Summary                         

Pharmacological and genetic studies over the past decade have established FSH as an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer’s disease (AD). Blocking FSH action prevents bone loss, fat gain and AD–like features in mice. This research group has recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody, MSHu6, and now reports the efficacy of MSHu6 in preventing obesity and osteoporosis in mice, and parameters of acute safety in monkeys.

Biodistribution studies using 89Zr–labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a β phase t½ of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. 215 variations of excipients were tested using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze–thaw and at different temperatures, with minimal aggregation, and without self–, cross–, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of “humanness” as human IgG1 in silico, and was non–immunogenic in ELISPOT assays for IL-2 and IFNγ in human peripheral blood mononuclear cell cultures. Results show that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.

Results from nanoScan® PET/CT

C57BL/6 or Tg32 mice (express the FCGRT transgene encoding the human FcRn receptor on chromosome 2 on a Fcgrt-/- background) were injected with 89Zr–MS-Hu6 as a single dose of ~250 μCi (~250 μg) into the retroorbital sinus. Timed blood (few drops drawn from the tail vein) and excreta collection was followed by weighing and γ–counting.

To study the biodistribution of 89Zr–MS-Hu6, we performed PET/CT scanning of mice at 24, 48 and 72 hours (N=3 mice). PET/CT scans were performed using nanoScan PET/CT. For whole body CT scans, the following parameters were used: energy, 50 kVp; current, 180 μAs; and isotropic voxel size, 0.25 mm - this was followed by a 30min long PET scan. Image reconstruction was performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from the Mediso Nucline software. The coincidences were filtered with an energy window between 400 and 600 keV. Voxel size was isotropic with 0.4–mm width, and the reconstruction was applied for four full iterations, six subsets per iteration. Image analysis was performed using Osirix MD. Namely, whole body CT images were fused with PET images and analyzed in an axial plane.

Regions of interest (ROIs) were drawn on various tissues. Testis, visceral WAT, subcutaneous WAT, kidneys, liver, and brain were traced in their entirety, and bone marrow uptake was assessed using three vertebrae in the lumbar spine. Mean standardized uptake values (SUVs, normalized to muscle) were calculated for each ROI. Subsequently, 89Zr–MS-Hu6 uptake of each tissue was expressed as the average of all mean SUV values per organ. After imaging, the mice were sacrificed and perfused with 20 mL of PBS and tissues of interest, namely brain, heart, kidney, pancreas, liver, lung, bone, bone marrow, BAT, subcutaneous WAT, visceral WAT, adrenal, blood, testis, spleen, and muscle, were isolated for γ–counting.

• The images show evidence of both decay of circulating radioactivity after 24 hours, and its persistence in multiple organs up to 72 hours (Fig. 5A).
• Maximal retention in terms of standardized uptake values (SUVs, normalized to muscle) was noted in the liver, with persistence in regions of interest, namely bone marrow, subcutaneous and visceral WAT depots, and the brain region (Fig. 5B).
• To determine the extent to which of 89Zr–MS-Hu6 persisted in individual tissues, we perfused the mice with 20 mL PBS before sacrifice and tissue isolation for γ–counting. Significant concentrations of 89Zr–MS-Hu6 were detected in multiple organs, including bone, bone marrow, subcutaneous WAT, visceral WAT, and BAT (Fig. 5C). Minimal amounts of 89Zr–MS-Hu6 were detected in isolated brain tissue at 72 hours—this is consistent with the low penetration of IgGs into the brain (0.05 to 0.1%) (Fig. 5C).
• To hone into the early events, we monitored the uptake of 89Zr–MS-Hu6 by dynamic PET/CT imaging over 240 minutes. At 10 minutes, radioactivity was detected mainly in large vessels, which was followed at 60 and 240 minutes by permeation into organs (Fig. 5D). As would be expected, radioactivity was not detected in the urine, but instead appeared in the feces (Fig. 5E).

To complement the 89Zr–based biodistribution studies, MS-Hu6 was labeled with Alexa-Fluor-750 (AF750), and injected to C57BL/6 mice (N=3 mice) intravenously through the tail vein with AF750–MS-Hu6 (200 μg), AF750 alone or PBS. At 16 hours post–injection, anaesthetized mice were imaged using the IVIS platform. We found significant soft tissue distribution of AF750–MSHu6 (Fig. 5F). The mice were then perfused with PBS, followed by IVIS imaging of isolated tissue. Consistent with the 89Zr-based studies, there was uptake of AF750–MS-Hu6 by liver, kidney, fat depots, bone, and brain (Fig. 5G). In contrast, in the AF750 (dye only) control group, localization was noted only in the kidney due to dye excretion, and not in other organs.

To understand MS-Hu6 biodistribution as it may apply to humans, 89Zr-MSHu6 was injected as a single bolus dose (1.3 mg, ~1.3 mCi) into the tail veins of two male Cynomolgus monkeys aged 14 and 15 years, respectively. Blood was drawn via tail vein at 5 minutes and at 48 and 20 hours. 89Zr-MS-Hu6 peaked in the blood at 5 minutes, with an expected decline, albeit with persistence in the serum, at 48 and 120 hours. PET/CT scanning revealed high SUV values in the liver and gall bladder, with lower SUVs in the kidney, spleen, fat depots, bone marrow, and the brain area.

Additional results show:

• increased food intake in mice injected with MS-Hu6 compared to those treated with human IgG. Despite this trend, there was a decline in body weight of the MS-Hu6 group, with statistically significant decrements at weeks 7 and 8. Quantitative PCR showed evidence for reduced expression of fat genes (Pparg, Fabp4 and Cebpa) in subcutaneous and gonadal WAT and BAT. Serum LH, GnRH and testosterone levels were unchanged after 8 weeks of MS-Hu6.
• significant increase in fractional bone volume and trabecular thickness, without an effect on trabecular number, revealed by histomorphometry of femoral metaphysis.
• that a single intravenous bolus injection into monkeys appeared to be generally safe: standard safety parameters were monitored in treated monkeys up to 100 minutes, and did not observe significant acute or delayed changes post–injection in heart rate, respiratory rate, mean arterial blood pressure, systolic or diastolic blood pressure, or rectal temperature. Blood samples were collected at day 0 (pre–injection) and at days 2 and 5 post–injection. No concerning deviations from normative values were noted.

Figure 5: Biodistribution and Excretion of MS-Hu6 in mice and monkeys. Representative PET-CT images of mice treated with a single bolus dose of 89Zr–labeled MS-Hu6 (250 μCi) at 24, 48 and 72 hours (A), together with quantitation in terms of standardized value uptake units (SUVs, normalized to muscle) in different organs (N=5, 4 and 2 mice for the three time points, respectively) (B). 89Zr–MS-Hu6 (γ–counts) in individual tissues isolated following perfusion of the mice with 20 mL PBS (N=5, 4 and 5 mice for the three time point, respectively) (C). Dynamic PET/CT images showing the uptake of 89Zr–MS-Hu6 over 240 minutes (D). Time course of excretion of 89Zr–labelled MS–Hu6 in feces (N=5 mice/time point) (E). Emitted whole body radiance on IVIS imaging of C57BL/6 mice injected with AF750-MS-Hu6 (200 μg) or PBS (F). IVIS imaging and quantitation (average radiance) of isolated perfused tissues, as shown. following AF750-MS-Hu6, AF750 or PBS injection (N=3 mice/group) (G).

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