WO2003032727A1 - Techniques et compositions permettant le ciblage de proteines sous-glycosylees a travers la barriere hemato-encephalique - Google Patents

Techniques et compositions permettant le ciblage de proteines sous-glycosylees a travers la barriere hemato-encephalique Download PDF

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Publication number
WO2003032727A1
WO2003032727A1 PCT/US2002/032968 US0232968W WO03032727A1 WO 2003032727 A1 WO2003032727 A1 WO 2003032727A1 US 0232968 W US0232968 W US 0232968W WO 03032727 A1 WO03032727 A1 WO 03032727A1
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igf
therapeutic
protein
targeting
fusion protein
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PCT/US2002/032968
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English (en)
Inventor
Jonathan H. Lebowitz
Wilian S. Sly
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Symbiontics Inc.
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Priority claimed from US10/136,639 external-priority patent/US20030072761A1/en
Priority claimed from US10/136,841 external-priority patent/US7396811B2/en
Application filed by Symbiontics Inc. filed Critical Symbiontics Inc.
Priority to EP02801739A priority Critical patent/EP1446007A4/fr
Priority to JP2003535542A priority patent/JP2005506340A/ja
Priority to AU2002362930A priority patent/AU2002362930A2/en
Priority to IL16135202A priority patent/IL161352A0/xx
Priority to CA002463473A priority patent/CA2463473A1/fr
Publication of WO2003032727A1 publication Critical patent/WO2003032727A1/fr

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention provides a means for specifically delivering proteins to the brain.
  • the ability to target proteins to the brain is of great utility in the treatment of neurological diseases.
  • Methods and compositions of the invention are useful to target proteins to cells across the blood brain barrier, and in particular, to target proteins to the lysosomes of cells in the CNS, including neuronal cells, macrophage cells, and other cell types.
  • the invention provides methods and compositions to deliver therapeutically useful proteins to treat lysosomal storage diseases ("LSDs") that affect the CNS.
  • LSDs lysosomal storage diseases
  • the blood-brain barrier maintains a homeostatic environment in the central nervous system (CNS).
  • CNS central nervous system
  • the capillaries that supply the blood to the brain have tight junctions which block passage of most molecules through the capillary endothelial membranes. While the membranes do allow passage of lipid soluble materials, water soluble materials such as glucose, proteins and amino acids do not pass through the blood brain barrier.
  • Mediated transport mechanisms exist to transport glucose and essential amino acids across the blood brain barrier. Active transport mechanisms remove molecules which become in excess, such as potassium, from the brain.
  • the blood brain barrier impedes the delivery of drugs to the CNS.
  • Many neurological diseases result from cellular defects in the CNS.
  • many lysosomal storage diseases affect cells of the CNS and result in mild to serious neurological symptoms.
  • the ability to deliver therapeutic compositions to the CNS is an important aspect of an effective treatment for many diseases, including many lysosomal storage diseases.
  • the present invention provides general methods and compositions for targeting compositions from the blood stream to the brain or CNS.
  • an IGF moiety is used to target a molecule from the blood stream to the brain parenchyma on the other side of the blood brain barrier.
  • Preferred molecules are therapeutic polypeptides.
  • the invention relates in one aspect to a protein including a therapeutic agent attached to an insulin-like growth factor (IGF) moiety or tag.
  • the protein is expressed as a fusion protein along with the IGF tag.
  • the IGF tag does not include IGF-II or a portion thereof
  • the fusion protein also includes a lysosomal targeting portion sufficiently duplicative of IGF-II such that the targeting portion binds the cation independent mannose-6-phosphate/IGF-IJ. receptor to mediate uptake by a lysosome.
  • the fusion protein also comprises mannose-6-phosphate in order to target the protein to the lysosomes.
  • IGF moieties or tags are IGF-I or IGF-II tags. Most preferred IGF tags are
  • the IGF tag is an intact IGF-I or IGF-II protein.
  • an IGF tag is a portion of an IGF-I or IGF-II protein that is sufficient for targeting through the blood brain barrier.
  • Preferred portions comprise at least one of the A, B, C, or D domains, or the C- terminal region or a portion thereof, of either IGF-I or IGF-II.
  • an IGF tag includes both an A and a B domain.
  • the A and B domains provide core structural features of a preferred IGF moiety.
  • the A and B domains may be linked by a linker peptide.
  • the A and B domains may be provided as separate peptides that dimerize to form an IGF tag.
  • a and B domains from the same IGF protein are used.
  • compositions of the invention include chimeric IGF-I/IGF-II molecules.
  • an A domain from one IGF protein can be joined to the C and B domains of another IGF protein.
  • a domain of one IGF protein can be joined directly to the domain of another IGF protein, for example by using an amino acid bridge such as a two amino acid bridge.
  • a most preferred IGF moiety comprises an IGF-I portion selected from the group consisting of IGF-I fragments from about residue 1 to about residue 25, IGF-I fragments from about residue 25 to about residue 40, IGF-I fragments from about residue 40 to about residue 65, and IGF-I fragments from about residue 65 to about residue 70 of the IGF-I sequence shown in Figure 1.
  • Alternative preferred regions of IGF-I and IGF-II comprise regions of homology between IGF-I and IGF-II such as those shown in Figure 1 for human IGF-I and IGF-II.
  • the sequences shown in Figure 1 relate to mature IGF-I and IGF-II proteins. Specific IGF variants described herein refer to the mature amino acid sequence numbering shown in Figure 1.
  • an IGF tag comprises the C-terminal fragment of an IGF protein, for example the region C-terminal to the D domain shown in Figure 2.
  • a preferred IGF tag includes an IGF-I C-terminal fragment.
  • IGF tags include peptide tags with a sequence that is sufficiently duplicative of the IGF tags described herein to effectively target compositions of the invention to the brain parenchyma across the blood brain barrier.
  • an IGF tag includes at least one peptide sequence from an IGF-I protein and one from an IGF-II protein.
  • IGF tags are based on human IGF proteins. However, IGF tags based on IGF proteins from other mammals, such as mouse, rabbit, monkey, and pig IGF proteins, are also useful according to the invention. Preferred IGF tags such as the IGF fragments, peptides, or domains described herein are between 1 and 100 amino acids long, more preferably between
  • IGF fragments, peptides, or domains are based on the mature IGF-I and IGF-II sequences.
  • IGF tags of the invention can be fused to a therapeutic peptide at its N-terminus, C- terminus, within the body of the therapeutic peptide, or a combination of the above.
  • an IGF signal peptide is preferably included in the expression construct.
  • an IGF signal peptide can also be included at the N-terminus when the IGF targeting moiety is located at the C-terminus or within the body of the therapeutic protein.
  • the IGF tag is fused to the C- terminal end of a peptide.
  • a first domain of an IGF tag is fused to a therapeutic peptide, and a second domain of the IGF tag is provided in a form that dimerizes with the first domain resulting in a protein that is targeted to the brain.
  • the therapeutic peptide can be fused to the A domain of an IGF protein, and dimerized with a B domain that is provided separately.
  • the therapeutic peptide can be fused to the B domain of an IGF protein
  • IGF protein and dimerized with an A domain that is provided separately.
  • the invention also relates to methods for identifying IGF-based peptide fragments that can reach neuronal tissue from blood and are useful to target an associated protein to the brain or CNS.
  • the effectiveness of IGF-based tags can be assayed using methods described herein, such as localization assays based on radioactive labels or histochemical staining.
  • the invention also relates to a nucleic acid (e.g., a DNA molecule) encoding an IGF tag or a protein fused to an IGF tag, and to a cell (e.g., a cell cultured in vitro including a mammalian cell culture such as a CHO cell culture, and/or a unicellular organism such as E. coli or Leishmania) containing such a nucleic acid.
  • a nucleic acid e.g., a DNA molecule
  • a cell e.g., a cell cultured in vitro including a mammalian cell culture such as a CHO cell culture, and/or a unicellular organism such as E. coli or Leishmania
  • the invention relates to a method of producing a therapeutic agent for targeting across the blood brain barrier, and in particular to the lysosomes of cells in the
  • the agent is produced by culturing a cell expressing a nucleic acid encoding a protein containing both a therapeutic agent and an IGF tag effective to target the protein across the blood brain barrier.
  • the protein is then harvested (e.g. from the milieu about the cell, or by lysing the cell).
  • the invention also relates to protein compositions described herein.
  • the invention relates in one aspect to a targeted therapeutic including a targeting moiety and a therapeutic agent that is therapeutically active and preferably active in a mammalian lysosome.
  • a therapeutically active encompasses at least polypeptides or other molecules that provide an enzymatic activity to a cell or a compartment thereof that is deficient in that activity.
  • “Therapeutically active” also encompasses other polypeptides or other molecules that are intended to ameliorate or to compensate for a biochemical deficiency in a cell, but does not encompass molecules that are primarily cytotoxic or cytostatic, such as chemotherapeutics.
  • the targeting moiety is a means (e.g. a molecule) for binding the extracellular domain of the human cation-independent M6P receptor in an M6P-independent manner when the receptor is present in the plasma membrane of a target cell.
  • the targeting moiety is an unglycosylated lysosomal targeting domain that binds the extracellular domain of the human cation-independent M6P receptor, hr either embodiment, the targeting moiety can include, for example, IGF-II; retinoic acid or a derivative thereof; a protein having an amino acid sequence at least 70% identical to a domain of urokinase-type plasminogen activator receptor; an antibody variable domain that recognizes the receptor; or variants thereof.
  • the targeting moiety binds to an IGF receptor (e.g., an IGF-I or an IGF-II receptor) with a submicromolar dissociation constant (e.g.
  • the means for binding binds to the extracellular domain of a receptor at least 10-fold less avidly (i.e. with at least a ten-fold greater dissociation constant) at or about pH 5.5 than at or about pH 7.4; in one embodiment, the dissociation constant at or about pH 5.5 is at least 10 "6 M.
  • association of the targeted therapeutic with the means for binding is destabilized by a pH change from at or about pH 7.4 to at or about pH 5.5.
  • a targeting moiety retains IGF-U-like binding affinity for the IGF-II receptor, but has reduced binding affinity for the IGF-I receptor
  • the invention also relates to methods of treating a patient (e.g. a patient with a disorder in the CNS, and preferably a CNS disorder resulting from a lysosomal storage disorder) by administering, for example, a protein including a therapeutic agent effective in the mammalian CNS and an IGF tag to target the protein to the CNS.
  • a protein including a therapeutic agent effective in the mammalian CNS and an IGF tag to target the protein to the CNS.
  • the protein also comprises a lysosomal targeting portion such as those described in U.S. Serial No. 10/136,841, filed on April 30, 2002; attorney docket number SYM-007CP entitled "Targeted Therapuetic
  • the invention relates to methods of treating a patient by administering a nucleic acid encoding such a protein and/or by administering a cell (e.g. a human cell, or an organism such as Leishmania) containing a nucleic acid encoding such a protein.
  • a cell e.g. a human cell, or an organism such as Leishmania
  • This invention also provides methods for producing therapeutic proteins that are targeted to lysosomes and/or across the blood-brain barrier and that possess an extended half-life in circulation in a mammal.
  • the methods include producing an underglycosylated therapeutic protein.
  • underglycosylated refers to a protein in which one or more carbohydrate structures that would normally be present if the protein were produced in a mammalian cell (such as a CHO cell) has been omitted, removed, modified, or masked, thereby extending the half-life of the protein in a mammal.
  • a protein may be actually underglycosylated due to the absence of one or more carbohydrate structures, or functionally underglycosylated by modification or masking of one or more carbohydrate structures that promote clearance from circulation.
  • a structure could be masked (i) by the addition of one or more additional moieties (e.g. carbohydrate groups, phosphate groups, alkyl groups, etc.) that interfere with recognition of the structure by a mannose or asialoglycoprotein receptor,
  • glycoprotein by covalent or noncovalent association of the glycoprotein with a binding moiety, such as a lectin or an extracellular portion of a mannose or asialoglycoprotein receptor, that interferes with binding to those receptors in vivo, or (iii) any other modification to the polypeptide or carbohydrate portion of a glycoprotein to reduce its clearance from the blood by masking the presence of all or a portion of the carbohydrate structure.
  • a binding moiety such as a lectin or an extracellular portion of a mannose or asialoglycoprotein receptor
  • the therapeutic protein includes a peptide targeting moiety (e.g.
  • IGF-I IGF-II, or a portion thereof effective to bind a target receptor) that is produced in a host
  • proteins produced by the host cell may lack terminal mannose, fucose, and or N-acetylglucosamine residues, which are recognized by the mannose receptor, or may be completely unglycosylated.
  • the therapeutic protein which may be produced in mammalian cells or in other hosts, is treated chemically or enzymatically to remove one or more carbohydrate residues (e.g. one or more mannose, fucose, and/or N-acetylglucosamine residues) or to modify or mask one or more carbohydrate residues. Such a modification or masking may reduce binding of the therapeutic protein to the hepatic mannose and/or asialoglycoprotein receptors.
  • one or more potential glycosylation sites are removed by mutation of the nucleic acid encoding the targeted therapeutic protein, thereby reducing glycosylation of the protein when synthesized in a mammalian cell or other cell that glycosylates proteins.
  • Figure 1 shows a sequence alignment of mature human IGF-I (SEQ ID NO: 1) and IGF-II (SEQ ID NO: 2), indicating regions of homology and the A, B, C, and D domains.
  • Figure 2 is a two-dimensional representation of an IGF protein showing the signal sequence, the A, B, C, and D domains, and the C terminal sequence.
  • Figure 3 shows protein (Figure 3A, SEQ ID NO: 3) and nucleic acid ( Figure 3B, SEQ ID NO: 4) sequences for human IGF-I mRNA.
  • Figure 4A depicts one form of a phosphorylated high mannose carbohydrate structure linked to a glycoprotein via an asparagine residue, and also depicts the structures of mannose and N-acetylglucosamine (GlcNAc).
  • Figure 4B depicts a portion of the high mannose carbohydrate structure at a higher level of detail, and indicates positions vulnerable to cleavage by periodate treatment. The positions of the sugar residues within the carbohydrate structure are labeled with circled, capital letters A-H; phosphate groups are indicated with a circled capital P.
  • Figure 5 shows several types of underglycosylation.
  • an IGF moiety is useful for targeting a composition, preferably a protein composition, to the CNS, across the blood brain barrier.
  • a composition may enter the CNS or brain parenchyma either directly across the blood-brain barrier (the BBB) or indirectly across the blood-cerebrospinal fluid barrier (the BCB).
  • the BBB is formed by capillary endothelial cells and the BCB is formed by epithelial cells of the choroid plexus. Transport across either barrier typically involves transcytosis.
  • a composition that is targeted across the BCB to the CSF can subsequently reach the brain parenchyma.
  • the CSF and brain parenchyma are separated by the ependyma, and diffusion or bulk flow can transport substances between these two compartments.
  • the invention exploits, in part, the recognition that [1251] IGF-I and IGF-II can be detected in the brain when infused into the carotid artery, and that IGF-I and analogs administered subcutaneously can be found in the cerebrospinal fluid. According to the invention, this suggests that both can traverse the BBB or BCB. According to the invention, the observed saturation of the transport process suggests that the process is carrier mediated.
  • preferred therapeutic compositions include a therapeutic peptide fused to an IGF tag.
  • the IGF tag does not include
  • IGF-II or a portion thereof.
  • Preferred therapeutic compositions include IGF-I tags that will direct LSD (lysosomal storage disease) proteins to which they are fused across the blood brain harrier. In this instance, the tag will not necessarily direct the protein to the lysosome of multiple cell types. However, by expressing such fusion proteins in mammalian cell culture systems, the invention exploits the endogenous M6P signal for lysosomal localization and uses the IGF-I tag to traverse the blood brain barrier. In preferred embodiments of the invention, a human IGF-I tag is used. In alternative embodiments, methods and compositions of the invention involve using allelic, species or other sequence variants of an IGF-I tag.
  • Preferred sequence variants include mutations that lessen binding of the IGF tag to the IGF-I receptor and/or IGF binding proteins such as Leu 60 -IGF-I, or Leu 24 IGF-I which have diminished binding to the IGF-I receptor or ⁇ l-3 IGF-I which has diminished binding to IGF-binding proteins. Additional useful sequence variants include IGF-I variants with amino acid replacements of Arg 5 and Arg 6 .
  • IGF-II based tags are also useful to target proteins to the brain. IGF-II has been reported to be transported across the blood brain barrier via transcytosis (Bickel et al. (2001)
  • preferred IGF-IJ-based tags target proteins to the brain and also target proteins to the lysosome via receptor binding in order to treat neurological symptoms associated with lysosomal storage diseases.
  • IGF-II have an amino acid replacement at Leu 24 .
  • chimeric tags are used that include fragments of
  • IGF-I and IGF-II conferring preferred functional properties of each protein.
  • the retained portion of IGF-II includes regions of IGF-II known to be critical for binding to the
  • This embodiment is particularly useful where IGF-I is more active as a tag for traversing the blood brain barrier.
  • the tag has optimized activity for lysosomal targeting in addition to brain targeting.
  • a recombinant form of this embodiment could be made in any expression system.
  • a useful recombinant LSD protein includes any one of the different IGF-based lysosomal targeting tags described U.S. Serial No. 10/136,841, filed on April 30, 2002; attorney docket number SYM-007CP entitled "Targeted Therapuetic
  • recombinant proteins of the invention including IGF-II tags are expressed in a mammalian expression system such as a CHO cell expression system.
  • a mammalian expression system such as a CHO cell expression system.
  • the endogenous M6P signal added in the mammalian cell culture enhances the lysosomal targeting that may be provided by an IGF-II tag.
  • useful minimal IGF tags and variant IGF tags can be identified based on known IGF-I and IGF-II sequences by testing minimal or variant IGF fragments in a CNS localization assay such as one described herein.
  • a preferred IGF tag is sufficiently duplicative of IGF-I to be targeted to the brain, but has reduced binding affinity for the IGF-I receptor thereby removing the mitogenic properties of
  • an IGF tag does bind to the IGF-13 receptor in order to be targeted to lysosomes. Accordingly, in one embodiment, an IGF tag is based on the IGF-I sequence but includes two hydrophobic IGF-II residues at positions 54 and 55 instead of the IGF-I Arg residues at these positions.
  • NMR structures of IGF-II have been solved by two groups (see, e.g. , Protein Data Bank record IIGL).
  • the general features of the IGF-II structure are similar to IGF-I and insulin.
  • the A and B domains of IGF-II correspond to the A and B chains of insulin.
  • Secondary structural features include an alpha helix from residues 11-21 of the B region connected by a reverse turn in residues 22-25 to a short beta strand in residues 26-28.
  • Residues 25-27 appear to form a small antiparallel beta sheet; residues 59-61 and residues 26-28 may also participate in intermolecular beta-sheet formation.
  • IGF-II In the A domain of IGF-II, alpha helices spanning residues 42-49 and 53-59 are arranged in an antiparallel configuration perpendicular to the B-domain helix. Hydrophobic clusters formed by two of the three disulfide bridges and conserved hydrophobic residues stabilize these secondary structure features. The N and C termini remain poorly defined as is the region between residues 31-40. [0036] IGF-II binds to the IGF-U/M6P and IGF-I receptors with relatively high affinity and binds with lower affinity to the insulin receptor. IGF-II also interacts with a number if serum
  • the lysosomal targeting portion is a protein, peptide, or other moiety that binds the cation independent M6P/IGF-II receptor in a mannose-6-phosphate- independent manner.
  • this embodiment mimics the normal biological mechanism for uptake of LSD proteins, yet does so in a manner independent of mannose-6- phosphate.
  • LSD gene cassettes fusion proteins are created that can be taken up by a variety of cell types and transported to the lysosome.
  • DNA encoding a precursor IGF-II polypeptide can be fused to the 3' end of an LSD gene cassette; the precursor includes a carboxyterminal portion that is cleaved in mammalian cells to yield the mature IGF-II polypeptide, but the IGF-II signal peptide is preferably omitted (or moved to the 5' end of the LSD gene cassette).
  • This method has numerous advantages over methods involving glycosylation including simplicity and cost effectiveness, because once the protein is isolated, no further modifications need be made.
  • IGF-I and IGF-II share identical sequences and structures in the region of residues 48-50 yet have a 1000-fold difference in affinity for the IGF-II receptor.
  • the NMR structure reveals a structural difference between IGF-I and IGF-JI in the region of IGF-II residues 53-58 (IGF-I residues 54-59): the alpha-helix is better defined in IGF-II than in IGF-I and, unlike IGF-
  • IGF-II binds to repeat 11 of the cation-independent M6P receptor.
  • a minireceptor in which only repeat 11 is fused to the transmembrane and cytoplasmic domains of the cation-independent M6P receptor is capable of binding IGF-II (with an affinity approximately one tenth the affinity of the full length receptor) and mediating internalization of
  • the putative IGF-II binding site is a hydrophobic pocket believed to interact with hydrophobic amino acids of IGF-II; candidate amino acids of IGF-II include leucine 8, phenylalanine 48, alanine 54, and leucine 55.
  • constructs including larger portions of the cation-independent M6P receptor generally bind IGF-II with greater affinity and with increased pH dependence (see, for example, Linnell et al. (2001) J. Biol. Chem.
  • the binding surfaces for the IGF-I and cation-independent M6P receptors are on separate faces of IGF-II.
  • functional cation-independent M6P binding domains can be constructed that are substantially smaller than human IGF-II.
  • the amino terminal amino acids 1-7 and/or the carboxy terminal residues 62-67 can be deleted or replaced.
  • amino acids 29-40 can likely be eliminated or replaced without altering the folding of the remainder of the polypeptide or binding to the cation- independent M6P receptor.
  • a targeting moiety including amino acids 8-28 and 41-61 can be constructed. These stretches of amino acids could perhaps be joined directly or separated by a linker.
  • amino acids 8-28 and 41-61 can be provided on separate polypeptide chains.
  • Comparable domains of insulin, wliich is homologous to IGF-II and has a tertiary structure closely related to the structure of IGF-II, have sufficient structural information to permit proper refolding into the appropriate tertiary structure, even when present in separate polypeptide chains (Wang et al. (1991) Trends Biochem. Sci. 279-281).
  • amino acids 8-28, or a conservative substitution variant thereof could be fused to a therapeutic agent; the resulting fusion protein could be admixed with amino acids 41-61, or a conservative substitution variant thereof, and administered to a patient.
  • analogous fragments of IGF-I are useful in fusions with a therapeutic.
  • IGF-II and related constructs can be modified to diminish their affinity for IGFBPs, thereby increasing the bioavailability of the tagged proteins.
  • the amino acid sequence of human IGF-I, IGF-II, or a portion thereof affecting transport into the brain may be used as a reference sequence to determine whether a candidate sequence possesses sufficient amino acid similarity to have a reasonable expectation of success in the methods of the present invention.
  • variant sequences are at least 70% similar or 60% identical, more preferably at least 75% similar or 65% identical, and most preferably 80% similar or 70% identical to human IGF-I or IGF-II.
  • the candidate amino acid sequence and human IGF-I or IGF-II are first aligned using the dynamic programming algorithm described in Smith and Waterman (1981) J. Mol. Biol. 147:195-197, in combination with the BLOSUM62 substitution matrix described in Figure 2 of Henikoff and Henikoff (1992) PNAS 89:10915- 10919.
  • an appropriate value for the gap insertion penalty is -12
  • an appropriate value for the gap extension penalty is -4.
  • Computer programs performing alignments using the algorithm of Smith- Waterman and the BLOSUM62 matrix such as the GCG program suite (Oxford Molecular Group, Oxford, England), are commercially available and widely used by those skilled in the art.
  • a percent similarity score may be calculated.
  • the individual amino acids of each sequence are compared sequentially according to their similarity to each other. If the value in the BLOSUM62 matrix corresponding to the two aligned amino acids is zero or a negative number, the pairwise similarity score is zero; otherwise the pairwise similarity score is 1.0.
  • the raw similarity score is the sum of the pairwise similarity scores of the aligned amino acids. The raw score is then normalized by dividing it by the number of amino acids in the smaller of the candidate or reference sequences. The normalized raw score is the percent similarity. Alternatively, to calculate a percent identity, the aligned amino acids of each sequence are again compared sequentially.
  • the pairwise identity score is zero; otherwise the pairwise identity score is 1.0.
  • the raw identity score is the sum of the identical aligned amino acids. The raw score is then normalized by dividing it by the number of amino acids in the smaller of the candidate or reference sequences. The normalized raw score is the percent identity. Insertions and deletions are ignored for the purposes of calculating percent similarity and identity. Accordingly, gap penalties are not used in this calculation, although they are used in the initial alignment.
  • the known atomic coordinates of IGF-II can be provided to a computer equipped with a conventional computer modeling program, such as INSIGHTII, DISCONER, or
  • DELPHI commercially available from Biosym, Technologies Inc.
  • QUANTA commercially available from QUANTA
  • CHARMM commercially available from Molecular Simulations, Inc.
  • these and other software programs allow analysis of molecular structures and simulations that predict the effect of molecular changes on structure and on intermolecular interactions.
  • the software can be used to identify modified analogs with the ability to form additional intermolecular hydrogen or ionic bonds, improving the affinity of the analog for the target receptor.
  • the software also permits the design of peptides and organic molecules with structural and chemical features that mimic the same features displayed on at least part of an IGF surface that is sufficient for targeting to the CNS.
  • a preferred embodiment of the present invention relates to designing and producing a synthetic organic molecule having a framework that carries chemically interactive moieties in a spatial relationship that mimics the spatial relationship of the chemical moieties disposed on the amino acid sidechains which are identified as associated with CNS targeting as described herein.
  • CAVEAT searches a database, for example, the Cambridge Structural Database, for structures which have desired spatial orientations of chemical moieties (Bartlett et al. (1989) in "Molecular Recognition: Chemical and
  • compositions of the invention are useful for producing and delivering any therapeutic agent to the CNS, the invention is particularly useful for gene products that overcome enzymatic defects associated with lysosomal storage diseases.
  • Preferred LSD genes are shown in Table 1. hi a preferred embodiment, a wild-type
  • LSD gene product is delivered to a patient suffering from a defect in the same LSD gene.
  • a functional sequence or species variant of the LSD gene is used.
  • a gene coding for a different enzyme that can rescue an LSD gene defect is used according to methods of the invention.
  • the therapeutic agent is glucocerebrosidase, currently manufactured by Genzyme as an effective enzyme replacement therapy for Gaucher Disease.
  • the enzyme is prepared with exposed mannose residues, which targets the protein specifically to cells of the macrophage lineage.
  • the primary pathology in type 1 Gaucher patients are due to macrophage accumulating glucocerebroside, there may be therapeutic advantage to delivering glucocerebrosidase to other cell types.
  • Targeting glucocerebrosidase to lysosomes using the present invention would target the agent to multiple cell types and may have a therapeutic advantage compared to other preparations.
  • the therapeutic portion and the targeting portion of compositions of the invention are necessarily associated, directly or indirectly.
  • the therapeutic portion and the targeting portion are non-covalently associated.
  • the targeting portion could be biotinylated and bind an avidin moiety associated with the therapeutic portion.
  • the targeting portion and the therapeutic portion could each be associated (e.g. as fusion proteins) with different subunits of a multimeric protein.
  • the targeting portion and the therapeutic portion are crosslinked to each other (e.g. using a chemical crosslinking agent).
  • the therapeutic portion is fused to the targeting portion as , a fusion protein.
  • the targeting portion may be at the amino-terminus of the fusion protein, the carboxy-terminus, or may be inserted within the sequence of the therapeutic portion at a position where the presence of the targeting portion does not unduly interfere with the therapeutic activity of the therapeutic portion.
  • the therapeutically active moiety is a heteromeric protein
  • one or more of the subunits may be associated with a targeting portion.
  • Hexosaminidase A for example, a lysosomal protein affected in Tay-Sachs disease, includes an alpha subunit and a beta subunit.
  • Either the alpha subunit, or the beta subunit, or both may be associated with a targeting portion in accordance with the present invention. If, for example, the alpha subunit is associated with a targeting portion and is coexpressed with the beta subunit, an active complex is formed and targeted to the lysosome.
  • a protein of the invention can be targeted to the CNS, and preferably taken up by lysosomes, whether it is expressed and isolated from Leishmania, baculovirus, yeast, bacteria, mammalian or other expression systems.
  • the invention permits great flexibility in protein production.
  • a protein to be produced includes one or more disulfide bonds
  • an appropriate expression system can be selected and modified, if appropriate, to further improve yield of properly folded protein.
  • one useful IGF targeting portion has three intramolecular disulfide bonds. Fusion proteins of the invention expressed in E. coli may be constructed to direct the protein to the periplasmic space.
  • IGF tags when fused to the C-terminus of another protein, can be secreted in an active form in the periplasm of E. coli (Wadensten, Ekebacke et al. 1991).
  • appropriate concentrations of reduced and oxidized glutathione are preferably added to the cellular milieu to promote disulfide bond formation.
  • any insoluble material is preferably treated with a chaotropic agent such as urea to solubilize denatured protein and refolded in a buffer having appropriate concentrations of reduced and oxidized glutathione, or other oxidizing and reducing agents, to facilitate formation of appropriate disulfide bonds (Smith, Cook et al. 1989).
  • a chaotropic agent such as urea to solubilize denatured protein and refolded in a buffer having appropriate concentrations of reduced and oxidized glutathione, or other oxidizing and reducing agents, to facilitate formation of appropriate disulfide bonds
  • a chaotropic agent such as urea to solubilize denatured protein and refolded in a buffer having appropriate concentrations of reduced and oxidized glutathione, or other oxidizing and reducing agents, to facilitate formation of appropriate disulfide bonds
  • a chaotropic agent such as urea to solubilize denatured protein and refolded in a
  • an IGF fusion protein is expressed in a symbiotic or parasitic organism that is administered to a host.
  • the expressed IGF fusion protein is secreted by the organism into the blood stream and delivered across the blood brain barrier.
  • CNS targeted proteins are delivered in situ via live Leishmania secreting the proteins into the lysosomes of infected macrophage. From this organelle, it leaves the cell and may be delivered across the blood brain barrier. Thus, the IGF tag and the therapeutic agent necessarily remain intact while the protein resides in the macrophage lysosome. Accordingly, when proteins designed for delivery to lysosomes in the CNS,are expressed in situ, they are preferably modified to ensure compatibility with the lysosomal environment.
  • therapeutic proteins of the invention can be delivered by expression in T. brucei wliich can penetrate the BCB. Nucleic acids and expression systems
  • Chimeric fusion proteins of the invention can be expressed in a variety of expression systems, including in vitro translation systems and intact cells. Since M6P modification is not a prerequisite for targeting, a variety of expression systems including yeast, baculovirus and even prokaryotic systems such as E. coli that do not glycosylate proteins are suitable for expression of targeted therapeutic proteins. In fact, an unglycosylated protein generally has improved bioavailability, since glycosylated proteins are rapidly cleared from the circulation through binding to the mannose receptor in hepatic sinusoidal endothelium.
  • chimeric targeted lysosomal enzymes in mammalian cell expression system produces proteins with multiple binding determinants for the cation- independent M6P receptor. Synergies between two or more cation-independent M6P receptor ligands (e.g. M6P and IGF-II, or M6P and retinoic acid) can be exploited: multivalent ligands have been demonstrated to enhance binding to the receptor by receptor crosslinking.
  • gene cassettes encoding the chimeric therapeutic protein can be tailored for the particular expression system to incorporate necessary sequences for optimal expression including promoters, ribosomal binding sites, introns, or alterations in coding sequence to optimize codon usage. Because the protein is preferably secreted from the producing cell, a
  • DNA encoding a signal peptide compatible with the expression system can be substituted for the endogenous signal peptide.
  • a signal peptide compatible with the expression system can be substituted for the endogenous signal peptide.
  • DNA encoding a signal peptide compatible with the expression system can be substituted for the endogenous signal peptide.
  • ⁇ -glucuronidase and ⁇ -galactosidase for expression of ⁇ -glucuronidase and ⁇ -galactosidase
  • DNA cassettes encoding Leishmania signal peptides are inserted in place of the DNA encoding the endogenous signal peptide to achieve optimal expression.
  • the endogenous signal peptide may be employed but if the IGF-I or IGF-II tag is fused at the 5' end of the coding sequence, it could be desirable to use the IGF-I or IGF-II signal peptide.
  • CHO cells are a preferred mammalian host for the production of therapeutic proteins.
  • DHFR dihydrofolate reductase
  • plasmid pcDNA3 uses the cytomegalovirus (CMV) early region regulatory region promoter to drive expression of a gene of interest and pSV2DHFR to promote DHFR expression.
  • CMV cytomegalovirus
  • a preferred plasmid for eukaryotic expression in this system contains the gene of interest placed downstream of a strong promoter such as CMV. An intron can be placed in the 3' flank of the gene cassette.
  • a DHFR cassette can be driven by a second promoter from the same plasmid or from a separate plasmid. Additionally, it can be useful to incorporate into the plasmid an additional selectable marker such as neomycin phosphotransferase, which confers resistance to G418.
  • Another CHO expression system (Ulmasov et al. (2000) PNAS 97(26): 14212-14217) relies on amplification of the gene of interest using G418 instead of the DHFR/methotrexate system described above.
  • a pCXN vector with a slightly defective neomycin phosphotransferase driven by a weak promoter permits selection for transfectants with a high copy number (>300) in a single step.
  • recombinant protein can be produced in the human HEK 293 cell line using expression systems based on the Epstein-Barr Virus (EBV) replication system.
  • EBV Epstein-Barr Virus
  • This consists of the EBV replication origin oriP and the EBV ori binding protein, EBNA-1. Binding of EBNA-1 to oriP initiates replication and subsequent amplification of the extrachromosomal plasmid. This amplification in turn results in high levels of expression of gene cassettes housed within the plasmid. Plasmids containing oriP can be transfected into EBNA-1 transformed HEK
  • 293 cells commercially available from Invitrogen
  • a plasmid such as pCEP4
  • EBV oriP can be employed.
  • the therapeutic proteins are preferably secreted into the periplasmic space.
  • LSD protein a nucleic acid cassette encoding a bacterial signal peptide such as the ompA signal sequence. Expression can be driven by any of a number of strong inducible promoters such as the lac, trp, or tac promoters.
  • pBAD/glll commercially available from Invitrogen which uses the Gene III signal peptide and the araBAD promoter.
  • a nucleic acid encoding a therapeutic protein can be advantageously provided directly to a patient suffering from a disease, or may be provided to a cell ex vivo, followed by administration of the living cell to the patient.
  • In vivo gene therapy methods known in the art include providing purified DNA (e.g. as in a plasmid) , providing the DNA in a viral vector, or providing the DNA in a liposome or other vesicle (see, for example, U.S. Patent No. 5,827,703, disclosing lipid carriers for use in gene therapy, and U.S. Patent No. 6,281,010, providing adenoviral vectors useful in gene therapy).
  • Methods for treating disease by implanting a cell that has been modified to express a recombinant protein are also well known. See, for example, U.S. Patent No. 5,399,346, disclosing methods for introducing a nucleic acid into a primary human cell for introduction into a human. Although use of human cells for ex vivo therapy is preferred in some embodiments, other cells such as bacterial cells may be implanted in a patient's vasculature, continuously releasing a therapeutic agent. See, for example, U.S. Patent Nos. 4,309,776 and 5,704,910. [0075] Methods of the invention are particularly useful for targeting a protein directly to a subcellular compartment without requiring a purification step.
  • an IGF-II fusion protein is expressed in a symbiotic or attenuated parasitic organism that is administered to a host.
  • the expressed IGF-II fusion protein is secreted by the organism, taken up by host cells and targeted to their lysosomes.
  • IGF fusion proteins such as GILT proteins are delivered in situ via live Leishmania secreting the proteins into the lysosomes of infected macrophage. From this organelle, it leaves the cell and is taken up by adjacent cells not of the macrophage lineage. Thus, the IGF tag and the therapeutic agent necessarily remain intact while the protein resides in the macrophage lysosome. Accordingly, when GILT proteins are expressed in situ, they are preferably modified to ensure compatibility with the lysosomal environment.
  • Human ⁇ -glucuronidase (human "GUS"), an exemplary therapeutic portion, normally undergoes a C-terminal peptide cleavage either in the lysosome or during transport to the lysosome (e.g. between residues 633 and 634 in GUS).
  • human GUS is preferably modified to render the protein resistant to cleavage, or the residues following residue 633 are preferably simply omitted from a GILT fusion protein.
  • any IGF tag of the invention is preferably modified to increase its resistance to proteolysis, or a minimal binding peptide (e.g. as identified by phage display or yeast two hybrid) is substituted for the wildtype IGF moiety.
  • Targeted therapeutic proteins are preferably underglycosylated: one or more carbohydrate structures that would normally be present if the protein were produced in a mammalian cell is preferably omitted, removed, modified, or masked, extending the half-life of the protein in a mammal.
  • Underglycosylation can be achieved in many ways, several of which are diagrammed in Figure 5. As shown in Figure 5, a protein may be actually underglycosylated, actually lacking one or more of the carbohydrate structures, or functionally underglycosylated through modification or masking of one or more of the carbohydrate structures.
  • a protein may be actually underglycosylated when synthesized, as discussed in Example 12, and may be completely unglycosylated (as when synthesized in E. coli), partially unglycosylated (as when synthesized in a mammalian system after disruption of one or more glycosylation sites by site- directed mutagenesis), or may have a non-mammalian glycosylation pattern.
  • Actual underglycosylation can also be achieved by deglycosylation of a protein after synthesis. As discussed in Example 12, deglycosylation can be through chemical or enzymatic treatments, and may lead to complete deglycosylation or, if only a portion of the carbohydrate structure is removed, partial deglycosylation.
  • the targeted therapeutics produced according to the present invention can be administered to a mammalian host by any route.
  • administration can be oral or parenteral, including intravenous and intraperitoneal routes of administration, h addition, administration can be by periodic inj ections of a bolus of the therapeutic or can be made more continuous by intravenous or intraperitoneal administration from a reservoir which is external (e.g., an i.v. bag).
  • the therapeutics of the instant invention can be pharmaceutical-grade. That is, certain embodiments comply with standards of purity and quality control required for administration to humans.
  • Veterinary applications are also within the intended meaning as used herein.
  • the formulations, both for veterinary and for human medical use, of the therapeutics according to the present invention typically include such therapeutics in association with a pharmaceutically acceptable carrier therefor and optionally other ingredient(s).
  • the carrier(s) can be "acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
  • Pharmaceutically acceptable carriers are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
  • Supplementary active compounds (identified according to the invention and/or known in the art) also can be incorporated into the compositions.
  • the formulations can conveniently be presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy/microbiology. In general, some formulations are prepared by bringing the therapeutic into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include oral or parenteral, e.g., intravenous, intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Ph can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Useful solutions for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's
  • Formulations for parenteral administration also can include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Suppositories for rectal administration also can be prepared by mixing the drug with a non- irritating excipient such as cocoa butter, other glycerides, or other compositions that are solid at room temperature and liquid at body temperatures.
  • Formulations also can include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Formulations for direct admimstration can include glycerol and other compositions of high viscosity.
  • Other potentially useful parenteral carriers for these therapeutics include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation admimstration can contain as excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9- lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Retention enemas also can be used for rectal delivery.
  • Formulations of the present invention suitable for oral administration can be in the form of discrete units such as capsules, gelatin capsules, sachets, tablets, troches, or lozenges, each containing a predetermined amount of the drug; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.
  • the therapeutic can also be administered in the form of a bolus, electuary or paste.
  • a tablet can be made by compressing or moulding the drug optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared by compressing, in a suitable machine, the drug in a free-flowing form such as a powder or granules, optionally mixed by a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered drug and suitable carrier moistened with an inert liquid diluent.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients.
  • Oral compositions prepared using a fluid carrier for use as a mouthwash include the compound in the fluid carrier and are applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose
  • a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or sacchar
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF,
  • Parsippany NJ or phosphate buffered saline (PBS).
  • the composition can be sterile and can be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the therapeutic which can be in microcrystallme form, for example, in the form of an aqueous microcrystallme suspension.
  • Liposomal formulations or biodegradable polymer systems can also be used to present the therapeutic for both intra-articular and ophthalmic administration.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, gels, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pasts; or solutions or suspensions such as drops.
  • Formulations for topical administration to the skin surface can be prepared by dispersing the therapeutic with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap.
  • a dermatologically acceptable carrier such as a lotion, cream, ointment or soap.
  • useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal.
  • the composition can include the therapeutic dispersed in a fibrinogen-thrombin composition or other bioadhesive.
  • the therapeutic then can be painted, sprayed or otherwise applied to the desired tissue surface.
  • the agent can be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • hydroxypropylcellulose or fibrinogen/thrombin solutions can be used to advantage.
  • tissue-coating solutions such as pectin-containing formulations can be used.
  • inhalation of powder (self-propelling or spray formulations) dispensed with a spray can a nebulizer, or an atomizer can be used.
  • Such formulations can be in the form of a finely comminuted powder for pulmonary administration from a powder inhalation device or self-propelling powder-dispensing formulations.
  • self-propelling solution and spray formulations the effect can be achieved either by choice of a valve having the desired spray characteristics (i.e., being capable of producing a spray having the desired particle size) or by incorporating the active ingredient as a suspended powder in controlled particle size.
  • the therapeutics also can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Nasal drops also can be used.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Nasal drops also can be used.
  • Systemic administration also can be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants generally are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and filsidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the therapeutics typically are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the therapeutics are prepared with carriers that will protect against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials also can be obtained commercially from Alza
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.
  • Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the therapeutics identified according to the invention can be formulated for parenteral or oral administration to humans or other mammals, for example, in therapeutically effective amounts, e.g., amounts which provide appropriate concentrations of the drug to target tissue for a time sufficient to induce the desired effect.
  • therapeutically effective amounts e.g., amounts which provide appropriate concentrations of the drug to target tissue for a time sufficient to induce the desired effect.
  • the therapeutics of the present invention can be administered alone or in combination with other molecules known to have a beneficial effect on the particular disease or indication of interest.
  • useful cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and antifungal agents and analgesics and anesthetics.
  • the effective concentration of the therapeutics identified according to the invention that is to be delivered in a therapeutic composition will vary depending upon a number of factors, including the final desired dosage of the drug to be administered and the route of administration.
  • the preferred dosage to be administered also is likely to depend on such variables as the type and extent of disease or indication to be treated, the overall health status of the particular patient, the relative biological efficacy of the therapeutic delivered, the formulation of the therapeutic, the presence and types of excipients in the formulation, and the route of administration.
  • the therapeutics of this invention can be provided to an individual using typical dose units deduced from the earlier-described mammalian studies using non-human primates and rodents.
  • a dosage unit refers to a unitary, i.e. a single dose which is capable of being administered to a patient, and which can be readily handled and packed, remaining as a physically and biologically stable unit dose comprising either the therapeutic as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.
  • organisms are engineered to produce the therapeutics identified according to the invention. These organisms can release the therapeutic for harvesting or can be introduced directly to a patient, hi another series of embodiments, cells can be utilized to serve as a carrier of the therapeutics identified according to the invention.
  • Therapeutics of the invention also include the "prodrug" derivatives.
  • the term prodrug refers to a pharmacologically inactive (or partially inactive) derivative of a parent molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release or activate the active component.
  • Prodrugs are variations or derivatives of the therapeutics of the invention which have groups cleavable under metabolic conditions. Prodrugs become the therapeutics of the invention which are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation.
  • Prodrug of this invention can be called single, double, triple, and so on, depending on the number of biotransformation steps required to release or activate the active drug component within the organism, and indicating the number of functionalities present in a precursor-type form.
  • Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam
  • prodrug derivatives according to this invention can be combined with other features to enhance bioavailability.
  • EXAMPLE 1 Fusion protein expressing constructs.
  • Nucleic acid constructs for expressing therapeutic protein fusions of the invention can be made recombinantly according to methods known in the art.
  • oligonucleotides complementary to genes encoding the different components described herein can be used to make synthetic genes or to amplify the natural genes and construct gene fusions.
  • proteins of the invention are expressed from a recombinant gene comprising a signal sequence.
  • useful nucleic acids include nucleic acids that encode IGF targeting moieties of the invention. Such nucleic acids can be based on the sequences of IGF- 1 shown in Figure 3.
  • Expression product can also be isolated from serum free media using other protozoa, including other Leishmania species.
  • the expression strain is grown in medium with serum, diluted into serum free medium, and allowed to grow for several generations, preferably
  • LSD proteins production of secreted recombinant LSD proteins can be isolated from Leishmania mexicana promastigotes that are cultured initially in 50 mL IX M199 medium in a 75 cm2 flask at 27° C. When the cell density reaches l-3x 10 7 /mL, the culture is used to inoculate 1.2 L of M199 media. When the density of this culture reaches about 5xl0 6 /mL, the cells were harvested by centrifugation, resuspended in
  • the initial cell density of this culture is typically about 5x 10 5 /mL.
  • This culture is expanded to a cell density of about 1.0 — 1.7 x 10e 7 cells/mL.
  • the cells are separated from the culture medium by centrifugation and the supernatant is filtered at 4°C through a 0.2 ⁇ filter to remove residual promastigotes.
  • the filtered media was concentrated from 12.0 L to 500 mL using a tangential flow filtration device (MILLIPORE Prep/Scale-TFF cartridge).
  • Preferred growth media for this method are Ml 99 and "Zima" growth media.
  • LSD proteins secreted from Leishmania and containing carbohydrate with terminal mannose residues can be purified as follows.
  • recombinant ⁇ -glucuronidase from Leishmania mexicana containing plasmsid pXSAPO-GUS was grown in Ml 99 culture medium with a small amount of serum proteins.
  • ConA-agarose column (4% cross-linked beaded agarose, Sigma).
  • the ConA-agarose column was pretreated with 1 M NaCl, 20 mM Tris pH 7.4, 5 mM each of CaCl 2 , MgCl 2 and MnCl 2 and then equilibrated with 5 volumes of column buffer (20 mM Tris pH 7.4, 1 mM CaCl 2 , and 1 mM MnCl 2 ).
  • a total of 179,800 units (nmol/hr) of GUS activity (in 2 L) in culture medium was loaded onto a 22 mL ConA agarose column. No activity was detectable in the flow through or wash.
  • the GUS activity was eluted with column buffer containing 200 mM methyl mannopyranoside. Eluted fractions containing the activity peak were pooled and concentrated: 143900 units of GUS activity were recovered from the column (80% recovery of activity loaded onto the column). This demonstrates that the recombinant J3-GUS secreted from L. mexicana possesses carbohydrate with terminal mannose residues and further points out the potential for using the interaction of mannose with ConA as the basis for an affinity purification step.
  • a useful model system to determine whether a protein, particularly an LSD protein, tagged with an IGF tag crosses the blood-brain barrier is the
  • recombinant human ⁇ -glucuronidase fused to an IGF tag can be produced in any convenient expression system such as Leishmania, yeast, mammalian, bacculovirus and other expression systems.
  • L. mexicana expressing and secreting ⁇ -GUS is grown at 26°C in 100 ml Standard
  • Promastigote medium (Ml 99 with 40 mM HEPES, pH 7.5, 0.1 mM adenine, 0.0005% hemin,
  • the 1 liter cultures are contained in 2 liter capped flasks with a sterile stir bar so that the cultures could be incubated at 26°C with gentle stirring.
  • the 1 liter cultures are aerated twice a day by moving them into a laminar flow hood, removing the caps and swirling vigorously before replacing the caps.
  • the cultures reach a density of 2-3 x 10 7 promastigotes/ml, the cultures are centrifuged as previously described except the promastigote pellet is discarded and the media decanted into sterile flasks.
  • the ammonium sulfate fraction is further purified on a
  • GUS minus mice generated by heterozygous matings of B6.C-H-2 bml /ByBTR- gus mps /+ mice are used to assess the effectiveness of GUS-IGF fusion proteins or derivatives in enzyme replacement therapy.
  • Two formats are used. In one format, 3-4 animals are given a single injection of 20,000U of enzyme in 100 ⁇ l enzyme dilution buffer (150 mM NaCl, 10 mM
  • mice are killed 72-96 hours later to assess the efficacy of the therapy, hi a second format, mice are given weekly injections of 20,000 units over 3-4 weeks and are killed 1 week after the final injection. Histochemical and histopathologic analysis of liver, spleen and brain are carried out by published methods. In the absence of therapy, cells (e.g. macrophages and
  • Kupffer cells of GUS minus mice develop large intracellular storage compartments resulting from the buildup of waste products in the lysosomes. It is anticipated that in cells in mice treated with GUS fusion constructs of the invention, the size of these compartments will be visibly reduced or the compartments will shrink until they are no longer visible with a light microscope.
  • newborn mice do not possess a complete blood brain barrier. However, by day 15 the blood brain barrier is formed to the point that ⁇ -glucuronidase no longer can be detected in the brain. Accordingly, the above experiments are preferably
  • mice that are at day 15 or greater.
  • experiments first assess the ability of complete IGF-I and IGF-II tags to direct proteins across the blood brain barrier. Next, specific mutant versions of the proteins that disrupt receptor or IGF binding protein binding are assayed. For domain swaps, the B domain of IGF-II (residues 1-28 of the mature protein) contains only two differences from IGF-I that could conceivably alter transport across the blood brain barrier Gl 1 and T16. Altering these residues in IGF-II would is essentially a domain B swap. Another swap of regions between residues 28 and 41 of IGF-II and the corresponding region of IGF-I can also be tested. This essentially swaps the C domains of the two proteins which contains the most divergent regions of the two proteins. An alternative swap switches the
  • EXAMPLE 4 Assays for protein accumulation in the brain or CNS.
  • Radioactive assays can be used to monitor the accumulation of protein product in the brain. For example, the uptake and accumulation of a radioactively labeled protein in the brain parenchyma can be assayed as disclosed in Reinhardt and Bondy (1994) Endocrinology
  • Enzyme assays can also be used to monitor the accumulation of protein product in the brain. Enzyme assays are particularly useful when the therapeutic protein moiety is an enzyme for wliich there is an assay that is applicable for histochemical staining. Useful enzyme assays for lysosomal storage disease proteins include assays disclosed in Sly at al. (2001)
  • EXAMPLE 5 In vivo therapy.
  • GUS minus mice generated by heterozygous matings of B6.C-H-2 bml /ByBIR- gus mps /+ mice (Birkenmeier, Davisson et al. 1989) are used to assess the effectiveness of compositions of the invention in enzyme replacement therapy. Two formats are used, hr one format, 3-4 animals are given a single injection of 20,000U of enzyme in 100 ⁇ l enzyme dilution buffer (150 mM NaCl, 10 mM Tris, pH7.5). Mice are killed 72-96 hours later to assess the efficacy of the therapy.
  • mice are given weekly injections of 20,000 units over 3-4 weeks and are killed 1 week after the final injection. Histochemical and histopathologic analysis of liver, spleen and brain are carried out by published methods (Birkenmeier, Barker et al. 1991; Sands, Vogler et al. 1994; Daly, Vogler et al. 1999). In the absence of therapy, cells
  • mice develop large intracellular storage compartments resulting from the buildup of waste products in the lysosomes. It is anticipated that in cells in mice treated with compositions of the invention, the size of these compartments will be visibly reduced or the compartments will shrink until they are no longer visible with a light microscope.
  • humans with lysosomal storage diseases will be treated using constructs targeting an appropriate therapeutic portion to their CNS and in particular to lysosomes within the CNS.
  • treatment will take the form of regular (e.g. weekly) injections of a fusion protein of the invention.
  • treatment will be achieved through administration of a nucleic acid to permit persistent in vivo expression of the fusion protein, or tlirough administration of a cell (e.g. a human cell, or a unicellular organism) expressing the fusion protein in the patient.
  • a protein the invention may be expressed in situ using a Leishmania vector as described in U.S. Patent No. 6,020,144, issued
  • 2x GUS reaction mix is prepared by adding 100 mg of 4- methylumbelliferyl- ⁇ -D glucuronide to 14.2 mL 200 mM sodium acetate, pH adjusted to 4.8
  • reaction volume 200 ⁇ L.
  • the reaction tubes are covered with parafilm and incubated in a
  • a standard curve is prepared using 1, 2, 5, 10, and 20 ⁇ L of a 166 ⁇ M 4-
  • methylumbelliferone standard in a final volume of 2 mL stop buffer methylumbelliferone standard in a final volume of 2 mL stop buffer.
  • the 4-methylumbelliferone standard solution is prepared by dissolving 2.5 mg 4- methylumbelliferone in 1 mL ethanol and adding 99 mL of sterile water, giving a concentration of approximately 200 nmol/mL. The precise concentration is determined spectrophotometrically. The extinction coefficient at 360 nm is 19,000 cm “1 M "1 . For example,
  • EXAMPLE 7 Protein production in mammalian cells. CHO cells [0111] In one example, GUS-GILT ⁇ l-7 (an IGF-II targeting portion with a deleton of
  • GUS ⁇ C18-GILT ⁇ 1-7 a fusion protein in which GUS ⁇ C18 is
  • the CHO cells were propagated in MEM media supplemented with 15% FBS, 1.2
  • the secreted enzyme produced The six highest producers secreted between 8600 and 14900 units/mL/24 hours. The highest producing line was selected for collection of protein.
  • GUS-GILT cassettes were cloned into pCEP4 (Invitrogen) for expression in HEK
  • Cassettes used included wild-type GUS-GILT; GUS-GILT ⁇ 1-7; GUS-GILTY27L;
  • GUS ⁇ C18-GILT ⁇ 1-7 GILTY27L, and GUS-GILTF19S/E12K.
  • HEK 293 cells were cultured to 50-80% confluency in 12-well plates containing
  • Chromatography including conventional chromatography and affinity chromatography, can be used to purify GUS fusion proteins such as GUS-GILT fusion proteins and other fusion proteins of the invention.
  • the column was washed with ConA column buffer (50 mM Tris pH 7.4, lmM CaCl 2 , lmM MnCl 2 ) before mannosylated proteins including GUS-GILT fusions were eluted using a gradient of 0-0.2M methyl- ⁇ -D- pyranoside in the ConA column buffer.
  • Fractions containing glucuronidase activity were pooled, concentrated, and the buffer exchanged to SP column buffer (25 mM sodium phosphate pH 6, 20 mM NaCl, 1 mM EDTA) in preparation for the next column.
  • SP column buffer 25 mM sodium phosphate pH 6, 20 mM NaCl, 1 mM EDTA
  • the GUS-GILT fusions were eluted from the column in two steps: 1) a gradient of 0-0.15 M glucuronic acid in 25 mM sodium phosphate pH 6 and 10% glycerol, followed by 0.2 M glucuronic acid, 25 mM sodium phosphate pH 6, 10% glycerol.
  • Fractions containing glucuronidase activity were pooled, and the buffer exchanged to 20 mM potassium phosphate pH 7.4. These pooled fractions were loaded onto an HA-ultrogel column equilibrated with the same buffer.
  • the GUS-GILT fusion proteins were eluted with an increasing gradient of phosphate buffer, from 145-340 mM potassium phosphate pH 7.4.
  • the fractions containing glucuronidase activity were pooled, concentrated, and stored at -80°C in 20 mM Tris pH 8 with 25% glycerol.
  • Mammalian cells overexpressing a GUS-GILT fusion protein are grown to confluency in Nunc Triple Flasks, then fed with serum-free medium (Waymouth MB 752/1) supplemented with 2% fetal bovine serum to collect enzyme for purification. The medium is harvested and the flasks are refed at 24 hour intervals. Medium from several flasks is pooled and
  • the resin When binding is complete, the resin is collected on a fritted glass funnel and washed with 750 mL of 10 mM Tris pH 9.0 in several batches. The resin is transferred to a 2.5 cm column and washed with an additional 750 mL of the same buffer at a flow rate of 120 mL/hour.
  • the DEAE column is eluted with a linear gradient of 0-0.4 M NaCl in 10 mM Tris pH 9.0.
  • the fractions containing the GUS-GILT fusion proteins are detected by 4-methylumbelliferyl- ⁇ -D glucuronide assay, pooled, and loaded onto a 600 mL column of Sephacryl S-200 equilibrated with 25 mM
  • the dialyzed enzyme is loaded at a flow rate of 36 mL/hour onto a 15 L column of CM-
  • fusion proteins are pooled; the pool is assayed for GUS activity and for protein concentration to determine specific activity. Aliquots are run on SDS-PAGE followed by Coomassie or silver staining to confirm purity. If a higher concentration of enzyme is required, Amicon
  • 30 units (30,000 molecular weight cutoff) can be used to concentrate the fusion protein.
  • fusion protein is stored at -80°C in the 10 mM Tris pH 7.5, 1 mM sodium ⁇ -glycerol phosphate,
  • Wash Buffer 10 mM Tris pH 7.5, 10 mM potassium phosphate, 0.5 M NaCl, 0.025% sodium
  • fusion protein is eluted with P6 buffer, fractions containing GUS activity are pooled, and the pooled fractions assayed for GUS activity and for protein. An SDS-PAGE gel stained with Coomassie Blue or silver stain is used to confirm purity.
  • the fusion protein is stored frozen at -80°C in P6 buffer for long-term stability.
  • lysate were assayed for protein concentration (Pierce Micro BCA protein assay, Pierce, IL).
  • Binding of GUS-GILT proteins to the M6P/IGF-II receptor on fibroblasts are measured and the rate of uptake is assessed similar to published methods (York et al. (1999) L Biol. Chem. 274(2): 1164-71).
  • GM4668 fibroblasts cultured in 12 well culture dishes as described above are washed in ice-cold media minus serum containing 1 % BSA.
  • Ligand (either GUS, GUS-GILT or GUS- ⁇ GILT, or control proteins) is added to cells in cold media minus serum plus 1% BSA. Upon addition of ligand, the plates are incubated on ice for 30 minutes.
  • the plate is then floated in a 37° water bath and 0.5 ml prewarmed media is added to
  • GUS minus mice can be used to assess the effectiveness of GUS-GILT and derivatives thereof in enzyme replacement therapy.
  • GUS minus mice are generated by heterozygous matings of B6.C-H-2 bml /ByBIR-gus ps /+ mice as described by Birkenmeier et al.
  • mice are tolerant to human ⁇ -GUS.
  • mice may carry a transgene with a defective copy of human ⁇ -GUS to induce immunotolerance
  • human ⁇ -GUS e.g.
  • GUS-GILT protein as a GUS-GILT protein
  • the initial experiment is to determine the tissue distribution of the targeted
  • At least three mice receive a CHO-produced GILT-tagged ⁇ -GUS protein
  • GUS ⁇ C18-GILT ⁇ 1-7 referred to herein as GUS ⁇ C18-GILT ⁇ 1-7, in which GUS ⁇ 18, a ⁇ -GUS protein omitting the last
  • mice receive either ⁇ -GUS, a
  • preferred doses are in the range of 0.5-7 mg/kg body weight.
  • the enzyme dose is 1 mg/kg body weight administered intravenously, and the enzyme concentration is about 1-3 mg/mL.
  • at least three mice receive a dose
  • mice are sacrificed and the following organs and tissues are isolated: liver, spleen, kidney, brain, lung, muscle, heart, bone, and blood. Portions of each
  • tissue are homogenized and the ⁇ -GUS enzyme activity per mg protein is determined as
  • mice receive weekly injections at a dose of 1 mg/kg body weight.
  • measurement of the half-life of the periodate-modif ⁇ ed enzyme is determined in comparison with untreated enzyme as described in Example 12.
  • Two other assay formats can be used.
  • 3-4 animals are given a single injection of 20,000U of enzyme in 100 ⁇ l enzyme dilution buffer (150 mM NaCl, 10 mM Tris, pH7.5). Mice are killed 72-96 hours later to assess the efficacy of the therapy, hi a second format, mice are given weekly injections of 20,000 units over 3-4 weeks and are killed 1 week after the final injection. Histochemical and histopathologic analysis of liver, spleen and brain are carried out by published methods (Birkenmeier et al. (1991) Blood 78(11):3081-92; Sands et al.
  • GILT protein can be expressed in situ using a Leishmania vector as described in U.S. Patent No. 6,020,144, issued February 1, 2000; U.S. Provisional Application No. 60/250,446, filed November 30,
  • Targeted therapeutic proteins of the invention can also be administered, and their effects monitored, using methods (enzyme assays, histochemical assays, neurological assays, survival assays, reproduction assays, etc.) previously described for use with GUS. See, for example, Vogler et al. (1993) Pediatric Res. 34(6):837-840; Sands et al. (1994) J. Clin. Invest. 93:2324-2331; Sands et al. (1997) J. Clin. Invest. 99:1596-1605; O'Connor et al. (1998) J. Clin.
  • Fabry's disease is a lysosomal storage disease resulting from insufficient activity of ⁇ -GAL A, the enzyme responsible for removing the terminal galactose from GL-3 and other neutral sphingolipids. The diminished enzymatic activity occurs due to a variety of missense and nonsense mutations in the x-linked gene. Accumulation of GL-3 is most prevalent in lysosomes of vascular endothelial cells of the heart, liver, kidneys, skin and brain but also occurs in other cells and tissues. GL-3 buildup in the vascular endothelial cells ultimately leads to heart disease and kidney failure.
  • Enzyme replacement therapy is an effective treatment for Fabry's disease, and its success depends on the ability of the therapeutic enzyme to be taken up by the lysosomes of cells in which GL-3 accumulates.
  • Genzyme product, Fabrazyme is recombinant ⁇ -GAL A produced in DUKX Bl 1 CHO cells that has been approved for treatment of Fabry's patients in Europe due to its demonstrated efficacy.
  • Fabrazyme The ability of Fabrazyme to be taken up by cells and transported to the lysosome is due to the presence of mannose 6-phosphate (M6P) on its N-linked carbohydrate. Fabrazyme is delivered to lysosomes through binding to the mannose-6-phosphate/IGF-lI receptor (M6P/IGF-Iir), present on the cell surface of most cell types, and subsequent receptor mediated endocytosis. Fabrazyme reportedly has three N-linked glycosylation sites at ASN residues 108, 161, and 184. The predominant carbohydrates at these positions are fucosylated biantennary bisialylated complex, monophosphorylated mannose-7 oligomannose, and biphosphorylated mannose-7 oligomannose, respectively.
  • the glycosylation independent lysosomal targeting (GILT) technology of the present invention directly targets therapeutic proteins to the lysosome via a different interaction with the M6P/IGF-Iir.
  • a targeting ligand is derived from mature human IGF-II, which also binds with high affinity to the M6P/IGF-Iir.
  • the IGF-JI tag is provided as a c-terminal fusion to the therapeutic protein, although other configurations are feasible including cross-linking.
  • GILT-modified enzymes for uptake into cells has been established using GILT-modified ⁇ -glucuronidase, which is efficiently taken up by fibroblasts in a process that is competed with excess IGF-II.
  • Advantages of the GILT modification are increased binding to the M6P/IGF-1T receptor, enhanced uptake into lysosomes of target cells, altered or improved pharmacokinetics, and expanded, altered or improved range of tissue distribution.
  • the improved range of tissue distributions could include delivery of
  • GILT-modified protein will be produced primarily in CHO cells.
  • the GILT tag will be placed at the c-terminus of ⁇ -GAL A although the invention
  • the efficacy of a targeted therapeutic can be increased by extending the serum half- life of the targeted therapeutic.
  • Hepatic mannose receptors and asialoglycoprotein receptors eliminate glycoproteins from the circulation by recognizing specific carbohydrate structures (Lee et al. (2002) Science 295(5561):1898-1901; Ishibashi et al. (1994) J. Biol. Chem.
  • the present invention permits targeting of a therapeutic to lysosomes and/or across the blood brain barrier in a manner dependent not on a carbohydrate, but on a polypeptide or an analog thereof.
  • Actual underglycosylation of these proteins is expected to greatly increase their half-life in the circulation, by minimizing their removal from the circulation by the mannose and asialoglycoprotein receptors.
  • functional deglycosylation e.g. by modifying the carbohydrate residues on the therapeutic protein, as by periodate/ sodium borohydride treatment
  • Any fusion protein of the invention for exampe, any IGF-I tagged protein; or any lysosomal enzyme using a peptide targeting signal such as IGF-JI can be chemically or enzymatically deglycosylated or modified to produce a therapeutic with the desirable properties of specific lysosomal targeting plus long serum half-life, hi the case of some lysosomal storage diseases where it might be important to deliver the therapeutic to macrophage or related cell types via mannose receptor, fully glycosylated therapeutics can be used in combination with underglycosylate targeted therapeutics to achieve targeting to the broadest variety of cell types.
  • the targeted therapeutic protein in some cases it will be preferable to produce the targeted therapeutic protein initially in a system that does not produce a fully glycosylated protein.
  • a targeted therapeutic protein can be produced in E. coli, thereby generating a completely unglycosylated protein.
  • an unglycosylated protein is produced in mammalian cells treated with tunicamycin, an inhibitor of Dol-PP-GlcNAc formation. If, however, a particular targeted therapeutic does not fold correctly in the absence of glycosylation, it is preferably produced initially as a glycosylated protein, and subsequently deglycosylated or rendered functionally underglycosylated.
  • Underglycosylated targeted therapeutic proteins can also by prepared by engineering a gene encoding the targeted therapeutic protein so that an amino acid that normally serves as an acceptor for glycosylation is changed to a different amino acid.
  • an asparagine residue that serves as an acceptor for N-linked glycosylation can be changed to a glutamine residue, or another residue that is not a glycosylation acceptor. This conservative change is most likely to have a minimal impact on enzyme structure while eliminating glycosylation at the site.
  • glycosylation acceptor can be modified, disrupting a recognition motif for glycosylation enzymes without necessarily changing the amino acid that would normally be glycosylated.
  • bacterial ⁇ -glucuronidase is naturally unglycosylated, and can be targeted to a mammalian lysosome and/or across the blood brain barrier using the targeting moieties of the present invention.
  • Such enzymes can be synthesized in an unglycosylated state, rather than, for example, synthesizing them as glycosylated proteins and subsequently deglycosylating them.
  • the targeted therapeutic is produced in a mammalian cell culture system, it is preferably secreted into the growth medium, wliich can be harvested, permitting subsequent purification of the targeted therapeutic by, for example, chromatographic purification protocols, such as those involving ion exchange, gel filtration, hydrophobic chromatography, ConA chromatography, affinity chromatography or immunoaffinity chromatography.
  • chromatographic purification protocols such as those involving ion exchange, gel filtration, hydrophobic chromatography, ConA chromatography, affinity chromatography or immunoaffinity chromatography.
  • Chemical deglycosylation of glycoproteins can be achieved in a number of ways, including treatment with trifluoromethane sulfonic acid (TFMS), or treatment with hydrogen fluoride (HF).
  • TFMS trifluoromethane sulfonic acid
  • HF hydrogen fluoride
  • reaction mix is cooled to below -20°C in a dry ice-ethanol bath
  • reaction is carried out in a closed reaction system such as can be obtained from Peninsula Laboratori.es, Inc. 10 mg GILT-GUS is vacuum dried and placed in a reaction vessel which is then connected to the HF apparatus. After the entire HF line is evacuated, 10 mL anhydrous HF is distilled over from the reservoir with stirring of the reaction vessel. The reaction is continued
  • Remaining traces of HF are removed under high vacuum.
  • the reaction mixture is dissolved in 2 mL 0.2M NaOH to neutralize any remaining HF and the pH is readjusted to 7.5 with cold 0.2M HCl.
  • N-linked carbohydrates can be removed completely from glycoproteins using protein N- glycosidase (PNGase) A or F.
  • PNGase protein N- glycosidase
  • a glycoprotein is denatured prior to treatment with a glycosidase to facilitate action of the enzyme on the glycoprotein; the glycoprotein is subsequently refolded as discussed in the "In vitro refolding" section above.
  • excess glycosidase is used to treat a native glycoprotein to promote effective deglycosylation.
  • protease-sensitive sites on the targeted therapeutic protein, which will diminish the half-life of the protein.
  • N-linked glycosylation is known to protect a subset of lysosomal enzymes from proteolysis (Kundra et al. (1999) J. Biol. Chem. 274(43):31039-46).
  • protease-sensitive sites are preferably engineered out of the protein (e.g. by site-directed mutagenesis).
  • the risk of revealing either a protease-sensitive site or a potential epitope can be minimized by incomplete deglycosylation or by modifying the carbohydrate structure rather than omitting the carbohydrate altogether.
  • the therapeutic protein is partially deglycosylated.
  • the therapeutic protein can be treated with an endoglycosidase such as endoglycosidase H, wliich cleaves N-linked high mannose carbohydrate but not complex type carbohydrate leaving a single GlcNAc residue linked to the asparagine.
  • a therapeutic protein treated in this way will lack high mannose carbohydrate, reducing interaction with the hepatic mannose receptor. Even though this receptor recognizes terminal GlcNAc, the probability of a productive interaction with the single GlcNAc on the protein surface is not as great as with an intact high mannose structure.
  • any complex carbohydrate present on the protein will remains unaffected by the endoH treatment and may be terminally sialylated, thereby diminishing interactions with hepatic carbohydrate recognizing receptors. Such a protein is therefore likely to have increased half-life.
  • steric hinderance by the remaining carbohydrate should shield potential epitopes on the protein surface from the immune system and diminish access of proteases to the protein surface (e.g. in the protease-rich lysosomal environment).
  • glycosylation of a therapeutic protein is modified, e.g. by oxidation, reduction, dehydration, substitution, esterification, alkylation, sialylation, carbon- carbon bond cleavage, or the like, to reduce clearance of the therapeutic protein from the blood.
  • the therapeutic protein is not sialylated.
  • treatment with periodate and sodium borohydride is effective to modify the carbohydrate structure of most glycoproteins. Periodate treatment oxidizes vicinal diols, cleaving the carbon-carbon bond and replacing the hydroxyl groups with aldehyde groups; borohydride reduces the aldehydes to hydroxyls.
  • a protein may be glycosylated on an asparagine residue with a high mannose carbohydrate that includes N-acetylglucosamine residues near the asparagine and mannose residues elsewhere in the structure.
  • the terminal mannose residues have three consecutive carbons with hydroxyl groups; both of the carbon-carbon bonds involved are cleaved by periodate treatment.
  • Some nonterminal mannose residues also include a vicinal diol, which would similarly be cleaved. Nevertheless, while this treatment converts cyclic carbohydrates into linear carbohydrates, it does not completely remove the carbohydrate, minimizing risks of exposing potentially protease-sensitive or antigenic polypeptide sites.
  • a ⁇ -glucuronidase construct (or other glycoprotein) coupled to a targeting moiety of
  • the invention when deglycosylated or modified by sequential treatment with periodate and sodium borohydride should enjoy a similar (e.g. more than twofold, more than fourfold, or more than tenfold) increase in halflife while still retaining a high affinity for the cation-independent
  • M6P receptor permitting targeting of the construct to the lysosome of all cell types that possess this receptor.
  • the construct is also predicted to cross the blood brain barrier efficiently.
  • Carbohydrate modification by sequential treatment with periodate and sodium borohydride can be performed as follows: Purified GILT-GUS is incubated with 40 mM NaIO 4
  • reaction mixture over a Sephadex G-25M column eluted with PBS at pH 7.5 terminates the reaction.
  • periodate oxidation modifies M6P sufficiently to abolish its ability to interact with the M6P receptor.
  • the carbohydrate can be modified by treatment with periodate and cyanoborohydride in a one step reaction as disclosed in Thorpe et al. (1985) Eur. J. Biochem.
  • a patient receiving a therapeutic protein also receives an immunosuppressive therapy in some embodiments of the invention.
  • An underglycosylated protein is expected to demonstrate reduced binding to ConA-sepharose when compared to the corresponding fully glycosylated protein.
  • An actually underglycosylated protein can also be resolved by SDS-PAGE and compared to the corresponding fully-glycosylated protein.
  • chemically deglycosylated GUS-GILT can be compared to untreated (glycosylated) GUS-GILT and to enzymatically deglycosylated GUS-GILT prepared with PNGase A.
  • the underglycosylated protein is expected to have a greater mobility in SDS-PAGE when compared to the fully glycosylated protein.
  • Underglycosylated targeted therapeutic proteins display uptake that is dependent on the targeting domain. Underglycosylated proteins should display reduced uptake (and, preferably, substantially no uptake) that is dependent on maimose or M6P. These properties can be experimentally verified in cell uptake experiments.
  • a GUS-GILT protein synthesized in mammalian cells and subsequently treated with periodate and borohydride can be tested for functional deglycosylation by testing M6P-dependent and mannose-dependent uptake. To demonstrate that M6P-dependent uptake has been reduced, uptake assays are performed using GM4668 fibroblasts. In the absence of competitor, treated and untreated enzyme will each display significant uptake.
  • E cells a mouse macrophage-like cell line bearing mannose receptors but few, if any, M6P receptors (Diment et al. (1987) J. Leukocyte Biol. 42:485-490). The cells are cultured in
  • DMEM low glucose
  • FBS fetal bovine serum
  • 4 mM glutamine 4 mM glutamine
  • antibiotic, antimycotic solution Sigma, A-5955.
  • Uptake assays with these cells are performed in a manner identical to assays performed with fibroblasts.
  • fully glycosylated enzyme will display significant uptake due to interaction with the mannose receptor.
  • Underglycosylated enzyme is expected to display substantially reduced uptake under these conditions.
  • the mannose receptor-dependent uptake of fully glycosylated enzyme can be competed by the addition of excess (100 ⁇ g/mL) mannan.
  • Pharmacokinetics of deglycosylated GUS-GILT can be determined by giving intravenous injections of 20,000 enzyme units to groups of three MPSVII mice per timepoint.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Cardiology (AREA)
  • Microbiology (AREA)
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Abstract

Cette invention a trait à des techniques et aux compositions correspondantes visant à apporter au cerveau des protéines thérapeutiques par ciblage. Ces techniques consistent à associer un fragment de facteur de croissance semblable à l'insuline (IGF) à une protéine thérapeutique, afin de cibler celle-ci sur le cerveau. Des protéines hybrides solubles renfermant un fragment d'IGF sont transportées par le sang vers le tissu nerveux du cerveau. Cette invention porte également sur des applications thérapeutiques visant au traitement de maladies lysosomales. Elle concerne, de surcroît, des acides nucléiques ainsi que des cellules exprimant ces protéines hybrides/IGF.
PCT/US2002/032968 2001-10-16 2002-10-16 Techniques et compositions permettant le ciblage de proteines sous-glycosylees a travers la barriere hemato-encephalique WO2003032727A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP02801739A EP1446007A4 (fr) 2001-10-16 2002-10-16 Techniques et compositions permettant le ciblage de proteines sous-glycosylees a travers la barriere hemato-encephalique
JP2003535542A JP2005506340A (ja) 2001-10-16 2002-10-16 血液脳関門をわたる過小グリコシル化タンパク質の標的化のための方法および組成物
AU2002362930A AU2002362930A2 (en) 2001-10-16 2002-10-16 Methods and compositions for targeting underglycosylated proteins across the blood brain barrier
IL16135202A IL161352A0 (en) 2001-10-16 2002-10-16 Methods and compositions for targeting underglycosylated proteins across the blood brain barrier
CA002463473A CA2463473A1 (fr) 2001-10-16 2002-10-16 Proteines therapeutiques ciblees

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US32965001P 2001-10-16 2001-10-16
US60/329,650 2001-10-16
US10/136,639 US20030072761A1 (en) 2001-10-16 2002-04-30 Methods and compositions for targeting proteins across the blood brain barrier
US10/136,841 2002-04-30
US10/136,639 2002-04-30
US10/136,841 US7396811B2 (en) 2001-04-30 2002-04-30 Subcellular targeting of therapeutic proteins
US38445202P 2002-05-29 2002-05-29
US60/384,452 2002-05-29
US38601902P 2002-06-05 2002-06-05
US60/386,019 2002-06-05
US40881602P 2002-09-06 2002-09-06
US60/408,816 2002-09-06

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AU (3) AU2002347910A1 (fr)
CA (1) CA2463473A1 (fr)
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WO2005078077A3 (fr) * 2004-02-10 2005-10-20 Zystor Therapeutics Inc Alpha-glucosidase acide et fragments de celle-ci
US7396811B2 (en) 2001-04-30 2008-07-08 Zystor Therapeutics, Inc. Subcellular targeting of therapeutic proteins
US7560424B2 (en) 2001-04-30 2009-07-14 Zystor Therapeutics, Inc. Targeted therapeutic proteins
WO2009137721A2 (fr) 2008-05-07 2009-11-12 Zystor Therapeutics, Inc. Peptides de ciblage lysosomial et leurs utilisations
US7629309B2 (en) 2002-05-29 2009-12-08 Zystor Therapeutics, Inc. Targeted therapeutic proteins
WO2010148253A2 (fr) 2009-06-17 2010-12-23 Zystor Therapeutics, Inc. Formulations pour enzymes lysosomales
US7981864B2 (en) 2001-10-16 2011-07-19 Biomarin Pharmaceutical Inc. Methods and compositions for targeting proteins across the blood brain barrier
WO2011163652A2 (fr) 2010-06-25 2011-12-29 Shire Human Genetic Therapies, Inc. Traitement du syndrome de sanfilippo de type b
US8545837B2 (en) 2010-06-25 2013-10-01 Shire Human Genetic Therapies, Inc. Methods and compositions for CNS delivery of iduronate-2-sulfatase
US9220677B2 (en) 2010-06-25 2015-12-29 Shire Human Genetic Therapies, Inc. Methods and compositions for CNS delivery of iduronate-2-sulfatase
US9283181B2 (en) 2010-06-25 2016-03-15 Shire Human Genetic Therapies, Inc. CNS delivery of therapeutic agents
US9320711B2 (en) 2010-06-25 2016-04-26 Shire Human Genetic Therapies, Inc. Methods and compositions for CNS delivery of heparan N-sulfatase
US9376480B2 (en) 2012-11-27 2016-06-28 Biomarin Pharmaceutical Inc. Targeted therapeutic lysosomal enzyme fusion proteins and uses thereof
WO2017147414A1 (fr) 2016-02-24 2017-08-31 Biomarin Pharmaceutical Inc. Protéines de fusion d'enzymes lysosomales thérapeutiques ciblées, formulations associées et leurs utilisations
US9770410B2 (en) 2010-06-25 2017-09-26 Shire Human Genetic Therapies, Inc. Methods and compositions for CNS delivery of arylsulfatase A
WO2019213180A1 (fr) * 2018-04-30 2019-11-07 Amicus Therapeutics, Inc. Constructions de thérapie génique et procédés d'utilisation
US10660944B2 (en) 2011-12-23 2020-05-26 Shire Human Genetic Therapies, Inc. Stable formulations for CNS delivery of arylsulfatase A
WO2020132452A1 (fr) 2018-12-20 2020-06-25 Shire Human Genetic Therapies, Inc. Purification d'une protéine de fusion d'immunoglobuline iduronate-2-sulfatase
US11097015B2 (en) 2018-10-10 2021-08-24 Amicus Therapeutics, Inc. Disulfide bond stabilized polypeptide compositions and methods of use
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JP2005506340A (ja) 2005-03-03
AU2010214643A1 (en) 2010-09-16
AU2002347910A1 (en) 2003-04-28
WO2003032913A2 (fr) 2003-04-24
CA2463473A1 (fr) 2003-04-24
WO2003032913A9 (fr) 2004-04-29
WO2003032913A3 (fr) 2004-08-12
AU2002362930A2 (en) 2003-04-28
IL161352A0 (en) 2004-09-27
JP2009203241A (ja) 2009-09-10
EP1446007A4 (fr) 2005-12-07
EP1446007A1 (fr) 2004-08-18

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