Biodistribution Study of Intravenously Injected Cetuximab-IRDye700DX in Cynomolgus Macaques.

PURPOSE: The use of receptor-targeted antibodies conjugated to photosensitizers is actively being explored to enhance treatment efficacy. To facilitate clinical testing, we evaluated cetuximab conjugated to IRDye700DX (IR700) in cynomolgus macaques. PROCEDURES: Total IR700 and intact cetuximab-IR700 were measured in 51 tissues at 2 and 14 days after intravenous injection of 40 and 80 mg/kg cetuximab-IR700, respectively, and compared with an unlabeled cetuximab-dosed control group (two each per sex per time point per group). RESULTS: The IR700 retrieved from all tissues at 2 and 14 days after dosing was estimated at 34.9 ± 1.8 and 2.53 ± 0.67% of the total dose, respectively. The tissues with the highest levels of intact cetuximab-IR700 at 2 days after dosing were the blood, lung, and skin. Formalin-fixed paraffin-embedded tissue sections at 2 days after dosing showed the highest IR700 signals in the axillary lymph node, mammary gland, and gall bladder. CONCLUSIONS: Both IR700 and intact cetuximab-IR700 biodistributions were consistent with known epidermal growth factor receptor (EGFR) expression, and changes between 2 and 14 days were consistent with rapid metabolism and excretion of the cetuximab-IR700.

A dual-reporter, diagnostic vector for prostate cancer detection and tumor imaging.

Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and as a strategy for tumor localization.

Systemic delivery of a breast cancer-detecting adenovirus using targeted microbubbles.

One of the major limitations of cancer gene therapy using recombinant human adenovirus (Ad) is rapid Ad inactivation from systemic delivery. To eliminate this, biotin-coated ultrasound contrast agents, or microbubbles (MBs), were streptavidin-coupled with biotinylated antibodies to three distinct tumor vasculature-associated receptors (α(V)ß(3) integrin, P-selectin and vascular endothelial growth factor receptor-2) for systemic targeting of a previously generated vector Ad5/3-Id1-SEAP-Id1-mCherry. This cancer-specific, dual-reporter vector was loaded in the targeted MBs and confirmed by confocal microscopy. MB loading capacity was estimated by functional assays as 4.72 ± 0.2 plaque forming unit (PFU) per MB. Non-loaded (free) Ad particles were effectively inactivated by treatment with human complement. The Ad-loaded, targeted-MBs were injected systemically in mice bearing MDA-MB-231 tumors (Grp 1) and compared with two control groups: Ad-loaded, non-targeted MBs (Grp 2) and free Ad (Grp 3) administered under the same conditions. Two days after administration the blood levels of secreted embryonic alkaline phosphatase (SEAP) reporter in Grp 1 mice (16.1 ng ml(-1) ± 2.5) were significantly higher (P<0.05) than those in Grp 2 (9.75 ng ml(-1) ± 1.5) or Grp 3 (4.26 ng ml(-1) ± 2.5) animals. The targeted Ad delivery was also confirmed by fluorescence imaging. Thus, Ad delivery by targeted MBs holds potential as a safe and effective system for systemic Ad delivery for the purpose of cancer screening.

Breast cancer metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice.

This study sought to determine if weekly X-ray exposure affected breast cancer cell metastasis to bone and to also evaluate the use of bioluminescent imaging (BLI) and microSPECT for detection of metastatic bone lesions. Five week old nude mice were randomly assigned to the CT exposed (n = 7) and no CT exposure (n = 6) treatment groups. Mice received an intracardiac injection of MDA-MB-435 human breast cancer cells transduced with luciferase, or a sham injection (saline). The CT exposed group of mice received CT irradiation once a week for 5 weeks. All mice underwent weekly BLI and select mice received Tc-99m-MDP followed by microSPECT imaging after 5 weeks. Pathological evaluation and histomorphometry were used to assess the affect of CT X-rays on bone metastasis and to evaluate BLI. BLI results found no significant difference in metastasis between animals that received CT and those that did not (P > 0.05); however, histomorphometry of the knee joints revealed a significant increase (P = 0.029) in tumor area of the leg bones in mice that received CT exposure (60% +/- 7%) compared to animals that did not receive CT scans (33% +/- 8%). Compared to histological analysis, BLI of the leg and spine was determined to have excellent sensitivity (100%), good specificity (80-90%) and accuracy (90-96%), a positive predictive value of 81-93% and a 100% negative predictive value. Thus, multi-modality imaging techniques can be very useful for monitoring bone metastasis, however microCT X-rays should be used judiciously in order to limit irradiation that may stimulate increased metastasis to specific regions of the skeleton. MicroSPECT imaging did not detect metastatic lesions in the legs of these young nude mice.

Noninvasive dual modality in vivo monitoring of the persistence and potency of a tumor targeted conditionally replicating adenovirus.

In clinical trials with cancer patients, the safety of conditionally replicating adenoviruses (CRAds) has been good. However, marginal data are available on the persistence or antitumor efficacy of these agents. The oncolytic potency of CRAds is determined by their capacity for entering target cells. Consequently, we constructed a retargeted CRAd featuring a secreted marker protein, soluble human carcinoembryogenic antigen (hCEA), which can be measured in growth medium or plasma. We found that virus replication closely correlated with hCEA secretion both in vitro and in vivo. Further, antitumor efficacy and the persistence of the virus could be deduced from plasma hCEA levels. Finally, using in vivo bioluminescence imaging, we were able to detect effective tumor cell killing by the virus, which led to enhanced therapeutic efficacy.

Site-specific delivery of acidic fibroblast growth factor stimulates angiogenic and osteogenic responses in vivo.

A major clinical problem in orthopedics is the healing of nonunion fractures. Limitations of this bone repair process include insufficient angiogenesis and mineralization. Integrating appropriate biomaterials with site-specific neovascularization and osteogenesis at the wound site has been the focus of several clinically relevant therapeutic strategies. As an extracellular protein, acidic fibroblast growth factor (FGF-1) induces, coordinates, and sustains site-specific molecular responses associated with angiogenesis and osteogenesis. To establish the ability of this growth factor to coordinate bone regenerative process in vivo, site-specific delivery of FGF-1, entrapped in a fibrin/hydroxyapatite composite, was evaluated. Kinetic analysis in vivo revealed the biocomposite was capable of delivering biologically active FGF-1. Release kinetics revealed an initial delivery of 87.5 ng/h of active FGF-1 in the first 20 h, followed by a reduced delivery of 28 ng/h during the next 20 h. In situ immunohistological analyses demonstrated that FGF-1-containing implants induced increased angiogenesis and infiltration of cells expressing osteogenic related markers (i.e., osteopontin, osteocalcin). Collectively, these efforts support that site-specific delivery of active FGF-1 in a fibrin/hydroxyapatite composite is competent to induce not only angiogenesis but also osteogenic cellular responses.

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus.

The effect of complement on transgene expression was evaluated in vivo and in vitro using mice lacking complement components. Complement component 3 (C3) deficient mice (C3-/-) and appropriate wild-type controls were intravenously injected with a replication incompetent, luciferase-expressing normal Ad5 (Ad5Luc1), or fibritin-fiber Ad5 (Ad5FFLuc1). Repeated, noninvasive bioluminescence imaging was conducted over 35 days. Our data show for the first time that C3 facilitates both short- and long-term hepatic expression of luciferase following systemic delivery. C3-/- mice showed significantly less (P < 0.05) luciferase expression in their liver than treatment-matched wild-type mice when 2.3 x 10(9) (Ad5Luc1) and 4.0 x 10(9) (Ad5Luc1 or Ad5FFLuc1) viral particles (v.p.) were infused. The maximal difference in luciferase activity between C3-/- and wild-type mice was 99-fold difference at 3 days for the 2.3 x 10(9) v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5FFLuc1). Preincubation of Ad5Luc1 with wild-type, C1q-/-, or factor B (FB) deficient mouse sera for 5 min significantly (P < 0.05) increased transduction of mouse liver cells, as compared to preincubation with C3-/- sera or PBS. These results suggest the classical or alternate complement pathway enhances Ad5-mediated liver transduction.

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.

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells.

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.

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.

Increased dietary salt accelerates chronic allograft nephropathy in rats.

BACKGROUND: Chronic allograft nephropathy (CAN), a major problem in renal transplantation, is related to both alloantigen-dependent and -independent processes. Because dietary salt intake modulated glomerular production of transforming growth factor-beta, which has been shown to play an important role in CAN, we hypothesized that dietary salt would directly enhance renal injury in a rodent model of CAN. METHODS: Dietary NaCl was increased from 1.0% (normal) to 8.0% in a group of Fisher/Lewis rats 25 days following orthotopic renal transplantation and was continued until 16 weeks after transplantation. RESULTS: Blood pressure, which was recorded using radiotelemetry in the first eight-weeks post-transplantation, did not differ between the groups, but allograft recipients on the 8.0% NaCl diet rapidly demonstrated increased urinary albumin excretion. Renal function determined by dynamic functional imaging was worse in allograft recipients on the 8.0% NaCl diet by six weeks following transplantation. Histologic examination at 16 weeks confirmed a significant increase in allograft damage in the 8.0% NaCl group compared with allografts from rats on 1.0% NaCl diet. These findings included glomerulosclerosis and tubulointerstitial injury that consisted of fibrosis, tubular atrophy and dilation, intratubular casts, and tubular epithelial cell damage. Small arteries and arterioles did not show evidence of damage from hypertension or other abnormality. CONCLUSIONS: In this model of CAN, renal allograft dysfunction preceded hypertension and was accelerated significantly by an increase in dietary salt.

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.

Imaging Tc-99m-labeled FGF-1 targeting in rats.

Recombinant human acidic fibroblast growth factor (FGF-1) was radiolabeled with (99m)Tc by the HYNIC method. The (99m)Tc-FGF-1 retained its representative molecular mass, heparin affinity, cellular binding to both low (Kd = 9.5 nM) and high (Kd = 125 pM) affinity sites, and mitogenic activity. Gamma camera imaging after intravenous dosing in rats confirmed high liver and kidney binding. Heparin significantly decreased (99m)Tc-FGF-1 liver uptake and increased urinary excretion. These studies illustrate a new method for imaging FGF-1 targeting under various conditions.

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.

Specific targeting of activated endothelium in rat adjuvant arthritis with a 99mTc-radiolabeled E-selectin-binding peptide.

OBJECTIVE: To determine the potential of an E-selectin-binding peptide (ESbp) to specifically bind activated endothelium in rheumatoid arthritis (RA) animal models. METHODS: ESbp (KYDGDITWDQLWDLMK; 2,027 daltons) was labeled with biotin and 99mTc. The affinity of ESbp derivatives for E-selectin was measured by enzyme-linked immunosorbent assay. The binding of biotin-ESbp was compared with that of an anti-E-selectin antibody, by immunohistochemical analyses of human synovial sections and sections from the Mycoplasma pulmonis MRL-lpr/lpr mouse arthritis model. 99mTc-ESbp was sequentially imaged in vivo with a gamma camera in the rat adjuvant-induced arthritis model. RESULTS: E-selectin expression was detected in human RA synovium and mouse arthritic synovium using biotin-ESbp. Both biotin-ESbp and 99mTc-labeled ESbp had high affinity for E-selectin (dissociation constant 2-5 nM). In vivo imaging showed specific binding of 99mTc-ESbp to the rat ankle joint prior to clinical manifestations of inflammation. CONCLUSION: These results demonstrate that activated endothelium can be targeted with 99mTc-ESbp. The specificity of targeting can be used to evaluate up-regulation of E-selectin in RA models, and to follow changes in this up-regulation during treatment trials.