Evaluation of a Novel Tc-99m Labelled Vitamin B12 Derivative for Targeting Escherichia coli and Staphylococcus aureus In Vitro and in an Experimental Foreign-Body Infection Model.

PURPOSE: Vitamin B12 (cyanocobalamin, Cbl) is accumulated by rapidly replicating prokaryotic and eukaryotic cells. We investigated the potential of a Tc-99m labelled Cbl derivative ([(99m)Tc]PAMA(4)-Cbl) for targeting infections caused by Escherichia coli and Staphylococcus aureus. In vitro binding assays were followed by biodistribution studies in a mouse model of foreign body infection. PROCEDURES: E. coli (ATCC 25922) and S. aureus (ATCC 43335) were used as test strains. [(57)Co]Cbl, [(67)Ga]citrate and [(99m)Tc]DTPA served as reference compounds. The in vitro competitive binding of [(57)Co]Cbl or [(99m)Tc]PAMA(4)-Cbl, and unlabeled Cbl, to viable or killed bacteria, was evaluated at 37 and 4 °C. A cage mouse model of infection was used for biodistribution of intravenous [(57)Co]Cbl and [(99m)Tc]PAMA(4)-Cbl in cage and dissected tissues of infected and non-infected mice. RESULTS: Maximum binding (mean ± SD) of [(57)Co]Cbl to viable E. coli was 81.7 ± 2.6 % and to S. aureus 34.0 ± 6.7 %, at 37 °C; no binding occurred to heat-killed bacteria. Binding to both test strains was displaced by 100- to 1000-fold excess of unlabeled Cbl. The in vitro binding of [(99m)Tc]PAMA(4)-Cbl was 100-fold and 3-fold lower than the one of [(57)Co]Cbl for E. coli and S. aureus, respectively. In vivo, [(99m)Tc]PAMA(4)-Cbl showed peak percentage of injected dose (% ID) values between 1.33 and 2.3, at 30 min post-injection (p.i.). Significantly higher retention occurred in cage fluids infected with S. aureus at 4 h and with E. coli at 8 h p.i. than in non-infected animals. Accumulation into infected cages was also higher than the one of [(99m)Tc]DTPA, which showed similar biodistribution in infected and sterile mice. [(57)Co]Cbl gradually accumulated in cages with peaks % ID between 3.58 and 4.83 % achieved from 24 to 48 h. Discrimination for infection occurred only in E. coli-infected mice, at 72 h p.i. [(67)Ga]citrate, which showed a gradual accumulation into cage fluids during 12 h, was discriminative for infection from 48 to 72 h p.i. (P < 0.05). CONCLUSION: Cbl displayed rapid and specific in vitro binding to test strains. [(99m)Tc]PAMA(4)-Cbl was rapidly cleared from most tissues and discriminated between sterile and infected cages, being a promising candidate for imaging infections in humans.

Tumor imaging in patients with advanced tumors using a new (99m) Tc-radiolabeled vitamin B12 derivative.

UNLABELLED: Targeting cancer cells with vitamin B12 (cobalamin) is hampered by unwanted physiologic tissue uptake mediated by transcobalamin. Adhering to good manufacturing practice, we have developed a new (99m)Tc-cobalamin derivative ((99m)Tc(CO)3-[(4-amido-butyl)-pyridin-2-yl-methyl-amino-acetato] cobalamin, (99m)Tc-PAMA-cobalamin). The derivative shows no binding to transcobalamin but is recognized by haptocorrin, a protein present in the circulation and notably expressed in many tumor cells. In this prospective study, we investigated cancer-specific uptake of (99m)Tc-PAMA-cobalamin in 10 patients with various metastatic tumors. METHODS: Ten patients with biopsy-proven metastatic cancer were included. Dynamic imaging was started immediately after injection of 300-500 MBq of (99m)Tc-PAMA-cobalamin, and whole-body scintigrams were obtained at 10, 30, 60, 120, and 240 min and after 24 h. The relative tumor activity using SPECT/CT over the tumor region after 4 h was measured in comparison to disease-free lung parenchyma. Patients 3-10 received between 20 and 1,000 µg of cobalamin intravenously before injection of (99m)Tc-PAMA-cobalamin. The study population comprised 4 patients with adenocarcinomas of the lung, 3 with squamous cell carcinomas of the hypopharyngeal region, 1 with prostate adenocarcinoma, 1 with breast, and 1 with colon adenocarcinoma. RESULTS: The median age of the study group was 61 ± 11 y. Six of 10 patients showed positive tumor uptake on (99m)Tc-PAMA-cobalamin whole-body scintigraphy. The scan was positive in 1 patient with colon adenocarcinoma, in 3 of 4 lung adenocarcinomas, in 1 of 3 hypopharyngeal squamous cell carcinomas, and in 1 breast adenocarcinoma. Renal uptake was between 1% and 3% for the left kidney. Predosing with cobalamin increased the tumor uptake and improved blood-pool clearance. The best image quality was achieved with a predose of 20-100 ug of cold cobalamin. The mean patient dose was 2.7 ± 0.9 mSv/patient. CONCLUSION: To our knowledge, we report for the first time on (99m)Tc-PAMA-cobalamin imaging in patients with metastatic cancer disease and show that tumor targeting is feasible.

Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin.

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.

Radiolabeling of rituximab with (188)Re and (99m)Tc using the tricarbonyl technology.

INTRODUCTION: The most successful clinical studies of immunotherapy in patients with non-Hodgkin's lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20(+) B-cell tumors. Rituximab radiolabeled with ß(-) emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the (99m)Tc- and (188)Re-tricarbonyl core (IsoLink technology). METHODS: The native format of the antibody (RTX(wt)) as well as a reduced form (RTX(red)) was labeled with (99m)Tc/(188)Re(CO)(3). The partial reduction of the disulfide bonds to produce free sulfhydryl groups (-SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts. RESULTS: The radiolabeling efficiency and kinetics of RTX(red) were superior to that of RTX(wt) ((99m)Tc: 98% after 3 h for RTX(red) vs. 70% after 24 h for RTX(wt)). (99m)Tc(CO)(3)-RTX(red) was used without purification for in vitro and in vivo studies whereas (188)Re(CO)(3)-RTX(red) was purified to eliminate free (188)Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37 °C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of (99m)Tc(CO)(3)-RTX(red) but not with pre-purified (188)Re(CO)(3)-RTX(red). Both conjugates revealed high binding affinity to the CD20 antigen (K(d) = 5-6 nM). Tumor uptake of (188)Re(CO)(3)-RTX(red) was 2.5 %ID/g and 0.8 %ID/g for (99m)Tc(CO)(3)-RTX(red) 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for (99m)Tc(CO)(3)-RTX(red) and for (188)Re(CO)(3)-RTX(red) 0.5 and 0.5 (24 h pi). CONCLUSION: Rituximab could be directly and stably labeled with the matched pair (99m)Tc/(188)Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmunodiagnosis and therapy.

Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.

In-line radiolabeling: a novel continuous-flow system for commercial-scale protein labeling.

UNLABELLED: A key limitation in developing radiotherapeutic proteins is the expense of manufacturing the drug in small batches using traditional reaction vessels. Removing limitations on the quantity of protein labeled at any one time significantly decreases the cost of production, and nowhere is the need for cost-effective radiotherapeutics more acute than in the treatment of cancer. METHODS: We describe a novel method that can rapidly radiolabel, theoretically, unlimited amounts of protein, without causing significant damage to binding potency or structural integrity. Our process controls the reaction rate for the isotope and reactants as they simultaneously flow through a reaction tube. RESULTS: We have demonstrated proof of principle by labeling nearly a gram of antibody with 481 GBq (13 Ci) of (131)I during a single 30-min reaction run. CONCLUSION: Simple to construct, our system is already used to manufacture a radiolabeled antibody, both in the United States and in India, as part of clinical trials to treat glioblastoma multiforme. Modified, this system may be also applicable for nonradioactive labeling.

New derivatives of vitamin B12 show preferential targeting of tumors.

Rapidly growing cells show an increased demand for nutrients and vitamins. The objective of our work is to exploit the supply route of vitamin B12 to deliver new derivatives of this vital vitamin to hyperproliferative cells. To date, radiolabeled ((57)Co and (111)In) vitamin B12 derivatives showed labeling of tumor tissue but also undesired high accumulation of radioactivity in normal tissue. By abolishing the interaction of a tailored vitamin B12 derivative to its transport protein transcobalamin II and therefore interrupting transcobalamin II receptor and megalin mediated uptake in normal tissue, preferential accumulation of a radiolabeled vitamin in cancer tissue could be accomplished. We identified transcobalamin I on tumors as a possible new receptor for this preferential accumulation of vitamin-mediated targeting. The low systemic distribution of radioactivity and the high tumor to blood ratio opens the possibility of a more successful clinical application of vitamin B12 for imaging or therapy.

Immunoscintigraphy of patients with head and neck carcinomas, with an anti-angiogenetic antibody fragment.

OBJECTIVE: In a phase I/II clinical study, we investigated tumor targeting in patients with head and neck squamous cell carcinomas (SCC), using an antibody directed against the extra-domain-B of fibronectin (EDB), a marker of angiogenesis and tissue remodeling. STUDY DESIGN AND SETTING: Five patients with SCC were injected with the 123-iodine-radiolabeled L19(scFv)2 antibody and underwent scintigraphic detection with single photon emission tomography with computerized tomography (SPECT/CT). In addition, 18F-fluorodeoxyglucose (18FDG) positron emission tomography with computerized tomography (PET/CT) was performed. RESULTS: Successful targeting of the primary tumor could be achieved in 4 of 5 patients and was comparable to PET imaging. No side effects were observed. CONCLUSIONS: Tumor targeting with the L19(scFv)2 antibody is also feasible for head and neck SCC. SIGNIFICANCE: These results may serve as a base for future therapeutical applications in human beings, with modified versions of the L19(scFv)2 antibody, designed to selectively deliver bioactive molecules into malignant tumors.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting.

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

The roles of chronic pressure and volume overload states in induction of arrhythmias: an animal model of physiologic sequelae after repair of tetralogy of Fallot.

OBJECTIVE: Sudden death occurs in as many as 8% of patients after repair of tetralogy of Fallot and has been attributed to arrhythmias. The purpose of this study was to establish an animal model to evaluate the individual contribution of different physiologic sequelae after tetralogy of Fallot repair in the development of late-onset arrhythmias. METHODS: Forty-nine piglets were divided into 5 groups: (1) pulmonary artery band; (2) pulmonary valvotomy; (3) pulmonary artery band plus pulmonary valvotomy; (4) infundibular scar; and (5) age-matched control animals. Baseline and follow-up electrocardiograms were obtained and recorded, as well as changes in QRS duration. A total of 45 animals underwent hemodynamic evaluation and programmed electrical stimulation at 5.6 months postoperatively. RESULTS: Sustained ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation) were induced in 31.1%, and atrial arrhythmias were induced in 33.3%. The pulmonary valvotomy group was 30 times more likely to evidence arrhythmias than control animals for sustained ventricular tachycardia/ventricular fibrillation, as well as atrial arrhythmias (P = .01). The pulmonary artery band group was 15 times more likely to evidence atrial arrhythmias than control animals (P = .02). Prolonged QRS duration was predictive of inducibility of both atrial arrhythmias (P < .01) and sustained ventricular tachycardia/ventricular fibrillation (P = .01). Mean right atrial (P = .01) and capillary wedge (P = .01) pressures predicted atrial arrhythmia inducibility. Right ventricular end-diastolic pressure predicted atrial arrhythmia (P= .01) and sustained ventricular tachycardia/ventricular fibrillation inducibility (P = .05). Right ventricular systolic pressure did not predict inducibility of either atrial arrhythmias (P = .10) or sustained ventricular tachycardia/ventricular fibrillation (P = .94). CONCLUSIONS: Chronic right ventricular volume overload resulted in an increased incidence of inducible ventricular and atrial arrhythmias.

Chronic hypoxemia increases myocardial cytochrome oxidase.

OBJECTIVE: Cyanotic patients have potentially decreased tissue oxygen tension. Cytochrome oxidase catalyzes the reduction of oxygen and is integral to adenosine triphosphate production. Cytochrome oxidase subunit I, the active site, is encoded by mitochondrial DNA. Using a newborn swine model of chronic hypoxemia, we evaluated ventricular cytochrome oxidase subunit I mRNA and protein expression and assessed cytochrome oxidase activity. METHODS: Thirty-two newborn piglets underwent thoracotomy and placement of a pulmonary artery-to-left atrium shunt or sham operation. Two weeks later, partial pressure of arterial oxygen, hematocrit, and left ventricular shortening fraction values were compared with baseline values. Northern blot hybridization and protein immunoblotting for ventricular cytochrome oxidase subunit I were performed. Cytochrome oxidase kinetic activity was measured. Heme a,a3 content and turnover number were determined. Significance was assessed with a t test. RESULTS: Baseline partial pressure of arterial oxygen and hematocrit values were similar. Hypoxemic piglets had a lower partial pressure of arterial oxygen of 38 +/- 10 mm Hg (P < .001) and higher hematocrit value of 31.4% +/- 2.9% (P < .001) compared with a partial pressure of arterial oxygen of 140 +/- 47 mm Hg and hematocrit value of 24.6% +/- 3.9% after the sham operation. Baseline and postprocedure left ventricular shortening fraction were similar within and between groups. Chronic hypoxemia increased right ventricular and left ventricular cytochrome oxidase I mRNA and protein by more than 1.4-fold. Cytochrome oxidase activity increased significantly in hypoxemia by 2.5-fold compared with that seen after the sham operation. Heme a,a3 content and turnover number increased by 1.5-fold during hypoxemia. CONCLUSIONS: Chronic hypoxemia increases cytochrome oxidase I message, protein expression, and activity. The increase in kinetics was due to increased enzyme content and catalytic activity. This is a possible adaptive mechanism that might preserve organ function during chronic hypoxemia.

Picolylamine-methylphosphonic acid esters as tridentate ligands for the labeling of alcohols with the fac-[M(CO)3]+ core (M = 99mTc, Re): synthesis and biodistribution of model compounds and of a 99mTc-labeled cobinamide.

[(Methyl-pyridin-2-ylmethyl-amino)-methyl]-phosphonic acid is a new bifunctional chelator for the fac-[(99m)Tc(CO(3))](+) core which can be linked to biomolecules via formation of phosphonic acid esters. Its synthesis and the coupling to model alcohols and to a bioactive molecule (cobinamide) are described. The rhenium complexes [Re(CO)(3)L] of the esters have been prepared and characterized, one of them by X-ray crystallography. The model esters could be labeled with [(99m)Tc(OH(2))(3)(CO)(3)](+) under mild conditions and relatively low ligand concentration with >97% yield and only one isomer formed. The (99m)Tc-labeled cobinamide analog was a mixture of four isomers. It bound strongly to transcobalamin I (TC I, haptocorrin) but only slightly to transcobalamin II (TC II) and intrinsic factor (IF), reflecting the binding abilities of cobinamide. Biodistribution studies in mice with B(16) melanoma exhibited fast clearance with no specific tissue binding.

Regional low-flow perfusion improves neurologic outcome compared with deep hypothermic circulatory arrest in neonatal piglets.

BACKGROUND: Regional low-flow perfusion is an alternative to deep hypothermic circulatory arrest, but whether regional low-flow perfusion improves neurologic outcome after deep hypothermic circulatory arrest in neonates remains unknown. We tested neurologic recovery after regional low-flow perfusion compared with deep hypothermic circulatory arrest in a neonatal piglet model. METHODS: Sixteen neonatal piglets underwent cardiopulmonary bypass, were randomized to 90 minutes of deep hypothermic circulatory arrest or regional low-flow perfusion (10 mL.kg(-1).min(-1)) at 18 degrees C, and survived for 1 week. Standardized neurobehavioral scores were obtained on postoperative days 1, 3, and 7 (0 = no deficit to 90 = brain death). Histopathologic scores were determined on the basis of the percentage of injured and apoptotic neurons in the neocortex and hippocampus by hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (0 = no injury to 4 = diffuse injury). Differences between groups were tested by using the Wilcoxon rank sum test, and results are listed as medians within a range. RESULTS: There were no significant differences between groups during cardiopulmonary bypass. Postoperative neurobehavioral scores were abnormal in 25% (2/8) of the regional low-flow perfusion animals versus 88% (7/8) of controls. Regional low-flow perfusion animals had significantly less neurologic injury compared with controls on postoperative day 1 (0.00 [range, 0-5] vs 12.5 [range, 0-52]; P <.008). There was a trend for less severe injury in the regional low-flow perfusion group (2.0 [range, 1-4] vs 0.0 [range, 0-50]; P =.08) on hematoxylin and eosin. The degree of apoptosis was significantly less in the regional low-flow perfusion group (0.0 [range, 0-1] vs 2.5 [range, 0-4]; P =.03). CONCLUSIONS: Regional low-flow perfusion decreases neuronal injury and improves early postoperative neurologic function after deep hypothermic circulatory arrest in neonatal piglets.

Design of multivalent complexes using the barnase*barstar module.

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.

A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity.

PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) is a tumor-associated antigen overexpressed in many solid tumors but shows limited expression in normal epithelial tissues. To exploit this favorable expression pattern for targeted cancer therapy, an Ep-CAM-specific recombinant immunotoxin was developed and its antitumor activity investigated. EXPERIMENTAL DESIGN: The immunotoxin 4D5MOCB-ETA was developed by genetically fusing a truncated form of Pseudomonas aeruginosa exotoxin A (ETA) (ETA(252-608)KDEL) to the highly stable humanized single-chain antibody fragment (scFv) 4D5MOCB. Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro. Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts. RESULTS: Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen-binding affinity. In vitro, 4D5MOCB-ETA potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC(50)s ranging from 0.005 to 0.2 pM. Upon systemic administration in mice, 4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumor:blood ratio of 5.4. The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon, or squamous cell carcinomas. CONCLUSIONS: We describe for the first time the development of a fully recombinant Ep-CAM-specific immunotoxin and demonstrate its potent activity against solid tumors of various histological origins. 4D5MOCB-ETA is currently being evaluated in a Phase I study in patients with refractory squamous cell carcinoma of the head and neck.

Versatile synthetic approach to new bifunctional chelating agents tailor made for labeling with the fac-[M(CO)(3)](+) core (M = Tc, (99m)Tc, Re): synthesis, in vitro, and in vivo behavior of the model complex [M(APPA)(CO)(3)] (APPA = [(5-amino-pentyl)-pyridin-2-yl-methyl-amino]-acetic acid).

A general synthetic approach for potent tridentate, bifunctional chelating agent (BFCA) for the [M(CO)(3)](+) fragment (M = (99g)Tc, (99m)Tc, and Re) has been elaborated. The strategy allows the facile preparation of BFCA with a pendent amino or carboxylic acid functionality for coupling to peptides and proteins via formation of an amide bond. [(5-amino-pentyl)-pyridin-2-yl-methyl-amino]-acetic acid (APPA) and [pyridin-2-yl-methyl-amino]-diacetic acid (PADA) were synthesized according to this protocol. The BFCA were labeled with the [M(CO)(3)](+) fragment, which resulted in formation of uniform products with a ligand to metal ratio of 1:1. The complexes have been fully characterized by means of mass spectrometry, IR, and NMR ((1)H, (13)C, (99)Tc) spectroscopy. Coordination of the tricarbonyl core with APPA and PADA was exclusively tridentate (via the acid function, the ternary amine, and the pyridine nitrogen). On the n.c.a. level the complexes were almost quantitatively formed (yield >90%) at ligand concentrations of 10+/-2 microM (PADA) or 50+/-4 microM (APPA) after 30 min at 70 degrees C. Chromatographic behavior of the (99m)Tc complexes is similar to that of the corresponding (99)Tc/Re complexes suggesting the identical chemical structure. Pharmacokinetic experiments with the (99m)Tc-APPA complex were performed in BALB/c mice and compared with previously published results of the (99m)Tc-PADA complex. The (99m)Tc-APPA complex revealed good clearance from the blood pool (0.29 +/- 0.03% ID after 24h p.i.) and a low uptake in the liver (2.41 +/- 0.14% ID/g), in the kidneys (2.81 +/- 0.12% ID/g) and other tissue and organs.

High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate.

To develop new recombinant monoclonal antibody fragments for therapy and imaging, it is indispensable to have a simple and easy procedure to handle the eukaryotic expression system for production of proteins in high amounts. Gene amplification techniques such as the dehydrofolate reductase (DHFR) system in Chinese hamster ovary cells or the glutamine synthase system in myeloma cells have a couple of disadvantages. The selection procedure is complex, time-consuming, and not fruitful in all cases. The toxic drug methotrexate (for the DHFR system) can increase the production rate but decreases the specific growth rate of the cells. The production rate is not always stable over a long-term cultivation period. To overcome these problems, we are using stably transfected human embryonic kidney (HEK-293) cells in combination with an efficient screening method. Sodium butyrate can increase the expression of recombinant antibody fragments in the transfectomas up to 500 micrograms/4.2 x 10(7) cells/24 h corresponding to 175 micrograms/mL culture medium. This strategy allows a rapid development of new recombinant monoclonal antibody fragments and allows one to proceed rapidly to in vivo testing.