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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 90Y-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 (90Y-DOTA injections. SJL leukemia mice treated with the bispecific 30F11-IgG1-C825 and 29.6 MBq (800 μCi) of 90Y-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.

Supplementary Materials, Methods and relevant AA sequences

Table S1 presents mouse survival data for in vivo experiments

Figures including: Supplemental Figure 1- Dose finding pharmacokinetics. Supplemental Figure 2- Dose finding tissue biodistribution. Supplemental Figure 3- body weight over time in a therapy study. Supplemental Figure 4- blood counts over time in a therapy study. Supplemental Figure 5- fusion protein binding to Ramos and Granta cell lines

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 90Y-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.

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 111In-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 90Y-DOTA-bis-biotin [tumor volumes after 11 days: 237 ± 66 mm3 with Y43A-SAv, 543 ± 320 mm3 with S45A-SAv, 1129 ± 322 mm3 with WT-SAv, and 1435 ± 212 mm3 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. Clin Cancer Res; 17(23); 7373–82. ©2011 AACR.

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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.

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Supplemental Figure 2. Cerenkov Light Images of excised organs. Groups of 5 mice were given 300 µCi on 100 µg of 90Y-DOTA-30F11 (A), 177Lu-DOTA-30F11 (B), 90Y-DOTA-Rat IgG (C), or 177Lu-DOTA-Rat IgG (D). Groups of mice were harvested and images collected at 4, 24, 48, and 72 hours post-injection, immediately prior to gamma counting of the tissues. By 24 hours post-injection, there was little or no detectable signal in non-target organs and tissues.

Supplementary Methods from Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the α-Emitting Radionuclides Bismuth-213 or Astatine-211

Supplemental Figure 1. In vitro phantom containing 100 μCi 177Lu. Phantom images were obtained by placing a minimal volume of activity source within a 1.5 mL polypropylene tube, which was then suspended in water inside a larger polypropylene tube. This allowed simulation of Cerenkov radiation through an aquatic medium in order to more closely mimic an activity source within tissue.

Enriched tungsten disulfide (186WS2) was evaluated at increasing proton beam currents (20–50 μA) and times (up to 4 h) on a GE PETtrace cyclotron for production of high specific activity (HSA) 186Re. The HSA 186Re was separated from the irradiated target as [186Re][ReO4]– by a liquid–liquid extraction method and radiolabeled with a new N2S2 ligand (222-MAMA-N-ethylpropionate). The enriched 186W was recovered from the extraction process, analyzed for purity and enrichment, and converted back to the disulfide (186WS2). The results demonstrate that the 186WS2 is an easily pressed target material that can withstand relatively high currents and can be readily recovered and recycled. The 186Re produced was isolated in high specific activity and readily formed the radiotracers [186Re][ReO(222-MAMA-N-ethylpropionate)] and [186Re][Re(CO)3(OH2)3] +.

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 (89Zr-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 89Zr-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 89Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard. Results In tumor-bearing mice, 89Zr-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. 89Zr-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 89Zr-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 89Zr-αGPC3 avidly accumulated in GPC3+ tumors with minimal off-target sequestration. 89Zr-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.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide with limited therapeutic options for advanced disease. Targeted alpha therapy (TAT) is an emerging class of targeted cancer therapy in which alpha-particle-emitting radionuclides, such as thorium-227, are specifically delivered 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 227Th-labeled GPC3 targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. METHODS: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3 targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring percentage of protein-bound 227Th by gamma-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2 cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 was evaluated in tumor bearing mice. Efficacy of 227Th-octapa-αGPC3 was assessed in tumor bearing animals with serial measurement of serum alpha-fetoprotein at 23 days after radionuclide injection. RESULTS: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, ≥97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate buffered saline. In HepG2 tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. 23 days after treatment, significant reduction in tumor burden was observed in mice receiving 500 kBq/kg 227Th-octapa-αGPC3 by tail vein injection. No acute off-target toxicity was observed and no animals died prior to termination of the study. CONCLUSION:227Th-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.

Targeted alpha therapy (TAT) is an emerging and powerful tool for treating late-stage cancers for which therapeutic options are limited. At the core of TAT is the development of targeted radiopharmaceuticals, where isotopes are paired with biological targeting vectors that enable tissue- or cell-specific delivery of alpha-emitters. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriamine pentaacetic acid (DTPA) are commonly used to chelate metallic radionuclides, but have several limitations. Significant efforts are underway to develop effective and stable chelators for alpha-emitters and are at various stages of development and community adoption. Isotopes such as 149Tb, 212/213Bi, 212Pb (for 212Bi), 225Ac, 226/227Th have identified suitable chelators, although further studies, especially in vivo studies, are required; while for others, including 223Ra, 230U, and arguably 211At an ideal chelate remains elusive. In this review, we will summarize the chelation chemistry reported to date for the incorporation of 149Tb, 211At, 212/213Bi, 212Pb (for 212Bi), 223Ra, 225Ac, 226/227Th and 230U into radiopharmaceuticals, with a focus on new discoveries and remaining challenges.

The alpha particle-emitting radionuclide astatine-211 (

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 211At-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 211At/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.

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 (

Purpose Thorium-226 (half-life 30.6 m) is a radionuclide of interest for use in targeted alpha therapy applications. Due to its short half-life, 226Th must be provided through a radionuclide generator system from its parent 230U (20.8 d). Furthermore, as the half-life of 226Th is very short, it should be provided in a form that is directly amenable to use in biomedical applications. Methods A reverse radionuclide generator system was developed employing a DGA extraction chromatography column. A 230U/226Th parent/daughter solution in equilibrium is added to a DGA column in >6 M HCl. The parent 230U is eluted first in 0.1 M HNO3 followed by elution of 226Th in 0.1 M citrate buffer pH 5. Results Thorium-226 was recovered from the radionuclide generator column with >96% yield. Greater than 99.5% of the 230U parent was isolated for reuse in the generator. Long term evaluation over six weeks demonstrated consistent supply of 226Th with greater than 99.5% radionuclidic purity. The only contaminant found in the final product was 230U ( Conclusions The reverse radionuclide generator described herein was shown to be a feasible method for providing 226Th in high yield, purity and in a chemical form that is amenable for direct use in biomedical applications.

Low-density aerogel materials are a promising platform for delivering lipophilic drugs with poor water solubility, but to date, the loading of aerogels with pharmaceuticals in crystalline form has not been reported other than by expensive supercritical loading techniques. Here, we demonstrate a simple and low-cost liquid-phase impregnation method to load the model drug coenzyme Q10 (CoQ10) within carbon aerogels. By controlling the chemical microstructure of the aerogel to exhibit π-conjugated (sp2) bonding via addition of graphene oxide, the rate of adsorption and total loading of CoQ10 is significantly enhanced. Furthermore, the adsorbed CoQ10 adopts an as-yet unreported crystalline phase that differs from the bulk material, not only offering the potential advantages of crystalline materials for drug delivery, but also a means to control the microstructure of this important lipophilic pharmaceutical.

Increasing access to the short-lived α-emitting radionuclide astatine-211 (211At) has the potential to advance targeted α-therapeutic treatment of disease and to solve challenges facing the medical...

Alzheimer's disease (AD) is a terminal neurodegenerative disease characterized by the buildup of amyloid fibrils, amorphous aggregates and tauopathies. Several treatment modalities, which rely on various biological processes to reduce disease burden, have been largely ineffective at treating Alzheimer's disease. Targeted alpha therapy (TAT) has demonstrated positive results in the treatment of cancer. Benzothiazole derivatives have been successfully shown to target these plaques and are used in several imaging applications. One such derivative, Flutemetamol (Vizamyl

The alpha particle-emitting radionuclide astatine-211 (211At) is of interest for targeted radiotherapy; however, low in vivo stability of many 211At-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 dPEG4-amino acid methyl ester derivatives were prepared as HPLC standards. The corresponding para-stannylbenzoic acid derivatives were also prepared and labeled with 125I and 211At. Oxidization of the [125I]iodo- and [211At]astato-benzamidyl-dPEG4-acid methyl ester derivatives provided materials for in vivo evaluation. A biodistribution was conducted in mice with coinjected oxidized 125I- and 211At-labeled compounds. The oxidized radioiodinated derivative was stable to in vivo deiodination, but unfortunately the oxidized [211At]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 [211At]NaAtO3 and [125I]NaIO3. Comparison of the tissue concentrations of the isolated material from the oxidized [211At]benzamide derivative with those of [211At]astatate indicated the species obtained after isolation was likely [211At]astatate.

Targeted α-therapy (TAT) is an emerging powerful tool treating late-stage cancers for which therapeutic options are limited. At the core of TAT are targeted radiopharmaceuticals, where isotopes are paired with targeting vectors to enable tissue- or cell-specific delivery of α-emitters. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and DTPA (diethylenetriamine pentaacetic acid) are commonly used to chelate metallic radionuclides but have limitations. Significant efforts are underway to develop effective stable chelators for α-emitters and are at various stages of development and community adoption. Isotopes such as (149)Tb, (212/213)Bi, (212)Pb (for (212)Bi), (225)Ac, and (226/227)Th have found suitable chelators, although further studies, especially in vivo studies, are required. For others, including (223)Ra, (230)U, and, arguably (211)At, the ideal chemistry remains elusive. This review summarizes the methods reported to date for the incorporation of (149)Tb, (211)At, (212/213)Bi, (212)Pb (for (212)Bi), (223)Ra, (225)Ac, (226/227)Th, and (230)U into radiopharmaceuticals, with a focus on new discoveries and remaining challenges.

The halogens bromine and iodine have similar chemical properties and undergo similar reactions due to their closeness in Group 17 of the periodic chart. There are a number of bromine and iodine radionuclides that have properties useful for diagnosis and therapy of human diseases. The emission properties of radiobromine and radioiodine nuclides with half-lives longer than 1 h are summarized along with properties that make radionuclides useful in PET/SPECT imaging and β/Auger therapy, such that the reader can assess which of the radionuclides might be useful for medical applications. An overview of chemical approaches that have been used to radiolabel molecules with radiobromine and radioiodine nuclides is provided with examples. Further, references to a large variety of different organ/cancer-targeting agents utilizing the radiolabeling approaches described are provided.

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 ((211)At) 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 (211)At to an anti-CD38 monoclonal antibody to create an (211)At-CD38 therapy. When examined in a bulky xenograft model of MM, single-dose (211)At-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 (211)At-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 (211)At-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 (211)At-CD38 to an autologous stem cell transplant (ASCT) conditioning regimen may improve ASCT outcomes for MM patients.

Encouraging results from targeted α-therapy have received significant attention from academia and industry. However, the limited availability of suitable radionuclides has hampered widespread translation and application. In the present review, we discuss the most promising candidates for clinical application and the state of the art of their production and supply. In this review, along with 2 forthcoming reviews on chelation and clinical application of α-emitting radionuclides, The Journal of Nuclear Medicine will provide a comprehensive assessment of the field. ispartof: J Nucl Med vol:62 issue:11 pages:1495-1503 ispartof: location:United States status: published

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 (89Zr) and yttrium-90 (90Y) 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 89Zr-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 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax 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 (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-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 (R2 = 0.87), and GTV of animals treated with 90Y-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 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Thorium-226 (half-life 30.6 m) is a radionuclide of interest for use in targeted alpha therapy applications. Due to its short half-life,A reverse radionuclide generator system was developed employing a DGA extraction chromatography column. AThorium-226 was recovered from the radionuclide generator column with96% yield. Greater than 99.5% of theThe reverse radionuclide generator described herein was shown to be a feasible method for providing

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 (t1/2 = 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, H4 octapa 3 (65%) and H4 py4pa 6 (87%). No reaction occurred with H4 neunpa-p-Bn-NO2 4, and the chelation reaction with another pa ligand H4 pypa 5 gave inconsistent yields with a very broad radio-HPLC peak. The ligands spermine-(Me-3,2-HOPO)4 1, H4 octapa 3, and H4 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 (t1/2 = 30.57 min) when heated to 80°C for 5 min.

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 (Ab)-streptavidin (SA) conjugate and radiolabeled DOTA-biotin. To overcome these limitations we designed anti-murine and anti-human CD45 bispecific antibody (Ab) constructs using 30F11 and BC8 Ab, 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 (1200 [Formula: see text] Ci) of (90)Y-DOTA-biotin survived at least 170 days after therapy, while all non-treated controls required euthanasia due to 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 (

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 (211At) to metastatic PCa. High kidney localization has been a problem with radiolabeled LuG derivatives, but has been adequately addressed in radiometal-labeled DOTA-LuG derivatives by linker optimization. Herein, we report an investigation of an alternate approach to diminishing the kidney concentrations of radiolabeled LuG-containing compounds. 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 [131I]16b, an unconjugated LuG derivative containing a dPEG4® linker, provided tumor concentrations of ~15% ID/g at 4 h pi. A second unconjugated LuG derivative with a similar structure, but containing a dPEG12® linker, [131I]16a had tumor concentrations of ~4%ID/g at 4 h pi. Those results suggest that long PEG linkers also affect tumor localization in a negative manner. 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 211At. Advances in knowledge and implications for patient care: This study is the first to evaluate PEGylated LuG and closo-decaborate (2-) moieties conjugated to proteins as potential methods for diminishing the kidney concentrations of radiolabeled LuG-containing compounds.

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 90Y, 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.

The purpose of this study is to develop a thorium-227 (227Th) antibody radioimmunoconjugate targeting glypican-3 (GPC3) and to test its therapeutic efficacy in a hepatocellular carcinoma (HCC) orthotopic xenograft model. GPC3 targeting antibody (αGPC3) was conjugated to bifunctional chelator p-SCN-Bn-H4octapa (octapa), and αGPC3-octapa binding affinity for GPC3 was evaluated by flow cytometry. 227Th radiolabeling of this conjugate was optimized, and in vitro stability of 227Th-αGPC3-octapa was evaluated in PBS with and without free radical scavenging agent over 14 days. For in vivo evaluation, an orthotopic xenograft model was generated by hepatic subcapsular injection of human HCC HepG2 cells. In vivo biodistribution was assessed in blood, tumor and organs at 1, 7 and 21 days after tail vein injection of 227Th-αGPC3-octapa (500 KBq/kg). To test therapeutic efficacy, tumor-bearing animals were injected with 227Th-αGPC3-octapa (250 kBq/kg or 500 kBq/kg) and compared to an irrelevant control antibody, 227Th-αBHV1-octapa (500 kBq/kg), and to a no-treatment control. Tumor burden was assessed with serial serum alpha-fetoprotein (AFP) measurements, a marker of tumor burden validated in this model. Toxicity to 227Th-αGPC3-octapa was measured by serum comprehensive metabolic panel obtained 21 days after injection. GPC3 binding affinity was highest after conjugation of αGPC3 with 10 equivalents of octapa. The protein recovery from the conjugation process was >85%, and mass spectral analysis indicated an average of 3.3 octapa moieties per molecule of αGPC3-octapa. After two weeks, >98% of αGPC3-octapa and αBHV1-octapa had 227Th bound in the presence of scavenging agent. αGPC3-octapa maintained high affinity for GPC3 as measured by flow cytometry, indicating the octapa conjugation reaction did not alter immunoreactivity of αGPC3. In vivo, 227Th-αGPC3-octapa accumulated in the tumor over time and cleared from normal tissues with a %ID/g of In conclusion, we report the development of a GPC3 targeted thorium conjugate and demonstrate its in vivo therapeutic efficacy in a murine orthotopic xenograft model of hepatocellular carcinoma. Citation Format: Kevin P. Labadie, Donald K. Hamlin, Aimee Kenoyer, Sara K. Daniel, Alan F. Utria, Andrew D. Ludwig, Heidi L. Kenerson, Delphine L. Chen, Johnnie Orozco, Raymond S. Yeung, Lily Li, Chris Orvig, Yawen Li, D Scott Wilbur, James O. Park. Glypican-3 targeted thorium-227 alpha therapy reduces tumor burden in an orthotopic xenograft model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 928.

In a canine model of pre-sensitization 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 pre-sensitization 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, which has a short path length, very high energy, and a short t 1/2 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, -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 seven 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 one year. Graft-versus-host disease (GVHD) 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 pre-sensitization 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.

Production of high specific activity 186gRe is of interest for development of theranostic radiopharmaceuticals. Previous studies have shown that high specific activity 186gRe can be obtained by cyclotron irradiation of enriched 186W via the 186W(d,2n)186gRe reaction, but most irradiations were conducted at low beam currents and for short durations. In this investigation, enriched 186W metal targets were irradiated at high incident deuteron beam currents to demonstrate production rates and contaminants produced when using thick targets. Full-stopping thick targets, as determined using SRIM, were prepared by uniaxial pressing of powdered natural abundance W metal or 96.86% enriched 186W metal encased between two layers of graphite flakes for target material stabilization. An assessment of structural integrity was made on each target preparation. To assess the performance of graphite-encased thick 186W metal targets, along with the impact of encasing on the separation chemistry, targets were first irradiated using a 22 MeV deuteron beam for 10 min at 10, 20, and 27 μA, with an estimated nominal deuteron energy of 18.7 MeV on the 186W target material (after energy degradation correction from top graphite layer). Gamma-ray spectrometry was performed post EOB on all targets to assess production yields and radionuclidic byproducts. The investigation also evaluated a method to recover and recycle enriched target material from a column isolation procedure. Material composition analyses of target materials, pass-through/wash solutions and recycling process isolates were conducted with SEM, FTIR, XRD, EDS and ICP-MS spectrometry. To demonstrate scaled-up production, a graphite-encased 186W target made from recycled 186W was irradiated for ~2 h with 18.7 MeV deuterons at a beam current of 27 μA to provide 0.90 GBq (24.3 mCi) of 186gRe, decay-corrected to the end of bombardment. ICP-MS analysis of the isolated 186gRe solution provided data that indicated the specific activity of 186gRe in this scaled-up production run was 2.6±0.5 GBq/μg (70±10 Ci/mg).

Introduction The availability of 211 At for targeted alpha therapy research can be increased by the 211 Rn/ 211 At generator system, whereby 211 At is produced by 211 Rn electron capture decay. This study demonstrated the feasibility of using generator-produced 211 At to label monoclonal antibody (BC8, anti-human CD45) for preclinical use, following isolation from the 207 Po contamination also produced by these generators (by 211 Rn α-decay). Methods 211 Rn was produced by 211 Fr electron capture decay following mass separated ion beam implantation and chemically isolated in liquid alkane hydrocarbon (dodecane). 211 At produced by the resulting 211 Rn source was extracted in strong base (2N NaOH) and purified by granular Te columns. BC8-B10 (antibody conjugated with closo -decaborate(2-)) was labeled with generator-produced 211 At and purified by PD-10 columns. Results Aqueous solutions extracted from the generator were found to contain 211 At and 207 Po, isolated from 211 Rn. High radionuclidic purity was obtained for 211 At eluted from Te columns, from which BC8-B10 monoclonal antibody was successfully labeled. If not removed, 207 Po was found to significantly contaminate the final 211 At-BC8-B10 product. High yield efficiencies (decay-corrected, n =3) were achieved for 211 At extraction from the generator (86%±7%), Te column purification (70%±10%), and antibody labeling (76%±2%). Conclusions The experimental 211 Rn/ 211 At generator was shown to be well-suited for preclinical 211 At-based research. Advances in knowledge We believe that these experiments have furthered the knowledge-base for expanding accessibility to 211 At using the 211 Rn/ 211 At generator system. Implications for patient care As established by this work, the 211 Rn/ 211 At generator has the capability of facilitating preclinical evaluations of 211 At-based therapies.

Astatine-211 (211At) is a promising cyclotron-produced radionuclide being investigated for use in targeted alpha therapy. The wet chemical isolation of trace quantities of 211At, produced within several grams of Bi metal deposited onto an aluminum cyclotron target assembly, involves a multi-step procedure. Because the 211At 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 211At/Bi mixture to the subsequent process step. The primary parameters involved in Bi metal solubilization (influent HNO3 concentration and flow rate) were optimized prior to evaluation of the system using replicate 211At-bearing cyclotron irradiated targets. The results indicate that the system performs in a predictable and reproducible manner, with cumulative Bi and 211At recoveries following a sigmoidal function.

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 211At isolation systems are of high interest for improving the availability of 211At. Based on the early studies of Bochvarova and co-workers involving isolation of 211At from irradiated thorium targets, we developed a method for 211At isolation from bismuth targets using tellurium-packed columns. Dissolution of irradiated bismuth targets is accomplished using HNO3; however, 211At is not captured on the Te column material in this matrix. Our method involves slow addition of aqueous NH2OH·HCl to the Bi target dissolved in HNO3 to convert to a HCl matrix. The amount of NH2OH·HCl was optimized because (1) the quantity of NH2OH·HCl used appears to affect the radiolabeling yield of phenethyl-closo-decaborate(2-) (B10)-conjugated antibodies and (2) reducing the volume of NH2OH·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 211At. High isolation yields (88–95%) were obtained. Radiochemical purity of the isolated 211At was assessed by radio-HPLC. Concentrations of Bi and Te contaminants in the 211At and the astatinated antibodies were evaluated using ICP-MS.

α-Emitting radionuclides have radiobiological properties that make them particularly attractive for the therapy of a number of difficult-to-treat diseases—such as cancers that are blood-borne or disseminated throughout the body—as well as residual cancer remaining after surgery. While there are many α-emitting radionuclides in nature, only ten have been identified as potentially useful for medical applications. And unfortunately, most of the members of this useful subset are difficult to obtain or are very expensive to produce. Despite these limitations, however, the enticing prospect of using these radionuclides to address medically unmet needs has prompted efforts to attach them to disease-targeting vectors and test their therapeutic efficacy in animal models of disease. Both metallic and non-metallic α-emitting radionuclides have been explored for targeted therapy, and as such, a variety of approaches to incorporating these radionuclides into targeting vectors have been developed, including direct covalent modification and the use of bifunctional chelators. While a fair number of bifunctional chelators have been developed, only a handful—e.g. CHX-A″-DTPA, DOTA, and HOPO—can be used for both α-emitters and radionuclides with imaging properties. In contrast, one of the α-emitting radionuclides that is not readily chelated, 211At, can be attached to disease-targeting agents through efficient electrophilic aromatic substitution reactions using trialkylstannyl intermediates or direct labeling on aromatic boron cage moieties. Radiopharmaceuticals containing α-emitting radionuclides are undergoing preclinical investigations for treatment of a number of cancers as well as the treatment of drug-resistant bacterial and viral infections. Furthermore, an α-emitting radiopharmaceutical—223RaCl2 (Xofigo™; Bayer HealthCare Pharmaceuticals, Inc., Whippany NJ, USA)—is an FDA-approved product, and several other radiopharmaceuticals containing α-emitting radionuclides have progressed to clinical trials.