EP0983357A1 - Modulateurs de la regeneration tissulaire - Google Patents

Modulateurs de la regeneration tissulaire

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Publication number
EP0983357A1
EP0983357A1 EP98923700A EP98923700A EP0983357A1 EP 0983357 A1 EP0983357 A1 EP 0983357A1 EP 98923700 A EP98923700 A EP 98923700A EP 98923700 A EP98923700 A EP 98923700A EP 0983357 A1 EP0983357 A1 EP 0983357A1
Authority
EP
European Patent Office
Prior art keywords
leu
ser
val
glu
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98923700A
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German (de)
English (en)
Inventor
Michele Sanicola-Nadel
Catherine A. Hession
Henry Wei
Richard L. Cate
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Biogen Inc
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Biogen Inc
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Application filed by Biogen Inc filed Critical Biogen Inc
Publication of EP0983357A1 publication Critical patent/EP0983357A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates generally to gene expression products, including nucleic acids (e.g., RNAs) and polypeptides that are upregulated in injured or regenerating tissues. Further, the invention relates generally to cDNAs and other nucleic acids encoding polypeptides that are upregulated in injured or regenerating tissues.
  • nucleic acids e.g., RNAs
  • polypeptides that are upregulated in injured or regenerating tissues.
  • cDNAs and other nucleic acids encoding polypeptides that are upregulated in injured or regenerating tissues.
  • tissue development during embryogenesis and of tissue repair and/or regeneration following injury or insult are presently the topic of intense investigation. Both processes involve a dynamic remodeling of tissue architecture, which is triggered and mediated by numerous biological interactions, including cell-cell contact, cell-matrix contact, release of soluble biological response modifiers, synthesis of structural components, and many other changes in cell phenotype, including changes in gene expression. Many of the factors involved in tissue genesis and in the response to tissue insult remain unknown or poorly understood.
  • a specific object of this invention is to identify products (e.g., RNAs, cDNAs, polypeptides) of genes that are upregulated in the context of healing tissue, or in tissue exposed to insult or injury.
  • a particular object is to identify products of genes that are upregulated in mammalian kidney tissue exposed to insult or injury, such as ischemia- reperfusion injury.
  • a second specific object of this invention is to identify products of genes that are upregulated in pathological conditions associated with abnormal responses to tissue insult or other stimulus, including autoimmune or other dysregulation pathologies, as well as pathologies associated with abnormal tissue growth including neoplastic growth.
  • a third specific object is to identify gene products that constitute appropriate targets for therapeutic intervention to stimulate, modulate, enhance, suppress or otherwise manipulate biological responses to tissue insult.
  • a particular object is to identify gene products for therapeutic intervention to manipulate responses of mammalian kidney tissue to tissue injury, such as ischemia-reperfusion injury.
  • a fourth specific object is to identify gene products that constitute appropriate targets for therapeutic intervention to manipulate abnormal or dysregulated responses to tissue insult or other stimulus, including abnormal tissue growth associated with cancer (neoplasia) and, conversely, abnormal tissue quiescence associated with degenerative diseases.
  • other particular objects of the invention include the identification of gene products as targets for therapeutic intervention in the clinical management (including prophylaxis, maintenance and treatment) of kidney diseases, including diseases involving renal failure, and of cancers derived from or affecting renal tissue.
  • the present invention rests on the discovery that the expression levels of numerous genes, including many genes heretofore unknown and or uncharacterized, are upregulated in mammalian kidney tissue that has been exposed to tissue insult or injury. More specifically, the present invention rests on the discovery of a novel class of Kidney Injury- associated Molecules (each member of the class is henceforth called a "KIM"). KIMs are polypeptides encoded by genes whose expression levels are upregulated in the mammalian kidney upon exposure to tissue injury, particularly after exposure to ischemia-reperfusion injury. Any KIM is useful as an indicator of tissue status (preferably renal tissue status) or of a change therein (e.g, exposure to tissue injury, or the occurrence or stage of a tissue repair process).
  • tissue status preferably renal tissue status
  • a change therein e.g, exposure to tissue injury, or the occurrence or stage of a tissue repair process.
  • any KIM further is useful as a target for therapeutic intervention to manipulate, whether by an agonist or an antagonist, a normal or abnormal tissue response to insult or other stimulus.
  • the invention accordingly provides, in a first aspect, purified and isolated nucleic acid molecules encoding all or a unique fragment of a KIM.
  • the present nucleic acids are RNAs.
  • they are DNA molecules, such as cDNAs.
  • the invention provides the complementary strands of nucleic acids encoding all or a unique fragment of a KIM.
  • the invention provides nucleic acids (preferably DNAs) that hybridize under low or, preferably, high stringency conditions to any of the foregoing nucleic acids.
  • the invention provides nucleic acids (preferably DNAs) which, but for the degeneracy of the genetic code, would hybridize to any of the foregoing nucleic acids.
  • a nucleic acid encoding all or a unique fragment of a KIM is an engineered (recombinant) nucleic acid, optionally in operative association with an expression control element or other regulatory element.
  • a nucleic acid encoding all or a unique fragment of a KIM is an antisense nucleic acid sufficient, when internalized within a cell, to disrupt expression of a cellular KIM gene.
  • Specific novel KIMs (and unique fragments thereof) of the present invention are coded for by nucleic acids having the sequences disclosed herein in TABLE 1 and in the Sequence Listing.
  • Other specific KIMs (and unique fragments thereof) are coded for by nucleic acids having sequences that are degenerate variants of any of the KIM sequences set forth in TABLE 1 and in the Sequence Listing.
  • Still other specific KIMs (and fragments) are coded for by nucleic acids that are substantially similar to (homologous to) any of the KIM sequences in TABLE 1 and in the Sequence Listing.
  • Such KIMs are defined herein as variants of the disclosed novel KIM sequences.
  • the present nucleic acid encodes a chimeric polypeptide comprising a novel KIM-encoding sequence (i.e., a sequence encoding part or all of a KIM) fused to a non-KIM sequence.
  • a novel KIM-encoding sequence i.e., a sequence encoding part or all of a KIM
  • the invention provides nucleic acids encoding novel KIM fusion proteins, non- limiting examples of which include KIM polypeptides fused to a secretable leader polypeptide, an immunoglobulin polypeptide, a binding pair partner (e.g., avidin, GST), a toxin or toxoid (e.g., ricin, tetanus), an enzyme (preferably one for which a detectable substrate is available, e.g., alkaline phosphatase, horseradish peroxidase, luciferase), or other detectable polypeptide (e.g., green fluorescent protein).
  • a binding pair partner e.g., avidin, GST
  • a toxin or toxoid e.g., ricin, tetanus
  • an enzyme preferably one for which a detectable substrate is available, e.g., alkaline phosphatase, horseradish peroxidase, luciferase
  • the invention provides a vector having a KIM-encoding nucleic acid inserted therein.
  • the vector is a biologically functional plasmid or viral DNA vector.
  • the vector is a retroviral vector.
  • the invention provides a prokaryotic or eukaryotic host cell comprising an internalized vector having a KIM-encoding nucleic acid insert.
  • the present host cell provides intracellular means for producing (synthesizing, folding, processing, or secreting) a KIM polypeptide of the present invention.
  • the invention provides a process for the production of a KIM polypeptide.
  • the present production process includes growing a host cell of the invention under culture conditions sufficient for the production of polypeptides of vector origin, and recovering an expressed KIM polypeptide.
  • the invention provides a purified and isolated novel KIM polypeptide, preferably substantially free of non-KIM polypeptides or proteins.
  • the present polypeptide is a full-length polypeptide, i.e., a polypeptide corresponding to the full-length open reading frame of a novel KIM-encoding cDNA.
  • the present polypeptide is a unique fragment of the full-length polypeptide.
  • novel KIM polypeptides of the present invention are variants of the disclosed novel KIM sequences, including without limitation splice variants, truncation variants, and substitution variants.
  • the invention provides a chimeric polypeptide comprising a novel KIM polypeptide fused (preferably via a peptide bond) to a non-KIM polypeptide.
  • novel KIM fusion proteins exemplified by the above-mentioned fusion constructs.
  • the invention also provides conjugated or derivatized novel KIM polypeptides, including without limitation detectable conjugates, imageable conjugates, radiolabeled conjugates, and toxin conjugates.
  • the invention provides chimeric polypeptides, fusion proteins and conjugates comprising a polypeptide appreciated herein as being a KIM (see TABLE 2).
  • the invention provides an antibody that binds selectively to a KIM polypeptide.
  • the antibody is a monoclonal antibody (or an engineered derivative thereof) produced by conventional means from a hybridoma derived from splenocytes of an animal immunized with a KIM polypeptide of the present invention.
  • the present antibody is conjugated or derivatized with a detectable moiety, toxin, imageable compound or radionuclide.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a KIM nucleic acid (e.g., an antisense nucleic acid); a KIM vector; a KIM polypeptide; a KIM fusion protein; a KIM-binding antibody (also referred to as an anti-KIM antibody), dispersed, dissolved or otherwise suspended in a physiologically acceptable carrier, vehicle, solvent or excipient.
  • a KIM nucleic acid e.g., an antisense nucleic acid
  • a KIM vector e.g., an antisense nucleic acid
  • KIM polypeptide e.g., a KIM polypeptide
  • KIM fusion protein e.g., a KIM fusion protein
  • KIM-binding antibody also referred to as an anti-KIM antibody
  • the invention provides methods and kits for detecting, either qualitatively or quantitatively, KIM expression and or KIM polypeptide.
  • KIMs are upregulated by exposure of mammalian kidney (renal) tissue to insult or injury, particularly ischemia-reperfusion injury.
  • any KIM (or combination or panel thereof) can be used as an indicator of tissue status (preferably renal tissue status), or of a change therein, including without limitation exposure to tissue injury, the occurrence or stage of an injury process, the occurrence or stage of a tissue repair or regeneration process, or the occurrence of an abnormal tissue response to injury or other stimulus, such as an autoimmune response or an abnormal proliferative response (e.g., a neoplastic response).
  • the invention provides a detection method and kit for diagnosis, prognosis, staging, or monitoring of renal injury or of renal disease or of the effectiveness of therapy therefor.
  • Some specific embodiments involve detecting and/or measuring the concentration of one or more KIM polypeptides in serum, urine, or urine sediment of an individual (a mammal, preferably a human) afflicted with or at risk of developing renal injury or an impairment of renal function.
  • Some such embodiments involve the use of at least one KIM polypeptide, anti-KIM antibody or conjugate thereof as elements of a reagent kit for immunoassay according to standard techniques.
  • any KIM disclosed herein or identified as such herein can be used as a kit element, along with other elements for KIM detection.
  • the kit can include reagents for detecting KIM activity, e.g., by enzyme assay.
  • Other specific embodiments involve detecting and/or measuring the level of expression of one or more KIM-encoding nucleic acids in renal cells in a kidney biopsy, or in cells shed into urine or urine sediment of an individual afflicted with or at risk of developing renal injury or an impairment of renal function.
  • Some such embodiments involve the use of a nucleic acid (e.g., RNA or DNA) encoding a KIM polypeptide, or a unique fragment thereof, or a probe nucleic acid capable of hybridizing to nucleic acid encoding a KIM, as elements of a reagent kit for hybridization assay according to standard techniques.
  • a nucleic acid e.g., RNA or DNA
  • a probe nucleic acid capable of hybridizing to nucleic acid encoding a KIM
  • any KIM nucleic acid disclosed herein or appreciated herein as such can be used as a hybridization kit element, along with other reagents for detection of hybridized nucleic acids.
  • the invention provides a method and kit for diagnosis, prognosis, staging or monitoring of an abnormal response of renal tissue to tissue injury or other stimulus, including an autoimmune response or an abnormal proliferative response, such as neoplasia arising from or affecting renal tissue.
  • Some specific embodiments involve detecting and/or measuring the concentration of one or more KIM polypeptides in serum, urine, or urine sediment of an individual (a mammal, preferably a human) afflicted with or at risk of developing renal injury or an impairment of renal function.
  • Some such embodiments involve the use of at least one KIM polypeptide, anti-KIM antibody or conjugate thereof as elements of a reagent kit for immunoassay according to standard techniques.
  • any K M disclosed herein or identified as such herein can be used as a kit element, along with other elements for KIM detection.
  • the kit can include reagents for detecting KIM activity, e.g., by enzyme assay.
  • Other specific embodiments involve detecting and or measuring the level of expression of one or more KIM-encoding nucleic acids in cells present in a kidney biopsy, or in cells shed into urine or urine sediment of an individual afflicted with or at risk of developing renal injury or an impairment of renal function.
  • Some such embodiments involve the use of a nucleic acid (e.g., RNA or DNA) encoding a KIM polypeptide, or a unique fragment thereof, or a probe nucleic acid capable of hybridizing to nucleic acid encoding a KIM, as elements of a reagent kit for hybridization assay according to standard techniques.
  • a nucleic acid e.g., RNA or DNA
  • a probe nucleic acid capable of hybridizing to nucleic acid encoding a KIM
  • any KIM nucleic acid disclosed herein or appreciated herein as such can be used as a hybridization kit element, along with other reagents for detection of hybridized nucleic acids.
  • the invention provides a method and reagent for imaging tissues, either in vitro or in vivo.
  • the invention provides a method and reagent for imaging the presence, extent or severity of kidney injury or of kidney tissue repair or regeneration.
  • the invention provides a method and reagent for imaging the presence, extent, severity or stage of an abnormal response to tissue injury or other stimulus, such as autoimmunity or neoplasia, particularly neoplasia arising from or affecting renal tissue.
  • the present invention also provides a method for targetting an imageable compound to cells or tissue expressing or producing a KIM.
  • the present method involves the step of contacting cells or tissue with a detectable (e.g., imageable) KIM-binding reagent, which in some embodiments is an anti-KIM antibody or conjugate thereof, or a KIM fusion protein.
  • the detectable KIM-binding reagent is a nucleic acid of the invention (e.g., a probe or antisense nucleic acid) labeled with a radionuclide or other imageable compound.
  • cells expressing or producing a KIM are visualized (imaged) by detecting the presence and/or location of an accumulation of the KIM-binding reagent.
  • the KIM- binding reagent is administered, by any appropriate route, to an individual (a mammal, preferably a human) suspected of harboring an imageable locus of KIM expression and/or production.
  • an individual a mammal, preferably a human
  • the present method can be used to detect the presence and/or location of a tissue mass (e.g., a tumor) abnormally producing or expressing a KIM.
  • the invention provides methods for treating an individual (a mammal, preferably a human) afflicted with or at risk of developing a disease or condition contributed to or associated with a dysfunction or dysregulation of a KIM gene or protein. Further, the invention provides methods for treating an individual afflicted with or at risk of developing a disease or condition beneficially affected by therapeutic administration of a KIM protein.
  • the present methods involve the step of administering to the individual a therapeutically effective amount of a KIM polypeptide, variant or fusion protein thereof, or, conversely, of an anti-KIM antibody. It is expected that such compounds will be useful in therapeutic methods which manipulate, e.g., stimulate or inhibit, biological responses that are dependent on KIM function.
  • the invention provides a method for inhibiting the growth of KIM-expressing tumor cells, involving the step of contacting the cells with an anti-KIM antibody, conjugated to a toxin or radionuclide.
  • the method involves the step of contacting the cells with an antisense KIM nucleic acid that is sufficient to suppress or disrupt expression of a KIM gene in the tumor cells.
  • the invention provides a method of gene therapy.
  • the present method involves the administration of a vector capable of directing the production of a KIM, to an individual afflicted with or at risk of a renal disorder, disease or injury.
  • a vector capable of directing the production of a KIM to an individual afflicted with or at risk of a renal disorder, disease or injury.
  • growth of new tissue, preferably renal tissue is stimulated, or survival of existing tissue, preferably renal tissue, is promoted.
  • Kidney Injury-related Molecules The discovery, reported herein, of a novel class of Kidney Injury-related Molecules (KIMs) was made by analyzing differences in mRNA expression between normal adult mammalian kidneys, and kidneys in the process of regenerating following exposure to tissue insult (specifically, to ischemia-reperfusion injury). Two established techniques were used for this purpose: representational difference analysis (RDA), and suppression subtractive hybridization (SSH). Both techniques were used to assess cDNAs isolated from various timepoints, e.g., 48 hours, after the onset of ischemia. In these studies, the normal adult kidney material was isolated from sham-operated subjects.
  • RDA representational difference analysis
  • SSH suppression subtractive hybridization
  • KIM protein is any protein or polypeptide encoded by mRNA which is selectively upregulated following injury to a kidney.
  • One group of KIM proteins of interest includes those coded for by mRNA which is selectively upregulated at any time within one week following any insult which results in injury to renal tissue. Examples of times at which such upregulation might be identified include 10 hours, 24 hours, 48 hours or 96 hours following an insult. Examples of tissue insults include toxin exposure, hypoxia, hyperoxia, hemodynamic disruption, ischemia, reperfusion, or mechanical compression.
  • proteins include cell surface proteins (e.g., ligands or counter-receptors involved in cell-cell or cell-matrix interactions), secreted proteins (e.g., diffusable biological response modifiers, such as growth factors, differentiation factors, survival factors and the like), intracellular proteins (e.g., elements of a signalling pathway), and nuclear proteins (e.g., transcription factors).
  • cell surface proteins e.g., ligands or counter-receptors involved in cell-cell or cell-matrix interactions
  • secreted proteins e.g., diffusable biological response modifiers, such as growth factors, differentiation factors, survival factors and the like
  • intracellular proteins e.g., elements of a signalling pathway
  • nuclear proteins e.g., transcription factors
  • a "KIM ligand” is any molecule which noncovalently and specifically binds to a KIM protein.
  • a ligand can be a protein, peptide, steroid, antibody, amino acid derivative, or other type molecule, in any form, including naturally-occurring, recombinantly produced, or otherwise synthetic.
  • a KIM ligand can be in any form, including soluble, membrane-bound, or part of a fusion construct with immunoglobulin, fatty acid, or other moieties.
  • the KIM ligand may be an integrin.
  • a membrane-bound KIM ligand can act as a receptor which, when bound to or associated with KIM, triggers a cellular response.
  • a KIM may associate with a plurality of KIM ligands, or may associate with a KJM ligand as part of a complex with one or more other molecules or cofactors.
  • the KIM may associate and react with KIM ligand which is bound to the same cell as the KIM, or it may associate and react with KIM ligand be bound to a second cell.
  • KIM ligation refers to the contact and binding of KIM with a KIM ligand.
  • a "unique fragment" of a nucleic acid means any fragment of sufficient length to have a sequence likely to be substantially unique in a mammalian genome. Thus, a unique fragment generally means an oligonucleotide at least 16 nucleotide bases in length.
  • a "unique fragment" of a polypeptide means any fragment of sufficient length to have an amino acid sequence likely to be substantially unique to a given mammalian protein, such as a KIM.
  • a unique peptide fragment generally means a peptide at least 7 amino acids in length.
  • KIM variant means a KIM whose sequence differs from a sequence disclosed herein by the presence of one or more internal or terminal insertions, deletions or substitutions of a nucleotide (when referring to KIM nucleic acids) or of an amino acid (when referring to KIM polypeptides).
  • the KIM variant is “substantially similar” to the corresponding disclosed KIM, or to a unique fragment thereof. That is, the KIM variant is “homologous” to the corresponding disclosed KIM.
  • “Substantially similar” or “homologous” variants are structurally similar to the corresponding disclosed KIM.
  • substantially similar or “homologous” variants have sufficient functional similarity to the corresponding disclosed KIM that they share one or more of the KIM's biological properties or functions (e.g., binding to a receptor or ligand, triggering of biological responses, transport of a metabolite, catalysis of a substrate, or the like).
  • a KIM variant can be naturally occurring or synthesized or produced by routine techniques, such as molecular engineering techniques.
  • alignment of sequences is meant the positioning of one sequence, either nucleotide or amino acid, with that of another, to allow a comparison of the sequence of relevant portions of one with that of the other.
  • sequences are aligned using the GAP and BESTFTT programs, which are based on the teachings of Needleman et al. (J. Mol. Biol. 48:443-453, 1970), Smith et al. (Adv. Appl. Math. 2:482-489, 1981), and Rechild et al. (CABIOS 5: 107-113, 1989).
  • homologous variant of a KIM when aligned with the corresponding KIM polypeptide, it will share amino acids that contribute to the KIM's three dimensional structure, such as cysteine residues.
  • cysteine residues Although the relative positions of cysteine residues is generally conserved in the variant sequence, homologous or functionally equivalent sequences can include functionally equivalent arrangements of the cysteines, including arrangements comprising amino acid insertions or deletions which alter the linear arrangement of the cysteines, but do not materially impair their relationship or ability to form disulfide bonds in the folded structure of the KIM protein. Therefore, minor internal gaps and amino acid insertions, such as occur in splice variants, are ignored when aligning sequences herein.
  • Sequence homology “percent (%) homology”, “sequence similarity” and “percent (%) similarity” are used interchangeably herein, and refer to the sum of the percentage of residues (whether nucleotides or amino acids) that are, when aligned with a reference sequence, the same as the corresponding reference residues, and those that are, when aligned, conservative substitutions for the corresponding reference residues.
  • Sequence identity and “percent (%) identity” also are used interchangeably herein, and refer to the percentage of residues (whether nucleotides or amino acids) that are, when aligned with a reference sequence, the same as the corresponding reference residues. Both sets of terms are used according to their definitions in Altschul et al. (1990), J. Mol. Biol. 215:403-410 and in the Basic Local Alignment Search Tool (BLAST) algorithm described therein. For present purposes, the algorithm gap weight is set at 3.0 and the length weight is set at 0.1.
  • Hybridization means the formation of a duplex nucleic acid molecule, in which nucleotide bases of a first polynucleotide strand bind noncovalently with cognate nucleotide bases of a second polynucleotide strand.
  • noncovalent binding occurrs in DNA only between adenosine (A) and thymidine (T) bases, and guanosine (G) and cytosine (C) bases.
  • a and uracil (U) uracil
  • G and C cytosine
  • hybridization conditions include 6.0X sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0X SSC at 50°C.
  • the salt concentration in the wash step can be selected from a low stringency wash of about 2.0X SSC at 50°C, to a high stringency was of about 0.2X SSC at 50°C.
  • the temperature in the wash step can be selected from a low stringency wash at room temperature, about 22°C, to a high stringency wash at about 65°C.
  • KIM agonist is a molecule which can specifically trigger a cellular response normally triggered by the interaction of KIM with a KIM ligand.
  • a KIM agonist can be a KIM variant, or a specific antibody to KIM, or a soluble form of the KIM ligand.
  • KIM antagonist is a molecule which can specifically associate with a KIM ligand or a KIM, thereby blocking or otherwise inhibiting KIM binding to the KIM ligand.
  • the antagonist binding blocks or inhibits cellular responses which would otherwise be triggered by ligation of the KIM ligand with KIM or with a KIM agonist.
  • KIM antagonists include certain KIM variants, KIM fusion proteins and specific antibodies to a KIM ligand or KIM.
  • isolated refers to a condition in which a nucleic acid or polypeptide of the present invention is essentially free of other nucleic acids, polypeptides, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • substantially pure refers to a condition in which a nucleic acid or polypeptide of the present invention is separated from other nucleic acids, polypeptides, or other contaminants, particularly naturally occurring contaminants, that interfere with the ability to detect, visualize or isolate the the nucleic acid or polypeptide of the invention, or that interfere with a biological function or property thereof.
  • a substantially pure nucleic acid or polypeptide of the invention is not generally found in nature.
  • a "chemical derivative" of another molecule contains one or more additional chemical or biochemical moieties not found naturally in association with the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences. 16th ed., Mack Publishing Co., Easton, Penn. (1980).
  • TABLE 1 lists KIMs for which potential full length (PFL) cDNA clones were obtained. Also shown are the predicted polypeptide sequences encoded in the open reading frames (ORFs) of many of the newly discovered KIMs.
  • ORFs open reading frames
  • TABLE 1 provides, where relevant, the subtracted amplified clone (SAC) designations and the PFL location(s) of partial sequences, many of which were reported in prior U.S. Provisional S.N. 60/047,490 and 60/047,491.
  • KIMs which are believed to be related to (e.g., substantially similar to) known molecules also were obtained through RDA or SSH techniques, and are set forth in TABLE 2 and in the Sequence Listing. It is believed that few to none of these molecules were previously appreciated to be KIMs, i.e., to have relevance to tissue injury or repair processes, particularly in the kidney. TABLE 2 lists these KIMs along with their clone designations. Partial sequences of some of the cDNA clones listed below were also set forth in the priority document. Also shown are the name(s) and GENBANK accession numbers of the known genes to which these KIMs may be related.
  • any of the foregoing KIM cDNAs can be inserted into a vector, liposome or other carrier vehicle for internalization and production in a host cell.
  • the invention encompasses derivatives and variants of each of the foregoing KIMs as listed in TABLES 1 and 2.
  • One embodiment of the invention provides soluble variants of a KIM protein that is usually synthesized as a membrane associated protein.
  • Soluble variants lack at least a portion of the transmembrane or intra-membrane section of a native KIM protein. In some examples, the soluble variant lacks the entire transmembrane or intra-membrane section of a native KIM protein.
  • Soluble variants include fusion proteins which encompass derivatives of KIM proteins that lack at least a portion of the transmembrane or intra- membrane section of a native KIM protein. All types of KIM fusion proteins are included, particularly those which incorporate his-tag, Ig-tag, and myc-tag forms of the molecule. These KIM fusions may have characteristics which are therapeutically advantageous, such as the increased half-life conferred by the Ig-tag. Also included are fusion proteins which incorporate portions of selected domains of the KIM protein.
  • Variants can differ from a naturally occurring KIM protein in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring KIM protein is substituted with a different natural amino acid, an amino acid derivative or non-natural amino acid. Particularly preferred substitution variants include naturally occurring KIM proteins, or biologically active unique fragments thereof, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide.
  • 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.
  • Other conservative substitutions are those which meet the criteria for an "accepted point mutation" as defined in the Atlas of Protein Sequence and Structure (Dayhoff et al., eds.), 1978. See also PCT publication no. WO97/44460.
  • Substitution variants can also have sequences which differ by one or more non- conservative amino acid substitutions, provided however that the substitution does not abolish the native KIM protein's biological activity, ligand- or receptor-binding characteristics, or other functional property of interest.
  • exemplary non-conservative substitutions are those in which: (I) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative charge, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g
  • KIM variants of this invention include truncation variants (comprising at least a unique fragment of the corresponding KIM), insertion variants, and splice variants.
  • KIM variants within the invention are those with modifications which increase polypeptide stability. Such variants can contain, for example, one or more non- peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D- amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Patent 5,219,990. KIM variants can be naturally-occurring, or produced through synthetic or molecular engineering techniques.
  • an engineered KIM variant can provide advantageous properties, e.g., in facilitating purification, improving stability, modulating a biological function, or the like.
  • a KIM variant will be desired that lacks a glycosylation site, or that has decreased aggregation potential due to elimination of a hydrophobic surface, or that has improved folding efficiency due to elimination of a cysteine residue, or the like.
  • KIM variants have at least fifty (50) % amino acid sequence homology or similarity with the corresponding KIM protein.
  • the variants Preferably, the variants have at least sixty-five (65) % sequence similarity, more preferably at least eighty (80) % similarity with the corresponding KIM protein.
  • the sequence similarity is at least ninety (90) , or most preferably, at least ninety-five (95) %.
  • Other preferred KIM variants are those which 1) share at least forty (40) % similarity to the corresponding KIM protein, and 2) share at least eighty (80) % of aligned cysteine residues with the corresponding KIM protein.
  • non-amino acid moieties can occur naturally in a given KIM as a result of post-translational processing, including acetylation, methylation, phosphorylation, carboxylation or glycosylation. As desired, such moieties can be removed or added by conventional synthetic or biochemical techniques. Further, non-natural moieties can be added to produce a derivative of a KIM or KIM variant of this invention. For example, polyethylene glycol (PEG) can be linked to a KIM to improve its stability or pharmacokinetic properties.
  • PEG polyethylene glycol
  • anti-KIM and anti-KIM variant antibodies can be produced by conventional techniques. Specifically contemplated are polyclonal and monoclonal antibodies, including antigen-binding fragments thereof and engineered derivatives thereof. Antigen-binding fragments of intact antibodies include complete Fab fragments, F(ab') compounds, V H regions, and F v regions. Engineered derivatives of antibodies of the invention include single chain antibodies (see, e.g., WO 96/23071), as well as human, humanized, primatized, or chimeric antibodies (see, e.g., PCT/US 95/16709).
  • Engineered derivatives of the present antibodies are produced, generally, using standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-99, 1991). These include "chimeric" antibodies, in which the antigen binding domain from an animal antibody is linked to a human constant domain.
  • an antibody with the desired KIM-binding selectivity is derived initially from a nonhuman mammal (e.g., a mouse, rat or hamster), and subjected to recombinant DNA manipulation to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobin light chain or heavy chain.
  • Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments.
  • Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed.
  • Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.
  • Fully human antibodies with KIM-binding specificity which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as is described in U.S. 5,569,825.
  • kidney ischemia-reperfusion model which similates acute renal failure, was employed.
  • the results of studies in this model system now have been published, and appear in Ichimura et al. (1998). J. Biol. Chem. 273:4135-4142, the teachings of which are incorporated by reference herein.
  • the kidney has the capacity for cell renewal (both structurally and funcitonally) after injury to tubular epithelial cells.
  • the nephron is damaged functionally by an ischemic reperfusion injury that results in regional areas of proximal tubule cell death.
  • the kidney proximal tubule epithelium undergoes a complex series of events including (1) cell death and cast formation in the tubule lumen (casts are aggregates of dead, semi viable and viable cells, as well as of cell debris); (2) proliferation of surviving proximal tubule epithelial cells; (3) formation of a poorly differentiated regenerative epithelium over the denuded basement membrane (this simplified epithelium expresses vimentin, a mesenchymal marker); and (4) differentiation of the regenerative epithelium to form fully functional proximal tubule epithelial cells.
  • Gene expression analysis of the kidney at various timepoints following the onset of ischemia revealed the upregulation of many KIMs anticipated to be involved in the injury and repair/regeneration processes.
  • Ischemic injured rat kidneys are generated as described by Witzgall et al. (J. Clin Invest. 93: 2175-2188, 1994). Briefly, the renal artery and vein from one kidney of an adult Sprague-Dawley rat are clamped for 40 minutes and then reperfused. Injured kidneys are harvested from the rats at 24 hours and at 48 hours after reperfusion. Kidneys from sham-operated, normal adult Sprague-Dawley rats are also harvested.
  • Post-ischemic rat kidneys are prepared as described by Witzgall et al. (J. Clin. Invest. 93: 2175-2188, 1994). Briefly, the renal artery and vein of the left kidney of an adult Sprague Dawley rat are clamped for 40 minutes and then reperfused. The ischemic kidneys are removed from rats either 24 hours or 48 hours after reperfusion. Normal rat kidneys are used as controls.
  • Total RNA is isolated with Trizol reagent (BRL catalog No. 1559-026). The kidney capsules are removed, then the kidneys are rinsed in PBS and immediately homogenized in Trizol solution. In some instances, the kidney is chopped into several pieces in PBS, frozen quickly in liquid nitrogen and kept at -70 °C until it is processed for total RNA isolation. The mRNA is purified from total RNA according to the manufacturer's instructions, using an mRNA purification kit (Pharmacia, Catalog No. 27-9258-02).
  • RDA Representational difference analysis
  • Double-stranded cDNA is synthesized using the Superscript Choice® system (BRL Catalog No. 18090), from mRNA isolated from rat kidney either 24 hours or 48 hours post-ischemia as a tester and normal rat kidney as a driver.
  • the tester and driver cDNA is digested with Dpnll and ligated to R- Bgl-12/24 oligonucleotides.
  • the adapter-ligated cDNA is amplified by PCR, and the PCR product is digested with Dpnll to remove the oligonucleotides.
  • the tester cDNA is ligated to J-Bgl- 12-24 oligonucleotides.
  • cDNA clones for Kim-1 and NMB contained in a plasmid vector are provided by T. Ichimura. They are digested with Sau3Al, purified and used as quenching reagents. cDNA fragments, 2-3 containing a fragment of annexin ⁇ , and 3-8 containing a fragment of alpha 2 macroglobulin (both provided by T. Ichimura) are amplified by PCR with R-Bgl-24 primer, digested with Dpnll to remove the oligonucleotides and used as quenching reagents. Tester cDNA is hybridized to an excess driver cDNA including the four quenching reagents described above. After mung bean nuclease digestion.
  • hybridization mixture is amplified by PCR to enrich the cDNA fragments upregulated by ischemia. Each of the PCR steps is tested with a range of template DNA concentrations, and the concentration giving the best signal is chosen for the next steps.
  • the DNA mixture containing tester and driver DNA for the subtractive hybridization step is washed twice with 70% EtOH by spinning for 3 minutes.
  • the second round cDNA subtraction is performed by hybridizing the first round cDNA subtraction product ligated to N-Bgl- 12-24 oligonucleotides to an excess of the same driver cDNA used for the first round subtraction.
  • the third round cDNA subtraction is performed by hybridizing the second round cDNA subtraction product ligated to J-Bgl- 12/24 oligonucleotides to an excess of the same driver cDNA used for the first round of cDNA subtraction at a ratio of 1: 14,000.
  • the final PCR product after the third round of subtraction is digested with Dpnll to remove the oligonucleotides and ligated to pCR- Script SK(+) vector. This represents a selected cDNA library by RDA.
  • SSH Suppression subtractive hybridization
  • SSH is perfomed according to the manufacturer's instructions (Clontech, Catalog No. Kl 804-1) with certain modifications. Briefly, double-stranded cDNA are synthesized from mRNA isolated from rat kidney 24 hours or 48 hours post-ischemia as a tester, and from nomal rat kidney as a driver with the Superscript ChoiceTM system for cDNA synthesis (BRL Catalog No. 18090). The cDNA is synthesized according to the manufacturer's instruction except that the first strand cDNA is synthesized at 42°C. cDNA is digested with RSAI. Tester cDNA after RSAI digestion is ligated to adaptors.
  • tester cDNA with adaptors is hybridized with excess driver cDNA at 68°C for 12 hours.
  • the mixture is hybridized at 68°C for 22 hours.
  • the cDNA mixture after the second hybridization is initially amplified by PCR for 27 cycles only. Then, a portion of the PCR product is re-amplified for 10 or 13 cycles.
  • the final subtracted PCR product is digested by RSAI to remove the adaptor, and separated on a gel. The three bands and the remaining smear are cut out separately and cloned to pCR- Script SK (+) vector (Stratagene, Catalog No. 211188). This represents a selected cDNA library by SSH.
  • Colonies are randomly picked from the selected RDA and SSH cDNA libraries, and plasmid DNA is isolated with Qiagen plasmid kit. Each cDNA clone, referred to as a Subtracted Amplified Clone (SAC) is sequenced with the vector primers. DNA sequences are checked against GeneBank EMBL databases for homology by the program BLASTTM.
  • Southern blots are prepared with the initial PCR products generated from normal and injured rat kidneys. The blots are hybridized to the inserts isolated from the SACs to confirm which SACs are induced by ischemia.
  • the ligated DNA is packaged and used to infect E. coli XL-1 Blue MRF.
  • the complexity of the library is 3.4xl0 6 independent recombinant plaques.
  • Another cDNA library is generated from 4 ug of polyA+ RNA from rat kidney 48 hours post-ischemia as described above.
  • pBluescript plasmid vector containing cDNA inserts are excised from Lambda ZAPII vector by in vivo excision. Inserts from the longest cDNA clones, referred to as Potential Full Length (PFL) clones, are sequenced. DNA sequences are checked by the program BLASTTM against the GenBank/EMBL databases for DNA homology. Predicted protein coding regions (open reading frames, or ORFs) from some of the PFL clones also were used to search the GenBank/EMBL databases. Diagnostic Uses of the Compounds of the Invention
  • Anti-KIM antibodies of the invention which specifically bind to a protein of the invention or a unique fragment thereof, are useful in several diagnostic methods. These agents can be labeled with detectable markers, such as fluoroscopically or radiographically opaque substances, and administered to an individual to allow imaging of tissues which express KIM protein. The agents can also be bound to (conjugated to) substances, such as horseradish peroxidase, which can be used as immunocytochemical stains to allow visualization of areas of KIM protein-positive cells on histological sections. A specific antibody could be used alone in this manner, and sites where it is bound can be visualized in a sandwich assay using an anti-immunoglobulin antibody which is itself bound to a detectable marker.
  • detectable markers such as fluoroscopically or radiographically opaque substances
  • the agents can also be bound to (conjugated to) substances, such as horseradish peroxidase, which can be used as immunocytochemical stains to allow visualization of areas of KIM protein-positive cells on histological sections.
  • the invention includes a method of diagnosing renal injury, or of monitoring a process of renal repair, by measuring the concentration of KIM or of KIM fragments in the urine, plasma or serum of a patient.
  • KIM can be measured in urine sediment, in particular in cellular debris in the urine sediment. Casts of renal tubule cells, which may be present in urine sediment from patients with ongoing renal disease, may contain elevated levels of KIM protein and mRNA.
  • Specific antibodies to KIM protein may also be bound to solid supports, such as beads or dishes, 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 post- translational modifications).
  • solid supports such as beads or dishes
  • Such characterization of an individual's KIM protein is expected to be useful in identifying deleterious mutants or processing defects which interfere with KIM function and are associated with abnormal phenotypes.
  • Additional imaging methods utilize KIM or KIM fragments, fused to imageable moieties, for diagnostic imaging of tissues that express KIM ligands, particularly tumors. Further diagnostic techniques are based on demonstration of upregulated KIM mRNA in tissues, as an indication of injury-related processes. (See, e.g., PCT publication no. WO 97/44460).
  • the therapeutic methods of the invention involve selectively promoting or inhibiting cellular responses that are dependent on the presence or concentration of any KIM, including without limitation KIM ligation, KIM enzyme activity, KIM secretion, KIM signalling, and KIM modulation of gene expression.
  • KIM any KIM
  • the signal transduction may occur in the KIM-expressing cell, in the KIM ligand-expressing cell, or in both.
  • KIM ligation-triggered response in a KIM ligand-expressing cell may be generated by contacting the cell with exogenous KIM, KIM fusion proteins or activating antibodies against KIM ligand, either in vitro or in vivo. Further, responses of the KIM ligand- expressing cell that would otherwise be triggered by endogenous KIM could be blocked by contacting the KIM ligand-expressing cell with a KIM ligand antagonist (e.g., an antagonist antibody that binds to KIM ligand), or by contacting the endogenous KIM with an anti- KIM antibody or other KIM-binding molecule which prevents the effective ligation of KIM with a KIM ligand.
  • a KIM ligand antagonist e.g., an antagonist antibody that binds to KIM ligand
  • KIM ligation- triggered response in a KIM-expressing cell may be generated by contacting the cell with a soluble KIM ligand, or certain anti-KIM activating antibodies.
  • responses of the KIM-expressing cell that would otherwise be triggered by interaction with endogenous KIM ligand could be blocked by contacting the KIM-expressing cell with an antagonist to KIM (e.g.., a blocking antibody that binds to KIM in a manner that prevents effective, signal-generating KIM ligation), or by contacting the endogenous KIM ligand with an anti- KIM ligand antibody or other KIM ligand-binding molecule which prevents the effective ligation of KIM with the KIM ligand.
  • an antagonist to KIM e.g., a blocking antibody that binds to KIM in a manner that prevents effective, signal-generating KIM ligation
  • an anti- KIM ligand antibody or other KIM ligand-binding molecule which prevents the effective ligation of KIM with the KIM ligand.
  • One therapeutic use of the KIM-related compounds of the invention is for treating a subject with renal disease, promoting growth of new tissue in a subject, or promoting survival of damaged tissue in a subject, and includes the step of administering to the subject a therapeutically effective amount of a KIM protein of the invention, or of a pharmaceutical composition which includes a protein of the invention.
  • the protein used in these methods can be a unique fragment of a full-length KIM protein, a soluble KM variant or a soluble ligand thereof, a KIM fusion protein, or a KIM agonist.
  • tissues of interest for these methods include any tissue, preferred tissues include renal tissue, liver, neural tissue, heart, stomach, small intestine, spinal cord, or lung.
  • Particular renal conditions which are expected to be beneficially treated with the compounds of the invention include acute renal failure, acute nephritis, chronic renal failure, nephrotic syndrome, renal tubule defects, kidney transplants, toxic injury, hypoxic injury, and trauma.
  • Renal tubule defects include those of either hereditary or acquired nature, such as polycystic renal disease, medullary cystic disease, and medullary sponge kidney. This list is not limited, and may include many other renal disorders (see, e.g., Harrison's Principles of Internal Medicine, 13th ed., 1994, which is herein incorporated by reference.)
  • the subject of the methods may be human.
  • a therapeutic intervention for inhibiting growth of undesirable, KIM- or KIM- ligand-expressing tissue in a subject includes the step of administering to the subject a therapeutically effective amount of a KIM antagonist (e.g.., an antibody blocks KIM ligation).
  • a KIM antagonist e.g.., an antibody blocks KIM ligation
  • the KIM antagonist or anti-KIM antibody can be used therapeutically to inhibit or block growth of tumors which depend on KIM protein for growth.
  • Other methods of the invention include killing KIM ligand-expressing tumor cells, or inhibiting their growth, by contacting the 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 can be within a subject, and the protein or the conjugated antibody is administered to the subject.
  • Also encompassed within the invention is a method for targeting a toxin or radionuclide to a cell expressing a KIM, comprising contacting the cell with a fusion protein comprising a KIM ligand and a toxin or radionuclide, or an anti-KIM antibody conjugated to a toxin or radionuclide.
  • Another embodiment includes the method of suppressing growth of a tumor cell which expresses KIM, comprising contacting the cell with a fusion protein of KIM ligand and a toxin or radionuclide or with an anti-KIM antibody conjugated to a toxin or radionuclide; the cell may be within a subject, and the protein administered to the subject.
  • subject as used herein is taken to mean any mammal to which KIM can be administered.
  • Subjects specifically intended for treatment with the method of the invention include humans, as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.
  • the KIM genes of the invention are introduced into damaged tissue, or into tissue where stimulated growth is desirable. Such gene therapy stimulates production of KIM protein by the transfected or transformed cells, promoting cell growth and/or survival of cells that express the KIM protein.
  • a gene coding for a KIM protein is be introduced into a renal target tissue.
  • the KIM protein is expected to be stably expressed and stimulate tissue growth, division, or differentiation, or to potentiate cell survival.
  • a KIM gene can be introduced into a target cell using a variety of well-known methods that use either viral or non-viral based strategies.
  • sequences of interest can be identified by conventional methods such as nucleic acid hybridization using probes comprising sequences that are homologous/complementary to the inserted gene sequences of the vector.
  • sequence(s) may be identified by the presence or absence of a "marker" gene function (e.g, thymidine kinase activity, antibiotic resistance, and the like) caused by introduction of the expression vector into the target cell.
  • a "marker" gene function e.g, thymidine kinase activity, antibiotic resistance, and the like
  • an exemplary carrier comprises normal physiologic saline (0.15M NaCl, pH 7.0 to 7.4).
  • Acceptable carriers can include biocompatible, inert or bioabsorbable salts, buffering agents, oligo- or polysaccharides, polymers, viscosity-improving agents, preservatives, and the like.
  • the term "carrier” encompasses liposomes and the HIV-1 tat protein (See Chen et al., Anal. Biochem. 227: 168-175, 1995) as well as any plasmid and viral expression vectors. Any KIM compound of this invention can be used in the form of a pharmaceutically acceptable salt.
  • KIM compounds of the invention are dispersed in the carrier to concentrations sufficient to deliver to the subject a therapeutically effective amount of the compound, which is an amount sufficient to produce a detectable, preferably medically beneficial effect in the subject.
  • Medically beneficial effects would include preventing, delaying or attenuating deterioration of, or detectably improving, the subject's medical condition. It is expected that the concentration or amount of a KIM compound that will produce a medically beneficial effect will vary considerably with the circumstances in which the invention is practiced.
  • An effective amount can be determined by an ordinarily skilled physician or other practitioner through no more than routine experimentation.
  • an indication of the status of renal injury or renal function can be monitored with one or more routine laboratory tests which measure the concentrations of relevant substances in blood or urine, other urine characteristics, or the rate of clearance of various substances from the blood into the urine. The parameters measured by these tests, either individually or in combination, can be used by a physician to assess renal function or damage.
  • Such parameters include the blood concentration of urea, creatinine or protein; the urine concentration of protein or of various blood cells such as erythrocytes or leucocytes; urine specific gravity; amount of urine; the clearance rates of inulin, creatinine, urea or p-aminohippuric acid; and the presence of hypertension or edema.
  • a KIM compound is formulated in a liposome delivery system, including without limitation any of a variety of unilamellar vesicles, multilamellar vesicles, or stable plurilamellar vesicles, all of which can prepared and administered according to methods well known to those of skill in the art, for example in accordance with the teachings of United States Patent 5,169,637, 4,762,915, 5,000,958 or 5,185,154.
  • treatment of human acute renal failure with liposome- encapsulated KIM protein may be performed in vivo by introducing a KIM protein into cells in need of such treatment using liposomes.
  • the liposomes can be delivered via catheter to the renal artery.
  • the recombinant KIM protein is purified, for example, from CHO cells by immunoaffinity chromatography or any other convenient method, then mixed with liposomes and incorporated into them at high efficiency.
  • the encapsulated protein may be tested in vitro for any effect on stimulating cell growth.
  • the compounds of the invention may be administered in any manner which is medically acceptable. Depending on the specific circumstances, local or systemic administration may be desirable.
  • the compound is administered via a parenteral route such as by an intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intratumor, intraorbital, intraventricular, intraperitoneal, subcapsular, intracranial, intraspinal, or intranasal injection, infusion or inhalation.
  • the compound also may be administered by implantation of an infusion pump, or a biocompatible or bioerodable sustained release implant, or by installation of a catheter (e.g., in a renal artery), into the subject.
  • certain compounds of the invention, or formulations thereof may be appropriate for oral or enteral administration. Still other compounds of the invention will be suitable for topical administration.
  • Determining appropriate dosage and frequency of treatment with any particular KIM compound to be administered to an individual is within the skills and clinical judgement of ordinary practitioners.
  • the general dosage and treatment schedule is established by preclinical and clinical trials, which involve extensive but routine studies to determine the optimal administration parameters of the compound. Even after such recommendations are made, the practitioner will often vary these dosages for different individuals based on a variety of considerations, such as the individual's age, medical status, weight, sex, and concurrent treatment with other pharmaceuticals. Determining the optimal dosage and administration regime for each KIM compound is a routine matter for those of skill in the pharmaceutical and medical arts.
  • ADDRESSEE Patent Administrator, Biogen, Inc.
  • CTGCAGCTCA CAAAACAAGC AGTGAAAACA TATAAACTAC CTATAATAGC CTCATTATAA 1920
  • CTGCCTTTGT TTCTTGTTAA TTTGCACATT ATATGCAAGT TTTCTCTTTT TAAAAAGATG 2820
  • AGCATTTAGC AAAATGAAAG ACATAGTGTC CCATGTGTCC TGAACATGTA TTGTTATGTG 3120
  • TTCTATATGC ATGACCCTTC TTATTAAAAA CAGATTGGGG AAAGGGGTCA ATTTTGTGTT 3180
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTAGGTTTGC CACTAGAAGG AAGCATACTT AAAACAATGG CTACTTGGAT CCTCAGGGAG 180
  • GCTAAACTCA AACCAGTTTA CAGAAACGAT GTATTTTGTG TATAGTAAAT TGTATATTCA 480
  • TATTCTCCAT TCTTGCTTAA TTGTATTAGC CATAAAGGAG TTCAGGTTCA TAGGACTCAG 120
  • TTCTAGTCCC TTTAGTCTTA GACAGTTTCT GTTTCTTGTA TTTATAAAGT TGGTATTAAA 600
  • CTCTTCACCT CTAATTAGCT CATACTGTTT CCCACTGACT CCCGTCTTTA ATGGCTTCAT 2520
  • CTATCTCCAC CTCCCTAATA ATGCTGGAGT TACAATCATG GACAACCATA CACACATGTT 947
  • AGA GCT GCC AGG AAA CAA GCT TCT CAA GTG TTT ACA GCG GAG AAG ATT 4159 Arg Ala Ala Arg Lys Gin Ala Ser Gin Val Phe Thr Ala Glu Lys He 340 345 350
  • GGCTACATCC CTTTGTGGTC ACATGGCCTG TAGCCTCTTG CTGACATTGC TGGTGTCTTC 240
  • TTCTCAGTTC ATCTTTCAGG TCCGTGTGTA CTCTGTGCTA GCAGCAGCCT TGGTGTTCAA 1320

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Abstract

L'invention concerne des protéines se développant dans les tissus lésés ou en cours de régénération, l'ADN codant ces protéines, ainsi que les compositions thérapeutiques renfermant lesdits composés et des traitements faisant intervenir ces composés.
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CA2290783A1 (fr) 1998-11-26
AU7592998A (en) 1998-12-11
WO1998053071A1 (fr) 1998-11-26

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