WO1998045708A1 - Etude et traitement des maladies liees a des fonctions cellulaires specifiques de proteines tyrosines kinases receptrices - Google Patents

Etude et traitement des maladies liees a des fonctions cellulaires specifiques de proteines tyrosines kinases receptrices Download PDF

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Publication number
WO1998045708A1
WO1998045708A1 PCT/US1998/006842 US9806842W WO9845708A1 WO 1998045708 A1 WO1998045708 A1 WO 1998045708A1 US 9806842 W US9806842 W US 9806842W WO 9845708 A1 WO9845708 A1 WO 9845708A1
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cells
ret
compounds
rptk
cell
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PCT/US1998/006842
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Douglas Clary
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Sugen, Inc.
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Priority to AU68876/98A priority Critical patent/AU6887698A/en
Publication of WO1998045708A1 publication Critical patent/WO1998045708A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the invention described herein relates generally to methods of determining receptor protein tyrosine kinase 0 function in cells or tissues, and to activating a receptor in a ligand independent manner.
  • the invention specifically relates to the discovery that activation of C-RET is necessary for neuronal cell survival.
  • the invention also relates to methods of preventing or treating disorders in patients 5 suffering from the effects of neuronal degeneration.
  • Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of amino acids regulates the activity of mature proteins by altering their structure and function.
  • Phosphate most often resides on the hydroxyl moiety of serine, threonine, or tyrosine amino acids in proteins. Enzymes that mediate phosphorylation of cellular effectors fall into two classes. While protein phosphatases hydrolyze phosphate moieties from phosphoryl protein substrates, protein kinases transfer a phosphate moiety from adenosine triphosphate to protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
  • Receptor protein kinases are divided further into two groups: receptor and non-receptor type proteins.
  • Receptor protein kinases comprise an extracellular region, a transmembrane region, and an intracellular region. Part of the intracellular regions of receptor protein kinases harbor a catalytic domain.
  • Receptor protein kinases are also divided into three classes based upon the amino acids they act upon. Some phosphorylate serine or threonine only, some phosphorylate tyrosine only, and some phosphorylate serine, threonine, and tyrosine.
  • Receptor protein tyrosine kinases are typically activated in the cell when a ligand binds to the extracellular region of the receptor.
  • a model for ligand mediated activation of RPTKs features the ligand bringing the receptors within close proximity to one another. Some ligands are dimers and thereby bring the receptors that bind them into close proximity with one another. By bringing two RPTKs together, ligands place RPTK intracellular catalytic regions in close proximity to one another such that they cross-phosphorylate. Cross phosphorylation requires not only the dimerization process but also the occurrence of a conformational change preceding phosphorylation. The necessity of the conformational change pre- ceding phosphorylation is illustrated by the fact that some RPTKs, such as the insulin receptor, are pre-dimerized and inactive before binding their activating ligands.
  • RPTK The presence of phosphate moieties on the RPTK intracellular regions constitutes a cellular signal that causes other signal transduction molecules to bind to the RPTK. In this manner, RPTKs propagate the extracellular signal to the cell nucleus, thereby generating messages encoding proteins that cause cellular affects.
  • RPTKs control a variety of cellular functions
  • any alteration in the normal function of an RPTK can result in an abnormal condition in an organism.
  • differentiation and survival of neuronal cells is dependent upon the proper function of RPTKs.
  • a signaling component such as SHC
  • Transgenic mice containing knockout mutations in genes encoding each of the known TRK receptors or the TRK ligand displayed severe neurological dysfunction and, in all cases, important types of neural tissue were absent (Smeyne et al . , 1994, Na ture 368 : 246-249; Klein et al . , 1994, Na ture 268 : 249-251, 1994).
  • glial-derived neurotrophic factor has been identified as a neuronal survival factor ( Jing et al . , 1996, Cell 85:1113-1124; Trupp et al . , 1996, Na ture 381:785-789; Durbec et al . , 1996, Nature 381:789-793) .
  • GDNF has been shown to bind to a complex of C-RET and another cell surface protein GDNFR- ⁇ , which has no intracellular domain.
  • a frequent hurdle to designing therapeutics for RPTK- related diseases is identifying the function of a particular receptor and relating its activity to a disease or condition.
  • One difficulty in identifying receptor function is that the ligands that activate the receptors are often unknown.
  • ligands can sometimes bind more than a single type of receptor and thus cause pleiotropic effects in an organism. Because alterations in the function of one RPTK can lead to a diseased state in an organism, determining the function of individual receptors is important for designing compounds that will prevent or treat these diseases.
  • RPTKs have also been activated by fusing their intracellular regions to other polypeptides. These fused polypeptide constructs are termed "chimeras" since they are single polypeptides constructed from multiple regions of different proteins (Fazioli et al . , 1991, Mol . Cell . Biol . 11 : 2040-2048; Seedorf et al . , 1992, Mol . Cell . Biol . 12: 4347- 4356) .
  • One such RPTK chimera comprises an intracellular region of a RPTK, fused to an epidermal growth factor (EGF) receptor extracellular region, and thus activatable with the EGF ligand.
  • EGF epidermal growth factor
  • RPTK intracellular regions have also been activated in a ligand independent fashion by fusing intracellular polypeptides to them that bind bifunctional organic compounds (Spencer et al . , 1993, Science 262: 1019-1024).
  • the activation of chimeric constructs of RPTK intracellular regions confers versatility (the intracellular regions of RPTKs can be readily interchanged using recombinant DNA techniques)
  • the ligands and organic compounds used to activate the chimeras can cause changes in cells or tissues other than those caused by the activated chimera.
  • the EGF ligand used to stimulate chimeric constructs containing the EGFR extracellular region can also activate endogenous EGFR expressed on the cell surface, sometimes existing at levels undetectable using currently known methods, and thereby interfere with the signal of the RPTK under study.
  • identifying compounds that modulate the function of RPTKs regulating neuronal survival in particular.
  • Many patients suffer from diseases caused by neuronal degeneration, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, for example.
  • neurotrophic factors have been proposed as therapeutic agents for neuronal diseases, they often cannot reach their target receptors since they rapidly degrade in the blood stream and cannot pass through cell membranes or the blood brain barrier.
  • identifying effective therapeutics for treating neuronal diseases lies in identifying non-peptide compounds which do not rapidly degrade in the blood stream and which can pass through cell membranes and the blood-brain barrier.
  • the invention relates to methods of evaluating the specific function of a receptor protein tyrosine kinase (RPTK) in cells or tissues by activating a receptor in a ligand- independent fashion.
  • the invention also relates to methods of preventing or treating disorders in patients suffering from the effects of neuronal degradation by administering compounds that modulate the function of C-RET to a patient afflicted with such a disorder.
  • the invention relates to _ pharmaceutical compositions of compounds that modulate the function of C-RET.
  • the invention features a method of evaluating RPTK function in cells.
  • the method comprises the following steps: (a) transfecting a nucleic acid vector into cells, where the nucleic acid vector encodes a chimera comprising an extracellular region and an intracellular region, where the intracellular region is from the receptor protein tyrosine kinase; (b) contacting the cells with an antibody, where the antibody has specific binding affinity to the extracellular region; and (c) monitoring an effect on the cells to evaluate the function of the receptor protein tyrosine kinase.
  • RPTK refers to receptor protein tyrosine kinase.
  • the invention includes any RPTK with an extracellular region that binds a ligand, a transmembrane region, and an intracellular region. RPTKs phosphorylate proteins on tyrosine residues.
  • the nucleic acid sequence encoding an RPTK of the invention can be isolated from eukaryotic organisms.
  • extracellular region refers to a polypeptide portion of a RPTK of the invention that exists outside the cell membrane. A requirement of the extracellular region of chimeras of the invention is that it has specific binding affinity to another molecule, preferably an antibody of the invention. The extracellular region of the chimeras must not bind to any other protein on the cell surface and not normally be expressed on the surface of the specific cell type used in the invention.
  • antibody refers to an antibody (e.g., a monoclonal or polyclonal antibody) , or antibody fragment, having specific binding affinity to RPTK' s or fragments thereof, preferably the extracellular region of the RPTK's.
  • specific binding affinity is meant that the antibody binds to target (RPTK) polypeptides with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies having specific binding affinity to a RPTK may be used in methods for detecting the presence and/or amount of a protein RPTK in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the RPTK.
  • Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope.
  • the diagnostic kit may also include notification of an FDA approved use and instructions therefor.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof, preferably an extracellular region of a RPTK.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al . , Nature 255:495-497 (1975), and U.S. Patent No. 4,376,110.
  • antibody fragment refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
  • ligand refers to a polypeptide molecule which binds to the extracellular region of a receptor.
  • ligands include, but are not limited to, nerve growth factor (NGF) , which binds to the extracellular region of the TRK receptor, and GDNF which binds to a complex of C-RET and GDNFR- ⁇ .
  • NGF nerve growth factor
  • transmembrane region refers to the regioin of a polypeptide that connects the extracellular region to the intracellular region, preferably in a chimera of the invention.
  • the transmembrane region can be isolated from any transmembrane protein and is preferably from the same polypeptide source as the intracellular region.
  • Transmembrane regions are often rich in hydrophobic amino acids, such as phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine, which interact with fatty acid moieties of the membrane lipids and thereby anchor the chimera to the cell membrane.
  • intracellular region of an RPTK defines any region existing on the inner side of the cell.
  • An intracellular region of a protein of the invention preferably includes the region of an RPTK with catalytic activity, and no other region of the RPTK.
  • DNA recombinant techniques known in the art provide the means of connecting an RPTK intracellular region to the extracellular region of another RPTK via a transmembrane region.
  • the intracellular region of an RPTK can be attached to the extracellular region of a protein other than an RPTK, such as a receptor protein phosphatase.
  • organism relates to any living being comprised of at least one cell.
  • An organism can be as simple as one eukaryotic cell or as complex as a mammal.
  • eukaryote refers to an organism comprised of cells that contain a nucleus. Eukaryotes are differentiated from "prokaryotes" which do not house their genomic DNA inside a nucleus. Prokaryotes include unicellular organisms such as bacteria, while eukaryotes are represented by, for example, yeast, invertebrates, and vertebrates.
  • mice refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans.
  • evaluating describes the process of determining the cellular effect of activating an RPTK intracellular region in cells.
  • the term "function" refers to the effect of activating the intracellular region of one type of RPTK.
  • the function of the intracellular region of one particular type of RPTK can be manifested in a change in the outward appearance of cells, or in a change in the interactions with proteins that bind to the intracellular region of the RPTK, and the like.
  • the term "transfecting” defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
  • nucleic acid vector relates to a single or double stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome.
  • a circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of nucleic acid vectors, -restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art.
  • a nucleic acid molecule encoding a chimeric receptor can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • the term "contacting" as used herein refers to mixing a solution comprising one or more test compounds with a liquid medium bathing the cells of the methods.
  • the solution comprising the one or more test compounds may also comprise another component, such as dimethylsulfoxide (DMSO) , which facilitates the uptake of the one or more test compounds into the cells of the methods.
  • DMSO dimethylsulfoxide
  • the solution comprising the test compounds may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
  • “Test compound” includes, but is not limited to, both antibodies and compounds of the invention.
  • compound refers to a peptide of less than twenty, preferably less than fifteen, preferably less than ten, or most preferably less than eight amino acids in length.
  • compound preferably refers to a peptidomimetic, more preferably refers to a non-peptide organic molecule, and most preferably refers to a non-peptide synthetic organic molecule. Examples of compounds are included in the Description of the Invention, herein.
  • peptidomimetic refers to an organic molecule with a structure related to a natural peptide, and which can exhibit or block the biological activities of the natural peptide.
  • monitoring refers to observing the effect of adding test compounds to the cells. The effect can be manifested in cell phenotype or in the interaction between an RPTK intracellular region and a natural binding partner.
  • effect preferably describes a change or an absence of a change in cell phenotype.
  • Effect can also describe a change or an absence of a change in the catalytic activity of the RPTK.
  • Effect can also describe a change or an absence of a change in an interaction between the RPTK and a natural binding partner.
  • cell phenotype refers to the outward appearance of a cell or tissue or the function of the cell or tissue.
  • Examples of cell phenotype include, but are not limited to, cell size (reduction or enlargement) , cell shape, cell proliferation (increased or decreased numbers of cells) , cell differentiation (changes in physiological state) , cell survival, apoptosis (cell death) , or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art.
  • cell proliferation refers to the rate at which a group of cells divides.
  • the number of cells growing in a vessel can be quantified by a person skilled in the art when that person visually counts the number of cells in a defined area using a common light microscope.
  • cell proliferation rates can be quantified by laboratory apparatae that optically measure the density of cells in an appropriate medium.
  • Cell differentiation and survival are phenomena simply measured by methods in the art. These methods can involve observing the number of cells or the appearance of cells under a microscope with respect to time (days) .
  • apoptosis refers to programmed cell death. It has been shown by example herein that activation of C-RET can prolong cell survival and decrease the probability of cell death.
  • catalytic activity in the context of the invention, defines the rate at which an RPTK intracellular region phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time.
  • Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate after a fixed period of time.
  • Phosphorylation of a substrate occurs at the active-site of a protein kinase.
  • the active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
  • interaction in the context of the invention, describes a complex formed between a RPTK and a natural binding partner or compound, preferably between a RPTK intracellular region and a natural binding partner or compound.
  • complex refers to an assembly of at least two molecules bound to one another.
  • Signal transduction complexes often contain at least two protein molecules bound to one another.
  • a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
  • Natural binding partner refers to polypeptides that bind to a RPTK. Natural binding partners can play a role in propagating a signal in a protein kinase signal transduction process. A change in the interaction between a RPTK and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the RPTK/natural binding partner complex.
  • a RPTK natural binding partner can bind to a RPTK intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of 10 "6 M or less.
  • a natural binding partner can also transiently interact with a RPTK intracellular region and chemically modify it.
  • Protein kinase natural binding partners are chosen from a group consisting of, but not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods of determining changes in interactions between protein kinases and their natural binding partners are readily available in the art.
  • the method of evaluating a function of a RPTK involves transfecting nucleic acid vectors that encode chimeras that contain an extracellular region of a RPTK selected from the group consisting of TRK, EGFR, PDGFR, and RET and intracellular regions of an orphan RPTK.
  • the orphan RPTK is selected from the group consisting of C-RET, SEK, MCK-10, AXL, TYR03, MER, EPH, ECK, EEK, ERK, ELK, EHK1 , EHK2, SEK, HEK, HEK2, MYK1, CEK9, MYK2, MDK1, IRR, CCK4, RYK, DDR, TYRO10, ROS, LTK, ALK, ROR1, ROR2, and TOR.
  • the orphan RPTK is C-RET.
  • orphan RPTK refers to any receptor whose primary amino acid sequence is closely related to the primary amino acid sequence of the RPTK family. It is called an orphan receptor because no ligand has been identified which directly activates it.
  • orphan receptor refers to an RPTK without a known function.
  • orphan RPTKs may be activated by a ligand known in the art, but the ligand has not yet been shown to directly activate the receptor of interest in the art.
  • the nucleic acid sequences of known RPTKs, and thereby their amino acid sequences can be readily determined by searching a sequence database with the three or four letter name of the RPTK. Examples of sequence databases are the EMBL and Genbank databases. In addition, the sequence database can be searched by the database accession number for an RPTK. Furthermore, the databases can be searched using sequences of the RPTKs themselves.
  • Genbank database accession numbers are designated in parenthesis for the sequence of the following RPTK genes: axl
  • an RPTK gene can be cloned from a cDNA library with nucleic acid probes designed from the nucleic acid sequence provided in the database.
  • RPTKs can be identified by designations other than those disclosed herein.
  • the RPTK AXL is also referred to in the art as ARK and UFO.
  • TYR03 is also referred to as
  • MER can be referred to as TYR012, EYK, and C-RYK.
  • ECK is also referred to as SEK2.
  • ERK is also referred to as HEK5, TYR05, CEK5, and NUK.
  • ELK can be referred to as CEK6 and HEK6.
  • EHK1 has been referred to as HEK7, CEK7, and BSK.
  • EHK2 is also referred to as HEK12.
  • SEK can be referred to as HEK8, CEK8, TYR01, and MPK3.
  • HEK has been referred to as HEK4, CEK4, TYR04, and MEK4.
  • HEK2 is also referred to as CEK10 and TYR06.
  • MYK1 is also referred to as TYROll, HPTK5, and HTK.
  • CEK9 has been also referred to as
  • MDK2 is also referred to as MDK1-T1 or T2, EHK-3, and
  • RPTKs like protein kinases in general, share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs can be (and have been) exploited using PCR-based cloning strategies well-known in the art to identify other RPTKs.
  • RPTK nucleic acid sequences can also be identified by: analyzing sequenced, but unidentified, DNA or RNA or polypeptide sequences from public databases; by probing DNA libraries (e.g., cDNA and genomic libraries) using degenerate probes to sequences conserved in various families and subfamilies of such kinases; or by PCR-based cloning using degenerate primers based on conserved sequences for various families and sub-families.
  • DNA libraries e.g., cDNA and genomic libraries
  • the genomic or cDNA insert in that clone can be at least partially sequenced by routine methods to confirm that the cloned sequence actually corresponds to a RTPK, and to provide a unique sequence identifying the full gene. Possession of such a unique sequence provides the full gene sequence by following routine techniques.
  • a probe sequence based on a specific homologous RTPK sequence from a different mammalian species can be used to detect the corresponding human version of that gene.
  • the cells of the invention and the extracellular region of the chimera are from different species.
  • the cells are mammalian, while the extracellular region of the RPTK is isolated from a chicken.
  • the invention also features methods of evaluating a cellular function of an RPTK, in which the antibodies of the invention have specific binding affinity to a TRK extracellular region isolated from a chicken and where the effect is a change in cell phenotype.
  • the invention features a method of identifying one or more compounds that modulate the function of a receptor protein tyrosine kinase in a cell.
  • the method comprises the following steps: (a) transfecting a nucleic acid vector into a cell, where the nucleic acid vector encodes a chimera comprising an extracellular region and an intracellular region, where the intracellular region is from the receptor protein tyrosine kinase; (b) contacting the cells with one or more compounds; (c) contacting the cells with an antibody, where the antibody has specific binding affinity to the extracellular region and (d) monitoring the effect on the cell to identify compounds that modulates the function of the receptor protein tyrosine kinase.
  • modulates refers to the ability of a test compound (or "modulator") to alter the function of a RPTK.
  • a modulator preferably activates or inhibits the activity of a RPTK depending on the concentration of the compound exposed to a RPTK.
  • a modulator can enhance or inhibit the catalytic activity of the RPTK by binding to the RPTK.
  • the compound may inhibit the catalytic activity of the RPTK by blocking interactions between the RPTK and a substrate that it phosphorylates.
  • a compound may activate the RPTK by bringing RPTKs into close proximity with one another such that they cross phosphorylate and thereby activate more effectively, or by increasing the probability that a catalysis-dependent conformational change occurs in the RPTK.
  • a modulator can inhibit the interaction between a RPTK and a natural binding partner by blocking interactions between amino acids at the interface of the complex.
  • a modulator may inhibit the activity of a natural binding partner which acts upon the RPTK.
  • a modulator can enhance the interaction between a RPTK and a natural binding partner by forming additional favorable interactions between the two molecules at the complex interface.
  • a modulator preferably inhibits the catalytic activity of a protein kinase, more preferably activates or inhibits the catalytic activity of a protein kinase depending on the concentration of the compound exposed to the protein kinase, or most preferably activates the catalytic activity of a protein kinase.
  • the term "activates” refers to increasing the cellular function of a RPTK.
  • the RPTK function is preferably the interaction with a natural binding partner or catalytic activity.
  • An activator of C-RET is a cellular factor that is responsible for the cellular response of C-RET after it binds to GDNF.
  • C-RET activators include, but are not limited to GRB2 and SOS. Compounds can enhance the interaction between C-RET and GRB2 or SOS and effectively activate the cellular function of C-RET.
  • the term "inhibit” refers to decreasing the cellular function of a RPTK.
  • the RPTK function is preferably the interaction with a natural binding partner or catalytic activity.
  • a hypothetical example of a protein that decreases the function of the RPTK, C-RET is a protein phosphatase that dephosphorylates the activated receptor and thereby decreases C-RET' s ability to recruit other proteins necessary for the C-RET activation response, such as GRB2 and SOS.
  • signal transduction pathway refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins) , proline-rich binding proteins (SH3 domain containing proteins) , nucleotide exchange factors, and transcription factors.
  • substrate refers to a molecule phosphorylated by a RPTK. RPTKs phosphorylate substrates on tyrosine amino acids.
  • the extracellular region of the chimera is an extracellular region of a RPTK selected from the group consisting of TRK, EGFR, PDGFR, and RET, and the intracellular region is an intracellular region of an orphan RPTK.
  • the orphan RPTK is selected from the group consisting of C-RET, SEK, MCK- 10, AXL, TYR03, MER, EPH, ECK, EEK, ERK, ELK, EHK1, EHK2, SEK, HEK, HEK2, MYK1, CEK9, MYK2, MDK1, IRR, CCK4 , RYK, DDR, TYRO10, ROS, LTK, ALK, ROR1, ROR2, and TOR.
  • the orphan RPTK is C-RET.
  • the cells of the invention and the extracellular region of the chimera are from different species.
  • the cells are mammalian, while the extracellular region of the RPTK is isolated from a chicken.
  • the invention also features methods of evaluating a cellular function of an RPTK, in which the antibodies of the invention have specific binding affinity to a TRK extracellular region isolated from a chicken and where the effect is a change in cell phenotype.
  • the change in cell phenotype includes, but is not limited to, a change in the catalytic activity of the RPTK intracellular region and a change in an interaction between the intracellular region and a natural binding partner.
  • the invention relates to methods of identifying compounds that modulate the function of C-RET RPTK comprising the following steps: (a) transfecting C-RET into cells; (b) contacting the cells with one or more compounds; and
  • the invention relates to a method of preventing or treating an abnormal condition in a mammal, where the abnormal condition is caused by an aberration in cell survival.
  • the method comprises the step of administering a pharmaceutical composition comprising one or more compounds to the mammal, where the one or more compounds modulate C-RET.
  • preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • therapeutic effect can refer to the inhibition of cell death causing or contributing to a neuronal disorder.
  • therapeutic effect can also refer to the inhibition or activation factors causing or contributing to the abnormal condition.
  • a therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.
  • a therapeutic effect refers to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of neuronal cells; (b) inhibition (i.e., slowing or stopping) of neuronal cell death; (c) inhibition of neuronal degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected neuronal population (e.g., increase in neurotrans- mitter synthesis) .
  • Compounds demonstrating efficacy against neuronal disorders can be identified as described herein.
  • abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
  • An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
  • Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
  • Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
  • a number of protein kinases are associated with the apoptosis pathways.
  • One RPTK associated with cell survival is C-RET. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.
  • the effect of administering a compound on organism function can then be monitored.
  • the organism is preferably a frog, more preferably a mouse, rat, rabbit, guinea pig, or goat, and most preferably a monkey or ape.
  • composition refers to a mixture of the compound with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administering the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.
  • Pharmaceutical compositions can also be obtained by reacting compounds with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, salicylic acid and the like.
  • pharmaceutically acceptable refers to solutions or components of the pharmaceutical composition that do not prevent the therapeutic compound from exerting a therapeutic effect and do not cause unacceptable adverse side effects.
  • pharmaceutically acceptable reagents are provided in The Uni ted Sta tes Pharmacopeia The Na tional Formulary, United States Pharmacopeial Convention, Inc., Rockville, MD 1990 and FDA Inactive Ingredient Guide 1990, 1996 issued by the Division of Drug Information Resources. Unacceptable side effects vary for different diseases. Generally, the more severe the disease the more toxic effects which will be tolerated. Unacceptable side effects for different diseases are known in the art.
  • physiologically acceptable defines a carrier or diluent that does not cause significant irritation to an organism and preferably does not abrogate the biological activity and properties of the compound.
  • carrier defines a chemical compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • carrier facilitates the uptake of many organic compounds into the cells or tissues of an organism.
  • diluent defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound.
  • Many salts dissolved in buffered solutions are utilized as diluents in the art.
  • One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Because buffer salts can control the pH of a solution at low concentrations, a diluent rarely modifies the biological activity of a compound.
  • the RPTK is C-RET.
  • the method at least partially reduces the symptoms associated with the abnormal condition, which is preferably caused by an aberration in cell survival.
  • Alzheimer's disease refers to a condition in which cell survival is enhanced or decreased with respect to cell survival parameters in normal cells.
  • Cell survival can be shortened in such disorders as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.
  • Cell survival can be increased in cancer related conditions.
  • Alzheimer's disease manifests itself as presenile dementia. The disease is characterized by confusion, memory failure, disorientation, restlessness, speech disturbances, and hallucination in mammals (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby) .
  • Parkinson's disease is a slowly progressive, degenerative, neurologic disorder characterized by resting tremor, loss of postural reflexes, and muscle rigidity and weakness (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby).
  • Amyotrophic lateral sclerosis is a degenerative disease of the motor neurons characterized by weakness and atrophy of the muscles of the hands, forearms, and legs, spreading to involve most of the body and face (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby).
  • the abnormal condition is a neurodegenerative disorder, preferably selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis.
  • Preferred methods at least partially prevent or reduce a symptom associated with the abonormal condition.
  • the term "neurodegenerative disorder” as used herein refers to an abnormality in a mammal in which neuronal integrity is threatened. Neuronal integrity can be threatened when neuronal cells display decreased survival or when the neurons can no longer propagate a signal.
  • the invention relates to the method of treating or preventing a abnormal condition in the mammal, where the mammal is identified as having said abnormal condition. Mammals can be identified as having, for example, a neurological disorder such as Alzheimer's disease,
  • Parkinson's disease or amyotrophic lateral sclerosis by monitoring the symptoms manifested in the mammals afflicted with these disorders. These symptoms are well documented in publications known to those skilled in the art.
  • the pharmaceutical composition is comprised of one or more compounds that are non-peptide molecules and one or more physiologically acceptable solvents, diluents, or carriers.
  • these compounds act intracellularly and can traverse the blood-brain barrier.
  • the compounds activate the catalytic activity of the RPTK, inactivates an inhibitor of the RPTK, or activates an activator of the RPTK.
  • the RPTK is C-RET.
  • non-peptide molecule refers to a compound that is not a polymer of amino acids.
  • a non-peptide molecule preferably does not contain chemical moieties that hydrolyze in physiological conditions, i . e. a peptidomometic.
  • solvent refers to a chemical compound that facilitates the solubilization of compounds of the invention.
  • solvents include, but are not limited to, pharmaceutically acceptable alcohols, such as ethanol and benzyl alcohol; polyoxyhydrocarbyl compounds, such as poly (ethylene glycol); pharmaceutically acceptable surfactants such as CREMOPHOOR EL; polyglycolized lipids, such as GELUCIRE and LABRASOL; and pharmaceutically acceptable oils, such as miglyol 812.
  • pharmaceutically acceptable alcohols such as ethanol and benzyl alcohol
  • polyoxyhydrocarbyl compounds such as poly (ethylene glycol)
  • pharmaceutically acceptable surfactants such as CREMOPHOOR EL
  • polyglycolized lipids such as GELUCIRE and LABRASOL
  • pharmaceutically acceptable oils such as miglyol 812.
  • pharmaceutically acceptable alcohol refers to alcohols which are liquids at about room temperature (approximately 20°C) . These include propylene glycol, ethanol, 2- (2-ethoxyethoxy) ethanol (TRANSCUTOL®, Gattefosse, Westwood, NJ 07675), benzyl alcohol, and glycerol.
  • polyoxyhydrocarbyl compound refers to a water soluble carbohydrate such as glucose, sucrose, maltotriose, and the like; water soluble carbohydrate derivatives such as gluconic acid and mannitol, and oligosaccharides; and water soluble polymers such as polyvinylpyrrolidone, poly (vinyl alcohol), and in particular, polyethers such as other polyoxyalkylenes including poly (ethylene glycol) or other water soluble mixed oxyalkylene polymers and the polymeric form of ethylene glycol.
  • polyoxyhydrocarbyl compounds preferably contain more than one carbon, oxygen, and hydrogen atom, some molecules such as poly (ethylene imine) are also included.
  • a particularly preferred class of solubilizing polyoxyhydrocarbyl moieties comprises poly (ethylene glycol) (PEG) and PEG derivatives, such as PEG monomethyl ether.
  • PEG derivatives include PEG-silicon derived ethers.
  • PEG-silicon derived ethers are commercially available in a variety of molecular weights. Others may be conveniently prepared from commercially available materials, such as by coupling of amino- PEG moiety to a haloalkyl silyl or silane moiety.
  • Suitable PEGs may vary in molecular weight from about 200 g/mol to about 20,000 g/mol or more, more preferably 200 g/mol to 5,000 g/mol, even more preferably 250 g/mol to 1,000 g/mol, and most preferably 250 g/mol to 500 g/mol.
  • the choice of a particular molecular weight may depend on the particular indolinone-based compound chosen and its molecular weight and degree of hydrophobicity, as well as the particular application for which the formulation is to be used.
  • pharmaceutically acceptable surfactant refers to a compound that can solubilize compounds of the invention into aqueous solutions.
  • the surfactant is a non-ionic surfactant.
  • examples of pharmaceutically acceptable surfactants include POLYSORBATE 80® and other polyoxyethylene sorbitan fatty acid esters, glyceryl monooleate, polyvinyl alcohol, ethylene oxide copolymers such as PLURONICTM (a polyether) and TETRONICTM (BASF) , polyol moieties, and sorbitan esters.
  • ethoxylated castor oils such as CREMOPHOR EL®, are used for the formulation of indolinone-based compounds .
  • ethoxylated castor oil refers to castor oil that is modified with at least one oxygen containing moiety.
  • the term refers to castor oil comprising at least one ethoxyl moiety.
  • pharmaceutically acceptable surfactant includes pharmaceutically acceptable non-ionic surfactants (for example polyoxyethylenepolypropylene glycol, such as POLOXAMER® 68
  • polyglycolized lipids refers to mixtures of monoglycerides, diglycerides, or triglycerides and polyethyleneglycol monoesters and diesters formed by the partial alcoholysis of vegetable oil using PEG of 200 g/mol to 2,000 g/mol or by the esterification of fatty acids using PEG 200 g/mol to 2,000 g/mol and glycerols.
  • these include GELUCIRE® 35/10, GELUCIRE® 44/14, GELUCIRE® 46/07, GELUCIRE® 50/13, GELUCIRE® 53/10, and LABRASOL®.
  • oils such as mineral oil or vegetable oil (including safflower oil, peanut oil, and olive oil), fractionated coconut oil, propylene glycol monolaurate, mixed triglycerides with caprylic acid and capric acid, and the like.
  • Preferred embodiments of the invention feature mineral oil, vegetable oil, fractionated coconut oil, mixed triglycerides with caprylic acid, and capric acid.
  • a highly preferred embodiment of the invention features Miglyol 812 (available from Huls America, USA) .
  • act intracellularly refers to a compound that can modulate the function of a RPTK by binding to the intracellular region of the RPTK.
  • the compound can bind to a natural binding partner of the RPTK intracellular region.
  • the "blood-brain" barrier is an anatomic feature of the brain that is thought to comprise walls of capillaries in the central nervous system combined with surrounding glial membranes (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby) .
  • a final aspect of the invention features pharmaceutical composition
  • pharmaceutical composition comprising one or more compounds and one or more physiologically acceptable solvents or diluents or carriers.
  • the one or more compounds of the pharmaceutical composition can modulate a RPTK cellular function, preferably activate or inhibit a RPTK cellular function, and most preferably modulates C-RET.
  • the invention relates to methods of evaluating the specific function of a receptor protein tyrosine kinase (RPTK) in cells or tissues by activating a receptor in a ligand- independent fashion.
  • RPTK receptor protein tyrosine kinase
  • the invention also relates to methods of preventing or treating disorders in patients suffering from the effects of neuronal degradation by administering pharmaceutical compositions comprised of compounds that modulate the function of C-RET to a patient afflicted with such a disorder.
  • the ligand-independent methods of evaluating the specific function of a RPTK overcome at least two problems associated with studying RPTKs and identifying compounds that modulate RPTK function. These problems are (1) lack of knowledge about and availability of the ligand that activates a RPTK, and (2) ligands may activate more than one type of RPTK or activate undetectable levels of endogenous receptor, and thereby cause effects in a test cell that are not truly associated with the receptor under study.
  • the invention features antibody-induced activation of chimeric RPTKs constructed from polypeptides of at least two different proteins.
  • the chimeras contain the extracellular and transmembrane region from one type of RPTK and an intracellular region from another type of RPTK. This chimeric approach confers both specificity and versatility towards evaluating RPTK cellular function.
  • the methods of the invention incorporate a novel cross species feature with respect to the chimeric constructs and cells utilized in these methods.
  • the cross species feature ensures that the only receptor appreciably activated in response to an antibody of the invention is the chimeric construct expressed on the surface of cells.
  • This cross species feature represents an improvement over the existing techniques which can activate multiple receptors on cell surfaces . Therefore, the methods set forth herein provide for test compound-induced activation of specific chimeric receptors expressed on the surface of cells under study.
  • the extracellular region of the chimeric construct may originate from chickens while the cells in which the chimeric construct is expressed originate from rats. Examples set forth herein demonstrate that antibodies raised specifically to the chicken extracellular region of the chimeric construct do not cross-react with other proteins expressed on rat cell surfaces.
  • RPTKs are essential regulatory molecules controlling a variety of cellular functions. For this reason, any alteration in the function of a RPTK can result in an abnormal condition in an organism.
  • Just two of the many functions controlled by RPTKs are cell proliferation and cell survival. Aberrations in cell survival can lead to neurological disorders.
  • the methods of preventing or treating disorders in patients suffering from the effects of neuronal degradation are based in part on the discovery that activation of C-RET is necessary for neuronal cell survival.
  • C-RET has been implicated in neuronal survival by methods of the invention that activate the receptor in a ligand independent fashion. These methods utilize antibodies specific for the TRK extracellular region of a TRK extracellular region/C-RET intracellular region chimeric receptor.
  • the antibodies specifically activate the chimeric receptor by bringing the C- RET intracellular regions in close proximity such that they cross phosphorylate and thereby activate. These methods were utilized to discover the function of C-RET before the ligand that activates this receptor, GDNF, was known to those skilled in the art.
  • Non-peptide compounds are preferred since they are often stable in a patient's blood stream.
  • non-peptide compounds can be selected such that they can pass through cell membranes and the blood-brain barrier. Although many of the non-peptide molecules discovered to modulate the function of RPTKs only inhibit their targets, activating compounds of RPTKs are being discovered.
  • Alterations in the function of an RPTK that normally regulates cell proliferation can lead to enhanced or decreased cell proliferative conditions evident in certain diseases.
  • Aberrant cell proliferative conditions include cancers, fibrotic disorders, mesangial disorders, abnormal angiogenesis and vasculogenesis, poor wound healing, psoriasis, restenosis, and inflammation.
  • Fibrotic disorders relate to the abnormal formation of the cellular extracellular matrix.
  • An example of a fibrotic disorder is hepatic cirrhosis.
  • Hepatic cirrhosis is characterized by an increased concentration of extracellular matrix constituents resulting in the formation of a hepatic scar.
  • Hepatic cirrhosis can cause diseases such as cirrhosis of the liver.
  • Mesangial cell proliferative disorders occur due to the abnormal proliferation of mesangial cells.
  • Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic micro angiopathy syndromes, transplant rejection, and glomerulopathies .
  • Angiogenic and vasculogenic disorders result from excess proliferation of blood vessels. Blood vessel proliferation is necessary in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. However, blood vessel proliferation is also essential in cancer tumor development.
  • blood vessel proliferative disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases (such as diabetic retinopathy) , where new capillaries in the retina invade the vitreous, bleed and cause blindness.
  • ocular diseases such as diabetic retinopathy
  • diseases related to the shrinkage, contraction or closing of blood vessels, such as restenosis are also implicated in adverse regulation of RPTKs.
  • Abnormalities in signal transduction pathways can lead to various diseases through both underactivity and over activity of the RPTKs involved in these pathways.
  • disorders which are characterized by underactivity of a RPTK in a signal transduction pathway include various neurodegenerative diseases such as myasthenia gravis, amyotrophic lateral sclerosis, cervical spondylosis, and Alzheimer's disease.
  • a neurological disease possibly characterized by over activity of an RPTK in a signal transduction pathway is neurofibromatosis. See, generally, The Merck Manual of Diagnosis and Therapy, 16th Edition, 1992.
  • neurotrophic factors which through activation of specific cell surface receptors generate differentiation and survival signals in neuronal cell types. Binding of the neurotrophic factor to its receptor initiates a cellular signal transduction cascade involving diverse cytoplasmic components which eventually results in a specific nuclear response.
  • Specific cellular responses in nerve cells can include, for example, neurite outgrowth, acquisition of Na+-based action potential and cell survival in serum-free medium.
  • the complex processes involved in cell survival are mediated by diverse and divergent signal transduction pathways.
  • the multiple phosphorylated tyrosines found in activated RPTKs serve as binding sites for different signaling components, which in turn modulate the transduction of a signal along a particular pathway.
  • SHC has been shown to bind to the GRB2/S0S complex, which in turn allows the activation of RAS.
  • Activation of PLC-gamma leads to the generation of phosphatidylinositol metabolites, such as inositol 1, 4, 5-triphosphate, which cause the release of calcium ions from intracellular compartments and the generation of diacylglycerol, the natural activator of PKC.
  • the following method which was used to demonstrate that C- RET regulates neuronal survival, can also be used to determine the function of other RPTK receptors.
  • This method specifically activates RPTKs in cells in a ligand-independent fashion.
  • the RPTKs are activated with antibodies specific to the extracellular region of a chimeric receptor.
  • the chimeric receptor can contain an extracellular region and an intracellular region from two different RPTKs.
  • a chimera can contain the extracellular region of the TRK receptor and an intracellular region of C-RET. Activation of such a chimeric construct resulted in the prolonged survival of various neuronal cells. Examples of such studies are provided herein.
  • a chimera as used in the context of the invention, defines a polypeptide constructed from polypeptides of at least two different proteins.
  • the chimeric polypeptides are encoded by nucleic acid molecules harbored by a nucleic acid vector transfected into cells.
  • a chimera can contain the extracellular and transmembrane region from one type of RPTK and the intracellular region from another type of RPTK.
  • the chimeric feature of the method provides versatility as the intracellular region of virtually any known RPTK can be incorporated readily into a chimeric construct using DNA recombinant techniques existing in the art.
  • the chimeras comprise a polypeptide extracellular region, a transmembrane region, and an intracellular region of a RPTK.
  • the chimeric polypeptides are encoded by nucleic acid molecules harbored by a nucleic acid vector transfected into cells or tissues.
  • the extracellular region of the chimera is a polypeptide region that exists outside of the cell and specifically binds to an antibody of the method.
  • the intracellular region of the _ chimera exists inside the cell and comprises any part of a RPTK intracellular region.
  • the chimeric feature of the method also confers specificity as chimeras can be activated in a specific manner by an antibody having specific binding affinity to the polypeptide extracellular region.
  • An antibody can specifically bind to a single type of extracellular region of a chimera on cell surfaces.
  • antibodies are "Y-shaped" molecules that can bind two polypeptide molecules, antibodies can bring the chimeras in close proximity to one another in the cell. Once the intracellular regions of the chimera are in close proximity to one another, they can cross phosphorylate one another and become activated.
  • the effect of a compound on the cellular function of a RPTK can then be measured by monitoring changes in cell phenotype, catalytic activity of the RPTK, or interactions of the RPTK with natural binding partners.
  • the method features a cross species chimera/cell system.
  • the extracellular region of the chimeric receptor is selected from an organism distantly related to the organism from which the cells expressing the chimeric receptor are derived.
  • This cross species strategy ensures that endogenous receptors expressed on the cell surface are not activated by the antibodies raised against the extracellular region of the chimeric construct.
  • the antibodies of the invention have specific binding affinity for the TRK extracellular region from chicken.
  • the antibodies having specific binding affinity for the chicken TRK extracellular region will consequently not cross react with the endogenous TRK receptor of rat or human cells when the chimeric constructs are expressed in these cells.
  • the antibodies of the invention prevent cross reactivity with endogenous receptors expressed on the surface of mammalian cells since the antibodies have specific binding affinity to a distinct TRK receptor of a different type of organism.
  • C-RET regulates cell survival.
  • Signal transduction molecules that form a complex with C-RET as a result of these phosphoryl moieties such as GRB2, SOS, RAS, and RAF, propagate a signal in the cell that promotes neural survival.
  • compounds that promote the interactions of these stimulatory molecules of C-RET would enhance the activity of C-RET.
  • protein phosphatases can remove the phosphoryl moieties placed on the intracellular region of C-RET in response to GDNF, and thus inhibit the signaling capability of C-RET.
  • compounds that inhibit phosphatases of C-RET will enhance the signaling capacity of C-RET.
  • C-RET is implicated in the development and survival of enteric, synaptic, and sensory neurons and neurons of the renal system upon stimulation by GDNF (Jing et al . , 1996, Cell 85:1113-1124; Trupp et al . , 1996, Nature 381:785-789; Durbec et al . , 1996, Nature 381:789-793). Lack of function mutations in C-RET can lead to Hirschsprung' s disease, for example, which manifests itself as a decrease in intestinal tract innervation in mammals.
  • compounds that activate C-RET are potential therapeutic agents for the treatment of neurodegenerative disorders, including, but not limited to, Hirschsprung' s disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.
  • Compounds that inhibit C-RET function are also possible anti-cancer agents as over- expression of RET in cells is implicated in cancers, such as cancer of the thyroid.
  • Modulation of C-RET activity may also be useful in treating cancers of the nerve tissue, such as neuroblastoma, even if an abnormality is not found the signaling pathway.
  • neurodegenerative diseases such as amyotrophic lateral sclerosis
  • exogenous recombinant neurotrophic factors such as BDNF
  • BDNF neurotrophic factor
  • administration of neurotrophic factors may stimulate the growth and differentiation of nerve cells grown on plates, the neurotrophic factors will likely be rapidly degraded in the blood stream by proteases since they are composed of amino acids.
  • the neurotrophic factors cannot pass through cell membranes or the blood brain barrier. In order to treat Parkinson's disease or Alzheimer's disease, these therapeutics need to reach the brain in order to be efficacious.
  • non-peptide compounds are likely candidates as therapeutics for neurological disorders. These compounds must be selected for their ability to activate C-RET in order to treat or prevent neurological diseases.
  • the methods set forth herein can select non-peptide molecules as activators of C-RET.
  • select compounds are probable activators of C-RET. These compounds are set forth in WO 96/40113 and its related priority documents. Examples of indolinone compounds are also set forth herein by example.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administra- tion; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the liposomes will be targeted to and taken up selectively by the tumor.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • compositions for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlo- rotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlo- rotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlo- rotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspend- ing, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water- soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspen- sions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semi- permeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Many of the PTK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counter- ions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sul- furic, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half- maximal inhibition of the PTK activity) . Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC) .
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vi tro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of admini- stration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
  • the WO 96/18738 provides disclosure describing the recombinant DNA techniques pertaining to the present invention, nucleic acid vectors, the nucleic acid elements of these vectors, the types of cells that can harbor these vectors, methods of delivering these vectors to cells or tissues, methods of producing and purifying antibodies, methods of constructing hybridomas that produce these antibodies, methods of detecting signaling molecule complexes, methods of detecting interactions with natural binding partners, antibodies to complexes, disruption of RPTK protein complexes, purification and production of complexes, transgenic animals containing nucleic acid vectors encoding a RPTK, antisense and ribozyme approaches, and gene therapy techniques.
  • nucleic acid vectors the nucleic acid elements of these vectors, the types of cells that can harbor these vectors, methods of delivering these vectors to cells or tissues, methods of producing and purifying antibodies, methods of constructing hybridomas that produce these antibodies, methods of detecting signaling molecule complexes, methods of detecting interactions with natural binding partners
  • the examples below are not limiting and are merely representative of various aspects and features of the present invention.
  • the examples below demonstrate methods of identifying compounds that modulate the function of C-RET.
  • the examples below demonstrate compounds that activate RPTKs.
  • the examples below demonstrate methods used to identify the role of C-RET in cell survival.
  • the examples also demonstrate methods of specifically activating RPTKs in a ligand-independent manner.
  • the examples demonstrate the specificity as well as the versatility of the methods.
  • the PC12CTAret cells were created by techniques well known to those skilled in the art.
  • the C-RET gene was subcloned from a nucleic acid vector described in the following examples into a nucleic acid vector suitable for stably transfecting PC12 cells. These nucleic vectors are commonly and commercially available for such purposes.
  • the methods of cloning the genes of interest into these nucleic acid vectors and the methods of transfecting these nucleic acid constructs into cells are also well known to those skilled in the art (Sambrook, Fritsch, Maniatis, Molecular Cloning, A Labora tory Manual 2nd Ed. , Cold Spring Harbor Laboratory Press, 1989).
  • NGF nerve growth factor
  • 25 [ ⁇ g/mL stock prepared in: 100 mM NaCl, 50 mM sodium acetate, pH 5.3) (Austral Biologicals Cat. # GF-022); h. dimethyl sulfoxide (100%) (Sigma Catalog No. D-8418) ; i. PBS, Ca ++ - and Mg ++ -free (Gibco Cat. # 14190-029; j. Culture medium for PC12CTAret: RPMI (Gibco Cat. # 11875-093)
  • HNTG Buffer (20 mM HEPES buffer pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, 5 mM EDTA, 1 mM sodium orthovanadate, 5 mM NaPyrophosphate) ; m. Anti-HA monoclonal antibody, clone 12CA5 (Boehringer
  • PC12CTAret was subcloned in a PC12 cell line that expresses a chimeric HA-tagged c-ret receptor under the CMV promoter (vector pcDNA3, Invitrogen) .
  • the cells were passed when they were 60-70% confluent (about 1 x 10 7 cells/plate) , usually 1:6 once every four days. Cells can be removed from the plates by pipetting medium over them several times.
  • NGF is diluted to 40 ng/mL in complete PC12CTAret medium. 7. Add NGF and drugs to the cells. 13 ⁇ L of diluted NGF is added per well, followed by 13 ⁇ L of 10 X concentrated drug.
  • EXAMPLE 2 Methods of Identifying Compounds That Modulate the Function of RPTKs
  • An enzyme linked immunosorbant assay was conducted to measure the catalytic activity of the FLK-1 receptor and more specifically, the inhibition or activation of indolinone compounds of the catalytic activity of the FLK-1 receptor.
  • the following assay was conducted to measure catalytic activity of the FLK-1 receptor in FLK-1/NIH3T3 cells.
  • One skilled in the art can adapt such a procedure to other receptor tyrosine protein kinases to identify compounds that modulate the function of the receptor.
  • This type of assay can be developed for nearly any RPTK using well known techniques utilized by persons of ordinary skill in the art.
  • the FLK-1/NIH3T3 cell lines were also created by techniques wee known to those skilled in the art.
  • the NIH3T3 cells were stably transfected with gene constructs containing the flk-1 gene.
  • the nucleic acid vectors used for this type of procedure are commonly and commercially available to those skilled in the art.
  • the methods of cloning the genes of interest into these nucleic acid vectors and the methods of transfecting these nucleic acid constructs into cells are also well known to those skilled in the art (Sambrook, Fritsch, Maniatis, Molecular Cloning, A Laboratory Manual 2nd Ed., Cold
  • EDTA 0.5 M (pH 7.0) as a 100X stock
  • h Sodium ortho vanadate (0.5 M as a 100X stock)
  • i Sodium pyro phosphate (0.2 M as a 100X stock)
  • NUNC 96 well V bottom polypropylene plates Applied Scientific Catalog No. AS-72092
  • k NIH3T3 C7#3 Cells FLK-1 expressing cells
  • 1 DMEM with IX high glucose L Glutamine catalog No. 11965-050
  • m FBS Gibco
  • n L-glutamine Gibco (catalog no. 25030-016)
  • o VEGF PeproTech, Inc. (catalog no. 100-20) (kept as 1 ⁇ g/100 ⁇ L stock in Milli-Q dH 2 0 and stored at -20°C;
  • ABTS ABTS solution
  • citric acid anhydrous
  • 250 mM Na 2 HP0 4 pH 4.0
  • 0.5 mg/mL ABTS Sigma catalog no. A-1888
  • solution should be stored in dark at 4 °C until ready for use
  • t. H 2 0 2 (30% solution) (Fisher catalog no. H325)
  • u. ABTS/H 2 0 2 (15 mL ABTS solution, 2 ⁇ L H 2 0 2 ) prepared 5 minutes before use and left at room temperature;
  • Protocol 1. Coat Corning 96-well ELISA plates with 1.0 ⁇ g per well Cappel Anti-rabbit IgG antibody in 0.1 M Na 2 C0 3 pH 9.6. Bring final volume to 150 ⁇ L per well. Coat plates overnight at 4 °C. Plates can be kept up to two weeks when stored at 4 °C.
  • media should be removed from the cells and washed one time with 200 ⁇ L/well PBS.
  • HNTG formulation includes sodium ortho vanadate, sodium pyro phosphate and EDTA.
  • Selected compounds are known to inhibit particular receptor protein tyrosine kinases with high affinity and specificity. For example, see International Patent Publication Number WO 96/22976 for descriptions of indolinone compounds. Most of the indolinone compounds specified inhibit RPTKs with high affinity, but many of them do not enhance the catalytic activity of these protein kinases.
  • EC 2 oo values were measured for the following indolinone compounds in the FLK-1 ELISA. EC 2 oo values are the concentration of the indolinone compound that yielded a two- hundred percent increase in catalytic activity in the FLK-1 ELISA.
  • the following example demonstrates that the intracellular region of a RPTK can be activated when fused to a TRK extracellular region isolated from chicken.
  • This chimeric construct was activated by an antibody that had specific binding affinity for the TRK extracellular region from chicken organisms.
  • the chimeric constructs were activated by polyclonal antibody preparation raised against the chicken TRKA receptor.
  • the antibody specifically activated the chimeric construct in cells, since the cells did not express any other proteins harboring regions homologous to the TRK extracellular region from chicken.
  • the antibodies distinguished between endogenous TRKA receptor expressed on the surface of the rat cells, and the chimeric construct containing the chicken TRKA extracellular region, also expressed on the surface of the cells.
  • CTA The antibody preparation used in the experiments, called CTA, was a rabbit antibody raised against the entire extracellular domain of chicken TRKA expressed in COS cells.
  • the antibody was specific for chicken TRKA and did not cross- react with the rat receptor, nor did it elicit biological activities in rat cells. It did, however, strongly recognize the chicken TRKA receptor by immunoblot or immunoprecipitation and acted as a ligand which activated the chicken receptor.
  • a useful control for these experiments was to activate the endogenous rat TRKA receptor without influencing the introduced chimeric receptor. This specific activation was achieved through the use of the RTA IgG preparation, which recognized the rat receptor but not the chicken receptor.
  • the sympathetic and sensory neurons used in the experiments were cultured in a defined medium (Awrot and Patterson, 1979, Methods Enzymol . 53: 574-584).
  • Sympathetic neurons were isolated from superior cervical ganglia dissected from E20 - E21 rat fetuses, while dorsal root ganglion sensory neurons were obtained from E16 - E18 rats.
  • the ganglia were treated with 0.25% trypsin for 10 minutes, washed, and triturated to obtain a single cell suspension.
  • Sensory neurons were pre-plated for 1 hour on tissue culture plastic to deplete adherent cells.
  • EXAMPLE 5 Construction of Adenoviral Vectors Expressing Receptor Constructs
  • a recombinant adenoviral system was developed, as adenovirus exhibits a wide host range, and can infect and direct gene expression in post-mitotic neurons. Recombinant adenoviruses were generated by in vivo ligation.
  • the transfer vector contained 5' adenovirus packaging sequences, the Rous Sarcoma Virus long terminal repeat promoter, a polylinker ending in the restriction site BstBI, the SV40 poly A region, 3' adenoviral sequences (which could be used for recombination) , and sequences for propagation in E. coli.
  • the viral DNA used for generation of recombinant viruses was derived from a virus which expressed the ⁇ -galactosidase gene driven by the RSV promoter, and which contained a BstBI site between the ⁇ - galactosidase gene and the poly A region.
  • This vector allowed screening for recombinant plaques based on the presence or absence of ⁇ -galactosidase activity, similar to a system described by Schaack and colleagues (Schaack et al . , 1995, J. Virol. 69: 3920-3923).
  • This virus lacks the E3 region.
  • the cells utilized for the generation of recombinant adenovirus were HEK293 cells. Early passage HEK293 cells (Graham et al . , 1977, J. Gen. Virol 36: 59-72) were maintained in Dulbecco's modified Eagles medium + 10% calf serum. Cells infected with recombinant adenovirus were detected as plaques in the cell monolayer.
  • HEK293 monolayers were transfected with the DNA and cultured from five to seven days to allow plaques to appear. The monolayers were then stained with 25 ⁇ g/mL X-GAL (5-bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside) for several hours to identify non-recombinant (stained) plaques. Putative recombi- nant plaques were screened for expression of the transgene by infection of HEK293 cultures followed by immunohistochemistry.
  • Viruses which were positive for transgene protein expression were picked and subjected to several rounds of plaque purification prior to amplification and purification on cesium chloride gradients. Banded viruses were diluted five-fold with dilution buffer (Curiel et al . , 1991, Proc. Natl. Acad. Sci. USA 88: 8850-8854) and stored at -80 °C. Approximate titers of the virus preparations were determined immunohistochemically on HEK293 cultures. Although recombinant viruses were generated through recombination into the viral genome or through co- transfection of cleaved DNAs, higher recovery of recombinants was achieved through the cleaved DNA method.
  • the putative recombinant viruses were screened for expression of the kinase transgene by immuno-histochemistry. Immuno-histochemistry techniques were performed on cultures of sympathetic neurons infected with the RSV KP or RSV c-ret viruses and fixed and stained as described (Lefcort et al . , 1996, J. Neurosci. 16: 3704-3713). The primary antibodies were the CTA IgG, and mouse anti-tyrosine hydroxylase. The primaries were detected with Cy3-anti rabbit IgG and fluorescein-anti mouse IgG.
  • EXAMPLE 6 Expression and Activation of C-RET, SEK, and MCK-10 Chimeras
  • DNA constructs were generated, which encoded the transmembrane and cytoplasmic domains from human MCK-10, C-RET, mouse SEK, and rat TRKA fused to the chicken TRKA extracellular domain. These chimeric receptor constructs were then inserted into the adenovirus transfer vector, and recombinant viruses were generated and purified. Rat C6 glioma cells were infected with the recombinant viruses, and the resulting chimeric receptors were analyzed via immunoblot analysis.
  • the chimeric ret protein appeared to be approximately 155 kDa as expected.
  • the chimeric SEK protein was approximately 150 kDa, which is very similar to the chimeric and non-chimeric TRKA constructs, which have a molecular weight of approximately 140 kDa, and consistent with a fully processed, mature receptor.
  • the MCK-10 chimeric protein appeared in three bands, of molecular weights of approximately 160, 120, and 105 kDa. The two lower molecular weight forms were quite abundant and probably represent immature forms of this chimera.
  • the chimeric receptors were expressed in the cells, their activity was determined by monitoring the degree of autophosphorylation after stimulation by ligand (e.g. the TRKA CTA antibody).
  • ligand e.g. the TRKA CTA antibody
  • the ability to activate the chimeras was evaluated by incubating the infected C6 monolayers with 50 ng/mL NGF for five minutes.
  • the receptors were immunoprecipitated with the CTA antibody, immunoblotted, and probed either with the anti- phosphotyrosine antibody 4G10 or with the CTA antibody preparation.
  • NGF-dependent activation of the C-RET and SEK chimeras was detected by the anti-phosphotyrosine antibody.
  • the chimeric C-RET receptor showed substantial activation in the absence of ligand addition.
  • Phosphotyrosine was not _ detected for the MCK-10 chimeric protein, either before or after incubation with NGF.
  • PC12 and C6 glioma cells used in the experiments were cultured as follows.
  • PC12 cultures Greene et al . , 1987, Methods Enzymol. 147: 207-216) were maintained in RPMI medium containing 10% horse serum and 5% fetal calf serum.
  • the medium was changed to RPMI containing IX N2 supplement and 0.1% BSA, and the cells were grown on a collagen I substrate.
  • PC12 cell survival the cells were grown in RPMI containing 0.1% BSA. All cultures also contained IX penicillin/streptomycin.
  • PC12 cells were incubated overnight with recombinant viruses at a multiplicity of infection (MOI) between 1 and 10. The cells were then washed and re-plated either into differentiation or survival conditions, with either no addition or addition of 50 ng/mL (NGF) or 20 ⁇ g/mL CTA IgG for two days.
  • MOI multiplicity of infection
  • the cell cultures were fixed with 2% paraformaldehyde and the percentage of cells bearing processes longer than 1 cell diameter was determined.
  • the cultures were incubated with 0.05% MTT for 1.5 hours to stain living cells, and the relative number of cells surviving in each condition was determined.
  • C6 glioma cells were grown in Ham's F10 medium containing 10% fetal calf serum; for receptor activation experiments the medium was changed to 0.5% fetal calf serum 12 hours prior to stimulation.
  • EXAMPLE 7 The C-RET Chimeric Receptor Promotes Differentiation and Survival of PC12 Cells The biological activities of the chimeric receptor constructs were tested after it was determined that these constructs were expressed and active when stimulated with the CTA antibody, as shown in Example 6. As a first test of the biological activities of these chimeric constructs, their effects on the differentiation and serum-free survival of the pheochromocytoma cell line PC12 were tested.
  • Lysates from PC12 cultures prepared two days after infection with viruses were probed for receptor expression with the CTA antibody. Strong expression of the full length and chimeric TRKA and MCK-10 receptors was observed. Lower levels of the C-RET and SEK receptors were expressed. As expected, the CTA antibody did not react with uninfected cultures or cultures infected with the ⁇ -galactosidase virus.
  • the PC12 cells were first treated with virus overnight, and then re-plated under conditions which promote cellular differentiation. The cultures were incubated either in the presence of CTA IgG, to activate the introduced receptors, or NGF, to activate both endogenous and exogenous pools.
  • CTA antibody preparation could promote extensive outgrowth of the PC12 cells infected with the c-ret chimeric virus as well as with the full length and chimeric trkA viruses. The antibody had no obvious effect on RSV sek or RSV mck-10.
  • RSV c-ret was able to promote differentiation of PC12 cells to a level comparable to that seen with either trkA construct.
  • RSV c-ret virus showed a much higher activity in the absence of ligand than the trkA viruses did, most likely due to its auto-activation. No detectable activity of the sek or mck-10 constructs was observed.
  • PC12 cells undergo programmed cell death in the absence of serum, and can be rescued by NGF.
  • PC12 cells infected with the recombinant adenoviruses were tested for the ability of CTA antibody to rescue these cells from serum deprivation.
  • the cells were infected as described above, and plated with and without CTA Antibody in serum free medium. MTT was used to detect surviving cells in the cultures two days later. The strongest effect on cell survival was observed for the two TRKA receptor constructs.
  • the C-RET chimeric receptor could prevent cell death as well, but its effect reached a plateau at a level below that of the trkA viruses.
  • the RSV sek and RSV mck-10 viruses had marginal if any effect on the rescue of cells from death.
  • the C-RET cytoplasmic domain had biological activities comparable to those of TRKA in promoting PC12 cell differentiation and PC12 cell survival, while these activities were not detected after transfection with the sek or mck-10 receptor chimeras.
  • EXAMPLE 5 Activation of the C-RET Chimera Promotes Survival of Embryonic Sympathetic Neurons
  • C-RET was activated in a ligand-independent manner and tested for its ability to prevent apoptosis in embryonic sympathetic neurons.
  • Sympathetic neurons extend processes and maintain viability and electrical excitability for weeks when cultured in the presence of NGF. In the absence of NGF, the neurons lose their processes, shrivel and undergo a classical program of apoptosis within hours.
  • Activation of the TRKA receptor can prevent apoptosis in sympathetic neurons without the addition of NGF.
  • chimeric receptor constructs were examined in rat sympathetic neurons by infecting the neurons, culturing them in the presence of RTA antibody, and lysing them 48 hours later.
  • the RTA antibody was used to maintain their viability in the event that expression of a TRKA chimera would inhibit the function of the endogenous rat TRKA when NGF was used as a ligand.
  • the lysates were immunoblotted and probed with the CTA antibody. Strong expression of RSV KP (TRKA chimera), RSV c-ret, and RSV sek was observed.
  • RSV mck-10 showed somewhat weaker expression, especially of the 160 kDa isoform.
  • the neurons were transfected and plated in order to examine the effects of the chimeric receptors on neuronal cell survival.
  • the infected neurons were cultured in the presence of RTA antibody to activate their endogenous TRKA receptor, CTA antibody to activate the introduced receptor, or no addition to examine the background of cell survival. After three days, the cultures were stained with the vital dye calcein AM, and the number of surviving neurons in each condition was determined.
  • Uninfected sympathetic neurons responded well to the RTA antibody preparation, while the CTA antibody was ineffective in promoting cell survival.
  • RTA could also sustain the survival of neurons infected with the RSV KP, RSV c-ret, RSV sek, or RSV mck-10 viruses.
  • the cells surviving through infection with the RSV c-ret virus were tested for the proper phenotype of a superior cervical ganglion neuron.
  • Neurons were cultured with or without NGF for two days following infection with either the RSV c-ret or the RSV KP virus. The cultures were then subjected to immunofluorescence. In all cases neurons which were strongly expressing the chimeric receptors exhibited a healthy phenotype.
  • the cells expressing the transgene comprised a subset of surviving neurons, while in the absence of NGF, all large healthy neurons were found to be expressing the chimeric receptor.
  • the neurons expressed comparable levels of tyrosine hydroxylase as compared to RSV KP controls.
  • EXAMPLE 6 Prevention of Sensory Neuron Programmed Cell Death by the C-RET Intracellular Region c-ret constructs were transfected into DRG embryonic rat sensory neurons to determine whether the RPTK could promote survival in these cells as well as the sympathetic neurons studied in example 5.
  • both the TRKA and C-RET chimeric receptors could promote a significant level of neuronal survival, while the sek receptor construct was inactive.
  • the level of survival promoted by C-RET in the presence of the CTA antibody was about 50% of that promoted by RSV KP, and again there was significant survival in the absence of the antibody (31% of that measured with RSV KP plus CTA antibody) . Therefore, the survival activity of the C-RET receptor intracellular region extends to at least two major cell types, sympathetic and sensory neurons, in the peripheral nervous system.
  • EXAMPLE 7 TRKA and C-RET Chimeric Receptors Promote MAPK Phosphorylation in Primary Neurons The molecular mechanisms behind cell survival were explored once it was observed that activation of the C-RET chimera stimulated cell survival, as demonstrated in Examples 5 and 6. In particular, the relationship between the C-RET intracellular region and the proteins that it potentially phosphorylated were explored.
  • TRKA downstream signal transduction effectors of the TRKA and C-RET receptors showed significant similarities in certain effector systems.
  • GRB2 has been shown to bind one isoform of the C-RET receptor at its cytoplasmic tail; this interaction is predicted to activate the RAS/RAF/MAPK pathway.
  • TRKA activates this pathway through interaction of the SHC adaptor protein with a phosphotyrosine located in the juxta membrane region. Experiments were performed which determined that survival-promoting receptors could activate the MAPK pathway in sympathetic neurons.
  • Neurons were infected with virus constructs carrying either active or inactive trkA receptor genes or the chimeric c-ret receptor gene.
  • the neurons were cultured in the presence of the RTA antibody for two days, and then starved for 3 hours. Half of the wells were stimulated for ten minutes with the CTA antibody, the cultures were lysed, and the resulting lysates immunoblotted.
  • the immunoblots were probed with antibodies recognizing MAPK p42 and p44 (ERK1 and ERK2) or probed with antibodies recognizing only the phosphorylated form of MAPK. CTA stimulation of the RSV KP and RSV c-ret receptors efficiently and equivalently promoted MAPK phosphorylation.
  • the invention is meant to also cover the final formulation formed by the combination of these excipients.
  • the invention includes formulations in which one to all of the added excipients undergo a reaction during formulation and are no longer present in the final formulation, or are present in modified forms.

Abstract

L'invention concerne des procédés relatifs à l'évaluation de la fonction spécifique d'une protéine tyrosine kinase réceptrice dans les cellules, qui consistent à activer le récepteur d'une manière indépendante du ligand. L'invention concerne aussi des procédés visant à identifier les composés qui modulent la fonction de protéine tyrosine kinase récepteur. Elle concerne enfin un procédé pour prévenir ou traiter tout état anormal résultant d'une aberration dans la fonction du récepteur C-RET, et spécifiquement pour traiter et prévenir les maladies neurodégénératives en administrant un composé qui module la fonction du récepteur considéré.
PCT/US1998/006842 1997-04-08 1998-04-07 Etude et traitement des maladies liees a des fonctions cellulaires specifiques de proteines tyrosines kinases receptrices WO1998045708A1 (fr)

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