[en] The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr ?-particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti-tenascin constructs with optimized properties for use in tandem with short half life radionuclides such as 211At (as well as 1.8-hr 18F for PET imaging) is warranted. Our specific aims are: (1) to construct a bivalent, anti-tenascin molecule containing murine 81C6 variable regions and the human IgG2 hinge region. Both the CH2 domain deletion construct (?CH2) and F(ab?)2 will be investigated; (2) to construct a single-chain Fv dimer or multimer with adequate stability, affinity and immunoreactivity for use in tandem with 211At for therapy and 18F for imaging; (3) to generate higher affinity scFv constructs reactive with the alternatively spliced fibronectin type III repeats CD of the tenascin molecule via phage display technology and site-directed mutagenesis; (4) to label promising anti-tenascin constructs with radioiodine, 211At, and 18F and evaluate their potential as radiodiagnostic and radiotherapeutic agents. The proposed studies include: characterization of affinity and immunoreactivity after labeling; evaluation of tissue distribution and projected dosimetry in normal mice, and athymic rodents with subcutaneous, intracranial and neoplastic meningitis xenografts; investigation of the nature of low and high molecular weight labeled catabolites generated in mice; and assessment of cytotoxicity in vitro and in vivo models of human glioma, and possibly, other tenascin expressing tumors; and (5) to investigate strategies for labeling scFv monomers and dimers which will minimize retention of the radiohalogen in the kidneys through the use of negatively charged templates

[en] Targeted radionuclide therapy is emerging as a viable approach for cancer treatment because of its potential for delivering curative doses of radiation to malignant cell populations while sparing normal tissues. Alpha particles such as those emitted by 211At are particularly attractive for this purpose because of their short path length in tissue and high energy, making them highly effective in killing cancer cells. The current impact of targeted radiotherapy in the clinical domain remains limited despite the fact that in many cases, potentially useful molecular targets and labeled compounds have already been identified. Unfortunately, putting these concepts into practice has been impeded by limitations in radiochemistry methodologies. A critical problem is that the synthesis of therapeutic radiopharmaceuticals provides additional challenges in comparison to diagnostic reagents because of the need to perform radio-synthesis at high levels of radioactivity. This is particularly important for α-particle emitters such as 211At because they deposit large amounts of energy in a highly focal manner. The overall objective of this project is to develop convenient and reproducible radiochemical methodologies for the radiohalogenation of molecules with the α-particle emitter 211At at the radioactivity levels needed for clinical studies. Our goal is to address two problems in astatine radiochemistry: First, a well known characteristic of 211At chemistry is that yields for electrophilic astatination reactions decline as the time interval after radionuclide isolation from the cyclotron target increases. This is a critical problem that must be addressed if cyclotrons are to be able to efficiently supply 211At to remote users. And second, when the preparation of high levels of 211At-labeled compounds is attempted, the radiochemical yields can be considerably lower than those encountered at tracer dose. For these reasons, clinical evaluation of promising 211At-labeled targeted radiotherapeutics currently is a daunting task. Our central hypothesis is that improvements in 211At radiochemistry are critically dependent on gaining an understanding of and compensating for the effects of radiolysis induced by 211At α-particles. Because of the widespread interest in labeling antibodies, antibody fragments and peptides with 211At, our proposed work plan will initially focus on reagents that we have developed for this purpose. Part of our strategy is the use of synthetic precursors immobilized on polymeric resins or perfluorous and triarylphosphonium supports. Their use could eliminate the need for a purification step to separate unreacted tin precursor from labeled product and hopefully provide a simple kit technology that could be utilized at other institutions. The specific aims of this project are: (1) To optimze methods for 211At production and isolation of 211At from cyclotron targets; (2) To develop convenient and reproducible methodologies for high activity level and high specific activity radiohalogenation of biomolecules with 211At; (3) to develop a procedure for extending the shelf-life of 211At beyond a few hours so that this radionuclide can be utilized at centers remote from its site of production; and (4) to work out high activity level synthesis methods for utilizing support immobilized tin precursors for 211At labeling. If we are successful in achieving our goals, the radiochemical methodologies that are developed could greatly facilitate the use of 211At-labeled targeted cancer therapeutics in patients, even at institutions that are distant from the few sites currently available for 211At production.

[en] To circumvent the in vivo instability of 5-iodo-2'-deoxyuridine (IUdR), a 2'-fluorine-substituted analogue, 5-iodo-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (FIAU) recently has been introduced. To facilitate the preparation of radioiodinated FIAU as well as its astatinated analogue, a tin precursor, 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (FTAU) was synthesized. Both [^125/131I]FIAU and 5-[²¹¹At]astato-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (FAAU) were prepared from FTAU in more than 85% radiochemical yield under mild conditions. The in vitro serum stability of both fluorine-substituted derivatives was higher than that of the corresponding unsubstituted parents. The enhanced stability of fluorinated derivatives was even more apparent in whole blood. The uptake of [¹²⁵I]FIAU in D-247 MG human glioma cells in vitro was 20-fold higher than that of [¹²⁵I]IUdR over an activity concentration range of 5-100 kBq/mL; the uptake of FAAU was not significantly different from that of 5-[²¹¹At]astato-2'-deoxyuridine (AUdR). Accumulation of radioiodine in mouse thyroid in vivo with [¹³¹I]FIAU was fivefold lower than [¹²⁵I]IUdR, indicating that the former was less susceptible to deiodination. The tissue uptake of FAAU was similar to that reported for AUdR

[en] A potent chemotactic peptide, formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine was derivatized by reaction with N-succinimidyl 4-fluorobenzoate. This derivatized peptide bound to human polymorphonuclear leukocytes in vitro and exhibited biological activity in a superoxide production assay. Peptide labeling using N-succinimidyl 4-[¹⁸F]fluorobenzoate was accomplished in reasonable yields with 10-15 mCi of labeled peptide available per 100 Ci of [¹⁸F]fluoride. With the exception of the gastrointestinal tract, clearance of activity from tissues following injection of this peptide in normal mice was rapid. Although preliminary in nature, these results suggest that ¹⁸F-labeled chemotactic peptides should be investigated as potential agents for positron emission tomographic imaging of bacterial infections

[en] No-carrier-added [¹²³I]MIBG was prepared from 3-(trimethylsilyl)benzylguanidine in 80-90% yield. Binding of this tracer to SK-N-SH human neuroblastoma cells maintained a constant level of > 50% over 2-3 log activity range. In comparison, the binding of [¹²³I]MIBG prepared by isotopic exchange steadily decreased with dose. Biodistribution studies in normal mice demonstrated maximal concentrations in heart and adrenals for both preparations. In heart, significant 1.5-3.0 times higher levels (P < 0.05) were seen for the no-carrier-added preparation. Radiation dosimetry calculations suggest that the no-carrier-added preparation would increase the dose received by several tissues, most notably the heart where a 91% increase in dose is predicted

[en] The α-melanocyte stimulating hormone (α-MSH) analogue [N1e⁴,D-Phe⁷]-α-MSH was labeled with ¹⁸F using N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) in >80% radiochemical yield. The IC₅₀ values of [N1e⁴,D-Phe⁷]-α-MSH and para-fluorobenzoyl-[N1e⁴,D-Phe⁷]-α-MSH ([N1e⁴,D-Phe⁷,Lys¹¹-(¹⁸F)PFB]-α-MSH) for inhibiting the binding of meta-[¹³¹I]iodobenzoyl-[N1e⁴,D-Phe⁷]-α-MSH ([N1e⁴,D-Phe⁷,Lys¹¹-(¹³¹I)MIB]-α-MSH) to B16-F1 murine melanoma cells were 89 ± 9 pM and 112 ± 22 pM, respectively, suggesting that addition of 4-fluorobenzoate did not compromise α-MSH receptor binding affinity. Binding of [N1e⁴,D-Phe⁷,Lys¹¹-(¹⁸F)PFB]-α-MSH was influenced by the specific activity of the preparation (400-1000 Ci/mmol). The normal tissue clearance of [N1e⁴,D-Phe⁷,Lys¹¹-(¹⁸F)PFB]-α-MSH in mice was quite rapid, with little evidence for defluorination

[en] Hyperthermia is a therapeutic modality under investigation for its ability to increase absolute levels of tumor uptake of radiolabeled monoclonal antibodies (MAbs). We have investigated whether hyperthermia may affect the binding parameters of MAbs. The effects of clinically relevant levels of hyperthermia on the kinetic binding parameters were investigated for 81C6, an antibody undergoing Phase I/II clinical trials for the treatment of brain tumors and neoplastic meningitis. No obvious effects of temperature in either the association or dissociation rate constants, nor in the equilibrium constants, were apparent between 37 deg. and 45 deg. C. The improved binding stability of the bivalent form of the MAb was apparent when compared with its monovalent Fab fragment

[en] Biotinyl-3-[²¹¹At]astatoanilide ([²¹¹At]AtBA) was prepared in more than 80% yield by destannylation. In vitro, [²¹¹At]AtBA exhibited a high affinity for streptavidin, and was stable after incubation in human serum, cerebrospinal fluid and distilled water, whereas it was rapidly degraded in mouse serum. HPLC analysis showed that the main degradation pathway in mouse serum was the cleavage of [²¹¹At]astatoaniline. In mice, [²¹¹At]AtBA and its ¹²⁵I-labeled analogue cleared rapidly from most tissues; however, there was some evidence for dehalogenation of both tracers

[en] In order to clarify the uptake and retention mechanisms of radioiodinated meta-iodobenzylguanidine (MIBG) in heart, the kinetics of no-carrier-added [¹²³I]MIBG were studied in the isolated working rat heart in interaction with pharmacologic agents. The tracer was administered in the perfusate as a 10-min pulse, followed by a 90-min washout period. Kinetic analysis of the externally monitored time-activity curves of control hearts showed avid uptake (K_i = 4.4 ± 0.7 mL/min/g), and monoexponential clearance (k_o 0.0056 ± 0.0017 1/min), indicating a distribution volume (V_d =K_i /k_o) of 834 ± 214 mL/g. Blocking experiments (n = 41) were performed with neuronal uptake (uptake-1) inhibitor desipramine (DMI; 50-100 nM) and the extraneuronal uptake (uptake-2) inhibitor N-(9-fluorenyl)-N-methyl-β-chloroethylamine (SKF550; 0.4-0.8 μM). Uptake rate was 27% reduced (P < 0.05) by 50 nM DMI but not significantly affected by 0.4 μM SKF550. Distribution volume was 88% reduced (P < 0.0005) by 50 nM DMI and 28% reduced (P < 0.05) by 0.4 μM SKF550. In DMI-blocked hearts, uptake rate was dramatically decreased (-80%, P < 0.0005) by SKF550 (0.4 μM), indicating uptake-2 transport contributed predominantly to the extraneuronal uptake of the tracer. The slow uptake rate seen with concomitant inhibition of uptake-1 and uptake-2 was further decreased by addition of unlabeled MIBG (1-10 μM) in a concentration-dependent manner, yet unaffected by addition of the vesicular uptake inhibitor Ro 4-1284 (1 μM). Thus, the uptake rate of [¹²³I]MIBG is primarily dependent on uptake-1 and uptake-2 activity. Other possible mechanisms of uptake such as passive diffusion in association with intracellular binding are significant only in conditions where uptake-1 and uptake-2 mechanisms are largely inhibited

[en] Normal tissue accumulation of ²¹¹At must be minimized during targeted radiotherapy with ²¹¹At-labeled compounds. Therefore, we investigated the ability of seven compounds to block normal organ uptake of [²¹¹At]astatide in mice: potassium iodide, sodium thiocyanate, sodium perchlorate, sodium periodate, cysteine, 2,3-dimercapto-1-propanesulfonic acid, and meso-2,3-dimercaptosuccinic acid. The monovalent anions I^-, SCN^-, and ClO₄^- reduced ²¹¹At uptake in stomach and thyroid, while thiocyanate and cysteine were the only compounds to significantly reduce activity levels in lungs and spleen. This study suggests that blocking agents may help reduce normal organ radiation doses in endoradiotherapeutic procedures with ²¹¹At-labeled radiopharmaceuticals