Vestronidase alfa: Recombinant human β-glucuronidase as an enzyme replacement therapy for MPS VII

Journal Pre-proof Vestronidase alfa: Recombinant human β-glucuronidase as an enzyme replacement therapy for MPS VII

Jaclyn Cadaoas, Gabrielle Boyle, Steven Jungles, Sean Cullen, Michel Vellard, Jeffrey H. Grubb, Agnieszka Jurecka, William Sly, Emil Kakkis PII:

S1096-7192(20)30059-7

DOI:

https://doi.org/10.1016/j.ymgme.2020.02.009

Reference:

YMGME 6619

To appear in:

Molecular Genetics and Metabolism

Received date:

25 October 2019

Revised date:

23 February 2020

Accepted date:

23 February 2020

Please cite this article as: J. Cadaoas, G. Boyle, S. Jungles, et al., Vestronidase alfa: Recombinant human β-glucuronidase as an enzyme replacement therapy for MPS VII, Molecular Genetics and Metabolism (2020), https://doi.org/10.1016/ j.ymgme.2020.02.009

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© 2020 Published by Elsevier.

Journal Pre-proof Vestronidase alfa: Recombinant Human β-glucuronidase as an Enzyme Replacement Therapy for MPS VII Jaclyn Cadaoas a* , Gabrielle Boyle a* , Steven Jungles a, Sean Cullen a, Michel Vellard Jeffrey H Grubb a,c , Agnieszka Jurecka a, William Sly d, Emil Kakkis a,** a

a,b

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Ultragenyx Pharmaceutical Inc., Novato, CA 94949, USA; bCurrently at Audacity Therapeutics PBC, France; c Currently at Saint Louis University School of Medicine, Department of Biochemistry and Molecular Biology, St. Louis, MO 63104, USA; dSaint Louis University School of Medicine, Department of Biochemistry and Molecular Biology, St. Louis, MO 63104, USA *These authors contributed equally to this manuscript.

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**Corresponding author: Emil Kakkis Ultragenyx Pharmaceutical Inc 60 Leveroni Ct Novato, CA 94949 Email: [email protected]

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Short Title: Vestronidase alfa (rhGUS) for MPS VII

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Key words: Mucopolysaccharidosis VII; MPS VII; vestronidase alfa; β-glucuronidase

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Abstract Mucopolysaccharidosis VII (MPS VII) is a rare lysosomal storage disease characterized by a deficiency in the enzyme β-glucuronidase that has previously been successfully treated in a mouse model with enzyme replacement therapy. Here, we present the generation of a novel, highly sialylated version of recombinant human β-glucuronidase (rhGUS), vestronidase alfa, that

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has high uptake, resulting in an improved enzyme replacement therapy for the treatment of

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patients with MPS VII. In vitro, vestronidase alfa has 10-fold more sialic acid per mole of rhGUS monomer than a prior rhGUS version (referred as GUS 43/44) and demonstrated very high

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affinity at ~1 nM half maximal uptake in human MPS VII fibroblasts. Vestronidase alfa has a

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longer enzymatic half-life after uptake into fibroblasts compared with other enzymes used as

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replacement therapy for MPS (40 days vs 3 to 4 days, respectively). In pharmacokinetic and tissue distribution experiments in Sprague-Dawley rats, intravenous administration of

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vestronidase alfa resulted in higher serum rhGUS levels and enhanced β-glucuronidase activity distributed to target tissues. Weekly intravenous injections of vestronidase alfa (0.1 mg/kg to

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20 mg/kg) in a murine model of MPS VII demonstrated efficient enzyme delivery to all tissues,

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including bone and brain, as well as reduced lysosomal storage of glycosaminoglycans (GAGs) in a dose-dependent manner, resulting in increased survival after 8 weeks of treatment. Vestronidase alfa was well-tolerated and demonstrated no toxicity at concentrations that reached 5-times the proposed clinical dose. In a first-in-human phase 1/2 clinical trial, a dose-dependent reduction in urine GAG levels was sustained over 38 weeks of treatment with vestronidase alfa. Together, these results support the therapeutic potential of vestronidase alfa as an enzyme replacement therapy for patients with MPS VII.

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Introduction Mucopolysaccharidosis VII (MPS VII, Sly Syndrome) is an inherited lysosomal storage disease caused by a deficiency of β-glucuronidase (GUS), a lysosomal hydrolase responsible for the removal of terminal glucuronic acid residues from non-reducing ends of certain glycosaminoglycans (GAGs) [1]. Reduced GUS activity in MPS VII leads to decreased

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degradation of three specific GAGs: dermatan sulfate (DS), chondroitin sulfate (CS), and

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heparan sulfate (HS), which accumulate progressively in diverse tissues, leading to systemic tissue and organ dysfunction within the heart, airways, lungs, liver, spleen, brain, and bone [2,

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3]. MPS VII spans a spectrum of disease severity, with substantial clinical heterogeneity among

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patients. Symptoms can include coarse facial features, enlarged spleen and liver, skeletal

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dysplasia, cardiac valve disease, and occasionally hydrops fetalis in newborns [4-7]. Historically, clinical improvement has been observed with hematopoietic stem cell transplant

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and enzyme replacement therapy (ERT) in other MPS disorders, including MPS I, MPS II, MPS

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IVA, and MPS VI [8, 9]. GUS 43/44 was first developed as an ERT in the murine model of MPS VII, showing decreased accumulation of GAGs in the lysosomes and improvement in diverse

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soft tissues and connective tissues, such as bone, as well as enhanced animal survival. In the brain, reduction in storage of GAGs was also observed [4-7]. Additional studies in adult mice showed that intravenous administration at 4 mg/kg after 13 weeks of treatment demonstrated a reduction in neuronal storage, as well as glial storage in the brain [5, 10]. Despite these successful proof of concept experiments, which were conducted 20 years ago, a recombinant human GUS enzyme (rhGUS) was not developed as an ERT for human patients, primarily because of the very small patient population with fewer than 200 known patients worldwide.

Journal Pre-proof We report that an rhGUS enzyme, referred to hereafter as vestronidase alfa, was produced in a genetically engineered Chinese hamster ovary (CHO) cell line and has an identical amino acid sequence and core protein structure to the naturally occurring GUS enzyme [11]. Interestingly, the large scale product optimization of vestronidase alfa serendipidously generated an enzyme with 27 times more sialic acid (1.1 mole/mole) than the earlier version GUS 43/44 (0.04 mole/mole) [5]. Although these effects are more often protein specific, sialylation has been

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shown to play a major role in the circulating half-life of glycoproteins due to the removal of asialo-glycoproteins in serum by liver asialo-glycoprotein receptors. Consequently, expression

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of terminal sialic acid residues may prevent serum glycoproteins from degradation [12] and

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thereby extend the in vivo half-life.

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The data described herein demonstrate that increased sialylation of rhGUS results in improved

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biochemical, pharmacokinetic, and pharmacodynamic properties, as well as in vivo efficacy and provide strong mechanistic rationale for the use of vestronidase alfa in the treatment of the

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many manifestations of MPS VII. Indeed, our preclinical work has translated well in patients as vestronidase alfa has been successfully developed as an ERT for the treatment of MPS VII and

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Results

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was recently approved in the United States, Europe, and Brazil [13].

Vestronidase Alfa and GUS 43/44 Protein Characteristics and Function In Vitro Recombinant human GUS was produced in a genetically engineered CHO cell line expressing complementary DNA encoding full-length native human GUS protein (GenBank accession no. NM_000181). The rhGUS protein was expressed as a 651 amino acid precursor with an Nterminal signal sequence of 22 amino acids [11]. After glycosylation at four N-linked glycosylation sites (asparagines 173, 272, 420, and 631) the approximate molecular weight for each monomer was 82 kDa.

Journal Pre-proof GUS 43/44 was expressed in CHO cells grown in a continuous perfusion system using microcarrier beads versus early preclinical lots of vestronidase alfa that were produced in CHO cells grown in suspension cultures using large scale bioreactors. GUS 43/44 and vestronidase alfa were purified using standard chromatography methods as a 332 kDa homotetramer, with two active sites per tetramer. Vestronidase alfa and GUS 43/44 were analyzed for physical and functional characteristics which are summarized in Table 1.

-400 99.2 97.7 12-14 1.1 3.6 1.2-1.7

-50 >95.0 99 12-14 0.04 3.7 1.4

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GUS 43/44

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Titer, mg/L Purity (Reducing SDS-PAGE), % Tetramer (SE-HPLC), % M6P N-Glycan Analysis, Mol-% Sialic Acid Content, moles/mole monomer Specific Activity, MU/mg Cellular Uptake, Kuptake nM

Vestronidase Alfa

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Characteristics

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Table 1. Comparison of Characteristics Between Vestronidase Alfa and GUS 43/44

Delivery of a lysosomal enzyme to various tissues during ERT is primarily dependent on the

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mannose-6-phosphate (M6P) content of the enzyme located on terminal ends of N-linked

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glycans [14, 15]. Analysis of the M6P content on N-linked glycans released from vestronidase alfa and GUS 43/44 determined that they were similar and confirms that the enzyme produced

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maintains the necessary phosphorylation for high uptake and delivery. To test this property experimentally, cellular uptake studies were performed in GUS-deficient, MPS VII patient-derived fibroblasts, GM-2784, which demonstrated that uptake of vestronidase alfa was saturable with a Kuptake of approximately 1.2 nM to1.7 nM, comparable to GUS 43/44 (Table 1). These results are consistent with other recombinant human ERTs with high cellular uptake (eg, rh-iduronidase Kuptake is 1 nM to 2 nM) [16]. While vestronidase alfa and GUS 43/44 exhibit similar physical and functional characteristics, one key difference is indicated in their degree of sialylation, with vestronidase alfa having 27 times higher sialic acid content than GUS 43/44 (Table 1).

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To investigate the effect of sialylation on the cellular half-life of vestronidase alfa, a pulse-chase experiment was performed to measure the decay of rhGUS over time after uptake in GM-2784 patient-derived fibroblasts. The half-life was measured using three different concentrations of vestronidase alfa (1, 2, or 4 µg/mL) and the resulting cell-associated GUS activity was assessed over the course of 0 to 42 days (Figure S1). For all drug concentrations, the intracellular half-life of vestronidase alfa in human MPS VII fibroblasts was determined to be approximately 40 days

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(1 µg/ml t1/2 = 39.3 days; 2 µg/ml t1/2 = 43.0 days; 4 µg/ml t1/2 = 40 days), which was nearly 8-fold

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longer than iduronidase (t1/2 = 3 to 5 days) [16]. The slight curvature of the plotted points underlying the best-fit line suggests that the rate of decline may be faster at higher enzyme

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concentrations and may diminish as levels reduce, which would further increase the

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substantially longer intracellular half-life of vestronidase alfa. When vestronidase alfa was

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compared with GUS 43/44, the similar M6P content of these two enzymes resulted in comparable t1/2, with vestronidase alfa CR015-21d t1/2 of 21.6 Days vs GUS 43/445-21d t1/2 of 20.5

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Days (Figure 1).

Journal Pre-proof Figure 1. Comparison of the t1/2 of vestronidase alfa CRO1 vs GUS 43/44 Activity After Uptake By Human MPS7 Fibroblasts

vestronidase alfa CR010-28d t1/2 = 22.6 Days vestronidase alfa CR015-21d t1/2 = 21.6 Days

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GUS 43/440-21d t1/2 = 18.9 Days GUS 43/445-21d t1/2 = 20.5 Days

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% of Cell Associated GUS B Activity Remaining

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Pharmacokinetics and Tissue Distribution in Sprague Dawley Rats

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It is well established that subtle modifications in the environmental growth conditions of CHO cultures can result in changes in the glycosylation pattern of secreted proteins [17]. To evaluate

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the pharmacokinetics and in vivo distribution, vestronidase alfa or GUS 43/44 was administered

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in a single 2-hour infusion in Sprague Dawley rats (5 animals per treatment). The concentration and dose of both vestronidase alfa and GUS 43/44 were equivalent, at 2 mg/ml and 2 mg/kg, respectively. This infusion protocol was intended to simulate the planned dosing regimen in patients which consisted of a slow infusion stage over the first hour (1/3 the total volume) and a fast infusion stage for the second hour (2/3 the total volume). In the course of the study, serum and tissue were collected at various timepoints and assayed for GUS activity. Rats infused with saline were used as non-treated controls (5 animals per group) to account for endogenous GUS activity, which was subtracted from the activity measured in rats infused with enzyme. The pharmacokinetic profiles for vestronidase alfa and GUS 43/44 were assessed during the

Journal Pre-proof infusion and 24 hours post infusion. Both profiles describe a time-dependent increase in enzyme activity levels that reached steady-state by the end of the slow infusion period, before increasing again at the start of the fast infusion period (Figure 2A). Both enzymes were rapidly cleared from the serum (t1/2: 5.30 minutes for vestronidase alfa; t1/2: 4.50 minutes for GUS 43/44) during the post-infusion clearance phase, which is generally characteristic of lysosomal enzymes [18]. The mean serum pharmacokinetics of vestronidase alfa and GUS 43/44 are

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provided in Table 2.

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Figure 2A. Pharmacokinetics of vestronidase alfa and GUS 43/44 in Sprague Dawley Rats Serum pharmacokinetic profile of vestronidase alfa and GUS 43/44. Sera was collected at various timepoints throughout the stages of infusion (slow, fast, post) and measured for GUS activity. The concentration and dose of both vestronidase alfa and GUS 43/44 were equivalent, at 2 mg/ml and 2 mg/kg, respectively.

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Table 2. Summary of Pharmacokinetic Analysis of Vestronidase Alfa and GUS 43/44

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Vestronidase alfa GUS 43/44 n=5 n=5 Cmax (Units/mL), mean 14800 4300 T max (h), mean 0 0.0133 AUC0-t (h*Units/mL), mean 18700 5580 AUC0-inf (h*Units/mL), mean 18800 5600 1st Phase Half-life (min), mean 5.30 4.50 2nd Phase Half-life (h), mean 1.1 0.967 Clearance (min*Units/mL)/(mg/kg) 1.8E06 6.23E-06 The concentration and dose of both vestronidase alfa and GUS 43/44 were equivalent, at 2 mg/ml and 2 mg/kg, respectively.

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Although the serum half-life of both enzymes was not substantially different, the highly sialylated

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vestronidase alfa showed slower clearance over time, leading to higher levels of rhGUS in

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serum compared with GUS 43/44. Specifically, serum levels of vestronidase alfa reached a level of GUS activity 2-fold higher at the end of the slow infusion period, and 3-fold higher at the end

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of the fast infusion period, compared with GUS 43/44 (Figure 2A and Table 2). Although tmax was similar for both groups, the mean C max for animals infused with vestronidase alfa was

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approximately 3- to 4-fold higher than the mean C max for GUS 43/44 (Table 2). This difference

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was also evident in the mean exposure measured by the area under the curve (AUC), which indicated a lower rate of serum clearance for animals dosed with vestronidase alfa than the animals dosed with GUS 43/44, suggesting that the enhanced sialylation of vestronidase alfa reduced its rate of clearance from serum and improved its pharmacokinetic properties over GUS 43/44. The lower exposure (AUC) of GUS 43/44 compared with vestronidase alfa may also be due to increased clearance of the asialo-enzyme in rats, resulting in decreased bioavailability of GUS 43/44 to be delivered to tissues. To assess delivery of both enzymes, tissue distribution after a single dose of 2 mg/kg of vestronidase alfa or GUS 43/44 was measured in the liver, spleen, heart, kidney, brain, and

Journal Pre-proof lung. Following necropsy 24 hours post infusion, homogenates were prepared from each tissue, assayed for GUS activity, and normalized to total protein. In rats infused with vestronidase alfa, increased GUS enzyme activity was readily observed over endogenous levels in the liver and spleen, with milder changes in enzyme activity observed in other tissues (Figure 2B). This increase in enzyme activity over normal endogenous levels confirmed successful delivery of vestronidase alfa to tissues following a single administration. In comparing the tissue distribution

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of both enzymes, vestronidase alfa demonstrated improved delivery versus GUS 43/44 across all tissue types, including those more difficult to perfuse, such as the heart and lung (Figure 2B).

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This suggests that the increased sialylation of vestronidase alfa may improve delivery to tissues

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that are more difficult to treat by delaying the clearance of enzyme in serum that otherwise

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receptors on reticuloendothelial cells.

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would have been rapidly removed via asialo-glycoprotein receptors of the liver or the mannose

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Figure 2B. Tissue Distribution of vestronidase alfa and GUS 43/44 in Sprague Dawley Rats Following 2-Stage infusion of vestronidase alfa and GUS 43/44, select peripheral organs were analyzed for GUS activity and normalized by total protein by BCA. Final GUS activity was adjusted to account for endogenous GUS activity from saline controls. Error bars represent standard deviation (SD).

Pharmacodynamic Activity and Efficacy of Vestronidase Alfa Administration in a Mouse Model of MPS VII A proof of concept study was performed in adult MPS VII/E540ATg mice to assess the distribution, safety, and efficacy of vestronidase alfa in reducing lysosomal storage of GAGs in a relevant disease model. These transgenic mice are deficient in murine GUS, but also express an inactive mutant version of human GUS that has an amino acid substitution at the active site

Journal Pre-proof nucleophile E540A, which enables these mice to be almost completely immunotolerant to infused human enzyme. This model retains the clinical, morphological, biochemical, and histopathological characteristics of the original, naturally occurring MPS VII (gus mps/mps ) mouse model and is phenotypically similar [19]. In this study, mice received vestronidase alfa by intravenous bolus tail vein injection once weekly for 8 consecutive weeks at doses of 0 (vehicle control), 0.1, 0.25, 1, 4, or 20 mg/kg (n=6 animals per dose). A second group of animals at the

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4 mg/kg dose remained on study for an additional week without treatment to investigate the recovery and accumulation of substrate. At the end of study, mice were necropsied and select

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tissue samples were collected and processed for histopathology and pharmacodynamic

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assessment based on lysosomal storage content and tissue distribution of vestronidase alfa.

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Complement-mediated anaphylactic and immune reactions are commonly observed in mice

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undergoing ERT through rapid infusion protocols and can result in poor tolerability [7, 19, 20]. Therefore, to manage allergic reactions, the study included an antihistamine (cyproheptadine)

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pretreatment beginning on week 4, prior to the next administration of vestronidase alfa. Following this modification, only one additional death (from the 0.1 mg/kg group) was observed

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in the treated animals from weeks 4 to 8, indicating that pretreatment with antihistamine was

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effective in preventing anaphylactic reactions to vestronidase alfa. Untreated, the MPS VII mouse model has a shortened life span of approximately 6 months. Thus, it is important to note that 2 of 6 mice in the control group succumbed to disease manifestations during the study, one at week 3 (age 3.5 months) and one at week 5 (age 4 months). Analysis of the overall survival in affected, treated animals showed an increased survival through the end of the recovery period compared with the control animals. Other than the observed anaphylactic immune responses in some mice, no significant adverse clinical observations were noted during the study. The majority of animals gained weight over the course of the study, and individual body weight gains at week 8 were similar between all

Journal Pre-proof groups (data not shown). Additionally, there were no apparent adverse changes in the serum chemistry and hematology parameters evaluated (data not shown). Vestronidase alfa was welltolerated in mice at doses up to 20 mg/kg and resulted in increased survival at all doses tested, without evidence of toxicological effects or pathology. Thus, our data demonstrate that vestronidase alfa is efficacious in a mouse model of MPS VII at doses that were well-tolerated. Dose-dependent Biodistribution of Vestronidase Alfa in Mouse Tissues After 8 weeks of

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ERT

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After 8 weeks of ERT with vestronidase alfa, serum and tissue extracts were prepared and

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assayed for GUS enzyme activity. Compared with the saline-treated controls, at 72 hours post final dose, vestronidase alfa treatment increased mean serum GUS activity in all groups, though

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this difference was only statistically significant in the 20 mg/kg cohort (mean 0.35 ± 0.1 SD

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units/mL for the saline controls compared with 13.14 ± 4.7 units/mL in the 20 mg/kg treated animals, p≤0.0001). Due to the short serum half-life of the enzyme, appreciable increases in

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vestronidase alfa.

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serum GUS activity 72 hours after an infusion were only anticipated for the highest dose of

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Enzyme activity was also evaluated in a variety of tissues, including the liver, spleen, lung, heart, kidney, brain, bone, muscle, thymus, thyroid, adrenal gland, testis, ovary/uterus, and prostate. Clinically relevant levels of enzyme, levels ≥ Kuptake, were attained in nearly all tissues in a dose-dependent manner (Figure 3), with some tissues in the 4 mg/kg and 20mg/kg groups nearing or exceeding GUS activity levels observed in wild-type mice. Levels as low as 1% to 3% are likely beneficial on the reduction in lysosomal storage. It is also important to recognize that fast bolus infusion, as required for mice, does not achieve the same breadth and degree of distribution that longer infusions over several hours can achieve in humans. Higher enzyme activity levels were attained in well-perfused tissues, such as the liver and spleen (1289% and

Journal Pre-proof 489%, respectively, in the 20 mg/kg dose compared with wild-type activity) while lesspenetrable tissues such as brain, thymus, and kidney took up less enzyme (4.1%, 10.8%, and 45.0%, respectively), though these were still likely above therapeutic levels. Increased GUS activity was maintained in the 4 mg/kg recovery group, demonstrating that vestronidase alfa

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activity persists in most tissues up to a week after infusion.

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Figure 3. Vestronidase alfa enhances GUS activity in MPSVII mice in a dose-dependent manner GUS activity levels after ERT for 8 Weeks with vestronidase alfa compared to activity levels in wild-type C57BL6/J mice. Tissue GUS activity was normalized to total protein and expressed as units/hr/mg. Vehicle control group (0 mg/kg dose) was n=4. All other dose groups were n=6.

Clearance of Storage Pathology in Mouse Tissues After 8-weeks of ERT with Vestronidase Alfa

Journal Pre-proof To assess the lysosomal storage in the MPS VII mice, fixed tissue sections were stained with Alcian Blue and a hematoxylin and eosin counterstain. The presence or absence of storage vacuoles in the stained tissue sections was observed by bright field microscopy and scored blindly by a board-certified pathologist at Charles River Laboratories. Representative microscopy images from control and treated (20 mg/kg) mice are depicted in Figure 4A. The untreated controls showed a large number of GAG-laden distended lysosomal vacuoles

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throughout the tissue. Treatment with vestronidase alfa resulted in a reduction in cytoplasmic

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vacuolation in most tissues evaluated.

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Figure 4A. Vestronidase alfa Reduces Lysosomal Storage in MPS VII Mice Tissues were stained with Alcian Blue and a hematoxylin and eosin counter stain and observed by bright field microscopy. Representative images (60X magnification) exhibit decreased cytoplasmic vacuolation following ERT for 8 weeks with 20 mg/kg vestronidase alfa (bottom row) compared with saline controls (top row).

To quantify the reduction in lysosomal storage, all tissues were blinded to the pathologist prior to scoring on a scale of 0 to 4, with zero representing normal tissue and increasing numbers indicating higher levels of cytoplasmic vacuolation. Compared with untreated controls, treatment with vestronidase alfa resulted in a reduction in cytoplasmic vacuolation in most tissues and tissue elements examined histologically; this reduction was often dose-dependent, starting at the lowest dose level (Figure 4B).

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Figure 4B. Vestronidase alfa Reduces Lysosomal Storage in MPS VII Mice Histopathological scores were attained based on the presence or absence of vacuoles in stained tissue sections of mice treated with vestronidase alfa. Tissues were scored (0 good, 4 worst) by a board-certified pathologist and plotted by increasing dose of vestronidase alfa. Blue dots represent individual mice. Red lines represent the mean of the group.

Journal Pre-proof Histopathology scores in tissues such as liver Kupffer cells, spleen macrophages, and bone sinusoidal lining cells, demonstrated that vestronidase alfa effectively reversed storage pathology, even at the lowest dose (0.1 mg/kg). In other more difficult to penetrate tissues, such as the kidney tubular epithelium and bone osteocytes, the reduction in histopathology scores followed a dose-dependent course, while other tissues like heart valvular and epicardial stromal cells and brain neurons, showed an overall reduction compared with untreated vehicle controls.

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Reduction in vacuolation for liver hepatocytes were not readily observed by the pathologist, despite being a known target of ERT. Further inspection of these slides showed that the

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lysosomes were, in fact, clear of storage but did contain swollen mitochondria, which may have

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arisen as an artifact of fixation and then mistaken for GAG accumulation. Additionally, except for

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glial cells (not shown) and perivascular brain cells, the reduced cytoplasmic vacuolation was maintained during the week-long recovery period, and did not recur, consistent with the 40-day

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enzyme half-life.

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Dose-dependent Reduction of Urinary GAGs Previous work has suggested that urinary GAG (uGAG) excretion can serve as an accurate

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biomarker to assess lysosomal storage in MPS diseases [21, 22]. Urinary GAG levels were measured by the non-reducing end (NRE) method [23], as shown in Figure 6, depicting the mean percent change in uGAG levels in mice treated for 8 weeks with vestronidase alfa compared with pre-treatment levels. The chondroitin sulfate/dermatan sulfate (CS/DS) levels represent most of the total GAG excreted in the MPS VII mouse and in human patients. In control, saline-treated MPS VII mice, CS/DS NREs remained at approximately 100% of baseline after 8 weeks. However, after vestronidase alfa treatment, CS/DS NREs decreased in a dose-dependent manner, with a greater than 80% reduction at the highest doses. The heparan sulfate (HS) NREs in the urine increased slightly in the untreated controls over the course of the

Journal Pre-proof study but decreased in the enzyme-treated mice in a dose dependent manner, reaching a greater than 80% reduction, as observed for the CS/DS NREs. Both CS/DS and HS uGAG reductions were sustained through the weeklong recovery period. More recently, an LC-MS/MS assay to measure GAGs was developed and selected for clinical sample analysis due to the higher throughput and increased reliability of this methodology.[24] Utilizing this method, we have shown the same correlation between GAG levels and doses in humans, supporting other

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evidence that GAGs represent a powerful pharmacodynamic biomarker of response to ERT.

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When comparing the dose response on tissue storage and uGAG results, maximal reduction of uGAG is correlated well with the reductions observed in a variety of difficult-to-treat tissues,

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such as neurons, osteocytes, and kidney tubular epithelium. In all three tissues, near maximal

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reduction is achieved at 4 mg/kg, with a modest pharmacological increase at 20 mg/kg, similar

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to the result observed with urinary CS/DS (Figure 5).

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Figure 5. Vestronidase alfa Reduces uGAG Levels in MPS VII Mice in a Dose-Dependent Manner Urinary GAG levels were measured by the non-reducing end (NRE) method and plotted as the percent change in uGAG levels after 8-week treatment with vestronidase alfa compared to individual pretreatment measurements. n.s. = not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbreviations: CS=chondroitin sulfate, DS=dermatan sulfate, HS=Heparan sulfate

Journal Pre-proof Reduction in uGAGs in Patients With MPS VII This first-in-human study included three subjects with MPS VII treated with intravenous vestronidase alfa every other week (QOW) for an initial 38 weeks at doses ranging from 1 mg/kg to 4 mg/kg (Table 3). Informed consent was obtained from all subjects prior to any study specific procedures. All three subjects experienced a rapid and sustained reduction in uGAG excretion (DS and CS) after treatment initiation at 2 mg/kg QOW (Figure 6a and 6b).

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Dose-responsive changes in DS and CS were observed during the forced-dose titration period,

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with a mean 42.18% reduction in DS at 1 mg/kg QOW, 61.76% reduction at 4 mg/kg QOW, and 52.4% reduction at 2 mg/kg QOW (Figure 6A). CS excretion was reduced by a mean of 47.7%

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at 1 mg/kg QOW, 58.97% at 4 mg/kg QOW, and 54.76% at 2 mg/kg QOW (Figure 6B).

Characteristic Age (years) At informed consent At diagnosis* History of hydrops fetalis Sex Race Ethnicity

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Table 3. Summary of Demographics and Baseline Characteristics in Three Subjects With MPS VII Treated With Intravenous Vestronidase Alfa in a Phase 1/2 Clinical Trial Subject 1

Subject 2

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5.5 9.4 0.6 0 Yes Yes Male Female White White Hispanic or Not Hispanic or Latino Latino Height (cm) 102.5 123.9 Weight (kg) 20.6 34.6 *Age at diagnosis was imputed when diagnosis date was unavailable

Subject 3 25.1 5.0 No Male Asian Not Hispanic or Latino 157.3 79.1

Journal Pre-proof Figure 6. Reduction in uGAGs in Patients With MPSVII Treated With Intravenous Vestronidase Alfa in a Phase 1/2 Clinical Trial

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A) Percent Change in uGAG DS Excretion (LC-MS/MS) from Week 0 (Baseline) to Week 38 by Subject

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Week 38 by Subject

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B) Percent Change in uGAG CS Excretion (LC-MS/MS) from Week 0 (Baseline) to

Journal Pre-proof Discussion Evidence of vestronidase alfa pharmacological activity has been established in in vitro and in vivo studies. Previous investigations have reported that weekly recombinant murine GUS treatment in newborn and adult MPS VII mice can improve the disease histopathology after 6 weeks [4-7]. Early studies with rhGUS as an enzyme replacement therapy in murine models of MPS VII successfully showed that the enzyme can be delivered to tissues in vivo and reduce

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the accumulation of GAGs in a dose dependent manner. Despite the demonstrated therapeutic

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potential of rhGUS as a treatment for MPS VII, no approved therapies existed due to the very small patient population. Recently, a new version of rhGUS, now referred to as vestronidase

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alfa, was generated in CHO cells and optimized to be highly sialylated, while still maintaining

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high affinity mannose-6-phosphate for efficient uptake via receptor mediated endocytosis

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characteristic of most lysosomal enzymes. We demonstrated that the increased sialylation of vestronidase alfa improved the biochemical and pharmacokinetic properties, enabling a longer

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cellular half-life, better delivery across most tissue types, sustained rescue of enzyme activity, and a reduction of GAG in a dose-dependent manner. In a proof-of-concept study in MPS VII

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mice, we demonstrated that repeat dosing of vestronidase alfa was able to attain clinically

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relevant levels of enzyme in nearly all tissues, with some tissues nearing or exceeding normal levels of GUS activity relative to wild-type mice, resulting in increased survival. This increase in GUS activity was well maintained in a recovery group treated with a mid-dose (4 mg/kg) of vestronidase alfa and suggests enhanced sialylation of vestronidase alfa may have contributed to its prolonged in vivo stability [25-27]. Reduced cytoplasmic vacuolation and sustained reductions in uGAG levels throughout this recovery period also support the durability of the treatment effect. These preclinical observations suggested that 4 mg/kg is the maximum efficacious dose in vivo. Given the greater cellular half-life of vestronidase alfa, and evidence that the one-week recovery did not diminish the pharmacodynamic effects observed in the

Journal Pre-proof murine model, dosing with vestronidase alfa may require less frequent administration than required for other ERTs, while still achieving a therapeutic benefit. Moreover, we demonstrate that a reduction in uGAG levels following treatment with vestronidase alfa correlated well with diminished tissue vacuolation in vivo, indicating that measuring uGAG levels in patients may be a predictive biomarker of vestronidase alfa activity. Indeed, in a Phase 1/2 open-label, dose exploration study in humans, we demonstrated rapid

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reduction in uGAG in all three subjects following treatment with vestronidase alfa. The results of

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this study demonstrated evidence of efficacy in patients with MPS VII and suggested that the optimal dose of vestronidase alfa for maximal uGAG reduction with comparable safety was

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4 mg/kg QOW. Altogether, the clinical data in humans support our preclinical observations that

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4 mg/kg of vestronidase alfa is an efficacious treatment dose. Also, the strong translatability of

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our preclinical data to what was achieved in early clinical trials further support the thesis that generation of a highly sialylated glucuronidase, such as vestronidase alfa, will have a significant

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impact on tissue storage throughout the body and has the potential to improve the clinical condition of patients with MPS VII. Enzyme replacement with rhGUS is thus a promising therapy

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Union, and Brazil.

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for the treatment of patients with MPS VII and is now approved in the United States, European

Materials and Methods

Vestronidase Alfa Cellular Half-life Determination in Human MPS VII Patient-derived Fibroblasts Human MPS VII fibroblast cell line GM-2784 (Coriell Genetic Mutant Cell Repository, Camden, NJ) was used for the cellular uptake studies described in this paper. UX003 (Vestronidase alfa, Lot CR01 and GUS lot 43/44) was diluted into fibroblast growth medium (MEM, L-Glutamine, penicillin/streptomycin, sodium pyruvate, 15% fetal bovine serum) then sterilized using 0.2µM

Journal Pre-proof syringe filters. Aliquots of sterile, diluted enzyme were added to 35mm dishes containing GM-2784 fibroblasts grown to confluence and incubated for 16 to 24 hours. Medium containing enzyme was removed from cells and replaced with fresh growth medium, and cells were allowed to recover for 4 hours to ensure delivery of enzyme to the lysosome. Cells were harvested following the recovery period to initiate time 0 and consequently harvested at various timepoints, concluding at 42 days. At each harvest point, the medium was aspirated, and cells

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were washed with ice-cold phosphate-buffered saline, then frozen dry at -20˚C. Following the completion of the chase period, all plates were thawed at room temperature and solubilized in

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1% sodium deoxycholate for 20 minutes on ice. The cell extracts were scraped from the plates

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using plastic policemen and transferred to tubes. β-glucuronidase activity was determined as

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described below. Activity was expressed as 1 unit = 1 nmole 4MU released/plate/hr at 37˚C. The results were plotted as the percent of cell-associated GUS activity remaining from Time 0.

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Half-lives were calculated using the linear fit model.

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Studies in Animals

All animal studies were approved by Ultragenyx Pharmaceutical Inc and conducted employing

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sound scientific practices and in accordance with Standard Operating Procedures. Animal

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welfare was in compliance with the U.S. Department of Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals. The animal facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) International. Non-GLP studies performed outside of Ultragenyx Pharmaceutical Inc. adhered to local guidelines, as described below. Pharmacokinetic Study of Vestronidase Alfa In Rats Vestronidase alfa (UX003 Lot CR01, 2 mg/mL) or non-enzyme control (0.9% sodium chloride; Baxter Healthcare, Marion, NC) were infused into male Sprague-Dawley rats (5 rats per group, weighing between 276 g to 307 g, Charles River Laboratories, Hollister, CA) at a dose of

Journal Pre-proof ~2 mg/kg body weight during a single intravenous infusion, consisting of two 1-hour phases into an indwelling jugular vein catheter. One-third of the dose was infused over the first hour, and two-thirds of the dose was infused during the second hour. Using an indwelling femoral vein catheter, blood samples were taken from each rat at pre-dose and 2, 10, 30, 60, 120 minutes during the infusion and at 2, 10, 30, 60, 120, 240, 480, and 1440 (24 hour) minutes post-end of infusion. Blood was allowed to clot on ice; serum was separated and stored frozen at -80˚C until

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assayed for GUS activity as described below. Twenty-four hours after dosing, tissues were collected at necropsy, snap frozen in liquid nitrogen and stored at -80°C until assayed for GUS

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activity.

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This non-GLP study was performed by Pacific BioLabs (Hercules, CA) in accordance with the

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Animal Welfare Act Regulations (9 CFR 1-3) and the guidelines of the Pacific BioLabs

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Institutional Animal Care and Use Committee (IACUC).

of 0 to 20 mg/kg

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Eight-Week ERT Study in MPS VII Mouse Model Using Vestronidase Alfa Weekly at Doses

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Male and female MPS VII/E540ATG transgenic mice tolerant to human GUS [19] were supplied

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by the laboratory of William S. Sly, MD at Saint Louis University School of Medicine. The mice ranged in age from approximately 8 to 12 weeks old at the start of the study and ranged in weight from 15.9 to 27.1 grams for males and 12.5 to 22.4 grams for females. This non-GLP study was carried out in the Department of Comparative Medicine at Saint Louis University School of Medicine in accordance with the standard operating procedures of that department. Analysis of all tissues and biological materials were carried out at Ultragenyx Pharmaceutical Inc. or their assigned contract research organizations. Mice were dosed with vestronidase alfa (UX003 Lot PR01) or saline control by bolus intravenous infusion into the tail vein weekly for 8 weeks by the weight-based doses shown in

Journal Pre-proof Table 1 (final volume of 125 μL). All animals were routinely evaluated for morbidity or mortality, and weekly body weights were recorded. Each week, collections of urine occurred 48 hours and 144 hours following each dosing day for uGAG analysis using Non-Reducing Ends SensiPro® Technology from Zacharon Pharmaceuticals Inc. (San Diego, CA). Baseline urine samples were collected on two separate days prior to the start of infusions and at 48 hours and 144 hours following each weekly infusion. To obtain a representative urine sample, the collections at each timepoint were performed every 2 to 3 hours over a 12–hour collection period, pooled, and

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frozen at -80˚C. The recovery (4 mg/kg) cohort had an additional urine collection 24 hours

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before sacrifice.

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At the end of the 8-week study, 72 hours after the final enzyme infusion, mice (Groups 1-6)

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were sacrificed and blood and tissue samples were collected (including adrenal gland, brain, bone [rib and sternum], epididymis [males], heart, kidney, liver, lung, muscle [quadriceps], ovary

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[females], pituitary gland, prostate [males], spleen, testis [males], thymus, thyroid, uterus

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[females], and serum). One-week post-final treatment, the recovery mice (Group 7) were sacrificed and samples collected. Blood samples were allowed to clot on ice, spun down, and

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serum collected and frozen at -80˚C until analyzed. Terminal blood collected from all animals

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was processed for serum chemistry and hematology evaluations. To provide normal mouse GUS activity values for comparison to the human GUS levels attained during ERT, frozen tissues collected from 3 males and 3 females of wild type C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). These tissues were thawed, and extracts were prepared and assayed for GUS and normalized to total protein levels in the same manner as the tissues collected for the study. rhGUS (Vestronidase Alfa) Activity in Rat or Mouse Tissues and Serum Whole or partial tissue specimens were thawed, weighed, and combined with 10 to 20 volumes of 25 mM Tris, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, pH 7.2. Tissue homogenates

Journal Pre-proof were prepared using a KinematicaPolytron (Luzerne, Switzerland) homogenizer for 30 seconds on ice; the resultant homogenates were freeze/thawed once (at -80o C) followed by sonication for 20 seconds with cooling on ice. A 25 µL total volume of each tissue homogenate (or serum) was measured for GUS using a synthetic 4MU-β-glucuronide substrate. Tissue extracts or serum were assayed in 0.1 M sodium acetate, pH 4.8, and 1 mg/mL crystalline BSA. 25 μL of extract were mixed with 50 µL of 10mM 4-MU-β-D-glucuronide

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substrate in 0.1 M sodium acetate, pH 4.8, 1 mg/mL crystalline BSA for 30 minutes at 37oC. The

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reactions were stopped by the addition of 200 µL glycine carbonate, pH 10.5, and read on a Molecular Devices M2e plate reader at excitation/emission wavelengths of 366/446 nm. Activity

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was expressed as 1 unit = 1 nmole 4MU released/ml/hr at 37oC. Protein concentration of the

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homogenates was determined by the bicinchoninic acid method. Tissue GUS levels were

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expressed as units/hr/mg.

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Histopathological Analysis

Collected tissue samples for each animal were split in half. The half designated for

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histopathology evaluation were fixed in 10% neutral-buffered formalin and transferred to 70%

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ethanol after 48 hours. The tissues were then shipped at ambient temperature for further processing and microscopic evaluation to Charles River Laboratories, Pathology Associates Inc. (Frederick, MD). Following staining with toluidine blue, hematoxylin and eosin (H&E), or Alcian blue counterstained with H&E as appropriate, the tissues were evaluated using light microscopy to determine lysosomal storage as indicated by cytoplasmic vacuolization. The tissues were scored by a board-certified veterinary pathologist blinded to the treatment group on a scale of 0 to 4, where 0 was normal and 4 represented the most severe pathology.

Journal Pre-proof Phase 1/2 ERT Study in Patients With MPS VII Using Vestronidase Alfa Three subjects were enrolled in the study: one 25.1 year-old Asian male, one 5.5 year-old white male, and one 9.4 year-old white female (Table 3). Vestronidase alfa was administered by intravenous infusion over approximately 4 hours, all dosing was QOW. During the initial treatment period, subjects received 2 mg/kg vestronidase alfa QOW for 14 weeks. During the forced-dose titration period that followed, subjects received vestronidase alfa for an additional 24 weeks at the following dose sequence:

1 mg/kg UX003 QOW for 8 weeks beginning on Week 14, then

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4 mg/kg UX003 QOW for 8 weeks beginning on Week 22, then

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2 mg/kg UX003 QOW for 8 weeks beginning on Week 30

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

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During the forced-dose titration period, the proposed dose of 2 mg/kg QOW (administered both

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before and after the other doses) was compared with a higher (4 mg/kg QOW) dose level and a lower (1 mg/kg QOW) dose level to evaluate differences in treatment response by assessing

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changes in uGAG excretion. The forced-dose titration was conducted after the initial treatment period to allow any immune response that might occur during the first 14 weeks of exposure to

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stabilize prior to testing different doses.

Urinary GAG Analysis in Patients The levels of three GAG species (CS, DS, and HS) in human urine were determined by LCMS/MS at the Greenwood Genetic Center (Greenwood, South Carolina). The intact GAGs were treated with methanolic acid to produce uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfoglucosamine dimers, which were then mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from CS, DS, and HS were separated by UPLC and analyzed by electrospray ionization MS/MS using selected reaction monitoring for each targeted GAG product and its corresponding internal

Journal Pre-proof standard. This assay reported the concentration of CS, DS, and HS separately, relative to the creatinine concentration in patient urine.

Statistical Analysis Statistical analysis was performed using GraphPad Prism Software version 7.0 for Windows (La Jolla, CA). Unpaired two-tailed Student’s t-tests were used to compare GUS activity in saline-

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and vestronidase alfa-treated rats. One-way ANOVA with Dunnett’s multiple comparison was used to statistically compare the means of each vestronidase alfa-treated group to the saline

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controls in all other experiments.

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Author Contributions

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Conceptualization, EK, WS and SJ; Methodology, JG, GB, JC, and WS; Validation, GB and MV;

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Investigation, JG, JC, GB, and SC; Writing-Original Draft, EK, JG, and MV; Writing-Review and Editing, all authors; Resources, JG, WS, SC, and SJ; Supervision, EK, SJ, MV, and GB; Data

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Author Disclosures

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Curation, MV, GB, and JC; Project Administration, SJ and MV.

JC, GB, and AJ are current employees and stockholders of Ultragenyx Pharmaceutical Inc. EK

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holds patents relevant to the work, and is the CEO and President, and is a stockholder of Ultragenyx Pharmaceutical Inc. MV, SC, SJ and JHG are former employees and stockholders of Ultragenyx Pharmaceutical Inc. WS and JHG report that their institution was reimbursed for costs associated with the portion of the work performed at their research site, and that the institution received licensing fees for one of the cell lines used to produce one of the two GUS enzymes included in this report.

Journal Pre-proof Acknowledgements Ultragenyx Pharmaceutical Inc. provided financial support for the conduct of this research. Ultragenyx Pharmaceutical Inc. funded the study design, and the collection, analysis and interpretation of data, and made the decision to submit the article for publication. James Ziobro, of Ultragenyx Pharmaceutical Inc., provided medical writing support in the preparation of this

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manuscript.

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