Evaluation of Acute Hematologic and Long-Term Pulmonary Toxicities of Radioimmunotherapy of Cryptococcus neoformans Infection in Murine Models

Abstract

We evaluated acute hematological and long-term pulmonary toxicity of radioimmunotherapy in murine models of Cryptococcus neoformans infection. Activities up to 250 μCi were well tolerated by healthy A/JCr mice for ²¹³Bi-18B7 and ¹⁸⁸Re-18B7 monoclonal antibodies. In infected mice, doses up to 150 μCi produced only transient toxicity. The lungs of treated mice had no evidence of radiation fibrosis.

Cryptococcus neoformans infection presents a serious therapeutic challenge in immunocompromised patients in whom antifungal therapy is not effective. It was recently demonstrated that treatment of C. neoformans-infected mice with a monoclonal antibody (MAb) to the capsular polysaccharide, labeled with either rhenium-188 (¹⁸⁸Re) or bismuth-213 (²¹³Bi), significantly prolonged the survival of lethally infected mice and reduced organ fungal burden (3). That study established a proof of principle for the usefulness of radioimmunotherapy (RIT) against an infection but invited questions about the safety of the approach. Here, we have evaluated the acute hematological and long-term pulmonary toxicity of RIT in murine models of C. neoformans infection.

To study the acute hematological toxicity of RIT, seven groups of five A/JCr female mice were infected intravenously with 10⁵ C. neoformans cells. The intravenous infection results in rapid death of infected animals and is a standard model for antifungal susceptibility testing (7). Twenty-four hours after infection, groups 1, 2, 3, and 4 received intraperitoneal injections of 100, 150, 200, and 250 μCi of ²¹³Bi-18B7 MAb, respectively; groups 5 and 6 received 100 and 200 μCi of ¹⁸⁸Re-18B7, respectively; and group 7 was left untreated. The amount of cryptococcal polysaccharide-specific MAb 18B7 per injection was 30 to 50 μg, and the injection volume was 0.2 ml of phosphate-buffered saline. Platelet counts were done to assay for potential bone marrow toxicity (6) on days 3, 7, and 14 posttreatment. To compare the hematological toxicity of this therapy in mice infected with C. neoformans and in healthy mice, eight groups of five healthy A/JCr mice were injected with 100, 150, 200, and 250 μCi of ²¹³Bi-18B7 or ¹⁸⁸Re-18B7 MAbs, with group 9 being left untreated, and the platelets were counted on days 3, 7, and 14 posttreatment. Student's t test for unpaired data was used to analyze differences in the number of platelets. Differences were considered statistically significant when P values were <0.05.

The serum platelet counts in healthy A/JCr mice and mice systemically infected with C. neoformans are presented in Fig. 1. Activities up to 250 μCi were well tolerated by healthy mice for both ²¹³Bi-18B7 and ¹⁸⁸Re-18B7 MAbs (Fig. 1a and c). A significant decrease in platelet counts was observed for all treatment groups at day 7 relative to untreated control mice and to the levels at day 3 (P < 0.01). This result is consistent with the reported nadirs in platelet and peripheral white blood cell counts, which are usually reached 1 week after radiolabeled antibody administration to tumor-bearing animals (1, 9). The platelet numbers recovered for all ²¹³Bi-18B7- and ¹⁸⁸Re-18B7-treated healthy animals on day 14. The tolerance of radiation was different when mice systemically infected with C. neoformans were treated with the same doses of radiolabeled MAb (Fig. 1b and d). For the ²¹³Bi-18B7 treatment, the 200- and 250-μCi doses proved to be radiotoxic, with all mice in these two groups dying by day 7. However, doses of 100 and 150 μCi did not produce mortality and did not cause significant decreases in platelet counts (P = 0.07). For the ¹⁸⁸Re-18B7-treated animals, the 200-μCi dose caused a significant drop in platelet counts (P = 0.02) at day 7, which did not normalize by day 14 and thus might attest to the possible radiotoxicity of this dose. The platelet number normalized by day 14 in mice treated with 100 μCi of ¹⁸⁸Re-18B7, consistent with transient toxicity. These measurements parallel our previous results (3) on therapy of C. neoformans-infected A/JCr mice with ²¹³Bi-18B7 and ¹⁸⁸Re-18B7 MAbs, where administration of 100-μCi doses of ²¹³Bi-18B7 and ¹⁸⁸Re-18B7 MAbs resulted in most of the animals surviving while a 200-μCi dose was apparently too toxic and caused death of treated animals.

FIG. 1.

Platelet counts in A/JCr mice. Healthy mice (a and c) and C. neoformans-infected mice (b and d) received various doses of ²¹³Bi-18B7 (a and b) or ¹⁸⁸Re-18B7 (c and d). A “0” indicates infected nontreated mice. C. neoformans-infected mice treated with 200 and 250 μCi of ²¹³Bi-18B7 (b) died by day 7 posttreatment. The platelet count in healthy nontreated A/JCr mice was (8.8 ± 2.4) × 10⁸ platelets/ml.

We considered the possibility that RIT may promote lung fibrosis in treated animals. Lungs are the target organ for C. neoformans infection and can develop fibrosis several months after treatment with external beam radiation therapy (5). To evaluate this potential complication we used a pulmonary model of C. neoformans where mice are infected intratracheally (IT). In this model, C. neoformans is mostly localized to the lungs on day 5 postinfection, and as a result up 10% of the injected dose/g is found in the lungs at 24 h after treatment with radiolabeled MAbs, versus 1.5% of the injected dose/g in the lungs of systemically infected mice (3). Eight groups of five BALB/c mice were infected IT with 10⁶ C. neoformans, and on day 5 postinfection, groups 1, 2, and 3 were treated with 50, 100, and 200 μCi of ²¹³Bi-18B7 MAb, respectively; groups 4, 5, and 6 were treated with the same activities of ¹⁸⁸Re-18B7, respectively, while groups 7 and 8 were left untreated. All mice were subsequently maintained on fluconazole to control infection (10 mg/kg in their drinking water). After 5 months the mice were sacrificed, and their lungs were removed, fixed with buffered formalin, sectioned, stained with hematoxylin and eosin, and analyzed histologically. There was no evidence of radiation fibrosis in the lungs of radiation-treated mice compared to control animals (Fig. 2). The absence of fibrosis can be explained by the relatively low doses of particulate radiation delivered by radiolabeled antibodies to the sites of the infection during RIT—the doses are in the range of 2.5 to 5 Gy (∼0.1 to 0.2 Gy/h) (2). For comparison, high-dose radiation, which is typical for external beam radiation therapy, delivers 60 Gy/h (8).

FIG. 2.

Micrographs of hematoxylin-and-eosin-stained lungs from BALB/c mice infected IT with C. neoformans and treated with radiolabeled antibodies. Mice were sacrificed 5 months after RIT. (a) Infected control for Bi group (no RIT); (b) 200 μCi of ²¹³Bi-18B7; (c) infected control for Re group (no RIT); (c) 200 μCi of ¹⁸⁸Re-18B7.

Important determinants of the extent and duration of myelosuppression following RIT in cancer patients include bone marrow reserve (based on prior cytotoxic therapy and extent of disease involvement), total tumor burden, spleen size, and radioimmunoconjugate stability (4). Clearly, the application of RIT to human cryptococcosis or other infectious diseases will require optimization of the dose to minimize toxicity. However, we are encouraged that these initial studies in mice suggest that RIT for infection is relatively well tolerated and may have a significantly higher therapeutic index than RIT for cancer. The results suggest a relatively high margin of safety for this novel antimicrobial therapy.