Preclinical Evaluation of Biodistribution and Toxicity of [211At]PSMA-5 in Mice and Primates for the Targeted Alpha Therapy against Prostate Cancer.

Astatine (211At) is a cyclotron-produced alpha emitter with a physical half-life of 7.2 h. In our previous study, the 211At-labeled prostate-specific membrane antigen (PSMA) compound ([211At]PSMA-5) exhibited excellent tumor growth suppression in a xenograft model. We conducted preclinical biodistribution and toxicity studies for the first-in-human clinical trial. [211At]PSMA-5 was administered to both normal male ICR mice (n = 85) and cynomolgus monkeys (n = 2). The mice were divided into four groups for the toxicity study: 5 MBq/kg, 12 MBq/kg, 35 MBq/kg, and vehicle control, with follow-ups at 1 day (n = 10 per group) and 14 days (n = 5 per group). Monkeys were observed 24 h post-administration of [211At]PSMA-5 (9 MBq/kg). Blood tests and histopathological examinations were performed at the end of the observation period. Blood tests in mice indicated no significant myelosuppression or renal dysfunction. However, the monkeys displayed mild leukopenia 24 h post-administration. Despite the high accumulation in the kidneys and thyroid, histological analysis revealed no abnormalities. On day 1, dose-dependent single-cell necrosis/apoptosis was observed in the salivary glands of mice and intestinal tracts of both mice and monkeys. Additionally, tingible body macrophages in the spleen and lymph nodes indicated phagocytosis of apoptotic B lymphocytes. Cortical lymphopenia (2/10) in the thymus and a decrease in the bone marrow cells (9/10) were observed in the 35 MBq/kg group in mice. These changes were transient, with no irreversible toxicity observed in mice 14 days post-administration. This study identified no severe toxicities associated with [211At]PSMA-5, highlighting its potential as a next-generation targeted alpha therapy for prostate cancer. The sustainable production of 211At using a cyclotron supports its applicability for clinical use.

Radioimmunotherapy with an 211 At-labeled anti-tissue factor antibody protected by sodium ascorbate.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti-TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody-drug conjugates (ADCs) and molecular imaging probes. We prepared an anti-TF mAb, clone 1084, labeled with astatine-211 (211 At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50-100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211 At-conjugated clone 1084 (211 At-anti-TF mAb) was disrupted by an 211 At-induced radiochemical reaction, we demonstrated that astatinated anti-TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF-expressing gastric cancer xenograft model, 211 At-anti-TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211 At-anti-TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211 At-radioimmunotherapy.

Labeling Monoclonal Antibody with α-emitting 211At at High Activity Levels via a Tin Precursor.

Background: In a previous clinical study, the authors evaluated the potential of antitenascin C monoclonal antibody (mAb) 81C6 labeled with 211At via the prosthetic agent N-succinimidyl 3-[211At]astatobenzoate (SAB) for the treatment of primary brain tumors. Although encouraging results were obtained, labeling chemistry failed while attempting to escalate the dose to 370 MBq. The goal of the current study was to develop a revised procedure less susceptible to radiolysis-mediated effects on 211At labeling that would be suitable for use at higher activity levels of this α-emitter. Materials and Methods: Addition of N-chlorosuccinimide to the methanol used to remove the 211At from the cryotrap after bismuth target distillation was done to thwart radiolytic decomposition of reactive 211At and the tin precursor. A series of 11 reactions were performed to produce SAB at initial 211At activity levels of 0.31-2.74 GBq from 50 µg of N-succinimidyl 3-trimethylstannylbenzoate (Me-STB), which was then reacted with murine 81C6 mAb without purification of the SAB intermediate. Radiochemical purity, immunoreactive fraction, sterility, and apyrogenicity of the 211At-labeled 81C6 preparations were evaluated. Results: Murine 81C6 mAb was successfully labeled with 211At using these revised procedures with improved radiochemical yields and decreased overall synthesis time compared with the original clinical labeling procedure. Conclusions: With 2.74 GBq of 211At, it was possible to produce 1.0 GBq of 211At-labeled 81C6 with an immunoreactive fraction of 92%. These revised procedures permit production of 211At-labeled mAbs suitable for use at clinically relevant activity levels.

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.

Labeling of Anti-HER2 Nanobodies with Astatine-211: Optimization and the Effect of Different Coupling Reagents on Their in Vivo Behavior.

The use of nanobodies (Nbs) as vehicles in targeted alpha therapy (TAT) has gained great interest because of their excellent properties. They combine high in vivo affinity and specificity of binding with fast kinetics. This research investigates a novel targeted therapy that combines the α-particle emitter astatine-211 (211At) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells. Two distinctive radiochemical methodologies are investigated using three different coupling reagents. The first method uses the coupling reagents, N-succinimidyl 4-(1,2-bis-tert-butoxycarbonyl)guanidinomethyl-3-(trimethylstannyl)benzoate (Boc2-SGMTB) and N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), which are both directed to amino groups on the Nb, resulting in random conjugation. The second method aims at obtaining a homogeneous tracer population, via a site-specific conjugation of the N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) reagent onto the carboxyl-terminal cysteine of the Nb. The resulting radioconjugates are evaluated in vitro and in vivo. 2Rs15d is labeled with 211At using Boc2-SGMTB, m-MeATE, and MSB. After astatination and purification, the binding specificity of the radioconjugates is validated on HER2+ cells, followed by an in vivo biodistribution assessment in SKOV-3 xenografted mice. α-camera imaging is performed to determine uptake and activity distribution in kidneys/tumors. 2Rs15d astatination resulted in a high radiochemical purity >95% for all radioconjugates. The biodistribution studies of all radioconjugates revealed comparable tumor uptake (higher than 8% ID/g at 1 h). [211At]SAGMB-2Rs15d showed minor uptake in normal tissues. Only in the kidneys, a higher uptake was measured after 1 h, but decreased rapidly after 3 h. Astatinated Nbs consisting of m-MeATE or MSB reagents revealed elevated uptake in lungs and stomach, indicating the presence of released 211At. α-Camera imaging of tumors revealed a homogeneous activity distribution. The radioactivity in the kidneys was initially concentrated in the renal cortex, while after 3 h most radioactivity was measured in the medulla, confirming the fast washout into urine. Changing the reagents for Nb astatination resulted in different in vivo biodistribution profiles, while keeping the targeting moiety identical. Boc2-SGMTB is the preferred reagent for Nb astatination because of its high tumor uptake, its low background signals, and its fast renal excretion. We envision [211At]SAGMB-2Rs15d to be a promising therapeutic agent for TAT and aim toward efficacy evaluation.

Enhancement of 211At Uptake via the Sodium Iodide Symporter by the Addition of Ascorbic Acid in Targeted α-Therapy of Thyroid Cancer.

211At is an α-emitter that has similar chemical properties to iodine and is used in targeted α-therapy. In the present study, we added ascorbic acid (AA) to 211At solution to increase the radiochemical purity of astatide and evaluated its efficacy against differentiated thyroid cancer, which is characterized by the expression of sodium/iodide symporter (NIS). Methods: Crude 211At solution (AA(-)) and 211At solution treated with AA (AA(+)) were prepared. Uptake by the thyroid was compared between the 2 solutions in normal male Wistar rats (n = 6). Cellular uptake in K1-NIS cells was analyzed under the AA(+) and AA(-) conditions. AA(+) was injected at 3 doses into K1-NIS xenograft mice: 1 MBq (n = 6), 0.4 MBq (n = 6), and 0.1 MBq (n = 6), and vehicle was injected into control mice (n = 6). The treatment effects were compared among the 4 groups. Results: Uptake by the thyroid was significantly enhanced in rats injected with the AA(+) as compared with those injected with AA(-). Cellular uptake analysis showed significantly increased uptake of 211At by the K1-NIS cells under the AA(+) condition as compared with the AA(-) condition. In the mouse xenograft model, the K1-NIS tumors showed significant accumulation of 211At at 3 and 24 h after administration (22.5 ± 10.4 and 12.9 ± 6.8 percentage injected dose, respectively). Tumor growth was immediately inhibited in a dose-dependent manner after administration of 211At. In the survival analysis, the 211At groups (0.1, 0.4, and 1 MBq) showed significantly better survival than the control group. Conclusion: Uptake of 211At was enhanced in differentiated thyroid cancer cells as well as the normal thyroid using 211At solution treated with AA. The method also showed dose-dependent efficacy against the K1-NIS xenografts, suggesting its potential applicability to targeted α-therapy.

Application of astatine-210: Evaluation of astatine distribution and effect of pre-injected iodide in whole body of normal rats.

We proposed use of astatine-210 in preclinical study. Astatine-210 has higher yield of production and is easier to quantify than astatine-211. We produced astatine-210 with Bi target and 40 MeV alpha beam accelerated by cyclotron, free astatine-210 was separated and injected to normal rats. Three male rats (blocking group) were injected non-radioactive iodide before injection of astatine-210. Compared with the control group, the astatine-210 accumulations in the blocking group decreased to 24% in the thyroid.

Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation.

INTRODUCTION: Derived from heavy chain only camelid antibodies, ~15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with 211At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap 211At in cancer cells after sdAb binding. This study evaluated two reagents, [211At]SAGMB and iso-[211At]SAGMB, for this purpose. METHODS: [211At]SAGMB and iso-[211At]SAGMB, and their radioiodinated analogues [131I]SGMIB and iso-[131I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [131I]SGMIB-5F7/[211At]SAGMB-5F7 and iso-[131I]SGMIB-5F7/iso-[211At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts. RESULTS: Radiochemical yields for Boc2-iso-[211At]SAGMB and Boc2-[211At]SAGMB synthesis, and efficiencies for coupling of iso-[211At]SAGMB and [211At]SAGMB to 5F7 were similar, with radiochemical purities of [211At]SAGMB-5F7 and iso-[211At]SAGMB-5F7 >98%. iso-[211At]SAGMB-5F7 and [211At]SAGMB-5F7 had immunoreactive fractions >80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding 211At- and 131I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[211At]SAGMB-5F7, 74.3 ± 2.8%, vs. [211At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[211At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [211At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[211At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [211At]SAGMB-5F7. CONCLUSION: These 211At-labeled sdAb conjugates, particularly iso-[211At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers.

Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients.

PURPOSE: Ovarian cancer is often diagnosed at an advanced stage with dissemination in the peritoneal cavity. Most patients achieve clinical remission after surgery and chemotherapy, but approximately 70% eventually experience recurrence, usually in the peritoneal cavity. To prevent recurrence, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation of (211)At in thyroid and other NIS-containing tissues. Mean absorbed doses to normal tissues were calculated from clinical data, including blood and i.p. fluid samples, urine, γ-camera images, and single-photon emission computed tomography/computed tomography images. Extrapolation of preclinical biodistribution data combined with clinical blood activity data allowed us to estimate absorbed doses in additional tissues. The equivalent dose was calculated using an RBE of 5 and the effective dose using the recommended weight factor of 20. All doses were normalized to the initial activity concentration of the infused therapy solution. RESULTS: The urinary bladder, thyroid, and kidneys (1.9, 1.8, and 1.7 mGy per MBq/L) received the 3 highest estimated absorbed doses. When the tissue-weighting factors were applied, the largest contributors to the effective dose were the lungs, stomach, and urinary bladder. Using 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective dose potentially corresponds to a risk of treatment-induced carcinogenesis, optimization may still be valuable.

α-Imaging Confirmed Efficient Targeting of CD45-Positive Cells After 211At-Radioimmunotherapy for Hematopoietic Cell Transplantation.

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.

Biodistribution and dosimetry of free 211At, 125I- and 131I- in rats.

131I is widely used for therapy in the clinic and 125I and 131I, and increasingly 211At, are often used in experimental studies. It is important to know the biodistribution and dosimetry for these radionuclides to determine potential risk organs when using radiopharmaceuticals containing these radionuclides. The purpose of this study was to investigate the biodistribution of 125I-, 131I-, and free 211At in rats and to determine absorbed doses to various organs and tissues. Male Sprague Dawley rats were injected simultaneously with 0.1-0.3 MBq 125I- and 0.1-0.3 MBq 131I-, or 0.05-0.2 MBq 211At and sacrificed 1 hour to 7 days after injection. The activities and activity concentrations in organs and tissues were determined and mean absorbed doses were calculated. The biodistribution of 125I- was similar to that of 131I- but the biodistribution of free 211At was different compared to 125I- and 131I-. The activity concentration of radioiodine was higher compared with 211At in the thyroid and lower in all extrathyroidal tissues. The mean absorbed dose per unit injected activity was highest to the thyroid. 131I gave the highest absorbed dose to the thyroid, and 211At gave the highest absorbed dose to all other tissues studied.

Sodium-iodide symporter positive cells after intracellular uptake of (99m)Tc versus α-emitter 211At. Reduction of clonogenic survival and characterization of DNA damage.

PURPOSE: We evaluated the DNA damaging potential of Auger electrons emitted in the decay of (99m)Tc compared to α-particles of 211At. MATERIAL AND METHODS: The impact of (99m)Tc and 211At was monitored in a NIS-expressing rat thyroid cell model PCCl3 with varying, yet defined intra- and extracellular radionuclide distribution (using ± perchlorate). The radiotoxicity of (99m)Tc and 211At was studied by the comet assay under neutral and alkaline conditions and colony formation. RESULTS: In the presence of perchlorate, the radioactivity yielding 37% cellular survival, A37, was estimated to be (0.27 ± 0.02) MBq/ml and (450 ± 30) MBq/ml for 211At and (99m)Tc, respectively. In absence of perchlorate, cellular radiotracer uptake was similar for both radionuclides (2.2%, 2.7%), yet the A37 was reduced by 82% for the α-emitter and by 95% for (99m)Tc. Cellular dose increased by a factor of 5 (211At) and 38 (99mTc). Comet assays revealed an increased DNA damage after intracellular uptake of both radiotracers. CONCLUSIONS: The data indicate damage to the cell to occur from absorbed dose without recognizable contribution from intracellular heterogeneity of radionuclide distribution. Comet assay under alkaline and neutral conditions did not reveal any shift to more complex DNA damage after radionuclide uptake. Cellular uptake of (99m)Tc and 211At increased cellular dose and reduced clonogenic survival.

Evaluation of effects on the peritoneum after intraperitoneal α-radioimmunotherapy with (211)At.

The introduction of the short-lived α-emitter (211)At to intraperitoneal radioimmunotherapy has raised the issue of the tolerance dose of the peritoneum. The short range of the α-particles (70 µm) and the short half-life (7.21 h) of the nuclide yield a dose distribution in which the peritoneum is highly irradiated compared with other normal tissues. To address this issue, mice were injected with (211)At-trastuzumab to irradiate the peritoneum to absorbed doses ranging between 0 and 50 Gy and followed for up to 34 weeks. The peritoneum-to-plasma clearance of a small tracer, (51)Cr-ethylenediamine tetraacetic acid, was measured for evaluation of the small solute transport capacity of the peritoneal membrane. The macroscopic status of the peritoneum and the mesenteric windows was documented when the mice were sacrificed. Biopsies of the peritoneum were taken for morphology and immunohistochemical staining against plasminogen activator inhibitor-1 and calprotectin. Peritoneum-to-plasma clearance measurements indicated a dose-dependent decrease in peritoneal transport capacity in irradiated mice. However, macroscopic and microscopic evaluations of the peritoneal membrane showed no difference between irradiated mice versus controls. The results imply that the peritoneal membrane tolerates absorbed doses as high as 30-50 Gy from α-particle irradiation with limited response.

Quantification of activity by alpha-camera imaging and small-scale dosimetry within ovarian carcinoma micrometastases treated with targeted alpha therapy.

Targeted alpha therapy (TAT) a promising treatment for small, residual, and micrometastatic diseases has questionable efficacy against malignant lesions larger than the α-particle range, and likely requires favorable intratumoral activity distribution. Here, we characterized and quantified the activity distribution of an alpha-particle emitter radiolabelled antibody within >100-µm micrometastases in a murine ovarian carcinoma model. Nude mice bearing ovarian micrometastases were injected intra-peritoneally with 211At-MX35 (total injected activity 6 MBq, specific activity 650 MBq/mg). Animals were sacrificed at several time points, and peritoneal samples were excised and prepared for alpha-camera imaging. Spatial and temporal activity distributions within micrometastases were derived and used for small-scale dosimetry. We observed two activity distribution patterns: uniform distribution and high stable uptake (>100% IA/g at all time points) in micrometastases with no visible stromal compartment, and radial distribution (high activity on the edge and poor uptake in the core) in tumor cell lobules surrounded by fibroblasts. Activity distributions over time were characterized by a peak (140% IA/g at 4 h) in the outer tumor layer and a sharp drop beyond a depth of 50 µm. Small-scale dosimetry was performed on a multi-cellular micrometastasis model, using time-integrated activities derived from the experimental data. With injected activity of 400 kBq, tumors exhibiting uniform activity distribution received <25 Gy (EUD=13 Gy), whereas tumors presenting radial activity distribution received mean absorbed doses of <8 Gy (EUD=5 Gy). These results provide new insight into important aspects of TAT, and may explain why micrometastases >100 µm might not be effectively treated by the examined regimen.

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.

In vivo distribution of avidin-conjugated MX35 and (211)At-labeled, biotinylated poly-L-lysine for pretargeted intraperitoneal α-radioimmunotherapy.

PURPOSE: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity. METHODS: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc). RESULTS: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose. CONCLUSIONS: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.

(211)At-antiCD33 in NMRI nu/nu mice. Biodistribution, in vivo stability and radiotoxicity.

UNLABELLED: The aim of this study is to verify the in vivo stability, to determine the biodistribution and to estimate the unspecific radiotoxicity of an (211)At-labelled CD33-antibody ((211)At-antiCD33) in mice with a view to therapeutic application in treating leukaemia. ANIMALS, METHODS: (211)At was produced via the (209)Bi(a,2n)(211)At reaction and was linked via 3-(211)At-succinimidyl-benzoate to the antiCD33-antibody. The biodistribution and the in vivo stability in serum were determined after i.v.-injection in NMRI nu/nu-mice. For toxicity experiments, mice received either three times 315-650 kBq (211)At-antiCD33 or unlabelled antibody and NaCl-solution respectively. RESULTS: (211)At-antiCD33 showed a characteristic biodistribution complying with the unspecific antibody retention in the reticular endothelial system. The largest proportion of radioactivity remained in blood and blood-rich tissues with a minor accumulation in the thyroid and stomach. After 21 h, >85% of activity in serum still represented intact antibody. Mice showed no difference in unspecific toxicity of (211)At-labelled antibodies over six months compared to those treated with unlabelled antibody and NaCl-solution respectively, with regard to histopathologic lesions, survival time, behaviour and haemograms. CONCLUSION: The radiolabelling method yielded adequate in vivo stability of (211)At-antiCD33. Biodistribution with rapid elimination of free (211)At via kidneys and urine complies with requirements for targeted therapy. Activity doses potentially required for treatment do not elicit radiotoxicity to normal organs in mice. Further development is required to enhance the apparent specific activity and to verify the efficacy in an adequate animal model before phase I clinical studies in leukaemia can be envisaged.

Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice.

Besides bone marrow, the kidneys are often dose-limiting organs in internal radiotherapy. The effects of high-linear energy transfer (LET) radiation on the kidneys after alpha-radioimmunotherapy (alpha-RIT) with the alpha-particle emitter, (211)At, were studied in nude mice by serial measurements of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals and animals bearing subcutaneous xenografts of the human ovarian cancer cell line, OVCAR-3, were used. The animals received approximately 0.4, 0.8, or 1.2 MBq in one, two, or three fractions. The mean absorbed doses to the kidneys ranged from 1.5 to 15 Gy. The renal function was studied by serial GFR measurements, using plasma clearance of (51)Cr-EDTA, up to 67 weeks after the first astatine injection. A dose-dependent effect on GFR was found and at the time interval 8-30 weeks after the first administration of astatine, the absorbed doses causing a 50% decrease in GFR were 16.4 +/- 3.3 and 14.0 +/- 4.1 Gy (mean +/- SEM), tumor- and non-tumor-bearing animals, respectively. The reduction in GFR progressed with time, and at the later time interval, (31-67 weeks) the corresponding absorbed doses were 7.5 +/- 2.4 and 11.3 +/- 2.3 Gy, respectively, suggesting that the effects of radiation on the kidneys were manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary dose-limiting organ in systemic alpha-RIT when using (211)At-MX35-F(ab')(2).

Reagents for astatination of biomolecules. 4. Comparison of maleimido-closo-decaborate(2-) and meta-[(211)At]astatobenzoate conjugates for labeling anti-CD45 antibodies with [(211)At]astatine.

An investigation was conducted to compare the in vivo tissue distribution of a rat antimurine CD45 monoclonal antibody (30F11) and an irrelevant mAbs (CA12.10C12) labeled with (211)At using two different labeling methods. In the investigation, the mAbs were also labeled with (125)I to assess the in vivo stability of the labeling methods toward deastatination. One labeling method employed N-hydroxysuccinimidyl meta-[(211)At]astatobenzoate, [(211)At]1c, and N-hydroxysuccinimidyl meta-[(125)I]iodobenzoate, [(125)I]1b, in conjugation reactions to obtain the radiolabeled mAbs. The other labeling method involved conjugation of a maleimido-closo-decaborate(2-) derivative, 2, with sulfhydryl groups on the mAbs, followed by labeling of the mAb-2 conjugates using Na[(211)At]At or Na[(125)I]I and chloramine-T. Concentrations of the (211)At/(125)I pair of radiolabeled mAbs in selected tissues were examined in BALB/c mice at 1, 4, and 24 h post injection (pi). The co-injected anti-CD45 mAb, 30F11, labeled with [(125)I]1b and [(211)At]1c targeted the CD45-bearing cells in the spleen with the percent injected dose (%ID) of (125)I in that tissue being 13.31 ± 0.78; 17.43 ± 2.56; 5.23 ± 0.50; and (211)At being 6.56 ± 0.40; 10.14 ± 1.49; 7.52 ± 0.79 at 1, 4, and 24 h pi (respectively). However, better targeting (or retention) of the (125)I and (211)At was obtained for 30F11 conjugated with the closo-decaborate(2-), 2. The %ID in the spleen of (125)I (i.e., [(125)I]30F11-2) being 21.15 ± 1.33; 22.22 ± 1.95; 12.41 ± 0.75; and (211)At (i.e., [(211)At]30F11-2) being 22.78 ± 1.29; 25.05 ± 2.35; 17.30 ± 1.20 at 1, 4, and 24 h pi (respectively). In contrast, the irrelevant mAb, CA12.10C12, labeled with (125)I or (211)At by either method had less than 0.8% ID in the spleen at any time point, except for [(211)At]CA12.10C12-1c, which had 1.62 ± 0.14%ID and 1.21 ± 0.08%ID at 1 and 4 h pi. The higher spleen concentrations in that conjugate appear to be due to in vivo deastatination. Differences in (125)I and (211)At concentrations in lung, neck, and stomach indicate that the meta-[(211)At]benzoyl conjugates underwent deastatination, whereas the (211)At-labeled closo-decaborate(2-) conjugates were very stable to in vivo deastatination. In summary, using the closo-decaborate(2-) (211)At labeling approach resulted in higher concentrations of (211)At in target tissue (spleen) and higher stability to in vivo deastatination in this model. These findings, along with the simpler and higher-yielding (211)At-labeling method, provide the basis for using the closo-decaborate(2-) labeling reagent, 2, in our continued studies of the application of (211)At-labeled mAbs for conditioning in hematopoietic cell transplantation.

Biodistributions, myelosuppression, and toxicities in mice treated with an anti-CD45 antibody labeled with the alpha-emitting radionuclides bismuth-213 or astatine-211.

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