WO1992020797A1 - Facteur neurotrophique, sa preparation et ses utilisations - Google Patents

Facteur neurotrophique, sa preparation et ses utilisations Download PDF

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Publication number
WO1992020797A1
WO1992020797A1 PCT/US1992/004260 US9204260W WO9220797A1 WO 1992020797 A1 WO1992020797 A1 WO 1992020797A1 US 9204260 W US9204260 W US 9204260W WO 9220797 A1 WO9220797 A1 WO 9220797A1
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dna sequence
neurotrophic factor
nucleotide number
dna
sequence
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PCT/US1992/004260
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Thomas H. Large
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Case Western Reserve University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to neurotrophic factors and particularly to a newly discovered factor, methods for its purification and production, and to diagnostic and therapeutic applications for the factor.
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's and Parkinson's diseases Huntington's chorea
  • multiple sclerosis by the ischemia resulting from stroke, by a traumatic injury, by the natural aging process, or by the accelerated degenerative effects of diabetes.
  • Neurotrophic factors are a class of molecules that have been initially identified as participants in the development of vertebrate nervous systems by facilitating the interaction of neurons with their target cells. It has been observed that competition among neurons for such target cells takes place and that only those neurons that achieve such interaction will survive (Leibrock et al., 1989, Nature. 3_4_1 149; Hohn et al., 1990, Nature, 344:339. Accordingly, such neurotrophic factors promote the survival and functional activity of nerve or glial cells. Evidence also exists to suggest that neurotrophic factors will be useful as treatments to prevent nerve or glial cell death or malfunction resulting from the conditions enumerated above (See, Appel, 1981, Ann. Neurology, _L0:499; U.S. Patent Nos.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • HDNF hippocampus-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • CNTF ciliary neurotrophic factor
  • a complication of the use of neurotrophic factors is their apparent specificity for only those subpopulations of nerve cells which possess the correct membrane receptors. Most nerve cells in the body lack NGF receptors and are apparently unresponsive to this neurotrophic factor. It is therefore of critical importance to discover new neurotrophic factors that can support the survival of different types of nerve or glial cells than are supported by NGF.
  • a new neurotrophic factor has been identified and the partial DNA sequence and corresponding amino acid sequence thereof have been determined.
  • the present neurotrophic factor appears to be distinguishable from the known factors NGF, BDNF and NT-3, although it shares certain homology with them, and has accordingly been designated neurotrophin-4, or NT-4.
  • NT-4 has thus far been identified as to the extent of its structural homology relative to the known neurotrophic factors NGF, BDNF and NT-3.
  • NT-4 may be characterized as follows: Amphibian NT-4 exhibits
  • the partial DNA sequence in Rana Pipiens is presented below in FIGURE 1 and in Sequence ID No. 1, or SEQ ID NO:l, and comprises a 170 bp segment, including a 5' primer comprised of nucleotides 1-27 and a 3' primer comprising nucleotides 147-170.
  • the alternate, primer- exclusive DNA sequence comprises a 120 bp segment that extends inclusively from nucleotide 28 to nucleotide 147 of SEQ ID N0:1. More particularly and as discussed in greater detail later on herein, a specific oligonucleotide probe was prepared from a DNA segment comprising 56 nucleotides extending inclusively from nucleotide 49 to nucleotide 104 of SEQ ID NO:l.
  • the corresponding primer-inclusive partial amino acid sequence in Rana Pipiens is also presented in SEQ ID N0:1, and comprises a single chain of 56 amino acids including a 5' primer comprised of 9 amino acids and a 3' primer comprised of 7 amino acids.
  • the primer-exclusive amino acid sequence comprises a chain of 40 amino acids commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 28-30, and terminating inclusively at the 3' end with glycine (Gly) defined by nucleotides 145-147.
  • amino acid sequence corresponding to the particular oligonucleotide probe prepared herein comprises a chain of 19 amino acids commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 49-51, and terminating inclusively at the 3' end with lysine (Lys) defined by nucleotides 100-102.
  • the partial DNA sequence in Xenopus is presented below in FIGURE 2 and in Sequence ID No. 2, or SEQ ID NO:2, and comprises a 173 bp segment, including a 5' primer comprised of nucleotides 1-27 and a 3' primer comprising nucleotides 150-173.
  • the alternate, primer-exclusive DNA sequence comprises a 123 bp segment that extends inclusively from nucleotide 28 to nucleotide 150 of SEQ ID NO:2.
  • the corresponding primer-inclusive partial amino acid sequence in Xenopus is also presented in SEQ ID NO:2, and comprises a single chain of 57 amino acids including a 5' primer comprised of 9 amino acids and a 3' primer comprised of 7 amino acids.
  • the primer-exclusive amino acid sequence comprises a chain of 41 amino acids commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 28-30, and terminating inclusively at the 3' end with glycine (Gly) defined by nucleotides 148-150.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes NT-4 or a fragment thereof, that possesses a partial DNA sequence selected from the primer-inclusive sequence set forth in SEQ ID NO:l, the primer-inclusive sequence set forth in SEQ ID NO:2, the primer-exclusive sequence extending inclusively from nucleotide 28 to nucleotide 147 of SEQ ID N0:1, and the primer-exclusive sequence extending inclusively from nucleotide 28 to nucleotide 150 of SEQ ID NO:2.
  • primer-inclusive sequence as determined in Xenopus and presented in SEQ ID NO:2 is set forth below.
  • the partial DNA sequence of the present invention or a portion thereof may be prepared as a probe to screen for complementary sequences and genomic clones in the same or alternate species, such as humans.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for NT-4.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector Ml3.
  • the present invention also includes the preparation of plasmids including such vectors.
  • the present invention also includes NT-4 proteins having the activities noted herein, and that display the partial amino acid seqences set forth and described above and selected from SEQ ID NO:l and SEQ ID NO:2.
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding NT-4, and more particularly, the complete DNA sequence determined from the partial sequences set forth above and in SEQ ID NO:l and SEQ ID NO:2.
  • NT-4 appears to be a member of a family of neurotrophic factors, the archetype of which is nerve growth factor (NGF) . Accordingly, many of the activities that have been determined and attributed to NGF, BDNF and NT-3 may find their cognates in the activity profile of the present neurotrophic factor, and it is anticipated that NT-4 is capable of therapeutic activity, for example, in the treatment of neuropathies of toxic or viral origin, diabetic neuropathy, and nerve damage resulting from physical trauma.
  • NGF nerve growth factor
  • the present neurotrophic factor plays in the activities of nerve cells and the particular neural cell subpopulation(s) with which it appears active are as yet largely undefined, its participation in the elicitation of certain of the activities and conditions associated with neural activation and regeneration may be strongly inferred.
  • the homology displayed by the present neurotrophic factor with BDNF suggests that the present factor may possess activity and effect in neuronal populations located either in the central nervous system, or that are directly connected to it.
  • the present invention also includes the preparation of frog, rat and human amino acid and nucleic acid NT-4 sequences.
  • a recombinant expression system is provided to produce biologically active animal or human NT-4.
  • the neurotrophic factor NT-4 may be prepared by isolation and purification from cells known to produce NT-4. Possible cellular sources for the production of NT-4 include the brain, heart, spleen or other organs, or peripheral tissues. The cells or active fragments likely to participate in NT-4 synthesis or to have NT-4 associated therewith may be subjected to a series of known isolation techniques, such as for example immunoprecipitation, whereupon the present neurotrophic factor may be recovered.
  • the present invention naturally contemplates alternate means for preparation of the neurotrophic factor, including stimulation of NT-4 producer cells with promoters of neurotrophic factor synthesis followed by the isolation and recovery of the neurotrophic factor as indicated above, as well as chemical synthesis, and the invention is accordingly intended to cover such alternate preparations within its scope.
  • the present invention also extends to antibodies to the neurotrophic factor NT-4 that would find use in a variety of diagnostic and therapeutic applications.
  • the antibodies could be used to screen expression libraries to obtain the gene that encodes NT-4.
  • those antibodies that neutralize NT-4 activity could initially be employed in intact animals to better elucidate the biological role that this factor plays.
  • Possible therapeutic applications would include administration in instances where NT-4 hyperactivity and correspondingly excessive neural development proves undesirable or physiologically or pathologically detrimental, or simply to better modulate nerve growth in certain situations.
  • the present invention also includes various diagnostic and therapeutic utilities predicated on the structure and activities of NT-4.
  • Diagnostic utilities include assays such as immunoassays with labeled NT-4, its antibodies, ligands and binding partners, receptor assays, and a drug screening assay to evaluate new drugs by their ability to promote or inhibit NT-4 production or activity, as desired.
  • the above assays could be used to detect the presence and activity of NT-4 or of invasive stimuli, pathology or injury the presence or absence of which would affect NT-4 production or activity.
  • the present invention also extends to therapeutic methods and corresponding pharmaceutical compositions based upon NT-4, and materials having the same or an antagonistic activity thereto.
  • Therapeutic methods would be based on the promotion of the activities of NT-4 and would extend to the treatment of neural debilitations or dysfunctions attributable to the absence of NT-4 activity, such as certain neuropathies and injury-related dysfunctions. This method could be effected with NT-4, its agonists or like drugs, or materials having a promotional effect on the production of NT-4 in vivo.
  • compositions comprising effective amounts of NT-4, its agonists, antibodies, antagonists, or like drugs, etc., and pharmaceutically acceptable carriers are also contemplated. Such compositions could be prepared for a variety of administrative protocols, including where appropriate, oral and parenteral administration. Exact dosage and dosing schedule would be determined by the skilled physician.
  • neurotrophin-4 a newly discovered neurotrophic factor in purified form, identified herein as neurotrophin-4, or NT-4.
  • FIGURE 1 depicts the partial NT-4 nucleic acid sequence and the deduced amino acid sequence derived from M13 clones prepared by screening frog (Rana pipiens. genomic DNA with degenerate oligonucleotide primers to conserved amino acid sequences in nerve growth factor (NGF) and pig brain-derived neurotrophic factor (BDNF) .
  • the nucleotides are numbered from 1 to 170, and the amino acids are numbered from 1 to 56.
  • This sequence is identically depicted in the SEQUENCE LISTING presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October 1, 1990, and is cumulatively and alternately referred to as SEQ ID NO:l.
  • FIGURE 2 depicts the partial NT-4 nucleic acid sequence and the deduced amino acid sequence prepared by screening frog (Xenopus) genomic DNA in the same manner as with the genomic DNA of Rana Pipiens.
  • the nucleotides are numbered from 1 to 173, and the amino acids are numbered from 1 to 57.
  • This sequence is identically depicted in the SEQUENCE LISTING presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October l, 1990, and is cumulatively and alternately referred to as SEQ ID NO:2.
  • FIGURE 3 depicts the alignment of the neurotrophin gene family.
  • the amino acid sequences (mature factor) of the known neurotrophin genes, from various species, are presented adjacent the partial sequences determined herein for NT-4.
  • the conserved cysteine residues are identified by solid circles. Residues conserved in every gene are identified with an asterisk.
  • the boxed sequences represent PCR fragments obtained by amplification with the two PCR primers shown at the bottom (solid arrows) .
  • the open arrows represent additional PCR primers that are being used to continue the screening for additional members of the family.
  • the black bar above the Rana Pipiens frog NT4 sequence represents the degenerate oligonucleotide used to screen the rat genomic library.
  • the hatched arrows represent primers used for "inverse PCR" cloning of DNA sequences flanking the frog NT4 PCR fragments.
  • FIGURE 4 depicts the partial amino acid sequences of Rana Pipiens NT-4 and the other putative members of the nerve growth factor family. Sequences were obtained from the literature and aligned via conserved cysteine residues. Amino acids enclosed in boxes are conserved between the various members of the nerve growth factor family.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide.
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • stimulation and its plural as used herein are intended to apply to invasive events such as infection, as well as conditions caused by wounding, and to idiopathic or spontaneous states that may for example, originate from cellular or metabolic derangements or other causes.
  • the present neurotrophic factor refers to protein material having the partial DNA and amino acid sequence data described herein and presented in FIGURE 1 (SEQ ID NO:l) and in FIGURE 2 (SEQ ID NO:2), and the profile of activities as are set forth herein and in the Claims. Accordingly, proteins having similar sequences to those set forth herein but displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are NT-4 producers. Also, the terms “the present neurotrophic factor”, “neurotrophin-4" and “NT-4" are intended to include within their scope the proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • neurotrophic effects means that the neurotrophic factor of the present invention has selective effects on specific neural elements that contribute to the survival, growth, maturation and regeneration of neurons present in the nervous tissue.
  • a “replicon” is any genetic element (e.g. , plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e.. capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g. , restriction fragments) , viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g. , restriction fragments
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e.. the strand having a sequence homologous to the mRNA) .
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N- ter inal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g. Maniatis et al. , supr ; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g. , a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • an “antibody” is any i munoglobulin, including antibodies and fragments thereof, such as Fab, F(ab') 2 or dAb, that binds a specific epitope.
  • the term encompasses, inter alia, polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • An antibody preparation is reactive for a particular antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen.
  • An antibody preparation is non-reactive for an antigen when binding of the individual immunoglobulin molecules in the preparation to the antigen is not detectable by commonly used methods.
  • any intron-free DNA provided by the present invention is novel, since it is believed that the naturally-occurring human genes contain introns.
  • the term "intron-free” excludes the DNA sequences which naturally occur in the chromosomes of human or bovine cells.
  • the present invention also encompasses the intron-free cDNA sequences derivable from the DNA sequences disclosed herein.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash.
  • the present invention concerns the isolation and identification of a newly discovered particular neurotrophic factor hereinafter referred to as neurotrophin-4 or NT-4.
  • the partial DNA sequence initally determined in frog comprises a 170 bp segment, including a 5' primer comprised of nucleotides 1-27 and a 3' primer comprising nucleotides 147-170.
  • the alternate, primer-exclusive DNA sequence comprises a 120 bp segment that extends inclusively from nucleotide 28 to nucleotide 147.
  • a specific oligonucleotide probe was prepared from a DNA segment comprising 56 nucleotides extending inclusively from nucleotide 49 to nucleotide 104.
  • the Rana Pipiens primer-inclusive partial amino acid sequence comprises a single chain of 56 amino acids including a 5' primer comprised of 9 amino acids and a 3' primer comprised of 7 amino acids.
  • the primer-exclusive amino acid sequence comprises a chain of 40 amino acids commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 28-30, and terminating inclusively at the 3' end with glycine (Gly) defined by nucleotides 145-147.
  • amino acid sequence corresponding to the particular oligonucleotide probe prepared from the Rana fragment comprises a chain of 19 amino acids commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 49-51, and terminating inclusively at the 3' end with lysine (Lys) defined by nucleotides 100-102.
  • the partial nucleotide sequence determined in Xenopus comprises a primer-inclusive chain of 173 nucleotides, and a primer exclusive chain of 123 nucleotides in length, the latter comprising inclusively nucleotides 28- 150.
  • a corresponding primer-inclusive segment of 57 amino acids is defined, with a primer-exclusive of 41 amino acids also commencing at the 5' end with the asparagine (Asn) corresponding to the codon defined by nucleotides 28-30, and terminating inclusively at the 3' end with glycine (Gly) defined by nucleotides 148-150.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes NT-4 or a fragment thereof, that possesses a partial DNA sequence selected from the primer-inclusive sequence set forth in SEQ ID NO:l; the primer-exclusive SEQ ID NO:l sequence extending inclusively from nucleotide 28 to nucleotide 147; the primer-inclusive sequence set forth in SEQ ID NO:2; and the primer- exclusive SEQ ID NO:2 sequence extending inclusively from nucleotide 28 to nucleotide 150.
  • SEQ ID NO:l is reproduced below.
  • the primer-inclusive sequence as determined in Xenopus frog is set forth below as SEQ ID NO:2.
  • the partial DNA sequences of the present invention or a portion thereof may be prepared as a probe to screen for complementary sequences and genomic clones in the same or alternate species, such as humans.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for NT-4.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector M13.
  • vector M13mpl9 was prepared and subcloned with the DNA fragment described above.
  • the present invention also includes the preparation of plasmids including such vectors.
  • the present neurotrophic factor may also be prepared in purified form from appropriate cells known to produce NT-4, for example, by isolating sufficient quantities of tissue composed of cells known to contain the neurotrophic factor, and extracting the neurotrophic factor therefrom. The extract may then be appropriately treated as by centrifuging, to recover a supernatant containing the crude factor. The factor may then be further treated as by filtration or precipitation. Thereafter, the crude factor may be subjected to a series of known isolation techniques, whereupon the purified neurotrophic factor may be recovered.
  • genomic DNA sequences including transcriptional promoters, for NT-4 genes may be isolated in alternate species.
  • a human genomic library may be screened with 32 P-labeled probes derived from the coding regions of the full sequence derived from the partial NT-4 DNA sequence defined herein. This would yield clones that would be anticipated to contain portions of the 5' untranscribed and untranslated regions of the NT-4 gene.
  • NT-4 transcriptional promoters have a number of uses. First, they are useful to construct vectors inducible by any agent found to induce expression of NT-4. Such vectors may be useful, for example, in gene transfer assays, wherein the inducible promoter is positioned so that it drives transcription of a reporter gene such as chloramphenicol acetyltransferase, beta-galactosidase, luciferase, etc. This construct will then be introduced transiently or stably into an appropriate mammalian cell line. Potential inhibitors or stimulators of induction can then be assayed by measuring their effect on induction by any or all of the inducers listed above.
  • a reporter gene such as chloramphenicol acetyltransferase, beta-galactosidase, luciferase, etc.
  • This construct will then be introduced transiently or stably into an appropriate mammalian cell line. Potential inhibitors or stimulators of induction can then be ass
  • Another feature of this invention is the expression of a full NT-4 DNA sequence derived from the partial sequences disclosed herein.
  • a DNA sequence may be expressed by operatively linking it to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g..
  • yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • any of a wide variety of expression control sequences may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5) , the promoters of the yeast ⁇ - mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the early and late promoters of SV40 or adenovirus include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well-known eukaryotic and prokaryotic hosts, such as strains of E. coli. Pseudomonas. Bacillus. Streptomyces. fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green
  • Monkey kidney cells e.g., COS 1, COS 7, BSC1, BSC40 and BMT10
  • insect cells e.g., Sf9
  • human cells and plant cells in tissue culture the foregoing list being illustrative and not limitative.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • the invention further includes a method for detecting idiopathic or invasive stimuli on the basis of their ability to elicit the production and activities affected by NT-4.
  • invasive stimuli could be identified and detected by their ability to stimulate the production of NT-4 by the relevant NT-4 producing cells.
  • samples of producer cells could be treated with/exposed to a number of materials known to stimulate the production of NT-4 as a control, while parallel cellular samples could be treated with or exposed to an extract of material from the situs of the suspected stimulus. All samples could then be incubated, and thereafter, in one embodiment, samples could be assayed for the presence of NT-4.
  • a receptor assay As neural activity may be measured by the binding of neural cells to corresponding receptors, an assay known as a receptor assay may be utilized.
  • a receptor assay the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of NT-4 may be radiolabeled, after which binding studies would be carried out using for example, neural cells and cell targets having a demonstrated NT-4 affinity. Solutions would then be prepared that contain various quantities of labeled and unlabeled NT-4 and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay system for screening of potential drugs effective to promote the synthesis and activity of NT-4, or to counteract NT-4 or to prevent its synthesis may be prepared.
  • the test drug could be administered to a sample of cells known to produce NT-4 or cells known to be rehabilitated by NT-4 stimulation.
  • a sample wherein the test drug is not present would serve as a control.
  • Measurements of NT-4 production, cellular rehabilitation and recovery could be compared as between the respective samples to ascertain the efficacy, if any, of the test drug.
  • the drug under test is a potential inhibitor of NT-4 production/activity
  • it could be administered to a cellular sample that is known to produce/respond to NT-4, to determine its effect upon the production and/or activity of the neurotrophic factor.
  • the test drug could be administered to a cellular sample or colony with an agonist that normally promotes NT-4 production. Measurement of the neurotrophic factor in the sample will determine if the test drug blocks NT-4 production, while measurement of neuronal cellular growth or rehabilitation will determine if NT-4 activity has been blocked.
  • the present invention also relates to a method for detecting the presence of stimulated, spontaneous, or idiopathic neuropathological states in mammals, by measuring the presence and activity of the neurotrophic factor of the present invention. More particularly, the activity of NT-4 may be followed directly by assay techniques such as those discussed herein, through the use of an appropriately labeled quantity of the neurotrophic factor. Alternately, NT-4 can be used to raise binding partners or antagonists that could in turn, be labeled and introduced into a medium to test for the presence and amount of NT-4 therein, and to thereby assess the state of the host from which the medium was drawn.
  • both NT-4 and any antibodies or other antagonists that may be prepared thereagainst are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antagonist to NT-4 that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • immunoassays such as a radioimmunoassay, using for example, an antagonist to NT-4 that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • a control quantity of the neurotrophic factor, an antibody or antagonist thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a neural cell-containing fluid sample of a mammal believed to be undergoing invasion. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, ⁇ , 125 I, 131 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro- photometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the neurotrophic factor NT-4.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the neurotrophic factor; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of NT-4 in a mammal.
  • one class of such kits will contain at least labeled NT-4 or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive", “sandwich”, “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence and activity of NT-4, comprising:
  • the diagnostic test kit may comprise: (a) a known amount of NT-4 as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each;
  • test kit (b) if necessary, other reagents; and (c) directions for use of said test kit.
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive”, “sandwich”, “double antibody”, etc.), and comprises:
  • the invention extends to antibodies including both polyclonal and monoclonal antibodies, and fragments thereof, that may be prepared and utilized as mentioned earlier for a variety of diagnostic nad therapeutic applications.
  • drugs that modulate the production or activity of the neurotrophic factor NT-4 include such agents that may antagonize the activity of NT-4 in the same fashion as antibodies and are included herein. All of these agents may possess certain therapeutic applications and may for example, be utilized for the purpose of treating the effects of hyperactive nerve growth attributable the action of the present neurotrophic factor.
  • NT-4 may be used to produce both polyclonal and monoclonal antibodies to itself in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • Panels of monoclonal antibodies produced against NT-4 peptides can be screened for various properties; i.e. , isotype, epitope, affinity, etc.
  • monoclonal antibodies that neutralize the activity of NT-4.
  • Such monoclonals can be readily identified in an NT-4 activity assay.
  • High affinity antibodies are also useful in im unoaffinity purification of native or recombinant NT-4.
  • the antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(V) portions of whole antibody molecules.
  • Preferred monoclonal antibodies should display an immunoreactivity for the antigen that is similar to that of those produced by the above-described hybridomas.
  • immunoreactivity in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of the NT-4 antigen. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both B 0 and B have been adjusted for background. See Robard, Clin. Chem.. 20:1255-1270 (1974).
  • the resulting antibodies could also be prepared in a suitable pharmaceutical composition and administered to avert or treat the abovenoted undesired condition.
  • suitable pharmaceutical compositions may vary in accordance with those known in the medical arts, and upon the specific instruction of a qualified physician or veterinarian.
  • the existence of antibodies against NT-4 makes possible another method for isolating other neurotrophins and ligands.
  • the method takes advantage of an antibody characteristic known as idiotypy.
  • Each antibody contains a unique region that is specific for an antigen. This region is called the idiotype.
  • Antibodies themselves contain antigenic determinants; the idiotype of an antibody is an antigenic determinant unique to that molecule.
  • Antibodies that recognize the idiotype of another antibody are called anti-idiotype antibodies.
  • Some anti- idiotypic antibodies mimic the shape of the original antigen that the antibody recognizes and are said to bear the "internal image" of the antigen.
  • antigen When the antigen is a ligand, certain anti-idiotypes can bind to that ligand's receptor. Investigators have identified several of these, including anti-idiotypes that bind to receptors for insulin, angiotensin II, adenosine I, ⁇ -adrenalin, and rat brain nicotine and opiate receptors. (Carlsson and Glad, 1989) .
  • Anti-idiotypes may be used to screen for molecules binding to the original antigen. For example, one may use this technique to identify other neurotrophin ligands.
  • the present invention relates to certain therapeutic methods which would be based upon the activity of NT-4, antibodies or other antagonists to NT- 4, or upon agents or other drugs determined to possess the same or an antagonistic activity.
  • a first therapeutic protocol is associated with the promotion of the manifestations of conditions following from the production and activity of the neurotrophic factor, and comprises administering either the neurotrophic factor or an agent capable of modulating the production and/or activity of the neurotrophic factor, either individually or in mixture with each other in an amount effective to promote neurotrophic factor activity in the host.
  • the method of promoting the synthesis of the neurotrophic factor contemplates facilitating the reaction/interaction between the neural cells or any active fragments thereof and the promoters of neurotrophic factor synthesis. Accordingly, this embodiment of the method is directed toward the facilitation or admininstration of agents that serve as neurotrophic factor synthesis promoters, such as agonists that may react with receptors on neural cells, intracellular signaling agents that initiate neurotrophic factor synthesis, enzymes that synthesize the neurotrophic factor, and agents that promote or participate in the synthesis or production of molecules that serve as metabolic precursors to the neurotrophic factor.
  • agents that serve as neurotrophic factor synthesis promoters such as agonists that may react with receptors on neural cells, intracellular signaling agents that initiate neurotrophic factor synthesis, enzymes that synthesize the neurotrophic factor, and agents that promote or participate in the synthesis or production of molecules that serve as metabolic precursors to the neurotrophic factor.
  • the method therefore comprises introducing to a medium or other sample or substrate, promoters or agonists to the aforementioned agents, in amounts effective to promote or cause the synthesis of the neurotrophic factor.
  • promoters or agonists to the aforementioned agents, in amounts effective to promote or cause the synthesis of the neurotrophic factor.
  • Representative intracellular signaling agents are selected from protein kinase C, cGMP, G-proteins and mixtures. Suitable agonists to the promoters of neurotrophic factor synthesis may be determined by appropriate assays.
  • a first therapeutic protocol in accordance with the present invention is predicated on the activation and mobilization of neural cells to respond to debilitation occasioned by invasion or injury in instances where the host exhibits neural incapacity or dysfunction.
  • appropriate compositions containing NT-4 or agents or drugs that mimic its activity could be administered to the patient or host in need of such treatment to promote neural cell rehabilitation.
  • the method of administration would include those known procedures, including parenteral techniques as are conventionally used by skilled medical personnel. Dosages and protocol of administration would likewise vary.
  • the second aspect of the therapeutic method generally referred to above includes a method for the treatment of neuropathies comprising cellular debilitation/dysfunction resulting from infection, injury, pathology such as diabetes, or other immunologically unknown causes, by the administration of pharmaceutical compositions that may comprise effective quantities of the neurotrophic factor NT-4, antibodies or other antagonists to NT-4, agonists to NT-4, promoters of NT-4 synthesis, inhibitors of NT-4 synthesis, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • a variant embodiment of this therapeutic method could include initially detecting the extent if any, of the presence and activity of the neurotrophic factor and thereafter administering the appropriate pharmaceutical composition, whether to augment or to limit its presence and activity.
  • NT-4 its analogs, antibodies, binding partner(s) or other ligands or agents exhibiting either mimicry or antagonism to NT-4 or control over its production or activity, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient having a particular neuropathy whether due to infection or other pathological derangement or physical injury, whether a consequence of NT-4 deficiency or excess, for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them oral admininstration of elixirs, tablets, capsules, and the like.
  • compositions may be administered by parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, including delivery in an irrigation fluid used to wash body wound areas, catheterizations and the like. Average quantities of the active ingredient may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • compositions useful in practicing the therapeutic methods of this invention may include, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of NT-4, an NT-4 synthesis promoters, an anti NT-4 antibody, an NT-4 synthesis inhibitor, or other antagonist, or analogs thereof, as described herein as an active ingredient.
  • a pharmaceutically acceptable excipient carrier
  • the amount of active ingredient in a unit dosage form may range up to about 4.0 g of the active per day for an adult male.
  • oligonucleotides were designed using the known sequences for Nerve Growth Factor (NGF) and pig Brain- Derived Neurotrophic Factor (BDNF) as presented in FIGURE 3.
  • the primers contained recognition sequences for EcoRl at either end to facilitate subcloning.
  • Frog genomic DNA (Rana pipiens) was provided by Martha Constantine-Paton at Yale University and was prepared for Polymerase Chain Reaction (PCR) by extensive phenol/chloroform extraction to remove endogenous proteins. PCR reactions were conducted as per instructions from Cetus and included 1 ⁇ g DNA and 1 ⁇ g of both primers or each primer alone (Control) .
  • PCR was performed at low stringency - 5 cycles at 37°C stringency, 5 cycles at 45°C stringency and 30 cycles at 55°C.
  • products were run on a 6% acrylamide/TBE gel.
  • the 190 bp fragment was eluted from the gel and amplified at high stringency (55 ⁇ C) . This product was then digested with EcoRl and subcloned into the vector M13 mp 19. Clones were obtained by transformation of XL-1 Blue cells and individual clones sequenced by the Sanger dideoxy method. Approximately two dozen M13 clones were sequenced and 50% were determined to be frog BDNF, 40% frog NGF and 10% were unique and subsequently named frog NT-4, the fourth member of the NGF family.
  • a full genomic DNA library was developed for the frog Xenopus and was screened with the primers and in accordance with the protocol set forth in Example 1, above.
  • the resulting clones were sequenced and clones corresponding to a putative NT-4 fragment in Xenopus were identified, having the nucleotide and amino acid sequences presented in FIGURE 2 and in SEQ ID NO:2.
  • FIGURES 1 and 2 represents a novel gene and not frog NT-3, is based on the analysis of sequence homology based upon the comparison presented in FIGURE 4, and in a direct comparison of the sequence of NT-4 and the known neurotrophins NGF, BDNF and NT-3 presented in Table 1, below.
  • NT-4 is unlikely to be the frog homolog of NT-3, and is instead a related member of the gene family. From the above, NT-4 appears slightly closer in sequence to BDNF (70%) than to NT-3 and more distantly related to NGF (50%) .

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Abstract

L'invention concerne un nouveau facteur neurotrophique appelé neurotrophine-4 ou NT-4. L'existence du facteur neurotrophique a été établie en analysant l'ADN génomique de la grenouille par la réaction de chaîne de polymérase (PCR) à l'aide d'amorces sur des séquences d'amino acides conservés dans le facteur de croissance des nerfs (NGF) et dans le facteur neurotrophique dérivé du cerveau (BDNF). On croit savoir que le nouveau facteur neurotrophique est différent dès lors qu'il présente une homologie structurelle limitée avec n'importe quel facteur neurotrophique connu. Des séquences d'ADN codant NT-4, des procédés pour leur production et la production de molécules d'ADN de recombinaison, ainsi que des procédés et substances diagnostiques et thérapeutiques sont décrits.
PCT/US1992/004260 1991-05-20 1992-05-20 Facteur neurotrophique, sa preparation et ses utilisations WO1992020797A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025684A1 (fr) * 1992-06-12 1993-12-23 Regeneron Pharmaceuticals, Inc. Procedes therapeutique et diagnostique bases sur l'expression de la neurotrophine-4
WO1994023736A1 (fr) * 1993-04-15 1994-10-27 Regeneron Pharmaceuticals, Inc. Methode de traitement de la depression a l'aide de neurotrophines
WO1996017865A2 (fr) * 1994-12-05 1996-06-13 Boehringer Mannheim Gmbh Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5571675A (en) * 1994-04-25 1996-11-05 Genentech, Inc. Detection and amplification of candiotrophin-1(cardiac hypertrophy factor)
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
US7935342B2 (en) 2006-02-02 2011-05-03 Rinat Neuroscience Corp. Methods for treating obesity by administering a trkB antagonist

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU654302B2 (en) * 1990-09-25 1994-11-03 Genentech Inc. Novel neurothrophic factor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991003568A1 (fr) * 1989-08-30 1991-03-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur neurotrophique derive du cerveau
WO1992005254A1 (fr) * 1990-09-25 1992-04-02 Genentech, Inc. Nouveau facteur neurotrophique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991003568A1 (fr) * 1989-08-30 1991-03-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur neurotrophique derive du cerveau
WO1992005254A1 (fr) * 1990-09-25 1992-04-02 Genentech, Inc. Nouveau facteur neurotrophique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NEURON vol. 6, no. 5, May 1991, pages 845 - 858; HALLB\\K, F. ET AL.: 'Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 89, no. 7, 1 April 1992, WASHINGTON US pages 3060 - 3064; IP, N.Y. ET AL.: 'Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity' *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025684A1 (fr) * 1992-06-12 1993-12-23 Regeneron Pharmaceuticals, Inc. Procedes therapeutique et diagnostique bases sur l'expression de la neurotrophine-4
WO1994023736A1 (fr) * 1993-04-15 1994-10-27 Regeneron Pharmaceuticals, Inc. Methode de traitement de la depression a l'aide de neurotrophines
US5624806A (en) * 1994-04-25 1997-04-29 Genentech, Inc. Antibodies to cardiac hypertrophy factor and uses thereof
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5571675A (en) * 1994-04-25 1996-11-05 Genentech, Inc. Detection and amplification of candiotrophin-1(cardiac hypertrophy factor)
US5627073A (en) * 1994-04-25 1997-05-06 Genentech, Inc. Hybridomas producing antibodies to cardiac hypertrophy factor
US5679545A (en) * 1994-04-25 1997-10-21 Genentech, Inc. Gene encoding cardiac hypertrophy factor
US5723585A (en) * 1994-04-25 1998-03-03 Genentech, Inc. Method of purifying cardiac hypertrophy factor
US6117650A (en) * 1994-04-25 2000-09-12 Genentech, Inc. Assay for cardiac hypertrophy
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
WO1996017865A3 (fr) * 1994-12-05 1996-09-19 Boehringer Mannheim Gmbh Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives
WO1996017865A2 (fr) * 1994-12-05 1996-06-13 Boehringer Mannheim Gmbh Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives
AU693982B2 (en) * 1994-12-05 1998-07-09 Boehringer Mannheim Gmbh Genes and gene products for use in the diagnosis of degenerative nerve damage
US7935342B2 (en) 2006-02-02 2011-05-03 Rinat Neuroscience Corp. Methods for treating obesity by administering a trkB antagonist

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