Aimee L. Kenoyer

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.

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

Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the α-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.

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

Pretargeting CD45 enhances the selective delivery of radiation to hematolymphoid tissues in nonhuman primates

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

Pretargeted Radioimmunotherapy using Genetically Engineered Antibody-Streptavidin Fusion Proteins for Treatment of Non-Hodgkin Lymphoma

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.

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 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.

Quantitative single‐particle digital autoradiography with α‐particle emitters for targeted radionuclide therapy using the iQID camera

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

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

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 211At-radioimmunotherapy, extending the analysis to include intraorgan 211At 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 211At-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 211At-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. 211At 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: 211At 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 211At 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: 211At-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 211At-B10-CA12.10C12 localization was achieved for efficient conditioning for HCT.