Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein.

Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy owing to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that both the SSTR2 and yCD were functional in binding assays, conversion assays and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy.

PET imaging of heat-inducible suicide gene expression in mice bearing head and neck squamous cell carcinoma xenografts.

The ability to achieve tumor selective expression of therapeutic genes is an area that needs improvement for cancer gene therapy to be successful. One approach to address this is through the use of promoters that can be controlled by external means, such as hyperthermia. In this regard, we constructed a replication-deficient adenovirus that consists of a mutated herpes simplex virus 1 thymidine kinase (mTK) fused to enhanced green fluorescent protein (EGFP) under the control of the full-length human heat shock (HS) 70b promoter. The virus (AdHSmTK-EGFP) was evaluated both in vitro and in vivo in oral squamous cell carcinoma SCC-9 cells for expression of both mTK and EGFP. The in vitro expression of mTK-EGFP was validated using both (3)H-penciclovir and fluorescence-activated cell sorting assays. These studies show that specific expression could be achieved by heating the cells at 41 degrees C for 1 h, whereas little expression was observed using high doses of virus without hyperthermia. The vector was also evaluated in vivo by direct intratumoral injection into mice bearing SCC-9 xenografts. These studies demonstrated tumor expression of mTK-EGFP after ultrasound heating of the tumors by radioactive biodistribution assays, histology and microPET imaging. These in vivo results, which demonstrate HS-inducible transgene expression using PET imaging, provide a means for noninvasive monitoring of heat-induced gene therapy in local tumors, such as oral squamous cell carcinomas.

Experience with a small animal hyperthermia ultrasound system (SAHUS): report on 83 tumours.

An external local ultrasound (US) system was developed to induce controlled hyperthermia of subcutaneously implanted tumours in small animals (e.g., mice and rats). It was designed to be compatible with a small animal positron emission tomography scanner (microPET) to facilitate studies of hyperthermia-induced tumour re-oxygenation using a PET radiopharmaceutical, but it is applicable for any small animal study requiring controlled heating. The system consists of an acrylic applicator bed with up to four independent 5 MHz planar disc US transducers of 1 cm in diameter, a four-channel radiofrequency (RF) generator, a multiple thermocouple thermometry unit, and a personal computer with custom monitoring and controlling software. Although the system presented here was developed to target tumours of up to 1 cm in diameter, the applicator design allows for different piezoelectric transducers to be exchanged and operated within the 3.5-6.5 MHz band to target different tumour sizes. Temperature feedback control software was developed on the basis of a proportional-integral-derivative (PID) approach when the measured temperatures were within a selectable temperature band about the target temperature. Outside this band, an on/off control action was applied. Perfused tissue-mimicking phantom experiments were performed to determine optimum controller gain constants, which were later employed successfully in animal experiments. The performance of the SAHUS (small animal hyperthermia ultrasound system) was tested using several tumour types grown in thighs of female nude (nu/nu) mice. To date, the system has successfully treated 83 tumours to target temperatures in the range of 41-43 degrees C for periods of 65 min on average.

Non-invasive gamma camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter.

A model epitope-tagged receptor was constructed by fusing the hemagglutinin (HA) sequence on the extracellular N-terminus of the human somatostatin receptor subtype 2 (hSSTr2) gene. This construct was placed in an adenoviral (Ad-HAhSSTr2) vector. This study evaluated Ad-HAhSSTr2 in vitro and in vivo using FACS, fluorescent microscopy, radioactive binding assays, and gamma camera imaging techniques. Infection of A-427 non-small cell lung cancer cells with Ad-HAhSSTr2 or Ad-hSSTr2 resulted in similar expression of hSSTr2 by FACS analysis and binding assays using a (99m)Tc-labeled somatostatin analogue ((99m)Tc-P2045). HAhSSTr2 expression in A-427 cells was specific for infection with Ad-HAhSSTr2. FITC-labeled anti-HA antibody (FITC-HA) confirmed surface expression in live A-427 cells and the absence of internalization. Gamma camera imaging and gamma counter analysis of normal mice showed significantly greater (P<0.05) liver uptake of (99m)Tc-labeled anti-HA antibody ((99m)Tc-anti-HA) in mice injected i.v. 48 h earlier with Ad-HAhSSTr2 (53.6+/-6.9% ID/g) as compared to mice similarly injected with Ad-hSSTr2 (9.0+/-1.3% ID/g). In a mouse tumor model, imaging detected increased tumor localization of (99m)Tc-anti-HA due to direct intratumor injection Ad-HAhSSTr2. Gamma counter analysis confirmed significantly greater (P<0.05) uptake of (99m)Tc-anti-HA in tumors injected with Ad-HAhSSTr2 (12.5+/-4.1% ID/g) as compared to Ad-hSSTr2-infected tumors (5.1+/-1.5% ID/g). These studies demonstrate the feasibility of using an epitope-tagged reporter receptor for non-invasively imaging gene transfer.

A noninvasive reporter system to image adenoviral-mediated gene transfer to ovarian cancer xenografts.

OBJECTIVE: Gene therapy trials for ovarian cancer would benefit from a noninvasive imaging modality to detect the location and extent of gene transfer. The human type 2 somatostatin receptor gene (hSSTr2) was evaluated as a reporter gene for imaging adenoviral (Ad) gene transfer to ovarian cancer. METHODS: A replication-incompetent Ad vector encoding hSSTr2 (Ad-hSSTr2) was used to infect SKOV3.ip1 cells in vitro and tumors growing in nude mice. Gamma camera imaging detected uptake of 99m-Tc-P2045 (a somatostatin analogue) due to expressed hSSTr2. RESULTS: Specific uptake of 99m-Tc-P2045 was imaged in Ad-hSSTr2-infected cells in vitro. Noninvasive in vivo imaging detected gene transfer to intraperitoneal tumors. Uptake of 99m-Tc-P2045 (percentage dose per gram of tumor) averaged 2.2 and 0.18 for Ad-hSSTr2-injected mice and controls, respectively. CONCLUSION: This study reports the first noninvasive imaging method for imaging gene transfer to ovarian cancer. A human gene therapy trial is planned.

An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression.

The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to alpha(v)beta(3) and alpha(v)beta(5) integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials.

Light-based imaging of green fluorescent protein-positive ovarian cancer xenografts during therapy.

OBJECTIVE: The purpose of the study was to develop a sensitive, noninvasive imaging method for monitoring ovarian xenografts during therapeutic intervention. METHODS: Human ovarian tumor cells (SKOV3.ip1) were infected with a replication-deficient adenoviral (Ad) vector encoding green fluorescent protein (GFP). The GFP-positive tumor cells were imaged in vitro and in vivo with a fluorescence stereomicroscope. Using appropriate filters, both GFP fluorescence and adriamycin were simultaneously detected. Nude mice implanted with GFP-positive cells were imaged repeatedly, in a noninvasive manner. RESULTS: SKOV3.ip1 cells infected with Ad-GFP showed high GFP fluorescence, which was eliminated after treatment with adriamycin. Loss of GFP fluorescence was confirmed to be dead cells. For in vivo imaging, intraperitoneal tumors as small as 0.2 mm in diameter were detected externally. Adriamycin uptake was detected in tumors by in vivo imaging, and reduction in tumor size was concurrent with decrease in GFP fluorescence. These findings were confirmed at necropsy. CONCLUSIONS: Fluorescence stereomicroscopy monitored the response of ovarian xenografts to adriamycin therapy. For the first time, GFP and adriamycin were imaged simultaneously.

Detection and measurement of in vitro gene transfer by gamma camera imaging.

The purpose of this work was to develop a high capacity method to image gene transfer to cancer cells growing as monolayers in cell culture plates. A sensitive and high capacity nuclear-imaging method for detection of gene transfer in vitro will allow rapid validation of vectors in different cell lines under various conditions. Human cancer cell lines (A-427 non-small cell lung, SKOV3.ip1 ovarian, MDA-MB-468 breast, and BxPC-3 pancreatic) were infected with a replication-incompetent adenoviral vector encoding the human type 2 somatostatin receptor (Ad-hSSTr2). Expression of the hSSTr2 reporter protein in cells was detected by imaging an internalized 99mTc-labeled, hSSTr2 binding peptide (P2045, Diatide, Inc.). Imaging provided an accurate measure of internally bound 99mTc as evidenced by equivalence of results for imaging region of interest (ROI) analyses and gamma counter measurements. Internally bound 99mTc-P2045 was linearly correlated (R2 = 0.98) with the percentage of hSSTr2-positive cells following gene transfer. Excess P2045 blocked binding and internalization of the 99mTc-P2045, indicating the specificity of the technique. Up to four 96-well plates could be imaged simultaneously, thereby demonstrating the high capacity of the system. This novel in vitro approach provides a new method to test enhanced gene transfer as new vectors are developed.

Simultaneous evaluation of dual gene transfer to adherent cells by gamma-ray imaging.

A gamma camera imaging method was developed to detect dual gene transfer to adherent cells growing as monolayers in cell culture plates. Human cancer cells were infected with replication-incompetent adenoviral vectors encoding the human type 2 somatostatin receptor (Ad-hSSTr2) and/or herpes virus thymidine kinase (Ad-TK). The hSSTr2 and TK reporter proteins were detected by imaging internally bound (99m)Tc-P2045 peptide (Diatide, Inc.) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Following gene transfer, expression of hSSTr2 and TK were accurately imaged in vitro.

Gene transfer strategies for improving radiolabeled peptide imaging and therapy.

Utilization of molecular biology techniques offers attractive options in nuclear medicine for improving cancer imaging and therapy with radiolabeled peptides. Two of these options include utilization of phage-panning to identify novel tumor-specific peptides or single chain antibodies and gene transfer techniques to increase the number of antigen/receptor sites expressed on malignant cells. Our group has focused on the latter approach for improving radiolabeled peptide imaging and therapy. The most widely used gene transfer vectors in clinical gene therapy trials include retrovirus, cationic lipids, and adenovirus. We have utilized adenovirus vectors for gene transfer because of their ability to accomplish efficient in vivo gene transfer. Adenovirus vectors encoding the genes for a variety of antigens/receptors (carcinoembryonic antigen, gastrin-releasing peptide receptor, somatostatin receptor subtype 2 (SSTr2)) have all shown that their expression is increased on cancer cells both in vitro and in vivo following adenovirus infection. Of particular interest has been the adenovirus encoding for SSTr2 (AdCMVSSTr2). Various radioisotopes have been attached to somatostatin analogues for imaging and therapy of SSTr2-positive tumors both clinically and in animal models. The use of these analogues in combination with AdCMVSSTr2 is a promising approach for improving the detection sensitivity and therapeutic efficacy of these radiolabeled peptides against solid tumors. In addition, we have proposed the use of SSTr2 as a marker for imaging the expression of another cancer therapeutic transgene (e.g. cytosine deaminase, thymidine kinase) encoded within the same vector. This would allow for non-invasive monitoring of gene delivery to tumor sites.

A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo.

Adenoviral (Ad) vectors are promising gene therapy vehicles due to their in vivo stability and efficiency, but their potential utility is compromised by their restricted tropism. Targeting strategies have been devised to improve the efficacy of these agents, but specific targeting following in vivo systemic administration of vector has not previously been demonstrated. The distinct aim of the current study was to determine whether an Ad-targeting strategy could maintain fidelity upon systemic vascular administration. We used a bispecific antibody to target Ad infection specifically to angiotensin-converting enzyme (ACE), which is preferentially expressed on pulmonary capillary endothelium and which may thus enable gene therapy for pulmonary vascular disease. Cell-specific gene delivery to ACE-expressing cells was first confirmed in vitro. Administration of retargeted vector complex via tail vein injection into rats resulted in at least a 20-fold increase in both Ad DNA localization and luciferase transgene expression in the lungs, compared to the untargeted vector. Furthermore, targeting led to reduced transgene expression in nontarget organs, especially the liver, where the reduction was over 80%. Immunohistochemical and immunoelectron microscopy analysis confirmed that the pulmonary transgene expression was specifically localized to endothelial cells. Enhancement of transgene expression in the lungs as a result of the ACE-targeting strategy was also confirmed using a new noninvasive imaging technique. This study shows that a retargeting approach can indeed specifically modify the gene delivery properties of an Ad vector given systemically and thus has encouraging implications for the further development of targetable, injectable Ad vectors.

Molecular modification of a recombinant anti-CD3epsilon-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model.

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT(390)) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT(390) anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 microg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells.

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.

Noninvasive monitoring of gene transfer using a reporter receptor imaged with a high-affinity peptide radiolabeled with 99mTc or 188Re.

UNLABELLED: Gene therapy protocols require better modalities to monitor the location and level of transferred gene expression. One potential in vivo mechanism to assess gene expression would be to image the binding of a radiolabeled peptide to a reporter receptor that is expressed in targeted tissues. This concept was tested in a tumor model using a replication-incompetent adenoviral vector encoding the human type 2 somatostatin receptor (Ad5-CMVhSSTr2). Expression of the hSSTr2 reporter was imaged using a radiolabeled, somatostatin-avid peptide (P829). METHODS: Bilateral subcutaneous A427 tumor xenografts were established on the flanks of athymic nude mice. These human-origin, non-small cell lung tumors are normally negative for hSSTr2 expression. One tumor was injected directly with Ad5-CMVhSSTr2, whereas the second tumor was injected directly with a control Ad5 vector. The mice were injected intravenously 48 h later with P829 peptide that was radiolabeled to high specific activity with 99mTc (half-life, 6 h) or 188Re (half-life, 17 h). Tumors were frozen and evaluated for somatostatin receptor expression using fluorescein-labeled somatostatin. RESULTS: The accumulation of radiolabeled P829 in hSSTr2-expressing tumors was easily visualized by gamma camera imaging 3 h after injection. Imaging region of interest analyses and biodistribution studies confirmed a 5- to 10-fold greater accumulation of both radiolabeled P829 peptides in the Ad5-CMVhSSTr2-injected tumors versus control tumors injected with control Ad5 vectors. Ad5-CMVhSSTr2-injected tumors accumulated 2.5-3.8 percentage injected dose per gram 3 h after injection. Only Ad5-CMVhSSTr2-injected tumors expressed somatostatin receptors, as determined by immunohistochemistry. CONCLUSION: These studies show the feasibility of imaging a 99mTc-labeled peptide's binding to a reporter receptor after in vivo gene transfer to tumor cells. The 188Re-labeled peptide worked equally well for this imaging approach and offers the additional advantage of energetic beta decay with potential therapeutic efficacy. 99mTc and 188Re are generator produced, an advantage for widespread availability and low cost, and both radioisotopes can be imaged with existing, high-resolution modalities. There is great potential for using 99mTc-labeled peptides for imaging gene transfer with the hSSTr2 reporter receptor, especially when the reporter correlates with the expression of therapeutic genes that can be included simultaneously in the gene therapy vector.

Targeting strategies for cancer radiotherapy.

Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.

In vivo localization of [(111)In]-DTPA-D-Phe1-octreotide to human ovarian tumor xenografts induced to express the somatostatin receptor subtype 2 using an adenoviral vector.

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 [(111)In]-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.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-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 [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-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. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-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 [(111)In]-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 [(111)In]-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.

Improved synthesis of the bifunctional chelating agent 1,4,7,10-tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N',N'',N'''-tri s(acetic acid)cyclododecane (PA-DOTA).

A concise synthesis of the bifunctional chelating agent 1,4,7,10-tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N',N'',N'' '-tris(acetic acid)cyclododecane (PA-DOTA) is reported. Difficulties involving the production of partially alkylated products and their removal have been addressed and obviated. After the pure nitro form of PA-DOTA was obtained, conversion to the isothiocyanato form PA-DOTA (1, conjugation to HuCC49 and HuCC49deltaCH2 monoclonal antibodies was achieved. Subsequent radiolabeling with 177Lu was performed, demonstrating a useful bifunctional chelating agent suitable for clinical radioimmunotherapy applications.

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.

A number of preclinical and human clinical gene therapy trials using adenoviral vectors have shown that the number of viral particles necessary to give adequate levels of gene transfer can be associated with significant vector-related toxicity. In an effort to reduce the number of adenoviral particles required for a given level of gene transfer, we sought to redirect adenoviral infection via a receptor that is highly expressed on the target cells. By using basic fibroblast growth factor (FGF2) as the targeting ligand, adenovirus-mediated gene transfer to the human ovarian cancer cell line SKOV3.ip1 was significantly enhanced, permitting the transduction of a greater number of target cells to be achieved by a given dose of virus. In a murine model of human ovarian carcinoma, an FGF2-redirected adenoviral vector carrying the gene for herpes simplex virus thymidine kinase (AdCMVHSV-TK) was shown to result in a significant prolongation of survival compared with the same number of particles of unmodified AdCMVHSV-TK. In addition, equivalent survival rates were achieved with a 10-fold lower dose of the FGF2-redirected AdCMVHSV-TK compared with the unmodified vector. To our knowledge, this is the first report demonstrating that strategies to enhance the efficiency of in vivo transduction of adenoviral vectors will be of clinical utility.

Tumor localization of a radiolabeled bombesin analogue in mice bearing human ovarian tumors induced to express the gastrin-releasing peptide receptor by an adenoviral vector.

BACKGROUND: The adenoviral vector, AdCMVGRPr, has been used to induce the expression of the murine gastrin-releasing peptide receptor (GRPr) both in vitro and in vivo. A bombesin analogue ([125I]-mIP-bombesin) has been shown to bind with high affinity to GRPr and to localize to intraperitoneal (i.p.) ovarian tumors 2 days after induction of GRPr in an athymic nude mouse model. The present study was conducted to determine the level of localization of [(125/131)I]-mIP-bombesin in the tumors at 2, 4, and 7 days after AdCMVGRPr administration and to determine the feasibility of giving multiple doses of [131I]-mIP-bombesin for therapy. METHODS: Human ovarian cancer cells (SKOV3.ip1) were infected in vitro with AdCMVGRPr and were assayed for receptor expression at 2, 4, and 7 days after infection by using a radiolabeled bombesin-binding assay. Biodistribution studies utilized athymic nude mice inoculated i.p. with SKOV3.ip1 cells. The tumors were induced to express GRPr with an i.p. injection of AdCMVGRPr followed by administration of [125I]-mIP-bombesin 2 days later (AdCMVLacZ or saline was used for negative controls). In addition, the tumor localization of [125I]-mIP-bombesin was determined 4 and 7 days after AdCMVGRPr administration. The tumor localization of [131I]-mIP-bombesin was compared with [125I]-mIP-bombesin in this in vivo model. RESULTS: SKOV3.ip1 cells infected with AdCMVGRPr resulted in 80.3 +/- 5.9% binding of [125I]-Tyr4-bombesin at 2 days after infection, which decreased to 46.8 +/- 0.4% at 4 days and to 17.7 +/- 0.1% at 7 days. The biodistribution study showed that the tumor localization (14.9 +/- 8.2% injected dose/gram; ID/g) of [125I]-mIP-bombesin 2 days after administration of AdCMVGRPr was significantly greater than its localization in other organs (P < 0.003) and was significantly greater than in AcCMVLacZ- and saline-treated mice (P < 0.003). Injections of [125I]-mIP-bombesin at 4 and 7 days after a single AdCMVGRPr administration showed tumor localization of 4.5 +/- 3.0% ID/g at Day 4 and 3.9 +/- 3.5% ID/g at Day 7. The decreased localization at longer times after AdCMVGRPr infection correlated with in vitro results. The tumor uptake of [125I]-mIP-bombesin was comparable to the uptake of [131I]-mIP-bombesin (21.2 +/- 8.3% ID/g versus 15.4 +/- 5.6% ID/g, respectively), as was the normal tissue biodistribution. CONCLUSIONS: The expression of GRPr in human ovarian cancer cells can be accomplished both in vitro and in vivo by using AdCMVGRPr, with the in vivo tumor localization of [125I]-mIP-bombesin being significantly greater than in control animals. The tumor localization of [125I]-mIP-bombesin and [131I]-mIP-bombesin at 2 days after AdCMVGRPr was comparable in a mouse model of human ovarian carcinoma. Injections of [125I]-mIP-bombesin at Days 4 and 7 after AdCMVGRPr infection resulted in tumor localization of [125I]-mIP-bombesin but at a level lower than 2 days. Thus, the total amount of radioactivity delivered to the tumor should be increased by multiple injections of [131I]-mIP-bombesin, which would be required for a therapeutic effect.

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.

UNLABELLED: The gastrin releasing peptide receptor (GRPr) has a high affinity for the 14 amino acid bombesin peptide. For this analysis, [125I]-Tyr4-bombesin was compared with [125I]-mIP-bombesin (a seven amino acid bombesin analog) for in vitro binding and internalization into tumor cells and for tumor localization in vivo. Also, a recombinant adenoviral vector (AdCMVGRPr) was used for gene transfer to induce the expression of GRPr in human ovarian cancer cells for binding and tumor localization with these radiolabeled peptides. METHODS: [125I]-mIP-bombesin was synthesized and compared with [125I]-Tyr4-bombesin in internalization assays using BNR-11 cells (mouse fibroblast cells stably transfected with GRPr) over a 24-hr period. In vitro binding assays used BNR-11, and A427, HeLa and SKOV3.ip1 human cancer cells, which were either uninfected or infected with AdCMVGRPr. Biodistribution studies were performed in normal BALB/c mice and in athymic nude mice bearing orthotopic SKOV3.ip1 ovarian cancer tumors. The SKOV3.ip1 tumors were induced to express GRPr with the AdCMVGRPr adenoviral vector. RESULTS: Internalization assays showed that [125I]-Tyr4-bombesin was rapidly internalized and catabolized at 37 degrees C with approximately 10% of the radioactivity remaining intracellularly at 4 hr, compared with approximately 30% with [125I]-mIP-bombesin. HeLa, A427 and SKOV3.ip1 cells were all induced to express levels of GRPr that were higher than those seen with the positive control BNR-11 cells. Normal mice showed a lower level of radioactivity in both the blood and thyroid for [125I]-mIP-bombesin [0.26% +/- 0.10% injected dose per gram (ID/g) and 0.24% +/- 0.05% ID] than for [125I]-Tyr4-bombesin (3.5% +/- 1.6% ID/g and 5.2% +/- 4.4% ID) at 4 hr postinjection. Mice bearing intraperitoneal (i.p.) SKOV3.ip1 tumors and given AdCMVGRPr i.p. 5 days after tumor cell inoculation followed by [125I]-mIP-bombesin i.p. at day 7 showed 16.5% +/- 4.8% ID/g in tumor compared with 5.9% +/- 3.0% ID/g with [125I]-Tyr4-bombesin at 4 hr postinjection. Tumor bearing mice given saline or a control adenovirus expressing the beta-galactosidase (LacZ) gene showed significantly lower tumor uptake values of both bombesin peptides. CONCLUSION: Internalization assays showed that [125I]-mIP-bombesin has favorable characteristics compared with [125I]-Tyr4-bombesin with regards to cellular internalization and retention. The results demonstrate successful in vitro and in vivo transduction of human tumor cells with a recombinant adenoviral vector-expressing GRPr. Additionally, tumors transduced in vivo to express GRPr demonstrated significantly greater localization of [125I]-mIP-bombesin when compared with [125I]-Tyr4-bombesin.