High-level production of alpha-particle-emitting (211)At and preparation of (211)At-labeled antibodies for clinical use.

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.

Astatine-211-labeled biotin conjugates resistant to biotinidase for use in pretargeted radioimmunotherapy.

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.

Synthesis and preliminary biological evaluation of (3-iodobenzoyl)norbiotinamide and ((5-iodo-3-pyridinyl)carbonyl)norbiotinamide: two radioiodinated biotin conjugates with improved stability.

A new class of radioiodinated biotin conjugated is described in which the amido bond between biotin and the labeled prosthetic group is reversed. One conjugate, (3-[125I]iodobenzoyl)norbiotinamide (4c, [125I]IBB) was labeled with Na125I in one step from (3-(tributylstannyl)benzoyl)norbiotinamide (4b, TBB) via a demetalation reaction. However, the analogous reaction with ((5-(tributylstannyl)-3-pyridinyl)carbonyl)norbiotinamide (6b, TPB) failed to yield ((5-[131I]iodo-3-pyridinyl)carbonyl)norbiotinamide (6c, [131I]IPB, necessitating a two-step approach for synthesizing [131I]IPB. The binding of [125I]IBB and [131I]IPB to streptavidin in vitro was identical to that of biotinyl-3-[125I]iodoanilide, a conjugate with an amido bond with normal configuration. Both [125I]IBB and [131I]IPB were stable in serum while the first-generation compound was rapidly degraded. The biodistribution patterns of [125I]IBB and [131I]IPB in mice are consistent with limited degradation of these conjugates by biotinidase and deiodinases.

Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate.

Monoclonal antibodies (mAbs) that internalize following binding to cell-surface receptors require radiolabeling approaches that minimize loss of radioactivity from the cell after intracellular processing. One class of internalizing mAbs of great interest for imaging and radioimmunotherapy are those specific for EGFRvIII, a truncated form of the epidermal growth factor receptor found on gliomas, non-small cell lung carcinomas, breast carcinomas, and ovarian carcinomas. Because lysosomes are known to retain positively charged compounds, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) might be ideal for radioiodination of these mAbs because of the positive charge on its pyridine ring. To investigate this hypothesis, the anti-EGFRvIII mAb L8A4 was labeled using SIPC, and internalization assays were performed using the EGFRvIII-positive cell lines HC2 20 d2 and NR6M. Compared with L8A4 labeled using Iodogen or N-succinimidyl 3-iodobenzoate, SIPC increased intracellular retention of activity by up to 65%. Reverse-phase high-performance liquid chromatography analyses indicated that a significantly higher fraction of the low molecular weight catabolites from mAbs labeled via SIPC were retained within cells (SIPC, 28.1%; Iodogen, 7.6% at 1 h). With SIPC, the primary labeled species in cell lysates was the 5-iodonicotinic acid (INA)-lysine conjugate, whereas in the supernatant, both INA-lysine and INA were seen. A 3-4-fold higher percentage of these catabolites were charged at lysosomal pH in comparison with those from mAb labeled using N-succinimidyl 3-iodobenzoate, in concert with the differences in cellular retention observed between these two labeling methods. In mice bearing HC2 20 d2 xenografts, a significant improvement in tumor retention of radioiodine and tumor:normal tissue ratios was seen when L8A4 was labeled using SIPC instead of the Iodogen method. These results suggest that SIPC is a promising reagent for the radioiodination of anti-EGFRvIII L8A4 and, possibly, other internalizing mAbs.

Enhanced binding and inertness to dehalogenation of alpha-melanotropic peptides labeled using N-succinimidyl 3-iodobenzoate.

Two peptides of potential utility for targeting melanoma cells, alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analogue [Nle4,D-Phe7]-alpha-MSH, were radioiodinated in 45-65% yield using N-succinimidyl 3-[125I]iodobenzoate (SIB). To determine whether this labeling method resulted in improved in vitro and in vivo characteristics, these peptides also were labeled with 131I by direct iodination with the iodogen method. For alpha-MSH, the rapid tissue clearance of both radionuclides in mice was consistent with rapid degradation of the peptide; however, significantly lower levels of 125I were observed in thyroid and stomach, reflecting a greater inertness to deiodination. More extensive comparisons were performed with [Nle4,D-Phe7]-alpha-MSH. The in vitro binding of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH (prepared using SIB) to the murine B-16 melanoma cell line, 34.1 +/- 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH (15.0 +/- 0.1%), and its KD was more than 10-fold lower than that for conventionally labeled peptide (10 +/- 5 versus 140 +/- 14 pM). The normal tissue clearance of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in mice was faster than that of [Tyr2(131I),-Nle4,D-Phe7]-alpha-MSH. The 19-40-fold lower activity concentrations of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in tissues accumulating free iodide (thyroid and stomach) suggest a greater inertness of this peptide to deiodination. The primary urinary catabolite of [Nle4,D-Phe7, Lys11-(125I)IBA]-alpha-MSH was the lysine conjugate of iodobenzoic acid, whereas radioiodide was the chief catabolite generated from [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH. We conclude that further evaluation of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH for targeting alpha-MSH receptors is warranted and that SIB may be a useful method for the radioiodination of peptides.

Catabolism of radioiodinated murine monoclonal antibody F(ab')2 fragment labeled using N-succinimidyl 3-iodobenzoate and Iodogen methods.

The F(ab')2 fragment of monoclonal antibody (MAb) Me1-14 was labeled with 125I using the Iodogen method and by reaction with N-succinimidyl 3-[125I]iodobenzoate (SIB). The labeled catabolites generated after exposure to tissue homogenates in vitro and following administration of labeled F(ab')2 into normal mice were investigated by size-exclusion HPLC, gel electrophoresis, and reverse-phase HPLC. Rapid conversion of F(ab')2 to Fab was observed with both labeling methods. With F(ab')2 labeled using the Iodogen method, the primary low molecular weight catabolites appeared to be [125I]iodide and, to a lesser extent, mono[125I]iodotyrosine. With SIB, [125I]iodide and [125I]iodobenzoic acid (IBA) as well as the glycine and lysine conjugates of IBA were all observed. Differences in low molecular weight catabolic products could explain the more rapid normal tissue clearance with MAbs and MAb fragments labeled with SIB compared with those labeled using iodogen.