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TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-alpha and a genetically engineered form of the Pseudomonas exotoxin, PE-38. After in vitro and in vivo animal studies that showed specific activity and defined the maximum tolerated dose (MTD), we investigated this agent in a Phase I trial. The primary objective of this study was to define the MTD and dose limiting toxicity of TP-38 delivered by convection-enhanced delivery in patients with recurrent malignant brain tumors. Twenty patients were enrolled in the study and doses were escalated from 25 ng/mL to 100 with a 40 mL infusion volume delivered by two catheters. One patient developed Grade IV fatigue at the 100 ng/mL dose, but the MTD has not been established. The overall median survival after TP-38 for all patients was 23 weeks whereas for those without radiographic evidence of residual disease at the time of therapy, the median survival was 31.9 weeks. Overall, 3 of 15 patients, with residual disease at the time of therapy, have demonstrated radiographic responses and one patient with a complete response and has survived greater than 83 weeks.

Purpose: The objective of this study was to determine the feasibility of exploiting the overexpression of somatostatin subtype-2 receptors (sstr(2)) on human medulloblastoma cells to develop targeted radiodiagnostics and radiotherapeutics for this disease., Experimental Design: The following radioiodinated peptides were prepared using chloramine-T and evaluated: [(131)I-Tyr(3)]octreotide ([(131)I]TOC), [(131)I-Tyr(3)]octreotate ([(131)I]TOCA), involving substitution of Thr(ol)(8) in TOC with Thr(8), and glucose-[(131)I-Tyr(3)]octreotide ([(131)I]Gluc-TOC) and glucose-[(131)I-Tyr(3)]octreotate ([(131)I]Gluc-TOCA), prepared by conjugation of glucose to the peptide NH(2) terminus. Specific internalization of the peptides by sstr(2)-expressing AR42J rat pancreatic carcinoma cells in vitro was evaluated in paired-label assays. The tissue distribution of i.v. administered [(131)I]TOC, [(131)I]TOCA, [(131)I]Gluc-TOC, and [(131)I]Gluc-TOCA was evaluated in athymic mice bearing s.c. D341 Med human medulloblastoma xenografts., Results: Compared with [(125)I]TOC, internalized radioiodine levels were higher for the other three peptides. For example, internalized counts were 1.9 +/- 0.2, 2.0 +/- 0.3, and 5.7 +/- 1.9 times higher for [(131)I]Gluc-TOC, [(131)I]TOCA, and [(131)I]Gluc-TOCA after a 3-h incubation, respectively, demonstrating that carbohydration and COOH-terminus modification significantly improved the retention of radioiodine activity in sstr(2)-expressing tumor cells. COOH-terminus modification significantly increased (131)I localization in D341 Med medulloblastoma xenografts [[(131)I]TOCA, 8.1 +/- 2.2% of injected dose/g (% ID/g); [(131)I]TOC, 3.9 +/- 0.5% ID/g at 1 h], whereas carbohydration of the NH(2) terminus resulted in even higher gains in tumor accumulation ([(131)I]Gluc-TOC, 11.1 +/- 1.8% ID/g; [(131)I]Gluc-TOCA, 21.4 +/- 7.3% ID/g). In addition, the three modified peptides exhibited liver activity levels that were less than half those of [(131)I]TOC. Uptake of the two glucose-peptide conjugates in this human medulloblastoma xenograft was blocked by coinjection of 100 micro g of octreotide, demonstrating that it was receptor-specific. Tumor:normal tissue uptake ratios for [(131)I]Gluc-TOCA generally were higher that those for [(131)I]Gluc-TOC. At 1 h, tumor:normal tissue ratios for [(131)I]Gluc-TOCA were 29:1, 15:1, 8:1, 8:1, 240:1, and 82:1 for blood, liver, kidney, spleen, brain, and muscle, respectively., Conclusions: Our findings suggest that additional investigation of radiolabeled Gluc-TOCA analogues for the imaging and targeted radiotherapy of medulloblastoma is warranted.

The objective of this study was to develop a method for labeling internalizing monoclonal antibodies (mAbs) such as those reactive to the anti-epidermal growth factor receptor variant III (EGFRvIII) with the alpha-particle emitting radionuclide (211)At. Based on previous work utilizing the guanidine-containing acylation agent, N-succinimidyl 4-guanidinomethyl-3-[(131)I]iodobenzoate ([(131)I]SGMIB), we have now investigated the potential utility of its astato analogue for labeling the anti-EGFRvIII mAb L8A4. N-succinimidyl 3-[(211)At]astato-4-guanidinomethylbenzoate ([(211)At]SAGMB) in its Boc-protected form was prepared from a tin precursor in 61.7 +/- 13.1% radiochemical yield, in situ deprotected to [(211)At]SAGMB, which was coupled to L8A4 in 36.1 +/- 1.9% yield. Paired-label internalization assays demonstrated that tumor cell retention of radioactivity for L8A4 labeled using [(211)At]SAGMB was almost identical to L8A4 labeled using [(131)I]SGMIB, and 3-4-fold higher than for mAb radioiodinated using Iodogen. Paired-label biodistribution of L8A4 labeled using [(211)At]SAGMB and [(131)I]SGMIB in athymic mice hosting U87MGdeltaEGFR xenografts resulted in identical uptake of both (211)At and (131)I in tumor tissues over 24 h. Although higher levels of (211)At compared with (131)I were sometimes seen in tissues known to sequester free astatide, these (211)At/(131)I uptake ratios were considerably lower than those seen with other labeling methods. These results suggest that [(211)At]SAGMB may be a useful acylation agent for labeling internalizing mAbs with (211)At.

Unlabelled: The purpose of this study was to evaluate the therapeutic efficacy and limitations of alpha-particle-emitting radiolabeled compounds by means of 2-dimensional histological images and distribution of activity on a microscopic level., Methods: A microdosimetric approach based on histological images is used to analyze the therapeutic effectiveness of alpha-particle-emitting (211)At and (213)Bi conjugated to 201B monoclonal antibody (mAb), which is reactive with murine lung blood vessels for the treatment of EMT-6 lung tumor colonies in nude mice. Autoradiography images were used to define the tissue morphology and activity distribution within lung tissues. Two animal groups were studied: Group A consisted of animals bearing small tumors (<130 micro m) and group B consisted of larger tumors (<600 micro m). Probability density functions (pdf) described the variability in average absorbed dose and survival probability among normal and tumor target cells and, in turn, were used to assess the survival fraction of tumor and normal tissue., Results: The average absorbed dose to tumor cells per unit cumulated activity concentration for animals in group A was 1.10 x 10(-3) and 1.37 x 10(-3) Gy g MBq(-1) s(-1) for (211)At and (213)Bi, respectively, and for animals in group B was 3.8 x 10(-4) and 5.6 x 10(-4) Gy g MBq(-1) s(-1) for (211)At and (213)Bi, respectively. The fraction of tumor cells that received a zero absorbed dose for animals in group A was 0.04% for (213)Bi and 0.2% for (211)At and for animals in group B was 25% for (213)Bi and 31% for (211)At. Both (213)Bi- and (211)At-labeled 201B mAb were effective therapies for animals with small tumors, where predicted therapeutic effectiveness was consistent with experimental findings; however, they were ineffective for animals with larger tumors., Conclusion: Microdosimetric methods based on knowledge of tissue morphology and activity distribution on a small-scale level can be a useful tool for evaluating a priori the therapeutic efficacy and limitations of targeted alpha-particle endoradiotherapeutic strategies.

An important criterion in design of acylation agents for the radioiodination of internalizing monoclonal antibodies (mAbs) is to maximize the retention of radioiodine in the tumor following mAb intracellular processing. We have previously shown that labeling methods that generate positively charged catabolites have enhanced tumor retention. Herein we have extended this strategy to investigate the potential utility of labeling internalizing mAbs with an acylation agent that yielded labeled catabolites that would be negatively charged at lysosomal pH. The negatively charged acylation agent, N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), was prepared from its tin precursor, N-succinimidyl 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoate (tBu-SPMTB), in 40% radiochemical yield. The free acid, 3-[(131)I]iodo-4-phosphonomethylbenzoic acid ([(131)I]IPMBA), was also prepared from the corresponding precursor, 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoic acid (tBu-PMTBA), in 80% radiochemical yield. The rapidly internalizing mAb L8A4 was conjugated to [(131)I]SIPMB in 25-40% yield with preservation of its immunoreactivity. Internalization and processing in the U87DeltaEGFR glioma cell line was studied in a paired label format with L8A4 labeled with (125)I using the Iodogen method. Retention of initially bound radioactivity in these cells at 24 h from [(131)I]SIPMB-labeled mAb was approximately 6-fold higher than that for directly labeled mAb. Catabolite analysis demonstrated that this difference reflected an order of magnitude higher retention of low molecular weight species in these cells. The [(131)I]SIPMB-L8A4 conjugate was intact over the first 2 h; thereafter, lysine-[(131)I]SIPMB was the predominant catabolite. In contrast, L8A4 labeled using Iodogen rapidly gave rise to mono-[(125)I]iodotyrosine within 2 h, which then cleared rapidly from the cells. These results suggest that SIPMB could be a potent candidate for labeling internalizing mAbs and warrant further study.

Purpose: To establish the theoretical framework and study the feasibility of (211)At-labeled anti-tenascin chimeric 81C6 monoclonal antibody (mAb) as anti-vascular endoradiotherapy for the treatment of glioblastoma multiforme (GBM) tumors., Methods and Materials: The morphology of blood vessels from histologic images was analyzed and used along with reaction-diffusion equations to assess the activity concentration of (211)At-labeled chimeric 81C6 mAb in GBM tumor and normal-brain tissue. Alpha particle microdosimetry was then used to assess the survival probability and average absorbed dose for tumor and normal tissue endothelial cells (ECs) per unit vascular cumulated activity concentration q(source) (MBq-s g(-1)). In turn, these survival probabilities were used to assess the probability of failure Phi for a single vessel. Furthermore, using the vessel density, the specific tumor control probability per unit mass of tumor tissue (tcp) and the specific normal-tissue complication probability per unit mass of normal-brain tissue (ntcp) were estimated. The specific tumor control probability, tcp, was used to assess the overall tumor control probability (TCP) as a function of tumor mass., Results: The levels of (211)At-labeled ch81C6 mAb cumulated activity concentration in GBM tumor tissue were approximately five times higher than that in normal-brain tissue. Thus, the average absorbed dose to tumor ECs was higher than that of normal tissue ECs, and the survival probability for GBM ECs was lower than for normal-brain tissue ECs. Consequently, the resulting vessel-failure probability, Phi, for GBM tumor and for normal-brain tissue differ considerably, yielding a q(source) range between 10(3) and 10(4) MBq-s g(-1)., Conclusions: This theoretical analysis demonstrated that (211)At-labeled chimeric 81C6 is an effective anti-vascular therapy for the treatment of GBM tumors, yielding a tcp higher than 0.999 for vascular cumulated activity concentrations q(source) higher than 1 x 10(4) MBq-s g(-1), while yielding a low probability for normal-brain tissue damage.

The cellular expression of the sodium iodide symporter (NIS) has been shown to confer iodide-concentrating capacity in non-thyroid cell types. We examined the role of NIS in the uptake of the alpha-particle emitting radiohalide [(211)At]astatide in the UVW human glioma cell line transfected to express NIS. [(211)At]Astatide uptake is shown to be NIS-dependent, with characteristics similar to [(131)I]iodide uptake. These studies suggest [(211)At]astatide as a possible alternative radionuclide to [(131)I]iodide for NIS-based endoradiotherapy, and provide a model for the study of [(211)At]astatide behavior at a cellular level.

Purpose: To assess the efficacy and toxicity of intraresection cavity (131)I-labeled murine antitenascin monoclonal antibody 81C6 and determine its true response rate among patients with newly diagnosed malignant glioma., Patients and Methods: In this phase II trial, 120 mCi of (131)I-labeled murine 81C6 was injected directly into the surgically created resection cavity of 33 patients with previously untreated malignant glioma (glioblastoma multiforme [GBM], n = 27; anaplastic astrocytoma, n = 4; anaplastic oligodendroglioma, n = 2). Patients then received conventional external-beam radiotherapy followed by a year of alkylator-based chemotherapy., Results: Median survival for all patients and those with GBM was 86.7 and 79.4 weeks, respectively. Eleven patients remain alive at a median follow-up of 93 weeks (range, 49 to 220 weeks). Nine patients (27%) developed reversible hematologic toxicity, and histologically confirmed, treatment-related neurologic toxicity occurred in five patients (15%). One patient (3%) required reoperation for radionecrosis., Conclusion: Median survival achieved with (131)I-labeled 81C6 exceeds that of historical controls treated with conventional radiotherapy and chemotherapy, even after accounting for established prognostic factors including age and Karnofsky performance status. The median survival achieved with (131)I-labeled 81C6 compares favorably with either (125)I interstitial brachy-therapy or stereotactic radiosurgery and is associated with a significantly lower rate of reoperation for radionecrosis. Our results confirm the efficacy of (131)I-labeled 81C6 for patients with newly diagnosed malignant glioma and suggest that a randomized phase III study is indicated.

p-[18F]Fluorobenzylguanidine ([18F]PFBG) is a norepinephrine analog that has been developed as a positron emission tomography (PET) imaging radiopharmaceutical. Myocardial sympathetic innervation, neuroendocrine structures, and tumors can be noninvasively imaged with [18F]PFBG. In this study, the uptake characteristics of [18F]PFBG were investigated in 2 dogs with a spontaneous pheochromocytoma. The extent of the pheochromocytoma was well documented in both dogs on the PET study. The standardized uptake values within the pheochromocytomas were greater than 25 by 10 min, and were 37 and 50 by 45 min in each dog. A third dog that was suspected to have an adrenal mass was also studied. In this dog, the [18F]PFBG study was normal. Surgical exploration and adrenal biopsy confirmed the [15F]PFBG imaging findings in both dogs. In each dog, there was rapid blood-pool clearance (within 10 min after intravenous administration of the [18F]PFBG), with high uptake specific within the myocardium and adrenal medulla. The results indicate that [18F]PFBG may be useful for imaging canine pheochromocytomas and aid in differentiating adrenal masses.

Monoclonal antibodies (mAbs) such as the tumor-specific anti-epidermal growth factor receptor variant III (EGFRvIII) that are internalized and degraded after cell binding necessitate the use of radioiodination methods that minimize the loss of radioactivity from the tumor cell after intracellular processing. The purpose of the current study was to determine the suitability of N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) for labeling this internalizing mAb. A series of paired-label biodistribution experiments were performed in athymic mice bearing subcutaneous, EGFRvIII-expressing, D-256 human glioma and U87 Delta EGFR xenografts. The tissue distribution of radioiodine activity following injection of anti-EGFRvIII mAb L8A4 labeled using N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) were compared to those for mAb labeled using Iodogen, N-succinimidyl 3-iodo-5-pyridinecarboxylate (SIPC) as well as the Boc-protected precursor of SGMIB. Tumor uptake of radioiodine activity for mAb labeled via SGMIB was significantly higher than co-administered L8A4 radioiodinated by other methods. For example, 3 days after injection, D-256 tumor uptake of L8A4 labeled via SGMIB was 20.4 +/- 4.6% ID/g compared with 11.7 +/- 5.5% ID/g when the SIPC method was used. Thyroid uptake for L8A4 (SGMIB) was up to 36 times lower than L8A4 (Iodogen) and less than 0.35% in all experiments, indicating a low degree of deiodination in vivo. These results suggest that SGMIB may be a useful reagent for the radioiodination of this internalizing anti-EGFRvIII mAb.

Receptor-mediated internalization of monoclonal antibodies (mAbs), such as those specific for the epidermal growth factor receptor variant III (EGFRvIII), can lead to rapid loss of radioactivity from the target cell. In the current study, the anti-EGFRvIII mAb L8A4 was radioiodinated using two methods -N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) and via a D-amino acid peptide LysArgTyrArgArg (D-KRYRR). Paired-label internalization assays performed on EGFRvIII-expressing U87DeltaEGFR cells in vitro demonstrated that labeling L8A4 using D-KRYRR resulted in significantly higher retention of radioiodine in the intracellular compartment. In athymic mice with D256 human glioma xenografts, tumor uptake was similar for both labeling methods through 24 hr. However, an up to fourfold higher tumor retention was observed for mAb labeled with the D-amino acid peptide at later time points. Radiation absorbed dose calculations based on these biodistribution data indicated that L8A4 labeled using D-KRYRR exhibited better tumor-to-normal-organ radiation dose ratios, suggesting that this labeling method may be of particular value for labeling internalizing mAbs.

Unlabelled: In vitro and in vivo studies in human glioma models suggest that the antitenascin monoclonal antibody 81C6 labeled with the 7.2-h-half-life alpha-particle emitter (211)At might be a valuable endoradiotherapeutic agent for the treatment of brain tumors. The purpose of this study was to develop methods for the production of high levels of (211)At and the radiosynthesis of clinically useful amounts of (211)At-labeled human/mouse chimeric 81C6 antibody., Methods: (211)At was produced through the (209)Bi(alpha, 2n)(211)At reaction using an internal target system and purified by a dry distillation process. Antibody labeling was accomplished by first synthesizing N-succinimidyl 3-[(211)At]astatobenzoate from the corresponding tri-n-butyl tin precursor and reacting it with the antibody in pH 8.5 borate buffer. Quality control procedures consisted of methanol precipitation, size-exclusion high-performance liquid chromatography (HPLC), and pyrogen and sterility assays, as well as determination of the immunoreactive fraction by a rapid procedure using a recombinant tenascin fragment coupled to magnetic beads., Results: A total of 16 antibody labeling runs were performed. Using beam currents of 50-60 microA alpha-particles and irradiation times of 1.5-4.5 h, the mean (211)At production yield was 27.75 +/- 2.59 MBq/microA.h, and the maximum level of (211)At produced was 6.59 GBq after a 4-h irradiation at 55 microA. The decay-corrected distillation yield was 67% +/- 16%. The yield for the coupling of the (211)At-labeled active ester to the antibody was 76% +/- 8%. The fraction of (211)At activity that eluted with a retention time corresponding to intact IgG on HPLC was 96.0% +/- 2.5%. All preparations had a pyrogen level of <0.125 EU/mL and were determined to be sterile. The mean immunoreactive fraction for these 16 preparations was 83.3% +/- 5.3%. Radiolysis did not interfere with labeling chemistry or the quality of the labeled antibody product., Conclusion: These results show that it is feasible to produce clinically relevant activities of (211)At-labeled antibodies and have permitted the initiation of a phase I trial of (211)At-labeled chimeric 81C6 administered directly into the tumor resection cavities of brain tumor patients.

A number of ring- and side-chain-substituted m-iodobenzylguanidine analogues were evaluated for their lipophilicity, in vitro stability, uptake by SK-N-SH human neuroblastoma cells in vitro, and biodistribution in normal mice. As expected, the lipophilicity of m-iodobenzylguanidine increased when a halogen was introduced onto the ring and decreased with the addition of polar hydroxyl, amino, and nitro substitutents. Most of the derivatives showed reasonable stability up to 24 h in PBS at 37 degrees C. While N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine and 3,4-dihydroxy-5-[(131)I]iodobenzylguanidine generated a more nonpolar product in addition to the free iodide, 3-[(131)I]iodo-4-nitrobenzylguanidine decomposed to a product more polar than the parent compound. The specific uptake of 4-chloro-3-[(131)I]iodobenzylguanidine, 3-[(131)I]iodo-4-nitrobenzylguanidine, and N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine by SK-N-SH human neuroblastoma cells in vitro, relative to that of m-[(125)I]iodobenzylguanidine, was 117 +/- 10%, 50 +/- 4%, and 12 +/- 2%, respectively. The specific uptake of the known m-iodobenzylguanidine analogues 4-hydroxy-3-[(131)I]iodobenzylguanidine and 4-amino-3-[(131)I]iodobenzylguanidine was 80 +/- 4% and 66 +/- 4%, respectively. None of the other m-iodobenzylguanidine derivatives showed any significant specific uptake by SK-N-SH cells. Heart uptake of 4-chloro-3-[(131)I]iodobenzylguanidine in normal mice was higher than that of m-[(125)I]iodobenzylguanidine at later time points (11 +/- 1% ID/g versus 3 +/- 1% ID/g at 24 h; p < 0.05) while uptake of 3-[(131)I]iodo-4-nitrobenzylguanidine and of N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine in the heart was lower than that of m-iodobenzylguanidine at all time points. In accordance with the in vitro results, none of the other novel m-iodobenzylguanidine derivatives showed any significant myocardial or adrenal uptake in vivo.

With the goal of developing MIBG analogues with improved targeting properties especially for oncologic applications, several radioiodinated ring- and side-chain-substituted MIBG analogues were synthesized. Except for 3-[(131)I]iodo-4-nitrobenzylguanidine and N-hydroxy-3-[(131)I]iodobenzylguanidine, the radioiodinated analogues were prepared at no-carrier-added levels from their respective tin precursors. The radiochemical yields generally were in the range of 70-90% except for 3-amino-5-[(131)I]iodobenzylguanidine for which a radiochemical yield of about 40% was obtained. While the silicon precursor N(1),N(2)-bis(tert-butyloxycarbonyl)-N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine did not yield 3-[(131)I]iodo-4-nitrobenzylguanidine, its deprotected derivative, N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine was radioiodinated in a modest yield of 20% providing 3-[(131)I]iodo-4-nitrobenzylguanidine. Exchange radioiodination of 3-iodo-4-nitrobenzylguanidine gave 3-[(131)I]iodo-4-nitrobenzylguanidine in 80% radiochemical yield. No-carrier-added [(131)I]NHIBG was prepared from its silicon precursor N(1)-hydroxy-N(3)-(3-trimethylsilylbenzyl)guanidine in 85% radiochemical yield.

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.

Even with novel chemotherapeutic agents and external beam radiation therapy, the prognosis of neoplastic meningitis secondary to malignant melanoma is still dismal. The authors report a case study of a 46-year-old white female who presented with progressive hearing loss, severe headaches, nausea, vomiting, and a rapid decline in neurologic status. She was referred to Duke University Medical Center after conventional chemotherapy for malignant melanoma failed. She was enrolled in a Phase I trial of (131)I-labeled monoclonal antibody Mel-14 F(ab')(2) fragment administered intrathecally. Within a year after her treatment, she recovered, having a normal neurologic exam except for residual bilateral hearing loss. The authors discuss dosimetry, preclinical, and clinical studies conducted with Mel-14 F(ab')(2) and introduce a potentially promising therapy option in the treatment of neoplastic meningitis in patients with malignant melanoma. Currently, the patient remains neurologically normal except for a mild bilateral hearing loss more than 4 years after treatment and has no radiographic evidence of neoplastic meningitis., (Copyright 2001 American Cancer Society.)

Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1. We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region. To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques. In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia. We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature. By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors.

Combinatorial variation of CDR3 of V(H) and V(L), followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma-bearing athymic mice. MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K(d) = 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impact in vivo glioma-specific targeting and immunotherapy., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment., Patients and Methods: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment., Results: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively., Conclusion: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).

Targeted radiotherapy or endoradiotherapy is an appealing approach to cancer treatment because of the potential for delivering curative doses of radiation to tumor while sparing normal tissues. Radionuclides that decay by the emission of alpha-particles such as the heavy halogen astatine-211 (211At) offer the exciting prospect of combining cell-specific molecular targets with radiation having a range in tissue of only a few cell diameters. Herein, the radiobiological advantages of alpha-particle targeted radiotherapy will be reviewed, and the rationale for using 211At for this purpose will be described. The chemistry of astatine is similar to that of iodine; however, there are important differences which make the synthesis and evaluation of 211At-labeled compounds more challenging. Perhaps the most successful approach that has been developed involves the astatodemetallation of tin, silicon or mercury precursors. Astatine-211 labeled agents that have been investigated for targeted radiotherapy include [211At]astatide, 211At- labeled particulates, 211At-labeled naphthoquinone derivatives, 211At-labeled methylene blue, 211At-labeled DNA precursors, meta-[211At]astatobenzylguanidine, 211At-labeled biotin conjugates, 211At-labeled bisphosphonates, and 211At-labeled antibodies and antibody fragments. The status of these 211At-labeled compounds will be discussed in terms of their labeling chemistry, cytotoxicity in cell culture, as well as their tissue distribution and therapeutic efficacy in animal models of human cancers. Finally, an update on the status of the first clinical trial with an 211At-labeled targeted therapeutic, 211At-labeled chimeric anti-tenascin antibody 81C6, will be provided.

The mutant epidermal growth factor receptor variant III (EGFRvIII) has been found on gliomas and other tumors but not on normal tissues, including those that express the wild-type receptor. Monoclonal antibodies (mAbs) specific for EGFRvIII are rapidly internalized and degraded after binding to EGFRvIII-expressing cells. If anti-EGFRvIII mAbs are to be useful for radioimmunotherapy, then methods for trapping radionuclides in target cells after mAb processing are required. Because lysosomes are known to retain positively charged molecules, we have evaluated a new reagent for this purpose that uses a polycationinc peptide composed of D-amino acids (D-Lys-D-Arg-D-Tyr-D-Arg-D-Arg; D-KRYRR). D-KRYRR was first labeled using lodogen and then coupled to the murine anti-EGFRvIII mAb L8A4 via maleimido bond formation in 60% yield. In vitro assays with the U87deltaEGFR cell line indicated that internalized and total cell-associated activity for the 125I-labeled D-KRYRR-L8A4 conjugate were up to 4 and 5 times higher, respectively, than for L8A4 labeled with 131I using Iodogen. Paired-label comparisons in athymic mice with s.c. U87deltaEGFR xenografts demonstrated up to 5-fold higher tumor uptake for mAb labeled using D-KRYRR. Higher levels of radioiodine activity also were observed in kidney when L8A4 was labeled using D-KRYRR. Another paired-label study directly compared L8A4 labeled using radioiodinated D-KRYRR and L-KRYRR, and confirmed the role of D-amino acids in enhancing tumor uptake. These results suggest that D-KRYRR is a promising reagent for the radioiodination of internalizing mAbs, such as the anti-EGFRvIII mAb L8A4.

Purpose: This study was designed to compare the cytotoxic effects of an alpha-emitting radioimmunoconjugate, which binds to osteosarcoma but not to bone marrow cells, with those of external gamma-irradiation., Materials and Methods: The human osteosarcoma cell line, OHS-s1, and mononuclear cells from bone marrow (BM) harvested from healthy donors, were used for these experiments. Cells in suspension were added to various activity concentrations of the anti-osteosarcoma monoclonal antibody TP-3 radiolabelled with 211At. Following incubation for 1 h, unbound radioactivity was washed off and cell survival was determined from clonogenic assays. Microdosimetry was calculated based on binding and retention kinetics of 211At to the cells, as well as cellular and nuclear diameters. For comparison, cell suspensions were irradiated with a single dose of 60Co gamma-rays., Results: 211At-labelled TP-3 showed heterogeneous binding to OHS-s1 cells, with a considerable variation among experiments. About 78% of the initially bound 211At decayed while associated with the OHS-s1 cells. D0 values estimated by microdosimetry were 0.33 (0.22-0.48, range) Gy and 1.18 (0.89-1.89) Gy for OHS-s1 and BM cells, respectively, whereas D0 values after external beam irradiation were 0.86+/-0.07Gy and 1.71+/-0.22Gy. The relative biological effectiveness (RBE) of 211At-labelled TP-3 at 37% survival was 3.43 for OHS-s1 and 1.55 for BM., Conclusions: High-LET targeted alpha-particle exposure killed osteosarcoma cells more effectively than bone marrow cells, although heterogeneous antigen expression among these tumour cells limited the magnitude of this effect.

Radionuclide conjugates or ricin A chain (RTA) immunotoxins that target pl85HER-2 have partially inhibited the growth of human ovarian cancer xenografts in athymic mice but generally have not cured mice bearing human tumor transplants. The present study was undertaken to explore whether a combination of ionizing radiation and an immunotoxin could exert additive or synergistic cytotoxicity in culture and in vivo against cancer cells that overexpress p185HER-2. In cell culture, treatment with 200-2000 cGy external beam irradiation followed by incubation with TA1-anti-pl85mHER2-RTA immunotoxin (TA1-RTA) produced synergistic inhibition of clonogenic growth of ovarian and breast cancer cells that expressed > 10(6) pl85HER-2 receptors/cell. The effect on cell survival correlated with an inhibition of DNA repair. A prior study (F. J. Xu et al, Nucl. Med. Biol., 24: 451-460, 1997) compared the biodistribution of radionuclide conjugates prepared with monoclonal antibodies that bind to different epitopes on the extracellular domain of pl85HER-2 and found optimal tumor uptake with the 520C9 antibody, which did not compete with TA1 for binding to the receptor. In this report, the TA1-RTA immunotoxin and the 131I-labeled 520C9 radionuclide conjugate could each inhibit the growth of clone-9002-18 xenografts in athymic mice but did not yield long-term survivors using maximally tolerated doses of each agent. When TA1-RTA and 131I-labeled 520C9 were used in combination, a greater inhibition of tumor growth was obtained than with either single agent. Similarly, survival with the combined treatment was significantly prolonged (P = 0.004) relative to treatment with immunotoxin or radionuclide conjugate alone. After treatment with an optimal combination of immunotoxin and radionuclide conjugate, 50% of mice survived >300 days, whereas controls succumbed with a median survival of 36 days. These results suggest that combinations of immunotoxins and radionuclide conjugates deserve further evaluation for the treatment of cancers that overexpress pl85HER-2.

Octreotide was coupled to 3-iodobenzoyl and 3-iodonicotinoyl moieties to obtain [N-(3-iodobenzoyl)-D-Phe(1)]octreotide (IBO) and [N-(3-iodonicotinoyl)-D-Phe(1)]octreotide (INO), respectively. The IC(50) values for the binding of IBO and INO to CA20948 rat pancreatic tumor membranes were 0.90 and 0.13 nM, respectively, compared with 0.35 nM for octreotide itself. Starting from N-succinimidyl 3-[(131)I]iodobenzoate and N-succinimidyl 5-[(131)I]iodopyridine-3- carboxylate, [(131)I]IBO and [(131)I]INO were prepared in overall radiochemical yields of 35%-50%. Likewise, ¿N-(3-[(211)At]astatobenzoyl)-D-Phe(1)¿octreotide ([(211)At]ABO) was prepared in similar yield from N-succinimidyl 3-[(211)At]astatobenzoate. In vitro assays with AR42J rat pancreatic tumor cells demonstrated a higher retention of cell-internalized radioiodine activity for [(131)I]INO compared with [(125)I]IBO. Tissue distribution studies with both conjugates revealed low levels of activity in the thyroid suggesting that dehalogenation of these peptides was minimal.

Purpose: The objective of this study was to perform the dosimetry and evaluate the dose-response relationships in newly diagnosed patients with malignant brain tumors treated by direct injections of (131)I-labeled 81C6 monoclonal antibody (MAb) into surgically created resection cavities (SCRCs)., Methods and Materials: Absorbed doses to the 2-cm-thick shell as measured from the margins of the resection cavity interface were estimated for 42 patients with primary brain tumors. MR images were used to assess the enhanced-rim volume as a function of time after radiolabeled MAb therapy. Biopsy samples were obtained from 15 patients and 1 autopsy., Results: The average absorbed dose [range] to the 2-cm shell region was 32 [3-59] Gy. For the endpoint of minimal time to MR contrast enhancement, the optimal absorbed dose and initial dose-rate were 43 +/- 16 Gy and 0. 41 +/- 0.10 Gy/h, respectively. There was a correlation between the absorbed dose and dose rate to the shell region and biopsy outcome (tumor recurrence, radionecrosis, and tumor recurrence and/or radionecrosis). In this Phase I study, the maximum tolerated dose (MTD) was 120 mCi. At this MTD, the estimated average absorbed dose and initial dose rate to the 2-cm shell were 41 [9-89] Gy and 0.51 [0.24-1.13] Gy/h, respectively. These values are in agreement with the optimal values based on the time to MR lesion rim enhancement., Conclusions: The average absorbed dose to the 2-cm shell region varied considerably and mainly depended on cavity volume. In future clinical trials, the administered activity of (131)I-labeled 81C6 MAb may be adjusted based on cavity volume in order to deliver the optimal absorbed dose of 43 Gy rather than giving a fixed administered activity.

Monoclonal antibody (MAb) internalization can have a major effect on tumor retention of radiolabel. Two anti-HER-2/neu MAbs (TA1 and 520C9) were radioiodinated using the iodogen, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), and tyramine-cellobiose (TCB) methods. Paired-label studies compared internalization and cellular processing of the labeled MAbs by SKOv3 9002-18 ovarian cancer cells in vitro. Intracellular radioiodine activity for 520C9 was up to 2.6 and 3.0 times higher for SIPC and TCB labeling, respectively, compared with iodogen. Likewise, intracellular activity for TA1 was up to 2.3 and 2.9 times higher with the SIPC and TCB methods compared with iodogen labeling. Unfortunately, similar advantages in tumor accumulation were not achieved in athymic mice bearing SKOv3 9008-18 ovarian cancer xenografts.

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

Purpose: The antitenascin human/mouse chimeric monoclonal antibody labeled with the alpha-particle-emitting radionuclide 211At is of interest as an endoradiotherapeutic agent for the treatment of brain tumors. To facilitate the investigation of 211At-labeled chimeric 81C6 in patients, the long-term radiotoxicity of this radiopharmaceutical has been evaluated., Methods and Materials: Antibody labeling was performed using N-succinimidyl 3-[211At]astato-benzoate. After an initial dose-finding experiment, a second toxicity study was carried out at 4 dose levels in groups of 30 nonthyroid blocked B6C3F1 mice per group (15 males, 15 females). Male mice received either saline or 15-81 kBq/g and females received either saline or 16-83 kBq/g of 211At-labeled antibody. Ten animals (5 males, 5 females) were followed for 6 months and the remainder for 1 year., Results: The lethal dose in 10% of animals (LD10) for 211At-labeled chimeric 81C6 was 46 kBq/g in females and 102 kBq/g in males. Toxic effects--perivascular fibrosis of the intraventricular septum of the heart, bone marrow suppression, splenic white pulp atrophy, and spermatic maturational delay--generally were confined to a few animals receiving the highest doses of labeled antibody., Conclusions: The LD10 of 211At-labeled chimeric 81C6 in this mouse strain was about half that of [211At]astatide. These results establish the preclinical maximum tolerated dose of 211At-labeled chimeric 81C6 and define in the mouse the target organs for toxicity. These studies will be useful for determining starting doses for clinical studies with 211At-labeled chimeric 81C6.

Unlabelled: Bisphosphonates were synthesized for use as carriers for astatine and iodine radioisotopes to target bone neoplasms., Methods: Radiohalogenated activated esters were coupled to the amino group in the side chain of the bisphosphonate. The bisphosphonate 3-amino-1-hydroxypropylidene bisphosphonate was combined with four different acylation agents: N-succinimidyl 3-[211At]astatobenzoate, N-succinimidyl 3-[131I]iodobenzoate, N-succinimidyl-5-[211At]astato-3-pyridinecarboxylate and N-succinimidyl-5-[131I]iodo-5-pyridinecarboxylate. The products, 3-[131I]iodobenzamide-N-3-hydroxypropylidene-3,3-bisphosphonate (IBPB), 3-[211At]astato-benzamide-N-3-hydroxypropylidene-3,3-bisphosphonat e (ABPB), 5-[131I]iodopyridine-3-amide-N-3-hydroxypropylidene-3,3-bisphospho nate (IPPB) and 5-[211At]astatopyridine-3-amide-N-3-hydroxypropylidene-3,3-bisphos phonate (APPB), were injected intravenously into Balb/c mice. MIRD and Monte Carlo methods were used on the basis of cumulated activity calculated from biodistribution data to estimate dose to organs and bone segments., Results: All 131I- and 211At-labeled analogs were strongly incorporated into osseous tissue and retained there at stable levels, while a rapid clearance from blood was observed. The bone uptake was found to be similar for 211At- and 131I-labeled bisphosphonate when compared in paired label experiments. Bone uptake and bone-to-tissue ratios were better for IBPB compared with IPPB, and ABPB compared with APPB. All four compounds appeared to be highly resistant to in vivo dehalogenation as indicated by low uptake of 131I/211At in the thyroid gland and stomach. According to dosimetric estimates, the bone surface-to-bone marrow ratio was three times higher with 211At than with 131I., Conclusion: Both the beta-particle- and alpha-particle-emitting compounds showed high in vivo stability and excellent affinity for osseous tissue. Further preclinical evaluation is therefore warranted.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

A method is described for preparing 211At- and radioiodinated amidobisphosphonates. The active esters N-succinimidyl 3-(tri-methylstannyl) benzoate (ATE) and N-succinimidyl 5-(tri-methylstannyl)-3-pyridinecarboxylate (SPC) were used as precursors. The isolated and purified radiolabeled intermediates were coupled to 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APB) in high yields ranging from 60% to 97%. The lipophilicity of the compounds was found to depend on the nature of the labeled template and the halogen. High in vitro stability in mouse, fetal calf, and human serum was documented by high performance liquid chromatography.

Monoclonal antibodies (MAbs) such as the anti-epidermal growth factor variant III (EGFRvIII) MAb L8A4 are rapidly internalized, which can lead to rapid loss of radioactivity from the tumor cell. The aim of this study was to evaluate the potential utility of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate ([211At]SAPC) for labeling murine L8A4 with 211At. SAPC was synthesized by astatodestannylation of N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate and then coupled to L8A4 in approximately 50% yield. The affinity and immunoreactive fraction for 211At-labeled L8A4 were comparable to those obtained when the MAb was labeled with 131I via N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). Paired-label comparisons of the 211At- and 131I-labeled MAbs demonstrated similar internalization and catabolism by EGFRvIII-positive cells in vitro, and with the exception of the stomach, similar tissue distribution in athymic mice with EGFRvIII-expressing U87MGdeltaEGFR xenografts. These results suggest that SAPC may be a useful reagent for labeling L8A4, and possibly other internalizing proteins, with 211At.

Unlabelled: The objective of this study was to perform the dosimetry of 131I-labeled 81C6 monoclonal antibody (MAb) in patients with recurrent malignant brain tumors, treated by direct injections of MAb into surgically created resection cavities (SCRCs)., Methods: Absorbed dose estimates were performed for nine patients. Dosimetry was performed retrospectively using probe counts (during patient isolation) and whole-body and SPECT images thereafter. Absorbed doses were calculated for the SCRC interface and for regions of interest (ROIs) 1 and 2 cm thick, measured from the margins of cavity interface. Also, mean absorbed doses were calculated for normal brain, liver, spleen, thyroid gland, stomach, bone marrow and whole body. The average residence time for the SCRC was 111 h (65-200h)., Results: The average absorbed dose per unit injected activity (range) to the SCRC interface and ROIs 1 and 2 cm thick from the cavity interface were 31.9 (7.8-84.2), 1.9 (0.7-3.6) and 1.0 (0.4-1.8) cGy/MBq, respectively. Average absorbed doses per unit administered activity to brain, liver, spleen, thyroid, stomach, bone marrow and whole body were 0.18, 0.03, 0.08, 0.05, 0.02, 0.02 and 0.01 cGy/MBq, respectively. The high absorbed dose delivered to the SCRC interface may have produced an increase in cavity volume independent of tumor progression., Conclusion: At the maximum tolerated dose of 3700 MBq 131I-labeled 81C6 MAb, the absorbed doses to the SCRC interface and ROIs of 1 and 2 cm thickness were estimated to be 1180, 71 and 39 Gy, respectively. The estimated average absorbed dose to the brain was 6.5 Gy. There was no neurological toxicity and minimal hematologic toxicity at this maximum tolerated administration level.

Substituting a pyridine ring for a benzene ring in the acylation agent N-succinimidyl 3-iodobenzoate has resulted in a useful approach for the radiohalogenation of monoclonal antibodies, peptides, and labeled biotin conjugates. It was hypothesized that such a substitution in m-iodobenzylguanidine (MIBG), a radiotracer used in the detection and treatment of neuroendocrine tumors, might result in an analogue with more rapid normal tissue clearance, thereby facilitating its use for tumor therapy. For the preparation of this analogue, 3-guanidinomethyl-5-iodopyridine (GMIP; 9b), the silicon precursor 4 was synthesized starting from 5-bromonicotinic acid. Attempts to convert 4 to 9b under various conditions were not successful. Radioiodinated 9b could be prepared by the iododestannylation of the tin precursor 8 in 65-70% radiochemical yield. A number of in vitro, in vivo, and ex vivo studies showed that pyridine-for-benzene substitution in MIBG yielded a compound that no longer was taken up by the uptake-1 pathway.

The in vivo properties of radiolabeled chimeric monoclonal antibodies (mAbs) with human IgG1 and IgG3 constant regions generally are similar to those of their corresponding murine construct. In contrast, we have observed that chimeric anti-tenascin mAb 81C6, which contains IgG2 constant regions, exhibits significantly higher localization in s.c. D-54 MG xenografts and prolonged retention in most normal tissues compared with its IgG2b murine parent. The purpose of the present study was to determine whether substitution of the murine IgG2b constant region domains in mAb 81C6 with those from human IgG2 enhanced the in vivo stability of the 81C6 mAb. Both mAbs were radioiodinated using Iodogen and administered to athymic mice bearing s.c. D-54 MG human glioma xenografts. The nature of the labeled species present in tumor and normal tissues over a 144-h period was investigated by trichloroacetic acid precipitation and SDS PAGE. In tumor and most normal tissues, a greater fraction of chimeric compared with murine 81C6 was present as intact IgG. For example, in tumor at 144 h, the fraction of radioactivity present as intact IgG was twice as high for chimeric compared with murine 81C6. A substantial fraction of murine but not chimeric 81C6 was present as a Mr 70,000-90,000 molecule, which could represent the generation of Fab/Fc monomers through the reduction of the interchain disulfide bonds in the murine IgG2b molecule. These results suggest that the higher tumor and normal tissue levels of chimeric compared with murine 81C6 can be attributed in part to the enhanced in vivo stability of the IgG2 chimeric mAb. The chimeric construct also was demonstrated to be more stable than murine after incubation with cyst fluid obtained from glioma resection cavity patients. Chimeric mAbs containing human IgG2 constant region domains could be of particular value for certain radioimmunotherapeutic applications.

Local hyperthermia has been shown to increase the tumor uptake and tumor:normal tissue ratios of radiolabeled monoclonal antibodies (mAbs) in athymic mouse xenograft models. The current study was undertaken to determine whether this behavior was related in part to alterations in mAb catabolism by local hyperthermia. Human/mouse chimeric 81C6 mAb reactive with tenascin and a nonspecific control mAb were labeled with 125I using Iodo-Gen and given to separate groups of athymic mice bearing s.c. D-54 MG human glioma xenografts. Half of the animals were then subjected to 4-h tumor-localized hyperthermia at 41.8 degrees C, a protocol previously shown to enhance the specific tumor uptake of the mAb in this xenograft model. The tumor, serum, liver, kidney, and urine were collected from heated as well as control animals 4 and 24 h after injection of the mAb and analyzed by SDS-PAGE and trichloroacetic acid precipitation. At 24 h, a significantly higher percentage of 81C6 was present as intact mAb in the tumors harvested from heated animals compared with those from controls. Unexpectedly, intact mAb was found in the urine of mice immediately after hyperthermia, but not in unheated control animals. We conclude that local hyperthermia decreases the catabolism of the mAb in the tumor and increases the urinary excretion of the mAb through a transient increase in glomerular permeability.

Asynchronous Chinese hamster V79 lung fibroblasts were incubated at 37 degrees C for 30 min with the thymidine analog 5-[211At]astato-2'-deoxyuridine (211AtdU, exposure from DNA-incorporated activity) or with [211At]astatide (211At-, exposure from extracellular activity), and DNA-incorporated activity was determined. The 211AtdU content in cellular DNA increased as a function of extracellular concentration. Incorporation of 211At- was less than 1% of that of 211AtdU. After exposure, cells were frozen in the presence of 10% DMSO. One month later, survival was determined by the colony-forming assay, and DNA double-strand breaks (DSBs) were measured by the neutral elution method (pH 9.6). The survival curve for 211AtdU was biphasic (D37 = 2.8 decays per cell), reflecting killing of 211At-DNA-labeled cells and of unlabeled cells irradiated by 211At in neighboring labeled cells. The toxicity of 211At- decaying outside the cell (30-min exposure) was negligible. Analysis of the survival curve produced a D0 of 1.3 decays/cell for 211At-labeled cells. The yield of DSBs from the decay of DNA-incorporated 211At was compared with that from DNA-incorporated 125I. Each decay of 211At produced at least 10 times the number of DSBs as that obtained per 125I decay. The extreme radiotoxicity of DNA-incorporated 211AtdU seems to be associated with considerable damage to the mammalian cell genome.

To circumvent the in vivo instability of 5-iodo-2'-deoxyuridine (IUdR), a 2'-fluorine-substituted analogue, 5-iodo-1-(2-deoxy-2-fluoro-beta-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-beta-D-arabinofuranosyl)ura cil (FTAU) was synthesized. Both [125/131I]FIAU and 5-[211At]astato-1-(2-deoxy-2-fluoro-beta-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.

Purpose: To determine the maximum-tolerated dose (MTD) of iodine 131 (131I)-labeled 81C6 monoclonal antibody (mAb) in brain tumor patients with surgically created resection cavities (SCRCs) and to identify any objective responses to this treatment., Methods: In this phase I trial, eligible patients were treated with a single injection of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating dosages of 131I (starting dose of 20 mCi with a 20-mCi escalation in subsequent cohorts) administered through an Ommaya reservoir in the SCRC. Patients were followed up for toxicity and response until death or for a minimum of 1 year after treatment. The SCRC patients, who were previously irradiated, were followed up without additional treatment unless progressive disease was identified., Results: We administered 36 treatments of 131I doses up to 120 mCi to 34 previously irradiated patients with recurrent or metastatic brain tumors. Dose-limiting toxicity was reached at 120 mCi and was limited to neurologic or hematologic toxicity. None of the patients treated with less than 120 mCi developed significant neurologic toxicity; one patient developed major hematologic toxicity (MHT). The estimated median survival for patients with glioblastoma multiforme (GBM) and for all patients was 56 and 60 weeks, respectively., Conclusion: The MTD for administration of 131I-labeled 81C6 into the SCRCs of previously irradiated patients with recurrent primary or metastatic brain tumors was 100 mCi. The dose-limiting toxicity was neurologic toxicity. We are encouraged by the minimal toxicity and survival in this phase I trial. Radiolabeled mAbs may improve the current therapy for brain tumor patients.

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.

Any immunotherapeutic approach to cancer cell eradication is based upon the specific recognition of neoplastic cells and the sparing of surrounding normal tissue; perhaps nowhere is this distinction more important than within the central nervous system, due to the diffuse infiltrative nature of primary glial tumor cell growth. Whether ultimate effect moieties are immunoglobulins, fragments and/or their constructs with drugs, toxins, radionuclides, or immune cells, the specificity of effector: cell surface marker is crucial. This review describes the identification, immunologic characterization, and biologic behavior of a transmembrane tumor-specific altered growth factor receptor molecule which may well serve as a mediator of multiple immunotherapeutic approaches: the class III variant of the epidermal growth factor receptor, EGFRvIII.

The high linear energy transfer, alpha-particle-emitting radionuclide astatine-211 (211At) is of interest for certain therapeutic applications; however, because of the 55- to 70-microm path length of its alpha-particles, achieving homogeneous tracer distribution is critical. Hyperthermia may enhance the therapeutic efficacy of alpha-particle endoradiotherapy if it can improve tracer distribution. In this study, we have investigated whether hyperthermia increased the cytotoxicity of an 211At-labelled monoclonal antibody (MAb) in tumour spheroids with a radius (approximately 100 microm) greater than the range of 211At alpha-particles. Hyperthermia for 1 h at 42 degrees C was used because this treatment itself resulted in no regrowth delay. Radiolabelled chimeric MAb 81C6 reactive with the extracellular matrix antigen tenascin was added to spheroids grown from the D-247 MG human glioma cell line at activity concentrations ranging from 0.125 to 250 kBq ml(-1). A significant regrowth delay was observed at 125 and 250 kBq ml(-1) in both hyperthermia-treated and untreated spheroids. For groups receiving hyperthermia, no increase in cytotoxicity was seen compared with normothermic controls at any activity concentration. These results and those from autoradiographs indicate that hyperthermia at 42 degrees C for 1 h had no significant effect on the uptake or distribution of this antitenascin MAb in D-247 MG spheroids.

The cytotoxicity of alpha-particle-emitting endoradiotherapeutic compounds is of increasing interest because clinical evaluation of these potential therapeutic agents is commencing. Astatine-211 is a radionuclide with a 7.2-h half-life that emits 5.87 and 7.45 MeV alpha particles. In the present work, we have investigated the in vitro cytotoxicity of 211At-labeled chimeric monoclonal antibodies (mAbs) in monolayers of D-247 MG human glioma cells and SK-MEL-28 human melanoma cells. The mAbs studied were 81C6, reactive with the extracellular matrix antigen tenascin, Mel-14, directed against the cell membrane antigen proteoglycan chondroitin sulfate, and a nonspecific control mAb, TPS3.2. Cell uptake increased as a function of activity concentration after a 1-h exposure to the 211At-labeled mAbs. The retention of activity was also measured to calculate cumulative activity associated with the cells and the medium. The clonogenic survival as a function of activity concentration was linear in all cases with no detectable shoulder. Microdosimetric analyses were performed based on measured cell geometry, cumulative activity and Monte Carlo transport of alpha particles. Using 18 kBq/ml activity concentration and 1 h of incubation, a two to five times higher activity bound to the microcolonies was found for the specific mAbs compared to the nonspecific mAb. These calculations indicated that a survival fraction of 0.37 was achieved with 0.24-0.28 Gy for D-247 MG cells and 0.27-0.29 Gy for SK-MEL-28 cells. The microdosimetric cell sensitivity, z0, for D-247 MG cells was significantly lower than for SK-MEL-28 cells (0.08 compared to 0.15 Gy). For both cell lines, reduction in survival to 0.37 required an average of only 1-2 alpha-particle hits to the cell nucleus.

We report herein the preparation and biological evaluation of two radioastatinated biotin conjugates, (3-[211At]astatobenzoyl)norbiotinamide and ((5-[211At]astato-3-pyridinyl)carbonyl)norbiotinamide. Both conjugates were stable in the presence of human serum and cerebrospinal fluid as well as murine serum, indicating a resistance to degradation to biotinidase. The normal tissue clearance of (3-[211At]astatobenzoyl)norbiotinamide and ((5-[211At]astato-3-pyridinyl)carbonyl)norbiotinamide was rapid, as observed previously with their iodo analogues. Also reported are the first syntheses of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate and 3-[211At]astatoaniline, two reagents of potential utility for labeling proteins and peptides with 211At.

The effects of specific activity of meta-[(131)I]iodobenzylguanidine (MIBG) were studied in uptake-2 blocked isolated perfused rat heart. [(131)I]MIBG was administered in the perfusate as an 8-min pulse, followed by an 80-min washout period. Kinetic analysis of the externally monitored time-activity curves gave estimates of uptake rate and multiexponential clearance. Uptake rate showed an MIBG concentration dependence that is sigmoidal, yielding Michaelis-Menten constants KM = 52 nM and Vmax = 0.23 nmol/min/g. Clearance rate was also dependent on loading MIBG concentrations; the primary effect of increasing loading concentration was an increase in the rate of the slowest clearance component, possibly reflecting nonspecific turnover. No effect of specific activity was observed on tissue uptake and retention of [(131)I]MIBG for loading concentrations of MIBG in the heart tissue under 0.5 nmol/g. Extrapolation of these results to human studies indicates that isotope-exchange-labeled [123I]MIBG has a specific activity sufficiently high to avoid mass effects on its heart retention.

A method is presented for calculating the small-scale dosimetry of 211At in red bone marrow using chord-length distributions obtained from digitized histological images. This study used histological samples of bone marrow from beagle dogs to convey morphological information about cell conglomerations within bone marrow. Two 211At activity distributions were considered within the extracellular fluid and the surface of red bone marrow cells. Results confirmed the influence of cell conglomeration and activity distribution in determining the microdosimetry of red bone marrow. Average S* values of 1.6 x 10(-9) and 1.90 x 10(-9) Gy g Bq(-1) s(-1) were calculated for activity distributions located within the extracellular fluid and the surface of red bone marrow cells, respectively. The cumulated activity required to reduce survival probability to 0.37 also was calculated as a function of cell sensitivity for both activity distributions. The activity distribution on the cell surface resulted in a higher cell-killing efficiency, requiring a lower activity concentration of approximately 25% when compared with activity located in the extracellular fluid. Of relevance to potential clinical studies with 211At, the probability for zero hits for red bone marrow cells was > 10% for cumulated activities of less than 5.5 x 10(8) Bq s g(-1) in bone marrow.

The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MG delta EGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MG delta EGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4.

A conjugation method has been developed for the radioiodination of proteins which should be adaptable to kit formulation. m-Hydroxybenzoic acid was converted to 3-hydroxy-4-[131I]iodobenzoic acid in 65% radiochemical yield using Chloramine-T as the oxidant. This intermediate was then converted to N-succinimidyl 3-hydroxy-4-[131I]iodobenzoate ([131I]mSHIB) in 75% yield by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide in a reaction time of only 10 min. Monoclonal antibody (mAb) 81C6 was labeled in 40-60% yield by reaction with [131I]mSHIB. Performing purifications of radioiodinated compounds using cartridges instead of HPLC did not alter conjugation efficiency, mAb immunoreactivity, or tissue distribution. Thyroid uptake of labeled mAb was low but up to 2.4 times higher than that seen when the mAb was labeled with N-succinimidyl 3-[125I]-iodobenzoate. These results suggest that [131I]mSHIB may be a useful reagent for the radioiodination of proteins, particularly in contexts when less complicated purification methods would be advantageous.

The HER-2/neu oncogene encodes a 185 kDa phosphoglycoprotein that is overexpressed in breast, ovarian and other cancers. Seven monoclonal antibodies reactive with oncoprotein were labeled with 131I. In vitro experiments with SKOv3 9002-18 cells determined binding affinity, internalization and degradation. The biodistribution of these antibodies in comparison to 125I-labeled nonspecific antibody was measured in athymic mice with SKOv3 9002-18 ovarian carcinoma xenografts. Antibody 520C9 exhibited the highest and most specific retention in tumor, peaking at 17.4 +/- 5.6% ID/g at 24 h.