Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination, as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of 211At-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with 125I, and that material was coinjected with the 211At-labeled Fab'. For comparison, direct labeling of the Fab' with 125I and 211At was conducted. Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[211At]At and Chloramine-T resulted in a yield of <1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[211At]astatobenzoate NHS ester, [211At]1c-Fab', and (separately) with p-[125I]iodobenzoate NHS ester, [125I]1b-Fab'. An evaluation in athymic mice demonstrated that [211At]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [125I]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [211At]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([125I]2-Fab'/ [211At]2-Fab') and the bis-nido-carborane (VFC) ([125I]3-Fab'/[211At]3-Fab') had very different in vivo distributions than the control [125I]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([125I]4-Fab'/[211At]4-Fab', [125I]6-Fab'/[211At]6-Fab', and [125I]7-Fab'/[211At]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [125I]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([125I]5-Fab'/[211At]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct 211At labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.

To lower treatment-related mortality and toxicity of conventional marrow transplantation, a nonmyeloablative regimen using 200 cGy total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for postgrafting immunosuppression was developed. To circumvent possible toxic effects of external-beam ␥ irradiation, strategies for targeted radiation therapy were investigated. We tested whether the short-lived (half-life, 46 minutes) ␣-emitter bismuth 213 ( 213 Bi) conjugated to an anti-CD45 monoclonal antibody (mAb) could replace 200 cGy TBI and selectively target hematopoietic tissues in a canine model of nonmyeloablative DLA-identical marrow transplantation. Biodistribution studies using iodine 123-labeled anti-CD45 mAb showed uptake in blood, marrow, lymph nodes, spleen, and liver. In a doseescalation study, 7 dogs treated with the 213 Bi-anti-CD45 conjugate ( 213 Bi dose, 0.1-5.9 mCi/kg [3.7-218 MBq/kg]) without marrow grafts had no toxic effects other than a mild, reversible suppression of blood counts. On the basis of these studies, 3 dogs were treated with 0.5 mg/kg 213 Bilabeled anti-CD45 mAb ( 213 Bi doses, 3.6, 4.6, and 8.8 mCi/kg [133, 170, and 326 MBq/kg]) given in 6 injections 3 and 2 days before grafting of marrow from DLAidentical littermates. The dogs also received MMF (10 mg/kg subcutaneously twice daily the day of transplantation until day 27 afterward) and CSP (15 mg/kg orally twice daily the day before transplantation until 35 days afterward). The therapy was well tolerated except for transient elevations in levels of transaminases in 3 dogs, followed by, in one dog, ascites. All dogs had prompt engraftment and achieved stable mixed hematopoietic chimerism, with donor contributions ranging from 30% to 70% after more than 27 weeks of follow-up. These results will form the basis for additional studies in animals and later the design of clinical trials using 213 IntroductionAllogeneic marrow transplantation provides a potential cure for a variety of hematologic and nonhematologic diseases. To avoid mortality and toxic effects associated with myeloablative marrow transplantation, a nonmyeloablative marrow transplantation regimen was developed in a canine model. The regimen uses 200 cGy total-body irradiation (TBI) before and administration of mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for immunosuppression after transplantation. 1 The combination of MMF and CSP given after transplantation controlled not only graft-versus-host disease (GVHD) but was also found to be essential for maintenance of the donor graft (ie, control of host-versus-graft reaction). The results of the preclinical studies have been successfully translated into the clinic to treat elderly or medically infirm patients with hematologic malignant diseases who were not eligible to receive high-dose conventional grafts. 2 Although the TBI dose used in these studies was low, there remains concern about the possible late toxic effects of ␥ radiation, especially in patients given transplan...

An investigation has been conducted to assess the in vivo stability of a series of astatinated benzamides and astatinated nido-carborane compounds in mice. It was hypothesized that the higher bond strength of boron-astatine bonds in the nido-carboranes might provide increased stability toward in vivo deastatination. Four tri-n-butylstannylbenzamides were prepared for radiohalogenation and evaluation in vivo. Those compounds were N-propyl-4-(tri-n-butylstannyl)benzamide 1a, N-propyl-3-(tri-n-butylstannyl)benzamide 2a, ethyl 4-tri-n-butylstannylhippurate 3a, and 4-tri-n-butylstannyl-hippuric acid 4a. Seven mono-nido-carboranyl derivatives were prepared for radiohalogenation and in vivo evaluation. Four of the seven mono-carboranyl derivatives (5a, 6a, 7a, 13a) contained a 3-(nido-carboranyl)propionamide functionality, and the remaining compounds (8a, 8g, 10a) contained a 4-(nido-carboranyl)aniline functionality. Two additional derivatives (11a, 12a) were prepared that contained bis-(nido-carboranylmethyl)benzene moieties (also referred to as Venus flytrap complexes (VFCs). All benzamide and nido-carborane compounds underwent facile iodination and radiohalogenation, except a 4-(nido-carboranyl)aniline derivative, 8a. Iodination of 8a resulted in a mixture, of which the desired iodinated product was a minor component. Therefore, radiohalogenation was not attempted. It is believed that the mixture of products is due to the presence of a thiourea bond. Previous studies have shown that thiourea bonds can interfere with halogenation reactions. In vivo comparisons of the compounds were conducted by co-injection of dual labeled (125/131I and 211At) compounds. Tissue distribution data were obtained at 1 and 4 h postinjection of the radiolabeled compounds, as that was sufficient to determine if astatine was being released. Stability of the astatinated compound was assessed by the difference in concentration of radioiodine and astatine in lung and spleen. All of the benzamides were found to undergo rapid deastatination in vivo. The nido-carborane derivatives appeared to be slightly more stable to in vivo deastatination; however, they had long blood residence times. The surprising finding was that the VFC derivatives did not release 211At in vivo, even though they rapidly localized to liver. This finding provides encouragement that stable conjugates of 211At may be attained if appropriate modifications of the VFC can be made to redirect their excretion through the renal system.

We are investigating the use of an 211At-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 211At-labeled anti-canine 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 123Ilabeled 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 123I-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 non-reducing 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 & 8), a new easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with co-administered 125I- and 211At-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.

Two major immunologic barriers, the hostversus-graft (HVG) and graft-versus-host (GVH) reactions, have to be overcome for successful allogeneic hematopoietic cell transplantation. T cells were shown to be primarily involved in these barriers in the major histocompatibility complex identical setting. We hypothesized that selective ablation of T cells using radioimmunotherapy together with postgrafting immunosuppression would suffice to ensure stable allogeneic engraftment. We

To reduce toxicity associated with external ␥-beam radiation, we investigated radioimmunotherapy with an anti-CD45 mAb labeled with the ␣-emitter, astatine-211 ( IntroductionWith the introduction of nonmyeloablative preparative regimens, allogeneic hematopoietic cell transplantation (HCT) has become a curative treatment option for a variety of malignant hematologic diseases in older and medically infirm patients. However, treatmentrelated toxicity and relapse are still major causes of morbidity and mortality. In an effort to increase the radiation dose delivered to the target organs while further reducing the late toxic effects of external beam ␥-irradiation, strategies using radioimmunotherapy (RIT) targeted toward hematopoietic tissues as a part of the conditioning regimen have been investigated. The efficacy and safety of this approach have been demonstrated in several clinical trials where antibody-coupled ␤-emitters, such as yttrium-90 ( 90 Y), rhenium-188 ( 188 Re), and iodine-131 ( 131 I), have been used to augment a variety of high-dose and reduced-intensity conditioning regimens. [1][2][3][4] The long path length of ␤-emitters, which makes them ideal in the setting of poorly perfused or bulky tumors, also makes them less optimal in situations with small volume tumors, minimal residual disease, or as part of an HCT conditioning regimen. 5 It has been estimated that only 1.5% and 17% of the energy from 90 Y-and 131 I-labeled mAbs, respectively, is deposited in tumors that are 200 m in diameter, whereas the rest is deposited in surrounding tissue resulting in nonspecific toxicity. 6 Alternative sources of radiation are available from the ␣-particle-emitting radionuclides. ␣-particles are characterized by short path lengths of 40 to 90 m in vivo, limiting the off-target toxicity to a few cell diameters. Furthermore, ␣-particles are more cytotoxic and have superior relative biologic effectiveness than ␤-particles because of a 400-fold greater linear energy transfer and the limited ability of tumor cells to repair ␣-particle-induced DNA damage. [7][8][9] Apart from our own preclinical experiences with the ␣-emitter bismuth-213 ( 213 Bi), the use of an ␣-emitter for RIT alone as conditioning in HCT has not been explored. We have previously demonstrated that 213 Bi-labeled mAb targeted toward the pan-hematopoietic antigen, CD45, or the T-cell receptor ␣␤ (TCR␣␤) could replace 200 to 300 cGy total body irradiation (TBI) as nonmyeloablative conditioning in dog leukocyte antigen (DLA)-identical or haploidentical bone marrow transplantation. [10][11][12][13] Although the treatment was successful in allowing sustained engraftment with minimal toxicity, obstacles, including short half-life (45.6 minutes), limited availability, and high cost of 213 Bi, made the translation of 213 Bi-labeled mAb into clinical studies impractical. Astatine-211 ( 211 At; t 1/2 ϭ 7.21 hours) is an alternative ␣-particle-emitting isotope. The advantage of 211 At is that it is available in quantities that can be scaled up for clinical stud...

Radioimmunotherapy (RIT) with ␣-emitting radionuclides is an attractive approach for the treatment of minimal residual disease because the short path lengths and high energies of ␣-particles produce optimal cytotoxicity at small target sites while minimizing damage to surrounding normal tissues. Pretargeted RIT (PRIT) using antibody-streptavidin (Ab-SA) constructs and radiolabeled biotin allows rapid, specific localization of radioactivity at tumor sites, making it an optimal method to target ␣-emitters with IntroductionNon-Hodgkin lymphoma (NHL) is the sixth most common type of cancer, with over 74 000 new cases diagnosed annually in the United States. 1 Following conventional treatment with chemotherapy or radiation therapy, patients with advanced stage indolent NHL inevitably relapse, with death occurring a median of 5 years after recurrence. 2 The introduction of rituximab, a monoclonal antibody against CD20, has led to improved survival in patients with NHL. [3][4][5] Despite the encouraging clinical results with anti-CD20 antibodies, however, the majority of patients with indolent NHL who respond to immunochemotherapy eventually relapse with recurrent lymphoma. 6,7 Recently, radioimmunotherapy (RIT) has emerged as a promising treatment option for lymphoma. RIT with iodine-131( 131 I) tositumomab or yttrium-90 ( 90 Y) ibritumomab tiuxetan as a single agent has yielded excellent overall response rates of 50% -80%, with complete response rates of 20% -40% in patients with relapsed or refractory indolent NHL. [8][9][10][11][12][13] Even more notable response rates have been observed when RIT is used as front-line treatment in patients with indolent NHL. 14 In a recent large phase 3 trial, the addition of 90 Y-ibritumomab tiuxetan in first remission after chemotherapy significantly improved response rates and remission durations in patients with advancedstage follicular lymphoma, 15 presumably by killing residual tumor cells that survived the induction chemotherapy. 16 Based on this data, 90 Y-ibritumomab tiuxetan has been approved by the FDA for first line consolidation therapy in follicular lymphoma. However, the ␤-emitting radionuclides used in current RIT schemes may not be ideal for irradiating microscopic tumors and isolated tumor cells present in the setting of minimal residual disease (MRD). It is estimated that the fraction of energy deposited in a tumor measuring 200 m in diameter is only 1.5% and 17% for 90 Y-labeled and 131 I-labeled antibodies (Abs), respectively. 17,18 The remainder of the ␤ energy is deposited in surrounding normal tissues, resulting in dose-limiting toxicities. Furthermore, the relatively low decay energies of ␤-particles result in suboptimal killing of tumor cells, ultimately contributing to relapse in the majority of treated patients. In contrast, ␣-emitting radionuclides impart high-linear-energytransfer radiation along densely ionized, linear tracks over relatively short distances (40 to 90 m or few cell diameters), which are highly effective in cell-killing. Alpha-particle...

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.