WO2001016169A2 - RET LIGAND 5 (Retl5) FROM HUMAN AND MOUSE - Google Patents

Abstract

This invention relates to Ret Ligand 5 (RetL5) compositions and uses thereof. Isolated nucleic acid and amino acid sequences for Ret Ligands are disclosed. Ret ligands encoded by the isolated nucleic acid sequences of the invention have a hydrophobic N-terminal signal sequence, a hydrophobic C-terminal sequence and a phosphatidylinositol glycan linkage motif. Vectors and host cells that includes Ret ligands encoded by the isolated nucleic acid sequences of the invention are also disclosed.

Description

RET LIGAND 5 (RetL5) COMPOSITIONS AND USES THEREOF

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial Number 60/152,024, filed September 1, 1999, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

One of the goals of current research on cell signaling and receptor activation is to enable therapeutic modulation of processes involved in cell growth and survival. Such processes determine outcome in diverse medical conditions, including organ failure, fetal development, and tumor growth, among others. Each of these conditions is of worldwide clinical importance, and has limited efficacious treatment options. It is an object of the invention to provide compositions and - methods for promoting regeneration or survival of damaged tissue, as well as for treating disorders involving the aberrant growth and development of tissues. Tissue loss or end-stage organ failure affects millions of people worldwide each year and adds substantially to health care costs. Organ or tissue loss is usually treated by transplanting organs from donors, by surgical reconstruction, or with mechanical devices. Each of these remedies has shortcomings. Transplantation is limited by donor shortage, surgical reconstruction can create other long-term problems, and mechanical devices cannot perform all the functions of a single organ, and therefore cannot prevent progressive deterioration. Thus, a real medical need exists for new solutions to these problems.

Protein factors that affect the growth, differentiation and/or survival of cells may be useful in the treatment of disorders of organs which contain responsive cells. Factors or ligands that interact with receptors of the receptor protein tyrosine kinase (RPTK) family are of particular interest in this regard. These receptors are involved in many cellular programs including cell growth and differentiation, and the genesis of many neoplasias. Thus the factors or ligands that interact with these receptors may prove useful in treating disorders of certain organs where the tissue has been damaged. Alternatively, it may be useful to block the interaction of these factors with their receptors in order to block tumor growth.

The Ret proto-oncogene encodes a receptor tyrosine kinase that is expressed during development in a variety of tissues, including the peripheral and central nervous systems and the kidney. The abnormalities present in Ret null mice suggest that Ret is critical for the migration and innervation of enteric neurons to the hindgut, and for proliferation and branching of the ureteric bud epithelium during kidney development (Nature 367; 380-383 (1994)). The search for a key component of the Ret signaling pathway, the Ret ligand, has been an area of intensive research.

SUMMARY OF THE INVENTION

This invention relates generally to novel Ret ligand 5 (RetL5) compositions and uses thereof.

The invention provides a purified and isolated DNA molecule coding for a novel Ret ligand, RetL5, having the nucleotide sequence of any RetL5, but specifically including murine RetL5 cDNA (SEQ ID NO:2). The invention further provides a RetL5 protein, with an amino acid sequence comprising that of murine RetL5 (SEQ ID NO:3).

In another embodiment, the invention includes a signal sequence in RetL5, preferably amino acids 1-21 of SEQ ID NO. 3 (which correspond to nucleotides 1- 63 of SEQ T NO. 2).

In another embodiment, the invention includes a first cysteine rich domain in RetL5, preferably comprising amino acids 22-112 of SEQ ID NO. 3 (which correspond to nucleotides 64-336 of SEQ ID NO. 2). The invention also includes a second cysteine rich domain in RetL5, preferably comprising amino acids 1 13-218 of SEQ ID NO. 3 (which correspond to nucleotides 337-654 of SEQ ID NO. 2).

In another embodiment, the invention includes a flexible hinge region in RetL5, preferably a first flexible hinge region comprising amino acids 219-247 of SEQ ID NO. 3 (which correspond to nucleotides 655-741 of SEQ ID NO. 2). In another embodiment, the invention includes a GPl cleavage/attachment site in RetL5, preferably comprising amino acids 248-250 of SEQ VD NO. 3 (which correspond to nucleotides 742-750 of SEQ ID NO. 2).

In another embodiment, the invention includes an amino linked glycosylation site in RetL5, preferably comprising amino acids 184-186 of SEQ ID NO. 3 (which correspond to nucleotides 550-558 of SEQ ID NO. 2).

In another embodiment, the invention incudes a RetL5 polypeptide having a domain structure including a signal sequence, two cysteine rich domains, a flexible hinge, a GPl cleavage/attachment site, and an N-linked glycosylation site. Preferably, the RetL5 polypeptide binds specifically to a GDNF family member polypeptide. In a specific embodiment, the domains of the present RetL5 polypeptide have the following approximate sites: signal sequence, 20 amino acids; cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids; GPl cleavage/attachment site, two amino acids; and an N-linked glycosylation site, two amino acids.

In another embodiment, the invention includes an extracellular RetL5 polypeptide having a domain structure including two cysteine rich domains, a flexible hinge, a GPl cleavage/attachment site, and an N-linked glycosilation site. Preferably, the RetL5 polypeptide binds specifically to a GDNF family member polypeptide. In a specific embodiment, the domains of the present RetL5 polypepetide have the following approximate sites: cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids; GPl cleavage/attachment site, two amino acids; and an N-linked glycosylation site, two amino acids. In another embodiment, the invention includes a soluble RetL5 polypeptide having a domain structure including two cysteine rich domains, a flexible hinge and an N-linked glycosilation site. Preferably, the RetL5 polypeptide binds specifically to a GDNF family member polypeptide. In a specific embodiment, the domains of the present RetL5 polypepetide have the following approximate sites: cysteine rich domains, at least 45 amino acids up to at most about 110 amino acids; flexible hinge, at least 25 amino acids, two amino acids; N-linked glycosylation site, two amino acids.

In another embodiment of the invention, a purified and isolated DNA molecule for use in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide product has at least a part of the primary structural conformation and the biological activity of RetL5; a) the DNA may be a DNA molecule which comprises murine RetL5 cDNA or the complementary strand of murine RetL5 cDNA; b) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in a) or unique fragments thereof; or c) DNA molecules which, but for the degeneracy of the genetic code, would hybridize to the DNA molecules defined in a) and b). A purified and isolated DNA molecule coding for a polypeptide fragment or variant of a human RetL5 having the biological activity of a RetL5 is also within the invention.

In yet another embodiment, the invention is an isolated nucleic acid comprising a nucleotide sequence complementary to or which hybridizes under conditions of medium stringency to high stringency to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or the complement of any of the foregoing. A complementary strand or complement (RNA or DNA) of a nucleic acid sequence encoding a RetL5 protein has a nucleic acid sequence which can form base pairs with the nucleic acid sequence encoding the RetL5 proteins.

In still another embodiment, the invention relates to nucleic acid sequences which hybridize to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 under conditions of moderate to high stringency. Any of the recombinant DNA molecules of the invention, if desired, can be operably linked to an expression control sequence.

Also included within the invention are vectors and delivery systems which encompass the DNA molecules or constructs defined elsewhere in this specification. The vector may encompass a DNA molecule encoding a RetL5 or a variant of a RetL5. The invention includes prokaryotic or eukaryotic host cells stably transformed or transfected by a vector comprising a DNA molecule encoding a native or variant RetL5.

A purified and isolated human RetL5 substantially free of other human proteins is specifically within the invention, as is a process for the production of a polypeptide product having part or all of the primary structural conformation and the biological activity of a RetL5. Such a process may include the steps of growing, under suitable culture conditions, prokaryotic or eukaryotic host cells transformed or transfected with any DNA molecule of the invention, in a manner allowing expression of such polypeptide product, and recovering a RetL5. The polypeptide product of the expression in a procaryotic or eukaryotic host cell of a DNA is also included.

The invention also includes proteins and protein fragments, variants and derivatives, whether soluble or membrane bound. In selected embodiments, the protein has an amino acid sequence which comprises murine RetL5, or is a variant thereof. In other embodiments, the protein is a fusion protein including a RetL5, fused to another molecule or molecular fragment, such as an immunoglobulin, toxin, imageable compound or radionuclide. Also included are chimeric molecules of RetL5. Other embodiments of the invention include specific monoclonal antibodies to a RetL5 of the invention. Such an antibody may be associated with a toxin, imageable compound or radionuclide.

The invention further includes a method of promoting growth of new tissue, or promoting survival of damaged tissue in a subject, including administering to the subject a therapeutically effective amount of a compound which interacts with cellular Ret and thereby induces autophosphorylation of Ret. The compound can be RetL5, a fragment of a full-length RetL5, or an antibody which binds to Ret. The compound can be administered concurrently with a therapeutically effective amount of a second compound, such as GDNF, neurturin or a GDNF family member molecule. While tissues of interest for these methods can include any tissue, preferred tissues include renal tissue, neural tissue, heart, stomach, small intestine, spinal cord, or lung. In one embodiment, the RetL5 is a soluble RetL5. The subject of the methods can be human.

In another method of the invention, Ret signal transduction between a first cell expressing a RetL5 and a second cell expressing Ret is inhibited by contacting the first cell with a soluble Ret ligand protein or with an antibody to the RetL5. The soluble Ret ligand protein may be a fusion protein.

The invention also includes a method for targeting a toxin, imageable compound or radionuclide to a cell expressing Ret, encompassing contacting the cell with a RetL5 fusion protein conjugated to a toxin, imageable compound or radionuclide. Preferably, the RetL5 fusion protein comprises a soluble RetL5 polypeptide. In another method, growth of a tumor cell which expresses Ret is suppressed, with a step of the method being contacting the cell with a fusion protein of a RetL5 and a toxin or radionuclide, conjugated to a toxin or radionuclide. The cell can be within a subject, and the protein or the conjugated antibody is administered to the subject.

Also encompassed within the invention is a method for targeting a toxin, imageable compound or radionuclide to a cell expressing a RetL5, comprising contacting the cell with an anti-RetL5 antibody conjugated to a toxin, imageable compound or radionuclide. Another embodiment includes the method of suppressing growth of a tumor cell which expresses a RetL5, comprising contacting the cell with a fusion protein of Ret and a toxin or radionuclide or with an anti- RetL5 antibody conjugated to a toxin or radionuclide; the cell can be within a subject, and the protein administered to the subject.

The RetL for any of the methods of the invention is RetL5, or a variant or unique fragment of RetL5.

Methods of gene therapy are also within the invention. One embodiment is a method of treating a subject with a disorder of Ret metabolism, comprising administering to the subject a vector comprising a DNA molecule encoding a RetL5, as well as a method of promoting growth of new tissue in a subject, comprising administering such a vector to the subject. Another embodiment includes a method of promoting survival of damaged tissue in a subject, one step of the method being administering a therapeutically effective amount of a vector encoding a RetL5 to the subject.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 is a murine RetL5 cDNA sequence predicted from DSW300 sequence by Visual Inspection Method (SEQ ID NO: 2)

FIGURE 2 is a murine RetL5 cDNA sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX (SEQ ED NO: 4).

FIGURE 3 is a murine RetL5 amino acid sequence predicted from DSW300 by Visual Inspection Method (SEQ ID NO: 3). The presumed signal sequence is underlined at the N-terminus of the protein and the presumed cleavage site/GPI attachment at the C-terminus is in bold and underlined.

FIGURE 4 is a murine RetL5 amino acid sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX (SEQ ED NO: 5). The presumed signal sequence is underlined at the N-terminus of the protein.

FIGURE 5 is a predicted human RetL5 cDNA sequence (SEQ ID NO: 6).

FIGURE 6 is a predicted human RetL5 amino acid sequence (SEQ ID NO: 7). The presumed signal sequence is underlined at the N-terminus of the protein and a possible GPl cleavage/attachment site is in red at the C-terminus FIGURE 7 is a murine RetL5 cDNA (SEQ ΣD NO: 8) alternatively spliced from the genomic sequence of SEQ ED NO: 1.

FIGURE 8 is a deduced murine RetL5 amino acid sequence (SEQ ED NO: 9) of SEQ ED NO: 8.

FIGURE 9 depicts murine RetL5-Fc fusion protein binding to the following GDNF family members. GDNF (Glial cell line-Derived Neurotrophic Factor), NTN (neurturin), NBN (neublastin), PSP (persephin).

FIGURE 10 depicts fusion proteins of rat RetLl (rLl-Ig), human RetL2 (rL2-Ig), murine RetL3 (mL3-Ig) and murine RetL5 (mL5-Ig) binding to rat neublastin (rNBN). FIGURE 11 depicts fusion proteins of rat RetLl (rLl-Ig), human RetL2 (rL2-Ig), murine RetL3 (mL3-Ig) and murine RetL5 (mL5-Ig) binding to neublastin and rat Ret AP (alkaline phosphatase).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.

Nucleotide and amino acid sequences refeπed to in the specification have been given the following sequence identification numbers: SEQ ED NO: 1 - mouse RetL5 genomic sequence. SEQ ED NO: 2 - mouse RetL5 cDNA predicted by Visual Inspection Method.

SEQ ED NO: 3 - mouse RetL5 amino acid predicted by Visual Inspection Method.

SEQ ED NO: 4 - mouse RetL5 cDNA sequence predicted from DSW300 sequence by GENESCAN/GENE ALEX. SEQ ED NO: 5 - mouse RetL5 - RetL5 amino acid sequence predicted from

DSW300 sequence by GENESCAN/GENE ALEX.

SEQ ED NO: 6 - predicted human RetL5 cDNA. SEQ ED NO: 7 - predicted human RetL5 amino acid sequence. SEQ ED NO: 8 - murine RetL5 cDNA alternatively spliced from SEQ ED NO: 1.

SEQ ED NO: 9 - deduced amino acid sequence of the murine RetL5 cDNA of SEQ ED NO: 8. Definitions

As used herein, the term "RetL" (also refeπed to herein as "GFRα" or "c- RetL") means any protein which specifically interacts with the receptor protein Ret, (also referred to herein as "c-Ret") and which, when it interacts with Ret, triggers Ret dimerization and/or autophosphorylation of the tyrosine kinase domain of Ret. The DNA sequences which code for RetL and for Ret are termed "RetL" and "Ret", respectively. A ligand optionally can be soluble, or present as a membrane-bound molecule on the same or on a different cell as the Ret molecule for which it is triggering autophosphorylation. In certain uses or interactions with Ret, the ligand may require additional molecules to trigger autophosphorylation. Ligands of the invention include co-receptors or accessory ligand cofactors. Ligands of the invention further include anti-Ret mAbs which act as Ret agonists, triggering Ret dimerization and autophosphorylation. The ligand can also be modified in various ways, such as incorporated as a portion of a fusion protein, or conjugated to a toxin or radionuclide.

By "alignment of sequences" is meant the positioning of one sequence, either nucleotide or amino acid, with that of another, to allow a comparison of the sequence of relevant portions of one with that of the other. An example of one method of this procedure is given in Needleman et al. (J. Mol. Biol. 48:443-453 (1970)). The method may be implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As will be understood by those skilled in the art, homologous or functionally equivalent sequences include functionally equivalent arrangements of positionally conserved amino acid residues such as the cysteine residues, within the sequence. "Functionally equivalent arrangements" can include amino acid insertions or deletions which alter the linear arrangement of conserved residues, but do not materially impair their relationship in the folded structure of the protein. Therefore, minor internal gaps and amino acid insertions in a candidate sequence are ignored for purposes of calculating the level of amino acid sequence homology or identity between the candidate sequence and a reference sequence (here, RetL5). One characteristic frequently used in establishing the homology of proteins is the similarity of the number and location of the cysteine residues between one protein and another.

By "unique fragment" of a polypeptide sequence is meant a peptide consisting of at least 8 contiguous amino acids of said polypeptide sequence, preferably at least about 12 contiguous amino acids. Similarly, a "unique fragment of a nucleic acid sequence" refers to at least 8 contiguous codons of a polypeptide encoding a sequence of a similar length of non-coding nucleic acid sequence.

By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.

By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

By "cDNA library" is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise a representation of the mRNA molecules present in an entire organism or tissue, depending on the source of the RNA templates. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra. Generally, RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines. Alternatively, RNA may be isolated from a tumor cell, derived from an animal tumor, and preferably from a human tumor. Thus, a library may be prepared from, for example, a human adrenal tumor, but any tumor may be used.

As used herein, the term "DNA polymorphism" refers to the condition in which individuals can have different nucleotide sequences at a particular site in the DNA of an organism. The differing sequences isolated from such individuals are thus "polymorphic variants."

"Expression vector" includes vectors which are capable of expressing DNA sequences contained therein, i.e., the coding sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. A useful, but not a necessary, element of an effective expression vector is a marker encoding sequence, which is a sequence encoding a protein which results in a phenotypic property (e.g. tetracycline resistance) of the cells containing the protein which permits those cells to be readily identified. In sum, "expression vector" is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified contained DNA code is included in this term, as it is applied to the specified sequence. Such vectors are frequently in the form of plasmids, so "plasmid" and "expression vector" are often used interchangeably. However, the invention is intended to include such other forms of expression vectors, including phage, which serve equivalent functions and which may from time to time become known in the art.

"GDNF-related molecule" means any molecule which is a GDNF family member, i.e., which has at least 40% amino acid sequence similarity to either GDNF or neurturin. Preferred GDNF family members are capable of specifically binding to RetL5.

The term "gene" means a polynucleotide sequence encoding a peptide. By "homogeneous" is meant, when referring to a peptide or DNA sequence, that the primary molecular structure (i.e., the sequence of amino acids or nucleotides) of substantially all molecules present in the composition under consideration is identical.

The term "oligonucleotide" as used herein in referring to probes, oligomer fragments to be detected, oligomer controls, unlabeled blocking oligomers and primers for amplification of sequences is defined as a molecule comprised of more than three deoxyribonucleotides or ribonucleotides. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.

The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to a target strand. "Recombinant host cells" refers to cells which have been transformed with vectors constructed using recombinant DNA techniques. As defined herein, the antibody or modification thereof produced by a recombinant host cell is by virtue of this transformation, rather than in such lesser amounts, or more commonly, in such less than detectable amounts, as would be produced by the untransformed host. As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes each of which cut double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term "restriction fragment length polymorphism" ("RFLP") refers to the differences among individuals in the lengths of a particular restriction fragment. Thus, an RFLP is one type of DNA polymorphism.

A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or omits residues that are found in the other, or if the sequence of amino acid residues is not identical. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Perm. (1980). By "vector" is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.

By "substantially pure" is meant any protein of the present invention, or any gene encoding any such protein, which is essentially free of other proteins or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.

Compounds of the Invention

The invention includes cDNA coding for a novel Ret ligand, RetL5, including for example the nucleotide sequence of murine RetL5 cDNA.

In addition, the compounds of the invention include sequences which are derivatives of these sequences. The invention also includes vectors, liposomes and other carrier vehicles which encompass one of these sequences or a derivative of one of these sequences. The invention also includes proteins transcribed and translated from RetL5 cDNA, preferably, murine RetL5 cDNA, including but not limited to murine RetL5 and derivatives and variants.

In another embodiment, the invention relates to the use of the nucleic acids and proteins of the present invention to design probes to isolate other proteins which have structural or functional properties of the RetL5 proteins of the invention. The probes can be a variety of base pairs in length. For example, a nucleic acid probe can be between about 10 base pairs in length to about 150 base pairs in length. Alternatively, the nucleic acid probe can be greater than about 150 base pairs in length. Exceptional methods are provided in Ausubel et al, "Current Protocols in Molecular Biology" J. Wiley (ed.) (1999), the entire teachings of which are herein incorporated by reference in their entirety.

The design of the oligonucleotide (also referred to herein as nucleic acid) probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any, and (b) It should be designed to have a T_m of about 80° C (assuming 2°C for each A or T and 4° for each G or C).

The oligonucleotide should preferably be labeled with γ- ^"P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4 x 10 dpm/pmole. The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at about 37°C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at about 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them. Highly stringent (also referred to herein as "high stringency") conditions are those that are at least as stringent as, for example, lx SSC at about 65°C, or lx SSC and 50%o formamide at about 42°C. Moderate stringency conditions are those that are at least as stringent as 4x SSC at about 65°C, or 4x SSC and 50% formamide at about 42°C. Moderate or reduced stringency conditions are those that are at least as stringent as 4x SSC at about 50°C, or 6x SSC and 50% formamide at 40°C.

The filter is then preferably incubated at about 65°C for 1 hour with gentle agitation in 6X SSC (20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to 1 x 10 dpm/mL. The filter is then preferably incubated at about 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2x SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2x SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with O.lx SSC/0.5% SDS at about 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed. The positive colonies are then picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.

In yet another embodiment, the invention relates to nucleic acid sequences (e.g., DNA, RNA) that hybridize to nucleic acids of RetL5 ligands. In particular, nucleic acids which hybridize to SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 4 or SEQ ED NO: 6 under high, moderate or reduced stringency conditions as described above.

In still another embodiment, the invention relates to a complement of nucleic acid of RetL5. In particular, it relates to complements of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 5 or SEQ ED NO: 5. In another embodiment, the invention relates to an RNA counterpart of the

DNA nucleic acid of RetL5. In particular, it relates to RNA counterparts of SEQ ED NO: 1, SEQ ED NO: 2, SEQ ED NO: 4 or SEQ ED NO: 6.

Another embodiment of the invention includes soluble variants of a RetL5. Soluble variants lack at least a portion of the intramembrane section of the native RetL5. In some examples, the soluble variant lacks the phosphatidylinositol glycan linkage ("GPl linkage") of the native RetL5. Soluble variants include fusion proteins which encompass derivatives of RetL5 that lack a phosphatidylinositol motif.

Variants can differ from naturally occurring RetL5 in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence ("sequence variants") are produced when one or more amino acids in naturally occurring RetL5 is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly preferred variants include naturally occurring RetL5, or biologically active fragments of naturally occurring RetL5, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences which differ by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the RetL5 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, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, 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 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 taken from the table below, and yet others are described by Dayhoff in the Atlas of Protein Sequence and Structure (1988).

TABLE 1 : CONSERVATIVE AMINO ACID REPLACEMENTS

Other variants within the invention are those with modifications which increase peptide stability. Such variants may contain, for example, one or more non- peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants 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 variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Patent 5,219,990. The peptides of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use. Splice variants are specifically included in the invention.

In addition to substantially full-length polypeptides, the present invention provides for biologically active fragments of the polypeptides. A RetL5 polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring RetL5. Such biological activities can include the ability to specifically bind the extracellular portion of Ret, with an affinity that is at least 50% of, and preferably at least equal to, the affinity of naturally occurring RetL5 for the extracellular portion of Ret. Similarly, RetL5 biological activity can include binding to a GDNF family member molecule (e.g., GDNF, neurturin, persephen, neublastin) with an affinity that is at least 50% of the affinity of naturally occurring RetL5 for binding to the GDNF family member molecule. Another biological activity is the ability to bind to an antibody which is directed at an epitope which is present on naturally occurring RetL5. Biologically active fragments of the RetL5 polypeptide can be predicted using computer programs known to those of skill in the art, including for example, the computer program P-SORT.

In other embodiments, variants with amino acid substitutions which are less conservative can also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics. Generally, substitutions that may be expected to induce changes in the functional properties of Ret polypeptides are those in which: (i) a hydrophilic residue, e.g., serine or threonine, is substituted by a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative charge, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine. Variants within the scope of the invention include proteins and peptides with amino acid sequences having at least sixty percent homology with murine RetL5 (SEQ ED NO:3). More preferably the sequence homology is at least eighty, at least ninety percent, or at least ninety-five percent. For the purposes of determining homology the minimum length of comparison sequences will generally be at least 8 amino acid residues, usually at least 12 amino acid residues. Variants of the compounds of the invention also includes any protein which 1) has an amino acid sequence which is at least forty percent homologous to a RetL5 protein of the invention, and also which 2) after being placed in an optimal alignment with the RetL5 sequence, has at least 80% of its cysteine residues aligned with cysteines in the RetL5 protein of the invention.

Just as it is possible to replace substituents of the scaffold, it is also possible to substitute functional groups which are bound to the scaffold with groups characterized by similar features. Such modifications do not alter primary sequence. These will initially be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization of portions of naturally occurring RetL5, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.

Also included within the invention are agents which specifically bind to a protein of the invention, or a fragment of such a protein. These agents include Ig fusion proteins and antibodies (including single chain, double chain, Fab fragments, and others, whether native, humanized, primatized, or chimeric). Additional descriptions of these categories of agents are in PCT application WO95/16709, the disclosure of which is herein incorporated by reference.

THERAPEUTIC USES OF THE COMPOUNDS OF THE INVENTION Native and variant RetL5, anti-RetL5 antibodies, and fusion proteins of Ret and of RetL5 may have therapeutic utility in situations where it is desirable to block or to activate the Ret signaling pathway, to stimulate renal and/or neuronal cell growth or survival in disease situations where these cells are lost or damaged, or to suppress growth of or to kill undesirable cells such as tumor cells that express Ret or a RetL5.

In general, compounds of the invention that bind to Ret, inducing dimerization and/or autophosphorylation of Ret, are useful for stimulating growth of or limiting damage to Ret-expressing tissues. The compounds of the invention are useful for stimulating renal tissue growth and/or survival, supporting renal function, and in minimizing damage to renal tissue after various insults. Particular conditions which may be beneficially treated with the compounds of the invention include acute renal failure, acute nephritis, chronic renal failure, nephrotic syndrome, renal tubule defects, kidney transplants, toxic injury, hypoxic injury, and trauma. Renal tubule defects include those of either hereditary or acquired nature, such as polycystic renal disease, medullary cystic disease, and medullary sponge kidney. This list is not limited, and may include many other renal disorders (see, e.g., Harrison's Principles of Internal Medicine, 13th ed., 1994, which is herein incorporated by reference.)

In other applications, the genes and proteins of the invention may be used to treat conditions where neural growth and regeneration is desirable. This would include any conditions involving disorders of neural degeneration, such as

Alzheimer's disease, Parkinson's, Huntington's, Tourette's, amyotrophic lateral sclerosis, as well as motor neuron disease, demyelinating diseases such as multiple sclerosis, bacterial diseases such as meningitis, abscess, or empyema, viral diseases such as HEV-associated myelopathy, prion diseases including Creutzfeldt-Jakob disease. Also included are disorders of damage to neural tissue, whether caused by neoplastic impingement, trauma, or cerebrovascular events such as hemorrhage or emboli. Diseases of the cranial nerves and of the spinal cord, including disorders involving traumatic, inflammatory, congenital or vascular etiologies, are specifically included, as are disorders affecting the autonomic nervous system. Also included are developmental neural disorders such as mental retardation, autism, fetal alcohol syndrome, Down's syndrome, and cerebral palsy. The compounds of the invention may also be used to treat syndromes involving the peripheral nervous system, particularly peripheral neuropathies. These disorders include those caused by any of the factors previously listed, and specifically include Lyme disease, HEV-associated neuropathies, polymyositis, muscular dystrophy, and myasthenia gravis. Anti-RetL5 antibodies and Ret fusion proteins of the invention, which specifically bind to the protein of murine RetL5 or fragments of thereof, are useful in several methods. The compounds may be used therapeutically to inhibit or block Ret receptor signaling, such as for blocking growth of tumors which depend on activation of Ret signaling for growth. These agents may also be fused to detectable markers, such as fluoroscopically or radiographically opaque substances, and administered to a subject to allow imaging of tissues which express a RetL5. The agents may also be bound to substances, such as horseradish peroxidase, which can be used as immunocytochemical stains to allow visualization of areas of RetL5- positive cells on histological sections. A specific antibody could be used alone in this manner, and sites where it is bound can be visualized in a sandwich assay using an anti-immuno globulin antibody which is itself bound to a detectable marker. Specific antibodies to any RetL5 are also useful in immunoassays to quantify the substance for which a given antibody has specificity. Specific antibodies to a RetL5 may also be bound to solid supports, such as beads or dishes, and used to remove the ligand from a solution, either for use in purifying the protein or in clearing it from the solution. Each of these techniques is routine to those of skill in the immunological arts.

While not intending to be limited to a single mechanism of action it is contemplated that RetL5 may act as an antagonist by interacting with Ret in a manner which blocks activation of Ret by a GDNF family member/Ret Ligand complex. This theory is based on the observation that RetL5 could interact with a GDNF family protein and this complex would not be able to interact with Ret. RetL5 would therefore bind to/tie up GDNF family proteins and prevent their proper interactions with other RetLigands. Alternatively, RetL5 is a truncated RetLigand that may be lacking the domain that would bind a GDNF family member yet still maintain the amino acids necessary to interact with.

Other methods of the invention include modulating Ret-RetL5 signaling by contacting Ret with an anti-Ret monoclonal antibody. The effect of such a mAb-Ret contact can be to either block or to stimulate activation of the Ret signaling pathway, depending on the characteristics of the interaction of each particular mAb with Ret. Certain mAbs interact with Ret as agonists, with the agonist mAb-Ret binding triggering the dimerization and autophosphorylation of Ret. Other mAbs act as Ret antagonists. The interaction of Ret with an antagonist mAb prevents Ret signaling activation by other RetL5, or by complexes comprising RetL5, which would otherwise activate the Ret signaling pathway. A RetL5 and/or antibodies to Ret or to a Ret fusion protein can be used to allow imaging of tissues which express Ret, or in the imrnunohisto logical or preparative methods described above for antibodies to a RetL5.

Fusion proteins encompassing a RetL5 and/or anti-Ret antibodies can be used to specifically target medical therapies against cancers and tumors which express Ret. Such tumors might include the several different tumor phenotypes which have been associated with mutations in Ret (N. Engl. J. Med. 335:943-951, 1996; Nature 367: 319-320, 1996; Trends Gen. 12:138-144, 1996). Therapeutic interventions against neoplasias which express a RetL5 utilize fusion proteins which incorporate Ret and/or an anti-RetL5 antibody. The anti-Ret antibody or anti-RetL5 antibody may be effective by itself through antibody-dependent and complement- dependent cytolysis mediated by the Fc domain. Such hybrid ligands and antibodies can be made more effective as cancer therapeutics by using them as delivery vehicles for antineoplastic drugs, toxins and cytocidal radionuclides, such as yttrium 90. Cytotoxic effector cells may be targeted to tumor cells using heteroconjugate antibodies, where an antibody specific for either Ret or for a RetL5 expressed by a tumor is covalently coupled to an antibody directed against a surface protein on cytotoxic effector cells, such as NK cells or CTLs.

One example of an anti-Ret antibody or RetL5 therapy is to conjugate the toxic A chain of ricin or a modified full-length form of ricin (which can no longer bind cells) to a RetL5 or to an antibody directed against the Ret polypeptide expressed on the surface of malignant cells. In another embodiment, a toxin is conjugated to Ret or to an anti-RetL5 antibody to selectively target and kill RetL5- positive cells, such as a tumor expressing a RetL5. Success with an analogous approach is reported in Grossbard et al., Blood 79:5761 (1992). 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.

The above approaches, using fusions of ricin or other toxins, are equally applicable to toxic conjugates of RetL5 or of an anti-Ret antibody; these are useful for selectively targeting and killing Ret-positive cells, such as tumor cells expressing Ret.

Another approach to such medical therapies is to use radioisotope labeled RetL5 or anti-Ret antibodies. Such radiolabeled compounds will preferentially target radioactivity to tumor sites in cells expressing Ret, sparing normal tissues. Depending on the radioisotope employed, the radiation emitted from a radiolabeled antibody bound to a tumor cell may also kill nearby malignant tumor cells that do not express Ret. A variety of radionuclides may be used. Isotopes that emit β particles (for example, ¹³¹I) have been successful when employed with monoclonal antibodies against CD20 present on B-cell lymphomas (Kaminski et al., N. Engl. J. Med. 329: 459 (1993); Press et al, N. Engl. J. Med. 329: 1219 (1993). Radionuclides emitting β particles generate radioactive emissions that are tumoricidal over distances spanning several cell diameters, permitting the eradication of antigen negative cells and diminishing the consequences of nonhomogenous deposition of antibody or ligand in tumors.

Radionuclides emitting particles may also be employed. The low dose rate irradiation generated by radionuclide labeled RetL5 or anti-Ret antibodies may be more therapeutically effective than the instantaneous irradiation delivered externally in conventional radiation therapy. Low dose rate irradiation can induce apoptosis (programmed cell death) in certain cell lines (Macklis et al., Radiat. Res. 130: 220 (1992); Maklis et al, Radiopharm. 5: 339 (1992).

The compounds of the invention are administered in therapeutically-effective amounts, which means an amount of a compound which produces a medically desirable result or exerts an influence on the particular condition being treated.

The term "subject" used herein is taken to mean any mammal to which Ret ligand or gene may be administered. Subjects specifically intended for treatment with the method of the invention include humans, as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well as the organs, tumors, and cells derived or originating from these hosts.

Use of Compounds of the Invention in Gene Therapy

The RetL5 genes of the invention are introduced into damaged tissue to stimulate production of a RetL5 by the transfected cells, to promote cell growth and/or survival of cells that express Ret.

In a specific embodiment of a gene therapy method a RetL5 gene may be introduced into a renal or neural target tissue of choice. A RetL5 would then be stably expressed and stimulate Ret receptor-positive cells to grow, divide, differentiate, and/or potentiate cell survival. Furthermore, RetL5 genes may be introduced into a target cell using a variety of well-known methods that use either viral or non-viral based strategies.

Non-viral methods include electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, and direct micro-injection into single cells. For instance, a RetL5 gene may be introduced into a cell by calcium phosphate coprecipitation (Pillicer et al., Science, 209: 1414-1422 (1980); mechanical microinjection and/or particle acceleration (Anderson et al., Proc. Natl. Acad. Sci. USA, 77: 5399-5403 (1980); liposome based DNA transfer (e.g., LEPOFECTIN-mediated transfection- Fefgner et al., Proc. Nat. Acad. Sci., USA, 84: 471-477, 1987; Gao and Huang, Biochim. Biophys. Res. Comm., 179: 280-285, 1991; DEAE Dextran-mediated transfection; electroporation (U.S. Patent 4,956,288); or polylysine-based methods in which DNA is conjugated to deliver DNA preferentially to liver hepatocytes (Wolff et al., Science, 247: 465-468, 1990; Curiel et al., Human Gene Therapy 3: 147-154, 1992). Target cells may be transfected with the genes of the invention by direct gene transfer. See, e.g., Wolff et al., "Direct Gene Transfer Into Moose Muscle In Vivo", Science 247:1465-68, 1990. In many cases, vector-mediated transfection will be desirable. Any of the methods known in the art for the insertion of polynucleotide sequences into a vector may be used. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, J. Wiley & Sons, NY (1992), both of which are incorporated herein by reference. Promoter activation may be tissue specific or inducible by a metabolic product or administered substance. Such promoters/enhancers include, but are not limited to, the native RetL promoter, the cytomegalovirus immediate-early promoter/enhancer (Karasuyama et al., J. Exp. Med.. 169: 13 (1989)); the human beta-actin promoter (Gunning et al, Proc. Nat. Acad. Sci. USA. 84: 4831 (1987); the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al, Mol. Ce \ Biol., 4: 1354 (1984)); the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al., RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1985)); the SV40 early region promoter (Bernoist and Chambon, Nature, 290:304 (1981)); the promoter of the Rous sarcoma virus (RSV) (Yamamoto et al., Cell, 22:787 (1980)); the heφes simplex virus (HSV) thymidine kinase promoter (Wagner et al., Proc. Nat. Acad. Sci. USA, 78: 1441 (1981)); the adenovirus promoter (Yamada et al., Proc. Nat. Acad. Sci. USA. 82: 3567 (1985)).

The RetL5 gene may also be introduced by specific viral vectors for use in gene transfer systems which are now well established. See for example: Madzak et al., J. Gen . Virol.. 73: 1533-36, 1992 (papovavirus SV40); Berkner et al., Curr. Top. Microbiol. Immunol. 158: 39-61, 1992 (adenovirus); Hofmann et al, Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Curr. Top. Microbiol. Immunol.. 158: 25-38, 1992 (vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol.. 158: 97-123, 1992 (adeno-associated virus); Margulskee, Curr. Top. Microbiol. Immunol.. 158: 67-93, 1992 (heφes simplex virus (HSV) and Epstein- Barr virus (HBV)); Miller, Curr. Top. Microbiol. Immunol.. 158: 1-24, 1992

(retrovirus); Brandyopadhyay et al., Mol. Cell. Biol.. 4: 749-754, 1984 (retrovirus); Miller et al, Nature. 357: 455-450, 1992 (retrovirus); Anderson, Science. 256: 808- 813, 1992 (retrovirus), Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., Eds.), Greene Publishing Associates, 1989, all of which are incoφorated herein by reference.

Preferred vectors are DNA viruses that include adenoviruses (preferably Ad- 2 or Ad-5 based vectors), baculovirus, heφes viruses (preferably heφes simplex virus based vectors), and parvoviruses (preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors). See, e.g., Ali et al., Gene Therapy 1: 367- 384, 1994; U.S. Patent 4,797,368 and 5,399,346 and discussion below.

The choice of a particular vector system for transferring, for instance, aRetL5 sequence will depend on a variety of factors. One important factor is the nature of the target cell population. Although retroviral vectors have been extensively studied and used in a number of gene therapy applications, they are generally unsuited for infecting cells that are not dividing but may be useful in cancer therapy since they only integrate and express their genes in replicating cells. They are useful for ex vivo approaches and are attractive in this regard due to their stable integration into the target cell genome. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. The general adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for gene therapy of Duchenne Muscular Dystrophy (DMD)and Cystic Fibrosis (CF). Both Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies.

Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. High titers (10¹³ plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (an adenovirus-transformed, complementation human embryonic kidney cell line: ATCC CRL1573) and cryo-stored for extended periods without appreciable losses. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders. See Y. Watanabe, Atherosclerosis. 36: 261-268 (1986); K. Tanzawa et al, FEBS Letters. 118(l):81-84 (1980); J.L. Golasten et al. New Engl. J. Med.. 309 (11983): 288-296 (1983); S. Ishibashi et al, J. Clin. Invest. 92: 883-893 (1993 V. and S. Ishibashi et al. J. Clin. Invest.. 93: 1889-1893 (1994), all of which are incoφorated herein by reference. Indeed, recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials. See, e.g., J. Wilson, Nature. 365: 691-692 (Oct., 21, 1993). Further support of the safety of recombinant adenoviruses for gene therapy is the extensive experience of live adenovirus vaccines in human populations.

Human adenoviruses are comprised of a linear, approximately 36 kb double- stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (El through E4) and late (LI through L5) regions, based on expression before or after the initiation of viral DNA synthesis. See, e.g., Horwitz, Virology, 2d edit., ed. B.N. Fields, Raven Press Ltd., New York (1990).

The first-generation recombinant, replication-deficient adenoviruses which have been developed for gene therapy of DMD and other inherited disorders contain deletions of the entire Ela and part of the Elb regions. This replication-defective virus is grown in 293 cells containing a functional adenovirus Ela gene which provides a transacting Ela protein. El -deleted viruses are capable of replicating and producing infectious virus in the 293 cells, which provide Ela and Elb region gene products in trans. The resulting virus is capable of infecting many cell types and can express the introduced gene (providing it carries its own promoter), but cannot replicate in a cell that does not carry the El region DNA unless the cell is infected at a very high multiplicity of infection. Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells such as neurons, and appear essentially non-oncogenic. Adenoviruses do not appear to integrate in to the host genome. Because they exist extrachromasomally, the risk of insertional mutagenesis is greatly reduced. Ali et al., supra, at 373. Recombinant adenoviruses (rAdV) produce very high titers, the viral particles are moderately stable, expression levels are high, and a wide range of cells can be infected. Their natural host cells are airway epithelium, so they are useful for therapy of lung cancers.

Baculovirus-mediated transfer has several advantages. Baculoviral gene transfer can occur in replicating and nonreplicating cells, and can occur in renal cells, as well as in hepatocytes, neural cells, spleen, skin, and muscle. Baculovirus is non-replicating and nonpathogenic in mammalian cells. Humans lack pre-existing antibodies to recombinant baculovirus which could block infection. In addition, baculovirus is capable of incoφorating and transducing very large DNA inserts.

Adeno-associated viruses (AAV) have also been employed as vectors for somatic gene therapy. AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering. Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep 62 and rep 40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene. See BJ. Carter, in Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome. The AAV life cycle is biphasic, composed of both latent and lytic episodes. During a latent infection, AAV virions enter a cell as an encapsilated ssDNA, and shortly thereafter are delivered to the nucleus where the AAV DNA stably integrates in to a host chromosome without the apparent need for host cell division. In the absence of a helper virus, the integrated AAV genome remains latent but capable of being activated and rescued. The lytic phase of the life cycle begins when a cell harboring an AAV provirus is challenged with a secondary infection by a heφesvirus or adenovirus which encodes helper functions that are recruited by AAV to aid in its excision from host chromatin (B.J. Carter, supra). The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or - ssDNA genomes following cell lysis. Adeno-associated viruses (AAV) have significant potential in gene therapy. The viral particles are very stable and recombinant AAVs (rAAV) have "drug-like" characteristics in that rAAV can be purified by pelleting or by CsCl gradient banding. They are heat stable and can be lyophilized to a powder and rehydrated to full activity. Their DNA stably integrates into host chromosomes so expression is long-term. Their host range is broad and AAV causes no known disease so that the recombinant vectors are non-toxic.

Once introduced into a target cell, sequences of interest can be identified by conventional methods such as nucleic acid hybridization using probes comprising sequences that are homologous/complementary to the inserted gene sequences of the vector. In another approach, the sequence(s) may be identified by the presence or absence of a "marker" gene function (e.g, thymidine kinase activity, antibiotic resistance, and the like) caused by introduction of the expression vector into the target cell.

Formulations and Administration

The compounds of the invention may be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants. Localized delivery is particularly contemplated, by such means as delivery via a catheter to one or more arteries, such as the renal artery or a vessel supplying a localized tumor.

The term "pharmaceutically acceptable carrier" means one or more organic or inorganic ingredients, natural or synthetic, with which the mutant proto-oncogene or mutant oncoprotein is combined to facilitate its application. A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. In this regard, the term "carrier" encompasses liposomes and the HEV-1 tat protein (See Chen et al., Anal. Biochem. 227: 168-175, 1995) as well as any plasmid and viral expression vectors. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

The liposome system may be any variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, and may be prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of United States Patents 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, it may be desirable to express the novel polypeptides of this invention, as well as other selected polypeptides, as lipoproteins, in order to enhance their binding to liposomes. As an example, treatment of human acute renal failure with liposome-encapsulated RetL5 may be performed in vivo by introducing a RetL5 into cells in need of such treatment using liposomes. The liposomes can be delivered via catheter to the renal artery. The recombinant RetL5 protein is purified, for example, from CHO cells by immunoaffinity chromatography or any other convenient method, then mixed with liposomes and incoφorated into them at high efficiency. The encapsulated protein may be tested in vitro for any effect on stimulating cell growth. This invention also contemplates that the novel polypeptide of this invention may be administered to an animal via liposome delivery system in order to enhance their stability and/or immunogenicity. Delivery of the novel polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in the treated animal. Such cells, upon ingesting the liposomal membrane and subsequently present the polypeptides to the immune system in conjunction with other molecules required to elicit a strong immune response.

Any of the novel RetL5 polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

EXEMPLIFICATION

EXAMPLE 1 : Functional Screening Assay for RetL5 Genomic DNA as well as a full length RetL5 cDNA (generated by overlap- extension PCR) is cloned into an expression vector for protein production in E293 cells. RetL5 peptide polyclonal antibodies are produced in rabbits using techniques known to those of skill in the art and are used to identify the expressed RetL5 proteins. A truncated Ret Ligand2, (RetL2c), is constructed for comparative puφoses. The isolated RetL5 proteins are assayed by ELISA to identify interactions with known members of the GDNF family of proteins. To test whether RetL5 is capable of interacting with cRET in the presence of any of the GDNF family members, E293 cells are transfected with the RetL5 cDNA clones and are assayed for a Ret-Fc fusion protein binding to the cells by a FACS (Flourescently Activated Cell Sorting) assay. RetL5 protein can also be used in a RET ternary complex ELIS A, wherein a GDNF family protein and Ret-AP (alkaline hosphatase) fusion protein are mixed in solution with a RetL5-Fc fusion protein. The protein mixture can then be captured on an antibody (anti Fc) coated ELISA well and the readout for the assay can be through the AP tag of the Ret-AP fusion protein. A positive signal indicates that a ternary complex is formed between the GDNF family protein, RetL5 fusion protein, and the Ret fusion protein.

EXAMPLE 2: RetL5 Northern Expression

Northern blot analysis of murine RL5 message in adult mouse tissues revealed expression in brain and testis. Two messages were observed in brain tissue and one message in testis. The size of the messages seen is consistent with the cDNA predicted from the genomic clone.

EXAMPLE 3: Human RetL5

A human mouse somatic cell hybrid cell line which contains human chromosome 20 in a murine A9 cell background was obtained. mRNA and cDNA libraries may be made from this cell line. The RNA and the cDNA can be assayed for the presence of known human genes surrounding the locus for RetL5. If they are present it should be assumed that RetL5 is also present. The A9/human chromosome 20 cDNA library is screened with a murine RetL5 probe to obtain clones which cross-hybridize. A radioactive probe generated by random priming a portion of the murine RetL5 coding region (Feinberg, A.P. and et al, Anal. Biochem., 132, 6-13, 1983; Feinberg, A.P. et al, Addendum Anal. Bioche,., 137, 266-267, 1984) is used to screen the library. In addition one can obtain a RetL5 cDNA by direct cDNA selection (Lovett, M. (1994). Direct selection of cDNAs using genomic contigs. In "Current Protocols in Human Genetics" (N.C. Dracopoli, et al, Eds.), Current

Protocols, New York.; A.D. Simmons and Lovett, M.(1999). Direct cDNA Selection Using Large Genomic DNA Targets. In "Methods in Enzymology" 303, [8],111- 126).

The RetL5 probe can also be used to screen other human cDNA libraries. The choice of cDNA libraries may be determined from results obtained by hybridizing the probe to commercially available Northern blots of human organs and cell lines.

EXAMPLE 4: Cloning of RetL5

The peptide sequence of murine RetL3, as disclosed in WO97/44356, incoφorated by reference herein, is used to search the GenBank database with the program BLAST in order to identify related proteins (i.e. isologs). BLAST, or Basic Local Alignment Search Tool, uses the method of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990) to search for similarities between a query sequence and all the sequences in the sequence database. The query sequence and the database to be searched can be either peptide or nucleotide in any combination. When the rat RetL3 peptide sequence is queried against the Expressed Sequence Tag (EST) nucleotide database, a novel sequence with GenBank accession# AU 035938 having an E value of 8e-l 8 was identified. Comparison of this sequence to the coding region of murine RetL3 indicated that this EST is likely to be produced from a splicing intermediate in which two introns are retained. One intron is found from NT 118-170 and the second intron begins at NT 416 and continues through the end of the sequence at 792. When aligned with the genomic structures of family members (Baloh, et al, Proc. Natl. Acad. Sci. USA 5/5801-5806 (1998); Eng, et al, Oncogene, 16:591-601 (1998), the teachings of which are incoφorated in their entirety), this clone appears to retain the intron between exons 4 and 5 and the intron after exon 6.

EXAMPLE 5: Identification and characterization of genomic clones for RetL5 An antisense oligonucleotide was generated from the AU035938 EST sequence: KD2-819 (AGC GCT CCA GGG GCT CCA GGC AAG AG (SEQ ED NO: 10); corresponding to the complement of nucleotides 69-95). lxlO⁶ plaque forming units from the Stratagene murine 129 SvJ lambda FIXE genomic library (cat# 946313) were screened in duplicate on Optitran™ filters. The filters were hybridized with ³²P-labeled oligonucleotide KD2-819 in 300 mis plaque screen buffer (50mM Tris pH 7.5, 1M NaCl, 0.1% sodium pyrophosphate, 0.2% Polyvinylpryrolidine and 0.2%o Ficoll) containing 10% Dextran sulfate and lOOμg/ml tRNA and 100 pmol ³² P-labeled oligonucleotide at 65Covernight. The filters were washed twice with 2X SSC/1%SDS and exposed to film. Four duplicate positives were purified. DNA from each of these clones was analyzed by restriction enzyme digestion followed by agarose gel electrophoresis and Southern blotting. The Southern filter was hybridized to KD2-819 to confirm the inserts hybridized to the probe. The 15914 bp insert (SEQ ED NO. 1, shown at pages 42 through 53) of one of these clones, DSW300, was completely sequenced. In addition, several restriction enzyme fragments from the insert of DSW300 were subcloned into the plasmid vector pBluescriptπ SK(+) (Stratagene) for sequence analysis. The following subclones spanned the entire insert of the lambda clone DSW300: DSW320 is a Notl/Hind H fragment spanning nucleotides 1-2551 DSW318 is a HindH/HindEH fragment spanning nucleotides 2552-5612 DSW319 is a HindH/Hindπi fragment spanning nucleotides 5613-5889 DSW316 is a HindE VBamHI fragment spanning nucleotides 5890-10948 DSW304 is a BamHI/Notl fragment spanning nucleotides 10949-15914.

DSW318, DSW319, DSW316, and DSW304 were entirely sequenced and the sequences obtained were 100%) identical to the corresponding portion of DSW300.

EXAMPLE 6: Protein coding region prediction The sequence of DSW300 was used to query all the public databases using

BLAST. This search indicated the presence of a novel member of the disintegrin family (ADAM) at the 5' end of DSW300 as well as sequence homology to GFRα family (RetL family). Based on homology to the Ret Ligand gene family, exons 4-8 for a novel family member were identified. Through visual alignment of DSW300 with the coding regions of RetLl, 2, 3 and 4, five exons can be identified and a putative coding region lacking an initiation codon and at least a portion of the signal sequence can be inferred from nucleotides 12443-12806 of SEQ ED NO. 1 (exonA) (the reading frame begins at nucleotide 12445 of SEQ ED NO. 1), nt 12860-12969 of SEQ ED NO. 1 (exon B), ntl3051-13185 of SEQ ED NO. 1 (exon C), ntl 3726- 13786 of SEQ ED NO. 1 (exon D), nt 13876-13993 of SEQ ED NO. 1 (exon E). The beginning of the putative secretion signal sequence with the initiating methionine beginning at nucleotide 11113 of SEQ ED NO. 1 and ending at nucleotide 11158 of SEQ ED NO. 1 was predicted using the program GeneALEX and Signal P. A polyadenylation signal is located at NT 14544-14549 of SEQ ED NO. 1.

DSW300 was analyzed by the programs GeneALEX and SignalP that predict a protein encoded by 6 exons. Exon 1 contains a putative secretion signal with the initiating MET beginning at NT 11113 of SEQ ED NO. 1. The 5' end of exonl is not predicted and the 3' end of exonl is at NT 11158 of SEQ ED NO. 1; exon2 is predicted at nt 12443-12810 of SEQ ED NO. 1; exon3 nt 12882-12969 of SEQ ED NO. l;exon 4 nt 13051-13185 of SEQ ED NO. 1; exon 5 nt 13726-13806 of SEQ ED NO. 1 ; and exon 6 nt 14078-14154 of SEQ ED NO. 1.

Analysis of DSW300 with the program GENSCAN: Burge, C, et al, J. Mol. Biol, 268, 78-94 (1997); Burge, C, et al, Curr. Opin. St ct. Biol. 8, 346-354 (1998); Salzberg, S., et al, Elsevier Science, Amsterdam, pp. 127-163; Burge, C. B., PhD thesis, Stanford University, Stanford, CA (1997); Burset, M., et al, Genomics, 34, 353-367 (1996), yielded a protein-coding region identical to that predicted by GeneALEX and Signal P. The corresponding nucleotide sequence and amino acid sequence obtained from the GENSCAN analysis are found in SEQ ED NO. 4 and SEQ ED NO. 5 and differ from the protein coding regions identified by visual inspection as disclosed in SEQ ED NO. 2 and SEQ ED NO. 3. The protein predicted by visual inspection and alignment with other family members maintains the cysteine spacing and number that is consistent within the family. RetL5 shares with the other members of the RetLigand family a hydrophobic N-terminus indicative of a signal sequence and a hydrophobic C-terminus indicative of a phosphatidylinositol glycan linkage motif. Although these methods vary in the protein -coding region predictions, in all predictions the putative protein domain structure of RetL5 differs from other family members in that domain 1 and the first flexible hinge region encoded by exons 2 and 3 of other family members is deleted. Other cases of truncated RetLigands have been reported in the literature. For example, alternatively spliced RetL2 transcripts have been detected in human brain and placenta, Wong, et.al, NeuroReport, Vol. 9, 3767-3773, (1998); Baloh, et.al, Neuron, Vol. 18, 793-802, May 1997. EXAMPLE 7: Murine RefL5-Alternative Splice Variant Murine Genomic DNA containing the entire murine RetL5 gene (DSW304- BamHI/Notl) was cloned into the expression vector CH269 for protein production in E293 cells. mRNA was isolated from these cells and subjected to RT-PCR to identify the RetL5 transcript. These cDNAs were then subcloned into the sequencing vector TOPO-TA(Invitrogen) for sequence analysis.

Murine RetL5-GPI2 cDNA Sequence: See Figure 7 (SEQ ED NO. 8)

Symbols: 1 to: 46 from: dsw30078.seq ck: 7775, 11113 to: 11158

Symbols: 47 to: 410 from: dsw30078.seq ck: 7775, 12443 to: 12806 Symbols: 411 to: 520 from: ds 30078.seq ck: 7775, 12860 to: 12969 Symbols: 521 to: 655 from: dsw30078.seq ck: 7775, 13051 to: 13185 Symbols: 656 to: 732 from: ds 30078.seq ck : 7775, 13726 to: 13802 Symbols: 733 to: 783 from: dsw30078.seq ck: 7775, 13943 to: 13993

EXAMPLE 8: Binding of Splice Variant

Briefly, the cDNA for the GPl splice variant of murine RetL4(5), (SEQ ED NO: 8) would be transiently transfected into E293 cells. Post transfection 48 hours the cells would be removed from the flask with EDTA and then washed with PBS. The cells are incubated with rat Ret-human Ig +/- GDNF family member (persephin; PSP) for 1.5 hours at RT. The cells are washed and are incubated with PE- conjugated anti-human Fc for 20 min at room temperature and read in the FACSCAN. If persephin (PSP) is a ligand for this RetL4/5 splice form, then it should form a ternary complex with Ret and the surface of the cell only in the presence of the GFL, in this case PSP. EXAMPLE 9: Human RetL5

From unordered sequences (Accession Numbers AC 055771, AC 013324, AL 356755) the human RetL5 gene was detected. While these sequences are labeled as being from three separate chromosomes, the RetL5 sequence is present on only one chromosome, three Bacterial Artificial Chromosome (BACs) RPl 1-561P16, RPl 1- 388K24, RP5-96457 used to obtain the genomic sequences had one gene in common: KJAA 0548. This has a corresponding locus clearly mapped to chromosome 20. It is known that chromosome assignments can be erroneous where specific tagged sequences (STSs) turn out to encompass moderately repetitive elements that reside on more than one chromosome; thus, the mapping data in public databases can be wrong or result from a chimeric BAC, derived from more than one chromosome.

The analysis of human RetL5 had been completed using the AC055771 sequence and hence all nucleic acid numbering is from that particular sequence.

Human RetL5 cDNA sequence (by Visual Inspection Method (with GENSCAN Initiating Sequence) See Figure 5 (SEQ ED NO. 6)

Symbols: 1 to: 46 from: ac055771.seq /rev ck: 3281, 90071 to: 90116

Symbols: 47 to: 393 from: ac055771.seq /rev ck: 3281, 87663 to: 88009

Symbols: 394 to: 503 from: ac055771.seq /rev ck: 3281, 87474 to: 87583

Symbols: 504 to: 638 from: ac055771.seq /rev ck: 3281, 87245 to: 87379 Symbols: 639 to: 730 from: ac055771.seq /rev ck: 3281, 86877 to: 86968

Symbols: 731 to: 850 from: ac055771.seq /rev ck: 3281, 86629 to: 86747

EXAMPLE 10: Murine RetL5 Binding Data Murine RetL5 can interact with NBN. The murine RetL5 used is a RetL5 fusion (amino acids 1-247) with the Fc portion of human IgG (Sanicola, et al, PNAS 94:6238-6243 (1997)). The fusion protein interacts with the recently decribed GDNF family member neublastin, NBN. (Figures 9 and 10).

Oligonucleotide sequence used in constructing the RetL5 fusion protein were as follows:

kd3-071: CAGCCGCCTG TGCCGGCCCC GTCTCCTTGC (SEQ ED NO: 11) kd3-072: GCAAGGAGAC GGGGCCGGCA CAGGCGGCTG (SEQ ED NO: 12) kd3-073: ACGCAGGCCT CATAGGCACC GTGGTCACCC (SEQ ED NO: 13) kd3-074: GGGTGACCAC GGTGCCTATG AGGCCTGCGT (SEQ ED NO: 14) kd3-075: GGAACCCCTG CTTGGATGGT GCCATACAAG (SEQ ED NO: 15) kd3-076:CTTGTATGGC ACCATCCAAG CAGGGGTTCC (SEQ ED NO: 16) kd3-077: GGACCAGACT GCTGGGCAAG GCACGAGTGG (SEQ ED NO: 17) kd3-079: ATAGTTTAGC GGCCGCTCAG AGCAGGGCCT GGAGAG (SEQ ED NO: 18) kd3-080: CCACTCGTGC CTTGCCCAGC AGTCTGGTCC (SEQ ED NO: 19) kd3-l 17: ATAAGAATGC GGCCGCGGTC CTAGGAAGAG ACATCT (SEQ ED NO: 20) kd3-l 18: CTGCTGCTGT TGTTGCTGCT GGGGTCTGCG AGCTTTACCG ACGG (SEQ ED NO: 21) kd3-119: CCGTCGGTAA AGCTCGCAGA CCCCAGCAGC AACAACAGCA GCAG (SEQ ED NO: 22) kd3-121: TTCCGCGGCC GCTATGGCCG ACGTCGACGA GCAGGGCCTG GAGAGCCAG (SEQ ED NO: 23)

PCR-SOE reactions were carried out using the Clontech Advantage-HF PCR kit according to manufacturer's directions:

Reaction A: oligos: kd3-l 17, kd3-l 19; template: DSW304-2 Reaction 4: oligos: kd3-l 18, kd3-072; template: DSW304-2 Reaction 5: oligos: kd3-071, kd3-074; template: DSW304-2 Reaction 6: oligos: kd3-073, kd3-076; template: DSW304-2 Reaction 7: oligos: kd3-075, kd3-080; template: DSW304-2 Reaction 8: oligos: kd3-077, kd3-079; template: DSW304-2 Reaction A/4: oligos: kd3-l 17, kd3-072; template: gel-purified products from Reaction A and Reaction 4 Reaction 5/6: oligos: kd3-071, kd3-076; template: gel-purified products from Reaction 5 and Reaction 6

Reaction 7/8: oligos: kd3-075, kd3-079; template: gel-purified products from Reaction 7 and Reaction 8

Reaction 5/8: oligos: kd3-071, kd3-079; template: gel-purified products from Reaction 5/6 and Reaction 7/8

Reaction A/8: oligos: kd3-l 17, kd3-079; template: gel-purified products from Reaction A/4 and Reaction 5/8

Reaction A/8 yielded the cDNA of murine RetL5 according to the visual inspection prediction. The cDNA of murine RetL5 was digested with Notl and inserted into the Notl site of the CH269 vector in a two-way ligation to produce construct DSW321.

The DSW322 (murine RetL5-human Fc fusion) construct was produced by amplifying DSW321 by PCR with oligos kd3-l 17 and kd3-121 and digested with Notl/Sall. The Notl /Sail murine RetL5 fragment was ligated to the Sail /Notl human Fc fragment from SAB144 (Sanicola, et al, PNAS 94:6238-6243 (1997)) and inserted into the Notl site of the CH269 vector in a three-way ligation to produce construct DSW322.

Amino acid sequence of murine RetL5-human Fc fusion:

MAHCMESALL LLLLLGSASF TDGNRCVDAA EACTADERCQ QLRSEYVARCLGRAAPGGRP GPGGCVRSRC RRALRRFFAR GPPALTHALL FCGCEGSACAERRRQTFAPA CAFSGPGLVP PSCLEPLERC ERSRLCRPRL LAFQASCAPAPGSRDRCPEE GGPRCLRVYA GLIGTVVTPN YLDNVSARVA PWCGCAASGNRREECEAFRK LFTRNPCLDG AIQAFDSLQP SVLQDQTAGQ GTSGLRRAVDKTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKP

REEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAP EEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ED NO: 24)

The murine RetL5 -human Fc fusion protein was expressed in EBNA293 cells by transient transfection. Murine RetL5-human Fc fusion protein conditioned media was purified over a protein A Sepharose column, pooled, and then purified over a Superdex 200 column. Protein A-purified material was concentrated to 500 μl using MiUipore 10k cutoff Ultrafree-4 centrifugal filter device (UFV4BGC25). The entire sample was applied that to a gel filtration column. The column was a prepacked Pharmacia 1 X 30 cm Superdex 200 column (17-1088-01). The protein was eluted with a 45 minute isocratic elution (25 mM HEPES, 150 mM NaCl, pH 7.5) at 0.5 mL/min on the BioCad. 500 uL fractions were collected and 10 uL of the fractions 11-30 applied to a 10-20% reducing SDS-PAGE gel.

EXAMPLE 11 : GFL (GDNF Family Ligand) ELISA Briefly, a Nunc-Immunoplate MaxiSoφ Surface plate were coated with 250 ng/ml GDNF family member (GFL) in 50 mM sodium bicarbonate/ carbonate, pH 9.6 overnight at 4° C. The plate is then blocked with 1% BSA in TBS + Tween (TBST) for 1 hour RT. The block (TBST) was removed and fresh TBST added to each well. Equal molar stock solutions of GFRα-human Ig fusions (2 nM) were prepared (Sanicola, et al, PNAS 94:6238-6243 (1997)) and serially diluted. Plate were incubated at room temperature. The plates were washed three times with TBST and incubated with an anti-human-HRP conjugate for 1 hour at RT. The plates were washed again and developed with developing solution (12.5 ml 0.1 M sodium acetate trihydrate + 0.1 M citric acid (monohydrate), 125 ul 42 mM TMB in DMSO, 1.95 ul 30% hydrogen peroxide). The reaction is allowed to continue for 5 minutes and stopped with an equal volume of 2 N H₂S0₄. The ELISA plate is then read in at 450 nm and the results plotted in Deltagraph. One of these splice variants, GFR 2c (RL2c), encodes a protein with a similar domain structure as RetL5 in that domain 1 and the first flexible hinge region are deleted.

EQUIVALENTS While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

<110> orley, Dane

<120> Novel RetLigand 5 (RETL5) Compositions and Uses Thereof

<130> Genomic Sequence

<140> <141> .

<160> 5

<170> Patentln Ver. 2.0

<210> 1 (SEQ ID NO. 1) <211> 15914 <212> DNA <213> Mouse

<400> 1 gcggccgcga gctcaattaa ccctcactaa agggagtcga ctcgatctct ctgctttccc 60 ttaatgggtc tctggtgtag ttaaagtccg cgggcacgcc ttgtcgtcct gctgcgactg 120 actgcgatct ccccgagttc tgcaccggca cctccccgta ttgccccgca gatgtttacc 180 tactggatgg ctcaccctgc gctgagggtc gcggctattg cctagacggc tggtgtccca 240 cgctggagca gcagtgccag cagctatggg ggcctggtga gaccgcacgc tggtcctggt 300 gccctgacca atactaaaac ctgcggtttt ctactgaggg caagctccac ccgtggaact 360 gaggcccgag ctgcccgctt cttactcccc ctccccccag ggtccaagcc ggccccagag 420 ccatgtttcc agcagatgaa ctccatgggg aattcgcaag ggaactgtgg ccaggaccac 480 aagggtagct tcctgccttg tgctcagagg tgaggtgtga tgctgagggt ctgcagctgt 540 aaagtagggc ggagcatgcg gagggaacac tccaagttgt tgaccacctt ccacttcctc 600 cccagggacg ctctgtgtgg gaaactgctg tgccagggag gggagccgaa cccactagtg 660 ccgcacatag tgactatgga ctccacaatt ctcctagagg gccgcgaagt ggtttgccga 720 ggggcctttg tgctcccaga tagtcacctg gaccagcttg acttgggtct ggtagagcca 780 ggcaccggct gtggacctag aatggtgagc cctgcccacc caacccctcc tggttattga 840 gtcctccatg ccaaagtgtt ctcctcactg cccagtgggc acaatgccca taggtgtgcc 900 aggacaggca ctgtcagaat gctacctccc aggagctgga acgttgcttg actgcctgcc 960 ataacggtgg ggtgagtagc ctaaggggtc agggtgacct tggaggtcct tgctacctgg 1020 tgacttttct atcctcatct taggtttgca atagcaatcg taactgtcac tgtgctgctg 1080 gctgggctcc acccttctgt gacaagcctg gcttgggtgg tagcgtggat agtggccctg .1140 cacagtctgc aagtatgcca gtggggtggg ggcaggcagg aaccacctgg gcagtagcct 1200 gcttgagact cagcacccct gccctccaca gaccgagatg ccttcccctt ggccatgctc 1260 ctcagcttcc tgctgcctct gctccctggg gctggcctag cctggtgcta ctaccagctc 1320 ccaacattct gtcatcgaag gggactgtgc tgcaggaggg accccctatg gaataggtag 1380 gttcggtgct caggtctctc ttcctgagcc tgcccccatg gctcctgctt ctcagaactc 1440 ttcagggctt tgtagagtga gaggctacag ggagctgggg ctttaggaag ctagatggga 1500 tccttattct ctagatgtag tgagagctcc caggctgtgg gaagaagtcc gtggtgtgta 1560 tcactgccct gaccagcact tggtggtgtg gtcctttcca tgggcttccc ttgatttttt 1620 tgttttatgt tttttgttgt tgttggtttt tttctgcttt aaagaaattc aacacagcct 1680 gtgctccgct ttgtcctgaa gacgactcta gcttccttgt ctcaatatca gccatctccc 1740 atggcctttg ccctaattac tcccttcagg tccctcggtg ctagccaagg acccctttgt 1800 gctccctata aactggccag ctatagtgtt gctctttctg tgccaggact ctgtgcctct 1860 gtcccctgta gatcacagtg otgtaaaccc aattttctgg caggcagaaa tgccacctga 1920 tagcacacat tatctaccga gggcagcctc ctcactaggc ccctcgggca agtcaggcac 1980 attatgtccc tgtctgaact tggaagtgtt ctagaccaat tgagggaagg gcagggttgg 2040 gttggagatg tgactagagg gcacctcagg ccagaaacag caccagcagg ccccaggagc 2100 cagtgagacg gtctggggaa agccaggtag tgcctggggg gtgggggcgg tgtctgcaga 2160 cagggaaaca ggtggagtga cagttgggag ggggcacttc agaggggtgg cagctgcaca 2220 ccgttatcgg gatagggtgt caaggacagt gggaatatct ggatgaacca tccaaagaaa 2280 tggcaaggtc tgtgagaaag gtcccggcag ttcagagtct agactggcag agcacggcta 2340 aacggagcat gggaaggcac agttcccacc aagggagaca tctctgcact tcagctctag 2400 gtgggccctc ggtgacgcct acatctagac tgagtggggg ctggagtgga gtcacctcgg 2460 gagaaaggat gcccacagcc ctggaggccc tgggacataa gcgaggtctg gacatcttaa 2520 ggacagaaca ggaatgtgga attcgaagct tgagtggaat ggagaagcaa tccccctttg 2580 tctcatacac ggtcactttc caaccttctg aactcttatc tggtctgtca ctggcccctg 2640 cagagacata cccctgggca gtgtgcatcc ggtggagttt ggctccatca tcactggaga 2700 gccctcgccc cctcgtaagt gtgccccttg ggacatggag agggaagcaa ggggtgggtg 2760 cttgccatct gcctccttct aaatgctctc cttaacacac cttcaatcct ctgccctgct 2820 cagccccatg gacctcttgc caacagcgtt cgcaccctcc atctcttgac ttgctctcag 2880 accctgcgaa ctctgagctt acctaagaac taccctctga agcagcctgg tctacagatt 2940 gagttccaga cctgccctat ccctatggta tggaagcacc ctgaggacct cctgttgccc 3000 agtcacctac ctctgtctca gtttgttgtc ccctcctcag atttacaggc ttgcatcaat 3060 aaagaaatga gacatgggcc tcagagaagc tgttgtcata gagaccatga tgctggaagc 3120 cctaggggca gggaagggag acactgtggt tcttcttggg tccttataga gggaggacaa 3180 atgtgccctg ccatgtgact tgcagtcctc agtttctcag acgcactctt ataattccta 3240 tgggctgtat gctgagctct tactcagcat aggaacccca gagcccgatc atgttgtatc 3300 ccgcctgccc tgagagctgt gctattctga aatgttagaa tgtatctaat aacaataaat 3360 ccacaagtta tatcagtgtt gttggctgtg acctgttaaa agggtctaag ttgtttatta 3420 aaaagatatg gagatggatt actgagaaag aaattagaaa caacatctgg acagtggagg 3480 agccagcact ggggaggaaa gggcagacag atctctgagt tcaaggccag cctggtctac 3540 agattgagtt ccaggatagc tagggccaca cagaggaaac cctgttttga aaaccaaaca 3600 gtcaaataat aaaaacaaac ttagtagcac cttgtacaga cagagagaaa ggttcccagg 3660 agagctcaga ccccagcacc agaggtggca aggcagagtc tcaaagccca ggtggcaaaa 3720 gtgggatctg ttagcataaa cccaaagggc gcgtgggaca ggcagggagt accctttgat 3780 gcaagcccac tctggtgagg gcccctcacc cctggatgtc tgttagcaag ggaatcagtc 3840 agtgtctcag tctgttaaca tctgtgagag gggaaaggct gctgcagaca tggcctgagc 3900 agcatctgga ttcgaacatt tgcactttag ggcctgctct ctcccctggg tggggcactc 3960 gccattcatt gcctttacga cagctgtgag agagaggttg ctgcaagtgt gtatggctgg 4020 gttagctcga gcccaccagg caatcatgat ttccctcgac atctagccat taacaaacac 4080 aggttctttt actacaattt taactaccaa tattaactaa taatgtgtat aaaatatata 4140 acacagtaca catatataag tacatagatg ttagtacata taaatattat atatatatat 4200 ataattttaa ttaatattac ttaattttat tttatcattt gatattattt tactgttagt 4260 tataacaatg tgcataatat atgtgtaata taaaatataa ttttattatt taatattatt 4320 atataaattt aattaatatt aattatacct atatatttag tacatataca taggttacag 4380 aatggctaca aaagtgccag gagccatcaa ggagaagcta aaagccagca agtgatcttc 4440 ctgagacggt tctgccatgg actgtacaat tagtgatgga tttgcttctg taggcaagga 4500 cgaggagatt tcattttagg aaagattcct gctattaata tgcttttcct ggtattatta 4560 aatatatata acaatcacta ggtattagcc caccgttttg aacagaatgt tctgcagaac 4620 aatgaagatg tactctcttg taatgatgct atatagacaa atagattatt tcttttttaa 4680 aaaagaaaaa agagccgggc gatggtggca catgccttta atcccagcac ttgggaggca 4740 gaggcaggca gatttctgag ttcaaggcca gcctggacta cagagtgagt tccaggacag 4800 ccagggctac tcagagaaac tctgtcttgg aaaaaaaaaa agaggaagaa agaaaaaaga 4860 tttatttatt tattttacac atatgagtac accatcagac acacaagaag agggcaccag 4920 accccattac agatggttgt gagccaccat gtggttgctg ggaattgaac tcaggacctc 4980 tggaagaaca gttggtgctc ttaacccctg agccatctct ccagcccaaa tagatgattt 5040 cttaattctt aaggatgatc ctataagaat tcctaaactt acattagtaa ttattaagct 5100 cttttacaat aggacttcta ttaagtcttc tctaatatga aaacttcaat aagaactctg 5160 ccagtcttca agtgtcatga gttagttgct tctgagatag caagtaggca tcaacaactt 5220 agagcacatt ctaggaggtt gtaaaaccat taaccagtgg tcttaaaaag ggaactaaca 5280 ataggctata ggtgcaagga cagaagataa aatattgact aggtttatca atacaaaatt 5340 tacccacaaa agttatgttt ttgacttttc ataaaaactc tttatgaacc tgtagaactg 5400 gtgaaagatg acgaatgctt agccagataa- ttactcctaa tagatatgca tgtgaatatt 5460 ctgtgctgta aacttattta tgtttgaact tccagtgaac ttttgtttaa aaaagggggg 5520 gggttgaaaa agccatgtga tctattctcc tagaaagggt acagaagact aagaaagatt 5580 acattggaga tgtaaccttg gagagaaagc tttgggagca agagcataga gagcaaggcc 5640 attgtggcat cagagcagga ggagagagca agattagaag gagaatgcag agtggaataa 5700 cttagaaact ataaggcaac ataaaaaatt aagagagcca tatgcagaat gcagagggaa 5760 agagaaaaaa aaaaaaaaaa agaagctgca gggagagcag aaggagcagg caggcttctc 5820 ctgaccatgg ggtagaacag ggcttttctt aataccaagg caggcttagt cttaaggata 5880 ataaagcttt tctttcttac agacttggtt ttaattcatt tagcaataaa agtgtaaaag 5940 tgttttcttt ccctatgcaa taaagattgg agcttatttt tcagccagaa tgagtgagtt 6000 ctctctgcaa cggtgcttgg tcttttgctt catatacaca cataagtgtg tgtgtgcgcg 6060 catgcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtaag tgtgcaatta tcagatggca 6120 tggaagctgg gctcaattgg ttcaaatggg gacttgtgag ggtatatgca tgaatctgta 6180 tatgaattca tgtgagctta tatatatttg cttgtgtaaa agtttttcct tctgtgagtg 6240 tgactctctt ctcctggttc aatagaggtt tattgcttca aacttccccc tagcctgaca 6300 gtcgagaggc atctggacaa σagagaaaag gctctagcca ttaatccttt tcttagatcc 6360 attttcttag agaactttct taggaaactg tttagagaga acatagaaac aggctgaaat 6420 cacttgtcaa actgtcccct tttcttccta aggacttcta ctagcagact gggagttaga 6480 gctgcacagt ccctgaggag atagaacaaa ggctgcttta ctgaatcccc tgctgtttta 6540 agatgaggtt ctaaaggaga ttgcagtttc tgacccccaa aaggaactca ggcaggtcag 66O0 ctacagtatc aaagtgactt aaacttaaga tagggaaatg ttttattatt aaacagcaac 6660 cctaaatatc tcataagatc aagtcttacc ccggctgaca cttccccctc tgttgcctca 6720 agaggaacca agcagaaaga accgccaggg ctggctcctg gcacaaatgg gttaaagatg 6780 ttgtagcatg gggaaatgaa gagatggctc agctattaag agaatatctt actcttccag 6840 aggaccagtg ttcaattccc agcaaacata tcaggcgcca caccatcact tgtagctcca 6900 gctgcagatc tgctacatct ggcctccata ggcacccaca cacaggtggc acccacaatt 6960 aaaaaaataa gataaatcta aaacagcaaa gttaaagcat gagctgaaac tagtaaagtg 7020 cttgtgtggc atagaccaag acctgggttt ggtccctacc tgttagaaat agtctcagta 7080 tcacacaaag gaacacccaa gcgaagcaaa agctcccagc aagacaaaac tacagtcttc 7140 attgagagtg tgcacgctga agaccgagca cactgggtgc aaaatgtact tggattctgt 7200 ttgcttgttt tgttccagac agggcttctc tgcataacag ccttggctgt ccctgaaatt 7260 cactgtgtag accaggctgg cctcaaaccc tcagagatcc gttcacccac gcttatctag 7320 gcttcagtct caccctgtga gatggcctga aagttgttag aaccgcgcgg gatctatttc 7380 tgacagactg gctggcatct tttccttctc tcagcatgag attcctgggg cgttcccatt 7440 tcagcatcaa gcatggtagc agagttggaa cctgagggct gagggctcag actcagacca 7500 taaactggaa gcagagagaa ctggagattg tgggaggctt tgaaacctca gctcctgccc 7560 cagcaaatac cttccagcaa ggccacacct cttaaacctc cccaaacagg gtcaccaact 7620 ggggacctaa tattcaaatg cccacaaata tgggagacat gacatccaaa ccgccaggac 7680 aggtgtatac ctccatgctt ggtttccgta gtaagaaaca ctaaacatta gcctttccta 7740 ataaacactg atataaagcc ctgctattct cgatgttttt cctctgttct gctcctcctt 7800 ctccacctgc ttctctgttc tctgacctct tctgtgtcac agatagccct gccatgtcca 7360 tctgccagcc atgttctgtc tacttgcccc tctctctgct ctggactctt ctanatgcct 7920 ctggctgttc tttctcatat ctacaataaa aacctggccc ttaattcata ccacagagat 7980 atcgagtctt ctttaaccaa attttcttgt agttctctna aaaggaagat ttaaatnaac 8040 agggctgagc acacaggagg atcatggcat attgacttgc ttgtgaggtt caagcaagat 8100 gttcaggctc tttgaataaa agagatactt ttcataaaaa ttntatcata cacacaacag 8160 ntaaagactt gcatgggaaa atactggtga gtaggccatg tccaggttac taatagcctc 8220 tgcacaggga gagcccaact gtggagaata ggccaaggac aggccgataa tcgccttatt 8280 tagatctgtt ctttaaaatg tgtgtgtgtg tgtgtgtgtg tgtctgtgtg tgtgtgtctg 8340 tgtgtgtgtg gtttatgtat atgagtacac tgtagctgtc ttcagacaca cagaagaggg 8400 catcacaccc cattacagat ggttgtgagc caccacatgg ttgctgagaa ttgaactcag 8460 gacctctgga agaacagtta gctcttaacc attgtaccat ctcttcagcc cttccttata 8520 tttgtatttt aaatagctta aaaaagaaaa agaaaccatg aatgtgaaaa ttagcataaa 8580 ctttagtgtt cagaaataga ttttttttgg aacacagcca σgttctttct tgtgttgact 8640 gtggagggca ttctgggaaa cagggtgtgg cctgggaagc tgtgaaggtg tacttctagt 8700 tccttctggc cctcctgcag acagtgcaag ccccacagta cactgctgca tgtctgggga 8760 ggttgttcca gctgctgaac aagttcttgt ggaacactgt caccctgccc tggggtcata 8820 atcatgacac tgctgctcct caatgtttga aggaaaatca cccttagtct caatgctgta 8880 taaataaacc ctctataaaa tcaaaaggcc gcattccata ggatgaaaca cgcggggaac 8940 attttcttcc atgcccaggc cttctcttcc tggcagggct ccaacatgtc gtggtctgcc 9000 tgccctgacg ccaggctacg gctggctgac tccttatcag gcacgtagcc ctccttggtc 9060 ctggttctac cagtccccaa gcctagtccc aggtcaatgc acagattcag cccctgtaat 9120 agtaatcttg ggggaggggg cgttctatgt ccctttttgc tcccaaataa ctgcagagtg 9180 tttctatacc cactcgagcc gtaaagatac agagacctgg catttttatt aacaagctat 9240 tagagtgtgt actcπaatat ccgggcagat tcttatctat cctaacccaa ttagactacc 9300 tactacccaa ccaaatgccc tggtacttgc cctctggtct tgccctgctt cactctggtc 9360 tatgtatgtt ctcatggctg ttttctcatg gcgaatcttc ctggtctctc cacgtggttt 9420 ctccacaccg tctcctcctc ctggtcctta tctactctct ggtctccttt gggaccccat 9480 gactgggatt ggaagtccct ccctatctct tctgctcagc taattggctg accagctctt 9540 ttattaacca atcagaggtg atggaaaaca atgtttacac aacattgaga tcgggagatg 9600 gctatcttcc agactgcaac cagatgtctg cggtaaagaa gtcagcatct gaacaacagt 9660 gcacaaaacc atcccccaac aagcccactc tggcctgccc tgccctgccc tgccctgccc 9720 cctccacacc tgcccagctc ttccagagga agagagtttg gtttccagca accagccttc 9780 cgagagctgg tgccctcttc tggcctcggg taacatgcat acagacacat aaaatctgct 9840 ttacaatgct gtcttaatct gaccagaaga aaatgtaaga ctaactcagt agaaatagtc 9900 caggaaataa ctagtgagaa ttcttatgaa aaatggggat aaggtgaagg agagtttaaa 9960 catccagggg aagttgggac gggcttccta aaagggttgc tccccttctc gagggaatgg 10020 tggcagggtg gaccttgaga aggtaaggga aatgggactc aggagtcaaa aatgcctctc 10O80 ttcgagccct caggaggact cccaactctt ggttaccctt cctatcctgg agttagatgt 10140 caactcgtgc tctgtaaaat ggttcccgac ggcctcttgt ttggtctgtt tttcctgggc 10200 agtcaattct ttccctttcc cacagaatga ccctggttaa caccaaggtt cccggtgcag 10260 agggcctggg gagatgacat cattggtgaa gttccacaca agcatgagaa cccgagttca 10320 acccccagca aaaccattaa aagctgagtg tagcaacccc acgtgtgtgt gtgtgtgtgt 10380 gtgtgtgtgt gtgtgtgtgt gtgtgtgtat gtgtgcatgt gcctgtgtgg tgagtgcatg 10440 ccttgtcaga ggcttcctga ggagagacaa cacacatgcc tcttcatctt agacaggcaa 10500 ctgacactgg gccagagaaa taaatccatc aaagttcaac ttggcaaata cttatttggg 10560 gggaatttgt tattattatt tttactcatt atttgagttt attggaatta cttacagggg 10620 tataattgtc aaaaatcacc cacttagaca tgggtggtga gcctagagca gtgcagggct 10680 ggcgggtagc ctgcctgctg ggatgtcttc tttcagccgt tgcctgtaac cctgaggaga 1O740 ggccttgtga atctcacatg tttcagctct tctggacttg agaggtttaa ttcttgctta 1O8O0 atttttgagt ctcaagaagt tcccctaccc ctgtccccag gagaaactaa ctgttcccac 1O860 taggagccaa tggcttgtgc acagcaggca gtccagtact ctggatctat ccagtaactt 1O920 caggccctgg cttagtgact ttggatccac ctgaaggtgc tgtccggggg tggagacaga 10980 gtgaagagag caggatgtta tttcctctct aaagctggga gtgcacctgt cagcaggtgc 11040 ctgctgcctt ccaggcttag ctggtcctag gaagagacat ctgtccaact ggggctctct 11100 ctgccaatcg tcatggccca ctgcatggag tctgcactgc tgctgttgtt gctgctgggt 11160 gagagcctgt ctcctccttg gccctggccc tggcccctgc cctctctagc cctaaccatc 11220 agggacaagg tgggattgta aaagtgacca aagaaaaggc tgggttgctg aagggatgct 11280 caaaattggc ttgtgctttg tggccgagga tctgtcgcaa tatgccaccc ccgctgtgca 11340 atgccttgta ccccatcttc ccattcagct gagccaccct tcccaaatct agccttgata 11400 cctgtgcccc ggggattttc tttttccttt cctccccctg accaaatgtg gtcctcagag 11460 ctcctgctct tgggagtccc ctactgattc atggtggctc ttagagcaga gacacgccat 11520 gttgagaaga gcacatttga tggatgagag gccaggtcag gcgattttcc tgggtctggg 11580 aagccagaga ggtaagaagg tgggaaaagg aagtgtcata aacctgaatt tggtgacttg 11640 gctggattta cgtatgtcca ctggcaagtt cggacacagc tgctactcca acagaggcag 11700 gcattctctt tccccggaat gggttgtcat ctgtgacata agttatataa gtcttcactg 11760 aaactactga taaacggtca ggtctgtctc aggacggtct gccatcaggg gtctggtctg 11820 tctgaatcca cttctggaag ccagggtctg aatgtgctgt tcaatataca atacctgtcg 11880 agaatccaat gctcacaatc cccttctgtg gtccggagcc agagtccctc tcattgagct 11940 gagttagacc aataaagaag catgtggaca ggggtcagga ggcacaggtc aggatgggag 12000 gagggccaga aacctgtaga tatactcacg gggtttccat gtgtcagaga agggaccatc 12O60 cgcgtgtcaa gaggagaatg gggagtgcta tgattaatgt gtccattaag cctcagtgtc 12120 ctgtcaggca gagctgggga ggatagtagg ggcccagtag cccctctgtg gatgcatggg 12180 ccatttctgg agatggtggt ggaggctgaa cctcagcttc tggggaaagg tcaggcccaa 12240 tctctgaata ctgcatccat tcacatcctg ggtagataga tatccctagt gcctgctcat 12300 gccccttctt caagatcaac gggtcacgcc tatgggccac actctctccc cttgggtttg 12360 ggtacccctt ccccaaaaat tcaagggtgt ggaatgtgga gaaccaagca cggaggactg 12420 cagcctgccc gcccttcacc agggtctgcg agctttaccg acgggaatcg ctgcgtggac 12480 gcggccgagg cgtgtacagc agacgagcgg tgccagcagc tgcgctctga gtacgtggca 12540 cgatgcctgg gccgggcagc gcccgggggc aggccgggac ccgggggctg cgtgcgctcc 12600 cgctgccgcc gagccctgcg ccgcttcttc gcgcgtgggc ctccggcgct cacgcatgcg 12660 ctgctcttct gcggctgcga aggctccgcg tgcgccgagc gccggcgcca gactttcgcg 12720 cccgcctgcg cgttctccgg cccggggttg gtgccgccct cttgcctgga gcccctggag 12780 cgctgcgagc gcagccgcct gtgccggtgc gtgcgtgcgg ggcgggctgg gccgctcacc 12840 cgcgtccggg cgcgcgcagg ccccgtctcc ttgccttcca ggcctcatgc gctcccgcgc 12900 ccggctcccg cgaccgctgc ccggaggagg ggggcccgcg ttgtctgcgc gtctacgcag 12960 gcctcatagg taggctgggc ggcctcgggc gggcgggacg gcggaggcag actcggggcg 13020 ccggtcacac gccctggggg tcccccgcag gcaccgtggt cacccccaac tacctggaca 13080 acgtgagcgc gcgcgttgcg ccctggtgcg gctgtgcggc cagtggaaac cggcgcgaag 13140 aatgcgaagc cttccgcaag ctctttacaa ggaacccctg cttgggtgag ggggcctgga 13200 ggtcccgggg aaccacggat gtctgtggcc caatccaagc tgcctggccc gtgggtctta 13260 tttacgtcgc atcatgtttg gtgtgggcga tggacagtgt gcacatgcca tggtacatgg 13320 gtggaagtca gcgttaaaac gtgtccaatg gcctgaagtt ggcctccctt ttgacactaa 13380 tggggtggcc ctttcttcca tgtgtgccca acttacctct gctgttcttg cctctgggtg 13440 gaatggctca gttccagatt tctgggggtc tgtttgaagc ctgtctctgc cacttagtag 13500 ctgagagtta aactcttatt aatcccgatt gtgttcacct gtaaagggag agggggcagc 13560 ccttgtcaac ctcactagca gtttgtgggt cattcttagc acagtgacct tccatctcag 13620 gccatgcctt gcaggttcca gggtgtctta ttttttgtcc caagggagtg gggtagtttc 13680 tagggtttcc tggccaagcc ttctctggat ctctgcactc cgcagatggt gccatacaag 13740 cctttgacag cttgcagcca tcagttctgc aggaccagac tgctgggtgc tgtttcccgc 13800 gggtaggtat ggggagagga cagggagttg acagtttccg aatccttccc atctgtgctc 13860 tcttaccctt cctaggcaag gcacgagtgg cctgagaaga gctggaggca gaaacagtcc 13920 ttgttttgtc ctaacgccca aggtgtcctg gctgtatgca ctcactgccc tggctctcca 13980 ggccctgctc tgattaggaa catgaaccgt ggacgacaca gctgactgcc atgtctcccg 14040 atgactgctc actgagctga aactcccttg ccctcaggtc tgctgccctt tgcaggcctg 14100 gacccctgtg tggctgtcct ctggattggg ggctggaggc tagggtctga ctgaaaagcc 14160 tgtgttcccg tcagtaggca tcttgtccat tttcttcccc atcccagagc tgagcaccca 14220 tagatgaggc ctcattgggt cccctgggct tacagagcag gacagagact agcccccgct 14280 cctagaattt ggaactgtcc ttttccaaga tgacaaggca ctaaggagat catatgaaca 14340 ggctgacaga caaggctgcc taaataccct cccagttagc cattactcac cattaagctt 14400 acccgtgtca cagcactgac gtggcttgtc acctatgaca cagtgtgtag acattaagga 14460 gagactgagg tccctcctgc tcagcacccc actggcttcc caggatctcc ctgccatggt 14520 ttccccagca cccgcagtgg ctcaataaac ccatgtgcac tgagaccaac ctggttttct 14580 gtcatttgac ctcgaaatgt ggagacctgt gtggccagaa ctactcttga gtctgagtac 14640 ctgagaatga atatagccat ctctggagca gcagccagca acggaaactg aatgcatgtg 14700 ttgcgattcc tttgcaaact caaagatggt gtttgctggc attatcagtg agctctaggc 14760 ttttgtcagg tcaaactggg ctcaggcaca tctgtctcaa gggttcaaag gaccttgtga 14820 tcacgagtcc tcacacagct gctcagtagc tagaaaaggc cccactgaag ggtgggtagg 14880 atgagcttca tactcagcat ctccacagag accgctaaga gaaagacacg cagccgggca 14940 gtggtgatgc acgcctttaa tcccagcact cgggaggcag aggcaggcgg agttctgagt 15000 tcgaggccag cctggtctac aaagcgagtt ccaggacagc cagggctata cagagaaact 15060 ctgtctcaaa aagaccaaaa gaaaaagaga gagagaggaa gacacgcttc cttttcagca 15120 tttggggatg atacattagt ggcgcagtgt gggtcacggg ccttttgctg aggagagtca 15180 cagccagcag gtcacctggg ataggagggt ttctcacagg aggtactggg cagagtaaga 15240 gtgaagctgg gtccaaactt ccaatttcac aaagagaatt ctcttcccca cctgcatgga 15300 cgaatgggtg gggagaggca gggggtgctg gggccagaga gtaatgaaaa catcactctg 15360 ccctgcttac tccagccaca aagtggctcc aacacaggat gaagtgtctg tctgatgatt 15420 tcctπatgtg agtgtgctca actccactgc tcgagtgact ttgatattta gcataaaaag 15480 tagacattaa aatttacacg tgtgcctgag tatacatata tgtgccatat gtatgtagtg 15540 cctatggaga ccagaagagg gtattggaac aagagttcca ggacactgtg agccacttga 15600 tgtgggtcct ggggaccaaa acccaagccc tctgcaatgc ttttaaccac agagactttt 15660 ttaaaagtag gaatgattct aaataaaaat acaattctgg atagaaagtg tgggtataag 15720 ccttctaaag ctagtaggaa gatgacgtgg acatagtcaa gttccacagt tggtggtggt 15780 cactgtccta aggacaggcg ccggcaaatg tattacacac tgggacggaa gcgtcacttc 15840 atacggtaga gatgtgatta tgttatagat cgagtcgacg ccctatagtg agtcgtatta 15900 gagctcgcgg ccgc 15914

<210> 2 (SEQ ID NO. 2)

<211> 834 <212> DNA <213> Mouse

<220>

<221> CDS

<222> (1) .. (834)

<220>

<221> rnisc_feature

<222> (1) .. (46)

<223> Corresponds to exon 1 (11113 to 11158) of seq ID 1

<220>

<221> misc_feature

<222> (47) .. (410)

<223> Corresponds to exon 2 (12443 to 12806)

<220>

<221> misc_feature

<222> (411) .. (520) <223> Corresponds to exon 3 (13051 to 13185)

<220>

<221> misc_feature

<222> (656) .. (716)

<223> Corresponds to exon 4 (13726 to 13786)

<220>

<221> misc_feature

<222> (717) .. (834)

<223> Corresponds to exon 5 (13876 to 13993)

<220>

<221> sig_peptide

<222> (1) .. (63)

<400> 2 atg gcc cac tgc atg gag tct gca ctg ctg ctg ttg ttg ctg ctg ggg 48

Met Ala His Cys Met Glu Ser Ala Leu Leu Leu Leu Leu Leu Leu Gly

1 5 10 15 tct gcg age ttt ace gac ggg aat cgc tgc gtg gac gcg gcc gag gcg 96 Ser Ala Ser Phe Thr Asp Gly Asn Arg Cys Val Asp Ala Ala Glu Ala 20 25 30 tgt aca gca gac gag egg tgc cag cag ctg cgc tct gag tac gtg gca 144 Cys Thr Ala Asp Glu Arg Cys Gin Gin Leu Arg Ser Glu Tyr Val Ala 35 40 45 cga tgc ctg ggc egg gca gcg ccc ggg ggc agg ccg gga ccc ggg ggc 192 Arg Cys Leu Gly Arg Ala Ala Pro Gly Gly Arg Pro Gly Pro Gly Gly 50 55 60 tgc gtg cgc tec cgc tgc cgc cga gcc ctg cgc cgc ttc ttc gcg cgt 240 Cys Val Arg Ser Arg Cys Arg Arg Ala Leu Arg Arg Phe Phe Ala Arg 65 70 75 80 ggg cct ccg gcg etc acg cat gcg ctg etc ttc tgc ggc tgc gaa ggc 288 Gly Pro Pro Ala Leu Thr His Ala Leu Leu Phe Cys Gly Cys Glu Gly 85 90 95 tec gcg tgc gcc gag cgc egg cgc cag act ttc gcg ccc gcc tgc gcg 336 Ser Ala Cys Ala Glu Arg Arg Arg Gin Thr Phe Ala Pro Ala Cys Ala 100 105 110 ttc tec ggc ccg ggg ttg gtg ccg ccc tct tgc ctg gag ccc ctg gag 384 Phe Ser Gly Pro Gly Leu Val Pro Pro Ser Cys Leu Glu Pro Leu Glu 115 120 125 cgc tgc gag cgc age cgc ctg tgc egg ccc cgt etc ctt gcc ttc cag 432 Arg Cys Glu Arg Ser Arg Leu Cys Arg Pro Arg Leu Leu Ala Phe Gin 130 135 140 gcc tea tgc get ccc gcg ccc ggc tec cgc gac cgc tgc ccg gag gag 480 Ala Ser Cys Ala Pro Ala Pro Gly Ser Arg Asp Arg Cys Pro Glu Glu 145 150 155 160 gQT ? ccg cgt tgt ctg cgc gtc tac gca ggc etc ata ggc ace gtg 528 Gly Gly Pro Arg Cys Leu Arg Val Tyr Ala Gly Leu lie Gly Thr Val 165 170 175 gtc ace ccc aac tac ctg gac aac gtg age gcg cgc gtt gcg ccc tgg 576 Val Thr Pro Asn Tyr Leu Asp Asn Val Ser Ala Arg Val Ala Pro Trp 180 185 190 tgc ggc tgt gcg gcc agt gga aac egg cgc gaa gaa tgc gaa gcc ttc 624 Cys Gly Cys Ala Ala Ser Gly Asn Arg Arg Glu Glu Cys Glu Ala Phe 195 200 205 cgc aag etc ttt aca agg aac ccc tgc ttg gat ggt gcc ata caa gcc 672 Arg Lys Leu Phe Thr Arg Asn Pro Cys Leu Asp Gly Ala lie Gin Ala 210 215 220 ttt gac age ttg cag cca tea gtt ctg cag gac cag act get ggg caa 720 Phe Asp Ser Leu Gin Pro Ser Val Leu Gin Asp Gin Thr Ala Gly Gin 225 230 235 240 ggc acg agt ggc ctg aga aga get gga ggc aga aac agt cct tgt ttt 768 Gly Thr Ser Gly Leu Arg Arg Ala Gly Gly Arg Asn Ser Pro Cys Phe 245 250 255 gtc eta acg ccc aag gtg tec tgg ctg tat gca etc act gcc ctg get 816 Val Leu Thr Pro Lys Val Ser Trp Leu Tyr Ala Leu Thr Ala Leu Ala 260 265 270 etc cag gcc ctg etc tga 834

Leu Gin Ala Leu Leu 275

<210> 3 (SEQ ID NO. 3) <211> 277 <212> PRT <213> Mouse

<400> 3 Met Ala His Cys Met Glu Ser Ala Leu Leu Leu Leu Leu Leu Leu Gly 1 5 10 15

Ser Ala Ser Phe Thr Asp Gly Asn Arg Cys Val Asp Ala Ala Glu Ala 20 25 30

Cys Thr Ala Asp Glu Arg Cys Gin Gin Leu Arg Ser Glu Tyr Val Ala 35 40 45

Arg Cys Leu Gly Arg Ala Ala Pro Gly Gly Arg Pro Gly Pro Gly Gly 50 55 60

Cys Val Arg Ser Arg Cys Arg Arg Ala Leu Arg Arg Phe Phe Ala Arg 65 70 75 80

Gly Pro Pro Ala Leu Thr His Ala Leu Leu Phe Cys Gly Cys Glu Gly 85 90 95

Ser Ala Cys Ala Glu Arg Arg Arg Gin Thr Phe Ala Pro Ala Cys Ala 100 105 110

Phe Ser Gly Pro Gly Leu Val Pro Pro Ser Cys Leu Glu Pro Leu Glu 115 120 125

Arg Cys Glu Arg Ser Arg Leu Cys Arg Pro Arg Leu Leu Ala Phe Gin 130 135 140

Ala Ser Cys Ala Pro Ala Pro Gly Ser Arg Asp Arg Cys Pro Glu Glu 145 150 155 160

Gly Gly Pro Arg Cys Leu Arg Val Tyr Ala Gly Leu lie Gly Thr Val 165 170 175

Val Thr Pro Asn Tyr Leu Asp Asn Val Ser Ala Arg Val Ala Pro Trp 180 185 190

Cys Gly Cys Ala Ala Ser Gly Asn Arg Arg Glu Glu Cys Glu Ala Phe 195 200 205

Arg Lys Leu Phe Thr Arg Asn Pro Cys Leu Asp Gly Ala lie Gin Ala 210 215 220

Phe Asp Ser Leu Gin Pro Ser Val Leu Gin Asp Gin Thr Ala Gly Gin 225 230 235 240

Gly Thr Ser Gly Leu Arg Arg Ala Gly Gly Arg Asn Ser Pro Cys Phe 245 250 255 Val Leu Thr Pro Lys Val Ser Trp Leu Tyr Ala Leu Thr Ala Leu Ala 260 265 270

Leu Gin Ala Leu Leu 275

<210> 4 (SEQ ID NO. 4) <211> 795 <212> DNA <213> Mouse

<220>

<221> CDS

<222> (1) .. (795)

<220>

<221> sig_peptide

<222> (1) .. (63)

<220>

<221> misc_feature

<222> (1) .. (46)

<223> Corresponds to exon 1 (11113 to 11158) of seq ID 1

<220>

<221> misc_feature

<222> (47) .. (414)

<223> Corresponds to exon 2 (12443 to 12810)

<220>

<221> misc_feature

<222> (415) .. (502)

<223> Corresponds to exon 3 (12882 to 12969)

<220>

<221> misc_feature

<222> (503) .. (637)

<223> Corresponds to exon 4 (13051 to 13185)

<220>

<221> misc_feature

<222> (638) .. (718)

<223> Corresponds to exon 5 (13726 to 13806)

<220>

<221> misc_feature

<222> (719) .. (795) WO 01/16169 PCTYUSOO/24111

-58-

<223> Corresponds to exon 6 (13876 to 13993)

<400> 4 atg gcc cac tgc atg gag tct gca ctg ctg ctg ttg ttg ctg ctg ggg 48 Met Ala His Cys Met Glu Ser Ala Leu Leu Leu Leu Leu Leu Leu Gly 1 5 10 15 tct gcg age ttt ace gac ggg aat cgc tgc gtg gac gcg gcc gag gcg 96 Ser Ala Ser Phe Thr Asp Gly Asn Arg Cys Val Asp Ala Ala Glu Ala 20 25 30 tgt aca gca gac gag egg tgc cag cag ctg cgc tct gag tac gtg gca 144 Cys Thr Ala Asp Glu Arg Cys Gin Gin Leu Arg Ser Glu Tyr Val Ala 35 40 45 cga tgc ctg ggc egg gca gcg ccc ggg ggc agg ccg gga ccc ggg ggc 192 Arg Cys Leu Gly Arg Ala Ala Pro Gly Gly Arg Pro Gly Pro Gly Gly 50 55 60 tgc gtg cgc tec cgc tgc cgc cga gcc ctg cgc cgc ttc ttc gcg cgt 240 Cys Val Arg Ser Arg Cys Arg Arg Ala Leu Arg Arg Phe Phe Ala Arg 65 70 75 80 ggg cct ccg gcg etc acg cat gcg ctg etc ttc tgc ggc tgc gaa ggc 288 Gly Pro Pro Ala Leu Thr His Ala Leu Leu Phe Cys Gly Cys Glu Gly 85 90 95 tec gcg tgc gcc gag cgc egg cgc cag act ttc gcg ccc gcc tgc gcg 336 Ser Ala Cys Ala Glu Arg Arg Arg Gin Thr Phe Ala Pro Ala Cys Ala 100 105 110 ttc tec ggc ccg ggg ttg gtg ccg ccc tct tgc ctg gag ccc ctg gag 384 Phe Ser Gly Pro Gly Leu Val Pro Pro Ser Cys Leu Glu Pro Leu Glu 115 120 125 cgc tgc gag cgc age cgc ctg tgc egg tgc gcc tea tgc get ccc gcg 432 Arg Cys Glu Arg Ser Arg Leu Cys Arg Cys Ala Ser Cys Ala Pro Ala 130 135 140 ccc ggc tec cgc gac cgc tgc ccg gag gag ggg ggc ccg cgt tgt ctg 480 Pro Gly Ser Arg Asp Arg Cys Pro Glu Glu Gly Gly Pro Arg Cys Leu 145 150 155 160 cgc gtc tac gca ggc etc ata ggc ace gtg gtc ace ccc aac tac ctg 528 Arg Val Tyr Ala Gly Leu lie Gly Thr Val Val Thr Pro Asn Tyr Leu 165 170 175 gac aac gtg age gcg cgc gtt gcg ccc tgg tgc ggc tgt gcg gcc agt 576 Asp Asn Val Ser Ala Arg Val Ala Pro Trp Cys Gly Cys Ala Ala Ser 180 185 190

gga aac egg cgc gaa gaa tgc gaa gcc ttc cgc aag etc ttt aca agg 624 Gly Asn Arg Arg Glu Glu Cys Glu Ala Phe Arg Lys Leu Phe Thr Arg 195 200 205

aac ccc tgc ttg gat ggt gcc ata caa gcc ttt gac age ttg cag cca 672 Asn Pro Cys Leu Asp Gly Ala lie Gin Ala Phe A.sp Ser Leu Gin Pro 210 215 220 tea gtt ctg cag gac cag act get ggg tgc tgt ttc ccg egg gta ggt 720 Ser Val Leu Gin Asp Gin Thr Ala Gly Cys Cys Phe Pro Arg Val Gly 225 230 235 240 ctg ctg ccc ttt gca ggc ctg gac ccc tgt gtg get gtc etc tgg att 768 Leu Leu Pro Phe Ala Gly Leu Asp Pro Cys Val Ala Val Leu Trp lie 245 250 255

ggSf SfS?c tgg agg eta ggg tct gac tga 795

Gly Gly Trp Arg Leu Gly Ser Asp

260 265

<210> 5 (SEQ ID NO. 5)

<211> 264 <212> PRT <213> Mouse

<400> 5

Met Ala His Cys Met Glu Ser Ala Leu Leu Leu Leu Leu Leu Leu Gly 1 5 10 15

Ser Ala Ser Phe Thr Asp Gly Asn Arg Cys Val Asp Ala Ala Glu Ala 20 25 30

Cys Thr Ala Asp Glu Arg Cys Gin Gin Leu Arg Ser Glu Tyr Val Ala 35 40 45

Arg Cys Leu Gly Arg Ala Ala Pro Gly Gly Arg Pro Gly Pro Gly Gly 50 55 60

Cys Val Arg Ser Arg Cys Arg Arg Ala Leu Arg Arg Phe Phe Ala Arg 65 70 75 80

Gly Pro Pro Ala Leu Thr His Ala Leu Leu Phe Cys Gly Cys Glu Gly Ser Ala Cys Ala Glu Arg Arg Arg Gin Thr Phe Ala Pro Ala Cys Ala 100 105 110

Phe Ser Gly Pro Gly Leu Val Pro Pro Ser Cys Leu Glu Pro Leu Glu 115 120 125

Arg Cys Glu Arg Ser Arg Leu Cys Arg Cys Ala Ser Cys Ala Pro Ala 130 135 140

Pro Gly Ser Arg Asp Arg Cys Pro Glu Glu Gly Gly Pro Arg Cys Leu 145 150 155 160

Arg Val Tyr Ala Gly Leu lie Gly Thr Val Val Thr Pro Asn Tyr Leu 165 170 175

Asp Asn Val Ser Ala Arg Val Ala Pro Trp Cys Gly Cys Ala Ala Ser 180 185 190

Gly Asn Arg Arg Glu Glu Cys Glu Ala Phe Arg Lys Leu Phe Thr Arg 195 200 205

Asn Pro Cys Leu Asp Gly Ala lie Gin Ala Phe Asp Ser Leu Gin Pro 210 215 220

Ser Val Leu Gin Asp Gin Thr Ala Gly Cys Cys Phe Pro Arg Val Gly 225 230 235 240

Leu Leu Pro Phe Ala Gly Leu Asp Pro Cys Val Ala Val Leu Trp lie 245 250 255

Gly Gly Trp Arg Leu Gly Ser A.sp

Claims

CLATMSWhat is claimed is:

1. An isolated nucleic acid that is selected from the group consisting of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 4, and SEQ ID No: 6, and complements and mRNA counterparts thereof.

2. An isolated nucleic acid which hybridizes to SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 4, and SEQ JD No: 6 under moderate stringency conditions.

3. An isolated nucleic acid encoding a Ret ligand polypeptide from the group consisting of murine RetL5 L SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, and amino acid substitution variants thereof.

4. An isolated nucleic acid of Claim 3, wherein said amino acid substitution variant, when aligned with the Ret ligand, shares at least 40% sequence similarity therewith, further wherein said substitution variant shares at least 80% of aligned cysteine residues with said Ret ligand.

5. An isolated nucleic acid of Claim 4, wherein said amino acid substitution variant, when aligned with the Ret ligand, shares at least 80% sequence similarity therewith.

6. An isolated nucleic acid encoding a soluble variant of a Ret ligand from the group consisting of murine RetL5 (SEQ JD No:3), murine RetL5 (SEQ ID No: 5), and human RetL5 (SEQ ID No: 7).

7. An isolated nucleic acid of Claim 6, wherein said soluble variant lacks a phosphatidylinositol glycan linkage motif.

8. An isolated nucleic acid having a nucleotide sequence SEQ ID No: 1.

9. A vector having the nucleic acid of Claim 1, 2, 3, 4, 5, 6, 7, or 8, present as an insert therein, said vector comprising an expression control sequence operably linked to said insert.

10. A vector of Claim 9, further comprising an expression control sequence operably linked to said insert.

11. A host cell comprising the vector of Claim 10.

12. A polypeptide production method, comprising the steps of: a) culturing host cells of Claim 11 in a cell culture medium; and b) recovering a Ret ligand polypeptide expressed from the vector insert within said host cells.

13. A Ret ligand polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, and amino acid substitution variants thereof.

14. A Ret ligand polypeptide of Claim 13, wherein the amino acid sequence is a substitution variant and wherein said Ret ligand polypeptide is soluble.

15. A Ret ligand polypeptide of Claim 14, which when bound to Ret, triggers dimerization or autophosphorylation thereof.

16. A soluble, truncated Ret ligand polypeptide comprising a substitution variant of an amino acid sequence selected from the group consisting of SEQ ID 3,

4, or 7 , said polypeptide being formed by the method comprising the steps of: a) removing a hydrophobic N-terminal sequence from said polypeptide; and b) removing a phosphatidylinositol glycan linkage motif from said polypeptide.

17. A fusion protein comprising a Ret ligand polypeptide of Claim 13, 14, 15, or 16, fused to an immunoglobulin polypeptide or a toxin polypeptide.

18. An antibody that binds specifically to a Ret ligand polypeptide of Claim 13, 14, 15, or 16.

19. An antibody that blocks binding of a Ret ligand polypeptide of Claim 13, 14, 15, or 16 to said Ret polypeptide.

20. A method of stimulating growth of, or limiting damage to, Ret expressing tissue in a subject, comprising the step of administering a therapeutically effective amount of a Ret ligand polypeptide of Claim 13, 14, 15, or 16, or an immunoglobulin fusion protein that includes said Ret ligand polypeptide, or an antibody that binds said Ret ligand polypeptide or fusion protein.

21. The method of Claim 20, wherein a Ret ligand polypeptide is administered, and said Ret ligand polypeptide is administered concurrently with a GDNF- related polypeptide.

22. The method of Claim 20, wherein said Ret expressing tissue is renal tissue or neural tissue.

23. A method of suppressing growth of a tumor cell that expresses Ret, comprising the step of contacting said cell with an effective amount of a Ret ligand polypeptide of Claim 16, a fusion protein that includes said Ret ligand polypeptide, or an antibody that binds said Ret ligand polypeptide.

24. A method of modulating Ret signal transduction involving a cell expressing either a Ret polypeptide or a Ret ligand polypeptide, comprising the step of contacting said cell with a Ret ligand polypeptide of Claims 13, 14, 15, or 16, or an immunoglobulin fusion protein that includes said Ret ligand polypeptide, or an antibody that binds said Ret ligand polypeptide or fusion protein.

25. A method of targeting a toxin, imageable compound, or radionuclide to a cell expressing either a Ret polypeptide or a Ret ligand polypeptide, comprising the step of contacting said cell with a Ret ligand polypeptide of Claims 13, 14, 15, or 16, or an immunoglobulin fusion protein that includes said Ret ligand polypeptide, or an antibody that binds said Ret ligand polypeptide or fusion protein.

26. A method of suppressing growth of a tumor cell expressing either a Ret polypeptide or a Ret ligand polypeptide, comprising the step of contacting said cell with a Ret ligand polypeptide of Claims 13, 14, 15, or 16, or an immunoglobulin fusion protein that includes said Ret ligand polypeptide, or an antibody that binds said Ret ligand polypeptide or fusion protein.

27. A method of treating a subject having a disorder of Ret metabolism, comprising the step of administering a vector of Claim 9 to said subject.

28. A method of stimulating growth of, or limiting damage to, Ret expressing tissue in a subject, comprising the step of administering a vector of Claim 9 to said subject.

29. The method of Claim 28, wherein the Ret expressing tissue is renal tissue or neural tissue.