JP2003510029A - EGFH2 gene and gene product - Google Patents

Abstract

(57) Abstract The present invention relates to mouse and human EGFH2, and variants thereof and polynucleotides encoding EGFH2. The invention also relates to therapeutic agents for polynucleotides and proteins. The invention also relates to an isolated nucleic acid molecule, comprising: (a) a polynucleotide encoding from about 1 to about 115 amino acids of SEQ ID NO: 2; (b) a polynucleotide encoding from about 2 to about 115 amino acids of SEQ ID NO: 2. (C) a polynucleotide encoding from about 1 to about 115 amino acids of SEQ ID NO: 4; (d) a polynucleotide encoding from about 2 to about 115 amino acids of SEQ ID NO: 4; (e) (a ), (B), (c) or (d) a polynucleotide complement; and (f) at least (a), (b), (c), (d) or (e) a polynucleotide. A polynucleotide selected from the group consisting of a polynucleotide that is 90% identical.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to novel nucleic acid sequences encoding many neuregulin families and polypeptides encoded by the nucleic acid sequences.

BACKGROUND OF THE INVENTION The ErbB / HER family of growth factor receptors plays an important role in cell signaling during development and proliferation. Some ErbB / HER
Family members are associated with human diseases, including cancer. Erb
A large number of ligands that bind and activate the B / HER receptor have been identified, including epidermal growth factor (EGF), epiregulin, amphiregulin and members of the neuregulin family. (Ben-Baruch et al., Hornones a
nd growth factors in development and
neoplasia. Dickson RB and Salomon DS (ed.) Kulwer Academic Publishers: Boston, p.
p. 145-168, 1988; Burden and Yarden, Neuro.
n 18: 847-855). Since the signal transduction pathway involving ErbB / HER family members is associated with human disease, there is a need in the art for ligands that signal through these receptors. Ligand proteins and the genes encoding them are useful for therapeutic purposes.

The present invention provides genes of the neuregulin family and polypeptides encoded by the genes. The present invention relates to mRNA expressed by a mammalian cell, a polynucleotide encoding a region corresponding to the mRNA, proteins and polypeptide products of the polynucleotide, and biological functions of the polypeptides and proteins.

SUMMARY OF THE INVENTION It is an object of the present invention to provide EGFH2 polynucleotides and polypeptides.

One embodiment of the invention provides a composition comprising an isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: (a) about 1 to about 115 of SEQ ID NO: 2. (B) a polynucleotide encoding from about 2 to about 115 amino acids of SEQ ID NO: 2; (c) a polynucleotide encoding from about 1 to about 115 amino acids of SEQ ID NO: 4 (D) a polynucleotide encoding amino acids from about 2 to about 115 of SEQ ID NO: 4; (e) a polynucleotide complement of the polynucleotide of (a), (b), (c) or (d); and (F) A polynucleotide which is at least 90% identical to the polynucleotide of (a), (b), (c), (d) or (e).

Another embodiment of the invention provides a composition comprising an isolated nucleic acid molecule comprising about 345 contiguous nucleotides from the coding region of SEQ ID NO: 1 or SEQ ID NO: 3.

[0007] In another embodiment, the invention provides a composition comprising an isolated nucleic acid molecule comprising a polynucleotide encoding a polypeptide, wherein at least one conservative amino acid substitution is removed, The polypeptide has an amino acid sequence selected from the group consisting of: (a) about 1 to about 115 amino acids of SEQ ID NO: 2; (b) about 2 to about 115 amino acids of SEQ ID NO: 2; (C) about 1 to about 115 amino acids of SEQ ID NO: 4; (d) about 2 to about 115 amino acids of SEQ ID NO: 4.

[0008]   In a preferred embodiment of the invention, the isolated nucleic acid molecule is DNA.

Another embodiment of the invention provides a method of making a recombinant vector comprising inserting a nucleic acid molecule into a vector in operable linkage with a promoter. The invention further provides a recombinant vector produced by this method.

In another embodiment, the invention provides a method of making a recombinant host cell comprising introducing the recombinant vector into the host cell. The invention further provides host cells made by this method.

A further object of the present invention is to provide a recombinant method for producing a polypeptide, which method comprises culturing a recombinant host cell under conditions in which the polypeptide is expressed and The step of recovering the polypeptide is included.

The invention provides an isolated polypeptide comprising an amino acid that is at least 95% identical to an amino acid selected from the group consisting of: (a) from about 1 to about 115 of SEQ ID NO: 2. (B) about 2 to about 115 amino acids of SEQ ID NO: 2; (c) about 1 to about 115 amino acids of SEQ ID NO: 4; and (d) about 2 to about 115 of SEQ ID NO: 4. amino acid.

A preferred embodiment of the present invention provides an isolated polypeptide, wherein
This polypeptide, except for at least one conservative amino acid substitution, is
), (B), (c) or (d) has an amino acid sequence selected from the group consisting of:

In the most preferred embodiment, the present invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of (a), (b), (c) or (d) above.

In the most preferred embodiment, the polypeptide is a biologically active EG
It includes an FH2 polypeptide. In a preferred embodiment, the polypeptide is
Includes precursor or mature EGFH2 protein. In another preferred embodiment, the polypeptide comprises the EGFH2 EGF-like domain.

A further embodiment of the invention provides a polypeptide comprising an epitope-bearing portion of the polypeptide selected from SEQ ID NO: 2 or SEQ ID NO: 4.

A preferred embodiment provides an epitope-bearing portion of a polypeptide comprising from about 10 consecutive amino acids to 100 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4.

A more preferred embodiment of the invention provides an epitope-bearing portion of a polypeptide comprising about 12 consecutive amino acids to 50 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4.

The most preferred embodiment of the invention provides an epitope-bearing portion of a polypeptide comprising about 15 to 25 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4.

The invention further provides a composition comprising an isolated antibody that specifically binds to a polypeptide of the invention.

In a preferred embodiment, the antibody is a monoclonal antibody, a polyclonal antibody or a single chain antibody.

It is an object of the invention to provide a method of detecting EGFH2 polypeptide or mRNA for diagnostic and prognostic purposes.

In a preferred embodiment, the method comprises an antibody that binds to a polynucleotide of the invention, a human-derived organism suspected of impairing EGFH2 protein modulation with the antibody under binding conditions that form a duplex. Contacting the biological sample and determining the amount of duplexes formed relative to the normal sample.

[0024] In another preferred embodiment, the method comprises the polynucleotide binding to an mRNA encoding a polypeptide of the invention under stringent conditions, a sample of which under binding conditions forming a duplex. Contacting the nucleic acid with the polynucleotide thereof and determining the amount of duplex formed by comparing to a normal sample.

Another object of the invention is to provide a method of modulating the amount of EGFH2 protein in a subject using the polypeptide and polynucleotide compositions of the invention.

[0026] In one embodiment, the method comprises the step of administering an effective amount of a composition comprising a polypeptide of the invention or an antibody that binds to a polypeptide of the invention.

In another embodiment, the method comprises the step of administering an effective amount of a composition comprising a polynucleotide of the invention.

A further object of the present invention is to provide methods of treating EGFH2 protein modulating disorders in a subject using the polypeptide and polynucleotide compositions of the present invention.

In one embodiment, the method comprises administering an effective amount of a composition comprising a polypeptide of the invention or an antibody that binds to a polypeptide of the invention, wherein the composition is In addition, it includes a pharmaceutically acceptable carrier.

In another embodiment, the method comprises the step of administering an effective amount of a composition comprising a polynucleotide of the invention, wherein the composition further comprises a pharmaceutically acceptable carrier. Including.

In a preferred embodiment, the method is accomplished by a transplant cell comprising a polynucleotide expressing a polypeptide of the invention in a patient, wherein the cell expresses an EGFH2 polypeptide in the patient.

[0032]   In the most preferred embodiment, the transplanted cells are encapsulated in a semipermeable membrane.

In another embodiment of the invention, the patient is treated with a therapeutically effective amount of a polynucleotide capable of hybridizing with a polynucleotide of the invention or a complement thereof.

In a preferred embodiment, the polynucleotide is an antisense construct or ribozyme.

In another preferred embodiment, the polynucleotide is a retroviral vector comprising a promoter and a polynucleotide of the invention or their complement.

In yet another embodiment, the patient is treated with a therapeutically effective amount of the polypeptide capable of binding the polypeptide of the invention.

[0037]   In the most preferred embodiment, the polypeptide is an antibody.

In another preferred embodiment, the polypeptide is the wild-type or mutant receptor for EGFH2.

In another embodiment of the invention, the patient is treated with a polypeptide of the invention.

DETAILED DESCRIPTION OF THE INVENTION Neuregulins are a family of structurally related polypeptides whose members modulate cell proliferation, differentiation, apoptosis and cell survival (Carrawaway and Burden, Curr. Opin. Neurobi
ol 5: 1-7 (1995)). In vivo studies reveal that neuregulin-1 disruption is associated with cardiac and neural abnormalities, indicating that neuregulin plays an important role in cardiac and neural development. (Meyer and Birchmeier, Nature
378: 386-390 (1995)). Furthermore, expression and signaling by certain neuregulin family members altered subsequent nerve damage and neurodegeneration (Carroll et al., J. Neurosc. 17: 1642-16).
59 (1997)). Thus, neuregulin has many applications (eg, cancer (breast cancer, prostate cancer, oral cancer, ovarian cancer, etc.); heart disease and disorders (myocardial infarction and ischemia, etc.); wound healing (fractures, tissue repair and regeneration, etc.). ); Skin conditions (burns, cuts, ulcers, etc.); Neurological conditions (neurodegenerative diseases and strokes, multiple sclerosis, peripheral neuropathy, amyotrophic lateral sclerosis, dementia, Alzheimer's disease, Parkinson's disease and Huntington's disease, brain Injury, acute spinal cord injury, nervous system injury and peripheral nerve injury etc.)
;, Including infections, dementia, epilepsy and acoustic trauma) are important therapeutic targets.

Neuregulin mediates cellular responses through interactions with their receptors (ErbB family of transmembrane tyrosine kinases) (Burden and Y).
Arden, Neuron 18: 847-855 (1997)). These receptors function as homodimers and heterodimers. Different receptor combinations exhibit different binding affinities for neuregulins and different abilities to activate downstream signaling complexes, and promote cell proliferation. Neuregulin-1 (NRG1) and neuregulin-2
(NRG2) binds directly to ErbB3 and ErbB4 receptors, but as a coreceptor ErbB1 and ErbB
2 can be replenished. Targeted disruption of the genes encoding ErbB2 and ErbB4 in mice results in congenital abnormalities of the heart and nervous system defects (Lee et al., N.
ature 378: 386-390 (1995); Gassman et al., Nat.
ure 378: 390-394 (1995)). However, certain nerve defects
Differences between ErbB2 and ErbB4 homozygous mutant mice (Caraway)
, BioEssays 18: 263-266 (1996)). These findings reveal that specific receptors play different roles during cell proliferation and development. Different receptor dimers bind preferentially to specific ligands, thus necessitating the identification of novel ligands of the neuregulin family.

Neuregulin constitutes a subfamily of the epidermal growth factor (EGF) family. Neuregulin contains a unique receptor-binding EGF-like motif with 6 conserved cysteine residues. Neuregulin is another extracellular motif (
Signal peptides, cysteine-containing N-terminal domains, either cysteine-rich domains (CRDs) or domains that are slightly related to the Kringle motif, including Ig-like loops, glycosyl domains and transmembrane domains). Burden and Yarden, Neuron
18: 847-855 (1997)). Depending on the presence or absence of the transmembrane domain, the neuregulin precursor can be either a transmembrane protein or a secreted protein. The most highly conserved regions of neuregulins are their EGF-like domains.

Three members of the neuregulin family (NRG1, NRG2 and NR
G3) have been identified. There are multiple isoforms of NRG1 and NRG2, some of which exert heterologous binding affinities for different ErbB receptors (Tzahar et al., J. Biol. Chem. 269: 25.
226-25233 (1994); Pinkas-Kramarski et al., Mo.
l. Cell Biol. 18: 6090-6101 (1998)). Different N
The RG1 isoform results from alternative splicing of a single gene (Ca
rraway, BioEssays 18: 263-266 (1996)). The present invention discloses polypeptides and polynucleotides that encode a novel member of the neuregulin family of proteins called EGFH2. EGFH
2 is expressed in specific tissues during development and promotes meiotic mutations in Xenopus oocytes. In humans, EGFH2 is expressed in skeletal muscle and pancreatic cells, and EGFH2 levels are elevated in pancreatic tumor tissue compared to normal pancreatic samples.

EGFH2 Nucleic Acids, Polypeptides and Antibodies The present invention provides full length mouse cDNA that encodes a predicted 115 amino acid protein homologous to a protein of the neuregulin subfamily of EGF proteins called mEGFH2. . The nucleotide sequence of mEGFH2 is shown in SEQ ID NO: 1. The putative polypeptide sequence encoded by mEGFH2 is shown in SEQ ID NO: 2.

The present invention also provides a human homologue of the mouse EGFH2 gene. Human EGF
The putative open reading frame of the H2 clone (hEGH2) encodes a 115 amino acid protein. The nucleotide sequence of hEGFH2 is shown in SEQ ID NO: 3. The amino acid sequence of the polypeptide encoded by hEGFH2 is shown in SEQ ID NO: 4. The mEGFH2 and hEGFH2 protein sequences are 76% identical (Figure 1). The percent identity between these sequences was performed in the MPSRCH program (Oxford Biomolecular) using the affine gap search with the following search parameters: gap open penalty, 12; gap extension penalty, 1 Smit.
Refers to the identity calculated using the h-Waterman homology search algorithm.

PCR amplification using an oligonucleotide that hybridizes to SEQ ID NO: 1 or SEQ ID NO: 3 also provides a mouse or human EGFH2 polynucleotide using either genomic DNA or cDNA as a template, Can be used. The polynucleotide molecules of the present invention can also be made using synthetic chemistry techniques which provide the sequences disclosed herein. The degeneracy of the genetic code allows for alternative nucleotide sequences that encode the amino acid sequences present in the synthesized SEQ ID NO: 2 and SEQ ID NO: 4. All such nucleotide sequences are within the scope of the invention.

The present invention includes nucleic acid sequences encoding mouse or human EGFH2. Also included within the scope of this specification are sequences that are substantially identical to the nucleic acid sequences encoding EGFH2. For example, such substantially identical sequences can be identified by a given E. coli according to well known and standard procedures. It may be replaced by a more easily expressed codon in a host cell such as E. coli. The present invention also relates to human or mouse EG
Nucleic acid sequences that hybridize to sequences encoding FH2 or its complement are included. Such nucleic acid sequences may be at least 90%, 91%, 92%, 93% or 94% identical, preferably 95%, 96%, 97%, 98% or 99%.
% Can be the same. The invention includes nucleic acid sequences that encode functional domains of EGFH2, such as EGF-like domains. Furthermore, the present invention relates to mEGFH2
Or a nucleic acid encoding a polypeptide that is recognized by an antibody that binds to any of the hEGFH2 polypeptides.

Also provided is an isolated gene corresponding to the cDNA sequences disclosed herein. Known methods can be used to isolate the corresponding gene using the provided cDNA sequences. These methods are for use in identifying or amplifying genes from genomic libraries or other sources of genomic DNA, using probes from the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. Includes primer preparation. Polypeptides encoded by the isolated genes are within the scope of the invention. These polypeptides include the polypeptides encoded by the cDNAs comprising SEQ ID NO: 1 and SEQ ID NO: 3 and isoforms of these polypeptides resulting from alternative splicing of the isolated gene. Not limited.

The present invention provides that any nucleic acid sequence within the scope of the present invention is operably linked,
Vectors containing expression control elements are included. Vectors can include, but are not limited to, parvoviruses including plasmids, episomes, retroviruses, lentiviruses, adenoviruses and adeno-associated viruses. The present invention also includes any type of host cell transformed with a vector containing an expression control element operably linked to any nucleic acid sequence within the scope of the present invention. Examples of host cells include bacterial cells, yeast cells, plants, tissue culture cells (including primary, immortalized, and transformed cell lines), and insect cells.

Testing of EGFH2 mRNA expression by Northern blot analysis was performed using EGFH
2 has been shown to be expressed in limited amounts of tissue in both mouse and human. Multiple mRNA species were detected (see Example 2), which indicates that multiple EGF
It is shown that the H2 polypeptide can be expressed. These polypeptides may be derived from the differential splicing of a single EGFH2 gene or the presence of one or more closely related EGFH2 genes. All EGFH2 isoforms from the differential splicing of a single EGFH2 gene and all related EGFH2 genes are within the scope of the invention.

The invention also includes polypeptide sequences that include sequences responsive to mouse and human EGFH2.

References herein to EGFH2 refer to growth factors of any origin, which are substantially homologous and bioequivalent to EGFH2 characterized and described herein. It is intended to be construed to include. Such substantially homologous growth factors can be native to any tissue or species,
And likewise biological activity can be characterized in any of a number of biological assay systems. For example, biological activity was assayed as described in Example 3 to determine the effect of a given polynucleotide or corresponding mRNA on the disrupted follicular follicles in Xenopus oocytes. Can be detected using.

The term “bioequivalent” refers to the composition of the present invention, E, as described herein.
It is intended to mean that it may exhibit some or all of the same growth characteristics in a similar fashion, but not necessarily to the same extent as GFH2 or recombinantly produced EGFH2 of the invention.

Sequence identity or percent identity refers to, for example, Lasergene biocomputer software (DNASTAR, INC, Madison, WI).
Clustal method for multiple sequence alignments in (Higgins et al., C
abios 8: 189-191, 1992), the alignment of two sequences is intended to mean the percentage of identical residues between the two sequences. In this method, multiple alignments are performed in a progressive fashion, in which increasingly larger groups of alignments are assembled using a similarity score calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score. The maximum alignment score is that of all scores between distinct residues in the alignment, determined from a residue weighting table, which indicates the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Average. Penalties for opening and widening gaps in the alignment contribute to the score. The default parameters used in this program are as follows: gap penalty for multiple alignments = 10; gap length penalty for multiple alignments = 10; k-tuples in pairwise alignments (k-tuple). )
Value = 1; Gap penalty in pairwise modal alignment = 3; Window value in pairwise modal alignment = 5; Diagonal = 5 conserved in pairwise modal alignment. The residue weighting table used for the alignment program is PAM250 (Dayhoff et al., A.
tras of Protein Sequence and Structu
Re, Dayhoff, NDRF, Washington, Volume 5, Separate Volume 3,
345, 1978).

Percent Conservation is the percentage of identical residues to the percentage of positions where two residues show conservative substitutions (defined as having a log odds value of 0.3 or greater in the PAM250 residue weighting table). Calculated from the above alignment by adding the percentages. Conservative amino acid changes that meet this requirement are as follows: RK; ED, YF, LM; VI, QH.

The present invention provides EGFH2 proteins or variants thereof having one or more polymers covalently linked to one or more reactive amino acid side chains. By way of example, and not limitation, such polymers may be conjugated with one or more free cysteine sulfhydryl residues, thereby blocking the formation of disulfide bonds and aggregates when the protein is exposed to oxidative conditions. Polyethylene glycol (PEG). Moreover, PEGylation of the EGFH2 protein and / or mutein is expected to provide such improved properties such as increased half-life, solubility and protease resistance. The EGFH2 protein and / or mutein may instead be modified by the covalent addition of polymers to free amino groups such as lysine epsilon or N-terminal amino groups. Preferred cysteines and lysines for covalent modification are
It is not involved in receptor binding. Those skilled in the art will appreciate that methods for assaying the biochemical and / or biological activity of EGFH2 can be used to determine whether modification of a particular amino acid residue affects the activity of the desired protein. That is clear.

It may be advantageous to improve the stability of EGFH2 by modifying one or more protease cleavage sites. Thus, the present invention provides that one or more protease cleavage sites, for example, to create EGFH2 variants with improved stability,
Provided are EGFH2 variants modified by one or more amino acid substitutions at the cleavage site. The stability of such improved proteins may be effective during protein production and / or therapeutic applications. Suitable protease cleavage sites for modification are well known in the art, and probably depending on the particular application considered,
Change.

A fusion protein comprising EGFH2 or a biologically active or antigenic fragment thereof can also be produced using methods known in the art. Such fusion proteins can be used therapeutically or can be produced to simplify isolation and purification procedures. Histidine residues can be incorporated to allow for immobilized metal affinity chromatography purification. EQKLISEEEDL
The residues contain the antigenic determinants recognized by the myc monoclonal antibody and can be incorporated to allow for myc monoclonal antibody-based affinity purification. Thrombin cleavage sites can be incorporated at selected sites to allow cleavage of the molecule; preferred thrombin cleavage sites are LVPRG residues. Purification of the molecule is achieved by binding SAWRHP to paramagnetic streptavidin beads.
This can be facilitated by incorporating sequences such as the QFGG residue. Such an embodiment is described in WO 97/25345, which is incorporated by reference.

Also as EGFH2, hybrid and modified forms of EGFH2, including fusion proteins and EGFH2 fragments, as well as hybrid and modified forms in which certain amino acids have been deleted or substituted, and one or more amino acids. A modification such as a modified amino acid or an abnormal amino acid,
As well as modifications such as glycosylation so long as the hybrid or modified form retains the biological activity of EGFH2. A polypeptide comprising a conserved structural or functional domain of EGFH2 (such as an EGF-like domain) is
Within the scope of the present invention. The EGF-like domain of neuregulin is specific for Erb
Can bind to B / HER receptor (Burden and Yarden, Neu
ron 18: 847-855 (1997)). Other neuregulin regions may serve to recruit receptor dimerization partners. The isolated EGFH2 EGF-like domain may affect the dimeric form and function of the receptor. For example, the isolated EGFH2 EGF-like domain is a full-length EGF
The isolated EGFH2 EGF-like domain, which is capable of directing the formation of specific receptor dimers different from the receptor dimers induced by H2, is responsible for receptor dimerization by not recruiting coreceptors. Can be prevented. Similarly, amino acid substitutions in the region of EGFH2 that are involved in coreceptor recruitment may affect dimer formation. EGFH affects receptor dimerization
2 and the ability of the mutant to be purified EGFH2 or epitope-tagged EGF
It can be tested by incubating cells expressing various ErbB / HER receptors with H2. Antibodies to EGFH2 or epitope tags are used to immunoprecipitate EGFH2 and related receptors according to well-established procedures in the art. Co-precipitated receptors can be identified by Western blot analysis using antibodies specific for the different receptors or by immunoprecipitation using radiolabeled cells. Isolated EGFH2 EGF-like domain and EG affecting receptor dimerization
FH2 variants are within the scope of the invention.

The present invention also includes fragments of EGFH2. Preferred fragments include about 9 to about 45 amino acids; about 4 to about 50 amino acids; about 4 to about 82 amino acids; about 1 or 2 to 50 amino acids; and about 1 or 2 to about 82 amino acids. . Such fragments can be prepared from the protein by standard biochemical methods or by expressing a polynucleotide encoding this fragment.

Also included within the scope of the invention are EGFH2 molecules that differ from native EGFH2 by the alteration of the biologically active site. EGFH2 has a putative transmembrane domain at amino acid residues 63-82. This transmembrane domain-free EGFH2 molecule can be prepared by expressing a DNA encoding EGFH2, in which some or all of the responsive codons at amino acid residues 63-82 have been deleted. DNA encoding EGFH2 with altered receptor binding can be produced as well. The EGF-like receptor binding domain of EGFH2 extends from approximately amino acid 4 to amino acid 50. For example, it may be desirable to alter EGFH2 receptor specificity by substituting a receptor binding region of a different neuregulin or other EGF-like domain for the receptor binding region of EGFH2.

Also included within the meaning of substantially homologous are the EGFs described herein.
Can be isolated by cross-reactivity with antibodies to H2 or genomic DNA
, MRNA, or cDNA, wherein the coding nucleotide sequence is any EGFH2 that can be isolated by hybridization of EGFH2 or a fragment thereof herein with the genomic or subgenomic nucleotide sequence or the complementary sequence of the cDNA. . Degenerate DNA sequence is human or mouse EG
It will also be appreciated by those of skill in the art that FH2 may be encoded, and these, as well as allelic variants of EGFH2, are intended to be included within the scope of the present invention.

Recombinant human EGFH2 or recombinant mouse EGFH2 can be made by expressing the DNA sequence encoding EGFH2 in a suitable transformed host cell. The DNA encoding EGFH2 can be ligated into an expression vector and transformed under established conditions suitable for expression of EGFH2 by cells transformed into host cells, using methods well known in the art.

The DNA encoding EGFH2 can be engineered to take advantage of the preferred codon usage of the host cell. For example, Pseudomonas aerugino
The frequency of codon usage in sa is as described by West et al., Nucleic Acids R.
es. 11: 9323-9335 (1988). For example, Sa
The codon usage in Ccharomyces cervisiae is Ll
Oyd et al., Nucleic Acids Res. 20: 5289-5295 (
1992). Codon preference in Corynebacterium and E. E. coli priority compared to Malubres et al., Gene 1
34: 15-24 (1993). For example, Drosophil
The frequency of codon usage in a melanogaster is determined by Akashi, Ge.
netics 136: 927-935 (1994).

Any suitable expression vector may be recombinant human EGFH2 or recombinant mouse EG
It can be used to produce FH2 (eg, an expression vector for use in insect cells). Baculovirus expression systems can also be used. A preferred method is expression in insect cells (eg Tr5 cells or Sf9 cells) using a baculovirus vector.

The present invention includes nucleic acid sequences that include sequences encoding human or mouse EGFH2. Also included within the scope of the invention are sequences that are substantially the same as the nucleic acid sequence encoding EGFH2. Such substantially identical sequences can be prepared according to well-known and standard procedures, eg, E. It may be replaced with a codon that is more easily expressed in a given host cell, such as E. coli. Such modified nucleic acid sequences are included within the scope of the invention.

Specific nucleic acid sequences can be modified by the person skilled in the art, and thus all nucleic acid sequences encoding the amino acid sequence of EGFH2 can likewise be modified in this way. Accordingly, the invention also includes a nucleic acid sequence (or, where appropriate, the complement of a nucleic acid sequence) that hybridizes to all such nucleic acid sequences, and an EFGH2 organism as disclosed herein. Encodes a polypeptide having a biologically equivalent activity.
The invention also includes nucleic acid sequences that encode a polypeptide having a biologically equivalent activity of EGFH2, and nucleic acid sequences that encode a polypeptide recognized by an antibody that binds to EGFH2.

The present invention also includes vectors that include expression control elements operably linked to any nucleic acid sequence included within the scope of the present invention. The present invention also includes any type of host cell transformed with a vector containing an expression control element operably linked to any nucleic acid sequence included within the scope of the present invention.

Methods for producing EGFH2 are also provided herein. Preparation can be by isolation from conditioned medium from various cell types, so long as the cell type produces EGFH2. The second and preferred method is to isolate or obtain a nucleic acid sequence encoding EGFH2, clone this sequence into an appropriate vector and cell type with appropriate regulatory sequences, and express this sequence to produce EGFH2. The use of recombinant methods by

EGFH2 polypeptides containing one or more amino acid substitutions are included in the present invention. Amino acid changes in the protein variants or derivatives of the invention can be conservative substitutions. A conservative amino acid change involves one amino acid substitution for another amino acid in a family of amino acids that have structurally related side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid) amino acids, basic (lysine, arginine, histidine) amino acids, nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, Methionine, tryptophan) amino acids, and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine)
amino acid. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. Non-naturally occurring amino acids can also be used to form the protein variants of the invention.

Whether an amino acid change results in a functional protein or polypeptide is
It can be readily determined by assaying the biological properties of the disclosed proteins or polypeptides. For example, as discussed in Example 3, the biological properties of mitogens can be measured by examining the effect of protein or polypeptide expression on Xenopus oocyte variants.

Standard biochemical methods may be used to isolate the protein of the invention from the tissue expressing the protein, or the protein, polypeptide or fusion protein may be used in recombinant host cells (in which DNA structures are present). Has been introduced). Protein purification methods (such as size exclusion chromatography, ammonium sulfate crystallization, focus electrophoresis or preparative gel electrophoresis) are well known,
And widely used in the field. Synthetic chemistry methods, such as solid phase peptide synthesis, may be used to synthesize the protein, fusion protein or polypeptide of the invention.

Recombinant EGFH2 can be produced by expressing a DNA sequence encoding mouse or human EGFH2 in a suitable transformed host cell. Using methods well known in the art, DNA encoding EGFH2 can be ligated into an expression vector and transformed under established conditions suitable for expression of EGFH2 by cells transformed into host cells. Any suitable expression vector, such as an expression vector for use in bacterial cells, yeast cells or insect cells, may be used for recombinant human EGFH.
2 or recombinant mouse EGFH2. Baculovirus expression systems can also be used. One preferred method is expression in insect cells (such as Tr5 cells or Sf9 cells) using baculovirus vectors.
Another method of producing EGFH2 is to isolate EGFH2 from conditioned medium from various cell types, as long as the cell type produces EGFH2. Such cells include primary cells, immortalized cells, transformed cells, or cells transiently or stably transfected with a vector expressing EGFH2 or EGF.
Examples include, but are not limited to, cells infected with H2 with a virus.

The resulting expressed protein can be purified from its culture medium or from extracts of its cultured cells using purification procedures known in the art. For example, for proteins that are completely secreted into the culture medium, cell-free medium can be diluted with sodium acetate and contacted with cation exchange resin, followed by hydrophobic interaction chromatography. Using this method, the desired polypeptide is typically greater than 95% pure. Further purification can be performed, for example, using any of the techniques listed above. The protein, fusion protein, or polypeptide may also be an epitope (eg, "Flag" epitope (Kodak)).
) And can be purified using an antibody that specifically binds to its epitope.

In order to obtain a functional protein, it may be necessary to modify the protein produced in yeast or bacteria, for example by phosphorylation or glycosylation at appropriate sites. Such covalent modifications can be made using known chemical or enzymatic methods.

The protein or polypeptide of the invention may also be expressed in cultured cells in a form that facilitates purification. For example, a secreted protein or polypeptide can be expressed as a fusion protein, including, for example, maltose binding protein, glutathione-S-transferase, or thioredoxin, and purified using commercially available kits. Kits for the expression and purification of such fusion proteins are available from companies such as New England BioLabs, Pharmacia and Invitrogen.

The present invention provides both full-length and mature forms of the disclosed proteins. The full length form of the protein has the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4. The full-length form of the protein can be enzymatically processed to remove the signal sequence, yielding the mature form of the protein. Signal sequences can be identified by examination of the amino acid sequences disclosed herein and comparison with amino acid sequences of known signal sequences (von Heigne (1985), "Sign.
al Sequences: The Limits of Variation
"J. Mol. Biol. 184: 99-105 and Kaiser and Bo.
Stein, (1986) Mol. Cell. Biol. 6: 2382. See).

Polypeptides consisting of less than the full-length protein of the invention are also provided. The polypeptides of the present invention can be linear or cyclized. The polypeptide can be used, for example, as an immunogen, diagnostic aid or therapeutic agent. Polypeptides less than the complete amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4 are also provided.

Polypeptide Fragments The present invention provides at least 8,10,12,15,18,19,20,25,50,75,1 selected from SEQ ID NO: 2 or SEQ ID NO: 4 of the disclosed proteins.
It may comprise 00, 105, 110 or 112 consecutive amino acids. All intermediate length fragments in this range (eg 21, 22, 23 etc .; 51, 52, 5
Also included are such as 3; and 76, 77, 78, etc. (these are exemplary only and not limiting).

Biologically Active Variants Variants of the proteins and polypeptides disclosed herein may also exist. Variants can be naturally occurring or non-naturally occurring. Naturally occurring variants include amino acid sequences found in humans or other species and that are substantially identical to the amino acid sequences set forth in SEQ ID NOs: 2 or 4. Species homologues of proteins may be prepared using the subgenomic polynucleotides of the invention to make suitable probes or primers, as described below, for other species (e.g., mouse, monkey, yeast,
Alternatively, it can be obtained by screening a cDNA expression library derived from bacteria), identifying this cDNA encoding a homologue of this protein, and expressing the cDNA as is known in the art.

Also included herein are non-naturally occurring variants that retain substantially the same biological activity as the naturally occurring protein variants. Preferably, the naturally occurring or non-naturally occurring variants have an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 2 or 4. More preferably, the molecule is at least 98% or 99%
It is the same. Percent identity is determined using any method known in the art. A non-limiting example is an affine gap with a gap open penalty of 12 and a gap extension penalty of 1.
A Smith-Waterman homology search algorithm that uses a search.
The Smith-Waterman homology search algorithm is based on Smith and W.
aterman, Adv. Appl. Math (1981) 2: 482-489.
Taught in.

Guidance in determining which amino acid residues can be substituted, inserted, or deleted without compromising biological or immunological activity can be found in computer programs well known in the art (eg, DNASTAR software). Fair) can be found. Preferably, the amino acid changes in the secreted protein variant are conservative amino acid changes, ie, substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions in one of the families of related amino acids in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid) amino acids, basic (
Lysine, arginine, histidine) amino acids, non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) amino acids, and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes co-classified as aromatic amino acids.

Independent substitutions of leucine with isoleucine or valine, isolated substitutions of aspartic acid with glutamic acid, isolated substitutions of threonine with serine, or similar substitutions of amino acids with structurally related amino acids result in the resulting modifications. It is reasonable to predict that it will not have a significant effect on the biological properties of the body.

Variants of EGFH2 proteins disclosed herein include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. Covalent variants can be prepared by linking functional groups to groups found in amino acid chains or N-terminal residues or C-terminal residues, as is known in the art.
Variants also include allelic variants, species variants, and muteins. Truncates or deletions of regions that do not affect the functional activity of the protein are also variants.

A subset of variants, called muteins, is a group of polypeptides in which neutral amino acids such as serine are replaced with cysteine residues that are not involved in disulfide bonds. These variants may be stable over a wider range of temperatures than the native secreted protein. See Mark et al., U.S. Pat. No. 4,959,314.

Preferably, the amino acid changes in variants of the EGFH2 protein or polypeptide are conservative amino acid changes (ie, substitution of similarly charged or uncharged amino acids). Conservative amino acid changes include substitutions in one of the amino acid families related to their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, Isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine,
Tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes co-classified as aromatic amino acids.

Independent replacement of leucine with isoleucine or valine, independent replacement of aspartic acid with glutamic acid, independent replacement of threonine with serine, or similar replacement of amino acids with structurally related amino acids. It is plausible to predict that does not have a significant effect on the biological properties of the resulting secreted protein or variant of the secreted polypeptide. The characteristics and functions of the variant of EGFH2 protein or polypeptide are the same type of characteristics and functions as the protein containing the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 3, but the characteristics and functions of the variant are Functions may vary to some extent.

EGFH2 protein variants include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. EGFH2 protein variants also include allelic variants, species variants, and muteins. Truncates or deletions of the region that do not lead to differential expression of the EGFH2 protein gene are also variants. Covalent variants can be prepared by linking functional groups to groups found in amino acid side chains or N-terminal residues or C-terminal residues, as is known in the art.

It is recognized in the art that some amino acid sequences of the EGFH2 proteins of the invention can be modified without significantly affecting the structure or function of the protein. If such differences in sequence are intended, it should be remembered that there are important regions on the protein that determine activity. Generally, if residues that serve a similar function are used, it is possible to replace the residues that form the tertiary structure. In other cases, the type of residue may not be fully important if the alteration occurs in a non-critical region of the protein. Amino acid substitutions also
The selectivity of binding to cell surface receptors can be altered. Ostade et al., Natu
Re 361: 266-268 (1993) describes specific mutations that result in the selective binding of TNF-α to only one of the two known TNF receptor types. Thus, the polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or artificial manipulations.

The invention further includes variants of EGFH2 polypeptides that exhibit comparable expression patterns or that contain antigenic regions. Such mutations include deletions, insertions, inversions, repeats, and type substitutions. Guidance regarding amino acid changes being phenotypically silent is given in Bowie, J. et al. U. Et al., “De
ciphering the Message in Protein Seq
uences: Tolerance to Amino Acid Subst
editions ", Science 247: 1306-1310 (1990).
) Can be found in.

Substitution of a charged amino acid with another charged amino acid and with neutral or negatively charged amino acids is of particular interest. The latter results in proteins with a reduced positive charge, improving the properties of the disclosed proteins. Prevention of agglomeration is highly desirable. Aggregation of proteins not only results in loss of activity, but can be problematic in preparing pharmaceutical formulations because they can be immunogenic (Pinckar).
d et al., Clin. Exp. Immunol. 2: 331-340 (1967);
Robbins et al., Diabetes 36: 838-845 (1987); C.
Leland et al., Crit. Rev. Therapeutic Drug Ca
rlier Systems 10: 307-377 (1993)).

Amino acids in the polypeptides of the invention that are essential for function can be generated by methods known in the art (eg, site-directed mutagenesis or alanine scanning mutagenesis (Cun).
Ningham and Wells, Science 244: 1081-108.
5 (1989)). The latter procedure introduces a single alanine mutation at every residue of the molecule. The resulting mutant molecules are then tested for biological activity, such as binding to natural or synthetic binding partners. Sites important for ligand-receptor binding have also been analyzed by structural analysis (eg crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224: 8).
99-904 (1992) and de Vos et al., Science 255: 3.
06-312 (1992)).

Antibodies to EGFH2 are provided by the present invention. The antibodies of the invention can be used, for example, to detect the polypeptides of the invention in culture medium or tissue samples. Antibodies can be polyclonal, monoclonal or single chain antibodies. Antibodies to mouse and human EGFH2 protein or epitopes thereof can be made by any of the many methods known in the art. Detailed methods for raising antibodies are described in Harlow and Lane, incorporated by reference,
Antibodies: A Laboratory Manual, Cold
Provided in Spring Harbor Laboratories, 1988. Methods of raising antibodies are also described in US patent application Ser. No. 08 / 988,671, which is incorporated by reference in its entirety. By epitope, reference is made to the antigenic determinant of a polypeptide. An epitope can include 3 amino acids in a spatial conformation unique to the epitope. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. Antibodies to EGFH2 are also raised against oligopeptides containing one or more conserved regions identified herein, such that known antibodies may cross-react with other species and / or family members. obtain.

Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting the antigen followed by subsequent boosts at appropriate intervals. Animal sera are assayed for immunoreactivity to EGFH2 by any of a number of methods including, for example, Western blot or ELISA. Monoclonal Antibodies by Fusing Splenocytes from Mice Immunized with Serially Replicating Tumor Cells
can be prepared according to the method of Lstein and Kohler (Milste
in and Kohler, Nature 256: 495-497, 1975).
.

Techniques for purifying antibodies are techniques available in the art. In a preferred embodiment, the antibody is affinity purified by passing the antibody through a column to which the amino acid sequence of the invention is attached. The bound antibody is then eluted. Any technique can be selected for antibody purification of the present invention.

The polypeptides encoded by EGFH2 can be used to screen peptide libraries to identify binding partners among the encoded polypeptides. Candidate binding partners for EGFH2 include:
EGFH2 to form cell surface receptors and multimeric protein complexes
Examples include proteins that bind proteins. Such binding partners are
It may be useful in treating cancer, hyperproliferation and related conditions. For example, peptides and antibodies that can bind to an EGFH2 binding partner or receptor can block EGFH2 binding to that partner or receptor, thereby inhibiting the biological activity of EGFH2. Polypeptide binding partners are identified by any method available in the art, such as expression cloning using a labeled EGFH2 polypeptide probe, or a yeast two-hybrid screen using a fusion protein containing the EGFH2 polypeptide as a bait. obtain. Yeast two-hybrid screens (also called interaction capture assays) are described, for example, by Gyuris et al., Cell 74: 791-803 (
1993). Binding partners can also be identified by low stringency immunoprecipitation using antibodies to the EGFH2 polypeptide, followed by sequencing the co-precipitated polypeptide.

Peptide agonists and antagonists are screened using any available method such as signal transduction, antibody binding, receptor binding, mitogen assay, chemotaxis assay and the like. A library of peptides can be synthesized according to the methods disclosed in US Pat. No. 5,010,175 and PCT WO91 / 1723. Assay conditions should ideally be similar to those in which native activity is shown in vivo, ie under physiological pH, temperature, and ionic strength. Suitable agonists or antagonists exhibit potent inhibition or enhancement of native activity at concentrations that do not cause toxic side effects in the subject.

The end result of such screens and experiments is at least a novel EGFH2 protein binding partner, such as a receptor, and at least one peptide agonist or antagonist of EGFH2. Such agonists and antagonists regulate EGFH2 protein function in cells,
It can be used to enhance or inhibit.

Therapeutic and Diagnostic Uses of EGFH2 Members of the neuregulin family affect cell proliferation, differentiation and apoptosis. For example, neuregulin-1 stimulates mitogenesis of mouse fibroblasts, human Schwann cells, epithelial cells and glial cells (Carr).
Away et al. Biol. Chem. 270: 7111-1116 (1995)
); Morrissey et al., Proc. Natl Acad. Sci. USA
92: 1431-1435 (1995); Peles and Yarden, Bi.
oesays 15: 815-824 (1993)). Neuregulin-1 also acts as a survival factor for astrocytes (Pinkas-Kramarski)
Et al., Prc. Natl Acad. Sci. USA 91: 9387-9391.
(1994)). Thus, neuregulin regulates cell proliferation and tissue homeostasis and is important for the treatment of abnormal cell proliferation, such as cancer, tumor progression, hyperproliferative cell proliferation or associated diseases with biological or physical manifestations. Equivalent to a good target. EGFH2 is a neuregulin family member that regulates cell proliferation. For example, EGFH2 induces meiotic maturation of Xenopus oocytes as illustrated in Figure 4.

Neuregulin is expressed in a tissue-specific and stage-specific pattern. Neuregulin-1 isoforms are predominantly expressed in nerve and muscle tissue (Carrawaway, Bioessays 18: 263-266 (1996).
) Will be discussed). Northern blot analysis of EGFH2 mRNA expression in various human tissues showed that EGFH2 is expressed predominantly in pancreatic and skeletal muscle tissues (see Example 2). In situ hybridization of samples from human pancreas detected elevated levels of EGFH2 expression in samples of human pancreatic tumors (see Example 2). Therefore, according to the present invention, EGFH2 may play a role in pancreatic cancer. Analysis of mouse EGFH2 expression in mouse embryos showed that this EGFH2 was widely expressed throughout the embryo except in the neural tube and limb regions. Therefore, EGFH2 may play a broad role in promoting cell proliferation during development. The differences in neuregulin-1 and EGFH2 mRNA expression patterns indicate that their corresponding polypeptide products have different roles in cell proliferation and development. Therefore, inactivating EGFH2 activity may have important biological effects. Given the sequence homology between neuregulin family members, the family members may share common biological properties. Therefore, EGF
It is possible that H2 may promote the growth of cells other than those normally expressed or contacted. Therefore, it is within the scope of the invention to use EGFH2 polynucleotides and polypeptides for the treatment of diseases or conditions that are not normally associated with EGFH2.

EGFH2 is a cell responsive to this molecule (cranial or peripheral nerves, sensory cells,
Treatment of hearing, olfactory or taste disorders due to loss / dysfunction of supporting cells and the like, and peripheral motility characterized by loss or function of neurons, nerves, other CNS or PNS cells, or supporting cells Or it may be useful in the treatment of sensory neurological disorders (amyotrophic lateral sclerosis, motor or sensory neuropathy (many causes: viral infection, metabolic disease, diabetes, autoimmune injury, idiopathic, degenerative) and trauma).

Cardiomyopathy characterized by functional deficits in the heart or cardiomyocyte or supporting cell death (congestive heart failure, myocarditis) can also be treated with EGFH2 such that correction of arrhythmias, ischemic heart disease and birth defects can be treated. Can be treated with. Musculoskeletal diseases characterized by loss of function of skeletal muscle cells, bone cells or supporting cells, inadequate function or death can also be treated with EGFH2. Examples include hereditary or acquired skeletal muscle disorders, bone disorders and arthritis. Other skeletal muscle conditions that can be treated include cancer,
AIDS, heart failure, wasting due to systemic diseases such as aging and inactivation, and rhabdomyosarcoma. The EGFH2 of the present invention can be a target for inhibitors or other modulators, or can be used as a differentiation factor to treat a direct condition. In diabetic patients, the EGFH2 of the invention may be useful in enhancing skeletal muscle glucose utilization.

Ischemic vascular disease can be treated with EGFH2. EGFH2 polynucleotides and polypeptides may help correct congenital defects resulting from defects in neuregulin or its function (heart, lungs, brain, kidneys, limbs, pancreas, etc.).

Pancreatic conditions that may be treated include pancreatic exocrine disorders related to exocrine insufficiency (especially malabsorption), pancreatic endocrine disorders (especially diabetes) and pancreatitis and related complications. Pancreatic cancer can be treated using inhibitors of EGFH2 or other modulators. Alternatively, or additionally, EGFH2 can be used as a differentiation factor to directly treat cancer.

Treatment of wound healing is yet another use for EGFH2 polypeptides and polynucleotides. Examples include cell regeneration due to liver regeneration, surgical wound healing, traumatic wound healing, ulcer healing, bone destruction, metabolic and inflammatory diseases.

The present invention includes methods of treating patients in need thereof with the polynucleotides and polypeptides of the present invention. Diseases and disorders that may be treated using the compositions of the invention include cancers including breast cancer, prostate cancer, pancreatic cancer, oral cancer and ovarian cancer, peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease. , Huntington's disease, ischemic stroke, brain injury, acute spinal cord injury, nervous system injury, multiple sclerosis,
Infections, dementia, epilepsy, peripheral nerve damage, acoustic trauma and tissue wounds,
It is not limited to these.

The present invention includes therapeutic or pharmaceutical compositions comprising the polynucleotides and polypeptides of the present invention. The pharmaceutical composition may include a polypeptide, antibody, small molecule or polynucleotide. A therapeutic agent, whether a polynucleotide or polypeptide, or a small molecule, is, for example, Bosland, Encyclope.
dia of Cancer, Volume II, pages 1283-1296 and 13
03-1313 (1997) Academic Press can be tested in the animal models and cell lines disclosed. The pharmaceutical composition can be designed to either reduce or increase EGFH2 activity, depending on the type of disease and disorder being treated. For example, cancer or a disease associated with hyperproliferation is
Decreasing EGFH2 activity (eg, therapeutically effective amount of antisense EG
FH2 RNA, EGFH2 ribozymes, inactivated antibodies, peptides or small molecule antagonists or inhibitors, EGFH2 variants or EGFH2 receptors or variants thereof). However, conditions or diseases that can be treated by promoting cell proliferation are treated, for example, by administering an expression vector comprising a biologically active EGFH2 polypeptide or an EGFH2 polynucleotide encoding a biologically active EGFH2 polypeptide. obtain.

The term “therapeutically effective amount” as used herein refers to a therapeutic agent that treats, ameliorates, or prevents a particular disease or condition, or that has a detectable therapeutic or prophylactic effect. The amount of drug. This effect can be detected by, for example, chemical markers or antigen levels. The effects also include reduction of physical symptoms. Effective doses for a given situation are determined by routine experimentation, and this is
It is at the clinician's discretion. The exact effective amount will vary depending on factors, including, but not limited to, the size and health of the subject, the nature and extent of the condition, and the therapeutic agent selected for administration. For purposes of this invention, an effective dose is about 0.01 mg / kg to 50 mg / kg, or 0. 0, for a polynucleotide, polypeptide, or antibody composition in the administered individual.
It is from 05 mg / kg to about 10 mg / kg.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of therapeutic agents (eg, antibodies, polypeptides, polynucleotides, and other therapeutic agents). Suitable carriers and pharmaceutically acceptable salts are well known to those of ordinary skill in the art. For a detailed discussion of pharmaceutically acceptable excipients, see Remington's Pharmaceut.
ical Sciences (Mack Pub. Co., N.J. 1991)
Is available at.

Once formulated, the polynucleotide and polypeptide compositions of the invention can be (1) directly administered to a subject; (2) delivered ex vivo to cells derived from the subject. Or; (3) can be delivered in vitro for expression of the recombinant protein. Direct delivery of the composition is generally accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or into the interstitial space of tissue (
interstitial space). The composition can also be administered within a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, as well as transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and are described, for example, in International Publication WO93 / 14778. In general, delivery of nucleic acids for both ex vivo and in vitro applications is
For example, dextran-mediated transfection, calcium phosphate precipitation, viral infection, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide encapsulation in liposomes, and direct microinjection of DNA into the nucleus, which can be achieved. all,
Well known in the art.

Examples of polynucleotide therapeutic agents include ribozymes, antisense RNAs and mammalian expression vectors. Trans-cleaving catalytic RNA (ribozyme) is an RNA molecule that has endoribonuclease activity. Ribozymes are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event destabilizes the mRNA and prevents protein expression. The design and therapeutic use of ribozymes is described in US
man et al., Current Opin. Struct. Biol. (1996)
6: 527-533. The EGFH2 polynucleotide sequence provides sequences suitable for constructing effective ribozymes. The target cleavage site is selected in the target sequence and the ribozyme is 5'and 3'consecutive to the cleavage site.
It is constructed based on the nucleotide sequence. The retrovirus vector is EGFH
Engineered to express monomeric and multimeric hammerhead ribozymes that target the mRNA of the two polynucleotide coding sequences. These monomeric and multimeric ribozymes are tested in vivo for their ability to cleave EGFH2 mRNA. The cell line is stably transduced with a retroviral vector expressing a ribozyme, the transduction being confirmed by Northern blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Cells are examined for inactivation of target mRNA by such indicators as reduced expression of disease markers or reduced gene product of target mRNA.

The present invention also relates to antisense oligonucleotides designed to interfere with the normal functioning of EGFH2 polynucleotides. Modifications or variations of any antisense molecule known in the art that are broadly applicable to antisense technology are included within the scope of the invention. Such modifications are described in US Pat. Nos. 5,536,821; 5,541,306; 5,550,111; 5,563,253.
No. 5,571,799; No. 5,587,361; No. 5,625.
Including adjustment of phosphorus-containing linkages, as disclosed in 050 and 5,958,773.

The antisense compounds of the present invention may include modified bases such as those disclosed in US Pat. No. 5,958,773 and the patents disclosed therein. The antisense oligonucleotides of the invention can also be modified by chemical linkage of the oligonucleotide with one or more moieties or conjugates to enhance activity, cellular distribution or cellular uptake of the antisense oligonucleotide. Such moieties or conjugates include lipids (eg cholesterol), cholic acid,
Thioether, aliphatic chain, phospholipid, polyamine, polyethylene glycol (P
EG), palmityl moieties and, for example, US Pat. Nos. 5,514,758 and 5;
, 565,552, 5,567,810, 5,574,142,
No. 5,585,481, No. 5,587,371, No. 5,597,69
No. 6, and others disclosed in No. 5,958,773.

Chimeric antisense oligonucleotides are also within the scope of the present invention, and include, for example, US Patents 5,013,830, 5,149,797, 5,403,711, No. 5,491,133, No. 5,565,350,
No. 5,652,355, No. 5,700,922 and No. 5,958,
It can be prepared from the oligonucleotides of the invention using the method described in 773.

In the antisense field, some routine experimentation is required to select the optimal antisense molecule for a particular target. Effectively, preferably the antisense molecule is targeted to accessible or bare sites on the target RNA molecule. Although in some cases information is available on the structure of the target mRNA molecule, current approaches to inhibition using antisense are empirical. Cellular mRNA levels include mRNA reverse transcription and cDNA
Assaying levels can be routinely measured in treated and control cells. Biological effects can be routinely determined by measuring cell proliferation or viability as known in the art.

Measuring the specificity of antisense activity by assaying and analyzing cDNA levels is an art-recognized method of confirming antisense results. For example, RNA from treated cells and control cells can be reverse transcribed and the resulting cDNA population can be analyzed (Branch, AD, T. et al.
I. B. S. 23: 45-50, 1998).

Antisense nucleic acids are RNA-DNA or RNs designed to specifically bind RNA and arrest DNA replication, reverse transcription or messenger RNA translation.
This produces a form of A-RNA hybrid. Antisense polynucleotides based on the selected sequence may interfere with expression of the responsive gene. Antisense polynucleotides are typically produced in cells by expression from an antisense construct containing the antisense strand as the transcribed strand. Antisense polynucleotides bind and / or prevent translation of the corresponding mRNA. The expression products of control cells and cells treated with antisense constructs are compared to detect the protein product of the gene corresponding to the polynucleotide. Proteins are isolated and identified using conventional biochemical methods.

Antisense treatments for various cancers are in the clinical stage and have been extensively discussed in the literature. In antisense therapy, given the broad background literature and clinical trials, one of skill in the art can use antisense EGFH2 polynucleotides as a therapy. The dosage and method of administration will be determined based on the specific characteristics of the composition, patient, disease stage and other relevant factors. Preferably, the therapeutic antisense composition comprises at least 12 of EGFH2 in the antisense orientation,
An expression construct comprising a promoter upstream of a polynucleotide segment of 22, 25, 30 or 35 contiguous nucleotides. The therapeutic antisense agent may be administered locally or systemically in a variety of ways known in the art. Examples cited are the methods and receptor-mediated targeted delivery referred to above.

Therapeutic compositions comprising antisense EGFH2 polynucleotides will range from about 100 ng for topical administration in gene therapy protocols, as discussed below.
The range of about 200 mg of polynucleotide is administered. In all cases, routine experimentation in clinical trials will determine the particular extent of optimal therapeutic effect.

Polypeptide compositions can also include antibodies and peptides. An effective amount of therapeutic composition comprising a protein, polypeptide or antibody is about 5 μm of patient weight.
g to about 50 μg / kg, about 50 μg to about 5 mg / kg, about 10 for patient weight
Ranges from 0 μg to about 500 μg / kg and about 200 μg to about 250 μg / kg.

Antibodies can be polyclonal, monoclonal or single chain antibodies prepared by methods known in the art. Specific antibodies to the polypeptide encoded by EGFH2 bind to the protein and prevent the protein from functioning in the cell. For example, the antibody may prevent EGFH2 from binding to protein partners or receptors. Such antibodies may also induce conformational changes in the binding polypeptide. The invention also relates to antibodies to protein partners and receptors of EGFH2 polypeptides. Such antibodies may interfere with the protein: protein interactions required for the cellular function of EGFH2. Antibodies also block downstream signaling by receptor proteins.

Also included in the invention are therapeutic compositions and methods that include peptide agonists and antagonists. The peptide can affect the function of the polypeptide encoded by EGFH2 or its binding partners and receptors. For example, may the peptide block protein: protein interactions,
Alternatively, it may result in a conformational change that diminishes or enhances the normal functional activity of EGFH2. The peptides may also modify the activity or specificity of EGFH2 in a therapeutically useful manner.

The therapeutic polynucleotides and polypeptides of the present invention can be used in gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (generally Jolly, Cancer Gene Therapy).
(1994) 1: 51-64; Kimura, Human Gene Ther.
apy (1994) 5: 845-852; Connelly, Human Ge.
ne Therapy (1995) 1: 185-193; and Kaplit.
, Nature Genetics (1994) 6: 148-153). Gene therapy vehicles for delivery of constructs containing the coding sequence for EGFH2 include:
It can be administered either locally or systemically. These constructs may utilize viral or non-viral vector approaches. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

Any method of gene delivery known in the art can be utilized. For example, the invention may use a recombinant retrovirus constructed to carry or express a selected nucleic acid molecule of interest. The invention also uses alphavirus-based vectors and parvoviruses that can function as gene delivery vehicles. Polycation-enriched DNA ligated or unligated only to killed adenoviruses, eg Curiel, Hum. Gene Ther. (1992) 3: 147-15.
4; ligand-ligated DNA, eg Wu, J. et al. Biol. Chem. (1989)
264: 16985-16987; see eukaryotic cell delivery vehicles, eg US Application No. 08 / 240,030, filed May 9, 1994 and US Application 08 / 404,796. Deposition of photopolymerized hydrogel material; hand-held gene transfer particle gun described in US Pat. No. 5,149,655; ionizing radiation described in US Pat. No. 5,206,152 and WO92 / 11033; Other gene delivery vehicles and methods can be used, including nuclear charge neutralization or cell membrane fusion. A further approach is described by Philip, Mol. C
ell Biol. (1994) 14: 2411-2418 and Woffen.
din, Proc. Natl. Acad. Sci. (1994) 91: 1158.
1-111585. Suitable packing cell lines for use with the retroviral vector constructs described above can be readily prepared (PCT
See WO 95/30763 and WO 92/05266), and producer cell lines (also referred to as vector cell lines) for the production of recombinant vector particles.

Non-viral delivery methods include mechanical delivery systems (eg Woffendin
Et al., Proc. Natl. Acad. Sci. USA (1994) 91 (24)
: 11581-11585) and naked DNA protocols. An exemplary method for introducing naked DNA is WO90
/ 11092 and US Pat. No. 5,580,859.

Members of the EGF signaling network are involved in human cancer. For example,
Some epithelial cancers overexpress ErbB-2 at levels 100-fold greater than normal, and in some types of cancer the extent of overexpression predicts disease outcome and response to treatment (Burden). And Yarden, Neuron 18
: 847-855 (1997)). The HER-2 / neu oncogene encodes a member of the ErbB / HER receptor family that is overexpressed in breast cancer. HER-2 / neu expression levels are used as diagnostic and prognostic factors in the diagnosis and prognosis of response to therapy (Ross and Flet).
cher, Oncologist 3: 237-252 (1998)).

Subgenomic EGFH2 polynucleotides and their complements can be used as markers for diagnosing and detecting the prognosis of cancer, tumor progression, hyperproliferating cell proliferation or associated biological and physical expression. . EGFH in the sample
The levels of 2 polynucleotides or polypeptides are compared to the levels in a normal control sample. A normal sample can include a pool of cells from a particular tissue or tissue and / or cells from the whole body. Immunoassays or nucleic acid assays can be used for such measurements. Any difference observed between the sample and normal controls may be indicative of the development of the disease or disorder. Typically, when the level of EGFH2 polynucleotide or polypeptide is higher than that found in normal controls, the result is cancer, tumor progression, hyperproliferative cell proliferation, and / or associated biological or Shows the occurrence of physical expression.

Nucleic acid assays utilize subgenomic polynucleotides that are capable of hybridizing under stringent conditions to EGFH2 polynucleotides or their complements. A polynucleotide probe comprising at least 10 contiguous nucleotides selected from the nucleotide sequence of EGFH2 can be labeled with, for example, a radioactive, fluorescent, biotinylated or chemiluminescent label, and is suitable for the particular label selected. It is detected by a known method. Subgenomic polynucleotides preferably do not contain introns. The polynucleotide corresponding to EGFH2 can be introduced into a vector and propagated in a suitable host. The plasmid can be introduced into the host cell using techniques available in the art. These techniques include, but are not limited to, electroporation and calcium phosphate mediated transfection. Plasmids are standard techniques (eg,
DNA by restriction enzyme digestion and gel electrophoresis or chromatography)
It can be isolated from the vector and purified. Polynucleotides can also be produced using the polymerase chain reaction according to techniques well known in the art.

Subgenomic polynucleotides can be used to compare related genes in normal control tissue and suspected diseased tissue by any means known in the art. For example, EGFH2 from suspected diseased tissue can be sequenced and compared to EGFH2 sequences in normal tissue. Polynucleotide-related genes of two tissues, or parts thereof, can be isolated using the polymerase chain reaction, eg
It is amplified using a nucleotide primer based on the nucleotide sequence of GFH2. The amplified gene or portion of the gene is hybridized with a nucleotide probe selected from the same nucleotide sequence and sequenced. Differences in the nucleotide sequences of polynucleotide-related genes in suspected diseased tissue when compared to normal nucleotide sequences suggest a role for the polynucleotide-encoded protein in disease, and for the preparation of therapeutic agents. Provide clues. Nucleotide probes or nucleotides that are incorporated during sequencing are labeled by a variety of methods, such as radioisotope labeling, biotinylation or labeling with fluorescent or chemiluminescent tags, and by standard methods known in the art. To be detected.

Alternatively, EGFH2 mRNA levels in normal tissue and cells suspected of being diseased are compared. PolyA ⁺ RNA is isolated from two tissues as is known in the art. For example, one skilled in the art will appreciate Northern blot analysis, primer extension, S1.
Nuclease protection, reverse transcription polymerase chain reaction (RT-PCR), or E
Differences in size or quantity of polynucleotide-related mRNA transcripts between two tissues can be readily determined by in situ hybridization using a polynucleotide probe that responds to GFH2 or its complement. Increased or decreased expression of polynucleotide-related mRNA in a tissue sample suspected of disease as compared to expression of the same polynucleotide-related mRNA in normal tissue indicates that the expressed protein plays a role in disease. Suggested and also provide clues for preparing therapeutic agents.

EGFH2 gene expression can also be tested using a polynucleotide array. Polynucleotide arrays provide a high throughput technique that can assay large numbers of polynucleotide sequences in a sample. This technique can be used as a diagnostic and as a tool for testing differential expression of encoded proteins. Techniques for constructing arrays and methods of using these arrays are described in European Patent No. 0.
799 897; PCT No. WO97 / 29212; PCT No. WO97 / 2
7317; European Patent 0 785 280; PCT WO 97/02357.
U.S. Patent No. 5,593,839; U.S. Patent No. 5,578,832; European Patent No. 0 728 520; U.S. Patent No. 5,599,695; European Patent No. 0.
721 016; US Pat. No. 5,556,752; PCT WO 95/2.
2058; and US Pat. No. 5,631,734, which are incorporated by reference.

Antibodies that bind EGFH2 and / or variant polypeptides can be used for the diagnosis and detection of cancer, tumor progression, hyperproliferative cell proliferation or associated biological and physical expression and prognosis. These antibodies can be monoclonal antibodies, polyclonal antibodies or single chain antibodies and are produced by methods well known in the art.

Any method known in the art can be used to compare EGFH2 encoded proteins from normal control and suspected disease samples. The protein size of the two tissues can be determined by eg EG
Comparisons can be made using an antibody to the polypeptide encoded by EGFH2 to detect FH2 polypeptides. Changes in the size of EGFH2 protein in suspected diseased tissue as compared to levels in normal control samples indicate that the protein is abnormal, probably due to shortened, deleted or altered post-translational modifications. Indicates. The size changes indicate that EGFH2 has a role in disease and provides clues for preparing therapeutic agents. Other changes such as protein expression levels and subcellular localization can also be detected immunologically using, for example, antibodies against the polypeptide encoded by EGFH2 for Western blots or immunofluorescence. High or low levels of EGFH2 protein in conditioned medium of cells suspected of having a disease or in cells derived from that tissue as compared to levels in a normal control sample indicate that EGFH2 has a role in disease. , And provide clues for preparing a therapeutic agent. Similarly, changes in subcellular localization of EGFH2 protein also indicate that EGFH2 has a role in disease.

Reagents specific to EGFH2 polynucleotides and polypeptides, such as antibodies and nucleotide probes, can be supplied in kits to detect the presence of expression products in biological samples. The kit also includes buffers or labeling components, detection reagents, instructions for using the reagents to detect and quantitate the expression products in the biological sample and the control normal biological sample. May be included. The normal biological sample can be in any form suitable for the particular detection method utilized by the kit. For example, a normal biological sample can be a polynucleotide, polypeptide, cell extract or tissue section.

Preferred embodiments of the invention are described in the examples below. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification, together with the examples, be considered as exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

(Example) (Example 1) (Identification of EGFH2) A clone derived from a mouse liver cDNA library was used as a putative mouse EGFH2 (
mEGFH2) was included. Sequencing of the mEGFH2 clone was performed using mEGFH
Two clones are full-length cDNAs encoding the predicted 115 amino acid protein
It was shown to contain. An in-frame stop codon was identified upstream of the initiator ATG.

A clone of mEGFH2 was used to identify a human homologue of the mouse EGFH2 gene. The sequence of the human clone contains 100 base pairs of unspliced intron sequence. The predicted open reading frame of the human EGFH2 clone (hEGFH2) encodes a 115 amino acid protein. m
Comparison of the EGFH2 and hEGFH2 sequences showed that the proteins were 75% identical (Figure 1).

EGFH2 does not appear to contain the predicted signal peptide or Ig domain. However, hydropathic analysis of the mEGFH2 protein revealed a cryptic transmembrane domain corresponding to the EGF-like domain at the N-terminus of EGFH2 (amino acids 4-50). mEGFH2 EGF-like domain is the EGF of neuregulin-1β
Is 32% identical to the like domain. In particular, both mEGFH2 and hEGFH2
Includes 6 conserved cysteine residue properties of EGF-like proteins. Furthermore, the expected E
The GFH2 amino acid sequence contains a C-terminal putative proteolytic cleavage site for the EGF-like domain. Therefore, the EGFH2 protein is a member of the EGF family.

Example 2 mRNA Expression Northern blot analysis of mouse tissues showed that mEGFH2 was expressed predominantly in liver tissues. Two dominant bands of 1.35 kb and 2.4 kb and three minor bands of 4.0 kb, 4.4 kb and 8 kb were observed. further,
Mouse heart, lung, skeletal muscle and kidney have 1.35 kb and 2.4 kb mRN
There was some expression of A. Northern blot analysis of various human tissues using the mouse EGFH2 probe confirmed a similar banding pattern in human skeletal muscle and pancreatic tissues.

Using the mEGFH2 probe, mice were given 8.5 and 9.5 days p. c.
In situ hybridization performed on embryos revealed that EGFH2 is widely expressed throughout the embryo except neural tube and limbs. Furthermore, the specific expression found in heart tissue became restricted to the atrium in the 11.5 day embryo. Thus, EGFH2 exhibits both tissue-specific and developmental stage-specific expression in mammals. Furthermore, in situ hybridization of human pancreatic samples using the mEGFH2 probe showed higher EGFH2 expression levels in human pancreatic tumor samples compared to normal pancreatic samples, indicating that EGFH2 has increased expression in cancer pancreatic cells. Indicated. Example 3 Biological Properties Secretion: The precursor form of the EGF family is proteolytically processed to yield secreted growth factors. To clarify that EGFH2 was secreted, C
The construct containing mEGFH2 with HA tag at the end was cloned into pCS2 expression vector and transfected into Cos cells. Transfected c
Supernatants of os cells and cell lysates were analyzed by Western blot using HA antibody. A protein of approximately 20 kD was detected in cell lysates, but not in Cos cell supernatants. However, since the putative proteolytic cleavage site is located at the C-terminus of the predicted secretion product containing the EGF-like domain, and the HA tag is located at the C-terminus of the precursor protein, EGFH2 It seems that the secreted form of was not detectable after the proteolytic treatment.

Furthermore, in order to test whether EGFH2 was secreted, HA labeled m
Co transfected with EGFH2 expression construct or vector control
s cells are radioisotopically labeled with [ ³⁵ S] -methionine. Cell lysates and supernatants are separated by SDS-PAGE and the presence of EGFH2 polypeptide is determined by autoradiography. The presence of a novel radioisotope labeled band in the cell supernatant indicates that EGFH2 is secreted. Cell lysates and supernatants are also tested by Western blot or immunoprecipitation of EGFH2 polypeptide using an antibody against the N-terminal region of the EGFH2 precursor protein. The presence of immunoprecipitated or Western blotted polypeptides specific for HA-tagged EGFH2 transfected cells in the cell supernatant indicates that EGFH2 was secreted. Mitogen Properties Members of the neuregulin family have a mitogenic effect on cell proliferation. To determine that EGFH2 possesses mitogenic properties, the effects of EGFH2 sense and antisense RNA on follicular disruption (GVBD) were tested. The EGFH2 cDNA insert was cloned into the pCDNA3 vector in the opposite direction of the CMV promoter. EGFH2 sense (Sample No. 3) and antisense (Sample No. 2) mRNA were added to pCDNA3 by EG
Sense of EGFH2 with HA tag prepared from FH2 gene (Sample No. 4
) MRNA was prepared from the EGFH2 gene of the pCS2 vector by in vitro transcription. Each mRNA sample was injected into Xenopus oocytes to evaluate the effect on GVBD. The results are tabulated in Figure 4. EGFH2 induces meiotic maturation of Xenopus oocytes.

The activity of neuregulin family members, mitogens, can be mediated by the MAP kinase pathway. By activating the MAP kinase pathway, EGFH2
To confirm that it induces meiotic maturation of Xenopus oocytes,
The ability of EGFH2 to induce GVBD was determined by dominant negative Raf or dominant negative MAP.
Test in the presence of kinase. Sense or antisense EGFH2 mRNA
Are injected into Xenopus oocytes in the presence or absence of varying amounts of sense or antisense dominant negative Raf or dominant negative Map kinase. The effect of various mRNA combinations on GVBD is measured.

From the foregoing, it will be understood that particular embodiments of the invention have been described herein for purposes of illustration and various modifications can be made without departing from the spirit and scope of the invention. . Accordingly, the invention is not limited except by the appended claims.

[Brief description of drawings]

FIG. 1 shows human EGFH2 (SEQ ID NO: 4) and mouse EGFH2 (SEQ ID NO: 2).
) Amino acid sequence alignments, with stars indicating conserved residues.

[Fig. 2]   Figure 2 shows the human EGFH2 polynucleotide sequence (SEQ ID NO: 3).

[Figure 3]   Figure 3 shows the mouse EGFH2 polynucleotide sequence (SEQ ID NO: 1).

FIG. 4 shows the effect of EGFH2 mRNA on Xenopus mature oocytes.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/395 A61K 48/00 4C084 A61P 9/10 4C085 48/00 17/02 4C086 A61P 9/10 19 / 08 4C087 17/02 21/04 4H045 19/08 25/00 21/04 25/08 25/00 25/14 25/08 25/16 25/14 25/28 25/16 31/00 25/28 35 / 00 31/00 43/00 111 35/00 C07K 14/485 43/00 111 16/22 C07K 14/485 C12N 1/15 16/22 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12P 21/02 5/00 A C12Q 1/68 A61K 37/02 (81) Designated country 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, MZ , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA , BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, 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, MA, MD, MG, MK, MN, MW, MX , MZ, 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 Lee, Paulin USA California 94608, Emilyville, Houghton Street 4560 F Term (reference) 4B024 AA01 BA21 BA44 CA04 DA02 EA04 FA02 GA11 HA01 HA14 4B063 QA01 QA18 QQ43 QR32 QR36 QR55 QS34 4B064 AG13 AG27 CA10 CA19 CC24 DA01 4B065 AA90X AA99Y AB01 AC14 BA02 CA24 CA25 CA44 CA46 4C076 AA53 BB32 CC01 CC11 CC18 CC21 CC27 CC29 CC31 FF63 4C084 AA02 AA03 AA06 AA07 AA13 BA02 BA22 BA23 CA53 DC50 NA14 ZA022 ZA062 ZA152 ZA162 ZA202 ZA342 ZA362 ZA892 ZA942 ZA962 ZB262 ZB312 ZB352 ZC022 4C085 AA13 AA14 BB11 CC04 CC05 DD22 DD33 ZA16 ZA26 ZA26 ZA16 ZA02 ZA16 ZA01 ZA16 ZA01 ZA02 ZA16 ZA01 MA16 ZA02 4C087 AA01 AA02 AA03 BB63 CA12 MA37 MA67 NA14 ZA02 ZA06 ZA15 ZA16 ZA18 ZA20 ZA34 ZA36 ZA89 ZA96 ZB26 ZB31 ZB35 ZC02 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA20 DA75 EA22 FEA28 EA28 A74

Claims (37)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) a polynucleotide encoding the amino acids of about 1 to about 115 of SEQ ID NO: 2;
(B) a polynucleotide encoding the amino acid of about 2 to about 115 of SEQ ID NO: 2;
(C) a polynucleotide encoding the amino acids of about 1 to about 115 of SEQ ID NO: 4;
(D) a polynucleotide encoding the amino acid of about 2 to about 115 of SEQ ID NO: 4;
(E) a polynucleotide complement of (a), (b), (c) or (d) polynucleotide; and (f) (a), (b), (c), (d) or (e). An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: a polynucleotide that is at least 90% identical to the polynucleotide of. 2. An isolated nucleic acid molecule, which is SEQ ID NO: 1 or SEQ ID NO: 3.
An isolated nucleic acid molecule comprising about 345 contiguous nucleotides from the coding region of. 3. An isolated nucleic acid molecule comprising a polynucleotide encoding a polypeptide, except for at least one conservative amino acid substitution,
The polypeptide comprises: (a) about 1 to about 115 amino acids of SEQ ID NO: 2; (b) about 2 to about 115 amino acids of SEQ ID NO: 2; (c) about 1 to about 115 of SEQ ID NO: 4. An isolated amino acid molecule having an amino acid sequence selected from the group consisting of: amino acids; and (d) about 2 to about 115 amino acids of SEQ ID NO: 4. 4. The isolated nucleic acid molecule of claim 1, which is DNA. 5. A method for producing a recombinant vector, which comprises the step of inserting the nucleic acid molecule according to claim 1 into a vector in an operably linked state with a promoter. 6. A recombinant vector produced by the method of claim 5. 7. A method for producing a recombinant host cell, which comprises the step of introducing the recombinant vector according to claim 6 into a host cell. 8. A recombinant host cell produced by the method of claim 7. 9. A recombinant method for producing a polypeptide, which comprises culturing the recombinant host cell according to claim 8 under conditions such that the polypeptide is expressed and recovering the polypeptide. A method comprising the step of: 10. An isolated polypeptide comprising: (a) about 1 to about 115 amino acids of SEQ ID NO: 2; (b) about 2 to about 115 amino acids of SEQ ID NO: 2; (c) A polypeptide comprising an amino acid that is at least 95% identical to an amino acid selected from the group consisting of: about 1 to about 115 amino acids of SEQ ID NO: 4; and (d) about 2 to about 115 amino acids of SEQ ID NO: 4. 11. An isolated polypeptide, wherein at least 1
Except for one conservative amino acid substitution, the polypeptide has: (a) about 1 to about 115 amino acids of SEQ ID NO: 2; (b) about 2 to about 115 amino acids of SEQ ID NO: 2; (c) a sequence. A polypeptide having an amino acid sequence selected from the group consisting of: about 1 to about 115 amino acids of SEQ ID NO: 4; and (d) about 2 to about 115 amino acids of SEQ ID NO: 4. 12. An isolated polypeptide, which comprises: (A) about 1 to about 115 amino acids of SEQ ID NO: 2; (B) about 2 to about 115 amino acids of SEQ ID NO: 2; (C) about 1 to about 115 amino acids of SEQ ID NO: 4; and (D) about 2 to about 115 amino acids of SEQ ID NO: 4, A polypeptide comprising an amino acid selected from the group consisting of: 13. A composition comprising an isolated polypeptide, wherein the polypeptide comprises: (a) about 4 to about 50 amino acids of SEQ ID NO: 2; (b) about 9 of SEQ ID NO: 2. (C) about 4 to about 50 amino acids of SEQ ID NO: 4; (d) about 9 to about 45 amino acids of SEQ ID NO: 4; and (e) (a), (b), (c) ), A polypeptide that is at least 86% identical to the polypeptide of (d), wherein the polypeptide has unaltered EGF receptor binding specificity, the amino acid sequence selected from the group consisting of: The composition. 14. A composition comprising an isolated polynucleotide encoding the polypeptide of claim 13. 15. An epitope-bearing portion of a polypeptide selected from SEQ ID NO: 2 or SEQ ID NO: 4. 16. The epitope-carrying portion of claim 15, comprising between about 10 and about 100 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. 17. An epitope-carrying portion according to claim 16, which comprises a contiguous number of amino acids between about 12 and about 50 of SEQ ID NO: 2 or SEQ ID NO: 4. 18. The epitope-carrying portion of claim 16, which comprises between about 15 and about 25 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. 19. An isolated antibody that specifically binds to the polypeptide of claim 10. 20. An isolated antibody that specifically binds to the polypeptide of claim 11. 21. An isolated antibody that specifically binds to the polypeptide of claim 12. 22. A method for diagnosing a EGFH2 protein modulating disorder using a biological sample from a human suspected of having the disorder, said method comprising: a) Providing an antibody that binds to the described polypeptide; b) contacting the sample with the antibody under binding conditions to form a duplex; and c) determining the amount of the duplex formed. Determining relative to a normal sample. 23. A method for diagnosing a EGFH2 protein-modulating disorder in a biological sample of human origin that is suspected of having the disorder, said method comprising: a) under severe conditions. Providing a polynucleotide that binds to the mRNA encoding the polypeptide of claim 10; b) contacting the polynucleotide with the nucleic acid of the sample under binding conditions to form a duplex; and c) the Determining the amount of duplexes formed relative to a normal sample. 24. A method for modulating the amount of EGFH2 protein in a subject, the method comprising: a) a polypeptide according to claim 10; and b) according to claim 10. An antibody that binds to a polypeptide, the method comprising administering an effective amount of a composition selected from the group consisting of: 25. A method for modulating the amount of EGFH2 protein in a subject, the method comprising administering an effective amount of a composition comprising the nucleotide sequence of claim 1. ,Method. 26. A method for treating an EGFH2 protein modulating disorder in a subject, the method comprising: a) a polypeptide according to claim 10; b) a poly according to claim 10. An antibody that binds to the peptide, comprising administering to the subject an effective amount of a composition selected from the group consisting of:
The method wherein the composition further comprises a pharmaceutically acceptable carrier. 27. A method for treating an EGFH2 protein modulating disorder in a subject, the method comprising administering to the subject an effective amount of a composition comprising the nucleotide sequence of claim 1. Wherein the composition further comprises a pharmaceutically acceptable carrier. 28. A method of providing cellular nutritional support in a patient in need of cellular nutritional support, the method comprising a polynucleotide encoding an EGFH2 polypeptide comprising SEQ ID NO: 2, SEQ ID NO: 4. A method comprising administering to said patient a composition selected from the group consisting of a polynucleotide encoding an EGFH2 polypeptide and the polypeptide of claim 10. 29. The method of claim 27, wherein said polynucleotide is administered by transplanting cells expressing said polynucleotide into said patient, wherein said cells are said A method of expressing an EGFH2 polypeptide in a patient. 30. The method of claim 29, wherein the transplanted cells are encapsulated in a semipermeable membrane. 31. The method of claim 27, wherein the mammal is
Breast cancer, prostate cancer, pancreatic cancer, oral cancer, ovarian cancer, peripheral neuropathy, amyotrophic lateral sclerosis,
Select from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemic stroke, brain injury, acute spinal cord injury, nervous system injury, multiple sclerosis, infection, dementia, epilepsy, peripheral nerve injury, acoustic trauma and skin wounds Suffering from a condition that has been treated. 32. The method of claim 27, wherein the polynucleotide is an antisense construct. 33. The method of claim 27, wherein the polynucleotide is a ribozyme. 34. The method of claim 27, wherein the polynucleotide is a retroviral vector containing a promoter. 35. A method of treating a patient having an EGFH2 protein modulating disorder, said method comprising administering a composition comprising a therapeutically effective amount of a polypeptide capable of binding EGFH2 or a variant thereof. A method comprising the steps of: 36. The method of claim 35, wherein the polypeptide is an antibody. 37. The method of claim 36, wherein the polypeptide is a wild-type or mutant receptor for EGFH2.