Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [111In]-DTPA-d-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ip1 human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1- octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 × 109 plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [111In]-DTPA-d-Phe1-octreotide 2 days later, showed a range of 15.3–60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [111In]-DTPA-d-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [111In]-DTPA-d-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [111In]-DTPA-d-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [111In]-DTPA-d-Phe1-octreotide tumor localization in animals administered AdCMVh-SSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [111In]-DTPA-d-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [111In]-DTPA-d-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [111In]-DTPA-d-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [111In]-DTPA-d-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.

Ovarian cancer is now the fifth leading cause of cancer deaths in United States women with ∼14,000 deaths/year (1). There have been advances in the treatment of ovarian cancer through various chemotherapeutic regimens; however, control of intraabdominal disease remains problematic. Some abdominal failures have been successfully treated with external-beam radiation or targeted radiotherapy (2, 3, 4, 5, 6, 7). Targeted radiotherapy has primarily been accomplished through the use of radiolabeled mAbs3(8, 9). However, the success of this approach has been limited because of bone marrow toxicity resulting from the long half-life of the radiolabeled mAb in the blood, poor tumor penetration of these large molecules, and low levels or heterogeneous distribution of tumor-associated antigens or receptors to which the mAb can bind (9). Therefore, novel strategies that can overcome one or all of these limitations will be important.

It has been shown previously that the levels of tumor-associated receptors or antigens can be increased both in vitro and in vivo through the use of gene transfer vectors (10, 11, 12, 13, 14, 15, 16, 17, 18). These vectors mediate gene transfer of the cDNA for the receptor or antigen of choice to the tumor cells, resulting in the cell surface expression of the target receptor or antigen. Our group has used adenoviral vectors to induce the expression of carcinoembryonic antigen and GRPr on a variety of cancer cells both in vitro and in vivo with subsequent binding and localization of radiolabeled peptides or mAbs (13, 14, 15, 16, 17). However, one of the drawbacks of using adenoviral vectors is their lack of specificity for transducing tumor cells. This problem can be overcome through the use of tropism-modified adenoviral vectors that target tumor cells or through the use of adenoviral vectors that contain tumor-specific promoters such that the receptor or antigen is only expressed on tumor cells (19, 20, 21, 22, 23, 24, 25, 26, 27). The receptor or antigen can then be targeted with a high affinity radiolabeled peptide or mAb. The use of radiolabeled peptides for therapy should help overcome the problems of bone marrow toxicity and limited tumor penetration experienced with radiolabeled mAbs because of their rapid blood clearance and small size. Further in this regard, recent studies have shown that radiolabeled peptides are effective for cancer therapy in vivo(28, 29, 30, 31, 32).

We have demonstrated previously that a radiolabeled peptide ([125I]-mIP-bombesin) will localize to human ovarian tumors induced to express the GRPr with an adenoviral vector, both in vitro and in vivo(15). To achieve an optimal therapeutic effect, the biological half-life of the peptide should match the physical half-life of the radioisotope (33, 34). Therefore, radiolabeling mIP-bombesin with 125I (60-day half-life) or 131I (8-day half-life) for therapeutic applications would not be ideal because of the 5.2-h biological half-life of [125I]-mIP-bombesin that was demonstrated in pharmacokinetic studies (17). Therefore, in this study, we investigated the somatostatin receptor/octreotide system, which has a variety of well-characterized radiolabeled octreotide analogues that can be used for therapy.

Octreotide is a derivative of the inhibitory peptide somatostatin and binds with highest affinity to hSSTr2 and lower affinity to hSSTr1, hSSTr3, hSSTr4, and hSSTr5 (35). Somatostatin binds with high affinity to all hSSTr subtypes and undergoes rapid in vivo degradation compared with octreotide (35). Octreotide has been labeled with 111In ([111In]-DTPA-d-Phe1-octreotide) or 123I ([123I]-Tyr3-octreotide) for imaging neuroendocrine tumors in patients (36, 37, 38, 39). [111In]-DTPA-d-Phe1-octreotide is also being evaluated as a radiotherapeutic agent (40, 41, 42, 43), and other octreotide analogues radiolabeled with 90Y, 188Re, and 64Cu have demonstrated therapeutic efficacy in animal models (28, 30, 31, 32).

In the present study, we describe the construction of a replication-incompetent adenoviral vector that encodes the hSSTr2 gene driven by the CMV promoter (AdCMVhSSTr2) and show that it can mediate the expression of hSSTr2 in various human tumor cell lines in vitro using a competitive binding assay with [125I]-Tyr1-somatostatin or [111In]-DTPA-d-Phe1-octreotide as the radioligands. The AdCMVhSSTr2 was then used to induce the expression of hSSTr2 in a mouse model of human ovarian carcinoma as demonstrated by tumor localization of [111In]-DTPA-d-Phe1-octreotide. Another objective of this study was to determine an optimal protocol for the in vivo expression of hSSTr2. The tumor localization and normal tissue distribution of [111In]-DTPA-d-Phe1-octreotide was investigated at multiple days after a single AdCMVhSSTr2 administration or after two injections of AdCMVhSSTr2. This will be important for determining the vector regimen necessary to maximize the radioactivity delivered to the tumor when using radiolabeled octreotide in therapy studies.

Cells.

The A427 human nonsmall cell lung carcinoma cells and the 293 human transformed primary embryonal kidney cells were obtained from American Type Culture Collection (Rockville, MD). The A427 and 293 cells were maintained in Eagle’s MEM containing 10% fetal bovine serum, 1% nonessential amino acids, and 1% sodium pyruvate. The human ovarian carcinoma cell line SKOV3.ip1 was obtained from Janet Price (Baylor University, Houston, TX) and maintained in DMEM containing 10% fetal bovine serum. All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2.

Construction of AdCMVhSSTr2.

A recombinant adenovirus encoding the human somatostatin receptor subtype 2 cDNA was prepared using standard techniques described by Graham and Prevec (44). This is similar to the method described previously for the construction of the adenovirus encoding the murine GRPr gene (17). Briefly, a cDNA fragment containing the hSSTr2 gene (obtained from the American Type Culture Collection) was subcloned into the pACCMVpLpARS(+) adenoviral shuttle vector (provided by R. Gerard, Katholieke Universiteit Leuven, Leuven, Belgium). This shuttle plasmid (pAC-hSSTr2) was cotransfected into the E1A transcomplementing cell line 293 with the adenoviral packaging plasmid pJM17 (provided by F. Graham, McMaster University, Hamilton, Ontario, Canada) using the DOTAP (Life Technologies, Inc., Gaithersburg, MD) cationic liposome vector. The recombinant adenovirus was plaque purified and validated by PCR. The AdCMVhSSTr2 was titered within the 293 cell line using plaque assay techniques for direct determination of viral pfu.

Competitive Binding Assay.

The induction of hSSTr2 in human carcinoma cell lines was evaluated using a competitive binding assay with membrane preparations of cells that had been infected with AdCMVhSSTr2. The assays were performed on membrane preparations from A427 cells and SKOV3.ip1 cells infected with 0, 10, 100, or 200 MOI AdCMVhSSTr2 using either [125I]-Tyr1-somatostatin or [111In]-DTPA-d-Phe1-octreotide. The cells were seeded such that they were ∼80% confluent at the time they were infected with AdCMVhSSTr2 and then harvested for membrane preparation 2 days after adenoviral infection. The AdCMVhSSTr2 was added to cells in Optimem (Life Technologies, Inc., Grand Island, NY) and incubated at 37°C in 5% CO2 for 2 h. The cells were then supplemented with complete media and incubated an additional 48 h at 37°C. Cell membranes were then prepared from the infected and uninfected cells using a protocol similar to that described previously (45, 46, 47, 48). Briefly, the cells were washed with PBS, scraped from the flask, and centrifuged at 90 × g for 5 min at 4°C. The cell pellet was resuspended in cold lysis buffer (10 mm Tris-HCl, 2 mm EDTA, and 2 mm MgCl2, pH 7.2) containing 0.5 mm phenylmethylsulfonyl fluoride and incubated on ice for 15 min. The mixture was vortexed and centrifuged at 600 × g for 15 min at 4°C, and the supernatant was removed and stored on ice. An additional lysis step was performed on the pellet, and the two supernatants were combined. The pooled supernatant was centrifuged at 28,000 × g for 30 min at 4°C, and the resulting supernatant discarded; the pellet was resuspended in 250 mm sucrose, 20 mm glycylglycine, and 1 mm MgCl2. A Bio-Rad (Hercules, CA) protein assay was performed to determined the protein concentration, and the samples were aliquoted and stored at −80°C.

For the competitive binding assays, the membrane preparations were thawed and diluted in buffer (10 mm HEPES, 5 mm MgCl2, 1 mm EDTA, 0.1% BSA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 0.5 μg/ml aprotinin, and 200 μg/ml bacitracin, pH 7.4) to 25 μg/sample (100 μl). The addition of these protease inhibitors to the buffer should prevent the rapid degradation of the radiolabeled peptides. Individual samples were added to Multiscreen Durapore filtration plates (type FB, 1.0 μm borosilicate glass fiber over 1.2 μm Durapore membrane; Millipore, Bedford, MA) and washed with buffer (10 mm HEPES, 5 mm MgCl2, 1 mm EDTA, and 0.1% BSA, pH 7.4). One-hundred μl of [125I]-Tyr1-somatostatin (∼0.05 nm; specific activity, 1100–2200 Ci/mmol; DuPont/NEN Research Products, Boston, MA) or [111In]-DTPA-d-Phe1-octreotide (∼0.05 nm; specific activity, 300–450 Ci/mmol; kindly provided by Mallinckrodt Medical, Inc., St. Louis, MO) were added to each well in triplicate along with various concentrations (0 to 7500 nm) of unlabeled Tyr1-somatostatin (Sigma Chemical Co., St. Louis, MO) or unlabeled DTPA-d-Phe1-octreotide (Mallinckrodt Medical, Inc.) and incubated for 90 min at room temperature. The samples were washed twice with ice cold buffer, the filters were allowed to dry, and the individual wells were punched out and counted in a gamma counter.

IC50s (the amount of cold ligand required to displace 50% of the radiolabeled ligand) were generated from experiments performed in triplicate and analyzed using nonlinear models of the DPM bound versus the concentration of blocking ligand. The IC50s were then used to derive the binding constants (Kd) and receptor density (Bmax), assuming homologous displacement (49). For each experiment, the appropriateness of the model was examined via mean squared error of the model and the degree of correlation between actual and predicted values. The experiments had a correlation between actual and predicted values of 90% or greater unless otherwise noted.

Analysis of hSSTr2 mRNA Expression.

A427 and SKOV3.ip1 cells were seeded at 3 × 106 cells and grown for 24 h before infection with 0, 10, 100, or 200 MOI of AdCMVhSSTr2. Cells were incubated with AdCMVhSSTr2 for 2 h, after which the virus was removed, and the cells were supplemented with complete media. The media was aspirated 18 h later, and the cells were lysed using a monophase solution of phenol and guanidinium thiocyanate (RNA STAT-60; Tel-Test “B”, Inc., Friendswood, TX). Total RNA was extracted according to the manufacturer’s protocol and resuspended in 1 mm sodium citrate, pH 6.4 (Ambion, Austin, TX). Total RNA concentrations were determined by UV spectrophotometry at 260 nm using a Beckman DU640B spectrophotometer (Beckman Instruments, Fullerton, CA). First-strand cDNA was generated from 1 μg of total RNA in a 20-μl volume using the oligo(dT) primer from a cDNA cycle kit (Invitrogen, Carlsbad, CA). Samples were also incubated without reverse transcriptase as a negative control. Aliquots (2 μl) of each reaction were amplified using 0.05 μm specific primers 5′-TGGATCCTTGGCCTTCCAG-3′ (sense) and 5′-ATTCTAGAAGCCAGGTGTGAG-3′ (antisense) for hSSTr2 in a total of 50 μl containing 60 mm Tris-HCl (pH 8.5), 15 mm (NH4)2SO4, 3.5 mm MgCl2, 1 mm deoxynucleotide triphosphates, and 2.5 units of Taq polymerase (Stratagene, La Jolla, CA). G3PDH primers (Clontech, Palo Alto, CA) were also used as controls to confirm the integrity of cDNA. The expected sizes of the amplified hSSTr2 and G3PDH DNA fragments were 1351 and 983 bp, respectively. The reaction mixture was heated to 94°C for 4 min and then subjected to 30 cycles of PCR (94°C, 1 min; 55°C, 2 min; and 72°C, 2 min). RT-PCR products were visualized with ethidium bromide after electrophoresis of 12 μl of sample on a 1% agarose gel.

Animal Biodistribution Studies.

Biodistribution studies were performed in groups of five athymic nude mice (National Cancer Institute, Frederick Research Laboratory, Frederick, MD) bearing i.p. SKOV3.ip1 tumors. The mice were injected i.p. with 2 × 107 SKOV3.ip1 cells. After 5 days, AdCMVhSSTr2 (1 × 109 pfu) was injected i.p., followed by i.p. administration of either [125I]-Tyr1-somatostatin (2 μCi) or [111In]-DTPA-d-Phe1-octreotide (2 μCi) 2 days later. The animals were killed 4 h after injection of the radiolabeled peptides, and the blood, lungs, liver, small intestine, spleen, kidney, muscle, tumor, abdominal lining, uterus, and pancreas were removed and weighed. The radioactivity was counted in a gamma counter. The tumors were ∼0.1 g in size and consisted of one nodule with no visible ascites. Control studies were performed in animals bearing i.p. SKOV3.ip1 tumors with either an adenovirus (AdCMVGRPr), which induces the expression of the GRPr or with no adenoviral injection, followed 2 days later by the administration of [111In]-DTPA-d-Phe1-octreotide. In addition, the biodistribution of a control peptide ([125I]-mIP-bombesin), which binds to the GRPr, injected 2 days after administration of AdCMVhSSTr2, was investigated. The Wilcoxon-Rank Sum test was used to compare uptake in varying tissues between [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide, whereas the Signed-Rank test was used to compare the amount of uptake between tissues for individual animals within each group.

The effect on the kidney uptake of [111In]-DTPA-d-Phe1-octreotide in this animal tumor model upon administration of l-lysine was also investigated. Mice bearing i.p. SKOV3.ip1 tumors administered AdCMVhSSTr2 as described above were given i.p. injections of l-lysine (2 mg/g body weight; Sigma Chemical Co.) at 30 min before and 1, 2, and 3 h after administration of [111In]-DTPA-d-Phe1-octreotide. The tumor and normal organs described above were harvested 4 h after administration of the radiolabeled peptide, weighed, and counted in a gamma counter. The Wilcoxon-Rank Sum test was used to compare uptake in varying tissues between mice administered [111In]-DTPA-d-Phe1-octreotide with or without l-lysine, whereas the Signed-Rank test was used to compare the amount of uptake between tissues for individual animals within each group.

The effect of two injections of AdCMVhSSTr2 on the tumor localization and biodistribution of [111In]-DTPA-d-Phe1-octreotide was also investigated. Five days after i.p. inoculation with SKOV3.ip1 cells, mice were administered AdCMVhSSTr2 (1 × 109 pfu; day 0), followed by an additional i.p. injection of AdCMVhSSTr2 (1 × 109) at day 1, 2, or 3. [111In]-DTPA-d-Phe1-octreotide was then given on day 2 when the second AdCMVhSSTr2 injection was given on day 1, or on day 4 when the second AdCMVhSSTr2 injection was given on day 2 or day 3. As a control, a single administration of AdCMVhSSTr2 (day 0), followed by an injection of [111In]-DTPA-d-Phe1-octreotide on day 1, 2, or 4, was evaluated. All animals were killed 4 h after injection of the radiolabeled peptide, and tumors and normal organs were harvested, weighed, and counted in a gamma counter. To assess the affect of the number of days after a single or double injection of AdCMVhSSTr2 on localization of [111In]-DTPA-d-Phe1-octreotide, the Kruskall-Wallis test was used. Pairwise comparisons were then performed using the Wilcoxon-Rank Sum test. These techniques were used because of the small sample sizes and slight skewness of the data.

The median localization as well as the 25th and 75th percentile was used to summarize the central tendency and variability for each tissue for the varying experiments. The interquartile range (75th percentile minus 25th percentile) is the appropriate measure of variability, and the median is the appropriate measure of central tendency when data are skewed and the sample sizes are small.

Competitive Binding Assay.

The A427 and SKOV3.ip1 cells were infected at a MOI of 0, 10, 100, or 200 with AdCMVhSSTr2 to induce the expression of hSSTr2. Membranes were then prepared from the cells 48 h after AdCMVhSSTr2 infection, and competitive binding assays using [125I]-Tyr1-somatostatin, unlabeled Tyr1-somatostatin, [111In]-DTPA-d-Phe1-octreotide, and unlabeled DTPA-d-Phe1-octreotide were performed to quantitate the number of receptors on the infected cells. Table 1 contains Bmax, Kd, and IC50s for each cell line when infected with AdCMVhSSTr2 as determined by the binding of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide and inhibition with unlabeled Tyr1-somatostatin and DTPA-d-Phe1-octreotide. As expected, both [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide had high affinity binding to the membrane preparations from cells induced to express hSSTr2 (Kd = 1.8–3.3 nm and 0.7–3.0 nm, respectively). The median Bmaxs of AdCMVhSSTr2-infected A427 cell membranes ranged from 930-5100 fmol/mg of protein, whereas the membranes from SKOV3.ip1 cells infected with AdCMVhSSTr2 had Bmaxs of 80–610 fmol/mg of protein. Thus, the A427 cells expressed more receptor when infected with AdCMVhSSTr2 than the SKOV3.ip1 cells, indicating that the A427 cells are more susceptible to adenoviral infection than the SKOV3.ip1 cells. This is in agreement with previous results with these cell lines after infection with AdCMVGRPr and binding with [125I]-bombesin (15, 17).

Analysis of hSSTr2 mRNA Expression.

To demonstrate that infection of A427 and SKOV3.ip1 cells with AdCMVhSSTr2 results in expression of the hSSTr2 gene, total RNA was isolated from uninfected and infected cells and subjected to RT-PCR analysis (Fig. 1). In A427 cells, hSSTr2 mRNA was detected after infection with 10, 100, and 200 MOI of AdCMVhSSTr2 but not when the cells were uninfected. The SKOV3.ip1 cells demonstrated hSSTr2 mRNA expression after infection with 100 and 200 MOI AdCMVhSSTr2 but not when the cells were uninfected or infected at 10 MOI. This correlates to what was observed in the competitive binding assays, in which Bmaxs were generated for A427 cells infected with 10, 100, and 200 MOI of AdCMVhSSTr2 and for SKOV3.ip1 cells infected with 100 and 200 MOI of AdCMVhSSTr2. Parallel RT-PCR reactions were carried out for each sample in the absence of reverse transcriptase to ensure that PCR products resulted from cDNA templates rather than genomic DNA (data not shown).

Animal Biodistribution Studies.

We next evaluated the in vivo induction of hSSTr2 in human ovarian cancer xenografts after regional administration of AdCMVhSSTr2 by the distribution of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide. The biodistribution of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide in athymic nude mice bearing i.p. SKOV3.ip1 tumors induced to express hSSTr2 with AdCMVhSSTr2 are shown in Fig. 2. The median tumor uptake of [111In]-DTPA-d-Phe1-octreotide (60.4% ID/g) was significantly greater than the median uptake of [125I]-Tyr1-somatostatin (3.5% ID/g) 4 h after i.p. administration of the radiolabeled ligands (P = 0.008). The median tumor concentration of [111In]-DTPA-d-Phe1-octreotide was 18.6% ID/g 24 h after the peptide was administered. When SKOV3.ip1 tumor-bearing mice were administered AdCMVGRPr as a control, the tumor localization of [111In]-DTPA-d-Phe1-octreotide (1.6% ID/g) at 4 h was significantly less than when AdCMVhSSTr2 was administered (P = 0.016). The [111In]-DTPA-d-Phe1-octreotide showed high uptake in the kidneys after AdCMVhSSTr2 (31.2% ID/g at 4 h) or AdCMVGRPr (35.3% ID/g at 4 h) administration. This shows that [111In]-DTPA-d-Phe1-octreotide demonstrated high tumor localization compared with [125I]-Tyr1-somatostatin and that this localization was specific, as evidenced by the low tumor localization when the negative control adenovirus (AdCMVGRPr) was administered.

In another experiment, the median SKOV3.ip1 tumor uptake 4 h after i.p. injection of [111In]-DTPA-d-Phe1-octreotide in mice administered AdCMVhSSTr2 was 15.3% ID/g, and the kidney uptake was 20.3% ID/g (Fig. 3). The tumor uptake of [111In]-DTPA-d-Phe1-octreotide was significantly greater than when mice bearing SKOV3.ip1 tumors administered AdCMVhSSTr2 were given [125I]-mIP-bombesin i.p. as a control peptide (0.3% ID/g; P = 0.008). Mice that were administered l-lysine showed a significant decrease in kidney uptake of [111In]-DTPA-d-Phe1-octreotide (7.6% ID/g) compared to when no l-lysine was given (20.3% ID/g; P = 0.008), whereas the tumor concentration was not significantly reduced (P = 0.42) in mice given the l-lysine (10.1% ID/g). Mice that received l-lysine and AdCMVhSSTr2 had significantly higher median tumor uptake of [111In]-DTPA-d-Phe1-octreotide (10.1% ID/g) than mice receiving l-lysine but not adenovirus (0.8% ID/g; P = 0.008). In addition, the [111In]-DTPA-d-Phe1-octreotide uptake in the uterus, abdominal lining, and liver was significantly increased when AdCMVhSSTr2 was administered compared to when no adenovirus was given (P = 0.008, 0.03, and 0.03, respectively), whereas the pancreas did not show a significant increase (P = 0.10). However, in no instance was the median % ID/g greater than 5%. The uptake of [111In]-DTPA-d-Phe1-octreotide in SSTr2-positive tissues such as the pancreas and small intestine when no AdCMVhSSTr2 was administered was low (0.6 and 0.5% ID/g, respectively). Thus, tumor localization was specific for [111In]-DTPA-d-Phe1-octreotide when AdCMVhSSTr2 was given, and the kidney concentration of [111In]-DTPA-d-Phe1-octreotide was significantly reduced when l-lysine was administered.

In a third experiment, the biodistribution of [111In]-DTPA-d-Phe1-octreotide was evaluated at 1, 2, and 4 days after a single administration of AdCMVhSSTr2 and at several times after two administrations of AdCMVhSSTr2 to determine the length of hSSTr2 expression and the effect of two AdCMVhSSTr2 injections on the tumor localization of [111In]-DTPA-d-Phe1-octreotide, respectively. The median tumor localization of [111In]-DTPA-d-Phe1-octreotide 4 h after injection was similar 1, 2, and 4 days after a single administration of AdCMVhSSTr2 (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88; Fig. 4). However, an overall difference in [111In]-DTPA- d-Phe1-octreotide uptake was observed in the small intestine (P = 0.007), spleen (P < 0.05), and pancreas (P < 0.05).

The median tumor localization of [111In]-DTPA-d-Phe1-octreotide after two injections of AdCMVhSSTr2 is shown in Fig. 5. The tumor uptake of [111In]-DTPA-d-Phe1-octreotide given 1 day after the second AdCMVhSSTr2 administration was significantly less (P = 0.009) when AdCMVhSSTr2 was administered on consecutive days (22.4% ID/g) than when the adenovirus was administered 2 days apart (53.2% ID/g). In addition, differences were observed in the lung (P = 0.006), spleen (P = 0.0006), kidney (P = 0.0003), uterus (P = 0.009), and pancreas (P = 0.006). There were not significant differences in [111In]-DTPA-d-Phe1-octreotide concentration in any tissues when two injections of AdCMVhSSTr2 were given either 1 or 2 days apart with [111In]-DTPA-d-Phe1-octreotide given 4 days after the first AdCMVhSSTr2 injection.

The median tumor localization of [111In]-DTPA-d-Phe1-octreotide 2 days after a single administration of AdCMVhSSTr2 (37.7% ID/g; Fig. 5) was significantly greater than when two injections of AdCMVhSSTr2 were given on consecutive days, followed by injection of [111In]-DTPA-d-Phe1-octreotide 2 days after the first adenoviral injection (22.4% ID/g; Fig. 5; P = 0.03). However, there was no difference in the median tumor uptake of [111In]-DTPA-d-Phe1-octreotide 4 days after a single AdCMVhSSTr2 injection (40.7% ID/g) and when two injections of AdCMVhSSTr2 were given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, followed by injection of [111In]-DTPA-d-Phe1-octreotide 4 days after the first adenoviral administration (P = 0.65). Thus, there is no advantage of two AdCMVhSSTr2 injections compared with one with regard to [111In]-DTPA-d-Phe1-octreotide tumor localization.

The objectives of this study were to construct a recombinant adenovirus encoding for the hSSTr2 and use it to induce the expression of hSSTr2 in human tumor cells lacking this receptor in vitro and in vivo as a target for binding and localization of [111In]-DTPA-d-Phe1-octreotide. In addition, the in vivo tumor localization of [111In]-DTPA-d-Phe1-octreotide was investigated at several intervals after a single AdCMVhSSTr2 injection, or after two injections of AdCMVhSSTr2, in an attempt to determine a dosing schema for achieving the maximum tumor localization of [111In]-DTPA-d-Phe1-octreotide. The results of this study indicate that [111In]-DTPA-d-Phe1-octreotide localized in vivo to human ovarian cancer xenografts induced to express hSSTr2 with an adenoviral vector and that two injections of AdCMVhSSTr2 did not increase the localization compared with one injection. Thus, a single adenoviral regimen will be the basis for a future therapeutic protocol using an octreotide analogue labeled with a therapeutic radioisotope.

Radiotherapy studies using somatostatin analogues labeled with 90Y, 188Re, or 64Cu in animals bearing SSTr2-positive tumors have shown tumor regressions (28, 30, 31, 32). Smith-Jones et al.(28) demonstrated that a single 0.48 mCi i.p. injection of a 90Y-labeled octreotide analogue in nude mice bearing s.c. AR42J pancreatic tumors resulted in a significant reduction in tumor growth. Stolz et al.(32) showed that a single dose of [90Y]-DOTA-Tyr3-octreotide led to reductions of 60 and 50% of the initial tumor volume in nude mice bearing AR42J and NCI-H69 human small cell lung cancer tumors, respectively. Complete remissions were observed in rats bearing s.c. CA20948 rat pancreatic tumors when a single 2.0 mCi dose of [90Y]-DOTA-Tyr3-octreotide was administered. Zamora et al.(31) labeled the somatostatin analogue RC-160 with 188Re and administered 7 doses of 0.2 mCi over a 14-day period intralesionally to nude mice bearing PC-3 human prostate cancer tumors. They showed that animals receiving [188Re]RC-160 had 60% survival compared to no survivors when control animals were injected with saline. Anderson et al.(30) demonstrated tumor growth inhibition of s.c. CA20948 tumors in Lewis rats using either a single or fractionated i.v. dose of [64Cu]TETA-octreotide. Thus, several octreotide analogues have shown potential as radiotherapeutic agents against SSTr2-expressing tumors in animals.

In an attempt to improve the therapeutic efficacy of these radiolabeled octreotide analogues, a recombinant adenoviral vector (AdCMVhSSTr2) encoding the hSSTr2 driven by the CMV promoter was constructed to induce the expression of hSSTr2 in vitro and in vivo. Bmaxs for membrane preparations from A427 cells infected with AdCMVhSSTr2 were shown to be dependent upon the viral dose used for the infection (Table 1), whereas Bmaxs for SKOV3.ip1 cell membrane preparations ranged from 80–610 fmol/mg of protein after infection with 100 or 200 MOI of AdCMVhSSTr2 but were undetectable after infection with 0 or 10 MOI. The uninfected A427 and SKOV3.ip1 cells do not express hSSTr2 or the other hSSTr subtypes (1, 3, 4, or 5), because [125I]-Tyr1-somatostatin binds with high affinity to all hSSTr subtypes (35), and binding to uninfected A427 and SKOV3.ip1 cells was not observed. Also, because the infection was carried out with an adenovirus containing hSSTr2, then any binding differences observed between uninfected and infected cells must be due to the presence of hSSTr2. Taylor et al.(47) reported Bmaxs that ranged between ∼5 and 720 fmol/mg of protein for various human tumor cell lines that naturally express hSSTr2. A recent paper by Virgolini et al.(18) reported Bmaxs ranging from 200–15,000 fmol/mg of protein for a variety of human tumor tissues that naturally express different hSSTr subtypes. Our results indicate that AdCMVhSSTr2 induces hSSTr2 expression on A427 and SKOV3.ip1 tumor cells in vitro that are otherwise negative for hSSTr2 to levels that are comparable with those reported for tumor cells natively expressing hSSTr2.

Athymic nude mice inoculated i.p. with SKOV3.ip1 cells were used for evaluating the in vivo induction of hSSTr2 with AdCMVhSSTr2. The tumor localizations of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide 2 days after i.p. administration of AdCMVhSSTr2 and 4 h after i.p. administration of the radiolabeled peptides were 3.5% ID/g and 60.4% ID/g, respectively (Fig. 2). This difference is likely due to metabolism and deiodination of [125I]-Tyr1-somatostatin, resulting in the faster clearance of 125I from the tumor compared with the metabolism of [111In]-DTPA-d-Phe1-octreotide, which leads to the sequestration of 111In within the tumor cell lysosomes (50). In addition, the [111In]-DTPA-d-Phe1-octreotide tumor localization 4 h after administration was greater than the tumor localization observed at 4 h in previous studies using the same animal tumor model, infection with AdCMVGRPr, and [125I]-mIP-bombesin administration (15, 16). It is difficult to compare the two studies, but one can speculate that the differences are due to differences in metabolism and clearance of the two radiolabeled peptides. Studies are under way to explain the differences observed between these two systems.

Control biodistribution studies demonstrated that the tumor localization of [111In]-DTPA-d-Phe1-octreotide was specific for expression of hSSTr2 because mice administered AdCMVGRPr (Fig. 2) or no virus (Fig. 3) had low tumor localization of [111In]-DTPA-d-Phe1-octreotide (1.6 and 0.8% ID/g, respectively). Also, the uptake of [111In]-DTPA-d-Phe1-octreotide was low in all normal tissues (<1.0% ID/g) when no AdCMVhSSTr2 was administered, except for the kidney, which is the clearance organ for [111In]-DTPA-d-Phe1-octreotide. Administration of AdCMVhSSTr2 (Fig. 3) increased the [111In]-DTPA-d-Phe1-octreotide uptake in the abdominal lining, liver, and uterus, although to levels <5% ID/g, which could be a result of tumor dissemination (which was observed in uterus; Ref. 51) or transduction of the normal tissues (52, 53) with AdCMVhSSTr2. If this increase is a result of transduction with AdCMVhSSTr2, the problem may be overcome through the use of tropism-modified adenoviral vectors that are targeted to the tumor or transcriptional targeting and the use of tumor-specific promoters such that the receptor is only expressed in tumor cells (19, 20, 21, 22, 24, 25, 26, 27). The pancreas and small intestine did not demonstrate a high level of [111In]-DTPA-d-Phe1-octreotide uptake without adenovirus administration, indicating that the level of somatostatin receptor expression on these tissues is low compared with the expression induced on the tumor with AdCMVhSSTr2. Administration of AdCMVhSSTr2 did not result in tumor localization of the control peptide [125I]-mIP-bombesin (0.3% ID/g). Kidney accumulation of [111In]-DTPA-d-Phe1-octreotide was reduced by administration of l-lysine (Fig. 3) as reported in previous studies (54). Thus, the tumor uptake of [111In]-DTPA-d-Phe1-octreotide was greater than the uptake in normal tissues after infection with AdCMVhSSTr2, except for the kidney, which is the clearance organ for [111In]-DTPA-d-Phe1-octreotide.

Methods to overcome the problem of transduction of normal organs are presently being investigated by several groups. Our group has used an immunological transductional targeting strategy to redirect adenoviral vectors to tumor cells overexpressing the folate, basic fibroblast growth factor, and epidermal growth factor receptors in vitro and in vivo(19, 20, 21, 22, 23). In addition, other investigators are using genetically modified adenoviral vectors to target adenovirus to heterologous cellular receptors (24, 25). Wickham et al.(24) demonstrated that an adenovirus containing a heparan-binding domain in the fiber portion of the adenovirus increased the gene transfer efficiency in cells containing heparan cellular receptors. Krasnykh et al.(25) showed that a serotype 5 adenovirus in which the fiber was modified to contain a serotype 3 knob bound to serotype 3 cellular receptors but not serotype 5 receptors. Tumor-specific promoters have also been used to limit protein expression to the tumor cells through a transcriptional targeting approach (26, 27). Stackhouse et al.(27) has demonstrated specific expression of GRPr in cells using the DF3 and erbB-2 promoters in adenoviral vectors encoding the GRPr gene. Thus, there are several strategies for overcoming the problem of receptor expression on normal tissues.

The tumor localization of [111In]-DTPA-d-Phe1-octreotide at day 1, 2, or 4 after a single administration of AdCMVhSSTr2 was similar (Fig. 4). This is in contrast to a previous study using the same animal tumor model, which showed a decrease in the tumor localization of [125I]-mIP-bombesin at day 2, 4, or 7 after a single administration of AdCMVGRPr (16). In addition, in vitro assays using adenoviral vectors coding for carcinoembryonic antigen or thyrotropin-releasing hormone receptor showed a decrease in radiolabeled ligand binding over time (16). Because the dynamics of adenoviral vector-encoded transgene expression should be the same in all of these instances, the differences in radiolabeled ligand binding over time are likely a reflection of the half-life of the receptor at the cell surface. Future studies will investigate this possibility by performing binding assays to membrane preparations of SKOV3.ip1 tumors at different times after adenoviral infection.

Biodistribution assays performed using two injections of AdCMVhSSTr2 showed tumor localization of [111In]-DTPA-d-Phe1-octreotide when the adenoviral injections were 1, 2, or 3 days apart, although the tumor uptake was significantly decreased when the AdCMVhSSTr2 injections were on consecutive days (Fig. 5). Previous studies in our laboratory have shown that multiple i.p. injections of AdCMVGRPr resulted in significantly less tumor uptake of [125I]-mIP-bombesin compared with a single AdCMVGRPr injection, regardless of the interval between adenoviral injections,4 in contrast to the results obtained in this study. Importantly, when AdCMVhSSTr2 was given 2 or 3 days apart, the tumor localization of [111In]-DTPA-d-Phe1-octreotide 4 days after the first adenoviral administration was not significantly greater than the tumor localization of [111In]-DTPA-d-Phe1-octreotide 4 days after a single administration of AdCMVhSSTr2. These results are not confounded by an immune response, because the studies were not performed in immunocompetent mice. Therefore, high tumor localization of [111In]-DTPA-d-Phe1-octreotide up to 4 days after a single injection of AdCMVhSSTr2 was accomplished and will be the regimen for future studies using a therapeutic analogue of octreotide. The advantage of using a single adenoviral administration relates to the potential immune response against a second injection of the adenovirus (55, 56).

Thus, the results of this study demonstrate that the adenoviral vector, AdCMVhSSTr2, can induce the expression of hSSTr2 both in vitro and in vivo as determined by [111In]-DTPA-d-Phe1-octreotide binding. The high level of tumor localization of [111In]-DTPA-d-Phe1-octreotide after AdCMVhSSTr2 infection led to a study evaluating the dosing schema necessary to maximize the tumor localization of [111In]-DTPA-d-Phe1-octreotide. Evaluation of administration of [111In]-DTPA-d-Phe1-octreotide at different times after a single AdCMVhSSTr2 injection or after two AdCMVhSSTr2 injections showed that the best dosing regimen consisted of a single AdCMVhSSTr2 injection, with [111In]-DTPA-d-Phe1-octreotide being administered anytime up to 4 days after AdCMVhSSTr2.

Future therapeutic studies will use a single dose of AdCMVhSSTr2 and a radiolabeled octreotide analogue to accomplish tumor regression in this model of human ovarian cancer. We envision this strategy as being directly translatable to the clinical treatment of ovarian cancer in which the primary lesion has been surgically debulked. To further extend this concept to the treatment of systemic disease, a s.c. tumor model will be established to determine the tumor localization of [111In]-DTPA-d-Phe1-octreotide after systemic administration. We are presently developing tropism-modified targeted adenoviral vectors (19, 20, 21) and adenoviral vectors with tissue-specific promoters (27), which will be necessary for tumor-restricted receptor expression in the treatment of metastatic disease.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

This work was supported by Department of Energy Grant DE-FG05-93ER61654 (to D. J. B.), NIH Grant R01 CA 73636 (to D. J. B.), NIH Grants R01 CA 68245 and R01 CA 74242 (to D. T. C.), and an American Cancer Society Institutional Grant and a Radiological Society of North America Seed Grant from the RSNA Research and Education Fund (to B. E. R.).

                
3

The abbreviations used are: mAb, monoclonal antibody; GRPr, gastrin-releasing peptide receptor; hSSTr2, human somatostatin receptor subtype 2; AdCMVhSSTr2, recombinant adenoviral vector encoding hSSTr2; CMV, cytomegalovirus; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; % ID/g, percent injected dose per gram of tissue; DTPA, diethylaminetriaminepentaacetic acid; mIP, meta-iodophenyl; pfu, plaque-forming unit; MOI, multiplicity of infection defined as pfu/cell; AdCMVGRPr, recombinant adenoviral vector encoding the GRPr; RT-PCR, reverse transcription-PCR.

        
4

B. E. Rogers, D. T. Curiel, and D. J. Buchsbaum, unpublished results.

Fig. 1.

RT-PCR analysis of hSSTr2 gene expression in uninfected and AdCMVhSSTr2-infected A427 and SKOV3.ip1 cells is shown in A. Lane M, 1-kb DNA markers; Lane 1, uninfected A427 cells; Lane 2, 10 MOI AdCMVhSSTr2-infected A427 cells; Lane 3, 100 MOI infected A427 cells; Lane 4, 200 MOI infected A427 cells; Lane 5, uninfected SKOV3.ip1 cells; Lane 6, 10 MOI AdCMVhSSTr2-infected SKOV3.ip1 cells; Lane 7, 100 MOI infected SKOV3.ip1 cells; Lane 8, 200 MOI infected SKOV3.ip1 cells; Lane 9, positive control DNA plasmid containing entire hSSTr2 gene fragment; Lane 10, negative control without DNA. G3PDH gene expression for each corresponding lane is shown in B.

Fig. 1.

RT-PCR analysis of hSSTr2 gene expression in uninfected and AdCMVhSSTr2-infected A427 and SKOV3.ip1 cells is shown in A. Lane M, 1-kb DNA markers; Lane 1, uninfected A427 cells; Lane 2, 10 MOI AdCMVhSSTr2-infected A427 cells; Lane 3, 100 MOI infected A427 cells; Lane 4, 200 MOI infected A427 cells; Lane 5, uninfected SKOV3.ip1 cells; Lane 6, 10 MOI AdCMVhSSTr2-infected SKOV3.ip1 cells; Lane 7, 100 MOI infected SKOV3.ip1 cells; Lane 8, 200 MOI infected SKOV3.ip1 cells; Lane 9, positive control DNA plasmid containing entire hSSTr2 gene fragment; Lane 10, negative control without DNA. G3PDH gene expression for each corresponding lane is shown in B.

Close modal
Fig. 2.

Biodistribution of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. Five days after tumor cell inoculation, the mice were injected i.p. with 1 × 109 pfu of AdCMVhSSTr2, followed by i.p. injection of the radiolabeled ligands 2 days later. Mice administered [125I]-Tyr1-somatostatin were killed 4 h later, and mice administered [111In]-DTPA-d-Phe1-octreotide were killed 4 and 24 h later. Negative control mice were administered AdCMVGRPr and [111In]-DTPA-d-Phe1-octreotide. BL, blood; LU, lung; LI, liver; SI, small intestine; SP, spleen; KI, kidney; MS, muscle; TU, tumor; AL, abdominal lining; UT, uterus; PA, pancreas. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile.

Fig. 2.

Biodistribution of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. Five days after tumor cell inoculation, the mice were injected i.p. with 1 × 109 pfu of AdCMVhSSTr2, followed by i.p. injection of the radiolabeled ligands 2 days later. Mice administered [125I]-Tyr1-somatostatin were killed 4 h later, and mice administered [111In]-DTPA-d-Phe1-octreotide were killed 4 and 24 h later. Negative control mice were administered AdCMVGRPr and [111In]-DTPA-d-Phe1-octreotide. BL, blood; LU, lung; LI, liver; SI, small intestine; SP, spleen; KI, kidney; MS, muscle; TU, tumor; AL, abdominal lining; UT, uterus; PA, pancreas. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile.

Close modal
Fig. 3.

Biodistribution of [111In]- DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. As described in Fig. 2, AdCMVhSSTr2 was injected i.p. 5 days after tumor cell inoculation, followed 2 days later by i.p injection of [111In]-DTPA-d-Phe1-octreotide or [125I]-mIP-bombesin as a control. Mice were killed 4 h later, and tissues were harvested and counted. The effect on kidney uptake of [111In]-DTPA-d-Phe1-octreotide after administration of l-lysine 30 min before and 1, 2, and 3 h after injection of [111In]-DTPA-d-Phe1-octreotide, either with or without AdCMVhSSTr2 administration, is shown. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Fig. 3.

Biodistribution of [111In]- DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. As described in Fig. 2, AdCMVhSSTr2 was injected i.p. 5 days after tumor cell inoculation, followed 2 days later by i.p injection of [111In]-DTPA-d-Phe1-octreotide or [125I]-mIP-bombesin as a control. Mice were killed 4 h later, and tissues were harvested and counted. The effect on kidney uptake of [111In]-DTPA-d-Phe1-octreotide after administration of l-lysine 30 min before and 1, 2, and 3 h after injection of [111In]-DTPA-d-Phe1-octreotide, either with or without AdCMVhSSTr2 administration, is shown. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Close modal
Fig. 4.

Biodistribution of [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. Five days after tumor cell inoculation, the mice were injected i.p. with 1 × 109 pfu of AdCMVhSSTr2, followed by i.p. injection of [111In]-DTPA-d-Phe1-octreotide 1, 2, or 4 days later. Mice were killed 4 h after [111In]-DTPA-d-Phe1-octreotide injection, and tissues were harvested and counted. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Fig. 4.

Biodistribution of [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells. Five days after tumor cell inoculation, the mice were injected i.p. with 1 × 109 pfu of AdCMVhSSTr2, followed by i.p. injection of [111In]-DTPA-d-Phe1-octreotide 1, 2, or 4 days later. Mice were killed 4 h after [111In]-DTPA-d-Phe1-octreotide injection, and tissues were harvested and counted. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Close modal
Fig. 5.

Biodistribution of [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells, followed by two administrations of 1 × 109 pfu of AdCMVhSSTr2. The first dose of AdCMVhSSTr2 was administered 5 days after inoculation with SKOV3.ip1 cells (day 0), and the second dose was administered at day 1, 2, or 3. [111In]-DTPA-d-Phe1-octreotide was administered either on day 2 (after AdCMVhSSTr2 injection on days 0 and 1) or on day 4 (after AdCMVhSSTr2 injection on days 0 and 2 or days 0 and 3). Mice were killed 4 h after [111In]-DTPA-d-Phe1-octreotide injection, and tissues were harvested and counted. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Fig. 5.

Biodistribution of [111In]-DTPA-d-Phe1-octreotide in athymic nude mice inoculated i.p. with 2 × 107 SKOV3.ip1 cells, followed by two administrations of 1 × 109 pfu of AdCMVhSSTr2. The first dose of AdCMVhSSTr2 was administered 5 days after inoculation with SKOV3.ip1 cells (day 0), and the second dose was administered at day 1, 2, or 3. [111In]-DTPA-d-Phe1-octreotide was administered either on day 2 (after AdCMVhSSTr2 injection on days 0 and 1) or on day 4 (after AdCMVhSSTr2 injection on days 0 and 2 or days 0 and 3). Mice were killed 4 h after [111In]-DTPA-d-Phe1-octreotide injection, and tissues were harvested and counted. Columns, median tissue concentration from a group of five animals; bars, range from the 25th percentile to the 75th percentile. Abbreviations are defined in Fig. 2.

Close modal
Table 1

Binding of [125I]-Tyr1-somatostatin and [111In]-DTPA-d-Phe1-octreotide to AdCMVhSSTr2-infected A427 and SKOV3.ip1 cellsa

[125I]-Tyr1-somatostatin[111In]-DTPA-d-Phe1-octreotide
Cell line (MOI)Kd (nm)Bmax (fmol/mg)IC50 (nm) (SST-14)bIC50 (nm) (DTPA-oct)cKd (nm)Bmax (fmol/mg)IC50 (nm) (SST-14)IC50 (nm) (DTPA-oct)
A427 (0) NDd ND ND ND ND ND ND ND 
A427 (10) 1.8 (2.1) 1362 (1258) 1.8 (2.1) 3.8 (3.3) 1.6 (0.7)e 933 (36)e 2.6 (3.3) 1.7 (0.7)e 
A427 (100) 2.9 (4.5) 2623 (5690) 3.0 (4.4) 4.4 (6.0) 2.3 (2.5) 3635 (2526) 2.0 (6.1) 2.4 (2.4) 
A427 (200) 3.3 (5.1) 4082 (10046) 3.3 (5.1) 9.6 (73.4) 3.0 (3.3) 5138 (4320) 3.8 (6.9) 3.0 (3.3) 
SKOV3.ip1 (0) ND ND ND ND ND ND ND ND 
SKOV3.ip1 (10) ND ND ND ND ND ND ND ND 
SKOV3.ip1 (100) 1.9 (0.3)e 246 (414)e 2.0 (0.3)e 2.1f 1.7 (1.1)e 100 (1)e 1.8f 1.7 (1.0)e 
SKOV3.ip1 (200) 2.7 (0.8)e 608 (1103)e 2.8 (0.9)e 2.5 (2.6)e 0.7 (0.1)e 82 (53)e 0.8 (0.2) 0.8 (3.0)e 
[125I]-Tyr1-somatostatin[111In]-DTPA-d-Phe1-octreotide
Cell line (MOI)Kd (nm)Bmax (fmol/mg)IC50 (nm) (SST-14)bIC50 (nm) (DTPA-oct)cKd (nm)Bmax (fmol/mg)IC50 (nm) (SST-14)IC50 (nm) (DTPA-oct)
A427 (0) NDd ND ND ND ND ND ND ND 
A427 (10) 1.8 (2.1) 1362 (1258) 1.8 (2.1) 3.8 (3.3) 1.6 (0.7)e 933 (36)e 2.6 (3.3) 1.7 (0.7)e 
A427 (100) 2.9 (4.5) 2623 (5690) 3.0 (4.4) 4.4 (6.0) 2.3 (2.5) 3635 (2526) 2.0 (6.1) 2.4 (2.4) 
A427 (200) 3.3 (5.1) 4082 (10046) 3.3 (5.1) 9.6 (73.4) 3.0 (3.3) 5138 (4320) 3.8 (6.9) 3.0 (3.3) 
SKOV3.ip1 (0) ND ND ND ND ND ND ND ND 
SKOV3.ip1 (10) ND ND ND ND ND ND ND ND 
SKOV3.ip1 (100) 1.9 (0.3)e 246 (414)e 2.0 (0.3)e 2.1f 1.7 (1.1)e 100 (1)e 1.8f 1.7 (1.0)e 
SKOV3.ip1 (200) 2.7 (0.8)e 608 (1103)e 2.8 (0.9)e 2.5 (2.6)e 0.7 (0.1)e 82 (53)e 0.8 (0.2) 0.8 (3.0)e 
a

Data represent the median of triplicate experiments (unless otherwise noted) with the range (maximum value minus minimum value) in parentheses. All data presented have a correlation between actual and predicted values of >90%.

b

SST-14, inhibited with unlabeled Tyr1-somatostatin.

c

DTPA-oct, inhibited with unlabeled DTPA-d-Phe1-octreotide.

d

ND, values not determined as correlation between actual and predicted values was <90%.

e

Data represent the median of duplicate experiments with the range between the two values in parentheses.

f

Data represent a single experiment.

We thank Christy Grimes, Barbara Krum, LaTasha Bulluck, and Sakib Hassan for excellent technical assistance. Also, Murray Stackhouse, Kevin Raisch, and Kim Laffoon are thanked for their advice in performing the mRNA studies. Mallinckrodt Medical is gratefully acknowledged for providing the [111In]-DTPA-d-Phe1-octreotide used in these studies. We also thank Sally Lagan for help in the preparation of the manuscript.

1
Landis S. H., Murray T., Bolden S., Wingo P. A. Cancer statistics.
CA Cancer J. Clin.
,
48
:
6
-29,  
1998
.
2
Markman M., Reichman B., Hakes T., Curtin J., Jones W., Lewis J. L., Jr., Barakat R., Rubin S., Mychalczak B., Saigo P., Almadrones L., Hoskins W. Intraperitoneal chemotherapy in the management of ovarian cancer.
Cancer (Phila.)
,
71
:
1565
-1570,  
1993
.
3
Hird V., Maraveyas A., Snook D., Dhokia B., Soutter W. P., Meares C., Stewart J. S., Mason P., Lambert H. E., Epenetos A. A. Adjuvant therapy of ovarian cancer with radioactive monoclonal antibody.
Br. J. Cancer
,
68
:
403
-406,  
1993
.
4
Fein D. A., Morgan L. S., Marcus R. B., Jr., Mendenhall W. M., Sombeck M. D., Freeman D. E., Million R. R. Stage III ovarian carcinoma: an analysis of treatment results and complications following hyperfractionated abdominopelvic irradiation for salvage.
Int. J. Radiat. Oncol. Biol. Phys.
,
29
:
169
-176,  
1994
.
5
Piver M. S., Recio F. O., Baker T. R., Driscoll D. Evaluation of survival after second-line intraperitoneal cisplatin-based chemotherapy for advanced ovarian cancer.
Cancer (Phila.)
,
73
:
1693
-1698,  
1994
.
6
Meredith R. F., Partridge E. E., Alvarez R. D., Khazaeli M. B., Plott G., Russell C. D., Wheeler R. H., Liu T., Grizzle W. E., Schlom J., LoBuglio A. F. Intraperitoneal radioimmunotherapy of ovarian cancer with lutetium-177-CC49.
J. Nucl. Med.
,
37
:
1491
-1496,  
1996
.
7
Alvarez R. D., Partridge E. E., Khazaeli M. B., Plott G., Austin M., Kilgore L., Russell C. D., Liu T., Grizzle W. E., Schlom J., LoBuglio A. F., Meredith R. F. Intraperitoneal radioimmunotherapy of ovarian cancer with 177Lu-CC49: a phase I/II study.
Gynecol. Oncol.
,
65
:
94
-101,  
1997
.
8
Knox S. J. Overview of studies on experimental radioimmunotherapy.
Cancer Res.
,
55 (Suppl.)
:
5832s
-5836s,  
1995
.
9
Buchsbaum D. J. Experimental radioimmunotherapy and methods to increase therapeutic efficacy Goldenberg D. M. eds. .
Cancer Therapy with Radiolabeled Antibodies
,
:
115
-140, CRC Press Boca Raton  
1995
.
10
Hand P. H., Robbins P. F., Salgaller M. L., Poole D. J., Schlom J. Evaluation of human carcinoembryonic-antigen (CEA)-transduced and non-transduced murine tumors as potential targets for anti-CEA therapies.
Cancer Immunol. Immunother.
,
36
:
65
-75,  
1993
.
11
Hinuma S., Hosoya M., Ogi K., Tanaka H., Nagai Y., Onda H. Molecular cloning and functional expression of a human thyrotropin-releasing hormone (TRH) receptor gene.
Biochim. Biophys. Acta.
,
1219
:
251
-259,  
1994
.
12
Falck-Pedersen E., Heinflink M., Alvira M., Nussenzveig D. R., Gershengorn M. C. Expression of thyrotropin-releasing hormone receptors by adenovirus-mediated gene transfer reveals that thyrotropin-releasing hormone desensitization is cell specific.
Mol. Pharmacol.
,
45
:
684
-689,  
1994
.
13
Raben D., Buchsbaum D. J., Khazaeli M. B., Rosenfeld M. E., Gillespie G. Y., Grizzle W. E., Liu T., Curiel D. T. Enhancement of radiolabeled antibody binding and tumor localization through adenoviral transduction of the human carcinoembryonic antigen gene.
Gene Ther.
,
3
:
567
-580,  
1996
.
14
Buchsbaum D. J., Raben D., Stackhouse M. A., Khazaeli M. B., Rogers B. E., Rosenfeld M. E., Liu T., Curiel D. T. Approaches to enhance cancer radiotherapy employing gene transfer methods.
Gene Ther.
,
3
:
1042
-1068,  
1996
.
15
Rogers B. E., Rosenfeld M. E., Khazaeli M. B., Mikheeva G., Stackhouse M. A., Liu T., Curiel D. T., Buchsbaum D. J. Localization of iodine-125-mIP-Des-Met14-bombesin(7–13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer.
J. Nucl. Med.
,
38
:
1221
-1229,  
1997
.
16
Rogers B. E., Curiel D. T., Mayo M. S., Laffoon K., Bright S., Buchsbaum D. J. Tumor localization of a radiolabeled bombesin analogue in mice bearing ovarian tumors induced to express the gastrin releasing peptide receptor by an adenoviral vector.
Cancer (Phila.)
,
80 (Suppl.)
:
2419
-2424,  
1997
.
17
Rosenfeld M. E., Rogers B. E., Khazaeli M. B., Mikheeva G., Raben D., Mayo M. S., Curiel D. T., Buchsbaum D. J. Adenoviral mediated delivery of gastrin releasing peptide receptor results in specific tumor localization of a bombesin analogue in vivo.
Clin. Cancer Res.
,
3
:
1187
-1194,  
1997
.
18
Virgolini I., Leimer M., Handmaker H., Lastoria S., Bischof C., Muto P., Pangerl T., Gludovacz D., Peck-Radosavljevic M., Lister-James J., Hamilton G., Kaserer K., Valent P., Dean R. Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829.
Cancer Res.
,
58
:
1850
-1859,  
1998
.
19
Douglas J. T., Rogers B. E., Rosenfeld M. E., Michael S. I., Feng M., Curiel D. T. Targeted gene delivery by tropism-modified adenoviral vectors.
Nat. Biotech.
,
14
:
1574
-1578,  
1996
.
20
Goldman C. K., Rogers B. E., Douglas J. T., Sosnowski B. A., Ying W., Siegal G. P., Baird A., Campain J. A., Curiel D. T. Targeted gene delivery to Kaposi’s sarcoma cells via the fibroblast growth factor receptor.
Cancer Res.
,
57
:
1447
-1451,  
1997
.
21
Rogers B. E., Douglas J. T., Sosnowski B. A., Ying W., Pierce G., Buchsbaum D. J., Della Manna D., Baird A., Curiel D. T. Enhanced in vivo gene delivery to human ovarian cancer xenografts utilizing a tropism-modified adenovirus vector.
Tumor Targeting
,
3
:
25
-31,  
1998
.
22
Rancourt C., Rogers B. E., Sosnowski B. A., Wang M., Piche A., Pierce G. F., Alvarez R. D., Siegal G. P., Douglas J. T., Curiel D. T. Basic fibroblast growth factor enhancement of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer.
Clin. Cancer Res.
,
4
:
2455
-2461,  
1998
.
23
Miller C. R., Buchsbaum D. J., Reynolds P. N., Douglas J. T., Gillespie G. Y., Mayo M. S., Raben D., Curiel D. T. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-dependent gene transfer.
Cancer Res.
,
58
:
5738
-5748,  
1998
.
24
Wickham T. J., Roelvink P. W., Brough D. W., Kovesdi I. Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types.
Nat. Biotech.
,
14
:
1570
-1573,  
1996
.
25
Krasnykh V. N., Mikheeva G. V., Douglas J. T., Curiel D. T. Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.
J. Virol.
,
70
:
6839
-6846,  
1996
.
26
Lan K. H., Kanai F., Shiratori Y., Ohashi M., Tanaka T., Okudaira T., Yoshida Y., Hamada H., Omata M. In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma.
Cancer Res.
,
57
:
4279
-4284,  
1997
.
27
Stackhouse, M. A., Buchsbaum, D. J., Kancharla, S. R., Grimes, C., Laffoon, K., Pederson, L. C., and Curiel, D. T. Specific membrane receptor gene expression targeted with radiolabeled peptide employing the erbB-2 and DF3 promoter elements in adenoviral vectors. Cancer Gene Ther., in press, 1999.
28
Smith-Jones P. M., Stolz B., Albert R., Ruser G., Macke H., Briner U., Tolcsvai L., Weckbecker G., Bruns C. Synthesis, radiolabeling, and evaluation of DTPA/octreotide conjugates for radiotherapy.
J. Labelled Compd. Radiopharm.
,
37
:
499
-501,  
1995
.
29
Wiseman G. A., Kvols L. K. Therapy of neuroendocrine tumors with radiolabeled MIBG and somatostatin analogues.
Semin. Nucl. Med.
,
25
:
272
-278,  
1995
.
30
Anderson C. J., Jones L. A., Bass L. A., Sherman E. L. C., McCarthy D. W., Cutler P. D., Lanahan M. V., Cristel M. E., Lewis J. S., Schwarz S. W. Radiotherapy, toxicity, and dosimetry of copper-64-TETA-octreotide in tumor-bearing rats.
J. Nucl. Med.
,
39
:
1944
-1951,  
1998
.
31
Zamora P. O., Gulhke S., Bender H., Diekmann D., Rhodes B. A., Biersack H. J., Knapp F. F., Jr. Experimental radiotherapy of receptor-positive human prostate adenocarcinoma with 188Re-RC-160, a directly-radiolabeled somatostatin analogue.
Int. J. Cancer
,
65
:
214
-220,  
1996
.
32
Stolz B., Weckbecker G., Smith-Jones P. M., Albert R., Raulf F., Bruns C. The somatostatin receptor-targeted radiotherapeutic [90Y-DOTA-DPhe1, Tyr3]octreotide (90Y-SMT 487) eradicates experimental rat pancreatic CA 20948 tumors.
Eur. J. Nucl. Med.
,
25
:
668
-674,  
1998
.
33
Volkert W. A., Goeckeler W. F., Ehrhardt G. J., Ketring A. R. Therapeutic radionuclides: production and decay property considerations.
J. Nucl. Med.
,
32
:
174
-185,  
1991
.
34
Yorke E. D., Beaumier P. L., Wessels B. W., Fritzberg A. R., Morgan A. C. Optimal antibody-radionuclide combinations for clinical radioimmunotherapy: a predictive model based on mouse pharmacokinetics.
Nucl. Med. Biol.
,
18
:
827
-835,  
1991
.
35
Woltering E. A., O’Dorisio M. S., O’Dorisio T. M. The role of radiolabeled somatostatin analogs in the management of cancer patients Ed. 4 DeVita V. T., Jr. Hellman S. Rosenberg S. A. eds. .
Principles and Practice of Oncology
,
Vol. 9
:
1
-16, Lippincott-Raven Philadelphia  
1995
.
36
Krenning E. P., Bakker W. H., Kooij P. P. M., Breeman W. A. P., Oei H. Y., de Jong M., Reubi J. C., Visser T. J., Bruns C., Kwekkeboom D. J., Reijs A. E. M., van Hagen P. M., Koper J. W., Lamberts S. W. J. Somatostatin receptor scintigraphy with indium-111-DTPA-d-Phe-1-octreotide in man: metabolism, dosimetry and comparison with iodine-123-Tyr-3-octreotide.
J. Nucl. Med.
,
33
:
652
-658,  
1992
.
37
Krenning E. P., Kwekkeboom D. J., Bakker W. H., Breeman W. A. P., Kooij P. P. M., Oei H. Y., van Hagen M., Postema P. T. E., de Jong M., Reubi J. C., Visser T. J., Reijs A. E. M., Hofland L. J., Koper J. W., Lamberts S. W. J. Somatostatin receptor scintigraphy with [111In-DTPA-d-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients.
Eur. J. Nucl. Med.
,
20
:
716
-731,  
1993
.
38
Krenning E. P., Kwekkeboom D. J., Pauwels S., Kvols L. K., Reubi J. C. Somatostatin receptor scintigraphy Freeman L. M. eds. .
Nuclear Medicine Annual
,
XI
:
1
-50, Raven Press, Ltd. New York  
1995
.
39
Briganti V., Sestini R., Orlando C., Bernini G., La Cava G., Tamburini A., Raggi C. C., Serio M., Maggi M. Imaging of somatostatin receptors by indium-111-pentetreotide correlates with quantitative determination of somatostatin receptor type 2 gene expression in neuroblastoma tumors.
Clin. Cancer Res.
,
3
:
2385
-2391,  
1997
.
40
Krenning E. P., Kooij P. P., Bakker W. H., Breeman W. A., Postema P. T., Kwekkeboom D. J., Oei H. Y., de Jong M., Visser T. J., Reijs A. E., Lamberts S. W. J. Radiotherapy with a radiolabeled somatostatin analogue, [111In-DTPA-d-Phe1]-octreotide. A case history.
Ann. NY Acad. Sci.
,
733
:
496
-506,  
1994
.
41
Fjalling M., Andersson P., Forssell-Aronsson E., Gretarsdottir J., Johansson V., Tisell L. E., Wangberg B., Nilsson O., Berg G., Michanek A., Lindstedt G., Ahlman H. Systemic radionuclide therapy using indium-111-DTPA-d-Phe1-octreotide in midgut carcinoid syndrome.
J. Nucl. Med.
,
37
:
1519
-1521,  
1996
.
42
Krenning E. P., Kooij P. P., Pauwels S., Breeman W. A., Postema P. T., De Herder W. W., Valkema R., Kwekkeboom D. J. Somatostatin receptor: scintigraphy and radionuclide therapy.
Digestion
,
57
:
57
-61,  
1996
.
43
McCarthy K. E., Woltering E. A., Espenan G. D., Cronin M., Maloney T. J., Anthony L. B. In situ radiotherapy with 111In-pentetreotide: initial observations and future directions.
Cancer J. Sci. Am.
,
4
:
94
-102,  
1998
.
44
Graham F. L., Prevec L. Manipulation of adenovirus vectors Murray E. J. eds. .
Methods in Molecular Biology: Gene Transfer and Expression Protocols
,
7
:
109
-128, The Humana Press, Inc. Clifton, NJ  
1991
.
45
Yamada Y., Post S. R., Wang K., Tager H. S., Bell G. I., Seino S. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.
Proc. Natl. Acad. Sci. USA
,
89
:
251
-255,  
1992
.
46
Weckbecker G., Liu R., Tolcsvai L., Bruns C. Antiproliferative effects of the somatostatin analogue octreotide (SMS 201–995) on ZR-75–1 human breast cancer cells in vivo and in vitro.
Cancer Res.
,
52
:
4973
-4978,  
1992
.
47
Taylor J. E., Theveniau M. A., Bashirzadeh R., Reisine T., Eden P. A. Detection of somatostatin receptor subtype 2 (SSTR2) in established tumors and tumor cell lines: evidence for SSTR2 heterogeneity.
Peptides
,
15
:
1229
-1236,  
1994
.
48
Buscail L., Delesque N., Esteve J. P., Saint-Laurent N., Prats H., Clerc P., Robberecht P., Bell G. I., Liebow C., Schally A. V., Vaysse N., Susini C. Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2.
Proc. Natl. Acad. Sci. USA
,
91
:
2315
-2319,  
1994
.
49
Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction.
Biochem. Pharmacol.
,
22
:
3099
-3108,  
1973
.
50
Duncan J. R., Stephenson M. T., Wu H. P., Anderson C. J. Indium-111-diethylenetriaminepentaacetic acid-octreotide is delivered in vivo to pancreatic, tumor cell, renal, and hepatocyte lysosomes.
Cancer Res.
,
57
:
659
-671,  
1997
.
51
Deshane J., Siegal G. P., Alvarez R. D., Wang M., Feng M., Cabrera G., Liu T., Kay M., Curiel D. T. Targeted tumor killing via an intracellular antibody against erbB-2.
J. Clin. Invest.
,
96
:
2980
-2989,  
1995
.
52
Brody S. L., Jaffe A., Han S. K., Wersto R. P., Crystal R. G. Direct in vivo gene transfer and expression in malignant cells using adenovirus vectors.
Hum. Gene Ther.
,
5
:
437
-447,  
1994
.
53
Smythe W. R., Kaiser L. R., Hwang H. C., Amin K. M., Pilewski J. M., Eck S. J., Wilson J. M., Albelda S. M. Successful adenovirus-mediated gene transfer in an in vivo model of human malignant mesothelioma.
Ann. Thorac. Surg.
,
57
:
1395
-1401,  
1994
.
54
de Jong M., Rolleman E. J., Bernard B. F., Visser T. J., Bakker W. H., Breeman W. A., Krenning E. P. Inhibition of renal uptake of indium-111-DTPA-octreotide in vivo.
J. Nucl. Med.
,
37
:
1388
-1392,  
1996
.
55
Dong J. Y., Wang D., Van Ginkel F. W., Pascual D. W., Frizzell R. A. Systematic analysis of repeated gene delivery into animal lungs with a recombinant adenovirus vector.
Hum. Gene Ther.
,
7
:
319
-331,  
1996
.
56
Worgall S., Wolff G., Falck-Pedersen E., Crystal R. G. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration.
Hum. Gene Ther.
,
8
:
37
-44,  
1997
.