WO2005117941A2 - Methods and compositions for the treatment of polycystic diseases - Google Patents
Methods and compositions for the treatment of polycystic diseases Download PDFInfo
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- WO2005117941A2 WO2005117941A2 PCT/US2005/014982 US2005014982W WO2005117941A2 WO 2005117941 A2 WO2005117941 A2 WO 2005117941A2 US 2005014982 W US2005014982 W US 2005014982W WO 2005117941 A2 WO2005117941 A2 WO 2005117941A2
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- This invention is related to the area of polycystic diseases and to the diagnosis and treatment of such diseases.
- ADPKD Autosomal Dominant Polycystic Kidney Disease
- This invention provides compositions and methods to diagnose and treat renal cystic disorders by modifying the biological activity of at least one gene identified in Tables 2 through 5, infra.
- renal cystic disorders is intended to include, but not be limited to, a large group of diseases, including polycystic kidney disease, vonHippel-Lindau, tuberosclerosis, nephronophthisis, autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), acquired cystic kidney disease (ACKD), and autosomal dominant polycystic liver disease (ARPKD).
- ADPKD autosomal dominant polycystic kidney disease
- ARPKD autosomal recessive polycystic kidney disease
- ACKD acquired cystic kidney disease
- ARPKD autosomal dominant polycystic liver disease
- CTGF CTGF gene or its expression product
- CTGF is an example of such a gene identified in Tables 2 through 5, infra. Accordingly, although the following discussion and examples are limited in most part to the CTGF gene and biological equivalents thereof, the invention is not so limited.
- the invention of this application encompasses any of the genes identified in Tables 2 through 5 as targets for therapeutic and pharmaceutical intervention; CTGF is but one member of this class of targets. Accordingly, it should be understood, although not explicitly stated, that any of the genes identified in Tables 2 through 5 can be substituted for the term "CTGF" as used herein.
- the invention provides a method of modifying the biological activity of at least one gene identified in Tables 2 through 5 by contacting an effective amount of modifying agent or molecule with the cell or tissue in need of treatment.
- Suitable modifying agents for use in the method include, but are not limited to a small molecule, a ribozyme, an antisense oligonucleotide, a double stranded RNA, a double-stranded interfering RNA (iRNA), a triplex RNA, an RNA aptamer, and at least a portion of an antibody molecule that binds to the gene product and inhibits its activity.
- the agent binds to the receptor and initiates signaling.
- antibody portions include, but are not limited to an intact antibody molecule, a single chain variable region (ScFv), a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.
- the antibodies can be generated in any appropriate in « vitro or in vivo system, e.g., simian, murine, rat or human. Suitable antibodies are commercially available from Torrey Pines Biolabs, Inc. (Cat.
- the antibody can optionally be bound to: a cytotoxic moiety, a therapeutic moiety, a detectable moiety, or an anti-cystic agent.
- the agent or molecule is isolated and then delivered.
- compositions and methods to treat or ameliorate abnormal cystic lesions and diseases associated with the formation of cysts in tissue treat and ameliorate pathological cyst formation in tissue by inhibiting, e.g., CTGF gene expression or the biological activity of its gene expression product.
- receptor activation is inhibited.
- receptor activity is initiated or augmented.
- a method of treating, inhibiting, or ameliorating the symptoms associated with Autosomal Dominant Polycystic Kidney Disease requires delivering to a subject in need thereof an effective amount of an agent or molecule, e.g., CTGF, that modulates the activity of the CTGF gene or its expression product.
- an effective amount of an agent that inhibits the biological activity of the CTGF receptor is delivered to the subject.
- U.S. Patent No. 6,555,322 discloses cDNA sequence encoding a receptor as well as its amino acid sequence.
- the agent or molecule is isolated and then delivered.
- delivery of a gene or polypeptide that augments expression is delivered.
- Such agents are known in the art and include, but are not limited to polynucelotides encoding the peptides or the polypeptides themselves.
- This invention also provides methods for aiding in the diagnosis of cystic abnormalities present in a tissue by detecting the expression level of the gene or its expression product. The method can be used for aiding in the diagnosis of ADPKD-associated renal cysts and cystic abnormalities in other organs, including the liver, pancreas, spleen and ovaries, that are commonly found in ADPKD. Additionally, by detecting overexpression or underexpression of the protein or polynucleotide prior to abnormal cyst formation, one can predict a predisposition to ADPKD and provide early diagnosis and/or treatment.
- kits for carrying out the diagnostic and prognostic methods contain compositions used in these methods and instructions for their use.
- This invention also provides compositions for use in the therapeutic and diagnostic methods.
- the composition comprises a molecule containing an antibody variable region which specifically binds to a CTGF protein (e.g., SEQ ID NO: 2) or its receptor so that binding is inhibited and/or blocked and the receptor is not activated or activation of the receptor is inhibited.
- the molecule can be, for example, an intact antibody molecule, a single chain variable region (ScFv), a monoclonal antibody, a chimeric or a humanized antibody.
- Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc.
- the molecule can optionally be bound to: a cytotoxic moiety, a therapeutic moiety, a detectable moiety or an anti-cystic agent.
- the invention provides nucleic acid molecules that inhibit the expression of the CTGF gene or the receptor to which CTGF protein binds, or the gene that encodes the receptor.
- nucleic acids include, but are not limited to a ribozyme, an antisense oligonucleotide, a double stranded RNA, iRNA, a triplex RNA or an RNA aptamer.
- the nucleic acid is delivered in an isolated form.
- the nucleic acid can be isolated from an animal or alternatively, recombinantly produced in any suitable recombinant system, e.g., bacterial, yeast, baculoviral or mammalian.
- the invention provides nucleic acid molecules that enhance, support, augment or increase expression of the gene or, its transcription and/or translation product or any ligand which activates the receptor.
- nucleic acids include, but are not limited to a ribozyme, an antisense oligonucleotide, a double stranded RNAs, iRNA, a triplex RNA or an RNA aptamer.
- the nucleic acid is delivered in an isolated form.
- the nucleic acid can be isolated from an animal or alternatively, recombinantly produced in any suitable recombinant system, e.g., bacterial, yeast, baculoviral or mammalian.
- Yet another aspect of the invention is a method to identify a CTGF binding ligand involved in CTGF-associated cyst formation.
- a test compound or agent such as an antibody or antibody derivative or variant is contacted with a CTGF protein or fragment thereof in a suitable sample under conditions that favor the formation of binding to CTGF or its receptor. Receptor-binding or CTGF- binding, if it occurred, is then detected.
- the therapeutic and diagnostic agents are used in combination with other agents. Co-administration of these agents or molecules with other agents or therapies can provide unexpected synergistic therapeutic benefit. In the co-administration methods, the agents or molecules are also useful in reducing deleterious side-effects of known therapies and therapeutic agents, as well as yet to be discovered therapies and therapeutic agents, by decreasing dosage.
- the use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component than may be required when each individual therapeutic method, compound or drug is used alone, thereby reducing adverse side effects.
- the present invention also includes methods involving co-administration of the compounds described herein with one or more additional active agents or methods. Indeed, it is a further aspect of this invention to provide methods for enhancing other therapies and/or pharmaceutical compositions by co-administering a compound of this invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In one embodiment, the compounds described herein are administered prior to the other active agent(s), therapy or therapies.
- the pharmaceutical formulations and modes of administration may be any of those described herein or known to those of skill in the art.
- a still further embodiment of the invention is a method to identify candidate drugs to treat cystic lesions by contacting cells which express the CTGF gene or its receptor with a test compound or agent.
- a test compound is identified as a candidate drug for treating cystic abnormalities if it increases or decreases expression of the CTGF gene or the gene encoding its receptor. Expression can be detected and quantified by any method known in the art, e.g., by hybridization of mRNA of the cells or tissue to a nucleic acid probe which is complementary to CTGF mRNA or receptor mRNA, where appropriate. Test compounds or agents which decrease expression are identified as candidates for treating abnormal CTGF cyst formation.
- Figure 1 shows the effect of an anti-CTGF antibody in an in vitro model of cystogenesis.
- Figure 2 shows that CTGF protein is expressed in jck mouse kidneys.
- Figure 3 is a 2D SDS gel profiling of cyst fluid from ADPKD patient.
- BRIEF DESCRIPTION OF THE TABLES Table 1A is a summary of SAGE libraries screened. It is a summary of total tags sequenced and unique tags.
- Table 1 B summarizes CTGF expression in normal and cystic kidneys.
- Table 2 identifies the top 20 up- and down-regulated genes in cystic liver (CL).
- Table 3 identifies the top 20 up- and down-regulated genes in cystic kidney (CK).
- the 20 most down- or up-regulated tags along with their counts in normal kidney (NK) or cystic kidney (CK) are represented as for the ones presented in Table 2.
- Table 4 identifies the up-regulated genes >5x common to liver and kidney epithelia.
- Table 5A identifies functional groups of genes up-regulated in CL.
- Table 5B identifies functional groups of genes up-regulated in CK.
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
- compositions consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about”.
- polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
- the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
- a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
- isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. In one aspect of this invention, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated with in its native or natural environment, e.g., on the chromosome.
- a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
- a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated” or less than "separated” than that of its naturally occurring counterpart.
- a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
- a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
- polynucleotide and “oligonucleotide” are used interchangeably, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or hbonucleotides, or analogs thereof. Polynucleotides may have any three - dimensional structure, and may perform any function, known or unknown.
- polynucleotides a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
- sequence of nucleotides may be interrupted by non-nucleotide components.
- a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
- the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
- a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for guanine when the polynucleotide is RNA.
- polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be ' input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
- a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated.
- a “gene product” or “expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
- "Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription.
- "Operatively linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
- a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
- Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- Gene delivery are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction.
- exogenous polynucleotide sometimes referred to as a "transgene”
- Such methods include a variety of well- known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked” polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
- the introduced polynucleotide may be stably or transiently maintained in the host cell.
- Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
- a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
- a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
- a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
- viral vectors examples include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
- Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky, Curr. Opin. Biotechnol. (1999) 5:434-439 and Ying, et al., Nat. Med. (1999) 5(7):823-827.
- a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
- retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
- the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
- retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
- Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
- the integrated DNA form is called a provirus.
- a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
- Ads adenoviruses
- Adenoviruses are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071.
- Ads are easy to grow and do not require integration into the host cell genome.
- Recombinant Ad derived vectors particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed.
- Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
- Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
- the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
- a "probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
- a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
- Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
- a “primer” is a short polynucleotide, generally with a free 3' -OH group that binds to a target or "template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
- a “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or a “set of primers” consisting of an "upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme.
- PCR A PRACTICAL APPROACH
- All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication.”
- a primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et al., supra.
- An expression “database” denotes a set of stored data that represent a collection of sequences, which in turn represent a collection of biological reference materials.
- cDNAs refers to complementary DNA, that is mRNA molecules present in a cell or organism made in to cDNA with an enzyme such as reverse transcriptase.
- a "cDNA library” is a collection of all of the mRNA molecules present in a cell or organism, all turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into “vectors” (other DNA molecules that can continue to replicate after addition of foreign DNA).
- vectors for libraries include bacteriophage (also known as "phage"), viruses that infect bacteria, for example, lambda phage. The library can then be probed for the specific cDNA (and thus mRNA) of interest.
- differentially expressed refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene.
- a differentially expressed gene may be overexpressed or underexpressed as compared to the expression level of a normal or control cell. In one aspect, it refers to a differential that is 2.5 times, or alternatively 5 times, or alternatively 10 times higher or lower than the expression level detected in a control sample.
- the term “differentially expressed” also refers to nucleotide sequences in a cell or tissue which are expressed where silent in a control cell or not expressed where expressed in a control cell.
- solid phase support or “solid support”, used interchangeably, is not limited to a specific type of support.
- Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels.
- solid support also includes synthetic antigen-presenting matrices, cells, and liposomes.
- a suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols.
- solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula
- a polynucleotide also can be attached to a solid support for use in high throughput screening assays.
- International PCT Application No. WO 97/10365 discloses the construction of high density oligonucleotide chips. See also, U.S. Patent Nos. 5,405,783; 5,412,087; and 5,445,934.
- the probes are synthesized on a derivatized glass surface also known as chip arrays.
- Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask, and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.
- expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins.
- expression may include splicing of the mRNA in an eukaryotic cell.
- “Overexpression” as applied to a gene refers to the overproduction of the mRNA transcribed from the gene or the protein product encoded by the gene, at a level that is 2.5 times higher, or alternatively 5 times higher, or alternatively 10 times higher than the expression level detected in a control sample.
- “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
- the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
- a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
- Hybridization reactions can be performed under conditions of different "stringency". In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature.
- a moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC.
- the reaction is called “annealing” and those polynucleotides are described as “complementary”.
- a double-stranded polynucleotide can be "complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
- “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
- a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
- This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.
- default parameters are used for alignment.
- a preferred alignment program is BLAST, using default parameters.
- composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
- pharmaceutical composition is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the compositions also can include stabilizers and preservatives.
- stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
- An "effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.
- a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, sport animals, and pets.
- a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or "negative”.
- a positive control a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease
- a negative control a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease.
- Therapeutic Methods This invention provides methods for treating and/or ameliorating the symptoms associated with cystic abnormalities present in a tissue.
- the cysts are a manifestation of Autosomal Dominant Polycystic Kidney Disease (ADPKD).
- ADPKD Autosomal Dominant Polycystic Kidney Disease
- the major manifestation of the disorder is the progressive cystic dilation of renal tubules which ultimately leads to renal failure in half of affected individuals.
- ADPKD-associated renal cysts may enlarge to contain several liters of fluid and the kidneys usually enlarge progressively causing pain.
- Other abnormalities such as hematuria, renal and urinary infection, renal tumors, salt and water imbalance and hypertension frequently result from the renal defect.
- Cystic abnormalities in other organs, including the liver, pancreas, spleen and ovaries are commonly found in ADPKD. Massive liver enlargement occasionally causes portal hypertension and hepatic failure.
- Biochemical abnormalities which have been observed have involved protein sorting, the distribution of cell membrane markers within renal epithelial cells, extracellular matrix, ion transport, epithelial cell turnover, and epithelial cell proliferation.
- the most carefully documented of these findings are abnormalities in the composition of tubular epithelial cells, and a reversal of the normal polarized distribution of cell membrane proteins, such as the Na + /K + ATPase.
- Carone, F.A. er a/., Lab. Inv. (1994) 70:437-448 provides methods for inhibiting, reducing or ameliorating the above- noted biochemical, structural and physiological abnormalities related to ADPKD.
- the method requires delivering to the tissue in need thereof an effective amount of an agent or molecule that modifies (inhibits or augments) expression of a gene identified in Tables 2 through 5 or its expression product, in affected tissue.
- an agent or molecule that modifies (inhibits or augments) expression of a gene identified in Tables 2 through 5 or its expression product in affected tissue.
- Applicants have discovered quite unexpectedly, that overexpression of the CTGF gene in tissue is related to cystic abnormalities and that downregulation of the gene or its expression product treats or ameliorates the symptoms associated with cystic abnormalities. Inhibiting the binding of CTGF to its receptor also treats or ameliorates symptoms associated with cystic abnormalities.
- CTGF has previously been postulated to be involved in conditions in which there is an overgrowth of connective tissue cells, such as systemic sclerosis, cancer, fibrotic conditions, and atherosclerosis.
- CTGF polypeptide The primary biological activities of CTGF polypeptide are reported to be related to its mitogenicity, or ability to stimulate target cells to proliferate and its role in the synthesis of the extracellular matrix. The ultimate result of this mitogenic activity in vivo, is the growth of targeted tissue.
- CTGF also is reported to possess chemotactic activity, which is the chemically induced movement of cells as a result of interaction with particular molecules. Elevated levels of CTGF are found in fibrotic lesions and suggested to be functionally involved in the development of fibrotic diseases and wound healing. Chih-Chiun, C. er a/., J. Biol. Chem. (2001 ) 276(13): 10443-10452; Hahn, A. et al., J. Biol.
- CTGF is a cysteine-rich monomeric peptide of M r 38,000. It is a member of the CCN family of growth regulators which includes the mouse (also known as fisp-12 or£IG-M2) and human CTGF, Cyr61 (mouse), Cef10 (chicken), and Nov (chicken). Based on sequence comparisons, it has been suggested that the members of this family all have a modular structure, consisting of (1 ) an insulin-like growth factor domain responsible for binding, (2) a von Willebrand factor domain responsible for complex formation, (3) a thrombospondin type I repeat, possibly responsible for binding matrix molecules, and (4) a C-terminal module found in matrix proteins, postulated to be responsible for receptor binding.
- CCN is the acronym for an emerging family of regulatory proteins which are reported to be involved in the regulation of cell proliferation, chemotaxis, angiogenesis and the formation of the extracellular matrix. Lin, CG. et al., J. Biol. Chem. (2003) 278(26):24200-24208; Lafont, J. er a/., J. Biol. Chem. (2002) 277(43):41220-41229; Li, CL. et al., J. Clin. Pathol. (2002) 55:250-261 ; Manara, M.C. et al., Am. J. Pathol. (2002) 160(3):849- 859.
- the family comprises both positive and negative regulators that share a common multi-modular organization.
- the cDNA for human CTGF has been reported to contain an open reading frame of 1047 nucleotides with an initiation site at position 130 and a TGA termination site at position 1177.
- the cDNA encodes a peptide of 349 amino acids. See, U.S. Patent Publ. US 2002/0115156A1.
- the cDNA sequence is also available at GenBank No.: NM_001901 , which is also reproduced as SEQ ID NO: 1.
- the gene is reported to contain 2312 nucleotides with the open reading frame represented by nucleotides 146 through 1195.
- the 572 amino acid polypeptide expressed from this sequence is available under GenBank No.: NP_001892.1 , which is also reproduced as SEQ ID NO: 2.
- GenBank No.: NP_001892.1 which is also reproduced as SEQ ID NO: 2.
- the cDNA sequence for rat CTGF is reported to contain an open reading frame of 2350 nucleotides with an initiation site at position 212 and a TAA termination site at position 1353 and encodes a peptide of 346 amino acids. See, paragraph 28 of U.S. Published Patent Doc. US 2002/0115156A1.
- the terms "treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
- the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
- Treating also covers any treatment of a disorder in a mammal, and includes: (a) preventing a disorder from occurring in a subject that may be predisposed to a disorder, but has not yet been diagnosed as having it; (b) inhibiting a disorder, i.e., arresting its development; or (c) relieving or ameliorating the disorder, e.g., cause regression of the disorder, e.g., ADPKD.
- to "treat” includes systemic amelioration of the symptoms associated with the pathology and/or a delay in onset of symptoms.
- Clinical and sub-clinical evidence of “treatment” will vary with the pathology, the individual and the treatment.
- These cells or tissue are identified by any method known in the art that allows for the identification of differential expression of the gene or its expression product. Exemplary methods are described herein.
- Therapeutic agents can be administered to suitable cells, tissues or to subjects as well as or in addition to individuals susceptible to or at risk of developing cystic abnormalities.
- the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To determine patients that can be beneficially treated, a regression of the cyst can be assayed. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy. Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.
- Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
- Suitable dosage formulations and methods of administering the agents are known in the art.
- the agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
- An agent of the present invention can be administered for therapy by any suitable route including nasal, topical (including transdermal, aerosol, buccal and sublingual), parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
- the polynucleotides useful for the methods of this invention can be replicated using PCR.
- PCR technology is the subject matter of United States Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: THE POLYMERASE CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) and references cited therein.
- one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA.
- this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
- these polynucleotides are further isolated.
- one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
- the DNA so amplified can be isolated from the cell by methods well known to those of skill in the art.
- RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
- the DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., Iiposome, plasmid or vector) or by electroporation.
- an appropriate gene delivery vehicle e.g., Iiposome, plasmid or vector
- electroporation When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) supra.
- mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook, er a/. (1989) supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures.
- Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific transcript RNA molecule. In the cell, the antisense nucleic acids hybridize to the corresponding transcript RNA, forming a double-stranded molecule thereby interfering with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded.
- Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules.
- the use of antisense methods to inhibit the in vitro translation of genes is known in the art. Marcus-Sakura, Anal. Biochem. (1988) 172:289 and De Mesmaeker, et al., Curr. Opin. Struct. Biol. (1995) 5:343-355.
- the information disclosed in these publications and known to those of skill in the art, in combination with Applicants' specification, enables one of skill in the art to make and use antisense DNA or RNA molecules as therapeutic agents.
- oligonucleotide Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix.
- Triplex compounds are designed to recognize a unique site on a chosen gene. Maher, et al., Antisense Res. and Dev. (1991) 1(3):227; Helene, C, Anticancer Drug Design (1991 ) 6(6):569.
- Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases.
- RNA molecules which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it.
- a major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
- U.S. Patent No. 6,458,559 discloses how to make and use RNA aptamer molecules to inhibit gene expression. The information disclosed in this patent, in combination with the Applicants' specification, enables one of skill in the art to make and use aptamers as CTGF inhibitory molecules.
- U.S. Published Patent Doc. US 20030180744 discloses methods to make and use high affinity oligonucleotide ligands to growth factors.
- U.S. Published Patent Doc. US 20030051263 discloses a process for introducing a double stranded RNA into a living cell to inhibit gene expression of a target gene in that cell. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical.
- the information disclosed in this published application, in combination with the Applicants' specification enables one of skill in the art to make and use double stranded RNA molecules as therapeutic agents. See, e.g., Elbashir, S.M.
- the agent when it is a nucleic acid, it can be added to the cell cultures by methods known in the art, which include, but are not limited to calcium phosphate precipitation, microinjection or electroporation. They can be added alone or in combination with a suitable carrier, e.g., a pharmaceutically acceptable carrier such as phosphate buffered saline. Alternatively or additionally, the nucleic acid can be incorporated into an expression or insertion vector for incorporation into the cells. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art.
- Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
- Stratagene La Jolla, CA
- Promega Biotech Promega Biotech
- consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
- vectors are viruses, such as baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- viruses such as baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
- non-viral vectors including DNA/liposome complexes, and targeted viral protein DNA complexes.
- the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface anti
- Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
- This invention also provides the targeting complexes for use in the methods disclosed herein.
- Polynucleotides are inserted into vector genomes using methods known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
- synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA.
- an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
- a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
- enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
- transcription termination and RNA processing signals from SV40 for mRNA stability transcription termination and RNA
- This invention also provides isolated polypeptides encoded by a gene identified in Tables 2 through 5, e.g., the CTGF gene.
- the CTGF polypeptide has the amino acid sequence shown in SEQ ID NO: 2.
- the polypeptide is modified by substitution with conservative amino acids.
- the polypeptide has the same function as the polypeptide of SEQ ID NO: 2 as determined using the examples set forth below and are identified by having more than 80% , or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98 or 99% sequence homology to SEQ ID NO: 2 as determined by sequence comparison programs such as BLAST run under appropriate conditions.
- the program is run under default parameters. Further provided are active fragments of these embodiments.
- the peptides used in accordance with the method of the present invention can be obtained in any one of a number of conventional ways.
- peptides can be prepared by chemical synthesis using standard techniques. Particularly convenient are the solid phase peptide synthesis techniques. Automated peptide synthesizers are commercially available, as are the reagents required for their use.
- isolated peptides of the present invention can be synthesized using an appropriate solid state synthetic procedure. Steward and Young, eds. (1968) SOLID PHASE PEPTIDE SYNTHESIS, Freemantle, San Francisco, Calif. One method is the Merrifield process.
- an isolated peptide of the invention may be purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
- chromatography e.g., ion exchange, affinity, and sizing column chromatography
- centrifugation e.g., centrifugation
- differential solubility e.g., differential solubility
- an epitope may be isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
- affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej er a/., Methods Enzymol. (1991 ) 194:508-509), and glutathione-S-transferase can be attached to the peptides of the invention to allow easy purification by passage over an appropriate affinity column.
- Isolated peptides can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
- the polynucleotides can be replicated using PCR or gene cloning techniques.
- this invention also provides a polynucleotide of this invention operatively linked to elements necessary for the transcription and/or translation of these polynucleotides in host cells.
- the polynucleotide is a component of a gene delivery vehicle for insertion into the host cells.
- the means by which the cells may be transformed with the expression construct includes, but is not limited to, microinjection, electroporation, transduction, transfection, lipofection, calcium phosphate particle bombardment mediated gene transfer or direct injection of nucleic acid sequences or other procedures known to one skilled in the art (Sambrook et al. (1989) supra).
- Host cells include eukaryotic and prokaryotic cells, such as bacterial cells, yeast cells, simian cells, murine cells and human cells.
- the cells can be cultured or recently isolated from a subject.
- the host cells are cultured under conditions necessary for the recombinant production of the polypeptide or recombinant replication of the polynucleotides. Recombinantly produced polynucleotides and/or polynucleotides are further provided herein.
- polypeptides that are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, crosslinking, acetylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand. Ferguson et al., Ann. Rev. Biochem. (1988) 57:285-320. This is achieved using various chemical methods or by expressing the polynucleotides in different host cells, e.g., bacterial, mammalian, yeast, or insect cells. Also provided by this invention are peptide fragments, e.g., immunogeneic or antigenic portions, alone or in combination with a carrier.
- An antigenic peptide epitope of the invention can be used in a variety of formulations, which may vary depending on the intended use.
- An antigenic peptide epitope of the invention can be covalently or non- covalently linked (complexed) to various other molecules, the nature of which may vary depending on the particular purpose.
- a peptide of the invention can be covalently or non-covalently complexed to a macromolecular carrier, including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, poly(amino acid), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids.
- a peptide can be conjugated to a fatty acid, for introduction into a Iiposome.
- a synthetic peptide of the invention can be complexed covalently or non-covalently with a solid support, a variety of which are known in the art.
- An antigenic peptide epitope of the invention can be associated with an antigen-presenting matrix with or without co-stimulatory molecules, as described in more detail below.
- protein carriers include, but are not limited to, superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin, myoglobulin, and immunoglobulin.
- Peptide-protein carrier polymers may be formed using conventional crosslinking agents such as carbodiimides.
- carbodiimides are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1 -ethyl-3-(3- dimethyaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide.
- CMC 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide
- EDC dimethyaminopropyl carbodiimide
- 1-ethyl-3-(4-azonia-44- dimethylpentyl) carbodiimide examples of other suitable crosslinking agents.
- any of a number of homobifunctional agents including a homobifunctional aldehyde, a homobifunctional epoxide, a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl halide, a homobifunctional hydrazide, a homobifunctional diazonium derivative and a homobifunctional photoreactive compound may be used.
- heterobifunctional compounds for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
- homobifunctional crosslinking agents include the bifunctional N-hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imidoesters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1 ,4-di-[3'-(2'-pyridyldithio) propion-amidojbutane, bismaleimidohexane, and bis-N-maleimido-1 , 8-octane; the bifunctional aryl halides 1 ,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'- dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4- azidosal
- Examples of other common heterobifu notional cross-linking agents that may be used to effect the conjugation of proteins to peptides include, but are not limited to, SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-( ⁇ -maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4- (N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidy
- Crosslinking may be accomplished by coupling a carbonyl group to an amine group or to a hydrazide group by reductive amination.
- Peptides of the invention also may be formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules may be done through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created using charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine) which contain numerous negative and positive charges, respectively.
- peptides may be non- covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. These biotin-binding proteins contain four binding sites that can interact with biotin in solution or be covalently attached to another molecule. Wilchek, Anal Biochem. (1988) 171 :1-32.
- Peptides can be modified to possess biotin groups using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts with available amine groups on the protein. Biotinylated peptides then can be incubated with avidin or streptavidin to create large complexes. The molecular mass of such polymers can be regulated through careful control of the molar ratio of biotinylated peptide to avidin or streptavidin. Also provided by this application are the peptides and polypeptides described herein conjugated to a detectable agent for use in the diagnostic methods.
- detectably labeled peptides and polypeptides can be bound to a column and used for the detection and purification of antibodies. They also are useful as immunogens for the production of antibodies, as described below.
- the peptides of this invention also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions.
- non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils.
- the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one.
- suitable adjuvants include, but are not limited to, Freund's Complete and Incomplete, mineral salts and polynucleotides.
- the proteins and polypeptides of this invention can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431 A, Foster City, CA, USA.
- the synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC).
- this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.
- Cellular differentiation can be monitored by histological methods or by monitoring for the presence or loss of certain cell surface markers.
- the reversal of pathological state in humans can be measured by the reduction in cystic (or renal) volume, using NMR.
- the method can also be practiced by delivering to the affected tissue an effective amount of therapeutic agent such as a blocking or inhibitory antibody or derivative thereof or small molecules.
- a therapeutic agent such as a blocking or inhibitory antibody or derivative thereof or small molecules.
- An exemplary antibody is described infra. These can be delivered alone or in combination with a carrier such as a pharmaceutically acceptable carrier.
- a carrier such as a pharmaceutically acceptable carrier.
- antibodies can be monoclonal or polyclonal. They can be chimeric, humanized, or totally human. Any functional fragment or derivative of an antibody can be used including Fab, Fab', Fab2, Fab'2, and single chain variable regions.
- Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc. So long as the fragment or derivative retains specificity of binding for the protein or fragment thereof it can be used.
- Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific.
- fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. See for example, Russel, N.D. er a/., Infection and Immunity (2000) April 2000:1820-1826; Gallo, M. L. er a/., European J. of Immun. (2000) 30:534- 540; Green, L. L., J. of Immun.
- Antibodies can also be made using phage display techniques. Such techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Single chain Fv can also be used as is convenient. They can be made from vaccinated transgenic mice, if desired. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc.
- Antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample. Antibodies can also be conjugated, for example, to a pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be linked to a cytokine, to a ligand, to another antibody.
- a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like.
- Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample.
- Antibodies can also be conjugated, for example, to a pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be linked to a cytokine, to a ligand, to another antibody.
- Suitable agents for coupling to antibodies to achieve an anti-tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 ( 131 l), yttrium-90 ( 90 Y), bismuth-212 ( 212 Bi), bismuth-213 ( 213 Bi), technetium-99m ( 99 Tc), rhenium-186 ( 186 Re), and rhenium-188 ( 188 Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria
- this invention provides methods for aiding in the diagnosis of cystic abnormalities present in a tissue.
- the pathological state of the cell or tissue is identified by differential expression of the CTGF gene, the gene for its receptor or their expression products.
- gene expression is determined by noting the amount (if any, e.g., altered) expression of the gene in the test system, e.g., differential expression is determined by an increase or in some aspects a decrease, by at least 1.5 fold, 2.5 fold, or alternatively at least 5 fold, or alternatively 10 fold, in the level of a mRNA transcribed from the gene.
- augmentation of the level of the polypeptide or protein encoded by the gene is indicative of the presence of the pathological condition of the cell.
- the method can be used for aiding in the diagnosis of ADPKD-associated renal cysts and cystic abnormalities in other organs, including the liver, pancreas, spleen and ovaries that are commonly found in ADPKD. Additionally, by detecting differential expression of protein or gene prior to abnormal cyst formation, one can predict a predisposition to cystic abnormalities and/or provide early diagnosis and treatment.
- Cell or tissue samples used for this invention encompass body fluid, solid tissue samples, tissue cultures or cells derived there from and the progeny thereof, and sections or smears prepared from any of these sources, or any other samples that may contain a cell having differential expression.
- a preferred sample is one that is prepared from a subject's renal tubules.
- the invention provides compositions and methods for diagnosing or monitoring cystic abnormalities, such as those associated with ADPKD disease by determining the expression level of the CTGF gene or its receptor and correlating the determined level of expression with said disease or its progression.
- Various methods are known for quantifying the expression of a gene of interest and include but are not limited to hybridization assays (Northern blot analysis) and PCR based hybridization assays. In assaying for an alteration in mRNA level, the nucleic acid contained in a sample is first extracted according to a standard method in the art.
- mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by the manufacturers.
- the CTGF mRNA contained in the extracted nucleic acid sample is then detected by hybridization (e.g., Northern blot analysis) and/or amplification procedures using nucleic acid probes and/or primers, respectively, according to standard procedures.
- Nucleic acid molecules having at least 10 nucleotides and exhibiting sequence complementarity or homology to the CTGF can be used as CTGF hybridization probes or CTGF PCR primers in the diagnostic methods. It is known in the art that a "perfectly matched" probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the hybridization specificity. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated.
- a probe useful for detecting CTGF mRNA is at least about 80% identical to the homologous region of comparable size contained in a previously identified sequence, e.g., see SEQ ID NO: 1.
- the probe is at least 85% or even at least 90% identical to the corresponding gene sequence after alignment of the homologous region.
- the total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments of the gene will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about 10 and about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect. Nucleotide probes having complementary sequences over stretches greater than about 10 nucleotides in length will increase stability and selectivity of the hybrid, and thereby improving the specificity of particular hybrid molecules obtained.
- nucleic acid molecules having gene-complementary stretches of more than about 25 and even more preferably more than about 50 nucleotides in length, or even longer where desired.
- Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology with two priming oligonucleotides as described in U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production.
- nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for detecting hybridization and therefore complementary sequences.
- indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
- a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents can also be used.
- enzyme tags colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. Hybridization reactions can be performed under conditions of different "stringency".
- the method comprises isolating multiple mRNAs from cell or tissue samples suspected of containing the transcript.
- the gene transcripts can be converted to cDNA.
- a sampling of the gene transcripts are subjected to sequence-specific analysis and quantified. These gene transcript sequence abundances are compared against reference database sequence abundances including normal data sets for diseased and healthy patients.
- the patient has the disease(s) with which the patient's data set most closely correlates and for this application, includes the differential of the transcript.
- the nucleotide probes of the present invention can also be used as primers for the amplification and detection of genes or gene transcripts.
- a primer useful for detecting differentially expressed mRNA is at least about 80% identical to the homologous region of comparable size of a gene or polynucleotide.
- amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase. General procedures for PCR are taught in MacPherson et al., PCR: A
- PCR conditions used for each application reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg 2+ ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides. After amplification, the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
- Probes also can be attached to a solid support for use in high throughput screening assays using methods known in the art.
- Photoprotected nucleoside phosphoramidites can be coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask, and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.
- the expression level of the gene is determined through exposure of a sample suspected of containing the polynucleotide to the probe-modified chip. Extracted nucleic acid is labeled, for example, with a fluorescent tag, preferably during an amplification step. Hybridization of the labeled sample is performed at an appropriate stringency level.
- the degree of probe-nucleic acid hybridization is quantitatively measured using a detection device, such as a confocal microscope. See, U.S. Patent Nos. 5,578,832 and 5,631 ,734. The obtained measurement is directly correlated with gene expression level.
- the probes and high density oligonucleotide probe arrays also provide an effective means of monitoring expression of a multiplicity of genes, one of which includes the gene.
- the expression monitoring methods can be used in a wide variety of circumstances including detection of disease, identification of differential gene expression between samples isolated from the same patient over a time course, or screening for compositions that upregulate or downregulate the expression of the gene at one time, or alternatively, over a period of time.
- Hybridized probe and sample nucleic acids can be detected by various methods known in the art.
- the hybridized nucleic acids can be detected by detecting one or more labels attached to the sample nucleic acids.
- the labels can be incorporated by any of a number of means known to those of skill in the art.
- the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acid.
- PCR polymerase chain reaction
- transcription amplification as described above, using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates a label in to the transcribed nucleic acids.
- a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after the amplification is completed.
- Means of attaching labels to nucleic acids are known to those of skill in the art and include, for example nick translation or end-labeling (e.g., with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).
- Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 l, 35 S, 14 C, or 32 P) enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
- Patents teaching the use of such labels include U.S.
- Radiolabels may be detected using photographic film or scintillation counters
- fluorescent markers can be detected using a photodetector to detect emitted light
- Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
- Patent Publication WO 97/10365 describes methods for adding the label to the target (sample) nucleic acid(s) prior to or alternatively, after the hybridization. These are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected.
- nucleic acid sample also may be modified prior to hybridization to the high density probe array in order to reduce sample complexity thereby decreasing background signal and improving sensitivity of the measurement using the methods disclosed in International PCT Application No. WO 97/10365.
- Results from the chip assay are typically analyzed using a computer software program. See, for example, EP 0 717 113 A2 and WO 95/20681.
- the hybridization data is read into the program, which calculates the expression level of the targeted gene(s). This figure is compared against existing data sets of gene expression levels for diseased and healthy individuals. A correlation between the obtained data and that of a set of diseased individuals indicates the onset of a disease in the subject patient.
- the invention provides methods and compositions for diagnosing or monitoring cystic abnormalities such as those associated with ADPKD disease by detecting and/or quantifying protein or polypeptide expressed from a gene identified in Tables 2 through 5, infra, present in a sample.
- a variety of techniques are available in the art for protein analysis and include, but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
- radioimmunoassays ELISA (enzyme linked immunoradiometric assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
- a diagnostic test includes a control sample derived from a subject (hereinafter "positive control"), that exhibits the pathological or abnormal expression level of the gene. It is also useful to include a "negative control" that lacks the clinical characteristics of the pathological state and whose expression level of the gene is within a normal range. A positive correlation between the subject and the positive control with respect to the identified alterations indicates the presence of or a predisposition to disease. A lack of correlation between the subject and the negative control confirms the diagnosis.
- the present invention also provides a screen for identifying leads and methods for reversing the pathological condition of the cells or tissues or selectively inhibiting growth or proliferation of the cells or tissues.
- the screen identifies lead compounds or biologies agents which are useful to treat cystic abnormalities or to treat or ameliorate the symptoms associated with ADPKD.
- the screens can be practiced in vitro or in vivo.
- test substances for screening can come from any source. They can be libraries of natural products, combinatorial chemical libraries, biological products made by recombinant libraries, etc. The source of the test substances is not critical to the invention.
- the present invention provides means for screening compounds and compositions which may previously have been overlooked in other screening schemes. To practice the screen or assay in vitro, suitable cell cultures or tissue cultures are first provided.
- the cell can be a cultured cell or a genetically modified cell which differentially expresses the gene.
- the cells can be from a tissue biopsy.
- U.S. Patent No.5,789,189 provides a method of producing a culture of polycystic kidney cells which form cysts in vitro. The cells are cultured under conditions (temperature, growth or culture medium and gas (CO2)) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. It also is desirable to maintain an additional separate cell culture; one which does not receive the agent being tested as a control.
- CO2 culture medium and gas
- suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting genotypic changes, phenotypic changes and/or cell death.
- the screen utilizes the compositions and methods of the MDCK cystic assay described infra.
- the agent is a composition other than a DNA or RNA nucleic acid molecule
- the suitable conditions may be by directly added to the cell culture or added to culture medium for addition.
- an "effective" amount must be added which can be empirically determined.
- the screen involves contacting the agent with a test cell differentially expressing the gene and then assaying the cell for the level of gene expression.
- test cell is a cultured cell from an established cell line that differentially expresses the CTGF gene .
- An agent is a possible therapeutic agent if gene expression is returned (reduced or increased) to a level that is present in a cell in a normal state.
- the test cell or tissue sample is isolated from the subject to be treated and one or more potential agents are screened to determine the optimal therapeutic and/or course of treatment for that individual patient. For example, kidney or liver tissue is suitable for this assay.
- an "agent” is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide.
- a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide.
- a vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent".
- various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen.
- the agents and methods also are intended to be combined with other therapies. They can be administered concurrently or sequentially.
- Use of the screen in an animal such as a rat or mouse the method provides a convenient animal model system which can be used prior to clinical testing of the therapeutic agent or alternatively, for lead optimization.
- a candidate agent is a potential drug, and may therefore be suitable for further development, if gene expression is returned to a normal level or if symptoms associated or correlated to the presence of cells containing differential expression of the CTGF gene are ameliorated, each as compared to untreated, animal having the pathological cells. It also can be useful to have a separate negative control group of cells or animals which are healthy and not treated, which provides a further basis for comparison.
- This invention also provides an antibody capable of specifically forming a complex with a protein or polypeptide of this invention, which are useful in the diagnostic and therapeutic methods of this invention.
- antibody includes polyclonal antibodies and monoclonal antibodies as well as derivatives thereof (described above).
- the antibodies include, but are not limited to mouse, rat, and rabbit or human antibodies.
- Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc.
- the antibodies are also useful to identify and purify therapeutic and/or diagnostic polypeptides.
- the monoclonal antibodies of this invention can be biologically produced by introducing protein or a fragment thereof into an animal, e.g., a mouse or a rabbit.
- the antibody producing cells in the animal are isolated and fused with myeloma cells or hetero-myeloma cells to produce hybrid cells or hybridomas.
- the hybridoma cells producing the monoclonal antibodies of this invention also are provided.
- the anti-CTGF antibody available under Catalog No. TP 143 (Torrey Pines Biolabs) or SC-14939 (Santa Cruz
- the antibody being tested competes with the monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope.
- the term "antibody” also is intended to include antibodies of all isotypes.
- Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in Steplewski, et al., Proc. Natl. Acad. Sci. USA (1985) 82:8653 or Spira, et al., J. Immunol. Methods (1984J 74:307.
- This invention also provides biological active fragments of the polyclonal and monoclonal antibodies described above. These "antibody fragments" retain some ability to selectively bind with its antigen or immunogen.
- Such antibody fragments can include, but are not limited to Fab; Fab'; F(ab') 2 ; Fv, and SCA.
- a specific example of "a biologically active antibody fragment” is a CDR region of the antibody. Methods of making these fragments are known in the art, see for example, Harlow and Lane (1988) supra.
- the antibodies of this invention also can be modified to create chimeric antibodies and humanized antibodies. Oi, et al., BioTechniques
- Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species.
- the isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can also be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies.
- An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant.
- an anti- idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the mirror image of the epitope bound by the first monoclonal antibody.
- the anti-idiotypic monoclonal antibody could be used for immunization for production of these antibodies.
- epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
- the antibodies of this invention can be linked to a detectable agent or label. There are many different labels and methods of labeling known to those of ordinary skill in the art. The coupling of antibodies to low molecular weight haptens can increase the sensitivity of the assay. The haptens can then be specifically detected by means of a second reaction.
- haptens such as biotin, which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See, Harlow and Lane (1988) supra.
- the antibodies of the invention also can be bound to many different carriers.
- this invention also provides compositions containing the antibodies and another substance, active or inert.
- examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
- the nature of the carrier can be either soluble or insoluble for purposes of the invention.
- compositions containing the antibodies, fragments thereof or cell lines which produce the antibodies are encompassed by this invention.
- compositions When these compositions are to be used pharmaceutically, they can be combined with a pharmaceutically acceptable carrier.
- the following experimental examples are intended to illustrate, not limit the invention. Experimental Methods Expression Analysis A combination of two different expression profiling technologies were used to identify CTGF as a therapeutic target: SAGE (Velculescu, V. et al. (1995) supra) and microarray.
- the second dimension was performed in 10% Duracryl polyacrylamide SDS gels run with the Investigator 2D electrophoresis system (Genomic Solutions, Ann Arbor, Ml, USA). Silver staining was performed as described in Rabilloud, "Methods in molecular biology: Proteom analysis protocols” (1999) 112:297- 305. Gels were scanned using intensity calibrated Phoretix Power Scan Software v. 3.0 and analyzed by Phoretix Advanced software v.5.0. (Non linear Dynamics Ltd, Durham, NC). For identification of proteins using peptide mass fingerprinting, gels were stained with SYPRO Ruby protein gel stain (Molecular Probes, Eugene, OR, USA) according to the manufacturer specifications. Results are shown in Figure 3.
- MDCK cells are grown in complete MEM (minimal essential media) containing 10% heat inactivated fetal bovine serum, penicillin/streptomycin (Gibco, Rockville, MD) until 80% confluent. MDCK cysts were grown as previously described in Pollack et al., Dev. Biol. (1998) 204:67-79. Briefly, MDCK cells were seeded into collagen matrix as follows: Day 1 : To ensure they are in a logarithmic growth phase MDCK cells are trypsinized and expanded into 100 mm petri dishes (BD Biosciences).
- Antigens were unmasked by incubating the section in trypsin solution (Sigma/Aldrich) for 30 min at room temperature, then washed with PBS 5 times for 5 min each. Sections were incubated in 20 ⁇ g/ml rabbit anti-CTGF (AbCam, Cambridge, MA) PBS/BSA for 2h at room temperature followed by 5 PBS washes of 5 min each. Sections were incubated with anti-rabbit antibody (Sigma/Aldrich) at a 1 :100 dilution (vol/vol) in PBS/BSA for 1 hr followed by 5 PBS washes of 5 min each.
- ADP ribosylatjon factor related protein 1 (ARFRP1) 1.00E-02
- MCAM Melanoma cell adhesion molecule
- TCTGCAAATT >5 >5 NM 032525 Tubulin B 5 ⁇ TUBB-5)
- MAP4K2 Mitogen-activated protein kinase kinase kinase kinase 2
- GACCTCCTGC 1 L32976 Mitogen-activated protein kinase kinase kinase 11 (MAP3K11)
- TATGACTTAA 1 7 2 2 AF031815 Potassium intermediate/small conductance calcium- activated channel, subfamily N, member 3 (KCNN3)
- HBA2 Hemoglobin- ⁇ 2
- MCAM Melanoma cell adhesion molecule
- VCAM1 Vascular cell adhesion molecule 1
- CD63 Melanoma 1 antigen
- TTCACTGCCG D49400 ATPase, H+ transporting, lysosomal 14kDa, V1 subunit F (ATP6V1F)
- ACAAACCCCC 2 6 0 2 W37827 ATPase, Na+/K+ transporting ⁇ 1 polypeptide (ATP1B1)
- CACAGTCAAA 1 5 0 1 R42029 (3-3 subunit voltage-dependent calcium channel (CACNB3)
- RBBP7 Retinoblastoma binding protein 7
- PGRMC1 Progesterone receptor membrane component 1
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Abstract
Description
Claims
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BRPI0510367-3A BRPI0510367A (en) | 2004-04-29 | 2005-04-29 | methods and compositions for the treatment of polycystic diseases |
CA002564092A CA2564092A1 (en) | 2004-04-29 | 2005-04-29 | Methods and compositions for the treatment of polycystic diseases |
EP05804799A EP1740221A4 (en) | 2004-04-29 | 2005-04-29 | Methods and compositions for the treatment of polycystic diseases |
MXPA06012446A MXPA06012446A (en) | 2004-04-29 | 2005-04-29 | Methods and compositions for the treatment of polycystic diseases. |
JP2007511046A JP2007535562A (en) | 2004-04-29 | 2005-04-29 | Methods and compositions for the treatment of polycystic disease |
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US56667004P | 2004-04-29 | 2004-04-29 | |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2007082733A1 (en) * | 2006-01-17 | 2007-07-26 | Universitaetsklinikum Freiburg | Method for diagnosing polycystic kidney disease |
WO2008051502A1 (en) * | 2006-10-19 | 2008-05-02 | Genzyme Corporation | Purine derivatives for the treatment of cystic diseases |
US8222375B2 (en) | 2005-12-08 | 2012-07-17 | Medarex, Inc. | Human monoclonal antibodies to protein tyrosine kinase 7 (PTK7) and methods for using anti-PTK7 antibodies |
US8252762B2 (en) | 2008-08-25 | 2012-08-28 | Excaliard Pharmaceuticals, Inc. | Antisense oligonucleotides directed against connective tissue growth factor and uses thereof |
US8946172B2 (en) | 2008-08-25 | 2015-02-03 | Excaliard Pharmaceuticals, Inc. | Method for reducing scarring during wound healing using antisense compounds directed to CTGF |
US9173894B2 (en) | 2011-02-02 | 2015-11-03 | Excaliard Pharamaceuticals, Inc. | Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF) |
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JP5995207B2 (en) * | 2014-11-28 | 2016-09-21 | 学校法人 学習院 | Boron-containing porphyrin derivatives |
KR102288447B1 (en) * | 2018-12-28 | 2021-08-10 | 서울대학교산학협력단 | Composition for diagnosing polycystic kidney disease comprising agent for detecting CTGF and use thereof |
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US5789189A (en) * | 1993-09-24 | 1998-08-04 | The Regents Of The University Of California | Inhibition of cyst formation by cytoskeletal specific drugs |
US6562618B1 (en) * | 1997-12-25 | 2003-05-13 | Japan Tobacco, Inc. | Monoclonal antibody against connective tissue growth factor and medicinal uses thereof |
US6875747B1 (en) * | 1999-05-24 | 2005-04-05 | Avi Bio Pharma, Inc. | Antisense to c-myc for treatment of polycystic kidney disease |
EP1434993B1 (en) * | 2001-09-18 | 2013-04-24 | Fibrogen, Inc. | Methods of assaying connective tissue growth factor |
US6965025B2 (en) * | 2001-12-10 | 2005-11-15 | Isis Pharmaceuticals, Inc. | Antisense modulation of connective tissue growth factor expression |
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- 2005-04-29 WO PCT/US2005/014982 patent/WO2005117941A2/en active Application Filing
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- 2005-04-29 CA CA002564092A patent/CA2564092A1/en not_active Abandoned
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
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US9505845B2 (en) | 2005-12-08 | 2016-11-29 | E. R. Squibb & Sons, L.L.C. | Treating lung cancer using human monoclonal antibodies to protein tyrosine kinase 7 (PTK7) |
US8222375B2 (en) | 2005-12-08 | 2012-07-17 | Medarex, Inc. | Human monoclonal antibodies to protein tyrosine kinase 7 (PTK7) and methods for using anti-PTK7 antibodies |
US9102738B2 (en) | 2005-12-08 | 2015-08-11 | E. R. Squibb & Sons, L.L.C. | Human monoclonal antibodies to protein tyrosine kinase 7 (PTK7) |
WO2007082733A1 (en) * | 2006-01-17 | 2007-07-26 | Universitaetsklinikum Freiburg | Method for diagnosing polycystic kidney disease |
JP2014169321A (en) * | 2006-10-19 | 2014-09-18 | Genzyme Corp | Purine derivative for therapy of cystic disease |
EP2617423A1 (en) * | 2006-10-19 | 2013-07-24 | Genzyme Corporation | Purine derivatives for the treatment of cystic diseases |
WO2008051502A1 (en) * | 2006-10-19 | 2008-05-02 | Genzyme Corporation | Purine derivatives for the treatment of cystic diseases |
US8772260B2 (en) | 2008-08-25 | 2014-07-08 | Isis Pharmaceuticals, Inc | Methods for inhibiting expression of connective tissue growth factor |
US8946172B2 (en) | 2008-08-25 | 2015-02-03 | Excaliard Pharmaceuticals, Inc. | Method for reducing scarring during wound healing using antisense compounds directed to CTGF |
US9096851B2 (en) | 2008-08-25 | 2015-08-04 | Excaliard Pharmaceuticals, Inc. | Antisense oligonucleotides directed against connective tissue growth factor and uses thereof |
US8252762B2 (en) | 2008-08-25 | 2012-08-28 | Excaliard Pharmaceuticals, Inc. | Antisense oligonucleotides directed against connective tissue growth factor and uses thereof |
US9688987B2 (en) | 2008-08-25 | 2017-06-27 | Excaliard Pharmaceuticals, Inc. | Antisense oligonucleotides directed against connective tissue growth factor and uses thereof |
US9173894B2 (en) | 2011-02-02 | 2015-11-03 | Excaliard Pharamaceuticals, Inc. | Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF) |
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EP1740221A4 (en) | 2009-01-07 |
WO2005117941A3 (en) | 2006-05-18 |
EP1740221A2 (en) | 2007-01-10 |
BRPI0510367A (en) | 2007-11-06 |
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