MXPA98009812A - Modulators of the regeneration of the tej - Google Patents

Abstract

The present invention relates to proteins that are promoted in damaged or regenerating tissues, as well as the DNA that codes for these proteins, as well as therapeutic compositions and treatment methods that comprise these components.

Description

$ MODULATORS OF TISSUE REGENERATION FIELD OF THE INVENTION The invention relates to proteins that are promoted in damaged or regenerating tissues, as well as to DNA that codes for these proteins. The invention also relates to therapeutic compositions and methods of treatment comprising these proteins.

BACKGROUND OF THE INVENTION A dynamic remodeling of the tissue architecture occurs during development and during tissue repair after damage. To study this process, he has focused on a model of kidney damage caused by an ischemia-reperfusion attack. The kidney is able to repair the damage to the proximal tubule epithelium through a complex series of events comprising cell death, the proliferation of surviving proximal tubule epithelial cells, the formation of poorly differentiated regenerative epithelium on the bare basement membrane, and the differentiation of the regenerative epithelium to form fully functional epithelial cells of the proximal tubule (Wallin et al., Lab. Invest. 66: 474-484, 1992; Witzgall et al., Mol. Cell Biol. 13: 1993- 1942, 1994; Ichimura et al., Am. J. Physiol. 269: F653-662, 1995; Thadhani et al., N. Engl. J. Med. 334: 1148-1460, 1996). Growth factors such as IGF, EGF, and HGF have been implicated in this repair process, since they have the endothelial cell adhesion molecule ICAM-1. However, the mechanisms by which tubular epithelial cells are restored are not yet understood. To identify the molecules involved in the process of damage and repair of the tubular epithelium, the difference in mRNA populations between damaged / regenerating kidneys and normal ones is analyzed using the figurative difference analysis (RDA, for its acronym in English) . RDA is a PCR-based method for subtraction that produces cDNA fragments specific to the target cell or tissue by subtraction and repetitive amplification (Hubank and Schutz, Nucí Acids Res. 22: 5640-5648, 1994).

SUMMARY OF THE INVENTION It is generally provided by molecules related to kidney damage (each of which is subsequently called a "KIM") that are promoted in renal tissue after damage to the kidney. The KIM proteins and peptides of the invention, as well as their antagonists and, agonists, and their corresponding ones are useful in a variety of therapeutic interventions. The invention provides a modified and isolated DNA molecule having a nucleotide sequence set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6. The invention also includes the complementary strands of these sequences, the DNA molecules that hybridize under severe conditions to the DNA molecules mentioned above, and the DNA molecules that, but for the degeneracy of the genetic code, they will hybridize to any of the DNA molecules defined above. These DNA molecules can be recombinant, and can be operably linked to an expression control sequence. The invention further provides a vector comprising a purified and isolated DNA molecule having a nucleotide sequence set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, or one of the other DNA molecules defined above. This vector can be a biologically functional plasmid or a viral DNA vector. One embodiment of the invention provides a host, prokaryotic or eukaryotic cell, transformed or stably transfected by a vector comprising a DNA molecule of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6. In P718 another embodiment of the invention, there is provided a process for the production of a KIM polypeptide product encoded with a DNA molecule as described above; the process comprises growth, under suitable growing conditions, of prokaryotic or eukaryotic host cells, transformed or transfected with the DNA molecule in a manner that allows the expression of the DNA molecule, and the recovery of the polypeptide product from expression. A purified, isolated, human KIM protein 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 biological activity of a protein of KIM. The KIM proteins of the invention may have an amino acid sequence comprising SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7, or may be a variant of SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7, or a purified and isolated encoded protein or DNA of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6. These proteins can be provided substantially free of other human proteins. The invention further includes variants of those proteins, such as soluble variants or fusion proteins. The KIM fusion proteins of the invention P718 may comprise an immunoglobulin, a toxin, an imageable compound or a radionuclide. The invention also provides a monoclonal antibody specific to the KIM proteins described above. The anti-KIM antibody can be associated with a toxin, an imageable compound or radionuclide. A hybridoma cell line that produces this specific antibody is also taught. Pharmaceutical compositions are also within the scope of the invention. A pharmaceutical composition of the invention may comprise a therapeutically effective amount of a KIM protein or an anti-KIM antibody of the invention, with a pharmaceutically acceptable carrier. Diagnostic methods are within the invention, such as the assessment of the presence or course of resolution of kidney damage by measuring the concentration of KIM in urine, serum, or urine sediment of patients who have or are in risk of developing kidney disease. The methods of treatment of the invention include the treatment of patients with therapeutically effective amounts of KIM, KIM variants, KIM analogs, KIM fusion proteins, KIM agonists and antibodies to KIM or to KIM ligands. Others P718 therapeutic compounds of the invention include KIM ligands, anti-KIM antibodies, and KIM ligand fusion proteins. These compounds may be useful in therapeutic methods that either stimulate or inhibit cellular responses that are dependent on KIM function. Additional methods of the invention inhibit growth in tumor cells expressing KIM by contacting the cells with a fusion protein of a KIM ligand and either a toxin or radionuclide, or with an anti-KIM antibody conjugated to a toxin or a radionuclide. Similarly, the growth of tumor cells expressing the KIM ligand can be inhibited by contacting the cells with a fusion protein of a KIM or either a toxin or a radionuclide, or with an anti-KIM ligand antibody. conjugated to a toxin or a radionuclide. The invention also encompasses methods of gene therapy. These include a method for treating a subject with a renal disorder, a method for promoting the growth of new tissue in a subject, and a method for promoting the survival of damaged tissue in a subject, which comprises administering to the subject a vector that includes the DNA comprising the nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6 The compounds of the invention are also useful for forming tissue images, either in vitro or in vivo. This method comprises directing an imageable compound to a cell that expresses a protein of SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7, which comprises contacting the cell with either a monoclonal antibody of the invention or a fusion protein comprising a protein as described above, fused to an imageable compound. For in vivo methods, the cell is within a subject, and the protein or monoclonal antibody is administered to the subject. The invention also includes diagnostic methods, such as a method for unifying renal cell regeneration damage in a subject, which comprises comparing the level of expression of either SEQ ID No. 1, SEQ ID No. 2, SEQ ID No 4 or SEQ ID No. 6, from renal cells of the subject to a level of control of expression of the sequence in the control renal cells. Another method of the invention includes identifying the promotion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6 in cells comprising contacting the cells with an antisense probe and inhibiting hybridization to the RNA within the cell. A further embodiment of the diagnostic methods of the invention includes the assessment of the presence or concentration of a molecule of the invention in either urine, serum, or other body fluids, or in the urine sediment or tissue samples. The molecule related to damage, measured, can be correlated with the presence, degree or course of a pathological process. This correlation can also be used to assess the efficacy of a therapeutic regimen.

BRIEF DESCRIPTION OF DRAWINGS OR FIGURES Figure 1 is the nucleotide sequence of the rat clone cDNA 3-2, with the putative protein reading frame of 615 to 1535. Figure 2 is a listing of the cDNA sequence of a rat clone 1-7, with the putative protein reading frame from 145 to 1065. Figure 3 is a listing of the cDNA sequence of a rat clone 4-7, with the putative protein reading frame of 107 to 1822. Figure 4 is a listing of the cDNA and reduced amino acid sequences of the human clone H13-10-85, with the putative protein reading frame from 1 to 1002. The upper line of the listing is the cDNA sequence (FIG. SEQ ID No. 6) and the lower line is the deduced amino acid line (SEQ ID No. 7). Figure 5 is a BESTFIT comparison of the nucleotide sequence of the human clone H13 -10-85 with that of the rat clone 3-2.

DETAILED DESCRIPTION OF A NATION KIM genes are identified by analyzing the differences in mRNA expression between regenerating and normal kidneys using the figurative difference analysis (RDA). The RDA is a PCR-based method for subtraction "that produces cDNA fragments specific to the cell or target tissue by subtraction and repetitive amplification. The 48-hr post-ischemic adult rat kidney RNA cDNA representation is subtracted from the normal adult rat kidney sample (operated with simulation). In this procedure, sequences "that are common to both kidney samples, both post-ischemic and normal, are removed, leaving those sequences that are significantly expressed only in damaged kidney tissue. These genes code for proteins that may be therapeutically beneficial for kidney disorders or be included in the damage process. Several clones have been obtained, sequenced and characterized. The clones are then investigated for their expression patterns during kidney repair, development and tissue distribution by Northern analysis and in situ RNA hybridization.

Sequence Identification Numbers The nucleotide and amino acid sequences referred to in the specification have been given the following sequence identification numbers: SEQ ID No. 1.- nucleotide sequence of the insert Rat 3-2 cDNA SEQ ID No. 2 .- nucleotide sequence of the rat 1-7 cDNA insert. SEQ ID No. 3 - amino acid sequence of rat KIM-1, encoded by rat DNAs 3-2 and 1-7 SEQ ID No. 4 - nucleotide sequence of the rat insert CDNA 4-7 of rat SEQ ID No. 5.- amino acid sequence encoded by the cDNA insert 4-7 SEQ ID No. 6.- nucleotide sequence of the human clone H13-10-85 of cDNA SEQ ID No. 1 . - amino acid sequence encoded by clone H13-10-85, human.

Definition of Terms A "KIM protein" used herein synonymously with "KIM", is a protein encoded by MRNA that is selectively promoted after damage to the kidney. A group of KIM proteins of interest include P718 those encoded by the mRNA that is selectively promoted at any time in the space of a week that follows after any attack that results in damage to the renal tissue. Examples of times in which the promotion can be identified include 10 hours, 24 hours, 48 hours or 96 hours after an attack. Examples of types of attack include those that result in types of ischemic, toxic or other types of damage. A "KIM agonist" is a molecule that can specifically trigger a cellular response normally triggered by interaction with KIM with a KIM ligand. A KIM agonist may be a variant of KIM, or an antibody specific to KIM, or a soluble form of the KIM ligand. A "KIM antagonist" is a molecule that can be specifically associated with a KIM ligand, or a KIM, thereby blocking or otherwise inhibiting the binding of KIM to the KIM ligand. The antagonist blocks the binding or inhibits cellular responses that would otherwise be triggered by the ligation of the KIM ligand with KIM or with a KIM agonist. Examples of KIM agonists include certain KIM variants, KIM fusion proteins, proteins and antibodies specific to a KIM or KIM ligand.

P718 A "KIM ligand" is any molecule that binds non-covalently and specifically to a KIM protein. This ligand can be a protein, peptide, steroid, antibody, amino acid derivative, or other type of molecule, in any form, including that it occurs in any natural way, produced recombinantly, or is otherwise synthesized. A KIM ligand may be in any form, including in soluble form, membrane bound, or as part of a fusion construct with immunoglobulin, fatty acid, or other portions. The KIM ligand can be an integrin. A ligand of KIM bound to the membrane can act as a receptor which, when bound, or is associated with KIM, triggers a cellular response. In some interactions, KIM may associate with more than one individual KIM ligand, or may associate with a KIM ligand as part of a complex with one or more different molecules or co-factors. In a situation where both the KIM and the KIM ligand bind to the cell membranes, the KIM can associate and react with the KIM ligand that binds to the same cell as the KIM, or it can associate with and react with the KIM ligand. KIM that is attached to a second cell. Where KIM ligation occurs between molecules bound to different cells, the two cells may be the same or different with respect to the cell type or origin, condition P718 phenotypic or metabolic, or type of cellular response (eg growth, differentiation or apoptosis) to a given stimulus. "KIM Ligation" refers to the contact and binding of KIM with a KIM ligand. By "sequence alignment" is meant the placement of a sequence, either of nucleotides or amino acids, with that of another, to allow a comparison of the sequence of the relevant portions of one with that of the other. An example of a method of this procedure is given in Needleman et al. (J. Mol. Biol. 48: 443-453, 1970). The method can be implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As will be understood by those skilled in the art, functionally equivalent homologous sequences that include functionally equivalent arrays of cysteine residues within the skeleton while retaining cysteine, include insertions or deletions of amino acids that alter the linear arrangement of these cysteines, but do not they impart their relationship materially to the folded structure of the protein. Therefore, internal separations and amino acid initiations in the candidate sequence are ignored for purposes of calculating the level of homology or identity of the amino acid sequence between the candidate and P718 reference. A feature frequently used in the establishment of protein homology is the similarity of the number and location of cysteine residues between one protein and another. An "antisense DNA" refers to the sequence of Chromosomal DNA that is transcribed. An "antisense probe" is a probe that comprises at least a portion of the antisense DNA for a portion of nucleic acid of interest. By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene and another DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods to generate DNA fragments, by joining the fragments to vector molecules, to introduce the compound DNA molecule into a host cell in which it can replicate, and the transductional plan which has the target gene from among the host, recipient cells. By "cDNA" is meant a complementary or copy DNA produced from an mRNA template by the action of an RNA-dependent DNA polymerase (inverted transcriptase). In this way, a "clone of CDNA "means a duplex DNA sequence complementary to the RNA molecule of interest, carried in a vector of P718 release. By "cDNA library" is meant a collection of recombinant DNA molecules containing cDNA inserts that together comprise a representation of the mRNA molecules present in a whole organism or tissue, depending on the source of the RNA templates. This cDNA library can be prepared by methods known to those skilled in the art, and described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra. In general, RNA is first isolated from the cells of an organism whose genome it is desired to clone a particular gene. Mammalian, and particularly human, cell lines are preferred for purposes of the invention. Alternatively, the RNA can be isolated from a tumor cell, derived from an animal tumor, preferably from a human tumor. In this way, a library of, for example, an adrenal tumor of human can be prepared, but any tumor can be used. As used herein, the term "DNA polymorphism" refers to the condition in which two or more different nucleotide sequences may exist at a particular site in the DNA. "Expression vector" includes vectors that are P718 capable of expressing DNA sequences contained therein, ie, in the coding sequences, other sequences capable of effecting their expression are operably linked. It is to be understood that although not always specifically pointed out, these expression vectors must be replicable in host organisms either as episomes or as an integral part of chromosomal DNA. A useful, but not necessary, element of a cash expression vector is a marker coding sequence, which is a sequence that codes for a protein that results in a phenotypic property (such as resistance to tetracycline) of the proteins. cells that contain the protein that allows these cells to be easily identified. In summary, the "expression vector" is given as a functional definition, and any DNA sequence that is capable of effecting the expression of a specific, contained DNA code is included in this term, since it is applied to the sequence specified As shown, these vectors are often in the form of plasmids, thus "plasmids" and "expression vector" are often used interchangeably. However, the invention is proposed to include other forms of expression vectors that serve equivalent functions and that may become known in the art, from time to time.

P718 By "functional derivative" is meant "fragments", "variants", "analogues", or "guimic derivatives" of a molecule. A "fragment" of a molecule, such as any of the antigens of the present invention, is meant to require any set of polypeptides of the molecule. A "variant" of these molecules is meant to refer to a molecule that is presented in a lateral manner substantially similar to either the entire molecule, or a fragment thereof. An "analogue" of a molecule is meant to refer to a natural molecule substantially similar to either the molecule or a fragment thereof. The term "gene" means a polynucleotide sequence that codes for a peptide. By "homogeneous" is meant, when referring to a peptide or a DNA sequence, with the primary molecular structure (i.e., the amino acid or nucleotide sequence) of substantially all of the molecules present in the composition under consideration being identical . "Isolated" refers to a protein of the present invention, or any gene that codes for this protein, that is substantially free of other proteins or genes, respectively, or of other contaminants with which nature could normally be found, and as It exists in a form not found in nature. The term "brand" refers to a molecular portion capable of detection, "Which includes, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, and dyes. The term "probe" refers to a ligand of known qualities capable of selectively binding to an objective antiligand. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to an objective strand. "Recombinant host cells" refers to cells that have been transformed with vectors constructed using recombinant DNA techniques. As described herein, the antibody or modification thereof produced by a recombinant host cell is by virtue of this transformation, rather than minor amounts, or more commonly, in less than detectable amounts, as would be produced by the non-transformed host . By "substantially pure" is meant any protein of the present invention, or any gene encoding this protein, which is essentially free of other foreign proteins, respectively, or other contaminants with which P718 could be found normally in nature, and as it exists in a form not found in nature. A molecule is said to be "substantially similar" to another molecule if the amino acid sequence in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, with the condition that two molecules have a similar activity, they are considered variants since how that term is used in the present 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 joint portions normally, not in a part of the molecule. These molecules can comprise the solubility of the molecule, absorption, via biological means, etc. The portions can alternatively decrease the toxicity of the molecule, eliminate or attenuate any side effect initiated from the molecule, etc. Molecules capable of mediating these effects are described, for example, in Reminqton's Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, Penn (1980). By "vector" is meant a DNA molecule, derived from a plasmid or bacteriophage, in which the DNA fragments can be inserted or cloned. A vector will have 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.

Compounds of the Invention The invention includes the cDNA of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6; as well as the sequences that include the sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, and derived from these sequences. The invention also includes vectors, liposomes and other carriers that span these sequences or derivatives thereof. The invention also includes protein transcribed from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, which includes but is limited to SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7, and its derivatives and variants. One embodiment of the invention includes soluble variants of a KIM protein that is usually synthesized as a membrane-associated protein, and which is promoted after damage. The soluble variants lack at least a portion of the transmembrane section. or intramembrane of a native KIM protein. In some examples, the soluble variant lacks the entire section Transmembrane or intramembrane P718 of a native KIM protein. Soluble variants include fusion proteins encompassing derivatives of KIM proteins that lack at least a portion of the transmembrane or intramembrane section of a native KIM protein. All types of KIM fusion proteins are included, particularly those that incorporate the his-tag, Ig-tag forms of the molecule. These KIM fusions may have characteristics that are therapeutically advantageous, such as the increased half-life conferred with the Ig-tag. Also included are fusion proteins that incorporate domain portions selected from the KIM protein. The variants may differ from the KIM protein that occurs naturally in the amino acid sequence or in ways that do not contain the sequences, or both. Variants in the amino acid sequence occur when one or more amino acids in the naturally occurring KIM protein are replaced with a different natural amino acid, an amino acid derivative or a non-native amino acid. Particularly preferred variants include the naturally occurring KIM protein, or biologically active fragment of the naturally occurring KIM protein, whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions, which P718 typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide. The variants may also have sequences that differ by one or more non-conservative substitutions, deletions and amino acid insertions that do not cancel the biological activity of the KIM protein. 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. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. 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 amino acids (acids) include aspartic acid and glutamic acid. Other conservative substitutions can be taken from the table below, and others are described below by Dayhoff in Atlas of Protein Sequence and Structure (1988).

P718 AMINO ACID CONSERVATIVE REPLACEMENTS P718 Other variants within the invention are those with modifications that increase the "stability P718 peptide. These variants may contain, for example, one or more non-peptide linkages (which replace the peptide linkages) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or amino acids that do not occur naturally or synthetically, such as beta- or gamma-amino acids and cyclic variants. The incorporation of the D-amino acids in place of the L-amino acids in the polypeptide can increase their resistance to proteases. See, for example, US Patent No. 5,219,990. In general, the substitutions that can be expected for those seeking changes in the functional properties of the KIM polypeptides are those in which: (I) a hydrophilic residue, for example, serine or threonine, is replaced by a hydrophobic residue, for example , leucine, isoleucine, phenylalanine or alanine; (ii) a cysteine residue that is replaced by (or by) any other residue; (iii) a residue having a natural electropositive chain, for example, lysine, arginine or histidine, is replaced by (or by) a residue having an electronegative charge, for example, glutamic acid or aspartic acid; or (iv) a residue having a side or bulky chain, for example, phenylalanine, P718 is replaced by (or by) one that does not have this side chain, for example, glycine. The peptides of this invention can also be modified by various changes such as insertions, deletions or substitutions, either conservative or non-conservative where these changes could provide certain advantages in their use. Binding variants are specifically included in the invention. In other embodiments, variants with amino acid substitutions "that are less conservative may also result in desired derivatives, for example, by causing changes in the face, with formation and other biological properties. These substitutions will include, for example, substitution of a hydrophilic residue for a hydrophobic residue, replacement of a cysteine or proline with another residue, substitution of a residue having a side chain attached to a residue having a bulky side chain or substitution of a residue. that has a net positive charge or a residue that has a net negative charge. When the result in a given substitution can not be predicted with certainty, the derivatives can be easily evaluated according to the methods described herein to determine the presence or absence of the desired characteristics. The variants within the scope of the invention P718 include proteins and peptides with amino acid sequences that have at least eighty percent homology to a KIM protein. More preferably, the homology of the sequence is at least ninety percent, or at least ninety-five percent. For purposes of determining the homology of the length of the separation sequences, it will generally be at least 8 amino acid residues, usually at least 20 amino acid residues. Variants of the compounds of the invention also include any protein that 1) has an amino acid sequence that is at least forty percent homologous to a KIM protein of the invention, and that also 2) after being placed in an optimal alignment with the KIM sequence (as depicted in Figure 1 for human and rat KIM-1) has at least 80% of its cysteine residues aligned with the cysteines of the KIM protein of the invention. As it is possible to replace substituents of the stage, it is also possible to replace functional groups that join the stage with groups characterized by similar characteristics. These substitutions will be initially conservative, that is, the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. The modification is not sequence may include, for example, the P718 chemical derivatization in vivo or in vitro of the portions of the KIM protein that occur naturally, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation. Also included within the invention are agents that specifically bind the protein, or a fragment of the protein (SEQ ID No. 3, 5 or 7). These agents include ligands and antibodies (including monoclonal, single chain, double chain, Fab fragments and others, whether native, human, humanized, primatized or chimeric). Additional descriptions of these agent categories are in PCT application 95/16709, the specification of which is incorporated herein by reference.

Experimental Procedures 1. Generation of RNA from adult, iscruemic, and normal rat kidneys Rat, damaged, ischemic kidneys were generated as described by itzgall et al.

(J. Clin Invest. 93: 2175-2188, 1994). Briefly, the renal artery and vein of a kidney of an adult rat Sprague-Dawley held on for 40 minutes and reperfused.

Damaged kidneys were collected from the rats 25 hours and 48 hours after reperfusion. I also know P718 collected the kidneys from normal Sprague-Dawley adult rats, operated with simulation. The total RNA is prepared from the organs based on the protocol of Glisin et al. (Biochemistry 13: 2633, 1974). Briefly, the harvested organs are placed immediately in CNG buffer (4M guanidine thiocyanate), 0.5% SDS, 25 mM sodium citrate, 0.1% Sigma antifoam) and destabilized on ice with a polytron. The cellular debris was removed. With a spin at low speed in a chemical centrifuge and the supernatant fluid was placed on a cushion with CsCl 5.7 M, 25 mM sodium acetate, 1 mM EDTA. The RNA was sedimented through the pad in a SW40TÍ rotor at 22 K for 15 hours. RNA was redispersed in water treated with sterile DEPC, precipitated twice with 3 M sodium acetate with 1/10 volume and 2.5 volumes of EtOH. Poly A + A N was isolated using an mRNA purification kit (Pharmacia, Catalog No. 27-91258-02). .- 2. Figurative Difference Analysis Method (RDA) to isolate RDA fragments 1-7, 3-2? _ 4.-1 Double-stranded cDNA is synthesized from poly A + RNA from the 48-h post-ischemic kidney , and of the operation with simulation, using the "Synthesis P718 Gibco BRL Superscript Cholee ™ System cDNA, Catalog No. 18090. The first strand is synthesized by priming with oligo dT and using the inverted transcriptase Superscript IIMR. The second strand is generated using DNA polymerase I and NRase from E. coli followed by T4-DNA polymerase using the conditions recommended by BRL. The RDA analysis is performed essentially as described by Hubank and Schatz (Nucleic Acid Research 22: 5640-48, 1994). Briefly, the cDNA of the 48-h post-ischemic kidney is digested with the restriction enzyme of Dpn II and ligated to the oligonucleotides R-Bgl-12/24 (see reference for exact sequence). PCR amplification (performed with Perkin-Elmer Taq polymerase and its corresponding PCR buffer) of the binder-linked cDNA is used to generate the initial representation. That PCR product is designated "tester amplicon". The same procedure is used to generate the "actuator amplicon" of the rat kidney cDNA operated with simulation. Hybridization of the tester and actuator amplicons followed by selective amplification is performed three times to generate the Differential Product One (DPI), Two (DP2) and Three (DP3). The DPI product generation is performed as described by Hubank and Schatz (Nuclei Acid Research 22: 5640-48, 1994). The products of DP2 and DP3 P718 is also generated as described by Hubank and Schatz (id.), Except that the ratio is of actuator: tester is changed to 5,333: 1 for DP2 and to 40,000: 1 to 4,000: 1 for DP3. Three RDA products are driven from DP3 in the cloning vector pUC18; the RDA product of 1-7 (252 bp) when the DP3 was generated using a ratio of 40,000: 1, and the RDA product of 3-2 (445 bp) and 4-7 (483 bp) when DP was generated using the ratio of 4,000: 1. The DNA fragments are subcloned using the Pharmacia Sureclone ™ kit (Catalog No. 27-9300-01) to repair the PCR ends with the Klenow enzyme and to facilitate ligation with blunt ends of the pUC18 vector fragments. 3_?. Northern Analysis Poly A + RNA (2.5 μg) of adult rat kidney, normal, (operated with simulation), adult kidney, post-ischemic damaged, 48 hours, and 18-day embryonic kidney are electrophoresed , and Northern (Cate, Cell 45: 685, 1986) is transferred to a GeneScreem ™ membrane (Dupont). Hybridization in PSB buffer (Tris 7.5 50 mM, 1 M NaCl, 0.1% sodium pyrophosphate, 0.2% PVP, 0.2% Ficoll, 0.2% BSA, 1% SDS, containing 10% dextran sulfate). % and 100 μg / ml of tRNA, is performed at 65 SC using three different P718 probes. Product of RDA 1-7, product of RDA 3-2, and product of RDA 4-7. They are then radiolabelled using the random priming marking kit, "Ready to Go" "from Pharmacia (Catalog No. 27-9551-01). The RDA products 1-7, 3-2 and 4-7 hybridize to mRNA present in all three samples, but more intensely to the mRNAs in the 48-h post-ischemic kidney mRNA samples. A Northern blot analysis of adult rat tissues indicates that the 1-7 gene is expressed at very low levels in the adult, normal, testes, basal and lung kidneys. The 3-2 gene is expressed in liver, kidney, basal, and brain. The 4-7 gene is expressed in baso, liver, lung, testis, heart, brain, liver and skeletal muscle. In the presence of mRNA of different size in some tissues in the transfer of 1-7 and 3-2 it indicates that the product and primary transcript of the gene of 1-7 and of the gene of 3-2 can undergo alternative binding and / or polyadenylation . 4. Isolation of cDNA clones from 3-2 and 4-7 A cDNA library is generated from 4 μg of poly A + RNA from the damaged 48-hour post-ischemic kidney using the BRL Supercript Choice ™ system reagents for cDNA synthesis, and the Stratagene ™ Lambda ZapII cloning kit (Catalog No. 236201), P718 according to the protocols recommended by the manufacturers. 105 clones are detected with the RDA 3-2 product as a probe (labeled, randomly primed as described above). Eight positive clones are selected and four are chosen at random for secondary analysis to obtain pure phage plaques. After tertiary detection, four dark phage clones are isolated. The cloned phage inserts are isolated by an in vivo excision procedure according to the Stratagene ™ * Lambda Zap II kit. The largest insert, approximately 2.6 kb (referred to as cDNA clone 3-2), is subjected to DNA sequencing. The sequence of the insert (SEQ ID No. 1) is shown in Figure 1. Clone 3-2 of cDNA (E. coli K-12, SOLR / p3-2 # 5-l) has been deposited as ATCC No. 98061. The sequence of clone 3-2 of cDNA is identical to that of clone 1-7 of cDNA (SEQ ID No. 2), except that nucleotides 136-605 of SEQ ID No. 1 represent an insertion. In this way, SEQ ID No. 2 represents a binding variant from SEQ ID No. 1. The clone for 1-7 (E. coli K-12, SOLR / pl-7 # 3-l) has been deposited as ATCC No. 98060. They are detected 105 clones with product 1-7 of RDA as a probe (radiolabelled, randomly primed as described above). Eight clones are selected P718 positive and four are chosen at random for secondary analysis to obtain pure phage plaques. After tertiary detection, four pure phage clones are isolated. Inserts cloned from the phage are isolated by the in vivo excision procedure according to the Stratagene "" Lambda Zap II kit. The larger insert of approximately 2.0 kb (referred to as probe 1-7 cDNA) is subjected to DNA sequencing; the sequence of the insert (SEQ ID No. 2) is shown in Figure 2. The clones are detected with the RDA product of 4-7 as a probe (labeled, randomly primed as described above and hybridized in PSB at 65 SC). Eight positive clones are selected and four are chosen at random for secondary analyzes to obtain pure phage plaques. After the secondary insertion, two clones of pure phage are isolated. Cloned phage inserts are isolated by the in vivo excision procedure according to the Stratagene ™ Lambda Zap II kit. The larger insert of approximately 2.4 kb (referred to as the 4-7 cDNA clone) is subjected to DNA sequencing. In the sequence of the insert (SEQ ID No. 4) is shown in Figure 3. Clone 4-7 of cDNA (E. coli K-12, SOLR / protein-7 # ll) has been deposited as ATCC No. 98062 .

. Characterization of clones 1-7, 3-2 and 4-7 cDNA P718 A) DNA sequences and. of the Protein The cDNA sequence 3-2 (Figure 1; SEQ ID No. 1) contains an open reading frame of 307 amino acids (Figure 1; SEQ ID No. 3). A signal sequence of 21 amino acids is inferred from the Von Heijne analysis (Von Heijne et al., Nucí Acid Res 14: 148683 (1986)); and a transmembrane region encompassing approximately 235-257 indicates that the product of 3-2 is a cell surface protein. The cDNA sequence of 1-7 (Figure 2; SEQ ID No. 2) contains an open reading frame of 307 amino acids, which is identical to the open reading frame contained in the 3-2 cDNA (SEQ ID No. 3). ). The cDNA sequence of 4-7 (Figure 3; SEQ ID No. 4) contains an open reading frame of 572 amino acids (SEQ ID No. 5). A transmembrane region is located approximately at amino acids 501-521.

B) In situ analysis of 1-7, 3-2 and 4-7 mRNAs in adult, contralateral and post-ischemic rat kidneys: In situ hybridization is carried out according to the method described by Finch et al. , Dev.

Dynamics 203: 223-240, 1995. Briefly, both ischemic and contralateral kidneys were fixed by perfusion with P718 4% paraformaldehyde in PBS. The kidneys were perfused overnight at 4SC and processed. The paraffin portions were dewaxed and rehydrated, fixed with 4% paraformaldehyde in PBS, digested with K proteins, reattached, then acetylated with acetic anhydride with triethanolamine buffer. The sections are then hydrated and hybridized with the 32P-labeled riboprobes at 55 aC at night, with the p-labeled radiosondes generated from the RDA 3-2 or RDA 1-7 products subcloned into the BamHl site. of pGEM-llZ After hybridization, the sections were washed under high stringency conditions (2 X SSC, 50% formamide at 65 SC) The sections are finally hydrated, the emulsion (NBT-2) is coated for autoradiography , and is exposed for at least a week.The silver grains rebel and the sections are counterstained with toluidine blue, microphotographs.The analysis of mRNA expression of 1-7 and 3-2 by in situ hybridization indicates that these genes are greatly promoted in damaged kidney cells compared to their expression in normal kidney sections.View expression is in regenerative cells of the cortex and outer medulla, the medulla of which appears to be proximal tubule cells. of the RNA expression pattern in P718 situ of 4-7 reveals abundant expression of this gene in the damaged ischemic kidney compared to the normal adult kidney. The site of expression appear to be infiltration cells. 6) Isolation of a human cDNA clone crue cross-hybridizes to rat 3-2 cDNA A 32P-labeled DNA probe comprising nucleotides 546-969 of the insert of clone 3-2 shown in Figure 1 is generates and is used to detect a gtlO, lambda, human embryonic liver cDNA library (Clontech Catalog #HL 5003a). 1 x 106 plates are detected in duplicate using normal conditions as described above, but the temperature for detection was 552C. For high security washing, the filters are washed in 2 X SSC at 55 aC. Fifty positive phages are identified and the plate is purified and the DNA prepared. The phage DNAs are subjected to Southern analysis using the same probe as before. The Southern transfer filter is subjected to a final wash with 0.5 X SSC at 55 SC. Two clones are identified as positive. The insert of clone H13-10-85 is sequenced and a region is found that codes for a protein with a high level of identity to the 3-2 protein shown in Figure 3. The nucleotide sequence (SEQ ID No. 6) and the P718 predicted amino acid sequence (SEQ ID No. 7) of the related human 3-2 protein are shown in Figure 4. As shown by the Bestfit analysis depicted in Figure 5, the human 3-2 related protein is 43.8% identical and 59.1% similar to rat protein 3-2. Both contain mucin, transmembrane and cytoplasmic IgG domain. The six cysteines within the IgG domains of both proteins are conserved. 7J_ Production of the KIM-1 Ig fusion protein An extracellular domain fusion protein from KIM and the Fc reaction of immunoglobulin (Ig) is a useful tool for the study of the molecular and cellular biology of the damaged kidney in regeneration and as a therapeutic molecule. To produce the KIM Ig fusion protein with the extracellular domain of the human and rat KIM-1 protein, an extracellular domain fragment of the KIM-1 DNA was amplified by PCR was cloned into the Biogen expression vector , pCA 125, for transient expression in COS cells. The expression vector pCA125 produces a fusion protein having structure from cloning in the N-terminus and an Fc reaction of human Ig in the C-terminus. COS cells were transfected with plasmid SJR 103 or 104; these plasmids express a fusion protein containing the P718 KIM sequence, human, 263-1147 (SEQ ID No. 6; SJR 103) or rat KIM sequences 599-1319 (SEQ ID No. 1; SJR 104) of the extracellular domain fused to the Ig Fc regions of human. The cells were cultured in 10% FBS in DMEM in the cell factory (Nunc, Naperville, II). The medium was collected after post-transfection, two to three days was concentrated using an Amicon concentrator, and the fusion protein was purified using the Protein-A-Sepharose column. After purification, the purity of the pressure protein was evaluated by SDS-PAGE.

Diagnostic Uses of the Compounds of the Invention The anti-KIM antibodies of the invention, which specifically bind to the protein of SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7 or a fragment thereof, are useful in various diagnostic methods. These agents can be labeled with detectable markers, such as fluoroscopically or radiographically opaque substances, administered to a subject to allow image formation of tissues that express the KIM protein. The agents can also be attached to substances, such as horseradish peroxidase, which can be used as immunocytochemical stains to allow the visualization of areas of KIM-protein-positive cells in histological sections. A specific antibody could be used alone P718 in this manner, and the sites where they bind can be visualized in an intercalation titration using an anti-immunoglobulin antibody that binds itself to a detectable label. The antibodies specific to the KIM protein are also useful in immunoassays to measure the presence or concentration of KIM in the sample of body tissues and fluids. These concentrations can be correlated with different disease states. As a particular modality of interest, the invention includes a method of diagnosis of renal damage, or of inspection of a renal repair process, by measuring the concentration of KIM or in the fragments of KIM in the urine, plasma or serum of a patient. . Similarly, KIM can be measured in the urine segment, particularly in cell debris in the urine sediment. Cylinders of renal tubular cells, which may occur in the urine sediment of patients with current kidney disease, may contain high levels of KIM protein and mRNA. Specific antibodies to the KIM protein can also be attached to solid supports, such as beads or disks, and used to remove the ligand from a solution, either for measurement or for purification and characterization of the protein or its attributes ( such as P718 post-transductional modifications). This characterization of a patient KIM protein may be useful in the identification of deleterious mutants for effects in procedures that interfere with KIM function and are associated with abnormal phenotypes of the patient. Each of these techniques is routine for those experts in immunological techniques. Additional methods of imaging are used KIM or fragments fused to imageable portions, for the formation of diagnostic images of tissues expressing KIM ligands, particularly tumors. Additional diagnostic techniques are based on the demonstration of KIM ARMNm promoted in tissues, as an indication of damage release processes. This technique has been tested and found workable in a model of ischemic damage in rats, as follows. To determine if the amount of KIM-1 protein increases after damage, homogenates of the contralateral and post-ischemic kidneys were examined, 24 and 48 hours after a 40-minute clamping of the renal artery and the vein of an individual kidney of each rat. Kidney homogenate was assessed for the presence of KIM-1 protein. Western blot analysis identifies three proteins detected P718 by two different antibodies after ischemic damage, which are not detectable in the homogenates from the contralateral kidneys that were not exposed to ischemic damage. At the apparent molecular weights of the bands are approximately 40 kDa, 50 kDa, and 70-80 kDa. The three protein species detected by Western blotting could represent glycosylated forms of the same protein given the presence of potential linked, N and 0 glycolization sites. The fact that each of these proteins reacts with two different sets of polyclonal antibodies supports the idea that they are related to KIM-1 and do not cross-react the bands. The confirmation of this prediction comes from the results of the partial CNBr excision of the three proteins that reveal shared, common CNBr excision fragments. Since the cytoplasmic domain of the KIM-1 protein is not predicted to contain any of the major post-transductional modifications, the two smaller products of digestion (4.7 kDa and 7.4 kDa) detected with antibodies directed against the cytoplasmic line of KIM-1 can be of the same size for the three different protein bands of KIM-1 if they originate from the same protein. It was observed that the proteins of 40 kDa and 70-80 kDa of KIM-1 produce fragments that look at the predicted size. The digestion P718 of the 50 kDa protein band also gave the same C-terminal signature band peptide. The KIM-1 sequence presents two putative sites for N-glycosylation and a mucin domain where O-glycosylation could cover the polypeptide chain. The three bands of KIM-1 detected in the post-ischemic should correspond to the glycosylation variants of the same core protein. Des-N-glycosylation with PNGase F resulted in a shift from all three bands to a lower molecular weight, corresponding to a loss of approximately 3 kDa, indicating that all three proteins were N-glycosylated. The differences in O-glycosylation could explain the differences in the sizes of these three bands.

Therapeutic Uses of the Compounds of the Invention Therapeutic methods of the invention comprise the selective promotion or inhibition of cellular responses that are dependent on KIM ligation. • Where the KIM and the KIM ligand bind on the membrane, they are expressed by different cells, signal transduction can occur in the cell expressing KIM, in the cell expressing the KIM ligand, or both. The response triggered by the KIM link in P718 A cell expressing the KIM ligand can be generated by contacting the cells with exogenous KIM, the KIM fusion proteins or the activation antibodies against the KIM ligand, either in vitro or in vivo. In addition, responses of the cell expressing the KIM ligand that could otherwise be activated by endogenous KIM could be blocked by contacting the cell expressing the KIM ligand with a KIM ligand antagonist (e.g., an antibody). antagonist that binds to the KIM ligand), or by contacting the endogenous KIM with a KIM antibody or another KIM binding molecule that prevents the effective binding of KIM to a KIM ligand. Similarly, the activated responses of the KIM ligation in the cell expressing KIM can be promoted or inhibited with exogenous compounds. For example, the response triggered by KIM ligation in a cell expressing KIM can be generated by contacting the cell with a soluble KIM ligand, or certain anti-KIM activation antibodies. In addition, responses of the KIM expressing cell that would otherwise be activated by interaction with the endogenous KIM ligand could be blocked by contacting the KIM expressing cell with a KIM antagonist (eg, a blocking antibody that is unites KIM in a way that prevents the KIM linkage from generating signals, effective), or by putting P718 contacts the endogenous KIM ligand with a KIM anti-ligand antibody or another KIM ligand binding molecule that prevents the effective binding of KIM to the KIM ligand. When the interventions described above are useful for particular therapeutic uses, it depends on the relevant etiological mechanism of either the pathological process to be inhibited, or the medically desirable process to be promoted, as is apparent to those skilled in the medical art. . For example, where the KIM ligation resulted in desirable cell growth, maintenance of the differentiated genotype, resistance to apoptosis induced by several attacks, or other clearly advantageous responses, one of the interventions described above that one of the interventions described above that promotes the response triggered by the ligation. Alternatively, one of the measurement interventions may be useful where KIM ligation causes undesirable consequences, such as neoplastic growth, deleterious loss of cell function, susceptibility to apoptosis, promotion of inflammation cases. The following are examples of the previously described therapeutic methods of the invention. The therapeutic use of the compounds related to KIM of the invention is for the treatment of this object with the P718 renal disease, which promotes the growth of new tissue of a subject, which promotes the survival of damaged tissue in a subject, and which includes the step of administering to the subject a therapeutically effective amount of a KIM protein of the invention, or a pharmaceutical composition that includes a protein of the invention. The protein used in these methods may be the fragment of a full-latitude KIM protein, and a soluble KIM binding protein, or an excision fragment, or a KIM agonist. These methods can also be practiced or administered to the subject a therapeutically effective amount of an agonist antibody of the invention, or a pharmaceutical composition that includes an agonist antibody of the invention. A KIM protein can be administered concurrently with a therapeutically effective amount of a second context that exerts a medically desirable adjunct effect. While the tissues of interest for these methods can include any tissue, preferred tissues include renal, liver, neural, heart, stomach, small intestine, bone, or lung tissue. Particular renal conditions that can 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, damage P718 toxic, hypoxic damage, and trauma. Kidney tubule defects include those either hereditary or acquired in nature, such as polycystic kidney disease, medullary cystic disease, and spinal cord attachment. This list is not limited, and may include many other renal disorders (see, for example, Harrison's Principles of Internal Medicine, 13th edition, 1994, which is incorporated herein by reference). The subject of the methods can be human. A therapeutic intervention to inhibit tissue growth that expresses the undesirable KIM ligand in a subject includes the step of administering to the subject a therapeutically effective amount of a KIM antagonist (eg, an antagonist antibody that binds to the ligand of KIM); or by administering a therapeutically effective amount of an anti-KIM antibody or other KIM binding molecule that blocks binding to KIM to the tissue expressing the KIM ligand. In an embodiment of interest, the KIM antagonist or the anti-KIM antibody can be used therapeutically to inhibit or block the growth of tumors that depend on the KIM protein for growth. Other methods of the invention include ligation of tumor cells expressing the KIM ligand, or inhibition of its growth, contacting P718 cells with a fusion protein of a KIM and a toxin or radionuclide or an anti-KIM ligand antibody conjugated to a toxin or radionuclide. The cell may be within a subject, and the protein or conjugated antibody is administered in the subject. Also encompassed within the invention is a method for targeting a toxin or radionuclide to a cell expressing a KIM, which comprises contacting the cell with a fusion cell comprising a KIM ligand and a toxin or radionuclide, or an antibody anti-KIM conjugated to a toxin, or radionuclide. Another embodiment includes the method of suppressing the growth of a tumor cell expressing KIM, which comprises contacting the cell with the fusion protein of the KIM ligand and a toxin or radionuclide or with an anti-KIM antibody conjugated to a toxin. or radionuclide; the cell placed inside a subject, and the protein is administered to the subject. The term "subject" used herein is taken to mean any mammal to which the KIM can be administered. Subjects proposed specifically for the treatment for the method of the invention include humans, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, gerbils, rats and mice, as well as organs, tumors, and cells derived or they originate from these hosts.

P718 Uses of the Gene Therapy Invention Compounds The KIM genes of the invention are introduced into the damaged tissue, or into the tissue where stimulated growth is desirable. This gene therapy stimulates the production of the KIM protein by the transfected cells; promoting cell growth and / or survival of cells expressing the KIM protein. In a specific embodiment of a gene therapy method, a gene encoding a KIM protein can be introduced into a renal target tissue. The KIM protein can be stably expressed and tissue growth, division, or differentiation, can be stimulated cell survival potential. In addition, a KIM gene can be produced in 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-speed bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate DNA precipitate, DEAE-dextran-mediated transrepression and direct microinjection in individual cells. For example, a KIM gene can be introduced into a cell by co-precipitation with calcium phosphate (Pillicer et al., Science, P718 209: 1414-1422 (1980); mechanical microinjection and / or acceleration of particles (Anderson et al., Proc. Nat. Acad. Sci. USA, 77: 5399-5403 (1980); DNA transfer on the basis of liposomes (for example, LIPOFECTIN-mediated transfection-Fefgner and collaborators, Proc. Nat. Acad. Sci., USA, 84: 471-477, 1987, Gao and Huang, Biochim, Biophys, Res. Comm., 179: 280-285, 1991; transfection mediated with DEAE-Dextran; electroporation (US Patent No. 4,956,288), or polylysine-based methods in which DNA is conjugated to distribute DNA preferentially to liver hepatocytes (Wolff et al., Science, 247: 465-468, 1990; Curiel et al. , Human Gen Therapy 3: 147-154, 1992.) The target cells can be transfected with the genes of the invention by direct gene transfer, see, for example, Wolff et al., "Direct Gene Transfer Into Moose Muscle In Vivo". Science 247-1465-68, 1990. In many cases, transfection mediated with vector Any of the methods known in the art for inserting the polynucleotide sequences into a vector can be used (see for example, Sambrook et al., Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor, Laboratory, Cold Spring Harbor , NY, 1989; and Ausubel et al., Current Protocols in Molecular Biology, J. Wiley & Sons, NY, 1992, P718 both of which are incorporated herein by refemecia). Activation by promoter can be tissue specific or inducible by a metabolic product or substance administered. These promoters / enhancers include, but are not limited to, the promoter of the native c-ret ligand protein, of the immediately preceding promoter / enhancer of cytomeg / alovirus (Karasuyama et al., J. Exp. Med., 169; 13, 1989); the human beta-actin promoter (Gunning et al., Proc. Nat. Acad. Sci. USA, 84: 4831, 1987), the glucocorticoid-inducible promoter present in the long terminal repeat of the mouse mammary tumor virus (MMTV LTR) (Klessig et al., Mol. Cell, Biol., 4: 1354, 1984), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al., RNA Tumor Viruses, cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985); the promoter of the previous SV 40 region (Bernoist and Cambon, Nature, 290: 304, 1981); the Rous sarcoma virus (RSV) promoter (Yamamoto et al., Cell 22: 787, 1980); the thymidine kinase promoter of herpes simplex virus (HSV) (Wagner et al., Proc. Nat. Acad. Sci. USA, 781: 1441, 1981); the adenovirus promoter (Yamada et al., Proc. Nat. Acad. Sci. USA, 82: 3567, 1985). You can also enter the KIM genes of P718 specific viral vectors for use in gene transfer systems that are now well established. See, for example: Madzak et al., J. Gen. Virol., 73: 1533-36, 1992 (SV40 papovavirus); Berkner et al., Curr. Op. Microbiol. Immunol. , 158: 39-61 1992 (adenovirus); Hofmann et al., Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Curr. Top. Microbiol. Immunol. , 158: 25-38, 1992 (vaccinia virus); Muzyczka, Curr. Top. Microbiol. Immunol., 158: 97-123, 1992 (adeno-associated virus); Marguleskee, Curr. Top. Microbiol. Immunol. , 158: 67-93 1992 (herpes simplex virus (HSV) and Epstein-Barr virus (HBV)); Miller, Curr. Top. Microbiol. Immunol. , 158: 1-24, 1992 (retrovirus); Brandyopadhyay et al., Mol. Cell. Biol., 4: 479-754, 1984 (retrovirus); Miller et al., Nature, 357: 455-450, 1992 (retrovirus); Anderson, Science, 256: 808-813, 1992 (retrovirus), Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., Eds.), Greene Publishing Associates, 1989, all of which are incorporated herein by reference. Preferred vectors are DNA viruses that include adenoviruses (preferably, vectors based on Ad-2 or Ad5), vaculoviruses, herpes viruses (preferably vectors based on herpes simplex virus) P718 and parvovivirus (preferably "defective" or non-autonomous parvovirus-based vectors, more preferably, adeno-associated virus-based vectors, most preferably AAV-2-based vectors). See, for example, Ali et al., Gene Therapy 1: 367,384, 1994; U.S. Patent No. 4,797,368 and U.S. Patent No. 5,399,346 and subsequent discussion. The choice of the particular vector system for the transfer of, for example, a KIM sequence will depend on a variety of factors. An important factor is the nature of the target cell population. Although a number of gene therapy applications have been studied extensively and used retrovirally, they are generally unsuitable for infecting cells that do not divide but may be useful in cancer therapy since they only integrate and express their genes in replication cells. They are useful for ex vivo approaches and are attractive in this respect, due to their stable integration into the genome of the target cell. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently distribute a therapeutic transgene or indicator to a variety of cell types. General adenovirus types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for the P718 gene therapy of Duchenne Muscular Dystrophy (DMD) and Cystic Fibrosis (CF). Both Ad2 and Ad5 correspond to a subclass of adenoviruses that are not consistent with human malignancies. Adenovirus vectors are capable of providing extremely high levels of transgenic distribution to virtually all cell types, despite the mitotic state. High titers (1013 plaque-forming units / ml) of recombinant virus can easily be generated in 293 cells (a line of human, embryonic kidney cells, complementation, transformed with adenovirus: ATCC CRL1573) and stored for prolonged periods without falls appreciable. The efficiency of this system in the distribution of a therapeutic transgene in vivo that complements a genetic inequilibrium has been demonstrated in animal models of several disorders. See Watanabe, Atherosclerosis, 36: 261-268, 1986; Tanzawa et al., FEBS Letters 118 (1): 81-84, 1980; Golasten et al., New Engl. J. Med. 309: 288-296, 1983: Ishibashi et al., J. Clin. Invest. 92: 883-893, 1993; and Ishibashi et al., J. Clin. Invest. 93: 1889-1893, 1994, all of which are incorporated herein by reference. In fact, the recombinant replication defective adenovirus is encoded for a cDNA for the transmembrane cystic fibrosis regulator P718 (CFTR) has been tested for use in at least two chemical assays of human CF. See, for example, Wilson, Nature 365: 691-692, 1993. Further support for the safety of recombinant adenoviruses for gene therapy is the extensive experience of live adenovirus vaccines in human populations. The recombinant, first generation, replication-deficient adenoviruses that have been developed for gene therapy of DMD and other inherited disorders contain deletions of the entire Ela and part of the Elb subregions. This defective replication virus is cultured in 293 cells that contain a functional Adenovirus Ela gene that provides a transa Ela protein. El suppressed viruses are capable of replicating and producing infectious viruses in 293 cells, which provide the gene products of the Ela and Elb in trans region. The resulting virus capable of infecting many cell types can be expressed in the introduced gene (which it provides by carrying its own promoter), but can not replicate in a cell that does not have the DNA of the El region unless the cell is infected at a time. very high multiplicity of infection. Adenoviruses have the advantage that they have a broad host range, they can infect quiescent cells or terminally differentiated neurons, and they seem essentially not P718 oncogenic. Adenoviruses do not seem to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. Ali et al., Supra, at 373. Recombinant adenoviruses (rAdV) produce very high titers, viral particles are moderately stable, expression levels are high, and a wide range of cells can be infected. Natural host cells are the epithelium of the airways, so they are useful for the therapy of lung cancers. Baculovirus-mediated transfer has several advantages. Vaculoviral gene transfer can occur in replication and non-replication, and can occur in renal cells, as well as in hepatocytes, neural cells, spleen, skin and muscle. The baculovirus is non-replicating and non-pathogenic in mammalian cells. Humans lack antibodies resistant to recombinant baculoviruses that could block infection. In addition, the baculovirus is capable of incorporating and transducing very large DNA grafts. Adeno-associated adenovirus (AAV) have also been used as vectors for somatic gene therapy. AAV is a small single-stranded DNA virus (ss) with a simple genomic organization (4-7 kb) P718 which makes it an ideal substrate for genetic management. Two open reading frames code for a series of rep and cap polypeptides. The rep (rep 78, rep68, rep62 and rep 40) polypeptides are included in the replication, rescue and integration of the AAV genome. The proteins (VP1, VP2 and VP3) form the capsid of the virion. With flanking of the open reading frames of rep and cap at the 5 'and 3' ends are inverted terminal repeats of 145 bp (ITRs), the first 125 bp of which are capable of forming Y or T-shaped duplex structures The importance for the development of AAV vectors, the complete rep and cap domains can be excised or replaced with a therapeutic gene or indicator. See B.J. Crankcase in Handbook of Parvoviruses, ed. , P. Tijsser, CRC Pres, pp. 155-167 (1990). It has been shown that the ITRs represent the minimum sequence required for the replication, rescue, packaging and integration of the AAV genome. Adeno-associated adenoviruses (AAV) have significant potential in gene therapy. Viral particles are very stable and recombinant AAVs (rAAV) 'have "drug-like" characteristics since rAAV can be encoded by sedimentation or by CsCl gradient grouping. They are heat stable and can be used to a powder and rehydrate to the activity P718 complete. DNA is stably integrated into the chromosomes of the host so that the expression is long term. Their range of hosts is broad and AAVs do not cause known disease so they are not non-toxic recombinant vectors. Once introduced into a target cell, the sequence of interest can be identified by conventional methods such as hybridization of the nucleic acid are the probes containing sequences that are homologous / complementary to the gene sequences inserted from the vector. In another approach, the sequence (s) can be identified by the presence or absence of a "marker" gene function (eg, thymidine kinase activity, antibiotic resistance, and the like) caused by the introduction of the vector of expression in the target cell.

Formulations and Administration The compounds of the invention are formulated in accordance with normal practice, such as preparations in a carrier vehicle. The term "pharmaceutically acceptable carrier" means one or more organic or inorganic ingredients, synthetic or natural, with which the mutant proto-oncogene or mutant oncoprotein is combined to facilitate its application. A suitable carrier includes P718 sterile saline although other sterile aqueous and non-aqueous isotonic solutions of its known sterile functions which are pharmaceutically acceptable are known to those skilled 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 plasmids and viral expression vectors. Any of the two polypeptides of this invention can be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases are capable of forming salts with the polypeptides of the present invention are well known to those skilled in the art, and include inorganic and organic base acids. A compound of the invention is delivered to a subject in a therapeutically effective amount, which means an amount of the compound that produces a medically desirable result or exerts an influence on the particular condition being treated. An effective condition of a compound of the invention is capable of improving or delaying the progression of the disease condition, degenerative, or damaged. The effective amount can be determined on an individual basis and will be based, in part, on the consideration of the physical attributes of the subject, symptoms to be treated and desired results.

P718 An effective amount can be determined by one skilled in the art by cooling these factors and not using more than routine experimentation. A liposome delivery system for a compound of the invention can be any of a variety of unilamellar vesicles, multilamellar vesicles, or plurilamellar vesicles, and can be prepared and administered according to methods well known to those skilled in the art, for example, in accordance with the teachings of U.S. Patent Nos. 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, such as polyproteins, in order to improve their binding to liposomes. As an example, the treatment of human acute renal failure with KIM protein encapsulated with liposome can be performed in vivo by introducing a KIM protein into cells and treatment needs using liposomes. Liposomes can be distributed via the catheter to the renal artery. The recombinant KIM protein is purified, for example, from CHO cells by immunoaffinity chromatography or by any other convenient method, then mixed with liposomes and incorporated therein at high efficiency. The encapsulated protein can be tested in vitro for any effect P718 in cell growth stimulation. The compounds of the invention can be administered in any manner that is medically acceptable. This may include injections, parenteral products such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intrahepitural, or other such as oral, nasal, ophthalmic, rectal or topical. Administration by sustained release is also specifically included in the invention, by means of reservoir injections or breakable implants. The localized distribution is particularly contemplated as a means of delivery via a catheter to one or more arteries, such as the renal artery, or a container supplying a localized tumor. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity and 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 for the scope of the appended claims.

P718 LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT Michele Sanicola-Nadel Joseph V. Bonventre Catherine A. Hession Takaharu Ichimura Henry Wei Richard L. Cate (ii) TITLE OF THE INVENTION: MODULATORS OF THE TISSUE REGENERATION (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Biogen, Inc. (B) STREET: 14 Cambrigde Center (C) CITY: Cambrigde (D) STATE: MA (E) COUNTRY: USA (F) ZIP CODE: 02142 (v) LEGIBLE FORM IN COMPUTER: P718 (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) NUMBER OF APPLICATION: (B) DATE OF SUBMISSION: MAY 23, 1997 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 60 / 018,228 (B) DATE OF SUBMISSION: MAY 24, nineteen ninety six (viii) INFORMATION ATTORNEY / AGENT: (A) NAME: Levine, Leslie M. (B) REGISTRATION NUMBER: 35,245 (C) REFERENCE NUMBER / DOCUMENT: PCO YEAR CIP P718 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) 679-2810 (B) TELEFAX: (617) 679-2838 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2566 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 615..1535 (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 1: P718 GCGGCCGCGT CGACGGTGCC TGTGAGTAAA TAGATCAGGG TCTCCTTCAC AGCACATTCT 60 CCAGGAAGCC GAGCAAACAT TAGTGCTATT TTACCCAGGA GGAAATCTAG GTGTAGAOAG 120 CTCTACGGAT CTAAGGTTTG GATCTGTACC CAGTGCTTTT TTAGGTGTCT TTAGACATTT 180 CTCAGGAAGA tGTAGTC-rct GTCACCATGT GTGGCTGAAT TC AGCTCJV3 TCCATCTTAT 240 TGTGTTTAAG GTAGTTGAAG TTTAGGAACC AACCAGTATG TCTCTGAGCA GAAGAGTACA 300 OTQTCCATCT TGAGGACAAG CTCATCTTTA CCATTAGAGG GCTGGCCTTG GCTTAGATTC 360 TACCGAGAAC? T? CTCTCTA ATGGCTGCCC TCAGTTTTCT CTGTTTGCTG TCTTATTTGT 420 GTCATGGCCA GAAGTCATAT GGATGGCTCT ATGTGAGCAA GGACCCAGAT AGAAGAGTGT 4B0 ATTTGGGGGA ACAGGTTGCC CTAACAGAGA GTCCTGTGGG ATTCATGCAG TCAGGATGAA 540 GACCTGATCA GACAGAGTGT GCTGAGTGCC ACGGCTAACC AGAGTGACTT GTCACTGTCC 600 TTCAGGTCAA CACC ATG GTT CAA CTT CAA GTC TTC ATT TCA GGC CTC CTG S50 Met Val Gln Leu Glp Val Phe He Ser Gly Leu Leu 1 5 10 CTG CTT CTT CCA GGC TCT GTA GAT TCT TAT GAA GTA GTG AAG GGG GTG S98?, Eu Leu Leu Pro Gly Ser Val Asp Ser Tyr Glu Val Val Lys ßly Val 15 20 25 GTG GGT CAC CCT GTC ACÁ ATT CCA TGT ACT TAC TCA ACÁ CGT GGA GGA 746 Val Gly His Pro Val Thr lie Pro Cys Thr Tyr Ser Thr Arg Gly Gly? O '35 40 ATC ACÁ ACG ACÁ TGT TGG GGC CGG GGG CAA TGC CCA TAT TCT AGT TGT 794 lie Thr Thr Thr Cys Trp Gly Arg sly Gln Cys Pro Tyr Ser Ser eye 4S 50 55 60 CAA AAT ATA CTT ATT TGG ACC AAT OGA TAC CAA GTC ACC TAT CG < 3 AGC 842 Gln Asn lie Leu lie Trp Thr Aßn Gly Tyr Glp Val Thr Tyr Arg Ser 65 70 75 AGC GGT CGA TAC AAC ATA AAG GGG CGT ATT TCA GAA GGA GAC GTA TCC 890 Ser Gly Arg Tyr Asn He Lys Gly Arg He Ser Glu Gly Asp Val Ser 80 '65 90 8 TTG ACÁ ATA GAG AAC CT GTT GAT AGT GAT AGT GGT CTG TAT TGT TGC 938 Leu Thr He Glu Asn Ser Val Asp Ser Asp Ser Gly Leu Tyr Cys Cys 95 100 105 CGA GTG GAG ATT CT GGA TGG TTC AAC GAT CAG AAA ATG ACC TTT TCA 986 Arg Val Glu He Pro Gly Trp Phe Asn Asp Gln Lya MeC Thr Phe Ser 110 115 120 TTG GAA GTT AAA CCA GAA ATT CCC ACÁ AGT CCT CCA ACÁ AGA CCC ACÁ 1034 Leu Glu Val Lys Pro Glu Lie Pro Thr Ser Pro Pro Thr Arg Pro Thr 125 130 135 140 ACT ACA AGA CCC ACÁ ACC AC AGG CCC ACÁ ACT ATT TCA ACÁ AGA TCC 1082 Thr Thr Arg Pro Thr Thr Thr Arg Thr Thr Thr Thr Thr Arg Ser 145 150 155 ACÁ CAT GTA CCA ACÁ TCA ACC AGA GTC TCC ACC TCT ACT CCA ACÁ CCA 1130 Thr His Val Pro Thr Ser Thr Arg Val Ser Thr Ser Thr Pro Thr Pro 160 165 170 GAA CAA ACA CAG ACT CAC AAA CCA GAA ATC rtCT ACA TTT TAT GCC CAT 1178 Glu Gln Thr Gln Thr His Lys Pro Glu He Thr Thr Phe Tyr Wing His 175 180 1B5 GAG ACA ACT GCT GAG GTG ACÁ GAA ACT CCA TCA TAT ACT CCT GCA GAC 1226 Glu Thr Thr Ala slu Val Thr Glu Thr Pro Ser Tyr Thr Pro Wing Asp 190 1S5 200 TGG AAT GGC ACT GTG ACA TCC TCA GAG GAG GCC TGG AAT AAT CAC ACT 1274 Trp Asn Gly Thr Val Thr Ser Ser Glu Glu Wing Trp Asn Asn His Thr 205 210 215 220 GTA AGA ATC CCT TTG AGG AAO CCG CAG AGA AAC CCG ACT AAG GGC TTC 1322 Val Arg He Pro Leu Arg Lys Pro Gln Arg Asn Pro Thr Lys Gly Phe 22S 230 235 TAT GTT GGC ATG TCC GTT GCA GCC CTG CTG CTG CTG CTG CTT GCG AGC 1370 Tyr Val Gly Met Ser Val Ala Ala Leu Leu Leu Leu Leu Leu Wing Ser 240 24S 250 ACC GTG GTT GTC ACC AGG TAC ATC ATT ATA AGA AAG AAG ATG GGC TCT 1418 Thr Val Val Val Thr Arg Tyr He He He Arg Lys Lyß Het Gly Ser 255 260 26S CTG AGT TTT GTT GCC TTC CAT GTC TCT AAG AGT AGA GCT TTG CAG AAC 1466 Leu Ser Phe Val Ala Phe His Val Ser Lys Ser? Rg Ala Leu Gln Asn 270 275 280 GCA GCG ATT GTG CAT CCC CGA GCT GAA GAC AAC ATC TAC ATT ATT GAA 1514 Ala Ala He Val Hia Pro Arg Ala Glu Asp Asn He Tyr He He Glu 285 290 295 300 GAT AGA TCT CGA GGT GCA GAA TGAGTCCCAG AGGCCTTCTG TGGGGCCTTC 1565 Asp Arg Ser Arg Gly Ala Glu 305 8 TGCCTGGGAT TACAGAGATC GTGACTGAT TCACAGAGTA AAAGACCCAG GCCAGCTCCT 1625 GGGAGATTTT GTGTTTTGGT TCTTCCAGCT GCAGTGGAGA GGGTAACCCT CTACCCTGTA 1685 TATGCAAAAC TCGAGGTTAA CATCATCCTA ATTCTTGTAT CAGCAACACC TCAGTGTCTC 1745 CACTCACTGC AGCGATTCTC TCAAATGTGA ACATTTTAGA AGTTTGTGTT TCCTTTTGTC 1805 CATGTAATCA TTGGTAATAC AAGAATTTTA TCTTGTTTAT TAAAACCATT AATGAGAGGO IB65 GAATAGGAAT TAAAAGCTGG TGGGAAGGGC CTCCTGAA? T TAGAAQCACT TCATGATTGT 1925 GTTTATCTCT TTTATTCTAA TTTGAAATGT TACTTCTATC CTTCCCAAGG GGCAAA? TCA 1985 TGGGAGCATG GAGGTTTTAA TTGCCCTCAT AGATAAGTAG AAGAAGAGAG TCTAATGCCA 2045 CCAATAGAGG TGGTTATGCT TTCTCACAQC TCTGGAAATA TGATCATTTA TTATGCAGTT 2105 GATCTTAGGA TGAGGATGGG TTTCTTAGGA GGAGAGGTTA CCATGGTGAG TGGACCAGGC 2155 ACACATCAGG GGAAGAAAAC AATGGATCAA GGGATTGAGT TCATTAGAGC CA ITCCACT 222S CCACTTCTGT CTTGATGCTC AGTGTTCCTA AACTCACCCA CTGAGCTCTG AATTAGGTGC 2285 AGGGAGGAGA CGTGCAGAAA CGAAAGAGGA AASAAAGGAS AGAGAGCAGG ACACAGGCTT 2345 TCTGCTGAGA GAAGTCCTAT TGCAGGTGTG ACAGTGTTTG GGACTACCAC GGGTTTCCTT 2405 CAGACTTCTA AGTTTCTAAA TCACTATCAT GTGATCATAT TTATTTTTAA AATTATTTCA 2465 GAAAGACACC ACATTTTCAA TAATAAATCA GTTTGTCACA ATTAATAAAA TATTTTGTTT 2525 GCTAAGAAGT AAAAAAAAAA AAAAAAAGTC GACGCGGCCG C 256S (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2084 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA P718! Ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 415..1065 (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 2 GCGGCCGCGT CGACGGTGCC TGTGAGTAAA TAGATCAGGG TCTCCTTCAC AGCACATTCT 60 CCAGGAAGCC GAGCAAACAT TAGTGCTATT TTACCCAGQA GGAAATCTAG GTGTAGAGAG 120 CTCTACGGAT CTAAGOTCAA CACC ATG GTT CAA CTT CAA GTC TTC ATT TCA 171 Mee Val Gln Leu Gln Val Phe He Ser 1 5 GGC CTC CTG CTG CTT CTT CCA GGC TCT GTA GAT TCT TAT GAA GTA GTG 219 Gly Leu Leu Leu Leu Leu Pro Gly Ser Val Asp Ser Tyr Glu Val Val 10 S 20 20 AAG GGG GTG GTG GGT CAC CCT GTC ACÁ ATT CCA TGT ACT TAC TCA ACÁ 267 Lys Gly Val Val Gly Hi3 Pro Val Thr He Pro Cys Thr Tyr Ser Thr 30 35 40 CGT GGA GGA ATC ACA ACG ACTA TGT TGG GGC CGG GGG CAA TGC CCA TAT 315 Arg Gly Gly lie Thr Thr Thr Cys Trp Gly Arg Gly Gln Cys Pro Tyr 45 50 S5 TCT AGT TGT CAA AAT ATA CTT ATT TGG ACC AAT GGA TAC CAA GTC ACC 3S3 Be Ser Cys Gln Asn He Leu He Trp Thr Aen Gly Tyr Gln val Thr 60 65 70 TAT CGG AGC AGC GGT CGA TAC AAC ATA AAG GGG CGT ATT TCA GAA GGA 411 Tyr Arg Ser Gly Arg Tyr Asn He Lys sly Arg He Ser Glu Gly 75 80 85 GAC GTA TCC TTG ACA ATA GAG AAC TCT GTT GAT AGT GAT AGT GGT CTG 59 Asp Val Ser Leu Thr He slu Asn Ser Val Asp Ser Asp Ser Gly Leu 90 95 100 105 TAT TGT TGC CGA GTG GAG ATT CCT GGA TGG TTC AAC QAT CAG AAA ATG 507 Tyr Cys Cys Arg Val slu He Pro Gly Trp Phe Asn Asp Gln Lys Het 110 115 120 ACC TTT 'TCA TTG GAA GTT AAA CCA GAA ATT CCC ACA AGT CCT CCA ACA 555 Thr Phe Ser Leu Glu Val Lys Pro Glu He Pro Thr Ser Pro Pro Thr 125 130 135 AGA CCC ACÁ ACT ACÁ AGA CCC ACÁ ACC AC AGG CCC ACÁ ACT ATT TCA 603 Arg Pro Thr Thr Thr Arg Pro Thr Thr Thr Arg Pro Thr Thr He Ser 140 145 150 P718 ACÁ AGA TCC ACÁ CAT GTA CCA ACÁ TCA ACC AGA GTC TCC ACC TCT ACT 65 Thr Arg Ser Thr His Val Pro Thr Ser Thr Axg Val Ser Thr Ser Thr 155 160 1-55 CCA ACÁ CCA GAA CAA ACÁ CAG ACT CAC AAA CCA GAA ATC ACT ACA TTT 699 Pro Thr Pro Glu Gln Thr G n Thr His Lys ro Glu lie Thr Thr Phe 170 175 180 185 TAT GCC CAT GAG ACE ACT GCT GAG GTG ACÁ GAA ACT CCA TCA TAT ACT 747 Tyr Ala His ßlu Thr Thr Wing Glu Val Thr Glu Thr Pro Ser Tyr Thr 190 193 200 CCT GCA GAC TGG AAT GGC ACT GTG ACA TCC TCA GAG GAG GCC TGG AAT 795 Pro Wing Asp Trp Asn Gly Thr Val Thr Ser Ser Glu Glu Wing Trp Asn 205 210 215 AAT CAC ACT GTA AGA ATC CCT TTG AGG AAG CCG CAG AGA AAC CCG ACT 843 Asn His Thr Val Arg He Pro Leu Arg Lyß Pro Gln Arg Asn Pro Thr 220 225 230 AAG GGC TTC TAT GTT GGC ATG TCC GTT GCA GCC CTG CTG CTG CTG CTG 891 Ly¿ Gly Phe Tyr Val Gly Met Ser Val Ala Ala Leu Leu Leu Leu Leu 235 240 245 CTT GCG AGC ACC GTG GTT GTC ACC AGG TAC ATC ATT ATA AGA AAG AAG 939 Leu Wing Being Thr Val Val Val Thr Arg Tyr He He He Arg Lys Lys 250 255 260 265 ATG GGC TCT CTG AGC TTT GTT GCC TTC CAT GTC TCT AAG AGT AGA GCT 987 Met Gly Ser Leu Ser Phe Val Wing Phe His Val Ser Lys Ser Arg Wing 270 275 280 TTG CAG AAC GCA GCG ATT GTG CAT CCC CG «GCT GAA GAC AAC ATC TAC 1035 Leu Gln Asn Ala Wing He Val His Pro Arg Wing Glu Asp Asn He Tyr 285 290 295 ATT ATT GAA GAT AGA TCT CGA GGT GCA GAA TGAGTCCCAG AGGCCTTCTG 10B5 He He Glu Asp Arg Ser? Rg Gly Ala Glu 300 305 TGGGGCCTTC TGCCTGGGAT TACAGAGATC GTGACTGATT TCACAGAGTA AAATACCCAT 1145 TCCAGCTCCT GGGAGATTTT GTGTTTTGGT TCTTCCAGCT GCAGTGGAGA GGGTAACCCT 1205 CTACCCTGTA TATGCAAAAC TCGAGGTTAA CATCATCCTA ATTCTTGTAT CAGCAACACC 1265 TCAGTGTCTC CACTCACTGC AGCGATTCTC TCAAATGTGA ACATTTTAGA AGTTTGTGTT 1325 TCCTTTTGTC CATGTAATCA TTGGTAATAC AAGAATTTTA TCTTGTTTAT TAAAACCATT 13B 5 P718 AATGAGAGGG GAATAGGAAT TAAAAGCTGs TGGGAAGsGC CTCCTGAATT TAGAAGCACT 1445 TCATGATTGT GTTTATCTCT TTTATTGTAA TTTGAAATGT TACTTCTATC CTTCCCAAGG 1505 GGCAAAATCA TGGGAGCATG GAGGTTTTAA TTGCCCTCAT AGATAAGTAG AAGAAGAGAG 1565 TCTAATGCCA CCAATAGAGG TGGTTATGCT TTCTCACAGC TCTGGAAATA TGATCATTTA 1625 TTATGCAGTT GATCTTAGGA TGAGGATGsG TITCTTAOGA GGAGAGsTTA CCATGGTGAs 1685 TGGACCAGGC ACACATCAGG ssAAGAAAAC AATGGATCAA GGsATTGAGT TCATTAGAGC 1745 CATTTCCACT CCACTTCTGT CTTGATGCTC AQTGTTCCTA AACTCACCCA CTGAsCTCTG 1805 AATTAGGTGC AGGGAGGAGA CGTGCAGAAA CGAAAGAGGA AAGAAAGGAG AGAGAGCAGG 1865 ACACAGGCTT TCTGCTGAGA GAAGTCCTAT TsCAGGTGTG ACAGTGTTTG GGACTACCAC 1925 GGGTTTCCTT CAGACTTCTA AGTTTCTAAA TCACTATCAT GTGATCATAT TTATTTTTAA 1985 AATTATTTCA GAAAGACACC ACATTTTCAA TAATAAATCA GTTTGTCACA ATTAATAAAA 2045 TATTTTQTTT GCTAAGAAGT AAAAAGTCGA CGCGGCCsC 2084 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 307 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 3 Mee Val Gln Leu Gln Val Phe He Ser Gly Leu Leu Leu Leu Leu Pro 1 5 10 15 Gly Ser Val Asp Ser Tyr Glu Val Val Lys Gly Val Val Gly Hi3 Pro 20 25 30 Val Thr He Pro Cys Thr Tyr Ser Thr Arg Gly Gly He Thr Thr Thr 35 40 45 Cys Trp Gly Arg Gly Gln Cys Pro Tyr Ser Ser Cys Gln Asn He Leu 50 55 60 He Trp Thr Asp Gly Tyr Gln Val Thr Tyr Arg Ser Gly Arg Tyr 65 70 75 80 Asn He Lys Gly Arg He Ser Glu Gly Asp Val Ser Leu Thr He Glu • 85 30 95 Asp Ser Val Asp be Asp Ser Gly Leu Tyr Cys Cys Arg Val Glu He 100 105 110? -rs Gly Trp Phe Even Asp Glp Lys Met Thr Phe Ser Leu Glu Val Lys US 120 12S Pro Glu He Pro Thr Ser Pro Pro Thr Arg Pro Thr Thr Thr Arg Pro 130 135 140 Thr Thr Thr Arg Pro Thr Thr Thr Ser Thr Arg Thr Th Hie Val Pro 145 150 155 160 Thr Ser Thr Arg Val Ser Thr Ser Thr Pro Thr Pro Glu aln Thr Gln 165 170 175 Thr His Lys Pro Glu He Thr Thr Phe Tyr Wing His Glu Thr Thr Wing 180 185 190 Glu Val Thr Glu Thr Pro Ser Tyr Thr Pro Wing Aep Trp Asn sly Thr 195 200 205 Val Thr Ser Ser Glu Glu Wing Trp Asn Asn Hie Thr Val Arg He Pro 210 215 220 Leu Arg Lys Pro Gln Arg Asn Pro Thr Lys Gly Phe Tyr Val Gly Met 223 230 235 240 Ser Val Ala Ala Leu Leu Leu Leu Leu Ala Ser Thr Val Val Val 245 250 2SS Thr Arg Tyr He He He Arg Lys Lys Met Gly Ser Leu Ser Phe Val 2á0 265 270 Wing Phe His Val Ser Lys Ser Arg Wing Leu Gln Asn Wing Wing Val 27S 280 285 His Pro Arg Wing Glu Asp Asn He Tyr Ha He slu Asp Arg Ser Arg 290 295 300 Gly Ala Glu 305 P718 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2303 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simplep D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 107. . 1822 (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 4: acaaceacst CGACTCGCAG GAOGCCGGCA CTCTGACTCC TGGTGGATGG GACTAGGGAG 6 O tCAGAGTCAA GCCCTGACTG GCTGAGGGCs GGCGCTCCGA GTCAGC ATO C3AA AGT 115 Met Glu Sar 1 CTC TGC < 3G3 GTC CTG GTA TTT CTG CTG CTO GCT GCA GGA CTG CCG CTC 163 Leu Cys Gly val Leu Val Phe Leu Leu Leu Wing Wing Gly Leu Pro Leu S 10 1S CAG GCG aCC AAG CGG TTC CGT GAT GTG CTG GGC CAT GAG CAG TAT CCG 211 Glp Wing Wing Lys Arg Phe Arg Asp Val Leu Gly His Glu Gln Tyr Pro 20 25 30 35 GAT CAC ATG AGG GAG AAC AAC CAA TTA CGT GGC TGG TCT TCA GAT GAA 259 Asp His Met Arg Glu Asn Asn Gln Leu Arg Gly Trp Ser As Asp slu 40 45 50 AAT GAA TGG GAT GAA CAG CTG TAT CCA GTG TGG AGG AGG GGA GAG GGC 307 Asn Glu Trp Asp Glu Gln Leu Tyr Pro Val Trp Arg Arg Gly Glu Gly 55 60 65 AGA TGG AAG GAC TCC TGG GAA GGA GGC CGT GTG CAG GCA GCC CTA ACC 355 Arg Trp Lys Asp be Trp Glu Gly sly Arg Val Glp Ala Wing Leu Thr 70 75 80 AGT GAT TCA CCG GCC TTG sTG GGT TCC AAT ATC ACC TTC GTA GTG AAC 403 Ser Asp Ser Pro Ala Leu Val Gly Ser Asn He Thr Phe Val Val Asn 85 90 95 CTG GTG. TTC CCC AGA TGC CAG AAG GAA GAT GCC AAC GGC AAT ATC GTC 451 LeU Go? Phe Pro Arg Cys Gln Lys Glu Asp Wing Asn Gly Asn lie Val 100 105 110 115 TAT GAO AGG AAC TGC AGA AGT GAT TTG GAG CTG GCT TCT CAC CCG TAT 499 Tyr Glu Arg Asn cye Arg Ser Asp Leu Glu Leu Wing Being Asp Pro Tyr 120 12S 130 GTC TAC AAC TGG ACC GGG GCA GAC GAC GAC GAG GAG TGG GAA GAC AGC 547 Val Tyr Asn Trp Thr Thr Gly Wing Asp Asp Glu Asp Trp Glu Asp Ser 135 140 145 ACC AGC CAA GGC CAG CAC CTC AGG TTC CCC GAC GGG AAG CCC TTC CCT 595 Thr Ser Gln Gly Gln His Leu Arg Phe Pro Asp Gly Lys Pro Phe Pro 150 155 160 CGC CCC CAC GGA CGG AAG AAA TGG AAC TTC GTC TAC GTC TTC CAC ACÁ 643 Arg Pro His Gly Arg Lys Lys Trp Asn Phe Val Tyr Val Phe His Thr 165 170 175 CTT GGT CAG TAT TTT CAA AAG CTG GGT CGG TOT TCA OCA CGA GTT TCT 691 Leu Gly Gln Tyr he Gln Lys Leu Gly Arg Cys Ser Wing Arg Val Ser 180 185 190 19S ATA AAC AC GTC AAC TTG ACÁ GTT GGC CCT CAG GTC ATG GAA GTG ATT 739 He Asn Thr Val Asn Leu Thr Val Gly Pro Gln Val Mee Glu Val He 200 205 210 GTC TTT CGA AGA CAC GGC CGG GCA TAC ATT CCC ATC TCC AAA GTG AAA 787 Val Phe Arg Arg His Gly Arg Ala Tyr He Pro He Ser Lys Val Lys 215 220 225 GAC GTG TAT GTG ATA ACÁ GAT CAG ATC CCT ATA TTC GTG ACC ATG TAC 835 Asp Val Tyr Val He Thr Asp Gln He Pro He Phe Val Thr Mßt Tyr 230 235 240 «CAG AA < 3 AAT GAC CGG AAC TCG TCT GAT GAA ACC TTC CTC AGA GAC CTC 883 Gln Lys Asn Asp Arg Asn Ser As Asp Glu Thr Pbe Leu Arg Asp Leu 245 250 255 18 CCC ATT TTC TTC GAT GTC CTC ATT CAC GAT CCC AGT CAT TTC CTC AAC 931 Pro He Phe Phe Asp val Leu He His Asp Pro Ser His Phe Leu Asn 260 265 270 275 TAC TCT GCC ATT TCC TAC AAG TGG AAC TTT GGG GAC AAC ACT GGC CTG 979 Tyr Ser Wing Ser Tyr Lys Trp Asn Phe Gly Asp Asn Thr Gly Leu 280 285 290 TTT OTC TCC AAC AAT CAC ACT TTG AAT CAC ACG TAT GTG CTC AAT GGA 1027 Phe val Ser Asn Asn His Thr Leu Asn His Thr Tyr Val Leu Asp Gly 295 300 305 ACC TTC AAC TTT AAC CTC ACC GTG CAA ACT GCA GTG CCG GGA CCA TGC 1075 Thr Phe- Asn Phe Asp Leu Thr Val Gln Thr Ala Val Pro Gly Pro Cys 310 315 320 CCC TCA CCC ACA CCT TCG CCT TCT TCT TCG ACT CT CCT CG CCT GCA 1123 Pro Ser Pro Thr Pro Pro Ser Ser Ser Thr Ser Pro Pro Pro Wing 325 330 335 CT TCG CCT TCA CCC ACÁ TTA TCA ACÁ CCT AGT CCC CT TTA ATG CCT 1171 Be Ser Pro Pro Pro Thr Leu Ser Thr Pro Pro Pro Ser Leu Met Pro 340 345 350 355 ACT GGC CAC AAA TCC ATG OAG CTG AGT GAC ATT TCC AAT GAA AAC TGC 1219 Thr Gly His Lys Ser Met siu Leu Ser Asp He Ser Asn siu Asn Cys 360 365 370 CGA ATA AAC AGA TAT GGT TAC TTC AGA GCC ACC ATC ACÁ ATT GTA GAT 1267 Arg He Asn Arg Tyr Gly Tyr Phe Arg Wing Thr He Thr He Val Aep 375 380 385 GGA ATC CTA GAA GTC AAC? TC? TC CAG GTA GCA GAT GTC CCA ATC CCC 1315 Gly He Leu Glu Val Asn He He Gln Val Wing Aep Val Pro He Pro 330 395 400 ACÁ ccs CAG CCT GAC AAC TCA CTG ATG GAC TTC ATT GTG ACC TGC AAA 1363 Thr Pro Gln Pro Asp Asn Ser Leu Met Asp Phe He Val Thr Cy3 Lys 405 410 415 GGG GCC ACT CCC ACG GAA GCC TGT ACG ATC ATC TCT GAC CCC ACC TGC 1411 Gly Wing Thr Pro Thr Glu Wing Cys Thr He He Ser Asp Pro Thr Cys 420 425 430 435 CAG ATC GCC CAG AAC AGG GTG TGC AGC CCG GTG GCT GTG GAT GAG CTG 1459 Gln He Wing Gln Asp Arg Val Cys Ser Pro Val Wing Val Asp slu Leu 440 445 450 TGC CTC CTQ CC GTG AGG AOA GCC TTC AAT GOQ CC ssc ACG TAC TGT 1507 Cya Leu Leu Ser Val Arg Arg Wing Phe Asn Gly Ser Gly Thr Tyr Cys 455 460 465 GTG AAT TTC ACT CTG GGA GAC GAT GCA AGC CTG GCC CTC ACC AGC scc 1555 Val Asn Phß Thr Leu Gly Asp Asp Wing Ser Leu Wing Leu Thr Ser Wing 470 475 480 P718 CTG ATC TCT ATC CCT GGC AAA GAC CTA GGC TCC CCT CTG AGA ACA GTG 1S03 Leu lie Ser He Pro Gly Lys Asp Leu Gly Ser Pro Leu Arg Thr Val 485 490 495 AAT GGT GTC CTG ATC TCC ATT GGC TGC CTG GCC ATG TTT GTC ACC ATG 1651 Asn Gly to Leu He Be He Gly Cys Leu Wing Met Phe Val Thr Met 500 505 S10 5a5 GTT ACC ATC TTO CTG TAC AAA AAA CAC AAG ACG TAC AAG CCA ATA GGA 1699 Val Tbr He Leu Leu Tyr Lya Lya His Lya Thr Tyr Lys Pro He Gly 520 525 530 AAC TGC ACC AGG AAC GTG GTC AAG GGC AAA GGC CTG AGT GTT TTT CTC 1747 Asp Cya Thr Arg Asn Val Val Lys Gly Lys Gly Leu Ser Val Phe Leu S3S 540 5 5 AGC CAT GCA AAA GCC CCG TTC TCC CGA GGA GAC CGG GAG AAG GAT CCA 1795 Ser His Wing Lys Wing Pro Phe Set Arg Gly Asp Arg slu Lys Asp Pro 5S0 SSS 560 CTG CTC CAG GAC AAG CCA TGG ATG CTC TAAGTCTTCA CTCTCACTTC I842 Leu Leu Gln Asp Lys Pro Trp Mee Leu 565 570 TGACTGGGAA CCCACTCTTC TGTGCATGTA TGTGAGCTGT GCAGAAGTAC ATGACTGGTA 1902 GCTGTTGTTT TCTACGGATT ATTGTAAAAT GTATATCATG GTTTAGGGAG CGTAGTTAAT 1962 TGGCATTTTA GTGAAGG5AT GGGAAGACAG TATTTCTTCA CATCTGTATT GTGGTTTTTA 2022 TACTGTTAAT AGGGTGGGCA CATTGTGTCT GAAGGGGGAG GGGGAGGTCA CTGCTACTTA 2082 AGGTCCTAGG TTAACTGGGA GAGGATGCCC CAGGCTCCTT AGATTTCTAC ACAAGATGTG 2142 CCTGAACCCA GCTAGTCCTG ACCTAAAGGC CATGCTTCAT CAACTCTATC TCAGCTCATT 2202 GAACATACCT GAGCACCTGA TGGAATTATA ATGGAACCAA GCTTGTTGTA TGGTGTGTGT 2262 GTGTACATAA GATACTCATT AAAAAGACAG TCTATTAAAA A 2303 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 572 amino acids (B) TYPE: amino acid P718 D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 5 Mee Glu Be Leu Cys Gly Val Leu Val Phe Leu Leu Leu Ala Wing Gly 1 5 10 15 Leu Pro Leu Gln Wing Wing Lys Arg Phe Arg Asp Val Leu Gly His Glu 20 25 30 Gln Tyr Pro Asp His Met Arg Glu Asn Asn Gln Leu Arg Gly Trp Ser 35 40 45 Ser Asp Glu Asn Glu Trp Asp Glu Gln Leu Tyr Pro Val Trp Arg Arg 50 55 60 Gly Glu Gly Arg Trp Lys Asp Ser Trp Glu Gly Gly Arg Val Gln Wing 65 70 75 80 Wing Leu Thr Being Asp being Pro Ala Leu Val Gly Being Asn He Thr Phe 85 90 95 Val Val Asn Leu Val Phe Pro Arg Cya Gln Lys Glu Asp Wing Asn Gly 100 105 lio Asn He Val Tyr Glu Arg Asn Cys Arg Ser Asp Leu Glu Leu Ala Ser 115 120 125? ßp Pro Tyr Val Tyr Asn Trp Thr Thr Gly Wing Asp Asp slu Asp Trp 130 135 140 Glu Asp Ser Thr Ser Gln sly Oln Hig Leu Arg Phe Pro Asp sly Lys 145 150 155 160 Pro Phe Pro Arg Pro His Gly Arg Lys Lys Trp Asn Phe at Tyr val 165 170 l7s Phe His Thr Leu Oly Oln Tyr Phe aln Lys Leu Gly Arg Cys Ser Wing 180 185 190 Arg Val Ser He Asn Thr val Asp Leu Thr Val Gly Pro Gln Val Met 195 200 205 P718 slu Val. I have val Phe Arg Arg His Gly Arg Ala Tyr He Pro He Ser 210 * 215 220 Lys Val Lys Asp Val Tyr Val He Thr Asp Gln He Pro He Phe Val 225 230 235 240 Thr Met Tyr Gln Lys Asp Aßp Arg Asn Ser Being Asp Glu Thr Phe Leu 245 250 255 Arg Asp Leu Pro He Phe Phe Asp Val Leu He His Asp Pro Ser His 260 265 270 Phe Leu Asn Tyr Ser Ala He Ser Tyr Lys Trp Aßn Phe Gly Aap Asn 275 280 285 Thr Gly Leu Phß Val Ser Asn Asn His Thr Leu Asn His Thr Tyr Val 290 295 300 Leu Asn Gly Thr Phe Asn Phe Asn Leu Thr Val Gln Thr Ala Val Pro 305 310 315 320 Gly Pro Cys Pro Pro Pro Thr Pro Ser Pro Ser Ser Ser Thr Ser Pro 325 330 335 Ser Pro Ala Ser Ser Pro Pro Pro Thr Leu Ser Thr Pro Ser Pro 340 345 350 Leu Met Pro Thr Gly His Lys Ser Met Glu Leu Ser Asp He Ser Asp 355 360 365 Glu Asn Cys Arg He Asp Arg Tyr Gly Tyr phe Arg Wing Thr He Thr 370 37S 380 He Val Asp Gly He Leu Olu Val Asn He He Gln Val Wing Asp Val 385 390 395 400 Pro He Pro Thr Pro Gln. Pro Asp Asn Ser Leu Met Asp Phe He Val 405 410 415 Thr Cys Lys Gly Wing Thr Pro Thr Glu Wing Cys Thr He He Ser Asp 420 425 430 Pro Thr Cys Gln He Wing Gln Asn Arg Val cys Ser Pro Val Wing Val 435 440 445 Asp Glu Leu Cys Leu Leu Ser Val Arg Arg Wing Phe Asn Gly Ser Gly 450 455 460 Thr Tyr Cys Val Asn Phe Thr Leu Gly Aßp Aep Ala Ser Leu Ala Leu 465 470 475 480 Thr Ser Ala Leu I have been Pro Gly Lys Asp Leu Gly Ser Pro Leu 485 490 495 Arg Thr Val Asn Gly Val Leu He Ser He Gly Cys Leu Ala Met Phe 500 505 510 P718 Val Thr Met Val Thr lie Leu Leu Tyr Lys Lys His Lys Thr T r Lys • '515 520 525 Pro He Gly Asn Cys Thr Arg Asn Val Val Lys Gly Lys Gly Leu Ser 530 535 540 Val Phe Leu Ser His Ala Lys Ala Pro Phe Ser Arg Gly Asp Arg Glu 545 550 555 5G0 Lys Asp Pro Leu Leu Gln Asp Lyß Pro Trp Met Leu 555 570 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1795 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 278..1279 (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 6: P718 GCGGCCGCGT CGACGAAGCT GGGAAGTCAG GGGCTGTTTC TGTGGGCAGC TTTCCCTGTC 60 CTTTGGAAGG CACAGAGCTC TCAGCTGCAG GGAACTAACA GAGCTCTGAA sCCGTTATAT 120 GTGGTCTTCT CTCATTTCCA GCAGAGCAGG CTCATATGAA TCAACCAACT GGGTGAAAAG 180 ATAAGTTGCA ATCTCAsATT TAAGACTTGA TCAGATACCA TCTGGTGOAG GGTACCAACC 240 AGCCTGTCTG CTCATTTTCC TTCAGGCTGA ATCC CAT CCT CAA GTG GTC 295 Met His Pro Gln Val Val 1 5 ATC TTA AQC CTC ATC CTA CAT CTG GCA GAT TCT GTA GCT GCT TCT GTA 343 He Leu Ser Leu He Leu His Leu Wing Asp Ser Val Wing Oly Ser Val 10 15 20 AAG GTT GGT GGA GAG GCA GGT CCA TCT GTC ACA CTA CCC TGC CAC TAC 391 Lys Val Gly Gly Glu Wing Gly Pro Ser Val Thr Leu Pro Cys His Tyr 25 30 35 AGT GGA GCT GTC ACÁ TCA ATG TGC TGG AAT AGA GGC TCA TGT TCT CTA 439 Ser Gly Wing Val Thr Ser Met Cys Trp Asn Arg Gly Ser Cys Ser Leu 40 45 50 TTC ACA TGC CAA AAT GGC ATT GTC TGG ACC AAT GGA ACC CAC GTC ACC 487 Phe Thr Cys Gln Asn Gly He Val Trp Thr Asn sly Thr His Val Thr 5S 60 65 70 TAT CGG AAG GAC ACÁ CGC TAT AAG CTA TTG GGs GAC CTT TCA AGA AGS 53S Tyr Arg Lys Asp Thr Arg Tyr Lye Leu Leu Gly Asp Leu Ser Arg Arg 75 80 85 GAT GTC TCT TTG ACC ATA GAA AAT ACÁ GCT GTG TCT GAC AGT GGC OTA 583 Asp Val Ser Leu Thr He Glu Asn Thr Wing Val Ser Asp Ser Gly Val 90 95 100 TAT TGT TGC CGT GTT GAG CAC CGT GGG TGG TTC AAT GAC ATG AAA ATC 631 Tyr Cys Cys Arg Val Glu His Arg Gly Trp Phe Asn Asp Met Lys He 105 110 115 P718 ACC GTA TCA TTG GAG ATT GTG CCA CCC AAG GTC ACG ACT ACT CCA ATT 679 Thr Val Ser Leu Glu He Val Pro Prc Lys Val Thr Thr Thr Pro He 120 125 130 GTC ACA ACT GTT CCA ACC GTC ACG ACT GTT CGA ACG AGC ACC ACT GTT 727 Val Thr Thr Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr Thr Val 135 140 145 150 CCA ACG ACÁ ACG ACT GTT CCA ACG ACÁ ACT GTT CCA ACÁ ACÁ ATG AGC 775 Pro Thr Thr Thr Thr Thr Thr Val Thr Pro Thr Thr Thr Thr Met Ser 1SS 160 165 ATT CCA ACG ACA ACG ACT GTT CCG ACG ACA ATG ACT GTT TCA ACG ACA 823 He Pro Thr Thr Thr Thr Thr Val Thr Thr Thr Thr Val Ser Thr Thr 170 175 180 ACG AGC GTT CCA ACG ACA ACG AGC ATT CCA ACÁ ACÁ ACÁ AGT GTT CCA 871 Thr Ser Val Pro Thr Thr Thr Ser He Pro Thr Thr Ser Val Pro 185 190 195 GTG ACÁ ACÁ ACG GTC TO 'ACC TTT GTT CCT CCA ATG CCT TTG CCC AGG 919 Val Thr Thr Thr Val Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg 200 205 210 CAG AAC CAT GAA CCA GTA GCC ACT TCA CCA TCT TCA CCT CAG CCA GCA 967 Gln Asn His Glu Pro Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Wing 215 220 225 230 GAA ACC CAC CCT ACG ACÁ CTG CAG GGA GCA ATA AGG AGA GAA CCC ACC 1015 Glu Thr His Pro Thr Thr Leu aln Gly Wing He Arg Arg Glu Pro Thr 235 240 245 AGC TCA CCA TTG TAC TCT TAC ACÁ ACÁ GAT CGG AAT GAC ACC GTG ACÁ 1063 Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr 250 255 260 GAG TCT TCA GAT GGC CTT TGG AAT AAC AAT CAA ACT CAA CTG TTC CTA 1111 Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr sln Leu Phe Leu 265 270 275 GAA CAT AGT CTA CTG ACG GCC AAT ACC ACT AAA GGA ATC TAT GCT GGA 1159 Glu His Ser Leu Leu Thr Ala Asp Thr .Thr Lys Ala Gly Tyr Gly He 280 285 290 GTC TGT ATT TCT GTC TTG GTG CTT CTT GCT CTT TTG GGT GTC ATC ATT 1207 Val Leu Val Cya I Ser Val Leu Leu Leu Leu Ala Val He He sly 295300 305 310 GCC AAA AAG TAT TTC TTC AAA AAG GAO GTT CAA CAA CTA AGA CCC CAT 1255 Ala Lyss Lys Tyr Phe Phe Lys Lyss slu Val Glp Gln Leu Arg Pro Hia 315 320 325 AAA TCC TGT ATA CAT CAA AGA GAA TAGTCCCTGG AAACATAGCA AATGAACTTC 1309 Lys Ser Cys He His Gln Arg Glu 330 P718 TATCTTGGCC ATCACAGCTG TCCAGAAGAG GGGAATCTGT CTTAAAAACC AGCAAATCCA 1369 ACGTQAGACT TCATTTGGAA scATTGTATs ATTATCTCTT GTTTCTATGT TATACTTCCA 1429 AATGTTGCAT TTCCTATGTT TTCCAAAGGT TTCAAATCGT GGGTTTTTAT TTCCTCCGTG 1489 GGGAAACAAA OTGAGTCTAA CTCACAGGTT TAGCTGTTTT CTCATAACTC TGGAAATGTG 1549 ATGCATTAAG TACTGGATCT CTGAATTOGG GTAGCTGTTT TACCAGTTAA AGAGCCTACA 1609 ATAGTATGGA ACACATAGAC ACCAGGGGAA sAAAATCATT TGCCAGGTGA TTTAACATAT 1669 TTATGCAATT TTTTTTTTTT TTTTTQAsAT GGAGCTTTGC TCTTGTTGCC CAGGCTGGAG 1729 TGCGATGGTG AAATCTCGGC TCACTGTAAC CTCCACCTTC CGGGTTCAAG CAATTCTCCC 1789 GTCGAC 1795 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 334 amino acids (B) TYPE: amino acid D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID No: 7 P718 Met Hie Pro Gln Val Val He Leu Ser Leu He Leu Kis Leu Ala Asp 1 5 10 15 ser Val Ala Gly Ser Val Lys Val Gly Oly Glu Ala Gly Pro Ser Val 20 2S 30 Thr Leu Pro Cys His Tyr Ser Gly Wing Val Thr Ser Met Cys Trp Asn 35 40 45 Arg Gly Ser Cys Ser Leu Phe Thr Cys Glp Asn Gly Ha Val Trp Thr 50 55 60 Aßn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu 65 0 75 80 Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr He slu Asn Thr Wing 85 90 95 Val Ser Asp Ser Gly Val Tyr Cya Cyß Arg Val Glu Hiß Arg Gly Trp 100 105 110 Phe Asn Asp Met Lys lie Thr Val Ser L * u Glu He Val Pro Pro Lys 115 120 125 Val Thr Thr Thr Pro He Val Thr Thr Val Pro Thr Val Thr Thr Val 130 135 140 Arg Thr Ser Thr Thr Thr Pro Thr Thr Thr Thr Thr Pro Thr Thr Thr 145 ISO 155 160 Val Pro Thr Thr Met Ser lie Pro Thr Thr Thr Thr Val Pro Thr Thr 165 170 175 Met Thr Val Ser Thr Thr Thr Ser Val pro Thr Thr Thr Ser He Pro 180 185 190 Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val Ser Thr Phe Val Pro 195 200 20 Pro Pro Pro Leu Pro Arg Gln Asn His Glu Pro Val Ala Pro Pro Thr 215 215 220 18 Ser Ser Pro Glp Pro Wing Glu Thr His Pro Thr Thr Leu Gln Gly Wing 225 230 235 240 He Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp 245 250 2S5 Gly Asn Asp Thr Val Thr Glu Ser Ser Aep Gly Leu Trp Asn Asn Aan 260 265 270 Gln Thr Gln Leu Phe Leu slu His Ser Leu Leu Thr Wing Asn Thr Thr 275 280 2T5 Lys Gly He Tyr Wing Gly Val Cys He Ser Val Leu Val Leu Leu Wing 290 295 300 Leu Leu Qly Val He lie Wing Lys Lys Tyr Pbe Phe Lys Lys slu Val 305 310 31S 320 Gln Gln Leu Arg Pro His Lys Ser Cys He His Gln Arg Glu 325 330 18

Claims (43)

1. CLAIMS: 1. Purified and isolated DNA, which codes for a kidney damage molecule (KIM), the KIM crue is a cell surface protein expressed selectively in mammalian kidney tissue, post-ischemic, DNA encoding either for: a) a 307 amino acid KIM polypeptide (KIM-1), having an IgG domain, a mucin domain, a transmembrane domain, and a cytoplasmic domain, wherein the IgG domain includes six conserved cysteines; or b) a KIM polypeptide, 572 amino acids, having a transmembrane domain. The DNA according to claim 1 (a), which codes for a polypeptide encoded by the insert of clone 3-2 (ATCC No. 98061), the insert of clone 1-7 (ATCC No. 98060), or the insert of clone H13-10-85. 3. The DNA according to claim 1 (b) "encoding a polypeptide encoded by the insert of clone 4-7 (ATCC No. 98062). 4. The DNA according to claim 1 (a) having a nucleotide sequence selected from: a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6; or P718 b) binding variants of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6; or c) degenerative variants of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6. 5. DNA having a sequence complementary to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 6 6. The DNA according to claim 1 (b) having a nucleotide sequence selected from: a) SEQ ID NO: 4; or b) A binding variant of SEQ ID NO: 4; or c) A degenerative variant of SEQ ID NO: 4. 7. DNA having a sequence complementary to SEQ ID NO: 4. 8. A purified and isolated DNA encoding a 307 amino acid KIM-1 polypeptide, which has an IgG domain, a mucin domain, a transmembrane domain, and a cytoplasmic domain, the sequence of the polypeptide that is selected from SEQ ID NO: 3 and SEQ ID NO: 7; or, of the amino acid substitution variants having at least 40% homology to, and sharing at least 80% of the cysteine residues of SEQ ID NO: 3 and SEQ ID NO: 7. 9. A purified DNA and isolate encoding a soluble variant of a 307 amino acid KIM-1 polypeptide, which has an IgG domain, a P718 mucin, a transmembrane domain, and a cytoplasmic domain, the polypeptide sequence that is selected from SEQ ID NO: 3 and SEQ ID NO: 7; or, of the amino acid substitution variants having at least 40% homology to, and sharing at least 80% of the cysteine residues of SEQ ID NO: 3 and SEQ ID NO: 7. 10. A purified DNA and isolate encoding a fusion protein, comprising: a) A KIM-1 polypeptide of 307 amino acids, having an IgG domain, a mucin domain, a transmembrane domain, and a cytoplasmic domain, the sequence of the polypeptide being selects from SEQ ID NO: 3 and SEQ ID NO: 7; or, of the amino acid substitution variants having at least 40% homology to, and sharing at least 80% of the cysteine residues of SEQ ID NO: 3 and SEQ ID NO: 7. b) an immunoglobulin, toxin , or a formable image compound. 11. A purified and isolated DNA encoding a 572 amino acid KIM polypeptide having a transmembrane domain, the sequence of the polypeptide that is selected from SEQ ID NO: 5 and the amino acid substitution variants that it has at least 40% homology with, and that share at least 80% of the cysteine residues of SEQ ID NO: 5. P718 12. A purified and isolated DNA encoding a soluble variant of a 572 amino acid KIM polypeptide, having a transmembrane domain, the sequence of the polypeptide selected from SEQ ID NO: 5 and variants of amino acid substitution having at least 40% homology to, and sharing at least 80% of the cysteine residues of SEQ ID NO: 5. 13. A purified and isolated DNA encoding a fusion protein, comprising: a) a 572 amino acid KIM polypeptide, having a transmembrane domain, the polypeptide sequence "which is selected from SEQ ID NO: 5 and from amino acid substitution variants having at least 40% homology with, and which share at least 80% of the residues of cysteine of SEQ ID NO: 5. b) an immunoglobulin, toxin, or an imageable formable compound. 14. A vector having the nucleic acid according to claim 8, 9, 10, 11, 12, or 13, present as an insert therein, the vector optionally comprising an expression control sequence operably linked to the insert. 15. A host cell comprising the vector of claim 14. 16. A method of polypeptide production, which P718 comprises the steps of: culturing a host cell of claim 15 in a cell culture medium; and, recovering, a KIM polypeptide expressed from the vector insert within the host cell. 17. A purified and isolated KIM protein, comprising: a) a KIM-1 polypeptide of 307 amino acids having an IgG domain, a mucin domain, a transmembrane domain and a cytoplasmic domain, the sequence of the polypeptide being selects from SEQ ID NO: 3 and SEQ ID NO: 7; or b) a 307 amino acid KIM-1 polypeptide having an IgG domain, a mucin domain, a transmembrane domain and a cytoplasmic domain, the polypeptide sequence that is selected from the amino acid substitution variants that it has minus 40% homology, and they share at least 80% of the cysteines of SEQ ID NO: 3 and SEQ ID NO: 7; or, c) a KIM polypeptide of 572 amino acids, having a transmembrane domain, the sequence of the polypeptide set forth in SEQ ID NO: 5; or d) a KIM polypeptide of 572 amino acids, having a transmembrane domain, the polypeptide sequence "which is selected from the amino acid substitution variants having at least 40% of P718 homology with, and that share at least 80% of the cysteines of SEQ ID NO: 5. 18. A soluble variant of a KIM protein of claim 17. 19. A fusion protein comprising a KIM protein of the claim 17; and an immunoglobulin, toxin, or imageable compound. 20. A KIM protein according to claim 17, conjugated to an immunoglobulin, toxin, imageable compound, or radionuclide. 21. An antibody that specifically binds to a KIM protein of claim 17. 22. An antibody according to claim 21, conjugated to a toxin, an imageable compound or radionuclide. 23. A hybridoma producing an antibody of claim 21. 24. The use in therapy of a vector of claim 14. 25. The use in therapy of a KIM protein of claim 17. 26. The use in therapy of a soluble variant KIM protein of claim 18. 27. The use in therapy of a KIM fusion protein of claim 19. P718 28. The use in therapy of a KIM conjugate of claim 20. 29. The use in therapy of an antibody of claim 21. 30. The use in therapy of an antibody conjugate of claim 22. 31. A A pharmaceutical composition comprising a physiologically acceptable carrier having dispersed therein, at a therapeutically effective concentration, a vector of claim 14. 32. A pharmaceutical composition comprising a physiologically acceptable carrier that has dispersed therein, at a therapeutically active concentration. effective: a KIM protein of claim 17, a soluble variant KIM protein of claim 18, a KIM fusion protein of claim 19 or a KIM conjugate of claim 20. 33. A pharmaceutical composition comprising a physiologically acceptable carrier having dispersed therein, at a therapeutically effective concentration, a The antibody of claim 21 or an antibody conjugate of claim 22. 34. A method for assessing the presence or course of resolution of kidney damage in a subject, comprising the step of measuring the concentration of an acid P718 KIM nucleic of claim 8 or 11, or a KIM polypeptide of claim 17, in the urine, serum, or pellet of the urine of a subject afflicted with, or at risk of developing, kidney damage or disease. 35. A method for image forming cells or tissue that produces a KIM polypeptide of claim 17, comprising the step of contacting the cells or tissue with an image-formable KIM antibody conjugate of claim 22 36. A method according to claim 35, wherein the cells or tissue are placed in vivo in a subject, and the imageable conjugate is administered to the subject. 37. A method for targeting a toxin, radionuclide or formable image compound to cells that produce a KIM polypeptide of claim 17, comprising the step of contacting the cells with a KIM antibody conjugate of claim 22. 38. A method according to claim 37, wherein the cells are tumor cells expressing KIM placed in vivo in a subject, the conjugate that is administered to the subject. 39. A method for treating a subject afflicted with, or at risk of developing, kidney disease, comprising the step of administering a composition Pharmaceutical P718 of claim 31, 32 or 33 to the subject. 40. A method for promoting tissue growth in a subject, comprising the step of administering a pharmaceutical composition of claim 31, 32, or 33 to the subject. 41. A method for promoting the survival of damaged tissue in a subject, comprising the step of administering a pharmaceutical composition of claim 31, 32, or 33 to the subject. 42. A method according to claim 41 or 41, wherein the tissue is a kidney tissue. 43. A method according to claim 34, 36, 38, 40 or 41, wherein the subject is a human. P718