WO1997044356A2 - RET LIGAND (RetL) FOR STIMULATING NEURAL AND RENAL GROWTH - Google Patents

RET LIGAND (RetL) FOR STIMULATING NEURAL AND RENAL GROWTH Download PDF

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Publication number
WO1997044356A2
WO1997044356A2 PCT/US1997/007726 US9707726W WO9744356A2 WO 1997044356 A2 WO1997044356 A2 WO 1997044356A2 US 9707726 W US9707726 W US 9707726W WO 9744356 A2 WO9744356 A2 WO 9744356A2
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Prior art keywords
leu
ser
human
ala
cys
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PCT/US1997/007726
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French (fr)
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WO1997044356A3 (en
Inventor
Michele Sanicola-Nadel
Catherine Hession
Richard L. Cate
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Biogen, Inc.
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Priority to IL126918A priority Critical patent/IL126918A/en
Priority to RO98-01550A priority patent/RO120343B1/en
Priority to BR9710665A priority patent/BR9710665A/en
Priority to SI9730750T priority patent/SI0914339T1/en
Priority to EE9800377A priority patent/EE9800377A/en
Priority to SK1524-98A priority patent/SK286115B6/en
Priority to NZ332706A priority patent/NZ332706A/en
Priority to DE69736428T priority patent/DE69736428T2/en
Priority to PL329946A priority patent/PL191248B1/en
Priority to EA199800990A priority patent/EA002544B1/en
Priority to CA002253871A priority patent/CA2253871C/en
Priority to JP54243197A priority patent/JP2002515743A/en
Priority to AU34729/97A priority patent/AU732392B2/en
Priority to EP97930981A priority patent/EP0914339B1/en
Application filed by Biogen, Inc. filed Critical Biogen, Inc.
Publication of WO1997044356A2 publication Critical patent/WO1997044356A2/en
Publication of WO1997044356A3 publication Critical patent/WO1997044356A3/en
Priority to IS4884A priority patent/IS2361B/en
Priority to US09/187,906 priority patent/US6677135B1/en
Priority to NO19985231A priority patent/NO323612B1/en
Priority to HK99105136A priority patent/HK1021821A1/en
Priority to US09/489,407 priority patent/US6861509B1/en
Priority to US10/668,936 priority patent/US20050080235A1/en
Priority to NO20065528A priority patent/NO324957B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to nucleotide sequences which encode a Ret ligand (RetL), as well as to methods of stimulating neural and renal growth by treating cells and mammalian subjects with RetL DNA or protein.
  • RetL Ret ligand
  • 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.
  • RPTK receptor protein tyrosine kinase
  • 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.
  • the invention provides a purified and isolated DNA molecule coding for a RetL. having the nucleotide sequence of any RetL, but specifically including rat retLl cDNA (SEQ ID NO:l), partial human retLl cDNA (SEQ ID NO:8), full-length human retLl cDNA (SEQ ID NO: 10), human retL2 cDNA (SEQ ID NO: 12), murine retL3 cDNA (SEQ ID NO: 16), partial human retL3 cDN A (SEQ ID NO: 18) or human retL3 cDNA (SEQ ID NO:20).
  • rat retLl cDNA SEQ ID NO:l
  • partial human retLl cDNA SEQ ID NO:8
  • full-length human retLl cDNA SEQ ID NO: 10
  • human retL2 cDNA SEQ ID NO: 12
  • murine retL3 cDNA SEQ ID
  • the invention further provides a RetL protein, with an amino acid sequence comprising that of rat RetLl (SEQ ID NO:2), partial human RetLl (SEQ ID NO:9), full-length human RetLl (SEQ ID NO: l 1 ), human RetL2 (SEQ ID NO: 13).
  • the invention includes a DNA sequence which encompasses the sequence (partial human retLl cDNA (SEQ ID NO:8)) of the insert DNA of clone HRL20, which is ATCC No. 97604, or the sequence of the insert DNA of clone #230-5A-86-17 (rat retLl cDNA (SEQ ID NO:l)), which is ATCC No. 98047.
  • 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 RetL;
  • the DNA may be a) a DNA molecule which comprises rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA, or the complementary strand of rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA; b) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in a) or fragments thereof; or c) DNA molecules which, but for the
  • Any of the recombinant DNA molecules of the invention may be operably linked to an expression control sequence.
  • the vector may encompass a DNA molecule encoding a RetL or a variant of a RetL.
  • the invention includes prokaryotic or eukaryotic host cells stably transformed or transfected by a vector comprising a DNA molecule encoding a native or variant RetL.
  • a purified and isolated human RetL 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 RetL.
  • 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 RetL.
  • 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.
  • the protein has an amino acid sequence which comprises rat RetLl, partial human RetLl, full-length human RetLl. human RetL2, murine RetL3, or human RetL3, or is a variant of one of these sequences.
  • the protein is a fusion protein including Ret or a RetL, fused to another molecule or molecular fragment, such as an immunoglobulin, toxin, imageable compound or radionuclide. Also included are chimeric molecules of RetL.
  • inventions include specific monoclonal antibodies to a RetL of the invention. Such an antibody may be associated with a toxin, imageable compound or radionuclide.
  • the invention also includes hybridoma cell lines which produce specific antibodies to Ret, including AA.FF9, AA.HE3, AF.E9, BA.B1, BB.B6, AA.GE7, CD.F11, AH.E3, CD.G4, AG.E7, BD.G6 and BH.G8, as well as subclones of these hybridomas, and the antibodies produced by these hybridomas or subclones of these hybridomas.
  • 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 may be RetLl, RetL2, or RetL3, a fragment of a full- length RetL, or an antibody which binds to Ret.
  • the compound may be administered concurrently with a therapeutically effective amount of a second compound, such as GDNF, neurturin or a GDNF-related molecule.
  • tissues of interest for these methods may include any tissue, preferred tissues include renal tissue, neural tissue, heart, stomach, small intestine, spinal cord, or lung.
  • the RetL is a soluble RetL.
  • the subject of the methods may be human.
  • Ret signal transduction between a first cell expressing a RetL and a second cell is inhibited by contacting the first cell with a soluble Ret protein or with an antibody to the RetL.
  • the soluble Ret 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 RetL fusion protein or an anti-Ret antibody conjugated to a toxin, imageable compound or radionuclide.
  • the RetL can be RetLl. RetL2 or RetL3.
  • 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 RetL and a toxin or radionuclide, or an anti-Ret antibody conjugated to a toxin or radionuclide.
  • the cell may 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 RetL, comprising contacting the cell with a fusion protein comprising Ret and a toxin, imageable compound or radionuclide, or an anti-RetL antibody conjugated to a toxin, imageable compound or radionuclide.
  • Another embodiment includes the method of suppressing growth of a tumor cell which expresses a RetL, comprising contacting the cell with a fusion protein of Ret and a toxin or radionuclide or with an anti-RetL antibody conjugated to a toxin or radionuclide; the cell may be within a subject, and the protein administered to the subject.
  • the RetL for any of the methods of the invention can be RetLl, RetL2 or RetL3, or a variant or fragment of RetLl, RetL2 or RetL3.
  • 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 RetL, 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 RetL to the subject.
  • FIGURE 1 is a cDNA sequence (SEQ ID NO: 1 ) and deduced amino acid sequence (SEQ ID NO:2) of rat RetLl .
  • the nucleotide sequence extends from base pair 201 through base pair 1700 of SEQ ID NO:l, and contains the entire open reading frame.
  • FIGURE 2A is a partial cDNA sequence (SEQ ID NO:8) and deduced amino acid sequence (SEQ ID NO:9) of human RetLl . This sequence is that of the insert of clone HRL20, deposited as ATCC No. 97604.
  • FIGURE 2B is a composite full-length DNA sequence (SEQ ID NO: 10) and deduced amino acid sequence (SEQ ID NO: 1 1 ) of human RetL 1.
  • FIGURE 3 A is a comparison of the nucleotide sequence of human RetLl (upper line of sequence) with that of rat RetLl sequence (lower line of sequence). Vertical lines between nucleotides show identity at a position, while a dot indicates a gap at that position.
  • FIGURE 3B is a comparison of the amino acid sequence of human RetLl (upper line of sequence) with that of rat RetLl sequence (lower line of sequence). Vertical lines between corresponding amino acids show identity at a residue, while a dot indicates a conservative substitution at that residue.
  • FIGURE 4A is a schematic diagram of a possible role for Ret and RetL in the interaction between a metanephric mesenchyme cell and a ureteric bud cell.
  • FIGURE 4B is a schematic diagram of a method of screening transfectants of a cDNA library for clones that express a RetL. The presence of expressed RetL on transfectants is detected by assessing the binding by those transfectants either of Ret/I gG fusion protein or of Ret/alkaline phosphatase fusion protein.
  • FIGURE 5 is a schematic diagram showing the construction of the plasmids used to express the rat Ret/I gG fusion protein.
  • FIGURE 6 is a schematic diagram showing the construction of the plasmids used to express the human Ret/IgG fusion protein.
  • FIGURE 7 is a cDNA sequence (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO: 13) of human retL2, as found in clone DS W240.
  • the protein reading frame is contained within nucleotides 25 to 1416.
  • FIGURE 8 is a comparison of the amino acid sequence of human RetL2 (upper line of sequence) with that of human RetLl sequence (lower line of sequence). Vertical lines between amino acids show identity at a position, while a dot indicates a gap at that position.
  • FIGURE 9 is a cDNA sequence (SEQ ID NO: 16) and deduced amino acid sequence (SEQ ID NO: 17) of murine RetL3.
  • FIGURE 10 is a cDNA sequence (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) of human RetL3. DETAILED DESCRIPTION OF THE INVENTION
  • RetL means any protein which specifically interacts with the receptor protein 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 may 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 antagonists, triggering Ret dimerization and autophosphorylation.
  • the ligand may also be modified in various ways, such as incorporated as a portion of a fusion protein, such as with a toxin or radionuclide.
  • 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.).
  • homologous or functionally equivalent sequences include functionally equivalent arrangements of the cysteine residues within the conserved cysteine skeleton, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the protein.
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • 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.
  • cDNA complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA moiecules 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 Laboratorv Manual, supra.
  • 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.
  • RNA may be isolated from a tumor cell, derived from an animal tumor, and preferably from a human tumor.
  • a library may be prepared from, for example, a human adrenal tumor, but any tumor may be used.
  • DNA polymorphism refers to the condition in which two or more different nucleotide sequences can exist at a particular site in DNA.
  • “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.
  • 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.
  • 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.
  • a “functional derivative” of a gene of any of the proteins of the present invention is meant to include “fragments”, “variants”, and “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity. ⁇ O
  • GDNF-related molecule means any moleule which is at least 40% homologous to either GDNF or neurturin, and is also capable of specifically binding a RetL.
  • gene means a polynucleotide sequence encoding a peptide.
  • 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.
  • 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.
  • probe refers to a ligand of known qualities capable of selectively binding to a target antiligand.
  • 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.
  • 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.
  • restriction fragment length polymorphism refers to the differences among individuals in the lengths of a particular restriction fragment.
  • 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 if the sequence of amino acid residues is not identical.
  • 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 I I
  • 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).
  • 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.
  • 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.
  • the invention includes cDNA coding for a RetL. such as the nucleotide sequence of rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA. human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA.
  • the compounds of the invention include sequences which include the above sequences, or are derivatives of one 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 rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA, including but not limited to rat RetL 1 , partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3, or human RetL3, and their derivatives and variants.
  • One embodiment of the invention includes soluble variants of a RetL. Soluble variants lack at least a portion of the intramembrane section of the native RetL.
  • the soluble variant lacks the phosphatidylinositol glycan linkage of the native RetL.
  • Soluble variants include fusion proteins which encompass derivatives of RetL that lack a phosphatidylinositol motif.
  • Variants can differ from naturally occurring RetL in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring RetL is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid.
  • variants include naturally occurring RetL, or biologically active fragments of naturally occurring RetL, 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 RetL 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.
  • 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.
  • 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.
  • the present invention provides for biologically active fragments of the polypeptides.
  • a RetL polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring RetL.
  • Such biological activities 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 RetL for the extracellular portion of Ret.
  • Another biological activity is the ability to bind to an antibody which is directed at an epitope which is present on naturally occurring RetL.
  • variants with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
  • 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.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
  • 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.
  • a cysteine residue is substituted for (or by) any other residue;
  • 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
  • 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 rat RetLl (SEQ ID NO:2), partial human RetLl (SEQ ID NO:9), full-length human RetLl (SEQ ID NO: 1 1), human RetL2 (SEQ ID NO: 13), murine RefL3 (SEQ ID NO: 17), partial human RetL3 (SEQ ID NO: 19) or human RetL3 (SEQ ID NO:21). 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 length of comparison sequences will generally be at least 8 amino acid residues, usually at least 20 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 RetL protein of the invenion, and also which 2) after being placed in an optimal alignment with the RetL sequence (as depicted for RetLl and RetL2 in Figure 8), has at least 80% of its cysteine residues alligned with cysteines in the RetL protein of the invention.
  • Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization of portions of naturally occurring RetL, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.
  • agents which specifically bind to a protein of the invention 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 95/16709, the specification of which is herein inco ⁇ orated by reference. n
  • RetLl The general strategy used to clone RetLl is shown in Figures 4 A and 4B.
  • Our strategy was based on the premise that at least a RetL is expressed on the metanephric mesenchyme of the developing kidney as a membrane protein (although it is possible that the ligand is also expressed in a soluble form; Figure 4A).
  • the RetL interacts with the Ret receptor on the ureteric bud cell, activating its tyrosine kinase cytoplasmic domain and sending a signal to the nucleus, which in turn activates genes involved in the growth and branching of the ureteric bud.
  • proteins containing the extracellular domain of Ret fused to either the F c portion of human immunoglobulin Gl (IgGl) or alkaline phosphatase (AP) can be used as part of a strategy to clone RetL as shown in Figure 4B.
  • the fusion proteins, the expression libraries and other reagents used in the cloning of RetLl are described below.
  • fusion proteins are generated consisting of the extracellular domains of either rat or human Ret fused to a protein, in one example the human Fc portion of IgGl, and in another example alkaline phosphatase. Both fusion partners can be easily assayed to detect cells which express the ligand as illustrated in Figure 4B.
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • a sense oligomer called kid-013 (SEQ ID NO:3; contains nucleotides 150-169 of Genbank sequence XI 5262) is chosen from the 5' end of the human ret cDNA sequence overlapping the ATG initiation codon. It includes nucleotides on its 5 ' end encoding a Not 1 restriction site for the purpose of cloning.
  • Two antisense oligomers called kid-014 (SEQ ID !%
  • RNA is isolated from the day 14 embryonic rat kidney and mRNA is purified using oligo-dT chromatography. mRNA is converted to cDNA using AMV reverse transcriptase and the cDNA is converted to double stranded cDNA and amplified using Taq polymerase in a standard polymerase chain reaction with oligomers kid-013 and kid-015. The synthesis of a
  • nucleotide sequence shown as that of extracellular rat ret cDNA may differ from the natural rat ret nucleotide and Ret peptide sequences in the regions of kid-013 and kid-015 sequences.
  • ret cDNA clones are isolated from a day 18 rat embryonic kidney cDNA library and a few nucleotide changes are observed in the primer regions resulting in two amino acid changes.
  • One change is in the signal sequence (arginine at position 5 to threonine) and one change is near the end of the extracellular domain (glutamic acid at position 633 to alanine). Both of these changes should not affect ligand binding.
  • Fusion proteins are generated consisting of the extracellular domains of the rat (aa residues #1-637) and human (aa residues #1-636) Ret receptors fused to the Fc portion of human IgGl .
  • Plasmid 2-4 was created previously via a three way ligation: a Notl - Sail fragment generated by PCR containing the extracellular domain of the rabbit TGF-beta type II receptor; a 693 bp Sail - Notl fragment from pSAB144 containing part of the Fc domain of human IgGl ; and Notl digested pSAB132.
  • a fragment containing the Fc domain can be released from the 2-4 plasmid as a 700 bp Sail fragment.
  • pJC012 is transfected into COS cells and the rat Ret/IgG fusion protein is purified from the medium 48 hrs later using Protein-A Sepharose chromatography.
  • the 2612 bp Notl fragment from pJC012 containing the entire rat Ret/IgG fusion protein is isolated and cloned into the Notl site of expression vector pMDR901.
  • the resulting plasmid is called pJC022.
  • Plasmid pJC022 is transfected into CHO cells to generate stable cell lines. The highest producing cell line is suspension adapted. Typical yields for the rat Ret/IgG CHO line are 75mg/L.
  • the construction of the plasmids used to express the human Ret/IgG fusion protein is shown schematically in Figure 6.
  • a gene encoding the human Ret/IgG fusion protein we obtain a plasmid containing a cDNA encoding the human Ret receptor from Dr. M.Takahashi (Department of Pathology, Nagoya University. School of Medicine. Nagoya, Japan).
  • a PCR fragment is generated from this plasmid using oligomers kid-013 and kid-014 .
  • the PCR fragment is treated with Klenow fragment followed by digestion with Notl to produce a PCR fragment with a sticky Notl end and one blunt end.
  • This fragment is cloned into the vector pGEMl lzf(+) previously digested with EcoRl, treated with Klenow fragment, and digested with Notl, in order to generate a sticky Notl end and one blunt end.
  • the resulting plasmid is called pJC013.
  • the 1916 bp Notl - Sail fragment from pJC013 is isolated after a complete digestion with Notl and a partial digestion with Sail, and ligated to the 693 bp Sail - Notl fragment from pSAB144 containing pan of the Fc domain of human IgGl, and the pSAB132 expression vector digested with Notl .
  • the resulting plasmid is called pJCOl 5.
  • the insert in plasmid pJC013 is sequenced and found to contain a single nucleotide difference which changes one amino acid in the extracellular domain of human Ret (Genbank sequence M57464 has a C at position 812, whereas pJC013 has a T at the corresponding position; this results in a change in amino acids from alanine to valine at position 294 of the human Ret protein sequence).
  • This nucleotide is corrected back to the C residue specified by Genbank sequence M57464 by site specific mutagenesis of plasmid pJC013, producing plasmid pJC023.
  • a 585 bp BstE2 ⁇ fragment from pJC023 containing the repaired nucleotide sequence is isolated and cloned into plasmid pJCOl 5 from which the 585 bp BstE2 fragment containing the variant nucleotide has been removed.
  • the new plasmid is called pJC024.
  • the 2609 bp Notl fragment from pJC024 containing the entire human Ret/IgG fusion protein is isolated and cloned into the Notl site of expression vector pMDR901.
  • the resulting plasmid is called pJC025.
  • Plasmid pJC025 is transfected into CHO cells to generate stable cell lines. The highest producing cell line is suspension adapted. Typical yields for the human Ret/IgG CHO line are 6 mg/L.
  • the organ culture assay consists of growing day 13-14 embryonic rat kidneys in organ culture for 3-5 days in the presence of the Ret/Ig fusion protein at a concentration of 50 ug/ml. Kidneys are also cultured in the presence of LFA-3TIP/IgG or vehicle buffer.
  • DB lectin Dolichos Biflorus Agglutinin
  • Paraffin sections are prepared from other kidneys for histological examination. Embryonic kidneys are treated with control buffer or with Ret/IgG, then stained with hematoxylin and eosin. The Ret/Ig-treated embryonic kidney exhibits less branching of the collecting ducts than the control buffer-treated embryonic kidneys. In addition, Ret/I gG-treated kidneys have fewer tubules. We have also observed this effect with the human Ret/IgG fusion protein. These observations are consistent with the fusion proteins blocking the inductive signal between the mesenchyme and the ureteric bud. Therefore we conclude that the fusion protein is a good reagent for cloning a RetL.
  • Receptor/alkaline phosphatase (AP) fusion proteins have been used successfully to identify and clone ligands for c-kit (Cell 63: 185, 1990), ligands for members of the eph family of o ⁇ han receptors (Cell 79:157. 1994), and recently to clone a receptor for leptin, the product of the ob gene (Cell 83:1263, 1995).
  • Plasmids encoding the rat Ret/AP fusion protein are constructed and the rat Ret/AP protein is produced in COS7 cells in cell factories. Subsequently, a stable NIH3T3 cell line is generated expressing on average 10 mg/L of fusion protein.
  • SDS- PAGE analysis of the rat Ret/AP protein indicates that its size is consistent with the predicted molecular weight, and gel filtration analysis indicates that it is produced as a dimer. Partial purification is achieved by affinity chromatography on an anti-AP column.
  • a rabbit polyclonal antibody is generated against the rat Ret/IgG fusion protein.
  • the antibody works on Western blots, FACS analysis of Ret positive cell lines, and immunohistochemistry of embryonic kidney sections.
  • a panel of hamster anti-rat Ret monoclonal antibodies is generated.
  • Rat Ret/IgG fusion protein coupled to Protein A Sepharose, is used to immunize Armenian hamsters.
  • 316 clones are obtained after the fusion and screened for their ability to bind rat Ret fusion proteins and/or human IgG in an ELISA assay.
  • 11 clones produce antibodies that bind only to rat Ret/IgG (and rat Ret/AP), but not human IgG.
  • the cross reactivity to human Ret is assayed by FACS; four clones produce antibodies that can bind to the Ret positive human cell line THP-1.
  • the following table summarizes the Ret binding properties of twelve monoclonal antibodies.
  • cDNA Expression Libraries from rat embryonic kidney, one in the CDM8 vector which utilizes the SV40 origin for amplification, and one in a modified InVitrogen vector, pCEP4, which utilizes the EBV origin for amplification.
  • This modified vector, CH269 has the EBNA-1 gene sequence removed.
  • the EBNA-1 protein interacts with the EBV origin, but the gene is not needed on the vector when cells are used which stably express the EBNA protein.
  • the library in the CDM8 vector contains 1.5 X 10 6 clones with an average inse ⁇ size of 1.18 kb, while the library in the CH269 vector contains approximately 1 XlO 6 clones with an average inse ⁇ size of 1.5 kb.
  • RetLl A number of direct expression methods have been tried to clone RetLl . All of these methods are based on the concept illustrated in Figure 4B. cDNAs from a cDNA library are introduced into mammalian cells; cells that receive RetLl can be identified using the Ret fusion proteins. Although the three approaches described below were unsuccessful, important knowledge and expertise was acquired, which was deployed in a subsequent approach that met with success.
  • a. Panning Method with Ret/IgG The rat Ret/IgG fusion protein is used in an attempt to isolate RetLl by direct expression cloning using a panning method (Aruffo and Seed. Proc. Natl. Acad. Sci. 84: 8753-8757 (1987)).
  • a day 18 embryonic rat kidney cDNA library in CDM8 is used for the panning effort. Pools of cDNAs from this library (5,000 - 10,000 cDNAs per pool) are introduced into COS cells using the DEAE-dextran method. After 48 hours, the cells are removed from the plates with EDTA, incubated with the fusion protein, and subsequently panned on plates coated with anti-human IgG, antibody.
  • DNA is recovered from cells that adhered, transformed back into E. coli, and subsequently isolated for a second round of panning. We are unable to see any cells bind after the third round of panning, and very few clones are obtained after transformation of the Hirt DNA back into E. Coli.
  • a VCAM cDNA used in conjunction with an anti-VCAM monoclonal antibody as a positive control, could only be diluted to a ratio of 1 : 100 and still be detected, indicating that our pool sizes are probably too large. Analysis of some of the clones that are obtained after the second round of panning, indicates that the clones are undergoing rearrangement and deletion.
  • Plasmids recovered from bacterial clones at the end of the second round are analyzed and found to contain large deletions and rearrangements.
  • c. Colorimetric Detection Method with Ret/AP - COS cells are transfected with 400 pools of the cDNA clones (1000 clones per pool) from the day 18 rat embryonic kidney cDNA library (CDM8 vector) and stained with the Ret/AP protein and a colorimetric substrate for alkaline phosphatase. The transfected cells are inspected under a microscope for positive signals. In one experiment, five potential positives were re-analyzed, but all were negative.
  • VCAM/AP protein As a control for the Ret/AP protein, a VCAM/AP protein is produced by fusing the first two domains of human VCAM to the N-terminus of placental AP. (VCAM binds to the integrin VLA4, which is composed of two chains, alpha-4 and beta-1). Transient transfections of COS cells produces sufficient VCAM/AP protein for control experiments.
  • the VCAM/AP protein is compared to VCAM/IgG directly coupled to AP, and to VCAM/lgG plus an AP coupled secondary antibody, in order to assess their ability to detect VLA4 on COS cells transfected with the alpha-4 chain cDNA (COS cells already express the beta-1 chain). The results show that while the VCAM/AP protein could detect VLA4 on transfected cells, the best detection is afforded by the VCAM/IgG protein in combination with an AP coupled secondary antibody.
  • the cells are washed two times with HBHA buffer (0.5 mg/ml BSA, 0.1% NaN 3, 20 mM HEPES (pH 7.0)) and incubated with 20 ug/ml rat Ret/IgG in Tris-buffered saline plus 1 mM MgCl 2 and CaCl 2 for 60-90 min at RT. Following this incubation, the cells are washed four times with HBHA buffer and then fixed with 60% acetone/3% formaldehyde/20 mM HEPES (pH 7.0) for 30 sec.
  • HBHA buffer 0.5 mg/ml BSA, 0.1% NaN 3, 20 mM HEPES (pH 7.0)
  • the cells are incubated with an AP-coupled secondary antibody (goat anti-human IgG Fc-gamma-specific F(ab') 2 (Jackson Immuno Research Laboratories; catalog # 109-056-098; 1 :5000 dilution in Tris-buffered saline plus 1 mM MgCl 2 and CaCl 2 ) for 60 min at RT.
  • an AP-coupled secondary antibody goat anti-human IgG Fc-gamma-specific F(ab') 2 (Jackson Immuno Research Laboratories; catalog # 109-056-098; 1 :5000 dilution in Tris-buffered saline plus 1 mM MgCl 2 and CaCl 2 ) for 60 min at RT.
  • the cells are then washed twice with HBS buffer and twice with AP substrate buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl 2 ) containing 2X Pierce Immuno Pure R Phosphatase suppressor (catalog #35002). The last wash is left for 15 min.
  • the AP substrates NBT (0.33 mg/ml) and BCIP (0.17 mg/ml) are then added in AP substrate buffer containing the Pierce AP inhibitor and incubated with the cells for 5-20 min.
  • the plates are then washed twice with water. The plates are then inspected under a dissecting microscope for the presence of pu ⁇ le stained cells.
  • Ret/IgG specific 10 out of the 17 positive pools only show staining with Ret/IgG fusion protein and not with another IgG fusion protein.
  • one of the above-described positive pools is broken down into smaller subpools in order to identify the cDNA within the pool that is conferring binding to the Ret/IgG fusion protein.
  • 600 cells from the glycerol stock for pool #230 are plated (10 times) and grown overnight. Colonies on these plates are scraped into medium: one tenth of the culture is used to generate a glycerol stock and the remaining portion is used for a DNA preparation.
  • the ten subpools of 600 clones are designated 230-1A through 230-5A and 230-1B through 230-5B. DNAs from these subpools are transfected into 293/EBNA cells and the procedure described above for staining with the Ret/IgG fusion protein is repeated.
  • One subpool #230-5A is positive for staining with the Ret/IgG protein.
  • Pool #230-5A is further broken down in order to identify the cDNA with this subpool that is conferring binding to the Ret/IgG fusion protein.
  • Cells from the glycerol stock of pool 230-5 A are plated and grown overnight. Colonies are picked into the wells of seven 96-well
  • Bioblocks ® and grown overnight. From each 96-well Bioblock, 4 pools of 20 clones and 1 pool of 16 clones are made. Thus 35 pools are generated from the seven Bioblocks* designated 230- 5A-71 through 230-5A-105. DNAs are prepared from each of these pools and transfected into 293/EBNA cells and re-assayed with the Ret/IgG fusion protein as described above. Pool #230- 5A-86 is positive.
  • Pool #230-5A-86 is broken down by going back to the Bioblock and identifying the 20 clones that were mixed together to make this pool. DNAs are made from all twenty clones and transfected individually into 293/EBNA cells and re-assayed for Ret/IgG as described above. Pool #230-5A-86-17 is found to be positive.
  • the hydrophobic C-terminus indicates that the protein may be linked to the cell via a phosphatidylinositol glycan linkage. There are three predicted N-linked glycosylation sites. These properties are consistent with those expected for a ligand for Ret.
  • rat RetLl protein
  • truncating the gene prior to the hydrophobic C terminus For example, this could be done by truncating after Lysine 435 (rat RetLl). Truncation upstream of this amino acid should also result in the expression of a soluble form of the rat RetLl protein.
  • the soluble rat RetLl protein can be expressed by itself or as a part of a fusion with human immunoglobulin. a histidine tag. or a small epitope that is recognized by an antibody.
  • a human embryonic kidney cDNA library in the vector lambda gtlO is purchased from Clontech (catalog #HL5004A).
  • Clontech catalog #HL5004A
  • One million plaque forming units from the phage stock are plated on 10 NuncTM plates.
  • Duplicate plaque lifts are made on Schleicher and Schuell OptitranTM filters.
  • a probe is generated by digesting plasmid rat RetL 1 with the restriction enzyme PvuII, followed by agarose gel isolation of a 1.34 kb fragment which corresponds to nt 242-1582 of the rat RetL nucleotide sequence (rat retLl cDNA).
  • This coding region probe is P 32 labeled by random pnming (Feinberg and Vogelstein. Anal. Biochem. 137:266-267. 1984).
  • the filters are hybridized overnight in 300 ml plaque screen PSB buffer (50 mM Tris pH 7.5, IM NaCl, 0.1 % sodium pyrophosphate, 0.2% PVP, and 0.2% Ficoll) containing 10% dextran sulphate.
  • Duplicate positives are cored from the master plates into SM (100 mM NaCl, 10 mM SO 4 , 50 mM Tris pH 7.5) plus gelatin. 24 of these positives are plaque purified. Lambda miniprep DNA from the purified candidate plaques is digested with Notl. electrophoresed on 1% agarose gel and Southern blotted. The Southern blot is hybridized with the rat rat RetLl coding region probe. Clone HRL20 has the longest insert (4.4 kb) which hybridizes intensely to the rat probe.
  • a human fetal kidney Marathon-ReadyTM cDNA kit is purchased from Clontech (catalog #7423-1 ).
  • Antisense oligonucleotides Kid- 155, corresponding to the complement of nucleotides 62-81 of SEQ ID NO: 8 (partial human retLl cDNA) and Kid- 154, corresponding to the complement of nucleotides 17-43 of SEQ ID NO: 8 (partial human retLl cDNA) are synthesized.
  • PCR is performed using the AdvantageTM cDNA PCR kit (Clontech catalog #8417-1) combined with MarathonTM cDNA reagents and the oligonucleotides Kid-155 or Kid-154.
  • PCR is carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following cycle conditions: 1 cycle of 94C for 1 min; 30 cycles of 94C for 30 sec. 55C for 30 sec. 68C for 4 min.
  • a nested PCR is performed using the product of the first PCR reaction. First, 5 ul of PCR product #1 is diluted 50 fold with TE (final volume 250 ul). The nested PCR reaction contains 35.5 ul H 2 O; 5.0 ul 10X KlenTaq Buffer; 1.0 ul lOmM dNTP mix: 1.0 ul 50X AdvantageTM KlenTaq Polymerase mix. These reagents are mixed as above.
  • the sequence obtained from clone HRL7G8 is found to overlap with the sequence of clone HRL20 (partial human retLl cDNA) and is used to generate a full-length sequence of human RetLl (full-length human retLl cDNA), also shown in Figure 2B.
  • the nucleotide sequence obtained from clone HRL7G8 represents nucleotides 1 to 502 of full-length human retLl cDNA; the nucleotide sequence from clone HRL20 represents nucleotides 460 to 1682 of full-length human retLl cDNA.
  • the sequence from clone HRL7G8 is confirmed by sequencing another cDNA clone (GJ102) isolated from the human embryonic kidney lambda gtlO cDNA library described above, using a probe derived from clone HRL7G6. Nucleotides 1 18 to 1497 comprise the protein reading frame of full-length human retLl cDNA.
  • rat RetL 1 The peptide sequence of rat RetL 1 (rat RetL 1 ) 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.
  • EST Expressed Sequence Tag
  • GenBank Accession # R02249 One is with GenBank Accession # R02249, a 229 bp EST from a combined human fetal liver and spleen cDNA library, and the other is with Genbank Accession # HI 2981, a 521 bp EST from a human infant brain cDNA library.
  • Genbank Accession # HI 2981 a Genbank Accession # HI 2981
  • 521 bp EST from a human infant brain cDNA library.
  • the two ESTs share 99% identity in a region of overlap indicating that they are from the same cDNA.
  • Oligonucleotides are generated from the HI 2981 EST: KID-228 (GAA TGA CAA CTG CAA GAA GCT GCG CTC CTC; corresponding to nucleotides 38-67 and also to nucleotides 534-563 of SEQ ID NO: 12), and antisense oligonucleotide KID-229 (GTG TAC TCG CTG GGC ACC CG; co ⁇ esponding to the complement of nucleotides 156-175 and also to the complement of nucleotides 652-671 of SEQ ID NO: 12).
  • IX 10 6 plaque forming units from a Clontech Human Fetal Liver 5' -Stretch Plus lambda
  • GT10 cDNA library (cat# HL5003a) are screened in duplicate on OPTITRANTM filters.
  • the filters are hybridized with 32 P-labeled oligonucleotides KID-228 and KID-229 in 400 mis plaque screen buffer (50mM Tris pH 7.5. IM NaCl, 0.1% sodium pyrophosphate, 0.2% Polyvinylpryrolidine and 0.2% Ficoll) containing 10% Dextran sulfate and 1 OOug/ml tRNA and 80 pmole each 32 P-labeled oligonucleotide at 65C overnight. They are washed twice with 2X SSC/1% SDS and twice with IX SSC/1% SDS and exposed to film. 11 duplicate positives are purified.
  • DNA from each of these clones is analyzed by restriction enzyme digest followed by agarose gel electrophoresis and Southern blotting.
  • the filters are hybridized to KID-228 and KID-229 to confirm that the inserts hybridize to the probe.
  • the insert of clone DSW240 is completely sequenced (human retL2 cDNA, SEQ ID NO: 12) and is shown in Figure 7.
  • Nucleotides 25-1416 comprise the protein reading frame of human retL2 cDNA. which encodes a protein of 464 amino acids (human RetL2; SEQ ID NO: 13), and is shown in Figure 7. As shown by the BESTFIT analysis depicted in Figure 8, the human RetL2 protein is 49.1% identical and 63.7% similar to the human RetLl protein. It shares in common with human
  • RetLl a hydrophobic N-terminus indicative of a signal sequence and a hydrophobic C-terminus indicative of a phosphatidylinositol glycan linkage motif.
  • 30 cysteines out of the 31 that are present in each protein are conserved.
  • RetL2 is a Ligand for Ret
  • RetL2 is a ligand for Ret by transfecting 293/EBNA cells with an expression plasmid that contains the insert of clone DSW240 and by showing that the cells can bind a soluble Ret/IgG fusion protein.
  • DSW240 is removed using Notl and cloned into the expression vector CH269 which contains an EBV origin and allows for high expression in EBNA positive cell lines. Restriction digests are performed to identify clones that have the correct orientation. Plasmid DNA is prepared from a clone having the correct orientation.
  • Plasmid DNAs (the retL2 expression plasmid, a retL 1 expression plasmid for a positive control, and an expression plasmid containing an unrelated protein for a negative control) are transfected into 293/EBNA cells (8 X 10 5 on a 60 mm plate) using the lipofection method. After 48 hr, the cells are washed two times with HBHA buffer (0.5 mg/ml BSA, 0.1 % NaN 3 20 mM HEPES (pH 7.0)) and incubated with 20 ug/ml rat Ret/IgG in Tris-buffered saline plus 1 mM MgCl 2 and CaCl 2 for 60-90 min at room temperature.
  • HBHA buffer 0.5 mg/ml BSA, 0.1 % NaN 3 20 mM HEPES (pH 7.0)
  • the cells are washed four times with HBHA buffer and then fixed with 60% acetone/3 % formaldehyde/20 mM HEPES (pH 7.0) for 30 sec. Following two washes with HBS buffer (150 mM NaCl, 20 mM HEPES (pH 7.0)), the cells are incubated with an AP-coupled secondary antibody (goat M
  • the AP substrates NBT (0.33 mg/ml) and BCIP (0.17 mg/ml) are then added in AP substrate buffer containing the Pierce AP inhibitor and incubated with the cells for 5-20 min. The plates are then washed twice with water. The plates are then inspected under a dissecting microscope for the presence of pu ⁇ le stained cells. The presence of purple stained cells indicates that the Ret/fusion protein has bound to the cells and that the RetL2 protein is a ligand for Ret. Pu ⁇ le stained cells are also observed after trasfection with the retLl expression vector but not with the negative control vector.
  • ESTs with homology to Ret ligands are AA049894, and AA050083 (which is partial murine retL3 cDNA, SEQ ID NO: 14). Plasmids encoding these ESTs are obtained from Genome Systems Inc. (Catalog# 475791 and #475497) as bacterial stabs. Plasmid DNA is prepared from single colonies obtained by streaking the stabs onto LB Amp plates. The inserts from these plasmids are sequenced in their entirety. Comparison of the two sequences demonstrates that AA049894, which has a 1.4 kb insert, is contained within AA050083. which has a 1.9 kb insert.
  • Marathon RACE Mouse 1 1-day embryo Marathon-ReadyTM cDNA (cat.#7458-l) and an AdvantageTM Kit (cat.# 8417-1 ) is purchased from Clontech.
  • Antisense oligonucleotides, Kid-366, corresponding to the complement of nucleotides 847-866 of SEQ ID NO: 14 and Kid-365, corresponding to the complement of nucleotides 589-615 of SEQ ID NO: 14 are synthesized.
  • PCR is performed using and an AdvantageTM cDNA PCR kit (Clontech cat.#8417-l) combined with MarathonTM cDNA reagents and oligonucleotide Kid-366.
  • PCR is carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following cycle conditions: 1 cycle of 94C for 1 min; 5 cycies of 94C for 30 sec, 72C for 4 min; 5 cycles of 94C for 30 sec, 70C for 4 min; 25 cycles 94C for 30 sec, 68C for 4 min.
  • a nested PCR is performed using the product of the first PCR reaction. First, 5 ul of PCR product#l is diluted 50 fold with TE( Final volume 250 ul).
  • the nested PCR reaction contains 35.5 ul H 2 O; 5.0 ul 10X KlenTaq Buffer; 1.0 ul lOmM dNTP mix; 1.0 ul 50X AdvantageTM KlenTaq Polymerase mix. These reagents are mixed as above. 5.0 ul diluted PCR product #1 ; 1.0 ul lOuM AP2 primer and 3.6 ul 2.8uM Kid-365 are then added. Cycle conditions are the same as above. The resultant product of approximately 665 bp is purified on a 1 % low-melt agarose gel and Qiaex II (Qiagen cat#20021) extracted.
  • the purified DNA is cloned into pNoTA/T7TM using PRIME PCR CLONERTM cloning system (5 Prime-> 3 Prime cat.# 1-725029). Sequence information is obtained from multiple isolates, including clones called DSW252 and DSW253.
  • DSW252 The sequence of DSW252 is found to overlap with SEQ ID NO:14 except that an additional T is present between NT 252 and NT 253 of the SEQ ID NO: 14 sequence. This T is also present in the other isolates DSW251 and DSW253. Insertion of this additional base corrects the ORF such that a single 1191 bp ORF (counting from the first Met) encoding 397 amino acids is obtained.
  • This ORF encodes a Met in the context of a canonical translation initiation consensus sequence (Kozak) and includes a signal sequence for surface expression/secretion. To obtain a full-length murine clone which is capable of being expressed, a 630 bp Notl-
  • DSW254 The insert of DSW254 is sequenced in its entirety (murine retL3; SEQ ID NO: 16) and the ORF is confirmed as encoding a protein of 397 amino acids (murine RetL3; SEQ ID NO: 17). These sequences are also shown in Figure 9.
  • the C-terminus of RetL3 is hydrophobic and indicative of a phosphatidylinositol glycan linkage motif.
  • RetL3 In order to find a candidate tissue source for cloning human RetL3, we utilize northern blots of mouse tissues to determine the expression pattern of murine RetL3. Of the tissues surveyed, expression of RetL3 is highest in heart tissue.
  • a human adult heart cDNA library in the vector lambda gtlO is purchased from Clontech (catalog# HL3026a). One million plaque forming units from the phage stock are plated on 10 Nunc plates. Duplicate plaque lifts are made on Schleicher and Schuell OptitranTM filters.
  • a probe is generated by PCR with primers Kid-366 and Kid-367. corresponding to nucleotides 397-420 of the AA05OO83 sequence.
  • PCR reaction is set up as follows: 10 ul 10X PFU Buffer. 2.0 ul lOmM dNTP mix, 72.1 ul H 2 O, 3.1 ul 13.2 uM Kid-367. 6.8 ul 5.88 uM Kid- 366, 5.0 ul 0.1 ug/ ul AA050083 DNA and 2.0 ul 2.5 Units/ul PFU (Stategene catalog # 600154) are mixed.
  • PCR is carried out in Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following conditions: 25 cycles of 94 C for 1 min, 53C for 1 min..
  • the product is purified by extraction with phenol, chloroform, isoamyl alcohol 50:49: 1 followed by low-melt agarose gel electrophoresis and QiaexII purification of the excised fragment.
  • This coding region probe is P 32 labeled by random priming (Feinberg and Vogelstein).
  • the filters are hybridized overnight in 200 ml Plaque screen buffer () containing 10% dextran sulphate, 100 ug/ml tRNA and 1.8 X 10 8 CPM of the mouse probe at 65C. They are washed twice with plaque screen buffer, twice with 2XSSC/1% SDS, twice with 1XSSC/1%SDS at 65C and exposed to film at - 70C with an intensifying screen.
  • Duplicate positives are plaque purified. Lambda miniprep DNA from the purified candidate plaques is digested with EcoRl, electrophoresed on a 1% agarose gel and Southern blotted. The Southern blot is hybridized with the mouse probe. Clone GJ128 , 3 V
  • Human RetL3 is 34.3% and 34.9% identical to human RetLl and human RetL2, respectively. It has 76.8% identity with murine RetL3.
  • RetL's. anti-RetL antibodies. anti-Ret antibodies, and fusion proteins of Ret and of RetL's 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 RetL.
  • 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.
  • 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 HIV -associated myelopathy, prion diseases including Creutzfeldt- Jakob disease.
  • 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.
  • 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. These disorders include those caused by any of the factors previously listed, and specifically include Lyme disease, HIV-associated neuropathies, polymyositis, muscular dystrophy, and myasthenia gravis.
  • Anti-RetL antibodies and Ret fusion proteins of the invention which specifically bind to the protein of rat RetL, partial human RetLl. full-length human RetLl. human RetL2. murine RetL3 or human RetL3, or fragments of these proteins, 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 RetL.
  • 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 RetL-positive cells on histological sections.
  • substances such as horseradish peroxidase, which can be used as immunocytochemical stains to allow visualization of areas of RetL-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-immunoglobulin antibody which is 3£
  • Specific antibodies to any RetL are also useful in immunoassays to quantify the substance for which a given antibody has specificity.
  • Specific antibodies to a RetL 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.
  • Other methods of the invention include modulating Ret- RetL 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 RetL's. or by complexes comprising RetL's, which would otherwise activate the Ret signaling pathway.
  • a RetL 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 immunohistological or preparative methods described above for antibodies to a RetL.
  • Fusion proteins encompassing a RetL 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 RetL utilize fusion proteins which inco ⁇ orate Ret and/or an anti-RetL antibody.
  • the anti-Ret antibody or anti-RetL 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 RetL 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.
  • an anti-Ret antibody or RetL 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 RetL or to an 2 1
  • a toxin is conjugated to Ret or to an anti-RetL antibody to selectively target and kill RetL-positive cells, such as a tumor expressing a RetL.
  • Ret or to an anti-RetL antibody to selectively target and kill RetL-positive cells, such as a tumor expressing a RetL.
  • ricin conjugated to a monoclonal antibody against the CD 19 antigen expressed on most neoplastic cells (Grossbard et al., Blood 79:576,1992).
  • 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.
  • Radioisotope labeled RetL or anti- Ret antibodies will preferentially target radioactivity to tumor sites in cells expressing Ret, sparing normal tissues.
  • 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 ⁇ particles (for example, 131 I) 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.
  • Radionuclides emitting ⁇ 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 RetL or anti-Ret antibodies may be more therapeutically effective than the instantaneous i ⁇ adiation delivered externally in conventional radiation therapy.
  • Low dose rate i ⁇ adiation 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.
  • 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.
  • the RetL genes of the invention are introduced into damaged tissue to stimulate production of a RetL by the transfected cells, to promote cell growth and/or survival of cells that express Ret.
  • a RetL gene may be introduced into a renal or neural target tissue of choice.
  • a RetL would then be stably expressed and stimulate Ret receptor-positive cells to grow, divide, differentiate, and/or potentiate cell survival.
  • RetL 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.
  • a RetL 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.
  • liposome based DNA transfer e.g., LIPOFECTTN-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, -3 ⁇
  • Promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • promoters/enhancers include, but are not limited to, the native RetL promoter, the cytomegalovirus immediate-early promoter/enhancer (Karasuyama et al., J. £x£. Med.. 169: 13 (1989)); the human beta-actin promoter (Gunning et al., Proc.
  • MMTV LTR mouse mammary tumor virus long terminal repeat
  • MoLV LTR Moloney murine leukemia virus
  • 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 he ⁇ es simplex virus (HSV) thymidine kinase promoter (Wagner et al., Proc. Nat. Acad. Sci. USA. 78: 1441 (1981)); the adenovirus promoter (Yamada et al., EXQC_. Nai. Acad. Sci. USA, 82: 3567 (1985)).
  • RSV Rous sarcoma virus
  • HSV Rous sarcoma virus
  • HSV Rous sarcoma virus
  • HSV Rous sarcoma virus
  • HSV Rous sarcoma virus
  • HSV Rous sar
  • the RetL genes 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., Cu ⁇ . Top. Microbiol. Immunol.. 158: 39-61. 1992 (adenovirus); Hofmann et al., Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Cu ⁇ . Top. Microbiol. Immunol.. 158: 25-38, 1992 (vaccinia virus); Muzyczka. Cu ⁇ . Top. Microbiol.
  • Prefe ⁇ ed vectors are DNA viruses that include adenoviruses (preferably Ad-2 or Ad-5 based vectors), baculovirus, he ⁇ es viruses (preferably he ⁇ es 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, c,0
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types.
  • Ad2 and Ad5 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 (10 13 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.
  • Ad2 and Ad5 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 Cys
  • adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator 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 M
  • DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication.
  • ITR inverted terminal repeats
  • 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 El a and part of the El b 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. AH 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 inco ⁇ orating and transducing very large DNA inserts.
  • Adeno-associated viruses 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
  • the cap proteins form the virion capsid.
  • ITRs inverted terminal repeats
  • the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene. See B.J. 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.
  • 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.
  • 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 he ⁇ esvirus or adenovirus which encodes helper functions that are recruited by AAV to aid in its excision from host chromatin (BJ. 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 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.
  • 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.
  • 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.
  • a "marker" gene function e.g, thymidine kinase activity, antibiotic resistance, and the like
  • 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.
  • 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.
  • carrier encompasses liposomes and the HIV-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.
  • treatment of human acute renal failure with liposome-encapsulated RetL may be performed in vivo by introducing a RetL into cells in need of such treatment using liposomes .
  • the liposomes can be delivered via catheter to the renal artery.
  • the recombinant RetL protein is purified, for example, from CHO cells by immunoaffinity chromatography or any other convenient method, then mixed with liposomes and inco ⁇ orated into them at high efficiency.
  • the encapsulated protein may be tested in vitro for any effect on stimulating cell growth.
  • novel polypeptide of this invention may be administered to an animal via liposome delivery system in order to enhance their stability and/or immuno genicity. Delivery of the novel polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in die 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 RetL 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 an, and include inorganic and organic acids and bases.
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • CTTCGTTCAC TTACGAGGTC ACATATAGAA CAAACATCAC CAACTATTAG CTTACCGTTA 2460 GCTTCCCAAC TATTAGCTTT CTATGTTTTG AAAGCAGTGT TGCTGACCCC ATGTTTTAAT 2520
  • CATCTTGCTT ACTAGCCACA CACCATCTCC TGGAGCCCTC CTCCTGACCT GGGCAGACCC 3180
  • Lys Asn Cys Leu Arg lie Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 100 105 110
  • GAG GTG AGC ACG CGG TGG GCA CTG GAT CGG GAG CTT CAG GAG AAG TAT 720
  • MOLECULE TYPE protein
  • GGT CTC GGT GCT TCC AGC CAC ATA ACC ACA AAA TCA ATG GCT GCT CCT 960 Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala Ala Pro 945 950 955
  • AAA GAA GGT CTC GGT GCT TCC AGC CAC ATA ACC ACA AAA TCA ATG GCT 1413 Lys Glu Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala 765 770 775
  • AAAAAAATAC AATATGGACA TGTAAAAAGA CAAAAACCAA GTTATCTGTT TCCTGTTCTC 1567
  • GGC CCC AGC AGA GCC AGA CCG TCG GCT GCC TTG ACC GTG CTG TCT GTC 1395 Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr Val Leu Ser Val 905 910 915
  • CTCCTGGAGC CCGCGACCTC CACTGCTGAT GATCCTGCTA CTGGTGCTGT CGTTGTGGCT 120
  • GCA GCA GAA CAA CTC AGG AAC AGC TCT CTG ATA GAC TGC AGG TGC CAT 343 Ala Ala Glu Gin Leu Arg Asn Ser Ser Leu He Asp Cys Arg Cys His 435 440 445
  • TCCTGTTTCC CAGGACCACC CAGAGGCTAA GGAATCAGTC ATTCCCTGTT GCCTTCTCCA 1621
  • CTCTGCCAGG CAATCCTGAA CATTTGGGCA TGAAGAGCTA AAGTCTTTGG GTCTTGTTTA 1801
  • AGA TGT CTA CGA GCA TAC 622 He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala Tyr 590 595 600

Abstract

This invention relates to nucleotide sequences which encode a Ret ligant (RetL9, as well as to methods of stimulating neural and renal growth by treating cells and mammalian subjects with RetL DNA or protein. The invention provides a purified and isolated DNA molecule coding for a RetL, having the nucleotide sequence of any RetL, but specifically including rat retL 1 cDNA (SEQ ID NO.1), partial human RetL1 cDNA (SEQ ID NO:8), full length human retL 1 cDNA (SEQ ID NO:10), human retL2 cDNA (SEQ ID NO:12), murine retL3 cDNA (SEQ ID NO:12) or human retL3 cDNA (SEQ ID NO;16), partial human retL3 cDNA (SEQ ID NO:18) or human retL3 cDNA (SEQ ID NO:20). The invention further provides a RetL protein, with an amino acid sequence comprising that of rat RetL1 (SEQ ID NO:2), partial human RetL1 (SEQ ID NO:9), full length human RetL (SEQ ID NO:11), human RetL2 (SEQ ID NO:13), murine RetL3 (SEQ ID NO:17), partial human RetL3 (SEQ ID NO:19) or human RetL3 (SEQ ID NO:21).

Description

RET LIGAND (RetL) FOR STTMULATTNG NEURAL AND RENAL GROWTH
FIELD OF THE INVENTION
This invention relates to nucleotide sequences which encode a Ret ligand (RetL), as well as to methods of stimulating neural and renal growth by treating cells and mammalian subjects with RetL DNA or protein.
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
The invention provides a purified and isolated DNA molecule coding for a RetL. having the nucleotide sequence of any RetL, but specifically including rat retLl cDNA (SEQ ID NO:l), partial human retLl cDNA (SEQ ID NO:8), full-length human retLl cDNA (SEQ ID NO: 10), human retL2 cDNA (SEQ ID NO: 12), murine retL3 cDNA (SEQ ID NO: 16), partial human retL3 cDN A (SEQ ID NO: 18) or human retL3 cDNA (SEQ ID NO:20). The invention further provides a RetL protein, with an amino acid sequence comprising that of rat RetLl (SEQ ID NO:2), partial human RetLl (SEQ ID NO:9), full-length human RetLl (SEQ ID NO: l 1 ), human RetL2 (SEQ ID NO: 13). murine RetL3 (SEQ ID NO: 17), partial human RetL3 (SEQ ID NO: 19) or human RetL3 (SEQ ID NO:21). In another embodiment, the invention includes a DNA sequence which encompasses the sequence (partial human retLl cDNA (SEQ ID NO:8)) of the insert DNA of clone HRL20, which is ATCC No. 97604, or the sequence of the insert DNA of clone #230-5A-86-17 (rat retLl cDNA (SEQ ID NO:l)), which is ATCC No. 98047.
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 RetL; the DNA may be a) a DNA molecule which comprises rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA, or the complementary strand of rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA; b) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in a) or 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 RetL having the biological activity of a RetL is also within the invention.
Any of the recombinant DNA molecules of the invention may 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 RetL or a variant of a RetL.
The invention includes prokaryotic or eukaryotic host cells stably transformed or transfected by a vector comprising a DNA molecule encoding a native or variant RetL.
A purified and isolated human RetL 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 RetL. 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 RetL. 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 rat RetLl, partial human RetLl, full-length human RetLl. human RetL2, murine RetL3, or human RetL3, or is a variant of one of these sequences. In other embodiments, the protein is a fusion protein including Ret or a RetL, fused to another molecule or molecular fragment, such as an immunoglobulin, toxin, imageable compound or radionuclide. Also included are chimeric molecules of RetL.
Other embodiments of the invention include specific monoclonal antibodies to a RetL of the invention. Such an antibody may be associated with a toxin, imageable compound or radionuclide. The invention also includes hybridoma cell lines which produce specific antibodies to Ret, including AA.FF9, AA.HE3, AF.E9, BA.B1, BB.B6, AA.GE7, CD.F11, AH.E3, CD.G4, AG.E7, BD.G6 and BH.G8, as well as subclones of these hybridomas, and the antibodies produced by these hybridomas or subclones of these hybridomas.
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 may be RetLl, RetL2, or RetL3, a fragment of a full- length RetL, or an antibody which binds to Ret. The compound may be administered concurrently with a therapeutically effective amount of a second compound, such as GDNF, neurturin or a GDNF-related molecule. While tissues of interest for these methods may include any tissue, preferred tissues include renal tissue, neural tissue, heart, stomach, small intestine, spinal cord, or lung. In one embodiment, the RetL is a soluble RetL. The subject of the methods may be human.
In another method of the invention, Ret signal transduction between a first cell expressing a RetL and a second cell is inhibited by contacting the first cell with a soluble Ret protein or with an antibody to the RetL. The soluble Ret 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 RetL fusion protein or an anti-Ret antibody conjugated to a toxin, imageable compound or radionuclide. The RetL can be RetLl. RetL2 or RetL3. 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 RetL and a toxin or radionuclide, or an anti-Ret antibody conjugated to a toxin or radionuclide. The cell may 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 RetL, comprising contacting the cell with a fusion protein comprising Ret and a toxin, imageable compound or radionuclide, or an anti-RetL antibody conjugated to a toxin, imageable compound or radionuclide. Another embodiment includes the method of suppressing growth of a tumor cell which expresses a RetL, comprising contacting the cell with a fusion protein of Ret and a toxin or radionuclide or with an anti-RetL antibody conjugated to a toxin or radionuclide; the cell may be within a subject, and the protein administered to the subject. The RetL for any of the methods of the invention can be RetLl, RetL2 or RetL3, or a variant or fragment of RetLl, RetL2 or RetL3.
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 RetL, 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 RetL to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a cDNA sequence (SEQ ID NO: 1 ) and deduced amino acid sequence (SEQ ID NO:2) of rat RetLl . The nucleotide sequence extends from base pair 201 through base pair 1700 of SEQ ID NO:l, and contains the entire open reading frame.
FIGURE 2A is a partial cDNA sequence (SEQ ID NO:8) and deduced amino acid sequence (SEQ ID NO:9) of human RetLl . This sequence is that of the insert of clone HRL20, deposited as ATCC No. 97604.
FIGURE 2B is a composite full-length DNA sequence (SEQ ID NO: 10) and deduced amino acid sequence (SEQ ID NO: 1 1 ) of human RetL 1.
FIGURE 3 A is a comparison of the nucleotide sequence of human RetLl (upper line of sequence) with that of rat RetLl sequence (lower line of sequence). Vertical lines between nucleotides show identity at a position, while a dot indicates a gap at that position.
FIGURE 3B is a comparison of the amino acid sequence of human RetLl (upper line of sequence) with that of rat RetLl sequence (lower line of sequence). Vertical lines between corresponding amino acids show identity at a residue, while a dot indicates a conservative substitution at that residue. FIGURE 4A is a schematic diagram of a possible role for Ret and RetL in the interaction between a metanephric mesenchyme cell and a ureteric bud cell.
FIGURE 4B is a schematic diagram of a method of screening transfectants of a cDNA library for clones that express a RetL. The presence of expressed RetL on transfectants is detected by assessing the binding by those transfectants either of Ret/I gG fusion protein or of Ret/alkaline phosphatase fusion protein.
FIGURE 5 is a schematic diagram showing the construction of the plasmids used to express the rat Ret/I gG fusion protein.
FIGURE 6 is a schematic diagram showing the construction of the plasmids used to express the human Ret/IgG fusion protein.
FIGURE 7 is a cDNA sequence (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO: 13) of human retL2, as found in clone DS W240. The protein reading frame is contained within nucleotides 25 to 1416.
FIGURE 8 is a comparison of the amino acid sequence of human RetL2 (upper line of sequence) with that of human RetLl sequence (lower line of sequence). Vertical lines between amino acids show identity at a position, while a dot indicates a gap at that position.
FIGURE 9 is a cDNA sequence (SEQ ID NO: 16) and deduced amino acid sequence (SEQ ID NO: 17) of murine RetL3.
FIGURE 10 is a cDNA sequence (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) of human RetL3. DETAILED DESCRIPTION OF THE INVENTION
Sequence Identification Numbers
Nucleotide and amino acid sequences referred to in the specification have been given the following sequence identification numbers: SEQ ID NO: 1 - rat retL 1 cDNA
SEQ ID NO:2 - rat RetLl aa
SEQ ID NO: 3 - oligomer kid- 13
SEQ ID NO:4 - oligomer kid- 14
SEQ ID NO: 5 - oligomer kid- 15 SEQ ID NO:6 - extracellular rat retL 1 cDNA
SEQ ID NO: 7 - extracellular rat RetLl aa
SEQ ID NO:8 - partial human retLl cDNA
SEQ ID NO:9 - partial human RetLl aa
SEQ ID NO: 10 - human retL 1 cDNA SEQ ID NO:l l - human RetLl aa
SEQ ID NO: 12 - human retL2 cDNA
SEQ ID NO: 13 - human RetL2 aa
SEQ ID NO: 14 - partial murine retL3 cDNA (EST AA50083)
SEQ ID NO: 15 - partial murine RetL3 aa SEQ ID NO: 16 - murine retL3 cDNA
SEQ ID NO:17 - murine RetL3 aa
SEQ ID NO:l 8 - partial human retL3 cDNA
SEQ ID NO: 19 - partial human RetL3 aa
SEQ ID NO:20 - human retL3 cDNA SEQ ID NO:21 - human retL3 aa
Definitions
As used herein, the term "RetL" means any protein which specifically interacts with the receptor protein 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 may 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 antagonists, triggering Ret dimerization and autophosphorylation. The ligand may also be modified in various ways, such as incorporated as a portion of a fusion protein, such as with 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 the cysteine residues within the conserved cysteine skeleton, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the protein. Therefore, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the level of amino acid sequence homology or identity between the candidate and reference sequences. 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 "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 moiecules 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 Laboratorv 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 two or more different nucleotide sequences can exist at a particular site in DNA.
"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.
Similarly, a "functional derivative" of a gene of any of the proteins of the present invention is meant to include "fragments", "variants", and "analogues" of the gene, which may be "substantially similar" in nucleotide sequence, and which encode a molecule possessing similar activity. ΛO
"GDNF-related molecule" means any moleule which is at least 40% homologous to either GDNF or neurturin, and is also capable of specifically binding a RetL.
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. 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 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 I I
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 RetL. such as the nucleotide sequence of rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA. human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA. In addition, the compounds of the invention include sequences which include the above sequences, or are derivatives of one 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 rat retLl cDNA, partial human retLl cDNA, full-length human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA, including but not limited to rat RetL 1 , partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3, or human RetL3, and their derivatives and variants. One embodiment of the invention includes soluble variants of a RetL. Soluble variants lack at least a portion of the intramembrane section of the native RetL. In some examples, the soluble variant lacks the phosphatidylinositol glycan linkage of the native RetL. Soluble variants include fusion proteins which encompass derivatives of RetL that lack a phosphatidylinositol motif. Variants can differ from naturally occurring RetL in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring RetL is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly preferred variants include naturally occurring RetL, or biologically active fragments of naturally occurring RetL, 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 RetL 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).
I S
TABLE 1: CONSERVATIVE AMINO ACID REPLACEMENTS
Figure imgf000015_0001
Figure imgf000016_0001
IS
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 RetL polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring RetL. Such biological activities 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 RetL for the extracellular portion of Ret. Another biological activity is the ability to bind to an antibody which is directed at an epitope which is present on naturally occurring RetL.
In other embodiments, variants with amino acid substitutions which are less conservative may 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 ceπainty, 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 rat RetLl (SEQ ID NO:2), partial human RetLl (SEQ ID NO:9), full-length human RetLl (SEQ ID NO: 1 1), human RetL2 (SEQ ID NO: 13), murine RefL3 (SEQ ID NO: 17), partial human RetL3 (SEQ ID NO: 19) or human RetL3 (SEQ ID NO:21). 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 length of comparison sequences will generally be at least 8 amino acid residues, usually at least 20 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 RetL protein of the invenion, and also which 2) after being placed in an optimal alignment with the RetL sequence (as depicted for RetLl and RetL2 in Figure 8), has at least 80% of its cysteine residues alligned with cysteines in the RetL 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 RetL, 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 95/16709, the specification of which is herein incoφorated by reference. n
EXPERIMENTAL PROCEDURE
Overview of Strategy
The general strategy used to clone RetLl is shown in Figures 4 A and 4B. Our strategy was based on the premise that at least a RetL is expressed on the metanephric mesenchyme of the developing kidney as a membrane protein (although it is possible that the ligand is also expressed in a soluble form; Figure 4A). The RetL interacts with the Ret receptor on the ureteric bud cell, activating its tyrosine kinase cytoplasmic domain and sending a signal to the nucleus, which in turn activates genes involved in the growth and branching of the ureteric bud. Therefore, proteins containing the extracellular domain of Ret fused to either the Fc portion of human immunoglobulin Gl (IgGl) or alkaline phosphatase (AP) can be used as part of a strategy to clone RetL as shown in Figure 4B. The fusion proteins, the expression libraries and other reagents used in the cloning of RetLl are described below.
We first isolate a cDNA for rat RetLl and then use it as a probe to isolate a cDNA for human RetL 1. cDNAs are subsequently isolated for RetL2 and RetL3.
Generation of Reagents Required for Direct Expression Cloning of Ret Ligands
1. Isolation of cDNA Encoding Rat Ret Extracellular Domain
To identify RetLl. fusion proteins are generated consisting of the extracellular domains of either rat or human Ret fused to a protein, in one example the human Fc portion of IgGl, and in another example alkaline phosphatase. Both fusion partners can be easily assayed to detect cells which express the ligand as illustrated in Figure 4B.
Since a cDNA coding for rat Ret has never been disclosed, we isolate a cDNA encoding the extracellular domain of the rat Ret receptor using the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) method. We compare the two nucleotide sequences for human (Genbank Accession numbers M57464 and XI 5262) and murine (Genbank Accession number X67812) ret and design oligonucleotide primers from regions of high identity between the two sequences. A sense oligomer called kid-013 (SEQ ID NO:3; contains nucleotides 150-169 of Genbank sequence XI 5262) is chosen from the 5' end of the human ret cDNA sequence overlapping the ATG initiation codon. It includes nucleotides on its 5' end encoding a Not 1 restriction site for the purpose of cloning. Two antisense oligomers called kid-014 (SEQ ID !%
NO:4; contains the complement of nucleotides 1819-1839 of Genbank sequence M57464) and kid-015 (SEQ ID NO:5; contains the complement of nucleotides 1894-1914 of Genbank sequence X67812) are chosen, respectively, from the human and murine cDNA sequences immediately 5' to the sequences that encode the transmembrane domains. Oligomers kid-014 and kid-015 contain additional nucleotides at their 5' ends that encode a Sail restriction site for the purpose of cloning.
Total RNA is isolated from the day 14 embryonic rat kidney and mRNA is purified using oligo-dT chromatography. mRNA is converted to cDNA using AMV reverse transcriptase and the cDNA is converted to double stranded cDNA and amplified using Taq polymerase in a standard polymerase chain reaction with oligomers kid-013 and kid-015. The synthesis of a
1942 bp PCR fragment is confirmed by running an aliquot of the PCR reaction on a 1% agarose gel. The rest of the PCR fragment is digested with Notl and Sail and cloned into pSAB132 previously digested with Notl and Sail. The resulting plasmid is called pJCOl 1. The entire insert of plasmid pJCOl 1 contained between the Notl and Sai l sites is sequenced, and is shown as extracellular rat retLl cDNA, SEQ ID NO:6. A translation of this sequence reveals the peptide sequence (SEQ ID NO:7) for extracellular rat RetL 1. Because oligomers for PCR were chosen from human and mouse sequences of ret. it is possible that the nucleotide sequence shown as that of extracellular rat ret cDNA, and the peptide sequence shown as that of extracellular rat Ret, may differ from the natural rat ret nucleotide and Ret peptide sequences in the regions of kid-013 and kid-015 sequences. Subsequently, ret cDNA clones are isolated from a day 18 rat embryonic kidney cDNA library and a few nucleotide changes are observed in the primer regions resulting in two amino acid changes. One change is in the signal sequence (arginine at position 5 to threonine) and one change is near the end of the extracellular domain (glutamic acid at position 633 to alanine). Both of these changes should not affect ligand binding.
2. Ret/IgG Fusion Proteins
Fusion proteins are generated consisting of the extracellular domains of the rat (aa residues #1-637) and human (aa residues #1-636) Ret receptors fused to the Fc portion of human IgGl .
The construction of the plasmids used to express the rat Ret/IgG fusion protein is shown schematically in Figure 5. In order to construct a gene encoding the rat Ret/IgG fusion protein, /c,
we digest pJCOl 1 (described above ) containing the rat Ret extracellular domain with Sail, and ligate it to a 700 bp Sail fragment from plasmid 2-4, to create plasmid pJC012. This Sail fragment contains part of the Fc domain of human IgGl originally derived from plasmid pSAB144. Plasmid 2-4 was created previously via a three way ligation: a Notl - Sail fragment generated by PCR containing the extracellular domain of the rabbit TGF-beta type II receptor; a 693 bp Sail - Notl fragment from pSAB144 containing part of the Fc domain of human IgGl ; and Notl digested pSAB132. As shown in Figure 5. a fragment containing the Fc domain can be released from the 2-4 plasmid as a 700 bp Sail fragment. pJC012 is transfected into COS cells and the rat Ret/IgG fusion protein is purified from the medium 48 hrs later using Protein-A Sepharose chromatography. In order to make a stable cell line producing the rat Ret/IgG protein, the 2612 bp Notl fragment from pJC012 containing the entire rat Ret/IgG fusion protein is isolated and cloned into the Notl site of expression vector pMDR901. The resulting plasmid is called pJC022. Plasmid pJC022 is transfected into CHO cells to generate stable cell lines. The highest producing cell line is suspension adapted. Typical yields for the rat Ret/IgG CHO line are 75mg/L.
The construction of the plasmids used to express the human Ret/IgG fusion protein is shown schematically in Figure 6. In order to construct a gene encoding the human Ret/IgG fusion protein, we obtain a plasmid containing a cDNA encoding the human Ret receptor from Dr. M.Takahashi (Department of Pathology, Nagoya University. School of Medicine. Nagoya, Japan). A PCR fragment is generated from this plasmid using oligomers kid-013 and kid-014 . The PCR fragment is treated with Klenow fragment followed by digestion with Notl to produce a PCR fragment with a sticky Notl end and one blunt end. This fragment is cloned into the vector pGEMl lzf(+) previously digested with EcoRl, treated with Klenow fragment, and digested with Notl, in order to generate a sticky Notl end and one blunt end. The resulting plasmid is called pJC013. The 1916 bp Notl - Sail fragment from pJC013 is isolated after a complete digestion with Notl and a partial digestion with Sail, and ligated to the 693 bp Sail - Notl fragment from pSAB144 containing pan of the Fc domain of human IgGl, and the pSAB132 expression vector digested with Notl . The resulting plasmid is called pJCOl 5. The insert in plasmid pJC013 is sequenced and found to contain a single nucleotide difference which changes one amino acid in the extracellular domain of human Ret (Genbank sequence M57464 has a C at position 812, whereas pJC013 has a T at the corresponding position; this results in a change in amino acids from alanine to valine at position 294 of the human Ret protein sequence). This nucleotide is corrected back to the C residue specified by Genbank sequence M57464 by site specific mutagenesis of plasmid pJC013, producing plasmid pJC023. A 585 bp BstE2 ■ fragment from pJC023 containing the repaired nucleotide sequence is isolated and cloned into plasmid pJCOl 5 from which the 585 bp BstE2 fragment containing the variant nucleotide has been removed. The new plasmid is called pJC024. The 2609 bp Notl fragment from pJC024 containing the entire human Ret/IgG fusion protein is isolated and cloned into the Notl site of expression vector pMDR901. The resulting plasmid is called pJC025. Plasmid pJC025 is transfected into CHO cells to generate stable cell lines. The highest producing cell line is suspension adapted. Typical yields for the human Ret/IgG CHO line are 6 mg/L.
Further details on production of the vectors employed in the methods of the invention are given in PCT applications 94/01456 and 92/02050, the specifications of which are herein incorporated by reference.
3. Bioactivity of the Ret/IgG Fusion Proteins To determine if the Ret/IgG fusion proteins that we produce are bioactive and therefore would be good screening reagents for the cloning of a RetL. we perform several organ culture assays for bioactivity. The organ culture assay consists of growing day 13-14 embryonic rat kidneys in organ culture for 3-5 days in the presence of the Ret/Ig fusion protein at a concentration of 50 ug/ml. Kidneys are also cultured in the presence of LFA-3TIP/IgG or vehicle buffer. After the culture period, some of the kidneys are stained with the fluorescent lectin Dolichos Biflorus Agglutinin (DB lectin) which stains the collecting duct tissues, which are epithelial cells derived from the ureteric bud. These "DB" positive cells mark the Ret- positive cells, since Ret is expressed in the ureteric bud and its epithelial derivatives. This provides a gross assessment of the Ret/IgG fusion protein on the growth and development of the embryonic kidney. There is a clear difference in collecting duct moφhology and growth between kidneys which have been cultured with LFA-3TIP and those cultured with the rat Ret/IgG fusion protein.. The Ret/I gG-treated kidneys have collecting ducts which show significantly less branching and are typically smaller overall.
Paraffin sections are prepared from other kidneys for histological examination. Embryonic kidneys are treated with control buffer or with Ret/IgG, then stained with hematoxylin and eosin. The Ret/Ig-treated embryonic kidney exhibits less branching of the collecting ducts than the control buffer-treated embryonic kidneys. In addition, Ret/I gG-treated kidneys have fewer tubules. We have also observed this effect with the human Ret/IgG fusion protein. These observations are consistent with the fusion proteins blocking the inductive signal between the mesenchyme and the ureteric bud. Therefore we conclude that the fusion protein is a good reagent for cloning a RetL.
4. Ret/alkaline phosphatase Fusion Protein
Receptor/alkaline phosphatase (AP) fusion proteins have been used successfully to identify and clone ligands for c-kit (Cell 63: 185, 1990), ligands for members of the eph family of oφhan receptors (Cell 79:157. 1994), and recently to clone a receptor for leptin, the product of the ob gene (Cell 83:1263, 1995). Plasmids encoding the rat Ret/AP fusion protein are constructed and the rat Ret/AP protein is produced in COS7 cells in cell factories. Subsequently, a stable NIH3T3 cell line is generated expressing on average 10 mg/L of fusion protein. SDS- PAGE analysis of the rat Ret/AP protein indicates that its size is consistent with the predicted molecular weight, and gel filtration analysis indicates that it is produced as a dimer. Partial purification is achieved by affinity chromatography on an anti-AP column.
5. Anti-Ret Antibodies
A rabbit polyclonal antibody is generated against the rat Ret/IgG fusion protein. The antibody works on Western blots, FACS analysis of Ret positive cell lines, and immunohistochemistry of embryonic kidney sections.
A panel of hamster anti-rat Ret monoclonal antibodies is generated. Rat Ret/IgG fusion protein, coupled to Protein A Sepharose, is used to immunize Armenian hamsters. 316 clones are obtained after the fusion and screened for their ability to bind rat Ret fusion proteins and/or human IgG in an ELISA assay. 11 clones produce antibodies that bind only to rat Ret/IgG (and rat Ret/AP), but not human IgG. The cross reactivity to human Ret is assayed by FACS; four clones produce antibodies that can bind to the Ret positive human cell line THP-1. The following table summarizes the Ret binding properties of twelve monoclonal antibodies. VL
Figure imgf000024_0001
6. cDNA Expression Libraries We prepare cDNA libraries from rat embryonic kidney, one in the CDM8 vector which utilizes the SV40 origin for amplification, and one in a modified InVitrogen vector, pCEP4, which utilizes the EBV origin for amplification. This modified vector, CH269, has the EBNA-1 gene sequence removed. The EBNA-1 protein interacts with the EBV origin, but the gene is not needed on the vector when cells are used which stably express the EBNA protein. The library in the CDM8 vector contains 1.5 X 106 clones with an average inseπ size of 1.18 kb, while the library in the CH269 vector contains approximately 1 XlO6 clones with an average inseπ size of 1.5 kb.
Expression Cloning of Ret Ligand RetLl
A. Cloning of Rat Ret Ligand RetLl 1. Initial Attempts at Expression Cloning of Ret Ligand RetLl / 2
A number of direct expression methods have been tried to clone RetLl . All of these methods are based on the concept illustrated in Figure 4B. cDNAs from a cDNA library are introduced into mammalian cells; cells that receive RetLl can be identified using the Ret fusion proteins. Although the three approaches described below were unsuccessful, important knowledge and expertise was acquired, which was deployed in a subsequent approach that met with success.
a. Panning Method with Ret/IgG - The rat Ret/IgG fusion protein is used in an attempt to isolate RetLl by direct expression cloning using a panning method (Aruffo and Seed. Proc. Natl. Acad. Sci. 84: 8753-8757 (1987)). A day 18 embryonic rat kidney cDNA library in CDM8 is used for the panning effort. Pools of cDNAs from this library (5,000 - 10,000 cDNAs per pool) are introduced into COS cells using the DEAE-dextran method. After 48 hours, the cells are removed from the plates with EDTA, incubated with the fusion protein, and subsequently panned on plates coated with anti-human IgG, antibody. DNA is recovered from cells that adhered, transformed back into E. coli, and subsequently isolated for a second round of panning. We are unable to see any cells bind after the third round of panning, and very few clones are obtained after transformation of the Hirt DNA back into E. Coli. A VCAM cDNA, used in conjunction with an anti-VCAM monoclonal antibody as a positive control, could only be diluted to a ratio of 1 : 100 and still be detected, indicating that our pool sizes are probably too large. Analysis of some of the clones that are obtained after the second round of panning, indicates that the clones are undergoing rearrangement and deletion.
b. Preparative FACS Method with Ret/IgG - 80,000 cDNA clones from the day 18 embryonic rat kidney library (CDM8 vector) are introduced into COS7 cells and subjected to preparative FACS using the rat Ret/IgG protein followed by a fluor-tagged secondary antibody. The top 0.5% and 0.9% of fluorescing cells are collected and the plasmid DNA is recovered by Hirt lysis. The DNA is electroporated back into E. coli: 228 clones are obtained for the 0.5% pool and 752 clones for the 0.9% pool. DNA is recovered from the bacterial clones and a second round of preparative FACS is performed. Plasmids recovered from bacterial clones at the end of the second round are analyzed and found to contain large deletions and rearrangements. c. Colorimetric Detection Method with Ret/AP - COS cells are transfected with 400 pools of the cDNA clones (1000 clones per pool) from the day 18 rat embryonic kidney cDNA library (CDM8 vector) and stained with the Ret/AP protein and a colorimetric substrate for alkaline phosphatase. The transfected cells are inspected under a microscope for positive signals. In one experiment, five potential positives were re-analyzed, but all were negative.
As a control for the Ret/AP protein, a VCAM/AP protein is produced by fusing the first two domains of human VCAM to the N-terminus of placental AP. (VCAM binds to the integrin VLA4, which is composed of two chains, alpha-4 and beta-1). Transient transfections of COS cells produces sufficient VCAM/AP protein for control experiments. The VCAM/AP protein is compared to VCAM/IgG directly coupled to AP, and to VCAM/lgG plus an AP coupled secondary antibody, in order to assess their ability to detect VLA4 on COS cells transfected with the alpha-4 chain cDNA (COS cells already express the beta-1 chain). The results show that while the VCAM/AP protein could detect VLA4 on transfected cells, the best detection is afforded by the VCAM/IgG protein in combination with an AP coupled secondary antibody.
d. Methodological Conclusions:
Three major conclusions emerged from these initial cloning efforts:
1) Methods which require that plasmid DNA be recovered for subsequent rounds (i.e. panning and preparative FACS) are not suitable when the abundance of the target cDNA is low, because of rearrangements and deletions that occur during these subsequent rounds. Based on the low expression of Ret, there is good reason to suspect that the expression of RetL 1 is also low. The preferred approach is to transfect in pools and use a detection method that allows a positive pool to be identified. The original pool can then be broken down, with no need to recover the transiently expressed DNA from transfected cells.
2) The Ret/IgG protein when coupled to a secondary reagent affords better detection capability than the Ret/AP protein.
3) Control experiments with a VCAM/IgG control protein (and an AP coupled secondary antibody) and the alpha-4 integrin cDNA (diluted into CDM8 vector and transfected into COS cells) indicate that our detection capability is just about one in a thousand (i.e. the pool size cannot exceed 1000 clones). To attain an improved level of sensitivity, we changed from an SV40 origin based vector (expressed in COS cells) to an EBV origin based vector (expressed in EBNA positive cell lines). EBV origin based vectors are maintained as episomes and are not as toxic to the cell as the SV40 origin based vectors after amplification. Considerable evidence exists that genes can be expressed at higher levels in these vectors and that cDNAs can be diluted much further (i.e. up to 1 to 80,000) and still be detected.
2. Screening of Pools from the EBV Origin Based cDNA library
We screen pools of clones from the day 18 rat embryonic kidney cDNA library (CH269 vector with the EBV origin) with the rat Ret/IgG fusion protein. In one experiment, 256 pools, each containing 5000 clones from the library, are generated. Briefly, an aliquot of the cDNA library is titered, 5000 cells are plated (256 times), and are allowed to grow overnight. The colonies are scraped into medium: part of the culture is used to generate a glycerol stock for the pool (stored at -70) and part is used for a plasmid preparation. DNAs from the 256 pools are individually transfected into 293/EBNA cells (8 X 105 on a 60 mm plate) using the lipofection method. After 48 hr. the cells are washed two times with HBHA buffer (0.5 mg/ml BSA, 0.1% NaN3, 20 mM HEPES (pH 7.0)) and incubated with 20 ug/ml rat Ret/IgG in Tris-buffered saline plus 1 mM MgCl2 and CaCl2 for 60-90 min at RT. Following this incubation, the cells are washed four times with HBHA buffer and then fixed with 60% acetone/3% formaldehyde/20 mM HEPES (pH 7.0) for 30 sec. Following two washes with HBS buffer (150 mM NaCl, 20 mM HEPES (pH 7.0)), the cells are incubated with an AP-coupled secondary antibody (goat anti-human IgG Fc-gamma-specific F(ab')2 (Jackson Immuno Research Laboratories; catalog # 109-056-098; 1 :5000 dilution in Tris-buffered saline plus 1 mM MgCl2 and CaCl2) for 60 min at RT. The cells are then washed twice with HBS buffer and twice with AP substrate buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2) containing 2X Pierce Immuno PureR Phosphatase suppressor (catalog #35002). The last wash is left for 15 min. The AP substrates NBT (0.33 mg/ml) and BCIP (0.17 mg/ml) are then added in AP substrate buffer containing the Pierce AP inhibitor and incubated with the cells for 5-20 min. The plates are then washed twice with water. The plates are then inspected under a dissecting microscope for the presence of puφle stained cells.
From an analysis of the 256 pools, 17 positive pools are identified in the primary screen. DNA from each positive pool is re-transfected into 293/EBNA cells and the above procedure repeated along with some additional control experiments to confirm that the staining observed is 2£
Ret/IgG specific. 10 out of the 17 positive pools only show staining with Ret/IgG fusion protein and not with another IgG fusion protein.
3. Breakdown of Pool #230
As an example, one of the above-described positive pools, designated #230, is broken down into smaller subpools in order to identify the cDNA within the pool that is conferring binding to the Ret/IgG fusion protein. 600 cells from the glycerol stock for pool #230 are plated (10 times) and grown overnight. Colonies on these plates are scraped into medium: one tenth of the culture is used to generate a glycerol stock and the remaining portion is used for a DNA preparation. The ten subpools of 600 clones are designated 230-1A through 230-5A and 230-1B through 230-5B. DNAs from these subpools are transfected into 293/EBNA cells and the procedure described above for staining with the Ret/IgG fusion protein is repeated. One subpool #230-5A is positive for staining with the Ret/IgG protein.
Pool #230-5A is further broken down in order to identify the cDNA with this subpool that is conferring binding to the Ret/IgG fusion protein. Cells from the glycerol stock of pool 230-5 A are plated and grown overnight. Colonies are picked into the wells of seven 96-well
Bioblocks® and grown overnight. From each 96-well Bioblock, 4 pools of 20 clones and 1 pool of 16 clones are made. Thus 35 pools are generated from the seven Bioblocks* designated 230- 5A-71 through 230-5A-105. DNAs are prepared from each of these pools and transfected into 293/EBNA cells and re-assayed with the Ret/IgG fusion protein as described above. Pool #230- 5A-86 is positive.
Pool #230-5A-86 is broken down by going back to the Bioblock and identifying the 20 clones that were mixed together to make this pool. DNAs are made from all twenty clones and transfected individually into 293/EBNA cells and re-assayed for Ret/IgG as described above. Pool #230-5A-86-17 is found to be positive.
4. Characterization of Clone #230-5A-86-17
Clone #230-5A-86-17 (called retL-17 or clone 17 and deposited as ATCC 98047) is further analyzed by DNA sequencing. The entire nucleotide sequence of the insert of this clone is SEQ ID NO: 1 (rat retLl cDNA). and part of the nucleotide sequence is shown in Figure 1. Within this nucleotide sequence, we find a reading frame coding for a protein of 468 amino acids (rat RetLl). The predicted protein has a signal sequence with a predicted cleavage after amino acid 24 (Von Heijne et al., Nucl. Acid Res. 14:14683 (1986)). The hydrophobic C-terminus indicates that the protein may be linked to the cell via a phosphatidylinositol glycan linkage. There are three predicted N-linked glycosylation sites. These properties are consistent with those expected for a ligand for Ret.
We can express soluble forms of the rat RetLl protein by truncating the gene prior to the hydrophobic C terminus. For example, this could be done by truncating after Lysine 435 (rat RetLl). Truncation upstream of this amino acid should also result in the expression of a soluble form of the rat RetLl protein. The soluble rat RetLl protein can be expressed by itself or as a part of a fusion with human immunoglobulin. a histidine tag. or a small epitope that is recognized by an antibody. B. Cloning of Human Ret Ligand RetLl
A human embryonic kidney cDNA library in the vector lambda gtlO is purchased from Clontech (catalog #HL5004A). One million plaque forming units from the phage stock are plated on 10 Nunc™ plates. Duplicate plaque lifts are made on Schleicher and Schuell Optitran™ filters.
A probe is generated by digesting plasmid rat RetL 1 with the restriction enzyme PvuII, followed by agarose gel isolation of a 1.34 kb fragment which corresponds to nt 242-1582 of the rat RetL nucleotide sequence (rat retLl cDNA). This coding region probe is P32 labeled by random pnming (Feinberg and Vogelstein. Anal. Biochem. 137:266-267. 1984). The filters are hybridized overnight in 300 ml plaque screen PSB buffer (50 mM Tris pH 7.5, IM NaCl, 0.1 % sodium pyrophosphate, 0.2% PVP, and 0.2% Ficoll) containing 10% dextran sulphate. 100 ug/ml tRNA, and 6.7 X 107 CPM of the rat probe, at 55C. They are washed twice with plaque screen buffer and twice with 2XSSC/1%SDS at 55C and exposed to film at -70C with an intensifying screen.
Duplicate positives are cored from the master plates into SM (100 mM NaCl, 10 mM SO4, 50 mM Tris pH 7.5) plus gelatin. 24 of these positives are plaque purified. Lambda miniprep DNA from the purified candidate plaques is digested with Notl. electrophoresed on 1% agarose gel and Southern blotted. The Southern blot is hybridized with the rat rat RetLl coding region probe. Clone HRL20 has the longest insert (4.4 kb) which hybridizes intensely to the rat probe. DNA sequence (partial human retLl cDNA; SEQ ID NO: 8; Figure 2A) and deduced peptide sequence (partial human RetLl; SEQ ID NO: 9; Figure 2A) have been obtained from this clone , confirming that it is the human homologue. This clone encodes most of the coding region, including the 3' end of the coding region.
To obtain the 5' end of the human cDNA, a human fetal kidney Marathon-Ready™ cDNA kit is purchased from Clontech (catalog #7423-1 ). Antisense oligonucleotides Kid- 155, corresponding to the complement of nucleotides 62-81 of SEQ ID NO: 8 (partial human retLl cDNA) and Kid- 154, corresponding to the complement of nucleotides 17-43 of SEQ ID NO: 8 (partial human retLl cDNA) are synthesized. PCR is performed using the Advantage™ cDNA PCR kit (Clontech catalog #8417-1) combined with Marathon™ cDNA reagents and the oligonucleotides Kid-155 or Kid-154. The first PCR reaction is set up as follows: 35.5 ul H2O; 5.0 ul IOX KlenTaq Buffer: 1.0 ul lOmM dNTP mix; 1.0 ul 50X AdvantageTM KlenTaq Polymerase mix. These reagents are combined and mixed. Then 5.0 ul Marathon-Ready™ Fetal Kidney cDNA; 1.0 ul lOuM API primer and 1.5 ul 6.4uM Kid-155 are added (final volume = 50 ul). PCR is carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following cycle conditions: 1 cycle of 94C for 1 min; 30 cycles of 94C for 30 sec. 55C for 30 sec. 68C for 4 min. A nested PCR is performed using the product of the first PCR reaction. First, 5 ul of PCR product #1 is diluted 50 fold with TE (final volume 250 ul). The nested PCR reaction contains 35.5 ul H2O; 5.0 ul 10X KlenTaq Buffer; 1.0 ul lOmM dNTP mix: 1.0 ul 50X Advantage™ KlenTaq Polymerase mix. These reagents are mixed as above. 5.0 ul diluted PCR product #1 : 1.0 ul lOuM AP2 primer and 1.5 ul 6.9uM Kid-154 are then added. Cycle conditions are the same as above. The resultant product of approximately 700 bp is purified on a 1% low-melt agarose gel and phenol extracted. The purified DNA is cloned into the EcoR5 site of pZErO™ (Invitrogen catalog #K2510-01). Sequence information is obtained from multiple isolates, including clones called HRL7G6 and HRL7G8. The sequence obtained from clone HRL7G8 is found to overlap with the sequence of clone HRL20 (partial human retLl cDNA) and is used to generate a full-length sequence of human RetLl (full-length human retLl cDNA), also shown in Figure 2B. The nucleotide sequence obtained from clone HRL7G8 represents nucleotides 1 to 502 of full-length human retLl cDNA; the nucleotide sequence from clone HRL20 represents nucleotides 460 to 1682 of full-length human retLl cDNA. The sequence from clone HRL7G8 is confirmed by sequencing another cDNA clone (GJ102) isolated from the human embryonic kidney lambda gtlO cDNA library described above, using a probe derived from clone HRL7G6. Nucleotides 1 18 to 1497 comprise the protein reading frame of full-length human retLl cDNA.
The complete amino acid sequence of human RetLl is also shown in Figure 2B. As shown by the BESTFIT analysis depicted in Figure 3A, the human retLl cDNA is 88.2% identical to the rat retLl cDNA. The peptide comparison (Figure 3B) shows the human putative peptide sequence to be 93.3% identical, and 97.2% similar, to that of the rat.
Cloning of Ret Ligand RetL2
A. Cloning of Human RetL2
The peptide sequence of rat RetL 1 (rat RetL 1 ) 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 RetLl peptide sequence is queried against the Expressed Sequence Tag (EST) nucleotide database, two significant matches are obtained. One is with GenBank Accession # R02249, a 229 bp EST from a combined human fetal liver and spleen cDNA library, and the other is with Genbank Accession # HI 2981, a 521 bp EST from a human infant brain cDNA library. The two ESTs share 99% identity in a region of overlap indicating that they are from the same cDNA. Oligonucleotides are generated from the HI 2981 EST: KID-228 (GAA TGA CAA CTG CAA GAA GCT GCG CTC CTC; corresponding to nucleotides 38-67 and also to nucleotides 534-563 of SEQ ID NO: 12), and antisense oligonucleotide KID-229 (GTG TAC TCG CTG GGC ACC CG; coπesponding to the complement of nucleotides 156-175 and also to the complement of nucleotides 652-671 of SEQ ID NO: 12). IX 106 plaque forming units from a Clontech Human Fetal Liver 5' -Stretch Plus lambda
GT10 cDNA library (cat# HL5003a) are screened in duplicate on OPTITRAN™ filters. The filters are hybridized with 32P-labeled oligonucleotides KID-228 and KID-229 in 400 mis plaque screen buffer (50mM Tris pH 7.5. IM NaCl, 0.1% sodium pyrophosphate, 0.2% Polyvinylpryrolidine and 0.2% Ficoll) containing 10% Dextran sulfate and 1 OOug/ml tRNA and 80 pmole each 32P-labeled oligonucleotide at 65C overnight. They are washed twice with 2X SSC/1% SDS and twice with IX SSC/1% SDS and exposed to film. 11 duplicate positives are purified. DNA from each of these clones is analyzed by restriction enzyme digest followed by agarose gel electrophoresis and Southern blotting. The filters are hybridized to KID-228 and KID-229 to confirm that the inserts hybridize to the probe. The insert of clone DSW240 is completely sequenced (human retL2 cDNA, SEQ ID NO: 12) and is shown in Figure 7.
Nucleotides 25-1416 comprise the protein reading frame of human retL2 cDNA. which encodes a protein of 464 amino acids (human RetL2; SEQ ID NO: 13), and is shown in Figure 7. As shown by the BESTFIT analysis depicted in Figure 8, the human RetL2 protein is 49.1% identical and 63.7% similar to the human RetLl protein. It shares in common with human
RetLl a hydrophobic N-terminus indicative of a signal sequence and a hydrophobic C-terminus indicative of a phosphatidylinositol glycan linkage motif. In addition, 30 cysteines out of the 31 that are present in each protein are conserved.
B. Demonstration that RetL2 is a Ligand for Ret We demonstrate that RetL2 is a ligand for Ret by transfecting 293/EBNA cells with an expression plasmid that contains the insert of clone DSW240 and by showing that the cells can bind a soluble Ret/IgG fusion protein.
The insert of DSW240 is removed using Notl and cloned into the expression vector CH269 which contains an EBV origin and allows for high expression in EBNA positive cell lines. Restriction digests are performed to identify clones that have the correct orientation. Plasmid DNA is prepared from a clone having the correct orientation.
Plasmid DNAs (the retL2 expression plasmid, a retL 1 expression plasmid for a positive control, and an expression plasmid containing an unrelated protein for a negative control) are transfected into 293/EBNA cells (8 X 105 on a 60 mm plate) using the lipofection method. After 48 hr, the cells are washed two times with HBHA buffer (0.5 mg/ml BSA, 0.1 % NaN3 20 mM HEPES (pH 7.0)) and incubated with 20 ug/ml rat Ret/IgG in Tris-buffered saline plus 1 mM MgCl2 and CaCl2 for 60-90 min at room temperature. Following this incubation, the cells are washed four times with HBHA buffer and then fixed with 60% acetone/3 % formaldehyde/20 mM HEPES (pH 7.0) for 30 sec. Following two washes with HBS buffer (150 mM NaCl, 20 mM HEPES (pH 7.0)), the cells are incubated with an AP-coupled secondary antibody (goat M
anti-human IgG Fc-gamma-specific F(ab')2 (Jackson Immuno Research Laboratories: catalog # 109-056-098; 1 :5000 dilution in Tris-buffered saline plus 1 mM MgCl2 and CaCl2) for 60 min at RT. The cells are then washed twice with HBS buffer and twice with AP substrate buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2) containing 2X Pierce Immuno Pure®Phosphatase suppressor (catalog #35002). The last wash is left for 15 min. The AP substrates NBT (0.33 mg/ml) and BCIP (0.17 mg/ml) are then added in AP substrate buffer containing the Pierce AP inhibitor and incubated with the cells for 5-20 min. The plates are then washed twice with water. The plates are then inspected under a dissecting microscope for the presence of puφle stained cells. The presence of purple stained cells indicates that the Ret/fusion protein has bound to the cells and that the RetL2 protein is a ligand for Ret. Puφle stained cells are also observed after trasfection with the retLl expression vector but not with the negative control vector.
Cloning of Ret Ligand RetL3 A. Murine RetL3 A search of the EST data base with rat RetLl amino acid sequence discloses two murine
ESTs with homology to Ret ligands. These ESTs are AA049894, and AA050083 (which is partial murine retL3 cDNA, SEQ ID NO: 14). Plasmids encoding these ESTs are obtained from Genome Systems Inc. (Catalog# 475791 and #475497) as bacterial stabs. Plasmid DNA is prepared from single colonies obtained by streaking the stabs onto LB Amp plates. The inserts from these plasmids are sequenced in their entirety. Comparison of the two sequences demonstrates that AA049894, which has a 1.4 kb insert, is contained within AA050083. which has a 1.9 kb insert. Translation of the DNA sequence from AA050083 indicates there is a continuous open reading frame from NT205 to NT1242 (partial murine RetL3; SEQ ID NO:15). This ORF had 37.5% identity to that of rat retLl and 40.2% identity to rat retL2. However, the open reading frame does not encode a Met or a signal sequence at the 5' end. We examine the 5' ORFs upstream of this region and find a Met in the context of a Kozak consensus sequence for translation iniation and a potential signal sequence for surface expression/secretion. This ORF is out of frame with the downstream ORF indicating that EST AA050083 contains a potential mutation, such as an insertion, deletion, intron or cloning artifact, at its 5' end. 3'Z.
In order to obtain the correct 5' end, we employ Marathon RACE. Mouse 1 1-day embryo Marathon-Ready™ cDNA (cat.#7458-l) and an Advantage™ Kit (cat.# 8417-1 ) is purchased from Clontech. Antisense oligonucleotides, Kid-366, corresponding to the complement of nucleotides 847-866 of SEQ ID NO: 14 and Kid-365, corresponding to the complement of nucleotides 589-615 of SEQ ID NO: 14 are synthesized. PCR is performed using and an Advantage™ cDNA PCR kit (Clontech cat.#8417-l) combined with Marathon™ cDNA reagents and oligonucleotide Kid-366. The first PCR reaction is set up as follows: 35.3 ul H2O; 5.0 ul IOX KlenTaq Buffer; l.Oul lOmM dNTP mix; 1.0 ul 50X Advantage™ KlenTaq Polymerase mix. These reagents are combined and mixed. Then 5.0 ul Marathon-Ready™ mouse 1 1-day embryo cDNA; 1.0 ul lOuM API primer and 1.7ul 5.88uM Kid-366 are added (final volume = 50ul). PCR is carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following cycle conditions: 1 cycle of 94C for 1 min; 5 cycies of 94C for 30 sec, 72C for 4 min; 5 cycles of 94C for 30 sec, 70C for 4 min; 25 cycles 94C for 30 sec, 68C for 4 min. A nested PCR is performed using the product of the first PCR reaction. First, 5 ul of PCR product#l is diluted 50 fold with TE( Final volume 250 ul). The nested PCR reaction contains 35.5 ul H2O; 5.0 ul 10X KlenTaq Buffer; 1.0 ul lOmM dNTP mix; 1.0 ul 50X Advantage™ KlenTaq Polymerase mix. These reagents are mixed as above. 5.0 ul diluted PCR product #1 ; 1.0 ul lOuM AP2 primer and 3.6 ul 2.8uM Kid-365 are then added. Cycle conditions are the same as above. The resultant product of approximately 665 bp is purified on a 1 % low-melt agarose gel and Qiaex II (Qiagen cat#20021) extracted. The purified DNA is cloned into pNoTA/T7™ using PRIME PCR CLONER™ cloning system (5 Prime-> 3 Prime cat.# 1-725029). Sequence information is obtained from multiple isolates, including clones called DSW252 and DSW253.
The sequence of DSW252 is found to overlap with SEQ ID NO:14 except that an additional T is present between NT 252 and NT 253 of the SEQ ID NO: 14 sequence. This T is also present in the other isolates DSW251 and DSW253. Insertion of this additional base corrects the ORF such that a single 1191 bp ORF (counting from the first Met) encoding 397 amino acids is obtained. This ORF encodes a Met in the context of a canonical translation initiation consensus sequence (Kozak) and includes a signal sequence for surface expression/secretion. To obtain a full-length murine clone which is capable of being expressed, a 630 bp Notl-
BamHl fragment of DSW252 and a 1308 bp BamHl-Notl fragment of AA050083 is purified S 3
and ligated to Not 1 digested expression vector CH269. The ligation is transformed into E.coli XL 1 -Blue (Strategene cat. #200236). Qiawell Ultra minipreps are performed on resultant transformants. These are analysed by restriction digest and gel electrophoresis for correct size and for orientation. This construct is called DSW254. The insert of DSW254 is sequenced in its entirety (murine retL3; SEQ ID NO: 16) and the ORF is confirmed as encoding a protein of 397 amino acids (murine RetL3; SEQ ID NO: 17). These sequences are also shown in Figure 9. The C-terminus of RetL3 is hydrophobic and indicative of a phosphatidylinositol glycan linkage motif.
B. Human RetL3 In order to find a candidate tissue source for cloning human RetL3, we utilize northern blots of mouse tissues to determine the expression pattern of murine RetL3. Of the tissues surveyed, expression of RetL3 is highest in heart tissue. A human adult heart cDNA library in the vector lambda gtlO is purchased from Clontech (catalog# HL3026a). One million plaque forming units from the phage stock are plated on 10 Nunc plates. Duplicate plaque lifts are made on Schleicher and Schuell Optitran™ filters.
A probe is generated by PCR with primers Kid-366 and Kid-367. corresponding to nucleotides 397-420 of the AA05OO83 sequence. PCR reaction is set up as follows: 10 ul 10X PFU Buffer. 2.0 ul lOmM dNTP mix, 72.1 ul H2O, 3.1 ul 13.2 uM Kid-367. 6.8 ul 5.88 uM Kid- 366, 5.0 ul 0.1 ug/ ul AA050083 DNA and 2.0 ul 2.5 Units/ul PFU (Stategene catalog # 600154) are mixed. PCR is carried out in Perkin-Elmer Cetus DNA Thermal Cycler 480 with the following conditions: 25 cycles of 94 C for 1 min, 53C for 1 min.. 72C for 4 min. The product is purified by extraction with phenol, chloroform, isoamyl alcohol 50:49: 1 followed by low-melt agarose gel electrophoresis and QiaexII purification of the excised fragment. This coding region probe is P32 labeled by random priming (Feinberg and Vogelstein). The filters are hybridized overnight in 200 ml Plaque screen buffer () containing 10% dextran sulphate, 100 ug/ml tRNA and 1.8 X 108 CPM of the mouse probe at 65C. They are washed twice with plaque screen buffer, twice with 2XSSC/1% SDS, twice with 1XSSC/1%SDS at 65C and exposed to film at - 70C with an intensifying screen. Duplicate positives are plaque purified. Lambda miniprep DNA from the purified candidate plaques is digested with EcoRl, electrophoresed on a 1% agarose gel and Southern blotted. The Southern blot is hybridized with the mouse probe. Clone GJ128 , 3 V
which has a 1.3 kb insert, hybridizes intensly to the mouse coding region probe. DNA sequence (partial human retL3 cDNA; SEQ ID NO:l 8) and deduced peptide sequence (partial human RetL3; SEQ ID NO: 19) are obtained from this clone, confirming that it is the human homologue. This clone encodes most of the coding region, including the 3' end of the coding region. The 1.3 kb insert from GJ128 is purified, labeled with P32 and used to screen the Clontech human adult heart library in order to obtain a clone with the 5' end. No clones containing the 5' end are obtained in a screen of 2 X 106 plaques from this library. Northern analysis of human adult tissue mRNA blots (Clontech catalog # 7760-1, 7759-1 and 7767-1) hybridized with the same probe, using protocols supplied by manufacturer, indicates that human RetL3 is expressed in human adult spinal cord, stomach, heart, pancreas, small intestine, colon, prostate and testis. A Clontech human adult spinal cord cDNA library (catalog # 5001a) is screened with GJ128 insert. 3 independant clones are purified and the longest, GJ135 is sequenced. The sequence of the insert of GJ135 overlaps with the insert of GJ128. allowing the generation of a composite sequence of the full-length human retL3 cDNA (SEQ ID NO:20) and the determination of the full-length human RetL3 (SEQ ID NO:21). These sequences are also shown in Figure 10.
Human RetL3 is 34.3% and 34.9% identical to human RetLl and human RetL2, respectively. It has 76.8% identity with murine RetL3.
THERAPEUTIC USES OF THE COMPOUNDS OF THE INVENTION
Native and variant RetL's. anti-RetL antibodies. anti-Ret antibodies, and fusion proteins of Ret and of RetL's 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 RetL.
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 incoφorated 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 HIV -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. These disorders include those caused by any of the factors previously listed, and specifically include Lyme disease, HIV-associated neuropathies, polymyositis, muscular dystrophy, and myasthenia gravis.
Anti-RetL antibodies and Ret fusion proteins of the invention, which specifically bind to the protein of rat RetL, partial human RetLl. full-length human RetLl. human RetL2. murine RetL3 or human RetL3, or fragments of these proteins, 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 RetL. 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 RetL-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-immunoglobulin antibody which is 3£
itself bound to a detectable marker. Specific antibodies to any RetL are also useful in immunoassays to quantify the substance for which a given antibody has specificity. Specific antibodies to a RetL 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.
Other methods of the invention include modulating Ret- RetL 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 RetL's. or by complexes comprising RetL's, which would otherwise activate the Ret signaling pathway.
A RetL 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 immunohistological or preparative methods described above for antibodies to a RetL.
Fusion proteins encompassing a RetL 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 RetL utilize fusion proteins which incoφorate Ret and/or an anti-RetL antibody. The anti-Ret antibody or anti-RetL 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 RetL 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 RetL 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 RetL or to an 2 1
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-RetL antibody to selectively target and kill RetL-positive cells, such as a tumor expressing a RetL. Such an approach has proved successful with blocked ricin conjugated to a monoclonal antibody against the CD 19 antigen expressed on most neoplastic cells (Grossbard et al., Blood 79:576,1992). 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. This approach should prove even more successful using a RetL or anti-Ret antibody, as contrasted to the known anti-CD 19 antigen approach, because Ret is expressed in a very limited number of tissues. The above approaches, using fusions of ricin or other toxins, are equally applicable to toxic conjugates of RetL 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 RetL 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 α particles may also be employed. The low dose rate irradiation generated by radionuclide labeled RetL or anti-Ret antibodies may be more therapeutically effective than the instantaneous iπadiation delivered externally in conventional radiation therapy. Low dose rate iπadiation 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 RetL genes of the invention are introduced into damaged tissue to stimulate production of a RetL 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 RetL gene may be introduced into a renal or neural target tissue of choice. A RetL would then be stably expressed and stimulate Ret receptor-positive cells to grow, divide, differentiate, and/or potentiate cell survival. Furthermore, RetL 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 RetL 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., LIPOFECTTN-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, -3π
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 incoφorated 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. £x£. Med.. 169: 13 (1989)); the human beta-actin promoter (Gunning et al., Proc. Nat. Acad. Sci. USA. 84: 4831 (1987); the glucocoπicoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al., Mfil- Cell. Bjol, 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., EXQC_. Nai. Acad. Sci. USA, 82: 3567 (1985)).
The RetL genes 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., Cuπ. Top. Microbiol. Immunol.. 158: 39-61. 1992 (adenovirus); Hofmann et al., Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Cuπ. Top. Microbiol. Immunol.. 158: 25-38, 1992 (vaccinia virus); Muzyczka. Cuπ. Top. Microbiol. Immunol.. 158: 97-123, 1992 ( adeno-associated virus); Margulskee, Cuπ. Top. Microbiol. Immunol.. 158: 67-93. 1992 (heφes simplex virus (HSV) and Epstein-Ban virus (HBV)); Miller, Cuπ. 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 iretrovirusl: Anderson. Science. 256: 808-813. 1992 (retrovirus), Cuπent 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.
Prefeπed 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, c,0
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, a RetL 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 SΆ 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); 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 M
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 El a and part of the El b 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. AH 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 B.J. 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 (BJ. 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 HIV-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 RetL may be performed in vivo by introducing a RetL into cells in need of such treatment using liposomes . The liposomes can be delivered via catheter to the renal artery. The recombinant RetL 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 immuno genicity. Delivery of the novel polypeptides via liposomes may be particularly advantageous because the liposome may be internalized by phagocytic cells in die 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 RetL 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 an, and include inorganic and organic acids and bases.
Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious to one skilled in the art that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.
U5
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(i) APPLICANT: doherty, jodi (ii) TITLE OF INVENTION: biogen patent application title (iii) NUMBER OF SEQUENCES: 21
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(D) SOFTWARE: Patentln Release #1.0, Version #1.30
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(A) APPLICATION NUMBER:
(B) FILING DATE: 07-MAY-1997
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(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/999,999
(B) FILING DATE: 01-JAN-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Levine, Leslie M.
(B) REGISTRATION NUMBER: 35,245
(C) REFERENCE/DOCKET NUMBER: A008 PCT CIP
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO
( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 257..1660
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCGGCCGCAG GTTGGGTCGG AACTGAACCC CTGAAAGCGG GTCCGCCTCC CGCCCTCGCG 60
CCCGCCCGGA TCTGAGTCGC TGGCGGCGGT GGGCGGCAGA GCGACGGGGA GTCTGCTCTC 120
ACCCTGGATG GAGCTGAACT TTGAGTGGCC AGAGGAGCGC AGTCGCCCGG GGATCGCTGC 180
ACGCTGAGCT CTCTCCCCGA GACCGGGCGG CGGCTTTGGA TTTTGGGGGG GCGGGGACCA 240
GCTGCGCGGC GGCACC ATG TTC CTA GCC ACT CTG TAC TTC GCG CTG CCA 289
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro 1 5 10
CTC CTG GAT TTG CTG ATG TCC GCC GAG GTG AGT GGT GGA GAC CGT CTG 337 Leu Leu Asp Leu Leu Met Ser Ala Glu Val Ser Gly Gly Asp Arg Leu 15 20 25
GAC TGT GTG AAA GCC AGC GAT CAG TGC CTG AAG GAA CAG AGC TGC AGC 385 Asp Cys Val Lys Ala Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser 30 35 40
ACC AAG TAC CGC ACA CTA AGG CAG TGC GTG GCG GGC AAG GAA ACC AAC 433 Thr Lys Tyr Arg Thr Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn 45 50 55
TTC AGC CTG ACA TCC GGC CTT GAG GCC AAG GAT GAG TGC CGT AGC GCC 481 Phe Ser Leu Thr Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala 60 65 70 75
ATG GAG GCC TTG AAG CAG AAG TCT CTG TAC AAC TGC CGC TGC AAG CGG 529 Met Glu Ala Leu Lys Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg 80 85 90
GGC ATG AAG AAA GAG AAG AAT TGT CTG CGT ATC TAC TGG AGC ATG TAC 577 Gly Met Lys Lys Glu Lys Asn Cys Leu Arg lie Tyr Trp Ser Met Tyr 95 100 105
CAG AGC CTG CAG GGA AAT GAC CTC CTG GAA GAT TCC CCG TAT GAG CCG 625 Gin Ser Leu Gin Gly Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro 110 115 120
GTT AAC AGC AGG TTG TCA GAT ATA TTC CGG GCA GTC CCG TTC ATA TCA 673 Val Asn Ser Arg Leu Ser Asp lie Phe Arg Ala Val Pro Phe lie Ser 125 130 135 0-7
GAT GTT TTC CAG CAA GTG GAA CAC ATT TCC AAA GGG AAC AAC TGC CTG 721 Asp Val Phe Gin Gin Val Glu His lie Ser Lys Gly Asn Asn Cys Leu 140 145 150 155
GAC GCA GCC AAG GCC TGC AAC CTG GAC GAC ACC TGT AAG AAG TAC AGG 769 Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Thr Cys Lys Lys Tyr Arg 160 165 170
TCG GCC TAC ATC ACC CCC TGC ACC ACC AGC ATG TCC AAC GAG GTC TGC 817 Ser Ala Tyr lie Thr Pro Cys Thr Thr Ser Met Ser Asn Glu Val Cys 175 180 185
AAC CGC CGT AAG TGC CAC AAG GCC CTC AGG CAG TTC TTC GAC AAG GTT 865 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Lys Val 190 195 200
CCG GCC AAG CAC AGC TAC GGG ATG CTC TTC TGC TCC TGC CGG GAC ATC 913 Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp lie 205 210 215
GCC TGC ACC GAG CGG CGG CGA CAG ACT ATC GTC CCC GTG TGC TCC TAT 961 Ala Cys Thr Glu Arg Arg Arg Gin Thr lie Val Pro Val Cys Ser Tyr 220 225 230 235
GAA GAA CGA GAG AGG CCC AAC TGC CTG AGT CTG CAA GAC TCC TGC AAG 1009 Glu Glu Arg Glu Arg Pro Asn Cys Leu Ser Leu Gin Asp Ser Cys Lys 240 245 250
ACC AAT TAC ATC TGC AGA TCT CGC CTT GCA GAT TTT TTT ACC AAC TGC 1057 Thr Asn Tyr lie Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys 255 260 265
CAG CCA GAG TCA AGG TCT GTC AGC AAC TGT CTT AAG GAG AAC TAC GCA 1105 Gin Pro Glu Ser Arg Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Ala 270 275 280
GAC TGC CTC CTG GCC TAC TCG GGA CTG ATT GGC ACA GTC ATG ACT CCC 1153 Asp Cys Leu Leu Ala Tyr Ser Gly Leu lie Gly Thr Val Met Thr Pro 285 290 295
AAC TAC GTA GAC TCC AGC AGC CTC AGC GTG GCA CCA TGG TGT GAC TGC 1201 Asn Tyr Val Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys 300 305 310 315
AGC AAC AGC GGC AAT GAC CTG GAA GAC TGC TTG AAA TTT CTG AAT TTT 1249 Ser Asn Ser Gly Asn Asp Leu Glu Asp Cys Leu Lys Phe Leu Asn Phe 320 325 330
TTT AAG GAC AAT ACT TGT CTC AAA AAT GCA ATT CAA GCC TTT GGC AAT 1297 Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala lie Gin Ala Phe Gly Asn 335 340 345
GGC TCA GAT GTG ACC ATG TGG CAG CCA GCC CCT CCA GTC CAG ACC ACC 1345 Gly Ser Asp Val Thr Met Trp Gin Pro Ala Pro Pro Val Gin Thr Thr 350 355 360 ACT GCC ACC ACT ACC ACT GCC TTC CGG GTC AAG AAC AAG CCT CTG GGG 1393 Thr Ala Thr Thr Thr Thr Ala Phe Arg Val Lys Asn Lys Pro Leu Gly 365 370 375
CCA GCA GGG TCT GAG AAT GAG ATC CCC ACA CAC GTT TTA CCA CCC TGT 1441 Pro Ala Gly Ser Glu Asn Glu lie Pro Thr His Val Leu Pro Pro Cys 380 385 390 395
GCG AAT TTG CAG GCT CAG AAG CTG AAA TCC AAT GTG TCG GGT AGC ACA 1489 Ala Asn Leu Gin Ala Gin Lys Leu Lys Ser Asn Val Ser Gly Ser Thr 400 405 410
CAC CTC TGT CTT TCT GAT AGT GAT TTC GGA AAG GAT GGT CTC GCT GGT 1537 His Leu Cys Leu Ser Asp Ser Asp Phe Gly Lys Asp Gly Leu Ala Gly 415 420 425
GCC TCC AGC CAC ATA ACC ACA AAA TCA ATG GCT GCT CCT CCC AGC TGC 1585 Ala Ser Ser His lie Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys 430 435 440
AGT CTG AGC TCA CTG CCG GTG CTG ATG CTC ACC GCC CTT GCT GCC CTG 1633 Ser Leu Ser Ser Leu Pro Val Leu Met Leu Thr Ala Leu Ala Ala Leu 445 450 455
TTA TCT GTA TCG TTG GCA GAA ACG TCG TAGCTGCATC CGGGAAAACA 1680 Leu Ser Val Ser Leu Ala Glu Thr Ser 460 465
GTATGAAAAG ACAAAAGAGA ACCAAGTATT CTGTCCCTGT CCTCTTGTAT ATCTGAAAAT 1740
CCAGTTTTAA AAGCTCCGTT GAGAAGCAGT TTCACCCAAC TGGAACTCTT TCCTTGTTTT 1800
TAAGAAAGCT TGTGGCCCTC AGGGGCTTCT GTTGAAGAAC TGCTACAGGG CTAATTCCAA 1860
ACCCATAAGG CTCTGGGGCG TGGTGCGGCT TAAGGGGACC ATTTGCACCA TGTAAAGCAA 1920
GCTGGGCTTA TCATGTGTTT GATGGTGAGG ATGGTAGTGG TGATGATGAT GGTAATTTTA 1980
ACAGCTTGAA CCCTGTTCTC TCTACTGGTT AGGAACAGGA GATACTATTG ATAAAGATTC 2040
TTCCATGTCT TACTCAGCAG CATTGCCTTC TGAAGACAGG CCCGCAGCCT AGTGTGAATG 2100
ACAAGTGGAG GTTGGCCTCA AGAGTGGACT TGGCAGACTC TACCTTGTAG TAATGTTCAC 2160
CTTTCCGTGT ATGGTCTCCA CAGAGTGTTT ATGTATTTAC AGACTGTTCT GTGATCCCCC 2220
AACAACAACA ACCACAAATT CCTTGGTCAC CTCCAAATGT AACCGGTCCT TTAGCCCAGT 2280
AGAGGAGGGT GGGTGTGGCC CTGGCACAGC TCCCGGATTG TTGATGGGCA CTCTCCTGAG 2340
CTTTGCTTGA GTGAGAAGCT GAATGTAGCT GAAAATCAAC TCTTCTTACA CTTCTTACTG 2400
CTTCGTTCAC TTACGAGGTC ACATATAGAA CAAACATCAC CAACTATTAG CTTACCGTTA 2460 GCTTCCCAAC TATTAGCTTT CTATGTTTTG AAAGCAGTGT TGCTGACCCC ATGTTTTAAT 2520
GATGGTTTAA TACATGCAGC CCTTTCCTCT CATCGGTAAC ACTAGCTCCA ACATCAACTT 2580
CATGCATGTG GCTCTCAAAA GCAGGCCCCA AGAAGCCCAG TTCTTTAGGA GAAAGCTGCG 2640
TCCTGTTTCT GTGGACAGGC AGGAGGAAAC AGAGCAGCCT GCCCGTGGTG TCTTTATCTG 2700
TTTTGAAATC AAGGCTGCCT GTGTGTAAGG AATGGTTCAA TTCTTATAAA GGGTGCCACT 2760
GTTGATGCCA CAACTGGCAG TTGGTCTAGC TCCAGGACAC CGGTTTCCAT GTTGCCTGGC 2820
AGAGACAGCT TTGATTGGGA CTGGCTGGCC ACAAGGGATG GGATGAAGAT GTGCTGCCCT 2880
CTCTTTCAAA GTTGAGCCCT GCCAGGGCAC ATAGAAGCAT CTTTGCTCCT GACCACAACG 2940
TAGAACAGCT TGGATTCAAG GTCATCAAGC GTCTCCTGTA CATTGCTCTG TGACCTTCAT 3000
AACAGACTGT CCCGCACAAA AGGAACGGCA GTTTATGGAT CTAGAGTGGG AGCACAGGGT 3060
CTGGAAAGGT GAACCGATTG GCAAAATACA CAGAACAGGA GGGAGAGTCT CAAGCCGAGA 3120
CATCTTGCTT ACTAGCCACA CACCATCTCC TGGAGCCCTC CTCCTGACCT GGGCAGACCC 3180
TTAGGTGTAT ATCTAAAGAC CTCTTCAATG TTCAGGTTCA GAATCTGTAA ATGGTTGCGT 3240
CCTGGCACCC ATTCCTGAAA ACTGAACAAA GGAGAGGATA TCTTTCCTCC ATTGAGCCCT 3300
GAAAGTATGA CTGGCTTCTC ACCCTCCCAC AGAGCAGGGA GCCCTGGTGC ACACAGTCTC 3360
CTGATATCCT CCCTGCTCTT TGAGGTTTGC CTTGGGAGAA AATGATTCAC CTCGGGAGGG 3420
GACGCTTTGG TGTCTGAAGT ACGTTTATAT CGAAATGTTA ATGAATACCC ATGTAAAATA 3480
CTCAATAGCC ACCTTTCTTC CCTTCACAAT GTTTTCGAGG GGAATGCATC CAACATCCAA 3540
GTGTACCTGG TCAGTGGGAA GTTCCATGAA GACTCATACA TTGAATAAAC ATATTCGATG 3600
TGCCGAAAGC GGCCGC 3616
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 Met Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30
Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45
Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Thr Ser 50 55 60
Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80
Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95
Lys Asn Cys Leu Arg lie Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125
Ser Asp lie Phe Arg Ala Val Pro Phe lie Ser Asp Val Phe Gin Gin 130 135 140
Val Glu His He Ser Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160
Cys Asn Leu Asp Asp Thr Cys Lys Lys Tyr Arg Ser Ala Tyr He Thr 165 170 175
Pro Cys Thr Thr Ser Met Ser Asn Glu Val Cys Asn Arg Arg Lys Cys 180 185 190
His Lys Ala Leu Arg Gin Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp He Ala Cys Thr Glu Arg 210 215 220
Arg Arg Gin Thr He Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Arg 225 230 235 240
Pro Asn Cys Leu Ser Leu Gin Asp Ser Cys Lys Thr Asn Tyr He Cys 245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gin Pro Glu Ser Arg 260 265 270
Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala 275 280 285
Tyr Ser Gly Leu He Gly Thr Val Met Thr Pro Asn Tyr Val Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320
Asp Leu Glu Asp Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335
Cys Leu Lys Asn Ala He Gin Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350
Met Trp Gin Pro Ala Pro Pro Val Gin Thr Thr Thr Ala Thr Thr Thr 355 360 365
Thr Ala Phe Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380
Asn Glu He Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gin Ala 385 390 395 400
Gin Lys Leu Lys Ser Asn Val Ser Gly Ser Thr His Leu Cys Leu Ser 405 410 415
Asp Ser Asp Phe Gly Lys Asp Gly Leu Ala Gly Ala Ser Ser His He 420 425 430
Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Ser Leu Ser Ser Leu 435 440 445
Pro Val Leu Met Leu Thr Ala Leu Ala Ala Leu Leu Ser Val Ser Leu 450 455 460
Ala Glu Thr Ser 465
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : AAGGAAAAAA GCGGCCGCCA TGGCGAAGGC GACGTCCGG 39
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGTTTTGTCG ACCGTGCGGC ACAGCTCGTC GCA 33
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : AGTTTTGTCG ACCGTGCGGC ACAGCGCATC ACA 33
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1926 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..1920
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :
GCGGCCGCC ATG GCG AAG GCG ACG TCC GGC GCC GCA GGG CTG GGG CTG 48
Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Gly Leu 470 475 480
AAG CTG TTT TTG CTG CTG CCG CTA CTG GGA GAA GCC CCG CTG GGT CTC 96 Lys Leu Phe Leu Leu Leu Pro Leu Leu Gly Glu Ala Pro Leu Gly Leu 485 490 495 TAC TTC TCA AGG GAT GCT TAC TGG GAG AGG CTG TAT GTG GAC CAG CCA 144
Tyr Phe Ser Arg Asp Ala Tyr Trp Glu Arg Leu Tyr Val Asp Gin Pro
500 505 510
GCT GGC ACA CCT CTG CTC TAT GTC CAT GCC CTA CGG GAT GCC CCT GGA 192
Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro Gly 515 520 525
GAA GTG CCC AGC TTC CGC CTG GGC CAG TAT CTC TAT GGC GTC TAC CGC 240
Glu Val Pro Ser Phe Arg Leu Gly Gin Tyr Leu Tyr Gly Val Tyr Arg 530 535 540 545
ACG CGT CTG CAT GAG AAT GAC TGG ATC CAC ATC GAT GCG GGC ACT GGC 288
Thr Arg Leu His Glu Asn Asp Trp He His He Asp Ala Gly Thr Gly 550 555 560
CTC CTC TAC CTC AAT CAG AGC CTG GAC CAT AGT TCC TGG GAG CAG CTC 336
Leu Leu Tyr Leu Asn Gin Ser Leu Asp His Ser Ser Trp Glu Gin Leu
565 570 575
AGC ATC CGA AAT GGC GGC TTC CCC TTG CTC ACC GTC TTC CTC CAG GTC 384
Ser He Arg Asn Gly Gly Phe Pro Leu Leu Thr Val Phe Leu Gin Val
580 585 590
TTC CTG GGG TCC ACA GCC CAG AGA GAG GGA GAG TGT CAT TGG CCA GGC 432
Phe Leu Gly Ser Thr Ala Gin Arg Glu Gly Glu Cys His Trp Pro Gly 595 600 605
TGT GCC CGT GTG TAC TTC TCC TTC ATC AAC GAC ACC TTC CCA AAT TGT 480
Cys Ala Arg Val Tyr Phe Ser Phe He Asn Asp Thr Phe Pro Asn Cys 610 615 620 625
AGC TCC TTC AAA GCC CGG GAT CTC TGC ACC CCA GAG ACG GGT GTG TCC 528
Ser Ser Phe Lys Ala Arg Asp Leu Cys Thr Pro Glu Thr Gly Val Ser 630 635 640
TTC CGC ATC AGG GAG AAC AGG CCC CCT GGC ACC TTC TAC CAG TTC CGC 576
Phe Arg He Arg Glu Asn Arg Pro Pro Gly Thr Phe Tyr Gin Phe Arg
645 650 655
ATG CTA CCT GTG CAG TTC CTT TGT CCT AAC ATC AGT GTG AAG TAC AAA 624
Met Leu Pro Val Gin Phe Leu Cys Pro Asn He Ser Val Lys Tyr Lys
660 665 670
CTC TTA GAA GGG GAC GGT CTG CCC TTC CGT TGT GAC CCC GAC TGT CTG 672
Leu Leu Glu Gly Asp Gly Leu Pro Phe Arg Cys Asp Pro Asp Cys Leu 675 680 685
GAG GTG AGC ACG CGG TGG GCA CTG GAT CGG GAG CTT CAG GAG AAG TAT 720
Glu Val Ser Thr Arg Trp Ala Leu Asp Arg Glu Leu Gin Glu Lys Tyr 690 695 700 705
GTG CTG GAG GCT GAG TGC GCA GTG GCA GGC CCT GGA GCC AAC AAG GAG 768
Val Leu Glu Ala Glu Cys Ala Val Ala Gly Pro Gly Ala Asn Lys Glu 710 715 720 AAG GTG GCC GTG TCC TTC CCG GTG ACG GTG TAT GAT GAA GAC GAC TCC 816 Lys Val Ala Val Ser Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser 725 730 735
CCG CCC ACC TTC TCC GGA GGT GTG GGC ACC GCC AGT GCT GTG GTG GAG 864 Pro Pro Thr Phe Ser Gly Gly Val Gly Thr Ala Ser Ala Val Val Glu 740 745 750
TTT AAG CGG AAG GAG GGC ACT GTG GTA GCC ACT CTG CAG GTG TTT GAT 912 Phe Lys Arg Lys Glu Gly Thr Val Val Ala Thr Leu Gin Val Phe Asp 755 760 765
GCA GAT GTG GTG CCA GCA TCT GGG GAG CTG GTG AGG CGG TAC ACA AGC 960 Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr Ser 770 775 780 785
ACA CTA CTC TCA GGG GAT TCC TGG GCC CAG CAG ACC TTC CGG GTG GAG 1008 Thr Leu Leu Ser Gly Asp Ser Trp Ala Gin Gin Thr Phe Arg Val Glu 790 795 800
CAC ACA CCC AAC GAG ACC TTG GTC CAG TCC AAC AAC AAC TCC GTG CGG 1056 His Thr Pro Asn Glu Thr Leu Val Gin Ser Asn Asn Asn Ser Val Arg 805 810 815
GCA ACC ATG CAC AAT TAC AAG CTG GTT CTC AAC AGG AGC CTG TCC ATC 1104 Ala Thr Met His Asn Tyr Lys Leu Val Leu Asn Arg Ser Leu Ser He 820 825 830
TCA GAG AGC CGA GTC CTG CAG CTA GTA GTC CTG GTC AAT GAC TCA GAC 1152 Ser Glu Ser Arg Val Leu Gin Leu Val Val Leu Val Asn Asp Ser Asp 835 840 845
TTC CAG GGG CCT GGG TCA GGT GTT CTC TTC CTC CAT TTC AAC GTG TCT 1200 Phe Gin Gly Pro Gly Ser Gly Val Leu Phe Leu His Phe Asn Val Ser 850 855 860 865
GTG CTG CCT GTC ACC CTG AAC CTA CCC ATG GCC TAC TCC TTC CCA GTG 1248 Val Leu Pro Val Thr Leu Asn Leu Pro Met Ala Tyr Ser Phe Pro Val 870 875 880
AAT AGG AGA GCC CGC CGT TAT GCC CAG ATT GGG AAA GTT TGC GTG GAG 1296 Asn Arg Arg Ala Arg Arg Tyr Ala Gin He Gly Lys Val Cys Val Glu 885 890 895
AAC TGC CAG GAG TTC AGC GGT GTC TCC ATC CAG TAC AAG CTG CAG CCC 1344 Asn Cys Gin Glu Phe Ser Gly Val Ser He Gin Tyr Lys Leu Gin Pro 900 905 910
TCC AGC ACC AAC TGC AGT GCC CTA GGT GTG GTC ACC TCA ACA GAA GAC 1392 Ser Ser Thr Asn Cys Ser Ala Leu Gly Val Val Thr Ser Thr Glu Asp 915 920 925 ACC TCA GGG ACC CTA TAT GTA AAT GAC ACG GAG GCC CTG CGG CGA CCT 1440 Thr Ser Gly Thr Leu Tyr Val Asn Asp Thr Glu Ala Leu Arg Arg Pro 930 935 940 945
GAG TGT ACC GAG CTT CAG TAC ACA GTG GTA GCC ACT GAC CGG CAG ACC 1488 Glu Cys Thr Glu Leu Gin Tyr Thr Val Val Ala Thr Asp Arg Gin Thr 950 955 960
CGC AGG CAG ACC CAA GCT TCG TTA GTC GTC ACA GTG GAG GGG ACA TAC 1536 Arg Arg Gin Thr Gin Ala Ser Leu Val Val Thr Val Glu Gly Thr Tyr 965 970 975
ATT GCA GAA GAA GTG GGC TGC CCC AAG TCC TGT GCA GTA AAC AAG AGG 1584 He Ala Glu Glu Val Gly Cys Pro Lys Ser Cys Ala Val Asn Lys Arg 980 985 990
CGA CCT GAG TGT GAG GAG TGT GGT GGC CTG GGT TCT CCA ACT GGC AGA 1632 Arg Pro Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg 995 1000 1005
TGT GAG TGG CGT CAG GGA GAT GGT AAA GGG ATC ACC AGG AAC TTC TCC 1680 Cys Glu Trp Arg Gin Gly Asp Gly Lys Gly He Thr Arg Asn Phe Ser 1010 1015 1020 1025
ACC TGT TCT CCT AGC ACC AGG ACC TGT CCT GAT GGC CAC TGT GAT GCT 1728 Thr Cys Ser Pro Ser Thr Arg Thr Cys Pro Asp Gly His Cys Asp Ala 1030 1035 1040
CTG GAG AGC CGG GAT ATC AAC ATT TGC CCC CAG GAC TGT CTC CGT GGC 1776 Leu Glu Ser Arg Asp He Asn He Cys Pro Gin Asp Cys Leu Arg Gly 1045 1050 1055
CCC ATT GTT GGC GGG CAT GAG CGA GGG GAG CGC CAG GGG ATT AAA GCC 1824 Pro He Val Gly Gly His Glu Arg Gly Glu Arg Gin Gly He Lys Ala 1060 1065 1070
GGC TAT GGC ATC TGC AAC TGT TTC CCT GAT GAG AAG AAG TGC TTC TGC 1872 Gly Tyr Gly He Cys Asn Cys Phe Pro Asp Glu Lys Lys Cys Phe Cys 1075 1080 1085
GAG CCA GAG GAC AGC CAG GGC CCA TTG TGT GAT GCG CTG TGC CGC ACG 1920 Glu Pro Glu Asp Ser Gin Gly Pro Leu Cys Asp Ala Leu Cys Arg Thr 1090 1095 1100 1105
GTCGAC 1926
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 637 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :
Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Gly Leu Lys Leu Phe 1 5 10 15
Leu Leu Leu Pro Leu Leu Gly Glu Ala Pro Leu Gly Leu Tyr Phe Ser 20 25 30
Arg Asp Ala Tyr Trp Glu Arg Leu Tyr Val Asp Gin Pro Ala Gly Thr 35 40 45
Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro Gly Glu Val Pro 50 55 60
Ser Phe Arg Leu Gly Gin Tyr Leu Tyr Gly Val Tyr Arg Thr Arg Leu 65 70 75 80
His Glu Asn Asp Trp He His He Asp Ala Gly Thr Gly Leu Leu Tyr 85 90 95
Leu Asn Gin Ser Leu Asp His Ser Ser Trp Glu Gin Leu Ser He Arg 100 105 110
Asn Gly Gly Phe Pro Leu Leu Thr Val Phe Leu Gin Val Phe Leu Gly 115 120 125
Ser Thr Ala Gin Arg Glu Gly Glu Cys His Trp Pro Gly Cys Ala Arg 130 135 140
Val Tyr Phe Ser Phe He Asn Asp Thr Phe Pro Asn Cys Ser Ser Phe 145 150 155 160
Lys Ala Arg Asp Leu Cys Thr Pro Glu Thr Gly Val Ser Phe Arg He 165 170 175
Arg Glu Asn Arg Pro Pro Gly Thr Phe Tyr Gin Phe Arg Met Leu Pro 180 185 190
Val Gin Phe Leu Cys Pro Asn He Ser Val Lys Tyr Lys Leu Leu Glu 195 200 205
Gly Asp Gly Leu Pro Phe Arg Cys Asp Pro Asp Cys Leu Glu Val Ser 210 215 220
Thr Arg Trp Ala Leu Asp Arg Glu Leu Gin Glu Lys Tyr Val Leu Glu 225 230 235 240
Ala Glu Cys Ala Val Ala Gly Pro Gly Ala Asn Lys Glu Lys Val Ala 245 250 255
Val Ser Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Pro Pro Thr 260 265 270 Phe Ser Gly Gly Val Gly Thr Ala Ser Ala Val Val Glu Phe Lys Arg 275 280 285
Lys Glu Gly Thr Val Val Ala Thr Leu Gin Val Phe Asp Ala Asp Val 290 295 300
Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr Ser Thr Leu Leu 305 310 315 320
Ser Gly Asp Ser Trp Ala Gin Gin Thr Phe Arg Val Glu His Thr Pro 325 330 335
Asn Glu Thr Leu Val Gin Ser Asn Asn Asn Ser Val Arg Ala Thr Met 340 345 350
His Asn Tyr Lys Leu Val Leu Asn Arg Ser Leu Ser He Ser Glu Ser 355 360 365
Arg Val Leu Gin Leu Val Val Leu Val Asn Asp Ser Asp Phe Gin Gly 370 375 380
Pro Gly Ser Gly Val Leu Phe Leu His Phe Asn Val Ser Val Leu Pro 385 390 395 400
Val Thr Leu Asn Leu Pro Met Ala Tyr Ser Phe Pro Val Asn Arg Arg 405 410 415
Ala Arg Arg Tyr Ala Gin He Gly Lys Val Cys Val Glu Asn Cys Gin 420 425 430
Glu Phe Ser Gly Val Ser He Gin Tyr Lys Leu Gin Pro Ser Ser Thr 435 440 445
Asn Cys Ser Ala Leu Gly Val Val Thr Ser Thr Glu Asp Thr Ser Gly 450 455 460
Thr Leu Tyr Val Asn Asp Thr Glu Ala Leu Arg Arg Pro Glu Cys Thr 465 470 475 480
Glu Leu Gin Tyr Thr Val Val Ala Thr Asp Arg Gin Thr Arg Arg Gin 485 490 495
Thr Gin Ala Ser Leu Val Val Thr Val Glu Gly Thr Tyr He Ala Glu 500 505 510
Glu Val Gly Cys Pro Lys Ser Cys Ala Val Asn Lys Arg Arg Pro Glu 515 520 525
Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg Cys Glu Trp 530 535 540
Arg Gin Gly Asp Gly Lys Gly He Thr Arg Asn Phe Ser Thr Cys Ser 545 550 555 560 Pro Ser Thr Arg Thr Cys Pro Asp Gly His Cys Asp Ala Leu Glu Ser 565 570 575
Arg Asp He Asn He Cys Pro Gin Asp Cys Leu Arg Gly Pro He Val 580 585 590
Gly Gly His Glu Arg Gly Glu Arg Gin Gly He Lys Ala Gly Tyr Gly 595 600 605
He Cys Asn Cys Phe Pro Asp Glu Lys Lys Cys Phe Cys Glu Pro Glu 610 615 620
Asp Ser Gin Gly Pro Leu Cys Asp Ala Leu Cys Arg Thr 625 630 635
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1223 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1038
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :
CTG CTG GAG GAT TCC CCA TAT GAA CCA GTT AAC AGC AGA TTG TCA GAT 48 Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp 640 645 650
ATA TTC CGG GTG GTC CCA TTC ATA TCA GTG GAG CAC ATT CCC AAA GGG 96 He Phe Arg Val Val Pro Phe He Ser Val Glu His He Pro Lys Gly 655 660 665
AAC AAC TGC CTG GAT GCA GCG AAG GCC TGC AAC CTC GAC GAC ATT TGC 144 Asn Asn Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp He Cys 670 675 680 685
AAG AAG TAC AGG TCG GCG TAC ATC ACC CCG TGC ACC ACC AGC GTG TCC 192 Lys Lys Tyr Arg Ser Ala Tyr He Thr Pro Cys Thr Thr Ser Val Ser 690 695 700
AAC GAT GTC TGC AAC CGC CGC AAG TGC CAC AAG GCC CTC CGG CAG TTC 240 Asn Asp Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe 705 710 715 s1
TTT GAC AAG GTC CCG GCC AAG CAC AGC TAC GGA ATG CTC TTC TGC TCC 288 Phe Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser 720 725 730
TGC CGG GAC ATC GCC TGC ACA GAG CGG AGG CGA CAG ACC ATC GTG CCT 336 Cys Arg Asp He Ala Cys Thr Glu Arg Arg Arg Gin Thr He Val Pro 735 740 745
GTG TGC TCC TAT GAA GAG AGG GAG AAG CCC AAC TGT TTG AAT TTG CAG 384 Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gin 750 755 760 765
GAC TCC TGC AAG ACG AAT TAC ATC TGC AGA TCT CGC CTT GCG GAT TTT 432 Asp Ser Cys Lys Thr Asn Tyr He Cys Arg Ser Arg Leu Ala Asp Phe 770 775 780
TTT ACC AAC TGC CAG CCA GAG TCA AGG TCT GTC AGC AGC TGT CTA AAG 480 Phe Thr Asn Cys Gin Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys 785 790 795
GAA AAC TAC GCT GAC TGC CTC CTC GCC TAC TCG GGG CTT ATT GGC ACA 528 Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu He Gly Thr 800 805 810
GTC ATG ACC CCC AAC TAC ATA GAC TCC AGT AGC CTC AGT GTG GCC CCA 576 Val Met Thr Pro Asn Tyr He Asp Ser Ser Ser Leu Ser Val Ala Pro 815 820 825
TGG TGT GAC TGC AGC AAC AGT GGG AAC GAC CTA GAA GAG TGC TTG AAA 624 Trp Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys 830 835 840 845
TTT TTG AAT TTC TTC AAG GAC AAT ACA TGT CTT AAA AAT GCA ATT CAA 672 Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala He Gin 850 855 860
GCC TTT GGC AAT GGC TCC GAT GTG ACC GTG TGG CAG CCA GCC TTC CCA 720 Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gin Pro Ala Phe Pro 865 870 875
GTA CAG ACC ACC ACT GCC ACT ACC ACC ACT GCC CTC CGG GTT AAG AAC 768 Val Gin Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg Val Lys Asn 880 885 890
AAG CCC CTG GGG CCA GCA GGG TCT GAG AAT GAA ATT CCC ACT CAT GTT 816 Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu He Pro Thr His Val 895 900 905
TTG CCA CCG TGT GCA AAT TTA CAG GCA CAG AAG CTG AAA TCC AAT GTG 864 Leu Pro Pro Cys Ala Asn Leu Gin Ala Gin Lys Leu Lys Ser Asn Val 910 915 920 925
TCG GGC AAT ACA CAC CTC TGT ATT TCC AAT GGT AAT TAT GAA AAA GAA 912 Ser Gly Asn Thr His Leu Cys He Ser Asn Gly Asn Tyr Glu Lys Glu 930 935 940 <?0
GGT CTC GGT GCT TCC AGC CAC ATA ACC ACA AAA TCA ATG GCT GCT CCT 960 Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala Ala Pro 945 950 955
CCA AGC TGT GGT CTG AGC CCA CTG CTG GTC CTG GTG GTA ACC GCT CTG 1008 Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu 960 965 970
TCC ACC CTA TTA TCT TTA ACA GAA ACA TCA TAGCTGCATT AAAAAAATAC 1058 Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 975 980
AATATGGACA TGTAAAAAGA CAAAAACCAA GTTATCTGTT TCCTGTTCTC TTGTATAGCT 1118
GAAATTCCAG TTTAGGAGCT CAGTTGAGAA ACAGTTCCAT TCAACTGGAA CATTTTTTTT 1178
TTTTCCTTTT AAGAAAGCTT CTTGTGATCC TTCGGGGCTT CTGTG 1223
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 :
Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp 1 5 10 15
He Phe Arg Val Val Pro Phe He Ser Val Glu His He Pro Lys Gly 20 25 30
Asn Asn Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp He Cys 35 40 45
Lys Lys Tyr Arg Ser Ala Tyr He Thr Pro Cys Thr Thr Ser Val Ser 50 55 60
Asn Asp Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe 65 70 75 80
Phe Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser 85 90 95
Cys Arg Asp He Ala Cys Thr Glu Arg Arg Arg Gin Thr He Val Pro 100 105 110
Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gin 115 120 125 Asp Ser Cys Lys Thr Asn Tyr He Cys Arg Ser Arg Leu Ala Asp Phe 130 135 140
Phe Thr Asn Cys Gin Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys 145 150 155 160
Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu He Gly Thr 165 170 175
Val Met Thr Pro Asn Tyr He Asp Ser Ser Ser Leu Ser Val Ala Pro 180 185 190
Trp Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys 195 200 205
Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala He Gin 210 215 220
Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gin Pro Ala Phe Pro 225 230 235 240
Val Gin Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg Val Lys Asn 245 250 255
Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu He Pro Thr His Val 260 265 270
Leu Pro Pro Cys Ala Asn Leu Gin Ala Gin Lys Leu Lys Ser Asn Val 275 280 285
Ser Gly Asn Thr His Leu Cys He Ser Asn Gly Asn Tyr Glu Lys Glu 290 295 300
Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala Ala Pro 305 310 315 320
Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu 325 330 335
Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 340 345
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1682 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS έ,2.
(B) LOCATION: 118..1497
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGGCGGCCAG AGCAGCACAG CTGTCCGGGG ATCGCTGCAT GCTGAGCTCC CTCGGCAAGA 60
CCCAGCGGCG GCTCGGGATT TTTTTGGGGG GGCGGGGACC AGCCCCGCGC CGGCACC 117
ATG TTC CTG GCG ACC CTG TAC TTC GCG CTG CCG CTC TTG GAC TTG CTC 165 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 350 355 360
CTG TCG GCC GAA GTG AGC GGC GGA GAC CGC CTG GAT TGC GTG AAA GCC 213 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 365 370 375
AGT GAT CAG TGC CTG AAG GAG CAG AGC TGC AGC ACC AAG TAC CGC ACG 261 Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser Thr Lys Tyr Arg Thr 380 385 390
CTA AGG CAG TGC GTG GCG GGC AAG GAG ACC AAC TTC AGC CTG GCA TCC 309 Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 395 400 405 410
GGC CTG GAG GCC AAG GAT GAG TGC CGC AGC GCC ATG GAG GCC CTG AAG 357 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 415 420 425
CAG AAG TCG CTC TAC AAC TGC CGC TGC AAG CGG GGT ATG AAG AAG GAG 405 Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 430 435 440
AAG AAC TGC CTG CGC ATT TAC TGG AGC ATG TAC CAG AGC CTG CAG GGA 453 Lys Asn Cys Leu Arg He Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 445 450 455
AAT GAT CTG CTG GAG GAT TCC CCA TAT GAA CCA GTT AAC AGC AGA TTG 501 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 460 465 470
TCA GAT ATA TTC CGG GTG GTC CCA TTC ATA TCA GTG GAG CAC ATT CCC 549 Ser Asp He Phe Arg Val Val Pro Phe He Ser Val Glu His He Pro 475 480 485 490
AAA GGG AAC AAC TGC CTG GAT GCA GCG AAG GCC TGC AAC CTC GAC GAC 597 Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp 495 500 505
ATT TGC AAG AAG TAC AGG TCG GCG TAC ATC ACC CCG TGC ACC ACC AGC 645 He Cys Lys Lys Tyr Arg Ser Ala Tyr He Thr Pro Cys Thr Thr Ser 510 515 520 GTG TCC AAC GAT GTC TGC AAC CGC CGC AAG TGC CAC AAG GCC CTC CGG 693 Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg 525 530 535
CAG TTC TTT GAC AAG GTC CCG GCC AAG CAC AGC TAC GGA ATG CTC TTC 741 Gin Phe Phe Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe 540 545 550
TGC TCC TGC CGG GAC ATC GCC TGC ACA GAG CGG AGG CGA CAG ACC ATC 789 Cys Ser Cys Arg Asp He Ala Cys Thr Glu Arg Arg Arg Gin Thr He 555 560 565 570
GTG CCT GTG TGC TCC TAT GAA GAG AGG GAG AAG CCC AAC TGT TTG AAT 837 Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn 575 580 585
TTG CAG GAC TCC TGC AAG ACG AAT TAC ATC TGC AGA TCT CGC CTT GCG 885 Leu Gin Asp Ser Cys Lys Thr Asn Tyr He Cys Arg Ser Arg Leu Ala 590 595 600
GAT TTT TTT ACC AAC TGC CAG CCA GAG TCA AGG TCT GTC AGC AGC TGT 933 Asp Phe Phe Thr Asn Cys Gin Pro Glu Ser Arg Ser Val Ser Ser Cys 605 610 615
CTA AAG GAA AAC TAC GCT GAC TGC CTC CTC GCC TAC TCG GGG CTT ATT 981 Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu He 620 625 630
GGC ACA GTC ATG ACC CCC AAC TAC ATA GAC TCC AGT AGC CTC AGT GTG 1029 Gly Thr Val Met Thr Pro Asn Tyr He Asp Ser Ser Ser Leu Ser Val 635 640 645 650
GCC CCA TGG TGT GAC TGC AGC AAC AGT GGG AAC GAC CTA GAA GAG TGC 1077 Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys 655 660 665
TTG AAA TTT TTG AAT TTC TTC AAG GAC AAT ACA TGT CTT AAA AAT GCA 1125 Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala 670 675 6B0
ATT CAA GCC TTT GGC AAT GGC TCC GAT GTG ACC GTG TGG CAG CCA GCC 1173 He Gin Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gin Pro Ala 685 690 695
TTC CCA GTA CAG ACC ACC ACT GCC ACT ACC ACC ACT GCC CTC CGG GTT 1221 Phe Pro Val Gin Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg Val 700 705 710
AAG AAC AAG CCC CTG GGG CCA GCA GGG TCT GAG AAT GAA ATT CCC ACT 1269 Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu He Pro Thr 715 720 725 730
CAT GTT TTG CCA CCG TGT GCA AAT TTA CAG GCA CAG AAG CTG AAA TCC 1317 His Val Leu Pro Pro Cys Ala Asn Leu Gin Ala Gin Lys Leu Lys Ser 735 740 745 AAT GTG TCG GGC AAT ACA CAC CTC TGT ATT TCC AAT GGT AAT TAT GAA 1365 Asn Val Ser Gly Asn Thr His Leu Cys He Ser Asn Gly Asn Tyr Glu 750 755 760
AAA GAA GGT CTC GGT GCT TCC AGC CAC ATA ACC ACA AAA TCA ATG GCT 1413 Lys Glu Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala 765 770 775
GCT CCT CCA AGC TGT GGT CTG AGC CCA CTG CTG GTC CTG GTG GTA ACC 1461 Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr 780 785 790
GCT CTG TCC ACC CTA TTA TCT TTA ACA GAA ACA TCA TAGCTGCATT 1507
Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 795 800 805
AAAAAAATAC AATATGGACA TGTAAAAAGA CAAAAACCAA GTTATCTGTT TCCTGTTCTC 1567
TTGTATAGCT GAAATTCCAG TTTAGGAGCT CAGTTGAGAA ACAGTTCCAT TCAACTGGAA 1627
CATTTTTTTT TTTTCCTTTT AAGAAAGCTT CTTGTGATCC TTCGGGGCTT CTGTG 1682
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15
Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30
Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45
Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 50 55 60
Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80
Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95
Lys Asn Cys Leu Arg He Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125
Ser Asp He Phe Arg Val Val Pro Phe He Ser Val Glu His He Pro 130 135 140
Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp 145 150 155 160
He Cys Lys Lys Tyr Arg Ser Ala Tyr He Thr Pro Cys Thr Thr Ser 165 170 175
Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg 180 185 190
Gin Phe Phe Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe 195 200 205
Cys Ser Cys Arg Asp He Ala Cys Thr Glu Arg Arg Arg Gin Thr He 210 215 220
Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn 225 230 235 240
Leu Gin Asp Ser Cys Lys Thr Asn Tyr He Cys Arg Ser Arg Leu Ala 245 250 255
Asp Phe Phe Thr Asn Cys Gin Pro Glu Ser Arg Ser Val Ser Ser Cys 260 265 270
Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu He 275 280 285
Gly Thr Val Met Thr Pro Asn Tyr He Asp Ser Ser Ser Leu Ser Val 290 295 300
Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys 305 310 315 320
Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala 325 330 335
He Gin Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gin Pro Ala 340 345 350
Phe Pro Val Gin Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg Val 355 360 365
Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu He Pro Thr 370 375 380
His Val Leu Pro Pro Cys Ala Asn Leu Gin Ala Gin Lys Leu Lys Ser 385 390 395 400 Asn Val Ser Gly Asn Thr His Leu Cys He Ser Asn Gly Asn Tyr Glu 405 410 415
Lys Glu Gly Leu Gly Ala Ser Ser His He Thr Thr Lys Ser Met Ala 420 425 430
Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr 435 440 445
Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 450 455 460
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1888 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..1416
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
AAAAAACGGT GGGATTTATT TAAC ATG ATC TTG GCA AAC GTC TTC TGC CTC 51
Met He Leu Ala Asn Val Phe Cys Leu 465
TTC TTC TTT CTA GAC GAG ACC CTC CGC TCT TTG GCC AGC CCT TCC TCC 99 Phe Phe Phe Leu Asp Glu Thr Leu Arg Ser Leu Ala Ser Pro Ser Ser 470 475 480 485
CTG CAG GGC CCC GAG CTC CAC GGC TGG CGC CCC CCA GTG GAC TGT GTC 147 Leu Gin Gly Pro Glu Leu His Gly Trp Arg Pro Pro Val Asp Cys Val 490 495 500
CGG GCC AAT GAG CTG TGT GCC GCC GAA TCC AAC TGC AGC TCT CGC TAC 195 Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn Cys Ser Ser Arg Tyr 505 510 515
CGC ACT CTG CGG CAG TGC CTG GCA GGC CGC GAC CGC AAC ACC ATG CTG 243 Arg Thr Leu Arg Gin Cys Leu Ala Gly Arg Asp Arg Asn Thr Met Leu 520 525 530
GCC AAC AAG GAG TGC CAG GCG GCC TTG GAG GTC TTG CAG GAG AGC CCG 291 Ala Asn Lys Glu Cys Gin Ala Ala Leu Glu Val Leu Gin Glu Ser Pro 535 540 545 CTG TAC GAC TGC CGC TGC AAG CGG GGC ATG AAG AAG GAG CTG CAG TGT 339
Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gin Cys 550 555 560 565
CTG CAG ATC TAC TGG AGC ATC CAC CTG GGG CTG ACC GAG GGT GAG GAG 387
Leu Gin He Tyr Trp Ser He His Leu Gly Leu Thr Glu Gly Glu Glu
570 575 580
TTC TAC GAA GCC TCC CCC TAT GAG CCG GTG ACC TCC CGC CTC TCG GAC 435
Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser Arg Leu Ser Asp 585 590 595
ATC TTC AGG CTT GCT TCA ATC TTC TCA GGG ACA GGG GCA GAC CCG GTG 483
He Phe Arg Leu Ala Ser He Phe Ser Gly Thr Gly Ala Asp Pro Val 600 605 610
GTC AGC GCC AAG AGC AAC CAT TGC CTG GAT GCT GCC AAG GCC TGC AAC 531
Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala Lys Ala Cys Asn 615 620 625
CTG AAT GAC AAC TGC AAG AAG CTG CGC TCC TCC TAC ATC TCC ATC TGC 579
Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr He Ser He Cys 630 635 640 645
AAC CGC GAG ATC TCG CCC ACC GAG CGC TGC AAC CGC CGC AAG TGC CAC 627
Asn Arg Glu He Ser Pro Thr Glu Arg Cys Asn Arg Arg Lys Cys His
650 655 660
AAG GCC CTG CGC CAG TTC TTC GAC CGG GTG CCC AGC GAG TAC ACC TAC 675
Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser Glu Tyr Thr Tyr 665 670 675
CGC ATG CTC TTC TGC TCC TGC CAA GAC CAG GCG TGC GCT GAG CGC CGC 723
Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys Ala Glu Arg Arg 680 685 690
CGG CAA ACC ATC CTG CCC AGC TGC TCC TAT GAG GAC AAG GAG AAG CCC 771
Arg Gin Thr He Leu Pro Ser Cys Ser Tyr Glu Asp Lys Glu Lys Pro 695 700 705
AAC TGC CTG GAC CTG CGT GGC GTG TGC CGG ACT GAC CAC CTG TGT CGG 819
Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp His Leu Cys Arg 710 715 720 725
TCC CGG CTG GCC GAC TTC CAT GCC AAT TGT CGA GCC TCC TAC CAG ACG 867
Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala Ser Tyr Gin Thr
730 735 740
GTC ACC AGC TGC CCT GCG GAC AAT TAC CAG GCG TGT CTG GGC TCT TAT 915
Val Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys Leu Gly Ser Tyr 745 750 755
GCT GGC ATG ATT GGG TTT GAC ATG ACA CCT AAC TAT GTG GAC TCC AGC 963
Ala Gly Met He Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Ser 760 765 770 (o o
CCC ACT GGC ATC GTG GTG TCC CCC TGG TGC AGC TGT CGT GGC AGC GGG 1011 Pro Thr Gly He Val Val Ser Pro Trp Cys Ser Cys Arg Gly Ser Gly 775 780 785
AAC ATG GAG GAG GAG TGT GAG AAG TTC CTC AGG GAC TTC ACC GAG AAC 1059 Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 790 795 800 805
CCA TGC CTC CGG AAC GCC ATC CAG GCC TTT GGC AAC GGC ACG GAC GTG 1107 Pro Cys Leu Arg Asn Ala He Gin Ala Phe Gly Asn Gly Thr Asp Val 810 815 820
AAC GTG TCC CCA AAA GGC CCC TCG TTC CAG GCC ACC CAG GCC CCT CGG 1155 Asn Val Ser Pro Lys Gly Pro Ser Phe Gin Ala Thr Gin Ala Pro Arg 825 830 835
GTG GAG AAG ACG CCT TCT TTG CCA GAT GAC CTC AGT GAC AGT ACC AGC 1203 Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp Ser Thr Ser 840 845 850
TTG GGG ACC AGT GTC ATC ACC ACC TGC ACG TCT GTC CAG GAG CAG GGG 1251 Leu Gly Thr Ser Val He Thr Thr Cys Thr Ser Val Gin Glu Gin Gly 855 860 865
CTG AAG GCC AAC AAC TCC AAA GAG TTA AGC ATG TGC TTC ACA GAG CTC 1299 Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys Phe Thr Glu Leu 870 875 880 885
ACG ACA AAT ATC ATC CCA GGG AGT AAC AAG GTG ATC AAA CCT AAC TCA 1347 Thr Thr Asn He He Pro Gly Ser Asn Lys Val He Lys Pro Asn Ser 890 B95 900
GGC CCC AGC AGA GCC AGA CCG TCG GCT GCC TTG ACC GTG CTG TCT GTC 1395 Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr Val Leu Ser Val 905 910 915
CTG ATG CTG AAA CTG GCC TTG TAGGCTGTGG GAACCGAGTC AGAAGATTTT 1446
Leu Met Leu Lys Leu Ala Leu 920
TGAAAGCTAC GCAGACAAGA ACAGCCGCCT GACGAAATGG AAACACACAC AGACACACAC 1506
ACACCTTGCA AAAAAAAAAT TGTTTTTCCC ACCTTGTCGC TGAACCTGTC TCCTCCCAGG 1566
TTTCTTCTCT GGAGAAGTTT TTGTAAACCA AACAGACAAG CAGGCAGGCA GCCTGAGAGC 1626
TGGCCCAGGG GTCCCCTGGC AGGGGAAACT CTGGTGCCGG GGAGGGCACG AGGCTCTAGA 1686
AATGCCCTTC ACTTTCTCCT GGTGTTTTTC TCTCTGGACC CTTCTGAAGC AGAGACCGGA 1746
CAAGAGCCTG CAGCGGAAGG GACTCTGGGC TGTGCCTGAG GCTGGCTGGG GGCAGGACAA 1806
CACAGCTGCT TCCCCAGGCT GCCCACTCTG GGGACCCGCT GGGGGCTGGC AGAGGGCATC 1866 GGTCAGCGGG GCAGCGGGGC TG 1888
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 464 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met He Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr 1 5 10 15
Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gin Gly Pro Glu Leu His 20 25 30
Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 35 40 45
Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu 50 55 60
Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala 65 70 75 80
Ala Leu Glu Val Leu Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 85 90 95
Arg Gly Met Lys Lys Glu Leu Gin Cys Leu Gin He Tyr Trp Ser He 100 105 110
His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 115 120 125
Glu Pro Val Thr Ser Arg Leu Ser Asp He Phe Arg Leu Ala Ser He 130 135 140
Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His 145 150 155 160
Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 165 170 175
Leu Arg Ser Ser Tyr He Ser He Cys Asn Arg Glu He Ser Pro Thr 180 185 190
Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe 195 200 205
Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys 210 215 220 Gin Asp Gin Ala Cys Ala Glu Arg Arg Arg Gin Thr He Leu Pro Ser 225 230 235 240
Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly 245 250 255
Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 260 265 270
Ala Asn Cys Arg Ala Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp 275 280 285
Asn Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gly Met He Gly Phe Asp 290 295 300
Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr Gly He Val Val Ser 305 310 315 320
Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 325 330 335
Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala He 340 345 350
Gin Ala Phe Gly Asn Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro 355 360 365
Ser Phe Gin Ala Thr Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 370 375 380
Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val He Thr 385 390 395 400
Thr Cys Thr Ser Val Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys 405 410 415
Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn He He Pro Gly 420 425 430
Ser Asn Lys Val He Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro 435 440 445
Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Leu Ala Leu 450 455 460
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1878 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 205..1242
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CGCGGCGCCC AGCGCAGGCA GAGCGCTGTC GCATCCCGGG CGTCCACCCG CCATGGGGCT 60
CTCCTGGAGC CCGCGACCTC CACTGCTGAT GATCCTGCTA CTGGTGCTGT CGTTGTGGCT 120
GCCACTTGGA GCAGGAAACT CCCTTGCCAC AGAGAACAGG TTTGTGAACA GCTGTACCCA 180
GGCCAGAAAG AAATGCGAGG CTAA TCC CGC TTG CAA GGC TGC CTA CCA GCA 231
Ser Arg Leu Gin Gly Cys Leu Pro Ala 465 470
CCT GGG CTC CTG CAC CTC CAG TTA AGC AGG CCG CTG CCC TTA GAG GAG 279 Pro Gly Leu Leu His Leu Gin Leu Ser Arg Pro Leu Pro Leu Glu Glu 475 480 485
TCT GCC ATG TCT GCA GAC TGC CTA GAG GCA GCA GAA CAA CTC AGG AAC 327 Ser Ala Met Ser Ala Asp Cys Leu Glu Ala Ala Glu Gin Leu Arg Asn 490 495 500 505
AGC TCT CTG ATA GAC TGC AGG TGC CAT CGG CGC ATG AAG CAC CAA GCT 375 Ser Ser Leu He Asp Cys Arg Cys His Arg Arg Met Lys His Gin Ala 510 515 520
ACC TGT CTG GAC ATT TAT TGG ACC GTT CAC CCT GCC CGA AGC CTT GGT 423 Thr Cys Leu Asp He Tyr Trp Thr Val His Pro Ala Arg Ser Leu Gly 525 530 535
GAC TAC GAG TTG GAT GTC TCA CCC TAT GAA GAC ACA GTG ACC AGC AAA 471 Asp Tyr Glu Leu Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys 540 545 550
CCC TGG AAA ATG AAT CTT AGC AAG TTG AAC ATG CTC AAA CCA GAC TCG 519 Pro Trp Lys Met Asn Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser 555 560 565
GAC CTC TGC CTC AAA TTT GCT ATG CTG TGT ACT CTT CAC GAC AAG TGT 567 Asp Leu Cys Leu Lys Phe Ala Met Leu Cys Thr Leu His Asp Lys Cys 570 575 580 585
GAC CGC CTG CGC AAG GCC TAC GGG GAG GCA TGC TCA GGG ATC CGC TGC 615 Asp Arg Leu Arg Lys Ala Tyr Gly Glu Ala Cys Ser Gly He Arg Cys 590 595 600
CAG CGC CAC CTC TGC CTA GCC CAG CTG CGC TCC TTC TTT GAG AAG GCA 663 Gin Arg His Leu Cys Leu Ala Gin Leu Arg Ser Phe Phe Glu Lys Ala 605 610 615 GCA GAG TCC CAC GCT CAG GGT CTG CTG CTG TGT CCC TGT GCA CCA GAA 711 Ala Glu Ser His Ala Gin Gly Leu Leu Leu Cys Pro Cys Ala Pro Glu 620 625 630
GAT GCG GGC TGT GGG GAG CGG CGG CGT AAC ACC ATC GCC CCC AGT TGC 759 Asp Ala Gly Cys Gly Glu Arg Arg Arg Asn Thr He Ala Pro Ser Cys 635 640 645
GCC CTG CCT TCT GTA ACC CCC AAT TGC CTG GAT CTG CGG AGC TTC TGC 807 Ala Leu Pro Ser Val Thr Pro Asn Cys Leu Asp Leu Arg Ser Phe Cys 650 655 660 665
CGT GCG GAC CCT TTG TGC AGA TCA CGC CTG ATG GAC TTC CAG ACC CAC 855 Arg Ala Asp Pro Leu Cys Arg Ser Arg Leu Met Asp Phe Gin Thr His 670 675 680
TGT CAT CCT ATG GAC ATC CTT GGG ACT TGT GCA ACT GAG CAG TCC AGA 903 Cys His Pro Met Asp He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg 685 690 695
TGT CTG CGG GCA TAC CTG GGG CTG ATT GGG ACT GCC ATG ACC CCA AAC 951 Cys Leu Arg Ala Tyr Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn 700 705 710
TTC ATC AGC AAG GTC AAC ACT ACT GTT GCC TTA AGC TGC ACC TGC CGA 999 Phe He Ser Lys Val Asn Thr Thr Val Ala Leu Ser Cys Thr Cys Arg 715 720 725
GGC AGC GGC AAC CTA CAG GAC GAG TGT GAA CAG CTG GAA AGG TCC TTC 1047 Gly Ser Gly Asn Leu Gin Asp Glu Cys Glu Gin Leu Glu Arg Ser Phe 730 735 740 745
TCC CAG AAC CCC TGC CTC GTG GAG GCC ATT GCA GCT AAG ATG CGT TTC 1095 Ser Gin Asn Pro Cys Leu Val Glu Ala He Ala Ala Lys Met Arg Phe 750 755 760
CAC AGA CAG CTC TTC TCC CAG GAC TGG GCA GAC TCT ACT TTT TCA GTG 1143 His Arg Gin Leu Phe Ser Gin Asp Trp Ala Asp Ser Thr Phe Ser Val 765 770 775
GTG CAG CAG CAG AAC AGC AAC CCT GCT CTG AGA CTG CAG CCC AGG CTA 1191 Val Gin Gin Gin Asn Ser Asn Pro Ala Leu Arg Leu Gin Pro Arg Leu 780 785 790
CCC ATT CTT TCT TTC TCC ATC CTT CCC TTG ATT CTG CTG CAG ACC CTC 1239 Pro He Leu Ser Phe Ser He Leu Pro Leu He Leu Leu Gin Thr Leu 795 800 805
TGG TAGCTGGGCT TCCTCAGGGT CCTTTGTCCT CTCCACCACA CCCAGACTGA 1292
Trp
810
TTTGCAGCCT GTGGTGGGAG AGAACTCGCC AGCCTGTGGA AGAAGACGCA GCGTGCTACA 1352 lb
CAGCAACCCG GAACCAACCA GGCATTCCGC AGCACATCCC GTCTGCTCCA GAAGAGGTCT 1412
TAGAAGTGAG GGCTGTGACC CTTCCGATCC TGAGCGGCTA GTTTTCAAAC CTCCCTTGCC 1472
CCTGCTTCCT TCTGGCTCAG GCTGCTCCTC CTTAGGACTT TGTGGGTCCA GTTTTGCCTT 1532
CTGTTCTGAT GGTGATTAGC GGCTCACCTC CAGCGCTTCT TCCTGTTTCC CAGGACCACC 1592
CAGAGGCTAA GGAATCAGTC ATTCCCTGTT GCCTTCTCCA GGAAGGCAGG CTAAGGGTTC 1652
TGAGGTGACT GAGAAAAATG TTTCCTTTGT GTGGAAGGCT GGTGCTCCAG CCTCCACGTC 1712
CCTCTGAATG GAAGATAAAA ACCTGCTGGT GTCTTGACTG CTCTGCCAGG CAATCCTGAA 1772
CATTTGGGCA TGAAGAGCTA AAGTCTTTGG GTCTTGTTTA ACTCCTATTA CTGTCCCCAA 1832
ATTCCCCTAG TCCCTTGGGT CATGATTAAA CATTTTGACT TAAAAA 1878
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ser Arg Leu Gin Gly Cys Leu Pro Ala Pro Gly Leu Leu His Leu Gin 1 5 10 15
Leu Ser Arg Pro Leu Pro Leu Glu Glu Ser Ala Met Ser Ala Asp Cys 20 25 30
Leu Glu Ala Ala Glu Gin Leu Arg Asn Ser Ser Leu He Asp Cys Arg 35 40 45
Cys His Arg Arg Met Lys His Gin Ala Thr Cys Leu Asp He Tyr Trp 50 55 60
Thr Val His Pro Ala Arg Ser Leu Gly Asp Tyr Glu Leu Asp Val Ser 65 70 75 80
Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met Asn Leu Ser 85 90 95
Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu Lys Phe Ala 100 105 110
Met Leu Cys Thr Leu His Asp Lys Cys Asp Arg Leu Arg Lys Ala Tyr 115 120 125 Gly Glu Ala Cys Ser Gly He Arg Cys Gin Arg His Leu Cys Leu Ala 130 135 140
Gin Leu Arg Ser Phe Phe Glu Lys Ala Ala Glu Ser His Ala Gin Gly 145 150 155 160
Leu Leu Leu Cys Pro Cys Ala Pro Glu Asp Ala Gly Cys Gly Glu Arg 165 170 175
Arg Arg Asn Thr He Ala Pro Ser Cys Ala Leu Pro Ser Val Thr Pro 180 185 190
Asn Cys Leu Asp Leu Arg Ser Phe Cys Arg Ala Asp Pro Leu Cys Arg 195 200 205
Ser Arg Leu Met Asp Phe Gin Thr His Cys His Pro Met Asp He Leu 210 215 220
Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala Tyr Leu Gly 225 230 235 240
Leu He Gly Thr Ala Met Thr Pro Asn Phe He Ser Lys Val Asn Thr 245 250 255
Thr Val Ala Leu Ser Cys Thr Cys Arg Gly Ser Gly Asn Leu Gin Asp 260 265 270
Glu Cys Glu Gin Leu Glu Arg Ser Phe Ser Gin Asn Pro Cys Leu Val 275 280 285
Glu Ala He Ala Ala Lys Met Arg Phe His Arg Gin Leu Phe Ser Gin 290 295 300
Asp Trp Ala Asp Ser Thr Phe Ser Val Val Gin Gin Gin Asn Ser Asn 305 310 315 320
Pro Ala Leu Arg Leu Gin Pro Arg Leu Pro He Leu Ser Phe Ser He 325 330 335
Leu Pro Leu He Leu Leu Gin Thr Leu Trp 340 345
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1889 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 41..1231
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16 :
CGCAGGCAGA GCGCTGTCGC ATCCCGGGCG TCCACCCGCC ATG GGG CTC TCC TGG 55
Met Gly Leu Ser Trp 350
AGC CCG CGA CCT CCA CTG CTG ATG ATC CTG CTA CTG GTG CTG TCG TTG 103 Ser Pro Arg Pro Pro Leu Leu Met He Leu Leu Leu Val Leu Ser Leu 355 360 365
TGG CTG CCA CTT GGA GCA GGA AAC TCC CTT GCC ACA GAG AAC AGG TTT 151 Trp Leu Pro Leu Gly Ala Gly Asn Ser Leu Ala Thr Glu Asn Arg Phe 370 375 380
GTG AAC AGC TGT ACC CAG GCC AGA AAG AAA TGC GAG GCT AAT CCC GCT 199 Val Asn Ser Cys Thr Gin Ala Arg Lys Lys Cys Glu Ala Asn Pro Ala 385 390 395
TGC AAG GCT GCC TAC CAG CAC CTG GGC TCC TGC ACC TCC AGT TTA AGC 247 Cys Lys Ala Ala Tyr Gin His Leu Gly Ser Cys Thr Ser Ser Leu Ser 400 405 410 415
AGG CCG CTG CCC TTA GAG GAG TCT GCC ATG TCT GCA GAC TGC CTA GAG 295 Arg Pro Leu Pro Leu Glu Glu Ser Ala Met Ser Ala Asp Cys Leu Glu 420 425 430
GCA GCA GAA CAA CTC AGG AAC AGC TCT CTG ATA GAC TGC AGG TGC CAT 343 Ala Ala Glu Gin Leu Arg Asn Ser Ser Leu He Asp Cys Arg Cys His 435 440 445
CGG CGC ATG AAG CAC CAA GCT ACC TGT CTG GAC ATT TAT TGG ACC GTT 391 Arg Arg Met Lys His Gin Ala Thr Cys Leu Asp He Tyr Trp Thr Val 450 455 460
CAC CCT GCC CGA AGC CTT GGT GAC TAC GAG TTG GAT GTC TCA CCC TAT 439 His Pro Ala Arg Ser Leu Gly Asp Tyr Glu Leu Asp Val Ser Pro Tyr 465 470 475
GAA GAC ACA GTG ACC AGC AAA CCC TGG AAA ATG AAT CTT AGC AAG TTG 487 Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met Asn Leu Ser Lys Leu 480 485 490 495
AAC ATG CTC AAA CCA GAC TCG GAC CTC TGC CTC AAA TTT GCT ATG CTG 535 Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu Lys Phe Ala Met Leu 500 505 510
TGT ACT CTT CAC GAC AAG TGT GAC CGC CTG CGC AAG GCC TAC GGG GAG 583 Cys Thr Leu His Asp Lys Cys Asp Arg Leu Arg Lys Ala Tyr Gly Glu 515 520 525 GCA TGC TCA GGG ATC CGC TGC CAG CGC CAC CTC TGC CTA GCC CAG CTG 631 Ala Cys Ser Gly He Arg Cys Gin Arg His Leu Cys Leu Ala Gin Leu 530 535 540
CGC TCC TTC TTT GAG AAG GCA GCA GAG TCC CAC GCT CAG GGT CTG CTG 679 Arg Ser Phe Phe Glu Lys Ala Ala Glu Ser His Ala Gin Gly Leu Leu 545 550 555
CTG TGT CCC TGT GCA CCA GAA GAT GCG GGC TGT GGG GAG CGG CGG CGT 727 Leu Cys Pro Cys Ala Pro Glu Asp Ala Gly Cys Gly Glu Arg Arg Arg 560 565 570 575
AAC ACC ATC GCC CCC AGT TGC GCC CTG CCT TCT GTA ACC CCC AAT TGC 775 Asn Thr He Ala Pro Ser Cys Ala Leu Pro Ser Val Thr Pro Asn Cys 580 585 590
CTG GAT CTG CGG AGC TTC TGC CGT GCG GAC CCT TTG TGC AGA TCA CGC 823 Leu Asp Leu Arg Ser Phe Cys Arg Ala Asp Pro Leu Cys Arg Ser Arg 595 600 605
CTG ATG GAC TTC CAG ACC CAC TGT CAT CCT ATG GAC ATC CTT GGG ACT 871 Leu Met Asp Phe Gin Thr His Cys His Pro Met Asp He Leu Gly Thr 610 615 620
TGT GCA ACT GAG CAG TCC AGA TGT CTG CGG GCA TAC CTG GGG CTG ATT 919 Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala Tyr Leu Gly Leu He 625 630 635
GGG ACT GCC ATG ACC CCA AAC TTC ATC AGC AAG GTC AAC ACT ACT GTT 967 Gly Thr Ala Met Thr Pro Asn Phe He Ser Lys Val Asn Thr Thr Val 640 645 650 655
GCC TTA AGC TGC ACC TGC CGA GGC AGC GGC AAC CTA CAG GAC GAG TGT 1015 Ala Leu Ser Cys Thr Cys Arg Gly Ser Gly Asn Leu Gin Asp Glu Cys 660 665 670
GAA CAG CTG GAA AGG TCC TTC TCC CAG AAC CCC TGC CTC GTG GAG GCC 1063 Glu Gin Leu Glu Arg Ser Phe Ser Gin Asn Pro Cys Leu Val Glu Ala 675 680 685
ATT GCA GCT AAG ATG CGT TTC CAC AGA CAG CTC TTC TCC CAG GAC TGG 1111 He Ala Ala Lys Met Arg Phe His Arg Gin Leu Phe Ser Gin Asp Trp 690 695 700
GCA GAC TCT ACT TTT TCA GTG GTG CAG CAG CAG AAC AGC AAC CCT GCT 1159 Ala Asp Ser Thr Phe Ser Val Val Gin Gin Gin Asn Ser Asn Pro Ala 705 710 715
CTG AGA CTG CAG CCC AGG CTA CCC ATT CTT TCT TTC TCC ATC CTT CCC 1207 Leu Arg Leu Gin Pro Arg Leu Pro He Leu Ser Phe Ser He Leu Pro 720 725 730 735
TTG ATT CTG CTG CAG ACC CTC TGG TAGCTGGGCT TCCTCAGGGT CCTTTGTCCT 1261 Leu He Leu Leu Gin Thr Leu Trp 740 CTCCACCACA CCCAGACTGA TTTGCAGCCT GTGGTGGGAG AGAACTCGCC AGCCTGTGGA 1321
AGAAGACGCA GCGTGCTACA CAGCAACCCG GAACCAACCA GGCATTCCGC AGCACATCCC 1381
GTCTGCTCCA GAAGAGGTCT TAGAAGTGAG GGCTGTGACC CTTCCGATCC TGAGCGGCTA 1441
GTTTTCAAAC CTCCCTTGCC CCTGCTTCCT TCTGGCTCAG GCTGCTCCTC CTTAGGACTT 1501
TGTGGGTCCA GTTTTGCCTT CTGTTCTGAT GGTGATTAGC GGCTCACCTC CAGCGCTTCT 1561
TCCTGTTTCC CAGGACCACC CAGAGGCTAA GGAATCAGTC ATTCCCTGTT GCCTTCTCCA 1621
GGAAGGCAGG CTAAGGGTTC TGAGGTGACT GAGAAAAATG TTTCCTTTGT GTGGAAGGCT 1681
GGTGCTCCAG CCTCCACGTC CCTCTGAATG GAAGATAAAA ACCTGCTGGT GTCTTGACTG 1741
CTCTGCCAGG CAATCCTGAA CATTTGGGCA TGAAGAGCTA AAGTCTTTGG GTCTTGTTTA 1801
ACTCCTATTA CTGTCCCCAA ATTCCCCTAG TCCCTTGGGT CATGATTAAA CATTTTGACT 1861
TAAAAAAAAA AAAAAAAAAA AAAAAAAA 1889
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 397 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Gly Leu Ser Trp Ser Pro Arg Pro Pro Leu Leu Met He Leu Leu 1 5 10 15
Leu Val Leu Ser Leu Trp Leu Pro Leu Gly Ala Gly Asn Ser Leu Ala 20 25 30
Thr Glu Asn Arg Phe Val Asn Ser Cys Thr Gin Ala Arg Lys Lys Cys 35 40 45
Glu Ala Asn Pro Ala Cys Lys Ala Ala Tyr Gin His Leu Gly Ser Cys 50 55 60
Thr Ser Ser Leu Ser Arg Pro Leu Pro Leu Glu Glu Ser Ala Met Ser 65 70 75 80
Ala Asp Cys Leu Glu Ala Ala Glu Gin Leu Arg Asn Ser Ser Leu He 85 90 95
Asp Cys Arg Cys His Arg Arg Met Lys His Gin Ala Thr Cys Leu Asp 100 105 110 He Tyr Trp Thr Val His Pro Ala Arg Ser Leu Gly Asp Tyr Glu Leu 115 120 125
Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met 130 135 140
Asn Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu 145 150 155 160
Lys Phe Ala Met Leu Cys Thr Leu His Asp Lys Cys Asp Arg Leu Arg 165 170 175
Lys Ala Tyr Gly Glu Ala Cys Ser Gly He Arg Cys Gin Arg His Leu 180 185 190
Cys Leu Ala Gin Leu Arg Ser Phe Phe Glu Lys Ala Ala Glu Ser His 195 200 205
Ala Gin Gly Leu Leu Leu Cys Pro Cys Ala Pro Glu Asp Ala Gly Cys 210 215 220
Gly Glu Arg Arg Arg Asn Thr He Ala Pro Ser Cys Ala Leu Pro Ser 225 230 235 240
Val Thr Pro Asn Cys Leu Asp Leu Arg Ser Phe Cys Arg Ala Asp Pro 245 250 255
Leu Cys Arg Ser Arg Leu Met Asp Phe Gin Thr His Cys His Pro Met 260 265 270
Asp He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala 275 280 285
Tyr Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe He Ser Lys 290 295 300
Val Asn Thr Thr Val Ala Leu Ser Cys Thr Cys Arg Gly Ser Gly Asn 305 310 315 320
Leu Gin Asp Glu Cys Glu Gin Leu Glu Arg Ser Phe Ser Gin Asn Pro 325 330 335
Cys Leu Val Glu Ala He Ala Ala Lys Met Arg Phe His Arg Gin Leu 340 345 350
Phe Ser Gin Asp Trp Ala Asp Ser Thr Phe Ser Val Val Gin Gin Gin 355 360 365
Asn Ser Asn Pro Ala Leu Arg Leu Gin Pro Arg Leu Pro He Leu Ser 370 375 380
Phe Ser He Leu Pro Leu He Leu Leu Gin Thr Leu Trp 385 390 395 ( 2 ) INFORMATION FOR SEQ ID NO : 18 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 1271 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..946
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
C GGC TAC TGT GAA ACA CCT CAA CTC AGG AAC AGC TCT CTG ATA GGC 46 Gly Tyr Cys Glu Thr Pro Gin Leu Arg Asn Ser Ser Leu He Gly
400 405 410
TGC ATG TGC CAC CGG CGC ATG AAG AAC CAG GTT GCC TGC TTG GAC ATC 94
Cys Met Cys His Arg Arg Met Lys Asn Gin Val Ala Cys Leu Asp He
415 420 425
TAT TGG ACC GTT CAC CGT GCC CGC AGC CTT GGT AAC TAT GAG CTG GAT 142
Tyr Trp Thr Val His Arg Ala Arg Ser Leu Gly Asn Tyr Glu Leu Asp 430 435 440
GTC TCC CCC TAT GAA GAC ACA GTG ACC AGC AAA CCC TGG AAA ATG AAT 190
Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met Asn
445 450 455 460
CTC AGC AAA CTG AAC ATG CTC AAA CCA GAC TCA GAC CTC TGC CTC AAG 238
Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu Lys
465 470 475
TTT GCC ATG CTG TGT ACT CTC AAT GAC AAG TGT GAC CGG CTG CGC AAG 286
Phe Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp Arg Leu Arg Lys
480 485 490
GCC TAC GGG GAG GCG TGC TCC GGG CCC CAC TGC CAG CGC CAC GTC TGC 334
Ala Tyr Gly Glu Ala Cys Ser Gly Pro His Cys Gin Arg His Val Cys
495 500 505
CTC AGG CAG CTG CTC ACT TTC TTC GAG AAG GCC GCC GAG CCC CAC GCG 382
Leu Arg Gin Leu Leu Thr Phe Phe Glu Lys Ala Ala Glu Pro His Ala 510 515 520
CAG GGC CTG CTA CTG TGC CCA TGT GCC CCC AAC GAC CGG GGC TGC GGG 430
Gin Gly Leu Leu Leu Cys Pro Cys Ala Pro Asn Asp Arg Gly Cys Gly
525 530 535 540 GAG CGC CGG CGC AAC ACC ATC GCC CCC AAC TGC GCG CTG CCG CCT GTG 478 Glu Arg Arg Arg Asn Thr He Ala Pro Asn Cys Ala Leu Pro Pro Val 545 550 555
GCC CCC AAC TGC CTG GAG CTG CGG CGC CTC TGC TTC TCC GAC CCG CTT 526 Ala Pro Asn Cys Leu Glu Leu Arg Arg Leu Cys Phe Ser Asp Pro Leu 560 565 570
TGC AGA TCA CGC CTG GTG GAT TTC CAG ACC CAC TGC CAT CCC ATG GAC 574 Cys Arg Ser Arg Leu Val Asp Phe Gin Thr His Cys His Pro Met Asp 575 580 585
ATC CTA GGA ACT TGT GCA ACA GAG CAG TCC AGA TGT CTA CGA GCA TAC 622 He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala Tyr 590 595 600
CTG GGG CTG ATT GGG ACT GCC ATG ACC CCC AAC TTT GTC AGC AAT GTC 670 Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe Val Ser Asn Val 605 610 615 620
AAC ACC AGT GTT GCC TTA AGC TGC ACC TGC CGA GGC AGT GGC AAC CTG 718 Asn Thr Ser Val Ala Leu Ser Cys Thr Cys Arg Gly Ser Gly Asn Leu 625 630 635
CAG GAG GAG TGT GAA ATG CTG GAA GGG TTC TTC TCC CAC AAC CCC TGC 766 Gin Glu Glu Cys Glu Met Leu Glu Gly Phe Phe Ser His Asn Pro Cys 640 645 650
CTC ACG GAG GCC ATT GCA GCT AAG ATG CGT TTT CAC AGC CAA CTC TTC 814 Leu Thr Glu Ala He Ala Ala Lys Met Arg Phe His Ser Gin Leu Phe 655 660 665
TCC CAG GAC TGG CCA CAC CCT ACC TTT GCT GTG ATG GCA CAC CAG AAT 862 Ser Gin Asp Trp Pro His Pro Thr Phe Ala Val Met Ala His Gin Asn 670 675 680
GAA AAC CCT GCT GTG AGG CCA CAG CCC TGG GTG CCC TCT CTT TTC TCC 910 Glu Asn Pro Ala Val Arg Pro Gin Pro Trp Val Pro Ser Leu Phe Ser 685 690 695 700
TGC ACG CTT CCC TTG ATT CTG CTC CTG AGC CTA TGG TAGCTGGACT 956
Cys Thr Leu Pro Leu He Leu Leu Leu Ser Leu Trp 705 710
TCCCCAGGGC CCTCTTCCCC TCCACCACAC CCAGGTGGAC TTGCAGCCCA CAAGGGGTGA 1016
GGAAAGGACA GCAGCAGGAA GGAGGTGCAG TGCGCAGATG AGGGCACAGG AGAAGCTAAG 1076
GGTTATGACC TCCAGATCCT TACTGGTCCA GTCCTCATTC CCTCCACCCC ATCTCCACTT 1136
CTGATTCATG CTGCCCCTCC TTGGTGGCCA CAATTTAGCC ATGTCATCTG GTGCCTGTGG 1196
GCCTTGCTTT ATTCCTATTA TTGTCCTAAA GTCTCTCTGG GCTCTTGGAT CATGATTAAA 1256
CCTTTGACTT AAAAA 1271 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Gly Tyr Cys Glu Thr Pro Gin Leu Arg Asn Ser Ser Leu He Gly Cys 1 5 10 15
Met Cys His Arg Arg Met Lys Asn Gin Val Ala Cys Leu Asp He Tyr 20 25 30
Trp Thr Val His Arg Ala Arg Ser Leu Gly Asn Tyr Glu Leu Asp Val 35 40 45
Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met Asn Leu 50 55 60
Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu Lys Phe 65 70 75 80
Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp Arg Leu Arg Lys Ala 85 90 95
Tyr Gly Glu Ala Cys Ser Gly Pro His Cys Gin Arg His Val Cys Leu 100 105 110
Arg Gin Leu Leu Thr Phe Phe Glu Lys Ala Ala Glu Pro His Ala Gin 115 120 125
Gly Leu Leu Leu Cys Pro Cys Ala Pro Asn Asp Arg Gly Cys Gly Glu 130 135 140
Arg Arg Arg Asn Thr He Ala Pro Asn Cys Ala Leu Pro Pro Val Ala 145 150 155 160
Pro Asn Cys Leu Glu Leu Arg Arg Leu Cys Phe Ser Asp Pro Leu Cys 165 170 175
Arg Ser Arg Leu Val Asp Phe Gin Thr His Cys His Pro Met Asp He 180 185 190
Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu Arg Ala Tyr Leu 195 200 205
Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe Val Ser Asn Val Asn 210 215 220 Thr Ser Val Ala Leu Ser Cys Thr Cys Arg Gly Ser Gly Asn Leu Gin 225 230 235 240
Glu Glu Cys Glu Met Leu Glu Gly Phe Phe Ser His Asn Pro Cys Leu 245 250 255
Thr Glu Ala He Ala Ala Lys Met Arg Phe His Ser Gin Leu Phe Ser 260 265 270
Gin Asp Trp Pro His Pro Thr Phe Ala Val Met Ala His Gin Asn Glu 275 280 285
Asn Pro Ala Val Arg Pro Gin Pro Trp Val Pro Ser Leu Phe Ser Cys 290 295 300
Thr Leu Pro Leu He Leu Leu Leu Ser Leu Trp 305 310 315
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 175..1374
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TGTGGACGCG CGCTTCGGAG TTGGAGGGCG GCGCCCAGGA CCCTGGTGGG AGAGTGTGTG 60
CGTCGCGCTG GAGGGCGGGA GGCGGGGGCG GGAGGTGCCG GTCGAGGGAG CCCCGCTCTC 120
AGAGCTCCAG GGGAGGAGCG AGGGGAGCGC GGAGCCCGGC GCCTACAGCT CGCC ATG 177
Met
GTG CGC CCC CTG AAC CCG CGA CCG CTG CCG CCC GTA GTC CTG ATG TTG 225
Val Arg Pro Leu Asn Pro Arg Pro Leu Pro Pro Val Val Leu Met Leu
320 325 330
CTG CTG CTG CTG CCG CCG TCG CCG CTG CCT CTC GCA GCC GGA GAC CCC 273
Leu Leu Leu Leu Pro Pro Ser Pro Leu Pro Leu Ala Ala Gly Asp Pro
335 340 345
CTT CCC ACA GAA AGC CGA CTC ATG AAC AGC TGT CTC CAG GCC AGG AGG 321
Leu Pro Thr Glu Ser Arg Leu Met Asn Ser Cys Leu Gin Ala Arg Arg 350 355 360 AAG TGC CAG GCT GAT CCC ACC TGC AGT GCT GCC TAC CAC CAC CTG GAT 369
Lys Cys Gin Ala Asp Pro Thr Cys Ser Ala Ala Tyr His His Leu Asp
365 370 375 380
TCC TGC ACC TCT AGC ATA AGC ACC CCA CTG CCC TCA GAG GAG CCT TCG 417
Ser Cys Thr Ser Ser He Ser Thr Pro Leu Pro Ser Glu Glu Pro Ser 385 390 395
GTC CCT GCT GAC TGC CTG GAG GCA GCA CAG CAA CTC AGG AAC AGC TCT 465
Val Pro Ala Asp Cys Leu Glu Ala Ala Gin Gin Leu Arg Asn Ser Ser 400 405 410
CTG ATA GGC TGC ATG TGC CAC CGG CGC ATG AAG AAC CAG GTT GCC TGC 513
Leu He Gly Cys Met Cys His Arg Arg Met Lys Asn Gin Val Ala Cys 415 420 425
TTG GAC ATC TAT TGG ACC GTT CAC CGT GCC CGC AGC CTT GGT AAC TAT 561
Leu Asp He Tyr Trp Thr Val His Arg Ala Arg Ser Leu Gly Asn Tyr 430 435 440
GAG CTG GAT GTC TCC CCC TAT GAA GAC ACA GTG ACC AGC AAA CCC TGG 609
Glu Leu Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp
445 450 455 460
AAA ATG AAT CTC AGC AAA CTG AAC ATG CTC AAA CCA GAC TCA GAC CTC 657
Lys Met Asn Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu 465 470 475
TGC CTC AAG TTT GCC ATG CTG TGT ACT CTC AAT GAC AAG TGT GAC CGG 705
Cys Leu Lys Phe Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp Arg 480 485 490
CTG CGC AAG GCC TAC GGG GAG GCG TGC TCC GGG CCC CAC TGC CAG CGC 753
Leu Arg Lys Ala Tyr Gly Glu Ala Cys Ser Gly Pro His Cys Gin Arg 495 500 505
CAC GTC TGC CTC AGG CAG CTG CTC ACT TTC TTC GAG AAG GCC GCC GAG 801
His Val Cys Leu Arg Gin Leu Leu Thr Phe Phe Glu Lys Ala Ala Glu 510 515 520
CCC CAC GCG CAG GGC CTG CTA CTG TGC CCA TGT GCC CCC AAC GAC CGG 849
Pro His Ala Gin Gly Leu Leu Leu Cys Pro Cys Ala Pro Asn Asp Arg
525 530 535 540
GGC TGC GGG GAG CGC CGG CGC AAC ACC ATC GCC CCC AAC TGC GCG CTG 897
Gly Cys Gly Glu Arg Arg Arg Asn Thr He Ala Pro Asn Cys Ala Leu 545 550 555
CCG CCT GTG GCC CCC AAC TGC CTG GAG CTG CGG CGC CTC TGC TTC TCC 945
Pro Pro Val Ala Pro Asn Cys Leu Glu Leu Arg Arg Leu Cys Phe Ser 560 565 570 s4/
GAC CCG CTT TGC AGA TCA CGC CTG GTG GAT TTC CAG ACC CAC TGC CAT 993 Asp Pro Leu Cys Arg Ser Arg Leu Val Asp Phe Gin Thr His Cys His 575 580 585
CCC ATG GAC ATC CTA GGA ACT TGT GCA ACA GAG CAG TCC AGA TGT CTA 1041 Pro Met Asp He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys Leu 590 595 600
CGA GCA TAC CTG GGG CTG ATT GGG ACT GCC ATG ACC CCC AAC TTT GTC 1089 Arg Ala Tyr Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe Val 605 610 615 620
AGC AAT GTC AAC ACC AGT GTT GCC TTA AGC TGC ACC TGC CGA GGC AGT 1137 Ser Asn Val Asn Thr Ser Val Ala Leu Ser Cys Thr Cys Arg Gly Ser 625 630 635
GGC AAC CTG CAG GAG GAG TGT GAA ATG CTG GAA GGG TTC TTC TCC CAC 1185 Gly Asn Leu Gin Glu Glu Cys Glu Met Leu Glu Gly Phe Phe Ser His 640 645 650
AAC CCC TGC CTC ACG GAG GCC ATT GCA GCT AAG ATG CGT TTT CAC AGC 1233 Asn Pro Cys Leu Thr Glu Ala He Ala Ala Lys Met Arg Phe His Ser 655 660 665
CAA CTC TTC TCC CAG GAC TGG CCA CAC CCT ACC TTT GCT GTG ATG GCA 1281 Gin Leu Phe Ser Gin Asp Trp Pro His Pro Thr Phe Ala Val Met Ala 670 675 680
CAC CAG AAT GAA AAC CCT GCT GTG AGG CCA CAG CCC TGG GTG CCC TCT 1329 His Gin Asn Glu Asn Pro Ala Val Arg Pro Gin Pro Trp Val Pro Ser 685 690 695 700
CTT TTC TCC TGC ACG CTT CCC TTG ATT CTG CTC CTG AGC CTA TGG 1374
Leu Phe Ser Cys Thr Leu Pro Leu He Leu Leu Leu Ser Leu Trp 705 710 715
TAGCTGGACT TCCCCAGGGC CCTCTTCCCC TCCACCACAC CCAGGTGGAC TTGCAGCCCA 1434
CAAGGGGTGA GGAAAGGACA GCAGCAGGAA GGAGGTGCAG TGCGCAGATG AGGGCACAGG 1494
AGAAGCTAAG GGTTATGACC TCCAGATCCT TACTGGTCCA GTCCTCATTC CCTCCACCCC 1554
ATCTCCACTT CTGATTCATG CTGCCCCTCC TTGGTGGCCA CAATTTAGCC ATGTCATCTG 1614
GTGCCTGTGG GCCTTGCTTT ATTCCTATTA TTGTCCTAAA GTCTCTCTGG GCTCTTGGAT 1674
CATGATTAAA CCTTTGACTT AAAAA 1699
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear % ^~
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Val Arg Pro Leu Asn Pro Arg Pro Leu Pro Pro Val Val Leu Met
1 5 10 15
Leu Leu Leu Leu Leu Pro Pro Ser Pro Leu Pro Leu Ala Ala Gly Asp 20 25 30
Pro Leu Pro Thr Glu Ser Arg Leu Met Asn Ser Cys Leu Gin Ala Arg 35 40 45
Arg Lys Cys Gin Ala Asp Pro Thr Cys Ser Ala Ala Tyr His His Leu 50 55 60
Asp Ser Cys Thr Ser Ser He Ser Thr Pro Leu Pro Ser Glu Glu Pro 65 70 75 80
Ser Val Pro Ala Asp Cys Leu Glu Ala Ala Gin Gin Leu Arg Asn Ser 85 90 95
Ser Leu He Gly Cys Met Cys His Arg Arg Met Lys Asn Gin Val Ala 100 105 110
Cys Leu Asp He Tyr Trp Thr Val His Arg Ala Arg Ser Leu Gly Asn 115 120 125
Tyr Glu Leu Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro 130 135 140
Trp Lys Met Asn Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp 145 150 155 160
Leu Cys Leu Lys Phe Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp 165 170 175
Arg Leu Arg Lys Ala Tyr Gly Glu Ala Cys Ser Gly Pro His Cys Gin 180 185 190
Arg His Val Cys Leu Arg Gin Leu Leu Thr Phe Phe Glu Lys Ala Ala 195 200 205
Glu Pro His Ala Gin Gly Leu Leu Leu Cys Pro Cys Ala Pro Asn Asp 210 215 220
Arg Gly Cys Gly Glu Arg Arg Arg Asn Thr He Ala Pro Asn Cys Ala 225 230 235 240
Leu Pro Pro Val Ala Pro Asn Cys Leu Glu Leu Arg Arg Leu Cys Phe 245 250 255
Ser Asp Pro Leu Cys Arg Ser Arg Leu Val Asp Phe Gin Thr His Cys 260 265 270 &4
His Pro Met Asp He Leu Gly Thr Cys Ala Thr Glu Gin Ser Arg Cys 275 280 285
Leu Arg Ala Tyr Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe 290 295 300
Val Ser Asn Val Asn Thr Ser Val Ala Leu Ser Cys Thr Cys Arg Gly 305 310 315 320
Ser Gly Asn Leu Gin Glu Glu Cys Glu Met Leu Glu Gly Phe Phe Ser 325 330 335
His Asn Pro Cys Leu Thr Glu Ala He Ala Ala Lys Met Arg Phe His 340 345 350
Ser Gin Leu Phe Ser Gin Asp Trp Pro His Pro Thr Phe Ala Val Met 355 360 365
Ala His Gin Asn Glu Asn Pro Ala Val Arg Pro Gin Pro Trp Val Pro 370 375 380
Ser Leu Phe Ser Cys Thr Leu Pro Leu He Leu Leu Leu Ser Leu Trp 385 390 395 400

Claims

What is claimed is:
1. A purified and isolated DNA molecule having a nucleotide sequence set forth as rat retLl cDNA, partial human retLl cDNA, human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA.
2. A DNA molecule coding for an amino acid sequence of rat RetLl, partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3 or human RetL3.
3. A purified and isolated DNA molecule for use in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide product having at least a part of the primary structural conformation and the biological activity of rat RetLl , partial human RetLl , full-length human RetLl, human RetL2, murine RetL3 or human RetL3, said DNA selected from among: a) the DNA molecule of rat retLl cDNA, partial human retLl cDNA, human retLl cDNA, human retL2 cDNA, murine retL3 cDNA, or human retL3 cDNA, or its complementary strand; b) DNA molecules which hybridize under stringent conditions to the DNA molecules defined in a) or fragments thereof; c) DNA molecules which, but for the degeneracy of the genetic code, would hybridize to the DNA molecules defined in a) and b).
4. A purified and isolated DNA molecule which is at least 80% homologous to rat retLl cDNA, partial human retLl cDNA, human retLl cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA.
5. The DNA of claim 4, further characterised by encoding a protein with the biological activity of rat RetL 1 , partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3 or human RetL3.
6. The recombinant DNA molecule according to claim 1, 2, 3, 4, or 5, operably linked to an expression control sequence.
7. A vector comprising a DNA molecule encoding rat RetL 1 , partial human RetL 1 , full- length human RetLl, human RetL2, murine RetL3, partial human RetL3 or full-length human RetL3.
8. A biologically functional plasmid or viral DNA vector comprising a DNA molecule according to one of claims 1 , 2, 3, 4 or 5.
9. A prokaryotic or eukaryotic host cell stably transformed or transfected by a vector comprising a DNA molecule encoding rat RetL 1 , partial human RetL 1 , full-length human RetLl, human RetL2, murine RetL3, or human RetL3.
10. A process for the production of a polypeptide product having part or all of the primary structural conformation and the biological activity of rat RetL 1 , partial human RetL 1 , full-length human RetLl , human RetL2, murine RetL3, partial human RetL3 or full-length human RetL3, said process comprising: growing, under suitable culture conditions, prokaryotic or eukaryotic host cells transformed or transfected with a DNA molecule according to claim 1 , 2, 3, 4 or 5 in a manner allowing expression of such polypeptide product, and recovering either rat RetLl, partial human RetLl, full-length human RetLl, human RetL2, murine RetL3 or human RetL3.
11. A polypeptide product of the expression in a procaryotic or eukaryotic host cell of a DNA according to claim 10.
12. Purified and isolated rat RetL 1 , partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3 or human RetL3, substantially free of other human proteins.
13. A protein having an amino acid sequence which comprises rat RetL 1 , partial human RetLl, full-length human RetLl, human RetL2, murine RetL3, partial human RetL3 or full- length human RetL3.
14. A protein which is a variant of rat RetL 1 , partial human RetL 1 , full-length human RetL 1 , human RetL2, murine RetL3, partial human RetL3 or full-length human RetL3.
15. A soluble variant of the protein according to claim 11, 12, 13 or 14.
16. An IgG fusion protein comprising rat RetL 1 , partial human RetL 1 , full-length human RetLl, human RetL2, murine RetL3, partial human RetL3 or full-length human RetL3.
17. The protein of claim 15, fused to a toxin, imageable compound or radionuclide.
18. A specific monoclonal antibody to a protein of claim 11 , 12, 13 or 14.
19. The antibody of claim 18, associated with a toxin, imageable compound or radionuclide.
20. A hybridoma cell line which produces a specific antibody to rat RetLl, partial human RetLl, full-length human RetLl, human RetL2, murine RetL3, partial human RetL3 or full-length human Retl 3.
21. An antibody produced by a hybridoma of claim 20.
22. A method of promoting growth of new tissue or promoting survival of damaged tissue in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide of rat RetLl, partial human RetLl, full-length human RetLl, human RetL2, murine RetL3, partial human RetL3 or full-length human RetL3.
23. The method of claim 22, further comprising administering to the subject a therapeutically effective amount of a second polypeptide.
24. The method of claim 23, wherein the second polypeptide is GDNF, neurturin or a GDNF-related polypeptide.
25. The method of claim 22, wherein the tissue is kidney or neural tissue.
26. The method of claim 22, wherein the polypeptide is membrane bound.
27. The method of claim 22, wherein the polypeptide is soluble.
28. The mediod of claim 27, wherein the polypeptide is a fusion protein.
29. The method of claim 22, 23, 24, 25, 26 or 27, wherein the subject is human.
30. A method of inhibiting signal transduction involving a cell expressing a RetL, comprising contacting the cell with an antibody to the RetL.
31. A method of inhibiting signal transduction involving a cell expressing a RetL, comprising contacting the cell with a soluble Ret protein.
32. The method of claim 31 , wherein the soluble Ret protein is a fusion protein.
33. A method for targeting a toxin, imageable compound or radionuclide to a cell expressing Ret, comprising contacting the cell with the RetL fusion protein of claim 17.
34. A method of suppressing growth of a tumor cell which expresses Ret, comprising contacting the cell with a fusion protein of RetL and a toxin or radionuclide.
35. The method of any of claims 30-34, wherein the cell is within a subject, and the protein is administered to the subject.
36. A method for targeting a toxin, imageable compound or radionuclide to a cell expressing a RetL, comprising contacting the cell with an anti-RetL antibody conjugated to a toxin, imageable compound or radionuclide.
37. A method of suppressing growth of a tumor cell which expresses a RetL, comprising contacting the cell with an anti-RetL antibody conjugated to a toxin or radionuclide.
38. The method of claim 36 or 37, wherein the cell is within a subject, and the conjugated antibody is administered to the subject.
39. A method for targeting a toxin, imageable compound or radionuclide to a cell expressing a RetL, comprising contacting the cell with a Ret fusion protein, wherein the Ret fusion protein comprises Ret and a toxin, imageable compound or radionuclide.
40. The method of claim 39, further comprising contacting the cell with GDNF, neurturin, or a GDNF-related polypeptide.
41. A method of suppressing growth of a tumor cell which expresses a RetL, comprising contacting the cell with a fusion protein of Ret and a toxin or radionuclide.
42. The method of claim 39, 40 or 41 , wherein the cell is within a subject, and the protein is administered to the subject.
43. A method of treating a subject with a disorder of RetL metabolism, comprising administering to the subject a vector of claim 7 or 8.
44. A method of promoting growth of new tissue in a subject, comprising administering to the subject a vector of claim 7 or 8.
45. The method of claim 44, wherein the tissue is renal tissue or neural tissue.
46. A method of promoting survival of damaged tissue in a subject, comprising administering a therapeutically effective amount of a vector of claim 7 or 8 to the subject.
47. The method of claim 46, wherein the tissue is renal tissue or neural tissue.
PCT/US1997/007726 1996-05-08 1997-05-07 RET LIGAND (RetL) FOR STIMULATING NEURAL AND RENAL GROWTH WO1997044356A2 (en)

Priority Applications (21)

Application Number Priority Date Filing Date Title
CA002253871A CA2253871C (en) 1996-05-08 1997-05-07 Ret ligand (retl) for stimulating neural and renal growth
BR9710665A BR9710665A (en) 1996-05-08 1997-05-07 Compounds that promote tissue development
SI9730750T SI0914339T1 (en) 1996-05-08 1997-05-07 RET LIGAND 3 (RetL3) FOR STIMULATING NEURAL AND RENAL GROWTH
EE9800377A EE9800377A (en) 1996-05-08 1997-05-07 Ret ligand (RetL) to stimulate nerve and kidney growth
SK1524-98A SK286115B6 (en) 1996-05-08 1997-05-07 Nucleic acid coding c-Ret ligand, polypeptide ligand c-Ret, process for the preparation thereof and use for stimulating neural and renal growth
NZ332706A NZ332706A (en) 1996-05-08 1997-05-07 Ret ligand (retl) for stimulating neural and renal growth
DE69736428T DE69736428T2 (en) 1996-05-08 1997-05-07 RET-LIGAND 3 (RetL3) TO STIMULATE NEURAL AND RENAL GROWTH
PL329946A PL191248B1 (en) 1996-05-08 1997-05-07 Ret ligand (retl) for stimulating growth of nerve and kidney tissue
RO98-01550A RO120343B1 (en) 1996-05-08 1997-05-07 C-ret polypeptide and nucleic acid encoding therefor
JP54243197A JP2002515743A (en) 1996-05-08 1997-05-07 Ret ligand for stimulating nerve and kidney proliferation (RetL)
EA199800990A EA002544B1 (en) 1996-05-08 1997-05-07 Ret ligand (retl) for stimulating neutral and renal growth
IL126918A IL126918A (en) 1996-05-08 1997-05-07 Ret ligand (retl) for stimulating neural and renal growth
EP97930981A EP0914339B1 (en) 1996-05-08 1997-05-07 RET LIGAND 3 (RetL3) FOR STIMULATING NEURAL AND RENAL GROWTH
AU34729/97A AU732392B2 (en) 1996-05-08 1997-05-07 Ret ligand (retL) for stimulating neural and renal growth
US09/187,906 US6677135B1 (en) 1996-05-08 1998-11-06 Ret ligand (RetL) for stimulating neutral and renal growth
IS4884A IS2361B (en) 1996-05-08 1998-11-06 Ret ligament to stimulate nerve growth and urinary tract growth
NO19985231A NO323612B1 (en) 1996-05-08 1998-11-09 Ret-ligandb3 (RetL3) for stimulation of neural and renal growth, nucleic acids encoding the polypeptide, fusion protein, and antibody that specifically binds the polypeptide.
HK99105136A HK1021821A1 (en) 1996-05-08 1999-11-09 Ret ligand 3 (retl3) for stimulating neural and renal growth
US09/489,407 US6861509B1 (en) 1996-05-08 2000-01-21 Antibodies to Ret and RetL3
US10/668,936 US20050080235A1 (en) 1996-05-08 2003-09-23 Ret ligand (RetL) for stimulating neural and renal growth
NO20065528A NO324957B1 (en) 1996-05-08 2006-11-30 Use of monoclonal antibody for the preparation of drug for the treatment of Ret-associated disorders.

Applications Claiming Priority (6)

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US1742796P 1996-05-08 1996-05-08
US60/017,427 1996-05-08
US1930096P 1996-06-07 1996-06-07
US60/019,300 1996-06-07
US2185996P 1996-07-16 1996-07-16
US60/021,859 1996-07-16

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TR199802252T2 (en) 1999-02-22
EP0914339A2 (en) 1999-05-12
CN1163509C (en) 2004-08-25
NO985231L (en) 1999-01-08
EA002544B1 (en) 2002-06-27
EP1757617A1 (en) 2007-02-28
DE69736428T2 (en) 2007-08-30
EE9800377A (en) 1999-04-15
IS4884A (en) 1998-11-06
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NO20065528L (en) 1999-01-08
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CA2253871C (en) 2005-04-26
DK0914339T3 (en) 2006-12-04
BR9710665A (en) 1999-08-17
WO1997044356A3 (en) 1998-02-19
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CN1624126A (en) 2005-06-08
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