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The α-emitter 211 At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211 At-labeled anti-CD45 monoclonal antibody (mAb) 211 At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 ( n = 14) or 0.75 ( n = 3) mg/kg dose of anti-CD45 mAb labeled with 211 At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/μL), lymphocytopenia (36-130 cells/μL), and thrombocytopenia (1.5-9 × 10 3 /μL) with prompt recovery were observed. The main adverse nonhematologic event related to 211 At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211 At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC., Methods: In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC., Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups., Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection., Competing Interests: Competing Interests: The authors have no relevant financial or non-financial interests to disclose.

Purpose: Hematopoietic cell transplantation (HCT) has curative potential for myeloid malignancies, though many patients cannot tolerate myeloablative conditioning with high-dose chemotherapy alone or with total-body irradiation (TBI). Here we report long-term outcomes from a phase I/II study using iodine-131 (131I)-anti-CD45 antibody BC8 combined with nonmyeloablative conditioning prior to HLA-haploidentical HCT in adults with high-risk relapsed/ refractory acute myeloid or lymphoid leukemia (AML or ALL), or myelodysplastic syndrome (MDS; ClinicalTrials.gov, NCT00589316)., Patients and Methods: Patients received a tracer diagnostic dose before a therapeutic infusion of 131I-anti-CD45 to deliver escalating doses (12-26 Gy) to the dose-limiting organ. Patients subsequently received fludarabine, cyclophosphamide (CY), and 2 Gy TBI conditioning before haploidentical marrow HCT. GVHD prophylaxis was posttransplant CY plus tacrolimus and mycophenolate mofetil., Results: Twenty-five patients (20 with AML, 4 ALL and 1 high-risk MDS) were treated; 8 had ≥ 5% blasts by morphology (range 9%-20%), and 7 had previously failed HCT. All 25 patients achieved a morphologic remission 28 days after HCT, with only 2 patients showing minimal residual disease (0.002-1.8%) by flow cytometry. Median time to engraftment was 15 days for neutrophils and 23 days for platelets. Point estimates for overall survival and progression-free survival were 40% and 32% at 1 year, and 24% at 2 years, respectively. Point estimates of relapse and nonrelapse mortality at 1 year were 56% and 12%, respectively., Conclusions: 131I-anti-CD45 radioimmunotherapy prior to haploidentical HCT is feasible and can be curative in some patients, including those with disease, without additional toxicity., (©2023 American Association for Cancer Research.)

Background: Early intrahepatic recurrence is common after surgical resection of hepatocellular carcinoma (HCC) and leads to increased morbidity and mortality. Insensitive and nonspecific diagnostic imaging contributes to EIR and results in missed treatment opportunities. In addition, novel modalities are needed to identify targets amenable for targeted molecular therapy. In this study, we evaluated a zirconium-89 radiolabeled glypican-3 (GPC3) targeting antibody conjugate ( 89 Zr-αGPC3) for use in positron emission tomography (PET) for detection of small, GPC3 + HCC in an orthotopic murine model. Athymic nu/J mice received hepG2, a GPC3 + human HCC cell line, into the hepatic subcapsular space. Tumor-bearing mice were imaged by PET/computerized tomography (CT) 4 days after tail vein injection of 89 Zr-αGPC3. Livers were then excised for the tumors to be identified, measured, bisected, and then serially sectioned at 500 μm increments. Sensitivity and specificity of PET/CT for 89 Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard., Results: In tumor-bearing mice, 89 Zr-αGPC3 avidly accumulated in the tumor within four hours of injection with ongoing accumulation over time. There was minimal off-target deposition and rapid bloodstream clearance. Thirty-eight of 43 animals had an identifiable tumor on histologic analysis. 89 Zr-αGPC3 immuno-PET detected all 38 histologically confirmed tumors with a sensitivity of 100%, with the smallest tumor detected measuring 330 μm in diameter. Tumor-to-liver ratios of 89 Zr-αGPC3 uptake were high, creating excellent spatial resolution for ease of tumor detection on PET/CT. Two of five tumors that were observed on PET/CT were not identified on histologic analysis, yielding a specificity of 60%., Conclusions: 89 Zr-αGPC3 avidly accumulated in GPC3 + tumors with minimal off-target sequestration. 89 Zr-αGPC3 immuno-PET yielded a sensitivity of 100% and detected sub-millimeter tumors. This technology may improve diagnostic sensitivity of small HCC and select GPC3 + tumors for targeted therapy. Human trials are warranted to assess its impact., (© 2023. The Author(s).)

Targeted radiopharmaceutical therapy with alpha-particle emitters (αRPT) is advantageous in cancer treatment because the short range and high local energy deposition of alpha particles enable precise radiation delivery and efficient tumor cell killing. However, these properties create sub-organ dose deposition effects that are not easily characterized by direct gamma-ray imaging (PET or SPECT). We present a computational procedure to determine the spatial distribution of absorbed dose from alpha-emitting radionuclides in tissues using digital autoradiography activity images from an ionizing-radiation quantum imaging detector (iQID). Data from 211 At-radioimmunotherapy studies for allogeneic hematopoietic cell transplantation in a canine model were used to develop these methods. Nine healthy canines were treated with 16.9-30.9 MBq 211 At/mg monoclonal antibodies (mAb). Lymph node biopsies from early (2-5 h) and late (19-20 h) time points (16 total) were obtained, with 10-20 consecutive 12-µm cryosections extracted from each and imaged with an iQID device. iQID spatial activity images were registered within a 3D volume for dose-point-kernel convolution, producing dose-rate maps. The accumulated absorbed doses for high- and low-rate regions were 9 ± 4 Gy and 1.2 ± 0.8 Gy from separate dose-rate curves, respectively. We further assess uptake uniformity, co-registration with histological pathology, and requisite slice numbers to improve microscale characterization of absorbed dose inhomogeneities in αRPT., (© 2022. The Author(s).)

The alpha particle-emitting radionuclide astatine-211 ( 211 At) is of interest for targeted radiotherapy; however, low in vivo stability of many 211 At-labeled cancer-targeting molecules has limited its potential. As an alternative labeling method, we evaluated whether a specific type of astatinated aryl compound that has the At atom in a higher oxidation state might be stable to in vivo deastatination. In the research effort, para-iodobenzoic acid methyl ester and dPEG 4 -amino acid methyl ester derivatives were prepared as HPLC standards. The corresponding para-stannylbenzoic acid derivatives were also prepared and labeled with 125 I and 211 At. Oxidization of the [ 125 I]iodo- and [ 211 At]astato-benzamidyl-dPEG 4 -acid methyl ester derivatives provided materials for in vivo evaluation. A biodistribution was conducted in mice with coinjected oxidized 125 I- and 211 At-labeled compounds. The oxidized radioiodinated derivative was stable to in vivo deiodination, but unfortunately the oxidized [ 211 At]astatinated benzamide derivative was found to be unstable under the conditions of isolation by radio-HPLC (post animal injection). Another biodistribution study in mice evaluated the tissue concentrations of coinjected [ 211 At]NaAtO 3 and [ 125 I]NaIO 3 . Comparison of the tissue concentrations of the isolated material from the oxidized [ 211 At]benzamide derivative with those of [ 211 At]astatate indicated the species obtained after isolation was likely [ 211 At]astatate.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227 Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227 Th-labeled GPC3-targeting antibody conjugate ( 227 Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p -SCN-Bn-H 4 octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227 Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227 Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227 Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227 Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227 Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227 Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227 Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227 Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227 Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion: 227 Th-octapa-αGPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia and higher-risk myelodysplastic syndrome (MDS). Of increasing interest are alpha-particle-emitting radionuclides such as astatine-211 ( 211 At) as they deliver large amounts of radiation over just a few cell diameters, enabling efficient and selective target cell kill. Here, we developed 211 At-based RIT targeting CD123, an antigen widely displayed on acute leukemia and MDS cells including underlying neoplastic stem cells. We generated and characterized new murine monoclonal antibodies (mAbs) specific for human CD123 and selected four, all of which were internalized by CD123+ target cells, for further characterization. All mAbs could be conjugated to a boron cage, isothiocyanatophenethyl-ureido-closo-decaborate(2-) (B10), and labeled with 211 At. CD123+ cell targeting studies in immunodeficient mice demonstrated specific uptake of 211 At-labeled anti-CD123 mAbs in human CD123+ MOLM-13 cell tumors in the flank. In mice injected intravenously with MOLM-13 cells or a CD123 NULL MOLM-13 subline, a single dose of up to 40 µCi of 211 At delivered via anti-CD123 mAb decreased tumor burdens and substantially prolonged survival dose dependently in mice bearing CD123+ but not CD123- leukemia xenografts, demonstrating potent and target-specific in vivo anti-leukemia efficacy. These data support the further development of 211 At-CD123 RIT toward clinical application., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In a canine model of presensitization using donor blood transfusions, 100% of historical control dogs receiving 9.2 Gy total body irradiation (TBI) conditioning before dog leukocyte antigen (DLA)-identical marrow grafts had graft rejection. In this presensitization model, we investigated whether the addition of monoclonal antibody (mAb)-based targeted radioimmunotherapy (RIT) with astatine-211 ( 211 At) to TBI could overcome graft rejection. 211 At is an alpha-particle-emitting isotope that has a short path length, very high energy, and a short t½ of 7.2 hours, which allowed targeting radiation to the T cells responsible for graft rejection. Normal canine recipients were given three preceding transfusions of unirradiated whole blood on days -24, -17, and -10 before transplant from their DLA-identical marrow donors. 211 At-anti-CD45 mAb was administered on day -3, and TBI followed by marrow grafts on day 0. Six of the 7 dogs (86%) achieved sustained engraftment as assessed by 100% donor chimerism in mononuclear cells, granulocytes, and CD3 + T cells. One dog receiving the lowest CD34 + cell content (0.35 × 10 6 cells/kg) rejected the graft. There were no late rejections in dogs followed up to 1 year. Graft-versus-host disease was seen in one dog. 211 At-anti-CD45 mAb in combination with TBI as conditioning was successful in abrogating graft rejection in 86% of dogs in this presensitization model. 211 At-anti-CD45 mAb conditioning with TBI may serve as a novel promising strategy to overcome graft rejection in heavily transfused patients with red cell disorders., (Copyright © 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 ( 89 Zr) and yttrium-90 ( 90 Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89 Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90 Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUV max by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R 2  = 0.90). Serum AFP was significantly lower 30 days after RIT in 90 Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R 2  = 0.87), and GTV of animals treated with 90 Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89 Zr and 90 Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Introduction: The 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 ( 211 At) 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., Methods: Our 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., Results: Covalently attaching LuG to succinylated SAv and HSA significantly reduced kidney localization, but unfortunately succinylation resulted in decreased tumor concentrations. In contrast, a potential metabolite [ 131 I]16b, an unconjugated LuG derivative containing a dPEG 4 ® linker, provided tumor concentrations of ~15% ID/g at 4 h pi. A second unconjugated LuG derivative with a similar structure, but containing a dPEG 12 ® linker, [ 131 I]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., Conclusion: Conjugation 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 211 At. 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., Competing Interests: Declaration of competing interest The Authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pretargeted radioimmunotherapy (PRIT) has been investigated as a multi-step approach to decrease relapse and toxicity for high-risk acute myeloid leukemia (AML). Relevant factors including endogenous biotin and immunogenicity, however, have limited the use of PRIT with an anti-CD45 antibody streptavidin conjugate and radiolabeled DOTA-biotin. To overcome these limitations we designed anti-murine and anti-human CD45 bispecific antibody constructs using 30F11 and BC8 antibodies, respectively, combined with an anti-yttrium (Y)-DOTA single-chain variable fragment (C825) to capture a radiolabeled ligand. The bispecific construct targeting human CD45 (BC8-Fc-C825) had high uptake in leukemia HEL xenografts [7.8 ± 0.02% percent injected dose/gram of tissue (% ID/g)]. Therapy studies showed that 70% of mice with HEL human xenografts treated with BC8-Fc-C825 followed by 44.4 MBq (1,200 μCi) of 90 Y-DOTA-biotin survived at least 170 days after therapy, while all nontreated controls required euthanasia because of tumor progression by day 32. High uptake at sites of leukemia (spleen and bone marrow) was also seen with 30F11-IgG1-C825 in a syngeneic disseminated SJL murine leukemia model (spleen, 9.0 ± 1.5% ID/g and bone marrow, 8.1 ± 1.2% ID/g), with minimal uptake in all other normal organs (<0.5% ID/g) at 24 hours after 90 Y-DOTA injections. SJL leukemia mice treated with the bispecific 30F11-IgG1-C825 and 29.6 MBq (800 μCi) of 90 Y-DOTA-biotin had a survival advantage compared with untreated leukemic mice (median, 43 vs. 30 days, respectively; P < 0.0001). These data suggest bispecific antibody-mediated PRIT may be highly effective for leukemia therapy and translation to human studies., (©2020 American Association for Cancer Research.)

One of the main challenges in targeted alpha therapy is assuring delivery of the α-particle dose to the targeted cells. Thus, it is critical to identify ligands for α-emitting radiometals that will form complexes that are very stable, both in vitro and in vivo. In this investigation, thorium-227 (t 1/2 = 18.70 days) chelation of ligands containing hydroxypyridinonate (HOPO) or picolinic acid (pa) moieties and the stability of the resultant complexes were studied. Chelation reactions were followed by reversed-phased HPLC and gamma spectroscopy. Studies revealed that high 227 Th chelation yields could be obtained within 2.5 h or less with ligands containing four Me-3,2-HOPO moieties, 1 (83%) and 2 (65%), and also with ligands containing pa moieties, H 4 octapa 3 (65%) and H 4 py4pa 6 (87%). No reaction occurred with H 4 neunpa-p-Bn-NO 2 4, and the chelation reaction with another pa ligand H 4 pypa 5 gave inconsistent yields with a very broad radio-HPLC peak. The ligands spermine-(Me-3,2-HOPO) 4 1, H 4 octapa 3, and H 4 py4pa 6 had high stability (i.e., 87% of 227 Th still bound to the ligand) in phosphate-buffered saline at room temperature over a 6-day period. Preliminary studies with ligand 6 demonstrated efficient chelation of thorium-226 (t 1/2 = 30.57 min) when heated to 80°C for 5 min., (© 2020 John Wiley & Sons, Ltd.)

Increasing access to the short-lived α-emitting radionuclide astatine-211 ( 211 At) has the potential to advance targeted α-therapeutic treatment of disease and to solve challenges facing the medical community. For example, there are numerous technical needs associated with advancing the use of 211 At in targeted α-therapy, e.g., improving 211 At chelates, developing more effective 211 At targeting, and characterizing in vivo 211 At behavior. There is an insufficient understanding of astatine chemistry to support these efforts. The chemistry of astatine is one of the least developed of all elements on the periodic table, owing to its limited supply and short half-life. Increasing access to 211 At could help address these issues and advance understanding of 211 At chemistry in general. We contribute here an extraction chromatographic processing method that simplifies 211 At production in terms of purification. It utilizes the commercially available Pre-Filter resin to rapidly (<1.5 h) isolate 211 At from irradiated bismuth targets (Bi decontamination factors ≥876 000), in reasonable yield (68-55%) and in a form that is compatible for subsequent in vivo study. We are excited about the potential of this procedure to address 211 At supply and processing/purification problems.

Cyclotron-produced astatine-211 ( 211 At) shows tremendous promise in targeted alpha therapy (TAT) applications due to its attractive half-life and its 100% α-emission from nearly simultaneous branched alpha decay. Astatine-211 is produced by alpha beam bombardment of naturally monoisotopic bismuth metal ( 209 Bi) via the (α, 2n) reaction. In order to isolate the small mass of 211 At (specific activity = 76 GBq·µg -1 ) from several grams of acid-dissolved Bi metal, a manual milliliter-scale solvent extraction process using diisopropyl ether (DIPE) is routinely performed at the University of Washington. As this process is complex and time consuming, we have developed a fluidic workstation that can perform the method autonomously. The workstation employs two pumps to concurrently deliver the aqueous and organic phases to a mixing tee and in-line phase mixer. The mixed phases are routed to a phase settling reservoir, where they gravity settle. Finally, each respective phase is withdrawn into its respective pump. However, development of a phase boundary sensor, placed in tandem with the phase settling reservoir, was necessary to communicate to the system when withdrawal of the denser aqueous phase was complete (i.e., the intersection of the two phases was located). The development and optimization of the autonomous solvent extraction system is described, and the 211 At yields from several ~1.1 GBq-level 211 At processing runs are reported.

Astatine-211 is an attractive radionuclide for use in targeted alpha therapy of blood-borne diseases and micrometastatic diseases. Efficient isolation methods that can be adapted to robust automated 211 At isolation systems are of high interest for improving the availability of 211 At. Based on the early studies of Bochvarova and co-workers involving isolation of 211 At from irradiated thorium targets, we developed a method for 211 At isolation from bismuth targets using tellurium-packed columns. Dissolution of irradiated bismuth targets is accomplished using HNO 3 ; however, 211 At is not captured on the Te column material in this matrix. Our method involves slow addition of aqueous NH 2 OH·HCl to the Bi target dissolved in HNO 3 to convert to a HCl matrix. The amount of NH 2 OH·HCl was optimized because (1) the quantity of NH 2 OH·HCl used appears to affect the radiolabeling yield of phenethyl-closo-decaborate(2-) (B10)-conjugated antibodies and (2) reducing the volume of NH 2 OH·HCl solution can effectively shorten the overall isolation time. A proof-of-concept semi-automated process has been demonstrated using targets containing ~0.96 GBq (~26 mCi) of 211 At. High isolation yields (88-95%) were obtained. Radiochemical purity of the isolated 211 At was assessed by radio-HPLC. Concentrations of Bi and Te contaminants in the 211 At and the astatinated antibodies were evaluated using ICP-MS.

Minimal residual disease (MRD) has become an increasingly prevalent and important entity in multiple myeloma (MM). Despite deepening responses to frontline therapy, roughly 75% of MM patients never become MRD-negative to ≤10-5, which is concerning because MRD-negative status predicts significantly longer survival. MM is highly heterogeneous, and MRD persistence may reflect survival of isolated single cells and small clusters of treatment-resistant subclones. Virtually all MM clones are exquisitely sensitive to radiation, and the α-emitter astatine-211 (211At) deposits prodigious energy within 3 cell diameters, which is ideal for eliminating MRD if effectively targeted. CD38 is a proven MM target, and we conjugated 211At to an anti-CD38 monoclonal antibody to create an 211At-CD38 therapy. When examined in a bulky xenograft model of MM, single-dose 211At-CD38 at 15 to 45 µCi at least doubled median survival of mice relative to untreated controls (P < .003), but no mice achieved complete remission and all died within 75 days. In contrast, in a disseminated disease model designed to reflect low-burden MRD, 3 studies demonstrated that single-dose 211At-CD38 at 24 to 45 µCi produced sustained remission and long-term survival (>150 days) for 50% to 80% of mice, where all untreated mice died in 20 to 55 days (P < .0001). Treatment toxicities were transient and minimal. These data suggest that 211At-CD38 offers the potential to eliminate residual MM cell clones in low-disease-burden settings, including MRD. We are optimistic that, in a planned clinical trial, addition of 211At-CD38 to an autologous stem cell transplant (ASCT) conditioning regimen may improve ASCT outcomes for MM patients., (© 2019 by The American Society of Hematology.)

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89 Zr-conjugated monoclonal anti-GPC3 antibody ( α GPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90 Y, a high-energy beta-particle-emitting radionuclide, to our α GPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to α GPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200  μ Ci/mouse or 300  μ Ci/mouse. Control mice received DOTA- α GPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200  μ Ci only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300  μ Ci experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90 Y- α GPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Andrew D. Ludwig et al.)

The objective of this study was to translate reaction conditions and quality control methods used for production of an astatine-211(211At)-labeled anti-CD45 monoclonal antibody (MAb) conjugate, 211At-BC8-B10, from the laboratory setting to cGMP production. Five separate materials were produced in the preparation of 211At-BC8-B10: (1) p-isothiocyanato-phenethyl-closo-decaborate(2-) (B10-NCS), (2) anti-CD45 MAb, BC8, (3) BC8-B10 MAb conjugate, (4) [211At]NaAt, and (5) 211At-BC8-B10. The 211At-labeling reagent, B10-NCS, was synthesized as previously reported. BC8 was produced, then conjugated with B10-NCS under cGMP conditions to form BC8-B10. [211At]NaAt was produced by α-irradiation of Bi targets, followed by isolation of the 211At using a "wet chemistry" method. The clinical product, 211At-BC8-B10, was prepared by reacting [211At]NaAt with BC8-B10 in NH4OAc buffer (pH 5.5) for 2 min at room temperature, followed by size-exclusion chromatography purification. Quality control tests conducted on the 211At-BC8-B10 included evaluations for purity and identity, as well as pyrogen and sterility tests. Stability of the 211At-BC8-B10 in 25 mg/mL sodium ascorbate solution was evaluated at 1, 2, 4, 6 and 21 h post isolation. For qualification, three consecutive 211At-BC8-B10 clinical preparations were successfully conducted in the cGMP suite, and an additional cGMP clinical preparation was carried out to validate each step required to deliver 211At-BC8-B10 to a patient. These cGMP preparations provided 0.80-1.28 Gbq (21.5-34.5 mCi) of 211At-BC8-B10 with radiochemical purity of >97%. The preparations were found to be sterile and have a pyrogen level <0.50 EU/mL. Cell binding was retained by the 211At-BC8-B10. 211At-BC8-B10 in ascorbic acid solution demonstrated a radiochemical stability of >95% for up to 21 h at room temperature. The experiments conducted have defined conditions for translation of 211At-BC8-B10 production from the laboratory to cGMP suite. This study has allowed the initiation of a phase I/II clinical trial using 211At-BC8-B10 (NCT03128034)., Competing Interests: The authors have declared that no competing interests exist.

Pretargeted radioimmunotherapy (PRIT) has demonstrated remarkable efficacy targeting tumor antigens, but immunogenicity and endogenous biotin blocking may limit clinical translation. We describe a new PRIT approach for the treatment of multiple myeloma (MM) and other B-cell malignancies, for which we developed an anti-CD38-bispecific fusion protein that eliminates endogenous biotin interference and immunogenic elements. In murine xenograft models of MM and non-Hodgkin lymphoma (NHL), the CD38-bispecific construct demonstrated excellent blood clearance and tumor targeting. Dosimetry calculations showed a tumor-absorbed dose of 43.8 Gy per millicurie injected dose of 90 Y, with tumor-to-normal organ dose ratios of 7:1 for liver and 15:1 for lung and kidney. In therapy studies, CD38-bispecific PRIT resulted in 100% complete remissions by day 12 in MM and NHL xenograft models, ultimately curing 80% of mice at optimal doses. In direct comparisons, efficacy of the CD38 bispecific proved equal or superior to streptavidin (SA)-biotin-based CD38-SA PRIT. Each approach cured at least 75% of mice at the highest radiation dose tested (1200 µCi), whereas at 600- and 1000-µCi doses, the bispecific outperformed the SA approach, curing 35% more mice overall ( P < .004). The high efficacy of bispecific PRIT, combined with its reduced risk of immunogenicity and endogenous biotin interference, make the CD38 bispecific an attractive candidate for clinical translation. Critically, CD38 PRIT may benefit patients with unresponsive, high-risk disease because refractory disease typically retains radiation sensitivity. We posit that PRIT might not only prolong survival, but possibly cure MM and treatment-refractory NHL patients., (© 2018 by The American Society of Hematology.)

Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Astatine-211 ( 211 At) is a promising cyclotron-produced radionuclide being investigated for use in targeted alpha therapy. The wet chemical isolation of trace quantities of 211 At, produced within several grams of Bi metal deposited onto an aluminum cyclotron target assembly, involves a multi-step procedure. Because the 211 At isolation method is labor-intensive and complex, automation of the method is being developed to facilitate routine processing at the University of Washington and to make it easier to transfer the process to other institutions. As part of that automation effort, a module useful in the initial step of the isolation procedure, dissolution of the Bi target, was designed and tested. The computer-controlled module performs in-line dissolution of Bi metal from the target assembly using an enclosed target dissolution block, routing the resulting solubilized 211 At/Bi mixture to the subsequent process step. The primary parameters involved in Bi metal solubilization (influent HNO 3 concentration and flow rate) were optimized prior to evaluation of the system using replicate 211 At-bearing cyclotron irradiated targets. The results indicate that the system performs in a predictable and reproducible manner, with cumulative Bi and 211 At recoveries following a sigmoidal function., (Copyright © 2017. Published by Elsevier Ltd.)

Streptavidin (SA)-biotin pretargeted radioimmunotherapy (PRIT) that targets CD20 in non-Hodgkin lymphoma (NHL) exhibits remarkable efficacy in model systems, but SA immunogenicity and interference by endogenous biotin may complicate clinical translation of this approach. In this study, we engineered a bispecific fusion protein (FP) that evades the limitations imposed by this system. Briefly, one arm of the FP was an anti-human CD20 antibody (2H7), with the other arm of the FP an anti-chelated radiometal trap for a radiolabeled ligand (yttrium[Y]-DOTA) captured by a very high-affinity anti-Y-DOTA scFv antibody (C825). Head-to-head biodistribution experiments comparing SA-biotin and bispecific FP (2H7-Fc-C825) PRIT in murine subjects bearing human lymphoma xenografts demonstrated nearly identical tumor targeting by each modality at 24 hours. However, residual radioactivity in the blood and normal organs was consistently higher following administration of 1F5-SA compared with 2H7-Fc-C825. Consequently, tumor-to-normal tissue ratios of distribution were superior for 2H7-Fc-C825 (P < 0.0001). Therapy studies in subjects bearing either Ramos or Granta subcutaneous lymphomas demonstrated that 2H7-Fc-C825 PRIT is highly effective and significantly less myelosuppressive than 1F5-SA (P < 0.0001). All animals receiving optimal doses of 2H7-Fc-C825 followed by 90 Y-DOTA were cured by 150 days, whereas the growth of tumors in control animals progressed rapidly with complete morbidity by 25 days. In addition to demonstrating reduced risk of immunogenicity and an absence of endogenous biotin interference, our findings offer a preclinical proof of concept for the preferred use of bispecific PRIT in future clinical trials, due to a slightly superior biodistribution profile, less myelosuppression, and superior efficacy. Cancer Res; 76(22); 6669-79. ©2016 AACR., (©2016 American Association for Cancer Research.)

Many patients with hematologic malignancies cannot tolerate hematopoietic cell transplantation (HCT), whereas others may not have a compatible human leukocyte antigen-matched donor. To overcome these limitations, we optimized a conditioning regimen employing anti-CD45 radioimmunotherapy (RIT) replacing total body irradiation (TBI) before haploidentical HCT in a murine model. Mice received 200 to 400 μCi (90)Y-anti-CD45 antibody (30F11), with or without fludarabine (5 days starting day -8), with cyclophosphamide (CY; days -2 and +2) for graft-versus-host disease prophylaxis, and 1.5 × 10(7) haploidentical donor bone marrow cells (day 0). Haploidentical bone marrow transplantation (BMT) with 300 μCi (90)Y-anti-CD45 RIT and CY, without TBI or fludarabine, led to mixed chimeras with 81.3 ± 10.6% mean donor origin CD8(+) cells detected 1 month after BMT, and remained stable (85.5 ± 11% mean donor origin CD8(+) cells) 6 months after haploidentical BMT. High chimerism levels were induced across multiple hematopoietic lineages 28 days after haploidentical BMT with 69.3 ± 14.1%, 75.6 ± 20.2%, and 88.5 ± 11.8% CD3(+) T cells, B220(+) B cells, and CD11b(+) myeloid cells, respectively. Fifty percent of SJL leukemia-bearing mice treated with 400 μCi (90)Y-DOTA-30F11, CY, and haploidentical BMT were cured and lived >200 days. Mice treated with 200 μCi (90)Y-DOTA-30F11 had a median overall survival of 73 days, while untreated leukemic mice had a median overall survival of 34 days (P < .001, Mantel-Cox test). RIT-mediated haploidentical BMT without TBI may increase treatment options for aggressive hematologic malignancies., (© 2016 by The American Society of Hematology.)

Unlabelled: α-radioimmunotherapy targeting CD45 may substitute for total-body irradiation in hematopoietic cell transplantation (HCT) preparative regimens for lymphoma. Our goal was to optimize the anti-CD45 monoclonal antibody (mAb; CA12.10C12) protein dose for (211)At-radioimmunotherapy, extending the analysis to include intraorgan (211)At activity distribution and α-imaging-based small-scale dosimetry, along with immunohistochemical staining., Methods: Eight normal dogs were injected with either a 0.75 (n = 5) or 1.00 (n = 3) mg/kg dose of (211)At-B10-CA12.10C12 (11.5-27.6 MBq/kg). Two were euthanized and necropsied 19-22 h after injection, and 6 received autologous HCT 3 d after (211)At-radioimmunotherapy, after lymph node and bone marrow biopsies at 2-4 and/or 19 h after injection. Blood was sampled to study toxicity and clearance; CD45 targeting was evaluated by flow cytometry. (211)At localization and small-scale dosimetry were assessed using two α-imaging systems: an α-camera and an ionizing-radiation quantum imaging detector (iQID) camera., Results: (211)At uptake was highest in the spleen (0.31-0.61% injected activity [%IA]/g), lymph nodes (0.02-0.16 %IA/g), liver (0.11-0.12 %IA/g), and marrow (0.06-0.08 %IA/g). Lymphocytes in blood and marrow were efficiently targeted using either mAb dose. Lymph nodes remained unsaturated but displayed targeted (211)At localization in T lymphocyte-rich areas. Absorbed doses to blood, marrow, and lymph nodes were estimated at 3.1, 2.4, and 3.4 Gy/166 MBq, respectively. All transplanted dogs experienced transient hepatic toxicity. Liver enzyme levels were temporarily elevated in 5 of 6 dogs; one treated with 1.00 mg mAb/kg developed ascites and was euthanized 136 d after HCT., Conclusion: (211)At-anti-CD45 radioimmunotherapy with 0.75 mg mAb/kg efficiently targeted blood and marrow without severe toxicity. Dosimetry calculations and observed radiation-induced effects indicated that sufficient (211)At-B10-CA12.10C12 localization was achieved for efficient conditioning for HCT., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Purpose: Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50-80 μm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments., Methods: The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ((211)At) activity distributions in cryosections of murine and canine tissue samples., Results: The highest spatial resolution was measured at ∼20 μm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10(-4) cpm/cm(2) (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the (211)At activity distribution was demonstrated at mBq/μg-levels., Conclusions: Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.

Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a β-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model.

α-Emitting radionuclides deposit a large amount of energy within a few cell diameters and may be particularly effective for radioimmunotherapy targeting minimal residual disease (MRD). To evaluate this hypothesis, (211)At-labeled 1F5 monoclonal antibody (mAb) (anti-CD20) was studied in both bulky lymphoma tumor xenograft and MRD animal models. Superior treatment responses to (211)At-labeled 1F5 mAb were evident in the MRD setting. Lymphoma xenograft tumor-bearing animals treated with doses of up to 48 µCi of (211)At-labeled anti-CD20 mAb ([(211)At]1F5-B10) experienced modest responses (0% cures but two- to threefold prolongation of survival compared with negative controls). In contrast, 70% of animals in the MRD lymphoma model demonstrated complete eradication of disease when treated with (211)At-B10-1F5 at a radiation dose that was less than one-third (15 µCi) of the highest dose given to xenograft animals. Tumor progression among untreated control animals in both models was uniformly lethal. After 130 days, no significant renal or hepatic toxicity was observed in the cured animals receiving 15 µCi of [(211)At]1F5-B10. These findings suggest that α-emitters are highly efficacious in MRD settings, where isolated cells and small tumor clusters prevail.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g) of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model.

The vast majority of patients with plasma cell neoplasms die of progressive disease despite high response rates to novel agents. Malignant plasma cells are very radiosensitive, but the potential role of radioimmunotherapy (RIT) in the management of plasmacytomas and multiple myeloma has undergone only limited evaluation. Furthermore, CD38 has not been explored as a RIT target despite its uniform high expression on malignant plasma cells. In this report, both conventional RIT (directly radiolabeled antibody) and streptavidin-biotin pretargeted RIT (PRIT) directed against the CD38 antigen were assessed as approaches to deliver radiation doses sufficient for multiple myeloma cell eradication. PRIT demonstrated biodistributions that were markedly superior to conventional RIT. Tumor-to-blood ratios as high as 638:1 were seen 24 hours after PRIT, whereas ratios never exceeded 1:1 with conventional RIT. (90)Yttrium absorbed dose estimates demonstrated excellent target-to-normal organ ratios (6:1 for the kidney, lung, liver; 10:1 for the whole body). Objective remissions were observed within 7 days in 100% of the mice treated with doses ranging from 800 to 1,200 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in treated mice compared with tumors that were 2,982% ± 2,834% of initial tumor volume in control animals) by day 23. Furthermore, 100% of animals bearing NCI-H929 multiple myeloma tumor xenografts treated with 800 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin achieved long-term myeloma-free survival (>70 days) compared with none (0%) of the control animals., (©2013 AACR.)

Despite aggressive chemotherapy combined with hematopoietic stem cell transplantation (HSCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using monoclonal antibodies labeled with β-emitting radionuclides has been explored to reduce relapse. β emitters are limited by lower energies and nonspecific cytotoxicity from longer path lengths compared with α emitters such as (211)At, which has a higher energy profile and shorter path length. We evaluated the efficacy and toxicity of anti-CD45 RIT using (211)At in a disseminated murine AML model. Biodistribution studies in leukemic SJL/J mice showed excellent localization of (211)At-anti-murine CD45 mAb (30F11) to marrow and spleen within 24 hours (18% and 79% injected dose per gram of tissue [ID/g], respectively), with lower kidney and lung uptake (8.4% and 14% ID/g, respectively). In syngeneic HSCT studies, (211)At-B10-30F11 RIT improved the median survival of leukemic mice in a dose-dependent fashion (123, 101, 61, and 37 days given 24, 20, 12, and 0 µCi, respectively). This approach had minimal toxicity with nadir white blood cell counts >2.7 K/µL 2 weeks after HSCT and recovery by 4 weeks. These data suggest that (211)At-anti-CD45 RIT in conjunction with HSCT may be a promising therapeutic option for AML.

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.

To reduce toxicity associated with external γ-beam radiation, we investigated radioimmunotherapy with an anti-CD45 mAb labeled with the α-emitter, astatine-211 ((211)At), as a conditioning regimen in dog leukocyte antigen-identical hematopoietic cell transplantation (HCT). Dose-finding studies in 6 dogs treated with 100 to 618 μCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) without HCT rescue demonstrated dose-dependent myelosuppression with subsequent autologous recovery, and transient liver toxicity in dogs treated with (211)At doses less than or equal to 405 μCi/kg. Higher doses of (211)At induced clinical liver failure. Subsequently, 8 dogs were conditioned with 155 to 625 μCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) before HCT with dog leukocyte antigen-identical bone marrow followed by a short course of cyclosporine and mycophenolate mofetil immunosuppression. Neutropenia (1-146 cells/μL), lymphopenia (0-270 cells/μL), and thrombocytopenia (1500-6560 platelets/μL) with prompt recovery was observed. Seven dogs had long-term donor mononuclear cell chimerism (19%-58%), whereas 1 dog treated with the lowest (211)At dose (155 μCi/kg) had low donor mononuclear cell chimerism (5%). At the end of follow-up (18-53 weeks), only transient liver toxicity and no renal toxicity had been observed. In conclusion, conditioning with (211)At-labeled anti-CD45 mAb is safe and efficacious and provides a platform for future clinical trials of nonmyeloablative transplantation with radioimmunotherapy-based conditioning.

Purpose: Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, "endogenous" biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that downmodulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem., Experimental Design: The single-chain variable region gene of the murine 1F5 anti-CD20 antibody was fused to the wild-type (WT) SAv gene and to mutant SAv genes, Y43A-SAv and S45A-SAv. FPs were expressed, purified, and compared in studies using athymic mice bearing Ramos lymphoma xenografts., Results: Biodistribution studies showed delivery of more radioactivity to tumors of mice pretargeted with mutant SAv FPs followed by (111)In-DOTA-bis-biotin [6.2 ± 1.7% of the injected dose per gram (%ID/gm) of tumor 24 hours after Y43A-SAv FP and 5.6 ± 2.2%ID/g with S45A-SAv FP] than in mice on normal diets pretargeted with WT-SAv FP (2.5 ± 1.6%ID/g; P = 0.01). These superior biodistributions translated into superior antitumor efficacy in mice treated with mutant FPs and (90)Y-DOTA-bis-biotin [tumor volumes after 11 days: 237 ± 66 mm(3) with Y43A-SAv, 543 ± 320 mm(3) with S45A-SAv, 1129 ± 322 mm(3) with WT-SAv, and 1435 ± 212 mm(3) with control FP (P < 0.0001)]., Conclusions: Genetically engineered mutant-SAv FPs and bis-biotin reagents provide an attractive alternative to current SAv-biotin PRIT methods in settings where endogenous biotin levels are high., (©2011 AACR.)

Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)-streptavidin (SA) conjugate and DOTA-biotin labeled with β-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. α-emitting radionuclides exhibit high cytotoxicity coupled with a short path length, potentially increasing the therapeutic index and making them an attractive alternative to β-emitting radionuclides for patients with acute myeloid leukemia. Accordingly, we have used (213)Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of (213)Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5% ± 1.1% of the injected dose of (213)Bi was delivered per gram of tumor. α-imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after (213)Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a β-emitting radionuclide ((90)Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of (213)Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 μCi of (213)Bi- or (90)Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for more than 100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an α-emitting radionuclide may be highly effective and minimally toxic for treatment of acute myeloid leukemia.

Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-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.

Radioimmunotherapy (RIT) with α-emitting radionuclides is an attractive approach for the treatment of minimal residual disease because the short path lengths and high energies of α-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 α-emitters with short half-lives, such as bismuth-213 (²¹³Bi). Athymic mice bearing Ramos lymphoma xenografts received anti-CD20 1F5(scFv)(4)SA fusion protein (FP), followed by a dendrimeric clearing agent and [²¹³Bi]DOTA-biotin. After 90 minutes, tumor uptake for 1F5(scFv)₄SA was 16.5% ± 7.0% injected dose per gram compared with 2.3% ± .9% injected dose per gram for the control FP. Mice treated with anti-CD20 PRIT and 600 μ Ci [²¹³Bi]DOTA-biotin exhibited marked tumor growth delays compared with controls (mean tumor volume .01 ± .02 vs. 203.38 ± 83.03 mm³ after 19 days, respectively). The median survival for the 1F5(scFv)₄SA group was 90 days compared with 23 days for the control FP (P < .0001). Treatment was well tolerated, with no treatment-related mortalities. This study demonstrates the favorable biodistribution profile and excellent therapeutic efficacy attainable with ²¹³Bi-labeled anti-CD20 PRIT.

Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv(4)-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a monobiotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv(4)-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents, good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-trigalactose blood clearance reagent, 2, was designed, synthesized, and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g., (111)In, (90)Y, (177)Lu) could be used in the pretargeting protocols employing scFv(4)-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [(125)I]scFv(4)-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 and [(111)In]4b) with standard reagents (1 and [(111)In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv(4)-SAv-S45A or scFv(4)-SAv-Y43A) were employed in combination with CA 2 and [(111)In]4b. Importantly, normal tissue concentrations of [(111)In]4b were similar to those obtained using the standard reagents (1 and [(111)In]3b), except that the blood and liver concentrations were slightly higher with the new reagents. While the reasons for the higher blood and liver concentrations are unknown, the differences in the galactose structures of the clearance agents 1 and 2 may play a role.

Background: A pilot study was conducted to determine whether conditioning using selective targeting of hematopoietic cells with an alpha-particle emitter, bismuth-213 ((213)Bi)-labeled anti-CD45 monoclonal antibody (mAb) is sufficient to overcome the major histocompatibility barrier in a canine model of dog leukocyte antigen-haploidentical hematopoietic cell transplantation (HCT)., Methods: Six dogs were administered 0.5 mg/kg (213)Bi-labeled anti-CD45 mAb (dose (213)Bi=2.26-4.9 mCi/kg) in six to eight injections. For postgrafting immunosuppression, all dogs received cyclosporine and mycophenolate mofetil., Results: All dogs had initial donor engraftment, with three of six dogs having sustained engraftment to last point of follow-up. Two dogs receiving 2.26 and 3.25 mCi/kg of (213)Bi rejected their grafts at day +127 and +125, respectively, whereas dogs receiving (213)Bi doses of 3.3 mCi/kg or greater achieved high level donor chimerism., Conclusion: The results suggest that nonmyeloablative conditioning with (213)Bi-labeled anti-CD45 mAb could be applicable to major histocompatibility haploidentical HCT without excessive nonhematologic regimen-related toxicity.

Introduction: In vivo deastatination of (211)At-labeled biomolecules can severely limit their use in endoradiotherapy. Our studies have shown that the use of closo-decaborate(2-) moiety for (211)At-labeling of biomolecules provides high in vivo stability towards deastatination. However, data from those studies have also been suggestive that some astatinated closo-decaborate(2-) catabolites may be retained in tissues. In this study, we investigated the in vivo distributions of several structurally simple closo-decaborate(2-) derivatives to gain information on the effects of functional groups if catabolites are released into the blood system from the carrier biomolecule., Methods: Thirteen closo-decaborate(2-) derivatives were synthesized and radioiodinated for evaluation. Tissue concentrations of the radioiodinated compounds were obtained in groups of five mice at 1 and 4 h postinjection (pi). Dual-label ((125)I and (131)I) experiments permitted evaluation of two compounds in each set of mice., Results: All of the target compounds were readily synthesized. Radioiodination reactions were conducted with chloramine-T and Na[(125/131)I]I in water to give high yields (75-96%) of the desired compounds. Biodistribution data at 1 and 4 h pi (representing catabolites released into the blood system) showed small differences in tissue concentrations for some compounds, but large differences for others. The results indicate that formal (overall) charge on the compounds could not be used as a predictor of tissue localization or retention. However, derivatives containing carboxylate groups generally had lower tissue concentrations. Acid cleavable hydrazone functionalities appeared to be the best candidates for further study., Conclusions: Further studies incorporating hydrazone functionalities into pendant groups for biomolecule radiohalogenation are warranted., ((c) 2010 Elsevier Inc. All rights reserved.)

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.

Pretargeted radioimmunotherapy (PRIT) is designed to enhance the directed delivery of radionuclides to malignant cells. Through a series of studies in 19 nonhuman primates (Macaca fascicularis), the potential therapeutic advantage of anti-CD45 PRIT was evaluated. Anti-CD45 PRIT demonstrated a significant improvement in target-to-normal organ ratios of absorbed radiation compared with directly radiolabeled bivalent antibody (conventional radioimmunotherapy [RIT]). Radio-DOTA-biotin administered 48 hours after anti-CD45 streptavidin fusion protein (FP) [BC8 (scFv)(4)SA] produced markedly lower concentrations of radiation in nontarget tissues compared with conventional RIT. PRIT generated superior target:normal organ ratios in the blood, lung, and liver (10.3:1, 18.9:1, and 9.9:1, respectively) compared with the conventional RIT controls (2.6:1, 6.4:1, and 2.9:1, respectively). The FP demonstrated superior retention in target tissues relative to comparable directly radiolabeled bivalent anti-CD45 RIT. The time point of administration of the second step radiolabeled ligand (radio-DOTA-biotin) significantly impacted the biodistribution of radioactivity in target tissues. Rapid clearance of the FP from the circulation rendered unnecessary the addition of a synthetic clearing agent in this model. These results support proceeding to anti-CD45 PRIT clinical trials for patients with both leukemia and lymphoma.

Relapsed B-cell lymphomas are currently incurable with conventional chemotherapy and radiation treatments. Radiolabeled antibodies directed against B-cell surface antigens have emerged as effective and safe therapies for relapsed lymphomas. We therefore investigated the potential utility of both directly radiolabeled 1F5 (anti-CD20), HD39 (anti-CD22), and Lym-1 (anti-DR) antibodies (Abs) and of pretargeted radioimmunotherapy (RIT) using Ab-streptavidin (SA) conjugates, followed by an N-acetylgalactosamine dendrimeric clearing agent and radiometal-labeled DOTA-biotin, for treatment of lymphomas in mouse models using Ramos, Raji, and FL-18 human lymphoma xenografts. This study demonstrates the marked superiority of pretargeted RIT for each of the antigenic targets with more complete tumor regressions and longer mouse survival compared with conventional one-step RIT. The Ab-SA conjugate yielding the best tumor regression and progression-free survival after pretargeted RIT varied depending upon the lymphoma cell line used, with 1F5 Ab-SA and Lym-1 Ab-SA conjugates yielding the most promising results overall. Contrary to expectations, the best rates of mouse survival were obtained using optimal single Ab-SA conjugates rather than combinations of conjugates targeting different antigens. We hypothesize that clinical implementation of pretargeted RIT methods will provide a meaningful prolongation of survival for patients with relapsed lymphomas compared with currently available treatment strategies.

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.

We previously investigated the potential of targeted radiotherapy using a bismuth-213 ((213)Bi)-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. Although this approach allowed sustained marrow engraftment, limited availability, high cost, and short half-life of (213)Bi induced us to investigate an alternative alpha-emitting radionuclide, astatine-211 ((211)At), for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either (213)Bi or (211)At. Mice were injected with 2 to 50 muCi on 10 microg or 20 muCi on 2 or 40 microg of 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167 +/- 23% to 417 +/- 109% injected dose/gram (% ID/g) after injection of the (211)At conjugate and 45 +/- 9% to 166 +/- 11% ID/g after injection of the (213)Bi conjugate. The higher concentrations observed for (211)At-labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. (211)At was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 muCi (211)At, but none with the same quantities of (213)Bi, had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 muCi (213)Bi, but not with lower doses of (213)Bi or with any dose of (211)At. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of (211)At-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less nonhematologic toxicity compared with (213)Bi-labeled antibody.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted (P)RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by a biotinylated clearing agent and radiolabeled 1,4,7,10-tetraazacylodode cane N,N',N",N'''-tetraacetic (DOTA)-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the PRIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled Ab for a prolonged time. Therapy experiments showed that (90)Y-DOTA-biotin significantly prolonged survival of mice treated with pretargeted anti-hCD45 Ab-SA compared with mice treated with conventional RIT using (90)Y-labeled anti-hCD45 Ab at 200 muCi. Because human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and PRIT using an anti-murine (m)CD45 Ab where the target antigen is present on normal hematopoietic tissues. After 24 h, 27.3% +/- 2.8% of the injected dose of activity was delivered per gram (% ID/g) of lymph node using (131)I-A20-Ab compared with 40.0 +/- 5.4% ID/g for pretargeted (111)In-DOTA-biotin. These data suggest that pretargeted methods for delivering RIT may be superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

We describe the use of pretargeted radioimmunotherapy (PRIT) using an anti-murine CD45 antibody-streptavidin (SA) conjugate followed by radiobiotin to deliver radiation selectively to murine hematolymphoid tissues, which may potentially augment the efficacy and decrease the toxicity of radioimmunotherapy for disseminated murine leukemia. Biodistribution and therapeutic results demonstrated high target organ to nontarget organ ratios of radioactivity and significant long-term survival in leukemic mice using PRIT. These data suggest that anti-CD45 PRIT using an anti-CD45-SA conjugate in a syngeneic murine model of disseminated leukemia may be more effective and less toxic than directly labeled monoclonal antibodies.

We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the short-lived alpha-particle-emitting radionuclides 213Bi ( t 1/2 = 45.6 min) and 211At ( t 1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides and also modified in a manner that diminishes its natural propensity for localization in the kidney. Modification for labeling with (213)Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A'' (CHX-A''), 3a. Modification for direct labeling with 211At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG/decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50-100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated rapidly radiolabeled with 213Bi (<5 min), providing a 72% isolated yield. 211At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66-71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in a much lower radiolabeling yield (18%). The 213Bi or 211At-labeled modified rSAv preparations were mixed with the corresponding 125 I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213Bi-labeled succinylated rSAv [ 213Bi] 6a has tissue concentrations similar to those of 125 I-labeled modified rSAv [ 125 I] 6b, suggesting that (213)Bi is quite stable toward release from the chelate in vivo. In vivo data also indicate that the (211)At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains an m-dPEG 12 moiety, has significantly decreased concentrations in blood and other tissues when compared with those of direct-labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A'' and succinylated (i.e., 5a) may be useful as a carrier of 213Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e., 5c) suggests that its further study as a carrier of 211At in pretargeting protocols is warranted.