WO2014194427A1 - Targeted iduronate-2-sulfatase fusion proteins - Google Patents

Abstract

The present invention is directed to polypeptides and peptidomimetics that include a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, and a lysosomal enzyme, methods for their production and precursors in their production. The polypeptides and peptidomimetics of the invention are produced using a method which utilizes a precursor comprising a tag. The reduces cleavage within the amino acid sequence of the polypeptide or peptidomimetic compared to levels of cleavage observed in the absence of the tag. These compounds are exemplified by IDS-Angiopep-2 polypeptides and peptidomimetics which can be used to treat MPS-I I. Where the targeting moiety transports the polypeptide or peptidomimetic across the blood brain barrier, these polypeptides and peptidomimetics can treat not only the peripheral disease symptoms, but may also be effective in treating CNS symptoms. Where the targeting moiety transports the polypeptide or peptidomimetic to the lysosomes, it is expected that the polypeptide or peptidomimetic will be more effective than IDS itself.

Description

TARGETED IDURONATE-2-SULFATASE FUSION PROTEINS

Cross-Reference to Related Applications

This application claims benefit of U.S. Provisional Application Nos. 61/831 ,913, filed June 6, 2013, which is hereby incorporated by reference in its entirety.

Background of the Invention

Lysosomal storage disorders are group of about 50 rare genetic disorders in which a subject has a defect in a lysosomal enzyme that is required for proper metabolism. These diseases typically result from autosomal or X-linked recessive genes. As a group, the incidence of these disorders is about 1 :5000 to 1 : 10,000.

Hunter syndrome or mucopolysaccharidosis Type I I (MPS-I I) results from a deficiency of iduronate-2-sulfatase (IDS; also known as idursulfase), an enzyme that is required for lysosomal degradation of heparan sulfate and dermatan sulfate. Because the disorder is X-linked recessive, it primarily affects males. Those with the disorder are unable to break down and recycle these mucopolysaccharides, which are also known as glycosaminoglycans or GAG. This deficiency results in the buildup of GAG throughout the body, which has serious effects on the nervous system, joints, and various organ systems including heart, liver, and skin. There are also a number of physical symptoms, including coarse facial features, enlarged head and abdomen, and skin lesions. In the most severe cases, the disease can be fatal in teen years and is accompanied by severe mental retardation.

There is no cure for MPS-I I. In addition to palliative measures, therapeutic approaches have included bone marrow grafts and enzyme replacement therapy. Bone marrow grafts have been observed to stabilize the peripheral symptoms of MPS-I I, including cardiovascular abnormalities,

hepatosplenomegaly (enlarged liver and spleen), and joint stiffness. This approach, however, does not stabilize or resolve the neuropsychological symptoms associated with this disease (Guffon et al. , J. Pediatr. 154:733-7, 2009).

Enzyme replacement therapy by intravenous administration of IDS has also been shown to have benefits, including improvement in skin lesions (Marin et al. , Pediatr. Dermatol. 29:369-370, 2012), visceral organ size, gastrointestinal functioning, and reduced need for antibiotics to treat upper airway infections (Hoffman et al. , Pediatr. Neurol. 45: 181 -4, 201 1 ). Like bone marrow grafts, this approach does not improve the central nervous system deficits associated with MPS-I I because the enzyme is not expected to cross the blood-brain barrier (BBB; Wraith et al. , Eur. J. Pediatr. 1676:267-7, 2008).

Methods for increasing delivery of IDS to the brain have been and are being investigated, including intrathecal delivery (Felice et al. , Toxicol. Pathol. 39:879-92, 201 1 ). Intrathecal delivery, however, is a highly invasive technique.

Less invasive and more effective methods of treating MPS-II that address the neurological disease symptoms, in addition to the other symptoms, would therefore be highly desirable.

Summary of the Invention

The present invention is directed to polypeptides and peptidomimetics that include a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, and a lysosomal enzyme, methods for their production and precursors in their production. The polypeptides and peptidomimetics of the invention are produced by recombinant expression technology using a method which utilizes a precursor comprising a tag moiety. The tag facilitates isolation of the precursor and also reduces cleavage within the amino acid sequence of the polypeptide or peptidomimetic compared to levels of cleavage observed in the absence of the tag.

Without being bound by theory, two possible mechanisms that aberrant cleavage is minimized are as follows. One possibility is that the tag enables the precursor to be isolated on a suitable resin and extensively washed, removing host cell proteases capable of cleaving the precursor within the amino acid sequence of the polypeptide or peptidomimetic. A second possibility is that the presence of the peptidic tag provides alternative sites of action for host cell proteases such that the tag effectively competes with any protease target sites within the amino acid sequence of the polypeptide or peptidomimetic product, thereby reducing cleavage at the sites within the polypeptide or peptidomimetic.

The polypeptides and peptidomimetics of the invention are exemplified by IDS-Angiopep-2 fusion proteins which can be used to treat MPS-I I. Where the targeting moiety transports the fusion protein across the blood brain barrier, these fusion proteins may be effective in treating CNS symptoms. Where the targeting moiety transports the fusion proteins to the lysosomes, the fusion proteins may be more effective than IDS itself, even in the treatment of peripheral symptoms.

Accordingly, in a first aspect, the invention features a composition comprising a polypeptide or peptidomimetic, which polypeptide or peptidomimetic comprises:

(a) a targeting moiety with an amino acid sequence with at least 70% identity to any one of SEQ ID NO:97, 1-69, 71 -73, 75-105, and 107-1 17; and

(b) iduronate-2-sulfatase (IDS), an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS;

wherein said targeting moiety is capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier,

wherein said polypeptide or peptidomimetic has IDS activity, and.

wherein said composition contains at least 90% polypeptide or peptidomimetic, measured as a percentage of the total protein content of the composition or less than 10% of an impurity generated by aberrant cleavage of the polypeptide or peptidomimetic.

In certain embodiments, the composition contains greater than 90% polypeptide or

peptidomimetic, measured as a percentage of the total protein content of the composition (e.g. , 95%, 96%, 98%, 99%, up to and including 100%). In certain embodiments, less than 10% (e.g. , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1 %, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1 %, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1 %, down to and including 0%) of the total protein content of the composition is comprised of impurities generated by aberrant cleavage of the polypeptide or peptidomimetic. In certain embodiments, less than 10% (e.g. , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1 %, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1 %, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1 %, down to and including 0%), relative to the amount (e.g., by weight) of the polypeptide or peptidomimetic in the composition, is comprised of impurities generated by aberrant cleavage of the polypeptide or peptidomimetic

The polypeptides and peptidomimetics of the invention may, in addition to (a) and (b) contain one or more additional amino acid residues N or C-terminal to the targeting moiety, the IDS moiety or both, provided that targeting activity and enzymatic activity is retained. In some embodiments, at least 90% of the polypeptide or peptidomimetic includes a glycine or serine at the N-terminus.

The polypeptides and peptidomimetics of the invention may include more than one targeting moiety, and/or more than one IDS moiety. The arrangement of the moieties may also vary. For example, in embodiments with just one targeting moiety and just one IDS moiety, the targeting moiety may be either N- or C-terminal to the IDS moiety. The skilled person will recognize that various arrangements are possible where the polypeptides and peptidomimetics comprise multiple targeting moieties and/or IDS moieties.

In a second aspect, the invention also features a pharmaceutical composition comprising the composition of the first aspect and a pharmaceutically acceptable carrier.

In another aspect, the invention features a method of treating or treating prophylactically a subject having a lysosomal storage disorder (e.g. , MPS-I I). The method includes administering to the subject a composition of the first aspect or a pharmaceutical composition described herein.

In another aspect, the invention features a method of producing a polypeptide or peptidomimetic comprising (a) a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, which targeting moiety exhibits an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-117; and (b) IDS, an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS; which method comprises:

(i) providing a host cell that is genetically engineered to express a precursor comprising (a) a targeting moiety exhibiting an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1-69, 71-73, 75-105 and 107-1 17; (b) IDS, an IDS fragment or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS; (c) a tag region; and (d) a cleavable sequence that separates said tag region from (a) and (b);

(ii) culturing said host cell under conditions in which the cell expresses said precursor;

(iii) isolating said precursor from the host cell culture; and

(iv) treating said precursor with a protease enzyme that recognizes and cleaves said cleavable sequence;

thereby producing a polypeptide or peptidomimetic comprising (a) a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, which targeting moiety exhibits an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-1 17; and (b) IDS, an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS.

The tag may be any amino acid sequence that binds with high affinity to a ligand, facilitating isolation of the precursor by chromatography or other known methods. In certain embodiments, the tag is sufficiently soluble that the precursor does not form inclusion proteins in the host cell. In some embodiments, the tag comprises more than 100 amino acids, more particularly, more than 150, or more than 200 amino acids. In a particular embodiment, the tag is an Ig Fc tag. In a more particular embodiment, the tag is an IgG Fc tag. In alternative embodiments, the tag is thioredoxin, nusA, SUMO(small ubiquitin-like modifier), glutathione-S-transferase, maltose binding protein-tag, strep-tag or chitin-binding domain. Where the tag is SUMO (small ubiquitin-like modifier), the optional cleavable sequence (d) is absent since SU MO protease self cleaves the SU MO tag. Where the tag is Ig Fc (e.g. IgG Fc), thioredoxin, nusA, SU MO(small ubiquitin-like modifier), glutathione-S-transferase, maltose binding protein-tag, strep-tag or chitin-binding domain, the cleavable sequence (d) is present.

In certain embodiments, the protease enzyme does not display proteolytic activity against said targeting moiety, IDS, IDS fragment, or IDS analog.

Protease enzymes capable of cleaving the cleavable sequence (d) are known in the art and are reviewed in http://wolfson. huii.ac. il/purification/pdf/literature/wauqh201 1. pdf . In certain embodiments, the protease may be TEV (tobacco etch virus), Factor XA, thrombin, enterokinase, SUMO (small ubiquitin-like modifier). The skilled person will appreciate that the cleavable linker (d) should include the recognition site for the appropriate protease enzyme. In a particular embodiment, the protease is Tobacco Etch Virus (TEV) protease and said cleavable sequence is recognized by TEV protease.

The step of isolating said precursor from the host cell culture may comprise a step of affinity purifying the precursor. The skilled person will appreciate that the tag enables affinity purification to take place utilizing an immobilized ligand of the tag. For example, a glutathione-S-transferase tag binds to immobilized glutathione, a maltose binding protein tag binds to amylase agarose, a strep-tag binds to immobilized streptavidin and the chitin-binding domain binds immobilized chitin. The Ig-Fc tag binds protein A agarose. This is very robust and permits the bound precursor to be washed under stringent conditions.

In particular embodiments, the method includes after step (iv), an additional step of gel filtration

(e.g. , on Superdex 75).

The precursor of the invention may, in addition to (a) to (d) contain one or more additional amino acid residues N or C-terminal to one or more of the targeting moiety, the IDS moiety, the tag or the cleavable sequence, provided that targeting activity and enzymatic activity of the polypeptide or peptidomimetic product is retained. In some embodiments, the polypeptide or peptidomimetic product includes a glycine or serine at the N-terminus.

The precursor of the invention may, contain more than one targeting moiety, and/or more than one IDS moiety and these may be arranged in any order. However, all targeting moieties and IDS moieties must be either N or C terminal to the cleavable sequence separating them from the tag moiety.

In another aspect, the invention features, a precursor comprising:

(a) IDS, an IDS fragment or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS;

(b) a targeting moiety exhibiting an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-117;

(c) a tag region; and optionally

(d) a cleavable sequence that separates said tag region from (a) and (b).

As described above, in particular embodiments said cleavable sequence is capable of being cleaved by TEV protease. In a more particular embodiment, said cleavable sequence comprises Glu-Asn-Leu-Tyr- Phe-Gln-Ser or Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

The precursor of the invention may, in addition to (a) to (d) contain one or more additional amino acid residues N or C-terminal to one or more of the targeting moiety, the IDS moiety, the tag or the cleavable sequence, provided that targeting activity and enzymatic activity of the polypeptide or peptidomimetic product is retained. In some embodiments, the polypeptide or peptidomimetic product includes a glycine or serine at the N-terminus.

The precursor of the invention may, contain more than one targeting moiety, and/or more than one IDS moiety and these may be arranged in any order. However, all targeting moieties and IDS moieties must be either N or C terminal to the cleavable sequence separating them from the tag moiety.

In any of the above aspects, the targeting moiety is capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier.

In one embodiment, the targeting moiety enables the polypeptide or peptidomimetic to efficiently cross the blood brain barrier (BBB). In a more particular embodiment, the targeting moiety may bind to receptors present on brain endothelial cells and thereby result in the polypeptide or peptidomimetic being transported across the BBB by transcytosis. The targeting moiety may lead to high levels of transendothelial transport without affecting the cell or BBB integrity.

In another embodiment, the targeting moiety enables the polypeptide or peptidomimetic to be efficiently transported into the lysosome.

In any of the above aspects,

By "subject" is meant a human or non-human animal (e.g. , a mammal).

By "lysosomal enzyme" is meant any enzyme that is found in the lysosome in which a defect in that enzyme can lead to a lysosomal storage disorder.

By "lysosomal storage disorder" is meant any disease caused by a defect in a lysosomal enzyme. Approximately fifty such disorders have been identified.

By "targeting moiety" is meant a portion of a peptide or a peptidomimetic that results in the entire peptide or peptidomimetic being transported the lysosome and/or across the BBB. In certain embodiments, the targeting moiety may bind to receptors present on brain endothelial cells and thereby be transported across the BBB by transcytosis. The targeting moiety may result inhigh levels of transendothelial transport without affecting the cell or BBB integrity. By "treating" a disease, disorder, or condition in a subject is meant reducing at least one symptom of the disease, disorder, or condition by administrating a therapeutic agent to the subject.

By "treating prophylactically" a disease, disorder, or condition in a subject is meant reducing the frequency of occurrence of or reducing the severity of a disease, disorder or condition by administering a therapeutic agent to the subject prior to the onset of disease symptoms.

By a polypeptide or peptidomimetic which is "efficiently transported across the BBB" is meant a polypeptide or peptidomimetic that is able to cross the BBB at least as efficiently as Angiopep-6 (i.e., greater than 38.5% that of Angiopep-1 (250 nM) in the in situ brain perfusion assay described in U.S. Patent Application No. 1 1/807,597, filed May 29, 2007, hereby incorporated by reference). Accordingly, a peptide which is "not efficiently transported across the BBB" is transported to the brain at lower levels (e.g. , transported less efficiently than Angiopep-6).

By "substantial identity" or "substantially identical" is meant an amino acid sequence that is identical to a reference sequence, or has a specified percentage of amino acid residues that are the same at the corresponding location within a reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence. For peptides or polypeptides, the length of comparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g. , a full-length sequence). Sequence identity may be measured using sequence analysis software on the default setting (e.g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, VVI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.

Brief Description of the Drawings

Figure 1 are the amino acid sequences of SEQ ID NO: 1 18-120.

Figure 2 is a schematic diagram showing the IgG Fc construct that was generated

Figure 3 is an image of a gel illustrating the enhanced purity of a fusion protein produced using an Fc fusion tag in contrast to fusion proteins produced using other tags.

Figure 4 is graph displaying the in situ brain perfusion of An2-IDS-Fc and IDS-Fc compared to

JR-032.

Detailed Description

The present invention relates to polypeptides and peptidomimetics including a targeting moiety and a lysosomal enzyme. The present disclosure relates to the finding that the use of a tag in precursors of the polypeptides and peptidomimetics reduces cleavage within the amino acid sequence of the polypeptide or peptidomimetic compared to levels of cleavage observed in the absence of the tag. This enables production of compositions of polypeptides and peptidomimetics of higher purity, due to lower aberrant cleavage. Methods for their production and methods for the treatment of MPS-I I by administering such polypeptides and peptidomimetics are also disclosed. The polypeptides and peptidomimetics are capable of targeting the lysosome and/or crossing the BBB. Such polypeptides and peptidomimetics are exemplified by IDS-AngioPep2 fusion proteins. These fusion proteins maintain IDS enzymatic activity both in an enzymatic assay and in a cellular model of MPS-I I. Accordingly, we believe that these fusion proteins can increase enzyme concentrations in the lysosome, thus resulting in more effective therapy, particular in tissues and organs that express the LRP-1 receptor, such as liver, kidney, and spleen.

These features overcome some of the biggest disadvantages of current therapeutic approaches because intravenous administration of IDS by itself does not treat CNS disease symptoms. In contrast to physical methods for bypassing the BBB, such intrathecal or intracranial administration, which are highly invasive and thus generally an unattractive solution to the problem of CNS delivery, the present invention allows for noninvasive brain delivery. In addition, improved transport of the therapeutic to the lysosomes may allow for reduced dosing or reduced frequency of dosing, as compared to standard enzyme replacement therapy.

Lysosomal storage disorders

Lysosomal storage disorders are a group of disorders in which the metabolism of lipids, glycoproteins, or mucopolysaccharides is disrupted based on enzyme dysfunction. This dysfunction leads to cellular buildup of the substance that cannot be properly metabolized. Symptoms vary from disease to disease, but problems in the organ systems (liver, heart, lung, and spleen), bones, as well as neurological problems are present in many of these diseases. Typically, these diseases are caused by rare genetic defects in the relevant enzymes. Most of these diseases are inherited in autosomal recessive fashion, but some, such as MPS-I I, are X-linked recessive diseases.

Lysosomal enzymes

The present invention may use any lysosomal enzyme known in the art that is useful for treating a lysosomal storage disorder. The polypeptides and peptidomimetics of the present invention have iduronate-2-sulfatase (IDS; also known as idursulfase) activity. The polypeptides or peptidomimetics may include IDS, a fragment of IDS that retains enzymatic activity, or an IDS analog which exhibits enzymatic activity.

To test whether particular fragment or analog has enzymatic activity, the skilled artisan can use any appropriate assay. Assays for measuring IDS activity, for example, are known in art, including those described in Hopwood, Carbohydr. Res. 69:203-16, 1979, Bielicki et al. , Biochem. J. 271 :75-86, 1990, and Dean et al. , Clin. Chem. 52:643-9, 2006. A similar fluorometric assay is also described below. Using any of these assays, the skilled artisan would be able to determine whether a particular IDS fragment or analog has enzymatic activity. These assays can also identify whether polypeptides or peptidomimetics have enzyme activity.

Three human isoforms of IDS are known, isoforms a, b, and c. Isoform a is a 550 amino acid protein, isoform b is a 343 amino acid protein which has a different C-terminal region as compared to the longer isoform a, and isoform c has changes at the N-terminal due to the use of a downstream start codon. Any of these isoforms may be used in the compounds of the invention. Recombinant iduronate- 2-sulfatase enzymes (e.g. , JR-032) are known in the art. JR-032 (SEQ ID NO: 118, Figure 1 ) is a recombinant human IDS full length isoform a (INN: idursulfase) manufactured as described in US Patent No. 5,932,21 1.

In certain embodiments, the IDS or the IDS fragment has the amino acid sequence of human IDS isoform a or a fragment thereof (e.g. , amino acids 26-550 of isoform a, which represents the mature form of isoform a). Where an IDS fragment is used, this may be at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.

IDS analogs have a different amino acid sequence to the human isoforms of IDS (or fragments thereof). IDS analogs may have sequences that are substantially identical (e.g. , at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence of human IDS isoform a, isoform b, isoform c, or to amino acids 26-550 of isoform a.

Naturally occurring residues are divided into groups based on common side chain properties:

(1 ) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu),

Isoleucine (lie), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe),

(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)

(3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu)

(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg)

(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);

(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), (7) polar: Ser, Thr, Asn, Gin

(8) basic positively charged: Arg, Lys, His, and;

(9) charged: Asp, Glu, Arg, Lys, His

Analogs may be generated by substitutional mutagenesis. To retain enzymatic activity, amino acids are typically substituted with others falling within the same group. Such substitutions are referred to as "conservative". Examples of substitutions identified as "conservative substitutions" are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1 may be considered.

Other amino acid substitutions are listed in Table 1.

Table 1 : Amino acid substitutions

In certain embodiments, the IDS portion of the polypeptide may be modified (e.g. , using any of the polypeptide modifications described herein) such that the compound of the invention is a peptidomimetic.

Targeting moieties

The targeting moiety may be less than 30, 25, 24, 23, 22, 21 , 20, or 19 amino acids in length. In one embodiment, the targeting moiety comprises an amino acid sequence that is substantially identical to any of the sequences of Table 2 (i.e. SEQ ID NOS. 1 -69, 71-73, 75-105 and 107-1 17), or a fragment thereof. More particularly, the targeting moiety comprises an amino acid sequence with at least 70% identity to any one of SEQ ID NO: 1-69, 71-73, 75-105 and 107-117. In certain embodiments, the targeting moiety comprises an amino acid sequence with at least 80%, 90%, 95%, 99%, or even 100% identity to to any one of SEQ ID NO: 1-69, 71-73, 75-105 and 107-117. The targeting moiety may have one to 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions relative to any one of SEQ ID NO: 1-69, 71-73, 75-105 and 107-117. The amino acid substitution(s) may be conservative or exemplary as described above in relation to the enzyme moiety. For example, the targeting moiety may have an arginine at one, two, or three of the positions corresponding to positions 1 , 10, and 15 of the amino acid sequence of any of SEQ ID NO: 1 , Angiopep-1 , Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7.

In one embodiment, the targeting moiety comprises the amino acid sequence of any one of SEQ

ID NO: 1-69, 71-73, 75-105 and 107-117. In certain embodiments, the targeting moiety comprises the amino sequence of Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97) (An2), Angiopep-3 (SEQ ID NO:107), Angiopep-4a (SEQ ID NO: 108), Angiopep-4b (SEQ ID NO: 109), Angiopep-5 (SEQ ID NO: 110), Angiopep-6 (SEQ ID NO: 111 ), Angiopep-7 (SEQ ID NO:112)) or reversed Angiopep-2 (SEQ ID NO: 117).

In certain embodiments, the targeting moiety has an amino acid sequence of any one of SEQ ID NO: 1-69, 71-73, 75-105 and 107-117 with one to 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid insertions or deletions. The insertions or deletions may be from either the N-terminus of the sequence, the C-terminus of the sequence, or a combination thereof. Other fragments include sequences where internal portions of the sequence are inserted or deleted.

Table 2: Exemplary targeting moieties

SEQ ID NO:

1 T F V Y G G C R A K R N N F K S A E D

2 T F Q Y G G C M G N G N N F V T E K E

3 P F F Y G G C G G N R N N F D T E E Y

4 s F Y Y G G C L G N K N N Y L R E E E

5 T F F Y G G C R A K R N N F K R A K Y

6 T -F F Y G G C R G K R N N F K R A K Y

7 T F F Y G G C R A K K N N Y K R A K Y

8 T F F Y G G C R G K K N N F K R A K Y

9 T F Q Y G G C R A K R N N F K R A K Y

10 T F Q Y G G C R G K K N N F K R A K Y

11 T F F Y G G C L G K R N N F K R A K Y

12 T F F Y G G S L G K R N N F K R A K Y

13 P F F Y G G C G G K K N N F K R A K Y

14 T F F Y G G C R G K G N N Y K R A K Y

15 P F F Y G G C R G K R N N F L R A K Y T F F Υ G G C R G K R N N F K R E K Y

P F F Υ G G C R A K K N N F K R A K E

τ F F Υ G G C R G K R N N F K R A K D τ F F Υ G G C R A K R N N F D R A K Y τ F F Υ G G C R G K K N N F K R A E Y

Ρ F F Υ G G C G A N R N N F K R A K Y

τ F F Υ G G C G G K K N N F K T A K Y τ F F Υ G G C R G N R N N F L R A K Y τ F F Υ G G C R G N R N N F K T A K Y τ F F Υ G G S R G N R N N F K T A K Y τ F F Υ G G C L G N G N N F K R A K Y τ F F Υ G G C L G N R N N F L R A K Y τ F F Υ G G C L G N R N N F K T A K Y τ F F Υ G G C R G N G N N F K s A K Y τ F F Υ G G C R G K K N N F D R E K Y τ F F Υ G G C R G K R N N F L R E K E τ F F Υ G G C R G K G N N F D R A K Y τ F F Υ G G S R G K G N N F D R A K Y τ F F Υ G G C R G N G N N F V T A K Y

Ρ F F Υ G G C G G K G N N Y V T A K Y

τ F F Υ G G C L G K G N N F L T A K Y

S F F Υ G G C L G N K N N F L T A K Y

τ F F Υ G G C G G N K N N F V R E K Y τ F F Υ G G C Μ G N K N N F V R E K Y τ F F Υ G G S Μ G N K N N F V R E K Y

Ρ F F Υ G G C L G N R N N Y V R E K Y

τ F F Υ G G C L G N R N N F V R E K Y τ F F Υ G G C L G N K N N Y V R E K Y τ F F Υ G G C G G N G N N F L T A K Y τ F F Υ G G C R G N R N N F L T A E Y τ F F Υ G G C R G N G N N F K s A E Y

Ρ F F Υ G G C L G N K N N F K T A E Y

τ F F Υ G G C R G N R N N F K T E E Y τ F F Υ G G C R G K R N N F K T E E D P F F Y G G C G G N G N N F V R E K Y

S F F Y G G C M G N G N N F V R E K Y

P F F Y G G C G G N G N N F L R E K Y

T F F Y G G C L G N G N N F V R E K Y

s F F Y G G C L G N G N N Y L R E K Y

T F F Y G G S L G N G N N F V R E K Y

T F F Y G G C R G N G N N F V T A E Y

T F F Y G G C L G K G N N F V s A E Y

T F F Y G G C L G N R N N F D R A E Y

T F F Y G G C L G N R N N F L R E E Y

T F F Y G G C L G N K N N Y L R E E Y

P F F Y G G C G G N R N N Y L R E E Y

P F F Y G G S G G N R N N Y L R E E Y

M R P D F C L E P P Y T G P C V A R I

A R I I R Y F Y N A K A G L C Q T F V Y G

Y G G c R A K R N N Y K S A E D c M R T C G

P D F c L E P P Y T G P C V A R I I R Y F Y

T F F Y G G c R G K R N N F K T E E Y

K F F Y G G c R G K R N N F K T E E Y

T F Y Y G G c R G K R N N Y K T E E Y c T F F Y G c C R G K R N N F K T E E

T F F Y G G c R G K R N N F K T E E Y c T F F Y G s C R G K R N N F K T E E

P F F Y G G c R G K R N N F K T E E Y

T F F Y G G c R G K R N N F K T K E Y

T F F Y G G K R G K R N N F K T E E Y

T F F Y G G c R G K R N N F K T K R Y

T F F Y G G K R G K R N N F K T A E Y

T F F Y G G K R G K R N N F K T A G Y

T F F Y G G K R G K R N N F K R E K Y

T F F Y G G K R G K R N N F K R A K Y

T F F Y G G c L G N R N N F K T E E Y

T F F Y G C G R G K R N N F K T E E Y

T F F Y G G R C G K R N N F K T E E Y 86 τ F F Y G G C L G N G N N F D T E E E

87 τ F Q Y G G C R G K R N N F K T E E Y

88 Υ Ν K E F G T F N T K G C E R G Y R F

89 R F K Y G G c L G N M N N F E T L E E

90 R F K Y G G c L G N K N N F L R L K Y

91 R F K Y G G c L G N K N N Y L R L K Y

92 Κ Τ K R K R K K Q R V K I A Y E E I F K

93 Κ Τ K R K R K K Q R V K I A Y

94 R G G R L S Y S R R F S T S T G R

95 R R L S Y S R R R F

96 R Q I K I W F Q N R R M K W K K

97 Τ F F Y G G S R G K R N N F K T E E Y

98 Μ R P D F C L E P P Y T G P c V A R I

I R Y F Y N A K A G L c Q T F V Y G G

C R A K R N N F K S A E D c M R T C G G

99 τ F F Y G G C R G K R N N F K T K E Y

100 R F K Y G G C L G N K N N Y L R L K Y

101 Τ F F Y G G C R A K R N N F K R A K Y

102 Ν A K A G L C Q T F V Y G G C L A K R N

Ε S A E D C M R T C G G A

103 Υ G G C R A K R N N F K S A E D C M R T

G A

104 G L C Q T F V Y G G C R A K R N N F K s

105 L C Q T F V Y G G C E A K R N N F K S A

107 Τ F F Y G G S R G K R N N F K T E E Y

108 R F F Y G G S R G K R N N F K T E E Y

109 R F F Y G G S R G K R N N F K T E E Y

110 R F F Y G G S R G K R N N F R T E E Y

111 Τ F F Y G G S R G K R N N F R T E E Y

112 Τ F F Y G G S R G R R N N F R T E E Y

113 C T F F Y G G S R G K R N N F K T E E Y

114 τ F F Y G G S R G K R N N F K T E E Y C

115 C T F F Y G G S R G R R N N F R T E E Y

116 τ F F Y G G S R G R R N N F R T E E Y C 1 17 Y E E T K F N N R K G R S G G Y F F T

In certain embodiments, the targeting moiety portion of the polypeptide may be modified (e.g. , using any of the polypeptide modifications described herein) such that the compound of the invention is a peptidomimetic.

In one embodiment, the targeting moiety comprises an amino acid sequence having the formula:

X1 -X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13-X14-X15-X16-X17-X18-X19 where each of X1 -X19 (e.g. , X1 -X6, X8, X9, X1 1 -X14, and X16-X19) is, independently, any amino acid (e.g. , a naturally occurring amino acid such as Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) or absent and at least one (e.g. , 2 or 3) of X1 , X10, and X15 is arginine. In some embodiments, X7 is Ser or Cys; or X10 and X15 each are independently Arg or Lys. In some embodiments, the residues from X1 through X19, inclusive, are substantially identical to any of the amino acid sequences of any one of SEQ ID NOS: 1 -69, 71-73, 75-105 and 107-1 17 (e.g. , Angiopep-1 , Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7). In some embodiments, at least one (e.g. , 2, 3, 4, or 5) of the amino acids X1 -X19 is Arg.

In one embodiment, the targeting moiety comprises the amino acid sequence Lys-Arg-X3-X4-X5- Lys (formula la), where X3 is Asn or Gin; X4 is Asn or Gin; and X5 is Phe, Tyr, or Trp; where the targeting moiety optionally includes one or more D-isomers of an amino acid recited in formula la (e.g., a D-isomer of Lys, Arg, X3, X4, X5, or Lys).

More particularly, the targeting moiety may comprise the amino acid sequence Lys-Arg-X3-X4- X5-Lys (formula la), where X3 is Asn or Gin; X4 is Asn or Gin; and X5 is Phe, Tyr, or Trp; where the targeting moiety is fewer than 19 amino acids in length (e.g. , fewer than 18, 17, 16, 15, 14, 12, 10, 1 1 , 8, or 7 amino acids, or any range between these numbers); and where the targeting moiety optionally includes one or more D-isomers of an amino acid recited in formula la (e.g. , a D-isomer of Lys, Arg, X3, X4, X5, or Lys).

In another embodiment, the targeting moiety comprises the amino acid sequence of Z1 -Lys-Arg- X3-X4-X5-Lys-Z2 (formula lb), where X3 is Asn or Gin; X4 is Asn or Gin; X5 is Phe, Tyr, or Trp; Z1 is absent, Cys, Gly, Cys-Gly, Arg-Gly, Cys-Arg-Gly, Ser-Arg-Gly, Cys-Ser-Arg-Gly, Gly-Ser-Arg-Gly, Cys- Gly-Ser-Arg-Gly, Gly-Gly-Ser-Arg-Gly, Cys-Gly-Gly-Ser-Arg-Gly, Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Tyr-Gly- Gly-Ser-Arg-Gly, Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Cys-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Phe-Phe-Tyr-Gly-Gly- Ser-Arg-Gly, Cys-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly, or Cys-Thr- Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly; and Z2 is absent, Cys, Tyr, Tyr-Cys, Cys-Tyr, Thr-Glu-Glu-Tyr, or Thr- Glu-Glu-Tyr-Cys; and where the targeting moiety optionally comprises one or more D-isomers of an amino acid recited in formula lb, Z1 , or Z2.

In certain embodiments, the targeting moiety comprises the amino acid sequence Lys-Arg-Asn- Asn-Phe-Lys. In other embodiments, the targeting moiety comprises the amino acid sequence of Lys- Arg-Asn-Asn-Phe-Lys-Tyr. In still other embodiments, the targeting moiety comprises the amino acid sequence of Lys-Arg-Asn-Asn-Phe-Lys-Tyr-Cys.

In certain embodiments, the targeting moiety comprises the amino acid sequence of X1 -X2-Asn-

Asn-X5-X6 (formula l la), where X1 is Lys or D-Lys; X2 is Arg or D-Arg; X5 is Phe or D-Phe; and X6 is Lys or D-Lys; and where at least one (e.g. , at least two, three, or four) of X1 , X2, X5, or X6 is a D-amino acid. In certain embodiments, the targeting moiety comprises the amino acid sequence of X1-X2-Asn- Asn-X5-X6-X7 (formula Mb), where X1 is Lys or D-Lys; X2 is Arg or D-Arg; X5 is Phe or D-Phe; X6 is Lys or D-Lys; and X7 is Tyr or D-Tyr; and where at least one (e.g., at least two, three, four, or five) of X1 , X2, X5, X6, or X7 is a D-amino acid.

In the above embodiments, the targeting moiety may comprise the amino acid sequence of Thr-

Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr (An2), where any one or more amino acids are D-isomers. For example, the targeting moiety can have 1 , 2, 3, 4, or 5 amino acids which are D-isomers. In a preferred embodiment, one or more or all of positions 8, 10, and 1 1 can be D- isomers. In yet another embodiment, one or more or all of positions 8, 10, 1 1 , and 15 can have D- isomers.

In certain embodiments, the targeting moiety may comprise Thr-Phe-Phe-Tyr-Gly-Gly-Ser-D-Arg- Gly-D-Lys-D-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr (3D-An2); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn- Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P1 ); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-Lys- Thr-Glu-Glu-Tyr-Cys (P1 a); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu- Glu-Tyr-Cys (P1 b); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu-Glu-D-Tyr- Cys (P1 c); D-Phe-D-Tyr-Gly-Gly-Ser-D-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu-D-Glu-D- Tyr-Cys (P1 d); Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P2); Ser-Arg-Gly- Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P3); Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P4); Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P5); D-Lys-D-Arg-Asn-Asn-D-Phe-Lys-Thr-Glu- Glu-Tyr-Cys (P5a); D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu-Glu-Tyr-Cys (P5b); D-Lys-D-Arg-Asn- Asn-D-Phe-D-Lys-Thr-Glu-Glu-D-Tyr-Cys (P5c); Lys-Arg-Asn-Asn-Phe-Lys-Tyr-Cys (P6); D-Lys-D-Arg- Asn-Asn-D-Phe-Lys-Tyr-Cys (P6a); D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Tyr-Cys (P6b); Thr-Phe-Phe-Tyr- Gly-Gly-Ser-D-Arg-Gly-D-Lys-D-Arg-Asn-Asn-Phe-D-Lys-Thr-Glu-Glu-Tyr; and D-Lys-D-Arg-Asn-Asn-D- Phe-D-Lys-D-Tyr-Cys (P6c); or a fragment thereof. In other embodiments, the targeting moiety may comprises a sequence of one of the aforementioned peptidomimetics having from 0 to 5 (e.g., from 0 to 4, 0 to 3, 0 to 2, 0 to 1 , 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, or 4 to 5)

substitutions, deletions, or additions of amino acids.

In certain embodiments, the targeting moiety may comprise Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys- Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu; Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu; Ser-Arg- Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu; Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu; Lys-Arg-Asn- Asn-Phe-Lys-Thr-Glu-Glu; or Lys-Arg-Asn-Asn-Phe-Lys, or a fragment thereof.

In any of the above aspects, the targeting moiety may comprise Thr-Phe-Phe-Tyr-Gly-Gly-Ser-D- Arg-Gly-D-Lys-D-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr (3D-An2); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-

14

RECTIFIED SHEET (RULE 91.1) Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys (P1 ); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe- Lys-Thr-Glu-Glu-Tyr-Cys (P1 a); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr- Glu-Glu-Tyr-Cys (P1 b); Phe-Tyr-Gly-Gly-Ser-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu-Glu-D- Tyr-Cys (P1 c); D-Phe-D-Tyr-Gly-Gly-Ser-D-Arg-Gly-D-Lys-D-Arg-Asn-Asn-D-Phe-D-Lys-Thr-Glu-D-Glu- D-Tyr-Cys (P1 d) or a fragment thereof (e.g. , deletion of 1 to 7 amino acids from the N-terminus of P1 ,

P1 a, P1 b, P1 c, or P1 d; a deletion of 1 to 5 amino acids from the C-terminus of P1 , P1 a, P1 b, P1 c, or P1 d; or deletions of 1 to 7 amino acids from the N-terminus of P1 , P1 a, P1 b, P1 c, or P1 d and 1 to 5 amino acids from the C-terminus of P1 , P1 a, P1 b, P1 c, or P1 d).

In any of the targeting moieties described herein, the moiety may include additions or deletions of 1 , 2, 3, 4, or 5 amino acids (e.g. , from 1 to 3 amino acids) from an amino acid sequence described herein (e.g. , from Lys-Arg-X3-X4-X5-Lys).

In any of the targeting moieties described herein, the moiety may have one or more additional cysteine residues immediately N- or C- terminal to the targeting moiety or both. In other embodiments, the targeting moiety may have one or more additional tyrosine residues immediately N- or C- terminal to the targeting moiety or both. In yet further embodiments, the targeting moiety has the amino acid sequence Tyr-Cys and/or Cys-Tyr immediately N- or C- terminal to the targeting moiety or both.

In certain embodiments of any of the above aspects, the targeting moiety may be fewer than 15 amino acids in length (e.g. , fewer than 10 amino acids in length). Peptidomimetics

In addition to polypeptides consisting exclusively of naturally occurring amino acids,

peptidomimetics are also encompassed by the present invention. Peptidomimetics include sequences containing amino acids not naturally encoded by DNA (e.g. , non-naturally occurring or unnatural amino acid) as well as modified amino acid sequences containing additional chemical moieties that do not normally form part of a polypeptide.

Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH₂(CH₂)_nCOOH wherein n is 2-6, sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, phenylglycine, citrulline, methionine sulfoxide, cysteic acid, ornithine and hydroxyproline.

Peptidomimetics containing certain non-naturally occurring amino acids can be produced by recombinant expression technology by known techniques including:

Altering the composition of growth media - deplete an amino acid from the growth media and force incorporation of a related but non-natural amino acid that has been added instead (this approach has been used for radioactive labelling of proteins (SILAC) and is described at http://www. ncbi. nlm. nih.gov/pubmed)

Utilising a cell based expression system that has engineered tRNA synthases/tRNAs that recognise non-natural amino acids and are recognised by redundant codons (for a review see http://www. ncbi. nlm. nih.gov/pubmed/20307192).

Recombinantly produced polypeptides can also be chemically modified to change the amino acid side chains of naturally occurring amino acids into non-naturally occurring amino acids. This may require a particular motif to be present in the polypeptide as is the case for modification of a cysteine to formylglycine by formylglycine generating enzymes (see http://www. ncbi. nlm. nih.gov/pubmed/17450134).

More generally, modifications include those by natural processes, such as posttranslational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a sequence including the polypeptide backbone, the amino acid side chains and the amino- or carboxy- terminus. The same type of modification may be present in the same or varying degrees at several sites in a given peptidomimetic, and a peptidomimetic may contain more than one type of modification.

Peptidomimetics may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic peptidomimetics may result from posttranslational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g. , fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

Examples of peptidomimetics include (1 ) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g. , acetyl, hexanoyl, octanoyl) an aroyl group (e.g. , benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy-terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to an antibody or other biological ligand and other types of derivatives, dd chemical groups at the polypeptide termini, such that the modified (poly)peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the

(poly)peptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of (poly)peptides in human serum (Powell et al., Pharm. Res. 10: 1268-1273, 1993). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified (poly)peptides consisting of polypeptides bearing an N-terminal acetyl group and/or a C-terminal amide group.

Isolation of the Precursor

Isolation may be carried out by methods well known in the art. Typically, this may comprise harvesting the culture supernatant by centrifugation/filtration followed by affinity purification. Where an Ig Fc tag is used, the supernatant may be applied to a column packed with a protein A resin (e. g. rProtein A sepharose fast flow available from GE healthcare) which has been pre-equilibrated into phopshate- buffered saline pH 7.4. Following application of the sample to the column, the resin may be washed with several column washes of neutral buffer, for example 50 mM tris pH 7.4 + 150. Note also that more stringent washes such as with a high salt concentration or a detergent included in the buffer may be performed at this point. The bound protein may then be eluted with several column volumes of a low pH buffer e. g. 100 mM glycine pH 2.0. The fraction(s) containing eluted protein should be immediately neutralised with a suitable buffer e. g. 1 M tris pH 8.0.

Following cleavage of the precursor, the resulting polypeptide or peptidomimetic may be further purified by any known method e.g. gel filtration. Concentrate the neutralised sample to an appropriate volume and then apply to a suitable column (e. g. Superdex75 available from GE Healthcare) which has been pre-equilibrated in a suitable buffer e. g. 50 mM tris pH 7.4. An isocratic elution should then be performed and protein-containing fractions should be retained and further analysed.

Treatment of lysosomal storage disorders

The present invention also features methods for treatment of lysosomal storage disorders such as MPS-I I. MPS-I I is characterized by cellular accumulation of glycosaminoglycans (GAG) which results from the inability of the individual to break down these products.

In certain embodiments, treatment is performed on a subject who has been diagnosed with a mutation in the IDS gene, but does not yet have disease symptoms (e.g. , an infant or subject under the age of 2). In other embodiments, treatment is performed on an individual who has at least one MPS-I I symptom (e.g. , any of those described herein).

MPS-I I is generally classified into two general groups, severe disease and attenuated disease.

The present invention can involve treatment of subjects with either type of disease. Severe disease is characterized by CNS involvement. In severe disease the cognitive decline, coupled with airway and cardiac disease, usually results in death before adulthood. The attenuated form of the disease general involves only minimal or no CNS involvement. In both severe and attenuated disease, the non-CNS symptoms can be as severe as those with the "severe" form.

Initial MPS-I I symptoms begin to manifest themselves from about 18 months to about four years of age and include abdominal hernias, ear infections, runny noses, and colds. Symptoms include coarseness of facial features (e.g. , prominent forehead, nose with a flattened bridge, and an enlarged tongue), large head (macrocephaly), enlarged abdomen, including enlarged liver (heptaomegaly) and enlarged spleen (slenomegaly), and hearing loss. The methods of the invention may involve treatment of subjects having any of the symptoms described herein. MPS-I I also results in joint abnormalities, related to thickening of bones.

Treatment may be performed in a subject of any age, starting from infancy to adulthood.

Subjects may begin treatment at birth, six months, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 15, or 18 years of age.

Administration and dosage

The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a polypeptide or peptidomimetic of the invention. The pharmaceutical composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. , 1985. For a brief review of methods for drug delivery, see, e.g. , Langer {Science 249: 1527-1533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides pharmaceutical compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g. , water, buffered water, saline, PBS, and the like. The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. The invention also provides pharmaceutical compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention provides pharmaceutical compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.

Pharmaceutical compositions for parenteral administration may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The pharmaceutical compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The pharmaceutical composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

The pharmaceutical compositions containing an effective amount can be administered for prophylactic or therapeutic treatments. In prophylactic applications, pharmaceutical compositions can be administered to a subject diagnosed as having mutation associated with a lysosomal storage disorder (e.g. , a mutation in the IDS gene). Pharmaceutical compositions of the invention can be administered to the subject (e.g. , a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of the disorder. In therapeutic applications, pharmaceutical compositions are administered to a subject (e.g. , a human) already suffering from a lysosomal storage disorder (e.g. , MPS-I I) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount adequate to accomplish this purpose is defined as a "therapeutically effective amount," an amount of a polypeptide or peptidomimetic sufficient to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of a lysosomal storage disease, an amount of a polypeptide or peptidomimetic that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of polypeptide or peptidomimetic is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.

Amounts effective for this use may depend on the severity of the disease or condition and the weight and general state of the subject. Idursulfase is recommended for weekly intravenous administration of 0.5 mg/kg. A polypeptide or peptidomimetic of the invention may, for example, be administered at an equivalent dosage (i.e. , accounting for the additional molecular weight of the fusion protein vs. idursulfase) and frequency. The polypeptide or peptidomimetic may be administered at an iduronase equivalent dose, e.g. , 0.01 , 0.05, 0.1 , 0.5, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, or 5 mg/kg weekly, twice weekly, every other day, daily, or twice daily. The therapeutically effective amount of the pharmaceutical compositions of the invention and used in the methods of this invention applied to mammals (e.g. , humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain polypeptides and peptidomimetics of the invention exhibit an enhanced ability to cross the BBB and to enter lysosomes, the dosage of the polypeptides and peptidomimetics of the invention can be lower than (e.g. , less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1 % of) the equivalent dose of required for a therapeutic effect of the corresponding enzyme. The agents of the invention are administered to a subject (e.g. , a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject (e.g. , reduction of GAG accumulation). Therapeutically effective amounts can also be determined empirically by those of skill in the art.

Single or multiple administrations of the pharmaceutical compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

The polypeptides or peptidomimetics of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy.

When the polypeptides or peptidomimetics of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a polypeptide or peptidomimetic of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

The following examples are intended to illustrate, rather than limit, the invention. Example 1

Design of IDS-Angiopep-2 fusion proteins

A IDS-Angiopep-2 fusion protein was designed. The IDS cDNA was obtained from Origene (Cat. No. RC219187). An N-terminal fusion (An2-IDS-Fc, SEQ ID NO: 120) was generated. A control without Angiopep-2 was also generated (IDS-Fc).

Human lgG Fc cDNA was custom synthesized, cloned into Nhe\ site of pcDNA3.1 plasmid and fully sequenced (GeneArt®, Life technologies™). IL2 secretion signal was included at the N-terminus to allow secretion of the Fc fusion protein in the cell media when expressed in CHO cells. A TEV recognition signal (ENLYFQG) and a short linker (GSS) were added at the C-terminus to allow tag removal after purification of the Fc fusion protein. Fc-IDS and Fc-An2-IDS were generated by inserting cDNA coding for the mature form of IDS and An2-IDS (without the native IDS peptide signal) into pcDNA3.1-HuFcTEV (Figure 2).

Example 2

Expression and activity of recombinant hIDS proteins in CHO-S cells

These constructs were then expressed in CHO-S cells grown in suspension. IDS constructs were expressed by transient transfection in Freestyle CHO-S cells (Invitrogen), using linear 25 KDa polyethyleneimine (PEI, Polyscience) as the transfection reagent. In one example, DNA ( 1 mg) was mixed with 70 ml Freestyle CHO Expression medium (Invitrogen) and incubated at room temperature for 15 min. PEI (2 mg) was separately incubated in 70 ml medium for 15 minutes, and then DNA and PEI solutions were mixed and further incubated for 15 min. The DNA/PEI complex mixture was added to 360 ml of medium containing 1 x 10⁹ CHO-S cells. After a four-hour incubation at 37°C, 8% C0₂ with moderate agitation, 500 ml of warm medium was added. CHO-S cells were further incubated for 5 days in the same conditions before harvesting.

Fc-IDS and Fc-An2-IDS were purified from CHO media using Protein A-agarose chromatography.

The Fc moiety was removed using TEV protease, and untagged IDS and An2-IDS (SEQ ID NO: 1 19) were further purified using size exclusion chromatography on a Superdex 75 column.

As can be seen in Figure 3, SDS-PAGE analysis shows that a >90% pure An2-IDS protein was produced with the Fc fusion partner (lane 3), similar to An2-IDS produced with a C-terminal (lane 1 ) or a N-terminal (lane 2) histidine-tag. The presence of intact angiopep-2 in the purified An2-IDS protein was confirmed by immunoblotting, mass spectrometry analysis (MALDI) and N-terminal aminoacid sequencing (EDMAN).

Example 3

In vitro enzymatic activity

Protocol for IDS Enzymatic Specific Activity

First, the concentration of proteins in JR-032 and fusion protein samples are determined by microBCA (bicinchoninic acid) (Smith, P. K. et al. , 1986, Anal. Biochem. , 150(1 ): 76-85). Test solutions are prepared by diluting JR-032 and fusion proteins 1/200 in Triton-X100 containing diluted buffer. A standard solution is prepared by diluting 1 mL 4-MU (4-methylumbelliferone) Stock Solution (0.01 mol/L) in 11.5 mL of Triton-X100 containing buffer (final concentration 800 μιτιοΙ/L), followed by preparation of serial dilutions of this standard solution by diluting 500 μΙ_ of 800 μηιοΙ/L in 500μΙ_ of Triton X100 containing buffer to make a 400 μηιοΙ/Ι_ solution. The process is repeated to have the following dilutions: 800, 400, 200, 100, 50, 25, 12.5 and 6.25 mol/L The solutions are distributed as follows: 10 μΙ_ each of the blank solution (Triton-X100 containing diluted buffer) in 2 wells (n=2), standard solution (6.25 μηιοΙ/Ι_ to 800 μηιοΙ/Ι_) in 2 wells (n=2) and the test solutions in 4 wells each (n=4) of a microplate, respectively. Subsequently to each well is added 100 μΙ_ of the substrate solution 4-methylumbelliferyl sulfate potassium salt (4-MUS) and the solutions are mixed gently. The plate is covered and placed in an incubator adjusted to 37^°C.

After 60 minutes, 190 μΙ_ of the stop solution is added to each well and mixed to stop the reaction. The plate is set in the fluorescence plate reader and the fluorescence intensity at excitation wavelength of 355 nm and detection wavelength of 460 nm is determined. The same measurement is performed with the reference material if comparison is required among tests.

Method of calculation:

Concentration of 4-MU produced from the test solution:

The concentration of 4-MU, Cu (μιτιοΙ/L), produced from the test solution was determined using the following formula.

w 10⁶

Cs

176.17 50 x 100

w: Amount (mg) of 4-MU ( 176.17: Molecular weight of 4-MU)

Cs: Concentration (μιτιοΙ/L) in the standard solution

Cu = Cs\

As

Au: Fluorescence intensity of the test solution

As: Fluorescence intensity of the standard solution

Specific activity of the sample solution:

The specific activity, B (mU/mg), of the sample solution was determined using the following formula.

C: Dilution factor of the desalted test substance

B: Specific activity (mU/mg)

P: Concentration (mg/mL) of proteins in the desalted test substance

Example 5

Brain perfusion

The brain perfusion of JR-032, An2-IDS and IDS was analyzed (Figure 3). As shown in the graph of Figure 3, An2-IDS-Fc showed greater volume of distribution in the brain, capillaries, and parenchyma when compared to the enzyme alone (JR-032). Protocol for in situ brain perfusion

The in situ mice brain perfusion method was established in the laboratory from the protocol described by Dagenais et al. , 2000. Briefly, the surgery was performed on sedated mice, injected intraperitoneal (i. p. ) with Ketamine / Xylazine (140/8 mg/kg). The right common carotid artery was exposed and ligated at the level of the bifurcation. The common carotid was then catheterized rostrally with polyethylene tubing (0.30 mm i.d. x 0.70 mm o.d. ) filled with saline/heparan (25 U/ml) solution mounted on a 26-gauge needle. The studied molecule was radiolabeled with ²⁵l in the days preceding the experiment using iodo-Beads from Pierce. Free iodine was removed on gel filtration column followed by extensive dialysis (cut-off 10 kDa). Radiolabeled proteins were dosed using the Bradford assay and JR-032 as the standard.

Prior to surgery, perfusion buffer consisting of KREBS-bicarbonate buffer - 9mM glucose was prepared and incubated at 37° C, pH at 7.4 stabilized with 95 % 0₂: 5% C0₂. A syringe containing radiolabeled compound added to the perfusion buffer was placed on an infusion pump (Harvard pump PHD2000; Harvard apparatus) and connected to the catheter. Immediately before the perfusion, the heart was severed and the brain was perfused for 2 min at a flow rate of 2.5 ml/min. All perfusions for IDS and An2-IDS conjugates were performed at a concentration of 5 nM. After perfusion, the brain was briefly perfused with tracer-free solution to wash out the blood vessels for 30s. At the end of the perfusion, the mice were immediately sacrificed by decapitation and the right hemisphere was isolated on ice and homogenized in Ringer/Hepes buffer before being subjected to capillary depletion. Capillary depletion

The capillary depletion method allows the measure of the accumulation of the perfused molecule into the brain parenchyma by eliminating the binding of tracer to capillaries. The capillary depletion protocol was adapted from the method described by Triguero et al. , 1990. A solution of Dextran (35%) was added to the brain homogenate to give a final concentration of 17.5%. After thorough mixing by hand the mixture was centrifuged (10 minutes at 10000 rpm). The resulting pellet contains mainly the capillaries and the supernatant corresponds to the brain parenchyma.

Determination of tracer signal

Aliquots of homogenates, supernatants, pellets and perfusates were taken to measure their contents in radiolabeled molecules. [ ²⁵l]-samples were counted in a Wizard 1470 Automatic Gamma Counter (Perkin-Elmer Inc, Woodbridge, ON). All aliquots were precipitated with trichloroacetic acid in order to get the radiolabeled precipitated protein fractions. Results are expressed in term of volume distribution (ml/100g/2min) for the different brain compartments.

Other embodiments

All patents, patent applications, and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

What is claimed is:

Claims

1. A composition comprising a polypeptide or peptidomimetic, which polypeptide or peptidomimetic comprises:

(a) a targeting moiety with an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-117; and

(b) iduronate-2-sulfatase (IDS), an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS;

wherein said targeting moiety is capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier,

wherein said polypeptide or peptidomimetic has IDS activity, and.

wherein said composition (i) contains at least 90% polypeptide or peptidomimetic, measured as a percentage of the total protein content of the composition or (ii) contains less than 10% of impurities generated by aberrant cleavage of the polypeptide or peptidomimetic, relative to either the total protein content in said composition or to the amount of said polypeptide or peptidomimetic present in said composition.

2. The composition of claim 1 , wherein said IDS or said IDS fragment has the amino acid sequence of full length human IDS isoform a or a fragment thereof, or wherein said IDS analog has at least 70% identity to the sequence of full length human IDS isoform a.

3. The composition of claim 2, wherein said IDS or said IDS fragment has the sequence of full length human IDS isoform a or the mature form of human isoform a (amino acids 26-550 of human isoform a).

4. The composition of any one of claims 1 -3, wherein said polypeptide or peptidomimetic is capable of efficiently crossing the blood brain barrier.

5. The composition of any one of claims 1 -3, wherein said polypeptide or peptidomimetic is capable of targetting the lysosome.

6. The composition of any one of claims 1 -5, wherein said targeting moiety comprises the sequence of AngioPep-2 (SEQ ID NO:97).

7. The composition of any one of claims 1 -6, wherein said polypeptide or peptidomimetic has at least 90% identity to SEQ ID NO: 1 19.

8. A pharmaceutical composition comprising the composition of any one of claims 1 -7 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, comprising one or more nanoparticles, wherein said nanoparticle is conjugated to the composition defined in any one of claims 1-7.

10. The pharmaceutical composition according to claim 8, which comprises a liposome formulation of the composition defined in any one of claims 1 -7.

1 1. A method of treating or treating prophylactically mucopolysaccharidosis Type I I (MPS-I I), said method comprising administering to a subject in need thereof a composition as defined in any one of claims 1 to 7 or a pharmaceutical composition as defined in any one of claims 8-10.

12. The method of claim 11 , wherein said subject has been diagnosed with the severe form of MPS- II.

13. The method of claim 11 , wherein said subject has been diagnosed with the attenuated form of MPS-I I.

14. The method of claim 1 1 , wherein said subject has neurological symptoms.

15. The method of claim 11 , wherein said subject starts treatment under 5 years of age.

16. The method of claim 15, wherein said subject starts treatment under 3 years of age.

17. The method of claim 11 , wherein said subject is an infant.

18. The method of claim 11 , wherein said administering comprises parenteral administration.

19. A method of producing a polypeptide or peptidomimetic comprising (a) a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, which targeting moiety exhibits an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-1 17; and (b) IDS, an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS; comprising:

(i) providing a host cell that is genetically engineered to express a precursor comprising (a) IDS, an IDS fragment or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS; (b) a targeting moiety exhibiting an amino acid sequence with at least 70% identity to any one of SEQ ID NO:97, 1 -96 and 98-117; (c) a tag region; and optionally (d) a cleavable sequence that separates said tag region from (a) and (b);

(ii) culturing said host cell under conditions in which the cell expresses said precursor;

(iii) isolating said precursor from the host cell culture; and

(iv) treating said precursor with a protease enzyme that recognizes and cleaves said cleavable sequence;

thereby producing a polypeptide or peptidomimetic comprising (a) a targeting moiety capable of transporting said polypeptide or peptidomimetic to the lysosome and/or across the blood brain barrier, which targeting moiety exhibits an amino acid sequence with at least 70% identity to any one of SEQ ID NO:97, 1-96 and 98-117; and (b) IDS, an IDS fragment, or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS.

20. The method of claim 19, further comprising, after step (iv), gel filtration.

21. The method of claim 19 or claim 20, wherein said IDS or said IDS fragment has the amino acid sequence of full length human IDS isoform a or a fragment thereof, or wherein said IDS analog has at least 70% identity to the sequence of full length human IDS isoform a.

22. The method of claim 21 , wherein said IDS or said IDS fragment has the sequence of full length human IDS isoform a or the mature form of human isoform a (amino acids 26-550 of human isoform a).

23. The method of any one of claims 19-22, wherein said targeting moiety comprises the amino acid sequence of AngioPep-2 (SEQ ID NO:97).

24. The method of any one of claims 19-23, wherein said tag is an Ig Fc tag.

25. The method of claim 24, wherein said tag is an IgG Fc tag.

26. The method of any one of claims 19-25, wherein said protease enzyme does not display proteolytic activity against said targeting moiety or said IDS, IDS fragment, or IDS analog.

27. The method of claim 26, wherein said protease enzyme is Tobacco Etch Virus (TEV) protease and said cleavable sequence is capable of being cleaved by TEV protease.

28. The method of claim 27, wherein said cleavable sequence comprises Glu-Asn-Leu-Tyr-Phe-Gln- Ser or Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

29. The method of any one of claims 19-28, wherein said polypeptide or peptidomimetic has at least 90% identity to SEQ ID NO: 1 19.

30. A polypeptide or peptidomimetic obtainable by any of the methods of claims 19-29.

31. A precursor comprising:

(a) IDS, an IDS fragment or an IDS analog which comprises an amino acid sequence having at least 70% identity to IDS;

(b) a targeting moiety exhibiting an amino acid sequence with at least 70% identity to any one of SEQ ID NOS. 1 -69, 71 -73, 75-105 and 107-117;

(c) a tag region; and optionally

(d) a cleavable sequence that separates said tag region from (a) and (b).

32. The precursor of claim 31 , wherein said IDS or said IDS fragment has the amino acid sequence of full length human IDS isoform a or a fragment thereof, or wherein said IDS analog has at least 70% identity to the sequence of full length human IDS isoform a.

33. The precursor of claim 32, wherein said IDS or said IDS fragment has the sequence of full length human IDS isoform a or the mature form of human isoform a (amino acids 26-550 of human isoform a).

34. The precursor of any one of claims 31-33, wherein said targeting moiety comprises the amino acid sequence of AngioPep-2 (SEQ ID NO:97).

35. The precursor of any one of claims 31-34, wherein said tag is an Ig Fc tag.

36. The precursor of claim 35, wherein said tag is an IgG Fc tag.

37. The precursor of any one of claims 31-36, wherein said cleavable sequence is capable of being cleaved by TEV protease.

38. The precursor of claim 36, wherein said cleavable sequence comprises Glu-Asn-Leu-Tyr-Phe- Gln-Ser or Glu-Asn-Leu-Tyr-Phe-Gln-Gly.

39. The precursor of any one of claims 31-38, wherein said precursor has at least 90% identity to SEQ ID NO: 120.