WO2019201855A1 - Protéine de fusion lysosomale - Google Patents

Protéine de fusion lysosomale Download PDF

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Publication number
WO2019201855A1
WO2019201855A1 PCT/EP2019/059675 EP2019059675W WO2019201855A1 WO 2019201855 A1 WO2019201855 A1 WO 2019201855A1 EP 2019059675 W EP2019059675 W EP 2019059675W WO 2019201855 A1 WO2019201855 A1 WO 2019201855A1
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polypeptide
lysosomal
fusion protein
seq
shielding
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PCT/EP2019/059675
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Joakim Nilsson
Erik Nordling
Anna SANDEGREN
Stefan SVENSSON GELIUS
Dominik POSSNER
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Swedish Orphan Biovitrum Ab (Publ)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01013—Sterol esterase (3.1.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06004—N-Acetylgalactosamine-6-sulfatase (3.1.6.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06008—Cerebroside-sulfatase (3.1.6.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06012—N-Acetylgalactosamine-4-sulfatase (3.1.6.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06013—Iduronate-2-sulfatase (3.1.6.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y310/00—Hydrolases acting on sulfur-nitrogen bonds (3.10)
    • C12Y310/01—Hydrolases acting on sulfur-nitrogen bonds (3.10) acting on sulfur-nitrogen bonds (3.10.1)
    • C12Y310/01001—N-Sulfoglucosamine sulfohydrolase (3.10.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • the present invention relates to proteins suitable for use in enzyme
  • 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.
  • LSDs lysosomal storage diseases
  • lysosomal storage diseases are characterized by a buildup of a metabolite (or metabolites) that cannot be degraded due to the insufficient degrading capacity.
  • lysosomes increase in size. How the accumulated storage material causes 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)).
  • the missing function caused by a mutated or missing protein may be restored by administration, and thus replacement of, the mutated/missing protein with a protein from a heterologous source.
  • administration of both enzymes, such as factor IX and factor VII, and proteins, such as factor VIII, that are part of activation complexes in the coagulation pathway have been successfully employed.
  • enzymes such as factor IX and factor VII
  • proteins, such as factor VIII proteins, such as factor VIII
  • lysosomal storage diseases In the field of lysosomal storage diseases, 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
  • M6PR mannose-6 phosphate receptors
  • ERT enzyme replacement therapies
  • ERT 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.
  • BBB blood-brain barrier
  • the endothelial cells of the BBB exhibit tight junctions which prevent paracellular passage, show limited passive
  • peripheral pathology is to some extent also sub-optimally addressed in current enzyme replacement treatment. Patients frequently suffer from arthropathy, clinically manifested in joint pain and stiffness resulting in severe restriction of motion. Moreover, progressive changes in the thoracic skeleton may cause respiratory
  • lysosomal protein after intravenous administration is dependent on the pattern of glycosylation of the protein.
  • N-glycans There are three general types of N-glycans: oligomannose (“high-mannose”), complex and hybrid, all of which are present on lysosomal proteins.
  • proteins directed to the lysosome carry one or more N-glycans which are phosphorylated.
  • the phosphorylation occurs in the Golgi and generates mannose-6-phospate (M6P) residues that are recognized by mannose-6-phosphate receptors (M6PRs) and initiate the transport of the lysosomal protein to the lysosome.
  • M6P mannose-6-phospate
  • M6PRs mannose-6-phosphate receptors
  • High-mannose glycans steer the protein to mannose receptor rich resident macrophages such as the Kuppfer cells in the liver
  • the mannose 6- phosphorylated glycans steer the protein to M6PR rich cells such as the hepatocytes in the liver.
  • the uptake of lysosomal proteins into cells is in most cases a rapid process and the half-life of a lysosomal protein in circulation is typically less than 1 hour. Consequently, in many tissues, e.g. tissues that are not well supplied by blood, therapeutically useful levels of lysosomal protein are difficult to achieve.
  • lysosomal storage diseases for instance, lysosomal storage diseases of cells of the central nervous system, and/or cells of bone and cartilage.
  • a fusion protein comprising
  • a lysosomal polypeptide i) a lysosomal polypeptide; and ii) a polypeptide moiety comprising 2-68 units, each unit being independently selected from the group consisting of all amino acid sequences according to SEQ ID NO: 1 :
  • X1 is P or absent
  • X2 is V or absent
  • X3 is P or T
  • X4 is P or T
  • X5 is T or V
  • X6 is D, G or T
  • X8 is A, Q or S
  • X9 is E, G or K
  • X10 is A, E, P or T;
  • XI I is A, P or T.
  • Fusing the lysosomal polypeptide to a polypeptide moiety as defined above to yield a fusion protein according to the invention has been found to reduce the rate of receptor-mediated cellular uptake of the lysosomal polypeptide in vivo, by shielding the lysosomal polypeptide from glycan-recognizing receptors.
  • the rate of uptake in tissues with abundant glycan-recognizing receptors is decreased, and the biological half-life of the lysosomal polypeptide is increased, which allows distribution also to tissues less abundant in glycan-recognizing receptors, and even
  • the fusion protein can retain the biological activity of the lysosomal
  • a fusion protein according to the invention can be transported across the blood-brain barrier in mammals and effect an enzymatic activity in the brain.
  • the reduced rate of receptor-mediated uptake of the fusion protein allows for better distribution to peripheral tissue involved in a peripheral pathology of a lysosomal storage disease is improved.
  • the present invention allows development of treatments that could potentially improve clinical outcome in a multitude of lysosomal storage diseases.
  • reduced clearance of the fusion protein may also advantageously allow for development of long-acting medicaments that can be administered to patients less frequently.
  • the high content of hydrophilic amino acid residues of the shielding polypeptide moiety serves to increase the solubility of the fusion protein relative to the lysosomal polypeptide as such.
  • the polypeptide moiety may improve the thermodynamic stability of the fusion protein relative to the lysosomal polypeptide alone.
  • the fusion protein may have a reduced tendency to aggregate when present in solution, and/or maintain its native structure (such as folding or dimerization) at elevated temperature.
  • An increased thermodynamic stability may, for instance, enable higher expression levels during production, facilitate protein purification and processing, allow formulation of compositions, including pharmaceutical compositions, of higher concentration of the active polypeptide, and/or provide better storage stability or increased shelf-life of a formulation including the fusion protein.
  • Lysosomal polypeptide means a polypeptide that exerts its biological activity in the lysosomal compartment of cells. Lysosomal polypeptides used in the present invention are typically degrading enzymes, such as sulfatases, glycoside hydrolases or proteases.
  • the polypeptide moiety that forms part of the fusion protein of the invention may be referred to as a“receptor mediated uptake reducing polypeptide” or as a“shielding polypeptide moiety”.
  • by“shielding polypeptide moiety” is meant a polypeptide that, by being fused to a polypeptide of interest, in particular a lysosomal polypeptide, reduces the interactions of the polypeptide of interest with glycan recognition receptors, thereby enabling, upon systemic administration to a subject, an altered in vivo distribution in terms of increased distribution of the polypeptide of interest to one or more diseased tissues or organs, such as a tissue or organ affected by a lysosomal storage disease.
  • amino acid sequences of the fusion partners of the fusion protein are referred to using the terms
  • polypeptide and“polypeptide moiety”. Notably, these terms are intended to include amino acid sequences as short as 18 amino acids, which effectively represents the smallest version of the shielding polypeptide moiety (2 units each of 9 amino acids).
  • An amino acid sequence of up to about 50 amino acids may sometimes be referred to as“peptide”; however for the sake of simplicity, in the present specification, the amino acid sequences of the fusion protein will be referred to as“polypeptide” or“polypeptide moiety” throughout.
  • glycan recognition receptors is meant receptors that recognize and bind to proteins mainly via glycan moieties of the proteins. For lysosomal proteins, such receptors can be exemplified by the mannose receptor, which
  • glycans selectively binds proteins where glycans exhibit exposed terminal mannose residues, and mannose 6-phosphate receptors, which are characteristic of lysosomal proteins.
  • 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.
  • embodiments of the invention had not only a fully retained biological activity, but in fact had an increased or improved biological activity.
  • the provision of a highly active protein offers a possibility of formulating an even more potent therapeutic for treatment of LSDs.
  • the lysosomal polypeptide is an enzymatically active lysosomal polypeptide.
  • the biological activity of the lysosomal polypeptide is typically an enzymatic activity.
  • the lysosomal polypeptide may be a sulfatase, a glycoside hydrolase, or a protease.
  • the lysosomal polypeptide may be selected from a sulfatase and a glucoside hydrolase.
  • the lysosomal polypeptide is a sulfatase.
  • the lysosomal polypeptide may be selected from the group consisting of: deoxyribonuclease-2-alpha; beta-mannosidase; ribonuclease T2; lysosomal alpha-mannosidase; alpha L-iduronidase; tripeptidyl-peptidase 1 ; hyaluronidase-3; cathepsin L2; ceroid-lipofuscinosis neuronal protein 5; glucosylceramidase; tissue alpha-L-fucosidase; myeloperoxidase; alpha- galactosidase A; beta-hexosaminidase subunit alpha; cathepsin D;
  • prosaposin beta-hexosaminidase subunit beta; cathepsin L1 ; cathepsin B; beta-glucuronidase; pro-cathepsin H; non-secretory ribonuclease; lysosomal alpha-glucosidase; lysosomal protective protein; gamma-interferon-inducible lysosomal thiol reductase; tartrate-resistant acid phosphatase type 5;
  • arylsulfatase A prostatic acid phosphatase; N-acetylglucosamine-6-sulfatase; arylsulfatase B; beta-galactosidase; alpha-N-acetylgalactosaminidase;
  • sphingomyelin phosphodiesterase ganglioside GM2 activator; N(4)-(beta-N- acetylglucosaminyl)-L-asparaginase; iduronate 2-sulfatase; cathepsin S; N- acetylgalactosamine-6-sulfatase; lysosomal acid lipase/cholesteryl ester hydrolase; lysosomal Pro-X carboxypeptidase; cathepsin O; cathepsin K; palmitoyl-protein thioesterase 1 ; arylsulfatase D; dipeptidyl peptidase 1 ;
  • alpha-N-acetylglucosaminidase galactocerebrosidase; epididymal secretory protein E1 ; di-N-acetylchitobiase; N-acylethanolamine-hydrolyzing acid amidase; hyaluronidase-1 ; chitotriosidase-1 ; acid ceramidase; phospholipase B-like 1 ; proprotein convertase subtilisin/kexin type 9; group XV
  • phospholipase A2 putative phospholipase B-like 2; deoxyribonuclease-2- beta; gamma-glutamyl hydrolase; arylsulfatase G; L-amino-acid oxidase; sialidase-1 ; legumain; sialate O-acetylesterase; thymus-specific serine protease; cathepsin Z; cathepsin F; prenylcysteine oxidase 1 ; dipeptidyl peptidase 2; lysosomal thioesterase PPT2; heparanase; carboxypeptidase Q; b-glucuronidase, N-sulfoglucosamine sulfohydrolase (sulfamidase), and sulfatase-modifying factor 1.
  • the lysosomal polypeptide may be selected from the group consisting of sulfamidase, iduronate 2-sulfatase, arylsulfatase A, arylsulfatase B, N-acetylgalactosamine-6-sulfatase, galactocerebrosidase, alpha-L-iduronidase, and beta-glucuronidase.
  • the lysosomal polypeptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 37, 49,232, 65, 78 and 82-149.
  • the fusion protein may comprise a lysosomal polypeptide that is glycosylated.
  • the lysosomal polypeptide may comprise high-mannose glycans, and/or at least one phosphorylated N-glycan, for example an N-glycan comprising at least one mannose-6-phosphate (M6P) moiety.
  • M6P mannose-6-phosphate
  • the lysosomal polypeptide of the fusion protein is capable of receptor-mediated uptake via a mannose-6-phosphate receptors (M6PRs), such as a calcium dependent M6PR and calcium independent M6PR.
  • M6PRs mannose-6-phosphate receptors
  • the polypeptide moiety of the fusion protein according to embodiments of the invention may be a shielding polypeptide moiety capable of reducing the rate of receptor-mediated uptake of the lysosomal polypeptide.
  • the uptake rate reduction may be due to steric hindrance or shielding of glycan moieties from glycan-recognizing receptors on cell surfaces.
  • the polypeptide moiety may form a contiguous sequence of 2-68 units of one or more sequence(s) as defined in SEQ ID NO: 1.
  • the polypeptide moiety which may also be referred to as a shielding polypeptide moiety, may comprise from 3 to 51 units as defined above.
  • the polypeptide moiety may comprise 3-34 units, such as from 3-17 units, or from 3 to 9 units, as defined above.
  • the fusion protein may comprise a plurality of polypeptide moieties, such as at least two polypeptide moieties, each of which may be as described herein, e.g. each comprising from 2 to 68 units, such as from 3-34 units, such as from 3-17 units, or from 3 to 9 units.
  • polypeptide moieties may be of the same length (having the same number of units), or may be of different lengths.
  • polypeptide moiety may be positioned N-terminally or C-terminally of said lysosomal polypeptide.
  • polypeptide moiety, or at least one of a plurality of polypeptide moieties may constitute an insertion into, or replacement of a part of, the amino acid sequence of the lysosomal polypeptide.
  • At least one of the residues X3 and X4 of SEQ ID NO:1 may be P. In some embodiments, at least one of X4 and X5 of SEQ ID NO:1 may be T. In some embodiments, at least one of X10 and X11 of SEQ ID NO:1 may be A or P. In some embodiments, X1 is P and X2 is V.
  • each unit of the polypeptide moiety may be selected from the group consisting of SEQ ID NOs: 2-11 and 159-230. That is, in embodiments of the invention, the polypeptide moiety may comprise 2-68 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 2-11 and 159-230. In some embodiments, each unit of the polypeptide moiety may be selected from the group consisting of SEQ ID NOs: 2-11. SEQ ID NOs: 2-11 sequences represent human variants of SEQ ID NO: 1.
  • the polypeptide moiety may correspond to a naturally occurring human amino acid sequence.
  • Such embodiments include, for instance, fusion proteins as defined above wherein the polypeptide moiety comprises, or consists of, at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12-27.
  • the polypeptide moiety comprises, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-77.
  • sequence of human origin may be advantageous as it is expected to contribute to a low immunogenicity in human subjects.
  • Amino acid sequences based on repeating units selected from SEQ ID NOs: 2-11 evaluated in vitro and in silico were found to have low immunogenic potential.
  • shielding polypeptide moieties consisting of such units are expected to be well tolerated, in terms of immune response, by human subjects.
  • the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-32, 34-36, 38-48, 50- 60, 62-64, 66-72, 79-81 , and 150-158.
  • the lysosomal polypeptides contemplated for use in the fusion protein of the invention often occur naturally in the form of multimeric protein complexes, such as dimers.
  • the invention relates to a multimeric protein complex comprising at least two fusion proteins according to the first aspect of the invention.
  • the multimeric protein complex may be a dimer, that is, it may comprise a dimer of the fusion protein.
  • the invention provides a composition, typically a pharmaceutical composition, comprising a fusion protein or a multimeric protein complex as defined herein and at least one carrier or ingredient, typically at least one pharmaceutically acceptable carrier, diluent, or ingredient.
  • the composition may be formulated as a pharmaceutical composition intended for administration to a subject in need thereof, in particular for treatment of a lysosomal storage disease.
  • the composition may be formulated e.g. for subcutaneous or intravenous administration.
  • the invention provides a fusion protein, a multimeric protein complex, or a composition according to the above-mentioned aspects of the invention, for use as a medicament, and particularly for use in treatment of a lysosomal storage disease, by enzyme replacement therapy.
  • the invention also provides a method of treating a lysosomal storage disease, comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein, the multimeric protein complex, or the composition according to the above-mentioned aspects.
  • the lysosomal storage disease to be treated may be manifested in one or more tissues and/or organs selected from: neural tissue, central nervous system, peripheral nervous system, brain, muscular tissue, endothelial tissue, heart, lungs, skeletal muscle, connective tissue, cartilage, bone, skeletal bone, and joints.
  • the organ may be the brain.
  • tissue may be cartilage and/or bone.
  • the lysosomal storage disease may be manifested in peripheral tissue.
  • peripheral tissue is intended to mean all tissues outside of the central nervous system.
  • peripheral tissue include muscular tissue, endothelial tissue, heart tissue, lung tissue, muscular tissue, connective tissue, cartilage, and bone.
  • the rate of receptor-mediated cellular uptake of the lysosomal polypeptide may be reduced.
  • the uptake may typically be mediated by mannose receptors and/or mannose-6-phosphate receptors (M6PR).
  • M6PR mannose-6-phosphate receptors
  • the distribution of the fusion protein or the multimeric protein complex to a target tissue or organ affected by the lysosomal storage disease may be increased, relative to the distribution of the lysosomal polypeptide as such (although possibly in multimeric form) without the polypeptide moiety.
  • the target tissue may in particular be selected from: neural tissue, central nervous system, peripheral nervous system, brain, muscular tissue, endothelial tissue, heart, lungs, skeletal muscle, connective tissue, cartilage, bone, skeletal bone, and joints.
  • Figure 1 is a schematic representation of a gene encoding a biologically active polypeptide (white) and one or more gene(s) encoding a shielding polypeptide moiety (shaded) according to embodiments of the invention.
  • Figure 2 is a photograph of an SDS-PAGE gel showing the results of electrophoretic separation of different fusions proteins of sulfamidase with shielding polypeptide moieties according to embodiments of the invention.
  • Figure 3 is a graph showing the aggregation propensity measured by static light scattering (counts) as function of increased temperature (°C), for sulfamidase (reference) as well as fusion proteins of sulfamidase with shielding polypeptide moieties, according to embodiments of the invention.
  • Figure 4 is a graph showing the mean and standard deviation serum concentration of sulfamidase (reference, filled boxes) and sulfamidase fusion proteins, according to embodiments of the invention, versus time following a 10 mg/kg i.v. administration in mice.
  • Figure 5 is a graph showing the relationship between the apparent molecular weight in solution in kDa (y-axis) of fusion proteins according to embodiments of the invention and the number of repeat units of the shielding polypeptide moiety and number of repeat units (x-axis).
  • Figure 6 is a graph showing the cellular uptake of iduronate-2-sulfatase and iduronate-2-sulfatase fusion proteins in the presence of increasing
  • Figure 7 is a photograph of an SDS-PAGE gel showing the results of electrophoretic separation of cell harvest media from production of GalN6S (reference) and GalN6S fusion proteins according to embodiments of the invention.
  • Figure 8 is a photograph of an SDS-PAGE gel showing the results of electrophoretic separation of ASB (reference) and the cell media harvest from production of ASB fusion protein according to an embodiment of the invention.
  • Figure 9 is a graph showing the mean and standard deviation serum concentration of iduronate 2-sulfatase (reference, SEQ ID NO: 37, filled boxes) and an iduronate 2-sulfatase fusion protein (SEQ ID NO: 46), according to embodiments of the invention, versus time following a 10 mg/kg i.v. administration in mice.
  • Figure 10 is a graph showing the normalised scattering signal as function of increased temperature (°C), for arylsulfatase A (PSI0590) represented by a solid black line, and fusion proteins of arylsulfatase A with shielding polypeptide moieties (PSI0681 , SEQ ID NQ:50; PSI0683, SEQ ID NO: 52; PSI0685, SEQ ID NO: 54; PSI0687, SEQ ID NO: 56; PSI0689, SEQ ID NO: 58; PSI0691 , SEQ ID NO: 60), each represented by a dashed line, according to embodiments of the invention.
  • PSI0590 arylsulfatase A
  • Figure 10 is a graph showing the normalised scattering signal as function of increased temperature (°C), for arylsulfatase A (PSI0590) represented by a solid black line, and fusion proteins of arylsulfatase A with shielding polypeptide moie
  • Figure 11 is a graph showing the normalised scattering signal as function of increased temperature (°C), for iduronidase (PSI0754, SEQ ID NO: 61 ) represented by a solid black line, and fusion proteins of iduronidase with shielding polypeptide moieties (PSI0753, SEQ ID NO: 62; PSI0755, SEQ ID NO: 63; PSI0756, SEQ ID NO: 64), each represented by a dashed line, according to embodiments of the invention.
  • iduronidase PSI0754, SEQ ID NO: 61
  • fusion proteins of iduronidase with shielding polypeptide moieties PSI0753, SEQ ID NO: 62; PSI0755, SEQ ID NO: 63; PSI0756, SEQ ID NO: 64
  • Figure 12 is a photograph of an SDS-PAGE gel showing the results of electrophoretic separation of the cell media harvest from production of GALC fusion protein according to an embodiment of the invention. Lane A:
  • Molecular weight marker SeeBlue plus 2 (Thermo Fisher Scientific)
  • lane B unrelated cell media harvest
  • lane C GALC-SPM1 -17 (PSI0817)
  • lane D SPM1 -17-GALC (PSI0818)
  • lane E SPM1 -17-GALC-SPM1 -17 (PSI0819).
  • the star indicates produced fusion protein in harvest media.
  • the present invention is based on the realization that fusion of a lysosomal polypeptide to a polypeptide moiety as defined hereinbelow provides a means of reducing the rate of receptor-mediated uptake of the lysosomal
  • polypeptide in particular reduce the rate of uptake in the liver, and thereby, through an increased biological half-life, enable distribution to tissues that have previously been poorly targeted.
  • Lysosomal proteins are usually rapidly cleared from circulation when administrated by intravenous injection. As described in more detail below, cellular uptake from the extracellular compartment is facilitated by receptors recognising the characteristic mannose and mannose 6-phosphate rich glycans of lysosomal proteins. Thus, distribution of lysosomal proteins is at least partly controlled by the density of these receptors on different cells. While the mannose recognizing receptors are abundantly present on tissue- resident macrophages and sinusoidal endothelial cells in the liver, the cation independent mannose 6-phosphate receptor is abundant on hepatocytes.
  • a major part of the dose of an intravenously administrated therapeutic lysosomal enzyme may distribute to the liver, which is sub-optimal for most therapeutic applications.
  • the two therapeutic a- galactosidase A preparations used as treatment for Fabry disease both show 60-70 % of the dose distributed to liver after a single dose in mice (Lee et al, Glycobiology 13: 305-313 (2003)).
  • cells in tissues that are not very well supplied by blood and/or have low abundance of receptors are not sufficiently targeted via these uptake mechanisms.
  • a fusion protein as disclosed herein may provide a better distribution of a lysosomal polypeptide in joints, connective tissue, cartilage and bone, when
  • Distribution of lysosomal protein after intravenous administration is highly dependent on the pattern of glycosylation of the protein, in particular N- glycosylation.
  • N-glycosylations can occur at an Asn-X-Ser/Thr sequence motif.
  • the initial core structure of the N-glycan is transferred by the glycosyltransferase oligosaccharyltransferase, within the reticular lumen.
  • This common basis for all N-linked glycans is made up of 14 residues: 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues.
  • This precursor is then converted into three general types of N-glycans; oligomannose, complex and hybrid, by the actions of a multitude of enzymes that both trims down the initial core and adds new sugar moieties.
  • Each mature N-glycan contains the common core Man(Man)2-GlcNAc-GlcNAc-Asn, where Asn represents the attachment point to the protein.
  • proteins directed to the lysosome carry one or more N-glycans which are phosphorylated.
  • the phosphorylation occurs in the Golgi and is initiated by the addition of N-acetylglucosamine-1 -phosphate to C-6 of mannose residues of oligomannose type N-glycans.
  • the N- acetylglucosamine is cleaved off to generate mannose-6-phospate (M6P) residues, that are recognized by mannose-6-phosphate receptors (M6PRs) and will initiate the transport of the lysosomal protein to the lysosome.
  • M6P mannose-6-phospate
  • the resulting N-glycan is then trimmed to the point where the M6P is the terminal group of the N-glycan chain.
  • the binding site of the M6PR requires a terminal M6P group that is complete, as both the sugar moiety and the phosphate group is involved in the binding to the receptor (Kim et al, Curr Opin Struct Biol 19(5):534-42 (2009)).
  • a lysosomal polypeptide is a polypeptide that exerts its biological activity in the lysosome.
  • Lysosomal polypeptides used in the present invention are typically degrading enzymes, such as sulfatases, glycoside hydrolases or proteases.
  • the lysosomal polypeptide is a lysosomal protein lacking transmembrane helices and having at least one N-glycosylation site.
  • lysosomal proteins examples include N-glycosylated lysosomal proteins. Some of the proteins might be known under other names. It should be understood that the protein listing above also encompasses any and all alternative names. Table I. Examples of lysosomal proteins
  • the lysosomal protein is selected from the group consisting of deoxyribonuclease-2-alpha; beta-mannosidase; ribonuclease T2; lysosomal alpha-mannosidase (Laman); tripeptidyl-peptidase 1 (TPP-1 ); hyaluronidase-3 (Hyal-3); cathepsin L2; ceroid-lipofuscinosis neuronal protein 5; glucosylceramidase; tissue alpha-L-fucosidase; myeloperoxidase (MPO); alpha-galactosidase A ;beta-hexosaminidase subunit alpha; cathepsin D; prosaposin; beta-hexosaminidase subunit beta; cathepsin L1 ; cathepsin B; beta-glucuronidase; pro-cathepsin H; cathepsin H;
  • ribonuclease lysosomal alpha-glucosidase; lysosomal protective protein; gamma-interferon-inducible lysosomal thiol reductase; tartrate-resistant acid phosphatase type 5 (TR-AP); arylsulfatase A (ASA); prostatic acid
  • PAP N-acetylglucosamine-6-sulfatase
  • ASB arylsulfatase B
  • beta-galactosidase alpha-N-acetylgalactosaminidase
  • sphingomyelin phosphodiesterase ganglioside GM2 activator
  • N(4)-(beta-N- acetylglucosaminyl)-L-asparaginase iduronate 2-sulfatase; cathepsin S; N- acetylgalactosamine-6-sulfatase; alpha-L-iduronidase; lysosomal acid lipase/cholesteryl ester hydrolase (Acid cholesteryl ester hydrolase) (LAL); lysosomal Pro-X carboxypeptidase; cathepsin O; cathepsin K; palmitoyl- protein thio
  • sulfamidase arylsulfatase D
  • ASD arylsulfatase D
  • dipeptidyl peptidase 1 alpha-N- acetylglucosaminidase; galactocerebrosidase (GALCERase); epididymal secretory protein E1 ; di-N-acetylchitobiase; N-acylethanolamine-hydrolyzing acid amidase; hyaluronidase-1 (Hyal-1 ); chitotriosidase-1 ; acid ceramidase (AC); phospholipase B-like 1 ; proprotein convertase subtilisin/kexin type 9; group XV phospholipase A2; putative phospholipase B-like 2;
  • deoxyribonuclease-2-beta gamma-glutamyl hydrolase; arylsulfatase G (ASG); L-amino-acid oxidase (LAAO) (LAO); sialidase-1 ; legumain; sialate O- acetylesterase; thymus-specific serine protease; cathepsin Z; cathepsin F (CATSF); prenylcysteine oxidase 1 ; dipeptidyl peptidase 2; lysosomal thioesterase PPT2 (PPT-2); heparanase; carboxypeptidase Q; b- glucuronidase, and sulfatase-modifying factor 1.
  • ASG arylsulfatase G
  • LAAO L-amino-acid oxidase
  • sialidase-1 ; legumain; sialate O- acetyleste
  • the lysosomal protein is a sulfatase.
  • Said sulfatase preferably has a FGIy residue at its active site.
  • said sulfatase is thus selected from arylsulfatase A; N- acetylglucosamine-6-sulfatase, arylsulfatase B; iduronate 2-sulfatase; N- acetylgalactosamine-6-sulfatase; N-sulfoglucosamine sulfohydrolase
  • sulfamidase arylsulfatase D, and arylsulfatase G.
  • said sulfatase is arylsulfatase A; N-acetylglucosamine-6-sulfatase; arylsulfatase B; iduronate 2-sulfatase; N-acetylgalactosamine-6-sulfatase or sulfamidase.
  • said sulfatase is arylsulfatase A.
  • said sulfatase is sulfamidase.
  • said sulfatase is iduronate-2- sulfatase. In one embodiment, said sulfatase is arylsulfatase B. In one embodiment, said sulfatase is N-acetylgalactosamine-6-sulfatase.
  • the lysosomal protein is a glycoside hydrolase.
  • said glycoside hydrolase is selected from alpha-galactosidase A; tissue alpha-L-fucosidase;
  • glucosylceramidase lysosomal alpha-glucosidase; beta-galactosidase; beta- hexosaminidase subunit alpha; beta-hexosaminidase subunit beta;
  • galactocerebrosidase lysosomal alpha-mannosidase; beta-mannosidase; alpha-L-iduronidase; alpha-N-acetylglucosaminidase; beta-glucuronidase; hyaluronidase-1 ; alpha-N-acetylgalactosaminidase; sialidase-1 ; di-N- acetylchitobiase; chitotriosidase-1 ; hyaluronidase-3, and heparanase.
  • said glycoside hydrolase is alpha-L-iduronidase, beta- glucuronidase or galactocerebrosidase.
  • said glycoside hydrolase is alpha-L-iduronidase.
  • said glycoside hydrolase is beta-glucuronidase.
  • said glycoside hydrolase is galactocerebrosidase.
  • the lysosomal protein is a protease.
  • said protease is selected from cathepsin D; cathepsin L2; cathepsin L1 ; cathepsin B; pro-cathepsin H; cathepsin S;
  • cathepsin O cathepsin K; dipeptidyl peptidase 1 ; cathepsin Z; cathepsin F; legumain; gamma-glutamyl hydrolase; tripeptidyl-peptidase 1 ;
  • said protease is tripeptidyl-peptidase 1.
  • the lysosomal protein comprises polypeptide consisting of an amino acid sequence selected from any one of SEQ ID NOs: 33, 37, 49, 232, 65, 78 and 82-149, or a polypeptide having at least 90 % sequence identity with an amino acid sequence selected from SEQ ID NOs: 33, 37, 49, 61 , 65, 78 and 82-149.
  • said polypeptide has at least 95 % sequence identity with an amino acid sequence selected from SEQ ID NOs: 33, 37, 49, 61 , 65, 78 and 82-149, such as at least 98 % sequence identity or at least 99 % sequence identity with an amino acid sequence selected from SEQ ID NOs: 33, 37, 49, 61 , 65, 78 and 82-149.
  • the lysosomal polypeptide is a sulfatase
  • polypeptide has an amino acid sequence is selected from SEQ ID NO: 49, SEQ ID NO: 109; and SEQ ID NO: 37.
  • the lysosomal protein is a glycoside hydrolase and comprises a polypeptide consisting of an amino acid sequence selected from any one of SEQ ID NO: 93; SEQ ID NO: 91 ; SEQ ID NO: 90; SEQ ID NO: 103; SEQ ID NO: 110; SEQ ID NO: 94; SEQ ID NO: 97; SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 83; SEQ ID NO: 61 ; SEQ ID NO: 123; SEQ ID NO: 100; SEQ ID NO: 127; SEQ ID NO: 111 ; SEQ ID NO: 138; SEQ ID NO: 125; SEQ ID NO: 128; SEQ ID NO: 87; and SEQ ID NO: 147.
  • said polypeptide has an amino acid sequence as set out in SEQ ID NO: 61 or SEQ ID NO:78.
  • the lysosomal protein is a protease and comprises a polypeptide consisting of an amino acid sequence selected from any one of SEQ ID NO: 95; SEQ ID NO: 143; SEQ ID NO: 86; SEQ ID NO:104; SEQ ID NO:131 ; SEQ ID NO:122; SEQ ID NO:119; SEQ ID NO:88; SEQ ID NO:98; SEQ ID NO:99; SEQ ID NO:101 ; SEQ ID NO:115; SEQ ID NO:118; SEQ ID NO:142; SEQ ID NO:139; SEQ ID NO:135; SEQ ID NO:117; SEQ ID NO:141 ; and SEQ ID NO:145.
  • said polypeptide has an amino acid sequence as set out in SEQ ID NO: 86.
  • the shielding polypeptide as described herein has the potential, when fused to a lysosomal protein, to decrease the rate of receptor-mediated uptake of the lysosomal protein, possibly by steric hindrance.
  • the shielding polypeptide is designed based on the C-terminal domain of human bile salt-stimulated lipase (BSSL).
  • Bile salt-stimulated lipase also referred to as bile salt-activated lipase (BAL) or carboxylic ester lipase (CEL) is a lipolytic enzyme produced by the human lactating mammary gland and pancreas. The protein is arranged in two domains, a large globular amino-terminal domain and a smaller but extended carboxy-terminal (C-terminal) domain (for a review, see e.g. Wang & Hartsuck (1993) Biochim. Biophys Acta 1166: 1 -19).
  • the present inventors have found that repetitive sequences based on or derived from the C-terminal domain of human BSSL can be successfully fused to various lysosomal polypeptides and confer reduced receptor-mediated uptake of the fusion partner, thereby extending its biological half-life and allowing for a different distribution pattern in vivo.
  • the C-terminal domain of human BSSL consists of repeating units of, or similar to, the formula“PVPPTGDSGAP”. Table 2 in Example 1 below lists the repeating units from human BSSL variants. The most common form of the C-terminal domain contains 18 repeating units (UniProt entry P19835).
  • each repeating unit has one site that may be O- glycosylated, increasing the hydrophilicity and size of the region (Stromqvist et al. Arch. Biochem. Biophys. 1997).
  • the C-terminal end of the domain is however hydrophobic, and has been shown to bind into the active site of BSSL and cause auto-inhibition of the enzyme.
  • the most frequent human sequence of this hydrophobic portion is“QMPAVIRF” (SEQ ID NO: 231 ) (Chen et al. Biochemistry 1998).
  • the C-terminal domain may be responsible for the stability of BSSL in vivo, for example its resistance to denaturation by acid and aggregation under physiological conditions (Loomes et al., Eur. J. Biochem. 1999, 266, 105-111 ).
  • the C-terminal domain which is enriched with Pro, Asp, Glu, Ser and Thr residues, is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that the protein may have a short half-life in vivo due to the repetitive sequences in the C-terminal domain (Kissel et al., Biochimica et Biophysica Acta 1989, 1006).
  • the extended biological half-life of a fusion protein comprising a shielding polypeptide moiety as defined herein, based on or derived from the C-terminal domain of human BSSL is believed to be due mainly to the decreased receptor mediated uptake of the protein.
  • other mechanisms may contribute to the increased biological half-life.
  • fusion protein refers to the joining of two or more portions of chemical entities of the same kind, such as peptides, polypeptides, proteins, or nucleic acid sequences.
  • a fusion protein as referred to herein typically comprises at least two polypeptide portions, which may be of different origin; for instance, a biologically active polypeptide, which is not BSSL, and a shielding polypeptide moiety, which may be derived from BSSL.
  • a fusion may contain the fused portions in any order and at any position; however, a fusion of genes is typically made in-frame (in-line), such that the open reading frames (ORFs) of the fused genes are maintained, as appreciated by persons of skill in the art.
  • both fusion partners of the fusion protein may be of human origin, but the fusion protein does not correspond to a naturally occurring human protein.
  • Figure 1 schematically illustrates nucleic acid constructs encoding a fusion protein according to embodiments of the present invention, comprising at least one gene encoding a biologically active polypeptide (Fig. 1 (a)-(g), white bar), such as a lysosomal polypeptide, and at least one gene encoding a shielding polypeptide moiety ((b)-(g), dashed bar).
  • a biologically active polypeptide Fig. 1 (a)-(g), white bar
  • a lysosomal polypeptide such as a lysosomal polypeptide
  • a shielding polypeptide moiety (b)-(g), dashed bar).
  • the gene encoding the biologically active polypeptide may be preceded by a signaling peptide for expression in mammalian cells. As shown in Fig.
  • the gene encoding the shielding polypeptide moiety may be located C-terminally, see Fig. 1 (b); N- terminally, see Fig. 1 (c); or both N- and C-terminally, Fig. 1 (d), to the gene(s) encoding at least one biologically active polypeptide.
  • a nucleotide sequence encoding a shielding polypeptide moiety may be positioned within the boundaries of the gene encoding the biologically active polypeptide (in-line positioning).
  • sequences encoding shielding polypeptide moieties may optionally be present at multiple sites, e.g. at three sites as shown in Fig.
  • the shielding polypeptide moiety is not necessarily located at the C-terminal of the biologically active polypeptide.
  • the at least one shielding polypeptide moiety may be located at the N-terminal of the biologically active polypeptide (Fig. 1 (c)), or shielding moieties may be located each at the N-terminal and C- terminal, respectively (Fig. 1 (d)).
  • one or more shielding polypeptides may be inserted at a position within the biologically active polypeptide (Fig. 1 (e)), for example in a position located in a surface-exposed loop of the biologically active polypeptide.
  • the shielding polypeptide moiety may replace a specific sequence segment of the biologically active polypeptide. For instance, when positioned as an insert, the shielding polypeptide moiety may replace a part of a surface-exposed loop on the biologically active
  • a shielding polypeptide may replace an entire domain, such as a N-terminal or a C-terminal domain, or an internal domain, of the biologically active polypeptide.
  • an in-line inserted shielding polypeptide moiety may be combined with either an N-terminal moiety, a C-terminal moiety, or both N-terminal and C-terminal shielding polypeptide moieties, see Fig. 1 (f).
  • each such shielding moiety may be independently defined as described herein. Otherwise stated, each such shielding moiety may comprise from 2 to 68 units of an amino acid sequence according to SEQ ID NO: 1.
  • the present invention is not limited to the use of a single biologically active polypeptide as fusion partner; rather, as illustrated in Fig. 1 (g), it is envisaged that in some embodiments the fusion protein may comprise multiple biologically active polypeptides, at least one being a lysosomal polypeptide, for instance two or more lysosomal polypeptides, or a
  • one or more shielding polypeptide moiety or moieties may also be located at the N- or C-terminal of the fusion protein.
  • the fusion protein may comprise two different biologically active polypeptides, at least one being a lysosomal polypeptide, optionally separated by a linker or spacer sequence and/or a shielding polypeptide moiety.
  • the fusion protein may comprise three different biologically active polypeptides, at least one being a lysosomal polypeptide. In embodiments where the fusion protein comprises more than one, e.g. two, lysosomal polypeptides, these may be the same or different.
  • biologically active polypeptides may be selected from the group consisting of growth factors, cytokines, enzymes and ligands, and that the remaining biologically active polypeptide(s) may be selected from antibodies or antibody fragments.
  • the shielding polypeptide moiety may be positioned as a linker between different antigen- binding regions.
  • the shielding polypeptide moiety used for fusion with a lysosomal polypeptide comprises an amino acid sequence comprising 2-68 repeating units, each unit being independently selected from the group of amino acid sequences defined by SEQ ID NO: 1 :
  • XI is P or absent
  • X2 is V or absent
  • X3 is P or T
  • X4 is P or T
  • X5 is T or V
  • X6 is D, G or T
  • X8 is A, Q or S
  • X9 is E, G or K
  • X10 is A, E P or T;
  • XI I is A, P or T.
  • a“unit” refers to an occurrence of an amino acid sequence of the general formula according to SEQ ID NO: 1 as defined above, including for instance any of the sequences according to SEQ ID NOs: 2-11 and 159- 230, such as any of the sequences according to SEQ ID NO: 2-11.
  • the shielding polypeptide comprises from 2 to 68 such units, which may be the same or different, within the definition set out above.
  • the units of the shielding polypeptide may also be referred to as“repeating units” (or“repeat units”) although there is some variation in the amino acid sequence between individual units, and hence“repeating units” is not to be understood exclusively as the repetition of one and the same sequence. Stated
  • the shielding polypeptide moiety comprises from 2 to 68 units, wherein each unit is an amino acid sequence independently selected from the group consisting of the individual sequences falling within the definition of SEQ ID NO:1.
  • the shielding polypeptide moiety may comprise at least 3 at least 4, at least 6, at least 8, at least 10, or at least 17 units of one or more amino acid sequence(s) according to SEQ ID NO: 1. Furthermore, in embodiments of the invention, the shielding polypeptide moiety may comprise up to 3, up to 5, up to 9, up to 10, up to 17, up to 18, up to 34, or up to 51 units of one or more amino acid sequence(s) according to SEQ ID NO: 1.
  • the shielding polypeptide moiety may comprise from 3 to 51 units of one or more amino acid sequence(s) according to SEQ ID NO: 1 , such as 3 to 34 units, such as 3 to 17 units, such as 5 to 17 units independently selected from the group consisting of SEQ ID NOs: 2-11 and 159-230, such as from the group consisting of SEQ ID NOs: 2-11.
  • the number of repeating units of the shielding polypeptide may be made with regards to the specific lysosomal polypeptide to which it is to be fused.
  • the number of N-glycosylation sites of the lysosomal polypeptide and their location in three-dimensional space may be considered when selecting the desired number and length of the shielding polypeptide(s). For instance, many N-glycosylation sites, which are positioned far from each other, may require use of a longer (higher number of units) shielding polypeptide, and/or the use of more than one shielding polypeptide, to achieve a certain shielding effect (reduced rate of receptor-mediated cellular uptake).
  • monomeric protein complexes may benefit from more and/or longer shielding polypeptide(s) to achieve a certain shielding effect, compared to dimeric protein complexes where the shielding polypeptides of the second fusion protein may to some extent shield the N- glycans of the lysosomal polypeptide of the first fusion protein.
  • the shielding polypeptide moiety comprises at least 17 contiguous repeats, and preferably a fusion protein of iduronate-2-sulfatase comprises two shielding polypeptide moieties, each of which comprises at least 17 contiguous units of SEQ ID NO: 1.
  • the shielding polypeptide moiety may comprise a contiguous sequence of at least 18 amino acids (corresponding to two units that are both 9-meric versions of SEQ ID NO:1 ), and typically up to 748 amino acids
  • the repeating units may be contiguous with one another, although it is also possible that the repeating units are separated by short spacing sequences. For instance, two repeating units may be separated by up to 10 amino acid residues that do not correspond to SEQ ID NO: 1 ; for instance, the short spacing sequence may be a peptide linker of the formula (G 4 S) 2 . In some embodiments, a spacing sequence may be up to 5 amino acid residues. In some embodiments one or more amino acid residue(s) may be positioned between two repeating units, e.g.
  • a linker such as one or more G 4 S linkers, may be used as spacing sequences between adjacent repeating units.
  • the contiguous sequence comprising up to 68 repeating units may be longer than 748 amino acids, for instance up to 800 amino acids.
  • the repeating units of the shielding polypeptide moiety are defined by SEQ ID NO: 1 , which is based on the repeating units of human variants of the BSSL C-terminal domain, and which allows some variation of amino acid residues in amino acid positions 3, 4, 5, 6, 8, 9, 10 and 11.
  • the residues at positions 1 (X1 ), 2 (X2) and 7 are fixed, although X1 and X2 may be absent.
  • a repeating unit consists of 9 amino acids only.
  • a shielding polypeptide moiety comprising 2 to 68 units typically comprises several variants of the amino acid sequence motif generally defined by SEQ ID NO:1 , such as at least two different variants according to SEQ ID NO:1.
  • the shielding polypeptide moiety may comprise at least one unit of each of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • these may be independently selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • the shielding polypeptide moiety may comprise SEQ ID NOs: 3-5 in this order, optionally preceded by SEQ ID NO: 2.
  • a unit according to SEQ ID NO: 2 may especially be located at the N-terminal end of the shielding polypeptide moiety, representing the first unit of the shielding polypeptide moiety. While other specific variations of the repeating units (e.g. the units according to SEQ ID NOs: 3-11 ) may appear repeatedly, SEQ ID NO: 2, if present, typically only appears once, as the first repeating unit of the shielding polypeptide moiety.
  • the conformation of the shielding polypeptide moiety is generally linear
  • the shielding polypeptide does not contribute to the a-helix and/or b-sheet content of the fusion protein as determined by circular dichroism or FTIR (Fourier Transform Infrared Spectroscopy).
  • a repeating unit defined by SEQ ID NO:1 is of human origin, and preferably all of the repeating units of the shielding polypeptide moiety correspond(s) to naturally occurring repeating units of a variant of the C-terminal domain of human BSSL. Such repeating units are represented by SEQ ID NOs: 2-11 (See also Example 1 , Table 1.2). In embodiments of the invention, all repeating units of the shielding polypeptide moiety are selected from the group consisting of SEQ ID NOs: 2-11 , e.g. SEQ ID NOs: 3-11.
  • the shielding polypeptide moiety may comprise 2-68 units, each independently selected from the group consisting of SEQ ID NO: 2-11 , or from the group consisting of SEQ ID NOs: 3-11.
  • the use of a sequence of human origin may be advantageous as it is expected to contribute to a low immunogenicity in human subjects.
  • the shielding polypeptide moiety comprises, or consists of, a sequence of repeating units that corresponds to a naturally occurring human sequence of repeating units. Examples of such natural human sequences of repeating units are presented in SEQ ID NO: 12-19 and 20-27.
  • Such sequences may comprise, as the first five repeating units, in this order: [SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5], or, alternatively, as the first four repeating units, in this order: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5].
  • the first repeating unit may be SEQ ID NO: 5.
  • the shielding polypeptide comprises only three repeating units, these may in particular be, in this order [SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] or [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5].
  • the shielding polypeptide moiety may in particular be: [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5]
  • the shielding polypeptide moiety may comprise, or consist of, 9 identical repeats SEQ ID NO: 5, which may be contiguous.
  • the shielding polypeptide moiety comprises an amino acid sequence according to any one of SEQ ID NOs: 12-27.
  • the shielding polypeptide moiety consists of a multiple of any one of the sequences of the group comprised of SEQ ID NOs: 12-27.
  • the shielding polypeptide moiety may consist of two or more, such as three or more, contiguous multiples, or copies, of an amino acid sequence according to any one of SEQ ID NOs: 12-27; for instance SEQ ID NO: 20.
  • SEQ ID NO: 20 comprises 17 units of an amino acid sequence according to SEQ ID NO: 1 , and thus a three-copy multiple of SEQ ID NO: 20 would comprise at least 51 units.
  • the shielding polypeptide moiety comprises an amino acid sequence that corresponds in part or in full to any one of SEQ ID NOs: 12-27, such as SEQ ID NO: 20.
  • the shielding polypeptide moiety may contain from 3 to 17 contiguous repeating units.
  • the shielding polypeptide moiety may have an amino acid sequence selected from among any one of SEQ ID NOs: 73-77.
  • the repeating units of the shielding polypeptide moiety can be independently selected from all units according to SEQ ID NO:1 and the invention is thus not limited to certain sequences of units being repeated. Accordingly, for instance a 51 -unit shielding polypeptide moiety is not necessarily formed of three copies of a 17-unit sequence, but may be formed of any combination of units according to SEQ ID NO:1 , and in particular of any combination of repeating units selected from SEQ ID NOs: 2-11 and 159- 230.
  • one or more shielding polypeptide moieties may completely lack glycosylation sites.
  • one or more shielding polypeptide moieties of the fusion protein may each comprise 2 to 68 units of one or more amino acid sequence(s) independently selected from the group consisting of SEQ ID NOs: 159-230, such as from the group consisting of SEQ ID NOs: 159-165.
  • the fusion protein may comprise a linker, typically a peptide linker, linking the lysosomal polypeptide to one or more shielding polypeptide moieties as described herein.
  • the fusion protein further comprises a peptide linker positioned between an amino acid sequence of the lysosomal polypeptide and an amino acid sequence of the shielding polypeptide moiety.
  • the peptide linker may be selected from -GS- and -(G4S) n -, wherein n is an integer from 1 to 5, typically from 1 to 3, or from 2 to 3.
  • the use of a linker may be
  • n may reduce the occurrence of, or, in the case of n being at least 2, prevent the formation of neo epitopes and subsequent binding of such neo epitopes by antigen-presenting cells of the immune system.
  • the fusion protein of the invention comprises a lysosomal polypeptide e.g. as described above, fused to a shielding polypeptide moiety as defined herein.
  • the fusion protein comprises an amino acid sequence has at least 95 % sequence identity with an amino acid sequence selected from SEQ ID NO: 28-32, 34-36, 38-48, 50-60, 62-64, 66-72, 79-81 , and 150-158, such as at least 98 % sequence identity, or at least 99 % sequence identity, with an amino acid sequence selected from SEQ ID NO: 28-32, 34-36, 38-48, 50-60, 62-64, 66-72, 79-81 , and 150-158.
  • said lysosomal polypeptide may be extended by one or more C- and/or N-terminal amino acid(s), making the actual lysosomal polypeptide sequence longer than in the sequence referred to above
  • the lysosomal protein may have an amino acid sequence which is shorter than the corresponding part of the relevant amino acid sequence referred to above, the difference in length e.g. being due to deletion(s) of amino acid residue(s) in certain position(s) of the sequence.
  • the fusion protein is isolated.
  • the lysosomal polypeptide is glycosylated.
  • the fusion proteins described herein can be produced by recombinant techniques using eukaryotic, such as mammalian (including human), expression systems using conventional methods known to persons of skill in the art. The examples below describe cloning and production of fusion proteins in which shielding polypeptide moieties are fused to biologically active polypeptides, represented by lysosomal proteins.
  • suitable cell lines for production of fusion proteins are known to persons of skill in the art, and examples include Pichia pastoris, Saccharomyces cerevisiae, algae, moss cells, plant cells such as carrot cells, and mammalian cells such as CHO, HEK-293, and HT1080.
  • said fusion protein is expressed in mammalian, Chinese hamster ovary, plant or yeast cells.
  • the resulting protein is thus glycosylated by one or more oligomannose N-glycans.
  • the fusion protein may be recombinantly produced in a continuous human cell line.
  • polypeptide moiety it may be advantageous to use synthetic genes which utilize the redundancy of the genetic code by including different, or all, codon variants for each amino acid that is to be encoded.
  • synthetic genes which utilize the redundancy of the genetic code by including different, or all, codon variants for each amino acid that is to be encoded.
  • the use of more variable DNA sequences may facilitate characterization of the nucleic acid
  • the shielding polypeptide moiety may provide increased solubility to the fusion protein.
  • the hydrophilic nature of the shielding polypeptide moiety may be beneficial in that it may increase the bioavailability of a fusion protein that is administered subcutaneously, relative to the bioavailability of the lysosomal polypeptide alone. In such cases, the increased solubility of the fusion protein may promote transfer to the blood stream rather than remaining in the tissue extracellular matrix after injection.
  • the fusion protein distributes to peripheral tissue when administered to a mammal.
  • peripheral tissue examples of peripheral tissue are given above.
  • the lysosomal protein may display biologic activity, such as retained enzymatic or catalytic activity, in said peripheral tissue.
  • the fusion protein according to aspects described herein may distribute to the brain when administered to a mammal, and may also display (retained) biological activity, such as retained enzymatic or catalytic activity, in the brain of said mammal. In one embodiment, the fusion protein has enzymatic activity in the brain.
  • the fusion protein may retain at least 25 %, preferably at least 50 %, and more preferably at least 75 %, of a biological activity or effect from the lysosomal polypeptide as such.
  • the fusion should be carried out carefully. The position of fusion must not alter the functional epitope or the active site of the lysosomal polypeptide such that the resulting fusion protein becomes inactive.
  • the fusion protein as disclosed herein may affect lysosomal storage in the brain, visceral organs or peripheral tissue of mammals, such as to decrease lysosomal storage, for example lysosomal storage of lipids, GAGs, glycolipids, glycoprotein, amino acids or glycogen.
  • a fusion protein according to some embodiments may affect lysosomal storage in the brain, visceral organs or peripheral tissue of mammals, such as to decrease lysosomal storage, for example lysosomal storage of lipids, GAGs, glycolipids, glycoprotein, amino acids or glycogen.
  • embodiments of the invention had not only a fully retained biological activity, but in fact had an increased or improved biological activity, compared to the lysosomal polypeptide as such.
  • an increased biological activity was observed for fusion proteins of iduronidase and iduronate-2- sulfatase, respectively, with shielding polypeptide moieties.
  • an increased biological activity may be due to increased solubility of the fusion protein and/or improved post-translational modification of the fusion protein, resulting in the production of a higher quality and/or more“activated” protein.
  • the provision of a highly active protein offers a possibility of
  • composition comprising a fusion protein where a lysosomal polypeptide is fused to one or several shielding
  • compositions may preferably be a pharmaceutical composition, suitable for administration to a patient (e.g. a mammal) for example by injection or orally.
  • the pharmaceutical composition may be formulated for any route of administration, including intravenous, subcutaneous, nasal, oral, and topical administration.
  • the pharmaceutical composition may be formulated, or intended to be formulated, for intravenous or subcutaneous administration.
  • composition aspect also applies to the composition aspect.
  • embodiments disclosed for other aspects also apply to the composition aspect.
  • embodiments related to content of glycan moieties, protein activity, and particular examples of lysosomal proteins are applicable also to this aspect.
  • the fusion protein of the invention may be used in a method of treatment of a lysosomal storage disease, comprising the step of administering to a patient suffering from a lysosomal storage disease a fusion protein comprising a lysosomal polypeptide fused to a shielding polypeptide moiety as described herein.
  • the patient is typically a mammal, such as a human.
  • the administering may be effected by intravenous infusion of a duration of at least 5 minutes, such as at least 15 minutes, and up to 6 hours, such as 4 hours or up to 2 hours.
  • administration may occur at a frequency of once daily to once monthly, for instance once weekly.
  • Example 1A Identification of repeating units of human origin
  • a blast search was performed with the catalytic domain of Bile salt-stimulated lipase (BSSL) versus the non-redundant protein sequence database at the National Institute of Health (NIH), USA and identified 10 reported protein sequences for the protein of human origin that contained the whole or part of the C-terminal repetitive unstructured domain.
  • BSSL Bile salt-stimulated lipase
  • Blast at NIH was used to search for proteins of human origin that match the catalytic domain of Bile salt stimulated lipase with UniProt ID P19835
  • the BLAST search resulted in finding 10 entries that contained both a significant portion of the catalytic domain and the C-terminal repetitive unstructured domain.
  • the number of the repeating units in the domains differed and some variability among the sequence of the repeating units was noted, see Table 1.1 for the different hits.
  • Each repeating domain is initiated by a truncated sequence of 9 residues, while the most prevalent repeating units are 11 residues long. In the table below, the repeating units are separated by a " ⁇ " sign for clarity. In the enclosed sequence listing, the repetitive portions are represented by SEQ ID NOs: 12-19. Table 1.1. Variants of human BSSL-CTD
  • Table 1.2 lists the unique sequences of repeating units of human origin, with reference to the sequence identity number in the enclosed sequence listing. Absent residues of the first sequence are marked by a dash. Table 1 .2. Units corresponding to repeating units found in human BSSL-CTD.
  • the most prevalent human form is made up of the combination of the following sequence of repeating units:[SEQ ID NO: 2] - [SEQ ID NO: 3] - [SEQ ID NO: 4] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 6] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO: 5] - [SEQ ID NO:
  • Example 1B Design of repeating units lacking O-glycosylation sites
  • This example describes sequences of repeating units without O-glycan sites in the sequence.
  • SEQ ID NO: 1 By utilizing the variable positions in SEQ ID NO: 1 the following sequences were designed that lacked serine or threonine that could be O-glycosylated during cultivation in eukaryotic expression systems such as CHO, HEK or yeast. The sequences are listed in Table 1.3 below. Table 1.3. Units corresponding to the general formula in SEQ ID NO: 1 without serine or threonine present.
  • Example 2 Expression and purification of N-sulfoglucosamine sulfohydrolase (sulfamidase) fusion proteins with the shielding polypeptide
  • DNA constructions DNA sequences encoding a set of fusion proteins including shielding polypeptide moieties were codon optimized for expression in Chinese hamster ovary (CHO) cells and synthesized by the Invitrogen GeneArt Gene Synthesis service at Thermo Fisher Scientific. The genes were cloned into expression vectors suitable for expression in mammalian cells. The encoded proteins are presented in Table 2.
  • Recombinant proteins were expressed using the ExpiCHO expression system (Thermo Fisher Scientific), essentially according to the manufacturer’s protocol.
  • ExpiCHO cells were transfected with expression vectors with synthetic genes for the encoded proteins listed in Table 2. Supernatants were harvested by centrifugation 6-9 days after transfection of expression vectors and stored at -70°C.
  • shielding polypeptide moiety is abbreviated“SPM”.“SPM1 - 3” denotes a shielding polypeptide moiety formed of the first three units (units).
  • “SPM1 -5” denotes a shielding polypeptide moiety formed of the first five units (units 1 -5) of a repetitive human BSSL-CTD sequence
  • “SPM1 -17” denotes a shielding polypeptide moiety formed of the first 17 units (units 1 -17) of a repetitive human BSSL-CTD sequence
  • “SPM4-12” denotes a shielding
  • polypeptide moiety formed of the fourth to the twelfth unit (units 4-12) of a repetitive human BSSL-CTD sequence.
  • Lane A is molecular weight marker SeeBlue plus 2 (Thermo Fisher Scientific)
  • lane B is sulfamidase-SPM1 -17 (PSI0438)
  • lane C is SPM1 -5-Sulfamidase-SPM1 -17 (PSI0618)
  • lane D is SPM1 -5-Sulfamidase-SPM4-12 (PSI0619)
  • lane E is SPM4-12-Sulfamidase-SPM4-12 (PSI0620)
  • lane F is sulfamidase-SPM4-12 (PSI0518)
  • lane G is sulfamidase (PSI0222).
  • Fusion proteins of sulfamidase with shielding polypeptide moieties can be produced by construction of synthetic genes, followed by expression in mammalian cell systems and purification to high purity using conventional techniques.
  • sulfohydrolase sulfamidase
  • This example describes the biophysical characterization of fusion proteins of sulfamidases with shielding polypeptide moieties, using unfused sulfamidase as reference.
  • Apparent size, molecular mass and Stokes radius in solution were determined by SEC/MALS, and aggregation propensity as a function of increased temperature by static light scattering (SLS).
  • the apparent size of the fusion proteins and unfused proteins in solution was assessed by analytical SEC on an AKTA Micro (GE Healthcare Life Sciences) using a calibrated Superdex 200 Increase 3.2/300 column (GE Healthcare Life Sciences).
  • the column was calibrated with Gel Filtration Calibration Kit LMW (code no. 28-4038-41 , GE Healthcare Life Sciences) and Calibration Kit HMW (code no. 28-4038-42, GE Healthcare Life Sciences), containing 8 globular proteins in the size range of 6 to 669 kDa and Blue Dextran 2000, using a running buffer of 25 mM NaP and 125 mM NaCI, pH 7.0 and a flow rate of 75 mI/min at a temperature of 25 °C.
  • the corresponding size and hydrodynamic radius in solution can be calculated from the elution volume of a protein on a calibrated column by the methods described in appendix 10 of Handbook of Size Exclusion Chromatography Principles and Methods (order no 18-1022-18, GE Healthcare Life Sciences).
  • the molecular mass of the proteins was determined by MALS using a static light scattering detector miniDAWN TriStar and the Astra 5 software (Wyatt Technology Europe, Germany) connected to an Akta Micro (GE Healthcare Life Sciences) with a Superdex 200 Increase 3.2/300 column (GE Healthcare Life Sciences) using a column temperature of 25 °C, a running buffer of PBS, pH 7.4 and a flow rate of 75 mI/min.
  • Aggregation propensity as function of increased temperature was determined by static light scattering (SLS) using UNcle (Unchained Labs). Thermal aggregation was measured through 90 degree (SLS) at two different wavelengths (266 and 473 nm). The samples were diluted to a concentration of 1 mg/ml with 50 mM Arg, 75 mM NaCI, 2% sucrose pH 7.8 and
  • Table 3 lists the values obtained for apparent size, molecular mass and number of repeat units in the fusion protein.
  • Figure 3 shows the aggregation propensity (SLS counts) as function of increased temperature (°C).
  • SLS counts aggregation propensity
  • the apparent size in solution was larger than expected for globular proteins of similar masses. A correlation between number of repeat units in the fusion proteins and their size in solution was observed. The increase in apparent size in solution was independent of the position of shielding polypeptide - the apparent size in solution to the same extent. From the aggregation propensity analysis it can be concluded that unfused sulfamidase (i.e., without shielding polypeptide) aggregates at a lower temperature compared to the fusion proteins.
  • Example 4 Retained formyl glycine content in active site of sulfamidase fusion proteins with shielding polypeptides
  • Sulfamidase activity is dependent on post-translational conversion of cysteine in the active site of the protein (Cys 50) into a formyl glycine.
  • Determination of relative amount of formyl glycine in active site of the protein was performed by LC-MS analysis.
  • Sulfamidase and sulfamidase fusion proteins with shielding polypeptides (20 mg) were reduced, alkylated and digested with trypsin.
  • the protein was dissolved in a denaturing buffer (6 M Gua-HCI, 0.2 M Tris, 3mM EDTA, pH 8.3) during the reduction and alkylation steps. Reduction of the protein was done by incubation with DTT (10 mM) at room temperature (RT) for 1 h. Subsequent alkylation with iodoacetamide (55 mM) was performed at RT and in darkness for 45 min.
  • the relative formyl glycine content of sulfamidase fusion proteins with shielding polypeptides is in a range of 0.8-2.2 of sulfamidase.
  • the relative formyl glycine content of sulfamidase fusion proteins to sulfamidase is tabulated in Table 4.
  • the sulfamidase fusion proteins with shielding polypeptides display a similar formyl glycine content as sulfamidase.
  • the formyl glycine conversion in the active site is not compromised by the fusion of shielding polypeptides to sulfamidase in the fusion proteins.
  • Example 5 Reduced In vitro glycan receptor mediated cellular uptake of sulfamidase fusion proteins with shielding polypeptides
  • This example demonstrates cellular uptake of sulfamidase and sulfamidase fusion proteins and effect on uptake by inhibition of the M6PR by addition of excess M6P.
  • Mouse embryonic fibroblasts (MEF-1 ) were seeded the day before treatment with proteins.
  • the proteins were diluted in cell media in a concentration range between 2 and 15 nM for sulfamidase, and between 2 and 75 nM for the sulfamidase fusion proteins.
  • the cells were incubated approximately 24 hours, washed in PBS and harvested in lysis buffer with protease and phosphatase inhibitors.
  • the inhibition experiments with M6P were performed according to the same procedure as described above.
  • the concentrations of sulfamidase and of the sulfamidase fusion proteins tested were 2 and 7.5 nM.
  • the concentration range of M6P was 0.3 - 2.5 mM.
  • electrochemiluminescence counts were proportional to the amount of sulfamidase in the samples and evaluated against a relevant sulfamidase standard.
  • the cellular uptake of the sulfamidase fusion proteins is tabulated in Table 5.1 as % decreased uptake compared to sulfamidase without shielding polypeptide.
  • the cellular uptake in the presence of increasing concentration of M6P is listed in Table 5.2.
  • Table 5.2 Cellular uptake of sulfamidase and sulfamidase fusion proteins in MEF-1 cells in the presence of M6P.
  • the sulfamidase fusion proteins showed lower cellular uptake compared to sulfamidase alone.
  • For fusion proteins with C-terminal shielding polypeptides the number of repeat units correlated to reduced uptake.
  • fusion proteins with both N- and C-terminal shielding polypeptides the cellular uptake was further reduced compared to fusion proteins with C-terminal shielding polypeptides only.
  • the M6PR is indicated as the major endocytotic receptor for sulfamidase uptake in MEF-1 cells.
  • the low uptake of fusion proteins indicates that the shielding
  • polypeptides reduce the M6PR-mediated uptake of sulfamidase fusion proteins.
  • M6P the uptake of unfused protein can be lowered to a level close to that of the fusion proteins without any M6P addition.
  • the remaining cellular uptake for sulfamidase fusion proteins can be further reduced by inhibiting the M6PR.
  • Example 6 Shielding polypeptides interfere with sulfamidase binding to mannose-6-phosphate receptor
  • This example demonstrates the binding of sulfamidase fusion proteins to M6PR in vitro. Binding properties are compared to properties of unfused sulfamidase (i.e., without shielding polypeptide).
  • M6PR Interactions between sulfamidase fusion proteins and M6PR were analyzed by SPR technology using a Biacore T200.
  • the extracellular part of M6PR (human ciM6PR, R&D systems) was used as ligand and was immobilized by amine coupling on a CM5 Biacore chip.
  • Analyte concentrations were kept constant at 500 nM with an association phase of 3 min and a dissociation phase of 2.5 min.
  • M6PR was coupled to the chip with a total coupling response of 7967 RU. All sulfamidase fusion proteins showed interaction with immobilized M6PR although with a significantly reduced response magnitude as compared to sulfamidase. By the end of the 3 minute association phase binding had reached equilibrium and responses at this time point is found in Table 6. Table 6. Interactions between M6PR and sulfamidase proteins with shielding polypeptides using SPR technology
  • Fusion proteins of sulfamidase with shielding polypeptides show significantly reduced interaction with the M6PR receptor as compared to sulfamidase alone. Fusion of shielding polypeptides to both N- and C-terminus of sulfamidase is particularly beneficial for effective blocking of the M6PR interaction driven by the phosphorylated high mannose glycans of
  • Example 7 Fusion proteins of sulfamidase with shielding polypeptides show increased serum exposure and improved distribution to the brain in mice Material and methods
  • mice serum exposure and distribution to CNS of sulfamidase and fusion proteins of sulfamidase with shielding polypeptides were investigated in mice (C57BL/6J).
  • the proteins were produced as described in Example 2.
  • the mice were given an intravenous single dose administration in the tail vein of 10 mg/kg.
  • Sulfamidase and fusion proteins of sulfamidase with shielding polypeptides, respectively, were formulated at 2 mg/mL in 50 mM arginine, 75 mM NaCI and 20 mg/mL sucrose, pH 7.8 and administered at 5 mL/kg.
  • Two non-terminal blood samples were taken from vena saphena at different time points with 3 mice per time point.
  • mice were anaesthetized by isoflurane and blood was collected from the orbital plexus. Blood samples were allowed to clot at room temperature, centrifuged at 1200 xg at 4 °C for 10 min. Serum samples were stored -70 °C until bioanalysis. Time-points collected were 5 min, 30 min, 1 , 2 (terminal), 4, 8, 24 (terminal), 32 and 48 (terminal) hours after the dose with a total of nine mice per fusion protein. At termination and after the last blood sample was taken, animals were subjected to cardiac perfusion with cold 0.9% saline. Brain was dissected, weighed and frozen rapidly in dry ice-chilled isopentane.
  • Brain homogenates were prepared in buffer (29 mM diethylbarbituric acid, 29 mM sodium acetate, 0.68 % (w/v) NaCI, pH 6.5) using a Lysing Matrix D device (MP Biomedicals, LLC, Ohio, US). Homogenization was performed for 25 s in a Savant
  • the serum and brain homogenate levels of sulfamidase and sulfamidase fusion proteins were analyzed by an immunoassay using the Meso Scale Discovery (MSD) platform. Streptavidin coated MSD plates were blocked with 5% Blocker A in PBS. The plates were washed and different dilutions of standard and samples were distributed on the plate. A mixture of a
  • biotinylated anti-Sulfamidase mouse monoclonal antibody (mAB) and Sulfo- Ru-tagged rabbit anti-Sulfamidase antibodies was added and the plates were incubated at RT.
  • Complexes of sulfamidase and labelled antibodies bind to the streptavidin coated plate via the biotinylated mAb.
  • the amount of bound complexes was determined by adding a read buffer to the wells and the plates were read in a MSD SI2400 instrument.
  • the recorded electrochemiluminescence counts were proportional to the amount of sulfamidase in the samples and evaluated against a relevant sulfamidase standard.
  • the area under the serum concentration versus time curve extrapolated to infinity (AUC ⁇ ) corrected for dose was calculated using Phoenix WinNonlin software version 8 (Certara, U.S.A.) by non- compartmental analysis.
  • Fusion proteins of sulfamidase with shielding polypeptides show increased AUC ⁇ as compared to sulfamidase, see Figure 4 and Tables 7.1 and 7.2.
  • Figure 4 plots the mean and standard deviation serum concentration versus time following a 10 mg/kg i.v. administration in male C57BL/6 mice of sulfamidase (PSI0222 as filled boxes) and sulfamidase fusion proteins (PSI0438: open boxes; PSI0618: open circles; PSI0619: filled triangles).
  • the fusion proteins showed concentrations in brain homogenates higher than the limit of quantification obtained for sulfamidase (PSI0222) at least at 2 hours post dose, with SPM1 -5-Sulfamidase-SPM1 -17 (PSI0618) up to 24 h post dose and SPM1 -5-Sulfamidase-SPM4-12 (PSI0619) up to 48 h post dose.
  • PSI0222 SPM1 -5-Sulfamidase-SPM1 -17
  • SPM1 -5-Sulfamidase-SPM4-12 PSI0619 up to 48 h post dose.
  • Table 7.1 Serum exposure (AUC ) and mean concentration in brain homogenate following a 10 mg/kg i.v. administration of sulfamidase and sulfamidase fusion proteins at 2, 24 and 48 hours after the dose
  • BLOQ be ow LOQ 0.26 nM for sulfamidase based on average brain weight in this group. If two or more values were below LOQ, no mean is reported.
  • BLOQ below LOQ, 1.4 g/L for sulfamidase based on average brain weight in this group. If two or more values were below LOQ, no mean is reported.
  • Fusion proteins of sulfamidase with shielding polypeptides show increased AUC ⁇ as compared to sulfamidase alone, at least partly due to the inhibition of receptor mediated uptake in peripheral tissue. Fusion proteins containing sulfamidase and shielding peptides showed significant improved distribution to brain.
  • Example 8 Expression and purification of fusion proteins of iduronate 2- sulfatase (idursulfase) with shielding polypeptides
  • DNA constructions were done as in Example 2. The encoded proteins are presented in Table 8. Table 8. Iduronate 2-sulfatase (idursulfase) and idursulfase fusion proteins
  • ExpiCHO cells were transfected with expression vectors and cultured as described in Example 2. Purifications were performed as in Example 2. Purified fusion proteins were formulated in PBS pH 7.4. Identity of idursulfase in the fusion proteins was determined by peptide map LC-MS analysis for a subset of proteins.
  • fusion proteins expressed well in ExpiCHO and purifications resulted in protein preparations with high purity as assessed by SDS-PAGE.
  • the correct identity of idursulfase in the fusion proteins was confirmed by high sequence coverage peptide map mass spectrometry analysis for the following proteins: PSI0593, PSI0692, PSI0693, PSI0694, PSI0695, PSI0696 and PSI0697.
  • Fusion proteins of idursulfase with shielding polypeptide moieties can be produced by construction of synthetic genes, followed by expression in mammalian cell systems and purification to high purity using conventional techniques.
  • Example 9 Altered biophysical properties of idursulfase fusion proteins with shielding polypeptides compared to un fused idursulfase
  • biophysical characterization of fusion proteins of idursulfase are described, using unfused idursulfase (i.e., without shielding polypeptide) as reference.
  • Apparent size, molecular mass and Stokes radius in solution were determined by SEC/MALS.
  • Example 3 Performed as in Example 3. For determination of molecular size and mass for idursulfase-SPM1 -5 (PSI0692), idursulfase-SPM1 -17 (PSI0693), SPM1 -5- idursulfase-SPM1 -5 (PSI0694), SPM1 -17-idursulfase-SPM1 -17 (PSI0695), SPM1 -5-idursulfase (PSI0696) and SPM1 -17-idursulfase (PSI0697), a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences), calibrated as in Example 3, was used.
  • Table 9 lists the values obtained for apparent size and molecular mass and the number of repeat units in the fusion proteins.
  • Figure 5 shows the relationship between the apparent molecular weight in solution (y-axis; in kDa) of the fusion proteins and number of repeat units of the shielding polypeptide moiety (x-axis).
  • Table 9 Biophysical characterization of idursulfase and idursulfase fusion proteins
  • the apparent size in solution was larger than expected for globular proteins of similar masses. A correlation between number of repeat units in the fusion proteins and their size in solution was observed. The increase in apparent size in solution was independent of the position of the shielding polypeptide(s) (N-terminal or C-terminal or both) - the apparent size in solution increased to the same extent.
  • Example 10 Retained catalytic activity of fusion proteins of id ursu If ase with shielding polypeptides
  • Idursulfase activity is dependent on post-translational conversion of a cysteine (cys 84) in the active site of the protein into a formyl glycine.
  • the specific activity of the protein can be measured by the method described in this example.
  • Idursulfase enzymatic activity was assessed using a specific substrate 4- methylumbelliferyl-a-L-iduronate 2-sulphate at 0.1 mM in 0.1 M sodium acetate, pH 4.5 at 37°C (Voznyi et al 2001 , J Inherit Metab Dis 24:675-680).
  • Aldurazyme iduronidase was added in an excess at 5 mU/mL as a coupling enzyme to release 4-methylumbelliferone (4-MU). The reaction was typically stopped after 35 minutes using an excess of 0.5 M sodium carbonate at pH 10.7 and the end product 4-MU formed was measured utilizing fluorescence intensity at X355/E460 nm.
  • the activity of fusion proteins of idursulfase with shielding polypeptides was found to be in a range of 0.5- 1.7 relative to idursulfase and is tabulated in Table 10 below.
  • the idursulfase fusion proteins display a similar in vitro activity as idursulfase.
  • the in vitro activity is not compromised by the fusion of shielding polypeptides to idursulfase.
  • Table 10 In vitro activity of idursulfase fusion proteins relative to idursulfase activity
  • Example 11 Reduced in vitro glycan receptor-mediated cellular uptake of fusion proteins of iduronate-2-sulfatase (idursulfase) with shielding
  • This example demonstrates cellular uptake of idursulfase and idursulfase fusion proteins and the effect on uptake by inhibition of the M6PR by addition of excess M6P.
  • Example 5 Experiments were performed as in Example 5 apart from the concentration of added protein to the cells and the immunoassay used for determining amount of protein taken up by the cells. Concentration of added protein was between 2 and 7.5 nM and the concentration of M6P was 0.2-1.3 mM. Cell lysates were analyzed for idursulfase and idursulfase fusion protein content by an immunoassay using the Meso Scale Discovery (MSD) platform. Streptavidin coated MSD plates were blocked.
  • MSD Meso Scale Discovery
  • the plates were washed in PBS-T and a biotinylated goat anti-idursulfase polyclonal antibodies (pAb) was added to the wells and the plates were incubated at room temperature followed by wash in PBS-T. Different dilutions of standard and samples were distributed on the plates and the plates were incubated at room temperature followed by wash in PBS-T. Sulfo-Ru-tagged goat anti-idursulfase polyclonal antibodies was added and the plates were incubated at room temperature. Complexes of idursulfase and labelled antibodies bind to the streptavidin coated plate via the biotinylated pAb.
  • pAb biotinylated goat anti-idursulfase polyclonal antibodies
  • the amount of bound complexes was determined by adding a read buffer to the wells and the plates were read in a MSD SI2400 instrument.
  • the recorded electrochemiluminescence counts were proportional to the amount of idursulfase in the samples and evaluated against a relevant idursulfase standard.
  • Concentration of M6P in mM is shown on the x-axis; intracellular concentrations of idursulfase proteins in pM is shown on the y- axis.
  • concentration of M6P in mM is shown on the x-axis; intracellular concentrations of idursulfase proteins in pM is shown on the y- axis.
  • the idursulfase fusion proteins showed decreased cellular uptake compared to unfused idursulfase, as shown in Table 11. A correlation of decreased cellular uptake with increasing number of repeat units was found. As demonstrated by M6P inhibition of idursulfase uptake, the M6PR is indicated as the major endocytotic receptor for idursulfase uptake in MEF-1 cells. The low uptake of fusion proteins indicates that the shielding polypeptides reduce the M6PR-mediated uptake of idursulfase fusion proteins. The remaining cellular uptake for idursulfase fusion proteins can be further reduced by inhibiting the M6PR.
  • Example 12 Shielding polypeptides interfere with idursulfase binding to mannose-6-phosphate receptor
  • This example demonstrates the binding of idursulfase fusion protein to M6PR in vitro. Binding properties are compared to properties of unfused idursulfase (i.e., without shielding polypeptide).
  • M6PR was coupled to the chip with a total coupling response of 7967 RU.
  • the idursulfase fusion proteins showed interaction with immobilized M6PR although with a significantly reduced response magnitude as compared to idursulfase.
  • binding had reached equilibrium and responses at this time point are found in Table 12.
  • Table 12 Interactions between M6PR and idursulfase protein with shielding polypeptides using SPR technology
  • the fusion protein of idursulfase with shielding polypeptides shows significantly reduced interaction with the M6PR receptor as compared to idursulfase alone.
  • Example 13 Fusion proteins of idursulfase with shielding polypeptides show increased serum exposure and improved distribution to the brain in mice
  • serum exposure and distribution to CNS of idursulfase (SEQ ID NO: 37) and a fusion protein of idursulfase with shielding polypeptides (SEQ ID NO:46) were investigated in mice (C57BL/6J). Idursulfase and fusion protein were produced as described in Example 2. The mice were given a single intravenous dose of 10 mg/kg in the tail vein.
  • Idursulfase or a fusion protein of idursulfase with shielding polypeptides were formulated at 2 mg/mL in 50 mM arginine, 75 mM NaCI and 20 mg/mL sucrose, pH 7.8 and administered at 5 mL/kg.
  • Two non-terminal blood samples were taken from the sublingual plexus at different time points with 3 mice per time point. At termination, the mice were anaesthetized by pentobarbital, blood was collected from the sublingual plexus and CSF was obtained by needle puncture of the cisterna magna.
  • Blood samples were allowed to clot at room temperature, centrifuged at 1200 x g at 4 °C for 10 min. Serum samples were stored -70 °C until bioanalysis. Time-points of blood sampling were 5 min, 30 min, 1 , 2 (terminal), 4, 8, 24 (terminal), 32 and 48 (terminal) hours after the dose with a total of nine mice per protein. At termination and after the last blood sample and the CSF sample were taken, animals were subjected to cardiac perfusion with cold 0.9% saline. Brain was dissected, weighed and snap frozen in an air-tight cryo tube in liquid nitrogen.
  • Brain homogenates were prepared in buffer (29 mM diethylbarbituric acid, 29 mM sodium acetate, 0.68 % (w/v) NaCI, pH 6.5) using tubes with Lysing Matrix D and a Homogenizer FastPrep-24TM 5G (MP Biomedicals, LLC, Ohio, US). The homogenates were subsequently centrifuged in an Eppendorf centrifuge 5417R at 10,000 ref at 10 °C for 10 min. and the supernatant was collected for concentration determinations.
  • MSD Streptavidin Gold plates were blocked, and samples or standards were incubated with polyclonal anti- idursulfase capture (BAF2449, R&D Systems) and detection (AF2449, R&D Systems, conjugated with MSD SULFO-TAG containing reutenium) antibodies. The following day the plates were washed with PBS-Tween, and read in a MSD QuickPlex SQ120 using electrochemiluminescence for detection. For the serum samples, 1 % mouse serum was added to the diluent (1 % fish gelatin in PBS-Tween) to compensate for matrix effects.
  • the recorded electrochemiluminescence counts were proportional to the amount of idursulfase in the samples and evaluated against the relevant idursulfase standard.
  • the area under the serum concentration versus time curve extrapolated to infinity (AUC ) corrected for dose was calculated using Phoenix WinNonlin software version 8 (Certara, U.S.A.) by non- compartmental analysis.
  • the fusion protein of idursulfase with shielding polypeptides showed an increased AUC ⁇ in serum as compared to idursulfase, see Figure 9 and Tables 13.1 and 13.2.
  • Figure 9 plots the mean and standard deviation serum concentration versus time following a 10 mg/kg i.v. administration in male C57BL/6 mice of (PSI0593, SEQ ID NO: 37, as filled boxes) and the idursulfase fusion protein (PSI0695, SEQ ID NO: 46, open boxes). Further, the idursulfase fusion protein (PSI0695) showed concentrations in CSF and brain homogenates higher than those obtained for idursulfase (PSI0593).
  • a fusion protein of idursulfase with shielding polypeptides showed an increased AUC ⁇ in serum as compared to idursulfase alone, at least partly due to the inhibition of receptor mediated uptake in peripheral tissue.
  • the fusion protein containing idursulfase and shielding peptides showed significant improved distribution to CSF and brain.
  • Example 14 Expression and purification of fusion proteins of arylsulfatase A (ASA) with shielding polypeptides
  • DNA constructions were done as in Example 2. The encoded proteins are presented in Table 12.
  • ExpiCHO cells were transfected with expression vectors and cultured as described in Example 2. Purifications were performed as in Example 2. Purified fusion proteins were formulated in 25 mM NaP, 125 mM NaCI, pH 7.0.
  • Fusion proteins of ASA with shielding polypeptide moieties can be produced by construction of synthetic genes, followed by expression in mammalian cell systems and purification to high purity using conventional techniques.
  • Example 15 Altered biophysical properties of ASA fusion proteins with shielding polypeptides compared to ASA
  • This example describes the biophysical characterization of fusion proteins of ASA with shielding polypeptide moieties, using unfused ASA as reference. Apparent size, molecular mass and Stokes radius in solution were determined by SEC/MALS. Aggregation propensity was determined by backlight scattering.
  • Example 3 Performed as in Example 3 with the exception that a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences), calibrated as in example 3, was used. Aggregation propensity as function of increased temperature was determined by backlight scattering using Prometheus NT.48 (NanoTemper). The samples were diluted to a concentration of 1 mg/ml in 15 ml using 25 mM NaP, pH 7.0; 125 mM NaCI and temperature was ramped from 20 to 90 °C at 2 °C/min.
  • a Superdex 200 Increase 10/300 GL column GE Healthcare Life Sciences
  • Aggregation propensity as function of increased temperature was determined by backlight scattering using Prometheus NT.48 (NanoTemper). The samples were diluted to a concentration of 1 mg/ml in 15 ml using 25 mM NaP, pH 7.0; 125 mM NaCI and temperature was ramped from 20 to 90 °C at 2 °C/min.
  • FIG. 10 shows the normalised scattering signal as function of increased temperature (°C).
  • arylsulfatase A (PSI0590, SEQ ID NO: 49) is represented by solid black line, whereas arylsulfatase A fusion proteins (PSI0681 ,
  • PSI0683, PSI0685, PSI0687, PSI0689, PSI0691 are represented each by a dashed line.
  • the apparent size in solution was larger than expected for globular proteins of similar masses. A correlation between number of repeat units in the fusion proteins and their size in solution was observed. The increase in apparent size in solution was independent of the position of the shielding polypeptides (the N-terminus, C-terminus or both) - the apparent size in solution increased to the same extent. From the aggregation propensity analysis it can be concluded that unfused arylsulfatase A (i.e., without shielding polypeptide) aggregates at a lower temperature compared to the fusion proteins.
  • Example 16 Retained catalytic activity of fusion proteins of ASA with shielding polypeptides
  • ASA activity is dependent on post-translational conversion of a cysteine (Cys 69) in the active site of the protein into a formyl glycine.
  • the specific activity of the protein can be measured by the method described in this example.
  • ASA enzymatic activity was assessed using para-nitrocatechol sulfate as substrate at 25 mM in 50 mM sodium acetate, pH 4.5 at 0°C. The reaction was typically stopped after 90 minutes using an excess of 0.5 M sodium carbonate at pH 10.7 and an end product nitrocatechol formed was measured utilizing absorbance at 515 nm, which is essentially based on Lee-Vaupel and Conzelmann 1987, Clin Chim Acta 164:171-180.
  • ASA fusion proteins The activity of ASA fusion proteins is in a range of 0.5-1.2 of ASA and is tabulated in Table 14.
  • the ASA fusion proteins display a similar in vitro activity as ASA.
  • the in vitro activity is not compromised by the fusion of shielding polypeptides to ASA.
  • Example 17 Reduced in vitro glycan receptor mediated cellular uptake of fusion proteins of arylsulfatase A (ASA) with shielding polypeptides
  • ASA arylsulfatase A
  • the ASA fusion proteins showed decreased cellular uptake compared to unfused ASA as shown in Table 15. The decrease in cellular uptake of the ASA fusion proteins was similar for all fusion proteins tested.
  • the fusion of shielding polypeptide(s) to ASA resulted in decreased cellular uptake of the proteins compared to unfused ASA. All fusion proteins showed similarly low cellular uptake, irrespective of the number of repeat units or their position (N- or C-terminal, or both).
  • Example 18 Expression and purification of fusion proteins of alpha-L-
  • DNA constructions were done as in Example 2. The encoded proteins are presented in Table 16. Table 16. Iduronidase and iduronidase fusion protein sequences.
  • ExpiCHO cells were transfected with expression vectors and cultured as described in Example 2. Purifications were performed as in Example 2. Purified fusion proteins were formulated in PBS, pH 7.4.
  • Fusion proteins of iduronidase with shielding polypeptide moieties can be produced by construction of synthetic genes, followed by expression in mammalian cell systems and purification to high purity using conventional techniques.
  • Example 19 Altered biophysical properties of fusion proteins of iduronidase with shielding polypeptides compared to iduronidase
  • This example describes the biophysical characterization of fusion proteins of iduronidase with shielding polypeptide moieties using unfused iduronidase (i.e., without shielding polypeptide) as reference. Apparent size, molecular mass and Stokes radius in solution were determined by SEC/MALS.
  • Example 3 Performed as in Example 3 with the exception that the unfused iduronidase was run at 0.58 mg/ml and a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences), calibrated as in example 3, was used. Aggregation propensity as function of increased temperature was determined by backlight scattering using Prometheus NT.48 (NanoTemper). The samples were diluted to a concentration of 1 mg/ml in 15 ul using 25 mM NaP, pH 7.0; 125 mM NaCI and temperature was ramped from 20 to 90 °C at 2 °C/min. Results
  • FIG. 11 shows the normalised scattering signal as function of increased temperature (°C).
  • iduronidase PSI0754, SEQ ID NO: 61
  • iduronidase fusion proteins PSI0753, SEQ ID NO: 62; PSI0755, SEQ ID NO: 63; PSI0756, SEQ ID NO: 64
  • PSI0753, SEQ ID NO: 62; PSI0755, SEQ ID NO: 63; PSI0756, SEQ ID NO: 64 are represented each by a dashed line.
  • the apparent size in solution was larger than expected for globular proteins of similar masses. A correlation between number of repeat units of the shielding polypeptide moieties of the and their size in solution was observed. The increase in apparent size in solution was independent of the position of the shielding polypeptide (N-terminus, C-terminus, or both) - the apparent size in solution increased to the same extent. From the aggregation propensity analysis it can be concluded that unfused iduronidase (i.e., without shielding polypeptide) aggregates at a lower temperature compared to the fusion proteins.
  • Example 20 Retained catalytic activity of fusion proteins of iduronidase with shielding polypeptides
  • the specific activity of iduronidase was measured by the method described in this example.
  • Iduronidase enzymatic activity was assessed using 4-methylumbelliferyl a-L- iduronide as substrate (Isemura et al 1978, J Biochem 84:627-632) at 0.1 mM in 0.2 M sodium formate, pH 3.5 at 37°C. The reaction was typically stopped after 20 minutes using an excess of 0.5 M sodium carbonate at pH 10.7 and an end product 4-MU formed was measured utilizing fluorescence intensity at X355/E460 nm. Results
  • iduronidase fusion proteins were in the range of 1.5-1.9 of iduronidase and are tabulated in Table 18.
  • the iduronidase fusion proteins display a similar in vitro activity as
  • iduronidase The in vitro activity was not compromised by the fusion of shielding polypeptides to iduronidase.
  • Example 21 Shielding polypeptides interfere with iduronidase binding to mannose-6-phosphate receptor
  • This example demonstrates the binding of iduronidase fusion proteins to M6PR in vitro. Binding properties are compared to properties of unfused iduronidase (i.e., without shielding polypeptide).
  • M6PR was coupled to the chip with a total coupling response of 7967 RU. All iduronidase fusion proteins showed interaction with immobilized M6PR although with a significantly reduced response magnitude as compared to iduronidase. By the end of the 3 minutes association phase binding had reached equilibrium and responses at this time point are found in Table 21.
  • Fusion proteins of iduronidase with shielding polypeptides show significantly reduced interaction with the M6PR receptor as compared to iduronidase alone. Fusion of shielding polypeptides to both N- and C-terminus of iduronidase blocks the M6PR interaction more efficiently than fusion of shielding polypeptides to either N- or C-terminus.
  • Example 22 Reduced In vitro glycan receptor mediated cellular uptake of iduronidase fusion proteins with shielding polypeptides
  • This example demonstrates effect on cellular uptake by iduronidase fusion proteins compared to iduronidase.
  • Mouse embryonic fibroblasts (MEF-1 ) were seeded the day before treatment with proteins.
  • the proteins were diluted in cell media in a concentration range between 78 pM and 10 nM.
  • the cells were incubated approximately 24 hours, washed in PBS and harvested in lysis buffer with protease and phosphatase inhibitors.
  • Cell lysates were analyzed for iduronidase and iduronidase fusion protein content by an immunoassay using the Meso Scale Discovery (MSD) platform. Streptavidin coated MSD plates were blocked with 5% Blocker A in PBS. The plates were washed in PBS-T and different dilutions of standard and samples were distributed on the plate.
  • MSD Meso Scale Discovery
  • a mixture of a biotinylated anti- iduronidase rabbit polyclonal antibody and Sulfo-Ru-tagged rabbit anti- idurondase antibodies were added and the plates were incubated at room temperature.
  • Complexes of iduronidase and labelled antibodies bind to the streptavidin coated plate via the biotinylated polyclonal antibody.
  • the amount of bound complexes was determined by adding a read buffer to the wells and the plates were read in a MSD SI2400 instrument. The recorded electrochemiluminescence counts were proportional to the amount of iduronidase in the samples and evaluated against a relevant iduronidase standard.
  • the cellular uptake of the iduronidase fusion proteins is tabulated in Table 23.1 as % decreased uptake compared to iduronidase without shielding polypeptide.
  • DNA constructions were done as in Example 2. The encoded proteins are presented in Table 19.
  • ExpiCHO cells were transfected with expression vectors and cultured as described in Example 2. Purifications were performed as in Example 2. Purified fusion protein was formulated in PBS pH 7.4
  • FIG. 7 shows the resulting cell media harvest of GalN6S and GalN6S fusion proteins, analyzed by SDS-PAGE.
  • Lane A Molecular weight marker SeeBlue plus 2 (Thermo Fisher Scientific)
  • lane B GalN6S (PSI0592)
  • lane C GalN6S-SPM1 -5 (PSI0777)
  • lane D GalN6S-SPM1 -17 (PSI0778)
  • lane E SPM1 -5-GalN6S-
  • Fusion proteins of GalN6S with shielding polypeptide moieties can be expressed in mammalian cell systems.
  • Example 24 Expression and purification of fusion protein of arylsulfatase B (ASB) with shielding polypeptide
  • DNA constructions DNA construction was carried out as set out in Example 2. The encoded protein sequence is presented in Table 20.
  • ASB and ASB fusion protein Cultivation and purification ExpiCHO cells were transfected with expression vector and cultured as described in Example 2. Purifications were performed as in Example 2. Purified fusion protein was formulated in PBS, pH 7.4.
  • FIG. 8 shows the resulting cell media harvest of ASB fusion protein, analyzed by SDS-PAGE and compared to ASB (Galsulfase; purchased).
  • Lane A Molecular weight marker SeeBlue plus 2 (Thermo Fisher Scientific)
  • lane B Galsulfase
  • lane C SPM1 -17-ASB (PSI0746). Star indicates produced protein of interest in harvest media.
  • ASB fusion proteins with shielding polypeptide moieties can be expressed in mammalian cell systems.
  • Example 25 Expression of fusion protein of galactocerebrosidase (GALC) with shielding polypeptide
  • DNA construction was done as in Example 2. The encoded protein sequences are presented in Table 20. Table 21 . GALC fusion protein
  • ExpiCHO cells were transfected with expression vectors and cultured as described in Example 2.
  • Figure 12 shows the resulting cell media harvest of GALC fusion protein.

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Abstract

L'invention concerne une protéine de fusion, comprenant i) un polypeptide lysosomal et ii) un fragment polypeptidique d'extension de 2 à 68 unités, chaque unité étant indépendamment sélectionnée dans le groupe formé par toutes les séquences d'acides aminés en fonction de SEQ ID NO:1 : dans laquelle, indépendamment : X1 représente P ou est absent ; X2 représente V ou est absent ; X3 représente P ou T ; X4 représente P ou T ; X5 représente T ou V ; X6 représente D, G ou T ; X8 représente A, Q ou S ; X9 représente E, G ou K ; X10 représente A, E, P ou T ; et X11 représente A, P ou T. La protéine de fusion présente une distribution améliorée au tissu cible et est utile dans le traitement par enzyme de substitution d'une maladie lysosomale.
PCT/EP2019/059675 2018-04-16 2019-04-15 Protéine de fusion lysosomale WO2019201855A1 (fr)

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Citations (2)

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