A Single Multipurpose FSH–Blocking Therapeutic for Osteoporosis and Obesity

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. We recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a GLP–compliant platform, we now report the efficacy of MS-Hu6 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. We tested 215 variations of excipients 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. We conclude that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.


INTRODUCTION
While obesity and osteoporosis are both diseases of public health concern, the paucity of therapies to prevent and treat them continues to represent a challenge (1,2). Accumulating clinical data suggest that the two disorders track together in women across the menopausal transition. Particularly during the late perimenopause, there is precipitous bone loss, onset of visceral obesity, dysregulated energy balance, and reduced physical activity (3)(4)(5)(6)(7)(8)(9). These aberrant physiologic changes across the menopausal transition are not fully explained by low estrogen, as estrogen levels are relatively unperturbed, while serum FSH levels rise to maintain estrogen secretion from an otherwise failing ovary (10)(11)(12).
The question has been whether a rising FSH level is a driver for post-menopausal obesity and osteoporosis. In 2006, we provided the first evidence for a direct action of FSH on bone (13).
Since then, despite controversy fueled mainly from the over-interpretation of clinical studies with GnRH agonists that suppress not only FSH, but also GnRH and LH (14), there is replicable evidence that the selective inhibition of FSH action in mice, for example by using novel FSHblocking antibodies or a GST-FSH fusion protein as a vaccine, protects against hypogonadal bone loss (15)(16)(17)(18)(19). Serum FSH, bone turnover, and bone mineral density also correlate well in women, particularly when FSH levels are rising during the late perimenopause [review: (20)].
Likewise, activating FSHR polymorphisms in postmenopausal women are linked to a high bone turnover and reduced BMD (21). Thus, it makes both biological and clinical sense to selectively inhibit FSH action to prevent bone loss.
We and our collaborators have also shown that inhibiting FSH by FSH-blocking antibodies reduces white adipose tissue (WAT) in every fat compartment, induces thermogenic (or beige) adipose tissue, and increases energy expenditure in mice (16). Reduced fat mass has also been documented with a vaccine containing tandem repeats of the 13-amino-acid-long FSH receptor-binding FSHβ sequence to which our antibodies were raised (22). An interventional study in treatment-naïve prostate cancer patients comparing orchiectomy versus triptorelin showed that, with near-zero testosterone, patients on triptorelin (reduced serum FSH and LH) had significantly lower body weight and fat mass compared to those post-orchiectomy (23). Even recognizing the constraints of using a GnRH agonist, this dataset suggests that lowering serum FSH could, in principle, have beneficial effects on body composition in people, despite concomitant reductions in GnRH and LH. There is also new evidence that selective FSH blockade lowers serum total and LDL cholesterol (24,25).
We hypothesize that blocking FSH action will reduce obesity and bone loss in people.
Towards this goal, we have developed our lead candidate, a first-in-class humanized FSHblocking antibody, MS-Hu6. The latter binds a 13-amino-acid-long epitope of human FSHβ (LVYKDPARPKIQK) with high affinity, and by doing so, blocks the interaction of FSH with its receptor (26). Here, we report a comprehensive characterization of MS-Hu6 in terms of its in vivo efficacy in mouse models of obesity and osteoporosis, acute safety in monkeys, a full evaluation of its pharmacokinetic, pharmacodynamic and biodistribution, and a compendium of its physicochemical properties. This new information provides the framework for first-in-human studies towards the future use of MS-Hu6 in obesity and, osteoporosis.

Efficacy of MS-Hu6 in Reducing Body Fat and Inducing Beige Adipose Tissue
In choosing MS-Hu6 as the lead candidate from an array of 30 humanized clones, we examined the electrostatic binding in silico and determined KD by surface plasmon resonance in vitro (26). MS-Hu6 had the best affinity (KD = 7.52 nM), approaching that of trastuzumab. We fine mapped the three top candidates to document subtle differences in binding modes (26). In addition, we established that MS-Hu6 blocked the binding of labeled recombinant human FSH to the FSHR, and in doing so, inhibited osteoclastogenesis and promoted beiging of adipocytes in vitro (26). Here, we studied the effect of MS-Hu6 on body composition in male ThermoMice fed ad libitum on a high fat diet. Mice were injected with MS-Hu6 or human IgG (7 µg/day, 5 daysa-week) for 8 weeks. The latter dose was based on the in vitro IC50 of MS-Hu6, which was ~30fold lower than our polyclonal Ab (26). We determined net food intake and measured body weight weekly. Of note was a trend towards an increase in food intake in MS-Hu6-treated mice compared with those given human IgG (Fig. 1A). Despite this trend, there was a decline in body weight, with statistically significant decrements at weeks 7 and 8 (Fig. 1A). We also performed quantitative nuclear magnetic resonance (qNMR) at week 8-this revealed a significant decrease in total mass and fat mass with an increase in lean mass (Fig. 1B). This data mimics FSHR haploinsufficiency in male Fshr +/mice (Fig. 1C). Reduced fat mass was notable on manual weighing in the mesenteric, renal, and gonadal compartments-and, expectedly not in brown adipose tissue (BAT) in MS-Hu6-injected mice (Fig. 1D) (16). Quantitative PCR showed evidence for reduced expression of fat genes, namely Pparg, Fabp4 and Cebpa-either significantly or with trends-in subcutaneous and gonadal WAT and BAT (Fig. 1E). Of note is that serum LH, GnRH and testosterone, levels were unchanged after 8 weeks of MS-Hu6, with an unexplained drop in in serum activin (Fig. 1F).
We explored the activation of interscapular BAT and beiging of WAT in vivo by IVIS imaging after 4 and 8 weeks of MS-Hu6 or human IgG treatment. ThermoMice harbor a Luc2-T2A-tdTomato dual reporter transgene on the Y chromosome driven by the Ucp1 promoter (27) ( Fig. 2A). Mice were injected with D-Luciferin (150 mg/kg), followed by imaging at 3, 5, 10 and 12 minutes to calculate average radiance. The peak radiance at 10 minutes was higher at 8 weeks, with a trend at 4 weeks in mice receiving MS-Hu6 compared with those given human IgG (Fig. 2B). The upper dorsal interscapular region showed profound BAT activation. WAT beiging and BAT activation were both confirmed by UCP1 immunohistochemistry in formalin-fixed, paraffin-embedded sections (Fig. 2C). Of note is that the UCP-1-high beige adipocytes in MS-Hu6-treated mice appeared highly condensed (Fig. 2C). This effect on cell size was confirmed by morphometry in hematoxylin/eosin stained sections (Fig. 2D). Certain beiging genes, including Ucp1, Cox7 and/or Cidea were upregulated in mice given MS-Hu6, again consistent with whiteto-beige transition (Fig. 2E).

Efficacy of MS-Hu6 in Improving Bone Mass
We examined the effect of MS-Hu6 on mouse bones in ThermoMice after 8 weeks of MS-Hu6 or human IgG treatment. Histomorphometry of femoral metaphysis showed significant increases in fractional bone volume (B.Ar/T.Ar) and trabecular thickness (Tb.Th), without an effect on trabecular number (Tb.N) (Fig. 3A). This increase in fractional bone volume was consistent with areal BMD measures (Fig. 3B). Dynamic histomorphometry showed evidence for increased mineral apposition rate (MAR) and bone formation rate (BAR) (Fig. 3C), confirming increased osteoblastic bone formation. In line with a predominately anabolic action of MS-Hu6, the expression of osteoblast genes in bone extracts, such as Col1a1, Alp and Runx2, were either significantly increased or showed trends (Fig. 3D). In contrast, while osteoclast surface (Oc.s/BS) was not reduced, there was a reduction in the expression of the osteoclast marker gene Acp5 (Fig. 3E). The latter finding is not surprising as male ThermoMice mice were not in a high bone turnover state, in which instance a further lowering of bone resorption from baseline would not normally be expected. However, the ex vivo reduction of Acp5 expression suggests that MS-Hu6 was inhibiting the effect of FSH on TRAP-positive osteoclasts.
To further explore the anabolic action of MS-Hu6, and to replicate our dataset in C.J.R.'s lab, 3-month-old female C57BL/6 mice were ovariectomized, followed 24 weeks later by injection of MS-Hu6 or human IgG, daily, at 100 µg/day, for 4 weeks and then 50 µg/day for a further 4 weeks. Total body and femoral BMD measured by Piximus was increased significantly in mice treated with MS-Hu6 (Fig. 3F). Micro CT of the femoral epiphysis showed increased BV/TV (P=0.079), Conn.D (P<0.05), and Tb.N (P=0.057), reduced Tb.Sp (P<0.05), and no change in Tb.Th (Fig. 3G). Expectedly, these anabolic effects were not seen in similarly treated C3H/HeJ mice, which are known to display a high bone mass phenotype (Supplementary Fig. 1) (28).
Overall, therefore, our data show that consistent with previous studies using our polyclonal antibody (17), MS-Hu6 displays an anabolic action in replenishing lost bone.

Pharmacokinetics and Pharmacodynamics of MS-Hu6
Pharmacokinetic studies were performed in three mouse models-C57BL/6, CD1 and Tg32 mice-using 89 Zr-labelled, unconjugated, or biotinylated MS-Hu6. For 89 Zr labeling, MS-Hu6 was incubated with the chelator DFO-p-NCS for 3 hours, followed by incubation with 89 Zroxalate for 1 hour at 37 o C, ultrafiltration (cut off 10 kDa), and thin layer chromatography for quality check (29) (see Methods). 89 Zr-MS-Hu6 was injected as a single dose of 250 µCi (~250 µg) into the retroorbital sinus of 3-month-old male C57BL/6 mice (N=5 mice). For γ-counting few drops of blood were drawn from the tail vein at 5, 30 and 60 minutes, and then at 2, 4, 24, 48, 72 and 120 hours. There was an increase in serum 89 Zr-MS-Hu6 levels to a Cmax of 29.6 µg/mL, which was followed by a gradual decay of radioactivity with a β phase t½ of 32 hours (Fig. 4A).
As C57BL/6 mice are inbred strains, we attempted to validate the pharmacokinetic studies in an outbred strain-CD1. The latter mice display genetic diversity reminiscent of the human population, and are used widely for toxicology and efficacy testing (30). For biotinylation, MS-Hu6 was incubated in the presence of NHS ester-biotin in NaHCO3 (pH 8) and the product was To enable mouse-to-human comparisons, we studied the pharmacokinetics of MS-Hu6 in Tg32 mice. These mice express the FCGRT transgene encoding the human FcRn receptor on chromosome 2 on a Fcgrt -/background. Tg32 mice show decreased plasma clearance of human IgG-based therapeutics--thus, more closely mimicking human pharmacokinetics than C57BL/6 mice. We injected 3-month-old male Tg32 mice with 89 Zr-MS-Hu6 (~250 µCi or 250 µg) into the retroorbital sinus followed by sampling at 1,18,24,48,72,144,192,216 and 240 hours (N=5 mice). Expectedly, the β phase t½ increased to 4 days (95 hours), with a Cmax of 12.2 µg/mL (Fig.   4C). We further studied the profile of i.p. administered unconjugated MS-Hu6 in Tg32 mice by injecting a single bolus dose of 200 µg, and measuring human IgG by an in-house sandwich ELISA in which anti-human Fc and Fab were used to capture and detect bound MS-Hu6, respectively (N=3 mice). This yielded Cmax of 20 µg/mL, with β phase t½ of 13 days (316 hours) ( Fig. 4D). Of note is that the β phase t½ for i.v. trastuzumab, currently in human use, is ~8.5 days in Tg32 mice (31)--this latter β decay translates into 21-day dosing intervals.
We looked for engagement of circulating MS-Hu6 with its target-FSH. For this, we injected groups of male and female C57BL/6 mice, intraperitoneally, with 200 µg MS-Hu6 or human IgG. Mice were bled 16 hours later and total IgG (mouse and human) was pulled down using protein A beads. The content of human IgG (control IgG or MS-Hu6) in the eluate, measured by a sandwich ELISA in which plates were coated with anti-human Fc and an antihuman IgG for capture, remained unchanged. We then subjected the same eluate to an sandwich ELISA, in which the plate was coated with anti-human Fc, and the MS-Hu6-FSHβα complex was captured by an antibody to the α subunit of FSH (incubation at 4 o C, overnight). We found that injected MS-Hu6, but control human IgG bound endogenous FSH in vivo (Fig. 4E).

Biodistribution and Excretion of MS-Hu6
To study the biodistribution of 89 Zr-MS-Hu6, we performed PET-CT scanning of the above treated mice at 24, 48 and 72 hours (N=3 mice). 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). Of note is that these values reflect the presence of 89 Zr-MS-Hu6 in both blood and tissues.
To determine the extent to which of 89 Zr-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 89 Zr-MS-Hu6 were detected in multiple organs, including bone, bone marrow, subcutaneous WAT, visceral WAT, and BAT (Fig. 5C). Minimal amounts of 89 Zr-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 89 Zr-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).
Consistent with the 89 Zr-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-in all, confirming organ retention of the AF750-MS-Hu6, and excretion of the unconjugated dye. Because of the expected minimal localization of MS-Hu6 in the brain, we further performed confirmatory studies by immunofluorescence. For this, we injected AF488-MS-Hu6 or unconjugated AF488 into the tail veins of C57BL/6 mice (200 µg per mouse). We detected immunofluorescence in liver, kidney and hippocampal sections in mice treated with AF488-MS-Hu6 (Fig. 5H). In contrast, AF488treated mice showed fluorescence in kidney sections, but not in liver or brain (Fig. 5H). Staining of hippocampal sections with an anti-human IgG confirmed localization (Fig. 5I).
To understand MS-Hu6 biodistribution as it may apply to humans, we injected 89 Zr-MS-Hu6 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 120 hours. 89 Zr-MS-Hu6 peaked in the blood at 5 minutes, with an expected decline, albeit with persistence in the serum, at 48 and 120 hours (Fig. 5J). 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 (Fig. 5K).

Acute and Chronic Safety of FSH Blockade
We monitored standard safety parameters 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 (Fig. 6A).
We also drew bloods at day 0 (pre-injection) and at days 2 and 5 post-injection. No concerning deviations from normative values were noted (Fig. 6B). This suggests that, albeit at a low dose, MS-Hu6 as a single intravenous bolus injection into monkeys appeared to be generally safe.
MS-Hu6 was generated by swapping the mouse framework region of our parent mouse monoclonal antibody Hf2 with the human IgG1 framework, keeping the CDR itself unaltered.
While both Fc and Fab region are human, mutations were introduced in the framework flanking the CDR region. Because MS-Hu6 is not fully "human", we first determined its "humanness" in silico by inputting the VL and VH sequences into abYsis. Comparison of Z-scores revealed a rightshift in comparison with the human-mouse chimeric antibody (26), and in correspondence with human IgG1 (Fig. 6C). In addition, we inputted the primary amino acid sequences of commercially utilized humanized ("zumab") and fully human ("mab") to find that the Z-scores fell in a narrow range away from our chimera or mouse IgGs (Fig. 6C, Table). We next tested immunogenicity experimentally using ELISPOT. The production of inflammatory cytokines IL-2 and IFNγ in human peripheral blood mononuclear cell cultures was unaltered by MS-Hu6, in comparison with a standard CEFT peptide pool (positive control, Immunospot) (Fig. 6D). Finally, we provide genetic evidence, using our Fshr +/mouse, that haploinsufficiency of the FSHR-which mimics the effect of FSH blockade on obesity and osteoporosis (13, 15, 16)-does not affect lifespan negatively in male or female mice (Fig. 6E).

Developability, Formulation and Physicochemistry of MS-Hu6
Therapeutic antibodies selected on the basis of affinity, potency, specificity, functionality and pharmacokinetics might not have unsuitable physicochemical attributes making it difficult to streamline optimal manufacturing. It is therefore imperative that, at an early stage, we determine physicochemical properties, after a rough in silico check for 'red flags' (32)(33)(34). We thus used a computational tool, Protein-Sol, based on machine learning of amino acid sequences and physicochemical variables from 48 FDA-approved antibodies and 89 antibodies in late-stage clinical development (https://protein-sol.manchester.ac.uk/abpred) (32)(33)(34). In the initial iteration, Protein-Sol provided predicted values for 12 separate physicochemical parameters that determine manufacturability. For all outputs, after inputting VH and VL regions, MS-Hu6 fell within acceptable thresholds, and was therefore deemed to be "safe" (Fig. 7). In essence, the physicochemical properties of MS-Hu6 were likely to be broadly similar to FDA-approved antibodies.
For validation, we inputted a version of MS-Hu6, wherein the CDR region was scrambled-5 of 12 outputs, namely affinity capture, cross interaction chromatography (CIC), polyspecificity reagent (PSR), expression titers in HEK cells (HEK), and differential scanning fluorescence (DSF), fell outside the respective thresholds-an early indication that the scrambled version was not manufacturable (Fig. 7). In fact, while 65% FDA-approved monoclonal antibodies show no 'red flags', those with up to 4 red flags have been FDA-approved (34). To complement data from individual outputs, we derived a meta value for both MS-Hu6 and its scrambled sequence (1=best; 100=worst) by averaging ranks for 8 experimental parameters. We found that meta value pairs fell within the lower left quadrant, suggesting overall acceptable physicochemical properties, even in comparison with certain FDA-approved antibodies in the upper right quadrant (Fig. 7).
Before testing the physicochemical characteristics of MS-Hu6 experimentally, we created an optimal formulation. To prevent deamidation and isomerization at neutral and basic pHs, therapeutic antibodies are generally formulated at pHs away from their isoelectric pH (pI) (35,36).
Using Expasy, we predicted the pI for MS-Hu6 as 8.58. Isoelectric focusing confirmed a pI pf 8.7 ( Fig. 8A). We tested 215 combinations of salt, detergent and sugars for their thermal stability (not shown). This yielded in a near-final formulation for MS-Hu6-stock solution of 2 mg/mL in 20 mM phosphate, 0.001% (v/v) Tween-20, 1 mM NaCl, and 260 mM sucrose (pH = 6.58). We found that both Fc and Fab regions of the formulated MS-Hu6 showed a thermal shift (Tm) compared with MS-Hu6 in PBS-and, hence, was confirmed as being more stable (Fig. 8B). We also examined the binding of formulated MS-Hu6 to purified human FSH. A Tm of 3.1 o C of the Fab, and not the Fc region, established greater thermal stability due to FSH binding (Fig. 8C).
Furthermore, and of note, is that compared with MS-Hu6 in PBS, formulated MS-Hu6 showed dampened peak signals (Fig. 8B). The latter finding indicates that a lower number of antibody molecules underwent unfolding-implying enhanced stability-despite identical added concentrations (20 µg/well). Highly soluble and hydrophilic monoclonal antibodies are expected to behave robustly during the manufacturing process. On the other hand, hydrophobic antibodies with high sensitivity to salt may display problems, such as poor expression, aggregation or precipitation during purification. Delayed retention times in a chromatographic assay with a hydrophobic matrix are indicative of a tendency for precipitation. We used a butyl sepharose column, and passed MS-Hu6 in 1.8 M NH2SO4 and 0.1 M Na2PO4 at pH 6.5 over 20 minutes at a flow rate of 1 mL/min. UV absorbance was monitored at 280 nm to yield a retention time of 7.1 minutes (Fig. 8F), which is below the theoretical threshold of 11.2 min (Fig. 7).
We stressed formulated or PBS-containing MS-Hu6 through three freeze-thaw cycles (from -80 o C to room temperature), and by incubation for 1 week at 4 o C, 37 o C or 50 o C, followed, in all cases, by SEC (Fig. 8G). We noted a major peak (#4) and three minor high molecular weight peaks (#1 to 3). Areas under peaks 1 and 2 were between 0 and 0.91% of the total eluate under all conditions for both PBS-containing and formulated MS-Hu6. Peak 3 remained generally low (<1.5%) with formulated MS-Hu6, but was considerably higher (up to 12.7%) during freeze-thaw in PBS. Furthermore, the major peak 4 was consistently >99% with formulated MS-Hu6, particularly when compared to PBS-containing MS-Hu6 under freeze thaw (87.2%). In all, the formulation protected against aggregation even under extreme stress conditions. Extending the protocol to 25 mL elution yielded no fragment peaks under any condition (Fig. 8G).
Therapeutic antibodies must undergo a test for polyreactivity to a panel of relevant antigens, including cardiolipin, hemocyanin (KLH), lipopolysaccharide (LPS), double and single stranded DNA, insulin, human albumin, flagellin, and baculovirus particles (BVP). The fold change for MS-Hu6 binding to every molecule in ELISAs, except hemocyanin and BVP, was below a threshold of 3, and was therefore acceptable (Fig. 8H). BVP scores greater than 5 have been shown to enhance antibody clearance, with potential effects on t½. In contrast. binding to hemocyanin, an arthropod protein, often used as a carrier for synthetic peptides during immunization (37), was likely due its homology with the 13-amino acid sequence of FSHβ that binds MS-Hu6 (Fig, 8H). However, as hemocyanin was not used during the production of the parent monoclonal antibody, Hf2, this is an irrelevant finding. It is now well recognized that targeting the receptor-binding epitope of FSHβ to block its interaction with the FSHR inhibits bone resorption, increases bone formation and bone mass, reduces body fat, and enhances thermogenesis (15)(16)(17)(18)(19)22). Notably, these effects are triggered not only with our polyclonal and monoclonal FSH-blocking antibodies (15)(16)(17)(18), but also through the use by others of vaccines, such as a GST-FSHβ fusion protein (19) or tandem repeats of the epitope (22). It is also important to note that even with high antibody doses, such as 200 µg/mouse/day, serum estrogen levels are unchanged (16,17), likely because of the abundance of FSHRs in the ovary that remain responsive to lowered levels of bioavailable FSH. These preclinical data in mice establishing FSH as an actionable target are reinforced by striking estrogen-independent correlations in women between serum FSH, rapid bone loss and visceral obesity during the menopausal transition, at which time serum estrogen is relatively normal and FSH levels are rising (4,5,(9)(10)(11)(12). This window is likely the most opportune to prevent both bone loss and obesity through selective FSH blockade. In this regard, while negative data with GnRH modulators are mostly confounded by concomitant changes in LH and GnRH (14), it is clear that low gonadotropin levels in triptorelin-treated men with prostate cancer are associated with lower fat mass and body weight than men undergoing orchiectomy, wherein gonadotropins are high (23). Overall, therefore, the data together lend credibility to the idea that FSH-driven changes, at the very least in body composition, in both sexes can be rescued by blocking FSH. The selective inhibition of FSH action therefore becomes a worthy imperative.

DISCUSSION
Menopause is also associated with dyslipidemia, which has long been thought to result from estrogen deficiency. However, there is compelling epidemiologic evidence that high serum FSH levels correlate with serum total and LDL cholesterol in post-menopausal women, and importantly, that total cholesterol rises across the perimenopausal transition, essentially tracking closely with bone loss and obesity (5,7,25,42). Impressively, exogenous FSH, in the presence of estrogen clamped at normal levels, increases serum total cholesterol in mice fed on a high cholesterol diet (24). And, consistent with the idea that FSH is an estrogen-independent driver of menopausal hypercholesteremia, an FSH-blocking antibody, which we have previously shown to be active in bone cells, lowers serum cholesterol (18,24). There is also human evidence that reducing serum FSH by >30% from its zenith in post-menopausal women through estrogen replacement therapy lowers serum cholesterol (25). As in the case of bone and fat cells (13,16), hepatocyte FSHRs couple with Gi2α, which signals through Akt to inhibit FoxO1 binding with the Srebp2 promoter and prevent its repression. Upregulated Srebp2, which drives de novo cholesterol biosynthesis, results in increased cholesterol accumulation and release (24). This action is in addition to the lowering of LDLR expression by FSH (25). Notwithstanding cholesterol-lowering mechanism(s), which are likely to be explored even further, it is possible that an FSH-blocking therapeutic, such as MS-Hu6, could have additional actions on lipid metabolism in people.
Furthermore, what underpins the preponderance of Alzheimer's disease (AD) in postmenopausal women, particularly in relation to disease risk, progression and severity, has remained unclear, now for decades. A role for post-menopausal hypoestrogenemia remains controversial, with improvement (43), no change (44,45) or worsening (46) of cognition with estrogen replacement therapy. In contrast, high serum FSH is strongly associated with the onset of AD, and has thus been suggested as a possible mediator (47,48). More importantly, certain neuropathologic features, including neuritic plaques, neurofibrillary tangles, and gliosis often begin during the perimenopausal transition (49)(50)(51)(52)(53). During this period, women also show a sharp decline in memory function and increased risk of mild cognitive impairment and dementia (11,52,53). We have recently documented exaggerated AD pathology and cognitive decline upon ovariectomy or exogenous FSH injection in three murine models of AD, even in the face of estrogen levels clamped in the normal range (41). This phenotype arises from the action of FSH on hippocampal and cortical neuronal receptors using a pathway involving CEBP/β and the δsecretase asparagine endopeptidase (41). Most notably, however, we found that our polyclonal FSH-blocking antibody, which shares the target epitope with MS-Hu6 (17,26), prevented the AD-like phenotype induced upon ovariectomy (41) In all, therefore, we and others have unraveled new actions of FSH that assign it as an actionable target requiring a highly specific approach to block its action in people. We believe that MS-Hu6 with an affinity approaching that of trastuzumab (54), which we have carefully characterized in terms of structure, biological actions, pharmacokinetics, target engagement, biodistribution, safety and manufacturability, is poised for future testing in human trials.
Admittedly ambitious, we envisage, in a best case scenario, and if mouse data translates into people, of treating four diseases that affect millions of women and men worldwide-namely obesity, osteoporosis, dyslipidemia and neurodegeneration-with a single multipurpose FSHblocking agent.

Animals
Colonies of male and female C57BL6 mice, and male Tg32, Fshr +/mice, and ThermoMice were obtained from Jackson Labs. Male CD1 mice were from Charles River Laboratories. Mice

Body Composition
To study the effects of MS-Hu6 on body composition, we used male SV129 ThermoMice that were allowed ad libitum access to a high-fat diet and injected with MS-Hu6 or human IgG (7 µg/day, 5 days-a-week, i.p.) for 8 weeks. The latter dose was based on the in vitro IC50 of MS-Hu6, which was ~30-fold lower than our polyclonal Ab (26). We determined net food intake and measured body weight weekly, and performed quantitative nuclear magnetic resonance (qNMR) at 8 weeks. For this, live mice were placed into a thin-walled plastic cylinder, with freedom to turn around. An Echo3-in-1 NMR analyzer (Echo Medical) was used to measure fat, lean and total mass, per manufacturer.
In ThermoMice, a luciferase reporter construct, Luc2-T2A-tdTomato, is inserted into the Ucp1 locus on the Y-chromosome (55). Activation of Ucp1 expression leads to upregulation of

Luc2, which can be quantitated in vivo by radiance measurements using IVIS Spectrum In Vivo
Imaging System (Perkin Elmer) following the injection of D-Luciferin (150 mg/kg). At 4 and 8 weeks, the mice were injected with D-Luciferin and radiance was captured from dorsal and ventral surfaces for optimal visualization of interscapular (BAT-rich) and inguinal (WAT-rich plus testes) regions, respectively. Isolated fat depots were weighed manually, and RNA extracted for quantitative PCR (qPCR) for fat (Pparg, Fabp4 and Cebpa) and beiging (Ucp1, Cox 8a, Cox7, Cidea, and Prdm16) genes using appropriate primer sets and Prism 7900-HT (Applied Biosystems Inc.) (56).

Histology and Immunodetection Methods
Tissues were subject to hematoxylin/eosin staining, immunohistochemistry for UCP1

Bone Phenotyping
At Maine Medical Center Research Institute, BMD was measured by dual energy X-ray absorptiometry (DXA) (Piximus, Lunar) with a precision of <1.5% (57). At Mount Sinai, we used Osteosys iNSIGHT and analyzed the DXA data using Insight v1.0.6. In both instances, anaesthetized mice were subject to measurements, with the cranium excluded. The instruments were calibrated each time before use per manufacturer's recommendation. (Sigma Aldrich, catalog # 387A-1KT). RNA was extracted from isolated whole femur for qPCR for osteoblast (Col1a1, Alpl, Runx2 and Bglap) and osteoclast (Acp5) genes using appropriate primer sets using Prism 7900-HT (Applied Biosystems Inc.) (56).

Pharmacokinetics and Biodistribution in Mice
We prepared 89 Zr-MS-Hu6 by incubating MS-Hu6 first with the chelator DFO-p-NCS for 3 hours at 37 o C (in steps of 5 µL until a 10-fold molar excess of chelator was achieved). The DFOfunctionalized MS-Hu6 was washed 3 times with PBS in a 10 kDa ultracentrifugation tube before radiolabeling. 89 Zr-oxalate was diluted with PBS and neutralized with 1M Na2SO4 before adding to functionalized MS-Hu6. This was followed by incubation with 89 Zr-oxalate for 1 hour at 37 o C, ultrafiltration (cut off 10 kDa), and thin layer chromatography (with 50 mM EDTA) for quality check (29). C57BL/6 or Tg32 mice were injected in separate experiments with 89 Zr-MS-Hu6 as a single dose of ~250 µCi (~250 µg, 250 ± 40 µCi) into the retroorbital sinus. Timed blood (few drops drawn from the tail vein) and excreta collection was followed by weighing and γ-counting For imaging, 89 Zr-MS-Hu6-treated mice (above) were anesthetized using 1% isoflurane in O2 at a flow rate of ∼1.0 L/minute. PET/CT scans were performed using a Mediso nanoScan PET/CT (Mediso, Budapest, Hungary). For whole body CT scans, we used the following parameters: energy, 50 kVp; current, 180 µAs; and isotropic voxel size, 0.25 mm)-this was followed by a 30-min PET scan. Image reconstruction was performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from the Mediso Nucline software (version 3.04).
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, version 11.0. 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, 89 Zr-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..
For biodistribution studies using AF750-labelled MS-Hu6, we first imaged the whole body 16 hours after injection using IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Mice were then perfused with PBS, sacrificed and organs, namely heart, thymus, brain, gastronemus and soleus muscle, BAT, adrenals, liver, gall bladder, spleen, kindey, subcutaneous WAT, visceral WAT gonadal WAT, testes and bone were removed and imaged using the same IVIS platform to calculate average radiance efficiency per square area.

Biodistribution and Safety Studies in Monkeys
After an overnight fast, two male Cynomolgus monkeys [Scott, aged 14 years, weight 9.8 kg; and Andy, aged 15 years, weight 6.15 kg] were anesthetized with ketamine (5.0 mg/kg) and dexemedetomidine (0.0075 to 0.0015 mg/kg). The monkeys were injected with 89 Zr-MS-Hu6, and blood was drawn at 30 minutes, and at 48 and 120 hours from the tail vein. Vitals, including mean arterial, systolic and diastolic blood pressure, respiratory rate, heart rate and rectal temperature, were recorded using Waveline Touch system (DRE) and Welch Allyn rectal thermometer. PET and MR images were acquired on a combined 3T PET/MRI system (Biograph mMR, Siemens Healthineers). Whole body MR images from each PET bed (head, thorax, pelvis) were automatically collated together with a scanner. MR parameters were as follows: acquisition plane, coronal; repetition time, 1000 ms; echo time, 79 ms; number of slices, 224; number of average, 2; spatial resolution of 0.6 mm x 0.6 mm x 1.0 mm; and acquisition duration, 29 min and 56 s per bed. After acquisition, PET raw data from each bed were reconstructed and collated offline using the Siemens proprietary e7tools with an ordered subset expectation maximization (OSEM) algorithm with point spread function (PSF) correction. A dual-compartment (soft tissue and air) attenuation map was used for attenuation. Image analysis was performed using Osirix MD, version 11.0. Whole-body MR images were fused with PET images and analyzed in an axial plane. Regions of interest (ROIs) were drawn on various tissues. The liver, kidney, BAT (interscapular region), subcutaneous WAT, visceral WAT, gonadal WAT, gallbladder, spleen, brain, and testes were traced in their entirety; bone marrow was imaged from the shoulder; and 3 lumbar vertebrae; and muscle was imaged from the quadriceps. Mean SUVs were calculated for each ROI. 89 Zr-MS-Hu6 uptake of each tissue was expressed as the average of all mean SUV values per organ. Serum was collected for blood chemistry analysis by IDEXX BioAnalytics.

ELISPOT Assay
Human peripheral blood mononuclear cells (PMBCs, obtained from Immunospot, Cellular Technology Ltd.) were cultured for 12 days in FBS-free DMEM with regular medium change, and plated at a density of 10 5 cells/well in ImmunoSpot ELISPOT plates. Cells were then exposed to MS-Hu6, CEFT or DMEM for 48 hours, following which IL-2 and IFNγ expressing cells were quantitated per manufacturer's instructions.

Target Engagement Assays
For pharmacodynamic studies-namely engagement of MS-Hu6 with FSH as its targetmale and female C57BL/6 mice were injected i.p. with MS-Hu6 or human IgG (200 µg, for each).
After 16 hours, mice were bled and total IgG (mouse and human) was pulled down using protein A beads (ThermoFisher, Catalog # 20333). An in-house sandwich ELISA was used to measure human IgG (control IgG or MS-Hu6) in the eluate. For this, plates were coated with anti-human Fc (Sigma, Catalog # FAB3700259) and goat anti-human HRP-conjugated IgG (H+L) (Invitrogen, Catalog # A18805) was used to for capture total human IgG. The same elute then underwent a second in-house sandwich ELISA, in which the plate was coated with anti-human Fc (Sigma, Catalog # FAB3700259), and after overnight incubation at 4 o C, the MS-Hu6-FSHβα complex was captured by an antibody to the α subunit of FSH. In a separate study, a commercial ELISA for FSH (Biotechnology Systems, Catalog # M7581) was used to determine whether MS-Hu6 binding to FSH interfered with its detection.

Protein Thermal Shift Assay
The thermal shift assay used a fluorescent reporter, Sypro-Orange (Protein Thermal Shift Dye Kit, ThermoFisher, Catalog # 4461146), to detect hydrophobic domains that are exposed following the heat-induced unfolding of globular proteins. MS-Hu6 (1.5 µg/µL), formulated or in PBS, was incubated with or without human FSH (0.5 µg/µL) at room temperature for 30 minutes, with fluorescence captured sequentially at 0.3 o C increments using a StepOne Plus Thermocycler (Applied Biosystems). Tm was calculated based on the inflection point of the melt curve, and thermal shift was derived from Tm = TmA-TmB.

Isoelectric Focusing
For determining the isoelectric pH (pI), two dimensional electrophoresis was performed by first rehydrating MS-Hu6 (500 µg) for 2 hours at room temperature in rehydration buffer (8M urea, 2% CHAPS, 0.5% IPG buffer, and trace of bromophenol blue) without DTT. The sample was then run on an 18 cm 3-10 strip using the Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare).
Four voltage steps (50 V for 10 hours; 500 V for 1 hour; 1000 V for 1 hour; 8000V for 4 hours) were followed by Coomassie blue staining.

Polyspecificity Testing
We adapted a previous method to antibody determine polyspecificity (34),

Statistical Methods
Statistically significant differences between any two groups were examined using a twotailed Student's t-test, given equal variance. P values were considered significant at or below 0.05. Data points were excluded if they were 2.5 standard deviations above or below means Ensuring Rigor and Reproducibility: There is a nascent movement to ensure that preclinical data is true and accurate (58)(59)(60)(61)(62)(63). M.Z. and C.J.R. coined the phrase 'contemporaneous reproducibility,' which refers to the synchronous reproduction of data in more than one laboratory.
As Zaidi's discovery of the effects of FSH on body fat were novel and unexpected, he reached out to C.J.R. for help in form of a reproducibility study. Key data sets were reproduced by C.J.R in a process that lasted over three years, as other validation studies were added by both laboratories. The term replicability refers to the ability of one or more independent groups to replicate a finding using a different technology or method--replicability is a measure of truth or significance of a given finding (64). Here, we have replicated a key finding that Hu6 increased bone mass in the M.Z. and C.J.R. labs, with µCT data independently produced by J.C. To further enhance transparency, we have hosted detailed procedures and raw datasets on our GLPcompliant MediaLab Document Control System that all investigators have access to. All data have undergone quality checks before the final product was signed off. Such practices requiring unfettered transparency remain fundamental to ensuring rigor.    Detection of the MS-Hu6-FSHβα complex was achieved using an in-house ELISA in which the plate was coated with anti-human Fc and the complex captured by an antibody to the α subunit of FSH (N=3 biologic replicates). Of note is that total human IgG (control IgG or MS-Hu6) captured by an in-house ELISA was not different across treatment groups (E , Table).  data (green) from: (65). In silico assessment of "humanness" using abYsis, with Z-scores comparing humanized MS-Hu6, mouse-human chimeric antibody (26), and human IgG1 (C).