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. Author manuscript; available in PMC: 2009 Nov 15.
Published in final edited form as: Clin Cancer Res. 2008 Nov 15;14(22):7488–7496. doi: 10.1158/1078-0432.CCR-07-5093

A Humanized Radioiodinated Minibody as an Imaging Agent for the Detection of Prostate Stem Cell Antigen (PSCA)-Expressing Tumors by microPET

Jeffrey V Leyton 1, Tove Olafsen 1, Eric J Lepin 1, Scott Hahm 2, Karl B Bauer 1, Robert E Reiter 2, Anna M Wu 1
PMCID: PMC2720761  NIHMSID: NIHMS75254  PMID: 19010866

Abstract

Purpose

Prostate Stem Cell Antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal microPET imaging using hu1G8, however, required a delay of 168 h post injection. In this study, 2B3 minibody (an 80 kDa engineered antibody fragment) has been produced for rapid targeting and imaging.

Experimental Design

A gene encoding a PSCA-specific minibody was assembled VL-linker-VH-hinge-huIgG1 CH3. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using 124I-labeled minibody was conducted at 4 and 21 h in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3 and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and Region of Interest analysis of the images was conducted.

Results

20 mg/L yields of purified 2B3 minibody were obtained that demonstrated specific binding to LNCaP-PSCA cells. Purified 2B3 minibody demonstrated specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nM. Radioiodinated 2B3 minibody demonstrated rapid non-target tissue and blood clearance kinetics (t1/2β = 11.2 h). MicroPET scanning using 124I-2B3 minibody showed both androgen-dependent and –independent tumors as early as 4 h and excellent high contrast images at 21 h post injection.

Conclusions

Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 h.

Keywords: minibody, PSCA, 124I, microPET, prostate cancer

INTRODUCTION

Prostate cancer is the most commonly diagnosed and second leading cause of cancer related deaths in American men according to the American Cancer Society. When diagnosed early the five year survival outlook in men with local and regional prostate cancer is 100%. Once a patient has been diagnosed with prostate cancer, appropriate staging and follow-up are required in order to select the most effective treatment. There are few means of detecting extra-prostatic sites of disease or recurrence, and an imaging agent to address these specific needs would contribute to patient management.

Several positron emission tomography (PET) 11C- and 18F-based radiotracers including 18F-fluoreodeoxyglucose (FDG) have been developed and evaluated for imaging of prostate cancer (1-4). However, 11C-based radiotracers are impractical without an on-site cyclotron and 18F-labeled small molecule tracers generally exhibit high urinary excretion. In addition, factors such as non-specific uptake, low glucose utilization, and inefficient radiolabeling and synthesis methods have made it difficult fully implement various radiotracers into the clinic (5). A recent pilot clinical study using the anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid (anti-18F-FACBC), synthetic l-leucine analog, has shown promise in imaging prostate cancer (6). The anti-18F-FACBC demonstrated localization to primary, metastatic and recurrent prostate carcinoma with variable, but relatively low renal excretion and bladder activity. However, this tracer shared some of the characteristics often observed with metabolic tracers, such as nonspecific uptake - in this case, incidental bowel uptake, occasional low-level uptake in benign inguinal lymph nodes, and regions of false positivity within the prostate.

An approach to overcome non-specific uptake and false positivity is to develop PET probes that can specifically target tumor cell surface antigens. High expression of receptors on cancer cells compared to normal tissues provides the molecular basis for using radiolabeled antigen-specific antibodies to visualize tumors. In prostate cancer, the 111In-labeled monoclonal antibody (mAb) capromab pendetide (Prostascint™; Cytogen Corp., Princeton, NJ ) specific for prostate specific membrane antigen (PSMA), has been extensively evaluated clinically and is primarily used for detecting advanced, recurrent local disease and lymph node metastates (7). However, the clinical utility of capromab pendetide is still highly debated due to a low sensitivity (62%−75%) caused by slow tumor uptake and poor clearance (8). In addition, capromab pendetide recognizes an internal epitope on PSMA (9), which may limit its utility in detecting viable cells. It also suffers from the general limitations of other radiolabeled intact antibodies; despite the ability to effectively deliver high activities to tumors, high background activity in the blood requires delays in imaging for optimal tumor evaluation. Furthermore, repeated administration is precluded due to the murine nature of the antibody.

Another prostate specific surface marker that is highly suitable for antibody-based therapy and imaging is the prostate stem cell antigen (PSCA), a cysteine-rich 123-aa glycosylphosphatidylinositol-anchored surface glycoprotein (10-12). We have shown in human prostate cancer specimens that more than 80% of local disease and all metastatic bone lesions examined express PSCA (11). Elevated PSCA expression correlates with increased tumor stage, grade and progression to androgen independence (11). Moreover, PSCA up-regulation has been detected in bladder and pancreatic cancers (13-15). The restricted PSCA expression in normal tissues makes PSCA an enticing target for immunoimaging and immunotherapy of cancerous lesions.

The murine 1G8 mAb, raised against an extracellular epitope of PSCA, has demonstrated anti-tumor growth properties (16). Targeting, imaging and therapy using a humanized version of the 1G8 mAb (hu1G8) has also been examined in preclinical models (17). The hu1G8 mAb exhibited significant inhibition of tumor take of low PSCA-expressing bladder carcinoma cells. When the antibody was evaluated by PET in LAPC-9 xenografted mice following radioiodination with the positron emitter 124I (t1/2 4.2 d), high contrast images were achieved. However, due to slow clearance kinetics of this intact humanized antibody, enhanced target-to-background images were not obtained until one week after administration of 124I-hu1G8.

Protein engineering techniques can be used to produce antibody fragments of various sizes, facilitating rapid clearance from the blood. These fragments demonstrate improved pharmacokinetics suitable for imaging while maintaining excellent tumor uptake, resulting in high tumor-to-background ratios at early time points (18-20). Tumor targeting with bivalent minibody (scFv-CH3 dimer, 80 kDa) fragments in mice bearing colorectal and breast cancer xenografts targeting carcinoembryonic antigen (CEA) and HER-2, respectively, has been achieved (21-23). These minibodies exhibited fast blood clearance and rapid tumor targeting that reached maximum uptake at 12 h (19, 22, 24). The 124I-labeled anti-CEA minibody was further evaluated by microPET imaging in mice bearing human colorectal cancer xenografts (21). This minibody provided excellent tumor uptake and high contrast images at 18 h.

The goal of this work was to develop and evaluate a minibody with specificity for PSCA for imaging prostate cancer xenograft-bearing mice. This minibody (referred to as 2B3 minibody) is derived from the intact hu1G8 antibody. The 2B3 minibody was labeled with 131I for tumor targeting studies and 124I for microPET evaluation in four different PSCA-expressing tumor models. The 2B3 minibody demonstrated fast blood clearance kinetics relative to the intact hu1G8 mAb, resulting in the ability to discern clear images of prostate cancer xenografts at 21 h versus 168 h. Moreover, the 2B3 minibody mimicked the tumor uptake of the intact hu1G8 mAb in the LAPC-9 model despite its rapid elimination from the circulation.. Finally, the ability of the 2B3 minibody to image androgen-independent (AI) xenografts as clearly as the androgen-dependent (AD) xenografts makes it a potential rapid imaging agent for prostate cancer patients stratified into high risk categories.

MATERIALS AND METHODS

Design and gene assembly of anti-PSCA minibody

The V genes from hu1G8 (17) were individually amplified by RT-PCR and and used in the assembly of a minibody gene consisting of a light chain leader sequence, a scFv fragment in the VL-VH orientation connected by an 18 amino acid GlySer rich linker followed by indirect fusion to the human IgG1 CH3 domain via the human IgG1 upper and middle hinge and a lower GlySer linker peptide of 10 residues (23). The 2B3 minibody gene was then transferred into the pEE12 mammalian expression vector as described (22).

Cell lines

NS0 mouse myeloma (25) and the human prostate cancer cell lines PC-3 (American Type Culture Collection [ATCC], Manassas, VA; #CRL-1435), PC-3-PSCA and LNCaP (ATCC #CRL-1740) transfected cells (LNCaP-PSCA) were maintained as described (17). The human B-cell lymphoma cell line SKW 6.4 (ATCC; #TIB-215) transfected with PSCA (SKW 6.4-PSCA) was maintained as recommended, supplemented with 1% β-Mercaptoethanol (1000×) (Gibco/BRL, Gaithersburg, MD).

Expression, screening, propagation and purification

The 2B3 minibody expression vector was transfected into NS0 cells as described (22). After two weeks of selection in glutamine deficient media, supernatants from individual cell clones were screened for 2B3 minibody expression by ELISA and Western blots as previously described (22). High expressing clones were expanded to terminal cultures and the level of 2B3 minibody expression was assayed by ELISA using serial dilutions of harvested supernatants with reference minibody of known concentration as a standard.

Cell culture supernatants containing 2B3 minibody were prepared as described (26) and concentrated down to 100 ml using a Labscale TFF system concentrator (Millipore, Billerica, MA) before being dialyzed against 50 mM acetic acid, pH 5.0. Dialyzed supernatant was then loaded onto a 1.6 ml POROS 20HS (Applied Biosystems, Foster City, CA) cation exchange chromatography column. Bound proteins were eluted with a NaCl step gradient ranging from 0−0.2 M in 50 mM acetic acid, pH 5.0 over 25 column volumes. Elution was monitored by UV absorption (280 nm). Eluted fractions containing the desired 2B3 minibody were pooled and diluted five-fold with 50 mM MES, pH 6.5, and reloaded onto the re-equilibrated 1.6 ml cation exchange column. Bound proteins were eluted with a NaCl gradient from 0−0.25 M in 50 mM MES, pH 6.5 over 30 column volumes. The 2B3 minibody-containing fractions were pooled and dialyzed against PBS with a porous membrane tubing (M.W. cut off: 30,000) (Spectrum Laboratories, Rancho Dominguez, CA) and concentrated with a Vivaspin 20 (M.W. cut off: 30,000) (Vivascience, Hannover, Germany). The concentration and total amount of protein obtained after purification were determined by ELISA and by UV absorbance at 280 nm using an extinction coefficient (ε) of 1.4 mg/ml.

Minibody size characterization

Purified proteins were analyzed by SDS-PAGE as described (22). Native size was determined by running 25−50 μg of purified protein isocratically in PBS on a size exclusion column (Superdex 75; GE Healthcare, Waukesha, WI) using a known 80 kDa minibody as standard.

The PSCA binding activity of the 2B3 minibody was determined by competition ELISA as described (17). Determination of relative affinity was calculated by linear extrapolation of percentage bound vs. 1/nM of murine 1G8, hu1G8 and 2B3 minibody. At 50% bound, relative affinities were calculated.

Cellular PSCA binding activity was assessed by flow cytometry. Briefly, 5 × 105 LNCaP and LNCaP-PSCA cells resuspended in 100 μl PBS/1% FBS were incubated on ice with 10 μl 2B3 minibody (0.1 μg/μl), washed and incubated with phycoerythrin(PE)-conjugated F(ab’)2 fragment (human IgG specific) (Jackson ImmunoResearch, West Grove, PA). Cells were washed, pelleted and resuspended in 0.5 ml PBS/1% FBS, and binding data was acquired on a FACScan (BD Biosciences, Franklin Lakes, NJ) and analyzed by using CellQuest software (BD Biosciences).

Radioiodination

Radioiodination with the positron emitting isotope 124I (Advanced Nuclide Technologies, Indianapolis, IN) and labeling efficiency were determined as described (20). Immunoreactivity was determined by incubating radioiodinated-2B3 minibody (∼100,000 cpm) with SKW 6.4-PSCA cells in PBS/1% FBS for 1 h at room temperature. Cells were pelleted and activity in an aliquot of the supernatant was counted in a Wizard 3” 1480 Automatic Gamma Counter (Perkin Elmer, Fremont, CA). A negative standard consisting of an equal amount of radiolabeled 2B3 minibody in PBS/1% FBS without cells was used as a total activity reference. The decrease in radionuclide activity relative to the standard determined the immunoreactivity (20).

124I microPET imaging and biodistribution

All animal handling was performed under an approved UCLA Chancellor's Animal Research Committee protocol. PC-3 (antigen negative) and LAPC-9 (antigen positive) xenografts were established as described (17). In another model, 2 × 106 PC-3 and PC-3-PSCA cells were injected in the left and right shoulders of 6-week old female athymic mice, respectively. LAPC-9 androgen independent (AI) xenografts were established in castrated 6-week old male mice as described (27). PC-3 tumors were allowed to grow for two weeks, whereas LAPC-9 tumors were allowed to grow for four weeks. Thyroid and stomach uptake of radioiodine was blocked as described (21).

For the PC-3 and LAPC-9 (AD and AI) models, mice (n = 16, four experimental sets of four mice) were each injected with approximately 50 μg of 124I-hu 2B3 minibody (specific activity of 1.8 ± 0.7 μCi/μg) in saline/1% human serum albumin (Sigma-Aldrich, St. Louis, MO) via the tail vein. In the irrelevant minibody experiment, 50 ug of 124I-labeled protein (specific activity of 1.5 μCi/μg) was also injected in LAPC-9 AD bearing mice (n = 4). At 4 and 21 h post injection, mice were anesthetized using 2% isoflurane, and imaged with a Focus microPET scanner (Concorde Microsystems Inc., Knoxville, TN) as previously described (26). Acquisition time was 10 minutes (1 bed position), and images were reconstructed using a filtered back projection (FBP) reconstruction algorithm (28) and displayed using the AMIDE software package (29). At the late time point, selected mice were also imaged by microCT and the images were co-registered with the microPET scans for anatomical reference and accuracy (30). Coronal projections were used to show the activity of the entire mouse at each given time point. Images were normalized for an individual mouse at different time points within the same experiment but not across different experiments.

Mice were euthanized following the 21 h scan. Tumors, liver, kidneys, lung, spleen and carcass were harvested, weighed and gamma counted. Radioactive uptake in organs was corrected for radioactive decay and converted to percentage of injected dose per gram (% ID/g) tissue.

Tumor targeting and pharmacokinetic biodistribution of 131I-2B3-minibody in SCID mice

Six week old male SCID mice (n = 4) were injected subcutaneously into the right shoulder region with 3 × 106 LNCaP-PSCA cells. Thirty days post cell injection, three mice were each injected with 8.5 μg (specific activity = 0.6 μCi/μg) of 131I-2B3 minibody via the tail vein. At 21 h post injection, mice were sacrificed and major organs were dissected, weighed and counted.

For biodistribution studies in non-tumor bearing mice, SCID mice (n = 18) were each injected with 11 μg (specific activity = 1.11 μCi/μg) of 131I-labeled 2B3 minibody via the tail vein. Groups of three mice were euthanized at 0, 2, 6, 12, 24 and 48 h post injection. Major organs and blood were harvested, weighed, and counted. Blood clearance curve was-calculated using a bi-exponential fit with the UCLA Kinetic Imaging System (KIS) (31).

MicroPET data analysis

Cylindrical regions of interest (ROIs) were drawn from 3-D FBP reconstructed PET images, co-registered with CT, at 4 and 21 h using AMIDE (29). In the sagittal orientation, 4 ROIs were drawn per tumor as well as in areas outside the animal termed “scatter.” In the coronal orientation, 4 ROIs were drawn over the low activity triceps muscle area adjacent to the tumors and termed “background.” The mean scatter-ROI values were subtracted from the tumor and background (muscle) ROI values, prior to calculation of tumor-to-background ratios. The positive tumor-to-negative tumor ratios were also determined after subtracting background scatter ROI values. Statistical analysis was performed using Excel 2000 (Microsoft, Redmond, WA). Student t-test with a 1-tailed distribution and 2-sample unequal variance parameter was used to assess the significance between tumor activity and background activity. All significance testing was determined at the P < 0.01 level.

RESULTS

Biochemical characterization of 2B3 minibody

Engineered 2B3 minibody was successfully expressed and yielded levels of ∼20 mg/L in terminal culture (approximately 300 ml batches). Analysis of purified 2B3 minibody by SDS-PAGE (Fig. 1A) showed that under reducing conditions, the minibody migrated as a monomer consistent with its predicted molecular weight of ∼ 40 kDa (lane 2), and as a covalent dimer of ∼ 80 kDa under non-reducing conditions (lane 1). Size exclusion chromatography verified that the 2B3 minibody eluted as a uniform peak at a time (∼29 min) corresponding to a correctly folded dimer of expected molecular weight (Fig. 1B) similar to a reference minibody (not shown).

Fig. 1.

Fig. 1

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A, SDS-PAGE analysis of anti-PSCA 2B3 minibody under non-reducing (Lane 1) and reducing (Lane 2) conditions. M, molecular weight markers. B, Size exclusion chromatography profile of purified hu1G8 minibody on a calibrated Superdex 75 column. C, Flow cytometry a) LNCaP and c) LNCaP-PSCA cells incubated with secondary anti-human (Fc specific) PE-F(ab’)2 only or b) LNCaP or d) LNCAP-PSCA cells incubated with purified 2B3 minibody (0.1 μg/μl). D, Competition ELISA binding assay against recombinant PSCA.

Functional characterization of 2B3 minibody

Following 2B3 minibody production, PSCA recognition and binding were evaluated. Flow cytometry (Fig. 1C) showed that 2B3 minibody could discriminate LNCaP-PSCA cells. Clear differential staining is evident for 2B3 minibody on PSCA-positive LNCaP cells and not on PSCA-negative LNCaP cells. The secondary antibody used for detecting the 2B3 minibody did not bind to either LNCaP or LNCaP-PSCA cells.

The affinity of the 2B3 minibody was evaluated by binding to recombinant soluble PSCA. Previously, the intact mouse 1G8 and hu1G8 antibodies were estimated to have an apparent KD of 2.6 and 16.7 nM (unpublished data). In the competition ELISA performed in this study, the relative affinities of intact 1G8 antibody, hu1G8 antibody and 2B3 minibody were 5 nM, 25 nM and 46 nM, respectively (Fig 1D). This represents a ∼9-fold loss in relative affinity to the mouse 1G8 antibody and a ∼2-fold relative loss to hu1G8 antibody for the engineered minibody fragment.

Binding to PSCA was also evaluated following radioiodination of 2B3 minibody using SKW 6.4-PSCA cells. The immunoreactivity of 2B3 minibody was 32(± 13)% (n = 5) following radioiodination with 124I. The immunoreactivity following two separate labeling reactions with 131I was 40 and 30%.

MicroPET imaging of LAPC-9 xenografts using 124I-2B3 minibody

MicroPET imaging was employed to evaluate the in vivo tumor targeting ability of the 2B3 minibody. For this purpose, the LAPC-9 model was selected as the target xenograft for its high level of endogenous PSCA expression (11). MicroPET images at 4 h of male SCID mice (n = 4) bearing xenografts of 218(± 190) mg revealed the ability of 124I-2B3 minibody to discern the xenograft suggesting rapid tumor localization (Fig. 2A). In addition, circulating activity in the thorax, abdomen and bladder was prominent. At 21 h, increased signal is seen in the tumor with reduced circulating activity in the thorax, abdomen and bladder resulting in enhanced contrast tumor image (Fig. 2B). The microPET/microCT overlay projection verifies the anatomical location of the tumor (Fig. 2C). The 124I-2B3 minibody exhibited similar biodistribution when evaluated by PET imaging in a set of larger [1.8(± 0.6) g] LAPC-9 tumors (n = 4). At 4 h, specific signal is seen in the tumor (Fig. 2D). Again, non-specific signal is present in the thorax, abdomen and bladder. At 21 h, the activity in the tumor is stronger with diminished activity in the non-specific areas (Fig. 2E). The microPET/microCT overlay again demonstrates signal coming from the xenograft (Fig. 2F).

Fig. 2.

Fig. 2

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Coronal microPET projections of mouse bearing an LAPC-9 xenograft of 251 mg (+ arrow) at 4 h (A) and 21 h (B) injected with 124I-2B3 minibody (∼ 50 μg/mouse). Panel C shows microPET/microCT projection overlay at 21 h. B = bladder. Color scale is indicated. Images in panels A and B were scaled to the same thresholds.

The biodistribution at 21 h for the mouse in Fig. 2A-C revealed that the tumor uptake reached 7.1% ID/g, while the blood, liver, spleen, lung and kidney each had uptakes of 4.7, 1.2, 1.3, 3.4 and 1.7% ID/g, respectively. The average tumor uptake in the group was 4.7% ID/g. Table 1 summarizes the collected uptakes of all the LAPC-9 tumors (n = 12), and other tissues of interest.

Table 1.

Biodistribution of radioiodinated-2B3 minibody in different PSCA-positive tumor bearing mice at 21 hours postinjection. Numbers represent mean uptake expressed in %ID/g ± SD.

Tissue LAPC-9 124 I (n = 12) LNCaP-PSCA 131 I (n = 4) PC-3-PSCA 124 I (n = 4) LAPC-9 AI 124 I (n = 4)
Positive tumor (T) 5.2 ± 1.6 9.3 ± 2.3 2.4 ± 0.2 2.5 ± 1.0
Negative tumor (PC-3) 2.6 ± 0.2*† ND 0.7 ± 0.3††† ND
Liver 1.3 ± 0.3† 1.1 ± 0.3†† 0.5 ± 0.1††† 0.9 ± 0.2
Spleen 1.5 ± 0.5† 0.7 ± 0.4†† 0.6 ± 0.2††† 0.8 ± 0.1
Kidney 1.9 ± 0.5† 2.0 ± 0.4†† 0.8 ± 0.2††† 1.3 ± 0.2
Lung 3.4 ± 1.0† 2.1 ± 0.3†† 1.2 ± 0.5 2.1 ± 0.4
Blood 4.9 ± 1.3 3.3 ± 1.6†† 2.3 ± 0.6 3.4 ± 0.5
Carcass 1.2 ± 0.1**† ND ND 0.6 ± 0.2
Ratios
    T/blood 1.1 2.8 1.0 0.7
    T/liver 4.0 8.5 4.8 2.8
    T/kidney 2.7 4.7 3.0 1.9
    T/neg tumor 2.0 ND 3.4 ND
Tumor weight (mg)
    PSCA-positive 910 ± 740 160 ± 40 150 ± 60 1640 ± 210
    PC-3 300 ± 130 ND 181 ± 64 ND
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*

n = 4

**

n = 8

†

Significantly different from LAPC-9 uptake (P < 0.01)

††

Significantly different from LNCaP-PSCA uptake (P < 0.01)

†††

Significantly different from PC-3-PSCA uptake (P < 0.01)

ND = Not determined

2B3 minibody PSCA-xenograft specificity

To evaluate whether targeting of 2B3 minibody was specific in vivo, a double tumor model was established with antigen positive (LAPC-9) and antigen-negative (PC-3) prostate cancer xenografts (Fig. 3 A-B). The average tumor weight in 4 mice was 759(± 419) and 298(± 34) mg for the LAPC-9 and PC-3 xenografts, respectively. Fig. 3A shows strong activity accumulation in the PSCA-positive LAPC-9 tumor and minimal uptake in the PSCA-negative xenograft at 21 h. Fig. 3B shows the microPET/microCT overlay for anatomical confirmation of the tumors. The average uptake in LAPC-9 for this set of mice was 5.5(± 0.7)% ID/g which was significantly higher (P < 0.01) than the non-specific tumor uptake in the PC-3 xenografts [2.9(± 0.3)% ID/g]. The liver, spleen, lung and kidney uptakes were 2.0, 2.2, 4.1 and 2.8% ID/g, respectively. The biodistribution at 21 h was consistent with the biodistribution seen with the single LAPC-9 tumor bearing mice and is therefore incorporated into Table 1.

Fig. 3.

Fig. 3

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Coronal microPET (A) and microPET/microCT (B) overlay projections of a SCID mouse bearing LAPC-9 (+ arrow) and PC-3 (− arrow) xenografts at 21h injected with 124I-2B3. Panel C shows coronal microPET/microCT overlay projection of LAPC-9 (+) bearing mouse at 21 h injected with irrelevant 124I-labeled minibody (∼ 50 μg). Scale bar has been placed for qualitative reference. Mouse in A - B is not scaled to mouse in C.

To further evaluate specificity, an irrelevant minibody fragment was labeled with 124I and assessed in mice bearing LAPC-9 xenografts [tumor size of 292(± 98) mg]. As seen in Fig. 3C, the microPET/microCT image at 21 h shows that the control minibody does not localize to the xenograft. Biodistribution at the time of sacrifice (28 h) revealed a tumor uptake of 0.6(± 0.2)% ID/g (n = 4). Thus, the uptake seen with 2B3 minibody in the LAPC-9 xenografts is specific and not due to non-specific tumor accumulation.

Minibody blood kinetics in non-tumor bearing SCID mice

To evaluate kinetics of distribution in the absence of tumor, 2B3 minibody was radiolabeled with 131I and biodistribution and blood clearance studies were conducted in SCID mice. The 131I-2B3 minibody showed a somewhat prolonged blood clearance pattern relative to that seen with other minibody fragments targeting CEA and HER2 (t1/2β = 7.0 and 5.6 h, respectively) (22, 24). For 131I-2B3 minibody, the distribution-phase half-life (t1/2α) was 1.3 h and the terminal phase half-life (t1/2β) was 11.2 h (Fig. 4). The liver, spleen and kidneys displayed low non-specific uptake that fell below 1% ID/g by 24 h, which is consistent with that observed for other minibody fragments. The blood activity was 2.4% ID/g at 24 h.

Fig. 4.

Fig. 4

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Biodistribution of 131I-2B3 minibody in non-tumor bearing male SCID mice. Groups of three mice were evaluated at each time point.

Targeting of LNCaP-PSCA xenografts using 131I-2B3 minibody

Many targeting studies for prostate cancer have been conducted with LNCaP xenografts. Therefore, using SCID mice bearing generated LNCaP-PSCA tumors, a 21 h time point biodistribution and tumor uptake of 131I-lableled 2B3 minibody was evaluated. At 21 h, high activity was present in the tumor with low blood and organ activity (Table 1). Tumor uptake was 9.3(± 2.3)% ID/g, while the blood was 3.3(± 1.6)% ID/g resulting in a tumor-to-blood ratio of 2.8.

MicroPET imaging of androgen independent (AI) xenografts using 124I-2B3 minibody

Targeting and imaging of AI xenografts was also explored in two separate experiments. The PC-3 cell line was engineered to stably express PSCA (16), thus an imaging study could be undertaken with a negative cell line that is a matched control. In this dual tumor model, 124I-2B3 minibody accumulation is clearly seen favoring the PSCA-positive (right shoulder) tumor at 21 h (Fig. 5A). Androgen independent LAPC-9 xenografts have also been developed and PET imaging of 124I-2B3 minibody in LAPC-9 AI tumor bearing mice is depicted in Fig. 5B. As seen with the LAPC-9 AD xenografts, the localization of the activity is in the tumor with minimal nonspecific activity in the midsection and typical bladder activity.

Fig. 5.

Fig. 5

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Coronal microPET/CT projections of a representative athymic mouse bearing PC-3-PSCA (+ arrow) and PC-3 (− arrow) xenografts (A) and a SCID mouse bearing a LAPC-9 AI xenograft (+) injected with 124I-2B3 minibody at 21 h. B = bladder.

Biodistribution of excised AI tumors showed moderate uptake compared to the AD xenografts (Table 1). Uptake in the PC-3-PSCA positive tumor was 2.4 (± 0.2)% ID/g which was significantly higher (P < 0.01) at 21 h than the uptake in the PC-3 tumor [0.7(± 0.3)% ID/g], resulting in a 3.4 positive-to-negative tumor ratio. This study demonstrates specific PSCA targeting in an AI tumor model. Activities in the liver, spleen, kidney and lung were at low levels.

Region-of-interest (ROI) data analysis

In the LAPC-9 AD xenograft model, ROI analysis at the early 4 h time point resulted in a tumor-to-background ratio of 1.3. At the later 21 h time point the contrast ratio improved to 5.3 resulting in a 4.1-fold improvement. In the LAPC-9 AI model a tumor-to-background ratio of 3.3 was obtained at 21 h. For the PC-3-PSCA model, the tumor-to-background ratio was 0.7 at 4 h, which improved to 3.7 at 21 h, resulting in a 5.3-fold improvement.

ROI analysis was also carried out to determine the ratio of PSCA positive-to-negative xenografts. In the LAPC-9 AD model, the LAPC-9-to-PC-3 ratio was 2 at 21 h. In the AI model with a matched PSCA-positive xenograft; the PC-3-PSCA-to-PC-3 ratio was also 2 at 21 h.

DISCUSSION

The present study introduces the genetically engineered radioiodinated (124I or 131I) 2B3-minibody as a novel antibody fragment for specific targeting and potential PET imaging agent for PSCA-expressing prostate cancers. The 2B3 minibody was evaluated by microPET in mice with different PSCA-expressing prostate cancer xenografts. Rapid uptake was noted by the ability to visually discriminate the various xenografts at 4 h while rapid blood clearance demonstrated the ability to produce enhanced contrast images at 21 h. Biochemical analysis demonstrated that the minibody was pure following chromatography, migrated as a covalent dimer, had high affinity and could discriminate PSCA in vitro. Furthermore, 124I-2B3 minibody was able to image and target PSCA-positive AI xenografts, revealing that androgen independent as well as androgen dependent tumors could be targeted. Pharmacokinetic studies showed a rapid yet slightly longer half-life compared to other tumor targeting minibody fragments. Nonetheless, the 2B3 minibody clearance proved rapid enough to provide high contrast images by 21 h. ROI determination of tumor-to-background ratios at different time points showed improved tumor-to-background ratios from 4 to 21 h. Importantly, in vivo specificity was demonstrated by minimal uptake in the PSCA-negative tumors and lack of tumor targeting by an irrelevant minibody. Thus, hu1G8 minibody displayed multiple positive xenograft targeting characteristics in vivo proving itself as a promising candidate for imaging prostate cancer.

The present 80 kDa anti-PSCA minibody fragment was derived from hu1G8 mAb which has a high relative affinity (KD = 25 nM) for PSCA (17). The hu1G8 mAb was humanized from the mouse monoclonal 1G8 antibody (KD = 1 nM) (16). The 124I-labeled intact murine 1G8 and hu1G8 mAbs were shown to successfully image PSCA-positive LAPC-9 xenografts in male SCID mice (17), reaching good tumor uptakes. However, a long delay was necessary in order to produce enhanced contrast tumor images. At times as late as 72 h, high activity remained in the blood and normal tissue, making tumor identification more difficult. In the current work, the smaller minibody tracer mimicked the large intact antibodies in LAPC-9 uptake, but reduced the time needed for optimal PET imaging to 21 h rather than 168 h. The LAPC-9 xenograft is one of the few available tumors that endogenously overexpresses PSCA. This xenograft line was established from a patient with metastatic prostate cancer (32) and has been a bench-mark for PSCA expression evaluation experiments (11, 12). In this model, 124I-2B3 minibody demonstrated similar tumor uptake (5.2 %ID/g) at 21 h to that of 124I-labeled murine 1G8 (5.8 %ID/g) and hu1G8 (6.6 %ID/g) mAbs at 168 h (17).

The LNCaP cell line of human prostate cancer has commonly been used for investigating antibody imaging and therapeutic approaches for prostate cancer (9, 33, 34). In the LNCaP-PSCA model, high tumor uptake (9.3% ID/g) with the 131I-2B3 minibody was achieved as well as a higher tumor-to-blood ratio (2.8) than that seen in the LAPC-9 model. Recently, we have determined that LNCaP-PSCA xenograft expresses PSCA at much higher levels than the LAPC-9 xenograft (unpublished data) suggesting a possible correlation between tumor uptake and antigen abundance. It will be of interest to formally evaluate the ability of the 2B3 minibody to images xenografts of various PSCA antigen expression levels, given the known heterogeneous nature of prostate cancer.

Patients with prostate cancer die from metastatic hormone refractory disease (35). Although the role of PSCA in progression to androgen independence is still under investigation, PSCA expression has been shown to be upregulated in a majority of prostate cancer metastases (12) and overexpressed in 83% of AI specimens (11). Therefore we asked whether the 2B3 minibody could be used to detect hormone refractory tumors in our xenograft model. In response to castration, LAPC-9 cells undergo growth arrest and persist in a dormant, androgen-responsive state for at least 6 months. After prolonged periods of androgen deprivation, spontaneous androgen-independent outgrowths develop (27). Using this xenograft model, 124I-2B3 minibody demonstrated the ability to target and produce high contrast tumor images at 21 h. In addition, 124I-2B3 minibody was able to image PC3-PSCA expressing xenografts. These animals contained PSCA-negative PC-3 xenografts which allowed evaluation of non-specific tumor uptake. The PSCA-positive-to-negative tumor ratio was 3.4 displaying clear evidence of PSCA specificity in this AI model.

The most extensively evaluated nuclear imaging agent for prostate cancer is Prostascint, which recognizes Prostate Specific Membrane Antigen (PSMA). In preclinical studies the radioiodinated parental antibody of Prostascint (7E11) was compared head-to-head with another radioiodinated anti-PSMA antibody (J591) for its tumor targeting potential in LNCaP xenografts (36). In a biodistribution study (tumor size ranging from 100 − 300 mg), the tumor uptake was 11.7 (± 4.3) and 11.2 (± 2.9) %ID/g for 7E11 and J591, respectively, at 48 h. However, the blood activity was 10.8 (± 3.5) %ID/g for 7E11and 8.6 (± 2.0) %ID/g for J591 resulting in a tumor-to-blood ratio of 1 which only rose to 2 by 144 h (34). In this work, uptake in LNCaP-PSCA tumors with the radioiodinated 2B3 minibody reached 9.3 (± 2.3) %ID/g at 21 h and the tumor-to-blood ratio was nearly 3. Thus, in the same xenograft model, the 2B3 minibody reached a comparable tumor uptake level with an improved tumor-to-blood ratio at 21 h, instead of 144 h. Protease derived 125I-J591 F(ab’)2 (110 kDa) and Fab (50 kDa) fragments were also evaluated for their ability to improve target-to-background ratios in LNCaP xenografts (37). The tumor and blood activity for 125I-J591 F(ab’)2 was 5.17 and 4.41 %ID/g, respectively, at 24 h. The tumor uptake with the 125I-J591 Fab reached a modest 1.85 %ID/g at 24 h but importantly a tumor-to-blood ratio of 3.44 was achieved (37). This study demonstrated that antibody fragments improved target-to-background ratios at an earlier time point than intact IgG.

Tumor targeting of cell surface antigens by using small peptides has also become a focus of interest due to their favorable properties. High receptor affinity and very fast blood clearance allows peptides to achieve high target-to-background ratios very early after administration injection [reviewed in (38)]. Two recent PET imaging studies using peptides labeled with the positron emitter 64Cu (t1/2 = 12.7 h) have shown promise as prostate cancer imaging agents (39, 40). In one study, the stability and pharmacokinetics of 64Cu-bombesin (BBN) peptide using either CB-TE2A or DOTA as metal chelators were compared in mice bearing PC-3 xenografts (39). The 64Cu- CB-TE2A-bombesin peptide exhibited better tumor targeting at 15 minutes after administration as well as improved in vivo stability that resulted in substantial decrease in liver uptake and improved clearance from the mouse at 24 h. In a more recent study, a vasoactive intestinal peptide (VIP) analogue (TP3939) targeting VPAC1 receptor, radiolabeled with 64Cu was evaluated in mice carrying PC3 xenografts (40). MicroPET images at 4 and 24 h showed specific localization to tumors with tumor to muscle and tumor to blood ratios of 5.2 and 2.5, respectively at 24 h. The 64Cu-TP3939 also demonstrated the ability to detect occult prostate cancer in the mouse prostate in natural TRAMP II model of prostate cancer development that was not detected by 18F-FDG.

The current study demonstrates the combnation of the high sensitivity and resolution of PET with the high specificity, selectivity and the suitable pharmacokinetics of the 2B3 minibody. An important next step will be evaluation of radioiodinated 2B3 minibody in pilot imaging studies in patients with known metastatic prostate cancer, to evaluate normal tissue distribution, pharmacokinetics, tumor localization, and assess immunological responses, if any. In addition, the studies reported here have been undertaken exclusively with radioiodine and it will be important to evaluate radiometal-labeled 2B3 minibody uptake and imaging in these same xenograft models. PET imaging using 64Cu could be an advantage since there is evidence of antibody internalization with PSCA (41) and 64Cu-labeled minibody demonstrated excellent tumor images in a different internalizing system (42).

In patients, by the time of diagnosis, only half of the tumors are clinically localized while the other half have detectable or undetectable extracapsular spread in patients with prostate cancer (43). Antibody-based PET (immunoPET) detection of PSCA provides a biological basis of imaging based on a tissue-specific marker, which would complement current anatomical and metabolic approaches to detection and monitoring of disease. As such, immunoPET could play an important role in staging at diagnosis, stratifying patients for PSCA-targeted therapies, monitoring treatment response, and in long-term follow-up. Future development of the 124I-labeled anti-PSCA 2B3 minibody for clinical PET imaging will provide an important new modality for detection and management of prostate cancer.

ACKNOWLEDGMENTS

The authors are thankful to Dr. D. Stout, J. Edwards and W. Ladno at UCLA for their assistance with the microPET/CT scans, and F.B. Salazar for providing excellent technical assistance. We are especially grateful to Dr. V.E. Kenanova for providing help with image analysis, Dr. Z. Gu and Dr. J. Yamashiro for critical analysis of data, and Dr. H. Huang for assistance with kinetic analysis. We also thank UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility.

Grant support: This work was supported by the UCLA SPORE in Prostate Cancer (NIH CA 92131), and a career development award from the UCLA ICMIC (In Vivo Centers for Molecular Imaging of Cancer) (NIH CA 86306). Additional support was provided by NIH P01 CA 43904. R.E.R. and A.M.W. are members of the Jonsson Comprehensive Cancer Center at UCLA (NIH CA 16042).

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