Development of [211At]astatine-based anti-CD123 radioimmunotherapy for acute leukemias and other CD123+ malignancies

Abstract

Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia and higher-risk myelodysplastic syndrome (MDS). Of increasing interest are alpha-particle-emitting radionuclides such as astatine-211 (²¹¹At) as they deliver large amounts of radiation over just a few cell diameters, enabling efficient and selective target cell kill. Here, we developed ²¹¹At-based RIT targeting CD123, an antigen widely displayed on acute leukemia and MDS cells including underlying neoplastic stem cells. We generated and characterized new murine monoclonal antibodies (mAbs) specific for human CD123 and selected four, all of which were internalized by CD123+ target cells, for further characterization. All mAbs could be conjugated to a boron cage, isothiocyanatophenethyl-ureido-closo-decaborate(2-) (B10), and labeled with ²¹¹At. CD123+ cell targeting studies in immunodeficient mice demonstrated specific uptake of ²¹¹At-labeled anti-CD123 mAbs in human CD123+ MOLM-13 cell tumors in the flank. In mice injected intravenously with MOLM-13 cells or a CD123^NULL MOLM-13 subline, a single dose of up to 40 µCi of ²¹¹At delivered via anti-CD123 mAb decreased tumor burdens and substantially prolonged survival dose dependently in mice bearing CD123+ but not CD123- leukemia xenografts, demonstrating potent and target-specific in vivo anti-leukemia efficacy. These data support the further development of ²¹¹At-CD123 RIT toward clinical application.

Figure 1. Binding characteristics of new anti-CD123 mAbs.

A panel of anti-CD123 mAbs (11F11, 10C4, 7G3, 5G4, 1H8; used at 2 μg/mL) was tested flow cytometrically against parental RS4;11 cells (endogenously lacking CD123), RS4;11 cells transduced with human CD123, CD123-expressing human AML cells lines (TF-1, KG-1, MOLM-13), and MOLM-13 cells with CRISPR/Cas9-mediated knockout of the CD123 locus (CD123^KO). Secondary antibody only negative control is shown as well.

Figure 2. Internalization of anti-CD123 mAbs.

Flow cytometry-based in vitro internalization assays of anti-CD123 mAbs (1H8, 5G4, 10C4, 11F11) in CD123+ human acute leukemia cell lines (KG-1, MOLM-13, TF-1). Shown are mean±SEM values of 3-4 independent experiments.

Figure 3. In vitro cytotoxic properties of 10C4 and 10C4-B10.

CD123+ human acute leukemia cell lines (KG-1, MOLM-13, MV4;11, and TF-1) were incubated either alone or with unconjugated or B10-conjugated 10C4 at various concentrations as indicated. 3 days later, cell numbers and the percentage of dead cells were quantified by flow cytometry. Shown are mean±SEM values of 3 independent experiments.

Figure 4. In vivo CD123+ cell targeting with ²¹¹At-CD123 RIT.

10⁶ parental (CD123+) MOLM-13 cells were implanted into the flanks of NRG mice. One week later, animals (5/group) received 50 μg of either B10-conjugated anti-CD123 mAb (10C4-B10) or B10-conjugated murine IgG1 negative control mAb (BHV-1-B10) labeled with 5 μCi ²¹¹At. One hour, 4 hours, 7 hours, or 20 hours later, mice were euthanized, organs harvested, and tissues analyzed on a gamma counter to calculate the percent of injected dose/gram of organ tissue (% ID/g), and radiation absorbed doses for harvested organs calculated. Data are presented as mean±SD.

Figure 5. In vivo anti-AML efficacy of ²¹¹At-CD123 RIT.

(A) In vivo fluorescence imaging 7 and 14 days after administration of 10C4-B10 labeled with either 10 μCi, 20 μCi, or 40 μCi of ²¹¹At. Control group was left untreated. (B) Kaplan-Meier survival estimates. 8-11 animals per group. All deaths were attributed to progressive leukemia. P<0.0001 for Ctrl vs. 10 μCi, P<0.004 for 10 μCi vs. 40 μCi.

Figure 6. Target antigen specificity of ²¹¹At-CD123 RIT in vivo.

Two days after injecting NRG mice intravenously with either luciferase-transduced parental CD123+ MOLM-13 cells or a clonally derived subline in which CD123 was deleted via CRISPR/Cas9 (CD123KO MOLM-13), mice were left untreated or injected with 50 μg of 10C4-B10 (i.e. B10-conjugated 10C4 anti-CD123 mAb without radioisotope) or 50 μg of 10C4-B10 labeled with either 20 μCi or 40 μCi of ²¹¹At. (A) Average radiance values (mean±SEM) derived from in vivo fluorescence imaging. (B) Kaplan-Meier survival estimates. 8 animals per group. One early death was thought to be a sequelae of radiation exposure rather than leukemia (lack of weight returning to baseline after radiation exposure). All other deaths were attributed to progressive leukemia. For CD123⁺ cells: P=0.0014 for Ctrl vs. 10C4-B10, P=0.031 for 10C4-B10 vs. 20 μCi; P=0.0112 for 10C4-B10 vs. 40 μCi.