JP2002540795A - DSP-4 bispecific MAP kinase phosphatase - Google Patents

Abstract

  (57) [Summary] The compositions and methods of the invention are provided for the treatment of conditions involving cell proliferation, cell differentiation, and cell survival. In particular, there are provided bispecific phosphatases DSP-4 and polypeptide variants of DSP-4 that stimulate dephosphorylation of DSP-4 substrates. DSP-4 polypeptides can be used, for example, to identify antibodies and other agents that inhibit DSP-4 activity. DSP-4 polypeptides and agents can be used to modulate cell proliferation, cell differentiation, and cell survival.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates generally to compositions and methods useful for treating conditions associated with defects in cell proliferation, cell differentiation, and / or cell survival. The invention more particularly relates to bispecific protein phosphatases and polypeptide variants thereof. The present invention also relates to the use of such polypeptides for the identification of antibodies and other factors (including small molecules) that modulate signaling leading to a proliferative response, cell differentiation, and / or cell survival.

BACKGROUND OF THE INVENTION [0002] Mitogen-activated protein kinases (MAP kinases) exist as components of a conserved cell signaling pathway with various conserved members. MAP kinase is activated by phosphorylation (by MAP kinase kinase) on a double phosphorylation motif having the sequence Thr-X-Tyr. Here, phosphorylation at tyrosine and threonine residues is required for activity. Activated MAP kinase has several transduction targets.
target (including transcription factors). Inactivation of MAP kinase is caused by a dual specificity phosphatase called MAP kinase phosphatase.
It is mediated by dephosphorylation at this site. In higher eukaryotes, the physiological role of MAP kinase signaling has been correlated with intracellular events, such as proliferation, tumorigenesis, development, and differentiation. Thus, the ability to modulate signaling through these pathways may lead to the development of treatment and prophylactic therapies for human diseases (eg, cancer) associated with MAP kinase signaling.

[0003] Dual-specific protein tyrosine phosphatases (dual-specific phosphatases)
Is a phosphatase that dephosphorylates both phosphotyrosine and phosphothreonine / serine residues (Walton et al., Ann. Rev. Biochem).
. 62: 101-120, 1993). Several bispecific phosphatases that inactivate MAP kinase have been identified, including MKP-1 (WO
97/00315; Keyse and Emslie, Nature 59:64.
4-647, 1992), MKP-4, MKP-5, MKP-7, Hb5 (WO
97/06245), PAC1 (Ward et al., Nature 367: 651-).
654, 1994), HVH2 (Guan and Butch, J. Biol. C).
hem. 270: 7197-7203, 1995), and PYST1 (Gro
EM et al. 15: 3621-3632, 1996). Expression of certain bispecific phosphatases is induced by stress or mitogens, while other bispecific phosphatases appear to be constitutively expressed in certain cell types. Regulation of bispecific phosphatase expression and activity is important in controlling MAP kinase-mediated cellular functions, including cell proliferation, cell differentiation, and cell survival. For example, a bispecific phosphatase can function as a negative regulator of cell growth. There may be many such bispecific phosphatases with varying specificities for cell type or activation. However, the regulation of bispecific phosphatases remains poorly understood and only a relatively small number of bispecific phosphatases have been identified.

[0004] Accordingly, there is a need in the art for an improved understanding of MAP signaling and the regulation of bispecific phosphatases in the MAP kinase signaling cascade. Increased understanding of bispecific phosphatase regulation is
Developing methods for modulating the activity of proteins involved in the AP kinase cascade, and for treating conditions associated with such cascades, may be facilitated. The present invention fulfills these needs, and further provides other related advantages.

SUMMARY OF THE INVENTION Briefly, the present invention provides compositions and methods for identifying factors that can modulate a cell proliferative response. In one aspect, the present invention provides an isolated DSP-4 polypeptide having the sequence of DSP-4 listed in SEQ ID NO: 2 or a variant thereof, wherein the variant comprises It differs by the deletion, addition, insertion, or substitution of one or more amino acids at 50% or less of the residues of SEQ ID NO: 2, so as to retain the ability to dephosphorylate MAP kinase.

In a further aspect, the present invention provides an isolated polynucleotide encoding at least 10 contiguous amino acids of a polypeptide having a sequence corresponding to SEQ ID NO: 2. In certain embodiments, the invention provides an isolated polynucleotide encoding at least 15 contiguous amino acids of a polypeptide having the sequence corresponding to SEQ ID NO: 2. Certain such polynucleotides are:
Encodes a DSP-4 polypeptide. Still further, the polynucleotide may comprise DS
Comprising at least 15 contiguous nucleotides that are complementary to a portion of a P-4 polynucleotide and / or in 50 × 0.1 × SSC and 0.1% SDS.
An antisense polynucleotide that detectably hybridizes to the complement of the sequence recited in SEQ ID NO: 1 under conditions that include a wash for at least 15 minutes at ℃. Expression vectors comprising any of the foregoing polynucleotides, and host cells transformed or transfected with such expression vectors are also provided.

[0007] The invention further provides, in another aspect, the following steps: (a) culturing the host cell as described above under conditions that allow expression of the DSP-4 polypeptide; and (b) Isolating a DSP-polypeptide from the host cell culture as described above.

[0008] DSP-4 polypeptide (eg, a polypeptide having the sequence of SEQ ID NO: 2)
An antibody and an antigen-binding fragment thereof that specifically binds to
Provided by the present invention.

[0009] The invention further provides, in another aspect, a pharmaceutical composition comprising a polypeptide, polynucleotide, antibody or fragment thereof as described above in combination with a physiologically acceptable carrier.

[0010] In a further aspect, the present invention provides a method for detecting DSP-4 expression in a sample, the method comprising the steps of: (a) forming an antibody / DSP-4 complex; Contacting the sample with an antibody or antigen-binding fragment thereof as described above under sufficient conditions and for a time; and (b) detecting the level of the antibody / DSP-4 complex.

In yet another aspect, the invention provides a method for detecting DSP-4 expression in a sample, the method comprising: (a) contacting a sample with an antisense polynucleotide as described above. And (b) detecting in the sample the amount of DSP-4 polynucleotide that hybridizes to the antisense polynucleotide. For example, a polymerase chain reaction or hybridization assay can be used to determine the amount of a DSP-4 polynucleotide that hybridizes to the antisense polynucleotide.

[0012] The present invention also provides DSP-4 polypeptides useful in screening assays for modulators of enzyme activity and / or substrate binding. In another aspect, there is also provided a method for screening for an agent that modulates DSP-4 activity, the method comprising the steps of: (a) combining a candidate agent with a DSP-4 polypeptide as described above; Contacting the polypeptide under conditions and for a time sufficient to permit interaction between the polypeptide and the candidate agent; and (b) subsequently, in the absence of the candidate agent, the polypeptide Assessing the polypeptide's ability to dephosphorylate a DSP-4 substrate compared to a predetermined ability to dephosphorylate a DSP-4 substrate. Such methods can be performed in vitro or in a cellular environment (eg, in intact cells).

[0013] In a further aspect, there is provided a method for screening for an agent that modulates DSP-4 activity, the method comprising the steps of: (a) identifying a candidate agent with a detectable transcript or protein; Contacting a cell containing a DSP-4 promoter operably linked to a polynucleotide encoding the polynucleotide under conditions and for a time sufficient to allow for interaction between the promoter and the candidate agent; and (B) assessing the expression of the polynucleotide in comparison to a predetermined level of expression in the absence of the candidate agent.

[0014] Also provided are methods for modulating a proliferative response in a cell, the method comprising contacting the cell with an agent that modulates DSP-4 activity.

[0015] In a further aspect, there is provided a method for modulating the differentiation of a cell, the method comprising contacting the cell with an agent that modulates DSP-4 activity.

The invention further provides a method for modulating cell survival, the method comprising:
Contacting the cell with an agent that modulates SP-4 activity.

[0017] In a related aspect, the invention relates to DSP-4 activity (or
Provided is a method for treating a patient suffering from a disorder (treatable by administration of SP-4), the method comprising administering to the patient a therapeutically effective amount of an agent that modulates DSP-4 activity. Include. Such disorders include cancer, graft versus host disease, autoimmune disease, allergy, metabolic disease, abnormal cell growth, abnormal cell proliferation, and abnormal cell cycle.

In a further aspect, a DSP-4 substrate capture mutant polypeptide is provided. Such a polypeptide may be such that the polypeptide binds to the substrate with substantially no reduced affinity compared to DSP-4 and the ability of the polypeptide to dephosphorylate the substrate. Has a deletion, addition, insertion or substitution of one or more amino acids at 50% or less of the residues in SEQ ID NO: 2, as reduced compared to DSP-4, Differs from the described sequence. In certain embodiments,
The substrate capture variant polypeptide comprises a substitution at position 119 or 150 of SEQ ID NO: 2.

The invention further provides, in another aspect, a method for screening a molecule for the ability to interact with DSP-4, the method comprising the steps of: (a) identifying a candidate molecule; Contacting said polypeptide with said candidate molecule under conditions and for a time sufficient to allow said polypeptide to interact; and (b) the presence of binding of said candidate molecule to said polypeptide. Or detecting the absence. This detection step can include, for example, an affinity purification step, a yeast two-hybrid screen, or a phage display screen.

[0020] These and other aspects of the invention will become apparent by reference to the following detailed description and the accompanying drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually incorporated.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention generally relates to compositions and methods for modulating (ie, stimulating or inhibiting) cell proliferative responses in vitro and in vivo. . Specifically, the present invention relates to the dual specificity phosphatase DSP-4 (FIGS. 1-2;
SEQ ID NOS: 1 and 2), variants thereof, and antibodies that specifically bind to DSP-4. Also provided herein are methods for using such compounds for screening, detection assays, and related therapeutic applications.

(DSP-4 Polypeptide and DSP-4 Polynucleotide) As used herein, the term “DSP-4 polypeptide” refers to DSP-4 as provided herein. A sequence or a variant of such a sequence,
Refers to a polypeptide. Such polypeptides can dephosphorylate both tyrosine and threonine / serine residues in the DSP-4 substrate and have a substantially reduced activity compared to the activity of native full-length DSP-4. Not having activity. DSP
-4 substrates include activated (i.e., phosphorylated) MAP-kinase. Other substrates can be identified using a substrate capture mutant, as described herein, and have one or more phosphorylated tyrosine, threonine, and / or serine residues. , Polypeptides.

[0023] DSP-4 polypeptide variants within the scope of the invention may comprise one or more substitutions, deletions,
It may include additions and / or insertions. For certain DSP-4 variants, the ability of the variant to dephosphorylate tyrosine and threonine residues within the DSP-4 substrate is not substantially reduced. The ability of such DSP-4 variants to dephosphorylate tyrosine and threonine residues within the DSP-4 substrate depends on the native DS-4
It can be enhanced or unchanged compared to P-4, or can be reduced by less than 50% compared to native DSP-4, and preferably
It can be reduced by less than 20%. Such variants can be identified using the representative assays provided herein.

According to the present invention, modified forms of DSP-4 in which certain functions are disabled are also contemplated. For example, such proteins may be constitutively active or inactive, or may exhibit altered binding or catalytic properties.
Such altered proteins can be produced using well-known techniques, and the altered function can be confirmed using screening as provided herein. Certain modified DSP-4 polypeptides are known as "substrate capture variants." Such polypeptides retain the ability to bind a substrate (
That is, the K _m is not substantially reduced, but the ability to dephosphorylate the substrate is reduced (ie, k _cat is preferably reduced to less than 1 per minute).
Is shown. Furthermore, the stability of the substrate capture mutant / substrate complex should not be substantially reduced compared to the stability of the DSP-4 / substrate complex. Complex stability can be assessed based on the association constant (K _a ). K _m, determined in k _cat and K _a are standard techniques known in the art (e.g., WO 98/04712; Lehnin
ger, Biochemistry, 1975 Worth Publisher
rs, NY) and using the assays provided herein.
It can be easily achieved. Substrate capture variants can be obtained by modifying DSP-4, for example, by amino acid substitutions at positions 119 or 150 (eg, by substituting the amino acid aspartic acid at position 119 with an alanine residue, or at residue 15).
(Replacement of cysteine 0 by serine). Substrate capture variants can be used, for example, to identify DSP-4 substrates. In short,
The modified DSP-4 may be contacted with a candidate substrate (alone or within a mixture of proteins such as cell extracts) to allow formation of a substrate / DSP-4 complex. The complex can then be isolated by conventional techniques, allowing for isolation and characterization of the substrate. The preparation and use of substrate capture mutants is described, for example, in PCT
It is described in publication number WO 98/04712.

[0025] Preferably, the variant comprises a conservative substitution. “Conservative substitutions” are used by those of skill in the art of peptide chemistry to determine the secondary structure and hydrophilicity / hydrophobicity of a polypeptide.
is a substitution in which one amino acid has been substituted for another amino acid having similar properties, as would be expected for hy.). Amino acid substitutions can generally be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and uncharged polar group groups with similar hydrophilicity. ) Include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln,
asn, ser, thr; (2) cys, ser, tyr, thr; (3) va
l, ile, leu, met, ala, phe; (4) lys, arg, his
And (5) phe, tyr, trp, his. Variants may also or alternatively include non-conservative changes.

In general, alterations can be made more easily in non-critical regions, which are regions of the native sequence that do not substantially alter the activity of DSP-4. Non-critical regions can be identified by altering the DSP-4 sequence in specific regions, as described herein, and assessing the ability of the resulting variant in a phosphatase assay. A preferred sequence modification is the active site domain (
VHCNAGVSRAAAIV, SEQ ID NO: 3). In certain preferred embodiments, such modifications affect the interaction between DSP-4 and cellular components other than the DSP-4 substrate. However, substitutions can also be made in critical regions of the native protein, provided that the resulting variant substantially retains the ability to stimulate substrate dephosphorylation. In certain embodiments,
Variants include substitutions, deletions, additions and / or insertions of up to 50%, preferably up to 25%, of amino acid residues.

A variant may also (or alternatively) be modified, for example, by deletion or addition of an amino acid that has minimal effect on the activity of the polypeptide. In particular, variants may include additional amino acid sequences at the amino and / or carboxy termini. Such sequences can be used, for example, to facilitate purification or detection of the polypeptide.

[0028] DSP-4 polypeptides can be prepared using any of a variety of well-known techniques. The recombinant polypeptide encoded by the DNA sequence as described below can be prepared using any of a variety of expression vectors known to those of skill in the art.
It can easily be prepared from their DNA sequence. Expression can be achieved in any suitable host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes the recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells (including mammalian cells), and the different forms of glycosylation can be altered by altering the host cell or post-isolation processing. Can be generated. Supernatants from a suitable host / vector system that secretes the recombinant protein or polypeptide into the culture medium can be first concentrated using commercially available filters. After concentration, the concentrate can be applied to a suitable purification matrix, such as an affinity matrix or an ion exchange resin. Finally, the recombinant polypeptide may be further purified utilizing one or more reverse phase HPLC steps.

[0029] Portions and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be produced by synthetic procedures using techniques well known to those skilled in the art. Can be done. For example, such polypeptides can be synthesized using any of the commercially available solid phase techniques (eg, Merrifield solid phase synthesis), wherein the amino acids are consecutively extended amino acids. Appended to the chain. Merrifield, J .; Am. Chem. Soc. 85: 2149
See -2146,1963. The apparatus for automatic synthesis of polypeptides comprises
Perkin-Elmer, Inc. , Applied BioSystems
It is commercially available from suppliers such as Division (Foster City, CA) and can be operated according to the manufacturer's instructions.

A “DSP-4 polynucleotide” is any polynucleotide that encodes at least a portion of a DSP-4 polypeptide or variant thereof, or that is complementary to such a polynucleotide. It is. Preferred polynucleotides comprise at least 15 contiguous nucleotides, preferably at least 30 contiguous nucleotides, that encode the DSP-4 polypeptide or are complementary to such sequences. Certain polynucleotides encode DSP-4 polypeptides; other polynucleotides may find use as probes, primers or antisense oligonucleotides as described below. A polynucleotide may be single-stranded (coding or antisense strand) or double-stranded, and may be a DNA (genomic DNA, cDNA or synthetic DNA) molecule or an RNA molecule. Additional coding or non-coding sequences may, but need not, be present in the polynucleotides of the invention, and the polynucleotides may be, but need not be, linked to other molecules and / or support materials. Absent.

A DSP-4 polynucleotide has a native sequence (ie, endogenous DSP-
4 sequences or portions thereof or splice variants) or variants of such sequences. Polynucleotide variants, as described above,
One or more substitutions, additions, deletions and / or insertions may be included such that the activity of the encoded polypeptide is not substantially reduced. The effect on the activity of the encoded polypeptide can generally be assessed as described herein.
The variant preferably has at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to the polynucleotide sequence encoding native DSP-4 or a portion thereof. % Identity. The percent identity is determined by the Align or BLAST algorithm (Alt
Schul, J .; Mol. Biol. 219: 555-565, 1991; He.
nikoff and Henikoff, Proc. Natl. Acad. Sci
. USA 89: 10915-10919, 1992), and can be readily determined by comparing sequences using computer algorithms well known to those skilled in the art, which algorithm is available on the NCBI website (http: // www /
ncbi. nlm. nih. gov / cgi-bin / BLAST). Default parameters can be used. Certain variants are substantially homologous to the native gene. Such polynucleotide variants may hybridize under moderately stringent conditions to a naturally occurring DNA or RNA sequence encoding a native DSP-4 (or complementary sequence). Suitable moderately stringent conditions are, for example, 5 × SSC, 0.5
Pre-washing in a solution of% SDS, 1.0 mM EDTA (pH 8.0); hybridizing with 50 ° C. to 70 ° C., 5 × SSC for 1 to 16 hours (for example, overnight); Once or twice at 22-65 ° C. for 20-40 minutes, 0.
2 × SSC, 0.5 × SSC and 0.2 × SS with 05-0.1% SDS
Washing with one or more of each of C. For additional stringency, the conditions were 0.1 × SSC and 50 × 60 ° C. for 15-40 minutes.
This may include washing with 1% SDS. As is known to those skilled in the art, altering the stringency of hybridization conditions is achieved by altering the time, temperature and / or concentration of the solution used for the prehybridization, hybridization and washing steps. The appropriate conditions may also depend, in part, on the particular nucleotide sequence of the probe used, and the particular nucleotide sequence of the nucleic acid sample of the blotted proband. Thus, appropriately stringent conditions are such that the desired selectivity of the probe is
It is recognized that if identified, based on their ability to hybridize to one or more specific origin sequences but not to other specific origin sequences, they can be readily selected without undue experimentation. You.

[0032] It will also be recognized by one of skill in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides retain minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

[0033] Polynucleotides can be prepared using any of a variety of techniques. For example, polynucleotides can be amplified from cDNA prepared from a suitable cell or tissue type (eg, human thymus or skeletal muscle cells). Such polynucleotides can be amplified via the polymerase chain reaction (PCR). For this approach, sequence-specific primers can be designed based on the sequences provided herein and purchased or synthesized.

Using the amplified portions, full-length genes can be isolated from a suitable library (eg, human thymus or skeletal muscle cDNA) using well known techniques. Within such a technique, a library (cDNA or genome) is screened using one or more polynucleotide probes or polynucleotide primers suitable for amplification. Preferably, the library is sized to include larger molecules. Random prime libraries may also be suitable for identifying the 5 'and upstream regions of a gene. Genomic libraries are suitable for obtaining introns and for extending 5 'sequences.

For hybridization techniques, the subsequence can be labeled using well known techniques (eg, by nick translation or end labeling with ³² P). The bacterial library or bacteriophage library is then
Filters containing denatured bacterial colonies (or lawns containing phage plaques) can be screened by hybridizing with a labeled probe (eg, Sambrook et al., Molecular).
Cloning: A Laboratory Manual, Cold Spr
ing Harbor Laboratories, Cold Spring
See Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is analyzed for further analysis.
Is isolated. Clones can be analyzed to determine the amount of additional sequence, for example, by PCR using primers from the partial sequence and primers from the vector. Restriction maps and subsequences can be generated to identify one or more overlapping clones. By linking the appropriate fragments using well known techniques, full length cDNA molecules can be produced.

Alternatively, a number of amplification techniques exist for obtaining full-length coding sequences from partial cDNA sequences. In such techniques, amplification is generally performed via PCR. One such technique is "rapid amplification of cDNA ends", ie, RACE.
It is known as This technique uses an internal primer and an external primer that hybridizes to the polyA region or vector sequence to 5 'to a known sequence.
And identifying the sequence that is 3 ′. The amplification step can be performed using any of a variety of commercially available kits. For example, primers can be designed using software well known in the art. Primers are preferably 17-32 nucleotides in length, have a GC content of at least 40%, and
Anneal to the target sequence at a temperature of 2 ° C. The amplified regions can be sequenced as described above, and overlapping sequences can be assembled into a contiguous sequence.

The cDNA sequence encoding DSP-4 is provided in FIG. 1 (SEQ ID NO: 1), and the deduced amino acid sequence is provided in FIG. 2 (SEQ ID NO: 2). Active site VH of DSP-4
CNAGVSRAAAIV (SEQ ID NO: 3) is the nucleotide position 1 of SEQ ID NO: 1
Encoded by nucleotide bases located at 48-161. Using the sequence information immediately adjacent to this site, 5'RACE and 3'RACE reactions using human skeletal muscle cDNA were designed to produce various cell types (heart, testis, thymus, and skeletal muscle tissue). 651 base pair cDNA that could be expressed by This cDNA encodes a bispecific phosphatase-4, a protein of 217 amino acids, referred to herein as DSP-4. As shown by the sequence comparison shown in FIG. 3, DSP-4 shows significant homology to other MAP kinase phosphatases.

[0038] DSP-4 polynucleotide variants can generally be prepared by any method known in the art, including, for example, solid phase chemical synthesis. Modification of the polynucleotide sequence may also include oligonucleotide site-directed mutagenesis.
de-directed site-specific mutagenesis
s) can be introduced using standard mutagenesis techniques. Alternatively, an RNA molecule can be produced by in vitro or in vivo transcription of a DNA sequence encoding DSP-4 or a portion thereof, provided that the DNA is a vector having a suitable RNA polymerase promoter (eg, T7 or SP6). Incorporated in A particular polynucleotide may be used to prepare the encoded polypeptide as described herein. Additionally or alternatively, the polynucleotide may be
The encoded polypeptide can be administered to a patient such that it is produced in vivo.

[0039] Polynucleotides that are complementary to at least a portion of a coding sequence (eg, antisense polynucleotides or ribozymes) can also be used as probes or primers, or to modify gene expression.
Oligonucleotides and ribozymes for use as antisense agents,
As well as the identification of DNA encoding genes for their targeted delivery includes methods well known in the art. For example, the desired properties, length and other characteristics of such oligonucleotides are well-known. Antisense oligonucleotides are typically designed to resist degradation by endogenous nucleolytic enzymes by using linkages such as: phosphorothioate, methylphosphonate, sulfone, sulfate, Ketyl
) Linkages, phosphorodithioate linkages, phosphoramidite linkages, phosphate linkages, and other such linkages (eg, Agrwal et al., Tetrehed).
ron Lett. 28: 3539-3542 (1987); Miller et al.,
J. Am. Chem. Soc. 93: 6657-6665 (1971); Ste.
c et al., Tetrehedron Lett. 26: 2191-2194 (198
5); Moody et al., Nucl. Acids Res. 12: 4769-478
2 (1989); Uznanski et al., Nucl. Acids Res. (19
89); Letsinger et al., Tetrahedron 40: 137-14.
3 (1984); Eckstein, Annu. Rev .. Biochem. 54
: 367-402 (1985); Eckstein, Trends Biol.
Sci. 14: 97-100 (1989); Oligodeoxynucleo.
tides. Antisense Inhibitors of Gene E
xsession, Cohen, Ed., Macmillan Press, Lo
Stein in Ndon, pp. 97-117 (1989); Jager et al., Bio.
chemistry 27: 7237-7246 (1988)).

An antisense polynucleotide is a nucleic acid (eg, mR
NA or DNA). When bound to mRNA having a complementary sequence, antisense prevents translation of the mRNA (eg, US Pat. No. 5,168,053, Altman et al .; 5,190,933).
No. 1, Inouye, 5,135,917, Burch; 5,087
No. 617, Smith and Clusel et al. (1993) Nucal. Aci
ds Res. 21: 3405-3411 (this is a dumbbell shape)
11) describes antisense oligonucleotides). A triplex molecule refers to a single DNA strand that joins double-stranded DNA to form a collinear triplex molecule, thereby preventing transcription (see, for example, US Pat. No. 176,996, Hogan et al., Which describes a method for making synthetic oligonucleotides that bind to a target site on duplex DNA).

Particularly useful antisense nucleotides and triplex molecules include the sense strand of DNA or mRNA encoding a DSP-4 polypeptide or a protein that mediates any other process associated with expression of endogenous DSP-4; A molecule that is complementary or binds so as to result in inhibition of translation of the mRNA encoding the DSP-4 polypeptide. CDNA that can be transcribed into antisense RNA
Constructs can also be introduced into cells or tissues to facilitate production of antisense RNA. Antisense technology can be used to regulate gene expression via interfering with the binding of polymerases, transcription factors or other regulatory molecules (Hu
ber and Carr, Molecular and Immunolog
ic Approaches, Futura Publishing Co. (
Mt. Kisco, NY; 1994). Or,
Antisense molecules hybridize to regulatory regions (eg, promoters, enhancers or transcription start sites) of the DSP-4 gene and may block transcription of this gene; or may bind transcript binding to ribosomes. It can be designed to block translation by inhibiting.

The present invention also contemplates DSP-4-specific ribozymes. The ribozyme is R
An RNA molecule that specifically cleaves NA substrates (eg, mRNA), resulting in specific inhibition or interference of cellular gene expression. There are at least five known classes of ribozymes involved in RNA strand cleavage and / or ligation. Ribozymes can be targeted to any RNA transcript and can catalyze the cleavage of such transcripts (eg, US Pat. Nos. 5,272,262; 5,144).
No. 5,019,5,168,053, 5,180,818, 5,116,742 and 5,093,246 (Cech et al.). Any DSP-4 mRNA-specific ribozyme, or nucleic acid encoding such a ribozyme, can be delivered to a host cell to effect inhibition of DSP-4 gene expression. Thus, a ribozyme is a DNA encoding a ribozyme linked to a eukaryotic promoter (eg, a eukaryotic virus promoter).
Can be delivered to the host cell such that upon introduction into the nucleus, the ribozyme can be transcribed directly.

[0043] Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3'end; the use of phosphorothioate or 2'O-methyl bonds other than phosphodiester bonds in the backbone; and / or non-traditional May include non-raditonal bases (eg, inosine, queosine and wibutosin, and acetyl, methyl, thio, and other modified forms of adenine, cytidine, guanine, thymine and uridine). No.

A nucleotide sequence as described herein may comprise an established recombinant DNA
It can be combined with various other nucleotide sequences using techniques. For example, a polynucleotide can be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a suitable vector will include an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and one or more selectable markers. Other elements will depend on the desired use, and will be apparent to those skilled in the art.

[0045] In certain embodiments, the polynucleotide is capable of entering a cell of a mammal,
And may be formulated to allow expression therein. Such a formulation is particularly useful for therapeutic purposes as described below. One of skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and that any suitable method may be utilized. For example, a polynucleotide can be incorporated into a viral vector using well-known techniques. The viral vector is
In addition, transfer or incorporate genes for selectable markers (to aid in the identification or selection of transduced cells) and / or genes for targeting moieties (eg, genes encoding ligands for receptors on particular target cells). Can make the vector target specific. Targeting can also be achieved using antibodies by methods known to those skilled in the art.

Other formulations for therapeutic purposes include colloidal dispersion systems (eg, polymer complexes, nanocapsules, microspheres, beads), and lipid-based systems (oil-in-water emulsions, micelles, mixed Micelles, and liposomes). A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (ie, an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0047] In another aspect, the DSP-4 promoter can be isolated using standard techniques. The invention provides a nucleic acid molecule comprising such a promoter sequence or one or more cis- or trans-acting regulatory elements thereof. Such a regulatory element may enhance or suppress the expression of DSP-4. The 5 'flanking region can be generated using standard techniques, based on the genomic sequences provided herein. If necessary, additional 5 'sequences can be generated using PCR-based or other standard methods. This 5 'region can be subcloned using standard methods and sequenced. Primer extension and / or RNase protection analysis can be used to confirm the predicted transcription start site from the cDNA.

To define the boundaries of the promoter region, putative promoter inserts of various sizes can be subcloned into a heterologous expression system containing a suitable reporter gene without promoter or enhancer. Suitable reporter genes may include a gene encoding luciferase, a gene encoding β-galactosidase, a gene encoding chloramphenicol acetyltransferase, a gene encoding secreted alkaline phosphatase, or a green fluorescent protein gene. Suitable expression systems are well known and can be prepared using well known techniques or are commercially available. Internal deletion constructs may be generated using only one internal restriction site or by partial digestion of non-unique restriction sites. The construct can then be transfected into cells that exhibit high levels of DSP-4 expression. Generally, the construct in which the smallest 5 'flanking region shows the highest expression level of the reporter gene is defined as a promoter. Such a promoter region can be linked to a reporter gene and used to evaluate an agent for its ability to regulate DSP-4 transcription.

[0049] Once a functional promoter has been identified, cis- and trans-acting elements can be localized. Cis-acting sequences can generally be identified based on homology to previously characterized transcription motifs. Point mutations can then be generated within the identified sequences to assess the regulatory role of such sequences. Such mutations can be generated using site-directed mutagenesis techniques or PCR-based strategies. The altered promoter is then cloned into a reporter gene expression vector, as described above, and the effect of this mutation on reporter gene expression is assessed.

The present invention also relates to allelic variants of DSP-4 as well as DSPs from other organisms.
The use of the -4 sequence is contemplated. Such sequences will generally be based on similarity (eg, using hybridization techniques) to the sequences provided herein, and may be used to determine DSP-4 activity using the assays provided herein. Can be identified based on the presence of

In general, polypeptides and polynucleotides as described herein have been isolated. An "isolated" polypeptide or polynucleotide is a polypeptide or polynucleotide that has been removed from its original environment. For example, a naturally occurring protein has been isolated if it has been separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure. A polynucleotide is considered isolated if, for example, it has been cloned into a vector that is not part of its natural environment.

Assays for Detecting DSP-4 Activity In accordance with the present invention, DSP-4 substrates can be phosphorylated at full length tyrosine phosphorylated proteins and polypeptides, and tyrosine residues, and in certain preferred implementations. Forms may also include fragments (eg, moieties), derivatives or analogs thereof that may be capable of phosphorylating at serine or threonine residues. Such fragments, derivatives and analogs include, for example, by forming a complex with DSP-4, to at least maintain the biological function of interacting with DSP-4 as provided herein. Any naturally occurring or engineered DSP-4 substrate polypeptide is included. A fragment, derivative or analog of a DSP-4 substrate polypeptide (including a substrate that is a fusion protein) can be any of the following: (i)
One or more amino acid residues are replaced with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may be encoded by the genetic code. One that is an amino acid residue that may not be encoded, or (ii) one or more of the amino acid residues contains a substituent, or (iii) the substrate polypeptide is a different compound (eg, halving the polypeptide). Phase-increasing compound (eg, polyethylene glycol), or fused to a detectable moiety such as a reporter molecule, or (iv) used for purification of additional amino acids (substrate polypeptide or proprotein sequence) Amino acids) to the substrate polypeptide. Such fragments, derivatives and analogs are deemed to be within the skill of the art. In a preferred embodiment, the MAP kinase polypeptide is a substrate for use as provided herein.

[0053] DSP-4 polypeptide variants can be tested for DSP-4 activity using any assay suitable for MAP kinase phosphatase activity. Such assays can be performed in vitro or in cell-based assays. For example, MAP kinase can be used as an DSP-4 substrate as provided herein in Upstate Biotechnology (La).
ke Placid, NY; catalog no. 14-198) as an inactivated form. Phosphorylation of MAP kinase can be determined by well-known techniques (eg, Zheng and Guan, J. Biol. Chem. 268: 16116-16119, 1).
993) using the MAP kinase kinase MEK-1.
(Upstate Biotechnology; available from catalog number 14-206).

For example, a substrate (eg, MAP kinase) radiolabeled with [ ³² P] can be used for a kinase reaction that results in activated radiolabeled MAP kinase. The DSP-4 polypeptide then binds the DSP-4 polypeptide to MAP kinase under appropriate conditions (eg, Tris (pH 7.5) 1 mM EDTA,
1 mM dithiothreitol, 1 mg / mL bovine serum albumin, 30 ° C. for 10 minutes; or Zheng and Guan, J. et al. Biol. Chem. 268: 16
116-16119, 1993) can be tested for the ability to dephosphorylate activated MAP kinase. MAP kinase dephosphorylation can be performed using any of a variety of assays, such as a ligation kinase assay (using any assay commonly known in the art to assess MAP kinase substrate phosphorylation). Or directly ((1) loss of radioactive phosphate groups (eg, by gel electrophoresis followed by autoradiography);
(2) shift in electrophoretic mobility after dephosphorylation; (3) loss of reactivity with antibodies specific for phosphotyrosine or phosphothreonine; or (4) MAP
(Based on phosphoamino acid analysis of the kinase). Specific assays are:
In general, it can be performed as described by Ward et al., Nature 367: 651-654, 1994, or Alessi et al., Oncogene 8: 2015-2020, 1993. Generally, 500 pg to 50 ng of DSP-4
Contacting the polypeptide with 100 ng to 100 μg of active MAP kinase can result in M
Detectable dephosphorylation of AP kinase should typically occur within 20-30 minutes. In certain embodiments, 0.01-10 units / mL (preferably about 0.1 units / mL, where units are in an amount sufficient to dephosphorylate 1 nmol of substrate per minute. DSP-4 polypeptide is 0.1 to 10 μl
Contacting with M (preferably about 1 μM) of active MAP kinase can result in detectable dephosphorylation of MAP kinase. Preferably, the DSP-4 polypeptide comprises M
Phosphorylation substrate (eg, tyrosine-phosphorylated and / or serine-phosphorylated peptide) at least as good as dephosphorylation of AP kinase, or dephosphorylation observed in the presence of a corresponding amount of native human DSP-4 Is generated. Other substrates identified using mutation-trapping substrates as described herein include:
It is clear that MAP kinase can be replaced in such an assay.

Antibodies and Antigen Binding Fragments Peptides, polypeptides and other non-peptide molecules that specifically bind to DSP-4 are also contemplated by the present invention. As used herein, a molecule reacts with DSP-4 at a detectable level and does not detectably react with a peptide containing an unrelated sequence or a sequence of a different phosphatase. It is said to "specifically bind" to DSP-4. Preferred binding molecules include antibodies, which include, for example, polyclonal, monoclonal, single chain,
Chimeric, anti-idiotype, CDR-grafted immunoglobulins, or fragments thereof (eg, proteolytically produced or recombinantly produced immunoglobulin F (ab ') ₂ , Fab, Fv, and Fd fragments) Can be Certain preferred antibodies are those that inhibit or block the activity of DSP-4 in an in vitro assay as described herein. Antibody DSP
-4 binding properties can generally be assessed using immunodetection methods, including, for example, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, immunoblotting, and the like, which are readily performed by those skilled in the art. Can be done.

[0056] Antibodies, polyclonal antisera, or monoclonal antibodies specific for DSP-4 can be produced using methods well known in the art. Antibodies can also be genetically engineered immunoglobulins (Ig) designed to have desired properties.
Or it can be produced as an Ig fragment. For example, and not by way of limitation, an antibody may be a chimera having at least one variable (V) region domain from a first mammalian species and at least one constant region domain from a second different mammalian species. Antibodies that are fusion proteins can include recombinant IgG. Most commonly, chimeric antibodies have mouse variable region sequences and human constant region sequences. Such a mouse / human chimeric immunoglobulin can be "humanized" by grafting complementarity determining regions (CDRs) from a mouse antibody, which incorporates a human derived V region framework region and a human derived constant region into the antigen. Gives binding specificity to the region. Fragments of these molecules can be produced by proteolytic digestion or, if necessary, by mild disulfide bond reduction and alkylation following proteolytic digestion. Alternatively, such fragments may also be produced by recombinant genetic engineering techniques.

As used herein, when the antibody reacts with DSP-4 at a detectable level, preferably when the affinity constant Ka is about 10 ⁴ M ⁻¹ or more, more preferably about Above 10 ⁵ M ⁻¹ , more preferably above about 10 ⁶ M ⁻¹ , and even more preferably above about 10 ⁷ M ⁻¹ , the antibody is “immunospecific for DSP-4 polypeptide. "Or" specifically binds ". The affinity of the binding partner or antibody can be determined by conventional techniques (eg, Scatchard et al. (Ann. NY).
Acad. Sci. USA 51: 660 (1949)) or surface plasmon resonance (BIAcore, Biosensor, Pisc).
ataway, NJ). For example, Wolff et al.
Cancer Res. 53: 2560-2565 (1993).

[0058] Antibodies can generally be prepared by any of a variety of techniques known to those of skill in the art. See, for example, Harlow et al., Antibodies: A Laboratory M
annual, Cold Spring Harbor Laboratory (
1988). In one such technique, animals are immunized with DSP-4 as an antigen to produce polyclonal antisera. Suitable animals include, for example, rabbits, sheep, goats, pigs, cows, and may also include smaller mammalian species (eg, mice, rats, and hamsters) or other species.

The immunogen may consist of DSP-4, a purified or partially purified DSP-4 polypeptide or a variant or fragment (eg, a peptide) thereof, or a cell expressing the DSP-4 peptide. . The DSP-4 peptide is
It can be produced by proteolytic cleavage or can be chemically synthesized. For example, provided herein is a nucleic acid sequence encoding a DSP-4 polypeptide;
As a result, one skilled in the art can routinely prepare these polypeptides for use as immunogens. Useful polypeptides or peptides for immunization can also be used to determine amino acid sequences that are more likely to produce an immunogenic response in a host animal.
It can be selected by analyzing the primary, secondary and tertiary structure of DSP-4 according to methods known to those skilled in the art. For example, Novotny, 1991 Mol.
Immunol. 28: 201-207: Berzofsky, 1985 Sc
issue 229: 934-40.

The preparation of an immunogen for injection into an animal can be accomplished by using another immunogenic protein (eg, keyhole limpet hemocyanin (KLH)) of the DSP-4 polypeptide (or a variant or fragment thereof). Or a covalent bond to a carrier protein such as bovine serum albumin (BSA). Further, DSP-4 peptides, polypeptides, or cells expressing DSP-4 used as an immunogen can be emulsified in an adjuvant. See, for example, Harlow et al., Antibodies.
: A Laboratory Manual, Cold Spring Har
See bor Laboratory (1988). Generally, after the first injection, the animals will receive one or more booster immunizations, particularly according to the antigen, adjuvant (if present), and / or a preferred schedule that may vary according to the particular animal species. The immune response is determined by periodically bleeding the animals, separating serum from the collected blood, and analyzing the serum in an immunoassay (such as an ELISA or Octalony diffusion assay) to determine specific antibody titers. Can be monitored by Once antibody titers are established, animals can be bled periodically to collect polyclonal antisera. The DSP-4 polypeptide or polyclonal antibody that specifically binds to the peptide is then purified from such antisera (eg, affinity chromatography using Protein A or DSP-4 polypeptide immobilized on a suitable solid support). (By graphy)
Can be done.

[0061] Monoclonal antibodies that specifically bind to a DSP-4 polypeptide or fragment or variant thereof, and hybridomas of immortal eukaryotic cell lines that produce monoclonal antibodies with the desired binding specificity are also, for example, Kohl
er and Milstein (Nature, 256: 495-497; 197).
6 Eur. J. Immunol. 6: 511-519 (1976)) and improvements thereof. An animal (eg, a rat, hamster, or preferably a mouse) is immunized with a DSP-4 immunogen prepared as described above. Antibody-forming cells, typically lymphocytes, including spleen cells, are obtained from the immunized animal and are drug-sensitized myeloma (eg, plasmacytoma) cell fusion partners, preferably syngeneic with the immunized animal, and Other desired properties (if needed)
For example, it can be immortalized by fusion with a partner having no expression ability of the endogenous Ig gene product). Lymphoid (eg, spleen) cells and myeloma cells can be combined with a membrane fusion enhancer (eg, polyethylene glycol or a non-ionic detergent) for several minutes and then support the growth of hybridoma cells, Tumor cells can be plated at low density on a selective medium that does not fuse. A preferred selection medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time (usually about 1-2 weeks), colonies of cells are observed. One colony is isolated and the antibodies produced by the cells can be tested for binding activity to the DSP-4 polypeptide or a variant or fragment thereof. Hybridomas producing monoclonal antibodies that are high affinity and specific for the DSP-4 antigen are preferred. Thus, hybridomas producing monoclonal antibodies that specifically bind to a DSP-4 polypeptide or a variant or fragment thereof are contemplated by the present invention.

[0062] Monoclonal antibodies can be isolated from the supernatant of a hybridoma culture. An alternative method of producing mouse monoclonal antibodies is to inject the hybridoma cells into the peritoneal cavity of syngeneic mice (eg, mice that have been treated (eg, pristane-stimulated) to promote the formation of ascites containing the monoclonal antibodies). It is. By conventional techniques (eg, chromatography, gel filtration, precipitation, extraction, etc.)
Contaminants can be removed from the ascites fluid obtained subsequently (usually within 1 to 3 weeks). For example, antibodies can be purified by affinity chromatography using appropriate ligands selected based on the particular properties of the monoclonal antibody (eg, heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotype antibodies and DSP-4 polypeptides or fragments or variants thereof. No.

[0063] Human monoclonal antibodies can be produced by a number of techniques familiar to those of skill in the art. Such methods include, but are not limited to: Epstein-Barr virus (EBV) of human peripheral blood cells (including, for example, B lymphocytes).
) Transformation, in vitro immunization of human B cells, yeast artificial chromosome (YAC)
Fusion of spleen cells from an immunized transgenic mouse carrying the human immunoglobulin gene inserted by, isolation from a human immunoglobulin V region phage library, or known in the art based on the disclosure herein. Other steps.

For example, one method for producing human monoclonal antibodies includes EB
Immortalization of human peripheral blood cells by V transformation. For example, U.S. Pat.
See 464,456. Immortalized cell lines that produce monoclonal antibodies that specifically bind to a DSP-4 polypeptide (or variant or fragment thereof) are identified by an immunodetection method as provided herein (eg, ELISA). And can then be isolated by standard cloning techniques. Another method for producing human monoclonal antibodies involves stimulating human spleen B cells with an antigen, followed by fusion of the stimulated B cells with a heterologous hybrid fusion partner.
In vitro immunization. See, for example, Boerner et al. Immu
nol. 147: 86-95.

[0065] Yet another method for the production of human DSP-4 specific monoclonal antibodies and polyclonal antisera for use in the present invention involves transgenic mice. See, for example, U.S. Pat. No. 5,877,397; Bruggema
nn et al., 1997 Curr. Opin. Biotechnol. 8: 455-
58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci
. 764: 525-35. In these mice, human immunoglobulin heavy and light chain genes are artificially introduced into germline sequences by genetic manipulation, and the endogenous mouse immunoglobulin genes are inactivated. For example, B
Ruggemann et al., 1997 Curr. Opin. Biotechnol
. 8: 455-58. For example, a human immunoglobulin transgene is
It can be a minigene construct or it can be a translocus of a yeast artificial chromosome, which causes B cell-specific DNA translocation and hypermutation in mouse lymphoid tissue. See, for example, Bruggemann et al., 1997 Curr. Op
in. Biotechnol. 8: 455-58. Human monoclonal antibodies that specifically bind to DSP-4 can be used to immunize transgenic animals,
Fusion of spleen cells in myeloma, selection and cloning of cells producing antibodies as described above. Polyclonal sera containing human antibodies are also obtained from the blood of the immunized animal.

[0066] Chimeric antibodies specific for DSP-4 (including humanized antibodies) can also be produced according to the present invention. A chimeric antibody has at least one constant region domain from a first mammalian species and at least one variable region domain from a second different mammalian species. See, eg, Morrison et al., 1984, Proc. Natl. A
cad. Sci. USA 81: 6851-55. In a preferred embodiment, the chimeric antibody comprises a polynucleotide sequence encoding at least one variable region domain from a non-human monoclonal antibody, such as a variable region from a mouse, rat, or hamster monoclonal antibody, comprising at least one human constant It can be constructed by cloning into a vector containing the nucleic acid sequence encoding the region. For example, Shin et al., 1989 Methods Enzym.
ol. 178: 459-76; Walls et al., 1993 Nucleic Ac.
ids Res. 21: 2921-29. By way of example, a polynucleotide sequence encoding the light chain variable region of a mouse monoclonal antibody can be inserted into a vector comprising a nucleic acid sequence encoding a human kappa light chain constant region sequence. In a separate vector, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody can be cloned in frame with the sequence encoding the human IgG1 constant region. The particular human constant region selected may depend on the effector function desired for a particular antibody (eg, complementary immobilization, binding to a particular Fc receptor, etc.). Another method known in the art for producing chimeric antibodies is homologous recombination (eg, US Pat. No. 5,482,856). Preferably, the vector is transfected into eukaryotic cells for stable expression of the chimeric antibody.

[0067] Non-human / human chimeric antibodies can be further engineered to produce "humanized" antibodies. Such a humanized antibody may comprise a plurality of CDRs from an immunoglobulin of a non-human mammalian species, at least one human variable framework region, and at least one human immunoglobulin constant region. Humanization in certain embodiments includes, for example, a non-human monoclonal antibody from which a DSP-4 binding variable region is obtained,
Alternatively, antibodies may be provided that have reduced binding affinity for DSP-4 when compared to any of the chimeric antibodies having such a V region and at least one human C region as described above. Thus, useful strategies for designing humanized antibodies can include, for example, but not by way of limitation, identification of a human variable framework region that is most homologous to a non-human framework region of a chimeric antibody. While not wishing to be bound by theory, such a strategy is that humanized antibodies may be
May increase the likelihood of retaining a specific binding affinity for, in some preferred embodiments, this is substantially the same affinity for the DSP-4 polypeptide or variant or fragment thereof. Obtained and in certain other preferred embodiments, this may be of greater affinity for DSP-4. See, for example, Jones et al., 1986 Nature 321: 522-25;
See chmann et al., 1988 Nature 332: 323-27. Thus, the design of such humanized antibodies involves determining the CDR loop structure and structure determinants of the non-human variable region (eg, by computer modeling), and then the known human CDR loop structure and structure of the CDR loops and determinants. It may include a comparison with a determinant. For example, Padlan et al., 1995 FASEB 9:
133-39; Chothia et al., 1989 Nature, 342: 377-.
See 383. Computer modeling can also be used to compare human structural templates selected by sequence homology at non-human variable regions. See, eg, Bajorat et al., 1995 Ther. Immunol. 2: 9
5-103; see EP-0578515-A3. If humanization of a non-human CDR results in a decrease in binding affinity, computer modeling will optimally (ie, increase to a higher level than the non-humanized antibody) partially, completely, or exceed the affinity To aid in the identification of specific amino acid residues that can be altered by site-directed or other mutagenesis techniques. Those of skill in the art are familiar with these techniques and will readily appreciate many variations and modifications to such design strategies.

In certain embodiments, it may be preferable to use an antigen-binding fragment of the antibody. Such fragments include Fab fragments or F (ab ') ₂
Fragments, which can be prepared by proteolytic digestion with papain or pepsin, respectively. Antigen binding fragments can be separated from Fc fragments by, for example, affinity chromatography using immobilized Protein A or Protein G, or immobilized DSP-4 polypeptide, or a suitable variant or fragment thereof. One of skill in the art would routinely and without undue experimentation, using, for example, the immunodetection methods provided herein, obtain suitable variants or fragments based on the characteristics of the resulting affinity purified antibodies. Can decide. An alternative method for producing Fab fragments is F (ab ') ₂
Mild reduction of the fragment followed by alkylation. For example, Wier
, Handbook of Experimental Immunology
, 1986, Blackwell Scientific, Boston.

According to certain embodiments, the variable regions of any of the above non-human, human, or humanized heavy and light chains of an Ig molecule are constructed as single chain Fv (sFv) polypeptide fragments (single chain antibodies). Can be done. See, for example, Bird et al., 1988 Science.
ce 242: 423-426; Huston et al., 1988 Proc. Nat
l. Acad. Sci. USA 85: 5879-5883. Multifunctional sFv fusion proteins can be produced by joining a polynucleotide sequence encoding an sFv polypeptide in-frame to at least one polynucleotide sequence encoding any of a variety of known effector proteins. These methods are known in the art and are described, for example, in EP-B1-0318554,
It is disclosed in U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, and U.S. Patent No. 5,476,786. For illustrative purposes, effector proteins can include immunoglobulin constant region sequences. For example, Hollenb
Augh et al., 1995 J. Am. Immunol. Methods 188: 1-7
checking ... Another example of an effector protein is an enzyme. By way of non-limiting example, such enzymes may provide a biological activity for therapeutic purposes (eg,
Simers et al., 1977 Bioconjug. Chem. 8: 510-19
Or detectable activity, such as horseradish peroxidase-catalyzed conversion of any of a number of well-known substrates to a detectable product for diagnostic use. Still other examples of sFv fusion proteins include Ig
-A toxin fusion, or an immunotoxin, wherein the sFv polypeptide is conjugated to the toxin. Those of skill in the art understand a wide variety of polypeptide sequences that have been identified as toxic to cells under appropriate conditions. As used herein,
A toxic polypeptide included in an immunoglobulin-toxin fusion protein can be any polypeptide that can be introduced into a cell in a manner that impairs cell survival, for example, by direct interference with vital functions or by inducing apoptosis. . Thus, toxins include, for example, Pseudomonas aeruginosa exotoxin A,
Ribosome-inactivating proteins such as plant gelonin, bryodin from Bryonia dioica and the like can be mentioned. See, eg, Thrush et al., 1996 Annu. Rev .. Immunol
. 14: 49-71; Frankel et al., 1996 Cancer Res. 5
6: 926-32. Chemotherapeutic agents, antimitotic agents, antibodies, apoptosis-inducing factors (or "apoptogens" such as Green
and a number of other toxins, including Reed, 1998, Science 281: 1309-1321), are known to those of skill in the art, and the examples provided herein are within the scope of the present invention. And is intended as a non-limiting example.

[0070] In certain embodiments, the sFv comprises a fusion protein and an antigen (eg, a DSP).
-4) can be fused to a peptide or polypeptide domain that allows the detection of specific binding between For example, the fusion polypeptide domain can be an affinity tag polypeptide. Thus, s to a binding partner (eg, DSP-4)
Binding of the Fv fusion protein can be detected using any of a variety of techniques familiar to those skilled in the art, using an affinity polypeptide or peptide tag, such as an avidin, streptavidin or His (eg, polyhistidine) tag. Detection techniques also include, for example, binding of avidin or streptavidin fusion proteins to biotin or a biotin mimetic sequence (eg, Luo et al., 1998 J. Bio.
technology. 65: 225 and references listed therein).
Direct covalent modification of a fusion protein having a detectable moiety (eg, a labeling moiety), non-covalent attachment of the fusion protein to a specific labeled reporter molecule, detectable by a fusion protein including a moiety having enzymatic activity It may involve enzymatic modification of the substrate or immobilization of the fusion protein on a solid support (covalent or non-covalent).

Thus, an sFv protein of the present invention comprising a DSP-4 specific immunoglobulin-derived polypeptide fused to another polypeptide, such as an effector peptide having the desired affinity properties, for example, is that the effector peptide is glutathione It may include a fusion protein that is an enzyme such as -S-transferase. As another example, the sFv fusion protein may also be Staphylococcus aureus.
A DSP-4 specific Ig polypeptide fused to a Protein A polypeptide may be included; its use in constructing fusion proteins having affinity for protein A encoding nucleic acids and immunoglobulin constant regions is generally described in, for example, It is disclosed in U.S. Pat. No. 5,100,788. Other affinity polypeptides useful for constructing sFv fusion proteins are described, for example, in WO 89/03422; US Pat. No. 5,48.
No. 5,528,691; WO93 / 24631; U.S. Pat. No. 5,168,049; Strepavidin fusion protein disclosed in U.S. Pat. No. 5,272,254 and elsewhere. And an avidin fusion protein (see, for example, EP 511,747). As provided herein, an sFv polypeptide sequence can be fused to a fusion polypeptide sequence, including an effector protein sequence, which can include a full length fusion polypeptide and, alternatively, a variant or fragment thereof. .

A further method for selecting antibodies that specifically bind to a DSP-4 polypeptide or variant or fragment thereof is phage display. See, eg, Winter et al., 1994 Annul. Rev .. Immunol. 12
: 433-55; Burton et al., 1994 Adv. Immunol. 57:
See 191-280. The human or mouse immunoglobulin variable region gene combinatorial library comprises Ig fragments (Fab, Fv, s) that specifically bind to the DSP-4 polypeptide or a variant or fragment thereof.
Fv or multimers thereof) can be made in a phage vector that can be screened to select. See, for example, U.S. Pat. No. 5,223,409; Huse et al., 1989 Science 246: 1275-81; Kang.
Et al., 1991 Proc. Natl. Acad. Sci. USA 88: 436
3-66; Hoogenboom et al., 1992 J. J. Am. Molec. Biol. 2
27: 381-388; Schlebusch et al., 1997 Hybridom.
a 16: 47-52 and the references listed therein. For example, a library containing a plurality of polynucleotide sequences encoding an Ig variable region fragment can be used to make an M13 fusion protein to a filamentous bacteriophage gene, such as M13 or a variant thereof, eg, the gene III of M13.
Alternatively, it can be inserted in frame using a sequence encoding a phage coat protein such as gene VIII. The fusion protein can be a fusion of the coat protein with the light and / or heavy chain variable region domains.

According to a particular embodiment, an immunoglobulin Fab fragment may also be displayed on a phage particle as follows. A polynucleotide sequence encoding an Ig constant region domain can be inserted in frame into the phage genome using a coat protein. Thus, the phage coat fusion protein can be fused to an Ig light or heavy chain fragment (Fd). For example, from a human Ig library, a polynucleotide sequence encoding a human kappa constant region can be inserted into a vector in frame with a sequence encoding at least one phage coat protein. Additionally or alternatively, a polynucleotide sequence encoding a human IgG1 CH1 domain can be inserted in frame with a sequence encoding at least one other phage coat protein. The plurality of polynucleotide sequences encoding the variable region domains (eg, from a DNA library) are then combined using a constant region coat protein fusion to express a Fab fragment that fuses to the bacteriophage coat protein. It can be inserted into the vector in frame.

A phage displaying an Ig fragment (eg, Ig V region or Fab) that binds to a DSP-4 polypeptide can use this phage library as a DSP-4
Or by mixing with a variant or fragment thereof, or by contacting the phage library with a DSP-4 polypeptide immobilized on a solid matrix under conditions and for a time sufficient to bind. obtain. Unbound phage is removed by a wash, which typically removes low concentrations of salt (eg, NaCl), preferably less than 100 mM NaCl, more preferably less than 50 mM NaCl, most preferably less than 10 mM NaCl. The buffer may be a buffer containing salt or a buffer containing no salt. The specifically bound phage are then eluted with a buffer containing NaCl (eg, by increasing the salt concentration in a stepwise manner). Typically, phage that bind to DSP-4 with high affinity require higher salt concentrations to release. The eluted phage can be propagated to a suitable bacterial host and, in general, successive rounds of DSP-4 binding and elution are repeated to increase the yield of phage expressing DSP-4 specific immunoglobulins. Can be Combinatorial phage libraries can also be used for humanization of non-human variable regions. For example, Ros
Ok et al., 1996 J. Am. Biol. Chem. 271: 22611-18; Ra
der et al., 1998 Proc. Natl. Acad. Sci. USA 95:
See 8910-15. The DNA sequence of the immunoglobulin gene inserted into the phage thus selected can be determined by standard techniques. S
ambrook et al., 1989 Molecular Cloning: A La.
boratemanual, Cold Spring Harbor P
See less. The affinity-selected Ig coding sequence can then be cloned into another suitable vector for expression of the Ig fragment, or if necessary, for expression of whole immunoglobulin chains, the Ig constant Can be cloned into a vector containing the region.

[0075] Phage display technology can also be used to select polypeptides, peptides or single-chain antibodies that bind to DSP-4. Examples of suitable vectors having a multiple cloning site into which a candidate nucleic acid molecule (eg, DNA) encoding such a peptide or antibody can be inserted are described by McLaffertyr et al.
ene 128: 29-36, 1993; Scott et al., 1990 Science.
ce 249: 386-390; Smith et al., 1993 Methods E.
nzymol. 217: 228-257; Fisch et al., 1996, Proc.
Natl. Acad. Sci. USA 93: 7761-66. The inserted DNA molecule may comprise a randomly generated sequence or a known peptide or a peptide which specifically binds to a DSP-4 polypeptide or a variant or fragment thereof as provided herein. A variant of the polypeptide domain may be encoded. Generally, the nucleic acid insert is a peptide of up to 60 amino acids, more preferably a peptide of 3 to 35 amino acids, and even more preferably 6 to 20 amino acids.
Encodes a peptide of amino acids. The peptide encoded by the inserted sequence is displayed on the surface of the bacteriophage. A phage expressing a binding domain for a DSP-4 polynucleotide may comprise an immobilized DSP-4 as described above.
Selection can be based on specific binding to the polypeptide. As provided herein, well-known recombinant genetic techniques can be used to construct a fusion protein comprising the fragment. For example, the polypeptide that can be produced is a tandem of two or more similar or different affinity-selected DSP-4 binding peptide domains in order to maximize the binding affinity of the resulting product to DSP-4. Includes array.

In another particular embodiment, the invention contemplates a DSP-4 specific antibody that is a multimeric antibody fragment. Useful methodologies are generally described, for example, in Hayde
n et al., 1997, Curr Opin. Immunol. 9: 201-12; C
Oloma et al., 1997 Nat. Biotechnol. 15: 159-63
It is described in. For example, a multimeric antibody fragment can be a miniantibody (minia).
diabodies (U.S. Pat. No. 5,910,573) or diabodies (Hollinger et al., 1997, Cancer Im).
munol. Immunother. 45: 128-130). Multimeric fragments can be produced that are multimers of a DSP-4-specific Fv or a bispecific antibody comprising a DSP-4-specific Fv non-covalently associated with a second Fv with different plateau specificity. See, for example, Koelemij et al. Immunother. 22:51
See 4-24. As another example, a multimeric antibody may include two single-chain antibodies or a bispecific antibody having a Fab fragment. According to certain related embodiments, the first Ig fragment can be specific for a first antigenic determinant on a DSP-4 polypeptide (or variant or fragment thereof), while the second Ig fragment is -4 polypeptide may be specific for a second antigenic determinant. Alternatively, in other specific related embodiments, the first immunoglobulin fragment may be specific for an antigenic determinant on a DSP-4 polypeptide or variant or fragment thereof, and the second immunoglobulin fragment Can be specific for an antigenic determinant on a second, different (ie, non-DSP-4) molecule. Bispecific antibodies that specifically bind to DSP-4 are also contemplated, wherein at least one antigen binding domain is present as a fusion protein.

Introduction of an amino acid mutation into a DSP-4 binding immunoglobulin molecule can be accomplished by adding
4 may be useful to increase specificity or affinity for, or to alter effector function. Immunoglobulins with high affinity for DSP-4 can be produced by site-directed mutagenesis of specific residues. Computer-aided three-dimensional molecular modeling can be used to identify amino acid residues to be changed in order to improve affinity for the DSP-4 polypeptide.
See, for example, Mountain et al., 1992, Biotechnol. Genet.
Eng. 10: 1-142. Alternatively, a combinatorial library of CDRs can be produced in M13 phage and screened for immunoglobulin fragments with improved affinity. For example,
Glaser et al., 1992, J. Am. Immunol. 149: 3903-3913
Bardas et al., 1994 Proc. Natl. Acad. Sci. USA
91: 3809-13; see U.S. Patent No. 5,792,456.

[0078] Effector function can also be altered by site-directed mutagenesis. For example, D
uncan et al., 1988 Nature 332: 563-64; Morgan
Et al., 1995 Immunology 86: 319-24; Eghtedar.
Zedheh-Kondri et al., 1997 Biotechniques 23:
See 830-34. For example, mutations in the glycosylation site on the immunoglobulin Fc protein can alter the ability of the immunoglobulin to anchor to its complementary strand. For example, Wright et al., 1997 Trends Biotechnol.
. 15: 26-32. Other mutations in the constant region domain
It may alter the ability of the immunoglobulin to anchor to the complementary strand, or alter the effects of antibody-dependent cytotoxicity. See, for example, Duncan et al., 1988 Nature.
332: 563-64; Morgan et al., 1995 Immunology.
86: 319-24; Sensel et al., 1997 Mol. Immunol. 3
See 4: 1019-29.

[0079] Nucleic acid molecules encoding an antibody or fragment thereof that specifically binds to DSP-4 described herein may be prepared by any of a variety of well-known methods for nucleic acid excision, ligation, transformation and transfection. According to the procedure, it can be propagated and expressed. Thus, in certain embodiments, the expression of the antibody fragment is Esche
It may be preferred in prokaryotic hosts such as Richia coli (see, eg, Pluckthun et al., 1989 Methods Enzymol. 17).
8: 497-515). In other specific embodiments, expression of the antibody or fragment thereof is in yeast (eg, Saccharomyces cece).
revisiae, Schizosaccharomyces pombe and Pichia pastoris), animal cells (including mammalian cells) or eukaryotic host cells including plant cells. Examples of suitable animal cells include, but are not limited to, myeloma, COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells. According to techniques known to those of skill in the art and based on the present disclosure, nucleic acid vectors can be designed to express foreign sequences in a particular host system, and then encode a polypeptide encoding a DSP-4 binding antibody (or fragment thereof). A nucleotide sequence can be inserted. Regulatory elements vary according to the particular host.

The DSP-4 binding immunoglobulins (or fragments thereof) described herein can be enzymes, cytotoxic agents, or other reporter molecules (dyes, radionuclides,
(Including luminescent groups, fluorescent groups, or biotin, etc.). DSP-4 specific immunoglobulins or fragments thereof can be radiolabeled for diagnostic or therapeutic applications. Techniques for radiolabeling antibodies are known in the art. For example, Adams 1998 In Vivo
12: 11-21; Hiltunen 1993 Acta Oncol. 32
: 831-9. Therapeutic applications are described in detail below and may include the use of DSP-4 binding antibodies (or fragments thereof) in combination with other therapeutic agents. Antibodies or fragments can also be combined with cytotoxic agents known in the art and provided herein (eg, toxins such as ribosome-inactivating proteins, chemotherapeutic agents, antimiotic agents, antibiotics, etc.). Can be combined.

The present invention also contemplates the production of anti-idiotype antibodies that recognize an antibody (or antigen-binding fragment thereof) that specifically binds to DSP-4 or a variant or fragment thereof provided herein. I do. Anti-idiotypic antibodies can be produced as polyclonal or as monoclonal antibodies by the methods described herein using anti-DSP-4 antibodies (or antigen-binding fragments thereof) as immunogens. Anti-idiotype antibodies or fragments thereof can also be produced by any of the recombinant genetic manipulation techniques described above, or by phage display selection. The anti-idiotype antibody is DS-anti-DSP-4 antibody.
It can react with the antigen binding site of the anti-DSP-4 antibody such that binding to the P-4 polypeptide is competitively inhibited. Alternatively, an anti-idiotype antibody provided herein may not competitively inhibit binding of an anti-DSP-4 antibody to a DSP-4 polypeptide.

[0082] Polyclonal and monoclonal antibodies can be used for affinity isolation of DSP-4 polypeptides according to the methodology provided herein and well known in the art. See, for example, Hermanson et al., Immobilized A.
fiffinity Technology, Acadedmic
Press, Inc. See New York, 1992. Briefly, antibodies (or antigen-binding fragments thereof) can be immobilized on a solid support material, which is then contacted with a sample containing the polypeptide of interest (eg, DSP-4). You. Following separation from the rest of the sample, the polypeptide is then released from the immobilized antibody.

(Methods for Detecting DSP-4 Expression) A particular aspect of the present invention uses antibodies raised against DSP-4 or hybridizing polynucleotides for diagnostic and assay purposes. Provide a way to Certain assays involve the use of antibodies or other agents to detect the presence or absence of DSP-4, or a proteolytic fragment thereof. Alternatively, the nucleic acid encoding DSP-4 can be detected using standard hybridization and / or PCR techniques. Suitable probes and primers are based on the DSP-4 cDNA sequences provided herein,
Can be designed by one skilled in the art. Assays are generally performed on various samples obtained from biological sources such as eukaryotic cells, bacteria, viruses, extracts prepared from such organisms, and fluids found in living organisms. It can be done using either. Biological samples that can be obtained from a patient include blood samples, biopsy specimens, tissue explants, organ cultures and other tissue or cell preparations. The patient or biological source can be a human or non-human animal, primary cell culture or culture compatible cell line, including genetically engineered cell lines (chromosomally integrated or episomal groups). Recombinant nucleic acid sequences), immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiable cell lines, transformed cell lines, and the like. In certain preferred embodiments, the patient or biological source is a human, and in a particularly preferred embodiment, the biological source is a non-human animal (eg, a rodent (eg, a mouse) that is a mammal. , Rats, hamsters, etc.), ungulates (eg, cows) or non-human primates). In certain other preferred embodiments of the invention, the patient may be suspected of having, or being at risk for, a disease associated with altered cell signaling, or is not at risk for such a disease. Or its absence may be known.

For detecting DSP-4 protein, the reagent is typically an antibody, which can be prepared as described below. There are a variety of assay formats known to those skilled in the art for using antibodies to detect polypeptides in a sample. For example, Harlow and Lane, Antibodies: A Labo
rattro Manual, Cold Spring Harbor Lab
org., 1988. For example, the assay can be performed in a Western blot format, in which protein preparations from a biological sample are separated by gel electrophoresis, transferred to a suitable membrane, and reacted with an antibody. The presence of the antibody on the membrane can then be detected using a suitable detection reagent as described below.

In another embodiment, the assay involves using an antibody immobilized on a solid support, binding to the target DSP-4, and removing from the rest of the sample. The bound DSP-4 can then be detected using a second antibody or reagent that contains a reporter group. Alternatively, competition assays may be utilized. In this competition assay, the DSP-4 polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubating the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody indicates the reactivity of the sample with the immobilized antibody and, consequently, the level of DSP-4 in the sample.

[0086] The solid support can be any material known to those of skill in the art to which the antibody can be attached, such as a test well in a microtiter plate, a nitrocellulose filter or another suitable membrane. Alternatively, the support may be beads or discs (eg, glass), fiberglass, latex or plastic (eg, polystyrene or polyvinyl chloride). Various techniques known to those skilled in the art (
Antibodies can be immobilized on a solid support (described extensively in the patent and scientific literature).

[0087] In certain embodiments, the assay for detection of DSP-4 in a sample is a two antibody sandwich assay. The assay may be performed by first contacting an antibody immobilized on a solid support (typically a well of a microtiter plate) with a biological sample, such that the D
SP-4 is allowed to bind to the immobilized antibody (a 30 minute incubation time at room temperature is generally sufficient). The unbound sample is then removed from the immobilized DSP-4 / antibody complex and a second antibody (enzyme, dye, radionuclide, luminescent group, fluorescent group, Or containing a reporter group such as biotin). The amount of the second antibody that remains bound to the solid support is then determined using methods appropriate for the specific reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, luminescent groups, and fluorescent groups. Biotin can be detected using avidin, conjugated to a different reporter group (generally a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can be detected, generally by adding a substrate (generally for a specific period of time) and then spectroscopically or otherwise analyzing the reaction product. Standards and standard additives can be used to determine the level of DSP-4 in a sample using well-known techniques.

In a related aspect of the invention, there is provided a kit for detecting DSP-4 and DSP-4 phosphatase activity. Such a kit can be designed to detect the level of DSP-4 or a nucleic acid encoding DSP-4,
Alternatively, the phosphatase activity of DSP-4 can be detected in a direct phosphatase assay or a binding phosphatase assay. Generally, a kit of the invention will include one or more containers enclosing the components (eg, reagents or buffers) used in the assay.

A kit for detecting the level of DSP-4 or a nucleic acid encoding DSP-4 typically comprises a DSP-4 protein, DSP-4 DNA or DSP-
4 Contains reagents that bind to RNA. To detect a nucleic acid encoding DSP-4, the reagent may be a nucleic acid probe or a PCR primer. To detect DSP-4 protein, the reagent is typically an antibody. Such kits also include a reporter group suitable for direct or indirect detection of a reagent (ie,
The reporter group can be covalently linked to the reagent, or can bind to a second molecule (eg, protein A, protein G, immunoglobulin or lectin), which itself binds to the reagent. Can). Suitable reporter groups include, but are not limited to, enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, and biotin. Such reporter groups can be used to detect, directly or indirectly, the binding of the reagent to the sample components using standard methods known to those skilled in the art.

[0090] Kits for detecting DSP-4 activity typically include a DSP-4 substrate in combination with a suitable buffer. DSP-4 activity can be specifically detected by performing an immunoprecipitation step with a DSP-4 specific antibody before performing the phosphatase assay as described above. Other reagents for use in detecting dephosphorylation of a substrate may also be provided.

In certain diagnostic assays, based on the presence of altered DSP-4 or altered levels of DSP-4 expression, a proliferative disorder may be caused by a patient or another organism as provided herein. In biological source organisms. For example, the antibody may distinguish between wild type DSP-4 and an altered DSP-4 having variations in amino acid sequence. Such variations may indicate the presence of, or susceptibility to, a proliferative disorder. Hybridization and amplification techniques can be used to detect modified DSP-4 sequences as well.

[0092] Methods for Identifying Modulators of DSP-4 Activity In one aspect of the invention, DSP-4 polypeptides can be used to identify agents that modulate DSP-4 activity. Such agents can inhibit or enhance signal transduction through the MAP-kinase cascade and cause cell proliferation. Agents that modulate DSP-4 activity may include DSP-4 expression and / or stability, DSP-4 protein activity and / or DS that dephosphorylate substrates.
The ability of P-4 can be varied. Agents that can be screened in such assays include, but are not limited to: antibodies and antigen-binding fragments thereof, such as competitive substrates or peptides that present a catalytic site or a dual phosphorylation motif, DSP- Antisense polynucleotides and ribozymes that interfere with the transcription and / or translation of 4, and other natural and synthetic molecules that bind and inactivate DSP-4 (eg, small molecule inhibitors).

[0093] Candidate agents for use in a method of screening for a modulator of DSP-4 according to the present invention may be provided as a "library" or a collection of compounds, compositions or molecules. Such molecules are typically in the art known as "small molecule", and less than 10 ⁵ daltons, preferably less than 10 ⁴ daltons and still more preferably compounds having a molecular weight of less than 10 ³ daltons including. For example, a member of a library of test compounds can be administered to a plurality of samples, each containing at least one DSP-4 polypeptide as provided herein, and then DSP-4 mediated dephosphorylation of a substrate. Assays for their ability to enhance or inhibit oxidation or DSP-4 mediated binding to a substrate. Compounds so identified as being able to affect DSP-4 function (eg, phosphotyrosine and / or phosphoserine / threonine dephosphorylation) are:
Useful for therapeutic and / or diagnostic purposes. Because these are DSP
Because it allows treatment and / or detection of diseases associated with -4 activity. Such compounds are also useful in studies on the mechanisms of molecular signaling involved in DSP-4, and on improvements in the discovery and development of future DSP-4 compounds that show additional specificity.

[0094] Candidate agents may further be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a predetermined plurality of chemical reactions performed in a plurality of reaction vessels. For example, various starting compounds can be prepared utilizing one or more solid phase synthesis, recorded random mixing procedures, and recorded reaction resolution means, wherein a given configuration comprises multiple permutations and / or It allows the combination of reaction conditions to be attributed. The resulting products can be screened and then subjected to a library of iterative selection and synthesis procedures (eg,
Peptides (eg, PCT / US91 / 08694, PCT / US91 / 046)
66, which are incorporated herein by reference in their entirety) or other compositions that may include small molecules as provided herein (eg,
PCT / US94 / 08542, EP 0774664, US Patent 5,798
U.S. Patent No. 5,789,172; U.S. Patent No. 5,751,629.
Nos., Which are incorporated herein by reference in their entirety)
A synthetic combinatorial library). Various classes of such libraries can be prepared according to established procedures, and DSPs according to the present disclosure.
One of skill in the art will understand that can be tested using -4.

In certain embodiments, the modulatory agent combines the candidate agent with a DSP-4 polypeptide or a polynucleotide encoding such a polypeptide, in vitro or in vivo, and the candidate agent DSP-4 phosphatase The effect on activity can be identified, for example, by assessing using the representative assays described herein. An increase or decrease in phosphatase activity can be measured by performing the representative assays provided herein in the presence and absence of the candidate agent. Briefly, the candidate drug is an active DSP-4
A mixture of a polypeptide and a substrate (eg, phosphorylated MAP-kinase) can be included with or without pre-incubation with one or more components of the mixture. Generally, suitable amounts of antibodies or other agents for use in such assays range from about 0.01 μM to about 100 μM. Then the drug DSP
The effect on -4 activity can be assessed by quantifying the loss of phosphate from the substrate and comparing that loss to the loss achieved with DSP-4 without the addition of a candidate agent. Alternatively, a binding kinase assay can be used in which DSP-4 activity is measured indirectly based on MAP-kinase activity.

Alternatively, a polynucleotide comprising a DSP-4 promoter operably linked to a DSP-4 coding region or reporter gene can be a test compound, DSP-4.
Can be used to assess the effect on transcription. Such an assay is a DSP-
Cells endogenously expressing 4 (eg, human or other mammalian heart, testis,
Thymus or skeletal muscle cells) or cells transfected with an expression vector containing a DSP-4 promoter linked to a reporter gene.
The effect of the test compound can then be assessed by assaying the effect of DSP-4 or the reporter on transcription, for example, using Northern blot analysis or an appropriate reporter activity assay.

[0097] DSP-4 activity can also be measured in all cells transfected with a reporter gene whose expression depends on activation of the appropriate substrate. For example, suitable cells (ie, cells that express DSP-4) can be transfected with a substrate-dependent promoter linked to a reporter gene. In such a system,
Reporter gene expression, which can be easily detected using methods well known to those skilled in the art, depends on the activation of the substrate. Dephosphorylation of the substrate can be detected based on a decrease in reporter activity. Candidate modulating agents can be added to such systems as described above to evaluate their effect on DSP-4 activity.

The present invention further provides a method for identifying a molecule that interacts with or binds to DSP-4. Such molecules generally have at least 10 ⁴ , preferably at least 10 ⁵ , more preferably at least 10 ⁶ ,
Still more preferably, at least 10 ⁷ , and most preferably, at least 1
A 0 ⁸ affinity constants (K _a) associates with DSP-4. Affinity constants can be determined using known techniques. Methods for identifying interacting molecules are described, for example, as initial screens for modulatory drugs or in vivo with DSP-4.
It can be used to identify factors involved in the activity. Techniques for substrate capture (eg, using DSP-4 variants or substrate capture variants as described above)
It is contemplated according to the specific embodiments provided herein. In addition to standard binding assays, there are many other techniques well-known for identifying interacting molecules, including yeast two-hybrid screening, phage display and affinity techniques. Such techniques may be performed using conventional protocols, which are well known to those skilled in the art (eg, Bartel et al., Cellu).
lar Interactions in Development: A Pr
physical Approach, D.C. A. Harley (ed.), Oxfor
d University Press (Oxford, UK), 153-17
9, page 1993). In these and other techniques, a candidate interacting protein (eg, a putative DSP-4 substrate) can be phosphorylated before assaying for the presence of a DSP-4 binding protein or a DSP-4 interacting protein.

In another aspect, the invention provides an animal model in which the animal does not express functional DSP-4 or altered DSP-4. Such animals can be produced using standard homologous recombination strategies. Animal models produced in this manner can be used to study the activity of DSP-4 polypeptides and modulators in vivo.

(Method of Dephosphorizing a Substrate) In another aspect of the present invention, a DSP-4 polypeptide can be used to dephosphorylate a DSP-4 substrate provided herein. . In one embodiment, the substrate is treated with a suitable buffer (eg, Tris, pH 7.0) at 30 ° C. for 10 minutes.
5,1 mM EDTA, 1 mM dithiothreitol, 1 mg / mL bovine serum albumin) can be dephosphorylated in vivo by incubating the DSP-4 polypeptide with the substrate. Any compound that can be dephosphorylated by DSP-4, such as MAP kinase, can be used as a substrate. Generally, the amounts of reaction components can range from about 50 pg to about 50 ng of DSP-4 polypeptide, and from about 10 ng to about 10 μg of substrate. The dephosphorylated substrate can then be purified, for example, by affinity techniques and / or gel electrophoresis. The extent of dephosphorylation of a substrate is generally determined by adding a [γ- ³² P] -labeled substrate to a test aliquot and assessing the level of dephosphorylation of the substrate as described herein. Can be monitored.

Methods of Modulating Cell Responses Modulators modulate, modify or otherwise alter cell responses, such as cell proliferation, differentiation and survival, in a variety of contexts, both in vivo and in vitro (eg, , Increase or decrease). Generally, to modulate such a response (eg, increase or decrease in a statistically significant manner),
It is contacted with an agent that modulates DSP-4 activity for a time and under conditions sufficient to allow modulation of P-4 activity. An agent that modulates a cellular response can function in any of a variety of ways. For example, an agent may modulate a pattern of gene expression (ie, increase or inhibit expression of a gene family or gene that is expressed in a coordinated manner). A variety of hybridization and amplification techniques are available for assessing gene expression patterns. Alternatively or additionally, the agent may cause apoptosis or necrosis of the cell and / or modulate the function of the cell cycle within the cell. (See, for example, Ashkenazi et al., 1998 Sc.
ence, 281: 1305; Thornbury et al., 1998 Scie.
Evan et al., 1998 Science 281:
1317; Adams et al., 1998 Science 281: 1322; and the references listed therein. ) Cells treated as described above may exhibit the standard characteristics of cells with altered proliferation, differentiation or survival characteristics. In addition, such cells may (but need not) show changes in other detectable properties, such as contact inhibition of cell growth, anchorage-independent growth or altered cell-cell adhesion. Such a property can be easily detected using techniques familiar to those skilled in the art.

Therapeutic Methods One or more DSP-4 polypeptides, modulators (any agent that specifically binds DSP-4 (eg, an antibody or fragment thereof as provided herein)) ) And / or polynucleotides encoding such polypeptides and / or modulators may also be used to modulate DSP-4 activity in a patient. As used herein, a "patient" can be any mammal, including a human, and can be affected by a condition associated with DSP-4 activity or have a detectable disease. May not be. Thus, treatment can be treatment of an existing disease or can be prophylactic. Conditions associated with DSP-4 activity include cell proliferation (including cancer, graft versus host disease (GVHD), autoimmune diseases, allergies or other conditions that may involve immunosuppression), metabolic diseases, abnormal cells Includes any injury associated with proliferation or abnormal cell differentiation, and cell cycle abnormalities.
Such specific injuries are associated with loss of phosphatase activity of normal MAP kinase, leading to unregulated cell growth. DSP-4 polypeptides and polynucleotides encoding such polypeptides can be used to ameliorate such damage. Activators of DSP-4 can also be used to treat certain disorders.

For administration to a patient, one or more polypeptides, polynucleotides and / or
Alternatively, the modulator is generally formulated as a pharmaceutical composition. The pharmaceutical compositions may be sterile aqueous or non-aqueous solutions, suspensions or emulsions, which may also be a physiologically acceptable carrier, ie, a non-toxic substance that does not interfere with the activity of the active ingredient.
including. Such compositions may be in the form of a solid, liquid or gas (aerosol). Alternatively, the compositions of the invention can be formulated as a lyophilizate, or the compound can be encapsulated in liposomes using well known techniques. Pharmaceutical compositions within the scope of the present invention may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, buffers (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose or dextran). Amino acids such as mannitol, proteins, polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; stabilizers, pigments, flavoring agents, and suspending and / or preservatives.

[0104] Any suitable carrier known to those of skill in the art can be used in the pharmaceutical compositions of the invention. Carriers for therapeutic use are well known and are described, for example, in Remi
ngtons Pharmaceutical Sciences, Mack
Publishing Co. (A. R. Gennaro ed. 1985). Generally, the type of carrier will be selected based on the mode of administration. The pharmaceutical composition may be administered (eg, topically, orally, nasally, intrathecally, rectally, vaginally, sublingually, or parenterally (subcutaneously, intravenously, intramuscularly, intrasternally, intracavity). , Intratratal, or intraurethral injection or infusion), including administration). For parenteral administration, the carrier preferably includes water, saline, alcohol, fat, wax or a buffer. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethylcellulose, ethylcellulose, glucose , Sucrose, and / or magnesium carbonate) can be used.

The pharmaceutical compositions (eg, for oral administration or delivery by injection) can be in liquid form (eg, elixirs, syrups, solutions, emulsions or suspensions).
Liquid pharmaceutical compositions may include, for example, one or more of the following: water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride,
Non-volatile oils, such as synthetic mono- or diglycerides, which can serve as solvents or suspending media; sterile diluents, such as polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents, such as benzyl alcohol or methyl paraben Antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate and agents for tonicity adjustment such as sodium chloride or glucose. . The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. The use of saline is preferred, and injectable pharmaceutical compositions are preferably sterile.

The compositions described herein can be formulated for sustained release (ie, a formulation such as a capsule or sponge that provides for a slow release of compound following administration). Such compositions may be prepared, generally using well-known techniques, and may be administered, for example, by oral, rectal or subcutaneous implantation, or by implantation at a desired target site. Sustained-release formulations may include the drug dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use in such formulations may be biocompatible and / or biodegradable; preferably, the formulations provide a relatively constant level of active ingredient release. I do. The amount of active ingredient included in a sustained release formulation will depend upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

For a pharmaceutical composition comprising a polynucleotide encoding a DSP-4 polypeptide and / or a modulator (as the polypeptide and / or modulator is produced in situ), the polynucleotide may comprise Nucleic acids, and
It can be present in any of a variety of delivery systems known to those of skill in the art, including bacterial, viral and mammalian expression systems. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. This DNA is also described, for example, in Ulmer et al.
science 259: 1745-1749, 1993 and Cohen
, Science 259: 1691-1692, 1993. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads that are effectively transported to cells.

[0108] Within a pharmaceutical composition, a DSP-4 polypeptide, polynucleotide or modulator can be linked to any of a variety of compounds. For example, such an agent can be linked to a targeting moiety that facilitates delivery of the agent to a target site, such as a monoclonal or polyclonal antibody, protein or liposome. As used herein, a "targeting moiety" can be any substance (e.g., compound or cell) that, when linked to an agent that increases the transport of an agent to a target cell or tissue, Increases the local concentration of the drug. Targeting moieties include antibodies or fragments thereof, receptors, ligands as well as other molecules that bind to cells of the target tissue or cells that are proximal to the target tissue. The antibody targeting agent can be an intact (all) molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments include F (ab '
) ₂ , -Fab ', Fab and F [v] fragments, which can be produced by conventional methods or by genetic or protein engineering. The linkage is generally a covalent bond and may be achieved, for example, by direct condensation or other reactions, or by way of a bifunctional or multifunctional linker. The targeting moiety can be selected based on the cell or tissue where the agent is expected to exert a therapeutic benefit.

Pharmaceutical compositions can be administered in a manner appropriate to the disease to be treated (or prevented). The appropriate dosage and the appropriate duration and frequency of administration are determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of active ingredient and the mode of administration. In general, an appropriate dosage and treatment regimen will determine therapeutic and / or prophylactic benefits (eg, more complete or partial remission, or long-term disease-free and / or overall survival). The drug is provided in an amount sufficient to provide a clinical outcome (eg, to improve clinical outcome). For prophylactic use, the dose must be sufficient to prevent, delay, or reduce the severity of, the disease associated with cell proliferation.

[0110] Optimal dosages may generally be determined using experimental models and / or clinical trials. Generally, the amount of polypeptide present in a dose or the amount of polypeptide produced in situ by DNA present in a dose is from about 0.01 μg / kg host to about 100 μg / kg host, typically about 0
. It ranges from 1 μg to about 10 μg. The use of a minimal dosage sufficient to provide effective treatment is usually preferred. Patients can generally be monitored for therapeutic or prophylactic efficacy using appropriate assays for the condition to be treated or prevented, known to those of skill in the art. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for a 10-60 kg animal.
Range.

The following examples are presented for purposes of illustration, but not limitation.

EXAMPLES Example 1 Cloning and Sequencing of cDNA Encoding DSP-4 This example illustrates the cloning of a cDNA molecule encoding human DSP-4.

A conserved sequence motif around the active site domain of dual-specificity phosphatases was identified as follows: The bispecific phosphatases are a larger family of protein tyrosine phosphatases (PTPs). Belongs to. This family of phosphatases shares a conserved catalytic domain containing a cysteine residue located at the N-terminus of a five variable amino acid stretch followed by an arginine residue (Fauman et al., Trends In.
Bioch. Sci. 21: 413-417, 1996). DSPs typically contain a PTP active site motif, but lack sequence homology to PTP in other regions (Jia, Biochem. And Cell Biol.
75: 17-26, 1997). However, no consensus sequences conserved between DSPs have been reported, and no obvious consensus regions have been reported from testing of known DSP sequences as mentioned above. Multiple known human bispecific phosphatase sequences were aligned and compared to derive a longer consensus DSP amino acid sequence motif useful for the identification of new DSP family members. By alignment of the eight amino acid sequences from the eight human DSPs with MAP-kinase phosphatase activity, the two containing the PTP active site signature motif
A conserved region of homology consisting of a three amino acid peptide sequence was obtained. Therefore, using a candidate peptide having the following sequence: GRVLVHCQAGISRSGTNILAYLM (SEQ ID NO: 4), an expressed sequence tag (Expressed Sequence)
nce Tag) database (Nat. Center for Biol. I)
nformation. www. ncbi. nlm. nih. gov / dbES
T) was searched. This search used an algorithm (tblastn) that allows the reverse translation of the candidate peptide in an iteration that allows for the degeneracy of the genetic code in the default parameters. This search result is EST AI
031656 was identified as a candidate MAP-kinase phosphatase sequence.
This EST did not include the complete coding region of the expressed gene (eg, the gene encoding DSP-4 with MAP-kinase phosphatase activity). Neither the sense strand nor the open reading frame was identified.

To obtain the full-length coding sequence, use the 5 '/ 3' RACE kit (Boehringer Mannheim, Indianapolis, Indiana) according to the supplier's instructions.
IN) as described (Frohman et al., Proc. Nat. Aca).
d. Sci. USA 85: 8998, 1988; Ohara et al., Proc. N
at. Acad. Sci. USA 86: 5673, 1989; Loh et al., Sc.
issue 243: 217, 1989), 5 'and 3' RACE (rapi
The human thymus cDNA was screened in a d amplification of cDNA end (rapid amplification of cDNA ends) reaction. The sequence information immediately adjacent to the conserved sequence motifs is used for 5 'and 3'
Used in the RACE reaction. This includes the following primers (SEQ ID NOs: 5-11)
) Was used:

[0115]

Embedded image CDNA encoding a protein of 217 amino acids (FIG. 2A; SEQ ID NO: 2)
FIG. 1; SEQ ID NO: 1) was identified as DSP-4. This sequence had significant homology to other MAP-kinase phosphatases (FIG. 3). Identified cD
The NA contains a 651 base pair coding region, and related 5 'and 3' untranslated sequences. The active site domain for DSP-4 was localized to the region encoded by the nucleotide beginning at position 148 of SEQ ID NO: 1.

A semi-quantitative RT-PCR analysis was performed. These analyzes showed significantly higher levels of DSP-4 mRNA in thymic and skeletal muscle tissues.

Genomic PCR analysis (Research Genetics, Inc., Huntsv) to locate the DSP-4 coding sequence in human genomic DNA.
(ille, AL) was performed using standard methodology using the following DSP-4 specific oligonucleotide mapping primers:

[0118]

Embedded image The DSP-4 sequence was localized to human chromosome 2 in the region between D2S324 and D2S117 (for chromosome localization nomenclature, for example, Gyap
ay et al., 1994 Nature Genetics 7: 246-339).

Example 2 DSP-4 Expression in Human Tissues This example shows that DSP-4 encoding nucleic acid sequences hybridize to human poly A + RNA from various tissue sources. For use as a nucleic acid hybridization probe, full length DSP-4 encoding cDNA (SEQ ID NO: 1)
In Ausubel et al. (1998 Current Protocols in
Molecular Biology, Green Publ. Assoc
. Inc. & John Wiley & Sons, Inc. , Boston
Was ³² P-labeled by the random primer method according to MA). The probe was blotted containing human poly A + RNA from multiple human tissues, normalized for the amount of detectable β-actin mRNA (Figure 4, Catalog No. 7759-1;
Clontech, Inc. , Palo Alto, CA). Blots were prehybridized in Express Hyb ^™ solution (Clontech) for 30 minutes at 68 ° C, and then hybridized with the labeled probe in Express Hyb ^™ solution for 1 hour at 68 ° C. The blot is then washed in 2 × SSC, 0.5% SDS at room temperature for 40 minutes, then a second time in 0.1 × SSC, 0.1% SDS at 50 ° C. for 40 minutes. Was washed. The blots were air dried and then Hyperfilm MP ^™ autoradiographic film (Amersham Life Sciences, Ar.
(Lington Hts, IL). FIG. 4 shows the results. Here, human tissue sources of RNA were as follows: (FIG. 4A) lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6,
Lane 7, kidney; lane 8, pancreas; (FIG. 4B) lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4, testis; lane 5, ovary; lane 6, small intestine; Colon; lane 8, peripheral blood leukocytes.

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

[Brief description of the drawings]

FIG. 1 shows the cDNA sequence for DSP-4 (SEQ ID NO: 1), with start and stop codons shown in bold.

FIG. 2 shows the deduced amino acid sequence of DSP-4 (SEQ ID NO: 2).

FIG. 3 is a sequence alignment showing sequence similarity between DSP-4 and other MAP-kinase phosphatases.

FIG. 4 shows Northern blot hybridization using a ³² P-labeled full length DSP-4 encoding nucleic acid sequence as a probe. Blots contained human poly A + RNA from various tissue types, such as: (FIG. 4A) lane 1, heart; 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; Lane 7, kidney; lane 8, pancreas; (FIG. 4B) lane 1, spleen; lane 2, thymus; lane 3, prostate; lane 4, testis; lane 5, ovary; lane 6, small intestine; Colon; lane 8, peripheral blood leukocytes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 37/08 4B065 37/06 C07K 16/40 4C084 37/08 C12N 1/15 4H045 C07K 16 / 40 1/19 C12N 1/15 1/21 1/19 9/16 B 1/21 C12Q 1/02 5/10 1/42 9/16 1/68 A C12Q 1/02 G01N 33/15 Z 1 / 42 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 33/58 Z C12P 21/08 33/566 C12N 15/00 ZNAA 33/58 5/00 A / / C12P 21/08 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, 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, 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, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, Z F-term (reference) 2G045 BB20 CB01 CB26 DA12 DA13 DA14 DA20 DA36 FB02 FB03 FB04 FB07 4B024 AA01 AA11 BA11 BA44 CA04 CA09 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 FA06 GA03 GA11 HA01 Q03 DD03A03 Q03 DD03 QQ01 QQ33 QQ42 QQ52 QR08 QR13 QR33 QR42 QR55 QR57 QR59 QR62 QR74 QR80 QR82 QS05 QS15 QS25 QS33 QS34 QS36 QX02 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA93A02 A02A02 A02A02 A02A02 A02A02 A02A02 A02 A02 A ZB132 ZB262 ZC212 4H045 AA11 AA30 BA10 CA40 DA76 EA20 EA50 FA72 FA74

Claims (49)

[Claims] 1. An isolated polypeptide having the sequence of DSP-4 listed in SEQ ID NO: 2 or a variant thereof, wherein said variant deactivates activated MAP-kinase. An isolated polypeptide that differs by deletion, addition, insertion, or substitution of one or more amino acids of 50% or less of the residues in SEQ ID NO: 2 so as to retain the ability to phosphorylate. 2. An isolated polynucleotide encoding at least 10 contiguous amino acids of a polypeptide having the sequence corresponding to SEQ ID NO: 2. 3. An isolated polynucleotide encoding at least 15 contiguous amino acids of a polypeptide having the sequence corresponding to SEQ ID NO: 2. 4. An expression vector comprising the polynucleotide according to claim 2 or 3. 5. A host cell transformed or transfected with the expression vector according to claim 4. 6. An isolated polynucleotide encoding the polypeptide of claim 1. 7. The polynucleotide according to claim 6, comprising the sequence listed in SEQ ID NO: 1. 8. An expression vector comprising the polynucleotide according to claim 6. 9. A host cell transformed or transfected with the expression vector according to claim 8. 10. An antisense polynucleotide comprising at least 15 contiguous nucleotides complementary to the polynucleotide of claim 6. 11. The complement of the sequence listed in SEQ ID NO: 1 at 50 ° C.
An isolated polynucleotide that detectably hybridizes under conditions including a wash in 0.1 × SSC and 0.1% SDS for 5 minutes. 12. A polynucleotide comprising the polynucleotide according to claim 10 or 11,
Expression vector. 13. A host cell transformed or transfected with the expression vector according to claim 12. 14. The following steps: (a) culturing the host cell of claim 9 under conditions allowing expression of the DSP-4 polypeptide; and (b) culturing the host cell culture. A method of producing a DSP-4 polypeptide, comprising: isolating a DSP-4 polypeptide. 15. An isolated antibody or antigen-binding fragment thereof that specifically binds to a DSP-4 polypeptide having the sequence set forth in SEQ ID NO: 2. 16. The antibody or fragment thereof according to claim 15, wherein said antibody is a monoclonal antibody. 17. The combination according to claim 15, wherein the combination with a physiologically acceptable carrier.
Or a fragment thereof. 18. A method comprising: (a) contacting a sample with the antibody or antigen-binding fragment of claim 15 under conditions and for a time sufficient to form an antibody / DSP-4 complex;
And (b) detecting the level of the antibody / DSP-4 complex and detecting the presence of DSP-4 in the sample therefrom, for detecting DSP-4 expression in the sample. Method. 19. The method of claim 18, wherein said antibody is linked to a support material. 20. The antibody of claim 18, wherein said antibody is linked to a detectable marker.
The method described in. 21. The method of claim 18, wherein said sample is a biological sample obtained from a patient. 22. The following: (a) contacting a sample with the antisense polynucleotide according to claim 10 or 11; and (b) DSP- hybridizing to the antisense polynucleotide in the sample. Detecting the amount of the polynucleotide and detecting the DSP-4 expression in the sample therefrom. 4. A method for detecting DSP-4 expression in a sample. 23. The method of claim 22, wherein the amount of DSP-4 polynucleotide that hybridizes to said antisense polynucleotide is determined using a polymerase chain reaction. 24. The method of claim 22, wherein the amount of DSP-4 polynucleotide that hybridizes to said antisense polynucleotide is determined using a hybridization assay. 25. The method of claim 22, wherein said sample comprises an RNA or cDNA preparation. 26. The following steps: (a) contacting a candidate agent with the polypeptide of claim 1 under conditions and for a time sufficient to allow an interaction between the polypeptide and the candidate agent. And (b) subsequently, the ability of the polypeptide to dephosphorylate a DSP-4 substrate,
Assessing the polypeptide relative to a predetermined ability to dephosphorylate the DSP-4 substrate in the absence of the candidate agent; and identifying an agent that modulates DSP-4 activity therefrom; A method for screening for an agent that modulates DSP-4 activity, comprising: 27. The DSP-4 substrate is a MAP kinase.
The method described in. 28. The method of claim 26, wherein said candidate agent is a small molecule. 29. The method of claim 26, wherein said small molecule is in a combinatorial library. 30. The following steps: (a) a cell comprising a DSP-4 promoter operably linked to a polynucleotide encoding a detectable transcript or protein, comprising the steps of: Contacting under conditions and for a time sufficient to allow an interaction between; and (b) subsequently, determining the expression of said polynucleotide by a predetermined amount of expression in the absence of said candidate agent Evaluating relative to level; and then DSP-4
A method of screening for an agent that modulates DSP-4 activity, comprising the step of identifying an agent that modulates activity. 31. The method of claim 30, wherein said polynucleotide encodes a DSP-4 polypeptide. 32. The method of claim 30, wherein said polynucleotide encodes a reporter protein. 33. A method for modulating a proliferative response in a cell, comprising contacting the cell with an agent that modulates DSP-4 activity. 34. A method for regulating differentiation of a cell, comprising contacting the cell with an agent that modulates DSP-4 activity. 35. A method for regulating cell survival, comprising contacting a cell with an agent that modulates DSP-4 activity. 36. The agent of claim 33, wherein the agent modulates a pattern of gene expression.
36. The method according to any one of -35. 37. The cell of claim 33, wherein said cell exhibits contact inhibition of cell growth.
The method according to any one of claims 1 to 4. 38. The method of claim 33, wherein said cells exhibit anchorage-independent growth. 39. The cell of claim 33, wherein said cells exhibit altered cell-cell adhesion properties.
35. The method according to any one of 35. 40. The method of claim 35, wherein said agent modulates apoptosis. 41. The method of claim 35, wherein said agent modulates the cell cycle. 42. The method of claim 32, wherein said cells are present in a patient. 43. A method for treating a patient suffering from a disorder associated with DSP-4 activity, comprising administering to the patient a therapeutically effective amount of an agent that modulates DSP-4 activity. 44. The disorder is selected from the group consisting of Duchenne muscular dystrophy, cancer, graft-versus-host disease, autoimmune disease, allergy, metabolic disease, abnormal cell growth, abnormal cell proliferation, and abnormal cell cycle. 44. The method of claim 43, wherein the method is performed. 45. A DSP-4 substrate capture mutant polypeptide, wherein the polypeptide binds to the substrate with substantially less reduced affinity compared to DSP-4, and The sequence listed in SEQ ID NO: 2 and one or less than 50% of the residues in SEQ ID NO: 2, such that the polypeptide has a reduced ability to dephosphorylate the substrate as compared to DSP-4. A DSP-4 substrate capture mutant polypeptide that differs by the deletion, addition, insertion or substitution of the above amino acids. 46. The polypeptide according to claim 46, wherein the polypeptide is at position 118 or 150 in SEQ ID NO: 2.
46. The substrate capture mutant polypeptide of claim 45, comprising a substitution at the position. 47. The following steps: (a) contacting a candidate molecule with the polypeptide of claim 1 under conditions and for a time sufficient to cause the candidate molecule to interact with the polypeptide; and (b) ) Detecting the presence or absence of binding of said candidate molecule to said polypeptide and determining therefrom whether said candidate molecule interacts with DSP-4. A method for screening molecules for their ability to interact. 48. The method of claim 47, wherein said detecting comprises an affinity purification step. 49. The method of claim 47, wherein said detecting comprises yeast two hybrid screening or phage display library screening.