**2. Results**

On separate days, three dogs were intravenously administered 155.3, 142.5, or 133.2 MBq (1→3)-β-glucan-targeting antibody 400-2 labeled with <sup>213</sup>Bi, respectively. Figure <sup>1</sup> shows the values for red blood cell (RBC), white blood cell (WBC), and platelet counts throughout the study. These parameters are of particular importance for radiolabeled antibody-based treatment as they reflect possible reversible or nonreversible toxicity to the bone marrow. In all three dogs, these parameters were within laboratory reference intervals at all time points and generally within the reference change value (RCV) limits throughout the duration of the study.

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**Figure 1.** Red blood cell (RBC) counts (upper row), white blood cell (WBC) counts (middle row), and platelet counts (lower row) for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Shaded areas show reference change values (RCVs). **Figure 1.** Red blood cell (RBC) counts (**upper row**), white blood cell (WBC) counts (**middle row**), and platelet counts (**lower row**) for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. Shaded areas show reference change values (RCVs). **Figure 1.** Red blood cell (RBC) counts (upper row), white blood cell (WBC) counts (middle row), and platelet counts (lower row) for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Shaded areas show reference change values (RCVs).

In two of the patients (dogs 1 and 2), atypical cells, most consistent with cells of lymphoid origin, were noted on days 7 and 14, respectively (Figure 2). These cells were intermediate to large in size and round to mildly irregular in shape. Cytoplasm was of low to moderate volume, basophilic, and occasionally had few pinpoint clear vacuoles or pale areas. Nuclei were round, but in rare cells could appear binucleate or possibly lobular, with ropey, occasionally clumped, chromatin (Figure 2). In two of the patients (dogs 1 and 2), atypical cells, most consistent with cells of lymphoid origin, were noted on days 7 and 14, respectively (Figure 2). These cells were intermediate to large in size and round to mildly irregular in shape. Cytoplasm was of low to moderate volume, basophilic, and occasionally had few pinpoint clear vacuoles or pale areas. Nuclei were round, but in rare cells could appear binucleate or possibly lobular, with ropey, occasionally clumped, chromatin (Figure 2). In two of the patients (dogs 1 and 2), atypical cells, most consistent with cells of lymphoid origin, were noted on days 7 and 14, respectively (Figure 2). These cells were intermediate to large in size and round to mildly irregular in shape. Cytoplasm was of low to moderate volume, basophilic, and occasionally had few pinpoint clear vacuoles or pale areas. Nuclei were round, but in rare cells could appear binucleate or possibly lobular, with ropey, occasionally clumped, chromatin (Figure 2).

showing examples of atypical cells, most consistent with lymphoid origin. These cells are intermediate to large in size with low volumes of basophilic cytoplasm, occasionally containing few small clear vacuoles. Nuclei in these two cells are cleaved to binucleate or lobular with clumped chromatin. Modified Wright stain. Bar = 20.0 µm. **Figure 2.** Direct smears of peripheral blood from dogs 1 (left micrograph) and 2 (right micrograph) showing examples of atypical cells, most consistent with lymphoid origin. These cells are intermediate to large in size with low volumes of basophilic cytoplasm, occasionally containing few small clear vacuoles. Nuclei in these two cells are cleaved to binucleate or lobular with clumped chromatin. Modified Wright stain. Bar = 20.0 µm. **Figure 2.** Direct smears of peripheral blood from dogs 1 (**left micrograp**h) and 2 (**right micrograph**) showing examples of atypical cells, most consistent with lymphoid origin. These cells are intermediate to large in size with low volumes of basophilic cytoplasm, occasionally containing few small clear vacuoles. Nuclei in these two cells are cleaved to binucleate or lobular with clumped chromatin. Modified Wright stain. Bar = 20.0 µm.

**Figure 2.** Direct smears of peripheral blood from dogs 1 (left micrograph) and 2 (right micrograph)

the study (Figure 4).

These atypical cells were not a persistent finding, and they were not discernible in any of the follow-up complete blood counts (CBCs) in the two affected dogs. The mild decrease in lymphocyte numbers could reflect a transient elevation in cortisol (stress); however, given the brief appearance of atypical cells, a very transient influence of the treatment on lymphoid populations in the bone marrow and other organs leading to lympholysis cannot be entirely ruled out. The changes in monocyte numbers did not follow a consistent pattern between subjects, and while decreased numbers are typically considered clinically insignificant, the mild elevations could reflect an increased demand for tissue macrophages. Although within the RCV limits, mean corpuscular volume (MCV) showed a consistent downward trend from day 3 to day 14 in all three dogs, and continued to 30 days in dogs 1 and 3 (Figure 3). While there was an upward trend in all dogs by day 90, the values again decreased at 180 days. This finding can be associated with iron-limited erythropoiesis, although its connection to the test therapy is unclear. The dogs were all on a low-protein, plant-based diet which may have influenced iron stores independent of the test therapy. These atypical cells were not a persistent finding, and they were not discernible in any of the follow-up complete blood counts (CBCs) in the two affected dogs. The mild decrease in lymphocyte numbers could reflect a transient elevation in cortisol (stress); however, given the brief appearance of atypical cells, a very transient influence of the treatment on lymphoid populations in the bone marrow and other organs leading to lympholysis cannot be entirely ruled out. The changes in monocyte numbers did not follow a consistent pattern between subjects, and while decreased numbers are typically considered clinically insignificant, the mild elevations could reflect an increased demand for tissue macrophages. Although within the RCV limits, mean corpuscular volume (MCV) showed a consistent downward trend from day 3 to day 14 in all three dogs, and continued to 30 days in dogs 1 and 3 (Figure 3). While there was an upward trend in all dogs by day 90, the values again decreased at 180 days. This finding can be associated with iron-limited erythropoiesis, although its connection to the test therapy is unclear. The dogs were all on a lowprotein, plant-based diet which may have influenced iron stores independent of the test therapy. These atypical cells were not a persistent finding, and they were not discernible in any of the follow-up complete blood counts (CBCs) in the two affected dogs. The mild decrease in lymphocyte numbers could reflect a transient elevation in cortisol (stress); however, given the brief appearance of atypical cells, a very transient influence of the treatment on lymphoid populations in the bone marrow and other organs leading to lympholysis cannot be entirely ruled out. The changes in monocyte numbers did not follow a consistent pattern between subjects, and while decreased numbers are typically considered clinically insignificant, the mild elevations could reflect an increased demand for tissue macrophages. Although within the RCV limits, mean corpuscular volume (MCV) showed a consistent downward trend from day 3 to day 14 in all three dogs, and continued to 30 days in dogs 1 and 3 (Figure 3). While there was an upward trend in all dogs by day 90, the values again decreased at 180 days. This finding can be associated with iron-limited erythropoiesis, although its connection to the test therapy is unclear. The dogs were all on a lowprotein, plant-based diet which may have influenced iron stores independent of the test therapy.

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*Molecules* **2020**, *25*, x 4 of 10

**Figure 3.** Mean corpuscular volume (MCV) for the dogs on days 0–180 post treatment with 213Bi-400- 2 antibody. Shaded areas show reference change values (RCVs). X axis shows days post injection of radiolabeled antibody. **Figure 3.** Mean corpuscular volume (MCV) for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. Shaded areas show reference change values (RCVs). X axis shows days post injection of radiolabeled antibody. **Figure 3.** Mean corpuscular volume (MCV) for the dogs on days 0–180 post treatment with 213Bi-400- 2 antibody. Shaded areas show reference change values (RCVs). X axis shows days post injection of radiolabeled antibody.

A selection of serum biochemical analytes are shown in Figure 4, Figure 5, Figure 6 and Figure 7. With the exception of a temporary decrease in creatinine at day 90 for dog 3, and mild elevations in urea relative to the RCV at day 180 in dogs 1 and 2, urea and creatinine were within RCV throughout A selection of serum biochemical analytes are shown in Figures 4–7. With the exception of a temporary decrease in creatinine at day 90 for dog 3, and mild elevations in urea relative to the RCV at day 180 in dogs 1 and 2, urea and creatinine were within RCV throughout the study (Figure 4). A selection of serum biochemical analytes are shown in Figure 4, Figure 5, Figure 6 and Figure 7. With the exception of a temporary decrease in creatinine at day 90 for dog 3, and mild elevations in urea relative to the RCV at day 180 in dogs 1 and 2, urea and creatinine were within RCV throughout the study (Figure 4).

**Figure 4.** Renal analytes for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper **Figure 4.** Renal analytes for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—urea; lower row—creatinine. Shaded areas show reference change values (RCVs). **Figure 4.** Renal analytes for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. **Upper row**—urea; **lower row**—creatinine. Shaded areas show reference change values (RCVs).

row—urea; lower row—creatinine. Shaded areas show reference change values (RCVs).

Chloride, phosphorus, and potassium were concurrently elevated relative to the RCV in dog 2, and potassium was elevated relative to the RCV in dog 1, suggesting altered hydration status was the likely cause of the urea elevation in both dogs (Figure 5). Hydration status was also the likely cause of elevations in potassium at day 30 in dog 2 and day 90 in dogs 1 and 3, as again sodium showed a similar increase or was above the RCV at these same time points and was accompanied by a chloride level above the RCV in dog 3 at day 90. There was a slight decrease in potassium relative to the RCV in dogs 1 and 3 at day 30 and dog 2 on day 90, which may have been related to decreased intake. Despite these changes, relative to the RCV, potassium was within laboratory reference intervals throughout the study. Chloride, phosphorus, and potassium were concurrently elevated relative to the RCV in dog 2, and potassium was elevated relative to the RCV in dog 1, suggesting altered hydration status was the likely cause of the urea elevation in both dogs (Figure 5). Hydration status was also the likely cause of elevations in potassium at day 30 in dog 2 and day 90 in dogs 1 and 3, as again sodium showed a similar increase or was above the RCV at these same time points and was accompanied by a chloride level above the RCV in dog 3 at day 90. There was a slight decrease in potassium relative to the RCV in dogs 1 and 3 at day 30 and dog 2 on day 90, which may have been related to decreased intake. Despite these changes, relative to the RCV, potassium was within laboratory reference intervals throughout the study.

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**Figure 5.** Phosphorus, chloride, and potassium for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—phosphorous; middle row—chloride; lower row—potassium. Shaded areas show reference change values (RCVs). X axis shows days post injection of radiolabeled antibody. **Figure 5.** Phosphorus, chloride, and potassium for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. **Upper row**—phosphorous; **middle row**—chloride; **lower row**—potassium. Shaded areas show reference change values (RCVs). X axis shows days post injection of radiolabeled antibody.

With the exception of alkaline phosphatase (ALP) in dog 2, which significantly increased above RCV at day 30 but corrected by day 90, and slight increases in alanine aminotransferase ALT at day 180 in dogs 2 and 3, all hepatobiliary enzymes remained within the RCV limits throughout the study (Figure 6). With the exception of alkaline phosphatase (ALP) in dog 2, which significantly increased above RCV at day 30 but corrected by day 90, and slight increases in alanine aminotransferase ALT at day 180 in dogs 2 and 3, all hepatobiliary enzymes remained within the RCV limits throughout the study (Figure 6).

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**Figure 6.** Liver enzyme measurements for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—alkaline phosphatase; lower row—alanine aminotransferase. Shaded areas show reference change values (RCVs). **Figure 6.** Liver enzyme measurements for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. **Upper row**—alkaline phosphatase; **lower row**—alanine aminotransferase. Shaded areas show reference change values (RCVs). **Figure 6.** Liver enzyme measurements for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—alkaline phosphatase; lower row—alanine aminotransferase. Shaded areas show reference change values (RCVs).

The bilirubin and albumin were somewhat low, relative to laboratory reference intervals, in all three dogs at baseline and throughout the 6-month study (Figure 7). Total bilirubin was within RCV limits for all dogs throughout the study. Albumin was decreased relative to the RCV in dog 1 (at 90 days), dog 2 (at 30 days), and dog 3 (at 90 and 180 days). In dogs 1 and 2, these were transient changes. However, in dog 3, who had the lowest albumin at the start of the study, there was suspicion of a pre-existing protein-losing enteropathy (PLE). The albumin results were consistently and significantly lower than laboratory reference intervals at baseline and throughout the study in dog 3. Suspicion of PLE was further supported by the development of ascites, characterized as a pure transudate, at day 90 which was responsive to a hypoallergenic diet and prednisolone therapy. The bilirubin and albumin were somewhat low, relative to laboratory reference intervals, in all three dogs at baseline and throughout the 6-month study (Figure 7). Total bilirubin was within RCV limits for all dogs throughout the study. Albumin was decreased relative to the RCV in dog 1 (at 90 days), dog 2 (at 30 days), and dog 3 (at 90 and 180 days). In dogs 1 and 2, these were transient changes. However, in dog 3, who had the lowest albumin at the start of the study, there was suspicion of a pre-existing protein-losing enteropathy (PLE). The albumin results were consistently and significantly lower than laboratory reference intervals at baseline and throughout the study in dog 3. Suspicion of PLE was further supported by the development of ascites, characterized as a pure transudate, at day 90 which was responsive to a hypoallergenic diet and prednisolone therapy. The bilirubin and albumin were somewhat low, relative to laboratory reference intervals, in all three dogs at baseline and throughout the 6-month study (Figure 7). Total bilirubin was within RCV limits for all dogs throughout the study. Albumin was decreased relative to the RCV in dog 1 (at 90 days), dog 2 (at 30 days), and dog 3 (at 90 and 180 days). In dogs 1 and 2, these were transient changes. However, in dog 3, who had the lowest albumin at the start of the study, there was suspicion of a pre-existing protein-losing enteropathy (PLE). The albumin results were consistently and significantly lower than laboratory reference intervals at baseline and throughout the study in dog 3. Suspicion of PLE was further supported by the development of ascites, characterized as a pure transudate, at day 90 which was responsive to a hypoallergenic diet and prednisolone therapy.

**Figure 7.** Bilirubin and albumin concentrations for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—bilirubin; lower row—albumin. Shaded areas show reference change values (RCVs). **Figure 7.** Bilirubin and albumin concentrations for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—bilirubin; lower row—albumin. Shaded areas show reference change values (RCVs). **Figure 7.** Bilirubin and albumin concentrations for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. **Upper row**—bilirubin; **lower row**—albumin. Shaded areas show reference change values (RCVs).

Cholesterol was decreased concurrently, relative to RCV, in dog 1 at 90 days and in dog 3 at 180 days (Figure 8). There was a mild increase in cholesterol in dog 2 at 30 days, with a concurrent mild elevation of ALP relative to the RCV. This likely reflected a transient cholestasis, although insufficient to influence bilirubin concentrations. In the same dog, cholesterol had decreased relative to the RCV at 90 days and showed a slight elevation above the RCV at 180 days. Cholesterol was decreased concurrently, relative to RCV, in dog 1 at 90 days and in dog 3 at 180 days (Figure 8). There was a mild increase in cholesterol in dog 2 at 30 days, with a concurrent mild elevation of ALP relative to the RCV. This likely reflected a transient cholestasis, although insufficient to influence bilirubin concentrations. In the same dog, cholesterol had decreased relative to the RCV at 90 days and showed a slight elevation above the RCV at 180 days.

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**Figure 8.** Cholesterol and glucose concentrations for the dogs on days 0–180 post treatment with 213Bi-400-2 antibody. Upper row—cholesterol; lower row—glucose. Shaded areas show reference change values (RCVs). **Figure 8.** Cholesterol and glucose concentrations for the dogs on days 0–180 post treatment with <sup>213</sup>Bi-400-2 antibody. **Upper row**—cholesterol; **lower row**—glucose. Shaded areas show reference change values (RCVs).

ALP corrected by 90 days. Elevations in cholesterol, relative to the RCV, were also noted in dog 3 at 30 and 90 days. These elevations were transient and nonspecific. Glucose was found to decrease relative to the RCV in dog 1 on days 90 and 180 (Figure 8). Delayed serum separation was thought to be the most likely explanation for this change. Mild elevations in glucose relative to the RCV were noted in dog 2 at day 90 and dog 3 at days 30 and 90, which could be explained by a cortisol response, further supported in dog 3 at 30 days by a concurrent decrease in lymphocyte numbers. However, in dog 3, the baseline glucose was low relative to the laboratory reference intervals (likely storage artifact). As the RCV was based on this artifactually low value, any future sample not influenced by storage artifact would be expected to exceed the upper limit of the RCV. CK was elevated above the RCV, but remained within laboratory reference intervals, at day 30 in dog 1, likely reflecting mild myocyte leakage related to restraint during sample collection. ALP corrected by 90 days. Elevations in cholesterol, relative to the RCV, were also noted in dog 3 at 30 and 90 days. These elevations were transient and nonspecific. Glucose was found to decrease relative to the RCV in dog 1 on days 90 and 180 (Figure 8). Delayed serum separation was thought to be the most likely explanation for this change. Mild elevations in glucose relative to the RCV were noted in dog 2 at day 90 and dog 3 at days 30 and 90, which could be explained by a cortisol response, further supported in dog 3 at 30 days by a concurrent decrease in lymphocyte numbers. However, in dog 3, the baseline glucose was low relative to the laboratory reference intervals (likely storage artifact). As the RCV was based on this artifactually low value, any future sample not influenced by storage artifact would be expected to exceed the upper limit of the RCV. CK was elevated above the RCV, but remained within laboratory reference intervals, at day 30 in dog 1, likely reflecting mild myocyte leakage related to restraint during sample collection.

#### **3. Discussion 3. Discussion**

Overall, throughout the study, changes outside the RCV were few, mild, and transient, attesting to the absence of systemic toxicity of 213Bi-400-2 antibody. In regard to the radiation safety, the research and veterinary staff administering the 213Bi-labeled antibody and providing animal care received a combined dose of no more than 6 µSv personal dose equivalent at the depth of 10 mm (Hp(10)) per handled dog as measured by electronic dosimeters due to the 440 keV gamma emissions from the 213Bi. The staff exposures indicate that, with proper precautions, personnel exposure will not be limiting in a clinical trial setting. The short half-life of 213Bi makes the required isolation period less burdensome than using longer-lived radionuclides. Overall, throughout the study, changes outside the RCV were few, mild, and transient, attesting to the absence of systemic toxicity of <sup>213</sup>Bi-400-2 antibody. In regard to the radiation safety, the research and veterinary staff administering the <sup>213</sup>Bi-labeled antibody and providing animal care received a combined dose of no more than 6 µSv personal dose equivalent at the depth of 10 mm (Hp(10)) per handled dog as measured by electronic dosimeters due to the 440 keV gamma emissions from the <sup>213</sup>Bi. The staff exposures indicate that, with proper precautions, personnel exposure will not be limiting in a clinical trial setting. The short half-life of <sup>213</sup>Bi makes the required isolation period less burdensome than using longer-lived radionuclides.

*B. dermatitidis* has relatively high prevalence in different areas in Canada and the United States [2,9], and the endemic regions may be increasing, as evidenced by reports in New York, Vermont,

*B. dermatitidis* has relatively high prevalence in different areas in Canada and the United States [2,9], and the endemic regions may be increasing, as evidenced by reports in New York, Vermont, Texas, Nebraska, and Kansas [10], which makes it a relevant fungal pathogen to be treated with radiolabeled antibodies to a pan-antigen beta-glucan. Treatment of mice infected intratracheally with *B. dermatitidis* by <sup>213</sup>Bi-400-2 antibody given as a single 5.6 MBq (277.5 MBq/kg body weight) intraperitoneal injection reduced the fungal burden in the lungs by two logs [8]. For treatment of the dogs in this study, approximately 18.5 MBq <sup>213</sup>Bi-400-2 antibody per kg body weight was administered, reflecting the difference in body weight to body surface area ratios between mice and dogs. In this regard, the studies of T-cell ablation in dogs with <sup>213</sup>Bi-labeled anti-TCRαβ antibody showed that the injected activities in the range of 137 to 207 MBq/kg did not cause normal organ damage [11,12]. In addition, because (1→3)-β-glucan is a fungal antigen with no homology to human proteins, the antibodies to (1→3)-β-glucan are expected to have very low cross-reactivity with human and canine cells, thus decreasing the potential side effects from off-target antibody mediated injury associated with radiolabeled antibody administration. <sup>213</sup>Bi-labeled whole antibodies have been successfully used in treatment of patients with acute myeloid leukemia and melanoma [3]. Currently, there is a worldwide effort to increase the availability of <sup>213</sup>Bi, which will enable the expansion of <sup>213</sup>Bi-labeled whole antibodies' use for a variety of oncological and infectious disease applications.
