Oxidation of p-[125I]Iodobenzoic Acid and p-[211At]Astatobenzoic Acid Derivatives and Evaluation In Vivo.

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.

The α-emitter astatine-211 targeted to CD38 can eradicate multiple myeloma in a disseminated disease model.

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.

CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies.

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.

A Solid-State Support for Separating Astatine-211 from Bismuth.

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 community. For example, there are numerous technical needs associated with advancing the use of 211At in targeted α-therapy, e.g., improving 211At chelates, developing more effective 211At targeting, and characterizing in vivo 211At 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 211At could help address these issues and advance understanding of 211At chemistry in general. We contribute here an extraction chromatographic processing method that simplifies 211At production in terms of purification. It utilizes the commercially available Pre-Filter resin to rapidly (<1.5 h) isolate 211At 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 211At supply and processing/purification problems.

Thorium chelators for targeted alpha therapy: Rapid chelation of thorium-226.

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.

Anti-CD45 radioimmunotherapy without TBI before transplantation facilitates persistent haploidentical donor engraftment.

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.

Astatine-211 conjugated to an anti-CD20 monoclonal antibody eradicates disseminated B-cell lymphoma in a mouse 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.

Anti-CD45 radioimmunotherapy using (211)At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model.

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

Durable donor engraftment after radioimmunotherapy using α-emitter astatine-211-labeled anti-CD45 antibody for conditioning in allogeneic hematopoietic cell transplantation.

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

Targeted Radiation Delivery before Haploidentical HCT for High-risk Leukemia or MDS Patients Yields Long-term Survivors.

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.

211At-Labeled Anti-CD45 Antibody as a Nonmyeloablative Conditioning for Canine DLA-Haploidentical Stem Cell Transplantation.

The α-emitter 211At 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 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-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 211At (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 × 103/µL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-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 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma.

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.

Anti-CD45 pretargeted radioimmunotherapy using bismuth-213: high rates of complete remission and long-term survival in a mouse myeloid leukemia xenograft model.

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

Design and synthesis of astatinated benzothiazole compounds for their potential use in Targeted Alpha Therapy (TAT) strategies to treat Alzheimer's disease-associated amyloid plaques.

PURPOSE: 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 (VizamylTM) is an FDA approved diagnostic tool for PET imaging of AD-associated plaques. We report the radiolabeling of benzothiazole derivatives with 211At, a 7.2 h alpha emitting radionuclide, using a copper catalyzed reaction with a boronic acid precursor molecule. Our final compound [211At]3'-At-PIB-OMe had a radiochemical yield of 55% and was found to be stable for at least 3 h in phosphate buffered saline.

Glypican-3 targeted positron emission tomography detects sub-centimeter tumors in a xenograft model of hepatocellular carcinoma.

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.

Reagents for astatination of biomolecules. 6. An intact antibody conjugated with a maleimido-closo-decaborate(2-) reagent via sulfhydryl groups had considerably higher kidney concentrations than the same antibody conjugated with an isothiocyanato-closo-decaborate(2-) reagent via lysine amines.

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

Conventional and pretargeted radioimmunotherapy using bismuth-213 to target and treat non-Hodgkin lymphomas expressing CD20: a preclinical model toward optimal consolidation therapy to eradicate minimal residual disease.

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

Harnessing α-Emitting Radionuclides for Therapy: Radiolabeling Method Review.

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 149Tb, 212/213Bi, 212Pb (for 212Bi), 225Ac, and 226/227Th have found suitable chelators, although further studies, especially in vivo studies, are required. For others, including 223Ra, 230U, and, arguably 211At, the ideal chemistry remains elusive. This review summarizes the methods 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.

Small-scale (sub-organ and cellular level) alpha-particle dosimetry methods using an iQID digital autoradiography imaging system.

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.

Development and biodistribution studies of 77As-labeled trithiol RM2 bioconjugates for prostate cancer: Comparison of [77As]As-trithiol-Ser-Ser-RM2 vs. [77As]As-trithiol-Glu-Ser-RM2.

INTRODUCTION: Recent progress with the production of 72As (2.49 Mev ß+max (64%), 3.33 Mev ß+max (16%), 834 keV γ (81%), t1/2: 26 h) and 77As (0.683 Mev ß-max (97%), 239 keV γ (1.59%), t1/2: 38.8 h) has facilitated their evaluation as a potential "theranostic pair" for PET imaging and radiotherapy. Our 3rd generation trithiol chelate with two carboxylic acid groups was further developed as a bifunctional chelate for radioarsenic. METHODS: The As complex with the trithiol chelate was synthesized and characterized. No carrier added (nca) [77As][H2AsO4-] was used for radiolabeling studies. The trithiol chelate was conjugated to the RM2 peptide (DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) via solid phase peptide synthesis with two different linkers, Ser-Ser and Glu-Ser. The trithiol chelate and its RM2 bioconjugates were radiolabeled with nca 77As, and the RM2 bioconjugates were compared in initial biodistribution studies. RESULTS: The As diacid trithiol complex was characterized by 1H NMR, 13C NMR and HR-ESI-MS. The trithiol-RM2 precursor and As trithiol bioconjugates were characterized by HR-ESI-MS and/or LC-ESI-MS. Radiolabeling of the RM2 bioconjugates with 77As resulted in over 85% radiochemical yield for [77As]As-trithiol-Ser-Ser-RM2 ([77As]8) and 90% for [77As]As-trithiol-Glu-Ser-RM2 ([77As]9). Both radiotracers demonstrated excellent in vitro stability (≥ 90% remaining intact through 24 h in PBS buffer) and were more hydrophilic than previous analogues based on log D7.4 values. Biodistribution results of the two radiotracers in healthy CF-1 male mice demonstrated blockable pancreatic uptake at 1 h (82% for ([77As]8 and 78% for [77As]9) indicating specific gastrin-releasing peptide receptor (GRPR) uptake. The primary route of excretion was through the gastrointestinal system for both radiotracers. CONCLUSIONS: A new trithiol chelate with improved hydrophilicity was successfully conjugated to the RM2 peptide via two linkers, and high radiolabeling yield with nca 77As was achieved. In vivo biodistribution studies with both radiotracers demonstrated blockable pancreatic uptake suggestive of specific receptor uptake.