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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.