Lebowitz, Jonathan H. (Frontenac, MO, US)
Beverley, Stephen M. (Clayton, MO, US)

Plaque It! Sponsored by: Flash of Genius

10/136841

07/08/2008

04/30/2002

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ZyStor Therapeutics, Inc. (Milwaukee, WI, US)

514/8

514/2, 424/185.1

A01N37/18; A61K38/16

514/8, 424/143.1, 424/192.1, 514/2, 424/198.1

4309776	Intravascular implantation device and method of using the same	January, 1982	Berguer	3/1
4522811	Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides	June, 1985	Eppstein et al.	514/2
4749570	Targeting conjugates of albumin and therapeutic agents	June, 1988	Poznansky	424/85
4801575	Chimeric peptides for neuropeptide delivery through the blood-brain barrier	January, 1989	Pardridge	514/4
4902505	Chimeric peptides for neuropeptide delivery through the blood-brain barrier	February, 1990	Pardridge et al.	424/85
5236838	Enzymatically active recombinant glucocerebrosidase	August, 1993	Rasmussen et al.
5258453	Drug delivery system for the simultaneous delivery of drugs activatable by enzymes and light	November, 1993	Kopecek et al.	525/54.1
5356804	Cloning and expression of biologically active human α-galactosidase A	October, 1994	Desnick et al.	435/208
5405942	Prepro insulin-like growth factors I and II	April, 1995	Bell et al.	536/23.1
5470828	Peptide analogs of insulin-like growth factor II	November, 1995	Ballard et al.	514/12
5476779	DNA encoding insulin-like growth factor II isolated from rainbow trout	December, 1995	Chen et al.	435/325
5549892	Enhanced in vivo uptake of glucocerebrosidase	August, 1996	Friedman et al.
5580757	Cloning and expression of biologically active α-galactosidase A as a fusion protein	December, 1996	Desnick et al.	435/69.7
5633234	Lysosomal targeting of immunogens	May, 1997	August et al.	514/44
5633235	Triciribine and analogs as antiviral drugs	May, 1997	Townsend et al.	514/49
5704910	Implantable device and use therefor	January, 1998	Humes	604/52
5736363	IGF-II analogues	April, 1998	Edwards et al.	435/69.4
5798366	Method for treatment of CNS-involved lysosomal storage diseases	August, 1998	Platt et al.
5817623		October, 1998	Ishii	514/3
5817789	Chimeric proteins for use in transport of a selected substance into cells	October, 1998	Heartlein et al.
5827703	Methods and composition for in vivo gene therapy	October, 1998	Debs et al.	435/172.3
5854025	IGF-II analogues	December, 1998	Edwards et al.	435/69.4
5977307	Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins	November, 1999	Friden et al.	530/350
5981194	Use of p97 and iron binding proteins as diagnostic and therapeutic agents	November, 1999	Jefferies et al.	435/7.1
6020144	Sustained delivery device comprising a Leishmania protozoa and methods of making and using the same	February, 2000	Gueiros-Filho et al.	435/7.22
6027921	Chimeric proteins for use in transport of a selected substance into cells and DNA encoding chimeric proteins	February, 2000	Heartlein et al.
6066626	Compositions and method for treating lysosomal storage disease	May, 2000	Yew et al.
6083725	Tranfected human cells expressing human α-galactosidase A protein	July, 2000	Selden et al.	435/69.8
6118045	Lysosomal proteins produced in the milk of transgenic animals	September, 2000	Reuser et al.	800/14
6226603	Method for the prediction of binding targets and the design of ligands	May, 2001	Freire et al.	703/11
6235874	Production of biologically active recombinant insulin-like growth factor II polypeptides	May, 2001	Wu et al.	530/303
6262026	Chimeric proteins for use in transport of a selected substance into cells	July, 2001	Heartlein et al.
6270989	Protein production and delivery	August, 2001	Treco et al.	435/69.1
6273598	Computer system and methods for producing morphogen analogs of human OP-1	August, 2001	Keck et al.	364/578
6281010	Adenovirus gene therapy vehicle and cell line	August, 2001	Gao et al.	435/325
6284875	Method for recovering proteins from the interstitial fluid of plant tissues	September, 2001	Turpen et al.
6329501	Methods and compositions for targeting compounds to muscle	December, 2001	Smith et al.
6344436	Lipophilic peptides for macromolecule delivery	February, 2002	Smith et al.	514/2
6348194	Tumor specific internalizing antigens and methods for targeting therapeutic agents	February, 2002	Huse et al.	424/143
6441147	Method for recovering proteins from the interstitial fluid of plant tissues	August, 2002	Turpen et al.
6451600	Enzymatically active recombinant glucocerebrosidase	September, 2002	Rasmussen et al.
6455494	Use of p97 and iron binding proteins as diagnostic and therapeutic agents	September, 2002	Jefferies et al.	514/2
6472140	α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.	October, 2002	Tanzi et al.	435/4
6537785	Methods of treating lysosomal storage diseases	March, 2003	Canfield	424/94.61
6566099	Nucleic acid encoding a chimeric polypeptide	May, 2003	Selden et al.	435/69.8
6569661	Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof	May, 2003	Qin et al.
6596500	Binding of retinoids to M6P/IGF-II receptor	July, 2003	Kang et al.	435/7.2
20010006635	USE OF HEPARINASE TO DECREASE INFLAMMATORY RESPONSES	July, 2001	Bennett et al.	424/94.1
20010025026	Chimeric proteins for use in transport of a selected substance into cells	September, 2001	Heartlein et al.	514/12
20020013953	Compositions and methods for treating enzyme deficiency	January, 2002	Reuser et al.	800/14
20020081654	Targeting hydrolase enzymes	June, 2002	Sandrin et al.	435/69.1
20020110551	Treatment of glycogen storage disease type II	August, 2002	Chen	424/94.61
20020142299	PTD-modified proteins	October, 2002	Davidson et al.	435/6
20030004236	Magnetic resonance imaging agents for detection and delivery of therapeutic agents and detection of physiological substances	January, 2003	Meade	524/98
20030021787	Method and devices for delivery of agents to breast milk ducts	January, 2003	Hung et al.	424/155.1
20030077806	Treatment of alpha-galactosidase a deficiency	April, 2003	Selden et al.	435/207
20030082176	Subcellular targeting of therapeutic proteins	May, 2003	LeBowitz et al.
20040029779	Methods of enhancing lysosomal storage disease therapy by modulation of cell surface receptor density	February, 2004	Zhu et al.	514/3
20040081645	Treatment of pompe's disease	April, 2004	Van Bree et al.	424/94.61
20040248262	Constructs for expressing lysomal polypeptides	December, 2004	Koeberl
20050026823	Use of the chaperone receptor-associated protein (RAP) for the delivery of therapeutic compounds to the brain and other tissues	February, 2005	Zankel et al.	514/12
20050058634	Methods for introducing mannose-6-phosphate and other oligosaccharides onto glycoproteins and its application thereof	March, 2005	Zhu	424/94.61

EP0599303	June, 1994			Peptide conjugate.
WO/1991/004014	April, 1991			METHOD FOR TRANSPORTING COMPOSITIONS ACROSS THE BLOOD BRAIN BARRIER
WO/1993/006216	April, 1993			FUSION PROTEINS TARGETED TO LYSOSOMES, FOR THE TREATMENT OF AIDS
WO/1995/002421	January, 1995			TRANSFERRIN RECEPTOR SPECIFIC LIGAND-NEUROPHARMACEUTICAL AGENT FUSION PROTEINS
WO/2000/053730	September, 2000			MEDICAL PREPARATIONS FOR THE TREATMENT OF ALPHA-GALACTOSIDASE A DEFICIENCY
WO/2001/019955	March, 2001			LYSOSOMAL TARGETING PATHWAY ENZYMES
WO/2002/044355	June, 2002			PROTOZOAN EXPRESSION SYSTEMS FOR LYSOSOMAL STORAGE DISEASE GENES
WO/2002/056907	July, 2002			MOLECULAR ANTIGEN ARRAY PRESENTING AMYLOID BETA
WO/2002/087510	November, 2002			SUBCELLULAR TARGETING OF THERAPEUTIC PROTEINS
WO/2003/032727	April, 2003			METHODS AND COMPOSITIONS FOR TARGETING UNDERGLYCOSYLATED PROTEINS ACROSS THE BLOOD BRAIN BARRIER
WO/2003/032913	April, 2003			METHODS AND COMPOSITIONS FOR TARGETING PROTEINS ACROSS THE BLOOD BRAIN BARRIER
WO/2003/057179	July, 2003			USE OF P97 AS AN ENZYME DELIVERY SYSTEM FOR THE DELIVERY OF THERAPEUTIC LYSOSOMAL ENZYMES
WO/2003/102583	December, 2003			TARGETED THERAPEUTIC PROTEINS

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Stucker, Jeffrey

Standley, Steven H.

Choate, Hall & Stewart LLP

This application claims the benefit of U.S. Ser. No. 60/287,531, filed Apr. 30, 2001; U.S. Ser. No. 60/304,609, filed Jul. 10, 2001; U.S. Ser. No. 60/329,461, filed Oct. 15, 2001, and U.S. Ser. No. 60/351,276, filed Jan. 23, 2002, the contents of which are incorporated by reference.

What is claimed is:

1. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only at a position selected from the group consisting of amino acid 9, amino acid 19, amino acid 26, and amino acid 27, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

2. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 1-7, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

3. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 62-67, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

4. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 29-40, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

5. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by an amino acid substitution selected from the group consisting of Tyr27Leu, Leu43Val, and Ser26Phe, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

6. The therapeutic fusion protein of claim 2, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

7. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Pompe Disease.

8. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Fabry Disease.

9. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Gaucher Disease.

10. A therapeutic fusion protein comprising: a lysosomal enzyme, and a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 1-7 and a substitution of Tyr27Leu, wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

11. The therapeutic fusion protein of claim 10, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

12. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Pompe Disease.

13. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Fabry Disease.

14. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Gaucher Disease.

This invention provides a means for specifically delivering proteins to a targeted subcellular compartment of a mammalian cell. The ability to target proteins to a subcellular compartment is of great utility in the treatment of metabolic diseases such as lysosomal storage diseases, a class of over 40 inherited disorders in which particular lysosomal enzymes are absent or deficient.

BACKGROUND

Enzyme deficiencies in cellular compartments such as the golgi , the endoplasmic reticulum, and the lysosome cause a wide variety of human diseases. For example, lysyl hydroxylase, an enzyme normally in the lumen of the endoplasmic reticulum, is required for proper processing of collagen; absence of the enzyme causes Ehlers-Danlos syndrome type VI, a serious connective tissue disorder. GnT II, normally found in the golgi , is required for normal glycosylation of proteins; absence of GnT II causes leads to defects in brain development. More than forty lysosomal storage diseases (LSDs) are caused, directly or indirectly, by the absence of one or more proteins in the lysosome.

Mammalian lysosomal enzymes are synthesized in the cytosol and traverse the ER where they are glycosylated with N-linked, high mannose type carbohydrate. In the golgi , the high mannose carbohydrate is modified on lysosomal proteins by the addition of mannose-6-phosphate (M6P) which targets these proteins to the lysosome. The M6P-modified proteins are delivered to the lysosome via interaction with either of two M6P receptors. The most favorable form of modification is when two M6Ps are added to a high mannose carbohydrate.

Enzyme replacement therapy for lysosomal storage diseases (LSDs) is being actively pursued. Therapy, except in Gaucher's disease, generally requires that LSD proteins be taken up and delivered to the lysosomes of a variety of cell types in an M6P-dependent fashion. One possible approach involves purifying an LSD protein and modifying it to incorporate a carbohydrate moiety with M6P. This modified material may be taken up by the cells more efficiently than unmodified LSD proteins due to interaction with M6P receptors on the cell surface. However, because of the time and expense required to prepare, purify and modify proteins for use in subcellular targeting, a need for new, simpler, more efficient, and more cost-effective methods for targeting therapeutic agents to a cellular compartment remains.

SUMMARY OF THE INVENTION

The present invention facilitates the treatment of metabolic diseases by providing targeted protein therapeutics that localize to a subcellular compartment of a cell where the therapeutic is needed. The invention simplifies preparation of targeted protein therapeutics by reducing requirements for posttranslational or postsynthesis processing of the protein. For example, a targeted therapeutic of the present invention can be synthesized as a fusion protein including a therapeutic domain and a domain that targets the fusion protein to a correct subcellular compartment. (“Fusion protein,” as used herein, refers to a single polypeptide having at least two domains that are not normally present in the same polypeptide. Thus, naturally occurring proteins are not “fusion proteins” as used herein.) Synthesis as a fusion protein permits targeting of the therapeutic domain to a desired subcellular compartment without complications associated with chemical crosslinking of separate therapeutic and targeting domains, for example.

The invention also permits targeting of a therapeutic to a lysosome in an M6P-independent manner. Accordingly, the targeted therapeutic need not be synthesized in a mammalian cell, but can be synthesized chemically or in a bacterium, yeast, protozoan, or other organism regardless of glycosylation pattern, facilitating production of the targeted therapeutic with high yield and comparatively low cost. The targeted therapeutic can be synthesized as a fusion protein, further simplifying production, or can be generated by associating independently-synthesized therapeutic agents and targeting moieties.

The present invention permits lysosomal targeting of therapeutics without the need for M6P addition to high mannose carbohydrate. It is based in part on the observation that one of the 2 M6P receptors also binds other ligands with high affinity. For example, the cation-independent mannose-6-phosphate receptor is also known as the insulin-like growth factor 2 (IGF-II) receptor because it binds IGF-II with high affinity. This low molecular weight polypeptide interacts with three receptors, the insulin receptor, the IGF-I receptor and the M6P/IGF-II receptor. It is believed to exert its biological effect primarily through interactions with the former two receptors while interaction with the cation-independent M6P receptor is believed to result predominantly in the IGF-II being transported to the lysosome where it is degraded.

Accordingly, the invention relates in one aspect to a targeted therapeutic including a targeting moiety and a therapeutic agent that is therapeutically active in a mammalian lysosome. “Therapeutically active,” as used herein, 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.

In one embodiment, 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. In another embodiment, the targeting moiety is an unglycosylated lysosomal targeting domain that binds the extracellular domain of the human cation-independent M6P receptor. In 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. In some embodiments, the targeting moiety binds to the receptor with a submicromolar dissociation constant (e.g. less than 10 ⁻⁸ M, less than 10 ⁻⁹ M, less than 10 ⁻¹⁰ M, or between 10 ⁻⁷ M and 10 ⁻¹¹ M) at or about pH 7.4 and with an dissociation constant at or about pH 5.5 of at least 10 ⁻⁶ M and at least ten times the dissociation constant at or about pH 7.4. In particular embodiments, the means for binding binds to the extracellular domain 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. In a further embodiment, 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.

In another embodiment, the targeting moiety is a lysosomal targeting domain that binds the extracellular domain of the human cation-independent M6P receptor but does not bind a mutein of the receptor in which amino acid 1572 is changed from isoleucine to threonine, or binds the mutein with at least ten-fold less affinity (i.e. with at least a ten-fold greater dissociation constant). In another embodiment, the targeting moiety is a lysosomal targeting domain capable of binding a receptor domain consisting essentially of repeats 10-15 of the human cation-independent M6P receptor: the lysosomal targeting domain can bind a protein that includes repeats 10-15 even if the protein includes no other moieties that bind the lysosomal targeting domain. Preferably, the lysosomal targeting domain can bind a receptor domain consisting essentially of repeats 10-13 of the human cation-independent mannose-6-phosphate receptor. More preferably, the lysosomal targeting domain can bind a receptor domain consisting essentially of repeats 11-12, repeat 11, or amino acids 1508-1566 of the human cation-independent M6P receptor. In each of these embodiments, the lysosomal targeting domain preferably binds the receptor or receptor domain with a submicromolar dissociation constant at or about pH 7.4. In one preferred embodiment, the lysosomal targeting domain binds with an dissociation constant of about 10 ⁻⁷ M. In another preferred embodiment, the dissociation constant is less than about 10 ⁻⁷ M.

In another embodiment, the targeting moiety is a binding moiety sufficiently duplicative of human IGF-II such that the binding moiety binds the human cation-independent M6P receptor. The binding moiety can be sufficiently duplicative of IGF-II by including an amino acid sequence sufficiently homologous to at least a portion of IGF-II, or by including a molecular structure sufficiently representative of at least a portion of IGF-II, such that the binding moiety binds the cation-independent M6P receptor. The binding moiety can be an organic molecule having a three-dimensional shape representative of at least a portion of IGF-II, such as amino acids 48-55 of human IGF-II, or at least three amino acids selected from the group consisting of amino acids 8, 48, 49, 50, 54, and 55 of human IGF-II. A preferred organic molecule has a hydrophobic moiety at a position representative of amino acid 48 of human IGF-II and a positive charge at or about pH 7.4 at a position representative of amino acid 49 of human IGF-II. In one embodiment, the binding moiety is a polypeptide including a polypeptide having antiparallel alpha-helices separated by not more than five amino acids. In another embodiment, the binding moiety includes a polypeptide with the amino acid sequence of IGF-I or of a mutein of IGF-I in which amino acids 55-56 are changed and/or amino acids 1-4 are deleted or changed. In a further embodiment, the binding moiety includes a polypeptide with an amino acid sequence at least 60% identical to human IGF-II; amino acids at positions corresponding to positions 54 and 55 of human IGF-II are preferably uncharged or negatively charged at or about pH 7.4.

In one embodiment, the targeting moiety is a polypeptide comprising the amino acid sequence phenylalanine-arginine-serine. In another embodiment, the targeting moiety is a polypeptide including an amino acid sequence at least 75% homologous to amino acids 48-55 of human IGF-II. In another embodiment, the targeting moiety includes, on a single polypeptide or on separate polypeptides, amino acids 8-28 and 41-61 of human IGF-II. In another embodiment, the targeting moiety includes amino acids 41-61 of human IGF-II and a mutein of amino acids 8-28 of human IGF-II differing from the human sequence at amino acids 9, 19, 26, and/or 27.

In some embodiments, the association of the therapeutic agent with the targeting moiety is labile at or about pH 5.5. In a preferred embodiment, association of the targeting moiety with the therapeutic agent is mediated by a protein acceptor (such as imidazole or a derivative thereof such as histidine) having a pKa between 5.5 and 7.4. Preferably, one of the therapeutic agent or the targeting moiety is coupled to a metal, and the other is coupled to a pH-dependent metal binding moiety.

In another aspect, the invention relates to a therapeutic fusion protein including a therapeutic domain and a subcellular targeting domain. The subcellular targeting domain binds to an extracellular domain of a receptor on an exterior surface of a cell. Upon internalization of the receptor, the subcellular targeting domain permits localization of the therapeutic domain to a subcellular compartment such as a lysosome, an endosome, the endoplasmic reticulum (ER), or the golgi complex, where the therapeutic domain is therapeutically active. In one embodiment, the receptor undergoes constitutive endocytosis. In another embodiment, the therapeutic domain has a therapeutic enzymatic activity. The enzymatic activity is preferably one for which a deficiency (in a cell or in a particular compartment of a cell) is associated with a human disease such as a lysosomal storage disease.

In further aspects, the invention relates to nucleic acids encoding therapeutic proteins and to cells (e.g. mammalian cells, insect cells, yeast cells, protozoans, or bacteria) comprising these nucleic acids. The invention also provides methods of producing the proteins by providing these cells with conditions (e.g. in the context of in vitro culture or by maintaining the cells in a mammalian body) permitting expression of the proteins. The proteins can be harvested thereafter (e.g. if produced in vitro) or can be used without an intervening harvesting step (e.g. if produced in vivo in a patient). Thus, the invention also provides methods of treating a patient by administering a therapeutic protein (e.g. by injection, in situ synthesis, or otherwise), by administering a nucleic acid encoding the protein (thereby permitting in vivo protein synthesis), or by administering a cell comprising a nucleic acid encoding the protein. In one embodiment, the method includes synthesizing a targeted therapeutic including a therapeutic agent that is therapeutically active in a mammalian lysosome and a targeting moiety that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, and administering the targeted therapeutic to a patient. The method can also include identifying the targeting moiety (e.g. by a recombinant display technique such as phage display, bacterial display, or yeast two-hybrid or by screening libraries for requisite binding properties). In another embodiment, the method includes providing (e.g. on a computer) a molecular model defining a three-dimensional shape representative of at least a portion of human IGF-II; identifying a candidate IGF-II analog having a three-dimensional shape representative of at least a portion of IGF-II (e.g. amino acids 48-55), and producing a therapeutic agent that is active in a mammalian lysosome and directly or indirectly bound to the candidate IGF-II analog. The method can also include determining whether the candidate IGF-II analog binds to the human cation-independent M6P receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of the human IGF-II open reading frame (SEQ ID NO:1) and its encoded protein (SEQ ID NO:2). Mature IGF-II lacks the signal peptide and COOH-cleaved regions.

FIG. 2 is a Leishmania codon-optimized IGF-II depicted in the XbaI site of pIR1-SAT; the nucleic acid is SEQ ID NO:3 and the encoded protein is SEQ ID NO:4.

FIG. 3 is a depiction of a preferred embodiment of the invention, incorporating a signal peptide sequence, the mature human β-glucuronidase sequence, a bridge of three amino acids, and an IGF-II sequence. The DNA sequence is SEQ ID NO:5 and the protein sequence is SEQ ID NO:6.

FIG. 4 depicts β-glucuronidase (GUS) activity in human mucopolysaccharidosis VII skin fibroblast GM4668 cells exposed to GUS, a GUS-IGF-II fusion protein (GILT-GUS), GILT-GUS with Δ1-7 and Y27L mutations in the IGF-II portion (GILT ² -GUS), or a negative control (DMEM).

FIG. 5 depicts GUS activity in GM4668 cells exposed to GUS (+β-GUS), GUS-GILT (+GILT), GUS-GILT in the presence of excess IGF-II (+GILT+IGF-II), or a negative control (GM4668).

FIG. 6 is an alignment of mature human IGF-I (SEQ ID NO:7) and IGF-II (SEQ ID NO:8), showing the A, B, C, and D domains.

FIG. 7 depicts GUS in GM4668 cells exposed to GUS, GUS-GILT, GUS-GILT, GUS-GILT with a deletion of the seven amino-terminal residues (GUS-GILT Δ1-7), GUS-GILT in the presence of excess IGF-II, GUS-GILT Δ1-7 in the presence of excess IGF-II, or a negative control (Mock).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “glycosylation independent lysosomal targeting” and “GILT” refer to lysosomal targeting that is mannose-6-phosphate-independent.

As used herein, “GILT construct” refers to a construct including a mannose-6-phosphate-independent lysosomal targeting portion and a therapeutic portion effective in a mammalian lysosome.

As used herein, “GUS” refers to β-glucuronidase, an exemplary therapeutic portion.

As used herein, “GUS-GILT” refers to a GILT construct with GUS coupled to an IGF-II targeting portion.

All references to amino acid positions in IGF-II refer to the positions in mature human IGF-II. Thus, for example, positions 1, 2, and 3 are occupied by alanine, tyrosine, and arginine, respectively.

The present invention facilitates treatment of metabolic diseases by providing targeted therapeutics that, when provided externally to a cell, enter the cell and localize to a subcellular compartment where the targeted therapeutic is active. The targeted therapeutic includes at least a therapeutic agent and a targeting moiety, such as a subcellular targeting domain of a protein, or, for lysosomal targeting, a means (e.g. a protein, peptide, peptide analog, or organic chemical) for binding the human cation-independent mannose-6-phosphate receptor.

Association Between Therapeutic Agent and Targeting Moiety

The therapeutic agent and the targeting moiety are necessarily associated, directly or indirectly. In one embodiment, the therapeutic agent and the targeting moiety are A non-covalently associated. The association is preferably stable at or about pH 7.4. For example, the targeting moiety can be biotinylated and bind avidin associated with the therapeutic agent. Alternatively, the targeting moiety and the therapeutic agent can each be associated (e.g. as fusion proteins) with different subunits of a multimeric protein. In another embodiment, the targeting moiety and the therapeutic agent are crosslinked to each other (e.g. using a chemical crosslinking agent).

In a preferred embodiment, the therapeutic agent is fused to the targeting moiety as a fusion protein. The targeting moiety can be at the amino-terminus of the fusion protein, the carboxy-terminus, or can be inserted within the sequence of the therapeutic agent at a position where the presence of the targeting moiety does not unduly interfere with the therapeutic activity of the therapeutic agent.

Where the therapeutic agent is a heteromeric protein, one or more of the subunits can 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. The alpha subunit, the beta subunit, or both can be associated with a targeting moiety in accordance with the present invention. If, for example, the alpha subunit is associated with a targeting moiety and is coexpressed with the beta subunit, an active complex is formed and targeted appropriately (e.g. to the lysosome).

For targeting a therapeutic to the lysosome, the therapeutic agent can be connected to the targeting moiety through an interaction that is disrupted by decreasing the pH from at or about 7.4 to at or about 5.5. The targeting moiety binds a receptor on the exterior of a cell; the selected receptor is one that undergoes endocytosis and passes through the late endosome, which has a pH of about 5.5. Thus, in the late endosome, the therapeutic agent dissociates from the targeting moiety and proceeds to the lysosome, where the therapeutic agent acts. For example, a targeting moiety can be chemically modified to incorporate a chelating agent (e.g. EDTA, EGTA, or trinitrilotriacetic acid) that tightly binds a metal ion such as nickel. The targeting moiety (e.g. GUS) can be expressed as a fusion protein with a six-histidine tag (e.g. at the amino-terminus, at the carboxy-terminus, or in a surface-accessible flexible loop). At or about pH 7.4, the six-histidine tag is substantially deprotonated and binds metal ions such as nickel with high affinity. At or about pH 5.5, the six-histidine tag is substantially protonated, leading to release of the nickel and, consequently, release of the therapeutic agent from the targeting moiety.

Therapeutic Agent

While methods and compositions of the invention are useful for producing and delivering any therapeutic agent to a subcellular compartment, the invention is particularly useful for delivering gene products for treating metabolic diseases.

Preferred LSD genes are shown in Table 1, and preferred genes associated with golgi or ER defects are shown in Table 2. In a preferred embodiment, a wild-type LSD gene product is delivered to a patient suffering from a defect in the same LSD gene. In alternative embodiments, a functional sequence or species variant of the LSD gene is used. In further embodiments, a gene coding for a different enzyme that can rescue an LSD gene defect is By used according to methods of the invention.

TABLE 1
Lysosomal Storage Diseases and associated enzyme defects
		Substance
Disease Name	Enzyme Defect	Stored
A. Glycogenosis
Disorders
Pompe Disease	Acid-a1, 4-	Glycogen α 1-4 linked
	Glucosidase	Oligosaccharides
B. Glycolipidosis
Disorders
GM1 Gangliodsidosis	β-Galactosidase	GM 1 Ganliosides
Tay-Sachs Disease	β-Hexosaminidase A	GM 2 Ganglioside
GM2 Gangliosidosis:	GM 2 Activator	GM 2 Ganglioside
AB Variant	Protein
Sandhoff Disease	β-Hexosaminidase	GM 2 Ganglioside
	A & B
Fabry Disease	α-Galactosidase A	Globosides
Gaucher Disease	Glucocerebrosidase	Glucosylceramide
Metachromatic	Arylsulfatase A	Sulphatides
Leukodystrophy
Krabbe Disease	Galactosylceramidase	Galactocerebroside
Niemann-Pick, Types	Acid	Sphingomyelin
A and B	Sphingomyelinase
Niemann-Pick, Type	Cholesterol	Sphingomyelin
C	Esterification Defect
Nieman-Pick, Type D	Unknown	Sphingomyelin
Farber Disease	Acid Ceramidase	Ceramide
Wolman Disease	Acid Lipase	Cholesteryl
		Esters
C. Mucopolysaccha-
ride Disorders
Hurler Syndrome	α-L-Iduronidase	Heparan &
(MPS IH)		Dermatan
		Sulfates
Scheie Syndrome	α-L-Iduronidase	Heparan &
(MPS IS)		Dermatan, Sulfates
Hurler-Scheie	α-L-Iduronidase	Heparan &
(MPS IH/S)		Dermatan
		Sulfates
Hunter Syndrome	Iduronate Sulfatase	Heparan &
(MPS II)		Dermatan
		Sulfates
Sanfilippo A	Heparan N-Sulfatase	Heparan
(MPS IIIA)		Sulfate
Sanfilippo B	α-N-	Heparan
(MPS IIIB)	Acetylglucosaminidase	Sulfate
Sanfilippo C	Acetyl-CoA-	Heparan
(MPS IIIC)	Glucosaminide	Sulfate
	Acetyltransferase
Sanfilippo D	N-Acetylglucosamine-	Heparan
(MPS IIID)	6-Sulfatase	Sulfate
Morquio A	Galactosamine-6-	Keratan
(MPS IV A)	Sulfatase	Sulfate
Morquio B	β-Galactosidase	Keratan
(MPS IVB)		Sulfate
Maroteaux-Lamy	Arylsulfatase B	Dermatan
(MPS VI)		Sulfate
Sly Syndrome	β-Glucuronidase
(MPS VII)
D. Oligosaccharide/
Glycoprotein
Disorders
α-Mannosidosis	α-Mannosidase	Mannose/
		Oligosaccharides
β-Mannosidosis	β-Mannosidase	Mannose/
		Oligosaccharides
Fucosidosis	α-L-Fucosidase	Fucosyl
		Oligosaccharides
Asparylglucosaminuria	N-Aspartyl-β-	Asparylglucosamine
	Glucosaminidase	Asparagines
Sialidosis	α-Neuraminidase	Sialyloligosaccharides
(Mucolipidosis I)
Galactosialidosis	Lysosomal Protective	Sialyloligosaccharides
(Goldberg Syndrome)	Protein Deficiency
Schindler Disease	α-N-Acetyl-
	Galactosaminidase
E. Lysosomal Enzyme
Transport Disorders
Mucolipidosis II (I-	N-Acetylglucosamine-	Heparan Sulfate
Cell Disease)	1-Phosphotransferase
Mucolipidosis III	Same as ML II
(Pseudo-Hurler
Polydystrophy)
F. Lysosomal
Membrane
Transport Disorders
Cystinosis	Cystine Transport	Free Cystine
	Protein
Salla Disease	Sialic Acid Transport	Free Sialic Acid and
	Protein	Glucuronic Acid
Infantile Sialic Acid	Sialic Acid Transport	Free Sialic Acid and
Storage Disease	Protein	Glucuronic Acid
G. Other
Batten Disease	Unknown	Lipofuscins
(Juvenile Neuronal
Ceroid
Lipofuscinosis)
Infantile Neuronal	Palmitoyl-Protein	Lipofuscins
Ceroid Lipofuscinosis	Thioesterase
Mucolipidosis IV	Unknown	Gangliosides &
		Hyaluronic Acid
Prosaposin	Saposins A, B, C or D

TABLE 2
Diseases of the golgi and ER
	Gene and
Disease Name	Enzyme Defect	Features
Ehlers-Danlos	PLOD1 lysyl	Defect in lysyl hydroxylation
Syndrome Type	hydroxylase	of Collagen; located in ER
VI		lumen
Type Ia glycoge	glucose6 phosphatase	Causes excessive
storage disease		accumulation of Glycogen in
		the liver, kidney, and
		Intestinal mucosa; enzyme is
		transmembrane but active site
		is ER lumen
Congenital Disorders of Glycosylation
CDG Ic	ALG6	Defects in N-glycosylation ER
	α1,3	lumen
	glucosyltransferase
CDG Id	ALG3	Defects in N-glycosylation ER
	α1,3	transmembrane protein
	mannosyltransferase
CDG IIa	MGAT2	Defects in N-glycosylation
	N-acetylglucosaminyl-	golgi transmembrane protein
	transferase II
CDG IIb	GCS1	Defect in N glycosylation
	α1,2-Glucosidase I	ER membrane bound with
		lumenal catalytic domain
		releasable by proteolysis

One particularly preferred therapeutic agent is glucocerebrosidase, currently manufactured by Genzyme as an effective enzyme replacement therapy for Gaucher's Disease. Currently, the enzyme is prepared with exposed mannose residues, which targets the protein specifically to cells of the macrophage lineage. Although the primary pathology in type 1 Gaucher patients are due to macrophage accumulating glucocerebroside, there can 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 can have a therapeutic advantage compared to other preparations.

Subcellular Targeting Domains

The present invention permits targeting of a therapeutic agent to a lysosome using a protein, or an analog of a protein, that specifically binds a cellular receptor for that protein. The exterior of the cell surface is topologically equivalent to endosomal, lysosomal, golgi , and endoplasmic reticulum compartments. Thus, endocytosis of a molecule through interaction with an appropriate receptor(s) permits transport of the molecule to any of these compartments without crossing a membrane. Should a genetic deficiency result in a deficit of a particular enzyme activity in any of these compartments, delivery of a therapeutic protein can be achieved by tagging it with a ligand for the appropriate receptor(s).

Multiple pathways directing receptor-bound proteins from the plasma membrane to the golgi and/or endoplasmic reticulum have been characterized. Thus, by using a targeting portion from, for example, SV40, cholera toxin, or the plant toxin ricin, each of which coopt one or more of these subcellular trafficking pathways, a therapeutic can be targeted to the desired location within the cell. In each case, uptake is initiated by binding of the material to the exterior of the cell. For example, SV40 binds to MHC class I receptors, cholera toxin binds to GM1 ganglioside molecules and ricin binds to glycolipids and glycoproteins with terminal galactose on the surface of cells. Following this initial step the molecules reach the ER by a variety of pathways. For example, SV40 undergoes caveolar endocytosis and reaches the ER in a two step process that bypasses the golgi whereas cholera toxin undergoes caveolar endocytosis but traverses the golgi before reaching the ER.

If a targeting moiety related to cholera toxin or ricin is used, it is important that the toxicity of cholera toxin or ricin be avoided. Both cholera toxin and ricin are heteromeric proteins, and the cell surface binding domain and the catalytic activities responsible for toxicity reside on separate polypeptides. Thus, a targeting moiety can be constructed that includes the receptor-binding polypeptide, but not the polypeptide responsible for toxicity. For example, in the case of ricin, the B subunit possesses the galactose binding activity responsible for internalization of the protein, and can be fused to a therapeutic protein. If the further presence of the A subunit improves subcellular localization, a mutant version (mutein) of the A chain that is properly folded but catalytically inert can be provided with the B subunit-therapeutic agent fusion protein.

Proteins delivered to the golgi can be transported to the endoplasmic reticulum (ER) via the KDEL receptor, which retrieves ER-targeted proteins that have escaped to the golgi . Thus, inclusion of a KDEL motif at the terminus of a targeting domain that directs a therapeutic protein to the golgi permits subsequent localization to the ER. For example, a targeting moiety (e.g. an antibody, or a peptide identified by high-throughput screening such as phage display, yeast two hybrid, chip-based assays, and solution-based assays) that binds the cation-independent M6P receptor both at or about pH 7.4 and at or about pH 5.5 permits targeting of a therapeutic agent to the golgi ; further addition of a KDEL motif permits targeting to the ER.

Lysosomal Targeting Moieties

The invention permits targeting of a therapeutic agent to a lysosome. Targeting may occur, for example, through binding of a plasma membrane receptor that later passes through a lysosome. Alternatively, targeting may occur through binding of a plasma receptor that later passes through a late endosome; the therapeutic agent can then travel from the late endosome to a lysosome. A preferred lysosomal targeting mechanism involves binding to the cation-independent M6P receptor.

Cation-independent M6P Receptor

The cation-independent M6P receptor is a 275 kDa single chain transmembrane glycoprotein expressed ubiquitously in mammalian tissues. It is one of two mammalian receptors that bind M6P: the second is referred to as the cation-dependent M6P receptor. The cation-dependent M6P receptor requires divalent cations for M6P binding; the cation-independent M6P receptor does not. These receptors play an important role in the trafficking of lysosomal enzymes through recognition of the M6P moiety on high mannose carbohydrate on lysosomal enzymes. The extracellular domain of the cation-independent M6P receptor contains 15 homologous domains (“repeats”) that bind a diverse group of ligands at discrete locations on the receptor.

The cation-independent M6P receptor contains two binding sites for M6P: one located in repeats 1-3 and the other located in repeats 7-9. The receptor binds monovalent M6P ligands with a dissociation constant in the μM range while binding divalent M6P ligands with a dissociation constant in the nM range, probably due to receptor oligomerization. Uptake of IGF-II by the receptor is enhanced by concomitant binding of multivalent M6P ligands such as lysosomal enzymes to the receptor.

The cation-independent M6P receptor also contains binding sites for at least three distinct ligands that can be used as targeting moieties. The cation-independent M6P receptor binds IGF-II with a dissociation constant of about 14 nM at or about pH 7.4, primarily through interactions with repeat 11. Consistent with its function in targeting IGF-II to the lysosome, the dissociation constant is increased approximately 100-fold at or about pH 5.5 promoting dissociation of IGF-II in acidic late endosomes. The receptor is capable of binding high molecular weight O-glycosylated IGF-II forms.

An additional useful ligand for the cation-independent M6P receptor is retinoic acid. Retinoic acid binds to the receptor with a dissociation constant of 2.5 nM. Affinity photolabeling of the cation-independent M6P receptor with retinoic acid does not interfere with IGF-II or M6P binding to the receptor, indicating that retinoic acid binds to a distinct site on the receptor. Binding of retinoic acid to the receptor alters the intracellular distribution of the receptor with a greater accumulation of the receptor in cytoplasmic vesicles and also enhances uptake of M6P modified β-glucuronidase. Retinoic acid has a photoactivatable moiety that can be used to link it to a therapeutic agent without interfering with its ability to bind to the cation-independent M6P receptor.

The cation-independent M6P receptor also binds the urokinase-type plasminogen receptor (uPAR) with a dissociation constant of 9 μM. uPAR is a GPI-anchored receptor on the surface of most cell types where it functions as an adhesion molecule and in the proteolytic activation of plasminogen and TGF-β. Binding of uPAR to the CI-M6P receptor targets it to the lysosome, thereby modulating its activity. Thus, fusing the extracellular domain of uPAR, or a portion thereof competent to bind the cation-independent M6P receptor, to a therapeutic agent permits targeting of the agent to a lysosome.

IGF-II

In a preferred embodiment, 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. Advantageously, this embodiment mimics the normal biological mechanism for uptake of LSD proteins, yet does so in a manner independent of mannose-6-phosphate.

For example, by fusing DNA encoding the mature IGF-II polypeptide to the 3′ end of LSD gene cassettes, fusion proteins are created that can be taken up by a variety of cell types and transported to the lysosome. 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-II is preferably targeted specifically to the M6P receptor. Particularly useful are mutations in the IGF-II polypeptide that result in a protein that binds the M6P receptor with high affinity while no longer binding the other two receptors with appreciable affinity. IGF-II can also be modified to minimize binding to serum IGF-binding proteins (Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76) to avoid sequestration of IGF-II/GILT constructs. A number of studies have localized residues in IGF-1 and IGF-II necessary for binding to IGF-binding proteins. Constructs with mutations at these residues can be screened for retention of high affinity binding to the M6P/IGF-II receptor and for reduced affinity for IGF-binding proteins. For example, replacing PHE 26 of IGF-II with SER is reported to reduce affinity of IGF-II for IGFBP-1 and -6 with no effect on binding to the M6P/IGF-II receptor (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Other substitutions, such as SER for PHE 19 and LYS for GLU 9, can also be advantageous. The analogous mutations, separately or in combination, in a region of IGF-I that is highly conserved with IGF-II result in large decreases in IGF-BP binding (Magee et al. (1999) Biochemistry 38(48):15863-70).

An alternate approach is to identify minimal regions of IGF-II that can bind with high affinity to the M6P/IGF-II receptor. The residues that have been implicated in IGF-II binding to the M6P/IGF-II receptor mostly cluster on one face of IGF-II (Terasawa et al. (1994) EMBO J. 13(23):5590-7). Although IGF-II tertiary structure is normally maintained by three intramolecular disulfide bonds, a peptide incorporating the amino acid sequence on the M6P/IGF-II receptor binding surface of IGF-II can be designed to fold properly and have binding activity. Such a minimal binding peptide is a highly preferred targeting portion. Designed peptides based on the region around amino acids 48-55 can be tested for binding to the M6P/IGF-II receptor. Alternatively, a random library of peptides can be screened for the ability to bind the M6P/IGF-II receptor either via a yeast two hybrid assay, or via a phage display type assay.

Blood Brain Barrier

One challenge in therapy for lysosomal storage diseases is that many of these diseases have significant neurological involvement. Therapeutic enzymes administered into the blood stream generally do not cross the blood brain barrier and therefore cannot relieve neurological symptoms associated with the diseases. IGF-II, however, has been reported to promote transport across the blood brain barrier via transcytosis (Bickel et al. (2001) Adv. Drug Deliv. Rev. 46(1-3):247-79). Thus, appropriately designed GILT constructs should be capable of crossing the blood brain barrier, affording for the first time a means of treating neurological symptoms associated with lysosomal storage diseases. The constructs can be tested using GUS minus mice as described in Example 7, infra. Further details regarding design, construction and testing of targeted therapeutics that can reach neuronal tissue from blood are disclosed in U.S. Ser. No. 60/329,650, filed Oct. 16, 2001, and in U.S. Ser. No. 10/136,639 filed Apr. 30, 2002.

Structure of IGF-II

NMR structures of IGF-II have been solved by two groups (Terasawa et al (1994) EMBO J. 13(23):5590-7; Torres et al. (1995) J. Mol. Biol. 248(2):385-401) (see, e.g., Protein Data Bank record 1IGL). 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. 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.

IGF-II binds to the IGF-II/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 IGFBPs.

Binding to the IGF-II/M6P Receptor

Substitution of IGF-II residues 48-50 (Phe Arg Ser) with the corresponding residues from insulin, (Thr Ser Ile), or substitution of residues 54-55 (Ala Leu) with the corresponding residues from IGF-I (Arg Arg) result in diminished binding to the IGF-II/M6P receptor but retention of binding to the IGF-I and insulin receptors (Sakano et al. (1991) J. Biol. Chem. 266(31):20626-35).

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-II 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-I, there is no bend in the backbone around residues 53 and 54 (Torres et al (1995) J. Mol. Biol. 248(2):385-401). This structural difference correlates with the substitution of Ala 54 and Leu 55 in IGF-II with Arg 55 and Arg 56 in IGF-I. It is possible either that binding to the IGF-II receptor is disrupted directly by the presence of charged residues in this region or that changes in the structure engendered by the charged residues yield the changes in binding for the IGF-II receptor. In any case, substitution of uncharged residues for the two Arg residues in IGF-I resulted in higher affinities for the IGF-II receptor (Cacciari et al. (1987) Pediatrician 14(3):146-53). Thus the presence of positively charged residues in these positions correlates with loss of binding to the IGF-II receptor.

IGF-II binds to repeat 11 of the cation-independent M6P receptor. Indeed, 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 IGF-II and its delivery to lysosomes (Grimme et al. (2000) J. Biol. Chem. 275(43):33697-33703). The structure of domain 11 of the M6P receptor is known (Protein Data Base entries 1GP0 and 1GP3; Brown et al. (2002) EMBO J. 21(5):1054-1062). 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. Although repeat 11 is sufficient for IGF-II binding, constructs including larger portions of the cation-independent M6P receptor (e.g. repeats 10-13, or 1-15) generally bind IGF-II with greater affinity and with increased pH dependence (see, for example, Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991).

Binding to the IGF-I Receptor

Substitution of IGF-II residues Tyr 27 with Leu, Leu 43 with Val or Ser 26 with Phe diminishes the affinity of IGF-II for the IGF-I receptor by 94-, 56-, and 4-fold respectively (Torres et at (1995) J. Mol. Biol. 248(2):385-401). Deletion of residues 1-7 of human IGF-II resulted in a 30-fold decrease in affinity for the human IGF-I receptor and a concomitant 12 fold increase in affinity for the rat IGF-II receptor (Hashimoto et al. (1995) J. Biol. Chem. 270(30):18013-8). The NMR structure of IGF-II shows that Thr 7 is located near residues 48 Phe and 50 Ser as well as near the 9 Cys-47 Cys disulfide bridge. It is thought that interaction of Thr 7 with these residues can stabilize the flexible N-terminal hexapeptide required for IGF-I receptor binding (Terasawa et at (1994) EMBO J. 13(23)5590-7). At the same time this interaction can modulate binding to the IGF-II receptor. Truncation of the C-terminus of IGF-II (residues 62-67) also appear to lower the affinity of IGF-II for the IGF-I receptor by 5 fold (Roth et al. (1991) Biochem. Biophys. Res. Commun. 181(2):907-14).

Deletion Mutants of IGF-II

The binding surfaces for the IGF-I and cation-independent M6P receptors are on separate faces of IGF-II. Based on structural and mutational data, functional cation-independent M6P binding domains can be constructed that are substantially smaller than human IGF-II. For example, the amino terminal amino acids 1-7 and/or the carboxy terminal residues 62-67 can be deleted or replaced. Additionally, 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. Thus, 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. Alternatively, amino acids 8-28 and 41-61 can be provided on separate polypeptide chains. Comparable domains of insulin, which 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). Thus, for example, 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.

Binding to IGF Binding Proteins

IGF-II and related constructs can be modified to diminish their affinity for IGFBPs, thereby increasing the bioavailability of the tagged proteins.

Substitution of IGF-II residue phenylalanine 26 with serine reduces binding to IGFBPs 1-5 by 5-75 fold (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Replacement of IGF-II residues 48-50 with threonine-serine-isoleucine reduces binding by more than 100 fold to most of the IGFBPs (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54); these residues are, however, also important for binding to the cation-independent mannose-6-phosphate receptor. The Y27L substitution that disrupts binding to the IGF-I receptor interferes with formation of the ternary complex with IGFBP3 and acid labile subunit (Hashimoto et al. (1997) J. Biol. Chem. 272(44):27936-42); this ternary complex accounts for most of the IGF-II in the circulation (Yu et at (1999) J. Clin. Lab Anal. 13(4):166-72). Deletion of the first six residues of IGF-II also interferes with IGFBP binding (Luthi et al. (1992) Eur. J. Biochem. 205(2):483-90).

Studies on IGF-I interaction with IGFBPs revealed additionally that substitution of serine for phenylalanine 16 did not effect secondary structure but decreased IGFBP binding by between 40 and 300 fold (Magee et al. (1999) Biochemistry 38(48):15863-70). Changing glutamate 9 to lysine also resulted in a significant decrease in IGFBP binding. Furthermore, the double mutant lysine 9/serine 16 exhibited the lowest affinity for IGFBPs. Although these mutations have not previously been tested in IGF-II, the conservation of sequence between this region of IGF-I and IGF-II suggests that a similar effect will be observed when the analogous mutations are made in IGF-II (glutamate 12 lysine/phenylalanine 19 serine).

IGF-II Homologs

The amino acid sequence of human IGF-II, or a portion thereof affecting binding to the cation-independent M6P receptor, 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. Preferably, 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-II.

To determine whether a candidate peptide region has the requisite percentage similarity or identity to human IGF-II, the candidate amino acid sequence and human 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 FIG. 2 of Henikoff and Henikoff (1992) PNAS 89:10915-10919. For the present invention, an appropriate value for the gap insertion penalty is −12, and 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.

Once the alignment between the candidate and reference sequence is made, 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. If the amino acids are non-identical, 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.

IGF-II Structural Analogs

The known structures of human IGF-II and the cation-independent M6P receptors permit the design of IGF-II analogs and other cation-independent M6P receptor binding proteins using computer-assisted design principles such as those discussed in U.S. Pat. Nos. 6,226,603 and 6,273,598. For example, the known atomic coordinates of IGF-II can be provided to a computer equipped with a conventional computer modeling program, such as INSIGHTII, DISCOVER, or DELPHI, commercially available from Biosym, Technologies Inc., or QUANTA, or 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. For example, 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 the surface of the cation-independent M6P receptor binding face of IGF-II. Because a major contribution to the receptor binding surface is the spatial arrangement of chemically interactive moieties present within the sidechains of amino acids which together define the receptor binding surface, 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 constitute the cation-independent M6P receptor binding face of IGF-II. Preferred chemical moieties, include but are not limited to, the chemical moieties defined by the amino acid side chains of amino acids constituting the cation-independent M6P receptor binding face of IGF-II. It is understood, therefore, that the receptor binding surface of the IGF-II analog need not comprise amino acid residues but the chemical moieties disposed thereon.

For example, upon identification of relevant chemical groups, the skilled artisan using a conventional computer program can design a small molecule having the receptor interactive chemical moieties disposed upon a suitable carrier framework. Useful computer programs are described in, for example, Dixon (1992) Tibtech 10: 357-363; Tschinke et al. (1993) J. Med. Chem 36: 3863-3870; and Eisen el al. (1994) Proteins: Structure, Function, and Genetics 19: 199-221, the disclosures of which are incorporated herein by reference.

One particular computer program entitled “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 Biological Problems” (Roberts, S. M., ed) pp 182-196). The CAVEAT program has been used to design analogs of tendamistat, a 74 residue inhibitor of α-amylase, based on the orientation of selected amino acid side chains in the three-dimensional structure of tendamistat (Bartlett et al. (1989) supra).

Alternatively, upon identification of a series of analogs which mimic the cation-independent M6P receptor binding activity of IGF-II, the skilled artisan may use a variety of computer programs which assist the skilled artisan to develop quantitative structure activity relationships (QSAR) and further to assist in the de novo design of additional morphogen analogs. Other useful computer programs are described in, for example, Connolly-Martin (1991) Methods in Enzymology 203:587-613; Dixon (1992) supra; and Waszkowycz et al. (1994) J. Med. Chenm. 37: 3994-4002.

Targeting Moiety Affinities

Preferred targeting moieties bind to their target receptors with a submicromolar dissociation constant. Generally speaking, lower dissociation constants (e.g. less than 10 ⁻⁷ M, less than 10 ⁻⁸ M, or less than 10 ⁻⁹ M) are increasingly preferred. Determination of dissociation constants is preferably determined by surface plasmon resonance as described in Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991. A soluble form of the extracellular domain of the target receptor (e.g. repeats 1-15 of the cation-independent M6P receptor) is generated and immobilized to a chip through an avidin-biotin interaction. The targeting moiety is passed over the chip, and kinetic and equilibrium constants are detected and calculated by measuring changes in mass associated with the chip surface.

Nucleic Acids and Expression Systems

Chimeric fusion proteins 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 glycosyla