JP2002526077A - Methods and compositions for polypeptides containing EGF repeats - Google Patents

Abstract

(57) Abstract The present invention provides novel polynucleotides, and proteins encoded by the polynucleotides, and further relates to therapeutic, diagnostic, and research uses for the polynucleotides and proteins. In particular, a polypeptide of the invention comprises an amino acid sequence that is similar to an EGF-repeat domain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

1. FIELD OF THE INVENTION The present invention provides novel polynucleotides, and the proteins encoded by those polynucleotides, and further relates to therapeutic, diagnostic, and research applications for those polynucleotides and proteins.

[0002]

Problems to be solved by the prior art and the invention

2. BACKGROUND OF THE INVENTION The technology targeted at the discovery of protein factors, including, for example, cytokines such as lymphokines, interferons, CSF and interleukins, has matured rapidly over the last decade. By currently used hybridization cloning and expression cloning techniques, novel polynucleotides can be used to generate information directly related to the discovered protein (ie, in the case of hybridization cloning, the partial DNA / amino acid sequence of the protein; expression cloning). In this case, it is cloned "directly" in the sense that it depends on the activity of the protein. More recently, `` indirect '' cloning techniques, such as signal sequence cloning, are techniques that isolate DNA based on the presence of the now well-recognized secretory leader sequence motif. What
Along with cloning techniques using PCR and low stringency hybridization cloning techniques, depending on the secretion characteristics in the case of leader sequence cloning, or in the case of PCR techniques, depending on the source of cells or tissues.
The state of the art has evolved until a large number of DNA / amino acid sequences for proteins known to have biological activity are available. The present invention
It relates to these proteins and the polynucleotides that encode them.

[0003]

[Means for Solving the Problems]

The compositions of the present invention include a novel isolated polypeptide, particularly a polypeptide comprising a novel EGF-repeat, an isolated polynucleotide encoding the polypeptide, i.e., a recombinant DNA molecule, Producing a naturally occurring variant, such as a cloned gene or a degenerate variant thereof, particularly an allelic variant, and an antibody that specifically recognizes one or more epitopes present on the polypeptide, and producing the polypeptide. Hybridomas are included.

[0004] In addition to the compositions of the invention, vectors, including expression vectors containing the polynucleotides of the invention, cells genetically designed to contain the polynucleotides, and expression of the polynucleotides, And cells that are genetically designed to

[0005] SEQ ID NO: 1 shows a portion of a nucleotide sequence encoding a polypeptide containing an EGF-repeat designated as EGF-Hy1. The deduced amino acid sequence of SEQ ID NO: 1 is set forth in SEQ ID NO: 2. The 5 'sequence of EGF-Hy1 was further obtained and the resulting sequence is set forth in SEQ ID NO: 10. The deduced amino acid sequence of SEQ ID NO: 10 is set forth in SEQ ID NO: 11. The polypeptide set forth in SEQ ID NO: 11 is 467 amino acids long, and the amino acid at position 38 in the sequence or another amino acid further upstream is the starting methionine. The EGF-repeat region is located at about amino acid residues 349-385, 387-423, and 425-467 of SEQ ID NO: 11, respectively, and the Prosite program (http: // www .expasy.ch / prosite /
) Is encoded by the nucleotides at positions 1048-1158, 1162-1270 and 1276-1404 of SEQ ID NO: 10. A cDNA or genomic DNA corresponding to SEQ ID NO: 1 or 10 containing another 5 'cDNA sequence can be further obtained by methods well known in the art. For example, a suitable cDNA or genomic DNA library can be screened under suitable hybridization conditions using SEQ ID NO: 1 or 10, or a portion thereof, as a probe. Alternatively, SEQ ID NO: 1 or 10 can be used as a suitable primer material to allow identification and / or amplification of the gene in a suitable genomic DNA or cDNA library. The authentic N-terminus of a mature protein can be confirmed by methods well known in the art (eg, through expression of DNA in a mammalian or other host cell that produces the polypeptide properly).

[0006] Polynucleotides of the present invention include naturally occurring DNA, or wholly or partially synthetic DNA, such as cDNA and genomic DNA, and RNA, such as mRNA. The isolated polynucleotide of the present invention has SEQ ID NO: 2 or
A polynucleotide encoding a polypeptide comprising the amino acid sequence of 11,
(Corresponding to amino acid residues 374 to 387 of SEQ ID NO: 11)
A polynucleotide encoding a polypeptide comprising the amino acid residue at position 14,
(Corresponding to amino acid residues 388 to 427 of SEQ ID NO: 11)
A polynucleotide encoding a polypeptide comprising the amino acid residues from position 54 to position 54 (corresponding to amino acid residues 428 to 467 of SEQ ID NO: 11);
Including, but not limited to, a polynucleotide encoding a polypeptide comprising the amino acid residues at positions 55-94, or a polypeptide comprising one or more of the EGF repeat regions of SEQ ID NO: 11. It is not limited.

[0007] The isolated polynucleotide of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 10, SEQ ID NO: (corresponding to nucleotides 1123-1404 of SEQ ID NO: 10). A polynucleotide comprising nucleotides 3 to 284 of SEQ ID NO: 1; a polynucleotide comprising nucleotides 3 to 44 of SEQ ID NO: 1 (corresponding to nucleotides 1123 to 1164 of SEQ ID NO: 10) nucleotide,(
(Corresponding to nucleotide positions 1165 to 1284 of SEQ ID NO: 10) No. 45 of SEQ ID NO: 1
A polynucleotide comprising the nucleotides at positions 164164, (128 of SEQ ID NO: 10)
A polynucleotide comprising the nucleotides at positions 165-284 of SEQ ID NO: 1, or a polynucleotide comprising one or more of the EGF repeat coding regions of SEQ ID NO: 10, (corresponding to nucleotides 5-1404). Including, but not limited to.

[0008] The polynucleotide of the present invention was also disclosed on September 18, 1998, in 10801 Universi.
ty Blvd., Manassas, Virginia, 20110-2209, American
A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited at the Type Culture Collection and given the accession number of ATCC 203198, ATCC
A polynucleotide containing the nucleotide sequence of the cDNA insert of clone pEGF-HY1 deposited under accession number 203198, including the full-length protein coding sequence of the cDNA corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198 Polynucleotide, cDN of clone pEGF-HY1, deposited under accession number ATCC 203198
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence corresponding to the A insert, or clone pEGF-HY deposited under accession number ATCC 203198.
Includes, but is not limited to, a polynucleotide encoding the mature protein corresponding to one cDNA insert.

The polynucleotide of the invention hybridizes with the complement of the nucleotide sequence of SEQ ID NO: 1 or 10 under stringent hybridization conditions,
And preferably a polynucleotide having EGF-Hy1 activity; a polynucleotide that is an allelic variant of any of the above-described polynucleotides; a polynucleotide that encodes a species homolog of any of the above-described proteins; or SEQ ID NO: 1 Or
Includes, but is not limited to, a polynucleotide encoding a polypeptide comprising a domain unique to 10 polypeptides or a truncated EGF repeat region.

[0010] The polynucleotides of the present invention additionally include the complement of any of the above polynucleotides.

The isolated polypeptide of the present invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 11; SEQ ID NO: (corresponding to amino acid residues 374 to 387 of SEQ ID NO: 11) : A polypeptide comprising the 1st to 14th amino acid residues of 2; (SEQ ID NO:
A polypeptide comprising the amino acid residues at positions 15-54 of SEQ ID NO: 2 (corresponding to the amino acid residues at positions 388-427 of 11); (Corresponding) comprises a polypeptide comprising amino acid residues 55-94 of SEQ ID NO: 2, or a polypeptide comprising one or more of the EGF repeat regions of SEQ ID NO: 11,
It is not limited to these.

[0012] The polynucleotide of the present invention further includes a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198, clone pEGF deposited under accession number ATCC 203198. -A polypeptide comprising the full-length protein encoded by the cDNA corresponding to the cDNA insert of -HY1, a polypeptide comprising the mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198, or ATCC 203198 But not limited to, the mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under Accession No.

[0013] Preferred embodiments include those wherein the protein produced by such a method is a mature form of the protein.

[0014] The protein composition of the present invention may further comprise an acceptable carrier, such as a hydrophilic, for example , pharmaceutically acceptable carrier.

The present invention also relates to a method for producing a polypeptide, comprising growing a cell culture of the present invention in a suitable culture medium and purifying the protein from the culture. including.

[0016] The polynucleotides of the present invention can be widely applied by various techniques known to those skilled in the field of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, and antisense DNA or RNA,
Uses in the production of their chemical analogs and the like are included. For example, if mRNA expression is greatly limited to a particular cell or tissue type, the polynucleotides of the invention may be used, for example, in in situ hybridization, to determine the presence of the particular cell or tissue mRNA in the sample. It can be used as a hybridization probe for detection.

As an example of another embodiment, the polynucleotide is used as an expressed sequence tag to identify an expressed gene or as described in Vollath et al., Science , 258, 52-59 (
1992) is used in diagnostics as an expressed sequence tag for physical mapping of the human genome as known in the art.

The polypeptides of the present invention can be used in a variety of conventional techniques and methods currently applied to other proteins. For example, the polypeptide of the present invention can be used to produce an antibody that specifically binds to the polypeptide. The polypeptide of the present invention can be used as a molecular weight marker and also as a food supplement.

A method for preventing, treating or alleviating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier. Provided.

In particular, the polypeptides and polynucleotides of the present invention may be used, for example, in epithelial tissues.
growth,For exampleIt can be adopted as a part of the method of promoting skin regeneration. Therefore
The polypeptides and polynucleotides of the invention can be used, for example, in tissue repair and regeneration.
Raw, corneal transplant therapy, burn treatment, skin graft manufacture and use, and wound healing, For example For treating ulcers, such as surgical incisions, gastric ulcers or diabetic ulcers
Can be adopted as part of In addition, polypeptides and polypeptides of the invention
Nucleotides may be involved in diseases involving cell necrosis and differentiation, such as leukemia.
Prevention and / or treatment, and part of a method for treating a neurological disease
You can enter.

[0021] The method of the invention further relates to a method for detecting the presence of a polynucleotide or polypeptide of the invention in a sample. The method can be used, for example, as part of the predictive and diagnostic evaluation of the disorders described above and to identify subjects who are predisposed to such conditions. In addition, the present invention provides methods for assessing the efficacy of a drug and for monitoring the progress of a patient's condition, involving clinical trials for the treatment of the aforementioned disorders.

The EGF-Hy1 protein of the present invention may be useful in certain cancer cells, such as tumors in the brain, lung, colon, gastrointestinal, breast, liver, prostate, kidney, urology, ovary, uterus, heart, bone, leukemia And can be expressed in blood cell malignancies such as, and skin cancers such as malignant melanoma. Other EGF motifs, including molecules, have been implicated in the progression of various cancers. Expression of EGF-Hy1 in tumor cells suggests that this protein is a potential marker for malignancy and a potential candidate in the development of small molecule therapies for the treatment of certain tumors It is.

Thus, the prognostic and diagnostic methods contemplated according to this aspect of the invention include tissue samples (eg, biological tissue obtained from brain, lung or other tissue) and body fluid samples (eg,
For example, methods of detecting or quantifying EGF-Hy1 polypeptide in cerebrospinal fluid, pleural fluid, sputum, ascites, blood, urine or other body fluids, particularly methods of diagnosing, prognosing or monitoring cancer.

The present invention also provides a method for identifying a compound that modulates the expression of a polynucleotide and / or polypeptide of the present invention. Such a method
For example, it can be used to identify compounds that can alleviate the symptoms of the disorders as described above. Such methods include, but are not limited to, assays for identifying compounds and other substances that interact (eg, bind) with a polypeptide of the invention.

The method of the present invention also includes a method for treating a disorder as described above, including the step of administering the compound to an individual exhibiting symptoms or constitution associated with the disorder. Good. In addition, the present invention is directed to methods for treating the disease or disorder by administering compounds and other agents that modulate the overall activity of the target gene product. Compounds and other substances are those that can provide such a modulation, either on the expression level of the target gene / protein or on the activity of the target protein.

The link between cancer and EGF-HY1 indicates that inhibitors of that activity (either by inhibiting the expression of the gene product or inhibiting the activity of the gene product itself) may be useful in treating cancer conditions. It suggests that there is. Such inhibitors include antisense polynucleotides, antibodies, and libraries or mixtures of, for example, inorganic or organic compounds (such as bacteria, fungi, mammals, insects or plant products, peptides, pseudopeptides and pseudoorganics). Other modulators have been identified through library screening. Such modulators can be administered parenterally, including into CSF, or topically via an implant or device.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions The term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide" are also referred to as heteropolymers of nucleotides, and are used interchangeably herein. In general, the nucleic acid segments provided by the present invention are combined with a genome to provide a synthetic nucleic acid that can be expressed in a microbial or viral operon or a recombinant transcription unit containing regulatory elements derived from eukaryotic genes. It may be assembled from fragments of short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides.

“Oligonucleotide fragment” or “polynucleotide fragment”, “portion”,
Or the term `` segment, '' has the length sufficient to identify or amplify the same or related portion of an mRNA or DNA molecule, for use in the polymerase chain reaction (PCR) or various hybridization methods, Refers to a stretch of polypeptide nucleotide residues.

“Oligonucleotides” or “nucleic acid probes” are prepared based on the polynucleotide sequences provided herein. Oligonucleotides are those that comprise a portion of a polynucleotide sequence, such as having at least about 15 nucleotides, and usually at least about 20 nucleotides. Nucleic acid probes include those portions of the polynucleotide that have less than about 6 kb, usually less than about 1 kb. After appropriate testing to rule out false positives, these probes can be used, for example, to determine whether a specific mRNA molecule is present in a cell or tissue, or by Walsh et al (Walsh, PS et al. ., 1992, PCR Methods
Appl 1 , pp. 241-250) may be used to isolate similar nucleic acid sequences from chromosomal DNA.

The term “probe” includes single- or double-stranded nucleic acids, either naturally occurring or recombinantly or chemically synthesized. Probes may be labeled by nick translation, Klenow-fill-in reaction, PCR or other methods well known in the art. Probes of the invention, their preparation and / or labeling are described in Sambrook, J. et al., 1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F
M. et al., 1989, Current Protocols in Molecular Biology, John Wiley &
Sons, New York, NY, the disclosure of which is incorporated by reference in its entirety.

The term “stringent” refers to a condition commonly understood in the art as stringent. Stringent conditions include high stringency conditions (ie, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS),
Hybridize to the bound DNA at 65 ° C. with 1 mM EDTA and wash with 0.1 × SSC / 0.1% SDS at 68 ° C.) and medium stringency conditions (ie, 42 ° C. Wash with 0.2 × SSC / 0.1% SDS).

As regards the hybridization of deoxyoligonucleotides, further exemplary hybridization conditions include 37 ° C. (for a 14 base oligo), 48 ° C. (for a 17 base oligo), 55 ° C. (for a 17 base oligo). Washing with 6 × SSC / 0.05% sodium pyrophosphate at 60 ° C. (for a 20 base oligo) and at 60 ° C. (for a 23 base oligo) is included.

The term “recombinant”, when used herein to refer to a polypeptide or protein, means that the polypeptide or protein is derived from a recombinant (eg, microbial or mammalian) expression system. I do. “Microorganism” shall refer to a recombinant polypeptide or protein produced in a bacterial or fungal (eg, yeast) expression system. As a product, a "recombinant microorganism" is defined as a polypeptide or protein that is substantially free of native endogenous material and with inherent concomitant glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., cultures of E. coli, should be free of glycosylation, and polypeptides or proteins expressed in yeast may be modified in mammalian cells. It should have glycosylation that is generally different from that expressed.

The term “recombinant expression vehicle or vector” shall refer to a plasmid or phage or virus or vector for expressing a polypeptide from a DNA (RNA) sequence. Expression vehicles include (1) promoters, enhancers, etc., one or more genetic elements that have a regulatory role in gene expression, (2) structural sequences that are transcribed into mRNA and translated into proteins, or It can include a transcription unit comprising a combination of a coding sequence, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence which enables extracellular secretion of proteins translated by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not subsequently be excised from the expressed recombinant protein, yielding the final product.

The term “recombinant expression system” refers to a host cell in which the recombinant transcription unit is stably integrated into chromosomal DNA, or a host cell that carries the recombinant transcription unit extrachromosomally. A recombinant expression system as defined herein should express a heterologous polypeptide or protein upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. The term also includes host cells that have stably integrated one or more recombinant genetic elements that have a regulatory role in gene expression, such as, for example, promoters or enhancers. The recombinant expression system as defined herein is an endogenous DN to be expressed.
Upon induction of a regulatory element linked to the A segment or gene, it should express a polypeptide or protein endogenous to the cell. The cells can be prokaryotic or eukaryotic.

The term ORF of "open reading frame" refers to a sequence of nucleotide triplets that does not contain any stop codons, encodes an amino acid, and is transcribable to a protein.

The term “expression modulating fragment” EMF refers to a series of nucleotides that modulate the expression of an operably linked ORF or other EMF.

As used herein, a sequence is said to “modulate the expression of an operably linked sequence” if the expression of the sequence is altered by the presence of EMF.
EMFs include, but are not limited to, promoters and sequences that modulate the promoter (inducible elements). Certain classes of EMF are operably linked in response to specific modulators or physiological events
ORF or a fragment that induces expression.

As used herein, the UMF of “uptake modulating fragment” is:
A sequence of nucleotides that mediates the uptake of a ligated DNA fragment into a cell. UMF can be easily identified using UMF, known as a target sequence or target motif, in the following computer-based system.

The presence and activity of UMF can be confirmed by attaching a candidate UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with the appropriate host under appropriate conditions and the incorporation of the marker sequence is determined. As described above, UMF should increase the frequency of incorporation of linked marker sequences.

The term “active form” refers to the biological and / or biological properties of various naturally occurring polypeptides.
Alternatively, it refers to a form of the polypeptide that retains immunological activity.

The term “naturally occurring polypeptide” refers to a polypeptide produced by a cell that is not genetically engineered, and in particular, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and It includes various polypeptides in which post-transcriptional modification of the polypeptide is found, including but not limited to acylation.

The term “derivative” refers to ubiquitin binding, labeling (eg, labeling with radionuclides or various enzymes) that would not normally occur in human proteins.
, Pegylation (derivatization with polyethylene glycol), and chemical synthesis of amino acids such as ornithine.

The term “recombinant variant” refers to various polypeptides created using recombinant DNA technology that differ from naturally occurring polypeptides by amino acid insertions, deletions and substitutions. And The strategy for determining which amino acid residue should be substituted, added or deleted without impairing the target activity such as cell trafficking is to change the sequence of a specific polypeptide to the sequence of a homologous peptide. It can be found by comparing and minimizing the number of amino acid sequence changes made to regions of high homology.

Preferably, the amino acid “substitution” replaces one amino acid with a similar structural and / or
Alternatively, it may be a result of substitution with another amino acid having a chemical property, that is, a conservative amino acid substitution. Amino acid substitutions may be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine;
Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; positively charged (basic) amino acids include
Arginine, lysine and histidine are included, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insert" or "
"Deletions" typically range from about 1 to 5 amino acids. Using recombinant DNA technology and assaying the activity of the resulting recombinant variants, systematically inserting, deleting or substituting amino acids in a polypeptide molecule to experimentally determine acceptable changes. You may decide.

Alternatively, if a modification in function is desired, insertions, deletions or non-conservative changes can be made to produce a modified polypeptide. Such modifications can, for example, alter one or more of the biological functions or biochemical properties of a polypeptide of the invention. For example, such modifications may include ligand binding affinity,
The properties of the polypeptide, such as interchain affinity, or degradation / rotation rates, may be altered.
In addition, such modifications can be selected to produce polypeptides that are more suitable for expression, scale-up, etc., in the host cell selected for expression. For example, to remove disulfide bridges, delete cysteine residues,
Alternatively, it can be replaced with another amino acid.

As used herein, the term “substantially equivalent” refers to the nucleotide sequence and amino acid sequence, including, for example, variant sequences, between the reference sequence and the subject sequence. It shall be possible to refer to sequences which differ from the reference sequence by one or more substitutions, deletions or additions, which do not cause a functional disparity in which the overall effect has an adverse effect. Usually, such substantially equivalent sequences will have no more than 20% difference from one of the sequences disclosed herein ( ie , the replacement of an individual residue in a substantially equivalent sequence). , The number of additions and / or deletions divided by the total number of residues in substantially equivalent sequences as compared to the corresponding reference sequence is less than or equal to about 0.2)
. Such sequences are said to have 80% sequence identity with the sequences listed above. In one embodiment, a substantially equivalent, eg , variant, sequence of the invention has no more than 10% (90% sequence identity) difference from the disclosed sequences, and in a further embodiment, 5%. Not more than (95% sequence identity), and in a further embodiment 2
% (98% sequence identity). A substantially equivalent, eg , variant, amino acid sequence of the present invention will generally have at least about 95% sequence identity with the amino acid sequence set forth above, while a substantially equivalent nucleotide sequence of the present invention will comprise: For example, lower percentages (eg, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, or at least about 65%, taking into account duplication or degeneracy of the genetic code) ).
For purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. To determine equivalence, truncation of the mature sequence (eg, due to mutations creating a false stop codon) should be ignored.

A nucleic acid sequence encoding such a substantially equivalent sequence, eg , a sequence having the above-mentioned identity, can be prepared by standard hybridization methods known to those skilled in the art.
It can be isolated and identified as predetermined.

If desired, the expression vector may be designed to include a “signal or leader sequence” that directs the polypeptide through the cell membrane. Such sequences may be naturally occurring in the polypeptides of the invention, or may be supplied from heterologous protein sources by recombinant DNA technology.

A polypeptide “fragment”, “portion” or “segment” is at least about 5 amino acids, optionally at least about 7 amino acids, usually at least about 9-13 amino acids, and many more. In embodiments, the stretch is at least about 17 or more amino acid residues. To be active, any polypeptide must have sufficient chain length to exhibit biological and / or immunological activity.

Alternatively, recombinant variants encoding the same or similar polypeptides may be synthesized or selected by taking advantage of the “degeneracy” of the genetic code. Various codon substitutions, such as silent changes creating various restriction sites, may be introduced to optimize cloning or expression into a plasmid or viral vector in a particular prokaryotic or eukaryotic system. . Mutations in the polynucleotide sequence can be added to the polypeptide to modify the properties of any portion of the polypeptide to alter properties such as ligand binding affinity, interchain affinity, or degradation / rotation rate. And the domain of the polypeptide or other polypeptide to be expressed.

As used herein, “activated” cells are those that are involved in extracellular or intracellular membrane transport, including the transport of neurosecretory or enzymatic molecules that are part of normal or disease processes. is there.

As used herein, the term “purified” refers to a nucleic acid or polypeptide,
It indicates that other biological macromolecules such as polynucleotides, proteins and the like are substantially absent. In certain embodiments, the polynucleotide or polypeptide is purified to comprise at least 95%, more preferably at least 99.8%, by weight of the foregoing biological macromolecules, provided that water, buffer and other Small molecules, especially those having a molecular weight of less than 1000 Daltons may be present).

As used herein, the term “isolated” refers to at least one other component (eg, a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide in a natural source. Nucleic acids or polypeptides isolated from According to one embodiment, the nucleic acid or polypeptide is a solvent, if any,
It is in the presence of only buffers, ions or other components normally present in the solution. The terms "isolated" and "purified" are not intended to include nucleic acids or polypeptides that are present in their natural source.

The term “infection” is used by using a virus or viral vector.
Refers to introducing a nucleic acid into a suitable host cell.

The term “transformation” refers to the introduction of DNA into a suitable host cell such that the DNA becomes replicable, either as an extrachromosomal element or by integration into the chromosome.

The term “transfection” refers to the expression vector being carried by a suitable host cell, whether or not any coding sequences are actually being expressed.

The term “intermediate fragment” refers to a nucleic acid having a chain length of 5 to 1000 base pairs, preferably 10 to 40 base pairs.

A “secreted” protein is a protein that, when expressed in a suitable host cell, is transported across or across a membrane, including the resulting transport of its amino acid sequence by a signal sequence. Name. “Secreted” proteins include, but are not limited to, proteins that are entirely (eg, soluble proteins) or partially (eg, receptors) secreted from the expressing cells. “Secreted” proteins also include proteins that are transported across the membrane of the endoplasmic reticulum.

Each of the above terms is intended to encompass all of the above unless otherwise stated.

Polynucleotides and nucleic acids of the present invention The nucleotide and nucleic acid sequences of the present invention are shown below. Fragments of the proteins of the present invention that can exhibit biological activity are also encompassed by the present invention. Protein fragments may be single-stranded or, for example, HU Saragovi et al., Bio / Technology 10, 773-778 (1992), which is incorporated herein by reference.
) And RS McDowell et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992). Such fragments may be fused to a carrier molecule such as an immunoglobulin for many purposes, including increasing the valency of the protein binding site. For example, a fragment of a protein may be fused to the Fc portion of an immunoglobulin via a "linker" sequence. For bivalent forms of the protein, such fusions may be made to the Fc portion of an IgG molecule. Other immunoglobulin isotypes can also be used to make such fusions. For example, a protein-IgM fusion would be made with a divalent form of the protein of the invention.

The present invention further provides both the full-length and mature forms of the disclosed proteins corresponding to SEQ ID NO: 2 or 11. Mature forms of such proteins are obtained by expressing the disclosed full-length polynucleotides in a suitable mammalian cell or other host cell. The sequence of the mature form of such a protein can also be determined from the amino acid sequence of the full-length form.

[0063] The present invention also provides genes corresponding to the cDNA sequences disclosed herein.
The corresponding gene can be isolated according to known methods, using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information for identification and / or amplifying the gene with an appropriate genomic library or other source of genetic material.

When the protein of the invention is membrane-bound (eg, a receptor), the invention also provides a soluble form of such a protein. In such forms, the intracellular and transmembrane domains of the protein are deleted so that the protein expressing it is completely secreted. The intracellular and transmembrane domains of the proteins of the invention can be identified by known techniques for determining such domains from sequence information.

[0065] Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs can be isolated and identified by making a suitable probe or primer from the sequences provided herein and screening for a suitable nucleic acid source from the desired species.

The present invention further relates to allelic variants of the disclosed polynucleotides or proteins, ie, isolated polynucleotides which also encode proteins identical, homologous or related to those encoded by the polynucleotide. Also encompasses naturally occurring alternative forms.

The compositions of the present invention include recombinant DNA molecules, cloned genes or degenerate variants thereof, particularly naturally occurring variants such as allelic variants, isolated novel polypeptides, and the like. Includes antibodies that specifically recognize one or more epitopes present on the polypeptide.

[0068] Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs can be isolated by making suitable probes or primers from the sequences provided herein and screening for a suitable nucleic acid source from the desired species.

The present invention also provides allelic variants of the disclosed polynucleotides or proteins,
That is, it includes naturally occurring alternative forms of the isolated polynucleotide that also encode a protein identical, homologous or related to that encoded by the polynucleotide.

5.2. Nucleic Acids of the Invention Isolated polynucleotides of the present invention include polynucleotides encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 11, SEQ ID NO: 2 or 11
A polynucleotide encoding a full-length or mature protein corresponding to
Includes, but is not limited to, a polynucleotide encoding an EGF repeat region or a specific domain thereof. For example, in the polynucleotide of the present invention,
(Corresponding to amino acid residues 374 to 387 of SEQ ID NO: 11)
A polynucleotide that encodes a polypeptide comprising the amino acid residues at positions -14, (corresponding to amino acid residues 388-427 of SEQ ID NO: 11)
A polynucleotide encoding a polypeptide comprising the amino acid residues 15-54, corresponding to amino acids 55-94 of SEQ ID NO: 2 (corresponding to amino acid residues 428-467 of SEQ ID NO: 11) It further includes a polynucleotide encoding a polypeptide comprising an amino acid residue, or a polypeptide comprising one or more of the EGF repeat regions of SEQ ID NO: 11.

[0071] In one embodiment, the isolated polynucleotide of the present invention comprises SEQ ID NO:
A polynucleotide comprising 1 or 10 nucleotide sequences, (SEQ ID NO: 10
A polynucleotide comprising nucleotides 3 to 284 of SEQ ID NO: 1 (corresponding to nucleotides 1123 to 1404 of SEQ ID NO: 10); SEQ ID NO: (corresponding to nucleotides 123 to 1164 of SEQ ID NO: 10) A polynucleotide comprising nucleotides 3 to 44 of SEQ ID NO: 1; nucleotides 45 to 164 of SEQ ID NO: 1 (corresponding to nucleotides 1165 to 1284 of SEQ ID NO: 10). A polynucleotide, a polynucleotide comprising nucleotides 165-284 of SEQ ID NO: 1 (corresponding to nucleotides 1285-1404 of SEQ ID NO: 10), or SEQ ID NO:
Includes, but is not limited to, a polynucleotide comprising one or more of the ten EGF repeat coding regions.

The polynucleotide of the present invention further includes a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198, ATCC 203
A polynucleotide comprising the nucleotide sequence of the cDNA insert of clone pEGF-HY1 deposited under accession number 198, clone pEG deposited under accession number ATCC 203198
A polynucleotide comprising the full-length protein coding sequence of the cDNA corresponding to the cDNA insert of F-HY1, a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198 Or c of clone pEGF-HY1 deposited under accession number ATCC 203198
Includes, but is not limited to, a polynucleotide encoding the mature protein corresponding to the DNA insert.

The polynucleotide of the present invention preferably has EGF-Hy1 biological activity and hybridizes under stringent hybridization conditions to the complement of the nucleotide sequence of SEQ ID NO: 1 or 10. A polynucleotide that encodes a species homologue of any of the above proteins; or a domain or a unique to the polypeptide of SEQ ID NO: 2 or 11. Includes, but is not limited to, a polynucleotide encoding a polypeptide that includes a truncated region.

The polynucleotides of the present invention additionally include the complement of any of the above polynucleotides.

The polynucleotide of the present invention also includes a nucleotide sequence substantially equivalent to the aforementioned polynucleotide. A polynucleotide of the present invention is one that can have at least about 80%, more preferably usually at least about 90%, and even more preferably usually at least about 95% sequence identity with the above-described polynucleotides. The invention further includes a nucleotide sequence having at least about 80%, more preferably usually at least about 90%, and even more preferably usually at least about 95% sequence identity with the polynucleotide encoding the polypeptide described above. A complement of the polynucleotide is also provided. A polynucleotide can be DNA (genomic, cDNA, amplified or synthesized DNA) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art, and are used, for example, to determine hybridization conditions that can routinely isolate a polynucleotide of the desired sequence. A method may be included.

The polynucleotides of the present invention can be linked to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
See NY). Useful nucleotide sequences for linking polypeptides include:
Vectors well known in the art, such as plasmids, cosmids,
Combinations of lambda phage derivatives, phagemids and the like are included. Accordingly, the present invention also provides a vector comprising the polynucleotide of the present invention, and a host cell comprising the polynucleotide. Generally, the vector will include at least one origin of replication functional in the organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. The vector of the present invention includes an expression vector, a replication vector, a vector for producing a probe, and a sequencing vector.
A host cell of the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or a part of a multicellular organism.

The sequences included within the scope of the present invention are not limited to the specific sequences described,
It also includes its allelic variants. The allelic variant is SEQ ID NO: 1 or
The sequence shown at 10, a representative fragment thereof, or a nucleotide sequence that is at least 99.9% identical to SEQ ID NO: 1 or 10 can be readily determined by comparing to a sequence from another isolate of the same species. it can. Furthermore, to accommodate codon variability, the present invention also encompasses nucleic acid molecules that encode amino acid sequences similar to those encoded by the particular ORFs disclosed herein. In other words, it is clearly contemplated, for example, to replace one codon with another codon encoding the same amino acid in the coding region of the ORF. By performing rearrangement (ie, sequencing of both strands) of particular fragments, such as ORFs, in both directions, any of the particular sequences disclosed herein can be easily screened for errors.

The present invention further provides a recombinant construct comprising a nucleic acid having the sequence of SEQ ID NO: 1 or 10 or a fragment thereof. The recombinant constructs of the present invention include a vector such as a plasmid or viral vector, into which the sequence of SEQ ID NO: 1 or 10 or a fragment thereof has been inserted in the forward or reverse direction. Of the present invention
In the case of a vector comprising one of the ORFs, the vector may further comprise regulatory sequences operably linked to the ORF, including, for example, a promoter. For vectors comprising EMF and UMF of the present invention, the vector may further comprise a marker sequence or a heterologous ORF operably linked to EMF or UMF. Numerous suitable vectors and promoters are known by those of skill in the art and are commercially available for making recombinant constructs of the present invention. The following vectors are given as examples. Bacteria: pBs, phagescript, PsiX174, pBluescript SK
, PBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3
, PKK233-3, pDR540, pRIT5 (Pharmacia). Fungi: pWLneo, pSV2cat, pOG44,
PXTI, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotides of the present invention may be used to produce pMT2 or pED expression as disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), for recombinantly producing proteins. It may be operably linked to an expression control sequence such as a vector. Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known; R. Kaufman,
Methods in Enzymology, 185, 537-566 (1990). As used herein, the term "operably linked" refers to a linked polynucleotide /
It means that the isolated polynucleotide of the present invention and the expression control sequence are adapted to a vector or a cell so that the protein is expressed by a host cell transformed (transfected) with the expression control sequence. .

The promoter region is a CAT (chloramphenicol transferase) vector
Or other vectors having a selectable marker to provide various desired
Can be selected from genes. Two suitable vectors are pKK232-8 and pCM7
. Particularly preferred bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R And trc are included. Eukaryotic promoters include early CMV and HSV thymidine
Kinase, early and late SV40, LTR from retrovirus, and mouse
Tallothionein-I is included. Selection of the appropriate vector and promoter
, Within the ordinary skill level of the art. Generally, a recombinant expression vector
Is the origin of replication, for exampleE. coliAmpicillin resistance gene andS. cerevisi ae Selectable markers that enable transformation of host cells, such as the TRP1 gene
Promoters from highly expressed genes that result in direct transcription of downstream structural genes
And Such promoters are, inter alia, 3-phosphoglycerate
Sugars such as kinases (PGKs), a-factors, acid phosphatases or heat shock proteins
Those derived from the operon encoding a degrading enzyme are preferred. The heterologous structural sequence is
Transcription initiation and termination sequences and, preferably, the cell membrane periphery of the translated protein.
With a leader sequence that can provide direct secretion into the marginal space or extracellular medium
In the appropriate phase. Optionally, the heterologous sequence is, for example, the set to be expressed.
Providing desired characteristics, such as stabilizing the replacement product or facilitating its purification
To encode a fusion protein including an N-terminal identification peptide
You. A useful expression vector for bacteria is an operable read with a functional promoter.
In the reconstitution phase, the desired protein is coded, along with the appropriate transcriptional start and stop
It is constructed by inserting a structural DNA sequence. The vector is
One or more expressions to ensure retention and to provide for amplification in the host, if desired.
It contains a type selectable marker and an origin of replication. Prokaryotes suitable for transformation
For cellular hosts,E. coli,Bacillus subtilis,Salmonella typhimuriumand
Various in Pseudomonas, Streptomyces, and Staphylococcus spp.
Species, but other options are possible.

As a representative but non-limiting example, expression vectors useful for bacteria include selections from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Mention may be made of those containing a possible marker and a bacterial origin of replication. Such commercially available vectors include, for example, pK
K223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Pr
omega Biotec, Madison, WI, USA). These pBR
The 322 "backbone" section is combined with an appropriate promoter and the structural sequence to be expressed. After transforming a suitable host strain and growing the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (eg, temperature shift or chemical induction), and The cells are cultured for an additional period. Usually, cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is collected and further purified.

Included within the scope of the nucleic acid sequences of the present invention are nucleic acid sequences that hybridize under stringent conditions to fragments of the DNA sequence shown in FIG. 1 or the complement thereof, wherein said fragment is about It should be more than 10 base pairs, preferably about 20-50 base pairs, or even more than 100 base pairs. According to the present invention, a polynucleotide sequence encoding a novel nucleic acid, or a functional equivalent thereof, is used to create a recombinant DNA molecule that results in the expression of such a nucleic acid, or a functional equivalent thereof, in a suitable host cell. May be.

The nucleic acid sequences according to the invention furthermore relate to sequences which code for variants of said nucleic acids.
These mutated amino acid sequences can be prepared by methods known in the art by introducing appropriate nucleotide changes into a natural or mutated polynucleotide. The construction of a mutated amino acid sequence involves two variables:
There is the location of the mutation and the nature of the mutation. The mutated amino acid sequence of the nucleic acid is preferably constructed by mutating a polynucleotide to give an amino acid sequence that does not occur in nature. These amino acid changes may be made at different sites (variable sites) in nucleic acids from different species, or at highly conserved regions (constant regions). Sites at such positions are preferably, for example, first conservatively selected (eg, hydrophobic amino acids as different hydrophobic amino acids) and then more separated (eg, hydrophobic amino acids as charged amino acids). , May be modified sequentially by making substitutions, followed by deletions or insertions at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are preferably contiguous. Amino acid insertions include amino and / or carboxy terminal fusions ranging in chain length from 1 to 100 residues or more, as well as insertion of one or more amino acid residues into the sequence. Insertions into the sequence may range from about 1 to 10 amino acid residues, preferably 1 to 5 residues. Examples of terminal insertions include heterologous signal sequences required for secretion in different host cells or for intracellular targeting.

In a preferred method, the polynucleotide encoding the novel nucleic acid is altered by site-directed mutagenesis. In this method, an oligonucleotide sequence encoding the polynucleotide sequence of the desired mutated amino acid and, in addition, sufficient both sides of the altered amino acid to form a stable duplex on either end of the altered site. Use adjacent nucleotides. In general, the technique of site-directed mutagenesis is well known to those skilled in the art, and this technique is described in Edelman et al., DNA
, 2 , p. 183 (1983). A versatile and effective way to create site-specific alterations in polynucleotide sequences has been described by Zoller and
Smith, Nucleic Acids Res. 10 , 6487-6500 (1982). PCR may be used to generate a mutated amino acid sequence of a novel nucleic acid. When a small amount of template DNA is used as a starting material, a desired mutant amino acid sequence can be prepared using primers that are slightly different in sequence from the corresponding region in the template DNA.
PCR amplification results in a population of DNA fragment products that differ from the polynucleotide template encoding the polypeptide at the position specified by the primers. The product of the DNA fragment replaces the corresponding region of the plasmid and results in the desired mutated amino acid sequence.

Additional techniques for generating variant amino acid sequences include the cassette mutagenesis technique described in Wells et al., Gene 34 , 315 (1985); and, for example, Sambroo
There are other mutagenesis techniques well known in the art, such as those in K et al., supra , and Current Protocols in Molecular Biology , Ausubel et al.
Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially similar or functionally equivalent amino acid sequences are used in practicing the present invention for the cloning and expression of these novel nucleic acids. You can also. Such DNA sequences include those that are capable of hybridizing under stringent conditions to the appropriate novel nucleic acid sequence.

5.3. Hosts The present invention further provides host cells that are genetically engineered to contain a polynucleotide of the present invention. For example, such a host cell comprises a nucleic acid of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention further provides host cells that are genetically engineered to express a polynucleotide of the present invention, wherein the polynucleotide drives expression of the polynucleotide in a cell. It is in operative association with a heterologous regulatory sequence.

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into host cells can be accomplished by calcium phosphate transfection, DEAE, transfection using dextran or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
). Host cells containing one of the polynucleotides of the present invention can be used in conventional ways to produce the gene product encoded by the isolated fragment (in the case of ORFs) or can be heterologous under the control of EMF. It can be used to produce proteins.

A variety of host / vector systems can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cells, COS cells and Sf9 cells, as well as prokaryotic hosts such as E. coli and B. subtilis. . Most preferred cells are those that do not normally express a given polypeptide or protein, or that express the polypeptide or protein at low native levels. The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. In order to produce such a protein, a cell-free translation system using RNA derived from the DNA construct of the present invention can also be used. Cloning and expression vectors suitable for use in prokaryotic and eukaryotic hosts are described in Samb, Inc., the disclosure of which is incorporated herein by reference.
rook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spr
ing Harbor, New York (1989).

[0089] Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian cell expression systems include Gluzman, Cell 23, 175 (1981
COS-7 cells of monkey kidney fibroblasts according to), and for example, C127, 3T3, CHO, HeLa
And other cell lines capable of expressing the compatible vector, such as the BHK cell line. The mammalian expression vector will include an origin of replication, a suitable promoter, and any necessary ribosome binding proteins, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'flanking non-transcribed sequences. Like. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splice and polyadenylation sites, may be used to provide the required non-transcribed genetic elements. Recombinant polypeptides and proteins produced in bacterial cultures
Usually, it is first extracted from the cell pellet and then subjected to one or more salting out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can also be used as needed to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for the final purification step. Bacterial cells used for protein expression can be disrupted by a variety of convenient methods, including freeze-thaw cycling, sonication, mechanical disruption, or use of fine lysing agents.

A number of cell types can serve as suitable host cells for expression of the protein. Mammalian host cells, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epithelial A431 cells, human Colo205 cells, 3T3 cells,
CV-1 cells, other transformed primate cell lines, normal diploid cells, primary tissue in
In vitro culture derived cell lines, primary grafts, HeLa cells, mouse L cells, BHK, HL-60
, U937, HaK or Jurkat cells and the like.

Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida or various yeast strains capable of expressing heterologous proteins are included. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhim
urium, or various bacterial strains capable of expressing a heterologous protein are included. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced there to obtain a functional protein, such as by phosphorylation or glycosylation at the appropriate site. Such covalent attachment can be performed using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising a polynucleotide of the present invention under the control of an inducible regulatory element, wherein The regulatory sequence of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace regulatory regions present in genes with regulatory sequences isolated from different genes or novel regulatory sequences, which are synthesized by genetic engineering techniques. Such regulatory sequences may include promoters, enhancers, scaffold attachment regions, natural regulatory elements, transcription initiation sites, regulatory protein binding sites, or combinations of such sequences. Alternatively, sequences that affect the structure or stability of the RNA or manufactured protein may be replaced, removed, added or polyadenylation signals,
Other modifications by targeting, including mRNA stabilization elements, splice sites, leader sequences to enhance or modify protein transport or secretion, or other sequences that alter or improve the function or stability of the protein or RNA molecule. May be done.

A targeting event is simply the insertion of regulatory sequences, such as inserting a new promoter or enhancer or both upstream of the gene.
It may be to place the gene under the control of a new regulatory sequence. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as a deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may be to replace an existing element, e.g., an enhancer with a broader cell type specificity than the naturally occurring element, or a cell type specificity different from the naturally occurring element Can replace the tissue-specific enhancer. Here, the naturally occurring sequence is deleted and a new sequence is added. In all cases, the identification of the targeting event is such that the targeting DNA is such that the exogenous DNA permits selection of cells that have integrated into the host cell genome.
This can be done without difficulty by using one or more selectable marker genes adjacent to A. The identification of the targeting event can also be such that the negative selectable marker is linked to the exogenous DNA, but the negative selectable marker is flanked by the targeting sequence, and to the sequence contained in the host cell genome. Stable integration of the negatively selectable marker as a result of the correct homologous recombination event of E. coli does not occur, and can be done without difficulty by using one or more marker genes that exhibit the characteristics of negative selection. . Markers useful for this purpose include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

Gene targeting or gene activation techniques that can be used with such features of the present invention are described in US Pat. No. 5,272,071 to Chappel, US Pat.
5,578,461, Selden et al., International Application No. PCT / US92 / 09627 (WO93 / 09222), Skoultchi
These are described in more detail in International Application No. PCT / US90 / 06436 (WO91 / 06667), the entire disclosure of which is incorporated herein by reference.

5.4. Polypeptides of the Invention Isolated polypeptides of the invention include, but are not limited to, polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or 11. The polypeptides of the present invention may further comprise one or more EGF repeat regions or specific regions in the amino acid sequence of SEQ ID NO: 2 or 11, such as (at amino acid residues 374-387 of SEQ ID NO: 11). (Corresponding to) a polypeptide comprising amino acid residues at positions 1-14 of SEQ ID NO: 2, (corresponding to amino acid residues at positions 388-427 of SEQ ID NO: 11) A polypeptide comprising the amino acid residues at positions 54 to 54, including the amino acid residues at positions 55 to 94 of SEQ ID NO: 2 (corresponding to the amino acid residues at positions 428 to 467 of SEQ ID NO: 11) Or polypeptide or one or more other EGFs identified herein
There are polypeptides that comprise a repeat region.

The polynucleotide of the present invention further includes a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198, clone pEGF deposited under accession number ATCC 203198. -A polypeptide comprising the full-length protein encoded by the cDNA corresponding to the cDNA insert of -HY1, a polypeptide comprising the mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198, or ATCC 203198 But not limited to, the mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under Accession No.

[0097] Preferred embodiments include those wherein the protein produced by such a method is a mature form of the protein.

[0098] The protein compositions of the present invention further include an acceptable carrier, such as a hydrophilic, eg , pharmaceutically acceptable carrier.

The present invention further relates to a method for producing a polypeptide, comprising growing a cell culture of the present invention in a suitable culture medium and purifying the protein from the culture. including. For example, the method of the present invention comprises producing a polypeptide by culturing a host cell containing a suitable expression vector containing the polynucleotide of the present invention under conditions permitting the expression of the polypeptide of interest. Process is included. The polypeptide is recovered from the culture, conveniently from the culture medium, and can be further purified.

The present invention further provides a polypeptide comprising an amino acid sequence substantially equivalent to the amino acid sequence of SEQ ID NO: 2 or 11. A polypeptide according to the present invention can have at least about 95%, and preferably usually at least about 98, sequence identity with SEQ ID NO: 2 or 11.

The invention further provides an isolated polypeptide encoded by a nucleic acid fragment of the invention or by a degenerate variant of a nucleic acid fragment of the invention. By the term "degenerate variant", a nucleic acid fragment of the invention (e.g., ORF)
), But due to the degeneracy of the genetic code, nucleotide fragments encoding the same polypeptide sequence are contemplated. A preferred nucleic acid fragment of the present invention is an ORF encoding a protein. Various methodologies known in the art can be utilized to obtain either an isolated polypeptide or protein of the present invention. At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. This is particularly useful for producing small peptides or fragments of larger peptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In yet another method, the polypeptide or protein is purified from bacterial cells that naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins to obtain one of the isolated polypeptides or proteins of the present invention. These include immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography, and the like,
It is not limited to these. For example, Scopes, Protein Purification: Pr inciples and Practice, Springer-Verlag (1994); Sambrook et al, Molecular Clo ning: A Laboratory Manual ; Ausubel et al., See Current Protocols in Molecular Bio logy.

[0102] The polypeptides and proteins of the present invention may be purified from cells that have been modified to express the desired polypeptide or protein. As used herein, when a cell has been genetically engineered to produce a polypeptide or protein that is not normally produced, or usually produced only at low levels, the desired polypeptide is obtained. It is said to have been modified to express the peptide or protein. One of skill in the art will appreciate methods for introducing or expressing either recombinant or synthetic sequences to produce cells that produce one of the polypeptides or proteins of the invention, either prokaryotic or eukaryotic cells. Can be applied without difficulty. Purified polypeptides can be used in in vitro binding assays, which are known techniques for identifying molecules that bind to a polypeptide. These molecules include, but are not limited to, for example, small molecules, molecules from combinatorial libraries, antibodies or other proteins.
Molecules identified in the binding assays are then tested for antagonists or agonists in in vivo tissue culture or animal models known in the art. Briefly, molecules are titrated in multiple cell cultures or animals and then examined for animal / cell death or animal / cell survival prolongation.

In addition, the binding molecule may be conjugated to a toxin, for example, ricin or cholera toxin, or other compound that has toxicity to cells. The toxin-binding molecule conjugate is then targeted to tumor cells or other cells depending on the specificity of the binding molecule of SEQ ID NO: 2 or 11.

The protein of the present invention may be, for example, a transgenic bovine, goat, pig or sheep milk component characterized by somatic or germ cells containing the nucleotide sequence encoding the protein. It may be expressed as an animal product.

Proteins may be produced by conventional, known chemical synthesis. The method for constructing the protein of the present invention by a synthetic method is well known to those skilled in the art. By sharing primary, secondary or tertiary and / or tertiary structural and / or conformational properties with proteins, protein sequences constructed by synthetic methods may possess biological properties in common with them, including their protein activity. It is. Thus, they can be used as biological or immunological substitutes for naturally occurring purified proteins in screening therapeutic compounds and in immunological processes for producing antibodies.

The proteins provided by the present invention are proteins characterized by an amino acid sequence similar to the purified protein, but also include those in which the modifications are naturally occurring or intentionally manipulated. I do. For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques. Of interest in modifying protein sequences may be alterations, substitutions, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with other amino acids to alter the configuration of the molecule. Techniques for such alterations, substitutions, insertions or deletions are well known in the art (see, for example, US Pat. No. 4,51,51).
8,584). Preferably, such alterations, substitutions, insertions or deletions will retain the desired activity of the protein.

Other fragments and derivatives of the protein sequence which are expected to retain all or a portion of the activity of the protein, and which may be useful for screening and other immunological methods, are also described herein. It can be done without difficulty by those skilled in the art based on the contents disclosed in the present document. Such modifications are considered to be encompassed by the present invention.

The protein may also be produced by operably linking the isolated polynucleotide of the invention to a suitable control sequence in one or more insect expression vectors and utilizing an insect expression system. Can be. Materials and methods for the baculovirus / insect cell expression system are available in kits such as, for example, those commercially available from Invitrogen, San Diego, California, USA (MaxBat. RTM. Kits) and such methods. Is Summer, which is incorporated herein by reference.
s and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1
It is well known in the art as described in 987). As used herein, an insect cell capable of expressing a polynucleotide of the present invention has been "transformed."

The proteins of the present invention are prepared by culturing the transformed host cells under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein is then purified from such a culture (ie, culture medium or extract) using known purification processes such as gel filtration and ion exchange chromatography. Purification of the protein is further achieved by affinity columns containing reagents that bind the protein; Concanavalin A agarose, Heparin-Toyopearl® R® or Shibachrome Blue 3GA Sepharose® R
One or more steps using an affinity resin such as ®, one or more steps utilizing hydrophobic interaction chromatography using a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity It may also include chromatography and the like.

Alternatively, the proteins of the present invention may be expressed in a form that facilitates purification. For example, it may be expressed as a fusion protein such as a maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) fusion protein. Kits for expressing and purifying such fusion proteins are available from New England BioLab (Beverly, Mass.), Pharmac.
ia (Piscataway, NJ) and In Vitrogen, respectively. Proteins can also be tagged with an epitope and then purified using a specific antibody directed against such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).

Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps utilizing a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups, It may be performed for further protein purification. Some or all of the above purification steps may be performed in various combinations to obtain a substantially homogeneous isolated recombinant protein. A protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as an "isolated protein".

5.4. Clone Deposit The following clone, pEGF-HY1, was deposited with the American Type Culture Collection (ATCC) Manassas, Virginia on September 18, 1998, and was assigned the following accession number: . This clone is typically a plasmid clone as described in the Examples below.

[0113]

[Table 1]

5.5. Uses and Biological Activities The polynucleotides and proteins of the present invention exhibit one or more of the uses or biological activities listed below, including those associated with the assays cited herein. It is expected. The uses and activities described for the proteins of the invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, gene therapy, or
A suitable vector for the introduction of DNA).

5.5.1. Research Uses and Utility The polynucleotides provided by the present invention can be used by a research community for a variety of purposes. A polynucleotide is used to express a recombinant protein for analysis, characterization or therapeutic use; the corresponding protein is preferentially expressed (constitutive expression or a specific stage of tissue differentiation or development or disease). State) as a marker for tissue; as a molecular weight marker for Southern gels; as a chromosomal marker or tag (if labeled) to identify chromosomes or to map the location of related genes; To compare with the patient's endogenous DNA to identify certain genetic disorders; as a probe to hybridize and thus discover novel related DNA sequences; to induce PCR primers for genetic fingerprinting Sources in the process of discovering other novel polynucleotides As a probe to "subtract" the known sequence; for the purpose of selecting and producing oligomers for attachment to a "gene chip" or other support, including expression pattern experiments; It can be used to raise protein antibodies; and to raise anti-DNA antibodies or as an antigen to elicit another immune response.

If the polynucleotide binds to another protein or encodes a protein with potential binding (eg, binding at a receptor-ligand interaction), the polynucleotide may further bind to To identify polynucleotides that encode proteins or to identify inhibitors of binding interactions, interaction capture assays (see, e.g., Gyuris et al., Cell 75, 791-803 (
1993)).

The proteins provided by the present invention have high throughput (high throughput)
In a panel of multiple proteins for screening; to produce antibodies or to elicit another immune response; designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid (Including constitutive expression or tissue differentiation or expression at a particular stage of development or disease state) in which the corresponding protein is preferentially expressed as reagents (including labeled reagents) in the assay. And, of course, can be used in assays to determine biological activity, including, of course, various purposes or uses, such as to isolate relevant receptors or ligands. If the protein binds to or has potential binding to another protein (eg, in a receptor-ligand interaction), to identify other proteins where binding occurs, or Proteins can be used to identify inhibitors of the interaction of. Proteins involved in these binding interactions can also be used to screen binding interacting peptides or small molecules for inhibitors or agonists.

Any or all of these research applications can be developed into reagent or kit formats for commercialization as research results.

[0119] Methods for performing the above uses are well known in the art. References disclosing such a method include "Molecular Cloning: A Laboratory Manu".
al ", 2nd edition, Cold Spring Harbor Laboratory Press, Sambrook, J., EF F
Edited by ritsch and T. Maniatis, 1989, and Methods in Enzymology: Guide t
o Molecular Cloning Techniquies, Acadmic Press, Berger, SL and A.R.
Edited by Kimmel, 1987.

5.5.2. Nutritional Uses The polynucleotides and proteins of the present invention can also be used as a nutritional source or supplement. Such uses include, but are not limited to, use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the invention can be added to the diet of a particular organism, or as a solid, solid or solid form, such as a powder, pill, solution, suspension or capsule. It can also be administered as a liquid preparation.
In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

5.5.3. Cytokines and Cell Proliferation / Differentiation Activity The proteins of the invention may exhibit cytokine, cell proliferation (either induction or inhibition) or cell differentiation (either induction or inhibition) activity, or Other cytokines in specific cell populations can be induced. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Many protein factors discovered to date, including any known cytokines, exhibit activity in one or more factor-dependent cell proliferation assays, which serve as a convenient method of confirming cytokine activity. The activity of the protein of the present invention is 32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M +), 2E8
RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK by any of a number of routine factor-dependent cell proliferation assays for cell lines, including but not limited to Proven.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for T-cell or thymocyte proliferation include Current Protocols in Imm
unology, JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach,
Edited by W. Strober, published by Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch.
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3
500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Bertagnolli et al.
, Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., I. Immunol. 149
: 3778-3783, 1992; including, but not limited to those described in Bowman et al, I. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and / or spleen cell, lymph node cell or thymocyte proliferation include polyclonal T cell stimulation, Kruisbeek AM and Sh
evach, EM, Current Protocols in Immunology. Edited by JEea Coligan, V
ol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and measurement of mouse and human interleukin-γ., Schreiber, RD, Current Pro.
tocols in Immunology. JE ea Coligan Editing Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include measurement of mouse and human interleukin-2 and interleukin-4, Bottomly
, K., Davis, LS and Lipsky, PE, Current Protocols in Immunology. J
E. ea Coligan Editing Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 1991; deVries et al., J. Exp.Med. 173: 1205-1211, 1991; Moreau et al., Nature.
336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. USA 80: 2931-
2938, 1983, Measurement of mouse and human interleukin-6-Nordan, R., Curr
ent Protocols in Immunology. Edited by JE ea Coligan Vol.1 pp. 6.6.1-6.6.
5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U
.SA 83: 1857-1861, 1986; Measurement of human interleukin-11--Bennett, F., Gi
annotti, J., Clark, SC and Turner, KJ, Current Protocols in Immuno
logy. JE ea Coligan Editing Vol 1 pp. 6. 15.1 John Wiley and Sons, Tor
onto. 1991; Measurement of mouse and human interleukin-9--Ciarletta, A., G
iannotti, J., Clark, SC and Turner, KJ, Current Protocols in Immun
ology. JE ea Coligan Editing Vol 1 pp. 6.13.1, John Wiley and Sons, Tor
including but not limited to those described in onto. 1991.

Assays for T-cell clonal responses to antigens, among others identifying proteins that affect APC-T cell interactions, and identifying direct T-cell effects by measuring proliferation and cytokine production Things), Current
Protocols in Immunology, JE Coligan, AM Kruisbeek, DH Margulies
, EM Shevach, W Strober, Greene Publishing Associates and Wiley-I
nterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; C
hapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-60.
95, 1980; Weinberger et al., Eur.J. Immun. 11: 405-411, 1981; Takai et al., J. Imm.
unol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

5.5.4. Immunostimulatory or Suppressive Activity The proteins of the present invention may also exhibit immunostimulatory or immunosuppressive activity in assays, including but not limited to the activities described herein. .
The polynucleotide of the present invention can encode a polypeptide exhibiting such an activity. Proteins are used in the treatment of various immunodeficiencies and disorders, including severe combined immunodeficiency (SCID), for example, in regulating the growth and proliferation of T and / or B lymphocytes (up-regulation or down-regulation). And may also be useful in providing cytolytic activity of NK cells and other cell populations. These immunodeficiencies can be genetic, caused by a virus (eg, HIV), bacterial or fungal infection, or caused by an autoimmune disorder. Is also good. More particularly, infectious diseases caused by viruses, bacteria, fungi, including various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania subspecies, malaria subspecies, and candidiasis , Or other infections can be treated using the proteins of the present invention. Of course, in this regard, the proteins of the invention may generally be desired to enhance the immune system, ie,
It may also be useful in treating cancer.

Autoimmune disorders that can be treated using the proteins of the invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome Autoimmune thyroiditis, insulin-dependent diabetes, myasthenia gravis, graft-host disease and autoimmune inflammatory eye diseases. Such proteins of the invention may also be useful in treating allergic reactions and conditions, such as asthma (particularly allergic asthma), or other respiratory disorders. Other conditions in which immunosuppression is desired (including, for example, organ transplantation) may also be treatable using the proteins of the invention.

The proteins of the present invention may also be used to immunoreact in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response that is already in progress, or may involve preventing the induction of an immune response. The function of the activated T cells may be to inhibit the function of the activated T cells by suppressing the response of the T cells or by inducing specific tolerance in the T cells, or both. Immunosuppression of a T cell response is usually an active, non-antigen-specific process that requires continuous exposure of the T cell to the inhibitor. Tolerance involves inducing unresponsiveness or inapparentness in T cells and is usually distinguishable from immunosuppression in that it is antigen-specific, resulting in termination of exposure to a tolerizing agent. It persists after Operationally, tolerance can be demonstrated by a lack of response of T cells upon exposure to a specific antigen in the absence of a tolerizing agent.

Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen function (eg, B7), eg, high level lymphokine synthesis by activated T cells Prevention and the like may be useful in the context of tissue, skin and organ transplantation, and in graft-versus-host disease (GVHD). For example, blocking T cell function should result in reduced tissue destruction in tissue transplantation. Usually, in tissue transplantation, rejection of the transplant is triggered through recognition by the T cells as foreign and subsequent immune reactions that destroy the transplant. Prior to transplantation, by administering a molecule that inhibits or blocks the interaction between B lymphocyte antigens and their natural ligand (s) on immune cells, the immune response can be reduced without transmitting the corresponding costimulatory signal. It can direct the binding of the molecule to the natural ligand (s) on the cell. Inhibiting B lymphocyte antigen function in this way prevents the synthesis of cytokines by immune cells such as T cells, thereby acting as a suppressor of immunity. In addition, the lack of costimulation may also be sufficient to render T cells unresponsive, which may induce tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen inhibitors may obviate the need for repeated administration of these inhibitors. In order to achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to inhibit the function of the combination of multiple B lymphocyte antigens.

The efficacy of a particular inhibitor in preventing organ transplant rejection or GVHD can be evaluated using animal models that predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart transplantation in rats and xenogeneic islet cell transplantation in mice, both of which are described in Lenschow et al., Sc.
ience 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, 89:
As described in 11102-11105 (1992), it has been used to test the immunosuppressive effect of CTLA4Ig fusion protein in vivo. In addition, a mouse model of GVHD (P
aul Editing, Fundamental Immunology, Raven Press, New York, 1989, pp. 846-8
47), the effect of B lymphocyte antigen function on the development of the disease
Can be used to examine in vivo.

Inhibition of antigen function may further be therapeutically useful for treating an autoimmune disease. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive to their own tissues and promote the production of cytokines and autoantibodies involved in the pathology of the disease. Preventing activation of autoreactive T cells may reduce or eliminate symptoms. To disrupt the T lymphocyte co-stimulation by disrupting the receptor: ligand interaction of the B lymphocyte antigen, administration of a reagent to inhibit T cell activation
And it can be used to prevent the production of autoantibodies or T cell derived cytokines that may be involved in the disease process. In addition, inhibitors may induce antigen-specific tolerance of autoreactive T cells, which may lead to long-term disease relief. The effectiveness of inhibitors in preventing or alleviating autoimmune disorders is
A number of well-characterized animal models of human autoimmune disease can be examined. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in NOD and BB rats, and murine experimental myasthenia gravis. (Paul edit, Fundamental Immunology, Raven Pre
ss, New York, 1989, pp. 840-856).

Up-regulation of antigen function (preferably B lymphocyte antigen function) as a means of up-regulating the immune response may also be useful in therapy. Up-regulation of the immune response may take the form of enhancing an existing immune response or eliciting an early immune response. For example, enhancing autoimmunity through stimulating B lymphocyte antigen function may be useful in case of viral infection. In addition, systemic viral diseases such as influenza, cold and encephalitis may be alleviated by systemic administration of a stimulating form of B lymphocyte antigen.

Alternatively, T cells may be removed from an infected patient and inoculated with APCs pulsed with a viral antigen expressing any of the polypeptides of the invention, or with a stimulated form of a soluble peptide of the invention. By co-stimulating T cells in vitro and reintroducing activated T cells in vitro into the patient, the anti-living immune response can be enhanced in infected patients. Another way to enhance the antiviral immune response is
Isolating infected cells from a patient and transfecting the cells with a nucleic acid encoding a protein of the invention, according to the disclosure herein, such that such cells express the entire protein or a portion thereof on the cell surface. Then, there is a method of introducing the transfected cells back into the patient. The infected cell should then be able to transmit a costimulatory signal to the T cell in vivo, thereby activating the T cell.

The presence of the peptide of the invention having the activity of the B lymphocyte antigen (s) on the surface of the tumor cells provides a costimulatory signal required for T cells, An immune response is elicited by the T cells against the infected tumor cells. In addition, if there is a lack of MHC class I or MHC class II molecules, or sufficient tumor cells can not be re-express MHC class I or MHC class II molecules in an amount, MHC class Iα chain protein and beta _{2-microglobulin} protein, Alternatively, it is transfected with all or part of the nucleic acid of the MHC class II α-chain protein and MHC class II β-chain protein (for example, the part lacking the cytoplasmic domain), so that the MHC class I or MHC class II is present on the cell surface. The protein can be expressed. Appropriate class I or class II MHC can be obtained using B lymphocyte antigen (
For example, when expressed in combination with a peptide having the activity of B7-1, B7-2, B7-3), an immune response is induced by T cells against the transfected tumor cells.
Optionally, a gene encoding an antisense construct that inhibits expression of MHC class II-related proteins, such as an invariant chain, may also be co-transfected with DNA encoding a peptide having B lymphocyte antigen activity. Can enhance the presentation of tumor-associated antigens and elicit tumor-specific immunity. In this way, by eliciting an immune response by T cells in a human subject, it is possible to sufficiently overcome tumor-specific usage in the subject.

The activity of the protein of the present invention can be measured by the method described below among various means.

Suitable assays for cytotoxicity on thymocytes or spleen cells include Current
Protocols in Imrnunology, JE Coligan, AM Kruisbeek, DH Margulie
s, EM Shevach, edited by W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Func
tion 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Pr.
oc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128.
1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., I.
Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988; He.
Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J.
Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985;
Takai et al., J. Immunol. 137: 3494-3500, 1986; Bowman et al., J. Virology 61: 1992-
1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Im
munology 133: 327-341, 1991; including, but not limited to those described in Brown et al., J. Immunol. 153: 3079-3092, 1994.

Assays for T cell-dependent immunoglobulin responses and isotope switching, especially those that identify proteins that modulate T cell-dependent antibody responses and that affect Th1 / Th2 profiles, include Maliszews
ki, J. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: I
n vitro antibody production, Mond, JJ and Brunswick, M., Current Prot
ocols in Immunology. JE ea Coligan Editing Vol 1 pp. 3.8.1-3.8.16, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994.

The lymphocyte mixed reaction (MLR) assay, which identifies, among other things, proteins that preferentially generate Th1 and CTL responses, includes Current Protocols in Immunology, J.
E. Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strobe.
r Editing, Pub.Green Publishing Associates and Wiley Interscience (Chapter
3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, I
mmunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986;
Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Immunol. 149:
3778-3783, 1992, but are not limited thereto.

Dendritic cell-dependent assays, among others that identify proteins expressed by dendritic cells that activate natural T cells, include Guery et al., J. Immunol. 134: 5.
36-544, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgador et al., J
ournal of Experimental Medicine 182: 255-260, 1995; Nair et al., Journal of Vi.
rology 67: 4062-4069, 1993; Huang et al., Science 264: 961-965, 1994; Macatonia
Et al., Journal of Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al., Jo
urnal of Clinical Investigation 94: 797-807, 1994; and Inaba et al., Journal
of Experimental Medicine 172: 631-640, 1990, including, but not limited to.

Assays for lymphocyte survival / apoptosis, among others identifying proteins that prevent apoptosis following superantigen induction and proteins that regulate lymphocyte homeostasis, include Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyc
a et al., Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1951,
1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of Immunology.
145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., In
Includes, but is not limited to, those described in the ternational Journal of Oncology 1: 639-648, 1992.

Assays for proteins that affect T cell orientation and its initial steps include Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology.
155: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Natl.
Acad. Sci. USA 88: 7548-7551, 1991, including, but not limited to.

5.5.5. Hematopoietic Modulating Activity The proteins of the present invention may be useful in regulating hematopoiesis and, consequently, in treating bone marrow or lymphoid cell defects. Involvement in hematopoietic regulation with minimal biological activity supporting colony forming cells or factor-dependent cell lines, e.g., supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, For example, useful in combination with radiation therapy / chemotherapy to treat various anemias, or to stimulate production of erythroid progenitor cells and / or red blood cells; bone marrows such as granulocytes and monocytes / macrophages Combining cell growth and proliferation (ie, traditional CSF activity) with, for example, chemotherapy to prevent the resulting myelosuppression; in support of megakaryocyte growth and proliferation, and consequently platelet growth and proliferation Support the prevention and treatment of various platelet disorders, such as thrombocytopenia, and generally replace or supplement platelet infusion Intended use; and / or can mature into any and all of said hematopoietic cells, and thus have various stem cell disorders, including but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria Therapeutic use in disorders that are usually treated by transplantation, including disease, and even as normal cells or cells that have been genetically engineered for gene therapy, either in-vivo or ex-vivo (ie, Involvement in Hematopoietic Stem Cell Growth and Proliferation with Bone Marrow Transplantation or Peripheral Progenitor Cell Transplantation (Allogeneic or Allogeneic) in Repopulation of Stem Cell Compartments Following Radiation / Chemotherapy Things.

The activity of the protein of the present invention can be measured by the following method, among other means.

Suitable assays for proliferation and differentiation of various hematopoietic cell lines are as described above.

Assays for fetal stem cell differentiation, particularly those that identify proteins that affect fetal differentiated hematopoiesis, include Johansson et al. Cellular Biology 15: 141-
151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486, 1993; Mc
Includes, but is not limited to, those described in Cianahan et al., Blood 81: 2903-2915, 1993.

Assays for stem cell survival and differentiation, especially those that identify proteins that regulate lympho-hematopoiesis, include Methylcellulose colony forming ass
ays, Freshney, MG In Culture of Hematopoietic Cells.RI Freshney,
Edit Vol. 265-268, Wiley-Liss, Inc., New York, NY 1994; Hirayama
Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoiet.
ic colony forming cells with high proliferative potential, McNiece, I. K
and Briddell, RA In Culture of Hematopoietic Cells.RI Freshney,
Edit Vol pp. 23-39, Wiley-Liss, Inc., New York, NY 1994; Neben et al., E
xperimental Hematology 22: 353-359, 1994; Cobblestone area forming cell a
ssay, Ploemacher, RE in Culture of Hematopoietic Cells.RI Freshne
y, et al. Edit Vol pp. 1-21, Wiley-Liss, Inc., New York, NY 1994; Long term
bone marrow cultures in the presence of stromal cells, Spooncer, E., De
xter, M. and Allen, T. In Culture of Hematopoietic Cells.RI Freshney
, Et al. Edit Vol pp. 163-179, Wiley-Liss, Inc., New York, NY 1994; Long te
rm culture initiating cell assay, Sutherland, HJ In Culture of Hemato
poietic Cells.RI Freshney et al. Edit Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, those described in New York, NY 1994.

5.5.6. Tissue Growth Activity The proteins of the present invention may also be used for the growth or regeneration of bone, cartilage, tendons, ligaments and / or nerve tissue, as well as for wound healing and tissue repair and replacement, and for burns. , Incisions and ulcers also have utility in compositions used in the treatment.

The proteins of the present invention, which induce cartilage and / or bone growth when bone is not formed normally, are applicable to the healing of fractures and cartilage damage or defects in humans and other animals. Such preparations using the proteins of the invention have prophylactic use in reducing closed and open fractures, as well as improving fixation of artificial joints. New bone formation induced by osteogenic agents contributes to the repair of cranial defects induced by congenital, traumatic or oncological resection, and is also useful in cosmetic surgery.

The proteins of the present invention may also be used in the treatment of periodontal disease and other dental restoration processes. Such agents may provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells, or induce differentiation of osteogenic cell precursors. The proteins of the present invention may further be through stimulation of bone and / or cartilage repair, or by inhibiting inflammation, or by inhibiting the process of tissue destruction mediated by the inflammatory process (collagenase activity, osteoclast activity, etc.). It is also useful in the treatment of osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can contribute to the proteins of the invention is tendon / ligament formation. Proteins of the invention that induce the formation of tendon / ligament-like or other tissue under conditions where such tissue is not formed normally include rupture, deformation, and other changes in tendons or ligaments in humans and other animals. For healing tendon or ligament defects. Preparations using proteins that induce tendon / ligament-like tissue may be used in prophylactic applications to prevent damage to tendon or ligament tissue, as well as to improve fixation of tendons or ligaments to bone or other tissue, or tendons or ligaments. It also has applications for repairing defects in ligament tissue. Neoplastic tendon / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of tendon or ligament defects due to congenital, traumatic or other causes, and for attachment or repair of tendons or ligaments. It is also useful in cosmetic surgery. The composition of the present invention provides an environment for inducing tendon-forming cells or ligament-forming cells, an environment for stimulating the growth of tendon-forming cells or ligament-forming cells, an environment for inducing differentiation of precursors of tendon-forming cells or ligament-forming cells, Alternatively, it may provide an environment to induce growth of tendon / ligament cells or precursors ex vivo for in vivo return to affect tissue repair. The compositions of the present invention are also useful in treating tendinitis, carcinomatosis and other tendon or ligament defects. These compositions may also contain suitable matrices and / or sequestering agents well known in the art as carriers.

The proteins of the present invention may also be used for the growth of nerve cells and the regeneration of nerve and brain tissue, ie, for the treatment of central and peripheral nervous system diseases and neurological disorders, and in mechanical and traumatic disorders, It may also be useful for treating those involving degeneration, death or trauma to nerve tissue. More particularly, proteins are used in diseases of the peripheral nervous system such as peripheral nerve injury, peripheral and focal neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy. -It may be useful in treating central nervous system diseases such as Dräger syndrome. Additional conditions that can be treated by the present invention include mechanical and traumatic disorders, such as spinal cord disorders, cerebrovascular disorders such as head trauma and stroke. Peripheral neuropathy resulting from chemotherapy or other medical therapy can also be treated using the proteins of the present invention.

The proteins of the present invention may be better or faster in progressive wounds, including but not limited to pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, etc. It can also be used to facilitate blockade.

The proteins of the present invention may be found in organs (eg, including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal, or cardiac) and vascular (including vascular endothelium) tissues. , Or an activity for the development or regeneration of other tissues, or an activity for promoting the growth of cells containing such tissues. Part of the desired effect is due to inhibition or modulation of fibrous scars, which allows normal tissue to regenerate. The proteins of the present invention may also exhibit angiogenic activity.

The proteins of the present invention may also be useful for gut protection or regeneration and lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

The proteins of the present invention may also be useful in promoting or inhibiting differentiation of the tissue from progenitor cells or cells, or in inhibiting the growth of the tissue.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for tissue producing activity include International Patent Publication No. WO95 / 16035 (bone, cartilage, tendon); International Patent Publication No. WO95 / 05846 (neural, neuron); International Patent Publication Mo.
Including, but not limited to, those described in WO91 / 07491 (skin, endothelium).

Assays for wound healing activity include Winter, Epidermal Wound Healing, pps
71-112 (Maibach, HI and Rovee, DT, Editing), Year Book Medical Publ.
ishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. De
rmatol 71: 382-84 (1978), including, but not limited to.

5.5.7. Activin / Inhibin Activity The proteins of the present invention may also exhibit activin-related or inhibin-related activity. The polynucleotide of the present invention preferably encodes a polypeptide exhibiting such properties. Inhibin is characterized by its ability to inhibit follicle stimulating hormone (FSH) release, while activin is characterized by its ability to promote follicle stimulating hormone (FSH) release. Thus, the proteins of the invention, alone or in a heterodimer with a member of the inhibin α-family, reduce fertility of inhibin in female mammals and contraception based on the ability to reduce spermatogenesis in dominant mammals. May be useful as a medicine. Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, a protein of the invention may be a fertilizer, based on the ability of an activin molecule to stimulate FSH release from cells of the anterior pituitary, either in a homodimer or in a heterodimer with other protein subunits of the inhibin β group. It may also be useful as an induction therapeutic. For example, U.S. Pat.
See 4,798,885. The proteins of the present invention may also be useful for increasing the reproductive capacity of livestock such as cattle, sheep, pigs, etc., and for hastening the onset of fertility in sexually immature mammals.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for actin / inhibin activity include Vale et al., Endocrinology 91
: 562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nature 321: 776.
-779, 1986; Mason et al., Nature 318: 659-663, 1985; Forage et al., Proc. Natl. Aca
d. Sci. USA 83: 3091-3095, 1986, including, but not limited to.

5.5.8. Chemotaxis / chemokine activity The proteins of the present invention include, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial and / or endothelial cells, and the like. It may have chemotactic or chemokine activity (eg, acting as a chemokine) on mammalian cells, including. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Chemotaxis and chemokine proteins can be used to move or attract a desired cell population to a desired site of action. Chemotaxis or chemokine proteins offer particular advantages in the treatment of wounds and other trauma to tissues, as well as in the treatment of local infections. For example, the attraction of lymphocytes, monocytes or neutrophils to the tumor or site of infection can result in an improved immune response to the tumor or infectious agent.

A protein or peptide will have chemotactic activity on a particular cell population if it can directly or indirectly stimulate the directed orientation or movement of that cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a population of cells can be determined without difficulty by using such a protein or peptide in various known assays for cell chemotaxis.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for chemotactic activity (assays that identify proteins that induce or prevent chemotaxis) are based on the ability of the protein to induce cells to migrate across a membrane, and the ability of one cell population to It consists of an assay that measures the ability of the protein to induce adhesion to the population. Suitable assays for migration and adhesion include Current Protocols in Immunology, JE Coligan. AM Kruisbeek, DH
Edited by Marguiles, EM Shevach, W. Strober, Pub. Greene Publishing Associ
ates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95: 1370-1376, 1995;
Lind et al. APMIS 103: 140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-17
48; Gruber et al. J. of Immunol. 152: 5860-5867, 1994; Johnston et at. J. Immu
nol. 153: 1762-1768, 1994, including, but not limited to.

5.5.9. Hemostasis and Thrombolytic Activity The proteins of the present invention may also exhibit hemostatic or thrombolytic activity. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Such proteins are expected to be useful in the treatment of various coagulation disorders, including genetic diseases such as hemophilia, or in the treatment of wounds such as those caused by trauma, surgery or other cases. It is expected to enhance coagulation and other hemostatic phenomena. The proteins of the present invention are also useful for inhibiting the dissolution or formation of thrombi, and for treating and preventing conditions resulting therefrom, such as infarction of the heart and central nervous system blood vessels, such as stroke. Can be useful.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for hemostatic and thrombolytic activity include those described by Linet et al., J. Clin. Pharmaco.
l. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphr.
ey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 467-474, 19
88, including, but not limited to.

5.5.10. Receptor / Ligand Activity Proteins of the present invention may also exhibit activity as receptors, receptor ligands or inhibitors or agonists of receptor / ligand interactions. The polynucleotide of the present invention can encode a polypeptide exhibiting such properties. Examples of such receptors and ligands include cytokine receptors and their ligands,
Receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (such as cell adhesion molecules (such as selectins, integrins and their ligands)) and antigen presentation, antigen recognition and (Including but not limited to, receptor / ligand pairs involved in the development of cellular and humoral immune responses).
Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors of the relevant receptor / ligand interaction. The proteins of the invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interactions.

The activity of the protein of the present invention may be measured by the following method, among other means.

Suitable assays for receptor-ligand activity include Current Protocols in I
mmunology, JE Coligan, AM Kruisbeek, DH Margulies, EM Shevac
h, edited by W. Strober, Pub. Greene Publishing Associates and Wiley-Interscie
nce (Chapter 7.28, Measurement of Cellular Adhesion under static conditi
ons 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6864-6868, 1
987; Bierer et al., J. Exp.Med. 168: 1145-1156, 1988; Rosenstein et al., J. Exp.M.
ed. 169: 149-160; Stoltenborg et al., J. Immunol. Methods 175: 59-68, 1994; Sti.
tt et al., Cell 80: 661-670, 1995, but are not limited thereto.

5.5.11. Anti-inflammatory activity The proteins of the invention may also exhibit anti-inflammatory activity. Anti-inflammatory activity provides stimulation to cells involved in the inflammatory response, inhibits or promotes cell-cell interactions (eg, cell adhesion, etc.), inhibits or promotes chemotaxis of cells involved in the inflammatory process, It can be achieved by inhibiting or promoting cell extravasation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Proteins exhibiting such activity include, but are not limited to, infection-related suggestions (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemic reperfusion injury, lethal endotoxin, arthritis Chronic or acute, including complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or diseases caused by overproduction of cytokines such as TNF or IL-1 It can be used to treat any inflammatory condition. The proteins of the invention may also be useful in treating anaphylaxis and hypersensitivity to antigenic substances or materials.

5.5.12. Leukemia Leukemia and related disorders can be treated or prevented by administering a therapeutic agent that promotes or inhibits the function of the polynucleotides and / or polypeptides of the invention. Such leukemias and related disorders include acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic leukemia, For a review of myeloid (granulocytic) leukemia and chronic lymphocytic leukemia (disorders listed above, see Fishman et al., 1985, Medicine , 2nd ed.
B. Lippincott Co., Philadelphia).

5.5.13. Nervous System Disorders Nervous system disorders are compounds that modulate the activity of the polynucleotides and / or polypeptides of the invention and involve cell types that can be tested for the efficiency of intervention. Thus, it can be treated by observing such signs of therapeutic use, including injuries to the nervous system, and any disconnection of axons, loss or degeneration of neurons, or demyelination. Includes, but is not limited to, the disease or disorder that causes it. Nervous system injuries in patients (including human and non-human mammalian patients) that can be treated according to the present invention include central (spinal cord,
(Including but not limited to, the brain) or the peripheral nervous system, including but not limited to: (i) traumatic injuries, including injuries caused by physical injuries, or surgical injuries such as, for example, amputation of a part of the nervous system or compression injuries; (ii) injuries to a part of the nervous system Ischemic injury in which lack of oxygen causes neuropathy or death, including cerebral infarction or cerebral ischemia, or spinal cord infarction or ischemia; (iii) malignancy from nervous or non-nervous tissue associated with malignancy Malignant tumor damage in which part of the nervous system has been destroyed or damaged by malignant tissue that is any of the tumors; (iv) as a result of infection, eg, by an abscess or human immunodeficiency virus, herpes zoster, or simple (V) limited infectious damage to parts of the nervous system that have been destroyed or damaged by infection with the herpes virus or by Lyme disease, tuberculosis, syphilis; , But is not intended to be a degenerative injury to a part of the nervous system that has been destroyed or damaged as a result of a degenerative process, including Parkinson's disease, Alzheimer's disease, Huntington's chorea or amyotrophic lateral sclerosis; vi) Nutritional disorders, including but not limited to vitamin B12 deficiency, folate deficiency, Wernicke's disease, tobacco-alcoholic amblyopia, Marchiafava-Vignami's disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration Or a nutritional disorder or disorder associated with a nutritional disorder in a part of the nervous system that has been destroyed or damaged by a metabolic disorder; (vii) without limitation, diabetes (diabetic neuropathy, Bell's palsy) For systemic disease, including systemic lupus erythematosus, carcinoma, or sarcoidosis (Viii) Injury caused by toxic substances, including alcohol, lead, or certain neurotoxins; and (ix) Without limitation, multiple sclerosis, human immunodeficiency virus Demyelinating diseases or diseases of various etiologies, including myelopathy involved, transverse spinal cord injury, progressive multifocal leukoencephalopathy, and demyelinating injury in parts of the nervous system destroyed or damaged by central pontine myelinolysis.

Therapeutic agents of the invention useful for the treatment of nervous system disorders can be selected by testing for biological activity that promotes survival or differentiation of neurons. For example,
The following effects: (i) increase the survival time of neurons in culture; (ii) increase the sprouting of neurons in culture or in vivo; (iii) neuron-related molecules such as exercise in culture or in vivo. Therapeutic agents that exhibit either increased production of choline acetyltransferase or acetylcholinesterase for neurons; or (iv) reduced symptoms of neuronal dysfunction in vivo are useful in the present invention, but are not limited to them . Such an effect can be measured by methods known in the art. In a preferred non-limiting embodiment, the increase in neuronal survival is
(1990, J. Neurosci. 10: 3507-3515); increased neuronal sprouting can be measured by Pestronk et al. (1980, Exp. Neurol. 70: 65-82) or Brown et al. (1981, A.
nn. Rev. Neurosci. 4: 17-42); the increase in the production of neuron-associated molecules can be measured by a bioassay, enzyme assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; Motor neuron impairment function can then be measured, for example, by assessing physical symptoms of motor neuron impairment, such as weakness, motor neuron conduction velocity, or impaired functional ability.

In certain embodiments, the motor neuron disorder that can be treated according to the present invention can affect infarction, infection, exposure to toxins, trauma, surgical injury, motor neurons and other components of the nervous system. Includes disorders that selectively affect neurons, such as degenerative diseases or malignancies, and amyotrophic lateral sclerosis, and progressive spinal muscular atrophy, progressive medullary palsy, primary amyotrophic lateral sclerosis, childhood and Includes disorders such as juvenile muscular atrophy, progressive medullary palsy in children (Fathio-Ronde syndrome), gray myelitis and post-pediatric syndrome, and hereditary motor sensory neuropathy (Charcot-Marie-Tooth disease), Not limited to them.

5.5.14. Other Activities Proteins of the present invention may have one or more of the following other activities or effects: bacteria, viruses, fungi, and parasites, without limitation. Inhibiting the growth, infection or function of infectious agents, including insects, or killing with such agents; height, weight, hair color, eye color, skin, leanness, without limitation Causing changes in physical characteristics, including the degree of obesity or other tissue pigmentation, or organ or body part size or appearance (eg, increased or decreased breast, altered bone morphology or appearance, etc.) Inhibiting or enhancing); causing a change in the biorhythm or the caricadic cycle or rhythm; causing a change in the fertility of a male or female subject; Altering the metabolism, catabolism, assimilation, processing, utilization, saving or elimination of fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors or components (s) of matter; Unintentionally altering behavioral characteristics, including appetite, sexual impulses, stress, cognition (including cognitive impairment), decline (including depressive disorders) and violent behavior; analgesic effects or other Providing a pain-relieving effect; promoting the differentiation and growth of embryonic stem cells in cell lines other than hematopoietic cell lines; hormonal or endocrine activity; in the case of enzymes, correcting and eliminating the deletion of said enzymes Treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antigen or complement) And the ability to act as an antigen in a vaccine composition to generate an immune response to such a protein, or another material or entity that is cross-reactive with such a protein.

5.5.15. Gene Therapy Mutations in the EGF-Hy1 gene may cause loss of the normal function of the encoded protein. Thus, the present invention provides gene therapy for restoring normal EGF-Hy1 activity or treating disease states involving EGF-Hy1 (eg, various forms of cancer described herein). Induction of a functional EGF-Hy1 gene into appropriate cells can be performed ex vivo, in situ, or in vivo using a vector, more specifically, a viral vector (eg, an adenovirus, an adeno-associated virus, or a retrovirus).
Alternatively, it is effectively performed ex vivo using a physical DNA transfer method (eg, liposome or chemical treatment). For example, Anderson, Nature, Vol. 392, no.
See Addendum 6666, pp. 25-20 (1998). Further, regarding gene therapy techniques, Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific Ameri
See: can: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it is contemplated that preventing expression of EGF-Hy1 or inhibiting the activity of EGF-Hy1 in other human disease states would be useful in treating the disease state. It is also contemplated that antisense therapy or gene therapy may be used to down-regulate EGF-Hy1 expression.

5.5.16. Transgenic Animals In a method for determining the biological function of EGF-Hy1 in vivo, the gene containing one or more EGF-repeats can be used to replicate the animal using homologous recombination. It is overexpressed or inactivated in cell lines [Capecchi, Science 244: 1288-1292 (1989)]. Animals in which the gene is overexpressed under the control of endogenous or exogenous promoter elements are known as transgenic animals. Animals in which the endogenous gene has been inactivated by homologous recombination have been termed "knock-out" animals. Knockout animals, preferably non-human animals, can be prepared as described in US Pat. No. 5,557,032, which is incorporated herein by reference. Transgenic animals are useful in determining the role of EGF-Hy1 in biological processes, preferably in disease states. Transgenic animals are E
It is useful as a model system for identifying compounds that modulate GF-Hy1 activity. Transgenic animals, preferably non-human animals, can be produced as described in US Pat. No. 5,489,743 and PCT Publication No. WO 94/28122, incorporated herein by reference.

To alter the level of expression of the EGF-Hy1 protein, transgenic animals in which all or part of the EGF-Hy1 promoter has been activated or inactivated can be prepared. Inactivation can be performed by homologous recombination as described above. Activation activates a homologous promoter that results in increased protein expression,
It can be achieved by replenishment or replacement. Recruitment of homologous promoters can be achieved by inserting one or more heterologous enhancer elements known to confer promoter activity on certain tissues.

[0182] Knowledge of the EGF-Hy1 DNA sequence provides the cell with modifications that permit or enhance expression of endogenous EGF-Hy1. EGF-Hy1 is expressed (for example, by homologous recombination, ) The cells can be modified. A heterologous promoter is inserted in such a way as to be operably linked to the sequence encoding EGF-Hy1. See, for example, PCT International Publication No. WO94 / 12650, PCT International Publication No. WO92 / 20808, and PCT International Publication No. WO91 / 09955. In addition to the heterologous promoter DNA, amplifiable marker DNA (eg, ada, dhfr, and carbamyl phosphate synthase,
It is also contemplated to insert a multifunctional CAD gene encoding aspartate transcarbamylase and dihydroorotase) and / or intron DNA along with the heterologous promoter DNA. When binding to the EGF-Hy1 coding sequence,
Amplification of the marker DNA by standard selection methods co-amplifies the EGF-Hy1 coding sequence in the cell.

5.6. Pharmaceutical Formulations and Routes of Administration Proteins of the invention, including those from recombinant and non-recombinant sources,
Without limitation, from a variety of sources) can be administered to a patient in need thereof at a dose to treat or ameliorate various disorders, by the protein itself or by a suitable carrier or carrier. It may be administered in a pharmaceutical composition mixed with excipient (s). Such compositions may also contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Is also good. The term "pharmaceutically acceptable" means non-toxic material (s) that do not interfere with the effect of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. Further, the pharmaceutical composition of the present invention, cytokines, rifokines, or M-CSF, GM-CSF, TNF, IL
-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12
Other hematopoietic factors such as, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin may also be included. In addition, the pharmaceutical compositions of the invention may contain other agents that either enhance the activity of the protein or enhance its activity or use in therapy. Such additional factors and / or agents may be included in pharmaceutical compositions to produce a synergistic effect with the proteins of the invention or to minimize side effects. Conversely, the proteins of the present invention may include certain cytokines, rifokines, other hematopoietic factors, or other hematopoietic factors, to minimize the side effects of thrombolytic or antithrombotic factors, or anti-inflammatory agents. , A thrombolytic or antithrombotic factor, or an anti-inflammatory agent formulation. A protein of the present invention may be active in multimers (eg, heterodimers or homodimers) or complexes with itself or with other proteins. As a result, a pharmaceutical composition of the invention may include a protein of the invention in such a multimerized or complexed form.

Techniques for the formulation and administration of the present compounds are described in “Remington's Pharmaceutical Scienc”.
es, "Mack Publishing Co., Easton, PA, latest edition. Further therapeutically effective doses can be found, for example, in treating, curing, preventing or ameliorating the associated medical condition, or improving such conditions, or such conditions. Reference is made to an amount of the compound that is sufficient to result in an increase in the rate of treatment, cure, prevention or amelioration of the compound. When applying an individual active ingredient administered alone, the therapeutically effective dose will be the said ingredient When applied in combination, it refers to the combined amount of the active ingredients that produce the therapeutic effect, whether the therapeutically effective dose is administered sequentially or simultaneously, in combination.

In practicing the methods of treatment or use of the present invention, a therapeutically effective amount of a protein of the present invention is administered to a mammal having the condition to be treated. The protein of the present invention
In accordance with the methods of the present invention, it may be administered either alone or in combination with other therapies, such as treatment with cytokines, rifokines or other hematopoietic factors. When administered with one or more cytokines, rifokines or other hematopoietic factors, the proteins of the invention may be treated with the cytokine (s), rifokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic. At the same time as sex factors,
Alternatively, they may be administered continuously. When administered continuously, the attending physician administers the protein of the invention in combination with the cytokine (s), rifokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic factors. Will determine the appropriate order to do.

5.6.1. Routes of Administration Suitable routes of administration are, for example, oral, rectal, transmucosal or intestinal; intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal In addition to injection or intraocular injection, parenteral delivery may include intramuscular, subcutaneous, intramedullary injection. The pharmaceutical composition or the protein of the present invention used to carry out the method of the present invention may be used in various forms, such as oral ingestion, inhalation, topical application or dermal injection, subcutaneous injection, intraperitoneal injection, parenteral injection or intravenous injection. Administration can be carried out in any conventional manner. Intravenous administration to a patient is preferred.

Alternatively, the compound may be administered by systemic techniques, for example, in the form of a depot or sustained release formulation, for example, by injection of the compound into an arthritic joint or directly into fibrotic tissue. Rather, it may be administered in a local manner. The compounds may be administered topically, for example, as eye drops, in order to prevent the scarring process that often occurs as a complication of glaucoma surgery. Furthermore, in drug-targeted drug delivery systems, for example, targeting arthritic or fibrotic tissue,
For example, they may be administered in the form of liposomes coated with specific antibodies.
The liposomes will be targeted and will be selectively absorbed by the affected tissue.

5.6.2. Compositions / Formulations The pharmaceutical compositions used according to the invention therefore comprise excipients and auxiliaries which facilitate the processing of the active compounds into pharmaceutically usable preparations. It may be formed in a conventional manner using one or more physiologically acceptable carriers, including. These pharmaceutical compositions may be manufactured in a manner known per se, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of a protein of the present invention is administered orally, the protein of the present invention can be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical compositions of the invention may additionally include a solid carrier such as gelatin or an adjuvant. The tablets, capsules and powders comprise about 5-95
% Of the protein of the invention, preferably about 25-90%. When administered in liquid form, a liquid carrier such as water, petroleum, peanut oil, mineral oil, animal or vegetable oils such as soybean oil or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical preparation may further contain physiological salt solutions, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical preparation contains about 0.5-90% by weight of a protein of the present invention, preferably about 1-50% by weight of a protein of the present invention.

When a therapeutically effective amount of a protein of the present invention is administered by intravenous, dermal or subcutaneous injection, the protein of the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. There will be. Preparations of such parenterally acceptable protein solutions with well considered pH, isotonicity, stability, etc., will exist in the art. Preferred pharmaceutical compositions for intravenous, cutaneous or subcutaneous injection are, in addition to the protein of the present invention, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, Ringer's lactate injection, or Includes other vehicles known in the art. The pharmaceutical composition of the invention may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those skilled in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally well-known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. With such carriers, the compounds of the invention may be formulated as tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. Can be Pharmaceutical preparations for oral use can contain solid excipients, crush the resulting mixture to obtain the core of tablets or dragees, and optionally add optional auxiliaries, if necessary After that, it is processed into a mixture of granules. Suitable excipients are fillers, such as sugars, including, inter alia, lactose, sucrose, mannitol or sorbitol; for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxy. Cellulose preparations such as sodium methylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or an alginate salt such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may be gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Classes are optionally included. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The press-fit capsule may contain the active ingredient as a mixture with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and any stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the invention may employ a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is conveniently delivered in the form of an aerosol spray jet from a pressurized pack or nebulizer. In the case of a pressurized aerosol the dosage unit may be determined by providing a value to deliver a measured amount. Cartridges and capsules, for example, for gelatin, for use in inhalers or insufflators, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsifiers in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. .

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the stability of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such depot preparations may be administered (eg, subcutaneously or intramuscularly), by implantation, or by intramuscular injection. Thus, for example, the compound may be a suitable polymer or hydrophilic material (eg, as an emulsifier in an acceptable oil)
Or together with an ion exchange resin, or as a slightly sparingly soluble derivative, for example,
It may be formulated as a slightly hardly soluble salt.

The pharmaceutical carrier for the hydrophilic compounds of the present invention is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic solvent and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution of 65% w / v polyethylene glycol 300, 8% w / v% nonpolar surfactant polysorbate 80, and 3% w / v benzyl alcohol, as measured in absolute ethanol. VPD co-solvent system (VPD: 5W) is a 1: 1 dilution of VPD in 5% dextrose in aqueous solution
Consists of This co-solvent system dissolves hydrophilic compounds well and itself produces only low toxicity upon systemic administration. Of course, the proportions of a co-solvent system may be varied without destroying its solubility and toxicity characteristics. Further, the identity of the co-solvent components is
For example, other low toxicity non-polar surfactants may be used in place of polysorbate 80; the fraction size of polyethylene glycol may be changed; other biocompatible polymers may be polyethylene glycol, For example, it may be replaced with polyvinylpyrrolidone; and dextrose may be replaced with another saccharide or polysaccharide. Alternatively, other delivery systems for hydrophilic pharmaceutical compounds may be used. Liposomes and emulsifiers are well-known examples of delivery vehicles or carriers for hydrophilic drugs. Usually, greater toxicity is at the expense, but certain organic solvents such as dimethyl sulfoxide may be used. In addition, the compounds may be delivered using a depot system, such as semipermeable matrices of solid hydrophilic polymers containing the therapeutic agent. Various long-lasting materials have been established and are well known in the art. Sustained-release capsules may release the compounds for several weeks to more than 100 days, depending on their chemical nature. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization may be used.

The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients. Specific examples of these carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many proteinase inhibiting compounds of the present invention may be provided as salts with pharmaceutically compatible counterions. Such pharmaceutically acceptable base addition salts are salts that retain the biological effectiveness and properties of the free acids, such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamines, dialkylamines. , Monoalkylamines, dibasic amino acids, sodium acetate, potassium benzoate, triethanolamine, and other inorganic or organic bases.

The pharmaceutical composition of the invention may be in the form of a complex of the protein (s) of the invention and a protein or peptide antigen. Said protein and / or peptide antigen will induce a stimulatory signal for both B and T lymphocytes. B lymphocytes will react with antigen through their surface immunoglobulin receptor. T lymphocytes are activated by THC following antigen presentation by MHC proteins.
Will react with antigen through the cell receptor (TCR). MHC and structurally related proteins, including those encoded by class I and class II MHC genes on host cells, will serve to present peptide antigen (s) to T lymphocytes . The antigen component could be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule capable of signaling directly to T cells. Alternatively, antibodies capable of binding to surface immunoglobulins and other molecules on B cells, in addition to antibodies capable of binding to TCRs and other molecules on T cells,
It can be combined with the pharmaceutical composition of the present invention. The pharmaceutical compositions of the present invention may comprise, in addition to other pharmaceutically acceptable carriers, amphiphilic drugs such as lamellar layers in aqueous solution, liquid crystals, lipids which are present in aggregated form such as insoluble monolayers or micelles. And the protein of the present invention may be in the form of a liposome. Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. For example, U.S. Patent No. 4,235,8, all of which are incorporated herein by reference.
71; 4,501,728; 4,837,028; and 4,737,323,
Preparation of such liposome formulations is at the level of skill in the art.

The amount of a protein of the present invention in a pharmaceutical composition of the present invention will depend on the type and severity of the condition to be treated and on the type of prior treatment experienced by the patient. . Ultimately, the attending physician will decide the amount of protein of the present invention to treat each individual patient. First, the attending physician will administer the protein of the invention at a low dose and observe the patient's response. Larger doses of the protein of the present invention may be administered to achieve optimal therapeutic effects for the patient and further increase the dosage. Various pharmaceutical compositions used to carry out the methods of the present invention comprise from about 0.01 μg to about 100 mg of protein of the present invention per kg body weight.
(Preferably from about 0.1 μg to about 10 mg, more preferably from about 0.1 μg to about 1 mg). For compositions of the invention useful for bone, cartilage, tendon or ligament regeneration, methods of treatment include administering the composition locally, systemically, or locally as an implant or device. When administered, the therapeutic composition used in the present invention is, of course, in a pyrogen-free, physiologically acceptable form. In addition, the composition may be desirably injected or encapsulated in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Agents useful for the treatment other than the protein of the present invention, which are optionally contained in the composition as described above, may be selectively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Good. Preferably, for bone and / or cartilage formation, the composition comprises bone and / or cartilage.
Alternatively, it will include a matrix that can deliver the protein-containing composition to the site of cartilage injury, provide a structure for developing bone and cartilage, and can be optimally resorbed by the body. Such matrices may be formed of materials currently used for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, physical properties, surface appearance and interface properties. The particular application of the composition will limit the suitable formulation. Possible matrices for the composition are biodegradable,
It may be chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are bioerodible and biologically well defined bone or skin collagen. Further matrices comprise pure proteins or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, hydroxyapatite, bioglass,
Such as aluminates or other ceramics. The matrix may comprise a combination of any of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics may be altered in calcium-aluminate-phosphate composition and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred are 50:50 (molar weight) copolymers of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150-800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or orthologous clot, to prevent the protein composition from dissociating from the matrix.

A preferred family of said sequestering agents is methylcellulose, ethylcellulose,
Cellulosic materials such as alkylcelluloses (including hydroxyalkylcellulose), including hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, most preferably cationic salts of carboxymethylcellulose (CMC) is there. Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (
Ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestering agent useful herein is from 0.5 to 20% by weight, preferably from 1 to 10% by weight, based on the total formulation weight, which prevents desorption of the protein from the polymer matrix and reduces the composition Indicates the amount needed to provide proper handling of the material, but not enough to prevent the primary cells from entering the matrix, thereby providing an opportunity for the protein to assist the primary cells with the osteogenic activity of the primary cells. Is given. In further compositions, the proteins of the invention may be combined with other agents that are beneficial in treating bone and / or cartilage defects, wounds, or the tissue in question. These drugs include epidermal growth factor
Various growth factors such as (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

At present, the therapeutic composition is also of value for veterinary applications. In addition to humans, especially domesticated animals and thoroughbred horses are medical objects for which such treatment with the proteins of the present invention is desired. The dosage regime of the protein-containing pharmaceutical composition to be used for tissue regeneration depends on various factors that modify the behavior of the protein, such as the amount of tissue weight desired to be formed, the site of the injury, the condition of the damaged tissue, Determined by the attending physician considering the size, type of tissue damaged (eg, bone), age, sex, and diet, severity of infection, number of doses, and other clinical factors of the medical subject Will. Dosage may vary with the type of matrix used for reconstitution, and may also vary with the inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF1 (insulin-like growth factor I) to the final composition, may result in a change in dosage. Progress can be monitored by periodic assessment of tissue / bone growth and / or repair, for example, x-ray, histomorphometry, and tetracycline labeling.

The polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells for expression in a mammalian subject, either in vivo or ex vivo. The polynucleotides of the present invention may also be administered by other known methods for introducing nucleic acids into cells or organs, including, but not limited to, in the form of viral vectors or naked DNA. The cells may be cultured ex vivo in the presence of a protein of the present invention in order to proliferate or to produce the desired effect or activity on the cells. The treated cells can then be introduced in vivo for therapeutic purposes.

5.6.3. Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions that contain an effective amount of the active ingredient to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount that is effective to prevent or alleviate the progression of the existing condition of the subject being treated. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, the dose
Can be formulated in animal models to achieve circulating concentrations that include the IC ₅₀ as determined in cell culture (ie, the concentration of the test compound that achieves half-maximal inhibition of the desired activity)
. Such information can be used to more accurately determine useful doses in humans.

[0204] A therapeutically effective dose refers to that amount of the compound that results in an improvement or prolongation of the patient's symptoms. Toxicity and therapeutic efficacy of the compounds are, for example, LD ₅₀ (50% of the population)
Can be determined by standard pharmaceutical procedures in cell culture or experimental animals to determine the ED ₅₀ (the amount that will kill) and the ED ₅₀ (the amount that produces a therapeutic effect in 50% of the population).
Dose ratio between toxic and therapeutic effects is the therapeutic index can be expressed as the ratio between LD ₅₀ and ED _50. Compounds that exhibit a high therapeutic index are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosage of such compounds lies preferably within a range of circulating concentrations that almost include the ED ₅₀ with absolutely no toxicity or no toxicity. Dosage may vary within this range depending on the dosage form employed and the route of administration used. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition. See, for example, Fingl et al., 1975, "The Pha
rmacological Basis of Therapeutics ", Chapter 1, page 1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to maintain therapeutic or minimal effect concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data.The dosage required to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0205] Dosage intervals can also be determined using MEC value. The compound has a duration of 10
Over 90%, preferably 30-90%, and most preferably 50-90% of the MEC
It should be administered using a regimen that maintains these plasma levels. For local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight,
It will depend on the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

5.6.4. Packaging The composition may, if desired, be contained in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

5.7. Antibodies Another aspect of the invention is an antibody that specifically binds a polypeptide of the invention.
Such antibodies include monoclonal and polyclonal antibodies, single-chain antibodies, chimeric antibodies, bifunctional / bispecific antibodies, humanized antibodies, human antibodies, which specifically recognize the polypeptides of the present invention. And complementarity determining region (CDR) -grafted antibodies, as well as antibodies containing compounds that include CDRs and / or antigen binding sequences. Preferred antibodies of the present invention are human antibodies produced and identified according to the method described in WO 93/11236, issued June 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments comprising Fab, F (ab) 2 and Fv are also provided by the present invention. The term "specific" refers to the variable regions of the antibodies of the invention that recognize and bind only to EGF-Hy1 polypeptide (ie, sequence identity, homology, or within a family of polypeptides). Despite the similarities observed in
Refers to the ability to distinguish EGF-Hy1 from polypeptides containing other EGF repeats), but to interact with other proteins (eg, S. aureu
s protein A or other antibodies found in ELISA techniques), especially also the constant regions within the molecule. Screening assays for determining binding specificity of the invention are well known in the art and are routinely performed. For a comprehensive description of such assays, see Harlow et al. (Eds), Antibodies A La
boratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY
(1988), Chapter 6. Antibodies that recognize and bind to fragments of the EGF-Hy1 polypeptides of the present invention are also contemplated, provided that the antibodies exhibit specificity for an EGF-Hy1 polypeptide as described above. As an antibody specific for the full length of a polypeptide comprising an EGF repeat, a family of polypeptides comprising an EGF repeat, despite identity, homology, or similarity found within the family of proteins, in a unique sequence There is an antibody of the present invention that recognizes an EGF-Hy1 fragment that can distinguish an EGF-Hy1 polypeptide from EGF-Hy1 polypeptide. The antibodies of the present invention are well known in the art,
And it could be produced using any of the methods routinely practiced.

[0209] Non-human antibodies will be humanized by methods well known in the art.
According to one method, non-human CDRs are inserted into a human or consensus antibody framework sequence. Additional modifications can be made to the antibody framework to modulate affinity or immunogenicity.

[0210] The proteins of the present invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies that specifically react with the proteins. Such antibodies may be obtained using either the entire protein or a fragment thereof as an immunogen. The peptide immunogen may further contain a cysteine residue at the carboxy terminus and conjugates to a hapten such as keyhole limpet hemocyanin (KLH). For example, RP Merrifield, J. Amer. Chem. Soc. 85, 2149
Methods for synthesizing such peptides are known in the art, as described in -2154 (1963); JL Krstenansky, et al., FEBS Lett. 211, 10 (1987). A monoclonal antibody that binds to a protein of the invention will be a useful diagnostic agent for immunodetection of said protein. Neutralizing monoclonal antibodies that bind to the protein may also be useful therapeutic agents, both for conditions associated with the protein and for the treatment of some forms of cancer involving aberrant expression of the protein. In the case of cancerous or leukemia cells, neutralizing monoclonal antibodies against the protein may increase the metastatic spread of cancer cells mediated by the protein,
It may be useful for detection and prevention. In general, techniques for preparing polyclonal and monoclonal antibodies, as well as hybridomas that are capable of producing the desired antibody, are well known in the art (Campbell.AM, Monoclon al Antibodies Technology; Laboratory Techniques in Biochemistry and Mole culer Biology, Elsevier Science Publishers, Amsterdam, Netherlands (19
. 84); St Groth et al, J. Immunol 35:. 1-21 ( 1990); Kohler and Milstein, Natur e 256: 495-497 ( 1975)), the trioma technique, the human B cell hybridoma technique (Kozbo
r et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

Any animal known to produce antibodies (mouse, rabbit, etc.)
It can be immunized with a peptide or polypeptide of the present invention. Methods of immunization are well-known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One of skill in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary depending on the animal immunized, the antigenicity of the peptide and the site of injection. The protein used as an immunogen may be administered or modified in an adjuvant to increase the antigenicity of the protein. Methods for increasing the antigenicity of a protein are well known in the art, and include antigens and heterogas proteins (globulins and β
-Galactosidase) or by the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from immunized animals were removed and SP
It is fused with a myeloma cell such as a 2 / 0-Ag14 myeloma cell to obtain a monoclonal antibody-producing hybridoma cell. Hybridoma cells producing antibodies with the desired characteristics can be identified by any one of a number of methods well known in the art. These include screening hybridomas using ELISA assays, Western blot analysis, or radioimmunoassays (Lutz et al . , Exp. Cell Research. 175: 109-124 (1988)). Hybridomas secreting the desired antibody are cloned and subclassed using procedures well known in the art (Campbell. AM, Monoclonal Antibodies Technology; Laboratory Techniques in Biochemistry and Moleculer Biology, Elsevier Science Publishers). , Amsterdam, the Netherlands (1984)). The techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to the proteins of the invention.

For polyclonal antibodies, antibodies, including serum, are isolated from the immunized animal and screened for the presence of an antibody with the desired specificity using one of the procedures described above. The invention further provides the antibody in a detectably labeled form. Antibodies include radioisotopes, affinity labels (such as biotin and avidin), and enzyme labels (such as horseradish peroxidase and alkaline phosphatase)
Fluorescent labels (such as FITC and rhodamine), paramagnetic atoms and the like can be used to detectably label. Such labeling procedures are well known in the art and are described, for example, in (Sternberger, LA et al., J. Histochem. Cytoch em. 18: 315 (1970); Bayer, EA et al ., Meth.Enzym. 62: 308 (1979); Engval, E. et al.
, Immunol. 109: 129 (1972); Goding, JW J. Immunol. Meth. 13: 215 (1976)).

The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues, for expressing fragments of a polypeptide of interest. The antibodies may also be used for direct therapy or other diagnostics. The present invention further provides the antibody immobilized on a solid support. Specific examples of the solid support include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, and polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, DM et al.,
" Handbook of Experimental Immunology " 4th edition, Blackwell Scientific Publi
cations, Oxford, UK, Chapter 10 (1986);.. Jacoby, WD et al., Meth Enzy m 34 Academic Press, New York (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays and immunoaffinity purification of the proteins of the present invention.

5.8. Computer- Readable Sequences In one application of this embodiment, the nucleotide sequences of the present invention can be recorded on a computer-readable medium. As used herein, "computer-readable medium" refers to any medium that can be read and accessed directly by a computer. Such media include magnetic storage media such as floppy disks, hard disk storage media and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and ceramic / optical storage. These include, but are not limited to, hybrids of these categories, such as media. One of ordinary skill in the art would be aware of any of the currently known computer readable media for making a product comprising the computer readable media having recorded thereon the nucleotide sequence of the present invention. It can be easily recognized how to use. As used herein, "recorded" refers to the step of storing on a computer-readable medium. One of skill in the art would readily employ any of the currently known methods for storing information on a computer readable medium to produce a product comprising the nucleotide sequence information of the present invention. can do.

A variety of data storage structures are available to one of skill in the art to create computer readable media having the nucleotide sequences of the present invention recorded thereon. The choice of data storage structure will generally be based on the means chosen to access the stored information. In addition, various data processor programs and formats can be used to store the nucleotide sequence information of the present invention on a computer-readable medium. The sequence information is WordPe
It can be shown as a word processing text file formatted with commercial software such as rfect and Microsoft Word, or as an ASCII file stored in a database application such as DB2, Sybase, Oracle, etc. Those skilled in the art can readily adapt to any number of data processor structural formats (eg, text files or databases) to obtain a computer readable medium having recorded thereon the nucleotide sequence information of the present invention. By providing a nucleotide sequence that is at least 99.9% or more identical to SEQ ID NO: 1 or 10 or a representative fragment thereof, or SEQ ID NO: 1 or 10 in computer readable form, one of ordinary skill in the art can routinely determine The sequence information can be accessed for various purposes. Computer software is publicly available and one skilled in the art can access sequence information provided on computer readable media. By way of example described below, BLAST (A) on the Sybase system to identify open reading frames (ORFs) in nucleic acid sequences.
ltschul et al., J. Mol. Biol. 215: 403-410 (1990)) and BLAZE (Brutlag et al . , Comp. Chem. 17: 203-207 (1993)). Is shown. Such ORFs may be fragments-encoding proteins and may be useful in fermentation reactions and in the production of commercially important proteins such as enzymes used in the production of commercially useful metabolites.

As used herein, “computer-based system” refers to hardware means, software means, and data storage means used for analyzing nucleotide sequence information of the present invention. The minimum hardware means of the computer-based system of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Those skilled in the art will readily recognize that any one of the currently available computer-based systems is suitable for use in the present invention. As described above, the computer-based system of the present invention comprises a data storage means for storing the nucleotide sequence of the present invention, and hardware means and software means necessary to support and execute the search means. Including.
As used herein, the term "data storage means" refers to a memory capable of storing the nucleotide sequence information of the present invention or a memory access means capable of accessing a product storing the nucleotide sequence information of the present invention.

As used herein, “search means” refers to one or more of the computer-based systems executed to compare the target sequence or target structural motif with the sequence information stored in the data storage means. Refers to a program. Search means are used to identify fragments or regions of a known sequence that match a particular target sequence or target motif. Various known algorithms are publicly disclosed, and there are various commercially available software for processing search means, which can be used in the computer-based system of the present invention. As a specific example of such software, MacP
attern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). One of ordinary skill in the art will readily recognize that any one of the available algorithms or software packages for performing the homology search can be adapted for use in this computer-based system. As used herein, a “target sequence” can be a nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. One skilled in the art will readily recognize that the longer the target sequence, the less likely the target sequence will be present as a random occurrence in the database. The most preferred sequence length of the target sequence is about 10-100 amino acids or about 30-300 nucleotide residues. However, it will be appreciated that searches for commercially important fragments, such as sequence fragments related to gene expression and protein processing, may be of shorter length.

As used herein, “target structural motif” or “target motif”
A rationally selected sequence or combination of sequences, which is a sequence (s) selected based on the three-dimensional configuration formed by the folding of the target motif. There are various target motifs known in the art. Protein target motifs include, but are not limited to, an enzyme active site and a signal sequence. As a nucleic acid target motif, a promoter sequence,
There are, but are not limited to, hairpin structures and inducible expression elements (protein binding sequences).

5.9. Triple Helix In addition, the fragments of the present invention can be used to control gene expression by triple helix or antisense DNA or RNA, as described broadly, either method. It is based on the binding of the polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20-40 bases in length and are designed to be complementary to regions of the gene involved in transcription (triple helix-Lee et al . , Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 15241: 456 (1
988); and Dervan et al., Science 251: 1360 (1991)) or mRNA itself (antisense-Olmno, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, Florida (19
88)). While the translation of mRNA molecules into polypeptides is blocked by antisense RNA hybridization, the triple helix structure advantageously results in -blocking of RNA transcription from DNA. Both techniques have proven effective in model systems. The information contained in the sequences of the invention is necessary for the design of antisense or triple helix oligonucleotides.

5.10. Diagnostic Assays and Kits The present invention provides a method for identifying the presence or expression of an ORF of the invention or one of its homologs in a test sample using a nucleic acid probe or antibody of the invention. Provide further.

In general, the method of detecting a polynucleotide of the present invention comprises the steps of: contacting a sample with a compound that binds to the polynucleotide to form a complex for a period sufficient to form a complex; The method includes a step of detecting the complex, and if the complex is detected, the polynucleotide of the present invention in the sample can be detected. Such methods also include, under stringent hybridization conditions, contacting the sample with a nucleic acid primer that anneals to the polynucleotide of the invention under those conditions, and amplifying the annealed polynucleotide. If the polynucleotide is amplified, the polynucleotide of the present invention can be detected in the sample.

In general, the method for detecting a polypeptide of the present invention comprises the steps of contacting a sample with a compound that binds to the polypeptide to form a complex for a period of time sufficient to form a complex; The method includes a step of detecting a complex, and if the complex is detected, the polypeptide of the present invention in the sample can be detected.

In particular, such a method may comprise testing a test sample with one or more antibodies or one or more antibodies of the invention.
Incubating with one or more nucleic acid probes and assaying the binding of a component in a test sample to said nucleic acid probes or antibodies.

[0225] Depending on the test sample, the conditions for incubating the nucleic acid probe or antibody may vary. Incubation conditions will depend on the format used in the assay, the detection method used, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that commonly available hybridization, amplification or immunological assay formats can be readily adapted for use with the nucleic acid probes or antibodies of the present invention. Specific examples of these assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands.
(1986); Bullock, GR et al., Techniques in Immunocytochemistry, Academic P.
ress, Orlando, Florida, Volume 1 (1982), Volume 2 (1983), Volume 3 (1985); Tijssen, P., P ractice and Theory of immunoassays: Laboratory Techniques in Biochemistr y and Moleculer Biology, Elsevier Science Publishers, Amsterdam, It can be found in the Netherlands (1985). Test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the method will depend on the assay format, the nature of the detection method, and the tissue, cells or extracts used as the sample being assayed. Methods for preparing protein or membrane extracts of cells are well known in the art and can be readily adapted to obtain samples that are compatible with the system utilized.

In another embodiment of the present invention, there is provided a kit comprising the necessary reagents for performing the assays of the present invention. In particular, the present invention provides, under strict monitoring, (a) a first container containing one of the probes or antibodies of the present invention; and (b) one or more other containers containing one or more of the following: That is, a compartment kit for containing one or more containers comprising a washing reagent, a reagent capable of detecting the presence of a bound probe or antibody, is provided.

In particular, compartment kits include any kit that includes the reagents in separate containers. Such containers include small glass containers, plastic containers,
Or including a strip of plastic or paper. Such containers do not cause cross-contamination of the sample and reagent, and the efficiency of one compartment to another compartment such that the drug or solution in each container can be added from one compartment to another in a quantitative manner. Transport is possible. Such containers would receive the test sample, containers containing the antibodies used in the assay, containers containing the washing reagents (phosphate buffer, Tris buffer, etc.), and detect bound antibodies or probes. Containers containing the reagents used for this purpose. Types of detection reagent include a labeled nucleic acid probe, a labeled second antibody, or, optionally, an enzyme- or antibody-binding reagent capable of reacting with the labeled antibody if the first antibody is labeled. Probes and antibodies disclosed in the present invention,
One skilled in the art will readily recognize that it can be easily incorporated into one of the established kit formats well known in the art.

5.11. Screening Assays Using the isolated proteins and polynucleotides of the present invention, the present invention provides
A polypeptide encoded by an ORF obtained from a polynucleotide having the sequence of SEQ ID NO: 1 or 10, of a polypeptide encoded by such a nucleic acid
Binds to an EGF repeat domain or specific domain, or SEQ ID NO: 1 or 10
Further provided is a method for obtaining and identifying a substance that binds to a nucleic acid having the sequence of Specifically, the method comprises the following steps: (a) contacting the substance with an isolated protein encoded by the ORF of the present invention or the nucleic acid of the present invention; and (b) Determining whether to bind to the protein or the nucleic acid.

Generally, such methods for identifying a compound that binds to a polynucleotide of the invention include contacting the compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide / compound complex. If the polynucleotide / compound complex is detected, a compound that binds to the polynucleotide of the present invention can be identified.

Similarly, generally, therefore, such methods for identifying a compound that binds to a polypeptide of the present invention include providing a compound to a polypeptide of the present invention to form a polypeptide / compound complex. If the polypeptide / compound complex is detected, the compound that binds to the polynucleotide of the present invention can be identified.

A method for identifying a compound that binds to a polypeptide of the invention comprises contacting a compound with a polypeptide of the invention in a cell for a period of time sufficient to form a polypeptide / compound complex. The complex drives the expression of the reporter gene sequence in the cell, and then binds to the polynucleotide of the invention if the polypeptide / compound complex is detected by detecting expression of the reporter gene sequence. Detecting the complex so that the compound is identified can also be included.

Compounds that modulate the activity of a polypeptide of the present invention (ie, that increase or decrease its activity relative to the activity observed in the absence of the compound) include compounds identified by such methods. Can be included. Alternatively, compounds identified by such methods include compounds that modulate the expression of a polynucleotide of the invention (ie, increase or decrease expression relative to the level of expression observed in the absence of the compound). can do. Compounds, such as those identified by the methods of the invention, can be tested for their ability to modulate activity / expression using standard assays well known in the art.

[0233] Substances screened in the above assays can include, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical substances. Agents can be randomly selected and screened, or can be rationally selected or designed using protein modeling techniques.

For random screening, substances such as peptides, carbohydrates, pharmaceutical substances are randomly selected and assayed for the ability to bind to the protein encoded by the ORF of the present invention. Alternatively, the materials can be rationally selected or designed. As used herein, a substance is said to be "rationally selected or designed" if it is selected based on the configuration of a particular protein. For example, those skilled in the art will recognize that rationally designed peptides, such as Hurby et al., Appl.
ication of Synthetic Peptides: Antisense Peptides, " Synthetic Peptides, A User's Guide , WH Freeman, NY (1992), pp. 289-307, and Kaspczak et al.
See Biochemistry 28: 9230-8 (1989), or presently available techniques for making peptides, drug substances, etc., that can bind to specific peptide sequences to make drug substances, etc. Can be applied without difficulty.

In addition to the aforementioned substances, one class of substances of the present invention broadly defines the OR of the present invention.
Can be used to control gene expression through binding to one of F or EMF.
As noted above, such substances may be randomly screened, or have rational design /
You can make a choice. By targeting ORFs and EMFs, one skilled in the art can modulate the expression of any sequence-specific or element-specific substance, either a single ORF or multiple ORFs that rely on the same EMF expression control. Can be designed. One class of DNA binding substances is those that contain base residues that form a hybridized or triple helical structure by binding to DNA or RNA. Such materials can be based on traditional phosphodiesters, ribonucleic acid backbones, or various sulfhydryl or polymer derivatives that have base attachment capabilities.

[0236] Suitable materials for use in these methods usually contains 20 to 40 bases, and complementary to a region of the gene involved in transcription (triple helix -Lee et, Nucl. Aci ds Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,
Science 251: 136 (1991)) or complementary to the mRNA itself (antisense-Okano. J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Ant isense Inhibitors of Gene Expression , CRC Press, Boca Raton. , FL (1988))
It is designed to be. The formation of a triple helix optimally results in DNA
Blockade of RNA transcription from, while antisense RNA hybridization inhibits the transcription of mRNA molecules into polypeptides. Both techniques have been proven to be effective in model systems. The information contained in the sequences of the present invention is essential for designing antisense or triple helix oligonucleotides, and other DNA binding agents. A substance that binds to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic reagent in controlling bacterial infection by modulating the activity of the protein encoded by the ORF. it can. A substance that binds to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques for making pharmaceutical compositions.

5.12. Use of Nucleic Acids as Probes Another feature of the present invention is to provide polypeptide-specific nucleic acid hybridization probes that are capable of hybridizing to naturally occurring nucleic acid sequences. The hybridization probe of the present invention may be derived from the nucleic acid sequence of SEQ ID NO: 1 or 10. Since the corresponding gene is expressed only in a limited number of tissues, especially adult tissues, the hybridization probe derived from SEQ ID NO: 1 or 10 requires that the RNA of the cell type of such tissue be present in the sample. Can be used as an index to indicate.

[0238] Suitable hybridization techniques, such as in situ hybridization, can be employed. PCR as described in US Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based on nucleotide sequences. Probes such as those used in PCR may be of recombinant origin, chemically synthesized, or a combination thereof. Probes comprising separate nucleotide sequences for the detection of matching sequences,
Alternatively, it would include a degenerate pool with potential for identification of closely related genomic sequences.

[0239] Other means for preparing specific hybridization probes for nucleic acids include cloning the nucleic acid sequence into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used as RNA probes in vitro by adding RNA polymerase as a T7 or S6 RNA polymerase and appropriate radioactively labeled nucleotides. Can be used to synthesize The nucleotide sequence can be used to construct a hybridization probe to map the corresponding genomic sequence. The nucleotide sequences provided in the present invention can be mapped to chromosomes or specific regions of chromosomes using well-known genetic and / or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis for known chromosome markers, hybridization screening using libraries or flow-selected chromosome preparations specific for known chromosomes. The technique of fluorescence in situ hybridization of chromosome seeds is described in particular in Verma et al., (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York NY.

Fluorescence in situ hybridization of chromosome preparations and other physical chromosome mapping techniques can correlate with other genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981 f). A correlation between the location of a nucleic acid on a physical chromosome map and a specific disease (or predisposition to a specific disease) can help define regions of DNA involved in the genetic disease. The nucleotide sequences of the present invention are used to detect differences in gene sequences between normal, carrier or affected individuals. The nucleotide sequence is recombined
Purified polypeptides can be produced using well known methods in DNA technology. After a gene has been isolated, there are many publications teaching methods for its expression, one of which is described in Goeddel (1990) Gene Expression Technology, Met.
hods and Enzymology, Vol 185, Academic Press, San Diego. Polypeptides can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cell may be from the same species as the species from which the nucleotide sequence of the particular polypeptide was isolated, or may be from a different species. Advantages of producing polypeptides by recombinant DNA technology include the ability to obtain appropriate amounts of protein for purification and the simplification of purification methods.

Each sequence so obtained was developed by Applied Biosystems and
The sequences were compared to GenBank sequences using a search algorithm built into the NHERIT ™ 670 sequence analysis system. In this algorithm, regions of homology were examined using a pattern specific language (developed by TRW, Los Angeles, CA). The three parameters that determine how to perform a sequence comparison were window size, window correction, and allowable measurement error. Using a combination of these three parameters, DNA databases were searched for sequences containing regions homologous to the query sequence, and appropriate sequences were scored with initial values. Subsequently, these homologous regions were examined using a dot matrix plot to distinguish homologous regions from accidental matches. A Smith-Waterman alignment was used to show the results of the homology search. Peptide and protein sequence homology was confirmed using an INHERIT ™ 670 sequence analysis system by methods similar to those used for DNA sequence homology. Using a pattern specific language and parameter window, protein databases were searched for sequences containing regions of homology scored by default. Dot-matrix homology plots were examined to distinguish areas of significant homology from chance matches.

Alternatively, using BLAST, which represents a basic local alignment search tool, local sequence alignments (Altschul SF (1993) J Mol Evol 36: 290-300; Altschul
, SF et al. (1990) J Mol Biol 215: 403-10). BLAST creates an alignment of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignment, BLAST is particularly useful in finding exact matches or in identifying homologs. Matches that do not include gaps are notional to perform a motif-style search because they are conceptual. The basic unit of output of the BLAST algorithm is a high scoring segment pair (HSP). An HSP is an optional, but two sequence fragment of the same length, whose alignment is locally maximal and whose alignment score matches or matches the threshold or blocking score set by the user. Consists of more than The BLAST approach searches for HSPs between the query and database sequences, evaluates the statistical significance of all matches found, and only matches that meet a user-selected significance threshold Is to report. Parameter E establishes a statistically significant threshold for reporting database sequence matches. E is interpreted as an upper limit of the expected frequency of occurrence of an HSP (or a combination of a plurality of HSPs) included in the contents of the entire database search. All database sequences whose match satisfies E are reported in the program output.

In addition, BLAST analysis was used to search for related molecules contained in a library in the LIFESEQ ™ database. This process, an "electronic northern" analysis, is similar to a northern blot analysis in that it uses the sequence of one selbrevin at a time to search for matching or homologous molecules at the set stringency. . Electronic Northern stringency is based on a "product score." The product score is 100 (the [% of the nucleotide or nucleic acid in Blast [between the query and control sequences] multiplied by the maximum% of the possible BLAST score [based on the length of the query and control sequences]). Defined as divided. At a product score of 40, the match is 1-2% accurate, and at 70, the match is accurate. Homologous or related molecules can be identified by selecting those that exhibit a product score between approximately 15-30.

[0244]

【Example】

The present invention is illustrated by the following examples. In view of the disclosure herein,
Those skilled in the art will be able to make various embodiments and modifications to the scope of the present invention. Accordingly, it is to be understood that a wide range of embodiments of the present invention are intended and should not be construed in a limiting sense by the following examples. 6. Example: A novel polynucleotide encoding a polypeptide containing an EGF-repeat obtained from a fetal liver spleen cDNA library. Was obtained using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques as described in Bonaldo et al., Genome Res. 6: 791-806 (1996). Inserts from the library were amplified by PCR using primers specific for the vector sequence flanking this insert.
The samples were spotted on a nylon membrane and interrogated with an oligonucleotide probe to obtain a sequence signature. These clones were assembled into groups of similar or identical sequences, and a representative single clone was selected from each clone for gel sequencing.

The 5 'sequence of the amplified insert was then deduced using primers for reverse M13 sequencing in a typical Sanger sequencing protocol. The PCR product was purified and subjected to fluorochrome termination cycle sequencing. Single application gel sequencing was performed using a 377 Applied Biosystems (ABI) sequencing device. Of these inserts identified as novel sequences, those not previously obtained from this library and not yet reported in public databases were inserts of clone pEGF-HY1. This clone was deposited with the ATCC under ATCC Deposit No. 203198.

The sequence was further determined using gene-specific primers (5′-ATGAGTGTAGCTCCAGTCCTTG-3 ′ (SEQ ID NO: 3) and 5′-CACCATCAACACAGATTCCTT-3 ′ (SEQ ID NO: 4)) to obtain the sequence shown in FIG. The nucleotide sequence set forth in SEQ ID NO: 1 was obtained.

The polypeptide encoded by nucleotides 3-284 of SEQ ID NO: 1 is also set forth as SEQ ID NO: 2 in FIG. This amino acid sequence has similarity to the epidermal growth factor repeat of the Notch protein, a protein involved in the cell life control mechanism that regulates multicellular growth. 7. Example: Expression of a novel gene containing SEQ ID NO: 1 To examine the role of SEQ ID NO: 1 and the polypeptide it expresses, expression of a gene containing SEQ ID NO: 1 was determined by semi-quantitative polymerase chain reaction. Analyzed using the technology used. The human cDNA library can be used to convert tissues of interest (adult brain, adult heart, adult kidney, adult lymph nodes, adult liver, adult lung, adult ovary, adult placenta, adult spleen, adult testis, bone marrow, fetal kidney, fetal liver, Fetal liver-spleen, fetal skin, fetal brain, fetal leukocytes and macrophages). Gene-specific primers (5'-CCCAAACCCCTCAGTGTG-3 ', SEQ ID NOs: 5 and 5'-GTCTCTGTCCACTTG-3 to amplify the 420 nt portion of the sequence of SEQ ID NO: 1 from the sample
', SEQ ID NO: 6) was used. Amplification products were separated on an agarose gel.

[0248] SEQ ID NO: 1 was expressed in limited human tissues. Of the 18 human tissues tested, PCR amplification products were found in fetal liver, fetal spleen liver lung, fetal skin, fetal brain and adult lung, indicating that the expression of SEQ ID NO: 1 was not ubiquitous. , Suggesting that it is limited to almost mature organizations. In addition, tests have been performed on various cancer tissues. 8. Example: The polypeptide comprising SEQ ID NO: 2 comprises an amino acid sequence homologous to the epidermal growth factor (EGF) -repeat The nucleotide sequence designated SEQ ID NO: 1 in FIG. 1 is also described in FIG. Encodes the translated amino acid sequence of SEQ ID NO: 2. In particular, nucleotides 3-284 of SEQ ID NO: 1 encodes the 94 amino acid sequence of SEQ ID NO: 2. Sequence analysis of the amino acid sequence of SEQ ID NO: 2 showed that this sequence contained three EGF-repeats.

In particular, FIG. 2 shows that the sequence of SEQ ID NO: 2 and Notch, CD97 and EGF
The amino acid alignment with the EGF-repeat is shown. This alignment is
It shows high homology between the repeats. The most prominent feature of this homology is
The cysteine and glycine amino acid residues are conserved within the repeat (underlined in FIG. 2). 9. Example: Other sequence of EGF-Hy1 Nucleotides 1 to 193 of SEQ ID NO: 10 were obtained using RACE.
Nucleotides 193-1120 of SEQ ID NO: 10 are replaced by another RAC in a three step process
Obtained by E reaction. Using the nucleotides at positions 882 to 1120 as templates for RACE using fetal brain marathon ready cDNA (CLONETECH, Palo Alto, CA), an adapter primer AP2 (CLONETECH) and a gene-specific primer (5'-GTAA
AGACCTAGTTGGGGAATTC-3 ', SEQ ID NO: 12). The resulting PCR product was cloned and sequenced. The nucleotides at positions 674 to 881 were replaced with an adapter primer AP2 and a gene-specific primer (5′-CAGCGGCAGAGGAA
AGTGTGGTG-3 ′ (SEQ ID NO: 13) was similarly obtained. The nucleotides at positions 193 to 673 were replaced with an adapter primer AP2 and a gene-specific primer (5′-GAGCTGCC
TCTCGTGTCCAGA-3 ′ (SEQ ID NO: 14) was similarly obtained.

The present invention has been described in terms of the embodiments, which are merely intended to illustrate one aspect of the invention, and functionally equivalent compositions and methods are intended to cover the scope of the invention. And its disclosure should not be construed as limiting its scope. Indeed, many modifications and variations in the practice of the present invention will occur to those skilled in the art in view of the preferred embodiments of the invention. Accordingly, the scope of the invention should be limited only by the claims provided in the appended claims. All documents cited herein are hereby incorporated by reference in their entirety.

[0251]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of SEQ ID NO: 1 at the top, and the amino acid sequence of SEQ ID NO: 2 at the bottom. These sequences represent embodiments of the novel polynucleotides and polypeptides of the present invention.

FIG. 2 is an EGF-repeat alignment. EGF-repeat at the amino acid sequence of SEQ ID NO: 2 with Notch (SEQ ID NO: 7), CD97 (SEQ ID NO: 8) and (labeled (C)) EGF receptor (SEQ ID NO: 9) proteins Is aligned with the consensus sequence. The Notch protein is involved in the cell fate control mechanism that regulates multicellular growth; CD97 is an activity-inducing antigen for leukocytes belonging to the group of seven-span transmembrane (7-TM) molecules named EGF-TM7. ; EG
F is epidermal growth factor. Gaps are indicated by spaces and non-conserved residues are indicated by dashes. This alignment shows that the amino acid sequence of SEQ ID NO: 2 contains two and a half EGF-repeat domains as shown. The position of the amino acid residue representing the head of each EGF-repeat domain of SEQ ID NO: 2 was entered. One letter amino acid notation was used. In each sequence, conserved cysteine, glutamic acid and glycine residues are underlined.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) / 06 11/00 9/00 11/06 11/00 17/00 11/06 17/02 17/00 19/00 17/02 19/02 19/00 19/10 19/02 21/00 19/10 21/04 21/00 25/00 21/04 101 25/00 25/14 101 25/16 25/14 27/02 25/16 29/00 27/02 101 29/00 31/00 101 31/04 31 / 00 31/10 31/04 31/12 31/10 31/16 31/12 31/18 31/16 31/22 31/18 33/00 31/22 35/00 33/00 35/02 35/00 37/06 35/02 43/00 105 37/06 C07K 14/485 43/00 105 16/22 C07K 14/485 C12N 1/15 16/22 1/19 C12N 1/15 1/21 1/19 C12Q 1 / 68 A 1/21 C12N 15/00 ZNAA 5/10 5/00 A C12Q 1/68 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ) , TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Yun, George United States 94043 San Mateo, California Magnolia Lane 102 (72) Inventor Montgomery, Julie United States 95033 California Santa Cruz Glenwood Drive 23411 F-term (reference) 4B024 AA01 AA11 BA01 BA44 CA01 FA01 GA11 HA14 HA15 4B063 QA01 QA18 QQ42 QQ52 QR32 QR55 QS34 QX01 4B065 AB01 AC14 BA02 CA02 ACA4 ACA4 BA02 BA08 BA19 BA20 BA22 BA23 CA25 DB53 NA14 ZA012 ZA022 ZA152 ZA162 ZA222 ZA332 ZA362 ZA402 ZA442 ZA512 ZA532 ZA542 ZA552 ZA592 ZA662 ZA892 ZA942 ZA962 ZA97 BAZ2 ZB012 ZB08 ZB212 ZB30 ZB12 ZB12 ZB12 ZB20 DA76 EA28 EA50 FA74

Claims (18)

[Claims] (1) a polynucleotide comprising the amino acid sequence of SEQ ID NO: 2; (2) a polynucleotide comprising the amino acid sequence of SEQ ID NO: 2; A polynucleotide containing amino acid residues at positions 15 to 54 of SEQ ID NO: 2; (4) a polynucleotide containing amino acid residues at positions 55 to 94 of SEQ ID NO: 2; (5) a polynucleotide comprising the amino acid sequence encoded by the cDNA insert of clone PEGF-HY1 deposited under ATCC Accession No. 203198; (6) a polynucleotide comprising the amino acid sequence of SEQ ID NO: 11; (7) A polynucleotide comprising the amino acid residues of positions 349 to 385 of SEQ ID NO: 11; (8) a polynucleotide comprising the amino acid residues of positions 387 to 423 of SEQ ID NO: 11; Poly containing the amino acid residue at position 467 Or a nucleotide sequence encoding a polypeptide selected from the group consisting of: (b) a nucleotide sequence, ie, (1) a nucleotide sequence of SEQ ID NO: 1; (2) a nucleotide sequence of positions 3-284 of SEQ ID NO: 1. Nucleotides: (3) nucleotides 3-44 of SEQ ID NO: 1; (4) nucleotides 45-164 of SEQ ID NO: 1; (5) nucleotides 165-284 of SEQ ID NO: 1; (7) Nucleotide sequence of SEQ ID NO: 10; (8) Nucleotide sequence at positions 4-1407 of SEQ ID NO: 10; (7) Nucleotide sequence of cDNA insert of clone PEGF-HY1 deposited under ATCC Accession No. 203198; 9) nucleotides at positions 1048 to 1158 of SEQ ID NO: 10; (10) nucleotides at positions 1162 to 1270 of SEQ ID NO: 10, and (11) nucleotides at positions 1276 to 1404 of SEQ ID NO: 10. Isolated nucleotide sequence, comprising Polynucleotide. 2. An isolated polynucleotide which hybridizes under stringent hybridization conditions to the complement of the polynucleotide of claim 1 and encodes a polypeptide having EGF-HY1 activity. 3. An isolated polynucleotide comprising the complement of the polynucleotide of claim 1. 4. A vector comprising the isolated polynucleotide according to claim 1 or 2. 5. An expression vector comprising the isolated polynucleotide according to claim 1 or 2. 6. A host cell genetically engineered to contain the polynucleotide of claim 1 or 2. 7. A host cell genetically engineered to contain the polynucleotide of claim 1 or 2, wherein the host cell has been engineered into an operable relationship with a regulatory sequence that controls expression of the polynucleotide in the host cell. Host cells. 8. An isolated polypeptide, that is: (1) the amino acid sequence of SEQ ID NO: 2; (2) the amino acid residue at positions 1 to 14 of SEQ ID NO: 2; Amino acid residues at positions 15-54; (4) amino acid residues at positions 55-94 of SEQ ID NO: 2; (5) encoded by the cDNA insert of clone PEGF-HY1 deposited at ATCC Accession No. 203198 (6) the amino acid sequence of SEQ ID NO: 11; (7) the amino acid residue of positions 349 to 385 of SEQ ID NO: 11; (8) the amino acid residue of positions 387 to 423 of SEQ ID NO: 11, or (9) An isolated polypeptide comprising the amino acid residue of positions 425 to 467 of SEQ ID NO: 11. 9. A composition comprising the polypeptide of claim 8 and a carrier. 10. An antibody against the polypeptide according to claim 8. 11. A method for detecting the polynucleotide according to claim 1 or 2 in a sample, comprising the steps of: (a) binding a sample to the polynucleotide to form a complex; Contacting the compound to be formed with the compound for a period of time sufficient to form a complex; and (b) detecting the complex, wherein the complex is detected. The method by which the polynucleotide is detected. 12. A method for detecting the polynucleotide according to claim 1 or 2 in a sample, comprising the steps of: (a) subjecting the sample to stringent hybridization conditions under stringent hybridization conditions. Contacting a nucleic acid primer that anneals to the polynucleotide of claim 1 or 2 under conditions, and (b) amplifying the polynucleotide of claim 1 or 2, wherein the polynucleotide is amplified. A method for detecting the polynucleotide according to claim 1 or 2, for example. 13. The method of claim 8, wherein the polynucleotide is an RNA molecule encoding the polypeptide of claim 8, and the method comprises the steps of:
13. The method according to claim 12, further comprising the step of reverse transcription into a polynucleotide. 14. A method for detecting a polypeptide according to claim 8 in a sample, comprising: (a) binding a sample to said polypeptide to form a complex; Contacting the compound with the compound for a period of time sufficient to form a complex, and (b) detecting the complex, wherein if the complex is detected, the polypeptide of claim 8 is detected. Way. 15. A method for identifying a compound that binds to the polypeptide according to claim 8, comprising the following steps: (a) adding the compound to the polypeptide according to claim 8; 9. The polypeptide of claim 8, comprising contacting for a period sufficient to form a peptide / compound complex; and (b) detecting the complex, wherein the polypeptide / compound complex is detected. A method wherein a compound that binds to a peptide is identified. 16. A method for identifying a compound that binds to the polypeptide of claim 8, comprising the steps of: (a) converting the compound to the polypeptide of claim 8 in a cell. , Polypeptide /
Contacting for a sufficient time to form a compound complex, wherein the complex drives expression of the reporter gene sequence in the cell, and (b) detecting the complex by detecting reporter gene sequence expression 9. A method for identifying a compound that binds to a polypeptide according to claim 8, comprising detecting a polypeptide / compound complex. 17. A method for modulating the activity of a polypeptide according to claim 8, wherein a compound that modulates the activity of the polypeptide is expressed in a cell that expresses the polypeptide. Contacting for a time sufficient to perform the method. 18. A method of modulating the activity of a polypeptide according to claim 8, wherein said polypeptide comprises a compound that modulates the activity of said polypeptide, sufficient to modulate said activity. For a long period of time.