Radioimmunotherapy of Blastomycosis in a Mouse Model With a (1→3)-β-Glucans Targeting Antibody

Abstract

Invasive fungal infections (IFI) cause devastating morbidity and mortality, with the number of IFIs more than tripling since 1979. Our laboratories were the first to demonstrate that radiolabeled microorganism-specific monoclonal antibodies are highly effective for treatment of experimental fungal, bacterial and viral infections. Later we proposed to utilize surface expressed pan-antigens shared by major IFI-causing pathogens such as beta-glucans as RIT targets. Here we evaluated in vivo RIT targeting beta-glucan in Blastomyces dermatitidis which causes serious infections in immunocompromised and immunocompetent individuals and in companion dogs. B. dermatitidis cells were treated with the 400-2 antibody to (1→3)-β-glucans radiolabeled with the beta-emitter 177Lutetium (¹⁷⁷Lu) and alpha-emitter 213Bismuth (²¹³Bi) and the efficacy of cell kill was determined by colony forming units (CFUs). To determine the antigen-specific localization of the 400-2 antibody in vivo, C57BL6 mice were infected intratracheally with 2 × 10⁵ B. dermatitidis cells and given ¹¹¹In-400-2 antibody 24 h later. To evaluate the killing of B. dermatitidis cells with RIT, intratracheally infected mice were treated with 150 μCi ²¹³Bi-400-2 and their lungs analyzed for CFUs 96 h post-infection. ²¹³Bi-400-2 proved to be more effective in killing B. dermatitidis cells in vitro than ¹⁷⁷Lu-400-2. Three times more ¹¹¹In-400-2 accumulated in the lungs of infected mice, than in the non-infected ones. ²¹³Bi-400-2 lowered the fungal burden in the lungs of infected mice more than 2 logs in comparison with non-treated infected controls. In conclusion, our results demonstrate the ability of an anti-(1-3)-beta-D-glucan antibody armed with an alpha-emitter ²¹³Bi to selectively kill B. dermatitidis cells in vitro and in vivo. These first in vivo results of the effectiveness of RIT targeting pan-antigens on fungal pathogens warrant further investigation.

Keywords: (1→3)-β-glucan; 213Bismuth; Blastomyces dermatitidis; mouse model; radioimmunotherapy.

FIGURE 1

Evaluation of the binding of ¹⁷⁷Lu-400-2 and ¹⁷⁷Lu-2G8 antibodies to (1→3)-β-glucans antigen after their incubation with acapsular C. neoformans cells for 3 h. ¹⁷⁷Lu-MOPC 21 was used as an IgG irrelevant control. (A) percentage of radiolabeled antibody in a sample bound to the cell pellet: (B) percentage of non-bound antibody. ^∗Indicates a significant difference between the groups (P value < 0.05).

FIGURE 2

Cytotoxicity of radioimmunotherapy (RIT) toward B. dermatitidis. (A) ²¹³Bi-400-2 mAb; (B) ¹⁷⁷Lu-400-2 mAb. Cells were incubated with the radiolabeled antibodies for 1 h at 37°C followed by plating for determination of CFUs. Samples treated with PBS, cold 400-2 mAb and ²¹³Bi- or ¹⁷⁷Lu–labeled MOPC-21 mAb were used as controls. *Indicates a significant difference between the groups (P value < 0.05).

FIGURE 3

Biodistribution of ¹¹¹In-400-2 mAb in B. dermatitidis-infected and in healthy mice. C57Bl6 mice were infected intratracheally with B. dermatitidis 2 × 10⁵ cells, injected IP with ¹¹¹In-400-2 mAb 24 h after infection, and euthanized at 24 or 72 h post antibody administration. Five animals per group were used. %ID/g represents the percentage of injected dose of the radiolabeled antibody per gram tissue (blood and lungs). *Indicates a significant difference between the groups (P value < 0.05).

FIGURE 4

Radioimmunotherapy (RIT) of B. dermatitidis infected mice. (A) CFUs in the lungs of the mice infected with 10⁶ B. dermatitidis cells for 24 h and then either left untreated (control) or treated intraperitoneally with 30 μg 400-2 cold antibody, 150 μCi ²¹³Bi-400-2, or 150 μCi ²¹³Bi-MOPC-21 and were euthanized at 96 h post-infection. * Indicates P value < 0.05; ** < 0.01; *** < 0.001. (B–F) images of representative plates seeded with the homogenates from lungs isolated from various treatment groups: (B) ²¹³Bi-400-2; (C) ²¹³Bi-MOPC-21; 1 (D) 400-2 cold antibody; (E) no treatment; (F) uninfected, untreated mice; (G) real time PCR results, showing B. dermatitidis BAD1 gene levels significantly (p = 0.001) lowered in the lungs of mice that received ²¹³Bi-400-2 treatment in comparison with untreated infected controls.