Research ArticleBasic Science Investigations

Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody

Scott M. Knowles, Kirstin A. Zettlitz, Richard Tavaré, Matthew M. Rochefort, Felix B. Salazar, David B. Stout, Paul J. Yazaki, Robert E. Reiter and Anna M. Wu
Journal of Nuclear Medicine March 2014, 55 (3) 452-459; DOI: https://doi.org/10.2967/jnumed.113.120873

Abstract

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%–100% of local prostate cancers and 87%–100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods: A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient–based partial-volume correction, and the results were compared with ex vivo biodistribution. Results: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I- and 89Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing 124I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing 89Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the 124I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor–to–soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 124I-A11 and 4.87 ± 0.52 89Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Conclusion: Both 124I- and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor–to–soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both 124I- and 89Zr-labeled A11 minibody.

ImmunoPET imaging is a powerful technique that has shown promise in noninvasive imaging of cell surface biomarkers with high specificity using radiolabeled antibodies and antibody fragments (1). Prostate stem cell antigen (PSCA) is a cell surface protein that has limited low-level expression in normal prostate, bladder, and stomach. PSCA, however, is expressed in 83%–100% of prostate cancers, where its expression correlates with the Gleason score and a poor prognosis (2–6). It is also highly expressed in most prostate cancer bone metastases (87%–100%), in many metastases to other sites (67%–95%), and in local bladder and pancreatic carcinoma (5–10). Our laboratory has previously shown that an 124I-labeled 2B3 anti-PSCA minibody, an engineered approximately 80 kDa antibody fragment (scFv-CH3 homodimer, Fig. 1A), can target and image PSCA-expressing xenografts with high contrast at earlier time points than the intact 2B3 anti-PSCA antibody because of the minibody’s faster clearance (11,12). Subsequent affinity maturation of the 2B3 minibody created the A11 anti-PSCA minibody, which showed improved immunoPET performance, and methods were established for current good manufacturing practices (cGMP) production and purification of this minibody (13,14). In the current work, we compare 124I and 89Zr radiolabels for immunoPET imaging using the affinity-matured cGMP-grade A11 anti-PSCA minibody and investigate quantitative immunoPET imaging with these alternative radionuclides. To assess the suitability of both 124I- and 89Zr-labeled A11 minibody for eventual use as clinical imaging agents capable of detecting both local prostate cancer and bone and soft-tissue metastases, we evaluated both the absolute tumor uptake and the tumor-to-nontumor contrast obtainable with each radiolabel in preclinical mouse models.

FIGURE 1.
FIGURE 1.

Cartoon representation of minibody, compared with intact antibody (A). SDS-PAGE (B) and size-exclusion chromatography (C) confirm that A11 minibody exists overwhelmingly as covalent scFv-CH3 dimer with minimal amounts of monomer present.

The slow clearance of most immunoPET radiotracers requires late imaging time points (≥20 h after injection) that are beyond the limits of the most frequently used PET radionuclide, 18F, because of its short physical half-life (t1/2 = 1.8 h, Table 1). Though strides have been made in optimizing fast-clearing immunoPET tracers that allow for 18F labeling, the 2 most promising positron-emitting radionuclides for mid- to late-time-point immunoPET are 124I and 89Zr because of their long physical t1/2 (4.2 and 3.3 d, respectively) (15). Both 124I and 89Zr have advantages and disadvantages for use as PET tracers and differ with regard to physical properties, conjugation and required chelators, clearance, and uptake (16).

View this table:
TABLE 1

Positron-Emitting Radionuclides for ImmunoPET

124I and 89Zr both suffer from low positron yields and emission of γ rays, which increase the absorbed dose to the patient. Quantitative PET imaging with 124I provides a challenge because of simultaneous emission of a 606-keV γ ray with 52% of positrons. This 606-keV γ ray falls into the energy window of most PET scanners and causes prompt γ coincidences, which increase the measured background in PET imaging by a factor ranging from 2.5 to 10 times that of 18F (17). 89Zr, on the other hand, emits a 909-keV γ ray with 100% of positrons but prompt γ coincidences do not occur both because the γ ray and positron emission are not simultaneous and because the γ ray is out of the scanner energy window (18,19).

PET imaging with 124I and 89Zr also suffers from lower effective resolution than 18F PET because the increased positron emission energy of 124I and 89Zr leads to a longer mean positron range (16). This increased positron range in turn contributes to a higher partial-volume effect (PVE) due to positrons traveling into voxels that neighbor where they were actually emitted. The PVE results in a lower measured activity in a hot region of interest (ROI) surrounded by a cold background than the true value due to positrons spilling out of the ROI, especially for small volumes (20). The reverse is also true: a cold ROI surrounded by a high-activity background will have a higher measured value than the true value due to activity spilling into the ROI. The PVE is not strictly a function of positron range: scanner resolution and the fact that an anatomic region may only partially occupy an image voxel also play a large role. However, the long positron range of 124I has been found to significantly increase the PVE in clinical PET scanners even in volumes 35 mm in diameter (21). The increased PVE due to positron range will especially affect small-animal PET scanning for which the scanner resolution (1.1–3.0 mm) is finer than the mean positron range of 124I and approaches the mean positron range of 89Zr, and the largest contributor to the PVE is the positron range (18,22,23).

In addition to their differences in the physical properties, 124I and 89Zr differ with regard to protein chemistry and biologic metabolism and can yield distinctly different results when used for immunoPET radiolabeling. 124I is generally randomly conjugated under oxidizing conditions to tyrosines present in the antibody. Once an 124I-labeled antibody is internalized and degraded, the 124I-iodide or 124I-iodotyrosine released is free to diffuse from the tissue, resulting in a loss of signal over time in target tissue but also low signal in background tissues and tissues of clearance (24). 89Zr, on the other hand, is most commonly conjugated onto antibodies in a 2-step process using the chelator desferrioxamine (DFO), which has previously been conjugated onto primary amines (N terminus and lysines) or free thiols (reduced cysteines) of the antibody (19,25). On internalization and catabolism, 89Zr-labeled radiometabolites are trapped in cellular lysosomes, allowing for higher retention of signal over time in target tissue than with 124I (19). Residualizing radiolabels such as 89Zr, however, often see higher uptake in organs of normal clearance (liver and kidney) and higher background uptake in other tissues that passively take up the antibody.

In the current study, we established a quantitative method of immunoPET imaging with 124I- and 89Zr-labeled A11 minibody that includes partial-volume correction to account for the long positron ranges of these isotopes. We then used quantitative immunoPET imaging to explore differences in tumor uptake and rates of tissue clearance with each of these radiotracers.

MATERIALS AND METHODS

Details of protein production and purification, cell lines and xenografts, quantitative flow cytometry, cell binding and affinity studies, radiolabeling with 89Zr and 124I, in vitro cellular uptake studies, and biodistributions can be found in the supplemental materials (available at http://jnm.snmjournals.org).

Small-Animal PET/CT

Approximately 25 μg (∼4 MBq) of 124I-A11 or 89Zr-A11 with a purity of 98% or more were administered to tumor-bearing mice via tail vein injection. Before 124I-A11 administration, thyroid and stomach uptake of radioiodine was blocked, respectively, with Lugol iodine and potassium perchlorate as previously described (13). At 6, 20, and 44 h after injection, mice were anesthetized with 1.5% isoflurane anesthesia and imaged with 10-min acquisitions on an Inveon small-animal PET scanner (Siemens Preclinical Solutions), followed by a micro-CT scan (microCAT II; Siemens Preclinical Solutions) (26). One mouse from each group also received a 2-h dynamic small-animal PET scan at the time of injection and additional scans at 4, 8, and 12 h after injection. Small-animal PET images were reconstructed by non–attenuation-corrected or scatter-corrected filtered backprojection and were analyzed and displayed using AMIDE (27). Coregistration of the small-animal PET and micro-CT scans was performed automatically using empirically determined scanner alignments.

Image Quantitation and Partial-Volume Correction

Small-animal PET cylinder factors were measured by scanning a 2.5-cm-diameter cylinder containing various concentrations of 124I or 89Zr measured by dose calibrator (Atom Lab 300; Biodex Medical Systems). The mean PET value of each scan was obtained from a 1.5-cm-diameter ROI drawn at the center of the cylinder to avoid PVEs, and the cylinder factor was calculated by linear regression of the dose calibrator and PET values. The generation of recovery coefficient (RC) curves for partial-volume correction was performed by imaging spheres of varying size filled with identical concentrations of either 124I or 89Zr. Spheres of 8.0-mL, 4.0-mL, 2.0-mL, 1.0-mL, 0.5-mL, 250-μL, 125-μL, 63-μL, and 31-μL volumes (Data Spectrum Corp.) were measured. In addition, 20-μL, 10-μL, and 5-μL volumes were measured in 0.5-mL microcentrifuge tubes because appropriately sized hollow spheres are not commercially available. The mean 124I or 89Zr concentration obtained for each diameter ROI was converted into an RC by dividing the mean value measured by small-animal PET by the known value. The resulting curve was fit to a 2-parameter sigmoid function (Eq. 1), modified from Jentzen et al., where a and b are fitting parameters and d is the diameter of the ROI in mm (21).Embedded ImageEq. 1

Tumor uptake values (%ID/gROI) and volumes were quantified by drawing elliptic ROIs over the entire tumor using the micro-CT. The ROI was subtlety repositioned (without changing the ROI dimensions), if required, to ensure placement over the tumor in the small-animal PET image because of small errors in small-animal PET/CT coregistration. The mean PET activity of the ROIs was converted to percentage injected dose per gram (%ID/g) using the decay-corrected injected dose and the cylinder factors for 89Zr and 124I, respectively, under the assumption that the density of the tumors was 1 g/mL. The RC of the ellipsoidal ROI was estimated by spheric approximation of the tumor volume (d = Embedded Image). The partial-volume–corrected %ID/g (%ID/gPVC) was then calculated using Equation 2, where %ID/gbkg is the background activity obtained from the mean of ROIs drawn in the nonspecific tissue around the tumor (28,29). The subtraction of %ID/gbkg before dividing by the RC allows for simultaneous correction for spill-in and spill-out effects with no partial-volume correction in the case in which the ROI is drawn in a uniform background (28). The partial-volume correction method was validated by comparison of small-animal PET values to ex vivo biodistribution data in mice sacrificed immediately after imaging. Percentage error for this comparison was calculated as (%ID/gROI − %ID/gBiodist)/%ID/gBiodist.Embedded ImageEq. 2

Data Analysis

Except where indicated otherwise, all values are reported as mean ± SD. Statistical analysis was performed using a 2-tailed Student t test at the 95% confidence level (P < 0.05). The P values obtained were adjusted for multiple comparisons via the Holm–Šidák method. Linear and nonlinear least-squares curve fitting was performed using GraphPad Prism 6.0. The linear fits of %ID/gROI versus %ID/gBiodist were weighted by 1/y2 because the uncertainty of the small-animal PET value is proportional to the uptake. All small-animal PET/CT images are displayed as full-thickness maximum-intensity projections.

RESULTS

Antibody and Cell Line Characterization

SDS-PAGE and size exclusion confirm the A11 minibody is assembled as a covalent scFv-CH3 80 kDa homodimer comprising 40 kDa subunits (Fig. 1; Supplemental Fig. 1). Quantitative flow cytometry with the murine 1G8 anti-PSCA antibody (n = 3) shows little or no expression of PSCA on 22Rv1 cells, expression of 2.2 × 106 PSCA antigens on 22Rv1×PSCA cells, and expression of 4.5 × 105 PSCA antigens on LAPC-9 cells (Fig. 2A). Flow cytometry shows specific binding of the A11 minibody to 22Rv1×PSCA cells with an apparent affinity of 13.7 ± 1.4 nM SEM (Fig. 2B). Measurement of A11 minibody binding on immobilized PSCA-mFc antigen using a quartz crystal microbalance shows an apparent affinity (KD) of 3.91 nM. No loss of affinity is seen with iodinated A11 minibody (KD = 3.43 nM) and only a small decrease in affinity is seen with DFO-conjugated A11 minibody (KD = 6.75 nM), allowing for a direct comparison of 124I and 89Zr radiolabels with minimal effects from differences in minibody affinity (Supplemental Fig. 2).

FIGURE 2.
FIGURE 2.

(A) Quantitative flow cytometry shows no expression of PSCA on 22Rv1 cells, high expression on 22Rv1×PSCA cells, and intermediate expression on disassociated LAPC-9 tumor cells (n = 3 each). (B) Binding of A11 minibody to 22Rv1×PSCA cells has KD of 13.7 ± 1.4 nM as measured by flow cytometry. (C) Incubation of 22Rv1×PSCA cells with 124I-A11 and 89Zr-A11 demonstrates that PSCA slowly internalizes and that on internalization 89Zr residualizes and accumulates in cells to higher degree than 124I. Error bars are shown as ± SEM.

Antibody Cell Binding and Uptake

In vitro antibody uptake experiments demonstrate antigen-specific binding and internalization of both 124I-A11 and 89Zr-A11 on 22Rv1×PSCA cells. However, 89Zr-A11 radiometabolites accumulate intracellularly to a higher degree than 124I-A11 radiometabolites over 44 h (Fig. 2C). These results are consistent with slow internalization of the PSCA, residualization of the 89Zr-A11 radiometabolites, and nonresidualization of the 124I-A11 radiometabolites as expected (30). 22Rv1 cells show no membrane binding or internalization of 89Zr-A11 or 124I-A11 at any time point (data not shown).

Radiolabeling

124I-A11 and 89Zr-A11 had mean specific activities of 141 ± 37 MBq/mg (3.8 ± 1.0 μCi/μg, n = 7) and 115 ± 37 MBq/mg (3.1 ± 1.0 μCi/μg, n = 3), respectively, with a radiochemical purity of 98% or more. Immunoreactivity of 124I-A11 and 89Zr-A11 were found to be 76.1% ± 9.7% (n = 7) and 52.0% ± 9.2% (n = 3), respectively, as measured by cellular association with excess 22Rv1×PSCA cells, with 5% or less binding to the negative control 22Rv1 cell line. Stability of 89Zr-A11 and 124I-A11 in both 1% fetal bovine serum/phosphate-buffered saline and mouse serum was 95% or more at 44 h.

In Vivo Characterization of 124I-A11 and 89Zr-A11 Minibody

Both 124I-A11 and 89Zr-A11 demonstrate specific uptake in antigen-positive 22Rv1×PSCA tumors, with uptake significantly higher than in 22Rv1 control tumors (P < 0.0001 for each, Fig. 3). LAPC-9 tumors showed similarly high levels of uptake, and high-contrast imaging was obtained with both radiotracers (Fig. 4). 89Zr-A11 demonstrates significantly higher tumor uptake and higher tumor-to-blood ratios than 124I-A11 in both 22Rv1×PSCA (Table 2) and LAPC-9 xenografts (Table 3) at 44 h after injection. However, 89Zr-A11 also demonstrates nonspecific background uptake, especially in the liver, kidneys, and spleen, but also in all other tissues measured, which all show activity higher than blood at 44 h after injection. Mice injected with 124I-A11, on the other hand, show uptake lower than blood in all organs other than the 22Rv1×PSCA and LAPC-9 tumors. 124I-A11 shows a 22Rv1×PSCA:22Rv1 tumor ratio of 13.3 and tumor-to-muscle ratio of 133.3 whereas with 89Zr-89 the 22Rv1×PSCA:22Rv1 tumor ratio was 4.9 and the tumor-to-muscle ratio was 15.9. In fact, other than the tumor-to-blood ratio, every tumor-to-nontumor ratio was higher for 124I-A11 than for 89Zr-A11 in both 22Rv1×PSCA–bearing nude mice and LAPC-9–bearing severe-combined immunodeficiency (SCID) mice, allowing for much better image contrast with 124I-A11 than with 89Zr-A11 (Figs. 3 and 4).

FIGURE 3.
FIGURE 3.

124I-A11 vs. 89Zr-A11 immunoPET in mice bearing 22Rv1 (left) and 22Rv1×PSCA (right) subcutaneous tumors. Small-animal PET images (A) and ex vivo biodistribution (B) show higher positive tumor contrast when imaging with 124I-A11 than with 89Zr-A11, despite lower absolute uptake.

FIGURE 4.
FIGURE 4.

124I-A11 vs. 89Zr-A11 immunoPET in SCID mice bearing bilateral LAPC-9 subcutaneous tumors at 44 h after injection shows higher contrast in tumors imaged with 124I-A11 despite higher absolute uptake in tumors imaged with 89Zr-A11. Small-animal PET images are shown as full-thickness maximum-intensity projections.

View this table:
TABLE 2

44-Hour Biodistribution of Male Nude Mice Bearing 22Rv1 (Negative) and 22Rv1×PSCA (Positive) Xenografts

View this table:
TABLE 3

44-Hour Biodistribution of Male SCID Mice Bearing Bilateral LAPC-9 Xenografts

Quantification of 124I-A11 and 89Zr-A11 Small-Animal PET and Comparison to Biodistribution

RC curves generated for 124I and 89Zr demonstrate that the higher positron emission energy and longer mean positron range of 124I has a detrimental effect on effective scanner resolution and increases the PVE, compared with 89Zr (Fig. 5). Comparison of non–partial-volume–corrected small-animal PET ROI analysis and biodistribution reveals a large underestimate of tumor uptake for both 124I and 89Zr, especially for small tumors (−43.7% ± 15.7% and −24.09% ± 19.49% mean percentage error, respectively; Figs. 5E and 5F in blue). RC-based partial-volume correction both improves correspondence with ex vivo biodistribution and reduces the variance for both 124I and 89Zr uptake, resulting in respective mean percentage errors of −0.4% ± 13.5% and 5.19% ± 8.45% after partial-volume correction (Figs. 5E and 5F in red).

FIGURE 5.
FIGURE 5.

RC curves for 124I and 89Zr show higher PVE for 124I (A) than for 89Zr (B). Quantitation of tumor uptake from small-animal PET images shows that non-PVC–corrected ROIs underestimate uptake of 124I-A11 (C and E) and 89Zr-A11 (D and F), especially for tumors of small size. Partial-volume–corrected small-animal PET data show better correspondence with biodistribution for both 124I-A11 and 89Zr-A11.

Dynamic imaging of both 124I-A11 and 89Zr-A11 shows peak uptake in 22Rv1×PSCA tumors at 12 h after injection. Tumor uptake then falls 49% from its peak over the next 32 h for the 124I-labeled minibody where catabolized radiometabolites do not residualize and can diffuse from the tumor. 89Zr-labeled radiometabolites, on the other hand, residualize and diffuse more slowly out of the tumor, and hence 22Rv1×PSCA tumor uptake decreases by only 19% between 12 and 44 h (Figs. 6A and 6B). Averages from serial imaging of 22Rv1×PSCA–bearing mice with 89Zr-A11 and 124I-A11 at 6, 20, and 44 h after injection similarly show significantly higher uptake and higher retention of 89Zr-A11 (n = 4) than 124I-A11 (n = 6) at all time points (Fig. 6D). Quantitation of dynamic imaging also demonstrates that while a left ventricular ROI can be used for estimating the activity in the blood pool with 124I-A11 by small-animal PET, the quantification of the blood activity with 89Zr-A11 using this method is confounded by nonspecific uptake in the heart wall in excess of that present in the blood. The spill-in from the heart wall results in an overestimation of blood activity of 89Zr-A11 at late time points and negates the ability to accurately quantify blood activity and tumor-to-blood ratios from a heart ROI (Figs. 6B and 6C). At 44 h after injection, comparison of blood activities by small-animal PET heart ROIs and ex vivo biodistribution shows excellent correspondence for 124I-A11 (0.56 ± 0.13 %ID/gBiodist vs. 0.59 ± 0.19 %ID/gPVC, n = 20, P = 0.593) but poor correspondence for 89Zr-A11, with the heart ROI significantly overestimating the activity in the blood pool (0.47 ± 0.27 %ID/gBiodist vs. 2.02 ± 0.47 %ID/gPVC, n = 7, P < 10−5). As a result, small-animal PET quantification of tumor-to-blood ratios using tumor and heart ROIs is accurate for 124I-A11 (mean tumor-to-blood ratio, 6.00 ± 2.39 biodistribution vs. 5.93 ± 2.75 small-animal PET, n = 28, P = 0.92) but greatly underestimates the tumor-to-blood ratio for 89Zr-A11 (mean tumor-to-blood ratio, 20.04 ± 9.67 biodistribution vs. 4.41 ± 0.93, n = 10, P < 0.0001).

FIGURE 6.
FIGURE 6.

Quantification of dynamic and serial imaging with 124I-A11 (A) and 89Zr-A11 (B) demonstrates higher uptake and retention of A11 minibody with 89Zr labeling than with 124I labeling. Blood uptake as quantified by heart ROI shows artificially elevated signal for 89Zr-A11 at late time points due to nonspecific uptake in the heart wall leading to underestimations of tumor-to-blood ratio when quantified with heart ROIs. (C) However, 124I-A11 blood activity and tumor-to-blood ratios can be accurately quantified by this method. (D) Serial imaging of 89Zr-A11 (n = 4) and 124I-A11 (n = 6) mice bearing 22Rv1×PSCA xenografts confirms results of dynamic scans. ***P < 0.0001.

DISCUSSION

In this work, we evaluated two alternative means of radiolabeling the A11 anti-PSCA minibody for imaging two PSCA-expressing tumors. 124I-A11 and 89Zr-A11 both show uptake in PSCA-expressing 22Rv1×PSCA and LAPC-9 tumors and result in high-contrast small-animal PET imaging. The rapid clearance of the minibody is advantageous for imaging with 124I-A11, for which excellent tumor contrast is seen, with uptake nearly an order of magnitude higher in the tumor than in any other organ at 44 h after injection. The residualizing nature of 89Zr-A11 results in 2- to 3-fold higher uptake and retention of tumor activity in both 22Rv1×PSCA and LAPC-9 tumor models, resulting in 2- to 3-fold higher tumor-to-blood ratios at 44 h than obtained with 124I-A11. 89Zr-A11, however, displays significantly increased background, compared with 124I-A11, in all nontumor tissue of both nude and SCID mice and results in lower tumor–to–soft-tissue contrast for 89Zr-A11 by both small-animal PET imaging and biodistribution. The nonspecific uptake in the bone and liver for 89Zr-A11 is especially problematic because these are common sites of prostate cancer metastases and may interfere with the detection of tumors in these locations.

Quickly clearing antibody fragments such as minibodies generally have lower uptake in tumors than intact antibodies because of shorter time allowed for extravasation from the vasculature and diffusion into the tissue. However, the minibody’s short residence time in the blood compensates for the lower absolute tumor uptake, allowing higher tumor-to-blood ratios and improved imaging contrast at early time points (12,31). The short t1/2 of the A11 minibody (t1/2β ≈ 6 h) also has the benefit of reducing the radioactivity exposure and absorbed dose to the patient.124I-A11 minibody and its radiometabolites clear the body quickly, and 124I-labeled probes are usually dose-limited only by bone marrow dose via cross-exposure from the blood, which will decrease when using quickly clearing 124I-labeled minibodies (32). The increased background uptake of 89Zr-A11 and its radiometabolites, on the other hand, will result in smaller improvements in patient dosimetry for the minibody than for an intact antibody. Dosimetry studies of other 89Zr-DFO–labeled antibodies have suggested that nonspecific accumulation of the radiometabolite in the liver or kidney will be dose-limiting, which will also likely be the case for 89Zr-A11 minibody (33–35).

Quantification of PET data from 89Zr and 124I is more challenging than with 18F, because of their lower positron yield and higher position emission energy, which leads to a long mean positron range, especially for 124I. Though the 603-keV γ ray of 124I falls into the energy window of the Inveon scanner and elevates measurements of relatively cold image areas, small-animal PET shows a remarkably linear response to varying concentrations of both 124I and 89Zr (Supplemental Fig. 3) (17). The increased positron range of 89Zr, and especially 124I, however, leads to a large PVE that cannot be ignored when quantifying small-animal PET imaging data with these isotopes. Measurements of in vivo tumor uptake without partial-volume correction underestimate the amount of uptake, especially for small tumors, and significant differences in uptake between tumor groups could easily be observed simply because of differences in the mean size of the tumors imaged. Correcting for the PVE is especially important when imaging response to therapy, in which what appears to be a downregulation of immunoPET uptake with treatment could simply be an artifact of a smaller average tumor in the treated group.

The partial-volume correction method used here requires fairly precise approximation of tumor volumes both for accurate determination of the mean PET uptake and for determination of the tumor volume from which the RC is calculated. Though precise determination of tumor volume is time-consuming and some intraoperator variability can be expected with any manual ROI drawing procedure, determination of tumor volumes from CT has been found to be more accurate than determination of tumor volume through the use of PET data or caliper measurements, with excellent correspondence between observers (r2 = 0.97) (36). The CT tumor volumes determined here correspond well with ex vivo tumor masses, with the CT volumes underestimating the tumor mass by only 8% on average (slope = 0.92, r2 = 0.96, Supplemental Fig. 4). Most of the underestimations of the tumor volume are due to measurements of larger tumors, which tend to be more irregularly shaped and therefore more difficult to approximate with an ellipsoid ROI. However, precise determination of tumor volume for partial-volume correction is less important for large tumors, as the RC curve approaches 1 at large diameters. Despite the limitations of manual ROI drawing, the partial-volume–corrected estimation of tumor activity is considerably more accurate and less noisy than other common methods of quantification, including the commonly used estimation from the maximum voxel in the image, which both exhibits a strong PVE and is more noisy than using the mean value, making partial-volume correction harder to apply (13.9% ± 30.8% 124I mean percentage error and 10.87% ± 26.57% 89Zr mean percentage error, Supplemental Fig. 5).

In summary, residualizing radiolabels such as 89Zr are essential for imaging rapidly internalizing antigens where the radiolabel can be accumulated and concentrated intracellularly, using nonresidualizing antibodies on rapidly internalizing antigens will result in low tumor uptake due to internalization and loss of signal through diffusion. When imaging noninternalizing antigens, on the other hand, image contrast is improved using a nonresidualizing radiolabel such as 124I because of lower nonspecific accumulation. With slowly internalizing antigens, such as PSCA, the choice of a residualizing or nonresidualizing radiolabel is a compromise between higher tumor uptake for the residualizing label and lower background for the nonresidualizing label that will vary with the choice of particular antigen and imaging agent t1/2. For imaging of PSCA expression with the A11 minibody, this balance clearly lies with the nonresidualizing 124I radiolabel and as such we have recently entered clinical evaluation of 124I-A11.

Quantitative small-animal PET imaging with 124I and 89Zr must take into account the long mean positron range of these isotopes. Partial-volume correction even with a simple RC-based method and a spheric approximation of the tumors greatly improves the correspondence between small-animal PET and ex vivo quantification. Further improvement can likely be seen with more advanced partial-volume correction methods (37).

CONCLUSION

This work showed rapid targeting and high-contrast imaging of PSCA-expressing prostate cancer xenografts using both 124I- and 89Zr-labeled A11 minibody. Although the 89Zr-labeled antibody achieved higher tumor uptake at 44 h after injection, the 124I-labeled antibody showed lower background and higher tumor–to–soft-tissue contrast. 124I was therefore determined to be the preferred radiolabel for A11 minibody immunoPET. Partial-volume correction greatly improved the correspondence of small-animal PET, and ex vivo biodistribution was found to be essential for quantification of small-animal PET data with both 89Zr and 124I radiolabels.

DISCLOSURE

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. This work was supported by grants CA092131 and CA016042 from the National Institutes of Health, W81WXH-08-1-0442 from the Department of Defense, DE-SC0001220 from the Department of Energy, NIH T32 GM008042 from the UCLA-Caltech Medical Scientist Training Program, and F30CA165824 NRSA Fellowship Award from the National Cancer Institute. Robert E. Reiter and Anna M. Wu are shareholders and consultants of ImaginAb Inc., which has licensed some of the technology described in this manuscript. No other potential conflict of interest relevant to this article was reported.

Acknowledgments

We thank Waldemar Ladno, Darin Williams, Melissa McCracken, Dr. John David, and Dr. Magnus Dahlbom for technical assistance with these experiments.

Footnotes

  • Published online Feb. 6, 2014.

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  • Received for publication May 30, 2013.
  • Accepted for publication November 1, 2013.
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