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Reagents for astatination of biomolecules. 3. Comparison of closo-decaborate(2-) and closo-dodecaborate(2-) moieties as reactive groups for labeling with astatine-211.

ABSTRACT: In vivo deastatination has been a major problem in the development of reagents for therapeutic applications of the alpha-particle emitting radionuclide (211)At. Our prior studies demonstrated that the use of a closo-decaborate(2-) ([closo-B(10)H(9)R](2-)) moiety for (211)At labeling of biomolecules provides conjugates that are stable to in vivo deastatination. In this investigation, the closo-decaborate(2-) moiety was compared with the structurally similar closo-dodecaborate(2-) ([closo-B(12)H(11)R](2-)) to determine if one has more favorable properties than the other for use in pendant groups as (211)At labeling molecules. To determine the differences, two sets of structurally identical molecules, with the exception that they contained either a closo-decaborate(2-) or a closo-dodecaborate(2-) moiety, were compared with regard to their synthesis, radiohalogenation, stability to in vivo deastatination and tissue distribution. Quite different rates of reaction were noted in the synthetic steps for the two closo-borate(2-) moieties, but ultimately the yields were similar, making these differences of little importance. Differences in radiohalogenation rates were also noted between the two closo-borate(2-) moieties, with the more electrophilic closo-decaborate(2-) reacting more rapidly. This resulted in somewhat higher yields of astatinated closo-decaborate(2-) derivatives (84% vs 53%), but both cage moieties gave good radioiodination yields (e.g., 79-96%). Importantly, both closo-borate(2-) cage moieties were shown to have high stability to in vivo deastatination. The largest differences between pairs of compounds containing the structurally similar boron cage moieties were in their in vivo tissue distributions. For example, [Et(3)NH](2)B(12)H(10)I-CONHpropyl, [(125)I]2b had high concentrations in kidney (1 h, 19.8%ID/g; 4 h, 26.5%ID/g), whereas [Et(3)NH](2)B(10)H(8)I-CONHpropyl, [(125)I]1e had much lower concentrations in kidney (1 h, 6.6%ID/g; 4 h, 0.27%ID/g). Interestingly, when another salt of the closo-decaborate(2-), [nBu(4)N](2)B(10)H(8)I-CONHpropyl, [(125)I]1b, was evaluated, the route of excretion appeared to be hepatobiliary rather than renal. Identical biotin derivatives containing the two closo-borate(2-) cage moieties had similar tissue distributions, except the closo-decaborate(2-) derivative had lower concentrations in kidney (1 h, 19.9%ID/g; 4 h, 24.4%ID/g vs 1 h, 38.9%ID/g; 4 h, 40.6%ID/g). In summary, the higher reactivity, faster tissue clearance, and lower kidney concentrations make the closo-decaborate(2-) more favorable for further studies using them in reactive groups for (211)At labeling of biomolecules.

SUBMITTER: Wilbur DS

PROVIDER: S-EPMC2668518 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Reagents for astatination of biomolecules. 3. Comparison of closo-decaborate(2-) and closo-dodecaborate(2-) moieties as reactive groups for labeling with astatine-211.

Wilbur D Scott DS Chyan Ming-Kuan MK Hamlin Donald K DK Perry Matthew A MA

Bioconjugate chemistry 20090301 3

In vivo deastatination has been a major problem in the development of reagents for therapeutic applications of the alpha-particle emitting radionuclide (211)At. Our prior studies demonstrated that the use of a closo-decaborate(2-) ([closo-B(10)H(9)R](2-)) moiety for (211)At labeling of biomolecules provides conjugates that are stable to in vivo deastatination. In this investigation, the closo-decaborate(2-) moiety was compared with the structurally similar closo-dodecaborate(2-) ([closo-B(12)H(1 ...[more]

PMID: 19236022

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Project description:An investigation was conducted to compare the in vivo tissue distribution of a rat antimurine CD45 monoclonal antibody (30F11) and an irrelevant mAbs (CA12.10C12) labeled with (211)At using two different labeling methods. In the investigation, the mAbs were also labeled with (125)I to assess the in vivo stability of the labeling methods toward deastatination. One labeling method employed N-hydroxysuccinimidyl meta-[(211)At]astatobenzoate, [(211)At]1c, and N-hydroxysuccinimidyl meta-[(125)I]iodobenzoate, [(125)I]1b, in conjugation reactions to obtain the radiolabeled mAbs. The other labeling method involved conjugation of a maleimido-closo-decaborate(2-) derivative, 2, with sulfhydryl groups on the mAbs, followed by labeling of the mAb-2 conjugates using Na[(211)At]At or Na[(125)I]I and chloramine-T. Concentrations of the (211)At/(125)I pair of radiolabeled mAbs in selected tissues were examined in BALB/c mice at 1, 4, and 24 h post injection (pi). The co-injected anti-CD45 mAb, 30F11, labeled with [(125)I]1b and [(211)At]1c targeted the CD45-bearing cells in the spleen with the percent injected dose (%ID) of (125)I in that tissue being 13.31 ± 0.78; 17.43 ± 2.56; 5.23 ± 0.50; and (211)At being 6.56 ± 0.40; 10.14 ± 1.49; 7.52 ± 0.79 at 1, 4, and 24 h pi (respectively). However, better targeting (or retention) of the (125)I and (211)At was obtained for 30F11 conjugated with the closo-decaborate(2-), 2. The %ID in the spleen of (125)I (i.e., [(125)I]30F11-2) being 21.15 ± 1.33; 22.22 ± 1.95; 12.41 ± 0.75; and (211)At (i.e., [(211)At]30F11-2) being 22.78 ± 1.29; 25.05 ± 2.35; 17.30 ± 1.20 at 1, 4, and 24 h pi (respectively). In contrast, the irrelevant mAb, CA12.10C12, labeled with (125)I or (211)At by either method had less than 0.8% ID in the spleen at any time point, except for [(211)At]CA12.10C12-1c, which had 1.62 ± 0.14%ID and 1.21 ± 0.08%ID at 1 and 4 h pi. The higher spleen concentrations in that conjugate appear to be due to in vivo deastatination. Differences in (125)I and (211)At concentrations in lung, neck, and stomach indicate that the meta-[(211)At]benzoyl conjugates underwent deastatination, whereas the (211)At-labeled closo-decaborate(2-) conjugates were very stable to in vivo deastatination. In summary, using the closo-decaborate(2-) (211)At labeling approach resulted in higher concentrations of (211)At in target tissue (spleen) and higher stability to in vivo deastatination in this model. These findings, along with the simpler and higher-yielding (211)At-labeling method, provide the basis for using the closo-decaborate(2-) labeling reagent, 2, in our continued studies of the application of (211)At-labeled mAbs for conditioning in hematopoietic cell transplantation.

Project description:Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the ?-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated.

Project description:We are investigating the use of an (211)At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using (211)At-labeled anticanine CD45 mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27-0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted in prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with (123)I-labeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with (123)I-labeled CA12.10C12 conjugated with an amine-reactive phenylisothiocyanato-CHX-A? derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and nonreducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g., 6 and 8), a new, easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with coadministered (125)I- and (211)At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.

Project description:IntroductionThe use of lysine-urea-glutamate (LuG) for targeting the PSMA antigen on prostate cancer (PCa) is a promising method for delivering the alpha particle-emitting radionuclide astatine-211 (211At) to metastatic PCa. High kidney localization has been a problem with radiolabeled LuG derivatives, but has been adequately addressed in radiometal-labeled DOTA-LuG derivatives by linker optimization. Herein, we report an investigation of an alternate approach to diminishing the kidney concentrations of radiolabeled LuG-containing compounds.MethodsOur approach involves PEGylated LuG moieties and closo-decaborate (2-) moieties conjugated to streptavidin (SAv) or human serum albumin (HSA). After preparing the LuG conjugates, SAv and HSA conjugates were succinylated to decrease their kidney localization and radioiodinated for evaluation in athymic mice bearing C4-2B osseous PCa tumor xenografts.ResultsCovalently attaching LuG to succinylated SAv and HSA significantly reduced kidney localization, but unfortunately succinylation resulted in decreased tumor concentrations. In contrast, a potential metabolite [131I]16b, an unconjugated LuG derivative containing a dPEG4® linker, provided tumor concentrations of ~15% ID/g at 4 h pi. A second unconjugated LuG derivative with a similar structure, but containing a dPEG12® linker, [131I]16a had tumor concentrations of ~4%ID/g at 4 h pi. Those results suggest that long PEG linkers also affect tumor localization in a negative manner.ConclusionConjugation of PEGylated LuG derivatives to proteins can be an effective approach to diminishing kidney localization of radiolabeled LuG reagents, but the protein, linker and the method of linkage need to be further studied. Additionally, modification of the unconjugated 16b to decrease kidney localization may provide PCa targeting agents for use with radiohalogens, including 211At. Advances in knowledge and implications for patient care: This study is the first to evaluate PEGylated LuG and closo-decaborate (2-) moieties conjugated to proteins as potential methods for diminishing the kidney concentrations of radiolabeled LuG-containing compounds.

Dataset Information

Reagents for astatination of biomolecules. 3. Comparison of closo-decaborate(2-) and closo-dodecaborate(2-) moieties as reactive groups for labeling with astatine-211.

Publications

Reagents for astatination of biomolecules. 3. Comparison of closo-decaborate(2-) and closo-dodecaborate(2-) moieties as reactive groups for labeling with astatine-211.

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