TECHNICAL FIELD

The present invention relates to modified sulfamidase for use in treatment of Mucopolysaccharidoses IMA (MPS IMA), which is also known as Sanfilippo Syndrome Type A.

BACKGROUND

Lysosomal storage disease

The lysosomal compartment functions as a catabolic machinery that degrades waste material in cells. Degradation is achieved by a number of hydrolases and transporters compartmentalized specifically to the lysosome. There are today over 40 identified inherited diseases where a link has been established between disease and mutations in genes coding for lysosomal proteins. These diseases are defined as lysosomal storage diseases (LSDs) and are characterized by a buildup of a metabolite (or metabolites) that cannot be degraded due to the insufficient degrading capacity. As a consequence of the excess lysosomal storage of the metabolite, lysosomes increase in size. How the accumulated storage material cause pathology is not fully understood but may involve mechanisms such as inhibition of autophagy and induction of cell apoptosis (Cox & Cachon-Gonzalez, J Pathol 226: 241-254 (2012)).

Enzyme replacement therapy

Storage can be reduced by administration of a lysosomal enzyme from a heterologous source. It is well established that intravenous administration of a lysosomal enzyme results in its rapid uptake by cells via a mechanism called receptor mediated endocytosis. This endocytosis is mediated by receptors on the cell surface, and in particular the two mannose-6 phosphate receptors (M6PR) have been shown to be pivotal for uptake of certain lysosomal enzymes (Neufeld; Birth Defects Orig Artie Ser 16: 77-84 (1980)). M6PR recognize phosphorylated oligomannose glycans which are characteristic for lysosomal proteins. Based on the principle of receptor mediated endocytosis, enzyme

replacement therapies (ERT) are today available for six LSDs, (Gaucher, Fabrys, Pompe and the Mucopolysaccharidosis type I, II IV, VI and VII). These therapies are efficacious in reducing lysosomal storage in various peripheral organs and thereby ameliorate some symptoms related to the pathology.

A majority of the LSDs however also cause lysosomal storage in the central nervous system (CNS) and consequently present a repertoire of CNS related signs and symptoms. A major drawback with intravenously administered ERT is the poor distribution to the CNS. The CNS is protected from exposure to blood borne compounds by the blood brain barrier (BBB), formed by the CNS endothelium. The endothelial cells of the BBB exhibit tight junctions which prevent paracellular passage, show limited passive endocytosis and in addition lack some of the receptor mediated transcytotic capacity seen in other tissues. Notably, in mice M6PR mediated transport across the BBB is only observed up to two weeks after birth (Urayama et al, Mol Ther 16: 1261 - 1266 (2008)).

Glvcan modification of lysosomal enzymes

Enzyme replacement therapy targeting the brain by glycan modification is a potential strategy to increase distribution of lysosomal enzyme to the CNS as has been disclosed in WO 2008/109677. In this published application, chemical modification of b-glucuronidase using sodium meta-periodate and sodium borohydride is described (see also Grubb et al, Proc Natl Acad Sci USA 105: 2616-2621 (2008)). Although the underlying mechanism of brain distribution is unclear, it was noted that the chemical modification disrupted glycan structure on b-glucuronidase and it was further demonstrated that receptor mediated endocytosis by M6PR was strongly reduced.

In addition, sulfamidase chemically modified according to the method disclosed in W02008/109677, displayed an increased half-life in mice, but no effect in the brain of MPS IMA mice was observed. The chemically modified sulfamidase did not distribute to the brain parenchyma when given repeatedly by intravenous administration (Rozaklis et al, Exp Neurol 230: 123-130 (2011 )).

In WO 2015/150490, chemical modification of sulfamidase is disclosed. The glycan structure of the sulfamidase has been modified, resulting in a modified sulfamidase displaying catalytic activity in the brain of a mammal.

Thus, there are still no clinically effective ERT for treatment of LSDs with neurological engagement, such as MPS IMA. Novel sulfamidase compounds that can be transported across the BBB while remaining enzymatically active would be of great value in the development of enzyme replacement therapies for the treatment of LSDs with CNS related pathology, such as MPS MIA.

A patient suffering from e.g. MPS MIA, should preferably stay on ERT treatment for life.

SUMMARY OF THE INVENTION

The present invention provides improved methods for safe and effective treatment of Mucopolysaccharidoses MIA (MPS MIA), which is also known as Sanfilippo Syndrome Type A. The present invention is, in part, based on the phase l/ll human clinical study demonstrating the safety, tolerability and efficacy in human MPS MIA patients.

The primary objective of such a phase l/ll human clinical study is an assessment of safety and tolerability; secondary objectives include

assessment of the pharmacodynamic (PD) effect of different dose levels and treatment duration on heparan sulfate (HS) levels in cerebrospinal fluid (CSF), serum and urine as an indicator of in vivo biological activity. Thus, among other things, the present invention provides methods of treating Mucopolysaccharidosis IMA (MPS IMA), comprising a step of administering, e.g. by i.v. infusion, to a subject in need thereof a replacement sulfamidase at a therapeutically effective dose and an administration interval with a treatment duration. In some embodiments, the replacement enzyme is administered for a period sufficient to decrease heparan sulfate (HS) levels in cerebrospinal fluid (CSF), serum and/or urine relative to baseline. Thus, some embodiments of the invention further comprise measuring levels of one or more

glycosaminoglycans (GAGs) (e.g., heparan sulfate) in CSF, urine, tissues and/or serum one or more times during the period, thereby determining a surrogate marker indicative of safety and/or therapeutic efficacy.

It should be understood that the present invention similarly provides a modified sulfamidase for use in methods of treatment as disclosed herein.

For a sulfamidase to modify the neuropathology of MPS MIA disease, it has to reach the lysosomes of cells in the brain. The sulfamidase used in the present invention may be a recombinant human sulfamidase, such as a modified sulfamidase. The modification of said sulfamidase may thus enable

transportation of said sulfamidase across the blood brain barrier of a mammal, wherein said sulfamidase has catalytic activity in the brain of said mammal, and may comprise a modification of the glycosylation of said sulfamidase, such that said modified sulfamidase comprises substantially no or reduced number of epitopes for glycan recognition receptors.

In one embodiment, natural glycan moieties of the modified sulfamidase used in the present invention are disrupted by single bond breaks and double bond breaks, the extent of single bond breaks per total bond breaks being at least 60 % in oligomannose glycans.

The sulfamidase used in the present invention may be a modified sulfamidase having a relative content of remaining intact natural glycan moieties of around 25 % of the content of natural glycan moieties in unmodified recombinant sulfamidase.

Sulfamidase used in the present invention may be a modified sulfamidase polypeptide consisting of an amino acid sequence as defined in SEQ ID NO:1 , or a polypeptide having at least 95 % sequence identity with an amino acid sequence as defined in SEQ ID NO:1 , wherein said epitopes are absent at at least four of the five N-glycosylation sites: N in position 21 (N(21 )), N in position 122 (N(122)), N in position 131 (N(131 )), N in position 244 (N(244)), and N in position 393 (N(393)) of SEQ ID NO:1.

Sulfamidase used in the present invention may be a modified sulfamidase comprising an oligomannose glycan at the N(131 ) site, said oligomannose being disrupted by single bond breaks and double bond breaks, the disruption being characterized by an extent of single bond breaks of at least 60 %.

Sulfamidase used in the present invention may be a modified sulfamidase comprising a Ca-formylglycine residue in position 50 of SEQ ID NO:1

(FGIy50) providing catalytic activity.

Sulfamidase used in the present invention may be a modified sulfamidase wherein in the therapeutically effective dose, the Ca-formylglycine (FGIy) to serine (Ser) ratio at the active site of sulfamidase is greater than 1.

Where sulfamidase is recombinantly produced, any expression system can be used. To give but a few examples, known expression systems include, for example, egg, baculovirus, plant, yeast, or mammalian cells.

In some embodiments, sulfamidase suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. , J. Gen Virol., 36:59,1977); human fibrosarcoma cell line (e.g., HT 1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980);

mouse sertoli cells (TM4, Mather, Biol. Reprod. , 23:243-251 , 1980); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO- 76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (WI38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather et al. , Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

In some embodiments, methods according to the present invention are used to deliver replacement enzymes produced from human cells. In some embodiments, methods according to the present invention are used to deliver replacement enzymes produced from CHO cells.

In some embodiments, human recombinant sulfamidase for use in the present invention may be conjugated or fused to a lysosomal targeting moiety that is capable of binding to a receptor on the surface of brain cells. A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP, p97, and variants, homologues or fragments thereof (e.g., including those peptides having a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a wildtype mature human IGF-I, IGF-II, RAP, p97 peptide sequence).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows SOBI003 concentration in serum, CSF and brain at 2 and 24 hours after a single 10 mg/kg i.v. dose in individual MPS IMA or wild type (WT) mice. Figure 2 shows mean ± standard deviation (SD) SOBI003 concentrations in serum, CSF and brain microdialysates corrected for recovery obtained from Sprague Dawley rats after a 30 mg/kg i.v. dose. Brain microdialysate was collected over 1 or 2 hour periods and the concentration is plotted at the midway point of the collection period.

Figure 3A shows mean ± standard deviation (SD) SOBI003 concentration in serum and CSF in cynomolgus monkey after a 15 minute 30 mg/kg i.v.

infusion.

Figure 3B shows individual plasma (left) and brain (right) exposure in cynomolgus monkeys at 72 hours after the fifth 15, 75 or 150 mg/kg 15 minute i.v. infusion dosed every third day. Different brain areas: frontal superior cortex (FC), mid inferior cortex (MC), hippocampus dentate gyrus (HP) and posterior cerebellum (CB).

Figure 4 shows relative levels of heparan sulfate (FIS) in CSF and brain homogenates from male MPS IMA mice at 29 weeks of age after i.v.

administration of vehicle or an active dose of SOBI003 at 3, 6, 12 or 17 mg/kg once weekly during 20 weeks (n=8). Mean data ± SEM are presented in the figures presenting FIS in CSF and brain. Statistics were calculated using one- way ANOVA, followed by Dunnett’s Multiple Comparison test versus vehicle group as post hoc analysis. P values >0.05 were considered non-significant, whereas ^* p<0.05, ^** p<0.01 , and ^*** p<0.001. In the bottom left graph individual FIS data in brain and CSF are presented and statistics were calculated using Pearson’s correlation, with r2 = 0.8494 and p < 0.0001.

DETAILED DESCRIPTION OF THE INVENTION

Among other things, the present invention provides methods for treating Mucopolysaccharidosis IMA (MPS MIA) based on administration, such as intravenous administration, of a modified sulfamidase at a therapeutically effective dose and an administration interval. In some embodiments, the modified sulfamidase is administered for a period sufficient to decrease heparan sulfate level in the cerebrospinal fluid (CSF) and/or urine relative to baseline. Modified sulfamidase is preferably administered to a human subject in need thereof. A human subject“in need thereof is to be understood as a human subject in need of such treatment.

Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

Sulfamidase used in the present invention may be a modified sulfamidase comprising substantially no or reduced number of epitopes for glycan recognition receptors, thereby enabling transportation of said sulfamidase across the blood brain barrier of a mammal, such as a human being, wherein said sulfamidase has catalytic activity in the brain of said mammal.

The modified sulfamidase for use according to the present invention may be a chemically modified recombinant human sulfamidase.

The modified sulfamidase may be modified in that epitopes for glycan recognition receptors have been removed, for example as compared to an unmodified sulfamidase (SEQ ID NO:1 ). Such a modified sulfamidase may be less prone to cellular uptake which is a consequence of removal of epitopes for glycan recognition receptors such as the two mannose-6 phosphate receptors (M6PR). The almost complete absence or reduced number of said epitopes reduces the affinity of the modified sulfamidase with respect to glycan recognition receptors. In particular, this might reduce the receptor mediated endocytosis of the modified sulfamidase in peripheral tissue, which in turn may result in a reduced clearance of modified sulfamidase from plasma when e.g. administrated intravenously to a mammal. This is probably at least partly due to the inhibition of receptor mediated uptake in peripheral tissue following chemical modification of the sulfamidase. By glycan recognition receptors is meant receptors that recognize and bind proteins mainly via glycan moieties of the proteins. Such receptors can, in addition to the mannose 6-phosphate receptors, be exemplified by the mannose receptor; which selectively binds proteins where glycans exhibit exposed terminal mannose residues. Lectins constitute another large family of glycan recognition receptors which can be exemplified by the terminal galactose recognizing asialoglycoprotein receptor 1 recognizing terminal galactose residues on glycans. Epitopes for glycan recognition receptors canthus be understood as (part of) glycan moieties recognized by such receptors.

In one embodiment, natural glycan moieties of the modified sulfamidase used in the present invention are disrupted by single bond breaks and double bond breaks, the extent of single bond breaks per total bond breaks being at least 60 % in oligomannose glycans.

Sulfamidase used in the present invention may be a modified sulfamidase having a relative content of remaining intact natural glycan moieties of around 25 % of the content of natural glycan moieties in unmodified recombinant human sulfamidase. A content of remaining intact natural glycan moieties of around 25 % could for example correspond to one remaining intact natural glycan moiety. Sulfamidase used in the present invention may be a modified sulfamidase polypeptide consisting of an amino acid sequence as defined in SEQ ID NO:1 , or a polypeptide having at least 95 % sequence identity with an amino acid sequence as defined in SEQ ID NO:1 , wherein said epitopes are absent at at least four of the five N-glycosylation sites: N in position 21 (N(21 )), N in position 122 (N(122)), N in position 131 (N(131 )), N in position 244 (N(244)), and N in position 393 (N(393)) of SEQ ID NO:1. Sulfamidase used in the present invention may be a modified sulfamidase comprising an oligomannose glycan at the N(131 ) site, said oligomannose being disrupted by single bond breaks and double bond breaks, the disruption being characterized by an extent of single bond breaks of at least 60 %.

Sulfamidase used in the present invention may be a modified sulfamidase comprising a Ca-formylglycine residue in position 50 of SEQ ID NO:1

(FGIy50) providing catalytic activity.

Sulfamidase used in the present invention may be a modified sulfamidase wherein the Ca-formylglycine (FGIy) to serine (Ser) ratio at the active site of sulfamidase is greater than 1.

One example of a modified sulfamidase for use in the present invention is disclosed in WO2015/150490, which is hereby incorporated by reference. As set out in WO2015/150490, a modified sulfamidase may be prepared in a method comprising: a) reacting a glycosylated sulfamidase with an alkali metal periodate, and b) reacting said sulfamidase with an alkali metal borohydride for a time period of no more than 2 h; thereby modifying glycan moieties of the sulfamidase and reducing the activity of the sulfamidase with respect to glycan recognition receptors, while retaining catalytic activity of said sulfamidase. The time period for the reaction of step a) may be no more than 4 hours, and alternatively or additionally, steps a) and b) may be performed in sequence without performing an intermediate step. The appended Reference Example gives further examples for preparing modified sulfamidase.

SOBI003 is an example of a modified sulfamidase for use according to the present invention.

SOBI003 is a chemically modified recombinant human sulfamidase. SOBI003 is suitable to be administered as a sterile solution for i.v. infusion after dilution. SOBI003 can for example be administered once weekly. By modification of the sulfamidase glycans, i.e. preparing a modified sulfamidase, the systemic uptake is strongly reduced. This translates into a reduced serum clearance as demonstrated in mice. A modified sulfamidase passes the blood-brain barrier (BBB) presumably by adsorptive mediated transcytosis, whereby the increased plasma exposure facilitates generation of pharmacological relevant levels of the modified sulfamidase in the central nervous system (CNS) compartment. Once the modified sulfamidase enters the target cells and reaches the lysosomal compartment, it shows enzymatic activity and stability comparable to that of unmodified sulfamidase, as demonstrated in fibroblasts of MPS IMA patients.

A sulfamidase suitable for use according to the present invention may be produced by any available means. For example, sulfamidase may be recombinantly produced by utilizing a host cell system engineered to express a replacement enzyme-encoding nucleic acid. Alternatively or additionally, sulfamidase may be produced by activating endogenous genes. Alternatively or additionally, replacement enzymes may be partially or fully prepared by chemical synthesis.

In one embodiment, the sulfamidase for use in the present invention is recombinant sulfamidase.

In one embodiment, the sulfamidase for use in the present invention is human sulfamidase.

Where sulfamidase is recombinantly produced, any expression system can be used. To give but a few examples, known expression systems include, for example, egg, baculovirus, plant, yeast, or mammalian cells.

In some embodiments, sulfamidase suitable for the present invention have been modified to enhance delivery or transport of such agents across the BBB and into the CNS, such as into the brain. Formulation, Dose, Administration interval and patients

Sulfamidase for use according to the present invention, such as a modified sulfamidase, is administered to a human subject in need thereof, and is preferably administered to a pediatric patient suffering from

Mucopolysaccharidosis IMA (MPS IMA).

The modified sulfamidase of the present invention is preferably administered by intravenous injection or infusion. The modified sulfamidase of the present invention is alternatively administered by intranasal, subcutaneous, or intrathecal administration.

In certain embodiments of the present invention, treatment is initiated at an age of the treated subject being less than 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 years of age. In certain

embodiments of the present invention, the subject being treated is about 1 year to 6 years of age. In certain embodiments of the present invention, the subject being treated is about 0 year to about 2.5 years of age. In certain embodiments of the present invention, the subject being treated is at least 1 year old, i.e. at least 12 months old. In certain embodiments, of the present invention, the subject being treated is between 0 and 12 months old. Patients with the S298P genotype, a mutation that is associated with a slower progression of disease, may also be treated.

Replacement sulfamidase according to the present invention, such as a modified sulfamidase, may be formulated as a sterile solution, with a concentration of about 20 mg/mL, by mixing with NaCI 0.9% infusion solution prior to administration.

The actual doses to be administered may be based on the patient’s

bodyweight at 4-week intervals (i.e., the actual dose administered at Weeks 1 to 4 is based on the bodyweight obtained pre-infusion at Week 1 , the actual dose administered at Weeks 5 to 8 is based on the bodyweight obtained pre- infusion at Week 5, etc.). The body weight is obtained either on the day prior to the infusion or the day of infusion. Sulfamidase for use according to the present invention, such as a modified sulfamidase, is mixed with NaCI 0.9% sterile infusion solution prior to administration. For a bodyweight < 25 kg, the targeted total infusion volume is about 100 ml_. For a bodyweight > 25 kg, the targeted total infusion volume is about 250 ml_. For lower bodyweights, such as < 10 kg, or < 5 kg, the targeted total infusion volume may be lower, such as 50 nriL or lower.

Sulfamidase for use according to the present invention, such as a modified sulfamidase, may be administered as a weekly i.v. infusions over a period of time, such as, about 15 minutes, 1 , 2, 3, 4, 5 or 6 hours, or even up to 24h. In particular, the administration may be a 4-hour i.v. infusion. The administration interval / treatment period comprises for example 24 weekly infusions.

Preferably, infusions should be administered every 7 ^th day. A +/- 1-day window may be applied after the 4 ^th infusion. Therefore, if the first infusion was administered for instance on a Wednesday, then the 5 ^th to the 24 ^th infusions can be administered on Tuesdays, Wednesdays or Thursdays.

Within 30 to 60 minutes prior to each infusion, a single dose of a non-sedative antihistamine may be administered. The antihistamine can either be e.g., cetirizine, levo-cetirizine, loratadine, des-loratadine or fexofenadine, and the administered doses will be in compliance with locally approved labeling.

Antipyretic medication may also be administered, if infusion-related reactions occur, then the infusion duration may be expanded up to 24 hours and supportive medication may be administered.

A slower infusion rate may be applied for the initial period / hour for all doses, targeting the delivery of 3% of the total dose/infusion volume during the first hour. The infusion rate may then be set to deliver the remaining 97% of the dose/infusion volume over the next hours. The treatment period according to the present invention is preferably 24 weekly infusions, but other interval / treatment periods such as 8, 12, 16, 20, 28, 32, 36, 40, 44, 48, 52, 104 or longer weekly infusions are also possible. It is also possible that the treatment with modified sulfamidase of the present invention will be lifelong.

Sulfamidase for use according to the present invention, such as a modified sulfamidase, may also be administered daily, biweekly or monthly.

Suitable doses according to the present invention are 0.1 , 0.3, 0.5, 1 , 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mg/kg based on the bodyweight of the patient. In particular, a suitable dose equals to or is greater than 3 mg/kg, equals to or is greater than 10 mg/kg, or equals to or is greater than 20 mg/kg. The body weight of the patient is preferably obtained either on the day prior to the infusion or the day of infusion.

The modified sulfamidase of the present invention may also be administered as a fixed-dose ranging from 0.3 g to 2 g. Such fixed dose may be selected based on, e.g. the age or length of the patient to be treated or the severity of the condition the patient is suffering from.

In various embodiments, the present invention relates to the use of a modified sulfamidase to improve, stabilize or reduce declining of one or more

neurocognitive or behavioral functions relative to a control or baseline or untreated behavioral or neurocognitive functions.

Example of such a neurocognitive function is

• one or more cognitive functions are assessed by the Bayley Scales of Infant Development (Third Edition);

• one or more cognitive functions are assessed by the Kaufman

Assessment Battery for Children (Second Edition);

• one or more behavioral functions are expressed as adaptive behavior age-equivalence score (AEq) as assessed by Vineland Adaptive Behavior Scales, Expanded Interview Form, Second edition (VABS-II); and • one or more cognitive functions are expressed as neurocognitive Development Quotient (DQ) as assessed by the Bayley Scales of Infant and Toddler Development, third edition (BSID-III) cognitive subtest or the Kaufman Assessment Battery for Children, Second edition (KABC-II);

In various embodiments, the present invention relates to the use of a modified sulfamidase to improve, stabilize or reduce declining of one or more physiological functions or properties relative to a control, baseline or untreated physiological functions.

Example of such a function is

• gray matter volume as assessed by brain volumetric magnetic

resonance imaging (MRI); and

• physiological functions and/or properties as assessed by Pediatric

Quality of Life Inventory (PedsQL) total score.

The terms, "improve",“stabilize”, "increase" or "reduce", as used herein, indicate values that are relative to a control or baseline. In some

embodiments, a suitable control is a baseline measurement, such as a measurement in the same subject/individual prior to initiation of the treatment described herein, or a measurement in a control subject/individual (or multiple control subjects/individuals) suffering from MPS IMA in the absence of the treatment described herein.

In some embodiments, treatment refers to decreased HS levels in the cerebrospinal fluid (CSF) and/or serum and/or in urine relative to a control or baseline.

The ability of a modified sulfamidase according to the present invention to improve, stabilize or reduce declining of one or more behavioral, physiological or neurocognitive functions relative to a control or baseline is usually expressed in percent (%). Example of possible values of such ability to improve, stabilize or reduce declining of one or more neurocognitive functions relative to a control or baseline is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% and 200%.

The ability of a modified sulfamidase according to the present invention to improve, stabilize or reduce declining of one or more physiological or neurocognitive functions relative to a control or baseline may also be expressed as a difference in units, such as for the Development Quotient and age-equivalence score (AEq). Example of possible values of such ability to improve, stabilize or reduce declining of one or more physiological or neurocognitive functions relative to a control or baseline would then be expressed as a number, such 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 and the like.

In some embodiments, treatment refers to decreased HS levels in

cerebrospinal fluid (CSF). In some embodiments, CSF FIS levels are reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to pretreatment or baseline levels. In some embodiments, CSF FIS levels are decreased by at least 1 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to pretreatment or baseline levels.

Mucopolysaccharidosis type IMA (MPS IMA; Sanfilippo Syndrome Type A) is characterized by a deficiency of the enzyme heparan N-sulfatase (FINS), an exosulfatase involved in the lysosomal catabolism of glycosaminoglycan (GAG) heparan sulfate (Neufeld EF, et al. The Metabolic and Molecular Bases of Inherited Disease (2001 ) pp. 3421 -3452). In the absence of this enzyme, GAG heparan sulfate (FIS) accumulates in lysosomes of neurons and glial cells. As a result, FIS accumulates significantly in the CSF of afflicted individuals. Thus, elevated levels of FIS in CSF indicate a subject in need of treatment, and reduction in FIS levels following administration of replacement sulfamidase, such as a modified sulfamidase, serves as a marker of therapeutic efficacy. In some embodiments, the subject in need of treatment has a HS level in the CSF greater than about 0.2 pg/ml (e.g., greater than about 0.3 Mg/ml, about 0.4 Mg/ml, about 0.5 Mg/ml, about 0.6 Mg/ml, about 0.7 pg/ml, about 0.8 pg/ml, about 0.9 pg/ml, about 1.0 pg/ml, about 1.1 pg/ml, about 1.2 Mg/ml, about 1.3 pg/ml, about 1.4 pg/ml, about 1.5 pg/ml, about 1.6 Mg/ml, about 1.7 pg/ml, about 1.8 pg/ml, about 1.9 pg/ml, or about 2.0 pg/ml before the treatment. In some embodiments, the HS levels of said subject has not prior to treatment been reduced by other methods for reducing HS levels.

A human being suffering from Mucopolysaccharidosis type IMA (MPS IMA; Sanfilippo Syndrome Type A) usually have a HS value in CSF of about 2.1 pg/ml (Naimy et al, Bioanalysis (2016), 8(4), 285-295), while literature reference value for non-MPS IMA CSF and serum levels of HS are 0.16 pg/ml, (Naimy et al, Bioanalysis (2016), 8(4), 285-295) or 0.14 pg/ml (Zhang et al, Clin. Chem. (2011 ), 57(7), 1005-1012).

In some embodiments, the method of treatment with modified sulfamidase further comprises a step of adjusting the dose and/or administration interval for administration based on the HS level in the CSF and/or the urine. This step of adjusting may in turn comprise a step of increasing the therapeutic effective dose for administration if the HS level in the CSF or urine fails to decrease relative to the control or baseline after 4 doses.

In some embodiments of the present invention the HS levels in CSF and / or urine might initially increase before a decrease is observed.

Abbreviations and definition of terms

ADA Anti-drug antibody

ADL Activities of daily living

AE Adverse event

AEq Age-equivalence

ALT Alanine transaminase

APTT Activated partial thromboplastin time AST Aspartate transaminase

AUC Area under curve

AUCl68h The area under the serum concentration-time curve from time 0 to 168 after dose

AUCT,SS Area under the plasma concentration-time curve during a dosage interval (T)

BBB Blood-brain barrier

BSID-III Bayleys Scales of Infant and Toddler

Development®, third edition

CDISC Clinical data interchange standards consortium

CL Clearance

CEnd of inf The observed serum concentration at the end of infusion of SOBI003

Cmax Maximum observed serum concentration

Clrough The minimum observed serum concentration

CNS Central nervous system

Cmax Maximum (peak) serum drug concentration

Cpre-dose The observed serum concentration immediately before the start of infusion of SOBI003

CRF Case report form

CRIM Cross reactive immunological material

CRO Contract research organization

CSF Cerebrospinal fluid

CSHQ Children ^' s sleep habits questionnaire

DMC Data monitoring committee

DQ Development quotient

ECG Electrocardiogram

ERT Enzyme replacement therapy

FAS Full-analysis set

GAG Glycosaminoglycan

GCP Good clinical practice

GLP Good laboratory practice

HS Heparan sulfate

i.v. Intravenous

ICF Informed consent form ICH International council for harmonisation

I EC Independent ethics committee

IL Interleukin

IMP Investigational medicinal product

IRB Institutional review board

KABC-II Kaufman assessment battery for children, second edition

LSD Lysosomal storage disease

MABEL Minimal anticipated biological effect level

MPS IMA Mucopolysaccharidosis type IMA

MRI Magnetic resonance imaging

MRSD Maximum recommended starting dose

MTD Maximum tolerated dose

NAb Neutralizing antibody

NCA Non-compartmental analysis

NCI CTC National cancer institute common terminology criteria

NOAEL No observed adverse effect level

NVI Nonverbal index

PAD Pharmacologically Active Dose

PD Pharmacodynamic

PedsQL Pediatric quality of life inventory

PK Pharmacokinetic

PT/INR Prothrombine time/international normalized ratio

SAE Serious adverse event

SAF Safety analysis set

SGSH N-Sulfoglucosamine sulfohydrolase

SRC Safety review committee

t Time

tEnd of inf The time of the end of the infusion of SOBI003 tmax The time at which the maximum serum

concentration is observed

tl/2 Half-life

TBV Total blood volume TEAE Treatment-emergent adverse event

TNF Tumor necrosis factor

ULN Upper limit of normal

VABS-II Vineland adaptive behavior scales, second

edition

Vd Volume of distribution

EXAMPLES

Example 1 : Clinical trial in pediatric MPSIII A patients to be performed

The primary objective is an assessment of safety and tolerability; secondary objectives is to include assessment of the pharmacodynamic (PD) effect of different dose levels and treatment duration of SOBI003 on heparan sulfate (HS) levels in cerebrospinal fluid (CSF), serum and urine as an indicator of in vivo biological activity.

SOBI003 is an example of a modified sulfamidase according to the present invention.

Type of study: Phase I/ll; Human pharmacology and Therapeutic

exploratory

STUDY

OBJECTIVES

Primary To evaluate the safety and tolerability of SOBI003 at objective: different dose levels.

Secondary 1. To characterize the pharmacokinetic (PK) objectives: properties of SOBI003 following single and

repeated administration by the use of non- compartmental analysis (NCA)

2. To assess the immunogenicity of SOBI003

3. To assess the pharmacodynamic (PD) effect of different dose levels and treatment duration of SOBI003 on heparan sulfate (HS) levels in cerebrospinal fluid (CSF), serum and urine

4. To assess the effect of SOBI003 at different dose levels on neurocognition

5. To assess the effect of SOBI003 at different dose levels on adaptive behavior

6. To assess the effect of SOBI003 at different dose levels on gray matter volume

7. To assess the effect of SOBI003 at different dose levels on Quality of Life

STUDY DESIGN AND METHODS

Study design: This is an open-label, non-controlled, parallel,

sequential ascending multiple-dose, multicenter study to assess the dose related safety, tolerability, PK and PD of SOBI003 in pediatric MPS IMA patients.

SOBI003 is administered as weekly i.v. infusions over a period of time of 4 hours. The study treatment period comprises 24 weekly infusions. Prior to initiation of each infusion, the patients are pretreated with a single dose of non-sedative antihistamine. If infusion-related reactions occur, then the infusion duration may be expanded up to 24 hours and supportive medication may be administered, at the discretion of the investigator.

Assessments Neurocognition is assessed by the BSID-III and/or for efficacy KABC-II at Baseline and at Week 24. Adaptive evaluation: behavior is assessed by the VABS-II at Baseline and at Week 24. Gray matter volume is assessed by MRIs at Baseline and at Week 24. The PedsQL is completed by the parent/primary caregiver at Baseline and at Weeks 12 and 24. Table 1 Investigational medicinal products

Actual dose levels to be determined by SRC

Baseline

Once patient eligibility is confirmed, including the central laboratory results of the patient’s sulfamidase activity and SGSH genotype, the patient is assigned a study specific Subject Number and the baseline procedures are initiated. Timing of doses for each patient

SOBI003 is administered as 4-hour i.v. infusions given once weekly for a duration of 24 weeks. Every effort should be made to have the infusions administered every 7 ^th day. A +/- 1-day window is applied after the 4 ^th infusion. Therefore, if the first infusion was administered for instance on a Wednesday, then the 5 ^th to the 24 ^th infusions can be administered on

Tuesdays, Wednesdays or Thursdays.

Within 30 to 60 minutes prior to each infusion, a single dose of a non-sedative antihistamine should be administered. The antihistamine can either be e.g., cetirizine, levo-cetirizine, loratadine, des-loratadine or fexofenadine, and the administered doses will be in compliance with locally approved labeling. The antihistamine agent is selected at the discretion of the investigator. Antipyretic medication may also be administered, at the discretion of the investigator. The actual doses of SOBI003 to be administered should be based on the patient’s bodyweight at 4-week intervals (i.e., the actual dose administered at Weeks 1 to 4 is based on the bodyweight obtained pre-infusion at Week 1 , the actual dose administered at Weeks 5 to 8 is based on the bodyweight obtained pre-infusion at Week 5, etc.). The body weight is obtained either on the day prior to the infusion or the day of infusion.

SOBI003 solution is mixed with NaCI 0.9% infusion solution prior to administration. For a bodyweight < 25 kg, the targeted total infusion volume is 100 ml_. For a bodyweight > 25 kg, the targeted total infusion volume is 250 ml_. If the infusion bag contains an overfill volume, the infusion should continue until the infusion bag is emptied.

Efficacy assessments

Adaptive behavior

The VABS-II is assessed at Screening, Baseline (within 2 weeks prior to first SOBI003 infusion) and at Week 24. The screening assessment is used to determine patient eligibility in terms of developmental age.

The patients’ ability to cope with changes, to demonstrate independence and to learn new skills are assessed by use of the Expanded Interview Form of the VABS-II. This measures five competence domains: communication, socialization, daily living skills, motor skills and an adaptive behavior composite of the first four domains.

Neurocoqnition

Neurocognition is assessed at Baseline and at Week 24, by use of the neurocognition domain of the BSID-III and/or the KABC-II Nonverbal Index (NVI).

The patient’s chronological age at the baseline assessment, in combination with the patient’s developmental age as established by VABS-II, will determine which neurocognitive test(s) to apply. The algorithm in Table 2 is applied and the test(s) administered at Baseline should also be used at Week Table 2 Selection of neurocog nitive assessment method

The neurocognitive developmental age (months) is derived on the basis of the BSID-III cognitive total raw score and BSID-III age-normative data.

The neurocognitive developmental age (months) is derived from the mean AEq scores on the NVI of the KABC-II.

The Development Quotient (DQ) is obtained by dividing the neurocognitive developmental age with chronological age.

Endpoints relating to the neurocognition, adaptive behavior, gray matter volume and Quality of Life are summarized using descriptive statistics. Quality of life and caregiver burden

The parent proxy-report format of the PedsQL questionnaire (Version 4.0 [26]) will be used to assess the child’s quality of life. The Family impact Module of the PedsQL (Version 2.0) will be used to assess the caregiver burden. The PedsQL will be completed at Baseline and at Weeks 12 and 24.

Pharmacodynamic assessments

Heparan sulfate

A method for determining heparan sulfate (HS) level in the cerebrospinal fluid (CSF) and/or serum and/or in urine is described by Naimy H, Powell KD, Moriarity JR, Wu J, McCauley TG, Haslett PAJ, et al., Bioanalysis.

2016;8(4):285-295 and Zhang H, Young SP, Millington DS, Curr. Protoc. Hum. Genet. 2013;17:17.12.. Example 2.

Prediction of HS levels in an in silico model

The dose, time, concentration and effect on HS in brain relationship was explored with an in silico model including a repeated i.v. dose regimen and the SOBI003 concentration in serum following a 2-compartment model. A fraction of serum SOBI003 passed the BBB and distributed to brain. Finally, a fraction of SOBI003 entered the target cells, with dynamics as determined in vitro in fibroblasts of a MPS IMA patient. Effect on biomarker was predicted with V _max /K _m kinetics. In the model, de novo synthesis of substrate was included. The model predicted the observed outcome of studies in MPS IMA mice. The mouse in silico model was translated to patient to predict HS biomarker reduction in brain. Repeated weekly administration of 3 mg/kg SOBI003 was predicted to gradually achieve a HS reduction in brain of ~ 35 % after 12 weeks and ~ 45 % after 24 weeks. Repeated weekly

administration of 20 mg/kg SOBI003 was predicted to achieve a brain HS reduction of ~ 80 % after 12 weeks months of treatment relative to baseline.

Table 3. Predicted PD parameters of HS reduction [%] in brain in patients following 3, 10 or 20 mg/kg 4-hour i.v. infusion once a week treatment regimen according to the present invention.

SOBI003 has improved chemical stability while retaining catalytic activity, and provides an enhanced plasma concentration due to reduced uptake in peripheral tissues.

Distribution of SQBI003 after intravenous administration

Concentration of SOBI003 in cerebrospinal fluid (CSF) and in brain homogenate of MPS IMA and C57BL/6 (wild type, WT) mice, Sprague Dawley rat and non-human primate was determined. Brains were perfused with cold saline before harvest to remove blood.

Concentration of SOBI003 in brain interstitial fluid was obtained by brain microdialys in rat. Rats were surgically fitted with microdialysis probes implanted bilaterally in the prefrontal cortex. After recovery, microdialysis probes with 4 mm open membrane surfaces (Brainlink, the Netherlands) were inserted into the guides the day before microdialysis sampling. SOBI003 concentrations in microdialysate were compensated for the in vitro evaluated recovery over the microdialysis probe.

SOBI003 was detected using confocal laser scanning microscopy on cryo- sections both by immunohistochemistry with an antibody against sulfamidase and by administration of fluorescently labelled SOBI003.

SOBI003 was detected in CSF and in brain homogenate of MPS IMA and wild type (WT) mice (Figure 1 ; a singlelO mg/kg i.v. dose), in CSF and brain interstitium in Sprague Dawley rats (Figure 2 and Table 4; a single 30 mg/ml i.v. dose) and in CSF and in brain homogenate of non-human primates (CSF in Figure 3A; a single 15 min 30 mg/ml i.v. infusion and brain homogenate in Figure 3B: after the fifth 15, 75 or 150 mg/kg 15 minute i.v. infusion dosed every third day).

For the determination of SOBI003 concentration in brain homogenate it was assumed that 1 gram corresponded to 1 ml_. Moreover, SOBI003 was found to be present in brain parenchyma as determined by immunohistochemistry and by a dose of fluorescently labeled SOBI003 followed by microscopy in mouse. Table 4. Mean ± SD serum and brain homogenate concentration in male Sprague Dawley rats after a 30 mg/kg i.v. dose.

In vitro human CNS model

CD34+ stem cells were differentiated into endothelial cells and grown for 15 to 20 days (Cecchelli R, et al. (2014) PLoS ONE 9(6): e99733). Cells were trypsinized and seeded onto coated polycarbonate filter inserts in co-culture with bovine brain pericytes for six days. Under these conditions, differentiated endothelial cells exhibit characteristics of a tight mature BBB including high transendothelial electric resistance, expression of tight junction markers, transporters and low permeability to non-permeant markers.

On the experimental day, filter inserts were moved from the pericyte co- culture, washed twice and inserted into Ringer-HEPES buffer. The tightness of the endothelial cells monolayer was evaluated by the parallel assessment of the permeability to the non-permeant fluorescent marker, Sodium fluorescein in each well.

The permeability of SOBI003 and recombinant human sulfamidase

(rhSulfamidase) across the in vitro BBB layer was assessed after a 3 hour incubation by determination of concentration in the donor and the receiver wells and evaluation of the mass balance.

SOBI003 and rhSulfamidase crossed the BBB in the in vitro human model. The endothelial permeability for SOBI003 and rhSulfamidase obtained in the human BBB model ranged from 0.005 to 0.022 *10-3 cm/min. The percentage of transport ranged from 0.1 to 1.2 % in the two experiments performed.

Intravenous treatment with SQBI003 in a mouse model

MPS IMA mice with a homozygous loss of function of the sulfamidase gene exhibit 3-4 % residual sulfamidase activity. This results in widespread lysosomal accumulation of HS in a variety of cell types in multiple organs, most prominently in neuronal tissue and liver. MPS IMA mice develop behavioral abnormalities, followed by neuroinflammation and neuro- degeneration, as well as cognitive problems. The life span of MPS MIA mice is significantly shortened.

MPS MIA mice received vehicle or an active dose of SOBI003 intravenously once weekly during 10 to 25 weeks. They were pretreated with methotrexate and chlorpheniramine to reduce anti-drug antibody responses and allergic reactions, respectively.

In the above mentioned MPS MIA mouse model, repeated administration of SOBI003 reduced HS storage in brain tissue and cerebrospinal fluid (CSF) in a dose- and time-dependent manner. The achieved reduction of HS showed a relation to the cumulated active dose of SOBI003 over the complete study period.

The relative levels of heparan sulfate (HS) in CSF and brain homogenates from male MPS MIA mice at 29 weeks of age after i.v. administration of vehicle or an active dose of SOBI003 at 3, 6, 12 or 17 mg/kg once weekly during 20 weeks (n=8) is shown in Figure 4. Mean data ± SEM are presented in the figures presenting HS in CSF and brain. Statistics were calculated using one-way ANOVA, followed by Dunnett’s Multiple Comparison test versus vehicle group as post hoc analysis. P values >0.05 were considered non-significant, whereas ^* p<0.05, ^** p<0.01 , and ^*** p<0.001. In the bottom left graph of Figure 4 individual HS data in brain and CSF are presented and statistics were calculated using Pearson’s correlation, with r2 = 0.8494 and p < 0.0001.

Immunohistological staining using antibodies specific for LIMP2, CD11 b, and GFAP was used to assess lysosomal swelling, microgliosis, and astrogliosis, respectively. Lysosomal autofluorescence, linked to secondary storage products of aggregated and oxidized lipids and proteins, was also assessed. SOBI003 was found to reduce the lysosomal swelling in brain and dampened the neuroinflammation in MPS IMA mice.

Efficacy of SOBI003 on behavioral abnormalities in MPS IMA mice was evaluated using a wire hanging test to assess neuromuscular function, an open field test to assess exploratory activity, a three chamber social interaction test to assess social ability, and a Barnes maze test to assess learning ability and short term memory. Trends towards beneficial effects of SOBI003 on behavioral impairments in these MPS MIA mice were also indicated. Treatment with SOBI003 increased the wire hanging performance as well as the exploratory activity. The sociability and learning behavior of the MPS MIA mice also showed tendencies to improvement.

Reference Example

SOBI003 is a modified sulfamidase comprising a polypeptide consisting of an amino acid sequence as defined in SEQ ID NO:1 , or a polypeptide having at least 95 % sequence identity with an amino acid sequence as defined in SEQ ID NO:1 and which has been chemically modified by any of the following modifying methods 1 to 5.

Modifying method 1 : Recombinant sulfamidase was oxidized by incubation with 20 mM sodium mefa-periodate at 0 degrees centigrade in the dark for 120 min in phosphate buffers having a pH of 6.0. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 degrees centigrade before sodium borohydride was added to the reaction mixture to a final concentration of 50 mM. After incubation at 0 degrees centigrade for 120 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 20 mM sodium phosphate, 100 mM NaCI, pH 6.0.

Modifying method 2: Performed as modifying method 1 with the exception that the concentration of sodium borohydride in the reduction step was 10 mM.

Modifying method 3: Recombinant sulfamidase was oxidized by incubation with 10 mM sodium mefa-periodate at 0 degrees centigrade in the dark for 180 min in acetate buffer having an initial pH of between 4.5 to 5.7. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 degrees centigrade before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 degrees centigrade for 60 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCI, pH 7.4.

Modifying method 4: Recombinant sulfamidase was oxidized by incubation with 10 mM sodium meta-periodate at 8 degrees centigrade in the dark for 60 min in acetate buffer having an initial pH of 4.5. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 degrees centigrade before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 degrees centigrade for 60 min in the dark, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCI, pH 7.4.

Modifying method 5: Recombinant sulfamidase was oxidized by incubation with 10 mM sodium meta-periodate at 8 degrees centigrade in the dark for 60 min in acetate buffer having an initial pH of 4.5. Glycan oxidation was quenched by addition of ethylene glycol to a final concentration of 192 mM. Quenching was allowed to proceed for 15 min at 6 degrees centigrade before sodium borohydride was added to the reaction mixture to a final concentration of 25 mM. After incubation at 0 degrees centigrade for 45 min in the dark and quenching the reaction with 0.1 M acetone, the resulting sulfamidase preparation was ultrafiltrated against 10 mM sodium phosphate, 100 mM NaCI, pH 7.4.