, in the 7-coordinate species the axial and equatorial coordination sites, 2-ax and 2-eq, are energetically inequivalent. Axial coordination (2-ax) is thermodynamically more favorable than equatorial coordination (2-eq) by around −41 kJ mol−1. The DFT calculations also suggest that the 8-coordinate complex with cis-coordination geometry with respect to the orientation of the H2O molecules (3-cis) is 95 kJ mol−1 more stable than the parent octahedral complex (1). Furthermore, complex 3-cis is 14 kJ mol−1 more stable than the sum of the thermodynamic stabilization achieved by complexes 2-ax and 2-eq (sum = −81 kJ mol−1). This additional stability of complex 3-cis arises because of structural relaxation from the cooperativity of the 2-coordinated H2O ligands, which allows the r(Zr-OH2(ax)) to decrease from 0.236 nm in complex 2-ax to 0.233 nm in complex 3-cis (Supplemental Table 6).Natural bond-order charge analysis (Supplemental Table 7) also supports the conclusion that thermodynamic stabilization of complex 3-cis arises from increased electrostatic interaction between the Zr4+ ion and axial H2O ligand (ligand-to-metal charge transfer). We expect that the coordinated H2O ligands would be kinetically labile and that species 1–3 were likely in rapid equilibrium at physiologic temperatures.
Basic Characterization of 89Zr Species In Vivo
Before full studies on 89Zr-radiolabeled mAbs are begun, it is important to understand the in vivo behavior of various 89Zr-labeled species, including the starting reagents, and potential impurities or metabolites. Therefore, we examined the biodistribution of 89Zr-chloride and 89Zr-oxalate and the complex 89Zr-DFO using PET (Fig. 2). Maximum-intensity-projection images of 89Zr-chloride and 89Zr-oxalate (11.1 MBq [300 μCi], 200 μL of sterile saline) were recorded at 24 h after injection in the tail vein of male athymic nu/nu mice (24). 89Zr-chloride was found to be sequestered in the liver, with little excretion (Supplemental Fig. 3). In contrast, administration of 89Zr-oxalate (most likely present as the thermodynamically stable species [89Zr(C2O4)4]4−) showed a high accumulation of 89Zr radioactivity in bones, joints, and potentially cartilage (Supplemental Fig. 4). Previous dynamic PET studies on 89Zr-DFO demonstrated that this complex is excreted rapidly within 20 min via a renal pathway, with a measured biologic half-life of 305 ± 6 s (Supplemental Figs. 5 and 6).
PET images showing maximum intensity projection of 89Zr-chloride and 89Zr-oxalate at 24 h after intravenous administration and dynamic PET images of 89Zr-DFO at 1 and 4 min after injection. For maximum-intensity-projection images, upper and lower intensity thresholds were set at 100% and 0%, respectively. Further details are presented in Supplemental Figures 3–6. MIP = maximum intensity projection.
Radiochemistry
J591 was functionalized with DFO using bioconjugation methods modified from the pioneering work of Verel et al. (23). The conjugation and purification chemistry was found to proceed in a moderate to high yield (65% ± 5%), with high chemical purity (>95%). Radiolabeling of DFO-J591 with 89Zr-oxalate was achieved at room temperature, in slightly alkaline solutions (pH 7.7–8.1), with crude radiochemical yields (>95%, n = 6). Facile purification of 89Zr-DFO-J591 from small-molecule radiolabeled impurities was achieved using either size-exclusion chromatography or spin-column centrifugation. The final radiochemical yield of the purified 89Zr-DFO-J591 was greater than 77%, and the product was formulated in 0.9% sterile saline with an RCP greater than 99% (n = 6) and a specific activity of 181.7 ± 1.1 MBq/mg (4.91 ± 0.03 mCi/mg) of mAb (Supplemental Figs. 7 and 8). The specific activity obtained in these studies compares favorably with the previously reported specific activities of other 89Zr-radiolabeled mAbs (22,26–32). Isotopic dilution assays revealed an average of 3.9 ± 0.3 accessible chelates per mAb.
89Zr-DFO-J591 Immunoreactivity and Stability Studies In Vitro
The immunoreactive fraction of the 89Zr-DFO-J591 formulations was measured by specific in vitro cellular association assays using LNCaP (PSMA-positive) cells before each in vivo experiment (Supplemental Fig. 9) (33). In studies using 213Bi-labeled J591, McDevitt et al. reported that the LNCaP cell line had an estimated 180,000 PSMA molecules per cell (11). However, in other studies using 111In- and 131I-labeled mAbs (including J415, J533, J591, and 7E11), we found higher PSMA copy numbers (600,000–800,000 sites/cell) for viable LNCaP cells (9). The average immunoreactive fraction of 89Zr-DFO-J591 was 0.95 ± 0.03 (n = 4). Control experiments (n = 4) using the PC-3 (PSMA-negative) cell line showed no binding, further demonstrating the specificity of 89Zr-DFO-J591 for PSMA-expressing cells.
Incubation of 89Zr-DFO-J591 in either saline or 1% bovine serum albumin for 7 d at 37°C revealed a less than 2% decrease in RCP (via demetalation), with an observed approximate 17% decrease in the immunoreactive fraction for the 1% bovine serum albumin experiment (0.78 ± 0.03, Supplemental Fig. 10). Therefore, in the absence of specific proteolysis or reductive or oxidative metabolism, 89Zr-DFO-J591 is expected to remain intact and immunoreactive in vivo on a time scale suitable for immunoPET.
Biodistribution Studies
The ability of 89Zr-DFO-J591 to target an extracellular epitope of the PSMA type II transmembrane glycoprotein receptor in vivo was initially assessed by conducting acute biodistribution studies in LNCaP tumor–bearing mice at 24, 48, 96, and 144 h after intravenous administration (Table 1; Supplemental Table 8; Fig. 3). The data reveal that high LNCaP tumor uptake was observed 24 h after injection (34.4 ± 3.2 percentage injected dose per gram [%ID/g]), with a steady increase through 48 (38.0 ± 6.2 %ID/g) and 96 h (40.4 ± 4.8 %ID/g) and reaching 45.8 ± 3.2 %ID/g at 144 h (P = 0.01 between LNCaP uptake at 24 and 144 h). This high accumulation of 89Zr-DFO-J591 is consistent with extraction of the activity from the blood pool (24 h, 21.8 ± 2.8 %ID/g; 48 h, 4.4 ± 1.9 %ID/g; and 96 h, 1.4 ± 0.8 %ID/g) and rapid internalization of the J591–PSMA complex, followed by sequestration of the 89Zr radioactivity inside the cell. In contrast, 89Zr-DFO-J591 uptake in the PC-3 (PSMA-negative) tumors at 48 (15.6 ± 2.1 %ID/g, P = 0.0025) and 96 h (24.0 ± 2.6 %ID/g, P = 0.0017) showed a statistically significant decrease in 89Zr accumulation, compared with uptake in PSMA-positive tumors. 89Zr-DFO-J591 activity in the blood remained 4-fold higher at 48 h (19.0 ± 1.1 %ID/g, P = 0.001) and 10-fold higher at 96 h (13.0 ± 1.8 %ID/g, P < 0.05) in mice bearing PC-3 tumors, compared with the corresponding LNCaP tumor–bearing mice (48 h, 4.4 ± 1.9 %ID/g; 96 h, 1.4 ± 0.8 %ID/g).
Biodistribution Data of 89Zr-DFO-J591, Administered Intravenously to Mice Bearing Subcutaneous LNCaP Tumors
Bar chart showing selected tissue biodistribution data (%ID/g) for uptake of either high- (181.7 ± 1.1 MBq/mg [4.91 ± 0.03 mCi/mg]; 3–4 μg of mAb per mouse) or low-specific-activity (60-fold decrease, 3.04 MBq/mg [0.082 mCi/mg]; 300 μg of mAb per mouse) formulations of 89Zr-DFO-J591 (0.55–0.74 MBq [15–20 μCi], in 200 μL of sterile saline for injection) in male athymic nu/nu mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors.
Competitive inhibition (blocking) studies using low-specific-activity formulations (60-fold decrease, 3.04 MBq/mg [0.082 mCi/mg]), compared with high-specific-activity formulations, revealed only 10.3 ± 0.8 %ID/g tumor uptake at 48 h after injection, an approximate 4-fold decrease (P < 0.002) (Tables 1 and 2; Fig. 3). Furthermore, in the low-specific-activity experiments, 89Zr-DFO-J591 activity in the blood remained high (2- to 3-fold higher at 48 h, 10.7 ± 0.4 %ID/g, P = 0.026), but 89Zr-accumulation in the liver showed a statistically significant decrease from 17.7 ± 1.6 %ID/g to 5.1 ± 0.4 %ID/g (P < 0.004). The competitive inhibition experiments concur with the in vitro data and further demonstrate the specificity of 89Zr-DFO-J591 for the PSMA in vivo.
Biodistribution Data of 89Zr-DFO-J591, Administered Intravenously to Mice Bearing Subcutaneous PC-3 Tumors (3–4 μg of mAb)
Interestingly, in the LNCaP tumor–bearing mice, 89Zr uptake in the bone was relatively high and increased between 24 and 96 h (24 h, 4.0 ± 0.8 %ID/g; 48 h, 8.2 ± 1.2 %ID/g; and 96 h, 8.7 ± 1.5 %ID/g) before decreasing slightly to 7.4 ± 1.3 %ID/g at 144 h. In contrast, bone accumulation of 89Zr activity in the PC-3 tumor–bearing mice was reduced by approximately 45% at 48 and 96 h (4.3 ± 0.6 %ID/g and 5.1 ± 0.5 %ID/g, respectively).
ImmunoPET with 89Zr-DFO-J591
Temporal immunoPET images of 89Zr-DFO-J591 (10.9–11.3 MBq [295–305 μCi], 60–62 μg of mAb, in sterile saline [200 μL]) recorded in LNCaP and PC-3 tumor–bearing mice between 3 to 144 h are presented in Figure 4. Time–activity curves generated from the immunoPET images showing the mean %ID/g radiotracer uptake in various tissues including the heart and blood pool, liver, and muscle in mice bearing LNCaP (n = 3) or PC-3 (n = 3) tumors are given in Figure 5. Radiotracer uptake in LNCaP tumors was observed less than 24 h after injection of 89Zr-DFO-J591, and high tumor-to-muscle (T/M) ratios (calculated using the mean %ID/g values derived from volume-of-interest analysis of the immunoPET images) were observed. At 48 h after injection, the immunoPET-measured mean and maximum %ID/g for radiotracer uptake in LNCaP tumor–bearing mice was 21.9 ± 0.6 and 38.2 ± 4.9 %ID/g, respectively, with a mean T/M ratio of 15.85 (Supplemental Table 8). By 120 and 144 h, the mean T/M ratio in LNCaP tumors increased to 22.49 and 25.89, respectively. The lower uptake observed in the quantitative immunoPET studies, compared with the biodistribution studies, is likely due to the different total masses of mAb administered (22).
Temporal immunoPET images of 89Zr-DFO-J591 (10.9–11.3 MBq [295–305 μCi], 60–62 μg of mAb, in 200 μL of sterile saline) recorded in LNCaP tumor–bearing (PSMA-positive, left shoulder) (A) and PC-3 tumor–bearing (PSMA-negative, right shoulder) (B) mice between 3 and 144 h after injection. Transverse and coronal planar images intersect center of tumors, and mean tumor-to-muscle ratios derived from volume-of-interest analysis of immunoPET images are given. Upper thresholds of immunoPET have been adjusted for visual clarity, as indicated by scale bars.
Time–activity curves derived by volume-of-interest analysis of immunoPET images showing mean %ID/g tissue uptake vs. time/h for 89Zr-DFO-J591 radiotracer accumulation in mice bearing subcutaneous LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors. Complete time–activity curve data for 89Zr-DFO-J591 immunoPET imaging is given in supplemental materials (Supplemental Tables 9 and 10; Supplemental Figs. 11–13).
In contrast to the high absolute tumor uptake and tumor-to-background contrast ratios observed in the LNCaP model, low accumulation and immunoPET contrast ratios for 89Zr-DFO-J591 uptake in PC-3 (PSMA-negative) tumors (Supplemental Tables 9 and 10; mean T/M ratios of 3.48, 4.36, and 4.19 at 48, 120, and 144 h, respectively) were observed. Uptake in these PC-3 tumors is in accordance with the enhanced permeation and retention mechanism (Supplemental Tables 9 and 10; Supplemental Figs. 11–13).
DISCUSSION
PET has distinct advantages over SPECT in terms of sensitivity and contrast resolution, especially for deep tissues, and these improved imaging characteristics are particularly important for radioimmunoimaging. Basic characterization of the in vivo behavior of several important 89Zr-labeled species, including the starting reagents 89Zr-chloride and 89Zr-oxalate and the key complex 89Zr-DFO, are reported. The nature of the aqueous-phase 89Zr species using PET was shown to dramatically affect the in vivo biodistribution, with 89Zr-chloride and 89Zr-oxalate sequestering for over 24 h in the liver and bones, respectively. In contrast, 89Zr-DFO is first-pass excreted through the kidneys and accumulates in the bladder, with a biologic half-life of 305 ± 6 s.
In this work, the novel radiopharmaceutical 89Zr-DFO-J591 has been characterized by a range of stability and cellular association assays in vitro. Previous studies have shown that although diethylenetriaminepentaacetic acid can be used for chelation and radiolabeling of mAbs with 89Zr4+ ions, demetalation occurs in vivo, and until new ligands are produced DFO remains the chelate of choice (24,34,35). Experiments on 89Zr-DFO mAbs have reported high in vivo stability with respect to demetalation or ligand dissociation and relatively low levels of radiotracer accumulation in background tissue in both animals and humans (26–28,30–32).
The nature of the electronic structure of the 89Zr–DFO complex has been explored using high-level DFT calculations. The computational results provide a rationale for the high experimentally observed in vitro and in vivo stability of the 89Zr-DFO–labeled radioimmunoconjugates. DFT studies suggest that the origin of the observed in vivo stability of 89Zr-DFO mAbs lies in a combination of the inherently high thermodynamic and kinetic stability of the 89Zr–DFO complex due to strong electrostatic interactions, coupled with the enhancement in thermodynamic stability induced by expansion of the first coordination sphere and geometry relaxation to give an 8-coordinate species. Indeed, in the case of 89Zr-DFO—and in contrast to the more familiar complexes with radionuclides of copper, gallium, indium, and yttrium—these calculations indicate that the presence of water or, for example, coordinating anions such as chloride, may actually increase the thermodynamic stability of the 89Zr–DFO complex in vivo.
The ability of 89Zr-DFO-J591 to target PSMA-expressing tissue was examined using acute biodistribution studies and immunoPET in vivo. The results demonstrate that 89Zr-DFO-J591 shows high specific uptake in LNCaP (PSMA-positive) tumors. Although a direct comparison with earlier work is made difficult because of the use of different models and murine-J591, the absolute tissue uptake values of 89Zr-DFO-J591 (humanized mAb) in most organs at various time points were found to be higher than those observed for either 111In-DOTA-labeled or 131I-labeled J591 (10). For example, at 48 h after administration the tumor uptake value was 13.6 ± 2.8 for 111In-DOTA-J591 and 11.2 ± 2.9 %ID/g for 131I-J591, with corresponding blood-pool activities of 8.98 ± 2.10 and 8.57 ± 2.04 %ID/g, respectively. In contrast, tumor uptake and concordant blood-pool activity of 89Zr-DFO-J591 at 48 h were 38.0 ± 6.2 and 4.4 ± 1.9 %ID/g, respectively. As revealed in the biodistribution data, the high immunoreactivity and specificity of 89Zr-DFO-J591 (Tables 1 and 2; Supplemental Table 8) led to a high uptake in the PSMA-positive tumors.
The degree of bone uptake is consistent with previously reported studies using various other 89Zr-labeled mAbs—including 89Zr-DFO-trastuzumab (22,32), for imaging HER2/neu expression, and 89Zr-DFO-bevacizumab, for imaging vascular endothelial growth factor (36). The nature of the radioactive species accumulating in the bone remains uncertain, but it is plausible that slow intratumoral metabolism and subsequent recirculation of 89Zr-labeled metabolites may occur. Full metabolic studies are beyond the scope of the current study and will be the subject of further investigations. However, in a recent clinical trial investigating the radiation dosimetry of 89Zr-DFO-U36 (a chimeric mAb directed against CD44v6) in 20 patients with head and neck squamous cell carcinoma, the liver was identified as the dose-limiting organ (28,29). Dosimetry studies based on the biodistribution data presented in this work suggest that for clinical patient studies, kidney uptake of 89Zr-DFO-J591 is the dose-limiting factor.
The immunoPET data demonstrate that 89Zr-DFO-J591 imaging provides high tumor–to–background tissue ratios and that this high uptake is specific for PSMA expression in tissue. Overall, the novel radiotracer 89Zr-DFO-J591 represents a promising candidate for translation to the clinic as an immunoPET agent for the noninvasive delineation of PSMA-positive primary and metastatic prostate cancers in vivo.
CONCLUSION
89Zr-DFO-J591 has been prepared with a high RCP (>99%) and specific activity (181.7 ± 1.1 MBq/mg). In vitro stability studies demonstrated that functionalization of J591 with 3.9 ± 0.3 accessible DFO chelates per mAb and subsequent radiolabeling do not compromise the immunoreactivity, and radiolabeled immunoconjugate remains active for up to 7 d at 37°C. Biodistribution and immunoPET experiments indicated that 89Zr-DFO-J591 shows high potential as a radiotracer for specific, noninvasive delineation of PSMA-positive PCs in vivo. Work toward the clinical translation of 89Zr-DFO-J591 and other 89Zr-labeled mAbs is under way.
Acknowledgments
We thank Drs. NagaVaraKishore Pillarsetty and Pat Zanzonico for informative discussions, Valerie M. Longo for assistance with the biodistribution experiments, Thomas Ku for advice with in vitro experiments, and Bradley Beattie for assistance with PET. We thank Professor Jennifer C. Green (Department of Chemistry, University of Oxford, United Kingdom) for access to computational facilities. We also thank the staff of the Radiochemistry/Cyclotron Core at the Memorial Sloan-Kettering Cancer Center (MSKCC). This study was funded in part by the Geoffrey Beene Cancer Research Center of Memorial Sloan-Kettering Cancer Center; the Office of Science (BER), U.S. Department of Energy (award DE-SC0002456); the Ludwig Center for Cancer Immunotherapy of the Sloan-Kettering Institute; and the Starr Cancer Consortium. Technical services provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by NIH grants R24 CA83084 and P30 CA08748. Dr. Neil Bander is the inventor on patents that are owned by Cornell Research Foundation (CRF) for the J591 antibody described in this manuscript. Dr. Neil Bander is a paid consultant to BZL Biologics, the company to which the patents were licensed by CRF for further research and development.
- © 2010 by Society of Nuclear Medicine
REFERENCES
- Received for publication February 10, 2010.
- Accepted for publication March 30, 2010.
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- Light-Induced Radiosynthesis of 89Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor
- First-in-Humans Imaging with 89Zr-Df-IAB22M2C Anti-CD8 Minibody in Patients with Solid Malignancies: Preliminary Pharmacokinetics, Biodistribution, and Lesion Targeting
- Exploitation of CD133 for the Targeted Imaging of Lethal Prostate Cancer
- Total-Body PET and Highly Stable Chelators Together Enable Meaningful 89Zr-Antibody PET Studies up to 30 Days After Injection
- A Novel Fully Human Antibody targeting Extracellular Domain of PSMA Inhibits Tumor Growth in Prostate Cancer
- Immuno-PET of Innate Immune Markers CD11b and IL-1{beta} Detects Inflammation in Murine Colitis
- Monosodium Glutamate Reduces 68Ga-PSMA-11 Uptake in Salivary Glands and Kidneys in a Preclinical Prostate Cancer Model
- Preclinical Evaluation of the Hsp70 Peptide Tracer TPP-PEG24-DFO[89Zr] for Tumor-Specific PET/CT Imaging
- Long-Half-Life 89Zr-Labeled Radiotracers Can Guide Percutaneous Biopsy Within the PET/CT Suite Without Reinjection of Radiotracer
- ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo
- First-in-Human Imaging with 89Zr-Df-IAB2M Anti-PSMA Minibody in Patients with Metastatic Prostate Cancer: Pharmacokinetics, Biodistribution, Dosimetry, and Lesion Uptake
- Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis
- Evaluation of Castration-Resistant Prostate Cancer with Androgen Receptor-Axis Imaging
- Molecular Imaging and Quantitation of EphA2 Expression in Xenograft Models with 89Zr-DS-8895a
- Targeted Imaging of the Atypical Chemokine Receptor 3 (ACKR3/CXCR7) in Human Cancer Xenografts
- New Strategies in Prostate Cancer: Prostate-Specific Membrane Antigen (PSMA) Ligands for Diagnosis and Therapy
- Immuno-PET Imaging of CD30-Positive Lymphoma Using 89Zr-Desferrioxamine-Labeled CD30-Specific AC-10 Antibody
- A Phase I/II Study for Analytic Validation of 89Zr-J591 ImmunoPET as a Molecular Imaging Agent for Metastatic Prostate Cancer
- Annotating STEAP1 Regulation in Prostate Cancer with 89Zr Immuno-PET
- CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma
- Preclinical Evaluation of Multistep Targeting of Diasialoganglioside GD2 Using an IgG-scFv Bispecific Antibody with High Affinity for GD2 and DOTA Metal Complex
- Glypican-3-Targeted 89Zr PET Imaging of Hepatocellular Carcinoma: Where Antibody Imaging Dares to Tread
- Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody
- Applying PET to Broaden the Diagnostic Utility of the Clinically Validated CA19.9 Serum Biomarker for Oncology
- Interrogating Tumor Metabolism and Tumor Microenvironments Using Molecular Positron Emission Tomography Imaging. Theranostic Approaches to Improve Therapeutics
- Phase II Study of Lutetium-177-Labeled Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody J591 for Metastatic Castration-Resistant Prostate Cancer
- Determination of the In Vivo Selectivity of a New {kappa}-Opioid Receptor Antagonist PET Tracer 11C-LY2795050 in the Rhesus Monkey
- Monitoring Afatinib Treatment in HER2-Positive Gastric Cancer with 18F-FDG and 89Zr-Trastuzumab PET
- Unmet Needs in the Prediction and Detection of Metastases in Prostate Cancer
- Imaging Tumor Burden in the Brain with 89Zr-Transferrin
- 64Cu-p-NH2-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma
- Advances in Immuno-Positron Emission Tomography: Antibodies for Molecular Imaging in Oncology
- PET of Signal Transduction Pathways in Cancer
- Imaging Androgen Receptor Signaling with a Radiotracer Targeting Free Prostate-Specific Antigen
- 2-(3-{1-Carboxy-5-[(6-[18F]Fluoro-Pyridine-3-Carbonyl)-Amino]-Pentyl}-Ureido)-Pentanedioic Acid, [18F]DCFPyL, a PSMA-Based PET Imaging Agent for Prostate Cancer
- Targeting the Internal Epitope of Prostate-Specific Membrane Antigen with 89Zr-7E11 Immuno-PET
- Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen
- Magnitude of Enhanced Permeability and Retention Effect in Tumors with Different Phenotypes: 89Zr-Albumin as a Model System