WO1992020797A1

WO1992020797A1 - Neurotrophic factor, preparation and uses thereof

Info

Publication number: WO1992020797A1
Application number: PCT/US1992/004260
Authority: WO
Inventors: Thomas H. Large
Original assignee: Case Western Reserve University
Priority date: 1991-05-20
Filing date: 1992-05-20
Publication date: 1992-11-26
Also published as: AU2010592A

Abstract

A new neurotrophic factor named herein neurotrophin-4 or NT-4 is disclosed. The neurotrophic factor was deduced from the screening of frog genomic DNA by polymerase chain reaction (PCR) with primers to conserved amino acid sequences in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The new neurotrophic factor is believed to be distinct as it exhibits limited structural homology with any of the known neurotrophic factors. DNA sequences encoding NT-4, methods for their production and the production of recombinant DNA molecules, and both diagnostic and therapeutic methods and materials are disclosed.

Description

NEUROTROPHIC FACTOR, PREPARATION AND USES THEREOF

TECHNICAL FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Severe mental and physical disabilities result from the death of nerve or glial cells in the nervous system. The death of nerve or glial cells can be caused by neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) , Alzheimer's and Parkinson's diseases, Huntington's chorea, and 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. 4,699,875 and 4,701,407 to Appel; U.S. Patent No. 4,923,696 to Appel et al.; European Patent Application Publication No. 0 082 612 published 29 June 1983 in the name of Baylor College of Medicine; and references cited therein) .

The best characterized of such neurotrophic factors is nerve growth factor (NGF) . NGF has been demonstrated to be a neurotrophic factor for the forebrain cholinergic nerve cells that die during Alzheimer's disease and with increasing age. The loss of these nerve cells is generally considered responsible for many of the cognitive deficits associated with Alzheimer's disease and with advanced age.

Experiments in animals demonstrate that NGF prevents the death of forebrain cholinergic nerve cells after traumatic injury and that NGF can reverse cognitive losses that occur with aging (Hefti & Weiner, 1986, Ann. Neurology, .20.:275; Fischer et al., 1987, Natur . 329;65. These results suggest the potential clinical utility in humans of this neurotrophic factor in the treatment of cognitive losses resulting from the death of forebrain cholinergic nerve cells through disease, injury or aging.

Other neurotrophic factors have been isolated and characterized, among them brain-derived neurotrophic factor (BDNF) (Leibrock et al. , supra.); a variant named hippocampus-derived neurotrophic factor (HDNF) (Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA. 87:5454) ; neurotrophin-3 (NT-3) (Hohn et al. , supra. ; Maisonpierre et al., 1990, Science, 247:1446; Rosenthal et al., 1990, Neuron , 4.:767) ; and a ciliary neurotrophic factor (CNTF) the last disclosed in PCT International Publication No. WO 90/07341 published 12 July 1990, in the name of Synergen, Inc. All of the foregoing are incorporated herein by reference.

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.

SUMMARY OF THE INVENTION

In accordance with the invention, 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.

The present neurotrophic factor 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. At present, NT-4 may be characterized as follows: Amphibian NT-4 exhibits

(A) from about 60% to about 70% sequence homology with mammalian NGF and NT-3, and (B) from about 70% to about 80% sequence homology with mammalian BDNF.

The extent of distinction reflected above is significant, as the degree of conservation between the amphibian and mammalian sequences of the known neurotrophins is characteristically greater than 90% on the average. Thus the limited degree of conservation exhibited by NT-4 strongly supports its independent existence.

Further structural distinction is present in the portion of the sequence of NT-4 that has been identified thus far. The partial DNA sequence was initially determined in the frog genus Rana Pipiens. and thereafter confirmed by a like determination in the frog genus Xenopus.

Accordingly, 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. Similarly, the 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.

The primer-inclusive sequence as determined in Rana Pipiens and depicted in SEQ ID NO:l, is reproduced below.

AAG CAA TAT TTT TAC GAG ACC AAG TGC AAC CCT CAA GGT AGC 42 Lys Gin Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Gin Gly Ser 1 5 10

ACA ACC AAT GGT TGC CGA GGG GTG GAC AAA AGA CAA TGG ATA 84 Thr Thr Asn Gly Cys Arg Gly Val Asp Lys Arg Gin Trp lie 15 20 25

TCA GAG TGT AAA CCA AAA CAG TCA TAT GTA CGG GCA CTA ACT 126 Ser Glu Cys Lys Pro Lys Gin Ser Tyr Val Arg Ala Leu Thr 30 35 40

GTG TTG GAC AAG ATG GTG GGC TGG AGG TTT ATC CGC ATT GAC 168 Val Leu Asp Lys Met Val Gly Trp Arg Phe lie Arg lie Asp 45 50 55

AC 170

The primer-inclusive sequence as determined in Xenopus and presented in SEQ ID NO:2 is set forth below.

AAA CAG TAT TTC TTT GAA ACT AAT TGC AAT CCA TCA GGC AGC 42 Lys Gin Tyr Phe Phe Glu Thr Asn Cys Asn Pro Ser Gly Ser 1 5 10

ACC ACT AGA GGA TGC CGA GGT GTA GAC AAA AAG CAA TGG ATA 84 Thr Thr Arg Gly Cys Arg Gly Val Asp Lys Lys Gin Trp lie 15 20 25

TCT GGG TGC AAA GCA AAA CAG TCT TAT GTA AGG GCT CTG ACC 126 Ser Gly Cys Lys Ala Lys Gin Ser Tyr Val Arg Ala Leu Thr 30 35 40

ATA GAT GCC AAC AAG CTT GTG GGT TGG AGA TGG ATT AGT ATA 168 lie Asp Ala Asn Lys Leu Val Gly Trp Arg Trp lie Ser lie 45 50 55

GAT AC 173 Asp 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. For example, the probes may be prepared with a variety of known vectors, such as the phage λ 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.

In a further embodiment of the invention, 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.

From analysis of the sequence information presented herein, 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.

Accordingly, while the exact role that 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. In this connection, 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.

According to other preferred features of certain preferred embodiments of the present invention, 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. For example, the antibodies could be used to screen expression libraries to obtain the gene that encodes NT-4. Further, 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.

Therapeutic 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.

Accordingly, it is a principal object of the present invention to provide a newly discovered neurotrophic factor in purified form, identified herein as neurotrophin-4, or NT-4.

It is a further object of the present invention to provide a method for the synthesis of the neurotrophic factor NT-4 as aforesaid by the stimulation of cells known to produce NT-4.

It is a further object of the present invention to provide probes which facilitate screening of cDNA and genomic libraries in order to clone the animal and human genes encoding NT-4.

It is a still further object of the present invention to provide the complete nucleic acid and corresponding amino acid sequences for animal and human NT-4.

It is yet a a further object of the present invention to provide recombinant expression systems in which the nucleic acid sequence for human or animal NT-4 can be used to produce human or animal NT-4 protein.

It is a still further object of the present invention to provide agonists, antibodies, antagonists, and analogs thereof to the neurotrophic factor as aforesaid, and methods for their discovery and preparation.

It is a still further object of the present invention to provide promoters of the synthesis of the neurotrophic factor as aforesaid, and methods for their preparation.

It is a further object of the present invention to provide a method for detecting the presence and amount of the neurotrophic factor in mammals in which invasive, spontaneous, or idiopathic pathological states injurious to relevant neural cells, such as infection or trauma are suspected to be present.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking or inhibiting the activity of the neurotrophic factor in mammals.

It is a still further object of the present invention to provide a method for the treatment of mammals to modulate the amount or activity of the neurotrophic factor, so as to control the consequences of such presence or activity.

It is a still further object of the present invention to provide a method for the treatment of mammals to promote the amount or activity of the neurotrophic factor, so as to treat or avert the adverse consequences of neural degeneration and/or dysfunction regardless of origin. It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the neurotrophic factor or its binding partner(s) , or upon agents or drugs that control the production and/or activities of the neurotrophic factor.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g.. Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982) ; "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985) ; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984) ; "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. 1985) ; "Transcription And Translation" (B.D. Hames & S.J. Higgins eds. 1984); "Animal Cell Culture" (R.I. Freshney ed. 1986) ; "Immobilized Cells And Enzymes" (IRL Press, 1986) ; B. Perbal, "A Practical Guide To Molecular Cloning" (1984) .

Therefore if appearing herein, the following terms shall have the definitions set out below.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, 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. In keeping with standard polypeptide nomenclature, J. Biol. Chem.. 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

It should be noted that all 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.

The term "stimulus" 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 terms "the present neurotrophic factor", "neurotrophin-4" and "NT-4" as used throughout the present application and claims refer 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.

The term "trophic 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. In any discussion of the structure of particular double-stranded DNA molecules, 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. For purposes of defining the present invention, 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. Within the promoter sequence will be found 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.

The term "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.

The term "primer" as used herein 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.

As used herein, the terms "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. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, 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. Thus, when the heterologous region encodes a mammalian gene, 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". Preferably, "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. Hence, 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.

The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash.

In its primary aspect, the present invention concerns the isolation and identification of a newly discovered particular neurotrophic factor hereinafter referred to as neurotrophin-4 or NT-4. Accordingly, the partial DNA sequence initally determined in frog (Rana Pipiens) 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. More particularly, 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. Similarly, the 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.

ACA ACC AAT GGT TGC CGA GGG GTG GAC AAA AGA CAA TGG ATA 84 Thr Thr Asn Gly Cys Arg Gly Val Asp Lys Arg Gin Trp He 15 20 25

GTG TTG GAC AAG ATG GTG GGC TGG AGG TTT ATC CGC ATT GAC 168 Val Leu Asp Lys Met Val Gly Trp Arg Phe He Arg He Asp 45 50 55

AC 170

The primer-inclusive sequence as determined in Xenopus frog is set forth below as SEQ ID NO:2.

ACC ACT AGA GGA TGC CGA GGT GTA GAC AAA AAG CAA TGG ATA 84 Thr Thr Arg Gly Cys Arg Gly Val Asp Lys Lys Gin Trp He 15 20 25

ATA GAT GCC AAC AAG CTT GTG GGT TGG AGA TGG ATT AGT ATA 168 He Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Ser He 45 50 55 GAT AC 173 Asp

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. For example, the probes may be prepared with a variety of known vectors, such as the phage λ vector M13. In particular, 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.

As mentioned earlier, genomic DNA sequences, including transcriptional promoters, for NT-4 genes may be isolated in alternate species. For example, a human genomic library may be screened with ³²P-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.

Another feature of this invention is the expression of a full NT-4 DNA sequence derived from the partial sequences disclosed herein. As is well-known in the art, 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, of course, 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, for example, 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 λ, e.g., NM989, and other phage DNA, e.g.. Ml3 and Filamentous single stranded phage DNA; yeast plasmids such as the 2μ 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. Such 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 λ, 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 α- mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

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) and human cells and plant cells in tissue culture, the foregoing list being illustrative and not limitative.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. 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.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

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. In particular, invasive stimuli could be identified and detected by their ability to stimulate the production of NT-4 by the relevant NT-4 producing cells. For example, in this method, 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.

Likewise, 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. In 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.

Accordingly, 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.

In similar fashion, 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. For example, 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.

In the instance where 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. Alternately, 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.

Thus, 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.

In an immunoassay, 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.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, γ , ¹²⁵I, ¹³¹I, and ¹⁸⁶Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, 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) .

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of NT-4 in a mammal. In accordance with the testing techniques discussed above, 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.

Accordingly, a test kit may be prepared for the demonstration of the presence and activity of NT-4, comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of NT-4 or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, 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;

(b) if necessary, other reagents; and (c) directions for use of said test kit.

In a further variation, the 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:

(a) a labeled component which has been obtained by coupling NT-4 to a detectable label;

(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:

(i) a ligand capable of binding with the labeled component (a) ; (ii) a ligand capable of binding with a binding partner of the labeled component (a) ;

(iii) a ligand capable of binding with at least one of the component(s) to be determined; and

(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between NT-4 and a specific binding partner thereto.

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. Likewise, 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.

In particular, 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.

The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody- producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g.. M. Schreier et al.. "Hybridoma Techniques" (1980) ; Hammerling et al. , "Monoclonal Antibodies And T-cell Hybridomas" (1981) ; Kennett et al. , "Monoclonal Antibodies" (1980) ; see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against NT-4 peptides can be screened for various properties; i.e. , isotype, epitope, affinity, etc. Of particular interest are 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. Further, 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. As used herein, the term "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/B₀ value of 0.5, where B₀ 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 B₀ 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. The exact quantities, intervals of administration and administrative techniques respecting such 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. By immunizing an organism with antibodies, one can raise "anti-antibodies" that recognize them, including antibodies that recognize the idiotype. 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. (Kennedy, 1986.) 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, β-adrenalin, and rat brain nicotine and opiate receptors. (Carlsson and Glad, 1989) .

Taking advantage of this phenomenon, other neurotrophins and ligands may be isolated using anti-idiotypic antibodies. 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.

In a further embodiment, 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. 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. 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. In such instances, 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.

As discussed earlier, 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. Also, such 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.

Representative therapeutic 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. By way of illustration and not limitation, 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.

The following examples set forth the details of the isolation and identification of the present neurotrophic factor, and its cloning, sequencing and expression. Naturally, the specific materials and techniques set forth hereinafter are exemplary only and may vary, so that the following is presented as illustrative but not restrictive of the present invention.

EXAMPLE 1

Degenerate 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 μg DNA and 1 μg of both primers or each primer alone (Control) . To identify novel gene fragments, PCR was performed at low stringency - 5 cycles at 37°C stringency, 5 cycles at 45°C stringency and 30 cycles at 55°C. To confirm the amplification of appropriately sized fragments (190 base pairs) , products were run on a 6% acrylamide/TBE gel.

The 190 bp fragment was eluted from the gel and amplified at high stringency (55^βC) . 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.

EXAMPLE 2

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.

EXAMPLE 3

Sequence comparison of the clones prepared in Examples 1 and 2 with known mammalian and avian sequences was made as depicted in FIGURE 4, and confirmed the determination that NT-4 appears to be a distinct gene. In particular, neither of the frog NT-4 genes appears to be the amphibian homolog of mammalian or avian NT-3 as both exhibit substantial amino acid changes from this gene and are most closely related to BDNF.

The conclusion reached herein that the material depicted in 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.

TABLE 1 FROG .Rana Pipiens)

From a review Table 1, it is clear that the deduced amino acid sequence is invariant for the mature (active) portion of all known mammalian NT-3 genes. If the gene product named herein NT-4 were the frog homolog of mammalian NT-3, one would expect a sequence conservation similar to that for BDNF and NGF, on the order of about 93-95%. In fact, NT-3 and BDNF, and to a lesser extent NGF, appear to be strongly conserved from amphibian to mammalian species. By contrast, the NT-4 sequences are only from 60% to about 70% conserved with mammalian NT-3. The particular conserved amino acids are partially set forth in FIGURE 4, and comprise the following:

Ser,Thr,Pro,Ala,Gly Asn,Asp,Glu,Gin His,Lys,Arg Met ,He ,Leu,Val Phe,Tyr,Trp Cys

This evidence supports the conclusion that 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%) .

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

2. A DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, wherein at least a portion of said DNA sequence exhibits the following structural homology: (A) from about 60% to about 70% sequence homology of amphibian NT-4 with mammalian NGF and NT-3; and (B) from about 70% to about 80% sequence homology of amphibian NT-4 with mammalian BDNF.

3. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

4. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, wherein at least a portion of said DNA sequence exhibits the following structural homology: (A) from about 60% to about 70% sequence homology of amphibian NT-4 with mammalian NGF and NT-3; and (B) from about 70 to about 80% sequence homology of amphibian NT-4 with mammalian BDNF.

5. The recombinant DNA molecule of either of Claims 3 or 4, wherein said DNA sequence is operatively linked to an expression control sequence.

6. The recombinant DNA molecule of Claim 5, wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast α-mating factors.

7. A probe capable of screening for the neurotrophic factor NT-4 in alternate species prepared from the DNA sequence of either of Claims 1 or 2.

8. A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; wherein said DNA sequence is operatively linked to an expression control sequence.

9. A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, wherein at least a portion of said DNA sequence exhibits the following structural homology: (A) from about 60% to about 70% sequence homology of amphibian NT-4 with mammalian NGF and NT-3; and (B) from about 70% to about 80% sequence homology of amphibian NT-4 with mammalian BDNF; wherein said DNA sequence is operatively linked to an expression control sequence.

10. The unicellular host of either of Claims 8 or 9, wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast α-mating factors.

11. The unicellular host of either of Claims 8 or 9 wherein the unicellular host is selected from the group consisting of E. coli, Pseudomonas. Bacillus_f Streptomyσes, yeasts, CHO, Rl.l, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

12. The neurotrophic factor NT-4 as defined by any of Claims 1-4, 8 or 9 labeled with a detectable label.

13. The probe of Claim 7 labeled with a detectable label.

14. The neurotrophic faactor NT-4 of Claim 11 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

15. The probe of Claim 13 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

16. A composition comprising mature neurotrophic factor- 4 (mNT-4) substantially free of other polypeptides.

17. A polypeptide comprising an amino acid sequence substantially homologous to the amino acid sequence of mNT-4 selected from the group consisting of the the sequence set forth in FIGURE 1, and the sequence set forth in FIGURE 2.

18. A DNA molecule comprising a replicon and a heterologous coding sequence which encodes mNT-4.

19. A DNA molecule comprising a coding sequence for mNT- 4 under the control of transcriptional and translational control sequences which are capable of effecting the expression of said coding sequence in a host cell, wherein at least one of said transcriptional and translational control sequences is heterologous to said coding sequence.

20. The DNA molecule of Claim 19 wherein said coding sequence is uninterrupted by introns.

21. A composition of cells transformed by the DNA molecule of Claim 16 substantially free of cells that are not transformed by said DNA molecule.

22. The ceils of Claim 21 which are prokaryotic cells.

23. The cells of Claim 21 which are eukaryotic cells.

24. The cells of Claim 21 which are mammalian cells.

25. A method for producing mNT-4 which comprises culturing the composition of cells transformed by a DNA molecule according to Claim 18 under conditions whereby said mNT-4 is expressed, and recovering the expressed mNT-4.

26. The method of Claim 25 wherein said cells are prokaryotic.

27. The method of Claim 25 wherein said cells are eukaryotic.

28. The method of Claim 26 wherein said cells are yeast.

29. A method for preparing neurotrophic factor NT-4 derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; said method comprising: i. gathering a sample of cells from a mammal known to produce said neurotrophic factor; ii. incubating a portion of said cells with a stimulator material known to stimulate the production of said neurotrophic factor; iii. inducing said cells to produce said neurotrophic factor; and iv. isolating said neurotrophic factor from a supernatant harvested from the mass of said cells.

30. A method for preparing neurotrophic factor NT-4 derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; said method comprising: i. gathering a biological sample selected from tissues and fluid, known to contain said neurotrophic factor; ii. extracting said neurotrophic factor from said biological sample; and iii. purifying the extract of Step ii. to obtain said neurotrophic factor NT-4.

31. A method for producing the neurotrophic factor NT-4 comprising culturing the transformed host of either of Claims 8 or 9.

32. An antibody to neurotrophic factor NT-4, the neurotrophic factor to which said antibody is raised derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

33. An antibody to neurotrophic factor NT-4, the neurotrophic factor to which said antibody is raised derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, wherein at least a portion of said DNA sequence exhibits the following structural homology: (A) from about 60% to about 70% sequence homology of amphibian NT-4 with mammalian NGF and NT-3; and (B) from about 70% to about 80% sequence homology of amphibian NT-4 with mammalian BDNF.

34. The antibody of either of Claims 32 or 33 comprising a polyclonal antibody.

35. The antibody of either of Claims 32 or 33 comprising a monoclonal antibody.

36. An immortal cell line that produces a monoclonal antibody according to Claim 35.

37. The antibody of either of Claims 32 or 33 labeled with a detectable label.

38. The antibody of Claim 37 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

39. A method for measuring the presence of neurotrophic factor NT-4 derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; wherein said neurotrophic factor is measured by: i. preparing at least one sample of said neurotrophic factor from animal cells that have been exposed to a corresponding number of distinct known invasive stimuli; ii. preparing at least one corresponding antibody or binding partner directed to said neurotrophic factor samples; iii. placing a detectible label on a material selected from the group consisting of said neurotrophic factor samples and said antibody or binding partners thereto; iv. immobilizing a material selected from the group consisting of the material from Step iii. that is not labeled, and a biological sample from a mammal in which said inflammatory cytokine is suspected, on a suitable substrate; v. placing the labeled material from Step iii. in contact with said biological sample, and in contact with the immobilized material; vi. separating the material from Step iii. that is bound to said immobilized material from material from Step iii. not bound to said immobilized material; and vii. examining said bound material for the presence of said labeled material.

40. A method for measuring the binding sites for neurotrophic factor NT-4 derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; wherein said binding sites are measured by: i. preparing at least one sample of said neurotrophic factor from a corresponding number of distinct known invasive stimuli; ii. placing a detectible label on said neurotrophic factor sample; iii. placing the labeled neurotrophic factor sample in contact with a biological sample from a mammal in which binding sites for said neurotrophic factor are suspected; and iv. examining said biological sample in binding studies for the presence of said labeled neurotrophic factor sample.

41. The method of Claim 39 comprising a method for determining the presence of invasive or idiopathic stimuli in mammals.

42. The method of Claim 39 comprising a method for determining the presence and extent of a neuropathy associated with the activity of said neurotrophic factor NT-4 in mammals.

43. A method of testing the ability of a drug to modulate the production and/or activity of a neurotrophic factor capable of reacting with a receptor for the neurotrophic factor on cells in the mammalian body which comprises culturing a colony of test cells which has said receptor in a growth medium containing the neurotrophic factor, adding the drug under test and thereafter measuring the reactivity of said drug with the said receptor on said colony of test cells, said neurotrophic factor comprising a protein material derived from a a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

44. An assay system for screening drugs and other agents for ability to modulate production and/or activity of a neurotrophic factor comprising an observable cellular test colony innoculated with a neurotrophic factor comprising a protein derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (A) the DNA sequence of FIGURE 1; (B) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (C) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (D) the DNA sequence of FIGURE 2; (E) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (F) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (G) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

45. A test kit for the demonstration of the neurotrophic factor NT-4 in tissue, serum or an aqueous medium, comprising: (A) a predetermined amount of at least one labeled immochemically reactive component obtained by the direct or indirect attachment of said neurotrophic factor or a specific binding partner thereto, to a detectable label, said neurotrophic factor comprising a protein derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (i) the DNA sequence of FIGURE 1; (ii) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (iϋ) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (iv) the DNA sequence of FIGURE 2; (v) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (vi) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (vii) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; (B) other reagents; and (C) directions for use of said kit.

46. A method of treating neural debilitations and/or dysfunctions resulting from infectious and noninfectious diseases, genetic abnormality or trauma in a mammal, comprising administering to said mammal a therapeutic amount of a material selected from the group consisting of a neurotrophic factor, an agent capable of promoting the production and/or activity of said neurotrophic factor, an agent capable of mimicking the activity of said neurotrophic factor, an antibody to said neurotrophic factor, an antagonist to said neurotrophic factor, an agent capable of inhibiting the production and/or activity of said neurotrophic factor, and mixtures thereof, said neurotrophic factor comprising a protein that is derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (i) the DNA sequence of FIGURE 1; (ii) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (iϋ) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (iv) the DNA sequence of FIGURE 2; (v) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (vi) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (vii) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences.

47. The method of Claim 46 comprising a method for treating amyotrophic lateral sclerosis.

48. The method of Claim 46 comprising a method for treating multiple sclerosis.

49. The method of Claim 46 comprising a method for treating Parkinson's disease.

50. The method of Claim 46 comprising a method for treating Huntington's chorea.

51. The method of Claim 46 comprising a method for treating Alzheimer's disease.

52. The method of Claim 46 comprising a method for treating the neurodegenerative effects of diabetes.

53. A pharmaceutical composition for the treatment of neural debilitations and/or dysfunctions resulting from infectious and noninfectious diseases, genetic abnormality or trauma in mammals, comprising: A. a therapeutically effective amount of a material selected from the group consisting of neurotrophic factor NT-4, an agent capable of promoting the production and/or activity of said neurotrophic factor, an agent capable of mimicking the activity of said neurotrophic factor, an antibody to said neurotrophic factor, an antagonist to said neurotrophic factor, an agent capable of inhibiting the production and/or activity of said neurotrophic factor, and mixtures thereof, said neurotrophic factor comprising a protein in purified form that is derived from a DNA sequence or degenerate variant thereof, which encodes the neurotrophic factor NT-4, or a fragment thereof, said DNA sequence selected from the group consisting of: (i) the DNA sequence of FIGURE 1; (ii) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 28 to nucleotide number 146; (iϋ) the DNA sequence of FIGURE 1 extending inclusively from nucleotide number 49 to nucleotide number 107; (iv) the DNA sequence of FIGURE 2; (v) the DNA sequence of FIGURE 2 extending inclusively from nucleotide number 28 to nucleotide number 150; (vi) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and (vii) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences; or specific binding partners thereto; and B. a pharmaceutically acceptable carrier.

54. The pharmaceutical composition of Claim 53 wherein said neurotrophic factor is present in an amount sufficient to deliver a unit dose ranging up to about 4.0 g per day for an adult male.