AU2005203058B2 - Hedgehog fusion proteins and uses - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Actual Inventor/s: Address for Service is: Curis, Inc.

Kathy Strauch and Ellen A. Garber and Frederick R. Taylor SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.

(02) 97771111 (02) 9241 4666 Invention Title: HEDGEHOG FUSION PROTEINS AND USES Details of Original Application No. 15838/01 dated 02 Nov 2000 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 35125AUP01 500642034_1.DOC/5844 00 -la-

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SHEDGEHOG FUSION PROTEINS AND USES The present application is a divisional application of Australian Application No.

15838/01, which is incorporated in its entirety herein by reference.

BACKGROUND OF THE INVENTION 0 O Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common Sgeneral knowledge in the field.

A peptide family which has been the focus of much research, and efforts to 10 improve its administration and bioavailability, is the hedgehog family of proteins. The hedgehog proteins are a family of extracellular signaling proteins that regulate various aspects of embryonic development both in vertebrates and in invertebrates (for reviews see Perrimon, N. (1995) Cell 80, 517-520 and Johnson, R. and Tabin, C. (1995) Cell 81, 313-316). The most well-characterized hedgehog protein is Sonic hedgehog (Shh), involved in anterior-posterior patterning, formation of an apical ectodermal ridge, hindgut mesoderm, spinal column, distal limb, rib development, and lung development, and in inducing ventral cell types in the spinal cord, hindbrain and forebrain (see Riddle, R. et al. (1993) Cell 75, 1401-1416; Echelard, Y. et al. (1993) Cell 75, 1417-1471; Roelink, et al. (1994) Cell 76, 761-775; and Roelink, et al. (1995) Cell 81,445- 455).

While the mechanism of action of hedgehog proteins is not understood fully, the most recent biochemical and genetic data suggest that the receptor for Shh is the product of the tumor suppressor gene, patched (Marigo, et al. (1996) Nature 384, 176-179; Stone, D. et al. (1996) Nature 384, 129-134) and that other proteins; smoothened (Alcedo, et al. (1996) Cell 86, 221-232), Cubitus interruptus or its mammalian counterpart gli (Dominguez, et al. (1996) Science 272, 1621-1625; Alexandre, et al. (1996) Genes Dev. 10, 2003-2013), andfused (Therond, P. et al. (1996) Proc.

Natl. Acad. Sci. USA 93, 4224-4228) are involved in the hedgehog signaling pathway.

Human Shh is synthesized as a 45 kDa precursor protein that is cleaved autocatalytically to yield: a 20 kDa N-terminal fragment that is responsible for all known hedgehog signaling activity (SEQ ID NOS. 6 and 24); and (II) a 25 kDa Cterminal fragment that contains the autoprocessing activity (Lee, J. et al. (1994) Science 266, 1528-1536; Bumcrot, et al. (1995) Mol. Cell Biol. 15, 2294-2303; 00 -2- 0 Porter, et al. (1995) Nature 374, 363-366). The N-terminal fragment of naturally Soccurring hedgehog consists of amino acid residues 24-197 of the full-length precursor sequence, of which the N-terminal amino acid residue is a cysteine.

t_ The N-terminal fragment remains membrane-associated through the addition of a cholesterol at its C-terminus (Porter, J. et al. (1996) Science 274, 255-258 Porter, J.

0 0 et al. (1995) Cell 86, 21-34) and a fatty acid at its N-terminus (Pepinsky et al., 0 (1998) J. Biol. Chem. 273, 14037-14045). These modifications are critical for restricting Sthe tissue localization of the hedgehog signal. The addition of the cholesterol is t catalyzed by the C-terminal domain during the processing step.

A major factor limiting the usefulness of proteinaceous substances such as hedgehog for their intended application is that, when given parenterally, they are eliminated from the body within a short time. This can occur as a result of metabolism by proteases or by clearance using normal pathways for protein elimination such as by filtration in the kidneys. The oral route of administration of these substances is even more problematic because in addition to proteolysis in the stomach, the high acidity of the stomach may inactivate them before they reach their intended target tissue. The problems associated with these routes of administration of proteins are well known in the pharmaceutical industry, and various strategies are being used in attempts to solve them.

A great deal of work dealing with protein stabilization has been published. One method of stabilization that has been widely used is the addition of an inert polymer to the protein. Numerous ways of conjugating selected amino acid residues of proteins (e.

cysteines, lysines, N-terminal residues) with polymeric materials are known, including use of dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications.

In the case of hedgehog, we have previously discovered that in certain cell types, the protein undergoes proteolytic clipping at various sites in the N-terminal domain.

Moreover, these N-terminally clipped forms of hedgehog are inactive in the 10T 1/2 assay (in which the cell line 10T1/2 exhibits upregulation of Alkaline phosphatase when cultured for five days in the presence of active Sonic Hedgehog protein). In particular, sonic hedgehog lacking the first 10 amino acids of its N- terminus is inactive and also antagonizes wild-type SHH when both forms are present in the assay.

00 -3- 0 SWO 2003/072036 and WO 2003/072736. Thus, if one wants to produce a fully active protein that can be further stabilized with a non-hedgehog moiety such as a polymer, one needs to prevent N-terminal proteolytic clipping.

c SUMMARY OF THE INVENTION This invention is based, in part, on our discovery that N-terminal clipping of 0 0 hedgehog during expression in certain cell types occurs intracellularly and appears to be C catalyzed by the KEX2 Golgi protease, or a similar KEX2-like intracellular protease.

SThe KEX2 recognition sites in Sonic Hedgehog were mutated in order to eliminate this intracellular proteolytic clipping and thus provide a hedgehog protein moiety capable of being linked to a non-hedgehog moiety an immunoglobulin domain). These mutant proteins were expressed as the N-terminal domain (codons Cys24-Gly197 of the Sonic Hedgehog coding sequence, corresponding to residues Cysl-Glyl74 of mature protein after signal sequence cleavage. Here we report on the stability and hedgehog activity of these mutants and on production of an active form of hedgehog-Fc fusion protein.

According to a first aspect, the present invention provides an isolated polypeptide having the amino acid sequence X-Y-Z, wherein X is a polypeptide comprising; an amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence, or an amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, wherein the N-terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence; Y is an optional linker moiety; and Z is a polypeptide comprising an immunoglobulin, or fragment thereof.

According to a second aspect, the present invention provides a fusion protein having an amino terminal region consisting of the amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, and a carboxy terminal region comprising an immunoglobulin, or fragment thereof comprising at least a portion of a constant region, wherein said portion 00 -3a- O of a constant region comprises at least one of a CH hinge, CH2, or CH3 domain, Swherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence or (ii) the hedgehog protein comprises a mutated KEX2 protease recognition Ssequence and the N-terminal cysteine of the hedgehog protein is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues.

0 According to a third aspect, the present invention provides an isolated polypeptide Shaving an amino acid sequence X-Y-Z, wherein X is a polypeptide comprising the Samino acid sequence of a Sonic hedgehog protein, wherein the Sonic hedgehog protein comprises: 10 a) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence, wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO:15) is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102, or b) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence wherein the N-terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog.

According to an fourth aspect, the present invention provides a fusion protein having an amino terminal region consisting of the amino acid sequence of a Sonic hedgehog protein and having a carboxy terminal region comprising at least a portion of a protein other than hedgehog, wherein the Sonic hedgehog protein comprises: a) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence, wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO:15) is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102, or b) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence wherein the N-terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO: is replaced with a sequence selected from one of SEQ ID NO: 88-94 or 99-102.

00 3b-

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O According to a fifth aspect, the present invention provides an isolated polypeptide Shaving the amino acid sequence X-Y-Z, wherein 3) X is a polypeptide comprising S(a) an amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, wherein the hedgehog protein 00 comprises a mutated KEX2 protease recognition sequence or S(b) an amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous Samino acids thereof that binds to a patched protein, wherein the N-terminal t cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog.

According to a sixth aspect, the present invention provides a fusion protein having an amino terminal region consisting of the amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, and a carboxy terminal region comprising at least a portion of a polypeptide other than hedgehog, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence or (ii) the hedgehog protein comprises a mutated KEX2 protease recognition sequence and the N-terminal cysteine of the hedgehog protein is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues.

According to a seventh aspect, the present invention provides an isolated nucleic acid sequence encoding the polypeptide or fusion protein according to the invention.

According to an eighth aspect, the present invention provides a recombinant nucleic acid comprising the nucleic acid sequence according to the seventh aspect, and an expression control sequence operatively linked thereto.

According to a ninth aspect the present invention provides a host cell transformed with the recombinant nucleic acid sequence according to the eighth aspect.

According to a tenth aspect the present invention provides a method of producing a recombinant polypeptide comprising: providing a population of host cells according to the ninth aspect; 00 -3cgrowing said population of cells under conditions whereby the polypeptide encoded by said recombinant nucleic acid is expressed; and isolating the expressed polypeptide.

Cc According to an eleventh aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the polypeptide or fusion protein 00 according to the invention.

According to a twelfth aspect, the present invention provides use of the Spolypeptide or fusion protein according to the invention in the manufacture of a medicament for treating a subject in need thereof.

S 10 According to a thirteenth aspect, the present invention provides use of the polypeptide or fusion protein according to the invention in the manufacture of a medicament for preventing and/or reducing the severity of a neurological condition deriving from: acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vessel injury, and deficits (such as the ischemia from stroke); (ii) infection and tumor-induced injury; (iii) aging of the nervous system including Alzheimer's disease; (iv) chronic Huntington's chorea, amylotrophic lateral sclerosis and the like; or chronic immunological diseases of the nervous system, including multiple sclerosis.

According to a fourteenth aspect, the present invention provides a method of treating a subject comprising administering an effective amount of an isolated polypeptide or fusion protein according to the invention.

According to a fifteenth aspect, the present invention provides a method of preventing and/or reducing the severity of a neurological condition deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vessel injury, and deficits (such as the ischemia from stroke); (ii) infection and tumor-induced injury; (iii) aging of the nervous system including Alzheimer's disease; (iv) chronic Huntington's chorea, amylotrophic lateral sclerosis and the like; or chronic immunological diseases of the nervous system, including multiple sclerosis comprising administering an effective amount of an isolated polypeptide or fusion protein according to the invention to a subject in need thereof.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an 00 -3d-

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O inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the Ssense of "including, but not limited to".

Further, we can exploit the advantages of an immunoglobulin hedgehog fusion Sprotein relative to non-fusion forms, whether or not the hedgehog protein is proteolytically clipped at the N-terminus. In particular however, we have developed an 0 0 hedgehog-Ig fusion composition with increased bioavailability relative to hedgehog lacking the Ig moiety and that further has the salutory properties of being unable to be Sclipped by intracellular proteases. Thus, modifications can be made to the hedgehog t moiety such that the products (hedgehog immunoglobulin fusion proteins) are either agonists or antagonists but retain all or most of their biological activities. The following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution g, ability to stay in the vasculature for longer periods of time) Such a formulation is a substantial advance in the pharmaceutical and medical arts and would make a significant contribution to the management of various diseases in which hedgehog has some utility, such as peripheral neuropathies and neurodegenerative diseases. In particular, the ability to remain for longer periods of time in the vasculature allows the hedgehog fusions to potentially cross the blood-brain barrier.

In particular, the invention relates to an isolated polypeptide having the amino acid sequence X-Y-Z, wherein X is a polypeptide having the amino acid sequence, or portion thereof, consisting of the amino acid sequence of hedgehog; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog. Preferably, X is human Sonic, Indian or Desert hedgehog. In the preferred embodiments, Z is at least a portion of a constant region of an immunoglobulin and can be derived from an immunoglobulin of the class selected from IgM, IgG, IgD, IgA, and IgE. If the class is IgG, then it is selected from one of IgG IgG2, IgG3 and IgG4. The constant region of human IgM and IgE contain 4 constant regions (CH 1, (hinge), CH2, CH3 and CH4, whereas the constant region of human IgG, o -4- O IgA and IgD contain 3 constant regions (CHI, (hinge), CH2 and CH3. In the most preferred fusion proteins of the invention, the constant region contains at least the hinge, CH2 and CH3 domains.

Another embodiment of the invention is a fusion protein having an amino terminal region consisting of the amino acid sequence of hedgehog or a portion thereof m and having a carboxy terminal region comprising at least a portion of a protein other c than hedgehog The carboxy portion is preferably at least a portion of a constant Sregion of an immunoglobulin derived from an immunoglobulin of the class selected Sfrom IgM, IgG, IgD, IgA, and IgE. In the most preferred fusion proteins, the constant C 10 region contains at least the hinge, CH2 and CH3 domains.

Another embodiment of the invention is a fusion protein whose hedgehog moiety X in the formula above) has been mutated to provide for muteins with an altered KEX2 protease recognition site.

Yet another embodiment of the invention is an isolated DNA encoding for the fusion proteins described above. The invention also pertains to a recombinant DNA comprising an isolated DNA encoding the fusion proteins described above and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. The scope of the invention also includes host cells transformed with the recombinant DNA sequences of the invention.

The invention further pertains to a method of producing a recombinant polypeptide comprising: providing a population of host cells according to the invention; growing the population of cells under conditions whereby the polypeptide encoded by the recombinant DNA is expressed; and isolating the expressed polypeptide.

A further aspect of the invention is a hedgehog fusion protein comprising hedgehog and an additional polypeptide with which it is not natively associated, in substantially purified form, the fusion having a bioavailability that is at least equal to, and preferably greater than, the bioavailability of hedgehog lacking the additional polypeptide.

Yet another aspect of the invention is a pharmaceutical composition comprising a ally effective amount of an hedgehog fusion protein.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. N-terminal sequences of Sonic, Indian and Desert Hedgehog FIGURE 2. Consensus Hedgehog Sequence FIGURE 3. N terminal Sequence of Sonic Hedgehog showing clip sites DETAILED DESCRIPTION All references cited in the Detailed Description are incorporated herein by references, unless stipulated otherwise. The following terms are used herein: I. Definitions The invention will now be described with reference to the following detailed description of which the following definitions are included: As used herein, the term hedgehog "antagonist" includes any compound that inhibits hedgehog from binding with its receptor. For the purposes of the invention a hedgehog antagonist also refers to an agent, a polypeptide such as an antihedgehog or anti-patched antibody which can inhibit or block hedgehog and/or patched-mediated binding or which can otherwise modulate hedgehog and/or patched function, by inhibiting or blocking hedgehog-ligand mediated hedgehog signal transduction. Such an antagonist of the hedgehog/patched interaction is an agent which has one or more of the following properties: it coats, or binds to, a hedgehog on the surface of a hedgehog bearing or secreting cell with sufficient specificity to inhibit a hedgehog-ligand/hedgehog interaction, the hedgehog/patched interaction; it coats, or binds to, a hedgehog on the surface of a hedgehog- bearing or secreting cell with sufficient specificity to modify, and preferably to inhibit, transduction of a hedgehog-mediated signal hedgehog/patched-mediated signaling; it coats, or binds to, a hedgehog receptor, patched) in or on cells with sufficient specificity to inhibit the hedgehog /patched interaction; it coats, or binds to, a hedgehog receptor patched) in or on cells with sufficient specificity to modify, and preferably to inhibit, transduction of hedgehog mediated hedgehog signaling, patched-mediated hedgehog signaling.

In preferred embodiments the antagonist has one or both of properties 1 and 2.

In other preferred embodiments the antagonist has one or both of properties 3 and 4.

Moreover, more than one antagonist can be administered to a patient, an agent which binds to hedgehog can be combined with an agent which binds to patched..

For example, antibody or antibody homolog-containing hedgehog proteins (discussed below) as well as other molecules such as soluble forms of the natural binding proteins for hedgehog are useful. Soluble forms of the natural binding proteins for hedgehog include soluble patched peptides, patched fusion proteins, or bifunctional o -6patched/Ig fusion proteins. For example, a soluble form of patched or a fragment thereof may be administered to bind to hedghog, and preferably compete for a hedgehog binding site on cells, thereby leading to effects similar to the administration of antagonists such as anti-hedgehog antibodies. In particular, soluble hedgehog mutants that bind patched but do not elicit hedgehog-dependent signaling are included 00 Swithin the scope of the invention Such hedgehog mutants can act as competitive Cc inhibitors of wild type hedgehog protein and are considered "antagonists".

CN As discussed herein, the hedgehog antagonists that can be fused or otherwise 0 conjugated to, for instance, an antibody homolog such as an immunoglobulin or C 10 fragment thereof are not limited to a particular type or structure of hedgehog or patched or other molecule so that, for purposes of the invention, any agent capable of forming a fusion protein and capable of binding to hedgehog antigens and which effectively blocks or coats hedgehog is considered to be an equivalent of the antagonists used in the examples herein.

As used herein, the term "antibody homolog" includes intact antibodies consisting of immunoglobulin light and heavy chains linked via disulfide bonds. The term "antibody homolog" is also intended to encompass a protein comprising one or more polypeptides selected from immunoglobulin light chains, immunoglobulin heavy chains and antigen-binding fragments thereof which are capable of binding to one or more antigens hedgehog or patched). The component polypeptides of an antibody homolog composed of more than one polypeptide may optionally be disulfide-bound or otherwise covalently crosslinked. Accordingly, therefore, "antibody homologs" include intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. Preferred fusion proteins of the invention may include portions of intact antibodies that retain antigen-binding specificity, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like.

The most preferred fusion proteins comprise a hedgehog moiety fused or otherwise linked to all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both. Thus, this invention features a molecule which includes: a hedgehog moiety, a second peptide, one which increases solubility or in vivo life time of the hedgehog moiety, a member of the immunoglobulin super family or fragment or portion thereof, a portion or a fragment of IgG, the human IgGI heavy chain constant region, CH2, CH3, and hinge regions; and a toxin moiety.

As used herein, a "humanized antibody homolog" is an antibody homolog, produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding have been substituted for the corresponding amino acids from a nonhuman mammalian immunoglobulin light or heavy chain. A "human antibody homolog" is an antibody homolog in which all the amino acids of an immunoglobulin light or heavy chain (regardless of whether or not they are required for antigen binding) are derived from a human source.

As used herein, the term hedgehog "agonist" includes any compound that activates the hedgehog receptor.

"amino acid"- a monomeric unit of a peptide, polypeptide, or protein. There are twenty amino acids found in naturally occurring peptides, polypeptides and proteins, all of which are L-isomers. The term also includes analogs of the amino acids and Disomers of the protein amino acids and their analogs.

A hedgehog protein has "biological activity" if it has at least one of the following properties: it has the ability to bind to its receptor, patched or it encodes, upon expression, a polypeptide that has this characteristic; and/or (ii) it may induce alkaline phosphatase activity in C3H10T1/2 cells. The hedgehog protein meeting this functional test of "biological activity" may meet the hedgehog consensus criteria as defined herein in Figure 2 (SEQ ID NO: 26) but it may also be a mutant form of hedghog. This term "biological activity" includes antagonists and agonists, as defined herein.

The term "bioavailability" refers to the ability of a compound to be absorbed by the body after administration. For instance, a first compound has greater bioavailability than a second compound if, when both are administered in equal amounts, the first compound is absorbed into the blood to a greater extent than the second compound.

As used herein, the term "covalently coupled" means that the specified moieties of the invention immunoglobulin fragment/hedgehog protein) are either directly covalently bonded to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties. The intervening moiety or moieties are called a "coupling group". The term "conjugated" is used interchangeably with "covalently coupled".

6 -8o "expression control sequence"- a sequence of polynucleotides that controls and regulates expression of genes when operatively linked to those genes.

I "expression vector"- a polynucleotide, such as a DNA plasmid or phage (among other common examples) which allows expression of at least one gene when the expression vector is introduced into a host cell. The vector may, or may not, be able to 00 Sreplicate in a cell.

The phrase "extracellular signaling protein" means any protein that is either secreted from a cell, or is associated with the cell membrane, and upon binding to the Sreceptor for that protein on a target cell, triggers a response in the target cell.

10 An "effective amount" of an agent of the invention is that amount which produces a result or exerts an influence on the particular condition being treated.

"functional equivalent" of an amino acid residue is an amino acid having similar reactive properties as the amino acid residue that was replaced by the functional equivalent; (ii) an amino acid of a ligand of a polypeptide of the invention, the amino acid having similar properties as the amino acid residue that was replaced by the functional equivalent; (iii) a non-amino acid molecule having similar properties as the amino acid residue that was replaced by the functional equivalent.

A first polynucleotide encoding hedgehog protein is "functionally equivalent" compared with a second polynucleotide encoding hedgehog protein if it satisfies at least one of the following conditions: the "functional equivalent" is a first polynucleotide that hybridizes to the second polynucleotide under standard hybridization conditions and/or is degenerate to the first polynucleotide sequence. Most preferably, it encodes a mutant hedgehog having the activity of an hedgehog protein; the "functional equivalent" is a first polynucleotide that codes on expression for an amino acid sequence encoded by the second polynucleotide.

The term "hedgehog" includes, but is not limited to, the agents listed herein as well as their functional equivalents. As used herein, the term "functional equivalent" therefore refers to an hedgehog protein or a polynucleotide encoding the hedgehog protein that has the same or an improved beneficial effect on the mammalian recipient as the hedgehog of which it is deemed a functional equivalent. As will be appreciated by one of ordinary skill in the art, a functionally equivalent protein can be produced by recombinant techniques, by expressing a "functionally equivalent DNA".

Accordingly, the instant invention embraces hedgehog proteins encoded by naturallyoccurring DNAs, as well as by non-naturally-occurring DNAs which encode the same protein as encoded by the naturally-occurring DNA. Due to the degeneracy of the nucleotide coding sequences, other polynucleotides may be used to encode hedgehog protein. These include all, or portions of the above sequences which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Such altered sequences are regarded as equivalents of these sequences. For example, Phe is coded for by two codons, TTC or TTT, Tyr is coded for by TAC or TAT and His is coded for by CAC or CAT. On the other hand, Trp is coded for by a single codon, TGG. Accordingly, it will be appreciated that for a given DNA sequence encoding a particular hedgehog there will be many DNA degenerate sequences that will code for it. These degenerate DNA sequences are considered within the scope of this invention.

"fusion"- refers to a co-linear linkage of two or more proteins or fragments thereof via their individual peptide backbones through genetic expression of a polynucleotide molecule encoding those proteins. It is preferred that the proteins or fragments thereof be from different sources. Thus, preferred fusion proteins include an hedgehog protein or fragment covalently linked to a second moiety that is not an hedgehog. Specifically, an "hedgehog protein/ Ig fusion" is a protein comprising an hedgehog protein of the invention, or fragment thereof linked to an N-terminus of an immunoglobulin chain wherein a portion of the N-terminus of the immunoglobulin is replaced with the hedgehog protein.

The term "fusion" or "fusion protein" refers to a co-linear, covalent linkage of two or more proteins or fragments thereof via their individual peptide backbones, most preferably through genetic expression of a polynucleotide molecule encoding those proteins. It is preferred that the proteins or fragments thereof are from different sources.

Thus, preferred fusion proteins include an hedgehog protein or fragment covalently linked to a second moiety that is not a hedgehog protein. Specifically, a "hedgehog/Ig fusion" is a protein comprising a biologically active hedgehog molecule of the invention Sonic hedgehog), or a biologically active fragment thereof linked to an N-terminus of an immunoglobulin chain wherein a portion of the N-terminus of the immunoglobulin is replaced with the hedgehog. A species of hedgehog/Ig fusion is an "hedgehog /Fc fusion" which is a protein comprising an hedgehog molecule of the invention hedgehog linked to at least a part of the constant domain of an immunoglobulin. A preferred Fc fusion comprises a hedgehog mutein of the invention linked to a fragment of an antibody containing the C terminal domain of the heavy immunoglobulin chains. Also, the term "fusion protein" means an hedgehog protein chemically linked via a mono- or hetero- functional molecule to a second moiety that is not an hedgehog protein and is made de novo from purified protein as described below.

"Heterologous promoter"- as used herein is a promoter which is not naturally 00 f associated with a gene or a purified nucleic acid.

3 "Homology"- as used herein is synonymous with the term "identity" and refers to I the sequence similarity between two polypeptides, molecules, or between two nucleic acids. When a position in both of the two compared sequences is occupied by the same N, 10 base or amino acid monomer subunit (for instance, if a position in each of the two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by a lysine), then the respective molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For instance, if 6 of 10 of the positions in two sequences are matched or are homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences CTGACT and CAGGTT share 50% homology (3 of the 6 total positions are matched). Generally, a comparison is made when two sequences are aligned to give maximum homology. Such alignment can be provided using, for instance, the method of Needleman et al., J. Mol Biol. 48: 443-453 (1970), implemented conveniently by computer programs described in more detail below. Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. In this regard, a "conservative substitution" of a residue in a reference sequence are those substitutions that are physically or functionally similar to the corresponding reference residues, that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" in Dayhoff et al., 5: Atlas of Protein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed. Res. Foundation, Washington, D.C. (1978).

"Homology" and "identity" each refer to sequence similarity between two polypeptide sequences, with identity being a more strict comparison. Homology and identity can each be determined by comparing a position in each sequence which may o -11- O be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same amino acid residue, then the polypeptides can be referred to as identical at that position; when the equivalent site is occupied by the same amino acid identical) or a similar amino acid similar in steric and/or electronic nature), then the molecules can be refered to as homologous at that position. A percentage of 00 homology or identity between sequences is a function of the number of matching or eC homologous positions shared by the sequences. An "unrelated" or "non-homologous" N sequence shares less than 40 percent identity, though preferably less than 25 percent 0identity, with an AR sequence of the present invention.

C 10 Various alignment algorithms and/or programs may be used, including FASTA, BLAST or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

A "hedgehog protein" of the invention is defined in terms of having at least a portion that consists of the consensus amino acid sequence of Figure 2 (SEQ ID NO: 26). The term also means a hedgehog polypeptide, or a functional variant of a hedgehog polypeptide, or homolog of a hedgehog polypeptide, or functional variant, which has biological activity.

The term "Hedgehog N-terminal fragment" is used interchangeably with "Hedgehog" and refers to the active mature sequence that is proteolytically cleaved from the hedgehog precursor.

The term "hydrophobic" refers to the tendency of chemical moieties with nonpolar atoms to interact with each other rather than water or other polar atoms.

Materials that are "hydrophobic" are, for the most part, insoluble in water. Natural products with hydrophobic properties include lipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes, isoprenoids, retenoids, biotin, and hydrophobic amino acids such as tryptophan, phenylalanine, isoleucine, leucine, valine, methionine, alanine, proline, and tyrosine. A chemical moiety is also hydrophobic or has -12hydrophobic properties if its physical properties are determined by the presence of nonpolar atoms.

The phrase "internal amino acid" means any amino acid in a peptide sequence that is neither the N-terminal amino acid nor the C-terminal amino acid.

"Isolated" (used interchangeably with "substantially pure")- when applied to nucleic acid polynucleotide sequences that encode polypeptides, means an RNA or DNA polynucleotide, portion of genomic polynucleotide, cDNA or synthetic polynucleotide which, by virtue of its origin or manipulation: is not associated with all of a polynucleotide with which it is associated in nature is present in a host cell as an expression vector, or a portion thereof); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature. By "isolated" it is further meant a polynucleotide sequence that is: amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized chemically; (iii) produced recombinantly by cloning; or (iv) purified, as by cleavage and gel separation.

"Isolated" (used interchangeably with "substantially pure")- when applied to polypeptides means a polypeptide or a portion thereof which, by virtue of its origin or manipulation: is present in a host cell as the expression product of a portion of an expression vector; or (ii) is linked to a protein or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature, for example, a protein that is chemically manipulated by appending, or adding at least one hydrophobic moiety to the protein so that the protein is in a form not found in nature.. By "isolated" it is further meant a protein that is synthesized chemically; or (ii) expressed in a host cell and purified away from associated and contaminating proteins. The term generally means a polypeptide that has been separated from other proteins and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) which are used to purify it.

"multivalent protein complex"- refers to a plurality of hedgehog proteins one or more). An antibody homology or fragment is attached to at least one of the plurality of hedgehog proteins. The hedgehog protein or the antibody homolog or fragement may be cross-linked or bound to another antibody homolog or fragment. Each protein may be the same or different and each antibody homolog or fragment may be the same or different.

o -13- O "mutant" any change in the genetic material of an organism, in particular any Schange deletion, substitution, addition, or alteration) in a wild type polynucleotide sequence or any change in a wild type protein. The term "mutein" is used interchangeably with "mutant".

"N-terminal end"- refers to the first amino acid residue (amino acid number 1) of 0n the mature form of a protein.

c "N-terminal cysteine"- refers to the amino acid number 1 as shown in Figures 1 and 2 (SEQ ID NOS. 23-26). In certain embodiments of the hedgehog protein, the N- Sterminal cysteine has been "modified". The term "modified" in this regard refers to C 10 chemical modification(s) of the N-terminal cysteine such as linkage thereof to another moiety such as a hydrophobic group and/or replacement of the N-terminal cysteine with another moiety, such as a hydrophobic group.

"operatively linked"- a polynucleotide sequence (DNA, RNA) is operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence. The term "operatively linked" includes having an appropriate start signal ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.

"protein"- any polymer consisting essentially of any of the 20 amino acids.

Although "polypeptide" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied. The term "protein" as used herein refers to peptides, proteins and polypeptides, unless otherwise noted.

The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein. The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein "Recombinant," as used herein, means that a protein is derived from recombinant, mammalian expression systems. Since hedgehog is not glycosylated nor contains disulfide bonds, it can be expressed in most prokaryotic and eukaryotic expression systems.

"Spacer" sequence refers to a moiety that may be inserted between an amino acid to be modified with an antibody homolog or fragment and the remainder of the o -14protein. A spacer is designed to provide separation between the modification and the rest of the protein so as to prevent the modification from interfering with protein function and/or make it easier for the modification to link with an antibody homolog moiety or any other moiety.

Thus, "substantially pure nucleic acid" is a nucleic acid which is not immediately 00 Scontiguous with one or both of the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic C, acid is derived. Substantially pure DNA also includes a recombinant DNA which is part Sof a hybrid gene encoding additional hedgehog sequences.

C 10 The phrase "surface amino acid" means any amino acid that is exposed to solvent when a protein is folded in its native form.

"standard hybridization conditions"- salt and temperature conditions substantially equivalent to 0.5 X SSC to about 5 X SSC and 65 o C for both hybridization and wash.

The term "standard hybridization conditions" as used herein is therefore an operational definition and encompasses a range of hybridization conditions. Higher stringency conditions may, for example, include hybridizing with plaque screen buffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mM Tris-HCI (pH 1 M NaCI; 0.1% sodium pyrophosphate; 1 SDS); 10% dextran sulfate, and 100 g/ml denatured, sonicated salmon sperm DNA at 65 C for 12-20 hours, and washing with 75 mM NaCI/7.5 mM sodium citrate (0.5 x SSC)/1% SDS at 650 C.

Lower stringency conditions may, for example, include hybridizing with plaque screen buffer, 10% dextran sulfate and 110 pg/ml denatured, sonicated salmon sperm DNA at C for 12-20 hours, and washing with 300 mM NaCI/30mM sodium citrate (2.0 X SSC)/1% SDS at 55 o C. See also Current Protocols in Molecular Biology, John Wiley Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989).

A "therapeutic composition" as used herein is defined as comprising the proteins of the invention and other biologically compatible ingredients. The therapeutic composition may contain excipients such as water, minerals and carriers such as protein.

"wild type" the naturally-occurring polynucleotide sequence of an exon of a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.

O Practice of the present invention will employ, unless indicated otherwise, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature. Unless stipulated otherwise, all references cited in the Detailed Description are incorporated herein by reference.

00 t II. General Properties of Isolated Hedgehog Proteins Cc The various naturally-occurring hedgehog proteins from which the subject C proteins can be derived are characterized by a signal peptide, a highly conserved N- Sterminal region (see Figure and a more divergent C-terminal domain. In addition to C 10 signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev. 2635-2645; Chang, D.E. et al. (1994) Development 120:3339-3353), hedgehog precursor proteins naturally undergo an internal autoproteolytic cleavage which depends on conserved sequences in the C-terminal portion (Lee et al. (1994) Science 266:1528-1537; Porter et al. (1995) Nature 374:363- 366). This autocleavage leads to a 19 kD N-terminal peptide and a C-terminal peptide of 26-28 kD. The N-terminal peptide stays tightly associated with the surface of cells in which it was synthesized, while the C-terminal peptide is freely diffusible both in vitro and in vivo. Cell surface retention of the N-terminal peptide is dependent on autocleavage, as a truncated form of hedgehog encoded by an RNA which terminates precisely at the normal position of internal cleavage is diffusible in vitro (Porter et al.

(1995) supra) and in vivo (Porter, J.A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown that the autoproteolytic cleavage of the hedgehog precursor protein proceeds through an internal thioester intermediate, which subsequently is cleaved in a nucleophilic substitution.

The vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene (reference). Three of these members, herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals.

A fourth member, herein referred to as tiggie-winkle hedgehog (Thh), appears specific to fish. Isolated hedgehog proteins used in the methods of this invention are naturally occurring or recombinant proteins of the hedgehog family and may be obtainable from either invertebrate or from vertebrate sources (see references below). Members of the vertebrate hedgehog protein family share homology with proteins encoded by the 0 -16- Drosophila hedgehog (hh) gene (Mohler and Vani, (1992) Development 115, 957-971).

Other members continue to be identified.

Mouse and chicken Shh and mouse Ihh genes (see, for example, U.S. Patent 5,789,543) encode glycoproteins which undergo cleavage, yielding an amino terminal fragment of about 20kDa and a carboxy terminal fragment of about 25kDa. The most ^t preferred 20kDa fragment has the consensus sequence SEQ ID NO: 26 which includes 3 the amino acid sequences of SEQ ID NOS: 23-25. Various other fragments that I encompass the 20kDa moiety are considered within the presently claimed invention.

SPublications disclosing these sequences, as well as their chemical and physical properties, include Hall et al., (1995) Nature 378, 212-216; Ekker et al., (1995) Current Biology 5, 944-955; Fan et al., (1995) Cell 81, 457-465, Chang et al., (1994) Development 120, 3339-3353; Echelard et al., (1993) Cell 75, 1414-1430 34-38); PCT Patent Application WO 95/23223 (Jessell, Dodd, Roelink and Edlund; PCT Patent Publication WO 95/18856 (Ingham, McMahon and Tabin). U.S. Patent 5,759,811 lists the Genbank accession numbers of a complete mRNA sequence encoding human Sonic hedgehog; a partial sequence of human Indian hedgehog mRNA, 5' end; and a partial sequence of human Desert hedgehog mRNA. The hedgehog therapeutic compositions of the subject method can be generated by any of a variety of techniques, including purification of naturally occurring proteins, recombinantly produced proteins and synthetic chemistry. Polypeptide forms of the hedgehog therapeutics are preferably derived from vertebrate hedgehog proteins, have sequences corresponding to naturally occurring hedgehog proteins, or fragments thereof, from vertebrate organisms.

However, it will be appreciated that the hedgehog polypeptide can correspond to a hedgehog protein (or fragment thereof) which occurs in any metazoan organism.

The vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene (SEQ ID No. 19). Three of these members, herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals. A fourth member, herein referred to as tiggie-winkle hedgehog (Thh), appears specific to fish. According to the appended sequence listing, (see also Table 1) a chicken Shh polypeptide is encoded by SEQ ID No:l; a mouse Dhh polypeptide is encoded by SEQ ID No:2; a mouse Ihh polypeptide is encoded by SEQ ID No:3; a mouse Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shh polypeptide is encoded by SEQ ID No:5; a human Shh polypeptide is encoded by SEQ ID No:6; a -17human Ihh polypeptide is encoded by SEQ ID No:7; a human Dhh polypeptide is encoded by SEQ ID No. 8; and a zebrafish Thh is encoded by SEQ ID No. 9.

Table 1 Guide to hedgehog sequences in Sequence Listing Nucleotide Amino Acid Chicken Shh SEQ ID No. 1 SEQ ID No. Mouse Dhh SEQ ID No. 2 SEQ ID No. 11 Mouse Ihh SEQ ID No. 3 SEQ ID No. 12 Mouse Shh SEQ ID No. 4 SEQ ID No. 13 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 14 Human Shh SEQ ID No. 6 SEQ ID No. Human Ihh SEQ ID No. 7 SEQ ID No. 16 Human Dhh SEQ ID No. 8 SEQ ID No. 17 Zebrafish Thh SEQ ID No. 9 SEQ ID No. 18 Drosophila HH SEQ ID No. 19 SEQ ID No. In addition to the sequence variation between the various hedgehog homologs, the hedgehog proteins are apparently present naturally in a number of different forms, including a pro-form, a full-length mature form, and several processed fragments thereof. The pro-form includes an N-terminal signal peptide for directed secretion of the extracellular domain, while the full-length mature form lacks this signal sequence.

As described above, further processing of the mature form occurs in some instances to yield biologically active fragments of the protein. For instance, sonic hedgehog undergoes additional proteolytic processing to yield two peptides of approximately 19 kDa and 27 kDa, the 19kDa fragment corresponding to an proteolytic N-terminal portion of the mature protein.

In addition to the sequence variation between the various hedgehog homologs, the proteins are apparently present naturally in a number of different forms, including a pro-form, a full-length mature form, and several processed fragments thereof. The proform includes an N-terminal signal peptide for directed secretion of the extracellular domain, while the full-length mature form lacks this signal sequence.

Family members useful in the methods of the invention include any of the naturally-occurring native hedgehog proteins including allelic, phylogenetic counterparts or other variants thereof, whether naturally-sourced or produced chemically including muteins or mutant proteins, as well as recombinant forms and b -18- O new, active members of the hedgehog family. Particularly useful hedgehog polypeptides have portions that include all or part of Figures 1 and 2 (SEQ ID NOS: 23-26).

Isolated hedgehog polypeptides used in the method of the invention have biological activity. The polypeptides include an amino acid sequence at least 00 ttn 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from Figures 1 c and/or 2 (SEQ ID NOS: 23-26). The polypeptide can also include an amino acid C sequence essentially the same as an amino acid sequence in Figures 1 and/or 2 (SEQ ID NOS: 23-26). The polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length and includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from Figures 1 and/or 2 (SEQ ID NOS: 23-26).

Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and posttranslational events. The polypeptide can be made entirely by synthetic means or can be expressed in systems, cultured cells, which result in substantially the same posttranslational modifications present when the protein is expressed in a native cell, or in systems which result in the omission of posttranslational modifications present when expressed in a native cell.

In one embodiment, isolated hedgehog is a hedgehog polypeptide with one or more of the following characteristics: it has at least 30, 40, 42, 50, 60, 70, 80, 90 or 95% sequence identity with amino acids of SEQ ID NOS: 23-26; (ii) it has a cysteine or a functional equivalent as the N-terminal end; (iii) it may induce alkaline phosphatase activity in C3H10T1/2 cells; (iv) it has an overall sequence identity of at least 50%, preferably at least more preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ ID NO; 23-26 it can be isolated from natural sources such as mammalian cells; (vi) it can bind or interact with patched; and (vii) it is modified at at least one amino acid residue by a polyalkylene glycol polymer attached to the residue or, optionally, via a linker molecule to the amino acid residue.

Preferred nucleic acids encode a polypeptide comprising an amino acid sequence at least 60% homologous or identical, more preferably 70% homologous or -19- Sidentical, and most preferably 80% homologous or identical with an amino acid sequence selected from the group consisting of Figures 1 and 2 (SEQ ID NOS: 23-26).

Nucleic acids which encode polypeptides at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology or identity with an amino acid sequence represented in one of SEQ ID Nos: 23-26 are also within the oO scope of the invention.

SIn another embodiment, the hedgehog protein is a polypeptide encodable by a C nucleotide sequence that hybridizes under stringent conditions to a hedgehog coding Ssequence represented in one or more of SEQ ID NOS: 1-9 or 19. Appropriate 1 10 stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45 degrees C, followed by a wash of 2.0 x SSC at 50 degrees C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 degrees C to a high stringency of about 0.2 x SSC at degrees C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 degrees C, to high stringency conditions at about 65 degrees C.

Preferred nucleic acids encode a hedgehog polypeptide comprising an amino acid sequence at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence selected from the group consisting of SEQ ID Nos:8-14. Nucleic acids which encode polypeptides at least about more preferably at least about 95%, and most preferably at least about 98-99% homology with an amino acid sequence represented in one of SEQ ID Nos:10-18 or are also within the scope of the invention.

Hedgehog polypeptides preferred by the present invention, in addition to native hedgehog proteins, are at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence represented by any of SEQ ID Nos:10-18 or 20. Polypeptides which are at least 90%, more preferably at least 95%, and most preferably at least about 98-99% homologous with a sequence selected from the group consisting of SEQ ID Nos:10-18 or 20 are also within the scope of the invention.

0 With respect to fragments of hedgehog polypeptide, preferred hedgehogs moieties include at least 50 amino acid residues of a hedgehog polypeptide, more preferably at least 100, and even more preferably at least 150.

Another preferred hedgehog polypeptide which can be included in the hedgehog therapeutic is an N-terminal fragment of the mature protein having a molecular weight 00 n of approximately 19 kDa.

c Preferred human hedgehog proteins include N-terminal fragments Scorresponding approximately to residues 24-197 of SEQ ID No. 15, 28-202 of SEQ ID No. 16, and 23-198 of SEQ ID No. 17. By "corresponding approximately" it is meant that the sequence of interest is at most 20 amino acid residues different in length to the reference sequence, though more preferably at most 5, 10 or 15 amino acid different in length.

Still other preferred hedgehog polypeptides includes an amino acid sequence represented by the formula A-B wherein: A represents all or the portion of the amino acid sequence designated by residues 1-168 of SEQ ID No: 21 or residues 1-167 of SEQ ID NO. 22; and B represents at least one amino acid residue of the amino acid sequence designated by residues 169-221 of SEQ ID No:21; (ii) A represents all or the portion of the amino acid sequence designated by residues 24-193 of SEQ ID and B represents at least one amino acid residue of the amino acid sequence designated by residues 194-250 of SEQ ID No: 15; (iii) A represents all or the portion of the amino acid sequence designated by residues 25-193 of SEQ ID No:13; and B represents at least one amino acid residue of the amino acid sequence designated by residues 194- 250 of SEQ ID No:13; (iv) A represents all or the portion of the amino acid sequence designated by residues 23-193 of SEQ ID No:11; and B represents at least one amino acid residue of the amino acid sequence designated by residues 194-250 of SEQ ID No:l 1; A represents all or the portion of the amino acid sequence designated by residues 28-197 of SEQ ID No: 12; and B represents at least one amino acid residue of the amino acid sequence designated by residues 198-250 of SEQ ID No:12; (vi) A represents all or the portion of the amino acid sequence designated by residues 29-197 of SEQ ID No:16; and B represents at least one amino acid residue of the amino acid sequence designated by residues 198-250 of SEQ ID No:16; or (vii) A represents all or the portion of the amino acid sequence designated by residues 23-193 of SEQ ID No.

17, and B represents at least one amino acid residue of the amino acid sequence designated by residues 194-250 of SEQ ID No. 17. In certain preferred embodiments, A -21- O and B together represent a contiguous polypeptide sequence designated sequence, A Srepresents at least 25, 50, 75, 100, 125 or 150 amino acids of the designated sequence, and B represents at least 5, 10, or 20 amino acid residues of the amino acid sequence designated by corresponding entry in the sequence listing, and A and B together preferably represent a contiguous sequence corresponding to the sequence listing entry.

00 SSimilar fragments from other hedgehog also contemplated, fragments which c correspond to the preferred fragments from the sequence listing entries which are C1 enumerated above.

O Generally, the structure of the a preferred conjugated hedgehog protein of this N 10 invention has the general formula: X-Y-Z, where wherein X is a polypeptide having the amino acid sequence, or portion thereof, consisting of the amino acid sequence of hedgehog; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog. Preferably, X is human Sonic, Indian or Desert hedgehog. In the preferred embodiments, Z is at least a portion of a constant region of an immunoglobulin and can be derived from an immunoglobulin of the class selected from IgM, IgG, IgD, IgA, and IgE. If the class is IgG, then it is selected from one of IgG, IgG2, IgG3 and IgG4. The constant region of human IgM and IgE contain 4 constant regions (CH1, (hinge), CH2, CH3 and CH4, whereas the constant region of human IgG, IgA and IgD contain 3 constant regions (CH1, (hinge), CH2 and CH3). In the most preferred fusion proteins of the invention, the constant region contains at least the hinge, CH2 and CH3 domains.

Another embodiment where A is a non-hedgehog moiety such as an immunoglobulin or fragment thereof; [Sp] is an optional spacer peptide sequence; B is a hedgehog protein (which optionally may be a mutein as described herein); and X is an optional hydrophobic moiety linked (optionally by way of the spacer peptide) to the hedgehog protein B or another residue such as a surface site of the protein.

III. Production of Recombinant Polypeptides The isolated hedgehog polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host.

In one embodiment of a recombinant method, a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding a wild type protein of interest.

-22- 0 Optionally, the sequence may be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, United States Patent 4,588,585. Another method of constructing a DNA sequence encoding a polypeptide of interest would be by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides may be preferably designed based on the amino acid sequence of the desired it polypeptide, and preferably selecting those codons that are favored in the host cell in c which the recombinant polypeptide of interest will be produced.

Standard methods may be applied to synthesize an isolated polynucleotide Ssequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence may be used to construct a back-translated gene. See Maniatis et al., supra. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide may be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or by another method), the mutant DNA sequences encoding a particular isolated polypeptide of interest will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations may be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Esherichia coli, including pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages. Preferred E. coli vectors include pL vectors containing the lambda phage pL promoter Patent 4,874,702), pET vectors containing the T7 polymerase promoter (Studier et al., Methods in Enzymology 185: 60-89, 1990 1) and the pSP72 vector (Kaelin et al., supra). Useful expression vectors for yeast cells, for example, include the 2 T and centromere plasmids. Further, within each specific expression vector, various sites may be selected for insertion of these DNA sequences. These sites are usually designated by the restriction endonuclease which cuts them. They are well-recognized by those of skill in the art. It will be appreciated that a given expression vector useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vector may be joined by the fragment by alternate means.

The expression vector, and the site chosen for insertion of a selected DNA fragment and operative linking to an expression control sequence, is determined by a variety of factors such as: the number of sites susceptible to a particular restriction enzyme, the size of the polypeptide, how easily the polypeptide is proteolytically degraded, and the like. The choice of a vector and insertion site for a given DNA is determined by a balance of these factors.

To provide for adequate transcription of the recombinant constructs of the invention, a suitable promoter/enhancer sequence may preferably be incorporated into the recombinant vector, provided that the promoter/expression control sequence is capable of driving transcription of a nucleotide sequence encoding a hedgehog protein.

Any of a wide variety of expression control sequences may be used in these vectors.

Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example pL, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast alpha-mating system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses, and various combinations thereof.

Promoters which may be used to control the expression of immunoglobulinbased fusion protein include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.

b -24- U.S.A. 78:144-1445), the regulatory sequences of the metallothionine gene (Brinster et al., 1982, Nature 296:39-42); plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res.

9:2871), and the promoter for the photosynthetic enzyme ribulose biphosphate 00 tn carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements 3 from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) C promoter, PGK (phosphoglycerol kinase) promoter, alkaline phophatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity C 10 and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425- 515); insulin gene enhancers or promoters which are active in pancreatic cells (Hanahan, 1985, Nature 315:115-122); immunoglobulin gene enhancers or promoters which are active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); the cytomegalovirus early promoter and enhancer regions (Boshart et al., 1985, Cell 41:521-530); mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268- 276); alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alphantitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1:161-171); -globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Any suitable host may be used to produce in quantity the isolated hedgehog polypeptides described herein, including bacteria, fungi (including yeasts), plants, insects, mammals, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More particularly, these hosts may include well known eukaryotic Sand prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, r fungi, yeast Hansenula insect cells such as Spodopterafrugiperda (SF9), and High Five T M (see Example animal cells such as Chinese hamster ovary (CHO), mouse cells such as NS/O cells, African green monkey cells COS1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells.

00 n It should be understood that not all vectors and expression control sequences

O

Swill function equally well to express a given isolated polypeptide. Neither will all hosts C1 function equally well with the same expression system. However, one of skill in the art 0 may make a selection among these vectors, expression control systems and hosts N 10 without undue experimentation. For example, to produce isolated polypeptide of interest in large-scale animal culture, the copy number of the expression vector must be controlled. Amplifiable vectors are well known in the art. See, for example, Kaufman and Sharp, (1982) Mol. Cell. Biol., 2, 1304-1319 and U.S. Patents 4,470,461 and 5,122,464.

Such operative linking of a DNA sequence to an expression control sequence includes the provision of a translation start signal in the correct reading frame upstream of the DNA sequence. If the particular DNA sequence being expressed does not begin with a methionine, the start signal will result in an additional amino acid (methionine) being located at the N-terminus of the product. If a hydrophobic moiety is to be linked to the N-terminal methionyl-containing protein, the protein may be employed directly in the compositions of the invention. Neverthless, since the preferred N-terminal end of the protein is to consist of a cysteine (or functional equivalent) the methionine must be removed before use. Methods are available in the art to remove such N-terminal methionines from polypeptides expressed with them. For example, certain hosts and fermentation conditions permit removal of substantially all of the N-terminal methionine in vivo. Other hosts require in vitro removal of the N-terminal methionine.

Such in vitro and in vivo methods are well known in the art.

Successful incorporation of these polynucleotide constructs into a given expression vector may be identified by three general approaches: DNA-DNA hybridization, presence or absence of "marker" gene functions, and expression of inserted sequences. In the first approach, the presence of the hedgehog gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to the inserted fusion protein gene. In the second approach, the recombinant vector/host system can be identified and selected -26-

O

C based upon the presence or absence of certain "marker" gene functions thymidine kinase activity, resistance to antibiotics such as G418, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the polynucleotide is inserted so as to interrupt a marker o 5 gene sequence of the vector, recombinants containing the insert can be identified by the t absence of the marker gene function. In the third approach, recombinant expression 3 vectors can be identified by assaying the foreign gene product expressed by the In recombinant vector. Such assays can be based, for example, on the physical or functional properties of the gene product in bioassay systems.

The preferred embodiment of the invention contemplates fusion proteins and DNA sequences coding for them. These fusion proteins have an amino-terminal region characterized by the amino acid sequence of hedgehog and a carboxy-terminal region comprising a domain of a protein other than hedgehog A preferred generic formula for such a protein is a protein having a primary amino acid sequence X-Y-Z, wherein X is a polypeptide having the amino acid sequence, or portion thereof, consisting of the amino acid sequence of human hedgehog; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than human hedgehog.

Moiety Z can include, for instance, a plurality of histidine residues or the Fc region of an immunoglobulin, "Fc" defined herein as a fragment of an antibody containing the C terminal domain of the heavy immunoglobulin chains.

In the most preferred fusion proteins, the hedgehog polypeptide is fused to at least a portion of the Fc region of an immunoglobulin. The hedgehog forms the aminoterminal portion, and the Fc region forms the carboxy terminal portion. In these fusion proteins, the Fc region is preferably limited to the constant domain hinge region and the CH2 and CH3 domains. The Fc region in these fusions can also be limited to a portion of the hinge region, the portion being capable of forming intermolecular disulfide bridges, and the CH2 and CH3 domains, or functional equivalents thereof. These constant regions may be derived from any mammalian source (preferably human) and may be derived from any appropriate class and/or isotype, including IgA, IgD, IgM, IgE and IgG1, IgG2, IgG3 and IgG4.

Recombinant nucleic acid molecules which encode the Ig fusions may be obtained by any method known in the art (Maniatis et al., 1982, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, or obtained from publicly available clones. Methods for the preparation of genes which O encode the heavy or light chain constant regions of immunoglobulins are taught, for example, by Robinson, R. et al., PCT Application, Publication No. W087-02671. The cDNA sequence encoding the hedgehog molecule or fragment may be directly joined to the cDNA encoding the heavy Ig contant regions or may be joined via a linker sequence. In further embodiments of the invention, a recombinant vector system may 00 trn be created to accommodate sequences encoding hedgehog in the correct reading frame Cc with a synthetic hinge region. Additionally, it may be desirable to include, as part of the CNI recombinant vector system, nucleic acids corresponding to the 3' flanking region of an 0immunoglobulin gene including RNA cleavage/polyadenylation sites and downstream C, 10 sequences. Furthermore, it may be desirable to engineer a signal sequence upstream of the immunoglobulin fusion protein-encoding sequences to facilitate the secretion of the fused molecule from a cell transformed with the recombinant vector.

The present invention provides for dimeric fusion molecules as well as monomeric or multimeric molecules comprising fusion proteins. Such multimers may be generated by using those Fc regions, or portions thereof, of Ig molecules which are usually multivalent such as IgM pentamers or IgA dimers. It is understood that a J chain polypeptide may be needed to form and stabilize IgM pentamers and IgA dimers.

Alternatively, multimers of hedgehog fusion proteins may be formed using a protein with an affinity for the Fc region of Ig molecules, such as Protein A. For instance, a plurality of hedgehog immunoglobulin fusion proteins may be bound to Protein Aagarose beads.

These multivalent forms are useful since they possess multiple hedgehog receptor binding sites. For example, a bivalent soluble hedgehog may consist of two tandem repeats of the amino acids of SEQ ID NO: 24 (or those encoded by nucleic acids of SEQ. ID. NO: 6) (moiety X in the generic formula) separated by a linker region (moiety the repeats bound to at least a portion of an immunoglobulin constant domain (moiety Alternate polyvalent forms may also be constructed, for example, by chemically coupling hedgehog -/Ig fusions to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran using conventional coupling techniques. Alternatively, hedgehog may be chemically coupled to biotin, and the biotin-hedgehog Fc conjugate then allowed to bind to avidin, resulting in tetravalent avidin/biotin/hedgehog molecules. Hedgehog/Ig fusions may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) 0 -28and the resulting conjugate precipitated with anti-DNP or anti-TNP-IgM, to form decameric conjugates with a valency of 10 for hedgehog receptor binding sites The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or 00 Sby any other standard technique for protein purification. For immunoaffinity 3 chromatography (See Example a protein such as Sonic hedgehog may be isolated by binding it to an affinity column comprising of antibodies that were raised against Sonic hedgehog, or a related protein and were affixed to a stationary support. For ,I 10 example, the hedgehog proteins and fragments may be purified by passing a solution thereof through a column having an hedgehog receptor immobilized thereon (see U.S.

Pat. No. 4,725,669). The bound hedgehog molecule may then be eluted by treatment with a chaotropic salt or by elution with aqueous acetic acid. The immunoglobulin fusion proteins may be purified by passing a solution containing the fusion protein through a column which contains immobilized protein A or protein G which selectively binds the Fc portion of the fusion protein. See, for example, Reis, K. et al., J. Immunol. 132:3098-3102 (1984); PCT Application, Publication No.

W087/00329. The chimeric antibody may then be eluted by treatment with a chaotropic salt or by elution with aqueous acetic acid. Alternatively the hedgehog proteins and immunoglobulin-fusion molecules may be purified on anti-hedgehog antibody columns, or on anti-immunoglobulin antibody columns to give a substantially pure protein. By the term "substantially pure" is intended that the protein is free of the impurities that are naturally associated therewith. Substantial purity may be evidenced by a single band by electrophoresis. Alternatively, affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be characterized physically using such techniques as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

An example of a useful hedgehog/Ig fusion protein of this invention is that of SEQ ID NO: 83, which is secreted into the cell culture by eukaryotic cells containing the expression plasmid PUB 116 (See Examples). This protein consists of the mature human hedgehog fused to a portion of the hinge region and the CH2 and CH3 constant -29- Sdomains of murine Ig. This contains a sufficient portion of the murine immunoglobulin to be recognized by the Fc binding protein, Protein A.

Other fusion proteins of the invention incorporating human hedgehog are shown in SEQ NOS: 80-82.

The preferred hedgehog proteins of the invention include the novel '"junction" 00 00DNA sequences which represent the 11 triplet codons on either side of the junction Cc between the hedgehog DNA and the DNA encoding the non-hedgehog moiety.

The DNA "junction" sequences can be used as DNA probes and may be the minimum DNA needed for hybridization under standard conditions to any DNA N 10 sequence encoding any hedgehog -/Ig fusion protein. Nevertheless, provided that the whole probe hybridizes to both sides of the junction and both sides of the hedgehog /constant region junction participate in the hybridization, smaller sequences may exist.

Furthermore, persons having ordinary skill in the art will understand that DNA sequences larger than these will be suitable for hybridization as well. One of ordinary skill in the art can test if a particular probe is capable of hybridizing on both sides of the junction by labelling the 5' end of either a single strand sense oligonucleotide or a single strand anti-sense oligonucleotide with an appropriately labelled phosphate of ATP using polynucleotide kinase. A sequence of the invention must hybridize to, and thus be labelled by both oligonucleotide probes. It is further understood that the invention encompasses fully degenerate sequences encoding junction sequences.

The most preferred hedgehog fusion proteins contain mutations in the putative KEX2 recognition site (See Table A. Production of Fragments and Analogs Fragments of an isolated protein fragments of SEQ ID NOS: 23-26) can also be produced efficiently by recombinant methods, by proteolytic digestion, or by chemical synthesis using methods known to those of skill in the art. In recombinant methods, internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a DNA sequence which encodes for the isolated hedgehog polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end nibbling" endonucleases can also generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated by random shearing, restriction digestion, or a combination or both.

Protein fragments can be generated directly from intact proteins. Peptides can be

I

0 cleaved specifically by proteolytic enzymes, including, but not limited to plasmin, thrombin, trypsin, chymotrypsin, or pepsin. Each of these enzymes is specific for the type of peptide bond it attacks. Trypsin catalyzes the hydrolysis of peptide bonds in which the carbonyl group is from a basic amino acid, usually arginine or lysine.

Pepsin and chymotrypsin catalyse the hydrolysis of peptide bonds from aromatic 00 in amino acids, such as tryptophan, tyrosine, and phenylalanine. Alternative sets of r cleaved protein fragments are generated by preventing cleavage at a site which is I suceptible to a proteolytic enzyme. For instance, reaction of the e-amino acid group of lysine with ethyltrifluorothioacetate in mildly basic solution yields blocked amino acid residues whose adjacent peptide bond is no longer susceptible to hydrolysis by trypsin. Proteins can be modified to create peptide linkages that are susceptible to proteolytic enzymes. For instance, alkylation of cysteine residues with 13haloethylamines yields peptide linkages that are hydrolyzed by trypsin (Lindley, 0956) Nature 178, 647). In addition, chemical reagents that cleave peptide chains at specific residues can be used. For example, cyanogen bromide cleaves peptides at methionine residues (Gross and Witkip, (1961) J. Am. Chem. Soc. 83, 1510). Thus, by treating proteins with various combinations of modifiers, proteolytic enzymes and/or chemical reagents, the proteins may be divided into fragments of a desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

Fragments can also be synthesized chemically using techniques known in the art such as the Merrifield solid phase F moc or t-Boc chemistry. Merrifield, Recent Progress in Hormone Research 23: 451 (1967) Examples of prior art methods which allow production and testing of fragments and analogs are discussed below. These, or analogous methods may be used to make and screen fragments and analogs of an isolated polypeptide hedgehog) which can be shown to have biological activity. An exemplary method to test whether fragments and analogs of hedgehog have biological activity is found in Example 3.

B. Production of Altered DNA and Peptide Sequences: Random Methods Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes the protein or a particular portion thereof. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate -31oligonucleotide sequences. Methods of generating amino acid sequence variants of a given protein using altered DNA and peptides are well-known in the art. The following examples of such methods are not intended to limit the scope of the present invention, but merely serve to illustrate representative techniques. Persons having ordinary skill in the art will recognize that other methods are also useful in this regard.

PCR Mutagenesis: See, for example Leung et al., (1989) Technique 1, 11-15.

Saturation Mutagenesis: One method is described generally in Mayers et al., (1989) Science 229, 242.

Degenerate Oligonucleotide Mutagenesis: See for example Harang, (1983) Tetrahedron 39, 3; Itakura et al., (1984) Ann. Rev. Biochem. 53, 323 and Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Symposium on Macromolecules, pp. 273-289 Walton, Elsevier, Amsterdam, 1981.

C. Production of Altered DNA and Peptide Sequences: Directed Methods Non-random, or directed, mutagenesis provides specific sequences or mutations in specific portions of a polynucleotide sequence that encodes an isolated polypeptide, to provide variants which include deletions, insertions, or substitutions of residues of the known amino acid sequence of the isolated polypeptide. The mutation sites may be modified individually or in series, for instance by: substituting first with conserved amino acids and then with more radical choices depending on the results achieved; (2) deleting the target residue; or inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

Clearly, such site-directed methods are one way in which an N-terminal cysteine (or a functional equivalent) can be introduced into a given polypeptide sequence to provide the attachment site for a hydrophobic moiety.

Alanine scanning Mutagenesis: See Cunningham and Wells, (1989) Science 244, 1081-1085).

Oligonucleotide-Mediated Mutagenesis: See, for example, Adelman et al., (1983) DNA 2, 183. We created a functional antagonist using oligonucleotide-directed mutagenesis by engineering an isolated DNA sequence that encodes a functional antagonist that has a mutation of the N-terminal cysteine to another amino residue, preferably a serine residue (SEQ ID NO: 17: Example 7).

Cassette Mutagenesis: See Wells et al., (1985) Gene 34, 315.

Combinatorial Mutagenesis: See, for example, Ladner et al., WO 88/06630 D. Other Variants of Isolated Polypeptides 0 -32- Included in the invention are isolated molecules that are: allelic variants, natural mutants, induced mutants, and proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide such as the Nterminal fragment of Sonic hedgehog (SEQ ID NO: 24) and polypeptides bound specifically by antisera to hedgehog peptides, especially by antisera to an active site or 00 I0 binding site of hedgehog. All variants described herein are expected to: retain the c biological function of the original protein and (ii) retain the ability to link to at least one CI non-hedgehog moiety an Ig).

SThe methods of the invention also feature uses of fragments, preferably CN 10 biologically active fragments, or analogs of an isolated peptide such as hedgehog.

Specifically, a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the peptide shown in SEQ ID NOS: 10-20 or 23- 26 or of other naturally occurring isolated hedgehog. Most preferably, the hydrophobically-modified fragment or analog has at least 10%, preferably 40% or greater, or most preferably at least 90% of the activity of Sonic hedgehog in any in vivo or in vitro assay.

Analogs can differ from naturally occuring isolated protein in amino acid sequence or in ways that do not involve sequence, or both. The most preferred polypeptides of the invention have preferred non-sequence modifications that include in vivo or in vitro chemical derivatization of their N-terminal end), as well as possible changes in acetylation, methylation, phosphorylation, amidation, carboxylation, or glycosylation.

Other analogs include a protein such as Sonic hedgehog or its biologically active fragments whose sequences differ from the wild type consensus sequence SEQ ID NO: 26) by one or more conservative amino acid substitutions or by one or more non conservative amino acid substitutions, or by deletions or insertions which do not abolish the isolated protein's biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, alanine and glycine; leucine and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

The non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The -33- O positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Other conservative substitutions can be readily known by workers of ordinary skill.

For example, for the amino acid alanine, a conservative substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine, and D-cysteine. For lysine, a 00 3 replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D-ornithine.

Other analogs used within the methods of the invention are those with modifications which increase peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic analogs. Incorporation of D- instead of L-amino acids into the isolated hedgehog polypeptide may increase its resistance to proteases. See, U.S. Patent 5,219,990 supra.

The term "fragment", as applied to an isolated hedgehog analog, can be as small as a single amino acid provided that it retains biological activity. It may be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit isolated hedgehog biological activity can be also assessed by methods known to those skilled in the art as described herein.

Hedgehog Proteins as Antagonists Isolated hedgehog proteins useful in the present invention may be antagonists such as recombinant fusion proteins containing additional sequences unrelated to hedgehog. Thus, the antagonist polypeptide may also include all or a fragment of an amino acid sequence from SEQ ID NOS: 10-20 or 23-26, fused, in reading frame, to additional amino acid residues. One version of the polypeptides of the invention is a protein having a first polypeptide portion and a hedgehog antagonist portion, the antagonist portion being fused or otherwise linked either 5' or 3' to the first polypeptide portion. Thus, first, additional polypeptide portion has an amino acid sequence unrelated to an antagonist polypeptide. The additional polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating o -34- 0 domain, a histidine tag. It is most preferably an immunoglobulin or portion thereof, fused or otherwise linked to either the N- or C-terminus of the antagonist portion.

A preferred antagonist has at least the following properties: the isolated protein binds the receptor patched-I with an affinity that may be less than, but is preferably at least the same as, the binding of mature hedgehog protein to patched-1; 00 and (ii) the isolated protein blocks alkaline phosphatase (AP) induction by mature r hedgehog protein when tested in an in vitro CH310T1/2 cell-based AP induction assay.

Antagonists of the invention may also have the additional properties of being (iii) unable to induce ptc-1 and gli-1 expression.

C 10 Persons having ordinary skill in the art can easily test any putative hedgehog antagonist for these properties. In particular, the mouse embryonic fibroblast line C3H10T1/2 is a mesenchymal stem cell line that is hedgehog responsive (as described in more detail below). Hedgehog treatment of the cells causes an upregulation of gli-1 and patched-1 (known indicators of hedgehog dependent signaling) and also causes induction of alkaline phosphatase activity, an indicator that the cells have differentiated down the chondrocyte/ bone osteoblast lineage. Several hedgehog variants are unable to elicit a hedgehog-dependent response on C3H10T1/2 cells, but they competed with mature hedgehog for function and therefore serve as functional antagonists. These functional antagonists are preferred as the hedgehog to which a non-hedgehog immunoglobulin) moiety is conjugated. In such a circumstance, it is not necessary to provide for muteins in which the KEX2-like intracellular protease recognition site is disabled. The synthesis and use of such hedgehog antagonist moieties are briefly described below.

A. N-Modified Hedgehog Polypeptides as Antagonists Certain hedgehog variants that contain N-terminal modifications can block hedgehog function because they lack the ability to elicit a hedgehog-dependent response but retain the ability to bind to hedgehog receptor, patched-1. The critical primary amino acid sequence that defines whether a hedgehog polypeptide a Sonic, Indian or Desert hedgehog) is a functional hedgehog antagonist is the N-terminal cysteine residue which corresponds to Cys-1 of the mature hedgehog. So long as the hedgehog polypeptide either lacks this N-terminal cysteine completely or contains this N-terminal cysteine in a modified form chemically modified or included as part of an N-terminal extension moiety), the resulting polypeptide can act as a functional hedgehog antagonist. In this regard, the fact that an N-terminal cysteine "corresponds 0 O to Cys-1" means: the N-terminal cysteine is the Cys-1 of mature Sonic, Indian or Desert hedgehog; or the N-terminal cysteine occupies the same position as Cys-1 of mature Sonic, Indian or Desert hedgehog. Provided that, for example, a Sonic hedgehog has an N-terminal cysteine corresponding to Cys-1 that is altered or otherwise modified as described herein, it can antagonize the action of any other 00 t member of the hedgehog family. Therefore, persons having ordinary skill in the art c will understand that it is possible to an Indian hedgehog protein that antagonizes the C activity of Sonic, Desert or Indian hedgehogs.

SExamples of these antagonists with N-terminal modifications are included c, 10 below and one skilled in the art can alter the disclosed structure of the antagonist, e.g., by producing fragments or analogs, and test the newly produced structures for antagonist activity. These examples in no way limit the structure of any related hedgehog antagonists, but are merely provided for further description. These, or analogous methods, can be used to make and screen fragments and analogs of a antagonist polypeptides. There are several variants that are able to function as antagonists.

1. N-terminal extensions Antagonist polypeptides of the invention may include a hedgehog polypeptide sequence in which the N-terminal cysteine is linked to an N-terminal extension moiety.

The isolated antagonist polypeptide can therefore be, as but one example, a recombinant fusion protein having: a first N-terminal polypeptide portion that can be 5' to the hedgehog polypeptide itself, and that contains at least one element an amino acid residue) that may be unrelated to hedgehog, linked to an N-terminal cysteine corresponding to Cys-1 of Sonic hedgehog that is part of a hedgehog antagonist of the invention, or a portion of hedgehog antagonist. This N-terminal extension moiety the first N-terminal polypeptide portion) can be a histidine tag, a maltose binding protein, glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. The functional antagonist may include an N-terminal extension moiety that contains an element which replaces the Cys-1 of mature hedgehog or an N-terminal cysteine that corresponds to Cys-1 of a mature Sonic hedgehog.

2. N-terminal deletions Another variation of a functional antagonist is a hedgehog protein that is missing no greater than about 12 amino acids beginning from that N-terminal cysteine Q corresponding to Cys-1 of a mature hedgehog. Deletions in more than the about the first 12 contiguous amino acid residues do not generate functional antagonists.

Preferably, deletions of about 10 contiguous amino acids will provide suitable functional antagonists. One can, however, remove fewer than 10 contiguous residues and still maintain antagonist function. Moreover, one can delete various combinations 00 in of non-contiguous residues provided that there are at least about 3 deleted residues in

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c total.

CI These structures highlight the importance of the N-terminus of hedgehog Sproteins for function and indeed, underscore the need to conjugate a hedgehog protein C1 10 at a site other than the N-terminal cysteine. All of the N-terminal deletion variants were indistinguishable from mature Sonic hedgehog (Shh) in their ability to bind patched-1, but were inactive in the in vitro C3H10T1/2 AP induction assay. All these N-terminal variants are unable to promote hedgehog-dependent signaling.

3. N-terminal mutations Yet another functional antagonist has a mutation of the N-terminal cysteine to another amino acid residue. Any non-hydrophobic amino acid residue may acceptable and persons having ordinary skill in the art following the teachings described herein will be able to perform the mutations and test the effects of such mutations. One example is Shh in which the N-terminal cysteine is replaced with a serine residue. This mutated form is indistinguishable from mature Shh in its ability to bind patched-1, but it blocks AP induction by mature Shh when tested for function in the C3H10T1/2 AP induction assay. Replacements with aspartic acid, alanine and histidine have also shown to serve as antagonists.

4. N-terminal cysteine modifications Because the primary amino acid sequence of hedgehog contains the Cys-1 that is important for biological activity, certain other modifications will result in inactive antagonist variants of hedgehog protein. Another antagonist is an isolated functional antagonist of a hedgehog polypeptide, comprising a hedgehog polypeptide containing an N-terminal cysteine that corresponds to Cys-1 of a mature Sonic hedgehog, except that the cysteine is in a modified form. Antagonist polypeptides of hedgehog may have non-sequence modifications that include in vivo or in vitro chemical derivatization of their N-terminal cysteine, as well as possible changes in acetylation, methylation, phosphorylation, amidation, or carboxylation. As an example, the functional antagonist can have an N-terminal cysteine in an oxidized form. Thus, a functional S-37- O antagonist can have an N-terminal cysteine that is effectively modified by including it as part of an N-terminal extension moiety.

B. Other Embodiments The functional antagonist polypeptides can include amino acid sequences that are at least 60% homologous to a hedgehog protein. The antagonist must exhibit at 00 Sleast the following functional antagonist properties: the isolated protein binds the 0C receptor patched-1 with an affinity that may be less than, but is preferably at least the same as, the binding of mature hedgehog protein to patched-1; and (ii) the isolated Sprotein blocks alkaline phosphatase (AP) induction by mature hedgehog protein when tested in an in vitro CH310T1/2 cell-based AP induction assay.

Antagonists useful in the present invention also include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and posttranslational events. The polypeptide can be made entirely by synthetic means or can be expressed in systems, cultured cells, which result in substantially the same posttranslational modifications present when the protein is expressed in a native cell, or in systems which result in the omission of posttranslational modifications present when expressed in a native cell.

In a preferred embodiment, isolated antagonist is a polypeptide with one or more of the following characteristics: it has at least 60, more preferably 90 and most preferably 95% sequence identity with amino acids of SEQ ID NOS: 23-26; (ii) it either has a modified N-terminal cysteine or lacks an N-terminal cysteine or has an N-terminal cysteine in a position different from the N-terminal cysteine corresponding to Cys-1 of the hedgehog; (iii) it blocks alkaline phosphatase induction by mature hedgehog in CH310T1/2 cells; (iv) it binds or interacts with its receptor patched-1 with an affinity that may be less than, but is preferably at least the same as, the binding of mature hedgehog protein to patched-1; it is unable to induce ptc-1 and gli-1 expression in vitro in CH310T1/2 cells; or (vi) it is unable to induce AP in CH310T1/2 assays.

Agonists of Hedgehog Biological Activity 00 -38-

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Other preferred hedgehog polypeptides of the invention are agonists that are derived Sfrom several sources of hedgehog protein. In one embodiment, the agonist is not Nterminally clipped (as described above) and contains a mutation in its KEX2-like recognition site. Other embodiments of a hedgehog agonist suitable for use in a fusion protein, moiety, are based, in part, on the discovery disclosed in WO 2003/072036 and 0 O WO 2003/072736 that human Sonic hedgehog, expressed as a full- length construct in either insect or in mammalian cells, has a hydrophobic palmitoyl group appended to the Salpha-amine of the N-terminal cysteine. This is the first example of an extracellular signaling protein being modified in such a manner, and, in contrast to thiol-linked 10 palmitic acid modifications whose attachment is readily reversible, this novel N-linked palmitoyl moiety is likely to be very stable by analogy with myristic acid modifications.

As a direct consequence of this initial discovery, it is known that increasing the hydrophobic nature of a hedgehog signaling protein can increase the protein's biological activity. Thus, the modified hedgehog acts as its own antagonist. In particular, appending a hydrophobic moiety to a signaling protein, such as a hedgehog protein, can enhance the protein's activity, and thus, act as an agonist. The N-terminal cysteine of biologically active proteins not only provides a convenient site for appending a hydrophobic moiety, and thereby modifying the physico-chemical properties of the protein, but modifications to the N-terminal cysteine can also increase the protein's stability. Additionally, addition of a hydrophobic moiety to an internal amino acid residue on the surface of the protein structure enhances the protein's activity. Use of these agonists in conjunction with one or more non-hedgehog conjugates an immunoglobulin or fragment thereof) will allow increased bioavailability of the hedgehog agonists in a therapeutic context.

Accordingly, the methods and compositions of the present invention include the use of the conjugated hedgehog agonists due to their increased biological activity and higher patched-1 binding affinity. Moreover, the subject methods can be performed on cells which are provided in culture (in vitro), or on cells in a whole animal (in vivo).

The agonists have at least one of the following properties the isolated protein binds the receptor patched-1 with an affinity that is at similar to, but is preferably higher than, the binding of mature hedgehog protein to patched-1 or (ii) the isolated protein binds to 00 -38a- 0 a hedgehog protein in such a way as to increase the proteins binding affinity to patched- S1 when tested in an in vitro CH310TI/2 cell-based AP 00

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-39induction assay. Agonists of the invention may also have the additional properties of being (iii) able to solely induce ptc-1 and gli-1 expression.

A. General Properties of Isolated Hedgehog Proteins Acting As Agonists The polypeptide portion of the hedgehog compositions of the subject method can be generated by any of a variety of techniques, including purification of naturally occurring proteins, recombinantly produced proteins and synthetic chemistry.

Polypeptide forms of the hedgehog proteins are preferably derived from vertebrate hedgehog proteins, have sequences corresponding to naturally occurring hedgehog proteins, or fragments thereof, from vertebrate organisms. However, it will be appreciated that the hedgehog polypeptide can correspond to a hedgehog protein (or fragment thereof) which occurs in any metazoan organism.

Family members useful in the methods of the invention include any of the naturally-occurring native hedgehog proteins including allelic, phylogenetic counterparts or other variants thereof, whether naturally-sourced or produced chemically including muteins or mutant proteins, as well as recombinant forms and new, active members of the hedgehog family.

The preferred agonists for use in conjugation with a non-hedgehog conjugate immunoglobulin or fragment thereof) include a derivitized hedgehog polypeptide sequence as well as other N-terminal and/or C-terminal amino acid sequence or it may include all or a fragment of a hedgehog amino acid sequence. Agonist polypeptides of the invention include those that arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and posttranslational events. The polypeptide can be made entirely by synthetic means or can be expressed in systems, cultured cells, which result in substantially the same posttranslational modifications present when the protein is expressed in a native cell, or in systems which result in the omission of posttranslational modifications present when expressed in a native cell.

In a preferred embodiment, the agonist to be conjugated is a hedgehog polypeptide with one or more of the following characteristics: it has at least 30, 40, 42, 50, 60, 70, 80, 90 or 95% sequence identity with a hedgehog sequence; (ii) it has a cysteine or a functional equivalent as the N-terminal end; (iii) it may induce alkaline phosphatase activity in C3H10T1/2 cells; 00

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O (iv) it has an overall sequence identity of at least 50%, preferably at least Smore preferably at least 70, 80, 90, or 95%, with a polypeptide of a hedgehog sequence; it can be isolated from natural sources such as mammalian cells (vi) it can bind or interact with patched; and 0 O (vii) it is hydrophobically-modified it has at least one hydrophobic moiety Sattached to the polypeptide).

SIncreasing the overall hydrophobic nature of a hedgehog protein increases the t biological activity of the protein. The potency of a signaling protein such as hedgehog 10 can be increased by: chemically modifying, such as by adding a hydrophobic moiety to, the sulfhydryl and/or to the alpha-amine of the N-terminal cysteine (see WO 2003/072036 and W02003/072736); replacing the N-terminal cysteine with a hydrophobic amino acid (see WO 2003/072036 and W02003/072736); or replacing the N-terminal cysteine with a different amino acid and then chemically modifying the substituted residue so as to add a hydrophobic moiety at the site of the substitution.

Additionally, modification of a hedgehog protein at an internal residue on the surface of the protein with a hydrophobic moiety by: replacing the internal residue with a hydrophobic amino acid; or replacing the internal residue with a different amino acid and then chemically modifying the substituted residue so as to add a hydrophobic moiety at the site of the substitution will retain or enhance the biological activity of the protein.

Additionally, modification of a protein such as a hedgehog protein at the Cterminus with a hydrophobic moiety by: replacing the C-terminal residue with a hydrophobic amino acid; or replacing the C-terminal residue with a different amino acid and then chemically modifying the substituted residue so as to add a hydrophobic moiety at the site of the substitution, will retain or enhance the biological activity of the protein.

For hydrophobically-modified hedgehog obtained by chemically modifying the soluble, unmodified protein, palmitic acid and other lipids can be added to soluble Shh to create a lipid-modified forms with increased potency in the C3HIOT1/2 assay.

Another form of protein encompassed by the invention is a protein derivatized with a variety of lipid moieties. The principal classes of lipids that are encompassed within this 00 40a 0 o invention are fatty acids and sterols cholesterol). Derivatized proteins of the Sinvention contain fatty acids which are cyclic, acyclic straight chain), saturated 00 e¢ unsaturated, mono-carboxylic acids. Exemplary saturated fatty acids have the generic formula: CH3 (CH2)n COOH. Table 2 below lists examples of some fatty acids that can be derivatized conveniently using conventional chemical methods.

TABLE 2: Exemplary Saturated and Unsaturated Fatty Acids Saturated Acids: CH3 (CH2)n COOH: Value of n 2 4 6 8 12 14 16 18 22 Common Name butyric acid caproic acid caprylic acid capric acid lauric acid myristic acid* palmitic acid* stearic acid* arachidic acid* behenic acid lignoceric acid crotonic acid myristoleic acid* palmitoleic acid* oleic acid* linoleic acid linolenic acid arachidonic acid Unsaturated Acids: CH3CH=CHCOOH CH3(CH2)3CH=CH(CH2)7COOH CH3(CH2)5CH=CH (CH2)7COOH CH3(CH2)7CH=CH(CH2)7COOH CH3(CH2)3(CH2CH=CH)2(CH2)7COOH CH3(CH2CH=CH)3(CH2)7COOH CH3(CH2)3(CH2CH=CH)4(CH2)3COOH The asterisk denotes fatty acids detected in recombinant hedgehog protein secreted from a soluble construct (Pepinsky et al., supra).

Other lipids that can be attached to the protein include branched-chain fatty acids and those of the phospholipid group such as the phosphatidylinositols phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5- biphosphate), phosphatidycholine, phosphatidylethanolamine, phosphatidylserine, and isoprenoids such as farnesyl or geranyl groups. Lipid-modified hedgehog proteins can be purified from either a natural source, or can be obtained by chemically modifying the soluble, unmodified protein.

For protein purified from a natural source, we showed that when full-length human Sonic hedgehog (Shh) was expressed in insect cells and membrane-bound Shh purified from the detergent-treated cells using a combination of SP-Sepharose 0 -42chromatography and immunoaffinity chromatography, that the purified protein migrated on reducing SDS-PAGE gels as a single sharp band with an apparent mass of kDa. The soluble and membrane-bound Shh proteins were readily distinguishable by reverse phase HPLC, where the tethered forms eluted later in the acetonitrile gradient. We then demonstrated that human Sonic hedgehog is tethered to cell 00 t membranes in two forms, one form that contains a cholesterol, and therefore is c analogous to the data reported previously for Drosophila hedgehog, and a second novel CI form that contains both a cholesterol and a palmitic acid modification. Soluble and tethered forms of Shh were analyzed by electrospray mass spectrometry using a triple NC 10 quadrupole mass spectrometer, equipped with an electrospray ion source as well as by liquid chromatography-mass spectrometry. The identity of the N-terminal peptide from endoproteinase Lys-C digested and hydrophobically modified Shh was confirmed by MALDI PSD mass spectrometric measurement on a MALDI time of flight mass spectrometer. The site of palmitoylation was identified through a combination of peptide mapping and sequence analysis and is at the N-terminus of the protein. Both modified forms were equally as active in the C3H10T1/2 alkaline phosphatase assay, but interestingly both were about 30-times more potent than soluble human Shh lacking the tether(s). The hydrophobic modifications did not significantly affect the apparent binding affinity of Shh for its receptor, patched.

For specific lipid-modified hedgehog obtained by chemically modifying the soluble, unmodified protein, palmitic acid and other lipids can be added to soluble Shh to create a lipid-modified forms with increased potency in the C3H10T1/2 assay.

Generally, therefore, the reactive lipid moiety can be in the form of thioesters of saturated or unsaturated carboxylic acids such as a Coenzyme A thioesters. Such materials and their derivatives may include, for example, commercially available Coenzyme A derivatives such as palmitoleoyl Coenzyme A, arachidoyl Coenzyme A, arachidonoyl Coenzyme A, lauroyl Coenzyme A and the like. These materials are readily available from Sigma Chemical Company (St. Louis, MO., 1998 catalog pp.

303-306).

There are a wide range of hydrophobic moieties with which hedgehog polypeptides can be derivatived. A hydrophobic group can be, for example, a relatively long chain alkyl or cycloalkyl (preferably n-alkyl) group having approximately 7 to carbons. The alkyl group may terminate with a hydroxy or primary amine "tail". To further illustrate, such molecules include naturally-occurring and synthetic aromatic -43and non-aromatic moieties such as fatty acids, esters and alcohols, other lipid molecules, cage structures such as adamantane and buckminsterfullerenes, and aromatic hydrocarbons such as benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, and naphthacene.

Particularly useful as hydrophobic molecules are alicyclic hydrocarbons,

OO

saturated and unsaturated fatty acids and other lipid and phospholipid moieties, waxes, Cr cholesterol, isoprenoids, terpenes and polyalicyclic hydrocarbons including adamantane CN and buckminsterfullerenes, vitamins, polyethylene glycol or oligoethylene glycol, (Cltn 0 C18)-alkyl phosphate diesters, -O-CH2-CH(OH)-O-(C12-C18)-alkyl, and in particular CN 10 conjugates with pyrene derivatives. The hydrophobic moiety can be a lipophilic dye suitable for use in the invention include, but are not limited to, diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan, Laurodan, Pyrene, Perylene, rhodamine, rhodamine B, tetramethylrhodamine, Texas Red, sulforhodamine, 1,l'-didodecyl- 3,3,3',3'tetramethylindocarbocyanine perchlorate, octadecyl rhodamine B and the BODIPY dyes available from Molecular Probes Inc.

Other exemplary lipophilic moieties include aliphatic carbonyl radical groups include 1- or 2-adamantylacetyl, 3-methyladamant-l-ylacetyl, 3-methyl-3-bromo-1adamantylacetyl, 1-decalinacetyl, camphoracetyl, camphaneacetyl, noradamantylacetyl, norbornaneacetyl, bicyclo[2.2.2.]-oct-5-eneacetyl, l-methoxybicyclo[2.2.2.]-oct-5-ene- 2-carbonyl, cis-5-norborene-endo-2,3-dicarbonyl, 5-norboren-2-ylacetyl, myrtentaneacetyl, 2-norboraneacetyl, anti-3-oxo-tricyclo[2.2.1.0<2,6> ]-heptane-7carbonyl, decanoyl, dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.

1. Chemical Modifications of the N-terminal cysteine of hedgehog If an appropriate amino acid is not available at a specific position, site-directed mutagenesis can be used to place a reactive amino acid at that site. Reactive amino acids include cysteine, lysine, histidine, aspartic acid, glutamic acid, serine, threonine, tyrosine, arginine, methionine, and tryptophan. Mutagenesis could also be used to place the reactive amino acid at the N- or C-terminus or at an internal position.

For example, it is possible to chemically modify an N-terminal cysteine of a biologically active protein, such as a hedgehog protein, or eliminate the N-terminal cysteine altogether and still retain the protein's biological activity. The replacement or modification of the N-terminal cysteine of hedgehog with a hydrophobic amino acid results in a protein with increased potency in a cell-based signaling assay. By replacing b -44the cysteine, this approach eliminates the problem of suppressing other unwanted modifications of the cysteine that can occur during the production, purification, formulation, and storage of the protein. The generality of this approach is supported by the finding that three different hydrophobic amino acids, phenylalanine, isoleucine, and methionine, each give a more active form of hedgehog, and thus, an agonist.

00 SThis is also important for conjugation with non-hedgehog moieties Cc immunoglobulin) as described below in which we introduce two isoleucine residues to the N-terminal cysteine end of Sonic and Desert hedgehog. This effectively allows us to Suse the thiol of C-terminal cysteine as the reactive site for covalent coupling. Thus, C 10 replacement of the N-terminal cysteine with any other hydrophobic amino acid should result in an active protein. Furthermore, since we have found a correlation between the hydrophobicity of an amino acid or chemical modification and the potency of the corresponding modified protein in the C3H10T1/2 assay Phe Met, long chain length fatty acids short chain length), it could be envisioned that adding more than one hydrophobic amino acid to the hedgehog sequence would increase the potency of the agonist beyond that achieved with a single amino acid addition. Indeed, addition of two consecutive isoleucine residues to the N-terminus of human Sonic hedgehog results in an increase in potency in the C3H10T1/2 assay as compared to the mutant with only a single isoleucine added. Thus, adding hydrophobic amino acids at the N- or Cterminus of a hedgehog protein, in a surface loop, or some combination of positions would be expected to give a more active form of the protein. The substituted amino acid need not be one of the 20 common amino acids. Methods have been reported for substituting unnatural amino acids at specific sites in proteins and this would be advantageous if the amino acid was more hydrophobic in character, resistant to proteolytic attack, or could be used to further direct the hedgehog protein to a particular site in vivo that would make its activity more potent or specific. Unnatural amino acids can be incorporated at specific sites in proteins during in vitro translation, and progress is being reported in creating in vivo systems that will allow larger scale production of such modified proteins.

There are many modifications of the N-terminal cysteine which protect the thiol and append a hydrophobic moiety. One of skill in the art is capable of determining which modification is most appropriate for a particular therapeutic use. Factors affecting such a determination include cost and ease of production, purification and formulation, solubility, stability, potency, pharmacodynamics and kinetics, safety, immunogenicity, and tissue targeting.

2 Chemical modification of other amino acids.

There are specific chemical methods for the modification of many other amino acids. Therefore, another route for synthesizing a more active form of hedgehog would be to chemically attach a hydrophobic moiety to an amino acid in hedgehog other than to the N-terminal cysteine. If an appropriate amino acid is not available at the desired position, site-directed mutagenesis could be used to place the reactive amino acid at that site in the hedgehog structure, whether at the N- or C-terminus or at another position. Reactive amino acids would include cysteine, lysine, histidine, aspartic acid, glutamic acid, serine, threonine, tyrosine, arginine, methionine, and tryptophan. Thus the goal of creating a better hedgehog agonist could be attained by many chemical means and we do not wish to be restricted by a particular chemistry or site of modification since our results support the generality of this approach.

The hedgehog polypeptide can be linked to the hydrophobic moiety in a number of ways including by chemical coupling means, or by genetic engineering. To illustrate, there are a large number of chemical cross-linking agents that are known to those skilled in the art. For the present invention, the preferred cross-linking agents are heterobifunctional cross-linkers, which can be used to link the hedgehog polypeptide and hydrophobic moiety in a stepwise manner. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating to proteins, thereby reducing the occurrences of unwanted side reactions such as homoprotein polymers. A wide variety of heterobifunctional cross-linkers are known in the art. These include: succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC), m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4succinimidyloxycarbonyl- a-methyl-a-(2-pyridyldithio)-tolune (SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo.

0 -46- 0 One particularly useful class of heterobifunctional cross-linkers, included above, contain the primary amine reactive group, N-hydroxysuccinimide (NHS), or its water soluble analog N-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilon groups) at alkaline pH's are unprotonated and react by nucleophilic attack on NHS or sulfo-NHS esters. This reaction results in the formation of an amide bond, and 00 in release of NHS or sulfo-NHS as a by-product.

Another reactive group useful as part of a heterobifunctional cross-linker is a thiol reactive group. Common thiol reactive groups include maleimides, halogens, and Spyridyl disulfides. Maleimides react specifically with free sulfhydryls (cysteine c 10 residues) in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodoacetyl functions) react with -SH groups at physiological pH's. Both of these reactive groups result in the formation of stable thioether bonds.

Testing for Biological Activity While many bioassays have been used to demonstrate hedgehog activity, the C3H10T1/2 cell line provides a simple system for assessing hedgehog function without the complication of having to work with primary cell cultures or organ explants. The mouse embryonic fibroblast line C3H10T1/2 is a mesenchymal stem cell line that, under defined conditions, can differentiate into adipocytes, chondrocytes, and bone osteoblasts (Taylor, and Jones, Cell 17: 771-779 (1979) and Wang, et al., Growth Factors 9: 57-71 (1993)). Bone morphogenic proteins drive the differentiation of C3H10T1/2 cells into the bone cell lineage and alkaline phosphatase induction has been used as a marker for this process (Wang et al., supra). Shh has a similar effect on C3H10T1/2 cells (Kinto, N. et al., FEBS Letts. 404: 319-323 (1997)) and we routinely use the alkaline phosphatase induction by Shh as a quantitative measure of its in vitro potency. Shh treatment also produces a dose-dependent increase in gli-1 and ptc-1 expression, which can be readily detected by a PCR-based analysis.

Preferred Muteins of the Invention The active N-terminal signaling domain of human Sonic Hedgehog protein (residues Cys24-197) can be expressed in many cell types (COS, insect cells, E. coli, yeast). In Baculovirus and yeast, the protein undergoes proteolytic clipping at various sites between Gly9 and Argl4 (See Figure 3 for the N terminal sequence of Sonic hedghog showing the clip sites). In the methylotropic yeast Pichia pastoris, strain GS115 (obtained from Invitrogen) this N-terminal clipping occurs exclusively at the Arg33- Arg34 bond, yielding N-10 Sonic Hedgehog protein (residues Arg34-Gly197). This d -47- Sclipping occurs intracellularly and appears to be catalyzed the KEX2 Golgi protease, or Sa similar KEX2-like intracellular protease.

The N-terminally clipped forms of SHH are inactive in the 10T1/2 assay (See Example N-10 SHH is inactive and also antagonizes wild-type SHH when both forms are present in the assay. Thus, under certain circumstances prevention of N- 00 t) terminal proteolytic clipping is necessary for production of fully active protein.

Cc Because of the N-terminal clipping, a monomeric form of SHH is expected to C, contain two protein species, intact SHH and N-10 SHH.

O In contrast, a dimeric fusion protein, such as a SHH-Fc (immunoglobulin) C, 10 protein, is expected to contain 3 species: a species with two intact SHH domains, a species with two clipped domains, and a species with one intact and one clipped SHH domain.

Monomeric SHH could be separated from N-terminally clipped SHH by standard protein purification techniques. A dimeric fusion protein, however, is a more difficult purification problem. In addition, a substantial proportion of N-terminal clipping would more severely reduce the proportion of dimeric molecules containg two intact SHH domains. Thus, efficient production of dimeric fusion proteins is particulary dependent on prevention of the N-terminal clipping.

The KEX2 protease has a recognition sequence at least 3 amino acid residues long of the form: [Arg or Lys]-Arg-[X] where X is not Pro This recognition sequence occurs twice in the N-terminal region of Sonic Hedgehog: at Lys9ArglOArgl 1 with cleavage between the two Arg residues (cleavage at this site is observed) and at ArglOArg1 lHisl2 with predicted cleavage between Arg and His (cleavage at this site is not observed). We presume that the Lys9ArglOArgl 1 site is preferred and cleavage at ArglOArg 1 destroys the ArglOArgllHisl2 site.) The KEX2 recognition sites in Sonic Hedgehog were mutated in order to eliminate this intracellular proteolytic clipping (See Example 1, Figure 3 and Table 3).

These mutant proteins were expressed as the N-terminal domain (codons Cys24- Gly197 of the Sonic Hedgehog coding sequence, corresponding to residues Cysl- Gly174 of mature protein after signal sequence cleavage.

Table 3: Summary of Mutations and their properties Mutation in Sonic Sequence of Clipping 10T1/2 Comments -48- Hedgehog basic Region activity Wt KRRHP KRRHP[32-36]RKRHP RKRHP (N-11) Some activity if Palmitoylated KRRHP[32-36]RKRPP RKRPP KRRHP[32-36]KKKHP KKKHP KRRHP[32-36]RQRHP RQRHP Maintains His RKKHP[32-36]RKKHP RKKHP Maintains His Indian-like GSRKRPPRK

GSRKRPPRK

2 KRRHP[32-36] QRKHP Maintains QRKHP central Arg KRRHP[32-36] QRRPP Maintains QRRPP central Arg Underlined residues are amino acid substitutions compared to wild type sequence.

2Underlined residues are amino acid substitutions compared to wild type Indian Sonic Hedgehog.

IV. UTILITY OF THE INVENTION The unique property of the preferred immunoglobulin fusion proteins of the invention for therapeutic applications of the present invention is their general biocompatibility. The fusion proteins of the invention are believed not toxic and they are believed non-immunogenic and non-antigenic and do not interfere with the biological activities of the hedgehog protein moiety when conjugated under the conditions described herein. They have long circulation in the blood and are easily excreted from living organisms.

The therapeutic fusions of the present invention may be utilized for the prophylaxis or treatment of any condition or disease state for which a hedgehog or patched protein constituent is efficacious. In addition, the constructs of the present invention may be utilized in diagnosis of constituents, conditions, or disease states in biological systems or specimens, as well as for diagnosis purposes in non-physiological systems.

-49- In therapeutic usage, the present invention contemplates a method of treating an animal subject having or latently susceptible to such condition(s) or disease state(s) and in need of such treatment, comprising administering to such animal an effective amount of a fusion protein of the present invention which is therapeutically effective for said condition or disease state. Subjects to be treated by the fusion proteins of the present invention include mammalian subjects and most preferably human subjects.

Depending on the specific condition or disease state to be combated, animal subjects may be administered constructs of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, and without undue experimentation.

Generally, the modified proteins described herein are useful for treating the same medical conditions that can be treated with the unmodified forms of the proteins.

As but one example of the application of the proteins of this invention in a therapeutic context, modified hedgehog proteins according to the invention can be administered to patients suffering from a variety of neurological conditions. The ability of hedgehog protein to regulate neuronal differentiation during development of the nervous system and also presumably in the adult state indicates that polymer conjugated hedgehog can reasonably be expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in lesioned cells; and prevention of degeneration and premature death which results from loss of differentiation in certain pathological conditions. In light of this, the present modified hedgehog compositions, by treatment with a local infusion can prevent and/or reduce the severity of neurological conditions deriving from: acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vessel injury, and deficits (such as the ischemia from stroke), together with infectious and tumor-induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like; and (iv) chronic immunological diseases of the nervous system, including multiple sclerosis. The modifed hedgehog proteins may also be injected into the cerebrospinal fluid, in order to address deficiencies of brain cells, or into the lymph system or blood stream as required to target other tissue or organ systemspecific disorders.

Hedgehog compositions of the invention may be used to rescue, for example, various neurons from lesion-induced death as well as guiding reprojection of these neurons after such damage. Such damage can be attributed to conditions that include, but are not limited to, CNS trauma infarction, infection, metabolic disease, nutritional deficiency, and toxic agents (such as cisplatin treatment). Certain hedgehog proteins 00 tt cause neoplastic or hyperplastic transformed cells to become either post-mitotic or n apoptotic. Such compositions may, therefore, be of use in the treatment of, for I instance, malignant gliomas, medulloblastomas and neuroectodermal tumors.

SModified proteins of the invention can be used to specifically target medical therapies against cancers and tumors which express the receptor for the protein. Such materials can be made more effective as cancer therapeutics by using them as delivery vehicles for antineoplastic drugs, toxins, and cytocidal radionuclides, such as yttrium A toxin may also be attached to the modified hedgehog to selectively target and kill hedgehog-responsive cells, such as a tumor expressing hedgehog receptor(s). Other toxins are equally useful, as known to those of skill in the art. Such toxins include, but are not limited to, Pseudomonas exotoxin, Diphtheria toxin, and saporin. This approach should prove successful because hedgehog receptor(s) are expressed in a very limited number of tissues. Another approach to such medical therapies is to use radioisotope labeled, modified protein. Such radiolabeled compounds will preferentially target radioactivity to sites in cells expressing the protein receptor(s), sparing normal tissues. Depending on the radioisotope employed, the radiation emitted from a radiolabeled protein bound to a tumor cell may also kill nearby malignant tumor cells that do not express the protein receptor. A variety of radionuclides may be used.

It is envisioned that subcutaneous delivery will be the primary route for therapeutic administration of the proteins of this invention. Local, intravenous delivery, or delivery through catheter or other surgical tubing may also be envisioned.

Alternative routes include tablets and the like, commercially available nebulizers for liquid formulations, and inhalation of lyophilized or aerosolized formulations. Liquid formulations may be utilized after reconstitution from powder formulations.

For neurodegenerative disorders, several animal models are available that are believed to have some clinical predicative value. For Parkinson's disease, models involve the protection, or the recovery in rodents or primates in which the nigral-striatal dopaminergic pathway is damaged either by the systemic administration of MPTP or -51the local (intracranial) administration of 6-hydroxydopamine [6-OHDA], two selective dopaminergic toxins. Specific models are: MPTP- treated mouse model (Tomac et al., (1995) Nature 373, 335-339); MPTP-treated primate (marmoset or Rhesus) model (Gash et al., (1996) Nature 380, 252-255) and the unilateral 6-OHDA lesion rat model (Hoffer et al., (1994) Neuroscience Lett. 182, 107-111). For ALS (Amyotrophic lateral sclerosis) models involve treatment of several mice strains that show spontaneous motor neuron degeneration, including the wobbler (Duchen, L.W. and Strich, (1968), J. Neurol. Neurosurg. Psychiatry 31, 535-542) and pmn mice (Kennel et al., (1996) Neurobiology of Disease 3, 137-147) and of transgenic mice expressing the human mutated superoxidase dismutase (hSOD) gene that has been linked to familial ALS (Ripps et al., (1995) Proc. Natl. Acad. Sci, USA, 92: 689-693).

For spinal cord injury, the most common models involve contusion injury to rats, either through a calibrated weight drop, or fluid (hydrodynamic) injury. For Huntington's models involve protection from excitotoxin (NMDA, quinolinic acid, kainic acid, 3nitro-propionic acid, APMA) lesion to the striatum in rats (Nicholson, L. et al., (1995) Neuroscience 66, 507-521; Beal, M.F. et al., (1993) J. Neuroscience 13, 4181-4192).

Recently, a model of transgenic mice overexpressing the human trinucleotide expanded repeat in the huntington gene has also been described (Davies, S. et al., (1997) Cell 537-548). For multiple sclerosis, EAE in mice and rats is induced by immunization with MBP (myelin basic protein), or passive transfer of T cells activated with MBP (Hebr-Katz, R. (1993) Int. Rev. Immunol. 9, 237-285). For Alzheimer's, a relevant murine model is a determination of protection against lesion of the fimbria-fornix in rats (septal lesion), the main nerve bundle supplying the cholinergic innervation of the hippocampus (Borg et al., (1990) Brain Res., 518, 295-298), as well as use of transgenic mice overexpressing the human beta-amyloid gene. For peripheral neuropathies, a relevant model is protection against loss of peripheral nerve conductance caused by chemtherapeutic agents such as taxol, vincristine, and cisplatin in mice and rats (Apfel et al., (1991) Ann. Neurol., 29, 87-90).

The products of the present invention have been found useful in sustaining the half life of hedgehog, and may for example be prepared for therapeutic administration by dissolving in water or acceptable liquid medium. Administration is by either the parenteral, aerosol, or oral route. Fine colloidal suspensions may be prepared for parenteral administration to produce a depot effect, or by the oral route while aerosol formulation may be liquid or dry powder in nature. In the dry, lyophilized state or in b -52solution formulations, the hedgehog protein -polymer conjugates of the present invention should have good storage stability. The thermal stability of conjugated hedgehog protein (data not shown) is advantageous in powder formulation processes that have a dehydration step.

The hedgehog proteins of the invention may be administered per se as well as 00 t in the form of pharmaceutically acceptable esters, salts, and other biologically Cc functional derivatives thereof. In such pharmaceutical and medicament formulations, the hedgehog protein preferably is utilized together with one or more pharmaceutically Sacceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) I 10 must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The hedgehog protein is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

The formulations include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration. Formulations suitable for oral, nasal, and parenteral administration are preferred.

When the hedgehog protein is utilized in a formulation comprising a liquid solution, the formulation advantageously may be administered orally or parenterally.

When the hedgehog protein is employed in a liquid suspension formulation or as a powder in a biocompatible carrier formulation, the formulation may be advantageously administered orally, rectally, or bronchially.

When the hedgehog protein is utilized directly in the form of a powdered solid, the hedgehog protein may advantageously be administered orally. Alternatively, it may be administered nasally or bronchially, via nebulization of the powder in a carrier gas, to form a gaseous dispersion of the powder which is inspired by the patient from a breathing circuit comprising a suitable nebulizer device.

The formulations comprising the present invention may conveniently be presented in unitdosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the active ingredient(s) into association with a carrier which constitutes one or more accessory 4 -53ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the active ingredient(s) into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.

Formulations of the present invention suitable for oral administration may be 00 Spresented as discrete units such as capsules, cachets, tablets, or lozenges, each Scontaining a predetermined amount of the active ingredient as a powder or granules; or Sa suspension in an aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a draught.

C 10o A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface active agent, or discharging agent. Molded tablets comprised of a mixture of the powdered polymer conjugates with a suitable carrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s). Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active conjugate, which preferably is isotonic with the blood of the recipient physiological saline solution). Such formulations may include suspending agents and thickening agents or other microparticulate systems which are designed to target the compound to blood components or one or more organs.

The formulations may be presented in unit-dose or multi-dose form.

Nasal spray formulations comprise purified aqueous solutions of the active conjugate with preservative agents and isotonic agents.

Formulations for rectal administration may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acid.

-54- 0 Ophthalmic formulations such as eye drops are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.

Topical formulations comprise the conjugates of the invention dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, 00 t or other bases used for topical pharmaceutical formulations.

n In addition to the aforementioned ingredients, the formulations of this invention C may further include one or more accessory ingredient(s) selected from diluents, buffers, O flavoring agents, disintegrants, surface active agents, thickeners, lubricants, C 10 preservatives (including antioxidants), and the like.

The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof. In particular, it will be understood that the in vivo, animal experiments described herein may be varied, so that other modifications and variations of the basic methodology are possible. These modifications and variations to the Examples are to be regarded as being within the spirit and scope of the invention.

EXAMPLE 1: MATERIALS AND METHODS Construction of pUB55, expression plasmid for Sonic Hedgehog in Pichia pastoris: (SEQ ID. NO. 80) contains the N-terminal domain of human Sonic Hedgehog (SEQ ID. MO. 37, Table 4) with the alpha factor PrePro region as the secretion signal.

was constructed in pCCM73, a derivative of pPIC9 (obtained from Invitrogen, San Diego, CA) with the Kanamycin gene (HinclI-HinclI fragment) of pUC4-K inserted at the Sphl site of pPIC9. The human Sonic hedgehog coding sequence from Earl-Notl was obtained from pEAG543 which has a stop codon and Notl site engineered following Gly197 in the coding sequence. Plasmid pCCM73 was cut with XhoI and NotI and was ligated with the Earl-Notl fragment of pEAG543 (containing the Sonic Hedgehog coding sequence, Table 4) and Oligonucleotides TCG AGA AAA GAT GCG GAC CGG GCA GGG GGT SEQ ID NO: 35 and 5' CGA ACC CCC TGC CCG GTC CGC ATC TTT TC SEQ ID NO: 36] that form a XhoI-EarI fragment and create the appropriate coding sequence for placing Sonic hedgehog adjacent to the alpha factor leader sequence in frame.

Construction of KEX2 cleavage site mutations in Sonic Hedgehog: pUB55 was digested with Xhol Bbsl and ligated with synthetic oligonucleotides (see Table 5 for oligonucleotides used for each mutation) that replace the XhoI-BbsI fragment which contins the N-terminal coding sequence of Sonic Hedgehog. [Note: although has multiple BbsI sites, each has a different 4 base-pair overhang, such that religation of the mixture recreates the pUB55 sequence outside of the novel oligonucleotides included in each ligation reaction.] Novel restriction sites were incorporated into the XhoI-BbsI fragment of each novel mutant.

Expression of Desert Hedgehog in Pichia pastoris and construction of KEX2 site mutations: The Desert Hedgehog coding region in plasmid pEAG680 was modified to incorporate'a BsrGI and an XmaI site site using the Stratagene QuikChange mutagenesis kit. With oligos HOG-711 and HOG-712 for BsrGI, pEAG680 was mutagenized yielding pMMC11. With Oligonucleotides HOG-720 and HOG-721 for XmaI, pMMC11 was mutagenized to yield pMMC13. An expression plasmid for wildtype Desert Hedgehog N-terminal domain was made by subcloning the XmaI-Notl fragment of pMMC13 to pKS314 at the same sites. [pKS314 contains the Sonic Hedgehog QRRPP mutant coding sequence of pKS310 (Table The XhoI-NotI fragment of pKS310 was subcloned to pWS 106, a derivative of pPic9 (Invitrogen) with the NcoI site in the HIS4 region destroyed by mutagenesis. The XmaI site in pKS314 lies within codons 3 and 4 (ProGly) sequence of Sonic Hedgehog. Because the first 4 residues of Sonic and Desert Hedgehog are identical, the Sonic Coding sequence can be used for the Desert Hedgehog constructs.) pKS310 contains a second XmaI site in the Kan gene, and was therefore unsuitable for this series of Desert Hedgehog constructions.] Mutations in the KEX2 site of Desert Hedgehog were constructed by a three way ligation with the BsrGI NotI fragment containing the DHH coding region from pMMC13, Oligonucleotides contining the XmaI-BsrGI region of DHH (Oligonucleotides as shown in Table K-2 and K-3) and the plasmid backbone from pKS314 (NotI-XmaI fragment).

Expression of Indian Hedgehog in Pichia pastoris and construction of KEX2 site mutations: Plasmid pEAG657 (SEQ ID. NO. 84) is pBluescript with the Indian Hedgehog coding sequence with a stop codon following codon GlyXXX. pEAG658 (SEQ ID. NO. 85) is pBluescript with the Indian Hedgehog coding sequence and a Sall site engineered within residues suitable for fusing the Indian Hedgehog coding sequence with Fc immunoglobulin coding sequences at the hinge region of immunoglobulins. To facilitate susequent manipulations, Spel and XmaI sites were introduced to pEAG658 by site-directed mutagenesis. pEAG658 was mutagenized -56with Oligonucleotides HOG-709 and Hog-710, introducing a Spel and yielding pM4MCIO. pMMCLO was subsequently mutagenized with Oligonucleotides HOG-722 and HOG-723, introducing an Xml site and yielding pMlCl2. The novel SpeI and XmaI sites were then subcloned to pEAG657 by ligating the small BbsI-DraIll fragment of pEAG657 and the large BbsI-DraIll of pMMC 12. An expression plasmid for wild-type Indian Hedgehog in Pichia pastoris (pMLMC 18) was constructed by subcloning the XmaI-NotI fragment of pMMC14 into pKS314 at the same sites.

Expression vectors for KEX2 site mutants (pMMC 19, RKRPP; and pMMvC2O, QRRPP) were constructed by ligatiing the SpeI-NotI fragment of pMMC 14, the X-maJ- NotI backbone of pKS3 14, and oligonucleotides forming an XmaI-Spel fragment that contains the KEX2 site mutation (as listed in Tables 5 and 6).

Table 4: DNA sequences of Hedgehog N-terminal domains and Immunoglobulin Fe Regions: Protein DNA Sequence human Sonic Hedgehog N- TGCGGACCGGGCAGcJGGGTTCGGGAAGAGGAGGCACCCC terminal Domain AAAAAGCTGACCCCTTI2AGCCTACAAGCAGT'1TATCCCCAA

TGTGGCCGAGAAGACCCTAGGCGCCAGCGGAAGGTATGAA

[SEQ ID NO: 37] GGGAAGATCTCCAGAAACTCCGAGCGATy1'AAGGAACTCA

CCCCCAATTACAACCCCGACATCATATAAG(JATGAAGA

AAACACCGGAGCGGACAGGCTGATGACTCAGAGGTGTAAG

GACAAGTTGAACGCTTUGGCCATCTCGJTGATGAACCAGT

GGCCAGGAGTGAAACTGCGGGTGACCGAGGGCTGGGACG

AAGATGGCCACCACTCAGAGGAGTCTCTGCACTACGAGGG

CCGCGCAGTGGACATCACCACGTCTGACCGGACCGyCAG AAGTACGGCATGCTGGCCCGCCTGGCGGTG(3AGGCICGGCT

TCGACTGGGTGTACTACGAGTCCAAGGCACATATCCACTG

CTCGGTGAAAGCAGAGAACTCGGTGGCGGCCAAATCGGGA

GGC

Human Indian Hedgehog N- TGCGGGCCGGGTCGGGTGGTGGGCAGCCGCCGGCGACCGC terminal Domain CACGCAAACTCGTGCCGCTCGCCTACAAGCAGTTCACCC CAATGTGCCCGAGAAGACCCTGGGGCCA3IyCGACGCTAT [SEQ ID NO: 38] GAAGGCAAGATCGCTCGCAGCTCCGAGCGCTTCAAJGGAGC

TCACCCCCAATTACAATCCAGACATCATCHTCAAGGACGA

GGAGAACACAGGCGCCGACCGCCTCATGACCCAGCGCTGY2 AAGGACCGCCTGAACTCGCTGGCTATCTCc3GTGATGAACC

AGTGGCCCGGTGTGAAGCTGCGGGTGACCGAGGGCTGGGA

CGAGGACGGCCACCACTCAGAGGAGTCCCTGCATTATGAG

GGCCGCGCGGTGGACATCACCACATCAGACCGCGACCGCA

ATAAGTATGGACTGCTGGCGCGCTTGGCAGTGGAGGCCGG '-57-

CTTTGACTGGGTGTATTACGAGTCAAAGGCCCACGTGCATT

GCTCCGTCAAGTCCGAGCACTCGGCCGCAGCCAAGACGGG

CGGC

Human Desert Hedgehog Nterminal Domain [SEQ ID NO: 39]

TGCGGGCCGGGCCGGGGGCCGGTTGGCCGGCGCCGCTATG

CGCGCAAGCAGCTCGTGCCGCTACTCTACAAGCAATTTGTG

CCCGOCGTGCCAGAGCGGACCCTGGGCGCCAGTGGGCCAG

CGGAGGGGAGGGTGGCAAGGGGCTCCGAGCGCTTCCGGG

ACCTCGTGCCCAACTACAACCCCGACATCATCTTCAAciGAT GAGGAGAACAGTGGAGCCGACCGCCTGATGACCGAGCG Fr

GTAAGGAGCGGGTGAACGCTTTGGCCATTGCCGTGATGAA

CATGTGGCCCGGAGTGCGCCTACGAGTGACTGAGGGCTGG

GACGAGGACGGCCACCACGCTCAGGATTCACTCCACTACG

AAGGCCGTGCTTTGGACATCACTACGTCTGACCGCGACCG

CAACAAGTATGGGTTGCTGGCGCGCCTCGCAGTGGAAGCC

GGCTLCGACTGGGTCTACTACGAGTCCCGCAACCACGTCCA

CGTGTCGGTCAAAGCTGATAACTCACTGGCGGTCCGGGCG

GGCGGC

I.-

Fc region of human IgGi with Asn-Gln glycosylation site mutation [SEQ ID. NO: 40]

GTCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG

AACTCCTGGGGGGACCGTCAGT =TCCTCTTCCCCCCAAAA

CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA

CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT

CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT

GCCAAGACAAAGCCGcgggaggagcagt accagagrcacgtaccgtgtggTCA

GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA

GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC

CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC

GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA

GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA

ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT

GTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA

CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC

ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG

CAGAAGAGCCTCTCCCTGTCTCCCGGGAAA

Fc region of murine IgG with GTCGACGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATG Asn-GIn glycosylation site TACAGTCCCAGAAGTATCATCTGTCTTCATC'ICCCCCCAA mutation AGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCI

ACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGL

00 00 [SEQ ID NO: 41]

TCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACA

GCCGCCA~CG~GGATCAACCT

CCCCGCGGATCCTAGACGATG

TCAGCAGGTAATCGGCAATCG

fl'CCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAA

GGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTC

CCAAGGAGCAGATGGCCAAGGATAAGTCAGTTGACCTG

CATGATAACAGACTTCTL'CCCTGAAGACATTACTGTGGAGT

GGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACA

CrCAGCCCATCATGGACACAGATGCT'iACTCGTCTAC AGCAAGCTCAATGTGCAGAAGAGCAACTGcYJAGGCAGGA

AAATTACGTTTTAAGGGCGAA

CCACCATACTGAGAAGAGCI'CCCACTCTCCTGGTAA

Fe region of murine IgG2a-- -with-Asn-Gln glycosylation sitemutation [SEQ IDNO: 42] i

I

GTICGACCCCAGAGGGCCCACAATCAGCCTGTCCTCCAT

GCAAATGCCCAGCACCTAACCTCYJ'GGGTGGACCATCCGTC

CCTGAGCCCCATAGTCACATGTGTGGTGY3TGGATGTGAGC

GAGGATGACCCAGATGTCCAGATCAGGGTGTGAACA

ACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGA

GGA LTACCAAAGTACaCTtCGGGTGGTCAGTGCrCCTCCCCAT

CCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAATGC

AAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAA

CCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGT

ATATGTCTTGCCTCCACCAGAAGAAGAGATGATAAGA

CAGGTCACTTGACCTGCATGGTGACAGACTCATCCTGA

AGACATTTACGTGGAGTGGACCAACACGGGAAAACAGA

GCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGAT

GTCTTACTTCATGTACAGCAAGTGAGAGTGGAAGA

AGAACTGGTGGAAAGAATAGTACTCCTGTCAGTGT

CCACGAGGGTCTGCACATCACCACACGATAAGAGCTTC

TCCCGGACTCCGGGTAAA

00 00 59 Table 5: KEX2 mutations and the Oligonucleotides for their construction Sonic Hedgehog mutations Oligos used plasmid name wt KR-RHP [SEQ ID NO: 87] KR-RPP [SEQ ID NO: 88] HOG-402 pKS285 HOG-403 HOG-404 HOG-403 KKKHP [SEQ ID NO: 89] HOG-402 pKS288 HOG-403 HOG-409 HOG-41 0 RQRHP [SEQ ID NO: 90] HOG-465 PKS301 HOG-466 HOG-462 HOG-4403 QRKHP [SEQ ID NO: 91] HOG-402 PKS309 HOG-403 HOG-565 HOG-566 QRRRP [SEQ ID NO: 92] HOG-402 PKS3 HOG-403 HOG-567 HOG-568 RKRHP [SEQ ID NO: 93] HOG-402 pKS287 HOG-403 HOG-406 HOG-407 RKKHP [SEQ ID NO: 94] HOG-463 pKS300 HOG-464 HOG-462 HOG-403 INDIAN-LIKE HOG-402 pKS289 00 -6 HOG-403 HOG-41 1 HOG-412 KKRHPKK [SEQ ID NO: 95] HOG-789 pMMC22 MMC86 HOG-799 MMC87 HOG-803 MMC88 HOG-808 RRRHPKK [SEQ ID NO: 96] HOG-791 pMMC23 MMC89 HOG-799 HOG-804 MMC9I HOG-808 QQQHPKK [SEQ ID NO: 97] HOG-795 pMMC25 MMC99 HOG-799 MMC 100 HOG-806 MMCIOI HOG-808 KRRHPQQ [SEQ ID NO: 98] HOG-797 pMMC26 MMC96 HOG-799 MMC97 HOG-807 MMC98 HOG-808 Indian Hedgehog mutations RKRPP [SEQ ID NO: 99] HOG-743 pMMC 19 MMC77 HOG-744 MMC78 QRRPP [SEQ ID NO: 100] HOG-745 pMMC2O MMC79 HOG-746 Desert Hedgehog mutation QRRPA [SEQ ID NO: 101] HOG-739 pMMCI16 MMC49 HOG-740 MMC51 RQRYA [SEQ ID NO: 102] HOG-741 pMMC 17 MMC52 HOG-742 MMC53 MMC54 60a Table 6: Sequences of Oligonucleotides used in Plasmid constructions of Table HOG-402 CTGACCCCTTTAGCCTACAAGCAGTTTATCCCCAATGTGG [SEQ ID NO: 43] CCGAGAAGACCC HOG-403 CCTAGGGTCTTCTCGGCCACATTGGGGATAAACTGCTTGT [SEQ ID NO: 44] AGGCTAAAGG HOG-404 TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGAAGA [SEQ ID NO: 45] GACCTCCCAAAAAG HOG-405 GGTCAGCTTTTTGGGAGGTCTCTTCCCGAACCCCCTGCCC [SEQ ID NO: 46] GGGCCGCATCTTTTC HOG-407 TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGAGGA [SEQ ID NO: 47] AGAGACACCCCAAAAAG HOG-408 GGTCAGCTTTTTGGGGTGTCTCTTCCTCCCGAACCCCCTGG [SEQ ID NO: 48] CCGGGCCGCATCTTTTC HOG-409 TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGAAGA [SEQ ID NO: 49] AGAAGCACCCCAAAAAG HOG-41 10 GGTCAGCTTTTTGGGGTGCTTCTTCTTCCCGAACCCCCTGC [SEQ ID NO: 50] CCGGGCCGCATCTTTTC HOG-411I TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGTCTA [SEQ ID NO: 5 1] GAAAGAGACCTCCCAGAAAG -61- HOG-4 12 GGTCAGCTTTrGGGAGGTCTCThtCTAGACCCGAACCCCCT (SEQ IID NO: 52] GCCCGGGCCGCATC='rC HOG-462 CTTACCCCTTfrAGCCTACAAGCAGTTTATCCCCAATGTGGCC [SEQ ID NO: 53] GAGAAGACCC HOG-463

TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGAJAGAA

[SEQ ID NO: 54] GAAGCACCCCAAAAAG HOG-464

GGTAAGCTTTTTGGGGTGCTTCTTCCTCCCGAACCCCCT(CC

(SEQ ID NO: 55] CGGGCCGCATCTTTTC HOG-465 TCGAGAAAAGATGCGCCCaGGCAGGGGGTTCG3cGcAGGCA [SEQ ID NO: 56] GAGACACCCCAAAAAG HOG-466 GGTaAGCTTTTTGGGGTGTCTCTGCCTCCCGAACCCCCTGCCt (SEQ ID NO: 57] GGGCCGCATC~rfrC HOG-565

TCGAGAAAAGATGCGGCCCGGGCAGGGGGTTCGGGCAGCG

[SEQ ID NO: 58] GAAGCACCCCAAAAAG HOG-566 GGTCAGCT=rrGGGGTGCTTCCGCTICCCGAACCCCCTGCC [SEQ ED NO: 59] CGGGCCGCATCTTTTC HOG-567 TCGAGAAAAGATGCGGCCCGGGCAGGGGGI'1TCGGGYCAGAG [SEQ ID NO: 60] AAGACCACCCAAAAAG HOG-568 GGTCAGCTTTTrGGGTGGTCTTCTTGCCGAACCCC1CTGCC [SEQ ID NO: 61] CGGGCCGCATCT'=C HOG-739

CCGGGCCGGGGGCCGGTTGGCCAACGCCGGCCGCCGCA

[SEQ ID NO: 62] AGCAGCTCGTGCCGCTACT HOG-740

GTACAGTAGCGGCACGAGCTGCTTGGCCGGCCGGCGTT

(SEQ ID NO: 63] GGCCAACCGGCCCCCGGC HOG-741

CCGCGGGCGTGCGCGGTTCCC

(SEQ ID NO: 64] AGCAGCTGGTGCCGCTACT HOG-742

GTCGACGACGTCTCCCTGGTC

[SEQ ED NO: 65] GGCCAACCGGCCCCCGGC HOG-743 CCGGGTCGGGTGGTGGGCAGCCGCAAGY2GGCCGCCACGCA (SEQ ID NO: 66] AA HOG-744

CTAGTTGCGTGGCGGCCGCTTGGTGCCCACCACCCGA

(SEQ IOD NO: 67] C HOG-745

CCGGGTCGGOTGGTOGGCAGCCAJACGTCGACCGCCACGCA

[SEQ ID NO: 68] AA HOG-746

CTGTGGGCGCAGTGTCCCACG

[SEQ ID NO: 69] C HOG-789

GCCGGGCAGGGGGTTCGGGAAGAAGAGGCACCCCAAAAA

[SEQ ID NO: 70] 7MCGACC HOG-79 1

GCCGCGGGTGGAGGAGACCAA

-62- [SEQ ID NO: 71] GCTGACC HOG-795 GCCCGGGCAGGGGGTTCGGGCAGCAGCAGCAcCCCAAAAA [SEQ ID NO: 72] GCTGACC HOG-797 GCCCGGGCAGGGGGTTCGGGAAGAGGAGGCACCCCCAGCcA [SEQ ID NO: 73] GCTGACC HOG-799 CCTTAGCCTACAAGCAGTTTATCCCCAAGGTGGCCGAGAA [SEQ ID NO: 74] GACC HOG-803 TAAAGGGGTCAGCTTTGGGGTGCCTCrrCTTCCCGAACCC [SEQ ID NO: 75] CCTGCCCG HOG-804 TAAAGGGGTCAGCT=rFGGGGTGCCTCCTCCTCCCGAACCC [SEQ ID NO: 76] CCTGCCCG HOG-806 TAAAGGGGTCAGCTTTTTGGGGTGCTGCTGCTGCCCGAACC [SEQ ID NO: 77] CCCTGCCCG HOG-807 TAAAGGGGTCAGCTGCTGGGGGTGCCTCCTCTTCCCGAACC [SEQ ID NO: 78] CCCTGCCCCJ HOG-808

CTAGGGTCTTCTCGGCCACATTGGGGAGAAACTGCTTGTAG

[SEQ ID NO: 79] GC Plasmid DNA sequence GATCTAACATCCAAAGACGAAAGGTGAATGAAACCTTnTTGCCATCCGACA

TCAAGCATTAAAAGGCACCAAGGGAA

(SEQ ID ACTAGCAGCAGACCGTGCAACGCAGGACTCCACTCCTCTTCCCTCAAC NO: 80] ACCCACTTTGCCATCGAAAAACCAGCCAGTTATTGGoTA'n'oAGyCT CGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTJIATTAG(JCr

GTCTATCCTGGCCCCCCTGGCGAGTTCATGTTTGA]CCGATGCAC

AAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTI'fCTGAGTGTG

GGGTCAAATAGTUCATGTTCCCCAAATGCCCAAAACTGACAGTTTIAAACG

CT~irGACATTAAAGGGTTACAGTACA

GTTGTGTAAGTAGCCGTGCAAGACTCA

AGTCGCCATACCG=GTTGTTGGTATTGAnGACGAATGCTCAAAAATA ATCTCATTAATGCTAGCGCAGTCrCTCTATCGCTTCTGAACCCCGGTGCACC

TGGCAAGAAGGAAACCCTTGAGTAGAT

TCCAATTTCTCAA7TGGGAATCGTGCA

CGTTCATGATCAAAATTACTGTTCTACCCCTACTTGACAGCATATATAA

ACAGAAGGAAGCTGCCCTGTCAACG'YrHATCATCATTAAGCT~

TATTAATGGCGTCAATGCACTTA=AGC

TTAACGACAACTTGAGAAGATCAAAAACTAATTATTCGAAGGATCCA

AACGATGAGATTTCCTTCAATTACTGCAGT=.ATTCGYJAGCATCCTCCG

CArGTCCATACCAAAAAGTAAGCCATC GGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTfrGCT GTTTGCCATTTrCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTAC TAT-rGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGC

GGACCGGGCAGGGGGTTCGGGAAGAGGAGGCACCCCAAAAAGCTGACCCCT

TTAGCCTACAAGCAGTrFATCCCCAATGTGGCCGAGAAGACCCTAGGCGCCA

GCGGAAGGTATGAAGGGAAGATCTCCAGAAACTCCGAGCGATTTAAGGAAC

TCACCCCCAATTACAACCCCGACATCATATTTAAGGATGAAGAAAACACCGG

AGCGGACAG3GCTGATGACTCAGAGGTGTAAGGACAAGTTGAACGCrnrGGC CATCTCGGTGATGAACCAGTGGCCAGGAGTGAAACTc3CGGGTGACCGAGGG

CTGGGACGAAGATGGCCACCACTCAGAGGAGTCTCTGCACTACGAGGGCCG

CGCAGTGGACATCACCACGTCTGACCGCGACCGCAGCAAGTACGGCATGyJTG

GCCCGCCTGGCGGTGGAGGCCGGCTTCGACTGGGTGTACTACGAGTCCAAGG

CACATATCCACTGCTCGGTGAAAGCAGAGAACTCGGTGGCGGCCAAJATCGG

GAGGCTGATTCGCGGCCGCGAATTAATTCGCCTAGACATGACTGTTCCTCA

GTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGA

GGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATr=nGATACmT TTTATTTGTAACCTATATAGTATAGGA TTTMGTCAGTTTCT-iTCG

TAGGTGTCGTACTTTGACGTATTTGGT

GGGGTTTGGGAAAATCATTCGAGTTTGATGTCTTGGTATCCCACTCC

T CAGAGTACAGAAGATTAAGTGAGAAGCGTTTGTCAAGCTTATCGA TAAGCTTTAATGCGGTAGTTATCACAGTTAATTCTACGyCAGTCAGGCA

CCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCT

GGATGCTGTAGGCATAGCTTGGUTATGCCGGTACTCCGGCCTCTTGCGG

GAACTCTCGCGACCCGCCAGCTCGTGG

TATATGCGTTGATGCAATTTCTATGCACCCGTTCTCGGAGCACTGTCCGAC

CGCTTTGGCCGCCGCCCAGTCCTTCCTCGCACTTGGAGCCACTATCGA

CTACGCGATCATGGCGACCACACCCGTCCTGTGGATdTATCGTTAATG

TAAGTTAAAATCTCTAAATAATAATAAGTCCCAGTCCCATACGA&ACC

TAACAGCATTGCGGTGAGCATCTAGACCTTCAAC

AGCAGCCAGATCCATCAC

TGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTGTGAAGTGATGA

ACTTCTGGAAGGTTGCAGTGTTAACTCCGTGTATTGACGGGCATATCCGTA

CGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTATCTCCACAATTCTGG-A

GAGTAGGCACCAACAAACACAGATCCAGGTGTGTArTGATCAACATAAG

AAAGATTGTTCGACAGGTAGGGATATG

CATTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGGGTTGTAGAGTGTGCA

ATCCTCTCATCACTTGACGAACTGTTA

CACTTCATTGAGTCTGCGCTTGCGCAA

AATCACCTGGGAATCAATACCATGTTCAGCTTGAGCAGAAGGTCTGAGGCA

CGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGACTTICAGAAGGCCC

AGCAGGCATGTCAATACTACACAGGCCTGATGTGTCATTnGACCATCATC

TTGGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAG

GACGTCGTCTACAACGTCGCGGGCCTC

AGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGG

TAGGACTCTTAGGAGTCAACATCTGAC

AGCAACTTrGGCAGGAACACCCAGCATCAGGAAGTGGAGGCAGATTGC GGTTCCACCAGGAATATAGAGGCCAATCTCAATAGGTCTyIAAAACGA 00 ~GAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTTCCGTTAGTTGAG

CTCTGATCTAGTTTTGGGTATGTTTAC

TTATCTGGCAATTGCATAAGCCTCTGGAAAGGAGCTACACAGGTG

0 ~~ACCATTTTGACGAACATTGTCGACAATTGG

GCATCCATATTGT

CCGTTTTCTGGATAGGACGACGAAGGGCATCTCAKH-1'CTGTGAGGAGGC

TTAGGTTTGGATTCTTCTTTAGGTTGTCCGGTGTATCCTGC'J-GGATCT

CCTTTC=TCTAGTGACCTTTAGGGACTTCATATCCAGGTnCTCTCCACCTCG

TCCAACGTCACACCGTACTTGGCACATCTCTTGCAATAATAAGT

CAGCACATTCCCAGGCTATATCTTCCTGGATAG C GGTCATCA

GCTTCCTCCCTAATTAGCGTTCAAACAAAACTTCGTCGTCTACCGTT

TGGTATAAGAACCTTCTGGAGCATTTTACGATCCCACAQTGJJTCC

ATGGCTCTAAGACC=~GATTGGCCAAACAGGAAGTGCGTTCCAAGTGAC

AGAAACCACACCTGrTGTTCAACCACAATTYCAGCAGTCCCATCACA ATCCAATTCGATACCCAGCAACTmGAGTCGTCCAGATGTAGCACCTTTAT

ACAAACTAGCAATGTGCCATTTTCTTG

CTCCGGAATAGACTTTGGACGAGTACACCAGGCCC&AACGAGTATTAGAA

GATACACAGATATGCACGGGTATGCA

GACGCCAACAAAATTTCACTGACAGGGAACGACATCTCAGAAAGTT

CGTATTCAGTAGTCAATTGCCGAGCATCAATAATGGGGATnATACCAGAAGC

AACAGTGGAAGTCACATCTACCAACTTTGCGGTCTCAGAGCATAAACA

GCCAAATCTAGGTCCAAATCACTTCATGATACCATTATGTACACTGA

GCATGCACGTCCATTGCTTTAGAGCCA

TTGCACATTAATGAAGCTCAGTCGATTGAGTGTGATCATGTGC

AGCTGGTCAGCAGCATAGGGAAACATTCTACCACCGGAAT

TACACCGACCTCTTGAGACAGAAGCTC

TGAAGTCGGACAGTGAGTGTAGCTGAGAAJATTCTGAAGCCGTATTmTAT

TACGGGCGCTAGGTCTTCCGAGACTGC

ACTCGTGCTACGGCAAGGGTGTGGCAA

CGCAACCGTGGAACGGTGCCTGGTAGG

GCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGACTGTTGG

GCGCCATCTCCTTGGACCTGCAGGGGGGGGGGGGGAAAGCCACGTTGTGTC

CAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAA

TAAAACTGTCTGGTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATAT

TCAACGGGAAACGTCTTGCTCAAGGCCGCGATFAAATTCCAACATGGATGCT

GATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGA

CAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACA

TGGCAAAGGTAGCGTTGCCAATGATGTACAGATGAGATGGTCAGACTAAAC

TGGCTGACGGAAT'ITATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCC

TGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAG

GTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAG

TGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTL=AACAGC

GATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGG

TTGATGCGAGTGATTTrGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGT

CTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACT

CATGGTGAT'MCTCACTTGATAACCTTFATTITGACGAGGGGAAATTAATAGG

TTGTATGATGTTGGACGAGTCGGAATC3CAGACCGATACCAGGATCTTGCC ATCCTATGGAACTGCCTCGGTGAGTTJ2TCTCCTTCATTACAGAAACGGCTTr TCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTrGA TGCTCGATGAGTTTTCTAATCAGAATTGc3TTAATTGGTTGTAACACTGGCAG AGCATTACGCTGACTTGACGGGACGGCGGC=rGTTGAATAAATCGAACTTT TGCTGAGTTGAAGGATCAciATCACGCATCTTCCCGACAACGCAGACCGTTCC GTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAciCT

CTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGG

CGTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCCCCCC

CCCCCCTGCAGGTCCCACGGCGGCGGTGCTCAACGGCCTCAACCTACTACTG

GGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGTATCTATGA

TTGGAAGTATGGGAATGGTGATACCCGCATTCTTCAGTGTCTTGAGGTCTCCT

ATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATT

TCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACTATCTCGGTTATG

ACAGCAGAAATGTCCTTCTrGGAGACAGTAAATGAAGTCCCACCAATAAAG AAATCCTTGTTATCAGGAACAAACTTCTTGTrTCGAACTTTICGGTGCCTrG

AACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAAATG

CTTACCTTCTGGACCTTCAAGAcK3TATGTAGGGTTTGTAGATACTGATGCCA

ACTTCAGTGACAACGTTGCTATTTCGTTCAAACCATTCCGAATCCAGAGAAA

TCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACA

ATAGTGTGC TCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAGTCTTTGA

TCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTT

TTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGC

TCGTAGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTVrTAGAGAAATT

TGCGGAGATGCGATATCGAGAAAGGTACGCTGATTVAACGTGAATT

ATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTGAJACCTCTGA

CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAGCGGATGCCGGGA

GCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG

CAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCITTAACTAT

GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT

CACTCAAAGGCGGTAATACGfJTTATCCACAGAATCAGGGGATAACGCAGGA

AAGAACATGTGAGCAAAGGCCAGCAAAAGGCCAGGAACCGTAAAGG

CGCGnrGCTGGCGTTTTTCCATAGGCTCCGCCCCCCGACGAGCATCACAAA CI

AATCGACGCTCAAGTCAGAGGTGGCGAAJ\CCCGACAGGACTATAAAGATAC

CAGGCGTrCCCCCTGGAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC GCTTACCGGATACCTGTCCGCCTCTCCCTTCGGGAACGTGCGCTThC

AAGTAGTTGTTTATCGTTGTG'CCCAGT

GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA

ACACTTGGCACCGAAAAGCTTGCCGCG

AGCCACTGGTAACAGGATTAGCAGAGCIGAGGTATGTAGGCGGTGCTACAGA

GTTCTTGAAGTGGTGGCCTAACTACGGTACACTAGAAGGACAGTATTTGGT

ATCTGCGCTCTGCTGAGCCAGTTACCCGGAAAGAGGGTACCU

GACGCACACACCGTGGTGT=TTCAC

GCAGATTACGCGCAGAAAAAGGATCTCAAGAAGATCCTTGATC'TTCT

ACGGGGTCTGACGCTCAGTGGAACGAACTCACGTTAGGGATTTTGGTCA

TGG~ACAAGACTACTGTCTTATAAAGA

TTAAATCAATCTAAAGTATATATGAGTAAACTTcYGTCTGACAGTACCAAT

GCTTAATCAGTGAGGCACCTATCTCAGATCTGTTATCG~CATCCATA

GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT

CTGGCCCCAGTGCTGATGATACCGCGAGACCCACGTCACCGCTCCAGA

TTATCAGCAATAAACCAGCCAGCCGGIAGGGCCGAGCGYCAGAAGTGGTCC

TGCAACTTTATCCGCCTCCATCCAGTTTATTGTTCCGGGAGCAGAG

TAAGTAGTCG

CAGTGCAGTGTTGCAGTGA

ATCGTGGTGTCACGCTCGTCGTT

GTATGGCTCATTCAGCTCCGGTTCCCA

ACGATCAAGGCGAGTACATGATCCCCCATGTTGTAAAAAGGTTAGC

TCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTCCGCIAGTGTTATCACT

CATGGTTATGGCAGCACTATCTACTGTCATGCATCCGTAGAT

GC=CTGTGACTGGTGAGTACTCACCAGTCATTCTIGAGATAGTGTATG

CGGCGACCGAGTGCTCTTCCGGCGTCACACGGGATAATACCGCCCAC

ATGAACTAAGGTACATGAAGTTCGGGA

ACTCTCAAGGATTTACCGGTGAGATCCAGTCGATGTACCCACTCGTG

CACCCAACTGATC-CAGCATCTTACTCACCAGCGTCTGGTGAGCA

-67-

AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA

ATGTTGAATACTCATACTCTTCCT'TCAATATTATTGAAGCATTTATCAGG

GTTATTGTCTCATGAGCGGATACATATTTGAATGTAT=AGAAAAATAAACA

AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAA

ACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT

ITCGTC=CAAGAATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGA

CTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATAACA

GTTATTATCGAGATC

pUB 114 GATCTAAGATCCAAAGACGAAAGGTTGAATGAAACCTTrGCCATCCGACA TCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAcyjGGATAC SEQ ID ACTAGCAGCAGACCGUTGCAAACGCAGGACCTCCACTCCTCT1'CCTCAC NO: 811 ACCCACTT11?GCCATCGAAAAACCAGCCCAGTTATTGGCTGATTGGAGCT

CGCTCATTCCAATTCCTCTATAGGCTACTAACACCATGACTATTAGCCT

GTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTATCCGAATCA

AAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTG

GGGTCAAATAGTTnCATGTCCCCAAATGGCCCAAAACTGACAGTTTAAACG

CTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAJGATGAACTAA

GTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTAAGAACTTCCA

AGTCGCCATACCGTTTGTCTGTTGGTATGATGACGAATCTCAAAAATA

ATCTCATTAATGCTrAGCGCAGTCTCTCTATCGC'FrCTGAACCCCGGTGCACC

TGGCAAGAAGGAAACCCTTGAGTAGAT

TCTCCACATTGTATGCTrCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAA

CGTTCATGATCAAAATTTACTGTTCTAACCCCTATTGACAGCAATATATA

ACGAGACGCTTTAA -iiiiiiMACTATTAC TACTT7CATAATTGCGACTGGTCCAATTGACAGCTTTTGATfTACGACT

TTACAACTAAGTAAAAACATATGAGTC

AACGATGAGATTTCCTTfCAAT1TTACTGCAG=yrATTCGCAGCyATCCTCCG

CATTAGCTGCCCAGTCAACACTACAACAGAJLGATGAAACGGCACAAATTCC

GGTAGTTACGTCCGATAAGGATCAGTC

GTmTGCCATTTCCAACAGCACAAATAACGGGATGATAATACTAC TATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAG&

AAGATGC

GGCGGAGGTCGAGGAGACCAAGTACC

ITrAGCCTACAAGCAGTrTATCCCCAATGTGGCCGAGAAGACCCTAGGCGCCA GCGGAAGGTATGAAGGGAAGATCTCCAGAAACTCCGAGCGA~rAGGAAC

TCACCCCCAATTACAACCCCGACATCATATTTAGGATGAAGAAAACACCGG

AGGAAGTAGCCGGTGAGAAGTACC~TG

CACCGGTACATGCGATAATCGTACAG

CTGGGACGAAGATGGCCACCACTCAGAGGAGTCTCTGCACTACGAGGGCCG

CGCAGTGGACATCACCACGTCTGACCGCGACCGCAGCAAGTACGGCATGCTG

GCCCGCCTGGCGGTGGAGGCCGGCTTCGACTGGGTGTACTACGAGTCCAAGG

Q--)CACATATCCACTGCTCGGTGAJLAGCAGAGAACTCGGTGGCGGCCAAATCGG

GAGGCGTCGACGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGT

CCCAGAAGTATCATCTGTCTTCATCTTCCCpCAAGCCCAAGGATGTGCTCA CCATTACTCTGACTCCTAAGGTCACGTGTGTT1GTGGTAGACATCAGCAAGGA

TGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACA

00GTAAGACaGGaAGATCAAC= CCCGC

GTGAACTTCCCATCATGCACCAGGACTGCTGGCAGGAGTTCAAATG

CAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAACCATCTCCAAA

N ~ACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAG

GAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATACAGACTTCTJ

TCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGA

ACTACAAGAACACTCAGCCCATCATGGACACAGATGGCCTTAC-1CGTCTA CAGCAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGAAATAC~rTCAC

CTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTC

TCCCACTCTCCTGGTAAATGATCCCAGTGTCCP.-GGAGCCCTCTGGTCCTACA

GCGCCATATCCTAACTATTCTATCATG

GCCTCAAGCGTTGTAATTACAAGTTAA

TGCTTCTAAAGAGTCTTTAATTTATGA

CCAAATTGATr=TAT

GTCTTGAGGTG

TCTACGCACCCGTATATTTGGTGGTTG

AAAATCATTCGAGTGATG'I

TGGTATTCCCACCCTCTTCAGAGT

ACAGAAGATTAAGTGAGAAGTTCGTGTGCAAATCGATAGC=AA

TGCGGTAGTTTATCACAGTTAJATTAACGYCAGTCAGGACCGTGTATGA

AATCTAACAATGCGCTCATCGTCATCCTCGCACCGTCACCCTGGATGCTGT

TGATGCAATr7CTATGCGCACCCGTTCGGAACTGTCCGACCGGG

CGCCGCCAGTCCTGCTCGCT'CGCTACTGGAGCCACATCGACTACGCGAT

CATGGCGACCACACCCGTCCTGTGGATCTATCGAATCTTGTAG~ThJ

ATCTCTAAATAATATAAGTCCCAGCCCATACGCTTCAGCAT

TGCGGTGAGCATCTAGACCAACAGCACCAGATCCATCACTTGGCC

AATATGTTCAGTCCCTCAGGAGTTACGTCGTGAGTGATGACTCGGA

AGTGATTACCGTTTTAGGAACGAGTGA

AGTGTGGTGGTACCGGAGGAGTATCTCCACACTCTCGGAGAGTAGGCA

CCAAAAAACACTTGTCTACAAAGAAGA

TCTCGATTTGCAGGATCAAGTGTTCAGGAGCGTACTGATGGACATTCCAA

AGCCTGCTCGTAGGTTGCAACCGATAGGGTGTAGAGTGTGCAATACACTTG

CGTACAATTCAACCCTTGCAACTGCACAGCTTGTGTGACAGCATCUTC

AATTCTGGCAACTCCTGTCTGTCATATCGACACCAGATCACCTGG

_____GAATCAATACCATGTTCAGCGAAGAAGGTCTGAGACGAATCTG

ATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATG

TCAATAGTACACAGGGCTGATGTGTCATTJGAACCATCATCTTGGCAGCAG

tAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAGGAACAGTTTCT

GTTCCGTAAGCCATAGCAGCTACTGCCTGGGCGCCTCCTGCTAGCACGATAC

ACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTACC

ATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCAACCAGCAACTTTG

00 GCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGCGGTTCCACCA

GGAATATAGAGGCCAACTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTA

CACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTTCATGGAA

TTTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCTGGCA

0 ATTGCATAAGTTCCTCTGGGAAAGGAGCTTCTAACACAGGTGTCTTCAAAGC CI GACTCCATCAAACTTGGCAGTTAGFrCTAAAAGGGCTTTGTCACCATTTTGAC GAACATTGTCGACAATTGG'TrGACTAATTCCATAATCTGTTCCGTTTCTGG ATAGGACGACGAAGGGCATCTTCAATCTTGTGAGGAGGCCrrAGAAACGT CAATTGCACAATTCAATACGACCTTCAGAAGGGACTT=rLAGGTTTGGAT

TCTTCTTTAGGTTGTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTCCTTCTA

GTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCCACCTCGTCCAACGTCAC

ACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGTCAGCACATTCC

CAGGCTATATCFTCCTTGGATTAGCTTCTGCAAGTTCATCAGCTTCCTCCCT

AATTFITAGCGTTCAAACAAAACTTCGTCGTCAAATAACCGTTTQGTATAAGA

ACCTTCTGGAGCATTGCTTTACGATCCCACAGTGCTTCCATGCTCTAAG

ACCCTTTGKITGGCCAAAACAGGAAGTGCGTTCCAAGTGACAGAAACCAAC

ACCTGTTTGTTCAACCACAAATITTCAAGCAGTCTCCATCACAATCCAATTCGA

TACCCAGCAACTT=GAGTTCGTCCAGATGTAGCACCTTTATACCACAAACC

GTGACGACGAGATTGGTAGACTCCAGTTTGTGTCCTTATAGCCTCCGGAATA

GACTTTTTGGACGAGTACACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCA

CCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACGCCAACA

AAATTTCACTGACAGGGAACTrI=GACATCTTCAGAAAGTTCGTATTCAGTA

GTCAATTGCCGAGCATCAATAATGGGGATTATACCAGAAGCAACAGTGGAA

GTCACATCTACCAACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTAC

CGCCATTAGTGAAACTrTCAAATCGCCCAGTGGAGAAGAAAAAGGCACAG

CGATACTAGCATTAGCGGGCAAGGATGCAACT=ATCAACCAGGGTCCTATA

GATAACCCTAGCGCCTGGGATCATCCTTTGACAACTCTTT1CT3CCAAATCTA GGTCCAAAATCACTTCATTGATACCATTATrGTACACTTGAGY2AAGTTGTCG

ATCAGCTCCTCAATTGGTCCTCTGTAACGGATGACTCAACTTGCJACATTAC

TTGAAGCTCAGTCGATTGAGTGAACTTGATCAGGTTGTGCAGCTGGTCAGCA

GCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTG

CAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACA

GTGAGTGTAGTGTTGAGAAATTCTGAAGCCGTATTTTL'NITATCAGTGAGTCA

c3TCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGACCTGCAGGTCGG

CATCACCGGCGCCACAGGTGCGGTTGTGGCGCTATATCGCCGACATCACC

GATGGGGAAGATCGGCTCGCCACTTCGGGCTCATGACGCTTGTTTCGGCG

TGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTT

__GGACCTGCAGGGGGGGGGGGGAAAGCCACGTGTGTCTCAAATCTCTGA

TGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTG

CTACATAAACAGTAATACAAGGGGTGTTATGACATATCACGGGAAC

GTCTTGCTCAAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGG

TGTATGGAAGCCCGATGCGCCAGAGTTGCGAACATGGAAAGGTAG

CGTTGCCAATGATGTTACAGATGAGATGGTCAACTCTGCTGACGGA

TTTATGCCTCrrCCGACCATCAAGCATflTTATCCGTACTCCTGATGATGCATG

GTTACTCACCACTGCGATCCCCGGGAAACAGCATTCCAGGTATTAGAAGAA

TACTATAGGAAAT~GTCCGCGGTCGGC

GTTGCATTCGATCCTGTTTGATGTCCT7AACAGCGATCGGTATTTIIC

GTCTCGCTCAGGCGCAATCACGAATGAT

4 &CGGTnGGTGATGCGAGTGA =GTAGGGATGTGCG7ACATTGAGAT

CAAGTTGCTCCCGATATGCCCTGGTTT

ACTTGATAACCTATTTrGACGAGGAAATAATAGGTTGTAn7GATGTTG

GACGAGTCGGAATCGCAGACCGATACCAGGATCCATCCTATGGACG

CCTCGGTGAGTCTCCTTCATTACAGACGGCAAATATGGTA

TTAATCGTTATATGATTATGTCCAGGT

TTCTAATCAGAATTGGTTAATTGGTTGTACACTGGCAGAGCATACGCTGA

CTGCGAGCGTTTGAAACAC'TGTATGA

GATCAGATCACGCATCTTCCCGACACGCAGACGTTCCGTGAAGCAAA

AGTTCAAAATCACCACTGGTCCACCTACAACAAAGCTCTCATCACCGTGG

CTCTATTTGTGTAGGGGTCGCTGAGGC

GCAACTTCCAGAACTACCCCCCCTCGT

CCCGGCGGTACGCCACTCATGCGTCTA

GCAGGAGTCGCATAAGGCAGAYGTCGAGTATCTATGATGGAAGTATGGG

AATGGTGATACCCGCATTCTTCAGTGTGAGGTTCCTATCAGATATGCC

CACAACACGGAGGTTCTGAATCCGCTT

GTACGAATGATTAACACCGTTAACGAT

TCClCTrGGAGACAGTAAATGAAGTCCCACCAATAAGAAATCCTGTTAT CAGAAATCTTTGA'~

GTCTGATTAAG

AGGGAAGCGTGATGACGCATCTCTCGA

CTTCAAGAGGTATGTAGGG

MGTAGATACTGATCCAACTCAGTGACAAC

GTGTTTGTAACTCGACAAAACAGTTTT

TATTGTCACATCGCYGACGCAATTCCT

TTTGAGGTCATTTGTATGAATAATCTAGTCTTTGATCTAATTCTTG

____ACGAGCCAAGGCGATAAATAACACACTCYAAACGTTA

-71-

GGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCC

TCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTGCGGAGATGCGA

TATCGAGAAAAAGGTACGCTGATTTTAAACGTGAAATTTATCTCAAGATCTC

TGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCC

CGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCC

GTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCC

AGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAG

CAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG

TAAGGAGAAAATACCGCATCAGGCGCTCTTCCGC11TCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG

GTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA

GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG

T1TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA GTCAGAGGTGGCGAAACCCGACAGciACTATAAAGATACCAGGCGTTTCCCC

CTGGAAGCTCCCTCGTGCGCTCTCCTG'ITCCGACCCTGCCGCTTACCGGATAC

CTGTCCGCCTTrCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTG TAGGTATCTCAGTTCGciTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT2GA GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGjTAAC

AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT

GCCTAACTACGGCTACACTAGAAGGACAGTATGGTATGGCTTGC

GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG*CAAJACAA

ACCACCGCTGGTAGCGGTGGTII11-1 GTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCrTTTACGGGGTCTGACGC TCAGTGGAACGAAAACTCACGTTAAGGGATTfrTGGTCATGAGATrATCAAAA

AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTAAATCAACTA

AAGTATATATGAGTAAACTTGGTCTGACAGTACCAATGCTTAATCAGTGAG

GCACCTATCTCAGCGATCTGTCTATTCGTTCATCCATAGTTGCCTGACTCCC

CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGyCCCCAQTGCT

GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA

ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAJACTTnATCCG CCCACATTTrATTGCGGACAATATG-CC AGTTAATAGTT1TGCGCAACGTTGTGCCATTGCTGCAGG.CATCGTGGTGTCAC

GCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA

GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCC

GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATYJ(A

GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC

TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGCGACCGAGT

TGCTCGCCCGGCGTCAACACGGGATAAACCGCGCCACATAGCAGAACTT

TAAAAGTGCTCATCATGGAAACGrCCGGGGGAAATCTCAAGGAT

CTTACCGCTGTTGAGATCCAGTCGATGTAACCCACTCGTGCACCCAACTGAT

CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG

GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT

CATACTCTTCCTTITTTCAATATTATTGAAGCATTATCAGGGTTATTGTCTCAT

GAGCGGATACATATTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG

CGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTAyrATCA in TGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGA

ATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGACTCATGTTGGTAT

GATC

pUB 115 GATCTAACATCCAAAGACGAAAGGTrGAATGAAACCTTTTTGCCATCCGACA

TCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGCATAC

[SEQ DD ACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAAC NO: 821 ACCCACTT=GCCATCGAAAAACCAGCCCAGTrATTGGGCTTGATTGGAGCT CGCTCATTCCAATTCCrrCTATrAGGCTACTAACACCATGAC=TAAGCCT GTCTATCCTGGCCCCCCTGGCGAGGTTCATGTrG=ATTTCCGJAJJyTC AAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTnCTGAGTGTG GGGTCAAATAGTTTCATGTT7CCCCAAATdGCCCAAAACTGACAGTTrA&ACG

CTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAGATGAATAA

GmGTCGTGAAATGCTAACGGCCAGTTGGTCAAAAAGAACTTCCA

AGTCGCCATACCGTTGTCTG=GGTATTGATTGACGAATGTCAAAATA

ATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTCTGAACCCCGGTGCACC

TGGCAAGAAGGAAACCC

TGAG~AGAT

TCCAATTTCTCAATTGGGAATCGTGCA

CGTTCATGATCAAAATTTAACTGTCTAACCCCTACTTGACAGCATATATAA

ACAGAAGGAAGCTGCCCTGTC17AAAC L-hI-lll ITATCATCATTATTAGCT

TACTTTCATAATTGCGACTGGTTCCAATTGAAAG=GA-=PACGACT

YrTAACGACAACTTGAGAAGATCAAAAAACAACTAAI-rATTCGAAGGATCCA AACGATGAGA=rCCTTCAAT1TACTGCAGTTTnCGAGCATCCTCCG

CATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAACGGCACAATCC

GGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTCGATGTTGCTj GTTTTGCCAmTCCAACAGCACAAATAACGGGTTAGTTATATACTAC

TATTGCCAGCATTGCTGCTAAAGAAGGGGTACTCGAGAAGATGC

GGACCGGGCAGGGGGTTCGGAAGAGAGGCACCCCAAGGACCCCT

YTAGCCTACAAGCAGTITATCCCCAATGTGCCGAGAAGACCCTAGGCGCCA

GCGGAAGGTATGAAGGGAAGATCTCCAGAACTCCGACGATTAGGAAC

TCACCCCCAATTACAACCCCGACATCATATTUAAGGATGAAGAAAACACCGG

AGCGGACAGGCTGATGACTCAGAGGTGTAGGACAAGTTGCY=G

CATCTCGGTGATGAACCAGTGGCCAGGAGTGAALCTGCGGGTGACCGAGGGJ

CTGGGACGAAGATGGCCACCACTCAGAGGAGTCTCTGCACTACGAGGGCJ(CG

CGCAGTGGACATCACCACGTCTGACCGCGACCGCAGCAAGTACGGCATGCTG

GCCCGCCTGGCGGTGGAGGCCGGCTTCGACTGGGTGTACTACGAGTCCAAGG

CACATATCCACTGCTCGGTGAAAGCAGAGAACTCGGTGGCGGCCAAATCGG

GAGGCGTCGACCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATG

CCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATC'CCCTCCAAAGA

TCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGT

00 trn GGATGTGAGCGAGGATGACCCAGATGTCCAG3ATCAGCTGGTTTGTGAACAAC

GTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACCAAAGT

ACaCTtCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGG

CAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGA

GAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGT

CTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGC

ATGGTGACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACG

GGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATG

GTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGG

AAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCA

CACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGAGCTCAGATCGATTCC

ATGGATCCTCACATCCCAATCCGCGGCCGCGAATTAATTCGCCTTAGACATG

ACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTC FrGCTAGAT

TCTAATCAAGAGGATGTGAGAATGCCATTTGCCTGAGAGATGCAGGCTTCAT

TTTTGATACTTrT=ATTTGTAACCTATATAGTATAGGATTTTII=GTCATTTf

TGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAA

TATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGrL=TCTTGGT

ATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGC

AAGCTTATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAAC

GCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGG

CACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCG

GGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCG

TGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGA

GCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGG

AGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCTAT

CGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTTrC TCCATACGAACC~rAACAGCATTGCGGTGAGCATCTAGACCTTCAACAGCAG CCAcIATCCATCACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCT TGTGAAGTGATGAACTTCTGGAAGGTTc3CAGTGTTAACTCCGCTGTATTGAC

GGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCC

ACAACTCTCTGGAGAGTAGGCACCAACAAACACAGATCCAGCGTGTT3TACT TGATCAACATAAGAAGAAGCATTCTCGATTTGCAGGATCAAGTGTrCAGGAG CGTACTGATTrGGACATTTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGGGT TGTAGAGTGTGCAATACACTTGCGTACAATTrCAACCCTTGGCAACTGCACA b GCTTGGTTGTGAACAGCATCTTCAATTCTGGY2AAGCTCCTTGTCTGTCATATC

GACAGCCAACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGCAGA

AGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGAA

CTTCAGAAGGCCCAGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATT

TTACACTTGCGATACACGTTCGACATT

TTGTCACATTAGGAACA=CTGTTCCGTGCATAGCAGCTACTGCCTG

GGCGCCTCCTGCTAGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAG

ATGACTTCTGGGGTAAGGGTACCATCCCAGTGGAGATCAAAACAA

AGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAACUTTCTCAATAGGT

CTrGCAAAACGAGAGCAGACTACACCAGQCJCAAGTCTCJAACTTGCAACGTCT

CCGTTAGTTGAGCTTCATGGAATTTCCTGACGTATCTATAGAGAGATCAATG

GCTCTCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAAGGACTTC

TAACACAGGTGTCTCAAAGCGACTCCATCAAACTTGCAGTTAGTTCTA

AGGGCTTGTCACCATTGACGAACATTGTCGACATTG=mGACTAATTC CATAATCTGTCCGThTCTGGATAGGACGACGAACATTTCAATTCTT GTAGGCTAAAGCA

=GAATCAAGCTCG

AGGGACTCrrTAGGTnGGAnTCTTCI

AGGTGCCGGTGTATCCTGG

CTTGGCATCTCCTTTCCTTCTAGTGACCTTTAGGGACTTCATATCCAGGTflT

CTCCTGCACTAACTATGCCTTATAGAA

TAAAATAAGTCAGCACACCCAGATATCTTCCTGGATTTACTTTGC

AAGTTCATCAGCTCCTCCCTAATTAGTTCACAAAAC~CGTCGTCA

AATAACCGTrGGTATAAGAACCTTCTGGAGCATTCTACGATCCCACA

AGTCTCTGTTAACTTGTGCAACGAGGG

TCCAAGTGACAGACCAACACCTGTT-GTFICAACCACAAAn7TCAAGCAGT

CTCCATCACAATCCAATTCGATACCCAGCACTTTTGAGTCGTCCAGATGTA

GCACCTTTATACCACAAACCGTGACGACGAGATGGTAGATCCAGTGTG

TCTAACTCGAAATTTGAGGAACGCCAG

GTAATTAGAAGAGTCAGCCACCAAGTAGTGAATAGACCATCGGGCGGTC

AGTAGTCAAAGACGCCAACAA~CACTGACAGAA

GACATCT

TCGAGTGATATGCATGCACTATAGGAT

TACCAGAAGCAACAGTGGAAGTCACATACCAAGGGTCTCAGAAAA

AGCATAAACAGTTCACTACCGCCATTAGTGAAA=CAATCGCCCAGT

GGAGAAGAAAAAGGCACAGCGATACTAGCATTACGGGCAGGATCAACT

TTTACAGTCAAAACCTGGCGGTACT7G

CAC=TCAACAGCAAACCTATAACTAT

TAACTACATGCACGCCTAATGCTTTAG

ATGACTCAACTGCACATTAACTGAAGCCAGTCGATTGAGTGAACTTGAT

CAGGTTGTGCAGCTGGTCAGCAGCATAGACACGC~TnCCTACCAAA

TCACCTCAACTCGAGAGTGAAGA

ATGTCATACTIGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCC

GTATT=A.TTATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGC

ATCGTGGCCGACCTGCAGGTCGGCATCACCGGCGCCACAGGTGCGGTTGCTG

GCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGG

GCTCATGAGCGCTTGTT1TCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGG

GGACTGTTGGGCGCCATCTCCTTGGACCTGCAGGGGGGGGGGGGGAAAGCC

00 ACGTTGTGTCTCAAAATCTCTGATGTTACATTGGACAAGATAAAAATATATC

ATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTI

ATGAGCCATATTCAACGGGAAACGTCTTGCTCAAGGCCGCGATTAAATrCCA ACATGGATGCTGA FflATATGGGTATAAATGGGCTCGCGATAATGTCGGGCA

ATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTG

YTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGG

TCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTT

ATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAA

CAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTG11TGA TGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTG TrGTAATTGTC

CTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAA

TAACGGTTTGUGTTGATGCGAGTGAT=hGATGACGAGCGTAATGGCTGGCCT

GTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATT

CAGTCGTCACTCATGGTGATITTCTCACTGATAACCTrTIIGACGAGGGG

AAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACC

AGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTCGTCCTTCATTACAG

AAACGGCT=IICAAAAATATGGTATlGATAATCCTGATATGAATAAATTGC AGTTTCATTrFGATGCTCGATGAG'T1CTAATCAGAATTGGTTAATI'GGTTG

TAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTUTGTTGAAT

AAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAAC

GCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCT

ACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGG

GOATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCT

CAGCGCCCCCCCCCCCCTGCAGGTCCCACGGCGGCGGTGCTCAACGGCCTCA

ACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCG

AGTATCTATGATTGGAAGTATGGOAATGGTGATACCCGCATTCFITCAGTGTC

TTGAGGTCTCCTATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATT

TCATGGTAAATTTCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACT

ATCTCGGTTATGACAGCAGAAATGTCCTTCTFGGAGACAGTAAATGAAGTCC

CACCAATAAAGAAATCCTTGnFATCAGGAACAAACTTCTTGTTTCGAACT=r

TCGGTGCCTTGAACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAG

CGGGCAAATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGTAGAT

ACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGTTCAAACCATTCCGAAT

CCAGAGAAATCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTT

-76-

GAAACTGACAATAGTGTGCTCGTGTTTGAGGTCATCTTTGTATGAATAAATC

TATTTACAAATTGCGGCAGGTATCCAT

TAAAACTCTTTTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACC

CCAAATCAGCTCGTAGTCTGATCCTCATCAACTTGAGGGGCACTATCTGTn' TAGAGAAATTTGCGGAGATGCGATATCGAGAAGGTACGCTGAT~rTAA

ACGTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTG

GGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTGGG

GTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACT

GGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG

GTGTGAAATACCGCACAGATGCGTAGGAGAAJAATACCGCATCAGGCGCTC

TTCCGCTTCCTCGTCACTGACTCGCTGCGCTCGTCGTCGGTGCGGCGAG

CGGTATCAGCTCACTCAAAGGCGGTAATACGGCTTATCCACAGATCAGGGGA

TAACGCAGGAAAGAACATGTGAGCAAAGGCCAGCAAAAGG3ICAGGAACC

GTAAGCGG'GTGG=CAAGTCCCCTAG

GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGYGAACCCGACAGGACT

ATAAAGATACCAGGCGTCCCCCTGGCCCCTCGTGGCTCCTGTTC

CGACCCTGCCGCTTACCGATACCTGTCCTCTCCCGGGAGGTG

GCGCTICTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG

CCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCACCCGACCGCTC

TATCCGGTACTATCGTCTTGAGTCCAACCCGGTAGACACGACTATCGC

CATGACGCCGTAAGTACGGGGTTTGC

GTCAAAT=AGGTGCTATCGTCCAAGA

AGTATTTGTATCTGCGCTCTTGCCAGTTACTTCGGAAAAGAGTT

GGTAGCTCTTGATCCGGCAAACAAACCACCTGGTAGGTGGTTTTTG

TTTGCAAGCAGCAGATTACGCGCAGAAAGGATCTCAGAAGATCCTTT

GATCTTrCTACGGGGTCTGACGCTCAGTGGACGAACTCACGTTAAGGG

ATTGTAGGTACAAGACTACAACTTAAT

AAAATGAAGTTrTAAATCAATCTAGTATATATGAGTAACTGGTCTGAC

AGTCATCTACGGGCCTTTACACGCATC

TCATCCATAGT-GCCTGACTCCCCGTCGTGTAGATAATACGATACGGGAG

GGCTTACCATCTGGCCCCAGTGCTGATGATACCGCGAGACCCACGCTCAC

CGGCTCCAGATnATCAGCAATACCAGCAGCCGGAGGCCGAGCGCA GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTJI-AUAATTGThTCCGG GAAGCTAGAGTAAGTAGTTCGCCAG TA~nGGCCGTTGTGCC-A TTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTATGCCAtCAG

TCCGGTTCCCAACGATCAGGCGAGTACATGATCCCCCATGGTAAAA

AAGCGGTAGCTCCTTCGGTCCTCCGATCGGTCAGAGTAGTTGGCCGC

AGGTTATAGTAGCGATCTATTTATTAG

CATCCGTAAGATGC=~CTGTGACTGTGAGTATCAACCAAGTCATTCTGA

GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATA

ATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTC

TTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATG

TAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT1AC1TTCACCAGCGT

TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG

GGCGACACGGAAATGTTGAATACTCATACTCTTrCCTTTTCAATATTATTGAA GCATTTATCAGGGTTrATTGTCTCATGAGCGGATACATATTrGAATGTATITAG

AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG

ACGTCTAAGAAACCATTArrATCATGACATTAACCTATAAAAATAGGCGTAT

CACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGF=GACAGCTTATCAT

CGATAAGCTGACTCATGTTGGTAT'TGTGAAATAGACGCAGATCGGGAACACT

GAAAAATAACAGTTATTATTCGAGATC

pUB 116 GATCTAACATCCAAAGACGAAAGGTTGAATGAAACC TITGCCATCCGACA

TCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATAC

[SEQ ID ACTAGCAGCAGACCCYFFGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAAC NO: 833 ACCCACTTTrGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATrGGAGCT

CGCTCATTCCAATTCCTTCTATAGGCTACTAACACCATGACTTTATTAGCCT

GTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTI'GTTTATTTCCGAATGCAAC

AAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTG

GGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTITAALCG

CTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAA

GTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTCCALJA

AGTCGCCATACCGTTTGTCrGTTGGTATTGATTGACGAATTCAAAAAT ATCTCATTAATG AGCGCAGTCTCTCTATCGCTTCTGAACCCCGYJTGCACC TGTGCCGAAACGCAAATGGGGAAACACCCGCTfTTGATGAUTATGCATTG

TCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAA

CGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAA

ACAGAAGGAAGCTGCCCTGTCTTAAACCTT=r~TTTATCATCATTATTAGCT~ TACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGA=1~AACGACT TTI7AACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAGGATCCA AACGATGAGATTTCCTrCAATflT-ACTGCAGTTTATTCGC~AGCATCCTCG

CATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAJATTCC

GGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGA'FITCGATGflT3CT GTTTTGCCATTLTCCAACAGCACAAATAACGc3GTTATTGTrATAAATACTAC TATTGCCAGCATTcJCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGATGC

GGACCGGGCAGGGGGTTCGGGAAGAGGAGGCACCCCAAAAAGCTGACCCCT

TTAGCCTACAAGCAGTIATCCCCAATGTGGCCGAGAAGACCCTAGGCGCCA

GCGGAAGGTATGAAGGGAAGATCTCCAGAAACTCCGAGCGAmAAinJAC

TCACCCCCAATTACAACCCCGACATCATATTTAAGGATGAAGAP.AACACCGG

AGCGGACAGGCTGATGACTCAGAGGTGTAAGGACAAGTTGACGCTTTGGC

b -78-

CATCTCGGTGATGAACCAGTGGCCAGGAGTGAAACTGCYGGGTGACCGAGGG

CTGGGACGAAGATGGCCACCACTCAGAGGAGTCTCTGCACTACGAGGGCCG

CGCAGTGGACATCACCACGTCTGACCGCGACCGCAGCAAGTACGGCATGCTG

GCCCGCCTGGCGGTGGAGGCCGGCTTCGACTGGGTGTACTACGAGTCCAAGG

CAAACATCCGGAGAAACCGGCGCATG

GAGGCGTCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT

GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG

ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGACGTGAGCCACGAG

ACCCTGAGGTCAAGTCAATGGTACGTGGACGGCGTGGAGTATAATGC

CAGCAGCcoaggatcaaga-acttgCGGCTACT

CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC

AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAGCCAGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGYGATGAGCTGACCA

AGAACCAGGTCAGCCTGACCTGCCTGGTCAApGCTTzCTATCCCAGCGACAT

CGCTGGGGGGATGCGCGGAACAAGCA

GCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG

TGACAAGAGCAGGTGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA

TGAGGCTCTGCACAACCACTACACGAGAAGACCTCTCCCTGTCTCCCGGG

AAATGAGTGCGGCGGCCGCGAATTAA-CGYCUAGACATGACTGnTCCTCA

GTCATGGATAGGAACGTTGTGTCATAG

GGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTGATACT

TTATGACTTTGAAGTTTTTTATTTTTCC

TACGAGCTTGCTCCTGATCAGCCTATCTCJCAG(JGATGATATCn7GTGGTA GGGGTTTGGGAAAATCATTCGAGTTGATG

=TJCTQTATTCCCACTCC

TCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTGTGCAAGCTTATCGA

TAAGCTTrAATGCGGTAGTTTATCACAGTTAATTGCTAAICGCAGTCAGGCA

CCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCT

GGATGTGTAGGCATAGGCTTGTATCGTACTGCCGGGCCTCTGGG

GATATCGTCCATCCGACAGCATCGCCAGTCACTATGTGCTGTAGI-.I-

TATATGCGTTGATGCATTCTATGCGCACCCGTCTCGGAGCACGTCCGAC

CGCTTGCCGCCGCCCAGTCCTCTCCTTA.TGGAGCACTATCGA

CTACGCGATCATGGCGACCACACCGTCCTGTGGATCTATCGTTATG

TAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTCTCCATACGAACCT

TAACAGCATrGCGGTGAGCATCTAGACCTTCACAGCAGCCAGATCCATCAC TGTGCATTTTATCTAGG7CTTGGATAG

ACTTCTGGAAGTGCAGTGTTACTCCGTGTATGACGGGCATATCCGTA

CGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCCACACTCTCTGGA

GAGTAGGCACCAACACACAGATCCACGTGTGTATGATCAACATAAG

AAAGATTGTTCGACATTCGACTCGTTG

CATTTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGG

TGTAGAGTGTGCA

ATACACTTGCGTACAATTTCAACCCTTGGCAACTGCACAGCTTG&ITGTGAA

CAGCATCTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCCAACAG

AATCACCTGGGAATCAATACCATGTTCAGCTTGAGGAGAAGGTCTGAGGCAA

CGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAAGGCCC

AGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATTh'TGAACCATCATC TrGGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAG

GAACAGTTTCTGTTCCGTAAGCCATAGCAGCTACTGCCTGGGCGCCTCCTGCT

AGCACGATACACI7TAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGG

TAAGGGTACCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTGCAAGC

AGCAACT11TGGCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGC

GGTTCCACCAGGAATATAGAGGCCAACTTTCTCAATAGGTCTTGCAAAACGA

GAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAG

CTTCATGGAAT'FTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACG

TTATCTGGCAATTGCATAAGTTCCTCTGGGAAAGGAGCTTCTAACACAGGTG

TCTTCAAAGCGACTCCATCAAACTTGGCAGTTAGTTCTAAAAGGGCTTTGTC

ACCAT71-TGACGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGTT

CCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTCTTGTGAGGAGGC

CTTAGAAACGTCAATMrGCACAATTCAATACGACCTTCAGAAGGGACTTCT TTAGGTTTGGATCTCTTTAGG1rGTTCCTTGGTGTATCCTGGCTTGGCATCT

CCTTTCCTTCTAGTGACCTTTAGGGACTTCAITATCCAGGTTTCTCTCCACCTCG

TCCAACGTCACACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGT

CAGCACATTCCCAGGCTATATCTTCCTTlGGATTAGCnTCTGCAAGTTCATCA

GCTTCCTCCCTAATTTTAGCGTTCAAACAAAACTTCGTCGTCAAATAACCGTT

TGGTATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAGGTGCTTCC

ATGGCTCTAAGACCCTTTGATTGGCCAAAACAGGAAGTGCGTTCCAAGTGAC

AGAAACCAACACCTGTTTG7JTCAACCACAAAT'FFCAAGCAGTCTCCATCACA

ATCCAATTCGATACCCAGCAACTTTTGAGTTCGTCCAGATGTAGCACCTTTAT

ACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTTTGTGTCCTTATAGC

CTCCcMJAATAGACTTTIGGACGAGTACACCAGGCCCAACGAGTAATTAGAA

GAGTCAGCCACCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAA

GACGCCAACAAAATITTCACTGACAGGGAACTTTTTGACATCTTCAGAAAGTT

CGTATTCAGTAGTCAATTGCCGAGCATCALTAATGGGGATTATACCAGAAGC

AACAGTGGAAGTCACATCTACCAACTT~rGCGGTCTCAGAAAAAGCATAAACA

GTTCTACTACCGCCATTAGTGAAACTTITTCAAATCGCCCAGTGGAGAAGAAA

AAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAACTTTATCAACCA

GGGTCCTATAGATAACCCTAGCGCCTGGGATCATCCTTTGGACAACTCTTT

GCCAAATCTAGGTCCAAAATCACTTCATTGATACCATTATTGTACAACTTGA

GCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACTCAAC

TTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAACTTGATCAc3GTTGTGC

AGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAAT

TATCAAACTCTGCAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACT

TGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCGTATTTTTAT

TATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCG

ACCTGCAGGTCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGaCt2CTATAT

CGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGC

00 GCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGATGTTGG kn ~GCGCCATCTCCTTGGACCTGCAGGGGGGGGGGGAGCCACGTGTTC

CAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAA

c-I TAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATAT

TCAACGGGAAACGTCTTGCTCAAGGCCGCGATTAAATTCCAACATGGATGCT

GATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGy2GA

CATTTGTGAGGACCGTCCAATGTCGAC

TGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAAC

TGCGCGATAGCCTCACTAGATTTCTCC

TGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAA&JACAGCATTCCAG

GTTAAGAACTATAGTAATTGTAGGTGA

TGTCGGCGTCTCATCGTGATGCTTACG

GATCGCGTATTCGTCTCGCTCAGGCGAATCACGAATGATAACGrjfl-1Gy

TTGATGCGAGTGATTTTGATGACGAGCGTAATGTGCCTGGACAGT

CTGGAAAGAAATGCATAAGC='GCCATTCTCACCGGAnCAGTCGTCACT

CATGGTGATTTCTCACTTGATAACCTTAT=IIGACGAGGGGAAATTAATAGG

TTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCC

ATCCTATGGAACTGCCTCGGTGAGTYTTCCTTCATTACAGACGGCTTTT

TCAATTGATAATCGTTATATGAMATG

TGTGTATI

CATAATTGTATGTTAATGA

AGCATTACGCTGACTTGACGGGACGGCGCTUGTP2GAATAAATCGAACTnT

TGTATGAGTAACCCACTCGCAGAACTC

GTGAACAATCAACCACGTCCTCAAAC

CTCATCAACCGTGGCTCCCTCACTCTGCGGATGATGGGCyGAp'TCAyJ CCTGGTATGAGTCAGCAACACCTTCrCACGAGGCAGACCTCAGCGYCCCCCC CCCCGAGCCCGGCGTCCAGCTACTCAr

GGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGTATCTATGA

TTGGAAGTATGGGAATGGTGATACCCGCATTCTTICAGTGTCTTGAGGTCTCCT

ATAATTCCATAGACCGGAGGTTAGTAT

TCTCTGACTTTrGGTCATCAGTAGACTCGACTGTGAGACTATCTCGGTTATG

ACAGCAGAAATGTCCTTCGGAGACAGTATGAGTCCCACCAATAAAG

AATCTTACGACACTCrTTGAT

CGGCT

AATTAAGAATGTTTGGAGAGACGCAT

CTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTPGTAGATA~rGATGCCA

ACTATAACTGTTTGTCACATCATCGGA

TCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACA

ATAGTGTGCTCGTGTTTTGAGGTCATC'ITTGTATGAATAAATCTAGTCTTTGA

TCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTT

TTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGC

TCGTAGTGTGATCCTCATCAACTTGAGGGGCACTATCTTGT=rAGAGAAATT TGCGGAGATGCGATATCGAGAAAAAGGTACGCTGATIrAAACGTGAAATrT

ATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA

CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGA

GCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCG

CAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGT.ATACTGGCTTAACTAT

GCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATAC

CGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTC

GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT

CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA

AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC

CGCGTTGCTGGCGTTTT[CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAh AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAJiGATAC CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTfCCGACCCTGCC GCTTACCGGATACCTGTCCGCCTTCTCCCTTCGGGAAGCGTGGCGC1'TCT~C

AATGCTCACGCGTAGGTATTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG

GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGyCGCCTTATCCGGTA ACTATCGTCT-rGAGTCCAACCCGGTAAGACACGACTTATCGCCACGG2AGCw

AGCCACTGGTAACAGGATL'AGCAGAGCGAGGTATGTAGGCGGTGCTACAGA

GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGT

ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAGAGTGTAGyCTCT' GACGCACACACCGTACGGTTTTTrCAC GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCy-rTTCT ACGGGQTCTGACGCTCAGTGGAACGAAAACTCACGTTAGGGcATrpy-joTCA TGAGArATCAAAAAGGATCTTCACCTAGATCCTyI'-rTTAAAATGAAG

TTTAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTH'ACCAAT

GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTCGTTCATCCATA

GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGTTACCAT

CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGA

TTTATCAGCAATAACCAGCCAGCCG4GGCCGAGCGCAGAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTAArGTTGCGGGAGCTAGAG

TAGATCCATATGTGGACTGTCATCGAG

ATCGTGGTGTCACGCTCGTCGTTTGGTATGcGCTTCATTCAGCTCCGGTTCCCA

ACACAGGGTCTACCCAGTTCAAACGTG

TCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACT

CATGGflATGGCAGCACTGCATATTCCTACTGTCATGCATCCGTAAGAT -82-

GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG

CGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCAC

ATAGCAGAACTTI2AAAAGTGCTCATCATTGAAAACGTTCTTCGGGGCGAAA

ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG

CACCCAACTGATCTTCAGCATCTTTTACTCACCACyGToCTGTGAGCA

AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA

ATGTTGAATACTCATACTCCCCATATTATTGAGCATTATCAGG

AATAGGGGTCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAG

ACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT

C) TT~CGTCTTCAAGAATTAATCTCATGTrGACACTATCATCGATAGCTGA

CTCATGTTGGTATGTGAATAGACGCAGATCGGACACTGAAAATAACA

GTTAYI'ATTCGAGATC

CTATGAGG7AATTTTAATGGTAT

GTA

7 TCAGCTCATT=TAACCAATAGGCCGAATCGGCAATCCCTTATAAATC

AAAAGAATAGACCGAGATAGGGTGAGTGTTGTCCAGTGGAACAAGAG

[SEQ ID TCATTAAACTGCCAAGCAGGGAACGCA NO: 841 CAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGT=J~TGGGGT

CGAGGTCCGTAAAGCACTATCCCTAAGGGAGCCCCGATTTA

GACTAGGAACGCACGGCAAAGAGAGA

GCGAAAGGAGCGGGCGCTAGGGCCTGAGTGTAGCGGTCACTCG

GTAACCACCACACCCGCCGCGC'AATGCGCGCTACAGGJCGCGTCCCA.JT

CGCCATTCAGGCTGCGCACTGTTGGGAAGGGGATCGGTGGGGCCTCTTC

GCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGYCAAGGCGATTAAGTTG

GGTAACGCCAGGGT1CCCAGTCACGACGTGTAACGACGGCAGTGAG

CGCGCGTAATACGACTCACTATAGGGCGATGGGTACCGGGCCCTCTAGAT

CCTTTCAGCTCCCTGCCCCGGACATGCCCAGTTGAGT~GTCCTCTC

AGCAGGAGACGCCCCAGGCGGTAGAGCAGGGGTACCATGACACCC

TCCCCCGGaGTCCAGCTGCCCCATCAAGTGAGAGTCTCAGGGGC

AGAGCATACAGGTGCGCCGCCAGAGT

CCACCACATCCTCCACCACCAGTGTCCCATCTTGTGAGI-GJ-GGCGTAGGC

CCGGGCCTTTGGCGTCAGGGAGTCGC

TGCCCACACGAGATGCAGTCCTGTGAA

GTGGCCCGGAAGCGGGCTGCCGGCTCCGTGTGATTGTCACCGTAAAGAGCA

GGTGAGCGGGTGTGAGTGCCAGGCGGCGTGcYGYJGTCCTGAGTCTCGATGA

CCGAGCCCGCGGGCTGGTCGAATACCT

GCTGAAGGTGGGGCTCCCATCCTCCCCCATGGCCAGCACACGGTCTCCCGGC

CTCACGGCTGACAAGGCCACACGCGCCCCACTCTcaggcgtacctgggctgcggccgcgaa tcgcccttgCGGiCATGTGATGCGGCAGAGG

_____GCCTTTGACTCGTAATACACCCAGTCAAAGCCGCCCACTGCCAAGCGCG

CCAGCAGTCCATACTTATTGCGGTCGCGGTCTGATGTGGTGATGTCCACCGC.

GCGGCCCTCATAATGCAGGGACTCCTCTGAGTGGTGGCCGTCCTCGTCCCAG

CCCTCGGTCACCCGCAGCTTCACACCGGGCCACTGGTTCATCACCGAGATAG

CCAGCGAGTTCAGGCGGTCCTTGCAGCGCTGGGTCATGAGGCGGTCGGCGCC

TGTGTTCTCCTCGTCC'FrGAAGATGATGTCTGGATTGTAATTGGGGGTGAGCT CCTTGAAGCGCTCGGAGCTGCGAGCGATC'rTGCCTTCATAGCGTCCGCTGGC 00

GCCCAGGGTCTTCTCGGGCACATTGGQGCTGAACTGCTTGTAGGCGAGCGGC

ACGAGTTTGCGTGGCGGTCGCCGGCGGCTGCCCACCACCCGACCCGGCCCGC

AGCCCCATGCCGCcGGCACCACCAGCAGCAGCAACAGGACCAGGCAGAAGT GCAGTCGGGGCCGGAGCCGggcgggagacatggcggccgcgacggtatcgataagcTTGATATC

GAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGG

CI TGGAGCTCCAGCT'ITTGTTCGCTITIAGTGAGGGTTAATTGCGCGCTTGGCGTA

ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC

ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG

TGAGCTAACTCACATTAAUrGCGTGCGCTCACTGCCCGCTITTCCAGTCGGGA AAGCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGGGGGAACi

GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC

GGTCGTCGGCTGCGGCGAGCGGTATCAGTCACTCAAAGGY2GGTAATpACGG TTATCCACAGAATCAGGGGATAACGCAGGAAJXGAACATGTGAGyCAAAAGGC CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGCCGm=CCATA

GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAJAGTCAGAGTG

GCAACGCGATTAGTACGCTTCCTGACC

CTCGTGCGCTCTCCTGTCCGACCCTGCCGCTACCGGATACCTGTCCGCCTT

TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCA

GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT

TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTQ"=GAGTCCAACCCG

GTAAAGCTTGCCGCACGCCGTAAGTAC

GAGCGAGGTATGTAGCGGTGCTACAGAGTCTGAAGTGGTGGCCTAACTA

CGGCTACACTAGAAGGACAGTATTTGGTATCTGCTCTCTGACCAGTT

ACCTTCGGAAAAGAGTTGGTAGCTCTGATCGAACAACCACCGCTG

GTAGCGGTGGTTmGTTTGCAAGCAGCAGATTACGCAGAAAAAGG

ATCTCAAGAAGATCCTTGATCTTTCTACGGGGTCTGACGTCAGTGGAC

GAAAACTCACGTTAAGGGATTTrGGTCATGAGATATAAAAGATCTTCA CCAAC= ATAAAGATTTATATTAGAAA GAGTAAA =TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT

CACACGCATCTCTCTGTCTATCCTGGA

ATACTACGATACGGGAGGGCTTACCATCTGCCCCAGTCTGAATGATAC

CGGGCCCCCCGCCAGTTTACAAACGCG

CGGAAGGGCCGAGCGCAGAAGTGGTCCGCYAACTTATCCGCCTCCATCCAG

TCTATTAATGTTGCCGGGAAGCTAGAGTAAGTAGTCCAGThATAGTTT

GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG

GTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATC

CCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCA

GAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTCATA

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTCTGTGACTGYJTGACGCGT

ATCATTGGAAAACGTrCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCp.ACTGATCTTCAGCATCT TTTACTnrCACCAGCG=rCTGGGTGAGCAAACAGGAAGGCAAAATGCCG 0 CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCT

TTITTCAATATTATTGAAGCATTATCAGGGTTATTGTCTCATGAGCGGATACA

TATTTGAATGTATTAGAAAAATAAACAAATAGGGGTCCGCGCACATTTCC

CCGAAAAGTGCCAC

pEAG6S CTAAATTGTAAGCGTITATATTrGTTAAAATTCGCGTTAAATTTTTGTTAAA 8 TCAGCTCATT=rAACCAATAGGCCGAATCGGCA&AATCCCTTATAAATC

AAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAG

[SEQ ID TCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAALJAACCGTCTAT NO: 85] CAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTT1rn'1.GGGGT

CGGTCGAACCAACGACTAGGGCCGTT

GAGCTTrGACGGGGAAAGCCGGCGAACGTc3GCGAGAAAGGAAGGGAAGAAA

GCAAGGGGGTGGGTGAGGACGCCCGG

GTACCAACGCCCTATCCGTCGGGGCCT

CGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGTGCGGCCTCHTC

GCTATTACGCCAGCTGGCGAAAGGGGATGTCTGAAGCJCGATTAAGTTG

GGTAACGCCAGGGTTCCCAGTCACGACGTTGTAAACGACGGY2CAGTGAG CGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCC~rCTAGAT CCTTT1CAGCTCCCTGCCCCGGACATGCCCAGTGGGTGGAAGCTGCCCTCTTCT

AGCAGGAGACGCCCCAGGCGGTAGAGCAGCTGGGGGTACCAJATGCACAC

TCCCCCGGaGTCCAGCTGCCCCATGCCAAGCTGTGAAAGAGTCTCAGGGGJCC AGAAGGCCAACTGAGCCAGGTGGTGGTCAGCCACGGCCGCGAAGyCAGGATG

CCACCACATCCTCCACCACCAGTGTCCCAGTTTGTAGCGGG~GCGTAGGC

CCCGAGGGCCACGTGTGTAGAGACAGCTGCCACGGCAGCGCAGGCC

TGGCACCCCAGCCACCAGCACGTACTGGCCATACGTGCTGAAAT

GTGGCCCGGAAGCGGGCTGCCGGCTCCGTGTGATTGTCAGCCGTAGAGCA

GGTGAGCGGGTGTGAGTGCCAGGCGGCGTGGGGf3GTCCTGAGTCTCGATGA

CCTGGAGGCTCTCAGCCTGTGGGGCTCGCGTCCAGGAAATGAGCACATC

GCTGAAGGTGGGGCTCCCATCCTCCCCCATGCJCCAGCYACACGGTCTCCCGGC

CTCACGGCTGACAAGGCCACACGCGCCCCACTCTCCAGGCGTACCTgggctccgg cagggtCgacgccgcccgtCttggCTGCGGCCGAGTGCTCGGACTTlGACG3QAGyAATGCyA

CGTGGGCCTITTGACTCGTAATACACCCAGTCAAAGCCGGCCTCCACTGCCAA

c3CGCGCCAGCAGTCCATACTTATTGCGGTCGCGGTCTGATGTGGTGATGTCC

ACCGCGCGGCCCTCATAATGCAGGGACTCCTCTGAGTGGTGGCCGTCCTCGT

CCCAGCCCTCGGTCACCCGCAGCTTCACACCGGGCCACTGGTTCATCACCGA

GATAGCCAGCGAGTTCAGGCGGTCCTTGCAGCGCTGGGTCATGAGGCGGTCG

c3CGCCTGTGTTCTCCTCGTCCTGAAGATGATGTCTGGATTGTAAFFGGGGGT

GAGCTCGTTGAAGCGCTCGGAGCTGCGAGCGATCTTGCCTTCATAGCGTCCG

CTGGCGCCCAGGGTCTTCTCGGGCACATTGGGGCTGAACTGCTTGTAGGCGA

GCGGCACGAGTTTGCGTGGCGGTCGCCGGCGGCTGCCCACCACCCGACCCGG

CCCGCAGCCCCATGCCGCcGGCACCACCAGCAGCAGCAACAGGACCAGGCA GAAGTGCAGTCGGGGCCGGAGCCGggcgggagacatggcggccgcgacggtatcgataagcTTG

ATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCAC

CGCGGTGGAGCTCCAGCTTTGTTCCCT1AGTGAGGGTTAATTGCGCGCTrG

GCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT

TCCACACAACATACGAcJCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA ATGAGTGAGCTAACTCACATTAATTrGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCAT'rAATGAATCGGCCAACGCGCGGGGA

GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG

CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA

TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAJLGAACATGTGAGCAA

AAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCrGGTT TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA4ATCGACGCTCAAGTCA GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT'ITCCCCCTGyJ

AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT

CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGG

TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC

CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC

AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG

ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTlGAAGTGGTGGC CTAACTACGGCTACACTAGAAGGACAGTATGTATCTGCCTCTGCiTGAJ

GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC

ACCGCTGGTAGCGGTGGrnIrGTTGCAAGCAGCAGATACGCYGCAGAA AAAAAGGATCTCAAciAAGATCCTTTGATC=rTCTACGGGTCTGAC3CTCA GTGGAACGAAAACTCACGTTAAGGGAT=rGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAATTAAATGAAGTmAAJATCAATJCTAAAG TATATATGAGTAAA =TGGTCTGACAGTTACCAJTGTTAATCAGTGAGGCA

CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT

CGGAAACAGTCGAGGTACTTGCCGGTC

ATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC

AGCGCGAGCGGGAAATGCTCATTTCCT

-86i

CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT

AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGTC

GTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA

CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATC

GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGAGCAC

TGCATAATTCTCTTAC'TlTr ATrr T Tr A A 'v4Trrr'mrA 00 tnGACGCGTCAACCAAGTCATTCTGAG AATAGTGTATGCGGC

AGGT

CTTGCCCGGCGTCAATACGGGATAAT ACCGCCACATAGAAnJA AGTGCTCATCATTGGAAAACGTTCTTCGGGGAAAACCT

GGATCA

CCGCTGFGAGATCCAGTCGATGTACCCACTCGTGACCCAACTGATCTC

AGCATCTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA

AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT

CTCTTCCTTTTCAATATTATTGAAGCATTTATCAGGGTATTGTCTCATGAGC

GGATACATATTTGAATGTATTTAGAAAAATAAACAAATAG

CATTTCCCCGAAAAGTGCCAC

Construction of Hedgehog-Ig Fusion Proteins Mutations with activity in the 1OT1/2 assay were subcloned to SHH- Fc(muIgGl) plasmid pUB1 14 (SEQ ID. No.: 81), which has the wild-type SHH domain fused to the CH2 and CH3 regions of murine IgGI.

The Fc region in pUB 114 contains a glycosylation site mutation [Asn297Gln, and pU 114 plasmids are identical outside of the region coding for the Fc domain fused to SHH. Plasmids identical to pUBi 14, but containing the human IgG1 or murine IgG2a Fc region are pUB 115 (SEQ 82) and pUB116 (SEQ ID. NO.: 83), respectively. (See also Table 4).

Consequently, mutations in SJH were subcloned to pUB114 from the Sad site upstream of SHH in the AOXI promoter to either the Avr2 site or the Sphl site (both downstream of the SHE mutations, but upstream of the SHH-Fc fusion joint).

For construction of yeast strains expressing protein, plasmids were digested with Stul and transformed into Pichia pastoris GS1 15 by electroporation in IM S-:Thitol (Invitrogen) or by a Li salt transformation procedure (Frozen EZ Yeast T -formation kit, Zymo Research, Orange, CA). His+ transformants were selected oh. AiiD agar. Colonies were purified on YPD agar and cultured for protein expression in 5 ml BMMY Methanol) medium. BvMY culture supernatants were harvested at 1 or 2 days (1-day harvests were concentrated by TCA precipitation) and were analyzed by SDS-PAGE and Coomassie blue staining to distinguish clipped and unclipped SHH.

Protein Purification Large scale preparations of protein for purification were prepared as follows: An inoculum in BMGY (late log to stationary phase) was added to 1 L BMGY in a Fembach flask and incubated at 150 rpm for 2-3 days. The stationary phase BMGY culture was centrifuged and the cell pellet from 1 L was resuspended in BMMY(2% Methanol) and incubated in a Fernbach flask at 30 C for 2-3 days. Pepstatin A (44 microM) was added to BMMY medium for expression of SHH-Fc fusion proteins.

A. Purification of Hedgehog N-terminal domain basic region mutants After removing the Pichia cells by centrifugation, the conditioned medium was diluted ten-fold with water to reduce the salt concentration and then re-concentrated using a 3K cutoff spiral filter (Amicon). The concentrate was applied to a CM-Poros® column (Perseptive Biosystems) equilibrated with 50 mM sodium phosphate, pH Elution with a gradient of 0-0.8 M NaCI separated two hedgehog peaks.

The first peak contained a mixture of full-length hedgehog as a disulfide with cysteine or glutathione and clipped hedgehog when a KEX2 proteolytic site was present. The second peak was the full-length disulfide-linked hedgehog homodimer.

This second peak of protein was used to assess bioactivity when the first peak contained significant amounts of clipped material.

The peaks were pooled separately, reduced with 10 mM DTT and dialyzed against 5 mM sodium phosphate, pH 5.5, 150 mM NaCI and 0.5 mM DTT. No DTT was used when the N-terminal cysteine of the protein was replaced with other amino acids. This single purification step is sufficient to achieve >95% purity owing to the low level of contaminating proteins in the conditioned medium. Purity was determined by SDS-PAGE on 4-20% gradient gels (Novex) stained with Coomassie Blue. Identity was confirmed by mass spectrometry, and potency was analyzed using a cell-based bioactivity assay (see below).

B. Purification of Hedgehog-Ig fusion protein constructs Pichia cells were removed from the conditioned medium by centrifugation before application to Protein A Fast Flow (Pharmacia). Protein from constructs utilizing human IgG1 (SEQ ID NO: 40) or murine IgG2A sequences (SEQ ID NO: 42) were applied directly to the Protein A. Constructs utilizing murine IgG1 sequences were diluted ten-fold with water to reduce the salt concentration, re-concentrated using a 3K cutoff spiral filter (Amicon) and the pH adjusted with the addition of sodium borate buffer, pH 8.5 to a final concentration of 50 mM.

0 -88- HHIg was eluted with 25 mM sodium phosphate, pH 2.8, and the fractions collected into tubes containing 0.1 volume of 0.5 M sodium phosphate pH 6 to readjust the pH. The Protein A eluant was then diluted eight-fold with 0.5 mM sodium phosphate, pH 6 and applied to a CM-Poros® column (Perseptive Biosystems) equilibrated with 50 mM sodium phosphate, pH 6.0. Elution with a gradient of 0-0.8 M 00 tt NaCI separated two HHIg peaks.

m The first is "one-armed" protein in which one of the HHIg polypeptides of the dimer is proteolytically cleaved at a sequence near the hinge and therefore this dimer Scontains only one HH N-terminal domain. The second peak is the dimer with two full- 10 length HHIg chains. The peaks were pooled separately, reduced with 10 mM DTT and dialyzed against 5 mM sodium phosphate, pH 5.5, 150 mM NaCI and 0.5 mM DTT.

No DTT was used when the N-terminal cysteine of the protein was replaced with other amino acids. These two purification steps achieve >95% purity. Purity was determined by SDS-PAGE on 4-20% gradient gels (Novex) stained with Coomassie Blue. Identity was confirmed by mass spectrometry, and potency was analyzed using a cell-based bioactivity assay (see below).

Mass spectrometry The molecular masses of the purified proteins were determined by electrospray ionization mass spectroscopy (ESI-MS) on a Micromass Quattro II triple quadrupole mass spectrometer. Samples were desalted using an on-line Michrom Ultrafast Microprotein Analyzer system with a Reliasil® C4 column (1 mm x 5 cm). All electrospray mass spectral data were processed using the Micromass MassLynx data system.

EXAMPLE 2: PHARMACOKINETICS AND PHARMACODYNAMICS Bioactivity assay.

Hedgehog proteins were tested for bioactivity in a cell-based assay measuring alkaline phosphatase induction in C3H10T1/2 cells (Pepinsky et al, JBC 273, 14037- 14045 (1998)).

Pharmacokinetics.

The hedgehog-Ig fusion proteins shown in Table 3 are compared to wt shh Nterminal domain in a screening pharmacokinetic study in mice as exemplified below. In this study, two female Balb/c mice were intraveneously injected with 50 lg of each protein. Occular bleeds were done at 5 minutes and at 5 or 7 hrs after injection for all proteins. The final bleed was done at 24 hrs after injection.

-89- O Serum prepared from all bleeds was frozen immediately on dry ice and stored at C. Hedgehog levels in the serum were determined by a sandwich ELISA where the protein was captured by coated anti-hedgehog mAb 5E1 followed by the secondary antibody (rabbit polyclonal against the 15 N-terminal amino acids of hedgehog) and detection with goat anti rabbit HRP conjugate. Values for various dilutions of the

O

t serum samples were backfited from a standard curve made with the specific protein being tested. The standard curves were validated by determining the concentration of C known levels of protein spiked into serum.

SResults RKRHP Mutation: This mutation was constructed to test whether the "N-11" clip site could be recognized by KEX2 if the N-10 clip does not occur. As expected, this mutant is expressed as a mixture of both intact and clipped SHH. We have not determined the exact clip site (by N-terminal sequencing of Mass Spectroscopy) but we presume that it occurs by cleavage of the Argll-Hisl2 bond. This protein is less extensively clipped than wild-type SHH, so we conclude that the N-11 site is indeed a poorer KEX2 site than the N-10 site. The N-11 KEX2 site must be eliminated by mutation to prevent KEX2 clipping of the Sonic Hedgehog protein.

RKRPP Mutation: This mutation destroys both KEX2 sites in the basic region of SHH.

We presumed that the conservative substituions of one basic residue for another [Lys9Arg and ArglOLys] would not be deleterious to activity. The Hisl2Pro substitution was chosen because the Indian Hedgehog homolog of Sonic Hedgehog has Pro in this position. When the protein was constructed it exhibited no clipping, as expected. When tested in the 10T1/2 assay, the RKRPP mutant protein showed no activity. As this mutation has three amino acid substitutions [Lys9Arg, ArglOLys, and Hisl2Pro], we cannot say which substitution(s) destroyed activity. However, Lys9 is an Arg residue in other homologs of Sonic Hedgehog, so it seemed unlikely to be responsible for inactivity. We postulate that the Hisl2Pro mutation is responsible for the inactivity of the protein.

GSRKRPPRK ("Indian-like" Sonic Hedgehog"). The Indian Sonic Hedgehog sequence has only one KEX2 site in the basic region, compared to Sonic Hedgehog, which has two. The Pro for His substitution is responsible for eliminating the second KEX2 site. Another distinction between Sonic and Indian in this region is the insertion of a Ser residue just upstream of the tribasic motif in Indian Hedgehog. We postulate that this Ser addition may compensate for the extra Pro residue in Indian Hedgehog Scompared to Sonic Hedgehog. Consequently a mutant was constructed that contains both the Ser insertion and the His to Pro mutation. The sequence GSRKRPPRK was substituted for the GKRRHPKK sequence in Sonic Hedgehog. Note that GSRKRPPRK differs from Sonic in five positions (GSRKRPPRK) but differs from the Indian sequence in only one position (GSRKRPPRK). This mutant exhibited no KEX2 00 t clipping and had measurable activity in the 10T1/2 assay, although it is a bit less active than wild-type Sonic Hedgehog.

KKKHP, RKKHP, RQRHP Mutants: These mutants were designed to maintain as Smuch positive charge as possible while maintaining the Hisl2 residue and eliminating C 10 the KEX2 recognition sites. All three of these mutants are inactive and thus demonstrate that the three residue Lys9ArglOArgl sequence in SHH contains a feature essential for activity. This data also raised the possibility that the Hisl2Pro mutation in the RKRPP mutant may be irrelevant to the loss of activity exhibited by the protein. As the RKRPP and RKRHP mutants differ only in the Hisl2 position, we tested the activity of the unclipped RKRHP mutant protein.

The unclipped RKRHP mutant protein was purified away from the clipped species and tested in the 10T1/2 assay, in which it had no detectable activity. As the RKRHP protein has two of the three substitutions present in the inactive RKRPP mutant, it demonstrates that the apparently conservative Lys9Arg and/or substitutions are sufficient to eliminate SHH activity in the 10T1/2 assay.

QRRPP and QRKHP Mutants: These mutants were constructed in order to destroy the KEX2 site while maintaining the ArglO residue and maximizing the number of positively charge residues. Both mutants had activity as high as the wild-type protein.

We conclude that maintaining the ArglO residue is critical for Sonic Hedgehog activity.

CONCLUSIONS

When the initial HHIg fusion construct with wild-type sonic hedgehog sequence was expressed and purified it was found to be clipped at the R10-R11 bond to give the protein. Therefore this protein was not suitable for development as an agonist because previous work had established that hedgehog proteins with truncated N-termini act as antagonists (see above). The sequence of the N-10 site suggested it might be the target of proteolysis by a KEX2-like protease that requires three residues K/R-R- X (where X is not proline). This hypothesis was verified by the construction of mutants of this cleavage site sequence which, when expressed and purified, yielded intact protein (Table Most mutants, however, were inactive in the C3H10T1/2 assay, and it was -91found that an arginine residue must be present at a critical position in the sequence in order to retain activity. The most potent and proteolytically resistant sequence was chosen for the construction of another series of HHIg fusion proteins, some of which also incorporated isoleucine substitutions of the N-terminal cysteine to increase potency and reduce oxidation problems. These fusion proteins have been expressed and purified and shown to be more potent in the C3H10T1/2 assay (Table 8).

Pharmacokinetic data in mice demonstrate that the fusion proteins have a substantial increase in serum half-life compared to the Sonic Hedgehog N-terminal domain (Table/9) Table 7. Hedgehog N-terminal domain basic region mutants.

Basic region sequence Clipping in basic Mass spectrum Potencyb sequence" (Found/Predicted) KRRH (wild type) -70% 19559/19560 1.2-4 l g/ml RKRH -20% 19560/19560 Inactive RKRP 19519/19520 Inactive RQRH <10% 19559/19560 Inactive RKKH 19530/19532 Inactive SRKRP ("Indian-like) <10% 19634/19635 3-6 gg/ml QR.KH 19532/19532 2 /g/ml QRR1P 19520/19520 1.3 g/ml "Estimate based on SDS-PAGE.

bPotency is expressed as the concentration of protein required to achieve 50% maximum alkaline phosphatase induction.

Table 8. HHIg constructs Construct Clipping in basic Mass spectrum Potency N-terminal domain/ sequence" Found/Predicted Fc sequence Wild-type Sonic/ 80% N-10 NDb ND hulgGI Wild-type Sonic/ ND ND ND muIgG1 Wild-type Sonic/ ND ND ND mulgG2a SRKRP Sonic/ 0% 45,533/45,533.6 1.3 gg/ml muIgG1 ^KP onc 18 igm KRRP Sonic/ muIgG1 1.8 gg/ml I I I QRRP Sonic, C24II/ Pending mulgGI QRRP Sonic, C2411 Pending mulgGI 'Determined by N-terminal sequencing.

bNot determined Table 9. Preliminary PK Studies in mice with Hedgehog Ig Fusion Proteins Construct Remaining in Serum hr 7 hr 24 hr Wild type Sonic N-terminal domain 0.3 0.1 0 Wild type Sonic/hu IgG NDa 4.2 0.4 Wild type Sonic/mu IgG Exp I ND 16.4 Exp 2 29 ND Wild type Sonic/mu IgG 2A ND 13.2 2.2 SRKRP Sonic/muIgGI 23 ND 3.2 aNot determined.

Example 3: Comparative Pharmacokinetics and Pharmacodynamics in Primates Comparative studies are conducted with hedgehog fusion and native hedgehog to determine their relative stability and activity in primates. In these studies, the pharmacokinetics and pharmacodynamics of the hedgehog fusion in primates is compared to that of native hedgehog and reasonable inferences can be extended to humans.

Animals and Methods Study Design This is a parallel group, repeat dose study to evaluate the comparative pharmacokinetics and pharmacodynamics of hedgehog fusion protein and nonfusion hedgehog.

Healthy primates (preferably rhesus monkeys) are used for this study. Prior to dosing, all animals will be evaluated for signs of ill health by a Lab Animal Veterinary on two occasions within 14 days prior to test article administration; one evaluation must be within 24 hours prior to the first test article administration. Only healthy animals will receive the test article. Evaluations will include a general physical examination and pre-dose blood draws for baseline clinical pathology and baseline b -93antibody level to hedgehog All animals will be weighed and body temperatures will be recorded within 24 hours prior to test article administrations.

Twelve subjects are enrolled and assigned to groups of three to receive hedgehog as either a fused or a non-fused, but otherwise identical hedgehog.

Administration is by either the subcutaneous (SC) or intravenous (IV) routes. Six male 00 t animals will receive test article by the IV route (3/treatment) and another 6 male Cc animals will receive test article by the SC route (3/treatment). All animals must be C naive to hedgehog treatment. Each animal will be dosed on two occasions; doses will

V')

Sbe separated by four weeks. The dose volume will be 1.0 mLkg.

Blood is drawn for pharmacokinetic testing at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours following each injection. Blood samples for measurements of the hedgehog are drawn at 0, 24, 48, 72, 96, 168, 336, 504 hours following administration of study drug.

Evaluations during the study period include clinical observations performed minutes and 1 hour post-dose for signs of toxicitiy. Daily cageside observations are performed and general appearance, signs of toxicity, discomfort, and changes in behavior will be recorded. Body weights and body temperatures will be recorded at regular intervals through 21 days post-dose.

Assay Methods Levels of hedgehog in serum are quantitated using a ELISA, as described above.

Pharmnacokinetic and Statistical Methods RstripTM software (MicroMath, Inc., Salt Lake City, UT) is used to fit data to pharmacokinetic models. Geometric mean concentrations are plotted by time for each group. Since assay results are expressed in dilutions, geometric means are considered more appropriate than arithmetic means. Serum hedgehog levels are adjusted for baseline values and non-detectable serum concentrations are set to 5 U/ml, which represents one-half the lower limit of detection.

For IV infusion data, a two compartment IV infusion model is fit to the detectable serum concentrations for each subject, and the SC data are fit to a two compartment injection model.

The following pharmacokinetic parameters are calculated: observed peak concentration, Cmx (U/ml); o -94- (ii) area under the curve from 0 to 48 hours, AUC using the trapezoidal rule; (iii) elimination half-life; and, from IV infusion data (if IV is employed): (iv) distribution half-life

OO

t clearance (ml/h) Cc (vi) apparent volume of distribution, Vd c, WinNonlin (Version 1.0, Scientific Consulting Inc., Apex, NC) software is 0used to calculate the elimination half-lives after SC and IM injection.

CN 10 For hedgehog, arithmetic means by time are presented for each group. Emax, the maximum change from baseline, is calculated. Cmx, AUC and Em, are submitted to a one-way analysis of variance to compare dosing groups. and AUC are logarithmically transformed prior to analysis; geometric means are reported.

Claims (9)

1. 2. The isolated polypeptide according to claim 1, wherein X is a polypeptide comprising an amino acid sequence of a hedgehog protein, or a fragment of contiguous amino acids thereof that binds to a patched protein, wherein the N-terminal cysteine is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence.
2. 3. The isolated polypeptide according to claim 1 or claim 2, wherein the hedgehog protein is selected from any of a Sonic hedgehog protein, Indian hedgehog protein, or Desert hedgehog protein.
3. 4. The isolated polypeptide according to any one of claims 1 to 3, wherein the hedgehog protein comprises an amino acid sequence at least 80% identical to any of SEQ ID NOs: 23-26. The isolated polypeptide according to claim 4, wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 23-26. 00 -96- O
4. 6. The isolated polypeptide according to any one of claims 1 to 5, wherein X is a >hedgehog agonist that binds to a patched protein and promotes hedgehog signaling. t 7. The isolated polypeptide according to any one of claims 1 to 6, wherein X is derivatized with a hydrophobic moiety. 00 S8. The isolated polypeptide according to any one of claims 1 to 7, wherein Z is at 0least a portion of a constant region of an immunoglobulin, wherein said portion of a tt constant region comprises at least one of a CH I, hinge, CH2, or CH3 domain. 0o10
5. 9. A fusion protein having an amino terminal region consisting of the amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof that binds to a patched protein, and a carboxy terminal region comprising an immunoglobulin, or fragment thereof comprising at least a portion of a constant region, wherein said portion of a constant region comprises at least one of a CH hinge, CH2, or CH3 domain, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence or (ii) the hedgehog protein comprises a mutated KEX2 protease recognition sequence and the N-terminal cysteine of the hedgehog protein is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues. The fusion protein according to claim 9, wherein the hedgehog protein is selected from a Sonic hedgehog protein, Indian hedgehog protein, or Desert hedgehog protein.
6. 11. The fusion protein according to claim 9 or claim 10, wherein the hedgehog protein comprises an amino acid sequence at least 80% identical to any of SEQ ID NOs:
7. 23-26. 12. The fusion protein according to claim 11, wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 23-26. 13. The fusion protein according to any one of claims 9 to 12, wherein the fusion protein binds to a patched protein and promotes hedgehog signaling. 00 -97- O o 14. The isolated polypeptide or fusion protein according to any one of claims 1 to 13, wherein the hedgehog protein is derivatized. O tI 15. The isolated polypeptide or fusion protein of claim 14, wherein the derivative is selected from a hydrophobic moiety and a polyalkyleneglycol polymer. 00 16. The fusion protein according to any one of claims 9 to 15, wherein comprises 0at least a portion of a constant region of an immunoglobulin comprising at least a hinge, In CH2 and CH3 domains. 10 0 io 17. The isolated polypeptide or fusion protein according to any one of claims 1 to 16, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence, and wherein the mutated KEX2 protease recognition sequence is selected from the sequences of Table 18. An isolated polypeptide having an amino acid sequence X-Y-Z, wherein X is a polypeptide comprising the amino acid sequence of a Sonic hedgehog protein, wherein the Sonic hedgehog protein comprises: a) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence, wherein a KEX2 recognition site corresponding to residues
8. 32-36 of a human Shh protein (SEQ ID NO:15) is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102 or b) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence wherein the N-terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO: 15) is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog. 00 -98- O 19. The isolated polypeptide according to claim 18, wherein a KEX2 recognition site >corresponding to residues 32-36 of a human Shh protein (SEQ ID NO:15) is replaced Swith SEQ ID NO: 92. 20. The isolated polypeptide according to claim 18 or 19, wherein X is a hedgehog 00 agonist that binds patched-1 with an affinity that is similar to or higher than the binding Saffinity of a mature naturally-occurring hedgehog protein to patched-1. (N, n 21. The isolated polypeptide according to any one of claims 18 to 20, wherein the O 10 Sonic hedgehog protein is derivatized with a hydrophobic moiety. 22. The isolated polypeptide according to any one of claims 18 to 21, wherein Z is a polypeptide comprising an immunoglobulin, or fragment thereof comprising at least a portion of a constant region, wherein said portion of a constant region comprises at least one of a CHI, hinge, CH2, or CH3 domain. 23. A fusion protein having an amino terminal region consisting of the amino acid sequence of a Sonic hedgehog protein and having a carboxy terminal region comprising at least a portion of a protein other than hedgehog, wherein the Sonic hedgehog protein comprises: a) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence, wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO:15) is replaced with a sequence selected from one of SEQ ID NO:88-94 or 99-102 or b) an N-terminal 20 kDa fragment of a full-length naturally-occurring Sonic hedgehog sequence wherein the N-terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO: 15) is replaced with a sequence selected from one of SEQ ID NO: 88-94 or 99-102. 00 -99- O 24. The fusion protein according to claim 23, wherein a KEX2 recognition site corresponding to residues 32-36 of a human Shh protein (SEQ ID NO: 15) is replaced Z with SEQ ID NO: 92. 25. The fusion protein according to claim 23 or 24, wherein the Sonic hedgehog 00 protein is derivatized. 26. The fusion protein according to claim 25, wherein the derivative is selected from *n a hydrophobic moiety and a polyalkylene glycol polymer. 27. An isolated polypeptide having the amino acid sequence X-Y-Z, wherein X is a polypeptide comprising an amino acid sequence of a hedgehog protein, or a fragment of contiguous amino acids thereof that binds to a patched protein, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence or an amino acid sequence of a hedgehog protein, or a fragment of contiguous amino acids thereof that binds to a patched protein, wherein the N- terminal cysteine is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than hedgehog. 28. The isolated polypeptide according to claim 27, wherein X is a polypeptide comprising an amino acid sequence of a hedgehog protein, or a fragment of contiguous amino acids thereof that binds to a patched protein, wherein the N-terminal cysteine is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues, and wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence. 29. The isolated polypeptide according to claim 27 or 28, wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 23-26. 00
9. 100- O 30. The isolated polypeptide according to any one of claims 27 to 29, wherein X is a hedgehog agonist that binds to a patched protein and promotes hedgehog signaling. O tQ 31. A fusion protein having an amino terminal region consisting of the amino acid sequence of a hedgehog protein, or a fragment of 50 contiguous amino acids thereof 00 that binds to a patched protein, and a carboxy terminal region comprising at least a 0portion of a polypeptide other than hedgehog, wherein the hedgehog protein comprises a mutated KEX2 protease recognition whsequence or the hedgehog protein comprises a mutated KEX2 protease recognition tt sequence or (ii) the hedgehog protein comprises a mutated KEX2 protease recognition sequence and the N-terminal cysteine of the hedgehog protein is absent or is substituted with phenylalanine, isoleucine, methionine, or two isoleucine residues. 32. The fusion protein according to claim 31, wherein the hedgehog protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NOs: 23-26. 33. The fusion protein according to claim 31 or 32, wherein the fusion protein binds to a patched protein and promotes hedgehog signaling. 34. The isolated polypeptide or fusion protein according to any one of claims 27 to 33, wherein the hedgehog protein comprises a mutated KEX2 protease recognition sequence, and wherein the mutated KEX2 protease recognition sequence is selected from the sequences of Table An isolated nucleic acid sequence encoding the polypeptide or fusion protein according to any one of claims 1 to 17 or 27 to 34. 36. A recombinant nucleic acid comprising the nucleic acid sequence according to claim 35 and an expression control sequence operatively linked thereto. 37. A host cell transformed with the recombinant nucleic acid sequence according to claim 36. 38. A method of producing a recombinant polypeptide comprising: 00 -101- S(a) providing a population of host cells according to claim 37; growing said population of cells under conditions whereby the polypeptide 0 Z encoded by said recombinant nucleic acid is expressed; and t isolating the expressed polypeptide. 00 39. A pharmaceutical composition comprising an effective amount of the polypeptide or fusion protein according to any one of claims 1 to 34. Use of the polypeptide or fusion protein according to any one of claims 1 to 34 in the manufacture of a medicament for treating a subject in need thereof. 41. Use of the polypeptide or fusion protein according to any one of claims 1 to 34 in the manufacture of a medicament for preventing and/or reducing the severity of a neurological condition deriving from: acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vessel injury, and deficits (such as the ischemia from stroke); (ii) infection and tumor-induced injury; (iii) aging of the nervous system including Alzheimer's disease; (iv) chronic Huntington's chorea, amylotrophic lateral sclerosis and the like; or chronic immunological diseases of the nervous system, including multiple sclerosis. 42. A method of treating a subject comprising administering an effective amount of an isolated polypeptide or fusion protein according to any one of claims 1 to 34. 43. A method of preventing and/or reducing the severity of a neurological condition deriving from: acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vessel injury, and deficits (such as the ischemia from stroke); (ii) infection and tumor-induced injury; (iii) aging of the nervous system including Alzheimer's disease; (iv) chronic Huntington's chorea, amylotrophic lateral sclerosis and the like; or chronic immunological diseases of the nervous system, including multiple sclerosis comprising administering an effective amount of an isolated polypeptide or fusion protein according to any one of claims 1 to 34 to a subject in need thereof. 00 -102- O 44. The isolated polypeptide or fusion protein according to any one of claims 8, 16, >22, or 23 to 26, wherein said at least a portion of the constant region is derived from an Z immunoglobulin of the class selected from classes IgM, IgG, IgD, IgA, and IgE. tn 45. The isolated polypeptide or fusion protein according to claim 44, wherein the 00 class is IgG. 46. The isolated polypeptide or fusion protein according to any one of claims 18 to l) 26, 44 or 45, wherein the at least a portion of the constant region of an immunoglobulin O 10 comprises at least a hinge, CH2 and CH3 domains. 47. An isolated polypeptide; a fusion protein; an isolated nucleic acid; a recombinant nucleic acid; a host cell or a pharmaceutical composition, substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. 48. A method or use substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.