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

Abstract

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.

Figure 1

Cell-binding assays evaluating the absolute (counts per minute [cpm]) binding of 1F5-B10 labeled with either ²¹¹At or ¹²⁵I with or without unlabeled blocking antibodies using Ramos and Jurkat cells confirm the antigen-specific cell binding of radiolabeled 1F5-B10 conjugates. (A) [²¹¹At]1F5-B10 binding to Ramos cells was blocked by preincubation with unlabeled anti-CD20 antibodies (2321.0 ± 475.1 vs 409.0 ± 27.5 CPM; 82% blocking). (B) Similar blocking was also observed when the 1F5-B10 was labeled with ¹²⁵I (14 642 ± 3621 vs 679 ± 60.6 CPM; 95% blocking). No difference was seen between the binding of 1F5-B10 antibody to the CD20-negative Jurkat cells in the presence or absence of blocking anti-CD20 antibodies. PBS, phosphate-buffered saline.

Figure 2

Analysis of tumor size and cumulative survival of mice bearing Ramos subcutaneous lymphoma xenografts treated with ²¹¹At-labeled 1F5-B10. (A) Ramos tumor xenografts were serially measured in athymic mice receiving [²¹¹At]1F5 (24, 36, or 48 µCi), [²¹¹At]HB8181-B10 (48 µCi), or no treatment (control) plotted as mean ± SD. Animals were killed based on the tumor size criteria (16 × 16 × 9 mm). (B) Kaplan-Meier survival curves of the same mice bearing Ramos subcutaneous lymphoma xenografts treated as indicated.

Figure 3

α-Camera imaging of subcutaneous Ramos xenografts. Images obtained 28 hours after IV injection of [²¹¹At]1F5-B10 (anti-CD20, left) or [²¹¹At]HB8181-B10 (control, right), demonstrating specificity of CD20 targeting but heterogeneous dose distributions. Images are color coded to express the intratumoral activity in pCi per voxel (17 × 17 × 16 μm). The white curve represents the activity variation along a line profile placed centrally in each tumor. The white bar (bottom center) indicates 1000 μm.

Figure 4

²¹¹At radiosensitivity of NOD/SCID mice. Non-tumor–bearing NOD/SCID mice (n = 5/group) received 230 µg of 1F5-B10 labeled with either 10 or 20 µCi of ²¹¹At followed by bone marrow rescue (1 × 10⁷ bone marrow cells IV from syngeneic donors) 2 days after treatment. The Kaplan-Meier survival curves demonstrates that 20-µCi [²¹¹At]1F5-B10 was universally lethal within in 5 days, whereas the 10-µCi dose was well tolerated.

Figure 5

Efficacy of ²¹¹At-B10-1F5 in an MRD model. Mice were inoculated via IV injection of Granta 519^Luc cells (1 × 10⁶) and monitored for tumor progression by BLI twice weekly for the duration of the study. On day 6, animals received 7.5 or 15 µCi of ²¹¹At-labeled 1F5-B10 (anti-CD20 mAb) or HB8181-B10 (nonbinding mAb control) or 1F5-B10 antibody alone or no therapy. All animals received stem cell rescue, either 2 days after the radiation dose (1F5-B10 or HB8181-B10 groups) or at the same time point without radiation (bone marrow transplant [BMT] control group). (A) Whole-body ventral BLI images on day 23 demonstrate diffuse signal corresponding with disease involvement in both control groups, with only 1 mouse demonstrating measurable disease in the 15-µCi anti-CD20–treated group. (B) Day-57 imaging of all surviving animals. (C) BLI plot demonstrating the mean ± standard error of the mean p/s for each group. The imaging data were normalized to the same scale for each figure.

Figure 6

Kaplan-Meier survival curves of mice bearing disseminated lymphoma. Groups of 10 mice bearing disseminated lymphoma after IV Granta-519^Luc (MCL) injections 6 days before treatment. Mice in the treatment groups were treated with 7.5 or 15 µCi of radiation via 1F5-B10 (anti-CD20), HB8181-B10 (nonbinding control), or 1F5-B10 (unlabeled mAb control). All animals received stem cell rescue either 2 days after the radiation dose (1F5-B10 or HB8181-B10 groups) or at the same time point without radiation (BMT control group).