JP2003510036A - Steroid hormone receptor polynucleotides, polypeptides, and antibodies - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel human steroid hormone receptor polypeptides and virally isolated nucleic acids comprising the coding region of the gene encoding such a polypeptide. The invention also provides vectors, host cells, antibodies, and recombinant methods for producing a human steroid hormone receptor polypeptide. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to the novel human steroid hormone receptor polypeptides.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to novel steroid hormone receptor proteins. More particularly, isolated nucleic acid molecules encoding novel steroid hormone receptor polypeptides are provided. Novel steroid hormone receptor polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human steroid hormone receptor polynucleotides and / or polypeptides.
The invention further relates to diagnostic and therapeutic methods useful in diagnosing, treating, preventing and / or predicting disorders involving these novel steroid hormone receptor polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the invention. The invention further relates to methods and / or compositions that inhibit the production and function of the polypeptides of the invention.

BACKGROUND OF THE INVENTION Physiological and therapeutic activities of steroid hormones are associated with the Steroid Hormone Receptor (SHR).
) Is mediated by a family of proteins. This protein is a member of the nuclear receptor family of proteins. These nuclear receptors include receptors for steroids, thyroid hormones, inducible hormones of vitamins A and D, and certain fatty acids (Kumar, R., and EB Thompson.
, Steroid, 64: 310-19 (1999)).

Members of this nuclear receptor family of proteins share some common structural features. All members of this family of proteins contain a variable N-terminal domain thought to be involved in transactivation of transcription. Moreover, all members of this family of proteins contain a well-conserved DNA-binding domain (which is believed to be important in DNA-binding and protein-protein interactions) and a C-terminal ligand-binding domain of the protein (hormone binding,
It is considered to be involved in protein-protein interaction and transactivation activity)
(Kumar, R., (1999)).

Steroid hormone receptors constitute a family of inducible transcription factors that translate the effects of small molecule lipophilic hormones into transcriptional responses. Activation of these receptors by binding of cognate ligands causes them to bind with high affinity to a specific response element (known as hormone responsive element t (HRE)) in the promoter region of the target gene. Induced, which induces transcription of specific genes (Klein-Hitpass, L. et al., J. Chem.
Mol Med, 76: 490-6 (1998)).

In addition to promoting transcription of specific genes, steroid hormone receptor proteins also function as transcriptional repressors. For example, increased expression of AP-1 subunit, or AP due to growth factors or phorbol esters
Activation of the -1 subunit results in inhibition of the hormone responsive element (HRE) promoter and interference with DNA binding (Beato, M et al., Cell, 8).
3: 851-7 (1995)).

Thus, there is a clear need to identify and utilize new members of the nuclear hormone receptor family of proteins. Although structurally related, such proteins may possess diverse and pleiotropic functions in various cell and tissue types. Receptor-type molecules should prove useful in target-based screening for small molecules and other such physiologically valuable agents. The purified steroid hormone receptor proteins of the present invention are useful for the identification, characterization, and purification of additional interacting proteins or receptor proteins, or other signaling pathway proteins that interact with steroid hormone receptor proteins. It is a search tool. In addition, assays designed to monitor the expression of steroid hormone receptor proteins can be useful as diagnostic tools for monitoring the presence or progression of certain types of cancer (eg breast cancer).

(Summary of the Invention) The present invention provides a polynucleotide sequence disclosed in the sequence listing and / or a polynucleotide sequence contained in the human cDNA plasmid described in Table 1, and Ameri.
It includes isolated nucleic acid molecules that comprise or consist of a polynucleotide sequence deposited at the can Type Culture Collection (ATCC). Fragments, variants and derivatives of these nucleic acid molecules are also encompassed by the present invention. The invention also includes an isolated nucleic acid molecule that comprises or is composed of a polynucleotide that encodes a steroid hormone receptor polypeptide. The invention further includes steroid hormone receptor polypeptides encoded by these polynucleotides. A steroid hormone receptor polypeptide disclosed in the Sequence Listing and / or a steroid hormone receptor polypeptide encoded by the human cDNA plasmid described in Table 1 and a steroid hormone receptor polypeptide deposited with the ATCC, or consisting thereof. Further provided are amino acid sequences that are: Antibodies that bind to these polypeptides are also encompassed by the present invention. Polypeptide fragments, variants and derivatives of these amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding these polypeptides and antibodies that bind to these polypeptides.

Detailed Description Table 1 Table 1 lists ATCC deposits, deposit dates, deposited with the ATCC in connection with the present application.
And ATCC designation numbers are summarized. Table 1 further summarizes the associated information for each "Gene No" listed below. This information includes the cdn
A clone identifier (ID), type of vector contained in cDNA clone identifier (ID), nucleotide sequence identifier number, nucleotide contained in disclosed sequence, position of 5'nucleotide of start codon of disclosed sequence, amino acid sequence It contains the identifier number and the last amino acid of the ORF encoded by the disclosed sequence.

Table 2 shows the official EST. At least 1, 2, 3, 4, 5, 10 or more of any one or more of these public EST sequences are optionally excluded from particular embodiments of the invention.

Table 3 summarizes the expression profile of the polynucleotides corresponding to the clones disclosed in Table 1. The first column is the cd for each contig sequence disclosed in Table 1.
A specific clone identifier "Clone number Z" is provided for the NA clone. The "library code" in the second column shows the expression profile of the library of tissues and / or cell lines expressing the polynucleotide of the present invention. The code for each library in the second column indicates the library code provided in Table 4 and the tissue / cell source identifier code corresponding to the library description. Expression of these polynucleotides was not observed in other tissue and / or cell libraries tested. Those of skill in the art will routinely use this information to identify tissues that exhibit a predominant expression pattern of the corresponding polynucleotides of the invention, or to identify polynucleotides that exhibit predominant and / or specific tissue expression. Can be identified.

The first column of Table 4 provides the library code disclosed in the second column of Table 3.
The second column provides details of the source of tissue or cells from which the corresponding library was derived.

Definitions The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, “isolated” refers to a substance taken from its original environment (eg, the natural environment if it is naturally occurring), and thus “from the natural state”. It has been modified by human hands. " For example, an isolated polynucleotide can be part of a vector or composition, or can be contained within a cell and still be "isolated." This is because the vector, composition, or particular cell is not the natural environment for the polynucleotide. The term "isolated" includes genomic or cDNA libraries, whole cell or mRNA preparations, genomic DNA preparations, including those separated by electrophoresis and transferred on blotting. A shared whole cell genomic DNA preparation,
Or do not refer to other compositions, which are indistinguishable to those skilled in the art from the features of the polynucleotides / sequences of the invention.

As used herein, “polynucleotide” refers to a molecule having the nucleic acid sequence contained in SEQ ID NO: X (described in column 5 of Table 1) or cDNA plasmid: Z (3rd of Table 1). Nucleic acid sequences listed in a column and contained within a pool of plasmids deposited with the ATCC). For example, a polynucleotide is a 5'and 3'untranslated sequence, a coding region with or without a natural or artificial signal sequence, a nucleotide sequence of a full-length cDNA sequence containing a protein coding region,
And fragments, epitopes, domains, and variants of this nucleic acid sequence. Further, as used herein, "polypeptide", as broadly defined, refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention (a poly A tail of a sequence corresponding to a cDNA. The poly-phenylalanine or polylysine peptide sequences resulting from the translation of E.

In the present invention, a representative plasmid containing the sequence of SEQ ID NO: X has been deposited with the American Type Culture Collection (“ATCC”) and / or listed in Table 1. As shown in Table 1, each plasmid is identified by its cDNA clone ID (identifier) and ATCC deposit number (ATCC deposit number: Z). Plasmids pooled and deposited as a single deposit have the same AT
Has a CC deposit number. ATCC is Virginia 20110-
2209, Boulevard University 10801 Manassas. ATC
The C deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure.

The “polynucleotide” of the present invention also includes, under stringent hybridization conditions, the sequence contained in SEQ ID NO: X, its complement (eg, any one of the polynucleotide fragments described herein, 2, 3, 4 or more complements) and / or sequences contained in the cDNA plasmid Z (eg any 1, 2, 3, 4 or more of the polynucleotide fragments described herein). Complementary) and those polynucleotides capable of hybridizing. "Stringent hybridization conditions" means 50% formamide, 5
SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 42 ° C. in a solution containing 20 μg / ml denatured sheared salmon sperm DNA.
Incubation overnight at 0 ° C., followed by washing the filters in 0.1 × SSC at about 65 ° C.

Nucleic acid molecules that hybridize to the polynucleotides of the invention under lower stringency hybridization conditions are also "polynucleotides" of the invention.
Is intended to be. Changes in hybridization stringency and signal detection are primarily achieved by manipulation of formamide concentrations (lower percentages of formamide produce reduced stringency); salt conditions, or temperature. For example, lower stringency conditions are 6 × SS
PE (20 × SSPE = 3M NaCl; 0.2M NaH ₂ PO ₄ ; 0.02M
EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100μ
Includes overnight incubation at 37 ° C. in a solution containing g / ml salmon sperm blocking DNA; followed by a wash with 1 × SSPE, 0.1% SDS at 50 ° C. Furthermore, in order to achieve even lower stringency, the washes performed after stringent hybridization can be performed at higher salt concentrations (eg 5xSSC).

Note that the changes in the above conditions can be achieved by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, and denatured salmon sperm DN.
A, and commercial proprietary formulations. Inclusion of specific blocking reagents may require modification of the above hybridization conditions due to compatibility issues.

Of course, the poly A + sequence (eg, any 3'of the cDNA shown in the sequence listing
A polynucleotide that hybridizes only to a terminal poly A + region (tract) or to a complementary stretch of T (or U) residues is such a polynucleotide is a poly (A) stretch or its complement (eg, Not hybridized to the definition of "polynucleotide" as it hybridizes to virtually any double stranded cDNA molecule (including virtually any double stranded cDNA clone) produced using the primer Tocitooligo dT.

The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DN.
It may be A or modified RNA or modified DNA. For example, a polynucleotide is a DNA that is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded regions.
Single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, single-stranded, or more typically double-stranded or a mixture of single-stranded and double-stranded regions. It can be composed of hybrid molecules, including possible DNA and RNA. Further, the polynucleotide may be composed of triple-stranded regions comprising RNA or DNA, or both RNA and DNA. Polynucleotides can also include one or more modified bases or DNA or RNA backbones that have been modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

In certain embodiments, the polynucleotide of the invention is at least 15, at least 30, at least 50, at least 100, at least 125, at least 500 or at least 1000 contiguous nucleotides, but 300 kb in length, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.
It is 5 kb, 5 kb, 2.5 kb, 2.0 kb or 1 kb or less. In a further embodiment, the polynucleotide of the invention, as disclosed herein, comprises part of the coding sequence but not all or part of any intron. In another embodiment, the polynucleotide comprising the coding sequence does not include the coding sequence of a genomic flanking gene (ie, 5'or 3'to the gene of interest in the genome). In another embodiment, the polynucleotide of the invention is 10
00, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
Does not contain more than 2 or 1 genomic flanking gene coding sequences.

“SEQ ID NO: X (SEQ ID NO: X)” refers to the polynucleotide sequence shown in the fifth column of Table 1, and “SEQ ID NO: Y (SEQ ID NO: Y)” means
Refers to the polypeptide sequences listed in column 10 of Table 1. SEQ ID NO: X is identified by the integer specified in the sixth column of Table 1. The polypeptide sequence of SEQ ID NO: Y comprises an open reading frame (encoded by the polynucleotide of SEQ ID NO: X
ORF) translation product. The polynucleotide sequence is shown in the sequence listing immediately followed by all polypeptide sequences. Therefore, the polypeptide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 2 is the first polypeptide sequence shown in the sequence listing. The second polypeptide sequence corresponds to the polynucleotide sequence shown in SEQ ID NO: 3 and the like.

The polypeptides of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. May be included. The polypeptide may be modified either by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well documented in basic textbooks and more detailed research articles, as well as in many research literature. Modifications include peptide backbones, amino acid side chains,
And can occur anywhere in the polypeptide, including the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides can be branched, for example, as a result of ubiquitination, and polypeptides can be cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond. , Cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation,
Such as GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation Transfer RNA-mediated addition of amino acids to various proteins, and ubiquitination. (For example, PROTEINS-STRUCTURE AND M
OLECULAR PROPERIES, 2nd Edition, T.S. E. Creig
hton, W.W. H. Freeman and Company, New
York (1993); POST TRANSLATIONAL COVALE
NT MODIFICATION OF PROTEINS, B.N. C. Jo
Hnson, Academic Press, New York, 1-
12 (1983); Seifter et al., Meth Enzymol 18
2: 626-646 (1990); Rattan et al., Ann NY Aca.
d Sci 663: 48-62 (1992)).

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides may include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or produced by a combination of these methods. Polypeptide is mentioned. Means for preparing such polypeptides are well understood in the art.

The polypeptide may be in the form of a secreted protein (including the mature form) or may be part of a larger protein (eg a fusion protein) (
See below). It is often the case to include additional amino acid sequences, including secretory or leader sequences, prosequences, sequences that aid in purification (eg, multiple histidine residues), or additional sequences for stability during recombinant production. It is advantageous.

The polypeptides of the present invention are preferably provided in isolated form and are preferably substantially purified. Recombinantly produced versions of polypeptides (including secreted polypeptides) are described herein or otherwise known in the art, eg, Smith and John.
Son, Gene 67: 31-40 (1988). The polypeptides of the invention may also be described by methods described herein or known in the art (eg, methods known in the art, inventions raised against the polypeptides of the invention). Antibodies) can be used to purify from natural or recombinant sources.

A polypeptide exhibiting “functional activity” is the full-length (complete) protein of the present invention.
By a polypeptide is meant a polypeptide capable of exhibiting one or more known functional activities associated with a polypeptide. Such functional activities include biological activity, antigenicity [ability to bind (or compete with the polypeptide of the invention for binding) to antibodies to the polypeptide of the invention], immunogenicity ( Ability to generate antibodies that bind to the polypeptides of the invention), the ability to form multimers with the polypeptides of the invention, and the ability to bind receptors or ligands for the polypeptides of the invention, including but not limited to: Not done.

A “polypeptide having a functional activity” is a polypeptide that exhibits an activity similar to that of the polypeptide of the present invention, but not necessarily identical to that activity (with or without dose dependence, Refers to the mature form, as measured in a particular assay (eg, biological assay). If a dose dependence is present, the dose dependence need not be identical to that of the polypeptide, but rather is substantially similar to the dose dependence in a given activity as compared to a polypeptide of the invention. Certain (ie, the candidate polypeptide exhibits higher activity as compared to the polypeptide of the invention, ie, about 1/25 or more, preferably 1/10 or more, and most preferably about 1/3 or more. .

The functional activity of polypeptides, and fragments, variants, derivatives and analogs thereof, can be assayed by a variety of methods.

For example, in one embodiment of assaying for the ability of an antibody to bind to a full-length polypeptide antibody for the ability to bind or compete with the full-length polypeptide of the invention, various immunoassay systems known in the art can be used, Assay systems include, for example, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg, colloidal gold, Using enzyme labels or radioisotope labels), Western blots, precipitation reactions, agglutination assays (eg gel agglutination assays, hemagglutination assays),
Includes, but is not limited to, competitive and non-competitive assay systems using techniques such as complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays and the like. In one embodiment, antibody binding is detected by detecting the label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of the secondary antibody or reagents to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In another embodiment, when a ligand is identified or the ability to multimerize a polypeptide fragment, variant, or derivative of the invention is assessed, binding is well known in the art, for example. It can be assayed by means such as reducing or non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. In general, Phiz
Icky, E. Et al., Microbiol. Rev. 59: 94-123 (199
See 5). In another embodiment, a physiologically correlated polypeptide of the invention that binds to its substrate (signal transduction) can be assayed.

Furthermore, the assays described herein (see Examples) and otherwise known in the art are routinely referred to as polypeptides of the invention and fragments, variants thereof, It can be applied to determine the ability of derivatives and analogs to induce polypeptide-related biological activity (either in vitro or in vivo). Other methods are known to those of skill in the art and are within the scope of the invention.

Polynucleotides and Polypeptides of the Invention (Features of Protein Encoded by Gene No. 1) In certain embodiments, the polypeptides of the invention include the following amino acid sequences, or alternatively: Consists of amino acid sequences:

[0034]

[Chemical 1] . Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the invention,
Antibodies that bind to these polypeptides are also included.

A preferred polypeptide of the invention is SEQ ID NO: 7 with residues: Thr-1 to S.
It comprises or consists of an immunogenic epitope designated as er-6. Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention, as well as antibodies that bind to these polypeptides.

This gene is found in many fetal tissues (eg, fetal lung tissue and fetal heart tissue),
And in placental tissue.

Accordingly, the polynucleotides and polypeptides of the present invention, including antibodies, are useful for differential identification of tissues or cell types present in a biological sample, as well as placenta and developing fetal disease and / or diseases. Or, it is useful as a reagent for diagnosis of diseases and conditions including, but not limited to, disorders. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher or lower than standard gene expression levels (ie expression levels in normal tissues or body fluids from individuals without this disorder) for many disorders of the tissues or cells, especially the fetus or placenta Levels of expression of this gene are determined by the specific tissue or cell type (eg, fetal tissue, placental tissue, cancerous tissue and wound tissue) or body fluid (eg, lymph, serum, etc.) taken from an individual having such a disorder. Plasma, urine, synovial fluid and spinal fluid) or another tissue or sample.

Tissue distribution in fetal and placental tissues indicates that polynucleotides, translation products and antibodies corresponding to this gene are useful in diagnosing, detecting and / or treating developing fetal and placental diseases and / or disorders. Indicates that there is.

This gene shares sequence homology to other steroid hormone receptors. Therefore, it is believed that the polynucleotides and translation products corresponding to this gene may share some biological activity of other steroid hormone receptors. Thus, the translation product corresponding to this gene may be involved in signal transduction of the specifically bound hormone, resulting in transcriptional activation of the target gene. Therefore, the translation product corresponding to this gene may be expressed in other specific tissues or organs,
In this tissue or organ, this translation product may play a functional role in connection with other processes such as hematopoiesis, inflammation, osteogenesis, and renal function, to name a few. In view of homology, translation products corresponding to this gene may also be involved in identifying regulatory factors for the control of reproductive and / or social behavior in order to increase fertility or to regulate hormone secretion. It is useful.

This tissue distribution suggests that polynucleotides, translation products and antibodies corresponding to this gene are useful in diagnosing and / or treating placental disorders. Specific expression in the placenta suggests that this gene product may play a role in the proper establishment and maintenance of placental function. Alternatively, the gene product can be produced in the placenta and then transported to the embryo, where it can play an important role in the development and / or survival of the developing embryo or fetus. Expression of this gene product in vascular-rich tissues such as placenta also suggests that this gene product may be more commonly produced in endothelial cells or circulation. In such cases, the gene product may play a more ubiquitous role in vascular function (eg, angiogenesis). This gene product can also be produced in the vasculature and have an effect on other cells in the circulation, such as hematopoietic cells. This gene product may serve to promote the proliferation, survival, activation, and / or differentiation of hematopoietic cells and other cells throughout the body. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 2) A translation product corresponding to this gene is a pheromone receptor isolated from Rattus norwegicus (International Publication No. WO97), which is believed to be involved in binding to pheromones. / 14790) and share sequence homology. Based on this homology, these proteins are believed to share at least some biological activity.

In a particular embodiment, the polypeptide of the invention comprises or consists of an amino acid sequence selected from the group consisting of:

[0043]

[Chemical 2] . Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the invention,
Antibodies that bind to these polypeptides are also included.

A preferred polypeptide of the present invention has the residues: His-1 to L in SEQ ID NO: 8.
It comprises, or consists of, one, two, or both immunogenic epitopes, designated as eu-11 and Ser-46 to Arg-51. Fragments and / or variants of these polypeptides (eg, such as fragments and / or variants as described herein)
Are encompassed by the present invention. These polypeptides (fragments and / or
Polynucleotides (including variants) are also encompassed by the present invention, as well as antibodies that bind to these polypeptides.

[0045]   This gene is expressed in hippocampal and testicular tumor tissues.

Accordingly, the polynucleotides and polypeptides (including antibodies) of the present invention are for differential identification of tissues or cell types present in a biological sample, as well as for neurological diseases and / or disorders, and / or Alternatively, they are useful as reagents for the diagnosis of diseases and conditions including, but not limited to, reproductive diseases and / or disorders. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie expression levels in normal tissues or fluids from individuals without this disorder) for many disorders of the tissues or cells, particularly the nervous and reproductive systems, or Lower levels of expression of this gene may be associated with specific tissues or cell types (eg, neural, reproductive, cancerous and wound tissues) or body fluids collected from individuals with such disorders (e.g.
For example, lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or sample.

Tissue distribution in hippocampal tissue and testicular tumor tissue, and homology to the pheromone receptor indicates that polynucleotides, translation products and antibodies corresponding to this gene are diagnostic of diseases and / or disorders of the nervous and / or immune system. ,detection,
And / or be useful in treatment.

The tissue distribution in hippocampal tissues is such that polynucleotides, translation products and antibodies corresponding to this gene are associated with neurodegenerative disease states and behavioral disorders (eg Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, schizophrenia, mania,
For the detection and / or treatment of dementia, paranoia, obsessive-compulsive disorder, fear disorder, learning disability, ALS, psychosis, autism, and behavioral changes (including impairment of nutritional supplementation, sleep patterns, balance, and perception) Suggest useful. In addition, the gene or gene product may also play a role in the treatment and / or detection of developmental or sexual disorders associated with the developing embryo. This protein may play a role in hormonal signal transduction, thus resulting in the activation of certain genes that may play roles associated with normal neural activation or development.

Alternatively, the tissue distribution in testicular tissue is such that the protein product of this clone is
It is shown to be useful in the treatment and diagnosis of conditions and cancers related to proper testicular function (eg endocrine function, sperm maturation). Therefore, this gene product is useful in the treatment of male infertility and / or impotence. This gene product is also useful in assays designed to identify binding agents. This is because such a drug (antagonist) is useful as a male contraceptive drug. Similarly, this protein is considered to be useful in the treatment and / or diagnosis of testicular cancer. The testis is also the site of active gene expression of transcripts that can be expressed at particularly low levels in other tissues of the body. Thus, again, this protein may be involved in signal transduction of hormones that specifically bind to it, resulting in transcriptional activation of the target gene. Thus, the gene product may be expressed in other specific tissues or organs where it is expressed by other processes (hematopoiesis, inflammation, bone formation, to name a few possible target indications). And renal function, etc.).

In view of homology, the translation products of this gene also identify regulatory factors for the control of reproductive and / or social behavior, either to increase fertility or to regulate hormone secretion. Is useful in. In addition, the translation products of this gene may be useful for recreational applications such as cosmetics or aphrodisiacs. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 3) A preferred polypeptide of the present invention has the residues: Asp-65 to 5 in SEQ ID NO: 9.
Gln-72, Arg-78 to Thr-92, and Lys-106 to Lys-.
It comprises, or consists of, one, two, three, or all three immunogenic epitopes, designated as 112. Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention, as well as antibodies that bind to these polypeptides.

This gene is present throughout pregnant uterine and fetal tissues, as well as in senescent fibroblasts and immune system tissues such as activated T cells, T cell lymphomas, and primary dendritic cells. Expressed.

Accordingly, the polynucleotides and polypeptides (including antibodies) of the invention are used for the differential identification of tissues or cell types present in a biological sample, as well as for diseases of the fetal and musculoskeletal system and / or It is useful as a reagent for the diagnosis of diseases and conditions, including but not limited to disorders. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. For many disorders of the tissues or cells, particularly the fetus, immune system, and musculoskeletal system, relative to standard gene expression levels (ie, expression levels in healthy tissues or fluids from individuals without this disorder). Significantly higher or lower levels of expression of this gene may result in specific tissue or cell types (eg musculoskeletal tissue, fetal tissue, immune tissue, cancerous tissue and wound tissue) taken from an individual having such a disorder. ) Or bodily fluids such as lymph, serum, plasma, urine, synovial fluid and spinal fluid or another tissue or sample.

Tissue distribution throughout pregnant uterus and fetal tissues, as well as in senescent fibroblasts and immune system tissues, includes polynucleotides corresponding to this gene, translation products,
And antibodies are useful in the diagnosis, detection, and / or treatment of developing fetal, immune system, and musculoskeletal diseases and / or disorders.

Expression in embryonic tissues and other cell sources characterized by proliferating cells suggests that the translation product corresponding to this gene may play a role in the regulation of cell division, and in cancer and other Can be useful in the diagnosis and treatment of proliferative disorders. Similarly, embryonic development also involves cell differentiation and / or patterning.
Or include a determination regarding apoptosis. Thus, the polynucleotides and translation products corresponding to this gene may also be involved in apoptosis or tissue differentiation and, in turn, may be useful in cancer therapy.

Alternatively, the tissue distribution in senescent fibroblasts can be determined by polynucleotides, translation products, and antibodies corresponding to this gene in which musculoskeletal diseases and / or disorders (eg, heart disease, restenosis, atherosclerotic arteries). Sclerosis, stroke, angina, thrombosis and wound healing), and disorders affecting connective tissue (eg, arthritis,
Diagnosis, detection and / or treatment of trauma, tendinitis, chondromalacia and inflammation, such as various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma and dermatomyositis, and dwarfs. , Spinal deformities, and certain joint abnormalities, as well as chondrodysplasia (ie, congenital vertebral epiphyseal dysplasia, familial arthritis, type II osteogenesis (Atelosteogenesis type)
II), Schmidt type metaphyseal cartilage dysplasia))).

Tissue distribution in immune system tissues (eg, T cells and primary dendritic cells) also indicates that the polynucleotides, translation products, and antibodies corresponding to this gene are used to diagnose diseases and / or disorders of the immune system, Shown to be useful for detection and / or treatment. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may have utility in treating cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, the gene or protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above. Therefore, it is also used as a drug for immunological disorders, including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. Can be done. Furthermore, this gene product may have commercial utility in expanding stem cells and committed progenitors of various blood lineages, and in differentiating and / or expanding different cell types.

This gene shares sequence homology to other steroid hormone receptors. Therefore, it is believed that the polynucleotides and translation products corresponding to this gene may share some biological activity of other steroid hormone receptors. Thus, the translation product corresponding to this gene may be involved in signal transduction of the specifically bound hormone, resulting in transcriptional activation of the target gene. Therefore, the translation product corresponding to this gene may be expressed in other specific tissues or organs,
In this tissue or organ, this translation product may play a functional role in connection with other processes such as hematopoiesis, inflammation, osteogenesis, and renal function, to name a few. In view of homology, translation products corresponding to this gene may also be involved in identifying regulatory factors for the control of reproductive and / or social behavior in order to increase fertility or to regulate hormone secretion. It is useful. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

FEATURES OF PROTEIN ENCODED BY GENE NO: 4 In certain embodiments, a polypeptide of the invention comprises or consists of the following amino acid sequence: HEPPSWKTKVTVFSFL.
KVLLRSVNDSQRNSTWVYLKLVFIHLC (SEQ ID NO: 15).
Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention, as well as antibodies that bind to these polypeptides.

A preferred polypeptide of the invention is the residue in SEQ ID NO: 10: Lys-43.
~ Comprises or consists of an immunogenic epitope designated as Arg-48. Fragments and / or variants of these polypeptides (such as, for example, fragments and / or variants as described herein) are encompassed by the present invention. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention, as well as antibodies that bind to these polypeptides.

This tissue distribution suggests that the protein product of this clone is useful in the diagnosis and / or treatment of placental disorders. Specific expression in the placenta suggests that this gene product may play a role in the proper establishment and maintenance of placental function. Alternatively, the gene product can be produced in the placenta and then transported to the embryo, where it can play an important role in the development and / or survival of the developing embryo or fetus. Expression of this gene product in vascular-rich tissues such as placenta suggests that this gene product may be produced more commonly in endothelial cells or circulation. In such cases, the gene product is
For example, it may play a more universal role in angiogenesis). This gene product can also be produced in the vasculature and have an effect on other cells in the circulation, such as hematopoietic cells. This gene product may serve to promote the proliferation, survival, activation, and / or differentiation of hematopoietic cells and other cells throughout the body. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 5) In a specific embodiment, the polypeptide of the present invention has the following amino acid sequence: A
Contains or consists of RAKIENENQNLYFRKYPEEEIFRFIPNSK (SEQ ID NO: 16). Fragments and variants of these polypeptides (fragments and / or variants described herein) are encompassed by this specification. Polynucleotides encoding these polypeptides (fragments and / or variants) are also included in the present invention and are antibodies that bind these polypeptides.

[0063]   This gene is mainly expressed in neutrophils.

Accordingly, the polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample and include, but are not limited to, immune and hematopoietic diseases and / or disorders. It is useful as a reagent for diagnosis of diseases and conditions without limitation. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie, expression levels in normal tissues or fluids from individuals without this disorder) for many disorders of the tissues or cells, particularly the immune system, or Low levels of expression of this gene result in specific tissues or cell types taken from individuals with such disorders, such as immune, hematopoietic, and cancerous and wound tissues.
Alternatively, it is routinely detected in body fluids (eg, lymph, serum, plasma, urine, synovial fluid, and spinal fluid) or another tissue or cell sample.

This gene is expressed in 9-week old human tissue, fetal brain tissue, and placental tissue.

Accordingly, the polynucleotides and polypeptides of the present invention, including antibodies, are useful for the differential identification of tissues or cell types present in a biological sample, as well as developmental diseases and / or disorders and placenta. It is useful as a reagent for diagnosis of diseases and conditions including, but not limited to, diseases and / or placental disorders. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher or lower than standard gene expression levels (ie expression levels in normal tissues or fluids from individuals without this disorder) for many disorders of the tissues or cells, especially the fetus and placenta Levels of this gene are expressed in specific tissues or cell types collected from individuals with such disorders (eg,
Fetal tissue, developmental tissue, placental tissue, cancerous tissue and wound tissue) or body fluid (eg lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or sample, routinely detected obtain.

Tissue distribution in 9-week old tissue, fetal brain tissue and placental tissue indicates that the polynucleotides, translation products and antibodies corresponding to this gene diagnose fetal, placental and developmental diseases and / or disorders, Shown to be useful for detection and / or treatment.

This gene shares sequence homology to other steroid hormone receptors. Therefore, it is believed that the polynucleotides and translation products corresponding to this gene may share some biological activity of other steroid hormone receptors. Thus, the translation product corresponding to this gene may be involved in signal transduction of the specifically bound hormone, resulting in transcriptional activation of the target gene. Therefore, the translation product corresponding to this gene may be expressed in other specific tissues or organs,
In this tissue or organ, this translation product may play a functional role in connection with other processes such as hematopoiesis, inflammation, osteogenesis, and renal function, to name a few. In view of homology, translation products corresponding to this gene may also be involved in identifying regulatory factors for the control of reproductive and / or social behavior in order to increase fertility or to regulate hormone secretion. It is useful.

The tissue distribution in neutrophils indicates that the polynucleotides, gene products and antibodies corresponding to this gene are useful in the diagnosis, detection and / or treatment of diseases and / or disorders of the immune system.

This gene shares sequence identity with other steroid hormone receptors, and thus the polynucleotides and translation products corresponding to this gene share some of the biological activity of other steroid hormone receptors. It is thought to get. Therefore, the translation product corresponding to this gene may include the step of transforming the signal of a hormone that specifically binds to obtain the transcription activity of the target gene. Therefore, the translation product corresponding to this gene may be expressed in other specific tissues or organs,
Here, it may play relevant functional roles in other processes such as hematopoiesis, inflammation, bone formation, and renal function. When homologous, the translation products corresponding to this gene identify modulators for the regulation of replication and / or social behavior,
It is useful in enhancing fertility or controlling hormone secretion.

The involvement of this gene product in the regulation of cytokine production, antigen presentation, or other processes may also suggest utility in treating cancer (eg, by boosting the immune response). Expression of this gene product in neutrophils also underscores a role for this protein in immune function and immune surveillance. This gene product, whose involvement in the regulation of cytokine production, antigen presentation, or other processes, has utility for the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of immunogenic origin, the gene or protein and antibodies directed against this protein may show utility as tumor markers and / or immunotherapeutic targets for the above tissues. Therefore, it is also useful as arthritis, asthma, immunodeficiency diseases (eg AIDS), leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, psoriasis. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or as targets for immunotherapy.

[0072]

[Table 1] Table 1 summarizes the information corresponding to each "gene number" above. The nucleotide sequence identified as "nucleotide sequence number X" is identified in Table 1 as "
cDNA clone ID "and, in some cases, additional partially related DNA clones from partially homologous (" overlapping ") sequences. The overlapping sequences were assembled into a highly redundant single contiguous sequence (usually 3-5 overlapping sequences at each nucleotide position) to give the final sequence identified as SEQ ID NO: X.

The cDNA Clone ID was deposited on that date, and the "ATCC Deposit No. Z
And the corresponding deposit numbers listed under "Dates". Some of the deposits contain multiple different clones corresponding to the same gene. "Vector" is
It refers to the type of vector contained in the cDNA clone ID.

“Total NT sequence” refers to the total number of nucleotides in the contig identified by the “gene number”. The deposited plasmid may contain all of these sequences, which are reflected by the nucleotide positions shown as "5'NT of clone sequence" and "3'NT of clone sequence" of SEQ ID NO: X. .. The NT position of the putative methionine start codon, SEQ ID NO: X, if present, is identified as the "5'NT of the start codon". Similarly, the nucleotide position of SEQ ID NO: X of the putative signal sequence (if present) is identified as "5'NT of the first amino acid of the signal peptide".

The translated amino acid sequence begins at the first translation codon of the polynucleotide sequence and is identified as "amino acid SEQ ID NO: Y", although other reading frames were also obtained using known molecular biology techniques. Can be easily translated. Polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

SEQ ID NO: X, where X can be any of the polynucleotide sequences disclosed in the Sequence Listing, and translated SEQ ID NO: Y, where Y is the polynucleotide disclosed in the Sequence Listing. (Which can be any of the peptide sequences) is sufficiently accurate and otherwise suitable for various uses well known in the art and further described below. For example, SEQ ID NO: X has uses including, but not limited to, designing a nucleic acid hybridization probe that detects the nucleic acid sequence contained in SEQ ID NO: X or the cDNA contained in the deposited plasmid. These probes also hybridize to nucleic acid molecules in biological samples, thereby enabling the various forensic and diagnostic methods of the invention. Similarly, the polypeptide identified from SEQ ID NO: Y includes uses including but not limited to producing antibodies that specifically bind to secreted proteins encoded by the cDNA clones identified in Table 1. Have.

Nevertheless, the DNA sequence produced by the sequencing reaction may contain sequencing errors. Errors are present as misidentified nucleotides or as insertions or deletions of nucleotides in the resulting DNA sequence. Incorrectly inserted or deleted nucleotides cause a frameshift in the reading frame of the deduced amino acid sequence. Although in these cases the resulting DNA sequence may be more than 99.9% identical (eg, a single base insertion or deletion in an open reading frame of more than 1000 bases) to the actual DNA sequence. , The deduced amino acid sequence differs from the actual amino acid sequence.

Therefore, for those applications requiring accuracy in the nucleotide or amino acid sequence, the present invention provides a generated nucleotide sequence identified as SEQ ID NO: X and a putative nucleotide sequence identified as SEQ ID NO: Y. Also provided is a sample of plasmid DNA containing the human cDNA of the invention deposited at the ATCC, as described in Table 1, as well as the translated amino acid sequence of The nucleotide sequence of each deposited plasmid can be readily determined by sequencing the deposited plasmid according to known methods. The deduced amino acid sequence can then be verified from such deposit. In addition, the amino acid sequence of the protein encoded by the particular plasmid also expresses the protein by peptide sequencing or in a suitable host cell containing the deposited human cDNA, the protein is collected, and the sequence is By determining, it can be determined directly.

The names of the vectors containing the cDNA plasmids are also provided in Table 1. Each vector is conventionally used in the art. The following additional information is provided for your convenience.

Vectors Lambda Zap (US Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (US Pat. No. 5,128,25).
6 and 5,286,636) and Zap Express (US Pat. Nos. 5,128,256 and 5,286,636) pBluescript.
t (pBS) (Short, JM, et al., Nucleic Acids Res).
． 16: 7583-7600 (1988); Alting-Mees, M .; A
． And Short, J. et al. M. , Nucleic Acids Res. 17
: 9494 (1989)) and pBK (Alting-Mees, MA).
Et al., Strategies 5: 58-61 (1992)) by Stratag.
ene Cloning Systems, Inc. , 11011 N.I. Tor
commercially available from rey Pines Road, La Jolla, CA, 92037. pBS contains the ampicillin resistance gene and pBK contains the neomycin resistance gene. Phagemid pBS is based on λZap vector and Un
can be excised from the i-Zap XR vector, and the phagemid pBK
It can be excised from the ap expression vector. Both phageimides were transformed into E. coli
Strain XL-1 Blue, which is also available from Stratagene, can be transformed.

The vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport3.0 are used in Life Technology.
es, Inc. , P .; O. Box 6009, Gaithersburg, MD
Obtained from 20897. All Sport vectors contain the ampicillin resistance gene, and E. coli. E. coli strain DH10B (again, Life Tech
(available from Nologies). For example, Grub
er, C.I. E. See Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia Uni
Versity, NY) contains the ampicillin resistance gene, and E. col
The i strain XL-1 Blue can be transformed. Vector pCR (registered trademark) 2.
1 (this is Invitrogen, 1600 Faraday Avenue,
Carlsbad, CA 92008) contains the ampicillin resistance gene, and E. coli strain DH10B (Life Technology)
(available from Ogies). For example, Clark, J
． M. , Nuc. Acids Res. 16: 9677-9686 (1988).
And Mead, D .; Et al., Bio / Technology 9: (1991).

The present invention also relates to the gene corresponding to SEQ ID NO: X, SEQ ID NO: Y, and / or the deposited plasmid (cDNA plasmid: Z). The corresponding gene is
It can be isolated according to known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequences, and identifying or amplifying the corresponding gene from a suitable source of genomic material.

Also provided in the present invention are allelic variants, orthologs or
and / or species homologues. Procedures known in the art correspond to SEQ ID NO: X, SEQ ID NO: Y, and / or cDNA plasmid: Z using the sequences disclosed herein or information from the ATCC deposited clones. It can be used to obtain full length genes, allelic variants, splice variants, full length coding portions, orthologs, and / or species homologs of a gene. For example, allelic variants and / or species homologs make suitable probes or primers from the sequences provided herein, and a suitable nucleic acid supply for the allelic variant and / or the desired homologue. It can be isolated and identified by screening the source.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO: X and / or the cDNA plasmid: Z. The invention also comprises, or alternatively consists of, the polypeptide sequence of SEQ ID NO: Y, the polypeptide encoded by SEQ ID NO: X, and / or the cDNA encoded by the cDNA in the plasmid: Z. Provide a polypeptide.
A polypeptide sequence of SEQ ID NO: Y, a polypeptide encoded by SEQ ID NO: X,
Polynucleotides comprising a polypeptide encoded by the cDNA in and / or cDNA plasmids: Z, or alternatively consisting thereof, are also encompassed by the present invention. The invention further encompasses polynucleotides that include, or alternatively consist of, the complement of the nucleic acid sequence of SEQ ID NO: X, and / or the complement of the coding strand of the cDNA in the cDNA plasmid: Z. Many polynucleotide sequences (eg, EST sequences) are publicly available,
It is then accessible through sequence databases and may have been publicly available before the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Listing all relevant sequences is very cumbersome for the present disclosure. Therefore, preferably SEQ ID NO: X is
The nucleotide sequence described by general formula ab, where a is 1 of SEQ ID NO: X
And the last nucleotide-15 of SEQ ID NO: X, and b is 15 to
Is an integer of the final nucleotide of SEQ ID NO: X, where both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: X, and where b is a + 14 or greater) Polynucleotides are excluded.

RACE Protocol for Recovery of Full Length Genes Partial cDNA clones are described in Frohman, M .; A et al., Proc. Nat '
l. Acad. Sci. USA, 85: 8998-9002 (1988), and the full length can be generated by utilizing the rapid amplification of cDNA ends (RACE) procedure. CDNA clones with deletions at either the 5'or 3'end can be reconstructed to contain deleted base pairs extending to the start or stop codon of translation, respectively. In some cases, the cDNA is therefore deleted at the start of translation. Below is a brief description of modifications of this original 5'RACE procedure. Polyscript A or total RNA was labeled with Superscript II (Gibc
o / BRL) and cDNA sequences and reverse transcribed with antisense or complementary primers. The primer was applied to Microcon Co
Remove from reaction using ncentrator (Amicon). First strand cDNA is then appended using dATP and terminal deoxynucleotide transferase (Gibco / BRL). Thus, an anchor sequence is produced, which is necessary for PCR amplification. Second strand dA-tail containing PCR
Buffer, Taq DNA Polymerase (Perkin-Elmer Cetus)
Synthesized from an oligo dT primer containing three flanking restriction sites (XhoI, SalI and ClaI) at the 5 ′ end and a primer containing these restriction sites correctly. This double-stranded cDNA is PCR amplified for 40 cycles using the same primers as well as the nested cDNA-specific antisense primer. This PC
The R product is size separated on an ethidium bromide agarose gel and the region of the gel containing the cDNA product of the estimated size of the missing protein-encoding DNA is removed. The cDNA is transferred to the Magic PCR Prep kit (Promega).
a) was purified from an agarose gel, digested with XhoI or SalI and digested with a plasmid (eg pBluescript SKII (St.
ratagene)) at the XhoI and EcoRV sites. This D
NA is transformed into bacteria and this plasmid clone is sequenced to identify the correct protein-coding insert. The correct 5'end is confirmed by comparing this sequence with the putatively identified homolog with the overlap with the partial cDNA clone. Amplify and recover the 3'end using similar methods and / or commercially available kits known in the art.

Several quality control kits are commercially available for purchase. Reagents and methods similar to those described above use 5'RACE and 3'for recovery of full-length genes.
For both'RACE's are supplied in kit form from Gibco / BRL. The second kit is available from Clontech. This kit is Du
mas et al., Nucleic Acids Res. , 19: 5227-32 (1
991), modification of related technology (SLIC (single-stranded cDNA,
Single-stranded linkage))). The main difference in the procedure is that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to ligate an anchor primer containing a restriction site to the first strand cDNA. This eliminates the need for dA tailing reactions that result in poly T elongation that has been difficult to sequence in the past.

An alternative to producing 5'cDNA or 3'cDNA from RNA is to use a cDNA library double-stranded DNA. Asymmetric PCR amplified antisense cDNA strands are synthesized using antisense cDNA specific primers and plasmid anchor primers. Remove these primers and
CR reactions are performed with nested cDNA-specific antisense primers and plasmid anchor primers.

(RN for producing a 5′-terminal sequence or a 3′-terminal sequence and obtaining a full-length gene
A Ligase Protocol) Once the gene of interest has been identified, several methods are available for the identification of the 5'or 3'parts of the gene which may not be present in the original cDNA plasmid. Become. These methods include, but are not limited to: filter probe probing, clonal enrichment using specific probes, 5'RACE.
And a protocol similar and identical to 3'RACE. The full-length gene may be present in the library and identified by searching, while the 5'end or 3
A useful method to generate the'ends is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5'RACE is available to generate the missing 5'end of the desired full-length gene (this method is described in Frommont-Racine et al., Nucleic Ac).
ids Res. , 21 (7): 1683-1684 (1993)). Briefly, a particular RNA oligonucleotide is ligated to the 5'end of a population of RNAs that probably contains the full-length gene RNA transcript, and the ligated RNA
5'of the desired full-length gene is prepared using a primer set containing a primer specific to the oligonucleotide and a primer specific to the known sequence of the gene of interest.
PCR amplify the portion. The amplified product can then be sequenced and used to generate the full length gene. The method starts with total RNA isolated from the desired source, and although not a requirement in this procedure, polyARN
A may be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5'phosphate groups on degraded or damaged RNA that can interfere with subsequent RNA ligase steps. It then inactivates the phosphatase, if used, and R
NA is treated with tobacco acid pyrophosphatase to remove the cap structure present at the 5'end of the messenger RNA. This reaction is followed by T4
It leaves a 5'phosphate group at the 5'end of the capped RNA that can be ligated to RNA oligonucleotides using RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using gene specific oligonucleotides. The first-strand synthesis reaction is then combined with the ligated RN.
A oligonucleotide-specific primer and the desired serpin (Serpi
n) can be used as a template for PCR amplification of the desired 5'end using primers specific for the known sequence. The resulting product is then sequenced and analyzed to confirm that the 5'end sequence belongs to the directly related serpin.

(Polynucleotide Fragment and Polypeptide Fragment) The present invention also relates to a polynucleotide fragment of the polynucleotide (nucleic acid) of the present invention. In the present invention, the "polynucleotide fragment" means
A polynucleotide having the following nucleic acid sequence: part of the cDNA contained in cDNA plasmid Z, or the cDNA contained in cDNA plasmid Z
A portion of a cDNA encoding a polypeptide encoded by: a portion of the polynucleotide sequence in SEQ ID NO: X or its complement; a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO: Y; A polynucleotide sequence that encodes a portion of an encoded polypeptide. The nucleotide fragments of the present invention are preferably at least about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt, at least about 50 nt, at least about 75 nt, at least about 100 nt. , At least about 125 nt, or at least about 150 nt in length. For example, a fragment "at least about 20 nt in length" is intended to include 20 or more contiguous bases derived from the sequence contained in the cDNA sequence of cDNA plasmid Z or the nucleotide sequence shown in SEQ ID NO: X or its complement. To be done. In this context “about” includes the specifically stated value or values that are greater or lesser by a few (5, 4, 3, 2, or 1) nucleotides. These nucleotide fragments have uses, including but not limited to, use as diagnostic probes and primers, as discussed herein. Of course, larger fragments (eg at least 150, 175, 200, 250, 500, 600
, 1000 or 2000 nucleotides in length) are also included in the invention.

Furthermore, typical examples of the polynucleotide fragment of the present invention include, for example,
Fragments containing or consisting of the following SEQ ID NO: X, or a sequence of approximately the number of nucleotides of its complementary strand, or consisting of: 1-50, 51-100
101-150, 151-200, 201-250, 251-300, 301
~ 350, 351-400, 401-450, 451-500, 501-550
, 551-600, 651-700, 701-750, 751-800, 800
~ 850, 851-900, 901-950, 951-1000, 1001-1
050, 1051 to 1100, 1101 to 1150, 1151 to 1200, 12
01-1250, 1251-1300, 1301-1350, 1351-140
0, 1401 to 1450, 1451 to 1500, 1501 to 1550, 1551
~ 1600, 1601-1650, 1651-1700 and 1701-173
4. In this context, "approximately" is a few (5, 4, 3, 2, or 1) nucleotides greater than this, particularly within the stated range, or at either or both termini. , Or including a small range. Preferably,
These fragments encode a polypeptide that has the functional activity (eg, biological activity) of the polypeptide encoded by the polynucleotide that is part of the sequence. More preferably, these fragments can be used as probes or primers as discussed herein above. Also included in the invention are polynucleotides that hybridize to one or more of these fragments under stringent hybridization conditions or under lower stringency conditions, as well as the polypeptides or fragments encoded by these polynucleotides. include.

Furthermore, typical examples of the polynucleotide fragment of the present invention include, for example:
The cDNA nucleotide sequence contained in the cDNA plasmid Z, or a sequence containing the following approximate number of nucleotides of its complementary strand, or a fragment consisting of the following is mentioned: 1-50, 51-100, 101-150, 151- Two
00, 201-250, 251-300, 301-350, 351-400, 4
01-450, 451-500, 501-550, 551-600, 651-7
00, 701-750, 751-800, 800-850, 851-900, 9
01-950, 951-1000, 1001-1050, 1051-1100,
1101-1150, 1151-1200, 1201-1250, 1251-1
300, 1301-1350, 1351-1400, 1401-1450, 14
51 to 1500, 1501 to 1550, 1551 to 1600, 1601 to 165
0, 1651-1700 and 1701-1734. In this context, "approximately" is a few (5, 4, 3, 2, or 1) nucleotides greater than this, particularly in the stated range, or at either or both termini. , Or including a small range. Preferably, these fragments are c
Encodes a polypeptide having the functional activity (eg, biological activity) of the polypeptide encoded by the cDNA nucleotide sequence contained in DNA plasmid Z. More preferably, these fragments can be used as probes or primers, as discussed herein. Also included in the invention are polynucleotides that hybridize to one or more of these fragments under stringent hybridization conditions or under lower stringency conditions, and the polypeptides encoded by these polynucleotides or fragments are also included. ,included.

In the present invention, the “polypeptide fragment” means a part of the amino acid sequence contained in SEQ ID NO: Y, a part of the amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: X, and / or the cDNA plasmid Z. Refers to an amino acid sequence which is a part of the amino acid sequence encoded by the cDNA contained therein. A protein (polypeptide) fragment may be "free-standing," or a larger polypeptide, which fragment forms a portion or region, most preferably as a single continuous region. )). Representative examples of polypeptide fragments of the present invention include, for example, fragments comprising the amino acid sequence of the approximate number of amino acids of the coding region of the following SEQ ID NO: or consisting of: 1-20, 21- 40, 41-60
, 61-80, 81-100, 102-120. Further, the polypeptide fragments of the present invention are at least about 10,15,20,25,30,35,40,4.
5, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110
Can be any amino acid length. In this context, "about" means the range or value specifically stated, or a few (5, 4, 3, 2, or 1) amino acids at either or both termini. Contains a range or value that is only greater or less than. Polynucleotides encoding these polypeptide fragments are also included in the invention.

Although deletion of one or more amino acids from the N-terminus of a protein results in modification of the loss of one or more biological functions of the protein, it may have other functional activities (eg, biological activity). , The ability to multimerize, the ability to bind ligand) can still be retained. For example, the ability of a truncated mutein to induce and / or bind to an antibody that recognizes the intact or mature form of the polypeptide is determined by , Is generally retained when removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunogenic activity is determined by conventional methods described herein and otherwise It can be readily determined by other methods known in the art. Muteins with multiple deleted N-terminal amino acid residues appear to be able to retain some biological or immunogenic activity. In fact, peptides consisting of as few as 6 amino acid residues can often elicit an immune response.

Accordingly, polypeptide fragments of the present invention include secreted proteins and mature forms. Further preferred polypeptide fragments include secreted proteins having a contiguous series of deleted residues from the amino or carboxy terminus or both, or the mature form. For example, any number of amino acids (range 1-60) can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids (range 1-30) may be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino-terminal and carboxy-terminal deletions is preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

The present invention provides a polypeptide disclosed herein (eg, a polypeptide of SEQ ID NO: Y, a polypeptide encoded by a polynucleotide sequence contained in SEQ ID NO: X, and / or a cDNA plasmid Z). Further provided is a polypeptide in which one or more residues have been deleted from the amino terminus of the amino acid sequence of the polypeptide encoded by the included cDNA). In particular, the deletion at the N-terminus is represented by the general formula mq
Can be described by q is a polypeptide of the invention (eg, SEQ ID NO: Y
Is a whole integer representing the total number of amino acid residues in the polypeptide disclosed in
Then, m is defined as an arbitrary integer in the range of 2 to q-6. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also included in the invention.

Also, as noted above, deletion of one or more amino acids from the C-terminus of a protein results in modification of the loss of one or more biological functions of the protein, although other functional The activity (eg, biological activity, ability to multimerize, ability to bind ligand) can still be retained. For example, the ability of a truncated mutein to induce and / or bind to an antibody that recognizes the complete or mature form of a polypeptide has fewer residues than most of the complete or mature polypeptide. Is generally retained when removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunogenic activity is determined by conventional methods described herein and otherwise. It can be readily determined by other methods known in the art. Muteins with multiple deleted C-terminal amino acid residues are likely to retain some biological or immunogenic activity. In fact, peptides composed of as few as 6 amino acid residues can often elicit an immune response.

Accordingly, the invention provides a polypeptide disclosed herein (eg, a polypeptide of SEQ ID NO: Y, a polypeptide encoded by a polynucleotide sequence contained in SEQ ID NO: X, and / or a cDNA plasmid). Further provided is a polypeptide having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide encoded by the cDNA contained in Z). In particular, the C-terminal deletion is
It may be described by general formula 1-n, where n is any whole integer in the range 6 to q-1, and n corresponds to the position of the amino acid residue in the polypeptide of the invention. . Polynucleotides encoding these polypeptides (including fragments and / or variants) are also included according to the invention.

In addition, any of the N-terminal or C-terminal deletions described above may be combined to produce N-terminal and C-terminal deleted polypeptides. The invention also provides polypeptides that have one or more amino acids deleted from both the amino and carboxy termini. This polypeptide is generally a polypeptide encoded by SEQ ID NO: X (including but not limited to the preferred polypeptide disclosed as SEQ ID NO: Y) and / or cDNA plasmid Z.
Of the polypeptide encoded by the cDNA, and / or the polypeptide encoded by their complements, wherein n and m are integers as described above. Is. These polypeptides (
Polynucleotides (including fragments and / or variants) are also included in the invention.

Any polypeptide sequence contained in the polypeptide of SEQ ID NO: Y, any polypeptide sequence encoded by the polynucleotide sequence set forth as SEQ ID NO: X, or any cDNA encoded by cDNA in cDNA plasmid Z. The polypeptide sequence of is, in order to determine certain preferred regions of that polypeptide,
Can be analyzed. For example, the amino acid sequence of the polypeptide encoded by SEQ ID NO: X or the polynucleotide sequence of the cDNA in cDNA plasmid Z is D
NASTAL Computer Algorithm (DNASTAR, Inc., 1228
S. Park St. , Madison, WI 53715 USA;
p: // www. dnastar. com /) default parameters.

Regions of the polypeptide that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to: Ga
α-region, β-region, turn region and coil region of rnier-Robson, α-region, β-region and turn region of Chou-Fasman, Kyt
Hydrophilic and hydrophobic regions of e-Doolittle, α-amphipathic and β-amphipathic regions of Eisenberg, flexible regions of Karplus-Schulz, surface-forming regions of Emini and Jameson with high antigenicity index.
-Wolf area. In this regard, some of the highly preferred polynucleotides of the invention include some structural features (eg, some of the features described above (eg, 1, 2, 3).
Alternatively, there is a polynucleotide encoding a polypeptide containing the region combined with 4)).

Furthermore, Kyte-Doolittle hydrophilic region and hydrophobic region, Emi
ni surface formation region, and Jameson-Wolf region (
That is, if identified using the default parameters of the Jameson-Wolf program, it is routinely used to include 4 or more consecutive amino acids with an antigenicity index of 1.5 or higher) The region of the polypeptide that represents the degree of potential is determined. Areas of high antigenicity are determined by DNASTAR analysis by selecting values that represent areas of the polypeptide that appear to be exposed on the surface of the polypeptide in the environment where antigen recognition may occur during the initiation process of the immune response. Determined from the data.

Preferred polypeptide fragments of the invention are those fragments which comprise or consist of an amino acid sequence which exhibits a functional activity (eg biological activity) of a polypeptide sequence whose amino acid sequence is a fragment. is there. A polypeptide exhibiting "functional activity" refers to one or more known functional activities associated with a full-length protein (eg, biological activity, antigenicity, immunogenicity, and / or high abundance, as described above). (Eg somatization).

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are fragments that exhibit an activity that is similar but not necessarily identical to that of the polypeptide of the invention. The biological activity of this fragment may include improved desired activity or reduced undesired activity. Polynucleotides encoding these polypeptide fragments are also included in the invention.

In a preferred embodiment, the polypeptide of the invention comprises one, two, three, four, five or more of the antigenic fragment of the polypeptide of SEQ ID NO: Y or a portion thereof. Or consist of them. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also included in the invention.

The present invention provides an epitope of the polypeptide sequence as set forth in SEQ ID NO: Y, or cd
An epitope of the polypeptide sequence encoded by the cDNA in NA plasmid Z, or an epitope of the polypeptide sequence encoded by a polynucleotide that hybridizes to the complement of the epitope coding sequence of SEQ ID NO: X, or as defined above. Polypeptide comprising or consisting of the epitope coding sequence contained in cDNA plasmid Z under different stringent or lower stringency hybridization conditions. The invention further provides a polynucleotide sequence that encodes an epitope of a polypeptide sequence of the invention (eg, the sequence disclosed in SEQ ID NO: X), a polynucleotide sequence of the complementary strand of a polynucleotide sequence that encodes an epitope of the invention, And a polynucleotide sequence that hybridizes to this complementary strand under stringent or lower stringency hybridization conditions as defined above.

The term “epitope” as used herein refers to that portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the invention includes a polypeptide that comprises an epitope, as well as a polynucleotide that encodes this polypeptide. As used herein, an "immunogenic epitope" is defined as the portion of a protein that elicits an antibody response in an animal and can be any method known in the art (e.g., antibody production as described below). Method). (For example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:
3998-4002 (1983)). As used herein, the term "antigenic epitope" means that an antibody, when measured by any method known in the art (eg, the immunoassays described herein), has its antigen. Is defined as the part of the protein that is capable of immunospecifically binding to. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not necessarily require immunogenicity.

Fragments that function as epitopes can be generated by any conventional means. (For example, Houghten, RA, Proc. Natl. Acad.
． Sci. USA 82: 5131-5135 (1985) (U.S. Pat. No. 4,6,6).
31, 211)).

In the present invention, an antigenic epitope preferably comprises a sequence of at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, more preferably at least 8 amino acids, at least 9 amino acids, at least 10 amino acids. A sequence of amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, and most preferably It comprises a sequence between about 15 and about 30 amino acids. Preferred polypeptides containing immunogenic or antigenic epitopes are at least 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
It is 75, 80, 85, 90, 95 or 100 amino acid residues long. Furthermore, non-exclusively preferred antigenic epitopes include the antigenic epitopes disclosed herein and portions thereof. Antigenic epitopes are useful, for example, to raise antibodies (including monoclonal antibodies) that specifically bind to the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, and any combination of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays. (For example, Wilson et al., Cell 37: 767.
-778 (1984); Sutcliffe et al., Science 219: 66.
0-666 (1983)).

Similarly, immunogenic epitopes can be used to induce, for example, antibodies according to methods well known in the art. (For example, Sutcliffe et al., Supra; Wilso
n et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 8
2: 910-914; and Bittle et al., J. Am. Gen. Virol. 66:
2347-2354 (1985)). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, and any combination of 2, 3, 4, 5 or more of these immunogenic epitopes. A polypeptide comprising one or more immunogenic epitopes can be presented to induce an antibody response in an animal system (eg, rabbit or mouse) with a carrier protein (eg, albumin), or the polypeptide is sufficient If very long (at least about 25 amino acids), the polypeptide can be presented to animal systems without a carrier. However, immunogenic epitopes containing as few as 8 to 10 amino acids have been shown to be sufficient to elicit at least an antibody capable of binding to a linear epitope in a denatured polypeptide (eg, , In Western blotting).

The epitope-bearing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art. These well-known methods include in vivo immunization,
In vitro immunization, and phage display methods are included, but are not limited to. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra; and Bittle et al., J. Chem. Gen. Virol. 66: 2347-2354 (19
See 85). When using in vivo immunization, animals can be immunized with free peptide; however, anti-peptide antibody titers will bind the peptide to macromolecular carriers such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. Can be boosted by For example, a peptide containing a cysteine residue is a maleimidobenzoyl-N-hydroxysuccinimide ester (MB
It may be attached to the carrier using a linker such as S). On the other hand, other peptides can be bound to the carrier using more common binding agents such as glutaraldehyde. Animals such as rabbits, rats, and mice have been prepared with, for example, emulsions (about 1
Immunization by intraperitoneal and / or intradermal injection of 00 μg of peptide or carrier protein and Freund's adjuvant, or any other adjuvant known to stimulate the immune response). Several booster injections may be needed to provide a useful titer of anti-peptide antibody, for example, at intervals of about 2 weeks. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on the solid surface. Titer of anti-peptide antibody in serum from immunized animals is increased by selection of anti-peptide antibody (eg, by adsorption of peptide on solid support and elution of selected antibody according to methods well known in the art). You can

As will be appreciated by those of skill in the art, and as discussed above, the polypeptides of the present invention and immunogenic and / or antigenic epitope fragments thereof are fused to other polypeptide sequences. obtain. For example, the polypeptides of the present invention may be fused to immunoglobulin (IgA, IgE, IgG, IgM) constant domains or portions thereof (CH1, CH2, CH3, or any combination thereof, and portions thereof). , Which results in a chimeric polypeptide. These fusion proteins can facilitate purification and increase half-life in vivo. This has been demonstrated for a chimeric protein consisting of the first two domains of human CD4 polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. For example, EP 394,82
7; Traunecker et al., Nature, 331: 84-86 (1988).
checking ... Enhanced delivery of antigen to the immune system across the epithelial barrier has been demonstrated by FcR
It has been demonstrated for an antigen (eg insulin) conjugated to an n-binding partner (eg IgG fragment or Fc fragment (see eg PCT publications WO 96/2024 and WO 99/04813)).
IgG fusion proteins with a disulfide-bridged dimer structure due to the gG moiety were also found to be more effective at binding and neutralizing other molecules than their monomeric polypeptides or fragments alone. Has been issued. For example, F
outertoukis et al. Biochem. , 270: 3958-396.
4 (1995).

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
Discloses a fusion protein comprising various portions of the constant region of an immunoglobulin molecule along with another human protein or part thereof. In many cases, the Fc portion in the fusion protein may be of therapeutic and diagnostic benefit, thus resulting in, for example, improved pharmacokinetic properties (EPA 0232 262). Thus, in certain embodiments, a fusion protein of the invention comprises or consists of amino acid residues 1-310 of SEQ ID NO: 2 fused to an Fc polypeptide sequence. Alternatively, it may be desirable to have the Fc portion deleted after the fusion protein has been expressed, detected, and purified. For example, the Fc portion may interfere with therapy and diagnosis if the fusion protein is to be used as an antigen for immunization. In drug discovery,
Human proteins, such as hIL-5, have been fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5. D. Bennett et al. Molec. Recognit
Ion 8: 52-58 (1995); Johnson et al. Biol
． Chem. 270: 9459-9471 (1995).

In addition, the polypeptides of the present invention can be fused to a marker sequence (eg, a peptide that facilitates purification of the fused polypeptide). In a preferred embodiment, the marker amino acid sequence is, among other things, a hexahistidine peptide (eg, pQE vector (QIAGEN, Inc., 9259 Eton Ave.
Nue, Chatsworth, CA, 91311))
And many of these are commercially available. Gentz et al., Proc. N
atl. Acad. Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of fusion proteins. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al.,
Cell 37: 767 (1984)).

Accordingly, any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention.

Nucleic acids encoding the above epitopes may also be expressed as an epitope tag (eg, a hemagglutinin (“HA”) tag or flag tag) of the expressed polypeptide with the gene of interest. It can aid in detection and purification.
For example, the system described by Janknecht et al. Allows for rapid purification of native fusion proteins expressed in human cell lines (Janknecht).
Et al., 1991, Proc. Natl. Acad. Sci. USA 88: 897.
2-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of 6 histidine residues. This tag serves as the membrane-bound domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni ²⁺ nitriloacetic acid-agarose columns, and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

Further fusion proteins of the invention may be produced through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). D
NA shuffling can be used to modulate the activity of the polypeptides of the invention, and such methods can be used to produce polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. . Generally, US Pat. Nos. 5,605,793, 5,811,238 and 5
, 830,721, 5,834,252 and 5,837,458.
And Patten et al., Curr. Opinion Biotechno
l. 8: 724-33 (1997); Harayama, Trends Bio.
technology. 16 (2): 76-82 (1998); Hansson et al., J.
． Mol. Biol. 287: 265-76 (1999); and Loren
zo and Blasco, Biotechniques 24 (2): 308-.
13 (1998), each of these patents and publications, hereby incorporated by reference in its entirety. In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: X and of the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. DN
A shuffling involves the assembly of two or more DNA segments by homologous recombination or site-specific recombination to generate variations in this polynucleotide sequence. In another embodiment, the polynucleotides or encoded polypeptides of the invention are labeled with error-prone (error-p) prior to recombination.
lone) PCR, random nucleotide insertion, or other methods that can be modified by subjecting it to random mutagenesis. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention are one or more components of one or more heterologous molecules. , Motifs, sections, parts, domains, fragments and the like.

Polynucleotide and Polypeptide Variants The present invention also includes steroid hormone variants. The present invention relates to a polynucleotide sequence disclosed in SEQ ID NO: X or a variant of a complementary strand thereto, and / or a variant of a cDNA sequence contained in a cDNA plasmid Z.

The invention also encodes a variant of the polypeptide sequence disclosed in SEQ ID NO: Y, a variant of the polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: X and / or the cDNA in cDNA plasmid Z. Variants of the polypeptide sequence are included.

A “variant” is different from a polynucleotide or polypeptide of the invention, but
A polynucleotide or polypeptide that retains those properties. In general, variants are closely similar and are in many regions identical to the polynucleotides or polypeptides of the invention.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a B) a nucleotide sequence encoding a steroid hormone receptor polypeptide having the amino acid sequence as shown in the sequence listing and as set forth in SEQ ID NO: X or the cDNA in the cDNA plasmid Z; (b) shown in the sequence listing and SEQ ID NO: X Or a nucleotide sequence encoding a mature steroid hormone receptor polypeptide having an amino acid sequence as described in cDNA in cDNA plasmid Z; (c) set forth in SEQ ID NO: or in cDNA in cDNA plasmid Z Amino acid sequence A nucleotide sequence encoding a biologically active fragment of a steroid hormone receptor polypeptide having: (d) a steroid having the amino acid sequence shown in the Sequence Listing and set forth in the cDNA of SEQ ID NO: X or cDNA plasmid Z A nucleotide sequence encoding an antigenic fragment of a hormone receptor polypeptide; (e) encodes a steroid hormone receptor polypeptide containing the complete amino acid sequence encoded by the human cDNA plasmid contained in the cDNA of SEQ ID NO: X or cDNA plasmid Z. Nucleotide sequence; (f) SEQ ID NO: X or cD in cDNA plasmid Z
A nucleotide sequence encoding a mature steroid hormone receptor polypeptide having an amino acid sequence encoded by the human cDNA plasmid contained in NA; A nucleotide sequence encoding a biologically active fragment of a steroid hormone receptor polypeptide having an amino acid sequence of: (h) SEQ ID NO: X or c in cDNA plasmid Z
A nucleotide sequence encoding an antigenic fragment of a steroid hormone receptor polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in DNA; (i) above (a), (b), (c), (d) ), (E
), (F), (g) or a nucleotide sequence complementary to any of the nucleotide sequences in (h).

The present invention also includes, for example, the above (a), (b), (c), (d), (e), (
f), (g), (h) or (i) with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 1 of the nucleotide sequences.
It relates to nucleic acid molecules which comprise or consist of nucleotide sequences which are 00% identical. Polypeptides encoded by these nucleic acid molecules are also included according to the invention. In another embodiment, the present invention provides the above (a) under stringent hybridization conditions or under lower stringency conditions.
), (B), (c), (d), (e), (f), (g), (h) or (i), or comprises a polynucleotide that hybridizes to the polynucleotide. Contains nucleic acid molecules. Polynucleotides that hybridize to the complements of these nucleic acid molecules under stringent hybridization conditions, or lower stringency conditions, are also according to the invention, like the polypeptides encoded by these polynucleotides. Included.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) shown in the Sequence Listing. And a nucleotide sequence encoding a steroid hormone receptor polypeptide having an amino acid sequence as shown in Table 1; (b) a mature steroid having the amino acid sequence shown in the Sequence Listing and as shown in Table 1 A nucleotide sequence encoding a hormone receptor polypeptide; (c) a nucleotide sequence encoding a biologically active fragment of a steroid hormone receptor polypeptide having the amino acid sequence shown in the Sequence Listing and set forth in Table 1. d) shown in the Sequence Listing and listed in Table 1 A nucleotide sequence encoding an antigenic fragment of a steroid hormone receptor polypeptide having the following amino acid sequence: (e) a complete amino acid sequence contained in the ACTT deposit and encoded by the human cDNA of the cDNA plasmid described in Table 1 A nucleotide sequence encoding a steroid hormone receptor polypeptide; (f) comprising an amino acid sequence contained in the ACTT deposit and encoded by the human cDNA of the cDNA plasmid set forth in Table 1.
A nucleotide sequence encoding a mature steroid hormone receptor polypeptide; (g) Biology of a steroid hormone receptor polypeptide having the amino acid sequence contained in the ACTT deposit and encoded by the human cDNA of the cDNA plasmid described in Table 1. Sequence encoding a biologically active fragment; (h) an antigenic fragment of a steroid hormone receptor polypeptide having the amino acid sequence contained in the ACTT deposit and encoded by the human cDNA of the cDNA plasmid described in Table 1. A nucleotide sequence encoding
(I) A nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g) or (h) above.

The present invention also includes, for example, (a), (b), (c), (d), (e) and (f
), (G), (h) or (i) at least 80
%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
It relates to nucleic acid molecules which comprise or consist of identical nucleotide sequences. Polypeptides encoded by these nucleic acid molecules are also included by the invention. In another embodiment, the present invention provides the above (a), (b), (c).
Polynucleotides which hybridize to the polynucleotides in, (d), (e), (f), (g), (h) or (i) under stringent hybridization conditions or under lower stringency conditions. Or includes a nucleic acid molecule. Polynucleotides that hybridize to the complements of these nucleic acid molecules under stringent hybridization conditions or under lower stringency conditions are also included by the invention, as are the polypeptides encoded by these polynucleotides. obtain.

The invention also includes, for example, the polypeptide sequence set forth in SEQ ID NO: Y, SEQ ID NO: X.
A polypeptide sequence encoded by the nucleotide sequence of, a polypeptide sequence encoded by the cDNA of cDNA plasmid Z, and / or a polypeptide fragment of any of these polypeptides (eg, described herein. At least 80%, 85%, 90%, 95%, 96%
, 97%, 98%, 99% or 100% identical amino acid sequences. Polynucleotides that hybridize to the complement of nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or under lower stringency conditions are also similar to the polypeptides encoded by these polynucleotides. Included by the present invention.

By a nucleic acid having a nucleotide sequence which is at least 95% “identical” to a reference nucleotide sequence of the present invention, the nucleotide sequence of the nucleic acid is 100 nucleotides each of the reference nucleotide sequence, the nucleotide sequence encoding a polypeptide. It is intended to be identical to a reference sequence, except that up to 5 point mutations can be included. In other words, at least 9 relative to the reference nucleotide sequence
Up to 5% of the nucleotides of the reference sequence can be deleted or replaced by another nucleotide in order to obtain a nucleic acid having a nucleotide sequence of 5% identity,
Alternatively, a large number of nucleotides, up to 5% of the total nucleotides in the reference sequence, can be inserted in the reference sequence. The query sequence can be the entire sequence referred to in Table 1, the ORF (open reading frame), or any fragment identified as described herein.

As a practical matter, any particular nucleic acid molecule or polypeptide will be at least 80%, 85%, 90%, 95%, 96%, 9% of the nucleotide sequences of the invention.
Whether 7%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall compatibility (also referred to as global sequence alignment) between a query sequence (sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App
． Biosci. 6: 237-245 (1990)) based on the FASTDB computer program. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA
Sequences can be compared by converting U to T. The results of this global sequence alignment are expressed as percent identity (%). Preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch P.
energy = 1, Joining Penalty = 30, Randomiz
ation Group Length = 0, Cutoff Score = 1,
Gap Penalty = 5, Gap Size Penalty 0.05,
Windows Size = 500 or the length of the nucleotide sequence of interest (whichever is shorter).

If the subject sequence is shorter than the query sequence due to the 5'or 3'deletions (as opposed to due to internal deletions), a manual correction must be made to the results. This is because the FASTDB program does not consider 5'and 3'truncations of the subject sequence when calculating percent identity. For a subject sequence that is cleaved at the 5'or 3'end, the percent identity to the query sequence is the 5'and 3'of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence. Corrected by calculating the number of bases in. Whether the nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the bases outside the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5'end of the subject sequence, so the FASTDB alignment shows no match / alignment at the first 10 bases at the 5'end. 1
0 unpaired bases represent 10% of the sequence (number of bases at the non-matching 5'and 3'ends / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases were an exact match, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, so that there are no bases at the 5'or 3'of the subject sequence that are not matched / aligned with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5'or 3'bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

A polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence of the present invention, for example, the amino acid sequence of the subject polypeptide is such that the subject polypeptide sequence corresponds to each of the query amino acid sequences. It is intended to be identical to the query sequence, except that changes of up to 5 amino acids per 100 amino acids may be included. In other words, up to 5% of the amino acid residues in the sequence of interest are inserted, deleted, (indels) in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence.
Or it may be replaced by another amino acid. These modifications of the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, either individually between the residues in the reference sequence or the reference sequence. Interspersed in any of one or more consecutive groups within.

As a practical matter, any particular polypeptide may be included in cDNA plasmid Z, eg, for the amino acid sequences in Table 1 or fragments thereof.
For amino acid sequences encoded by DNA or fragments thereof,
Whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall match (also referred to as global sequence alignment) between a query sequence (the sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App. Biosci). . 6: 237-
245 (1990)) based on the FASTDB computer program. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the above global sequence alignments are given in percent identity. Preferred parameters used for FASTDB amino acid alignment are: Mat
rix = PAM 0, k-tuple = 2, Mismatch Penalty
= 1, Joining Penalty = 20, Randomization
Group Length = 0, Cutoff Score = 1, Windows
Size = length of array, Gap Penalty = 5, Gap Size P
energy = 0.05, Window Size = 500 or the length of the target amino acid sequence (whichever is shorter).

If the subject sequence is shorter than the query sequence due to N-terminal or C-terminal deletions (and not due to internal deletions), then manual corrections must be made to the results. This is because the FASTDB program does not consider N-terminal and C-terminal truncations of the subject sequence when calculating the overall percent identity. Regarding the target sequence shortened at the N-terminus and C-terminus, with respect to the query sequence,
Percent identity is corrected by calculating the number of residues in the query sequence that are N- and C-terminal to the subject sequence that are not matched / aligned with the corresponding subject residues as a percentage of the total bases of the query sequence. . Whether the residues are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using specific parameters to arrive at the final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only those residues N- and C-terminal to the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only the query residue is the furthest N in the subject sequence.
Located outside the terminal and C-terminal residues.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. Deletions occur at the N-terminus of the subject sequence, and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (mismatched N-terminus and C
Represents the number of residues at the end / total number of residues in the query sequence, and the result is FAST
10% is subtracted from the percent identity score calculated by the DB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. In this case, the deletion is an internal deletion, so there are no residues at the N-terminus or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected.
No other manual correction is made for the purposes of the present invention.

Variants may include changes in the coding regions, non-coding regions, or both. Especially preferred are those that produce silent substitutions, additions or deletions,
Polynucleotide variants containing changes that do not alter the properties or activity of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Furthermore, 5 in any combination
Less than 0, less than 40, less than 30, less than 20, less than 10, or 5 to 50, 5 to 25
Variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added are also preferred. Polynucleotide variants optimize codon expression for a variety of reasons (eg, codon expression for a particular host (eg, those skilled in the art can change codons in human mRNA to those preferred by bacterial hosts such as E. coli). To obtain)).

Naturally occurring variants are referred to as “allelic variants” and refer to one of several alternative forms of a gene that occupy a given locus on the chromosome of an organism. (Genes II, Lewin, B., edited by John Wiley &
Sons, New York (1985)). These allelic variants are
It can vary at either the polynucleotide level and / or the polypeptide level and is included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants are
It can be made to improve or change the properties of the polypeptides of the invention. For example, as discussed herein, one or more amino acids can be deleted from the N-terminus or C-terminus of a polypeptide of the invention without substantial loss of biological function. . Ron et al. Biol. Chem. 268: 2984-2988 (1
993) reported a modified KGF protein that has heparin-binding activity even after deletion of 3, 8 or 27 amino-terminal amino acid residues. Similarly, interferon γ is 8-1 from the carboxy terminus of this protein.
After deleting 0 amino acid residues, it showed up to 10 times higher activity (Do.
beli et al. Biotechnology 7: 199-216 (1988).
)).

Furthermore, a wealth of evidence demonstrates that variants often retain activities similar to those of naturally occurring proteins. For example, Gayle and co-workers (J. Biol. Chem 268: 22105-22111 (1
993)) performed an extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a variants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were tested at every potential amino acid position. The investigators found that "the majority of molecules can be altered with little effect on either [binding activity or biological activity]." (See summary). In fact, of the over 3,500 nucleotide sequences tested,
Only 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type.

In addition, deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide may result in modification or deletion of one or more biological functions, but not other biological functions. Activity can still be retained. For example, the ability of a deletion variant to induce and / or bind an antibody that recognizes a secreted form is the ability to remove less than the majority of the secreted form from the N-terminus or C-terminus. Seems to be retained. Whether a particular polypeptide lacking the N-terminal or C-terminal residues of a protein retains such immunogenic activity depends on conventional methods described herein, and so on. If not, it can be easily determined by a conventional method known in the art.

Accordingly, the invention further includes polypeptide variants which exhibit functional activity (eg, substantial biological activity) of a polypeptide of the invention. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art such that they have little effect on activity.

The application is at least 80%, 85%, relative to the nucleic acid sequences disclosed herein (eg, encoding a polypeptide having an amino acid sequence with an N-terminal and / or C-terminal deletion). Nucleic acid molecules that are 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, whether or not they encode a polypeptide having functional activity. Because, even if a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still be familiar with methods of using the nucleic acid molecule (eg, as a hybridization probe or polymerase chain reaction (PCR) primer). Because I know. The use of the nucleic acid molecule of the present invention that does not encode a polypeptide having functional activity includes, in particular: (1) cDNA
Isolating the gene or allelic variant or splice variant thereof in the library; (2) Verma et al., Human Chromoso.
mes: A Manual of Basic Techniques, Per
As described in gamon Press, New York (1988),
In situ hybridizing to metaphase chromosomal spreads that provide the precise chromosomal location of the gene (eg, "FISH"); and (3) Northern blot analysis to detect mRNA expression in specific tissues. .

However, at least 80%, 85% relative to the nucleic acid sequences disclosed herein
, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleic acid molecules are preferred, which nucleic acid molecules actually encode a polypeptide having functional activity. .

Of course, due to the degeneracy of the genetic code, for example, at least 80 relative to the nucleic acid sequence of the deposited clone, the nucleic acid sequence referred to in Table 1 (SEQ ID NO: X) or a fragment thereof. %, 85%, 90%, 95%, 96%, 97%,
Those skilled in the art will immediately understand that many nucleic acid molecules having 98%, 99%, or 100% identical sequences encode polypeptides that have "functional activity." In fact, since all degenerate variants of any of these nucleotide sequences encode the same polypeptide, this will often be apparent to one of skill in the art without performing the comparative assay described above. Is. It is further understood in the art that, for nucleic acid molecules that are not degenerate variants, a reasonable number also encodes a polypeptide having functional activity. Amino acid substitutions that allow a protein to function significantly or are unlikely to function (eg, replacing one aliphatic amino acid with a second aliphatic amino acid), as described further below.
Because I know completely.

For example, for guidance on how to make phenotypically silent amino acid substitutions, see Bowie et al., “Deciphering the Message in”.
Protein Sequences: Tolerance to Amin
o Acid Substitutions, Science 247: 13.
06-1310 (1990), where the authors note that there are two major strategies for studying the tolerance of amino acid sequences to changes.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the course of evolution. Conserved amino acids can be identified by comparing amino acid sequences in different species. These conserved amino acids appear to be important for protein function. In contrast, the amino acid positions at which the substitutions were tolerated by natural selection indicate that these positions are not important for protein function. Thus, the positions that tolerate amino acid substitutions may be modified, yet still maintain the biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions in the cloned gene to identify regions important for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (
The introduction of one alanine mutation at each residue in the molecule) can be used. (Cuni
ngham and Wells, Science 244: 1081-1085 (
1989)). The resulting mutant molecule can then be tested for biological activity.

As the authors mention, these two strategies have revealed that the protein is surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be tolerated at particular amino acid positions in the protein. For example, amino acid residues that are mostly buried (within the tertiary structure of the protein) require non-polar side chains, but surface side chain characteristics are generally poorly conserved. In addition, tolerant conservative amino acid substitutions include the substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; the substitution of Ser and Thr for hydroxyl residues; the substitution of Asp and Glu for acidic residues; Amide residue Asn
And Gln, basic residues Lys, Arg, and His; aromatic residues Phe, Tyr, and Trp, and small-sized amino acids Ala, Ser, Thr, Met, and Gly. Including replacement. In addition to conservative amino acid substitutions, variants of the invention include (i) substitution with one or more non-conservative amino acid residues, wherein the amino acid residue to be replaced is encoded by the genetic code. It may or may not be an amino acid residue, or (i
i) Substitution with one or more amino acid residues having a substituent, or (iii) another compound (for example, a compound for increasing the stability and / or solubility of the polypeptide (for example, polyethylene glycol)) Fusion of the mature polypeptide with, or (iv) fusion of the polypeptide with an additional amino acid, such as an IgG Fc fusion region peptide, or a leader or secretory sequence, or a sequence that facilitates purification. . Such variant polypeptides are considered to be within the skill of those in the art given the teachings herein.

Polypeptide variants containing, for example, amino acid substitutions of charged amino acids with other charged amino acids or neutral amino acids are proteins that have improved properties (eg, less aggregation). Can be generated. Aggregation of pharmaceutical formulations results in both reduced activity and increased clearance due to the immunogenic activity of the aggregate. (Pinckard et al., Clin. Exp. Immunol. 2: 331.
-340 (1967); Robbins et al., Diabetes 36: 838-.
845 (1987); Cleland et al., Crit. Rev. Therapeu
tic Drug Carrier Systems 10: 307-377 (
1993)).

A further embodiment of the invention comprises at least one amino acid substitution, but 50
Amino acids of a polypeptide having an amino acid sequence of no more than amino acid substitutions, even more preferably no more than 40 amino acid substitutions, even more preferably no more than 30 amino acid substitutions, and even more preferably no more than 20 amino acid substitutions. A polypeptide comprising a sequence. Of course, the polypeptide comprises at least one amino acid substitution, in order of increasing preference, at least 10, 9, 8, 7,
The amino acid sequence of the polypeptide of SEQ ID NO: Y, the amino acid sequence encoded by SEQ ID NO: X, and no more than 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or
Alternatively, it is highly preferred to have an amino acid sequence that includes the amino acid sequence encoded by the cDNA contained in cDNA plasmid Z. In certain embodiments, the amino acid sequence of SEQ ID NO: Y or a fragment thereof (eg, the mature forms and / or other fragments described herein), the amino acid sequence encoded by SEQ ID NO: X or a fragment thereof, and / or The number of additions, substitutions, and / or deletions in the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. Z or a fragment thereof is 1-5, 5-10,
5 to 25, 5 to 50, 10 to 50, or 50 to 150, and conservative amino acid substitutions are preferred. As discussed herein, the polypeptides of the present invention can be used to produce fusion proteins. For example, the polypeptides of the present invention can be used as an antigenic tag when fused to a second protein. Antibodies raised against the polypeptides of the invention can be used to indirectly detect a second protein by binding to the polypeptide. Furthermore, since secreted proteins target cellular locations based on transport signals, the polypeptides of the invention shown to be secreted can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to the polypeptides of the invention include heterologous signal sequences, as well as other heterologous functional regions. This fusion need not necessarily be direct, but can occur via a linker sequence.

In certain preferred embodiments, the protein of the invention comprises a polypeptide of N
It includes fusion proteins that are truncation variants and / or C-terminal truncation variants.
In a preferred embodiment, the present application contemplates at least 90%, 95%, 96%, 97%, 98 nucleic acid sequences encoding a polypeptide having the amino acid sequences of particular N-terminal and C-terminal deletion variants. %, Or 99% identical nucleic acid molecules. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also included in the invention.

In addition, fusion proteins can also be engineered to improve the properties of the polypeptides of the invention. For example, regions of additional amino acids, especially charged amino acids,
It may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from host cells, or subsequent manipulation and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Addition of peptide moieties to facilitate manipulation of the polypeptide is a technique well known and routine in the art.

As will be appreciated by those in the art, the polypeptides of the invention described above, and epitope-bearing fragments thereof, can be fused to heterologous polypeptide sequences. For example,
The polypeptide of the present invention is an immunoglobulin (IgA, IgE, IgG, IgM)
Constant domain or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof), resulting in a chimeric polypeptide. Such fusion proteins may facilitate purification and increase half-life in vivo. This has been shown for a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. For example, EP 394,8
27; Traunecker et al., Nature, 331: 84-86 (1988).
)checking. IgG fusion proteins having a disulfide-bonded dimer structure due to disulfide bonds of the IgG moiety are also more effective in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone Was found. (Funtoulakis et al., J. Biochem.
, 270: 3958-3964 (1995)).

Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotides of the present invention, host cells, and the production of polypeptides by recombinant techniques. For example, the vector can be a phage vector, a plasmid vector, a viral vector, or a retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation generally will be complete (complementary).
only occurs in host cells.

The polynucleotide of the present invention may be ligated to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

The polynucleotide insert may be labeled with a suitable promoter (eg, the phage λPL promoter, the E. coli lac promoter, the trp promoter, the phoA promoter and the tac promoter, the SV40 early promoter and the SV40 late promoter, to name a few). Should be operably linked to the LTR promoter). Other suitable promoters are known to those of skill in the art. The expression construct further comprises sites for transcription initiation, transcription termination,
And in the transcribed region, it contains a ribosome binding site for translation. The coding portion of the transcript expressed by this construct is preferably a translation initiation codon initially, and a termination codon (U) appropriately located at the end of the polypeptide to be translated.
AA, UGA or UAG).

As indicated, the expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance genes for eukaryotic cell culture, as well as E. coli. It contains a tetracycline, kanamycin or ampicillin resistance gene for cultivation in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells (eg E. c.
oli, Streptomyces and Salmonella typhim
urium cells); fungal cells (eg yeast cells (eg Saccharom)
yces cerevisiae or Pichia pastoris (AT
CC Accession No. 201178))); insect cells (eg Drosophilia
S2 and Spodoptera Sf9 cells); animal cells (eg, CHO cells, COS cells, 293 cells, and Bowes melanoma cells); and plant cells, but are not limited thereto. Appropriate culture media and conditions for the above-described host cells are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE6
0 and pQE-9 (available from QIAGEN, Inc.); pBluesc
ript vector, Phagescript vector, pNH8A, pNH16
a, pNH18A, pNH46A (Stratagene Cloning S
systems, Inc. And ptrc99a, pKK223.
-3, pKK233-3, pDR540, pRIT5 (Pharmacia B
iotech, Inc. Available from). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (S
(available from tratagene); and pSVK3, pBPV, pMSG
And pSVL (available from Pharmacia). Preferred expression vectors for use in yeast systems include pYES2, pYD1, pTEF1
/ Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph,
pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.
5K, pPIC9K, and PAO815 (all from Invitrogen, C
(available from Arlbad, CA), but is not limited thereto. Other suitable vectors will be readily apparent to those of skill in the art.

Introduction of the construct into the host cell is carried out by calcium phosphate transfection, DE
It can be provided by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described by Davis et al., Basic Metho.
It is described in many standard laboratory manuals such as ds In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by host cells lacking the recombinant vector.

Polypeptides of the invention may be ammonium sulfate precipitated or ethanol precipitated, acid extracted, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectins. It can be recovered from recombinant cell culture and purified by well known methods, including chromatography. Most preferably high performance liquid chromatography (“HPLC”)
Is used for purification.

The polypeptides of the present invention may also be recovered from: purified products from natural sources, including body fluids, tissues and cells, whether directly isolated or cultured; chemical; Products of synthetic procedures; and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host used in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the present invention may also, in some cases, include an initial modified methionine residue as a result of host-mediated processes. Therefore, it is well known in the art that, in general, the N-terminal methionine encoded by the translation initiation codon is highly efficiently removed from any post-translational protein in all eukaryotic cells. N-terminal methionine is also effectively removed in most prokaryotes in most proteins, but for some proteins this prokaryotic removal process depends on the nature of the amino acids to which the N-terminal methionine is covalently attached. And is inefficient.

In one embodiment, the yeast Pichia pastoris is used to express a polypeptide of the invention in a eukaryotic cell line. Pichi
a pastoris is a methyltrophic yeast that can metabolize with methanol as its sole carbon source. The major step in the methanol metabolism pathway is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pas
toris produces high levels of alcohol oxidase, due in part to the relatively low affinity of alcohol oxidase for O ₂ . Consequently, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active in growth media that relies on methanol as the major carbon source. In the presence of methanol, the alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of total soluble protein in Pichia pastoris. Ellis, S .; B. Et al., Mol. Cell.
Biol. 5: 1111-21 (1985); Koutz, P .; J. Et al, Yea
st 5: 167-77 (1989); Tschopp, J .; F. Nucl
． Acids Res. 15: 3859-76 (1987). Thus, a transcriptionally regulated heterologous coding sequence (eg, a polynucleotide of the invention, etc.) under the transcriptional regulation of all or part of the AOX1 regulatory sequence is grown in the presence of methanol in Pich.
It is expressed exponentially at high levels in ia yeast.

In one example, the plasmid vector pPIC9K was transformed into "Pichia P
rotocols: Methods in Molecular Biolog
y ", D.I. R. Higgins and J.M. Edited by Cregg (The Humana
Press, Totowa, NJ, 1998), and is used to express DNA encoding a polypeptide of the invention as shown herein in the Pichea yeast system, essentially as described. This expression vector is a Pichia pastoris located upstream of the multiple cloning site.
The strong AOX1 promoter linked to the alkaline phosphatase (PHO) secretion signal peptide (ie leader) allows expression and secretion of the polypeptides of the invention.

Many other yeast vectors (eg pYES2, pYD1, pTEF1 / Ze
o, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pP
IC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K
, And PAO815) are arranged appropriately such that transcription, translation, secretion (if desired), etc., including an in-frame AUG when the proposed expression construct is required, as will be readily appreciated by those of skill in the art. Can be used in place of pPIC9K as long as it provides a good signal.

In another embodiment, high level expression of a heterologous coding sequence (eg, a polynucleotide of the invention, etc.) is achieved by cloning a heterologous polynucleotide of the invention into an expression vector (eg, pGAPZ or pGAPZalpha). , And can be achieved by growing the yeast culture in the absence of methanol.

In addition to including host cells containing the vector constructs discussed herein, the present invention also includes primaries of vertebrate origin, particularly mammalian origin.
y) Includes host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, coding sequences) and / or genetic material operably linked to a polynucleotide of the invention (eg, A heterologous polynucleotide sequence) and activates, alters, and / or amplifies the endogenous polynucleotide. For example, techniques known in the art can be used to operably link heterologous regulatory regions (eg, promoters and / or enhancers) and endogenous polynucleotide sequences via homologous recombination (eg, 199).
US Patent No. 5,641,670 issued June 24, 1995; International Publication No. WO 96/29411 published September 26, 1996; International published August 4, 1994 Publication WO 94/12650; Koller et al., Proc.
． Natl. Acad. Sci. USA 86: 8932-8935 (1989).
); And Zijlstra et al., Nature 342: 435-438 (19).
89). Each of these disclosures is incorporated by reference in their entirety).

In addition, the polypeptides of the invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Stru).
cultures and Molecular Principles, W.C. H.
Freeman & Co. , N .; Y. And Hunkapiller et al., Na
Tur., 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide can be synthesized by using a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, the normal amino acid D isomer, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g. -Abu, e-Ahx, 6-aminohexanoic acid,
Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b- Alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. Furthermore, the amino acid is D (dextrorotatory)
Or it may be L (levorotatory).

The present invention may be used during or after translation, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular agents. It includes polypeptides of the invention that are differentially modified, such as by binding to a ligand. Any of numerous chemical modifications, including the following may be carried out by known, but not limited to techniques: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage by NaBH _4; acetylation, Formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Further post-translational modifications encompassed by the present invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone. Attachment, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label such as an enzymatic label, a fluorescent label, an isotope label or an affinity label to allow detection and isolation of the protein.

The present invention also provides chemically modified derivatives of the polypeptides of the present invention that may provide additional advantages such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. (See US Pat. No. 4,179,337). The chemical moieties for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three or more attached chemical moieties.

The polymer can be a polymer of any molecular weight and can be branched or unbranched. For polyethylene glycol, a preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term "about" refers to some molecules in the preparation of polyethylene glycol Heavy, and some indicate lighter than the indicated molecular weight). The desired therapeutic profile (eg, desired duration of sustained release, effect on biological activity in some cases, ease of handling, degree of antigenicity or lack of antigenicity, and therapeutic protein or analog). Other sizes may be used, depending on other known effects of polyethylene glycol).

The polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domains of this protein. There are numerous coupling methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF to P, incorporated herein by reference).
EG)), Malik et al., Exp. Hematol. 20: 1028-
1035 (1992) (pegylation of GM-CSF with tresyl chloride).
See also report)))). For example, polyethylene glycol can be covalently attached via a amino acid residue by a reactive group such as a free amino or carboxyl group. Reactive groups are groups to which activated polyethylene glycol molecules can be attached. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Residues may be mentioned. Sulfhydryl residues can also be used as a reactive group to attach the polyethylene glycol molecule. Preferred for therapeutic purposes is a bond at the amino group, eg at the N-terminus or at the lysine group.

Proteins that are chemically modified at the N-terminus may be specifically desired. Using polyethylene glycol as an illustration of the composition of the invention, from various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, the pegs to be performed. The type of oxidization reaction and the method of obtaining the selected N-terminal pegylated protein can be selected. The method of obtaining the N-terminal PEGylated preparation (ie, separating this moiety from other mono-PEGylated moieties, if necessary) is performed by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. The selective protein chemically modified by N-terminal modification is
It can be achieved by reductive alkylation, which takes advantage of the differential reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in a particular protein. Under appropriate reaction conditions, substantially selective derivatization of proteins at the N-terminus with a carbonyl group-containing polymer is achieved.

Polypeptides of the invention can be monomeric or multimeric (ie, dimers, trimers,
Tetramers and higher multimers). Accordingly, the present invention relates to monomeric and multimeric polypeptides of the invention, their preparation and compositions (preferably therapeutic agents) containing them. In particular embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further embodiment, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the present invention can be homomers or heteromers. The term homomer, as used herein, is encoded by the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: Y or encoded by SEQ ID NO: X or the complement of SEQ ID NO: X, and / or by the cDNA plasmid Z. A multimer containing only amino acid sequences (including fragments, variants, splice variants and fusion proteins corresponding to those described herein). These homomers may include polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers that only include polypeptides that have the same amino acid sequence. In another particular embodiment, the homomers of the invention are
It is a multimer containing polypeptides having different amino acid sequences. In certain embodiments, the multimers of the invention are homodimers (eg, including polypeptides having the same or different amino acid sequences) or homotrimers (eg, polymers having the same and / or different amino acid sequences). (Including peptides). In a further embodiment, the homomeric multimers of the invention are at least homodimers, at least homotrimers or at least homotetramers.

The term heteromer, as used herein, refers to a multimer that comprises, in addition to a polypeptide of the invention, one or more heterologous polypeptides (ie, polypeptides of different proteins). In certain embodiments, the multimers of the invention are heterodimers, heterotrimers or heterotetramers. In a further embodiment, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer or at least a heterotetramer.

The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent attachment and / or may be indirectly linked, for example by liposome formation. Thus, in one embodiment, multimers of the invention (eg, homodimers or homotrimers, etc.) are formed when the polypeptides of the invention contact each other in solution. In another embodiment, a heteromultimer of the invention (eg, a heterotrimer or heterotetramer, etc.) is a polypeptide of the invention in solution in which the antibody to the polypeptide of the invention (of the invention Formed) when contacted with antibodies (including antibodies against heterologous polypeptide sequences in a fusion protein). In other embodiments, the multimers of the invention are formed by covalent attachment to and / or between polypeptides of the invention. Such covalent bonds are included in the polypeptide sequence (eg, listed in SEQ ID NO: Y or contained in a polypeptide encoded by SEQ ID NO: X and / or cDNA plasmid: Z,
(Polypeptide sequence) may comprise one or more amino acid residues contained within. In one example,
This covalent bond is a bridge between cysteine residues that are present between the interacting polypeptide sequences in the native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of chemical or recombinant manipulation. Alternatively, such a covalent bond may comprise one or more amino acid residues contained in the heterologous polypeptide sequence in the fusion protein. In one example, the covalent bond is between the heterologous sequences contained in the fusion protein of the invention (eg, US Pat. No. 5,47).
See No. 8,925). In a particular example, this covalent bond is between the heterologous sequences contained in the Fc fusion protein of the invention (as described herein).
In another particular example, the covalent attachment of a fusion protein of the invention is such that another protein capable of forming covalently linked multimers (eg, osteoprotegerin).
tegerin) (see, eg, WO 98/49305, the contents of which are incorporated herein by reference in its entirety). In another embodiment, two or more polypeptides of the invention are linked via a peptide linker. For example, US Pat. No. 5,0
73,627 (incorporated herein by reference). Proteins comprising multiple polypeptides of the invention separated by a peptide linker can be produced using conventional recombinant DNA techniques.

Another method of preparing a multimeric polypeptide of the invention involves the use of a polypeptide of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper domain and isoleucine zipper domain
They are polypeptides that promote multimerization of the proteins they were discovered. The leucine zipper was originally designed for several DNA-binding proteins (Landschul).
Z et al., Science 240: 1759 (1988)) and has since been found in a variety of different proteins. Known leucine zippers are naturally occurring peptides that dimerize or trimerize, and their derivatives. An example of a leucine zipper domain suitable for producing the soluble multimeric protein of the present invention is the leucine zipper domain described in PCT application WO94 / 10308, which is incorporated herein by reference. A recombinant fusion protein comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimers in solution is expressed in a suitable host cell and the resulting soluble multimeric fusion protein is , Harvested from the culture supernatant using techniques known in the art.

The trimeric polypeptides of the invention may provide the advantage of increased biological activity.
Preferred leucine zipper and isoleucine moieties are those that preferentially form trimers. One example is Hoppe et al. (FEBS Letters 3
44: 191, (1994)) and U.S. patent application Ser. No. 08 / 446,922 (
Leucine zipper derived from lung surfactant protein D (SPD), as described herein by reference). Other peptides derived from naturally occurring trimeric proteins can be used in the preparation of trimeric polypeptides of the invention.

In another example, a protein of the invention is bound by an interaction between the Flag® polypeptide sequences contained in a fusion protein of the invention that comprises a Flag® polypeptide sequence. In a further embodiment, a binding protein of the invention is bound by an interaction between a heterologous polypeptide sequence contained in a Flag® fusion protein of the invention and an anti-Flag® antibody.

The multimers of the invention can be produced using chemical techniques known in the art. For example, the polypeptides desired to be included in the multimers of the present invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art (eg, US Pat. , 478,925, which is incorporated herein by reference in its entirety). Furthermore, the multimers of the invention are known in the art to form one or more intermolecular bridges between cysteine residues located in the sequence of the polypeptide desired to be included in the multimer. (See, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). Furthermore, the polypeptides of the present invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art modified one or more of these. It can be applied to generate multimers containing polypeptides (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, techniques known in the art can be applied to produce liposomes containing the polypeptide component desired to be included in the multimers of the invention (see, eg, US Pat. No. 5,478,925). See
Which is hereby incorporated by reference in its entirety).

Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptides comprised in the multimers of the invention are recombinantly produced using fusion protein technology described herein or otherwise known in the art (eg, , US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In a particular embodiment, a polynucleotide encoding a homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention linked to a sequence encoding a linker polypeptide and then the original C-terminus. Produced by further ligation to a synthetic polynucleotide (lacking a leader sequence) encoding the translation product of the polypeptide in the reverse direction from N to the N-terminus (eg, US Pat. No. 5,478).
, 925, which is incorporated herein by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art have been applied to include a set of the invention comprising a transmembrane domain (or hydrophobic peptide or signal peptide). The modified polypeptide is produced and can be incorporated into liposomes by membrane reconstitution techniques (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). Is incorporated as).

Antibodies Further polypeptides of the invention may immunospecifically bind to a polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y of the invention, and / or an epitope of the invention (specific antibody). -Antibodies and T-cell antigen receptors (TCRs) as determined by immunoassays well known in the art for assaying antigen binding. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, Fab expression libraries produced by Fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the invention), and epitope binding fragments of any of the above antibodies. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (ie, molecules that contain an antigen binding site that immunospecifically binds an antigen). Say. The immunoglobulin molecules of the invention can be of any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, IgA, and I.
gY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1
And IgA2) or a subclass of molecule.

Most preferably, the antibody is a human antigen-binding antibody fragment of the invention and comprises Fab, Fab 'and F (ab') 2, Fd, single chain Fv (scFv), single chain antibody, disulfide linked Fv. (SdFv) and fragments containing either the VL domain or the VH domain are included, but are not limited to. Antigen-binding antibody fragments, including single chain antibodies, have variable regions either alone or in the following:
It may be included in combination with all or part of the hinge region, CH1 domain, CH2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of hinge region, CH1 domain, CH2 domain and CH3 domain and variable regions. Antibodies of the invention can be from any animal origin including birds and mammals. Preferably, the antibody is human, murine (
For example, mouse and rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, a "human" antibody includes an antibody having the amino acid sequence of human immunoglobulin and is transgenic and endogenous to a human immunoglobulin library or for one or more human immunoglobulins. Animals that do not express sex immunoglobulins (see below and, for example, Kucherlapati et al., US Pat.
939,598).

Antibodies of the invention may be monospecific, bispecific, trispecific or more multivalent multispecific. Multispecific antibodies can be specific for different epitopes of a polypeptide of the invention, or specific for both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Can be objective. For example, PCT Publication WO 93/17715; WO 92/08802;
WO 91/00360; WO 92/05793; Tutt et al., J. Am. Immu
nol. 147: 60-69 (1991); U.S. Pat. No. 4,474,893;
No. 4,714,681; No. 4,925,648; No. 5,573,92.
No. 0; 5,601, 819; Kostelny et al., J. Am. Immunol.
148: 1547-1553 (1992).

The antibodies of the present invention may be described or specified with respect to the epitopes or portions of the polypeptides of the present invention to which they recognize or specifically bind. Portions of this epitope or polypeptide can be identified as described herein, for example, by N-terminal and C-terminal positions, by size at contiguous amino acid residues. Antibodies that specifically bind any epitope or polypeptide of the invention can also be excluded. Accordingly, the present invention includes antibodies that specifically bind the polypeptides of the present invention and allow for the exclusion of the polypeptides of the present invention.

The antibodies of the invention may also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the invention are included. At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity to the polypeptides of the present invention. Also included in the invention are antibodies that bind a polypeptide having (as calculated using methods known in the art and described herein). In certain embodiments, the antibodies of the invention cross-react with human, mouse, rat and / or rabbit homologs of human proteins and their corresponding epitopes. 9 for polypeptides of the invention
Less than 5%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, 6
Binds polypeptides having less than 5%, less than 60%, less than 55% and less than 50% identity (as calculated using methods known in the art and described herein) Antibodies that do not are also included in the invention. In certain embodiments, the above cross-reactivity is any monospecific antigenic or immunogenic polypeptide disclosed herein, or 2, 3, 4, 5 or more specific. It relates to a combination of antigenic and / or immunogenic polypeptides. Further included in the invention are antibodies that bind the polypeptide encoded by the polynucleotide that hybridizes to the polynucleotide of the invention under stringent hybridization conditions (as described herein). . The antibody of the present invention also comprises
Their binding affinities for the polypeptides of the invention may be described or specified. Preferred binding affinity is less than 5 × 10 ⁻² M, less than 10 ⁻² M, 5 ×
Less than 10 ^-3 M, less than 10 ^-3 M, less than 5 × 10 ^-4 M, less than 10 ^-4 M, 5 × 10 ^-5 M
Less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁶ M, less than 5 × 10 ⁻⁷ M, 1
Less than 0 ⁻⁷ M, less than 5 × 10 ⁻⁸ M, less than 10 ⁻⁸ M, less than 5 × 10 ⁻⁹ M, less than 10 ⁻⁹ M, less than 5 × 10 ⁻¹⁰ M, less than 10 ⁻¹⁰ M, 5 × Less than 10 ^-11 M, less than 10 ^-11 M,
Less than 5 × 10 ^-12 M, less than 10 ^-12 M, less than 5 × 10 ^-13 M, less than 10 ^-13 M, 5 ×
Included are affinities with dissociation constants or Kd's less than 10 ^-14 M, less than 10 ^-14 M, less than 5 × 10 ^-15 M or less than 10 ^-15 M.

The present invention also includes antibodies to epitopes of the invention, as determined by any method known in the art for determining competitive binding (eg, immunoassays described herein). Antibodies that competitively inhibit the binding of In a preferred embodiment, the antibody is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% competitively bound to an epitope. Inhibit.

The antibodies of the present invention can act as agonists or antagonists of the polypeptides of the present invention. For example, the invention includes antibodies that disrupt the receptor / ligand interaction with a polypeptide of the invention either partially or completely. Preferably, the antibodies of the invention bind the antigenic epitopes disclosed herein, or portions thereof. The present invention features both receptor-specific and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie signal transduction) can be determined by the techniques described herein, or otherwise known in the art. For example, receptor activation can be determined by phosphorylation of the receptor (eg, tyrosine or serine / threonine), or immunoprecipitation followed by detection of its substrate by Western blot analysis (eg, as described above). . In certain embodiments, in the absence of this antibody, the ligand activity or receptor activity is at least 95% of its activity,
At least 90%, at least 85%, at least 80%, at least 75%,
Antibodies are provided that inhibit at least 70%, at least 60%, or at least 50%.

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, and, preferably, with unbound receptors or unbound ligands. It has the characteristics of a receptor-specific antibody that does not recognize. Similarly, the present invention binds ligands and neutralizing antibodies that prevent the binding of ligands to receptors, and ligands, thereby preventing receptor activation, but not ligand binding to receptors. Contains antibodies. Further, the invention includes antibodies that activate the receptor. These antibodies may act as receptor agonists, ie enhance or activate either all or a subset of the biological activation of ligand-mediated receptor activation by, for example, inducing receptor dimerization. obtain. The antibody may be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of the peptides of the invention disclosed herein. The antibody agonist can be produced using a method known in the art. For example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-19.
88 (1998); Chen et al., Cancer Res. 58 (16): 366
8-3678 (1998); Harrop et al., J. Am. Immunol. 161 (4
): 1786-1794 (1998); Zhu et al., Cancer Res: 58.
(15): 3209-3214 (1998); Yoon et al., J. Am. Immunol
． 160 (7): 3170-3179 (1998); Prat et al. Cell
． Sci. 111 (Pt2): 237-247 (1998); Pittard et al.,
J. Immunol. Methods 205 (2): 177-190 (199).
7); Liautard et al., Cytokine 9 (4): 233-241 (1).
997); Carlson et al. Biol. Chem. 272 (17): 11
295-11301 (1997); Taryman et al., Neuron 14 (4).
): 755-762 (1995); Muller et al., Structure 6 (
9): 1153-1167 (1998); Bartunek et al., Cytokin.
e 8 (1): 14-20 (1996), all of the above references are incorporated herein by reference in their entireties.

Antibodies of the invention can be used, for example, but not limited to, to purify, detect, and target the polypeptides of the invention. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays for qualitatively and quantitatively measuring the levels of polypeptides of the invention in biological samples. For example, Harlo
w et al., Antibodies: A Laboratory Manual, (C
See Old Spring Harbor Laboratory Press, 2nd Edition, 1988), which is hereby incorporated by reference in its entirety.

As discussed in further detail below, the antibodies of the invention can be used either alone or in combination with other compounds. The antibody can further be recombinantly fused to the heterologous polypeptide at the N-terminus or C-terminus to the polypeptide or other compound, or can be chemically linked (including covalent and non-covalent). For example, the antibodies of the present invention can be recombinantly fused or conjugated to effector molecules such as molecules and heterologous polypeptides, agents, radionuclides, or toxins that are useful as labels in detection assays. For example, PCT publication WO92 / 0849
5; WO91 / 14438; WO89 / 12624; US Pat. No. 5,314,9
95; and EP 396,387.

The antibodies of the invention are modified (ie, covalently attached).
attachment), by covalent attachment of any type of molecule to the antibody such that the antibody does not prevent it from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylations, acetylations, pegylations, phosphorylations.
n), amidation, known blocking / blocking groups
Antibodies modified by derivatization with, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the invention may be produced by any suitable method known in the art.
Polyclonal antibodies to the antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the invention may be administered to a variety of host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. . Various adjuvants can be used to increase the immunological response, depending on the host species, and Freund's (complete and incomplete) mineral gels such as aluminum hydroxide (
mineral gel), surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
Keyhole limpet hemocyanin, dinitrophenol, and BCG (bacillus Calmette-Guerin) and corynebacterium parvum
Include, but are not limited to, potentially useful human adjuvants such as Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using the hybridoma technology known in the art, including: Har
Low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
, 2nd edition, 1988); Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), which is incorporated by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods of producing and screening for specific antibodies using hybridoma technology are routine and well known in the art, and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or cells expressing such a peptide. Once an immune application is detected (eg, an antibody specific for the antigen is detected in mouse serum), the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg cells from cell line SP20 available from the ATCC). Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides a method for producing an antibody produced by a method comprising culturing a monoclonal antibody and a hybridoma cell secreting the antibody of the present invention, wherein, preferably, this A hybridoma is a hybridoma clone that fuses myeloma cells with splenocytes isolated from a mouse immunized with the antigen of the present invention and then secretes an antibody capable of binding to the polypeptide of the present invention.
Produced by screening the hybridomas resulting from the fusion.

Antibody fragments that recognize a particular epitope can be produced by known techniques. For example, Fab and F (ab ') 2 fragments of the invention may be papain (to produce Fab fragments) or pepsin (F (ab').
Enzymes) to produce 2 fragments) and can be produced by proteolytic cleavage of immunoglobulin molecules. F (ab ′) 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In certain embodiments, such phage may be cloned into a repertoire antibody library or a combinatorial antibody library (eg,
It can be used to display an antigen binding domain expressed from human or mouse). Phage expressing an antigen binding domain that binds an antigen of interest may be selected or identified with the antigen (eg, using labeled antigen or antigen bound or captured on a solid surface or solid beads). . Phage used in these methods are typically phage having the antibody domain of Fab, Fv or disulfide stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Is a filamentous phage containing the fd and M13 binding domains expressed from E. coli. Examples of phage display methods that can be used to generate the antibodies of the invention include those disclosed below: Brinkman et al.
Immunol. Methods 182: 41-50 (1995); Ames
Et al., J. Immunol. Methods 184: 177-186 (1995)
); Kettleborough et al., Eur. J. Immunol. 24:95
2-958 (1994); Persic et al., Gene 187 9-18 (19).
97); Burton et al., Advances in Immunology 5
7: 191-280 (1994); PCT published PCT / GB91 / 01134
PCT publication WO90 / 02809; WO91 / 10737; WO92 / 01
047; WO92 / 18619; WO93 / 11236; WO95 / 15982.
WO95 / 20401; and US Pat. No. 5,698,426;
No. 223,409; No. 5,403,484; No. 5,580,717; No. 5,427,908; No. 5,750,753; No. 5,821,047.
No. 5,571,698; No. 5,427,908; No. 5,516.
No. 637; No. 5,780,225; No. 5,658,727; No. 5,7.
33,743 and 5,969,108, each of which is incorporated by reference in its entirety.

As described in the above references, after phage selection, the region encoding the antibody derived from the phage may be used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment. And can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be used using methods known in the art. This method is, for example, the method disclosed below: PCT Publication WO 92/22324; Mullinax et al., Bio.
Techniques 12 (6): 864-869 (1992); and Sa.
wai et al., AJRI 34: 26-34 (1995); and Better et al.
Science 240: 1041-1043 (1998) (the above references are incorporated by reference in their entirety).

Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston.
Et al., Methods in Enzymology 203: 46-88 (19.
91); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some applications, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different portions of this antibody are derived from different animal species (eg, an antibody having variable regions derived from mouse monoclonal antibodies and human immunoglobulin constant regions). Methods for producing chimeric antibodies are known in the art. For example, Morrison, S
science 229: 1202 (1985); Oi et al., BioTechniq.
ues 4: 214 (1986); Gillies et al., (1989) J. Am. Imm
unol. Methods 125: 191-202; and US Pat. No. 5,8.
07,715; 4,816,567; and 4,816,397, which are hereby incorporated by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibodies that bind a desired antigen having one or more complementarity determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions have been identified by methods well known in the art, for example modeling interactions between CDRs and framework residues to identify framework residues important for antigen binding, as well as specific positions. By sequence comparison to identify unusual framework residues in. (For example, Queen et al., US Pat. No. 5,585,089; Riechm.
See Ann, et al., Nature 332: 323 (1988), which are incorporated herein by reference in their entirety). Antibodies can be humanized using various techniques known in the art including, for example: CDR-grafting (European Patent 239,400; PCT Publication WO 91).
/ 09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (European patent 592). 106; European Patent 519,596; Padlan, Molecular Immu.
28 (4/5): 489-498 (1991); Studnic
ka et al., Protein Engineering 7 (6): 805-814.
(1994); Roguska et al., PNAS 91: 969-973 (1994).
)), And chain shuffling (US Pat. No. 5,565,332).

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887 and 4,716
, 111; and PCT publications WO98 / 46645, WO98 / 50433.
, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO
See also 96/33735, and WO 91/10741, each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins but which are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region interfere with endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mouse is then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies against the antigen can be obtained from immunized, transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes are harbored by rearrangement of transgenic mice during B cell differentiation and then undergo class switching and somatic mutations. Thus, the use of such techniques allows for the production of therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lo
nberg and Huszar, Int. Rev. Immunol. 13:65
-93 (1995). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, eg, PCT Publication WO98 / 24893; WO92.
/ 01047; WO96 / 34096; WO96 / 33735; European Patent No. 0.
598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,0.
No. 16; No. 5,545,806; No. 5,814,318; No. 5,88.
5,793,5,916,771; and 5,939,598, which are incorporated herein by reference in their entireties. In addition, Abgenix, Inc. (Freemont, CA) and Genpha
Companies such as rm (San Jose, CA) may be engaged in providing human antibodies to selected antigens using techniques similar to those described above.

Human antibodies that fully recognize the selected epitope were "guided (gui).
ed) selection ”. In this approach,
Selected non-human monoclonal antibodies (eg, murine antibodies) are used to guide the selection of human antibodies that fully recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 (1988)).

Furthermore, antibodies to the polypeptides of the present invention can in turn be utilized to generate anti-idiotypic antibodies that "mimic" the polypeptides of the present invention, using techniques well known to those of skill in the art. (Eg Greenspan and Bona, FASE
BJ. 7 (5): 437-444 (1989); and Nissinoff.
J. Immunol. 147 (8): 2429-2438 (1991)). For example, binding and multimerization of polypeptides.
antibody and / or an antibody that competitively inhibits the binding of the polypeptide of the invention to a ligand is used to "mimic" the multimerization and / or binding domain of the polypeptide and, as a result, the polypeptide and / or
Alternatively, it may generate an anti-idiotype that binds to and neutralizes its ligand.
Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such an anti-idiotype antibody may be used to bind a polypeptide of the invention and / or to bind its ligand / receptor,
Thereby, its biological activity may be blocked.

Polynucleotides Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also provides that under stringent conditions or lower stringency hybridization conditions (
(For example, as described above), a polynucleotide encoding an antibody (preferably an antibody that specifically binds to the polypeptide of the present invention), preferably a polynucleotide having the amino acid sequence of SEQ ID NO: 2 and / or 4. A polynucleotide that encodes a binding antibody, and a polynucleotide that hybridizes to.

These polynucleotides can be obtained and the nucleotide sequence of these polynucleotides determined by any method known in the art. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, Kutmeier et al., BioTechniques 17: 242 (199).
4)), which, in brief, is the synthesis of overlapping nucleotides containing portions of the antibody-encoding sequence, the annealing and ligation of those oligonucleotides, and then this ligation by PCR. Including amplification of the oligonucleotide.

Alternatively, antibody-encoding polynucleotides can be made from nucleic acids from suitable sources. No clone containing a nucleic acid encoding a particular antibody is available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin is
It can be chemically synthesized or from a suitable source (eg, an antibody cDNA library, or any tissue or cell expressing the antibody (eg, a hybridoma cell selected for expression of an antibody of the invention)). The generated cDNA library, or a nucleic acid isolated from it (preferably poly A + RNA), can be used, for example, to identify a cDNA clone from a cDNA library encoding an antibody at the 3'end and 5 It can be obtained by PCR amplification using synthetic primers hybridizable to the'end or by cloning using an oligonucleotide probe specific for that particular gene sequence. PC
The amplified nucleic acid produced by R can then be cloned into a replicable cloning vector using any method known in the art.

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined,
Nucleotide sequences of antibodies can be prepared by methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL.
Laboratory Manual, 2nd Edition, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. Both are incorporated herein by reference in their entirety. )) Can be engineered with to produce antibodies with different amino acid sequences to generate, for example, amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains may be sequenced using methods well known in the art for identifying sequences of complementarity determining regions (CDRs), such as other methods. By comparing the known amino acid sequences of the variable regions of the heavy and light chains to determine the sequence hypervariable regions. Conventional recombinant DNA
Using techniques, one or more CDRs can be inserted within a framework region, eg, within a human framework region to humanize a non-human antibody, as described above. This framework region may be a naturally occurring or common framework region, and preferably a human framework region (for a list of human framework regions see, eg, Chothia et al., J. Mol. Bi.
ol. 278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of the framework regions and the CDRs is
Encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, one or more amino acid substitutions occur within the framework regions, as discussed above, and preferably the amino acid substitutions improve binding of the antibody to its antigen. In addition, such methods provide antibodies that lack one or more intrachain disulfide bonds, either for amino acid substitutions or by deleting one or more variable region cysteine residues that participate in intrachain disulfide bonds. It can be used to generate molecules. Other modifications to the polypeptide are accomplished by the present invention and techniques in the art.

Furthermore, by splicing a gene from a mouse antibody molecule of suitable antigen specificity with a gene from a human antibody molecule of suitable biological activity, "
Chimeric antibody "(Morrison et al., Proc. Natl. Acad. Sci.
81: 851-855 (1984); Neuberger et al., Nature 3
12: 604-608 (1984); Takeda et al., Nature 314:
Techniques developed for the production of 452-454 (1985)) can be used. As mentioned above, a chimeric antibody is a molecule in which different parts are derived from different animal species (
For example, a molecule having a variable region derived from a mouse mAb and a human immunoglobulin constant region), for example, a humanized antibody.

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946)
, 778; Bird, Science 242: 423-42 (1998);
Huston et al., Proc. Natl. Acad. Sci. USA 85:58
79-5883 (1998); and Ward et al., Nature 334: 54.
4-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via amino acid bridges to produce a single chain polypeptide. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Sk
erra et al., Science 242: 1038-1041 (1988)).

Methods of Antibody Production The antibodies of the invention may be produced by any method known in the art for the synthesis of antibodies, particularly by chemical synthesis, or preferably by recombinant expression techniques.

An antibody of the invention, or a fragment, derivative or analog thereof (eg,
Recombinant expression of the heavy or light chain of an antibody of the invention, or a single chain antibody of the invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody.
Once the polynucleotide encoding the antibody molecule or the heavy or light chain of the antibody, or a portion thereof (preferably comprising the variable domain of the heavy or light chain) of the invention is obtained, for the production of the antibody molecule. Vectors can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expression of a polynucleotide containing an antibody encoding a nucleotide sequence are described herein. Methods well known to those of skill in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain operably linked to a promoter. provide. Such a vector may comprise a nucleotide sequence encoding the constant region of an antibody molecule (eg PCT International Publication No. WO 86/05).
807; PCT International Publication No. WO 89/01036; and US Pat. No. 5,1.
22, 464), and the variable region of the antibody can be cloned into such a vector for full expression of the heavy or light chain.

The expression vector is transferred into a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention.
Accordingly, the invention includes a host cell containing a single chain antibody of the invention operably linked to a polynucleotide encoding an antibody of the invention, or its heavy or light chain, or a heterologous promoter. In a preferred embodiment for expression of double-chain antibodies, the vectors encoding both heavy and light chains are co-expressed in a host cell for expression of whole immunoglobulin molecules, as described in detail below. Can be done.

A variety of host expression vector systems may be utilized to express the antibody molecule of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest may be produced and subsequently purified, but also in situ when transformed or transfected with sequences encoding the appropriate nucleotides. 2 represents a cell capable of expressing an antibody molecule of the invention. These include, but are not limited to: Bacteria transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA expression vectors or cosmid DNA expression vectors containing antibody-encoding sequences (eg, Microorganisms such as E. coli, B. subtilis); yeast transformed with a recombinant yeast expression vector containing antibody-encoding sequences (eg, Saccharomyces, Pichia); recombinant viral expression containing antibody-encoding sequences Insect cell lines infected with vectors (eg baculovirus); Plant cell lines infected with recombinant viral expression vectors (eg cauliflower mosaic virus, CaMV; Tobacco mosaic virus, TMV) or recombinants containing sequences encoding antibodies. Plastic Bromide expression vector (for example,
A plant cell line transformed with a Ti plasmid), or a promoter derived from the genome of a mammalian cell (eg, metallothionein promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K). A mammalian cell line carrying a recombinant expression construct comprising a promoter (eg COS, CHO, BHK, 293, 3T3 cells). Preferably bacterial cells such as Escherichia coli, and more preferably eukaryotic cells, especially for the expression of whole recombinant antibody molecules, are used for the expression of the recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major immediate early gene promoter element from human cytomegalovirus are effective expression systems for antibodies. (Föcking et al., Gene 45:10.
1 (1986); Cockett et al., Bio / Technology 8: 2 (
1990)).

In bacterial systems, many expression vectors may be advantageously selected depending on the use intended for the expression of the antibody molecule. For example, if large quantities of such proteins are produced, vectors that direct high levels of expression of fusion protein products that are easily purified may be desired for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to: the antibody coding sequence in frame with the lacZ coding region in the vector.
Can be individually ligated together, resulting in the production of a fusion protein in E. coli. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (198).
3)); pIN vector (Inouye & Inouye, Nucleic Ac)
ids Res. 13: 3101-3109 (1985); Van Heeke.
& Schuster, J .; Biol. Chem. 24: 5503-5509 (1
989)) and so on. The pGEX vector can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector was designed to contain a thrombin or Factor Xa protease cleavage site so that the cloned target gene product is G
It can be released from the ST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express heterologous genes. This virus grows in Spodoptera frugiperda cells. The antibody coding sequences can be individually cloned into non-essential regions of the virus (eg the polyhedrin gene) and placed under control of the AcNPV promoter (eg the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, the sequence encoding the antibody of interest may be linked to the adenovirus transcription / translation control complex, such as the late promoter and a three-part leader sequence. Then
This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in nonessential regions of the viral genome (eg, E1 or E3 regions) result in recombinant viruses that are viable and capable of expressing antibody molecules in infected hosts (eg, Logan & Shenk).
, Proc. Natl. Acad. Sci. USA 81: 355-359 (1
984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals are ATG
It includes a start codon and adjacent sequences. In addition, this initiation codon ensures that translation of the entire insert is ensured by the reading frame of the desired coding sequence (
must be in phase with the reading frame). These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al. Methods i).
n Enzymol. 153: 51-544 (1987)).

In addition, a host cell line may be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired.
Such modifications (eg glycosylation) and processing of protein products (
Cleavage, for example) can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells can be used that have the cellular machinery for the precise processing of primary transcription, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and especially, for example, BT48
3, breast cancer cell lines such as Hs578T, HTB2, BT20 and T47D, and normal breast cell lines such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using an expression vector that includes a viral origin of replication, the host cell may employ suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites,
Etc.), and DNA controlled by the selectable marker. Following the introduction of exogenous DNA, the engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and the cell stably integrates the plasmid into its chromosome and proliferates to form cell foci, which in turn can be cloned into a cell line. To be able to be expanded to. This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Sz.
ybalski, Proc. Natl. Acad. Sci. USA 48:20
2 (1992)), and adenine phosphoribosyl transferase (Low
y et al., Cell 22: 817 (1980)), but without limitation, these genes may be used in tk-, hgprt- or aprt- cells, respectively. Antimetabolite resistance can also be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wig.
ler et al., Natl. Acad. Sci. USA 77: 357 (1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:15.
27 (1981)); gpt, which confers resistance to mycophenolic acid (
Mulligan & Berg, Proc. Natl. Acad. Sci. USA
78: 2072 (1981)); neo, which is the aminoglycoside G-418.
(Clinical Pharmacy 12: 488-
505; Wu and Wu, Biotherapy 3: 87-95 (199).
1); Tolstoshev, Ann. Rev. Pharmacol. Toxi
col. 32: 573-596 (1993); Mulligan, Science.
Ce 260: 926-932 (1993); and Morgan and A.
nderson, Ann. Rev. Biochem. 62: 191-217 (1
993); May, 1993, TIB TECH 11 (5): 155-21.
5); and hygro, which confers resistance to hygromycin (S
anterre et al., Gene 30: 147 (1984)). Methods well known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described below: eg Ausub.
el et al. (ed.), Current Protocols in Molecular
r Biology, John Wiley & Sons, NY (1993); K
riegler, Gene Transfer and Expression
, A Laboratory Manual, Stockton Press,
NY (1990); and Chapters 12 and 13, Dracopoli et al. (Eds.).
, Current Protocols in Human Genetics
, John Wiley & Sons, NY (1994); Colberre-G.
arapin et al. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (for review,
Bebington and Hentschel, The use of ve
ctors based on gene amplification fo
r the expression of cloned genes in
mammalian cells in DNA cloning, Vol. Three
． (See Academic Press, New York, 1987). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is linked to the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257.
(1983)).

A host cell may be co-transfected with two expression vectors of the invention, a first vector encoding a polypeptide from the heavy chain and a second vector encoding a polypeptide from the light chain. .. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used, which encodes and expresses both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Pr
audfoot, Nature 322: 52 (1986); Kohler, P.
roc. Natl. Acad. Sci. USA 77: 2197 (1980)).
. The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

Once the antibody molecule of the present invention has been produced by an animal, chemically synthesized, or recombinantly expressed, any of those known in the art for the purification of immunoglobulin molecules. Methods such as chromatography (eg, ion exchange,
It can be purified by affinity (particularly by affinity for protein A followed by specific antigen), and size column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.
Furthermore, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

The present invention provides a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 of this polypeptide of the invention.
Or 100 amino acids), recombinantly fused or chemically linked (including both covalent and non-covalent bonds) to produce a fusion protein. The fusion need not be direct, but can occur via a linker sequence. The antibody is a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70,
(80, 90, or 100 amino acids). For example, targeting a polypeptide of the invention to a particular cell type, either in vitro or in vivo, by fusing or conjugating the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Antibodies can be used to Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, eg, Harbor et al., Supra, and PCT Publication WO 93/21.
232; EP 439,095; Naramura et al., Immunol. Lett
． 39: 91-99 (1994); U.S. Patent No. 5,474,981; Gill.
ies et al., PNAS 89: 1428-1432 (1992); Fell et al., J.
． Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The invention further includes compositions comprising a polypeptide of the invention fused or linked to a domain other than the variable region of an antibody. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The part of this antibody fused to the polypeptide of the invention comprises the constant region, hinge region, CH1 domain, C
It may include the H2 domain, and the CH3 domain, or any combination of whole domains or portions thereof. These polypeptides can also be fused or conjugated to portions of the above antibodies to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the invention to antibody moieties are known in the art. For example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,04.
No. 6, No. 5,349,053; No. 5,447,851; No. 5,112.
, 946; EP 307,434; EP 367,166; PCT publication WO9.
6/04388; WO91 / 06570; Ashkenazi et al., Proc. N
atl. Acad. Sci. USA 88: 10535-10539 (1991).
); Zheng et al. Immunol. 154: 5590-5600 (199
5); and Vil et al., Proc. Natl. Acad. Sci. USA 89
: 11337-11341 (1992) (the above references are incorporated by reference in their entireties).

A polypeptide corresponding to a polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y, as discussed above, is used to increase the in vivo half-life of the polypeptide, or It can be fused or conjugated to the above antibody moieties for use in immunological assays using methods known in the art.
In addition, the polypeptide corresponding to SEQ ID NO: Y can be fused or conjugated to the antibody portion described above to facilitate purification. One reported example describes a chimeric protein consisting of the first two domains of the human CD4 polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins (E
P 394,827; Traunecker et al., Nature 331: 84-.
86 (1988)). Polypeptides of the invention fused or conjugated to an antibody having a disulfide-linked dimer structure (due to IgG) also bind to other molecules than monomeric secreted proteins or protein fragments alone. And may be more efficient at neutralizing (Funtoulakis et al., J. Bio.
chem. 270: 3958-3964 (1995)). In many cases, the Fc portion of fusion proteins may be beneficial in therapy and diagnosis, thus resulting, for example, in improved pharmacokinetic properties (EP A 232,262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 have been used for the purpose of high throughput screening assays to identify antagonists of hIL-5.
has been fused to the c moiety (Bennett et al., J. Molecular Rec.
ignition 8: 52-58 (1995); Johnson et al., J. Am. B
iol. Chem. 270: 9459-9471 (1995)).

Furthermore, the antibodies of the invention or fragments thereof may be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats.
The tag, provided by W. Worth, CA, 91311), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc
． Natl. Acad. Sci. Hexa-histidine provides convenient purification of fusion proteins, as described in USA 86: 821-824 (1989). Another peptide tag useful for purification is the "HA" tag (Wilson et al., Ce, which corresponds to an epitope derived from the influenza hemagglutinin protein.
ll 37: 767 (1984)), and "flag" tags.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically, for example, to monitor tumor development or progression as part of a clinical testing procedure (eg, to determine the efficacy of a given treatment regimen). Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances,
Positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance is either directly or indirectly directed to the antibody (or fragment thereof) by a mediator using techniques known in the art, such as linkers known in the art. Can be linked or conjugated via. See, eg, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; bioluminescent substances Examples of luciferase include luciferase, luciferin and aequorin; and examples of suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc.
Is mentioned.

In addition, the antibody or fragment thereof may be used in therapeutic moieties (eg cytotoxins (eg cytostatic or cytotoxic agents)), therapeutic agents or radioactive metal ions (eg α-emitters (eg 213Bi)). Etc.). Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (t
Enoposide), vincristine, vinblastine, colchicine (col)
chicin), doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
Glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof. The therapeutic agent may be an antimetabolite (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine (5
-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa (thioepa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide (cyclothosphamide), busulfan, dibromomannitol, streptozotocin, mitomycin C, And cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, mithramycin, And anthramycin (AMC),
And anti-mitotic agents such as vincristine and vinblastine, but are not limited thereto.

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or drug moiety is not construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that has the desired biological activity. Such proteins include, for example, toxins (eg, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin); proteins (eg,
Tumor necrosis factor, a-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg TNF-α, TNF-β, AIM I (International Publication No. WO97 / 338).
99), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol).
． 6: 1567-1574 (1994)), VEGI (International Publication No. WO99 / 2).
3105)), thrombotic or anti-angiogenic agents (eg, angiostatin or endostatin); or biological response modifiers (eg, lymphokines, interleukin-1 (“IL-1”)). , Interleukin-2 ("
IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CS ").
F "), or other growth factors).

Antibodies can also be attached to a solid support, which is particularly useful in immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moieties to antibodies are well known, eg Arn.
on et al., “Monoclonal Antibodies For Immun.
targeting Of Drugs In Cancer Therap
y ”Monoclonal Antibodies And Cancer T
cure, Reisfeld et al. (eds.), pp. 243-56 (Alan RL
iss, Inc. 1985); Hellstrom et al., "Antibodies.
For Drug Delivery "Controlled Drug D
release (2nd edition), Robinson et al. (eds.), pp. 623-53 (Ma.
rcel Dekker, Inc. 1987); Thorpe, "Antibo
dy Carriers Of Cytotoxic Agents In C
cancer Therapy: A Review "Monoclonal An
tibodies'84: Biological And Clinical
Applications, Pinchera et al. (Eds.), Pp. 475-506 (1
985); "Analysis, Results, And Future Pr.
Descendant Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Ther
apy ”Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (Ed.), 3
03-16 (Academic Press 1985), and Thorp
e, et al., "The Preparation And Cytotoxic Pr.
operations of Antibody-Toxin Conjugate
s ", Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a second antibody, and is described in US Pat. No. 4,676,980 (
This may form an antibody heteroconjugate as described by Segal in (incorporated herein in its entirety).

Antibodies, with or without a therapeutic moiety that binds to the antibody, administered alone or in combination with cytotoxic factors and / or cytokines, can be used as therapeutic agents.

Immunophenotyping The antibodies of the invention can be utilized for immunophenotyping cell lines and biological samples. The translation products of the genes of the present invention are useful as cell-specific markers, or more particularly as cell markers that are differentially expressed at various stages of differentiation and / or maturation of particular cell types. obtain. Monoclonal antibodies against specific epitopes, or combinations of epitopes, allow screening of cell populations that express the marker. Various techniques can be used with monoclonal antibodies to screen for cell populations that express markers, and include magnetic separation using antibody-coated magnetic beads, a solid matrix (i.e., "Panning" with antibodies attached to plates), as well as flow cytometry (see, eg, US Pat. No. 5,985,660; and Morrison et al., Cell, 96: 737-49 (1999)). To be

These techniques apply to hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And allows screening of specific populations of cells as may be found with "non-self" cells in transplantation to prevent graft versus host disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells that may undergo proliferation and / or differentiation as may be found in human cord blood.

Assays for Antibody Binding The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include Western blots, radioimmunoassays, to name just a few.
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A Included, but not limited to, competitive and non-competitive assay systems that use techniques such as immunoassays. Such assays are routine and well known in the art (eg, Ausubel et al. Eds., 1994, Current Protocol), which is incorporated by reference herein in its entirety.
ls in Molecular Biology, Volume 1, John Wil
ey & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1, 1% NP-40 or Triton X-100, supplemented with protein phosphatase inhibitors and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate). % Sodium deoxycholate, 0.1%
SDS, 0.15M NaCl, 0.01M sodium phosphate (pH 7.2)
) Lysing a population of cells in a lysis buffer such as 1% Transylol), adding the antibody of interest to the cell lysate, and incubating at 4 ° C. for a period of time (eg 1-4 hours) Adding protein A sepharose beads and / or protein G sepharose beads to the cell lysate, incubating at 4 ° C. for about 1 hour or more, washing the beads in lysis buffer, and SDS / sample buffer Resuspending the beads in. The ability of the antibody of interest to immunoprecipitate a particular antigen can be determined, for example, by Western blot analysis.
Can be evaluated. One of skill in the art will be aware of parameters that can be modified to increase binding of antibody to antigen and to reduce background (eg, preclearing cell lysates with Sepharose beads). obtain.
For further discussion on immunoprecipitation protocols, see, eg, Ausubel.
Et al., 1994, Current Protocols in Molecule.
ar Biology, Volume 1, John Wiley & Sons, Inc. ，
See New York, 10.16.1.

Western blot analysis generally involves the steps of preparing a protein sample, polyacrylamide gel (eg 8% -20% SDS-depending on the molecular weight of the antigen).
Electrophoresis of the protein sample in PAGE, transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking solution (eg 3% BSA or PBS with skim milk powder) ), Blocking the membrane in a wash buffer (eg,
Washing the membrane in PBS-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer , A secondary antibody conjugated to an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, 32P or 125I) diluted in blocking buffer (which recognizes the primary antibody, eg, anti-body). Human antibody) to block the membrane, wash the membrane in wash buffer, and detect the presence of the antigen. One of skill in the art will be aware of parameters that can be modified to increase the signal detected and to reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al., Eds., 1994, Current Protocols i.
n Molecular Biology, Volume 1, John Wiley & S
ons, Inc. , New York, 10.8.1.

ELISA involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, conjugating to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). The antibody of interest is added to the wells and incubated for a period of time, and the presence of the antigen is detected. In the ELISA, the antibody of interest need not be bound to a detectable compound; instead, a second antibody conjugated to the detectable compound (which recognizes the antibody of interest) will bind to the wells. Can be added. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated wells. One of skill in the art will be aware of parameters that can be modified to increase the signal detected and other variations of the ELISA known in the art. For further discussion on ELISA, see, eg, Ausu.
Bel et al., 1994, Current Protocols in Mole.
polar Biology, Volume 1, John Wiley & Sons, In
c. , New York, 11.2.1.

[0241] The binding affinity of the antibody for the antigen and the off-rate of the antibody-antigen interaction (of
f-rate) can be determined by competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which involves labeling an antigen (eg, 3H or 125) with an antibody of interest in the presence of increasing amounts of unlabeled antigen.
Incubation of I) and detection of antibody conjugated to labeled antigen. The affinity of the antibody of interest for a particular antigen, and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with the second antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest bound to a labeled compound (eg, 3H or 125I) in the presence of increasing amounts of unlabeled second antibody.

Therapeutic Uses The present invention further relates to antibody-based therapies for treating an animal, preferably a mammal, and most preferably for treating one or more of the disclosed diseases, disorders, or conditions. Preferably, the step of administering the antibody of the present invention to a human patient is included. The therapeutic compounds of the present invention include the antibodies of the present invention (including fragments, analogs and derivatives of the antibodies as described herein) as well as the antibodies of the invention (the antibodies as described herein). Nucleic acids, including fragments, analogs and derivatives thereof, and anti-idiotypic antibodies). Antibodies of the invention include diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the invention (including any one or more of the diseases, disorders, or conditions described herein). Can be used to treat, inhibit or prevent). Treatment and / or prophylaxis of diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the present invention comprises the step of alleviating the symptoms associated with those diseases, disorders or conditions. Not limited. The antibodies of the present invention are known in the art or can be provided in a pharmaceutically acceptable composition as described herein.

A summary of how the antibodies of the invention can be used therapeutically is mediated locally or systemically in the body (eg, mediated by complement (CDC), or by effector cells (ADCC)). By direct binding of the polynucleotide or polypeptide of the invention by the direct cytotoxicity of the antibody. Some of these approaches are described in more detail below. Given the teachings provided herein, one of ordinary skill in the art would be able to determine, without undue experimentation, how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes. Recognize.

The antibodies of the invention may be other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2 IL-3 and IL
(Such as −7).

The antibodies of the invention can be administered alone or in combination with other types of treatments such as radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents.
Generally, it is preferred to administer a species-sourced or species-reactive product that is the same species as the patient's species (in the case of antibodies). Thus, in a preferred embodiment, human antibodies,
The fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

A high affinity and / or high affinity for a polypeptide or polynucleotide of the invention, both for immunoassays for the polynucleotides or polypeptides of the invention, including fragments thereof, and for the treatment of disorders associated therewith. Alternatively, it is preferred to use potent in vivo inhibitory and / or neutralizing antibodies, fragments thereof, or regions thereof. Such antibodies, fragments or regions preferably have an affinity for the polynucleotides or polypeptides of the present invention, including fragments thereof. The preferred binding affinity is 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M,
10 ⁻⁴ M, 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M,
10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M
10 ^-10 M, ^{5x10 -11} M, 10 ^-11 M, ^{5x10 -12} M, 10 ^-12 M, ^{5x1
0 -13 M, 10 -13 M,} 5 × 10 -14 M, 10 -14 M, 5 × 10 -15 M, and 10 ^{- 15} binding affinity are exemplified with small dissociation constant or Kd than M.

Gene Therapy In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof treats a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention, Administered for the purpose of gene therapy to inhibit or prevent. Gene therapy refers to a therapy that is performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

Any method available in the art for gene therapy can be used in accordance with the present invention. An exemplary method is described below.

For a general review of methods of gene therapy, see Goldspiel et al., Cli.
Nal Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstos.
hev, Ann. Rev. Pharmacol. Toxicol. 32: 573
-596 (1993); Mulligan, Science 260: 926-.
932 (1993); and Morgan and Anderson, Ann.
Rev. Biochem. 62: 191-217 (1993); May, TIB.
See TECH 11 (5): 155-215 (1993). Methods generally known in the recombinant DNA art that can be used are described in Ausubel.
Et al., Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Express.
Ion, A Laboratory Manual, Stockton Pre
ss, NY (1990).

In a preferred aspect, the compound contains a nucleic acid sequence encoding an antibody, said nucleic acid sequence being an expression vector expressing the antibody, or a fragment or chimeric protein thereof, or a heavy or light chain thereof in a suitable host. Is part of. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, said promoter being inducible or constitutive, and optionally tissue-specific. In another particular embodiment, nucleic acid molecules are used that flank the region encoding the antibody and any other desired sequence that facilitates homologous recombination at the desired site in the genome, whereby the antibody is Provides intrachromosomal expression of a nucleic acid encoding (Koller and Smithies, Proc
． Natl. Acad. Sci. USA 86: 8932-8935 (1989).
); Zijlstra et al., Nature 342: 435-438 (1989).
). In a particular embodiment, the expressed antibody molecule is a single chain antibody; or the nucleic acid sequence comprises sequences encoding both the heavy and light chains of the antibody or fragments thereof.

Delivery of nucleic acids to a patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cells first use the nucleic acid in vitro). Transformed and then transplanted to the patient). These two approaches are known as in vivo gene therapy or as ex vivo gene therapy, respectively.

In certain embodiments, the nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This can be accomplished by a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector,
And by administering it intracellularly (eg, defective or attenuated retroviruses or other viral vectors (US Pat. No. 4,980,
286)), or by direct injection of naked DNA, or microparticle bombardment (eg gene gun; Bioli).
Stic, Dupont) or by coating with lipids or cell surface receptors or transfectants, encapsulation in liposomes, microparticles, or microcapsules, or with peptides known to enter the nucleus By administering in combination with a ligand that undergoes receptor-mediated endocytosis (see, eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)). (Which can be used to target cell types that specifically express the receptor) and the like. In another embodiment, a nucleic acid-ligand complex can be formed where the ligand comprises a fusogenic viral peptide that disrupts endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO92 / 06180; WO92 /). No. 22635; No. WO92 / 20316; No. WO93 /
14188, WO 93/20221). Alternatively, the nucleic acid can be introduced inside the cell and, by homologous recombination, for expression in the host cell DN.
Can be incorporated into A (Koller and Smithies, Proc. Na
tl. Acad. Sci. USA 86: 8932-8935 (1989); Z.
ijstra et al., Nature 342: 435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acid sequences that encode the antibodies of the invention are used. For example, a retroviral vector can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993).
checking). These retroviral vectors contain the necessary components for the correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequence encoding the antibody used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into patients. For more details on retroviral vectors, see Boesen et al., Biothe.
rapid 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make the stem cells more resistant to chemotherapy. Can be issued. Other references describing the use of retroviral vectors in gene therapy are: Clo
wes et al. Clin. Invest. 93: 644-651 (1994);
Kiem et al., Blood 83: 1467-1473 (1994); Salmo.
ns and Gunzberg, Human Gene Therapy 4: 1.
29-141 (1993); and Grossman and Wilson, C.
urr. Opin. in Genetics and Devel. 3: 110
-114 (1993).

Adenovirus is another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenovirus naturally infects respiratory epithelia where it causes mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, Curr
ent Opinion in Genetics and Developopm
ent 3: 499-503 (1993) provides an overview of adenovirus-based codon gene therapy. Bout et al., Human Gene Therapy 5:
3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are described by Rosenfeld et al., Science 252: 431-43.
4 (1991); Rosenfeld et al., Cell 68: 143-155 (1).
992); Mastrangeli et al., J. Am. Clin. Invest. 91: 2
25-234 (1993); PCT Publication No. WO94 / 12649; and Wa.
ng et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:
289-300 (1993); US Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves transfer of the selectable marker to the cells. The cells are then subjected to selection to isolate those cells that take up and express the transferred gene.
The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art, including but not limited to: transfection, electroporation, microinjection, viruses including nucleic acid sequences. Infection with vector or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Many techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Met.
h. Enzymol. 217: 599-618 (1993); Cohen et al., M.
eth. Enzymol. 217: 618-644 (1993); Cline,
Pharmac. Ther. 29: 69-92m (1985)),
And if the necessary developmental and physiological functions of the recipient cells are not disrupted, they can be used according to the invention. This technique should provide for stable transfer of the nucleic acid to the cell so that the nucleic acid is expressible by the cell, preferably heritable and expressible by the progeny of the cell.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient condition, etc. and can be determined by one of skill in the art.

Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type, including but not limited to: epithelial cells, endothelial cells,
Keratinocytes, fibroblasts, muscle cells, hepatocytes; T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, blood cells such as granulocytes; various stem cells or progenitor cells, In particular, hematopoietic stem cells or hematopoietic progenitor cells (eg cells such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver, etc.).

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments in which recombinant cells are used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cells such that the nucleic acid sequences are expressible by the cells or their progeny, and then the recombinant cells are , Administered in vivo for therapeutic effects. In a specific embodiment, stem cells or progenitor cells are used. Any stem and / or progenitor cells that can be isolated in vitro and maintained in vitro can potentially be used according to this embodiment of the invention (
For example, PCT Publication No. WO 94/08598: Temple and Ande.
rson, Cell 71: 973-985 (1992); Rheinwald.
, Meth. Cell Bio. 21A: 229 (1980); and Pit
telkow and Scott, Mayo Clinic Proc. 61: 7
71 (1986)).

In a specific embodiment, the nucleic acid introduced for gene therapy purposes contains an inducible promoter operably linked to the coding region so that expression of the nucleic acid is controlled by the appropriate transcription inducer. Can be controlled by controlling the presence or absence of.

Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro prior to use in humans and then in vivo for the desired therapeutic or prophylactic activity. To be done. For example, in vitro assays to demonstrate therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on cell lines and / or tissue samples can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cytolytic assays. In vitro assays that can be used in accordance with the present invention to determine if administration of a particular compound is indicated include in vitro cell culture assays, in which a patient tissue sample is grown in culture, The compound is then exposed or otherwise administered and the effect of such compound on the tissue sample is observed.

Therapeutic / Prophylactic Administration and Compositions The present invention provides for treatment by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention, Methods of inhibition and prevention are provided. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, and preferably mammals,
And most preferably human.

Formulations and methods of administration that can be used when the compound comprises nucleic acids or immunoglobulins are described above; additional suitable formulations and routes of administration are selected from among those described herein below. Can be done.

Various delivery systems are known and can be used to administer a compound of the invention (eg, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound). , Receptor-mediated endocytosis (eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1
987)), construction of nucleic acids as part of retroviruses or other vectors). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining, such as the oral, rectal and intestinal mucosa, and other It can be administered with a biologically active agent. Administration can be systemic or local. Further, the pharmaceutical compounds or compositions of the invention may be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection may be for example, intraventricular injection attached to a reservoir, such as the Ommaya reservoir. Introduction to the central nervous system by means of a catheter). Pulmonary administration may also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compound or composition of the invention locally to the area in need of treatment; this is for example, but not limiting, purpose. Local injection during surgery, topical application (eg, in combination with post-surgical wound dressing), injection, catheter, suppository, or implant (the implant being a membrane such as a sialastic membrane). Or a porous, non-porous, or glue-like material, including fibers). Preferably, in administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

In another embodiment, the compound or composition can be delivered into vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (
1990); Treat et al., Liposomes in the Therap.
y of Infectious Disease and Cancer, L
opez-Berestein and Fidler (ed.), Liss, New
York, pp. 353-365 (1989); Lopez-Berestein,
Ibid, pages 317-327; see ibid.).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Lang.
er (supra); Sefton, CRC Crit. Ref. Biomed. En
g. 14: 201 (1987); Buchwald et al., Surgery 88:
507 (1980); Saudek et al., N. et al. Engl. J. Med. 321: 5
74 (1989)). In another embodiment, polymeric materials may be used (Medical Applications of Control).
led Release, Langer and Wise (ed.), CRC Pre
s. , Boca Raton, Florida (1974); Control
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
1 (eds.), Wiley, New York (1984); Ranger and P.
eppas, J .; , Macromol. Sci. Rev. Macromol. C
hem. 23:61 (1983); Levy et al., Science.
228: 190 (1985); During et al., Ann. Neurol. 25:
351 (1989); Howard et al., J. Am. Neurosurg. 71: 105
(1989) also). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, thus requiring only a portion of the systemic dose (eg, Goodson, Medical Applica).
Tions of Controlled Release (supra), Volume 2,
115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

In a specific embodiment, wherein the compound of the invention is a nucleic acid encoding a protein, the nucleic acid constitutes it as part of a suitable nucleic acid expression vector and is such that it is intracellular. By administration (eg, by use of retroviral vectors (see US Pat. No. 4,980,286), or by direct injection, or microparticle bombardment (eg, gene gun; Biolis).
Tic, Dupont) or by coating with a lipid or cell surface receptor or transfecting agent, or in combination with a homeobox-like peptide known to enter the nucleus (eg,
Joliot et al., Proc. Natl. Acad. Sci. USA 88:18
64-1868 (see 1991)) and the like, so as to enhance the expression of the encoded protein in vivo. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for homologous recombination for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" has been approved by the Federal Regulatory Authority or the U.S. Government for use in animals, and more particularly in humans, or in the United States Pharmacopeia or other Means that it is listed in the generally accepted pharmacopoeia.
The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Can be. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene. , Glycol, water, ethanol and the like. The composition can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E.I. W. "Reming" by Martin
ton's Pharmaceutical Sciences ". Such a composition comprises a therapeutically effective amount of the compound, preferably in purified form,
It is included with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthesia such as lignocaine to relieve pain at the site of injection. Generally, the components are either separately or mixed together in a single dosage form, eg, in ampoules or sachets (s) showing a fixed amount of active agent.
supplied as a lyophilized powder or water-free concentrate in a sealed container such as an achette). Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, the ingredients may be mixed prior to administration,
Ampoules of sterile water for injection or saline can be provided.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
Mention may be made of salts formed with cations such as those derived from 2-ethylaminoethanol, histidine, procaine and the like.

The amount of a compound of the invention that is effective in this treatment (suppression and prevention of diseases or disorders associated with aberrant expression and / or activity of the polypeptides of the invention) will be determined by standard clinical techniques. Can be done. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. Precise dosages to be used in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg to 100 mg per kg patient body weight. Preferably, the dosage administered to a patient is between 0.1 mg and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, low dosages and infrequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies of the invention may be altered (eg,
It can be reduced by enhancing antibody uptake by lipidation and the like and tissue penetration (eg into the brain).

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Notifications in the form prescribed by governmental agencies regulating the manufacture, use or sale of pharmaceutical or biological products may optionally be accompanied by such containers. This notice
It reflects the agency's approval of manufacture, use or sale for human administration.

Diagnostic and Imaging Labeled antibodies that specifically bind to the polypeptide of interest, and their derivatives and analogs thereof, have been used for diagnostic purposes to detect abnormal expression and expression of the polypeptides of the invention. Diseases and / or disorders associated with activity can be detected, diagnosed or monitored. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which comprises (a) using one or more antibodies specific for the polypeptide of interest to target in a cell or body fluid of an individual. Assaying the expression of the polypeptide of
And (b) comparing the level of gene expression to the level of standard gene expression, whereby increasing or decreasing the level of assayed polypeptide gene expression compared to the standard level of expression. Shows abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, which assay comprises (
a) assaying the expression of the polypeptide of interest in cells or fluids of an individual using one or more antibodies specific for the polypeptide of interest, and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. With respect to cancer, the presence of relatively high amounts of transcripts in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the onset of actual clinical symptoms. Can be provided. A more definitive diagnosis of this type may allow health professionals to use preventative measures or early aggressive treatment, thereby preventing the development or further progression of cancer.

The antibodies of the invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those of skill in the art (eg, Jalk).
anen et al. Cell. Biol. 101: 976-985 (1985);
Jalkanen et al. Cell. Biol. 105: 3087-3096 (
1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and are enzymatic labels (eg glucose oxidase); radioisotopes (eg iodine (125I, 1, 1
21I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg luminol); and fluorescent labels (eg fluorescein and rhodamine), and Examples include biotin.

One aspect of the present invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the polypeptide of interest in animals, preferably mammals, and most preferably humans. In one embodiment, diagnosis is a) administering to a subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds to a polypeptide of interest. B) to allow the labeled molecule to preferentially concentrate at the site in the subject where the polypeptide is expressed (and to remove unbound labeled molecule to background levels) A) waiting for a time interval after administration; c) determining the background level; and d) detecting the labeled molecule in the subject, so that the label is above this background level. Detection of the activating molecule indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to standard values previously determined for a particular system.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally be 99mTc, in the range of about 5-20m Curie. The labeled antibody or labeled antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein. In vivo tumor imaging is described by S. et al. W. Burchiel et al.
Immunopharmacokinetics of Radiolabel
ed Antibodies and Their Fragments "(T
Umor Imaging, Chapter 13: The Radiochemical
Detection of Cancer, S.M. W. Burchiel and B
． A. Rhodes, Masson Publishing Inc. (19
82).

Depending on a number of variations, including the type of label used and the mode of administration, the labeled molecule is allowed to preferentially concentrate at a site in the subject and is bound. The time interval after administration for removal of unlabeled molecules to background levels is 6-48 hours or 6-24 hours or 6-12 hours. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, monitoring diseases and disorders is performed by repeating the method for diagnosing a disease or disorder (eg, one month after initial diagnosis, first 6 months after diagnosis, 1 year after initial diagnosis, etc.).

The presence of labeled molecule can be detected in a patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art can determine the appropriate method for determining a particular label. Methods and devices that may be used in the diagnostic methods of the present invention include computer tomography (CT), whole body scans (eg, protons).
n) emission tomography (PET)), magnetic resonance imaging (MRI), and ultrasound diagnostics, but not limited to these.

In a particular embodiment, the molecule is labeled with a radioisotope and is subjected to a radiation responsive surgical instrument.
(Thurston et al., US Pat. No. 5,441,050)
To detect in patients using. In another embodiment, the molecule is labeled with a fluorescent compound and detected in a patient using a fluorescence response scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

Kits The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a particular embodiment, the kit of the invention comprises a substantially isolated polypeptide comprising an epitope. This epitope specifically immunoreacts with the antibody contained in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kit of the invention comprises means for detecting binding of the antibody to a polypeptide of interest (eg, the antibody is a detectable substrate (eg, a fluorescent compound, an enzyme substrate). , Radioactive compounds or luminescent compounds), or a secondary antibody that recognizes the primary antibody can be conjugated with a detectable substrate).

In another particular embodiment of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. is there. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen containing the epitope. This epitope specifically immunoreacts with at least one anti-polypeptide antigen antibody. Further, such a kit comprises means for detecting the binding of the above antibody to an antigen (eg, the antibody is a fluorescent compound (eg, fluorescein or rhodamine that can be detected by flow cytometry) and a conjugate). Can be). In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be attached to a solid support.

[0289] In a more specific embodiment, the detection means of the kit comprises a solid support to which the polypeptide antigen is attached. Such a kit may also include a non-attached reporter labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody described above.

In a further embodiment, the invention comprises a diagnostic kit for use in screening serum containing antigens of the polypeptides of the invention. The diagnostic kit comprises a substantially isolated antibody that specifically immunoreacts with a polypeptide or polynucleotide antigen, and a means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. Prepare In one embodiment, the antibody is attached to a solid support. In a particular embodiment, the antibody may be a monoclonal antibody. This detection means of the kit may include a secondary labeled monoclonal antibody. Alternatively, or additionally, the detection means can include a labeled competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface bound antigen obtained by the method of the invention. After binding of the specific antigen antibody to this reagent and removal of unbound serum components by washing, this reagent is reacted with a reporter-labeled anti-human antibody to bind the bound anti-human antibody to the solid support. A reporter is bound to this reagent in proportion to the amount of antigen antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme, which is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent or colorimetric substrate (Sigma, St. Louis, MO). To be done.

The solid surface reagents in the above assay are used to convert protein material to solid support material (
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96-well plates or filtration material). These methods of attachment generally include non-specific adsorption of proteins to the support or chemically active groups (eg, activated carboxyl groups, hydroxyl groups, or aldehyde groups) on the solid support. Covalent attachment of proteins (typically via free amine groups). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

The invention therefore provides an assay system or kit for carrying out this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Uses of Polynucleotides Each of the polynucleotides identified herein can be used as reagents in a number of ways. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. Chromosome marking reagents based on actual sequence data (repeated polymorphisms) are currently scarcely available, so there remains a need to identify new chromosomal markers. Each sequence can be specifically targeted to and hybridized to a particular location on an individual human chromosome. Thus, each polynucleotide of the present invention may be conveniently used as a chromosomal marker using techniques known in the art.

Briefly, the sequence is derived from the sequence set forth in SEQ ID NO: X or a complement thereto to a PCR primer (preferably at least 15 bp (eg 15-25 bp.
bp)) can be mapped to the chromosome. The primer is
Primers can be optionally selected using computer analysis so that they do not span more than one predicted exon in the genomic DNA. These primers are then used to PCR somatic cell hybrids containing individual human chromosomes.
Used for screening. Only the hybrid containing the human gene corresponding to SEQ ID NO: X produces an amplified fragment.

[0297] Similarly, somatic cell hybrids include a polynucleotide P
It provides a rapid method for CR mapping. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotide can be accomplished using a panel of specific chromosomal fragments. Other gene mapping strategies that may be used are in situ hybridization, labeled flow sorted.
pre-screening on the flow sorted) chromosome, chromosome-specific cd
Preselection by hybridization to construct an NA library, and computer mapping techniques (eg, Shuller, Trends Biotechnol 16:45, which is incorporated herein by reference in its entirety).
6-459 (1998)).

The exact chromosomal location of the polynucleotide is also determined by the metaphase chromosomal spread (spr
ead) fluorescence in situ hybridization (FISH). This technique uses polynucleotides as short as 500 or 600 bases; however, 2,000-4,000 bp polynucleotides are preferred. For a review of this technology, see Verma et al., “Human Chromoso.
mes: a Manual of Basic Technologies ", Pe
See rgamon Press, New York (1988).

For chromosome mapping, polynucleotides may be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (to mark multiple sites and / or multiple chromosomes). Can be done.

Accordingly, the present invention also provides a method for chromosomal localization, which method comprises: (a) preparing a PCR primer from the polynucleotide sequence in Table 1 and SEQ ID NO: X, and (b A) screening somatic cell hybrids containing individual chromosomes.

The polynucleotides of the present invention can also be used for radiation hybrid mapping, HA.
Useful for PPY mapping and long range limited mapping. For an overview of these and other techniques known in the art, see, eg, Dear “Genom.
e Mapping: A Practical Approach "IRL P
pressure at Oxford University Press, London
on (1997); Aydin, J .; Mol. Med. 77: 691-694 (
1999); Hacia et al., Mol. Psychiatry 3: 483-49
2 (1998); Herrick et al., Chromosome Res. 7:40
9-423 (1999); Hamilton et al., Methods Cell B.
iol. 62: 265-280 (2000); and / or Ott, J. et al. He
red. 90: 68-70 (1999), each of which is incorporated herein by reference in its entirety.

Once the polynucleotide has been mapped to a precise chromosomal location, the physical location of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between the chromosomal location and the presentation of a particular disease. (Disease mapping data is described in, for example, V. McKusick, Mendel.
ian Inheritance in Man (Johns Hopkins
(Available online through the University Welch Medical Library)). Given 1 megabase mapping resolution and 1 gene per 20 kb, the cDNA precisely localized to the chromosomal region associated with disease could be one of 50-500 potential causative genes.

Thus, once co-inheritance is established, differences in the polynucleotides of the invention and corresponding genes between affected and unaffected individuals can be tested. First, visible structural changes in the chromosome (eg, deletions or translocations) are examined in chromosome spreads or by PCR. In the absence of structural changes, the presence of point mutations is confirmed. Mutations observed in some or all affected individuals but not in normal individuals indicate that this mutation may cause the disease. However, complete sequencing of polypeptides and the corresponding genes from some normal individuals is required to distinguish mutations from polymorphisms. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

In addition, increased or decreased expression of genes in affected individuals compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these changes (
Altered expression, chromosomal rearrangements, or mutations) can be used as a diagnostic or prognostic marker.

Accordingly, the invention also provides a diagnostic method useful during the diagnosis of disorders, which method comprises the step of measuring the expression level of a polynucleotide of the invention in cells or body fluids from an individual, and Comprising comparing the measured gene expression level to a standard polynucleotide expression level, whereby an increase or decrease in gene expression level relative to the standard is indicative of a disorder.

In yet another embodiment, the invention comprises a kit for analyzing a sample for the presence of proliferative and / or cancerous polynucleotides from a test subject. In a general embodiment, the kit comprises at least one polynucleotide probe containing a nucleotide sequence that specifically hybridizes to a polynucleotide of the invention, and a suitable container. In a particular embodiment, the kit comprises two polynucleotide probes defining an internal region of a polynucleotide of the invention, wherein each probe contains one 31 ′ mer end internal to this region. Has a chain. In a further embodiment, this probe may be useful as a primer for polymerase chain reaction amplification.

[0307] The present invention is useful as a prognostic indicator when the diagnosis of related disorders, including, for example, the diagnosis of tumors, has already been performed by conventional methods, and thus the polynucleotides of the present invention that are enhanced or suppressed thereby. Patients exhibiting expression experience poor clinical outcomes compared to patients expressing this gene at levels closer to normal levels.

By “measuring the expression level of the polynucleotide of the present invention”, the level of the polypeptide of the present invention or the mRNA encoding the polypeptide of the present invention
Is directly (eg, by determining or assessing absolute protein or mRNA levels) or relative (eg, polypeptide levels in the second biological sample) in the first biological sample. Qualitatively or quantitatively) or by comparison to mRNA levels). Preferably, the polypeptide or mRNA level in the first biological sample is measured or evaluated and compared to a standard polypeptide or mRNA level, the standard being an individual without an associated disorder. Determined from a second biological sample obtained from or by averaging the levels from a population of individuals without associated disorders. As will be appreciated in the art, once standard polypeptide or mRNA levels are known, this can be used repeatedly as a standard for comparison.

By “biological sample” is meant a polypeptide of the invention or the corresponding mRN.
Any biological sample obtained from an individual, body fluid, cell line, tissue culture or other source, including A, is contemplated. As shown, the biological sample is found to express the polypeptide of the invention in a body fluid (eg, semen, lymph, serum, plasma, urine, synovial fluid and spinal fluid) containing the polypeptide of the invention. Includes tissue sources issued. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, tissue biopsy is a preferred source.

The methods provided above can preferably be applied to diagnostic methods and / or kits in which the polynucleotides and / or polypeptides of the invention are bound to a solid support. In one exemplary method, the support is made according to US Pat. No. 5,837,
It may be a "gene chip" or a "biological chip" as described in 832, 5,874,219 and 5,856,174. Further, using such a gene chip to which the polynucleotide of the present invention is bound, the polymorphism between the isolated polynucleotide sequence of the present invention and the polynucleotide isolated from the test subject is confirmed. Can be identified. Knowledge of such polymorphisms (ie, the location of the polymorphism, and the presence of the polymorphism) can lead to many disorders (eg, neurological disorders, immune system disorders,
It is useful in identifying disease loci for muscle disorders, reproductive disorders, gastrointestinal disorders, lung disorders, cardiovascular disorders, renal disorders, proliferative disorders, and / or cancerous diseases and conditions. Such methods are described in US Pat. Nos. 5,858,659 and 5,856,104. The United States patents referenced above are incorporated herein by reference in their entireties.

The present invention includes the polynucleotides of the present invention, either chemically synthesized or reconstructed as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNA serves as a preferred form when the polynucleotide of the invention is incorporated into a solid support or gene chip. For the purposes of the present invention, peptide nucleic acid (PNA) is a DNA analogue of the polyamide type, and monomer units for adenine, guanine, thymine and cytosine are commercially available (Perceptive Biosystems). Certain components of DNA (eg, phosphorus, phosphorus oxide or deoxyribose derivatives) are labeled with PNA.
Does not exist inside. P. E. Nielsen, M .; Egholm, R.A. H. Ber
g and O. Buchardt, Science 254, 1497 (1997)
); And M. Egholm, O.I. Buchardt, L .; Christen
sen, C.I. Behrens, S .; M. Freier, D.F. A. Driver,
R. H. Berg, S .; K. Kim, B.H. Norden and P.M. E. Niel
As disclosed by Sen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than does DNA itself. This is probably because there is no electrostatic repulsion between the two chains,
And again because the polyamide skeleton is more flexible. By this, PN
A / DNA duplexes bind under a wider range of stringency conditions than DNA / DNA duplexes, making it easier to perform multiplex hybridization. Due to the strong binding, smaller probes can be used than with DNA. Furthermore, perhaps single base mismatches can be determined using PNA / DNA hybridizations. Because the single mismatch in the 15-mer PNA / DNA is 4 ° C to 16 ° C for the 15-mer duplex of DNA / DNA, whereas the melting point (T.sub.m) is 8-20. This is because the temperature is lowered by ° C. Also, the absence of charged groups in the PNA means that hybridization can be done at low ionic strength and reduce possible interference by salts during the analysis.

The present invention has applications including, but not limited to, detection of cancer in mammals. In particular, the invention is useful during the diagnosis of pathological cell proliferative neoplasia, including but not limited to: acute myeloid leukemia (acute monocytic leukemia, acute myeloblastic leukemia, acute Including promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia); and chronic myelogenous leukemia (chronic myelomonocytic leukemia, chronic granulocytic leukemia) Including leukemia). Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Especially preferred is human.

Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes (
Gelmann, E .; P. Et al., “The Etiology of Accue Leukemia: Molecular Genetics and Vir.
al Oncology ”, Neoplastic Diseases of
the Blood, Volume 1, Wiernik, P .; H. Et al., 161-182
(1985)). Neoplasia is now from qualitative alterations of the normal cellular gene product, by the insertion of viral sequences into the chromosome, chromosomal translocations of genes into more actively transcribed regions, or by some other mechanism, or of gene expression. It is believed to result from quantitative modification (Gelmann et al., Supra). Mutated or altered expression of certain genes appears to be involved in the pathogenesis of some leukemias, among other tissues and other cell types (Gelmann et al., Supra). In fact, human counterparts of some animal neoplastic oncogenes have been amplified or translocated in some cases of human leukemia and cancer (Gelmann et al., Supra).

For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. The levels of c-myc are found to be down-regulated when HL-60 cells are chemically induced to stop amplification (International Publication No. WO 91/15580). However, exposure of HL-60 cells to a DNA construct that is complementary to the 5'end of c-myc or c-myb down-regulates expression of the c-myc or c-myb protein.
It has been shown to block translation of NA, causing cell growth and arrest of differentiation of treated cells (International Publication No. WO 91/15580; Wickstro).
m, et al., Proc. Natl. Acad. Sci. 85: 1028 (1988);
Anfossi et al., Proc. Natl. Acad. Sci. 86: 3379 (
1989)). However, one of ordinary skill in the art will find the utility of the present invention in the treatment of proliferative disorders of hematopoietic cells and tissues, given the large number of cells and cell types of various origins known to exhibit a proliferative phenotype. Recognize that you are not limited.

In addition to the foregoing, the polynucleotides of the invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense technology is described in, for example, Okano, J. et al. Neurochem
． 56: 560 (1991); "Oligodeoxynucleotides
as Antisense Inhibitors of Gene Exp
", CRC Press, Boca Raton, FL (198).
8). Triple helix formation is described, for example, in Lee et al., Nucleic.
Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Sc.
ience 251: 1360 (1991). Both methods rely on the binding of the polynucleotide to complementary DNA or RNA. For these techniques, the preferred polynucleotides are usually oligonucleotides 20-40 bases in length, and the gene regions involved in transcription (triple helix-Le.
e et al., Nucl. Acids Res. 6: 3073 (1979); Cone
y et al., Science 241: 456 (1988); and Dervan et al.,
Science 251: 1360 (1991)) or mRNA.
As such (Antisense-Okano, J. Neurochem. 56: 560 (1
991); Oligodeoxy-nucleotides as Antis.
sense Inhibitors of Gene Expression, C
RC Press, Boca Raton, FL (1988)). Triple helix formation optimally results in the blockage of RNA transcription from DNA, whereas antisense RNA hybridisation results in mRN to polypeptide.
Block the translation of the A molecule. The oligonucleotides described above can also be delivered to cells such that antisense RNA or DNA can be expressed in vivo to inhibit polypeptide production of the antigens of the invention. Both techniques are effective in model systems, and the information disclosed herein provides antisense in an attempt to treat disease, particularly for the treatment of proliferative diseases and / or conditions. Or it can be used to design triple helix polynucleotides.

The polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism that has a defective gene in an attempt to correct the genetic defect. The polynucleotide disclosed in the present invention is
It provides a means to target such genetic defects in a highly accurate manner. Another goal is to insert new genes that were not present in the host genome, thereby creating new traits in the host cell.

The polynucleotides are also useful for identifying an individual from a small biological sample. For example, the United States military is considering the use of restriction fragment length polymorphisms (RFLPs) to identify its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed for Southern blots to generate unique bands to identify staff. This method does not suffer from the current limitations of the "Dog tag," which can be difficult to positively identify by being lost, exchanged, or stolen. The polynucleotide of the present invention can be used as an additional DNA marker for RFLP.

The polynucleotides of the present invention may also be used as an alternative to RFLP by determining the actual base-by-base DNA sequence of selected portions of the genome of an individual. These sequences can be used to prepare PCR primers to amplify and isolate such selected DNA, which can then be sequenced. Individuals can be identified using this technique because each individual has a unique set of DNA sequences. Once a unique ID database is established for an individual, whether that individual is alive or dead, positive identification of that individual can be made from very small tissue samples.

Forensic biology will also benefit from the use of DNA-based identification techniques as disclosed herein. Tissue (eg hair or skin) or body fluid (eg blood, saliva, semen, synovial fluid, amniotic fluid, milk, lymph, lung sp (pulmonary sp)
DNA) obtained from a very small biological sample, such as urium) or surfactant, urine, fecal material, etc.) can be amplified using PCR.
In one prior art, gene sequences amplified from polymorphic loci such as the DQa class II HLA gene are used in forensic biology to identify individuals. (Errich, H., PCR Technology, Freem.
an and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes. This results in an identified set of bands for Southern blots probed with DNA corresponding to the DQa class II HLA gene. Similarly, the polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents that can identify the source of a particular tissue. Such a need arises in forensic medicine, for example, if tissue of unknown origin is provided. Suitable reagents may include, for example, DNA probes or primers prepared from the sequences of the invention. Such a panel of reagents may identify tissues by species and / or organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contaminants.

The polynucleotides of the present invention are also useful as hybridization probes for the differential identification of tissues or cell types present in a biological sample. Similarly, the polypeptides and antibodies relating to the polypeptides of the invention provide immunological probes for the differential identification of tissues (eg, immunohistochemistry assays) or cell types (eg, immunocytology assays). Useful for. Furthermore, the polynucleotide / polypeptides of the invention against many disorders of the above-mentioned tissues or cells as compared to “standard” gene expression levels (ie expression levels of healthy tissues from individuals without disorders). Significantly higher or lower levels of gene expression of
Specific tissues (eg, tissues expressing the polypeptide and / or polynucleotide of the present invention, and / or cancerous tissues and / or wound tissues) or body fluids (eg, serum) collected from an individual having such a disorder. , Plasma, urine, synovial fluid or spinal fluid).

Accordingly, the present invention provides a method of diagnosing a disorder comprising the steps of: (a) assaying gene expression levels in cells or body fluids of an individual; (b) standard gene expression levels. Comparing this gene expression level, whereby an increase or decrease in the assayed gene expression level relative to a standard expression level is indicative of a disorder.

At a minimum, the polynucleotides of the present invention were "subtracted (s
(utract-out) "to raise anti-DNA antibodies using DNA immunization techniques to select and make oligomers for attachment to" gene chips "or other supports as probes to known sequences, And can be used as an antigen to elicit an immune response.

Uses of Polypeptides Each of the polypeptides identified herein can be used in a number of ways. The following description should be considered exemplary and utilizes known techniques.

Polypeptides and antibodies directed against the polypeptides of the invention can be expressed in tissue (
For example, an immunohistochemical assay such as ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29: 577-580 (198).
1)) or cell types (eg, immunocytochemistry assays) are useful for providing immunological probes for differential identification.

The antibodies may be used to assay levels of the polypeptide encoded by a polynucleotide of the invention in a biological sample using classical immunohistological methods known to those of skill in the art. (For example, Jalkanen et al., J. Cell. Bio.
l. 101: 976-985 (1985); Jalkanen et al. Cell
． Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting gene expression of proteins include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and are enzymatic labels (eg glucose oxidase); radioisotopes (eg iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C). ), Sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ I)
n), and technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Tl), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F). ), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh.
, ⁹⁷ Ru; luminescent labels (eg luminol); and fluorescent labels (eg fluorescein and rhodamine) and biotin.

In addition to assaying the levels of polypeptides of the invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of proteins include those detectable by radiography, NMR, or ESR. Labels suitable for radiography include radioisotopes such as barium or cesium, which emit detectable radiation, but are clearly not harmful to the subject. Suitable markers for NMR and ESR include markers with a detectable characteristic spin (eg deuterium), which are incorporated into the antibody by labeling the nutrients for the relevant hybridoma. obtain.

[0328]   Radioisotopes (eg,¹³¹I,¹¹²In,^99mTc, (¹³¹I,¹²⁵I,^{one two Three}I, ¹²¹ I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^115m In,^113mIn,¹¹²In,¹¹¹In), and technetium (⁹⁹Tc,^99mTc
),thallium(²⁰¹Tl), gallium (⁶⁸Ga,⁶⁷Ga), palladium (¹⁰³Pd
),molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷ Lu,¹⁵⁹Gd,¹⁴⁹Pm,¹⁴⁰La,¹⁷⁵Yb,¹⁶⁶Ho,⁹⁰Y,⁴⁷Sc,¹⁸⁶
Re,¹⁸⁸Re,¹⁴²Pr,¹⁰⁵Rh,⁹⁷Ru), radiopaque material, or nucleus
Labeled with a suitable detectable imaging moiety, such as a material detectable by magnetic resonance
In addition, protein-specific antibodies or antibody fragments have been tested for disorders of the immune system.
Introduced into the mammal to be investigated (eg parenterally, subcutaneously, or intraperitoneally)
It The size of the subject and the imaging system used will produce diagnostic images.
It is understood in the art to determine the amount of imaging moiety needed to achieve this. Radioactive isotope
In the case of body parts, for human subjects, the amount of radioactivity injected will typically be^99m
It is in the range of about 5 to 20 millicuries of Tc. Then, the labeled antibody or anti-antibody
The body fragment is a polypeptide encoded by the polynucleotide of the present invention.
Accumulates preferentially at the location of cells expressing. In vivo tumor imaging is described by S. W.
Burchiel et al., “Immunopharmacokinetics of
  Radiolabeled Antibodies and Their F
ragments "(Tumor Imaging Chapter 13: The Rad
iochemical Detection of Cancer, S.M. W. B
urchiel and B. A. Edited by Rhodes, Masson Public
ing Inc. (1982)).

In one embodiment, the invention is by administering a polypeptide of the invention (eg, a polypeptide and / or antibody encoded by a polynucleotide of the invention) that associates with a heterologous polypeptide or nucleic acid. , For the specific delivery of the compositions of the invention to cells. In one example, the invention provides a method for delivering therapeutic proteins into targeted cells. In another example, the invention provides a single-stranded nucleic acid (eg, an antisense or ribozyme) or a double-stranded nucleic acid (eg, a DNA that can integrate into the genome of a cell or replicate episomally and be transcribed). ) To targeted cells.

In another embodiment, the invention provides for specific destruction of cells (eg, by administering a polypeptide of the invention related to a toxin or cytotoxic prodrug, eg,
Tumor cell destruction).

A “toxin” is an endogenous cytotoxic effector system, radioisotope, holotoxin (
holotoxin), modified toxins, catalytic subunits of toxins, or one or more compounds that bind and activate any molecule or enzyme normally absent in or on the surface of a cell that causes cell death under defined conditions. means. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, compounds that bind to an intrinsic or induced endogenous cytotoxic effector system. (For example, antibody (or complement fixing-containing portion)), thymidine kinase, endonuclease, RNAse, α-toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin,
Momordin, gelonin, pokeweed antiviral protein, alpha sarcin and cholera toxin. "toxin"
Are also cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions (eg α-emitters (eg ²¹³ Bi)) or other radioisotopes (eg ¹⁰³ Pd, ¹³³ Xe, ¹³¹ I). , ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ³⁵ S
, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ yttrium, ¹¹⁷ tin, ¹⁸⁶ rhenium, ¹⁶⁶ holmium, and ¹⁸⁸ rhenium); luminescent labels (eg, luminol); And fluorescent labels (eg, fluorescein and rhodamine), and biotin.

Techniques known in the art can be applied to label the polypeptides (including antibodies) of the invention. One such technique is the use of bifunctional binders (
For example, US Pat. Nos. 5,756,065; 5,714,631; 5
, 696, 239; 5,652, 361; 5,505, 931;
No. 5,489,425; No. 5,435,990; No. 5,428,13.
No. 9; No. 5,342, 604; No. 5,274, 119; No. 4,994.
, 560; and 5,808,003; the content of each of which is incorporated herein by reference in its entirety), but is not limited thereto.

The invention thus provides a method of diagnosing a disorder, which comprises: (a) assaying the expression level of a polypeptide of the invention in cells or body fluids of an individual; and (b) an assay. Comparing the expression level of the expressed polypeptide with the expression level of the standard polypeptide, whereby an increase or decrease in the expression level of the assayed polypeptide relative to the standard expression level is indicative of an impairment. For cancer, the presence of relatively high amounts of transcripts in biopsied tissue from an individual may indicate a predisposition to the development of the disease, or the actual clinical manifestation of the disease before it becomes apparent. May be provided for detecting. A more definitive type of diagnosis may allow health professionals to use preventative measures or aggressive treatment earlier, thereby preventing the onset or further progression of cancer.

Furthermore, using the polypeptides of the present invention, diseases or conditions (eg neuropathy,
Immune system disorder, muscle disorder, reproductive disorder, gastrointestinal disorder, lung disorder, cardiovascular disorder, renal disorder,
Proliferative disorders and / or cancerous diseases and conditions, etc.) can be treated or prevented.
For example, the patient reverses the absence or reduced level of polypeptide (eg, insulin), supplements the absence or reduced level of a different polypeptide (eg, hemoglobin S vs. hemoglobin S, SOD, Catalase, DNA repair protein), inhibiting activity of polypeptide (eg oncogene or tumor suppressor), activating activity of polypeptide (eg by binding to receptor), membrane for free ligand Decreasing the activity of membrane-bound receptors by competing with bound receptors (eg, soluble TNF receptors used in reducing inflammation) or producing a desired response (eg, inhibition of blood vessel growth, proliferating cells) Or to enhance the immune response to tissues) There are, polypeptide can be administered in the present invention.

Similarly, antibodies directed against the polypeptides of the present invention may also be used to treat diseases (as described above and elsewhere herein). For example, administration of an antibody directed against a polypeptide of the invention may bind the polypeptide and / or neutralize the polypeptide and / or reduce overproduction of the polypeptide. Similarly, administration of the antibody can activate the polypeptide, for example, by binding to a membrane-bound polypeptide (receptor).

At a minimum, the polypeptides of the invention will be SDS-using methods well known to those of skill in the art.
It can be used as a molecular weight marker for PAGE gels or molecular sieve gel filtration columns. As a method of assessing transformation of host cells, polypeptides can also be used to raise antibodies, which are then used to measure protein expression from recombinant cells. In addition, the polypeptides of the present invention can be used to test the following biological activities.

(Gene Therapy Method) Another aspect of the present invention is a gene therapy method for treating or preventing disorders, diseases and conditions. This gene therapy method relates to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal in order to achieve the expression of the polypeptide of the present invention. This method requires a polynucleotide encoding a polypeptide of the invention operably linked to a promoter and any other genetic elements required for expression of this polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art (see, eg, WO 90/11092, incorporated herein by reference).

Thus, for example, cells from a patient may be prepared by ex vivo inducing a polynucleotide (DNA or RN) containing a promoter operably linked to a polynucleotide of the invention.
A) can be engineered with and the engineered cells are then provided to a patient to be treated with a polypeptide of the invention. Such methods are well known in the art. For example, Belldegrun, A .; Et al., J. Natl. Cancer
Inst. 85: 107-216 (1993); Ferrantini, M .;
Et al., Cancer Research 53: 1107-1112 (1993).
Ferrantini, M .; Et al., J. Immunology 153: 460
4-4615 (1994); Kaido, T .; Int. J. Cancer
60: 221-229 (1995); Ogura, H .; Et al, Cancer Re
search 50: 5102-5106 (1990); Santodonat
o, L. Et al., Human Gene Therapy 7: 1-10 (1996).
); Santodonato, L .; Et al, Gene Therapy 4: 124.
6-1255 (1997); and Zhang, J. et al. -F. Et Cancer
See Gene Therapy 3: 31-38 (1996). These are incorporated herein by reference. In one embodiment, the engineered cells are arterial cells. The arterial cells can be reintroduced into the patient via direct injection into the artery, the tissue surrounding the artery, or via catheter infusion.

As discussed in more detail below, the polynucleotide constructs can be used to deliver injectable substances to cells of an animal by any method (eg, tissue (heart, muscle, skin, lung, liver, etc.). Injection into the interstitial space). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0340] In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle (viral sequence, viral particle, liposomal formulation, lipofectin or precipitate) that acts to assist, facilitate or facilitate entry into cells. (Including agents) is not included. However, the polynucleotides of the invention may also be delivered, such as in liposomal formulations and lipofectin formulations that may be prepared by methods well known to those of skill in the art. Such a method is, for example,
US Pat. Nos. 5,593,972, 5,589,466 and 5,5
80,859, which are incorporated herein by reference.

The polynucleotide vector construct used in the gene therapy method is preferably one that does not integrate into the host genome or contains sequences that allow replication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; Pha
pSVK3, pBPV, pMSG and pSVL available from Rmcia;
And pEF1 / V5, pcDNA3., Available from Invitrogen.
1 and pRc / CMV2. Other suitable vectors will be readily apparent to those of skill in the art.

Any strong promoter known to those of skill in the art can be used to drive the expression of the polynucleotide sequence. Suitable promoters include adenovirus promoters (eg adenovirus major late promoter); or heterologous promoters (eg cytomegalovirus (CMV) promoter); RS virus (RSV) promoter; inducible promoters (eg MMT promoter, Metallothionein promoter); heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg, herpes simplex thymidine kinase promoter); retroviral LTR; b-actin promoter; and human growth hormone promoter. The promoter can also be the native promoter for the polynucleotides of the invention.

One major advantage of introducing naked nucleic acid sequences into target cells, unlike other gene therapy techniques, is the transient nature of polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for production of the desired polypeptide for a period of up to 6 months.

Polynucleotide constructs are used in tissues in animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis. , Ovary, uterus, rectum, nervous system, eye, gland, and connective tissue). The interstitial spaces of this tissue are the intercellular liquid mucopolysaccharide matrix between the reticulated fibers of the organ tissue, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue, or connective tissue sheath muscle cells (connective muscle cells). tissu
The same matrix within an eensheating muscle cell or in a bony tear. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue into the interstitial space is preferred for the reasons discussed below. The polynucleotide constructs of the present invention may be conveniently delivered by injection into tissues containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression may be in undifferentiated cells or cells that are not fully differentiated ( (Eg, blood stem cells or dermal fibroblasts). Muscle cells in vivo are particularly qualified for their ability to take up and express polynucleotides.

For naked nucleic acid sequence injections, an effective dosage of DNA or RNA is about 0.05.
It ranges from mg / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary depending on the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, including, for example, inhalation of aerosol formulations, especially for delivery to lung or bronchial tissues, throat or mucous membranes of the nose, among others. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in this procedure.

Naked polynucleotides can be any method known in the art including, but not limited to, direct needle injection at the site of delivery, intravenous injection, topical administration, catheter injection, and so-called “gene gun”. Delivered by. These methods of delivery are known in the art.

The constructs can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofectin, precipitants and the like. Such delivery methods are known in the art.

[0349] In certain embodiments, the polynucleotide construct is complexed in a liposome preparation. Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because they can form a robust charge complex between the cationic liposomes and the polyanionic nucleic acid. Cationic liposomes, in functional form, include plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. U, incorporated herein by reference).
SA (1987) 84: 7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. U., incorporated herein by reference).
SA (1989) 86: 6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1) incorporated herein by reference).
990) 265: 10189-10192) has been shown to mediate intracellular delivery.

Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTM
A) Liposomes are particularly useful, and G under the trademark Lipofectin
IBCO BRL, Grand Island, N .; Y. (Felgner et al., Proc. Natl. Acad. Sci.
USA (1987) 84: 7413-7416). Other commercially available liposomes include transfectase (trans).
surface) (DDAB / DOPE) and DOTAP / DOPE (Bo
ehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. DOTAP (1,2-bis (oleoyloxy)-
For a description of the synthesis of 3- (trimethylammonio) propane) liposomes, see, eg, PCT Publication No. WO 90/11092, which is incorporated herein by reference. The preparation of DOTMA liposomes is described in the literature and is described, for example, in P. et al. Felgner et al., Proc.
Natl. Acad. Sci. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic and neutral liposomes can be prepared, for example, by Avant.
It is readily available from i Polar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials.
Such substances include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC).
), Dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These substances also
It can be mixed with DOTMA starting material and DOTAP starting material in suitable proportions. Methods of using these materials to form liposomes are well known in the art.

For example, commercially, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) were used in various combinations, with the addition of cholesterol. Even without, conventional liposomes can be produced. Therefore,
For example, DOPG / DOPC vesicles can be prepared by drying each 50 mg of DOPG and DOPC under a stream of nitrogen gas into a sonicated vial. The sample is placed under vacuum pump overnight and hydrated the next day with deionized water. The sample was then placed in a capped vial using a Heat Systems Model 350 sonicator equipped with an inverted cup (bath type) probe at maximum setting, circulating the bath at 15 EC. Sonicate for 2 hours. Alternatively, negatively charged vesicles can be prepared without sonication to generate multilamellar vesicles or extruded through nucleopore membranes to separate unilamellar vesicles of different sizes. Can be generated. Other methods are known and available to those of skill in the art.

This liposome includes multilamellar vesicles (MLV), small unilamellar vesicles (SUV).
Alternatively, large unilamellar vesicles (LUVs) may be mentioned, SUVs being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example,
Straubinger et al., Method, incorporated herein by reference.
Sof Immunology, 101: 512-527 (1983). For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipids on the walls of glass tubes and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs, producing a homogeneous population of unilamellar vesicles. Pre-formed M to be encapsulated
Add to suspension of LV, then sonicate. When using liposomes containing cationic lipids, dry lipid membranes should be treated with sterile water or 10 mM Tris / N.
Resuspend in a suitable solution, such as an isotonic buffered solution such as aCl, sonicate, then mix the preformed liposomes directly with the DNA. Due to the binding of the positively charged liposomes to the cationic DNA, the liposomes and DNA form a very stable complex. SUVs find use with small nucleic acid fragments. LUVs are prepared by many methods well known in the art. A commonly used method is Ca ²⁺ -EDTA chelation (Papahadjopou).
los et al., Biochim. Biophys. Acta (1975) 394: 4.
83; Wilson et al., Cell (1979)) 17:77; ether injection (D.
eamer, D.E. And Bangham, A .; , Biochim. Biophy
s. Acta (1976) 443: 629; Ostro et al., Biochem. B
iophys. Res. Commum. (1977) 76: 836; Fulle.
y et al., Proc. Natl. Acad. Sci. USA (1979) 76:33.
48); detergent dialysis (Enoch, H. and Strittmatter,
P. , Proc. Natl. Acad. Sci. USA (1979) 76:14.
5); and reverse phase evaporation (REV) (Fraley et al., J. Biol.
． Chem. (1980) 255: 10431; Szoka, F .; And Pap
ahadjopuulos, D.A. , Proc. Natl. Acad. Sci. U
SA (1978) 75: 145; Schaefer-Ridder et al., Scie.
No. (1982) 215: 166), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes is from about 10: 1 to about 1:10. Preferably, the ratio is about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

US Pat. No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with cationic liposome carriers into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466.
No. 5,693,622, No. 5,580,859, No. 5,703.
055 and WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. No. 5,589,466,
No. 5,693,622, No. 5,580,859, No. 5,703,05
No. 5 and WO 94/9469, which are incorporated herein by reference, provide methods of delivering DNA-cationic lipid complexes to mammals.

In a particular embodiment, an RN comprising a sequence encoding a polypeptide of the invention.
The retroviral particles containing A are used to engineer cells ex vivo or in vivo. Examples of the retrovirus capable of inducing a retrovirus plasmid vector include Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus,
Harvey sarcoma virus, chicken leukemia virus, gibbon leukemia virus,
Human immunodeficiency virus, myeloproliferative sarcoma virus, and breast cancer virus are included, but are not limited to.

This retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaging cell lines that can be transfected include Miller, Human Gene Therapy, 1: 5-14 (199), which is incorporated herein by reference in its entirety.
0) PE501, PA317, R-2, R-AM, PA1 as described in
2, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP +
Examples include, but are not limited to, E-86, GP + envAm12 and DAN cell lines. The vector may transduce the packaging cells by any means known in the art. Such means, electroporation, the use of liposomes, and CaPO ₄ precipitation include, but are not limited to. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or attached to lipids and then administered to a host.

This producer cell line produces infectious retroviral vector particles which contain a polynucleotide encoding a polypeptide of the invention. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cell expresses a polypeptide of the invention.

In certain other embodiments, the polynucleotides contained in the adenovirus vectors are used to engineer cells ex vivo or in vivo. Adenovirus can be engineered so that it encodes and expresses the polypeptide of the invention, while at the same time being inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, resulting in less worry about insertional mutagenesis. In addition, adenovirus has been used for many years as a live vaccine for the intestine with excellent safety aspects (Schwartzet, AR et al., (197).
4) Am. Rev. Respir. Dis. , 109: 233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in a number of cases, including the transfer of α-1-antitrypsin and CFTR into the lungs of cotton rats (R
osenfeld, M .; A. Et al., (1991) Science, 252: 431.
~ 434; Rosenfeld et al., (1992) Cell 68: 143-15.
5). Furthermore, extensive research seeking to establish adenovirus as the causative agent in human cancers was uniformly negative (Green, M. et al. (1979) Pr.
oc. Natl. Acad. Sci. USA, 76: 6606).

Suitable adenovirus vectors useful in the present invention are, for example, Koz.
arsky and Wilson, Curr. Opin. Genet. Devel
． 3: 499-503 (1993); Rosenfeld et al., Cell, 68.
: 143-155 (1992); Engelhardt et al., Human Gen.
et. Ther. 4: 759-769 (1993); Yang et al., Nature.
e Genet, 7: 362-369 (1994); Wilson et al., Natu.
Re, 365: 691-692 (1993); and US Pat. No. 5,652,2.
24, which are hereby incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the adenovirus E1 region and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene deleted from this vector. . In addition to Ad2, a variety of other adenoviruses (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably the adenovirus used in the present invention is replication defective. Replication-defective adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest operably linked to a promoter, but is unable to replicate in most cells. A replication-defective adenovirus can be deleted at one or more of the following genes: E1a, E1b, E3, E4, E2a or all or part of L1 to L5.

In certain other embodiments, adeno-associated virus (AAV) is used to engineer the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (
Muzyczka, N .; Curr. Topics in Microbiol
． Immunol. , 158: 97 (1992)). AAV is also one of the few viruses that can integrate its DNA into cells that are not dividing.
Vectors containing AAV as small as 300 base pairs can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods of generating and using such AAV are known in the art. For example, US Pat. Nos. 5,139,941, 5,173,414 and 5,354,67.
No. 8, No. 5,436, 146, No. 5,474, 935, No. 5, 478.
, 745 and 5,589,377.

For example, suitable AAV vectors for use in the present invention include all of the sequences necessary for DNA replication, encapsidation, and host cell integration. Sam
Brook et al., Molecular Cloning: A Laborator.
y Manual, Cold Spring Harbor Press (19
The polynucleotide construct is inserted into this AAV vector using standard cloning methods, such as those found in 89). The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus,
Or herpes virus. Once the packaging cells are transfected and infected, they contain infectious AAV containing polynucleotide constructs.
Generates virus particles. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cell contains the polynucleotide construct integrated into its genome and expresses a polypeptide of the invention.

Another method of gene therapy is homologous recombination (see, eg, US Pat. No. 5,641,6).
70, published June 24, 1997; International Publication No. WO 96/29411, 19
Published September 26, 1996; International Publication No. WO94 / 12650, August 4, 1994
Published: Koller et al., Proc. Natl. Acad. Sci. USA, 8
6: 8932-8935 (1989); and Zijlstra et al., Nature.
e, 342: 435-438 (1989)) operably linking a heterologous regulatory region and an endogenous polynucleotide sequence (eg, a sequence encoding a polypeptide of the invention). including. The method involves activation of a gene that is present in the target cell but is normally not expressed in that cell or is expressed at a lower level than desired.

Polynucleotide constructs are made using standard techniques known in the art. This construct contains a promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is located sufficiently close to the 5'end of the desired endogenous polynucleotide sequence so that upon homologous recombination the promoter is operably linked to the endogenous sequence.

The promoter and targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains additional restriction enzyme sites at the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence contains the 3'end of the amplified promoter. Includes the same restriction sites as the termini. The amplified promoter and targeting sequences are digested and ligated together.

Liposomes, viral sequences, viral particles, whole viruses, lipofections, either as naked polynucleotides or as described in more detail above.
The promoter-targeting sequence construct is delivered to cells, either with a transfection facilitating agent such as a precipitating agent. Direct needle injection, intravenous injection,
The P promoter-targeting sequence may be delivered by any method, including local administration, catheter infusion, particle accelerators and the like. This method is described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination occurs between this construct and the endogenous sequence, so that the endogenous sequence is placed under the control of this promoter. This promoter then drives the expression of the endogenous sequence.

[0370] Preferably, the polynucleotide encoding the polypeptide of the present invention comprises a secretory signal sequence that promotes secretion of the protein. Typically, the signal sequence is located in the coding region of the polynucleotide, expressed towards or at the 5'end of the coding region of the polynucleotide. The signal sequence may be homologous or heterologous to the polynucleotide of interest and homologous or heterologous to the transfected cells. In addition, the signal sequence can be chemically synthesized using methods known in the art.

Any dosage form of any of the above polynucleotide constructs can be used, so long as the dosage form expresses one or more molecules in an amount sufficient to provide a therapeutic effect. . This includes direct needle injections, systemic injections, catheter injections, biolistic injectors, particle accelerators (ie "gene guns"), gel foam sponge depots, and other commercial depots.
pot) substances, osmotic pumps (eg Alza minipumps), solid (tablets or pills) pharmaceutical formulations for oral or suppository, and intra-operative decanting or topical application. For example, direct injection of calcium phosphate-precipitated naked plasmid into rat liver and rat spleen or direct injection of protein-coated plasmid into the portal vein resulted in gene expression of foreign genes in rat liver (Ka
neda et al., Science, 243: 375 (1989)).

The preferred method of local administration is by direct injection. Preferably, the recombinant molecule of the present invention complexed with the delivery vehicle is administered by direct injection within the arterial region or locally within the arterial region. Topical administration of the composition within the arterial region refers to injection of the composition into the artery several centimeters, preferably several millimeters.

Another method of topical administration is to contact the polynucleotide constructs of the invention within or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the tissue surface inside the wound or the construct can be injected into the tissue area inside the wound.

Therapeutic compositions useful for systemic administration include a recombinant molecule of the present invention complexed with a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use in systemic administration include liposomes containing ligands that target the vehicle to a particular site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injections may be performed using methods standard in the art. Aerosol delivery may also be performed using methods standard in the art (eg, Stribling, incorporated herein by reference).
Et al., Proc. Natl. Acad. Sci. USA, 189: 11277-1.
1281, 1992). Oral delivery can be accomplished by complexing the polynucleotide constructs of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery may be accomplished by mixing a lipophilic reagent (eg DMSO) that is capable of passing into the skin with a polynucleotide construct of the present invention.

Determining the effective amount of a substance to be delivered can be determined, for example, by the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and administration. It may depend on a number of factors, including the pathway. The frequency of treatment is
It depends on many factors such as the amount of polynucleotide construct administered per dose and the health and medical history of the subject. The exact amount, frequency of dosing and timing of dosing will be determined by the attending physician or veterinarian.

The therapeutic compositions of the present invention may be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats,
Rabbits, sheep, cows, horses and pigs are mentioned, with humans being particularly preferred.

Biological Activity The polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides of the invention, or agonists or antagonists, are active in a particular assay, then it is likely that these molecules may be involved in the disease associated with their biological activity. Thus, the polynucleotides and polypeptides, as well as agonists or antagonists, can be used to treat related diseases.

Members of the steroid hormone receptor family of proteins transduce hormone signals across cell membranes, which bind these receptors with high affinity and specific response elements in the promoter region of target genes (hormone response). (Known as the sex element (HRE)) and induces transcription of specific genes). Accordingly, the compositions of the present invention, including the polynucleotides, polypeptides and antibodies of the present invention, as well as fragments and variants thereof, are useful for diagnosing, detecting and / or diagnosing diseases and / or disorders associated with aberrant signaling activity. It can be used in treatment. In a preferred embodiment, the compositions herein, including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof, are associated with diseases and / or disorders related to cancer (eg, breast cancer and / or It may be used in the diagnosis, detection and / or treatment of "immune activity", "anti-angiogenic activity", and "cell level diseases"). Accordingly, the polynucleotides, translation products and antibodies of the present invention are diagnostic, detecting and / or treating diseases and / or disorders related to activity including, but not limited to, diseases and / or disorders involving signal transduction and / or cancer. Useful for.

More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful in diagnosing, detecting and / or treating diseases and / or disorders associated with the following systems.

(Immune Activity) The polynucleotide, polypeptide, antibody, and / or agonist or antagonist of the present invention is obtained, for example, by activating or inhibiting proliferation, differentiation, or recruitment (chemotactic) of immune cells. , May be useful in treating, preventing, and / or diagnosing diseases, disorders and / or conditions of the immune system. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, erythrocytes, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune diseases, disorders and / or conditions can be genetic, somatic (eg cancer and some autoimmune diseases), acquired (eg due to chemotherapy or toxins) or infectious. possible. In addition, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention can be used as markers or detectors of diseases or disorders of the particular immune system.

The polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention are for treating diseases, disorders and / or conditions of hematopoietic cells,
It may be useful in prevention and / or diagnosis. A polynucleotide of the present invention,
Polypeptides, antibodies, and / or agonists or antagonists include pluripotent stem cells in an attempt to treat or prevent diseases, disorders and / or conditions associated with the reduction of certain (or many) types of hematopoietic cells It can be used to increase the differentiation and proliferation of hematopoietic cells. Examples of immunodeficiency syndromes are blood protein diseases, disorders and / or conditions (eg, agammaglobulinemia, hypogammaglobulinemia), ataxia-telangiectasia, unclassified immunodeficiency,
Di George syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCID)
, Viscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria, but is not limited thereto.

Furthermore, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention may also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). . Polynucleotides or polypeptides of the invention and / or agonists or antagonists are used to increase the hemostatic or thrombolytic activity, for example, to treat diseases, disorders and / or conditions of blood coagulation (eg fibrinogen-free). Blood, platelet deficiency), blood platelet diseases, disorders and / or conditions (eg thrombocytopenia), or wounds resulting from trauma, surgery or other causes may be treated or prevented. Alternatively,
Polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention that can reduce hemostatic or thrombolytic activity can be used to inhibit or lyse blood clots. These molecules may be important in the treatment or prevention of heart attack (infarction), stroke or scarring.

The polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention may also be useful in treating, preventing and / or diagnosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign substances by immune cells. This improper recognition triggers an immune response that results in the destruction of host tissue. Therefore, administration of the polynucleotides and polypeptides of the present invention capable of inhibiting the immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective treatment in the prevention of autoimmune disorders.

Autoimmune diseases and disorders that can be treated, prevented and / or diagnosed with the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention include, but are not limited to, one or more of the following. Not: autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis , Relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg, IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmitis, multiple endocrine glands, purpura (eg, Henoch-Schoen) Line (Henloch-
Scoenlein), Reiter's disease, Stiff-Man syndrome, autoimmune lung inflammation, autism, Guean-Barre syndrome, insulin-dependent diabetes mellitus (me
and autoimmune inflammatory eyes, autoimmune thyroiditis, hypothyroidism (ie Hashimoto's thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, receptor autoimmunity (eg (a) Graves' disease, (B) myasthenia gravis, and (c) insulin resistance), autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies (s
cleroderma), mixed connective tissue disease, polymyositis / dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis (primary glomerulonephritis and IgA nephropathy), bullous pemphigoid, Sjogren's syndrome, Diabetes and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine disorders, vitiligo, vasculitis, myocardial infarction Posterior (post-MI), open heart syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy and other inflammatory disorders, granulomatous disorders,
Degenerative and atrophic disorders.

An additional, which may be treated, prevented and / or diagnosed by the composition of the invention,
Autoimmune disorders (which can occur) include, but are not limited to, rheumatoid arthritis (eg, often characterized by immune complexes in the joints), scleroderma with anti-collagen antibodies (scleroderma).
For example, often characterized by nucleolar and other nuclear antibodies, mixed connective tissue disease (eg, often characterized by antibodies to soluble nuclear antigen (eg, ribonucleoprotein)), polymyositis (eg, Often characterized by non-histone ANA), pernicious anemia (eg, often characterized by anti-parietal cells, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (eg, by fluid and cell-mediated adrenal cytotoxicity). Characterized)
, Infertility (eg, often characterized by anti-sperm antibody), glomerulonephritis (eg, often characterized by glomerular basement membrane antibody or immune complex), bullous pemphigoid (eg, in basement membrane) IgG and complement, often characterized, Sjogren's syndrome (eg, often characterized by multiple tissue antibodies, and / or certain non-histone ANA (SS-B)), diabetes (eg, cell-mediated and humoral) Often characterized by islet cell antibodies), as well as adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (eg, often characterized by beta adrenergic receptor antibodies).

Additional autoimmune disorders that may be treated, prevented, and / or diagnosed with the compositions of the present invention include, but are not limited to: Chronic active hepatitis. (Eg, often characterized by smooth muscle antibodies), primary biliary cirrhosis (eg, often characterized by mitochondrial antibodies), other endocrine disorders (eg, in some cases often by specific tissue antibodies) , Vitiligo (often characterized by melanocyte antibodies), vasculitis (often characterized by Ig and complement in the vascular wall and / or low serum complement), post MI (eg , Often characterized by myocardial antibodies, open heart syndrome (eg, to myocardial antibodies) Thus often characterized), urticaria (eg, often characterized by IgG and IgM antibodies to IgE), atopic dermatitis (eg, often characterized by IgG and IgM antibodies to IgE), asthma ( For example, often characterized by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative and atrophic disorders.

In a preferred embodiment, autoimmune diseases and disorders and / or conditions associated with the diseases and disorders listed above are for example antagonists, or agonists, polypeptides or polynucleotides, or antibodies of the invention. Are used to treat, prevent, and / or diagnose.

In a preferred embodiment, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention are for boosting immunoreactivity in B cell and / or T cell immunodeficient individuals. It can be used as a drug.

B cell immunodeficiencies that can be ameliorated or treated by administering a polynucleotide, polypeptide, and / or agonist of the invention include, but are not limited to, the following: Severe complex Type immunodeficiency (SCID) -X
Linkage, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infant agammaglobulinemia, acquired Agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, abnormal gammaglobulinemia, hypogammaglobulinemia, transient infant hypogammaglobulinemia, non-specific hypogammaglobulinemia , Agammaglobulinemia, unclassified immunodeficiency (CVI) (acquired), Viscott-Aldrich syndrome (W
AS), X-linked immune deficiency with excess IgM, non-X-linked immune deficiency with excess IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal Ig or elevated Ig Antibody deficiency, immune deficiency with thymoma,
Ig heavy chain deletion, κ chain deletion, B cell lymphoproliferative disorder (BLPD), selective Ig
M immune deficiency, regressive agammaglobulinemia (Swiss type), reticulohypoplasia, neonatal neutropenia, severe congenital leukopenia, thymic lymphodysplasia with immunodeficiency-hypothyroidism or Dysplasia, ataxia-telangiectasia, short limbs (sho
rt limbed) dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezerov's complex immune deficiency with Ig, purine nucleoside phosphorylase deficiency (PN)
P), MHC class II deficiency (deficient lymphocyte syndrome) and severe combined immunodeficiency.

T cell disorders that can be alleviated or treated by administering a polypeptide or polynucleotide of the invention, and / or an agonist thereof, include, but are not limited to, eg, DiGeorge malformation, Thymic hypoplasia, 3rd and 4th pharyngeal sac syndrome, 22q11.2 deficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4 + T lymphocyte depletion, immune deficiency with marked T cell deficiency ( Unspecified), and unspecified immune deficiency of cell-mediated immunity. In certain embodiments, the DiGeorge malformation or condition associated with the DiGeorge malformation is alleviated or treated, eg, by administering a polypeptide or polynucleotide of the invention, or an antagonist or agonist thereof.

Other immunodeficiencies that may be ameliorated or treated by administering a polypeptide or polynucleotide of the invention, and / or their agonists or antagonists include Severe Combined Immunodeficiency (SCID; eg X-Linked). S
CID, autosomal SCID, and adenosine deaminase deficiency), ataxia-telangiectasia, viscot-oldrich syndrome, short-limb (short-).
Liber) dwarfism, X-linked lymphocytosis syndrome (XLP), Nezerov's syndrome (eg, purine nucleoside phosphorylase deficiency), MHC class II deficiency, but is not limited thereto. In certain embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are ameliorated or treated by administering a polypeptide or polynucleotide of the invention, and / or an agonist thereof. .

In certain preferred embodiments, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention are used to treat, prevent, and / or diagnose rheumatoid arthritis. In another particular embodiment, systemic lupus erythematosus is treated, prevented and / or treated with a polynucleotide, polypeptide, antibody and / or agonist or antagonist of the invention.
Or is diagnosed. In another particular preferred embodiment, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention are used to treat, prevent and / or diagnose idiopathic thrombocytopenic purpura.
In another particular preferred embodiment, IgA nephropathy is treated, prevented and / or diagnosed with the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention. In a preferred embodiment, autoimmune diseases and disorders and / or conditions associated with the diseases and disorders described above are treated, prevented, and / or diagnosed with antibodies to the proteins of the invention.

Similarly, allergic reactions and conditions such as asthma (especially allergic asthma)
Or other respiratory disorders) may also be treated, prevented and / or diagnosed with the polypeptides, antibodies or polynucleotides of the invention, and / or their agonists or antagonists. In addition, these molecules can be used to treat, prevent and / or diagnose anaphylaxis, hypersensitivity to antigenic molecules, or blood group mismatches.

In addition, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention may also be used to treat, diagnose, and / or prevent inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, respiratory disorders such as asthma and allergies.
Gastrointestinal disorders (eg, inflammatory bowel disease, etc.); cancer (eg, gastric cancer, ovarian cancer, lung cancer,
Bladder cancer, liver cancer, breast cancer, etc .; CNS disorders (eg, multiple sclerosis, blood-brain barrier permeability, ischemic brain injury, and / or cerebral infarction, traumatic brain injury, neurodegenerative disorders (eg, Parkinson's disease and Alzheimer's disease), AIDS-related dementia, and prion disease); cardiovascular disorders (eg, atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); and characterized by inflammation Many additional diseases, conditions, and disorders (eg, chronic hepatitis (
B and C), rheumatoid arthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Graves' disease, systemic lupus erythematosus, diabetes mellitus (eg, type I diabetes), and allogeneic. System transplant rejection).

In certain embodiments, the polypeptides, antibodies, or polynucleotides of the invention, and / or agonists or antagonists thereof, are useful for transplant rejection, graft versus host disease, autoimmune and inflammatory diseases (eg, Immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, type II collagen-induced arteritis,
It is useful for treating, diagnosing, and / or preventing experimental allergic and hyperacute xenograft rejection, rheumatoid arthritis, and systemic lupus erythematosus (SLE). Organ rejection results from the destruction of host immune cells in the transplanted tissue by an immune response. Similarly, the immune response also involves GVHD, in which foreign transplanted immune cells destroy host tissue. The polypeptide, antibody, or polynucleotide of the present invention and / or its agonist or antagonist (which inhibits immune response, particularly T cell activation, proliferation, differentiation, or chemotaxis) is
It may be an effective treatment to prevent organ rejection or GVHD.

Similarly, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention may also be used to modulate and / or diagnose inflammation. For example, a polypeptide, antibody, or polynucleotide of the invention, and / or an agonist or antagonist of the invention may inhibit the activation, proliferation and / or differentiation of cells involved in the inflammatory response, and Used to relate to inflammation associated with infection (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), inflammation associated with ischemia-reperfusion injury, inflammation associated with endotoxin lethality, arthritis Inflammation, inflammation associated with complement-mediated hyperacute rejection, inflammation associated with nephritis, inflammation associated with lung injury induced by cytokines or chemokines, inflammation associated with inflammatory bowel disease, inflammation associated with Crohn's disease and cytokines Chronic, including but not limited to inflammation resulting from overproduction of (eg, TNF or IL-1) Treating inflammatory conditions of both acute state and may diagnose or prognose.

The polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the invention can be used to treat, detect, and / or prevent infectious agents. For example, by increasing the immune response, especially by increasing the proliferation, activation and / or differentiation of B and / or T cells,
Infectious diseases can be treated, detected, and / or prevented. The immune response can be augmented by either augmenting an existing immune response or eliciting a new immune response. Alternatively, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention may also directly inhibit infectious agents (as described in the Infectious Agents Application Section, etc.) without the inevitable elicitation of an immune response. You can

Further preferred embodiments of the present invention include, but are not limited to, the use of polypeptides, antibodies, polynucleotides and / or agonists or antagonists in the following applications: boosting the immune system, one or more. Increased the amount of antibodies (eg, IgG, IgA, IgM, and IgE) of, and produced high affinity antibodies (eg, IgG, Ig
A, IgM and IgE) and / or animals for increasing immune response (eg, mouse, rat, rabbit, hamster, guinea pig, pig, miniature pig, chicken, camel, goat, horse, cow, sheep, Administration to dogs, cats, non-human primates, and humans, most preferably humans).

A reconstituted or partially reconstituted immune system that is either unable to produce a functional endogenous antibody molecule or has an otherwise compromised endogenous immune system, but from another animal Administration to an animal, including but not limited to transgenic animals, including but not limited to the animals listed above, which is capable of producing a human immunoglobulin molecule by
For example, PCT publication number, WO98 / 24893, WO96 / 34096, WO
96/33735, and WO 91/10741).

[0401]   A vaccine adjuvant that enhances immune responsiveness to a specific antigen.

[0402]   Adjuvants for enhancing tumor-specific immune responses.

Adjuvants that enhance the antiviral immune response. Antiviral immune responses that can be enhanced using the compositions of the invention as adjuvants include viruses and virus-related diseases and conditions described herein or otherwise known in the art. In certain embodiments, the composition of the invention has the following: AI
Used as an adjuvant to enhance the immune response to a virus, disease or condition selected from the group consisting of DS, meningitis, dengue, EBV, and hepatitis (eg hepatitis B). In another particular embodiment, the composition of the invention comprises: HIV / AIDS, RS virus, dengue, rotavirus, Japanese encephalitis,
Enhances the immune response to a virus, disease or condition selected from the group consisting of influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin virus, chikungunya virus, rift valley fever, herpes simplex, and yellow fever Used as an adjuvant.

Adjuvants for increasing the anti-bacterial or anti-fungal immune response. Anti-bacterial or anti-fungal immune responses that may be augmented using the compositions of the present invention as an adjuvant include bacterial or fungal, and those described herein or otherwise known in the art. Included are bacteria or fungi associated with the disease or condition. In a particular embodiment, the composition of the invention comprises a bacterial or fungal, disease,
Alternatively, it is used as an adjuvant to increase the immune response to a condition, which is selected from the group consisting of tetanus, diphtheria, botulism, and meningitis B. In certain preferred embodiments, the composition of the invention comprises a bacterial or fungal, disease, or symptom, which is a Vibrio cholerae, Myco.
Bacterium leprae, Salmonella typhi, Sa
lmonella paraphyphi, Meisseria meningi
tidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp. , Enter
otoxigenic Escherichia coli, Enterohe
morrhagic E. coli, Borrelia burgdorfer
i, and Plasmodium (selected from the group consisting of malaria)) as an adjuvant for increasing the immune response.

Adjuvants for increasing antiparasitic immune response. Anti-parasitic immune responses that may be augmented using the compositions of the present invention as an adjuvant include parasites and diseases associated with parasites described herein or otherwise known in the art. Or there are symptoms. In a particular embodiment, the compositions of the invention are used as an adjuvant to increase the immune response against parasites. In another particular embodiment, the composition of the invention is used as an adjuvant for increasing the immune response to Plasmodium (malaria).

[0406]   As a stimulator of B cell responsiveness to pathogens.

[0407]   As an activator of T cells.

[0408]   As a drug that increases an individual's immune status before receiving immunosuppressive therapy.

[0409]   As a drug for inducing higher affinity antibodies.

[0410]   As a drug to increase serum immunoglobulin levels.

[0411]   As a drug to promote recovery of immunocompromised individuals.

[0412]   As a drug to boost immune responsiveness among older populations.

As an immune system enhancer before, during, or after bone marrow transplants and / or other transplants (eg, allogeneic or exogenous organ transplants). With respect to transplantation, the compositions of the present invention can be administered prior to, concurrently with, and / or after transplantation. In certain embodiments, the compositions of the invention are administered after transplantation and before the onset of recovery of the T cell population. In another specific embodiment, the composition of the invention is administered first after transplantation, after initiation of recovery of the T cell population, but prior to complete recovery of the B cell population.

As an agent for boosting immune responsiveness between individuals with an acquired defect in B cell function. HIV acquired infections, AIDS, bone marrow transplants, and B cells include conditions that result in an acquired loss of B cell function that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof. Examples include, but are not limited to, chronic lymphocytic leukemia (CLL).

As an agent for boosting immune responsiveness between individuals with transient immunodeficiency. Viral infections (eg, influenza) include conditions that result in a transient immunodeficiency that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof.
Recovery from malnutrition, malnutrition-related conditions, recovery from infectious mononucleosis or stress-related conditions, recovery from measles, recovery from blood transfusions, recovery from surgery, but not limited to these. Not done.

As a regulator of antigen presentation by monocytes, dendritic cells and / or B cells. In one embodiment, the polynucleotides, polypeptides, antibodies of the invention,
And / or the agonist or antagonist enhances or antagonizes antigen presentation in vitro or in vivo. Further, in a related embodiment, this enhanced antigen presentation or antagonization may be useful as an anti-tumor treatment or to modulate the immune system.

The individual's immune system is responsive to the humoral response (ie, TH, as opposed to the TH1 cellular response).
As a drug for directing the occurrence of 2).

As a means of inducing tumor growth and thus making the tumor more sensitive to anti-neoplastic agents. For example, multiple myeloma is a slow dividing cell division.
), And is therefore refractory to virtually all antitumor regimens. If these cells were grown more rapidly, their sensitivity profile would probably change.

AIDS, Chronic Lymphocytic Disorder and / or Nonclassified Immunodeficiency (Comm
as a stimulator of B cell production in pathologies such as on Variable Immunology.

As a treatment for the production and / or regeneration of lymphoid tissue following surgery, trauma or genetic defects.

As a gene-based therapy for inherited disorders that result in immunodeficiency as observed among SCID patients.

As an antigen for generating antibodies to inhibit or enhance an immune-mediated response to a polypeptide of the invention.

[0423]   As a means of activating T cells.

Parasitic diseases affecting monocytes (Leshmania)
A) as a means of activating monocytes / macrophages to protect against.

As a pretreatment of bone marrow samples prior to transplantation. Such treatment increases B cell presentation and thus accelerates recovery.

As a means of regulating secreted cytokines induced by the polypeptides of the present invention.

Furthermore, the inventive polypeptides or polynucleotides of the invention, and / or their agonists, can be used to treat or prevent an IgE-mediated allergic response. Such allergic reactions include, but are not limited to, asthma, rhinitis and eczema.

[0428]   All of the above applications may be applied to veterinary medicine.

Antagonists of the invention include, for example, binding and / or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the steroid hormone receptor (eg, steroid hormone receptor-Fc fusion protein) (eg, Example 9 See)). They neutralize many of the activities of the above ligands and are expected to find clinical or practical applications such as: As a means of blocking various aspects of the immune response to foreign factors or self . Examples include autoimmune disorders such as lupus and arthritis, as well as skin allergies, inflammation, bowel disease, injuries and immune responses to pathogens.

As a treatment to prevent B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

As an inhibitor of B cell and / or T cell migration in endothelial cells.
This activity disrupts histology or cognate responses and is useful, for example, in disrupting the immune response and blocking sepsis.

[0432]   As an inhibitor of graft-versus-host disease or graft rejection.

ALL, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma and EBV convertible (EBV-tr
Treatment for B cell and / or T cell malignancies such as disorders.

Monoclonal hypergammaglobulinemia of undetermined significance (monoclona)
lgammopathy of unterminated signature
ance) (MGUS), Waldenström's disease, associated idiopathic monoclonal hypergammaglobulinemia, and plasmacytoma,
Treatment for chronic hypergammaglobulinemia events.

[0435]   Treatment to reduce cell proliferation of large B cell lymphoma.

[0436]   A means of reducing the involvement of B cells and Ig associated with chronic myelogenous leukemia.

[0437]   Immunosuppressants.

The polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention can be used to modulate IgE levels in vitro or in vivo.

In another embodiment, the administration of the polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the invention treats or prevents IgE-mediated allergic reactions, including but not limited to asthma, rhinitis and eczema. Can be used to

Agonists and antagonists can be used, for example, in compositions containing a pharmaceutically acceptable carrier, as described above.

Agonists or antagonists are for example macrophages and their precursors, as well as neutrophils, basophils, B lymphocytes and some T cell subsets in certain autoimmune and chronic inflammatory and infectious diseases. (For example,
Can be used to inhibit polypeptide chemotaxis and activation of activated T cells and CD8 cytotoxic T cells as well as natural killer cells). Examples of autoimmune diseases described herein include multiple sclerosis and insulin-dependent diabetes. Antagonists or agonists can also be used to treat infectious diseases, including, for example, silicosis, sarcoidosis, idiopathic pulmonary fibrosis, by preventing recruitment and activation of mononuclear phagocytes. They can also be used to treat idiopathic eosinophilia syndrome, for example by interfering with eosinophil production and migration. The antagonist or agonist can also be
For example, it can be used to treat atherosclerosis by blocking monocyte infiltration in the arterial wall.

Antibodies to the polypeptides of the invention can be used to treat ARDS.

The agonists and / or antagonists of the invention also have use in stimulating wound and tissue repair, stimulating angiogenesis, stimulating repair of vascular or lymphatic diseases or disorders. In addition, the agonists and antagonists of the invention can be used to stimulate regeneration of mucosal surfaces.

In a particular embodiment, the polynucleotide or polypeptide, and / or
Alternatively, the agonists are used to treat or prevent disorders characterized by primary or acquired immunodeficiency, defective serum immunoglobulin production, recurrent infections and / or immune dysfunction. In addition, the polynucleotides or polypeptides, and / or their agonists may be used for infection of joints, bones, skin and / or parotid glands, blood-borne infections (eg sepsis, meningitis, septic arthritis and / or bone marrow). Inflammation), autoimmune diseases (eg, autoimmune diseases as disclosed herein), inflammatory disorders, and malignant diseases, and / or any of these infections, diseases and / or malignant diseases. Disease or disorder or condition (CVID, other primary immunodeficiency, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe zoster) Herpes) and / or pneumocystis, but is not limited thereto).

[0445] In another embodiment, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention are non-classified immunodeficiency (C
Ommon Variable Immunology ("CV
ID "; also known as" acquired agammaglobulinemia "and" acquired hypogammaglobulinemia ") or an individual having a subset of this disease may be used to treat and / or diagnose .

In certain embodiments, polynucleotides, polypeptides, antibodies of the invention,
And / or agonists or antagonists treat, diagnose and / or (1) cancers or neoplasms and (2) autoimmune cell or tissue related cancers or neoplasms
Or it can be used to prevent. In a preferred embodiment, a polynucleotide, polypeptide, antibody, and / or agonist or antagonist of the invention conjugated to a toxin or a radioactive isotope, as described herein, is used in acute bone marrow. It can be used to treat, diagnose, and / or prevent sex leukemia. In a further preferred embodiment, as described herein,
Polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention conjugated to toxins or radioactive isotopes can be used to treat chronic myelogenous leukemia, multiple myeloma, non-Hodgkin's lymphoma, and / or Hodgkin's disease. Can be used to treat, diagnose, and / or prevent.

In another specific embodiment, polynucleotides or polypeptides of the invention and / or agonists or antagonists are used to produce selective Ig
A deficiency, myeloperoxidase deficiency, C2 deficiency, ataxia-telangiectasia, Di
George malformation, unclassifiable immunodeficiency (CVI), X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), chronic granulomatosis (CGD), and Wisk
It can be used to treat, diagnose, prognose, and / or prevent Ott-Aldrich syndrome.

Examples of autoimmune disorders that may be treated or detected include Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture syndrome, Graves. Disease, multiple sclerosis, myasthenia gravis, neuritis, ocular pain, bullous pemphigoid, pemphigus, multiple endocrine disorders, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, Systemic lupus erythematosus, autoimmune lung inflammation, Guyan-Barre syndrome, insulin-dependent diabetes mellitus,
And autoimmune inflammatory eye diseases, but are not limited thereto.

In a preferred embodiment, the autoimmune diseases and disorders and / or conditions associated with the above diseases and disorders are steroid hormone receptor antibodies and / or anti-steroid hormone receptor antibodies and / or soluble steroid hormone receptor polypeptides of the invention. The peptides are used to be treated, prevented, and / or diagnosed.

In certain embodiments, the compositions of the present invention provide a drug for boosting immune responsiveness among B cell immunodeficient individuals (eg, those who experience partial or complete splenectomy). Used as.

In addition, the polynucleotides, polypeptides, and / or antagonists of the present invention can affect apoptosis and are therefore useful in treating a number of diseases associated with increased cell survival or inhibition of apoptosis. . For example, diseases associated with inhibition of increased cell survival or apoptosis that can be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the invention include cancer (eg, follicular lymphoma, carcinoma with a p53 mutation). , And hormone-dependent tumors, which are: colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, (Including but not limited to myxoma, myoma, lymphoma, endothelium, osteoblastoma, giant cell tumor of bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);
Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis,
Biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg herpesvirus, poxvirus and adenovirus), Inflammation, graft-versus-host disease, acute graft rejection,
And chronic graft rejection. In a preferred embodiment, the polynucleotides, polypeptides and / or antagonists of the invention are used to inhibit the growth, progression, and / or metastasis of cancers, especially those listed above.

Additional diseases or conditions associated with increased cell survival that can be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the present invention include malignant disease progression and / or metastasis and associations such as: Disorders including, but not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia). ) As well as chronic leukemia (eg, chronic myelogenous (including
Granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (eg Hodgkin's and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and Solid tumors (sarcomas and carcinomas (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma), lymphatic sarcoma, lymphatic endothelioma, periosteal tumor, Mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma , Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchoprogenitor carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung , Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, (Including but not limited to oligodendrogliomas, menangiomas, melanomas, neuroblastomas, and retinoblastomas)).

Diseases associated with increased apoptosis that may be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the invention include: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease). , Amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related disorders; autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, biliary cirrhosis, Behcet Disease (Behcet's disease
as)), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (eg, aplastic anemia), graft-versus-host disease, ischemic injury (myocardium). Infarction, stroke and reperfusion injury), liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver cancer);
Toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Examples of hyperproliferative diseases and / or disorders that can be detected and / or treated by the polynucleotides, polypeptides, and / or antagonists of the present invention include liver, abdomen, bone, breast, digestive system, pancreas , Peritoneum, endocrine (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, As well as neoplasms located in the genitourinary organs.

Similarly, other hyperproliferative disorders can also be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the invention. Examples of such hyperproliferative disorders include hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sézary's syndrome, Waldenström macroglobulinemia, Gaucher's disease. , Histiocytosis, as well as any other hyperproliferative disease, as well as neoplasms located in the organ systems listed above,
It is not limited to them.

Hyperproliferative Disorders Polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat or detect hyperproliferative disorders, including neoplasms. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may inhibit the growth of this disorder via direct or indirect interactions. Alternatively, the polynucleotides or polypeptides of the invention, or agonists or antagonists, can proliferate other cells that can inhibit hyperproliferative disorders.

For example, by increasing the immune response, particularly by increasing the antigenic quality of hyperproliferative disorders, or by expanding, differentiating, or mobilizing T cells.
Hyperproliferative disorders can be treated. This immune response can be augmented by either enhancing an existing immune response or initiating a new immune response. Alternatively, reducing the immune response may also be a method of treating hyperproliferative disorders, such as chemotherapeutic agents.

Examples of hyperproliferative disorders that may be treated or detected by the polynucleotides or polypeptides of the invention, or agonists or antagonists, include colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands. (Adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral)
, But not limited to neoplasms located in the lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and genitourinary organs.

Similarly, other hyperproliferative disorders can also be treated or detected with the polynucleotides or polypeptides of the invention, or agonists or antagonists. Examples of such hyperproliferative disorders include hypergammaglobulinemia, lymphoproliferative disorders, disorders, paraproteinemias, purpura, sarcoidosis, Sézary syndrome, Waldenstrom macroglobulinemia, Examples include, but are not limited to, Gaucher disease, histiocytosis, as well as any other hyperproliferative disease, as well as neoplasms located in the organ systems listed above.

One preferred embodiment utilizes the polynucleotides of the invention to utilize the invention,
And / or gene therapy with protein fusions or fragments thereof inhibits abnormal cell division.

The invention thus provides a method for treating a cell proliferative disorder by inserting a polynucleotide of the invention into an abnormally proliferating cell. here,
The above polynucleotide suppresses the above expression.

Another embodiment of the invention provides a method of treating a cell proliferative disorder in an individual, which method comprises adding one or more active gene copies of the invention to abnormally proliferating cells. Is administered. In a preferred embodiment, the polynucleotide of the present invention is a DNA construct containing a recombinant expression vector effective in expressing the DNA sequence encoding the above polynucleotide. In another preferred embodiment of the invention, a DNA construct encoding a polynucleotide of the invention is inserted into a cell and treated with a retrovirus (or, more preferably, an adenovirus vector). G J. Nave, incorporated herein by reference.
1 et al., PNAS 1999 96: 324-326). In the most preferred embodiment, the viral vector is deficient and does not transform non-proliferating cells, only proliferating cells. Further, in a preferred embodiment, the polynucleotide of the invention inserted into proliferating cells, alone or in combination with or in fusion with other polynucleotides, is then subjected to external stimulation (i.e., Magnetic properties, certain small molecules, chemicals, or drug administration, etc.). This stimulus acts on a promoter upstream of the above polynucleotide to induce expression of the encoded protein product. In this way, the beneficial therapeutic effects of the present invention can be clearly modulated (ie, to increase, decrease, or inhibit expression of the polynucleotides of the present invention) based on the external stimuli described above.

Polynucleotides encoding the polypeptides of the present invention may be administered with other polynucleotides encoding other angiogenic proteins. Angiogenic proteins include acidic and basic fibroblast growth factor, VEGF-1, VEG
F-2, VEGF-3, epidermal growth factor α and β, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulation Factors, granulocyte / macrophage colony stimulating factor, and nitric oxide synthase, but are not limited thereto.

The polynucleotides of the present invention may be useful in suppressing the expression of oncogenic genes or antigens. "Suppressing the expression of oncogenic genes" means suppression of gene transcription,
It is intended to degrade gene transcripts (pre-message RNA), inhibit splicing, disrupt messenger RNA, prevent post-translational modification of proteins, disrupt proteins, or inhibit normal function of proteins.

For topical administration to abnormally proliferating cells, the polynucleotides of the invention may be administered by any method known to those of skill in the art, including transfection, electroporation, cellular Microinjection, including, but not limited to, lipofectin, or naked polynucleotides in vehicles such as liposomes, or any other method described throughout this specification. The polynucleotides of the present invention can be used in known gene delivery systems (eg, retroviral vectors (Gilboa,
J. Virology 44: 845 (1982); Hocke, Nature.
320: 275 (1986); Wilson et al., Proc. Natl. Aca
d. Sci. USA. 85: 3014); vaccinia virus system (Chakra)
barty et al., Mol. Cell Biol. 5: 3403 (1985)) or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (
1985))). These references are exemplary only and incorporated herein by reference. Retroviruses or adenoviruses known to those of skill in the art to specifically deliver to, or transfect, and to leave non-dividing cells in cells that are abnormally proliferating (eg, in the art or It is preferred to utilize a delivery system (as described herein). Since host DNA replication requires retroviral DNA to integrate, this retrovirus cannot self-replicate due to the lack of the required retroviral genes for its life cycle. For the polynucleotides of the invention, such retroviral delivery systems are utilized to target the above genes and constructs to aberrantly proliferating cells and leave non-dividing normal cells.

The polynucleotides of the present invention can be used to direct cell-proliferative disorders in internal organs, body cavities, etc. by the use of imaging devices that are used to direct the injection needle directly to the site of disease.
It can be delivered directly to the disease site. The polynucleotides of the present invention may also be administered to the disease site during surgical intervention.

By “cell proliferative disorder” is meant that any human or animal disease or disorder affects any one or any combination of organs, cavities, or body parts. The disease, benign or malignant, is characterized by a single or multiple localized abnormal growth of cells, groups of cells, or tissues.

[0468] Any amount of the polynucleotide of the invention can be administered so long as the amount has a biologically inhibitory effect on the growth of treated cells. Moreover, more than one polynucleotide of the invention can be administered at the same site at the same time. "
By “biologically inhibitory” is meant partial or total growth inhibition as well as a decrease in the rate of proliferation or growth of cells. A biologically inhibitory dose may be used to assess the effect of a polynucleotide of the invention on the growth of abnormally proliferating cells in the target malignant or tissue culture, tumor growth in animals and cell culture, or It can be determined by any other method known to those skilled in the art.

The present invention further relates to antibody-based therapies, the method comprising:
Administering anti-polypeptide and anti-polynucleotide antibodies to a mammalian (preferably human) patient to treat one or more. Methods for producing anti-polypeptide and anti-polynucleotide antibodies (polyclonal and monoclonal antibodies) are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

An overview of the ways in which the antibodies of the present invention can be used therapeutically is to provide the polynucleotides or polypeptides of the present invention locally or systemically in the body or directly to the cytotoxicity of the antibody (eg, Binding via complement mediated (CDC) or effector cell mediated (ADCC). Some of these approaches are described in more detail below. Given the teachings provided herein, one of skill in the art would know how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the invention treat a subject having or developing a cell proliferative disorder and / or a cell differentiation disorder as described herein. Useful for. Such treatment involves the administration of single or multiple doses of the antibody or fragment, derivative or conjugate thereof.

The antibodies of the invention may be advantageously used, for example, in combination with other monoclonal or chimeric antibodies, or in combination with lymphokines or hematopoietic growth factors, which determines the number of effector cells that interact with the antibody or Helps to increase activity.

[0473]   High affinity for the polypeptide or polynucleotide of the invention and / or
Is a potent in vivo inhibitory and / or neutralizing antibody, fragment thereof or
Regions are defined as polynucleotides or polypeptides of the invention (fragments thereof).
Immunoassays for) and the treatment of disorders associated therewith.
It is preferable to use Such antibodies, fragments or regions are preferred.
More preferably, the polynucleotide or polypeptide (including fragments thereof)
) Has an affinity for. The preferred binding affinity is 5 x 10^-6M, 10 ^-6 M, 5 x 10^-7M, 10^-7M, 5 x 10^-8M, 10^-8M, 5 x 10^-9M, 10 ^-9 M, 5 x 10^-TenM, 10^-TenM, 5 x 10^-11M, 10^-11M, 5 x 10^-12M
10,^-12M, 5 x 10^-13M, 10^-13M, 5 x 10^-14M, 10^-14M, 5 x 1
0^-15M and 10^-15Binding affinities with dissociation constants less than M, or Kd
Be done.

Furthermore, as described elsewhere herein, the polypeptides of the present invention, either alone or as a fusion protein or in combination with other polypeptides, are either direct or indirect. And are useful in inhibiting angiogenesis of proliferating cells or tissues. In the most preferred embodiment, this anti-angiogenic effect is
It can be achieved indirectly, for example through the inhibition of hematopoietic cells, tumor-specific cells (eg tumor-associated macrophages) (Joseph IB et al., J Natl Ca.
ncer Inst, 90 (21): 1648-53 (1998), which is incorporated herein by reference). Antibodies directed against the polypeptides or polynucleotides of the invention may also directly or indirectly result in inhibition of angiogenesis (Wite L et al. Cancer Metastatis Re.
v. 17 (2): 155-61 (1998), which is incorporated herein by reference).

The polypeptides of the invention (including fusion proteins) or fragments thereof are
It may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. This polypeptide can be used as a death domain receptor (eg, tumor necrosis factor (T
In the activation of NF) receptor 1, CD95 (Fas / APO-1), TNF receptor-related apoptosis mediator protein (TRAMP) and TNF-related apoptosis inducing ligand (TRAIL) receptor 1 and receptor 2),
It can act either directly or indirectly to induce apoptosis in proliferative cells and tissues (Schulze-Osthoff K et al., Eur J.
Biochem 254 (3): 439-59 (1998), which is incorporated herein by reference). Furthermore, in another preferred embodiment of the invention, the polypeptide is administered through other mechanisms (eg, in activating other proteins that activate apoptosis), or alone or in small molecule drugs or adjuvants (eg, Either in combination with apoptonin, galectin, thioredoxin, anti-inflammatory protein),
Through stimulation of expression of this protein, apoptosis can be induced (eg M
utat Res 400 (1-2): 447-55 (1998), Med H.
yptheses. 50 (5): 423-33 (1998), Chem Bi.
ol Interact. Apr 24; 111-112: 23-34 (199.
8), J Mol Med. 76 (6): 402-12 (1998), Int.
J Tissue React; 20 (1): 3-15 (1998), all of which are incorporated herein by reference).

The polypeptides of the present invention, including fusion proteins or fragments thereof, therewith are useful in inhibiting metastasis of proliferating cells or tissues. Inhibition can be as a direct result of administration of the polypeptide, or an antibody directed against the polypeptide as described elsewhere herein, or indirectly (eg, known to inhibit metastasis. Activation of expression of a protein (eg, α4 integrin) that has been generated (eg, Curr Top Microbiol Imm)
unol 1998; 231: 125-41, which is incorporated herein by reference). Such therapeutic effects of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention comprises a composition comprising a polypeptide of the invention (eg, a polypeptide or polypeptide antibody associated with a heterologous polypeptide, a heterologous nucleic acid, a toxin, or a prodrug. To a target cell expressing the polypeptide of the present invention. A polypeptide or polypeptide antibody of the invention may associate with a heterologous polypeptide, heterologous nucleic acid, toxin, or prodrug via hydrophobic, hydrophilic, ionic and / or covalent interactions.

The polypeptides of the invention, their fusion proteins or fragments thereof can be directly vaccinated (eg, because the polypeptides of the invention are responsive to proliferative antigens and immunogens, an immune response). "As is the case" or indirectly (eg, as in the activation of expression of proteins (eg, chemokines) known to enhance the immune response to this antigen and immunogen). , Are useful in enhancing the immunogenicity and / or antigenicity of proliferating cells or tissues.

Cardiovascular Disorders Polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat cardiovascular disorders, including peripheral arterial disease such as limb ischemia.

Cardiovascular disorders include arterio-arterial fistulas.
stula), arteriovenous fistula, cerebral arteriovenous birth defects, congenital heart defects (congeni)
cardiovascular abnormalities such as tal heart defects, pulmonary valve atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, tricentric heart, coronary vessel anomalies
, Cross heart, right thoracic heart, patent ductus arteri
oss), Ebstein's anomaly, Eisenmenger complex, left ventricular underdevelopment syndrome, left thoracic heart, tetralogy of Fallot, transposition of the great arteries, bilateral right ventricle origin, tricuspid valve closure, residual arterial canal , And heart septal deficiency
ts) (e.g., aortopulmonary separation)
eptal defect, endocardial bed deficiency, Luthambache syndrome, trilogy of Fallot, ventricular heart septal defect (ventricular heart sep
tal defects)).

Cardiovascular disorders also include arrhythmias, carcinoid heart disease, high cardiac output (hi
gh cardiac output, low cardiac output (low cardiac)
output), cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm,
Cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction (post-inf)
arcion heart rupture), ventricular septal rupture, heart valve disease,
Myocardial disease, myocardial ischemia, endocardial effusion, epicarditis (including infarct and tuberculosis),
Pneumopericardial disease, postpericardiotomy syndrome, right heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications
and heart diseases such as scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

As the arrhythmia, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature contraction, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long)
QT syndrome), collateral contraction, Lone-Ganning-Levine syndrome, Maheme-type pre-excita
tion syndrome), Wolff-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. As tachycardia, paroxysmal tachycardia,
Supraventricular tachycardia, ventricular intrinsic rhythm promotion, atrioventricular nodal reentry tachycardia (atrioventri
circular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial nodal reentry tachycardia
dal reentry tachycardia), sinus tachycardia, torsade
De pointe, and ventricular tachycardia.

Heart valve diseases include aortic valve dysfunction, aortic stenosis, and murmur (hea).
r murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve dysfunction, mitral valve stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Includes atresia, tricuspid dysfunction, and tricuspid stenosis.

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restricted cardiomyopathy, Chagas cardiomyopathy, intracardiac Membrane fibroelasticity, endocardial myocardial fibrosis, Keens syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes angina, coronary aneurysm, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunnin.
g) such as coronary artery disease.

Cardiovascular diseases also include aneurysms, angiogenesis, hemangiomatosis, bacterial hemangiopathies, Hippel-Lindau Diseases.
), Klipel-Tornone-Weber syndrome, Sturgi-Weber syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Leuriche syndrome,
Arterial occlusive disease, arteritis, arteritis, polyarteritis nodosa, cerebrovascular disease, diabetic vasculopathy, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoids, liver Venous obstruction disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease, Raynaud's disease, CREST syndrome, Retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, ataxia tela
vascular diseases such as hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcers, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms. Is mentioned.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and occlusive thrombosis. Sexual vasculitis.

Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous birth defects, cerebral artery disease, cerebral embolism and thrombosis, Carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage
Morrage), cerebral infarction, cerebral ischemia (including transient), subclavian stealing syndrome, periventricular leukomalac
ia), vascular headache, cluster headache, migraine, and vertebro.
basallar) dysfunction.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, toe cyanosis syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. As thrombosis, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
Includes sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

As ischemia, there are cerebral ischemia, ischemic colitis, partition syndrome (compartme).
nt syndrome, anterior compar
ment syndrome), myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet.
t) Syndrome, Churg-Strauss Syndrome, Mucocutaneous Lymph Node Syndrome, Obstructive Thrombotic Vasculitis, Hypersensitivity Vasculitis, Schoenline-Henoho Purpura (Schoenl)
ein-Henoch purpura), allergic cutaneous vasculitis and Wegener's granulomatosis.

The polynucleotides or polypeptides of the invention, or agonists or antagonists, are particularly effective for the treatment of dangerous limb ischemia and coronary artery disease.

The polypeptide can be administered using any method known in the art including direct needle injection at the site of delivery, intravenous injection, topical administration,
Catheter injection, biolistic injection, particle accelerators, gel foam sponge depots, other commercial depot materials, osmotic pumps, solid pharmaceutical formulations for oral or suppository, intraoperative decanting or topical application, aerosol delivery. But is not limited to these. Such methods are known in the art.
Polypeptides are described in more detail below in Therapeutic Agents (Therapeuti).
It can be administered as part of c). Methods of delivering polynucleotides are described in more detail herein.

Anti-Angiogenic Activity The naturally occurring equilibrium between an endogenous stimulator and an inhibitor of angiogenesis is the equilibrium in which the inhibitory effects predominate. Rastinejad et al., Cell 5
6: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryogenesis, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal. It is specified. Conditions for pathological angiogenesis (eg, characterizing solid tumor growth)
Below, there is no control over these adjustments. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization, including the growth and metastasis of solid tumors, arthritis, some types of ocular disorders and psoriasis. For example, Mo
ses et al., Biotech. 9: 630-634 (1991); Folkman.
N. et al. Engl. J. Med. 333: 1757-1763 (1995);
Auerbach et al. Microvasc. Res. 29: 401-411
(1985); Folkman, Advances in Cancer Re.
search, Klein and Weinhouse, Academic P
less, New York, pp. 175-203 (1985); Patz, Am.
． J. Opthalmol. 94: 715-743 (1982); and Fol.
See review by Kman et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated that suggests that solid tumor growth depends on angiogenesis. Folkman and Klagsbrun, S
science 235: 442-447 (1987).

The present invention provides treatment of diseases or disorders associated with neovascularization by the administration of the polynucleotides and / or polypeptides of the present invention and the agonists or antagonists of the present invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the invention, or agonists or antagonists, include malignant diseases, solid tumors, and cancers described herein, as well as others known in the art. (For a review of such disorders, see Fishman
Et al., Medicine, 2nd Edition, J. Am. B. Lippincott Co. , Ph
iladelphia (1985)), but is not limited thereto. Accordingly, the present invention provides a method of treatment of angiogenesis-related diseases and / or disorders, which method comprises administering a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist of the present invention Administering to an individual in need of treatment. For example, the polynucleotides, polypeptides, antagonists and / or agonists can be utilized in a variety of additional ways to therapeutically treat cancer or tumors. Cancers that can be treated with polynucleotides, polypeptides, antagonists and / or agonists include prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver cancer, parotid cancer. Solid tumors, including cholangiocarcinoma, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, renal cancer, bladder cancer, thyroid cancer; primary tumors and metastases; melanoma; glioblastoma; Kaposi's sarcoma Leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and tumors arising from blood (eg, leukemia). For example, the polynucleotides, polypeptides, antagonists and / or agonists can be delivered locally to treat cancers such as skin cancer, head and neck tumors, breast tumors and Kaposi's sarcoma.

In yet another aspect, the polynucleotides, polypeptides, antagonists and / or agonists can be utilized to treat superficial forms of bladder cancer, eg, by intravesical administration. Polynucleotides, polypeptides, antagonists and / or agonists can be delivered directly to the tumor or near the tumor site via injection or catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary with the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and / or agonists may be useful in treating other disorders in addition to cancer, including angiogenesis. These disorders include, but are not limited to, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas); atherosclerotic plaques; ocular angiogenesis. Diseases (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, rubeosis, retinoblastoma, uveitis and pterygium of the eye (Pter).
ygia) (abnormal blood vessel growth)); rheumatoid arthritis; psoriasis; delayed wound healing;
Endometriosis; Angiogenesis; Granulation; Hyperplastic scar (keloid); Pseudoarthritis fracture; Scleroderma; Trachoma; Vascular adhesion; Myocardial angiogenesis; Coronary coronary
cerebral collaterals; arteriovenous malformations; ischemic extremity angiogenesis; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, in one aspect of the present invention, treating hyperplastic scars and keloids comprising the step of administering the polynucleotides, polypeptides, antagonists and / or agonists of the present invention to hyperplastic scars or keloids. A method for doing so is provided.

In one embodiment of the invention, polynucleotides, polypeptides, antagonists and / or agonists are directly injected into hyperplastic scars or keloids to prevent the progression of these lesions. This treatment is of particular value in the prophylactic treatment of conditions known to result in the development of hypertrophic scars and keloids (eg burns), and preferably at the time the proliferative phase progresses (of the initial injury). About 14 days later) but before the onset of hyperplastic scars or keloids. As mentioned above, the present invention also provides neovascularization disorders of the eye (eg,
Methods are provided for treating corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post lens fibroplasia and macular degeneration.

Furthermore, the polynucleotides and polypeptides of the invention (agonists and / or
Ocular disorders associated with neovascularization that can be treated with (including or antagonists) include, but are not limited to, neovascular glaucoma, diabetic retinopathy, retinoblastoma, posterior lens fibrosis. Hyperplasia, uveitis, immature retinopathy, macular degeneration, corneal transplant neovascularization, as well as other inflammatory diseases of the eye, eye tumors,
And diseases associated with choroidal or iris neovascularization. For example, Waltma
n et al., Am. J. Ophthal. 85: 704-710 (1978) and G.
artner et al., Surv. Ophthal. 22: 291 to 312 (1978)
) See the review article.

Accordingly, in one aspect of the invention, corneal neovascularization (comprising the step of administering to the patient a therapeutically effective amount of a compound (as described above) to the cornea such that blood vessel formation is inhibited. Methods for treating neovascularization disorders of the eye, including corneal transplant neovascularization) are provided. Briefly, the cornea is a tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries may extend from the limbal pericorneal plexus to the cornea. When the cornea becomes vascularized, it also becomes clouded, resulting in diminished vision of the patient. The cornea becomes completely opaque (opa
vision loss can be complete. A wide variety of disorders can result in corneal neovascularization, including, for example: corneal infections (eg trachoma,
Herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (eg, graft rejection and Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (of any cause), toxic and nutritional deficiencies, and As a complication of wearing contact lenses.

In a particularly preferred embodiment, the present invention may be prepared for topical administration in saline, in combination with any preservative and antibacterial agent commonly used in ophthalmic formulations, and It can be administered in the form of eye drops. Solutions or suspensions may be prepared in pure form and administered several times daily. Alternatively, the anti-angiogenic composition prepared as described above can also be administered directly to the cornea. In a preferred embodiment, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to the cornea. In a further embodiment, the anti-angiogenic factor or anti-angiogenic composition can be utilized as an adjunct to conventional steroid therapy. Topical treatment may also be prophylactically useful in corneal lesions that are known to have a high potential to induce an angiogenic response (eg, chemical burns). In these cases, treatment (possibly combined with steroids) may be started immediately to help prevent subsequent complications.

In another embodiment, the above compounds may be injected directly into the corneal stroma by an ophthalmologist under the guidance of a microscope. Although the preferred site of injection may vary in the form of individual lesions, the goal of administration is to place the composition on the advancing surface of the vasculature (ie, dispersed between blood vessels and normal corneas). It is). In most cases this is a peril to "protect" the cornea from advancing vessels.
imbic) Cornea injection is included. This method can also be utilized immediately after corneal injury to prevent corneal neovascularization prophylactically. In this situation, the substance may be injected between the corneal lesion and its undesired potential blood supply limbus and injected into the perilimbal cornea. Such methods can also be utilized in a similar fashion to prevent capillary infiltration of the transplanted cornea. In the sustained release form, the infusion is 2 to 1 year.
It may only be needed three times. Steroids may also be added to the infusion solution to reduce inflammation resulting from the injection itself.

In another aspect of the invention, angiogenesis comprising the step of administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye so as to inhibit the formation of blood vessels. Methods for treating glaucoma are provided. In one embodiment, the compound may be administered topically to the eye to treat the early forms of neovascular glaucoma. In other embodiments, the compound may be implanted by injection into the area of the anterior chamber angle. In other embodiments, the compound can also be placed in any location such that the compound is continuously released into the aqueous humor. In another aspect of the invention, the step of administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye such that blood vessel formation is inhibited,
Methods are provided for treating proliferative diabetic retinopathy.

In a particularly preferred embodiment of the invention, proliferative diabetic retinopathy affects the aqueous humor or the vitreous to increase local concentrations of polynucleotides, polypeptides, antagonists and / or agonists in the retina. It can be treated by injection. Preferably, this treatment should be initiated prior to the acquisition of the severe disease requiring photocoagulation.

In another aspect of the invention, the lens comprising administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye such that blood vessel formation is inhibited. Methods are provided for treating post-fibroproliferative disorders. The compounds may be administered locally via intravitreal injection and / or via intraocular implantation.

In addition, disorders that can be treated with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: hemangiomas, arthritis, psoriasis, angiofibromas, atherosclerotic plaques. , Delayed wound healing, granulation, hemophilic joints, hyperplastic scars, pseudo-articular fractures, Osler-Weber syndrome, pyogenic granulomas, scleroderma, trachoma,
And vascular adhesion.

Further, disorders and / or conditions that can be treated with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: solid tumors, blood borne. Tumors (eg leukemia), tumor metastases, Kaposi's sarcoma, benign tumors (eg hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas), rheumatoid arthritis,
Psoriasis, ocular angiogenic disorders (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, rubeosis, retinoblastoma, and uveitis), Delayed wound healing, endometriosis, angiogenesis, granulation, hyperplastic scar (keloid), pseudoarticular fracture, scleroderma, trachoma, vascular adhesion, myocardial angiogenesis, coronary collateral ( coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic extremity angiogenesis, Ausler-Weber syndrome,
Plaque neovascularization, telangiectasia, hemophilic joint, angiofibromas, fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control drug (
By controlling the angiogenesis necessary for embryo implantation that controls menstruation, pathogenic consequences (eg, cat scratch disease (Rochele minalia qu).
diseases with angiogenesis, such as intosa), ulcers (Helicobacter pylori), Bartonellosis and bacterial hemangiomas).

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after sexual intercourse and fertilization occur, thus providing an effective method of birth control, perhaps "post-treatment." (Morning after) method. Polynucleotides, polypeptides, agonists and / or agonists can also be used in controlling menstruation or can be administered either as peritoneal lavage fluid in the treatment of endometriosis or for peritoneal transplantation.

The polynucleotides, polypeptides, agonists and / or agonists of the invention can be incorporated into surgical sutures to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and / or agonists are
It can be utilized in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (eg, in the form of a spray or film) is used to separate normal surrounding tissue from malignant tissue and / or prevent the spread of disease to surrounding tissue. In order to do so, it can be utilized to coat or spray the area prior to tumor removal. In another aspect of the invention, the composition (eg, in the form of a spray) is delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. obtain. In yet another aspect of the invention, a surgical mesh coated with an anti-angiogenic composition of the invention can be utilized in any procedure in which a surgical mesh can be utilized. For example, in one embodiment of the invention, a surgical mesh laden with an anti-angiogenic composition.
en) may be utilized during abdominal cancer resection surgery (eg, after colectomy) to provide support for the structure and to release certain amounts of anti-angiogenic factors.

In a further aspect of the invention, polynucleotides, polypeptides, agonists and / or agonists are added to the tumor margin after resection so that local recurrence of cancer and formation of new blood vessels at the site are inhibited. Methods for treating a tumor excision site are provided that include administering. In one embodiment of the invention, the anti-angiogenic compound is administered directly to the site of tumor resection (eg, smearing, brushing, or otherwise ablated the tumor with the anti-angiogenic compound). Applied by coating). Alternatively, the anti-angiogenic compound is
It can be incorporated into known surgical pastes prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy and neurosurgery for malignancy.

[0513] In one aspect of the invention, the polynucleotides, polypeptides, agonists and / or agonists are administered to the margins of a wide variety of tumors including, for example, breast, colon, brain and liver tumors. Can be done. For example, in one embodiment of the invention, the anti-angiogenic compound is present at the site of the neurological tumor after resection.
It can be administered so that the formation of new blood vessels at the site is inhibited.

The polynucleotides, polypeptides, agonists and / or agonists of the invention can also be administered with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasive factors, retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2, Plasminogen activator inhibitor-1, plasminogen activator inhibitor-2 and the lighter "group d" transition metals of various forms.

Lighter "group d" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium and tantalum species. Such transition metal species are
A transition metal complex can be formed. Suitable complexes of the above transition metal species include oxo transition metal complexes.

[0516] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate complexes and vanadyl complexes. Suitable vanadate complexes include, for example, metavanadate complexes and orthovanadate complexes such as ammonium metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate, including vanadyl sulfate hydrates such as vanadyl sulfate monohydrate and vanadyl sulfate trihydrate.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxotungsten complexes include tungstate complexes and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI).
An oxide is mentioned. Suitable oxomolybdenum complexes include molybdate,
Included are molybdenum oxide and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide,
Included are molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, glycerol, tartaric acid and sugar derived hydroxo derivatives.

A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include: Platelet Factor 4; Protamine Sulfate; Sulfated Chitin Derivatives (prepared from queen crab shells) (Murata et al. Cancer Res. 51: 22-26, 1991)
Sulphated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids (eg, estrogen) and tamoxifen citrate); staurosporine; regulators of substrate metabolism (eg, (Including proline analogs, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, α, α-dipyridyl, aminopropionitrile fumarate)
4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone; methotrexate; mitoxantrone; heparin; interferon; 2 macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267).
: 17321-17326, 1992); chymostatin (Tomkinson et al., Biochem J. 286: 475-480, 1992); cyclodextrin tetradeca sulfate; eponomycin; camptothecin; fumagillin (
Ingber et al., Nature 348: 555-557, 1990); Sodium gold thiomalate ("GST"; Matsubara and Ziff, J.C.
lin. Invest. 79: 1440-1446, 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. Chem.
． 262 (4): 1659-1664, 1987); Bisantoren (Natio).
nal Cancer Institute); lobenzarit disodium (
N- (2) -carboxyphenyl-4-chloroanthronylic acid (chloroa)
nthoronic acid) disodium, or "CCA"; Tak
euchi et al., Agents Actions 36: 312-316, 199.
2); thalidomide; Angostatic steroids; AGM-1470; carboxynaminolmidazole.
); And metalloproteinase inhibitors (eg, BB94).

Diseases at the Cellular Level Diseases associated with increased cell survival or inhibition of apoptosis that can be treated or detected by the polynucleotides or polypeptides of the invention and antagonists or agonists include cancer (eg, follicular). Lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, which are: colon cancer, heart tumor,
Pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer,
Including neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, giant cell tumor of bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer,
Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, biliary cirrhosis, Behcet's d).
disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg, herpesvirus, poxvirus and adenovirus), inflammation, graft-versus-host disease, acute Graft rejection as well as chronic graft rejection are mentioned. In a preferred embodiment, the polynucleotides, polypeptides, and / or antagonists of the invention include cancer growth, progression, and / or metastasis (especially listed above).
It is used to inhibit the metabolism.

Additional diseases or conditions associated with increased cell survival that can be treated or detected by the polynucleotides or polypeptides of the present invention, or agonists or antagonists, include malignant disease progression and / or metastasis and associations such as: Disorders including, but not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia). Including)) and chronic leukemia (eg, chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's and non-Hodgkin's disease), multiple bone marrow Tumors, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (
Sarcomas and carcinomas (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphatic sarcoma, lymphatic endothelium, periostealoma, mesothelioma) , Ewing tumor, leiomyosarcoma,
Rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, pith Cancer, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal Somatoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma
, Melanoma, neuroblastoma, and retinoblastoma)).

Diseases associated with increased apoptosis that can be treated or detected by the polynucleotides or polypeptides of the invention and agonists or antagonists include: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease). , Amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related disorders; autoimmune disorders (eg, multiple sclerosis,
Sjogren's syndrome, Hashimoto thyroiditis, biliary cirrhosis, Behcet's disease (Behc
et's disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (eg, aplastic anemia), graft-versus-host disease, ischemic Injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (eg hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxicants Induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation In accordance with yet a further aspect of the present invention, a polynucleotide or polypeptide of the present invention, as well as an antagonist or agonist, are used to treat epithelial cells for therapeutic purposes, eg, for wound healing purposes. Processes are provided that are utilized to stimulate proliferation and basal keratinocytes, as well as to stimulate hair follicle production and skin wound healing. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may be clinically useful in stimulating wound healing, including: surgical wounds, excisional wounds, deep wounds (damage to the dermis and epidermis). ), Ocular tissue wounds, tooth tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and others Abnormal wound healing states (eg, uremia, malnutrition, vitamin deficiency, and complications associated with systemic treatment with steroids, radiation therapy and antitumor drugs and antimetabolites. Polynucleotides or Polynucleotides of the Invention. Peptides, as well as agonists or antagonists, can be used to promote skin recovery after skin loss.

Polynucleotides or polypeptides of the invention, as well as agonists or antagonists, are used to increase adhesion of skin grafts to the wound bed and to stimulate re-epithelialization from the wound bed. Can be done. The following are types of implants in which the polynucleotides or polypeptides, agonists or antagonists of the invention can be used to increase adhesion to the wound bed: autograft, artificial skin, allograft. , Autograft, autoepdermographic graft, avascular
r) Grafts, Blair-Brown grafts, bone grafts, embryonic tissue grafts, dermal grafts, delayed grafts, skin grafts, epidermal grafts, fascia grafts, full-thickness skin grafts, xenografts. (Heterologous graft), xenograft (xenogra
ft), allograft, proliferative graft, lamellar graft, reticular graft, mucosal graft, Olie-Tielsch graft, omental graft, patch graft. , Stem grafts, full-thickness grafts (p
enetrating graft, split skin graft
Raft), a split-thickness graft. The polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, can be used to promote skin strength and improve the appearance of aged skin.

The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may also induce hepatocyte proliferation and lung, breast, pancreas, stomach, small intestine (smal).
l intesting), and changes in epithelial cell proliferation in the large intestine. The polynucleotides or polypeptides of the invention, as well as the agonists or antagonists, can be used to bind epithelial cells (eg, sebocytes).
te), hair follicles, hepatocytes, type II alveolar epithelial cells (type II pneumo).
cyte), mucin-producing goblet cells, and other epithelial cells, and their precursors contained in the skin, lungs, liver, and gastrointestinal tract). The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, can also be used to reduce the toxic side effects of gut toxicity resulting from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may have a cytoprotective effect on the small intestinal mucosa. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may also stimulate healing of mucositis (mouth ulcers) resulting from chemotherapy and viral infections.

Polynucleotides or polypeptides of the invention, as well as agonists or antagonists, provide complete regeneration of skin (ie, hair follicles, sweat glands, and sweat glands, and Regrowth of the sebaceous glands), and other skin defects such as psoriasis. Polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, may affect epidermolysis bullosa, an epidermis to the underlying dermis that results in frequent open and painful blisters by promoting re-epithelialization of these lesions. Can be used to treat adhesion defects). The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, also treat gastric and duodenal ulcers and heal by scarring of the lining of the mucosa and regeneration of the lining of the glandular and duodenal mucosa. It can be used to help more quickly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases that cause destruction of the mucosal surface of the small intestine or large intestine, respectively. Thus, the polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, promote resurfacing of mucosal surfaces, aid in the faster healing of inflammatory bowel disease, and of inflammatory bowel disease. It can be used to prevent progression. Treatment with the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, is expected to have a significant effect on the production of mucus in the entire gastrointestinal tract, and either from ingested harmful substances or after surgery. , Can be used to protect the intestinal mucosa from harmful substances. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used to treat diseases associated with its underexpression.

In addition, the polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, can be used to prevent and cure damage to the lung due to various pathological conditions. The polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, can stimulate the growth and differentiation of alveolar and bronchiola epithelium and repair them to prevent or treat acute or chronic lung injury. Can be promoted. For example, alveolar
i) emphysema resulting in progressive loss, and inhalation injury resulting in bronchiolar epithelium and alveolar necrosis (ie resulting from smoke inhalation and burns) is a polynucleotide or polypeptide of the invention. , Agonists or antagonists can be used to effectively treat. Also, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used to stimulate the proliferation and differentiation of type II alveolar epithelial cells, which results in pulmonary hyalineosis in premature infants (e.g., It can help treat or prevent diseases such as infant respiratory distress syndrome and bronchopulmonary dysplasia.

Polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can stimulate hepatocyte proliferation and differentiation, and thus disease and pathology of the liver (eg, fulminant liver failure caused by cirrhosis, viral hepatitis). And toxic substances (ie, acetaminophen, carbon tetrachloride).
), and other liver toxins known in the art), which may be used to alleviate or treat liver damage).

Furthermore, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used to treat or prevent the onset of diabetes mellitus. In patients newly diagnosed with type I and type II diabetes, if some islet cell function remains, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, will result in permanent expression of the disease. , Can be used to maintain, maintain, retard, or prevent its islet function. Also, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used as an adjunct in islet cell transplantation to improve or promote islet cell function.

Neurological Disorders In accordance with yet a further aspect of the invention, a polynucleotide or polypeptide of the invention and an agonist for therapeutic purposes, eg, to stimulate neurological cell proliferation and / or differentiation. Alternatively, a process is provided for utilizing the antagonist. Thus, the polynucleotides, polypeptides, agonists and / or antagonists of the invention can be used to treat and / or detect neurological disorders. Furthermore, the polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used as markers or detectors of certain nervous system diseases or disorders.

Examples of neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the invention include: brain disorders (eg of maternal phenylketonuria). Metabolic brain diseases including phenylketonuria), pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke encephalopathy, cerebral edema, infratent (infr
cerebellar neoplasms such as cerebellar neoplasms including neoplasms, ventricular neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, tentative neoplasms, Canavan's disease, ataxia-telangiectasia Cerebellar diseases such as cerebellar ataxia including spinocerebellar ataxia, cerebellar dyskinesia, Friedreich's ataxia, Machado-Joseph disease, olive pontine cerebellar atrophy, cerebellar neoplasms such as subtentorial neoplasms , Diffuse periaxial encephalitis, bulbous cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy and subacute sclerosing panencephalitis, cerebrovascular disease (eg carotid thrombosis, carotid stenosis) And carotid artery disease including Moyamoya disease), brain amyloid angiopathy, cerebral aneurysm,
Cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, carotid thrombosis, sinus thrombosis and cerebral embolism and thrombosis such as Wallenberg syndrome, epidural hematoma, subdural hematoma And cerebral hemorrhage such as subarachnoid hemorrhage, cerebral infarction, transient cerebral ischemia,
Subclavian steal syndrome and vertebrobasilar insufficiency
cerebral ischemia such as insufficiency, vascular dementia such as multiple cerebral infarction dementia, periventricular leukomalac
ia), vascular headache such as cluster headache, migraine, dementia such as AIDS dementia complex, presenile dementia such as Alzheimer's disease and Creutzfeldt-Jakob disease, Alzheimer's disease and progressive supranuclear palsy Such as senile dementia, vascular dementia such as multiple cerebral infarction dementia, encephalitis such as diffuse periaxial encephalitis, epidemic encephalitis, Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis and West Nile fever Viral encephalitis, acute disseminated encephalomyelitis, uveal meningitis syndrome, meningoencephalitis such as post-encephalitis Parkinson's disease and subbulbar sclerosing panencephalitis, encephalomalacia such as periventricular leukoplakia, Epilepsy such as generalized epilepsy, including nasal spasms, Absence epilepsy (abs)
epilepsy), myoclonus epilepsy including MERRF syndrome, tonic / clonic epilepsy, partial epilepsy such as frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, persistent epilepticus such as continuous partial epilepsy , Hydrocephalus such as Hallerfolden-Spatz syndrome, Dandy-Walker syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy including cataplexy, medullary polio (bul)
Cerebral pseudotumor, Rett's syndrome, Reye's syndrome, thalamic disease, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger's syndrome, central nervous system infections such as AIDS dementia, brain abscess, subdural empyema. , Encephalomyelitis, such as equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
Visna, cerebral malaria, meningitis such as arachnoiditis, aseptic meningitis such as viral meningitis including lymphocytic choriomeningitis, bacterial meningitis including Haemophilus meningitis, Listeria meningitis, meningococcal meningitis such as Waterhouse-Friedericksen syndrome, pneumococcal meningitis and meningococcal disease, fungal meningitis such as cryptococcal meningitis, Subdural exudate, meningoencephalitis such as uveal meningoencephalitis syndrome, myelitis such as transverse myelitis, neurosyphilis such as spinal fistula, poliomyelitis including medullary polio and post-polio syndrome,
Prion diseases (eg Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, Gerstcon-Stroisler syndrome, kuru, scrapie) cerebral toxoplasmosis, brain neoplasms including cerebellar neoplasms such as subtentorial neoplasms Central nervous system neoplasms, choroid plexus neoplasms, ventricular neoplasms such as hypothalamic and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms including epidural neoplasms, such as Canavan's disease Diffuse, including demyelinating disease, adrenoleukodystrophy
cerebral cerebral disease, diffuse periaxial encephalitis, bulbous cell leukodystrophy, metachromatic leukodystrophy diffuse cerebral sclerosis, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal lesions Leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, scrapie, lordosis, chronic fatigue syndrome, visna, high pressure nerve syndrome
s Syndrome), spinal cord disorders such as meningopathy, congenital myotonia, amyotrophic lateral sclerosis, spinal muscle atrophy such as Verdnig-Hoffmann disease, spinal cord squeezing, epidural neoplasia Spinal neoplasms, syringomyelia, spinal fistula, Stiffman syndrome,
Maternal origin 15q-13 Minor deficiency such as micro deficiency, cat squealing syndrome, de Lange syndrome, Down syndrome, gangliosidosis such as gangliosidosis G (M1), Zanthoff's disease, Tay-Sachs disease, heart-up Disease, homocystinuria, Lawrence-Moon-Beedle syndrome, Lesch-Nyhan syndrome, maple syrup disease, mucolipidosis such as fucose storage disease, neuronal ceroid lipofuscinosis, ocular-brain renal syndrome, maternal phenylketonuria Nervous system abnormalities such as phenylketonuria, Prader-Villi syndrome, Rett syndrome, Rubinstein-Taby syndrome, tuberous sclerosis, WAGR syndrome, total forebrain, water incompetence (h
neural tube defects such as incompetence including dyrangenesis, arnold-chiari malformations, cerebral hernias, meningocele, meningocele, spinal cord fusion disorders such as cystic spina bifida and occult spina bifida, Charcot-Marie disease Hereditary sensory and autonomic disorders such as motor and sensory neuron disorders, including visual atrophy, Refsum disease, hereditary spastic paraplegia, Verdnig-Hoffmann disease, congenital analgesia and familial autonomic neuropathy Neuropathy, neurological manifestations (eg, agnosia including Gerstmann syndrome), amnesia such as retrograde amnesia, apraxia, neurogenic bladder, weakness, deafness, partial hearing loss and loud voice ( communication disorders such as deafness, including rehabilitation and tinnitus, ataxia, Broca aphasia and Wernicke loss Speech disorders such as aphasia, including dyslexia, dyslexia such as acute dyslexia, language development disorders, aphasia such as aphasia, aphasia including Broca aphasia and Wernicke aphasia, accumulation disorders, construction disorders, reverberant language, Information transmission disorders such as speech disorders including silence and stuttering, speech disorders such as aphonia and hoarseness,
Decerebrate rigidity, delirium, fasciculations, hallucinations, meningopathy, maternal origin 15 q-13
Minor deficiency, ataxia, athetosis, chorea, ataxia, hypokinesis, hypotonia, movement disorders such as myoclonus, tics, torticollis and tremor, muscle tension such as stiff man syndrome such as stiff man syndrome. Hyperparalysis, muscular spasticity, nerve palsy such as facial nerve palsy, including ear zoster, gastroparesis, hemiplegia, ophthalmoplegia such as diplopia, chronic progressive externals such as duene syndrome, horner syndrome, keens syndrome Paraplegia such as ophthalmoplegia, bulbar paralysis, tropical spastic paraparesis, Brown-Secar syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, taste palsy, phantom limbs, tastelessness and taste dysgeusia Disability, amblyopia, blindness, color blindness, diplopia, semi-blindness, visual disorders such as scotoma and subnormal vision, Kleine-Levin syndrome, Sleep disorders such as hypersomnia including insomnia and sleepwalking, spasticity such as trismus, coma, persistent vegetative state and loss of consciousness such as syncope and dizziness, neuromuscular such as congenital myotonia Disease, amyotrophic lateral sclerosis, Lambert-Eaton myasthenic syndrome, motor neuron disease, atrophy of the spine muscle, Charcot-Marie disease and muscular atrophy such as Verdnig-Hoffmann disease, post-polio syndrome, muscular dystrophy, severe muscle Asthenia, atrophic myotonia, congenital myotonia, nemarin myopathy, familial quadriplegia, multiple paramyloclonus (Multiplex P)
amyloclonus), tropical convulsive paraparesis and Stiffman syndrome, peripheral nervous system diseases such as acropacia, renal amyloidosis, Ardy syndrome, Barre-Lieou syndrome, familial autonomic neuropathy, Horner syndrome, reflex sympathy. Autonomic nervous system diseases such as neurodystrophy and Shy-Drager syndrome, cranial nerve diseases such as inner ear nerve diseases such as acoustic neuroma including neurofibromatosis 2, facial nerve diseases such as facial neuralgia, Merkerson-Rosen Tar syndrome, ocular motility disorders including amblyopia, nystagmus, ocular palsy, ophthalmoplegia such as Duane's syndrome, horner syndrome, chronic progressive external ophthalmoplegia including Keenes syndrome, strabismus and exotropia God like ocular atrophy, including strabismus, oculomotor nerve palsy, and hereditary eye atrophy Diseases, optic disc drusen, optic neuritis such as optic myelitis, papilledema, trigeminal neuralgia, vocal cord paralysis, demyelinating diseases such as neuromyelitis optica and lordosis, diabetic neuropathy such as diabetic foot, Neural squeezing syndrome such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve squeezing syndrome, causalgia, brachial neuralgia, neuralgia such as facial neuralgia and trigeminal neuralgia, experimental allergic Neuritis, optic neuritis, polyneuropathy, polyradiculoneuritis and radiculitis (eg polyradiculoneuritis, hereditary motor and sensory neuropathy (eg Charcot-Marie disease, hereditary eye atrophy) , Lefsum disease, hereditary spastic paraplegia and Wernig-Hoffmann disease, hereditary sensory and autonomic neuropathy (congenital analgesia and. Including sexual autonomic neuropathy), POEMS
Neuritis such as the syndrome, sciatica, taste sweating syndrome and tetany)).

Infectious Diseases Polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used to treat or detect infectious agents. For example,
Infectious diseases can be treated by increasing the immune response, especially by increasing the proliferation and differentiation of B and / or T cells. The immune response is
It can be elevated either by raising an existing immune response or by initiating a new immune response. Alternatively, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may also directly inhibit the infectious agent without necessarily eliciting an immune response.

Viruses are an example of infectious agents that can cause diseases or conditions that can be treated or detected by the polynucleotides or polypeptides of the invention, and / or agonists or antagonists. Examples of viruses include the following D
Viruses and viruses of NA and RNA include, but are not limited to: Arbovirus, Adenovirus, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Calciviridae, Circoviridae. , Coronavirus family, Dengue fever, EBV, HI
V, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (eg, cytomegalovirus, herpes simplex, herpes zoster), Mononegavirus (eg, paramyxoviridae, measles virus, rhabd) Viridae), Orthomyxoviridae (eg, influenza A, influenza B, and parainfluenza), papillomavirus, papovaviridae, parvoviridae, picornaviridae, poxviridae (eg, pox or vaccinia), reovirus. Family (for example, rotavirus), retroviridae (HTLV-I, HTLV-II, lentivirus),
And Togaviridae (eg, Rubivirus). Viruses that fall within these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, ocular infections (eg, conjunctivitis). , Keratitis), chronic fatigue syndrome,
Hepatitis (A, B, C, E, chronic activity, Delta), Japanese encephalitis, Argentine hemorrhagic fever, Chingnia, Rift Valley fever, yellow fever, meningitis, opportunistic infections (eg AIDS), Pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, colds, polio, leukemia, rubella, sexually transmitted diseases,
Skin diseases (eg capouge, warts), and viremia. Any of these conditions or diseases can be treated or detected using the polypeptides or polynucleotides of the invention, or agonists or antagonists. In certain embodiments, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used for the treatment of: Meningitis, Dengue, EBV, and / or Hepatitis (eg Hepatitis B). In a further specific embodiment,
The polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients who are non-responsive to one or more other commercially available hepatitis vaccines. In a more specific embodiment, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.

Similarly, bacterial or fungal agents that can cause a disease or condition and that can be treated or detected by the polynucleotides or polypeptides of the invention, and / or agonists or antagonists are the following Gram-negative and Gram-negative: Positive bacteria and bacteria, including but not limited to fungi: A
ctinomycetales (eg, Corynebacterium, M
ycobacterium, Norcardia), Cryptococcus
neoformans, Aspergillosis, Bacilracea
e (eg, Anthrax, Clostridium), Bacteroid
aceae, Blastomycosis, Bordetella, Borre
lia (eg, Borrelia burgdorferi), Brucel
loss, Candidiasis, Campylobacter, Cocc
ioidomycosis, Cryptococcosis, Dermat
ocycoses, E. coli (for example, Enterotoxigenic
E. E. coli and Enterohemorrhagic E. coli. coli), E
nterobacteriaceae (Klebsiella, Salmone
lla (eg, Salmonella typhi, and Salmonel
la paratyphi), Serratia, Yersinia), Ery
sipelothrix, Helicobacter, Legionellos
is, Leptospirosis, Listeria, Mycoplasma
tales, Mycobacterium leprae, Vibrio ch
olerae, Neisseriaceae (for example, Acinetobacter
er, Gonorrhea, Menigococcal), Meisseria
meningitidis, Pasteurellacea infections (eg, Actinobacillus, Hemophilus (eg, Heam)
opilius influenza B type), Pasteurella), P
sudomonas, Rickettsiaceae, Chlamydiac
eae, Syphilis, Shigella spp. , Staphyloc
occal, Meningiococcal, Pneumococcal and Streptococcal (eg, Streptococcus pneu
moniae and Group B Streptococcus). These bacterial or fungal families can cause diseases or conditions including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunism. Infections (eg, AIDS-related infections), periungual inflammation, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg, pertussis or pyometra), sepsis, Lyme disease, cat scratch disease, dysentery, Paratyphoid fever, Food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (eg meningitis type A and B), chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulinum poisoning, gangrene, tetanus, impetigo , Rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatomycosis)
), toxemia, urinary tract infection, wound infection. The polypeptides or polynucleotides, agonists or antagonists of the invention may be used to treat or detect any of these conditions or diseases. In a specific embodiment, the polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diphtheria, botulinum, and / or meningitis type B.

Further, diseases or conditions caused by a parasitic factor that can be treated or detected by the polynucleotides or polypeptides of the invention and / or agonists or antagonists include, but are not limited to, the following families or classes: Not to be done: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dinuclear amebiasis (Dientamoebiasis), quarantine, ectoparasite, giardia flagellosis, helminthosis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomes And Trichomonas (Tric
homonas), as well as sporozoan disease (Sporozoan) (eg Pl
asmodium virax, Plasmodium falcipariu
m, Plasmodium malariae and Plasmodium o
vale). These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, scrub typhus, ocular infections, intestinal diseases (eg, dysentery, giardia giardiasis), liver diseases, lungs. Diseases, opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may be used to treat or detect any of these conditions or diseases.

A polynucleotide or polypeptide of the invention, as well as an agonist or antagonist, administers to a patient an effective amount of the polypeptide, or removes cells from the patient and supplies the polynucleotides of the invention to the cells, Then the engineered cells can either be returned to the patient (ex vivo treatment). Moreover, the polypeptides or polynucleotides of the invention can be used as antigens in vaccines to elicit an immune response against infectious diseases.

Regeneration Polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used to differentiate, proliferate and attract cells to guide tissue regeneration (Science 276: 59-87 ( 1997)). Tissue regeneration can be used with birth defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis).
tis), periodontal disease, liver failure), surgery including cosmetic surgery, fibrosis, reperfusion injury, or tissue damaged by systemic cytokine damage may be repaired, replaced, or protected.

Tissues that can be regenerated using the present invention include the following: Organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, or myocardium),
Tissues of the vascular system (including blood vessels and lymph vessels), nerves, hematopoiesis, and skeleton (bones, cartilage, tendons, and ligaments). Preferably, regeneration occurs without or with reduced scarring. Regeneration can also include angiogenesis.

Furthermore, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, may increase the regeneration of tissues that are difficult to heal.
For example, increasing tendon / ligament regeneration speeds recovery time after injury. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can also be used prophylactically in an attempt to avoid injury.
Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Further examples of tissue regeneration of non-healing wounds include ulcers associated with decubitus ulcers, vascular insufficiency, surgical wounds, and traumatic wounds.

Similarly, nerve and brain tissue can also be regenerated by using the polynucleotides or polypeptides of the invention and agonists or antagonists to proliferate and differentiate nerve cells. Diseases that can be treated using the present methods include central and peripheral nervous system diseases, neuropathy, or mechanical and traumatic disorders (eg, spinal cord disorders, head trauma, cerebrovascular disorders, and stroke (
stock)). In particular, diseases associated with peripheral nerve injury, peripheral neuropathy (eg resulting from chemotherapy or other medical therapy), localized neuropathy,
And central nervous system disorders (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) are all using the polynucleotides or polypeptides of the invention and agonists or antagonists. Can be treated.

Chemotaxis The polynucleotides or polypeptides of the invention, as well as the agonists or antagonists, may have chemotactic activity. Chemotactic molecules allow cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to move to specific sites in the body (eg, Site of inflammation, infection, or hyperproliferation). The recruited cells may then repel and / or heal the particular trauma or abnormality.

The polynucleotides or polypeptides of the invention, as well as the agonists or antagonists, may increase the chemotactic activity of certain cells. These chemotactic molecules are then used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to specific locations in the body. obtain. For example, chemotactic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. The chemotactic molecules of the invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that the polynucleotides or polypeptides of the invention, as well as the agonists or antagonists, may inhibit chemotactic activity. These molecules can also be used to treat disorders. Thus, the polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can be used as chemotaxis inhibitors.

Binding Activity The polypeptides of the invention can be used to screen for molecules that bind to this polypeptide, or for molecules to which this polypeptide binds. The binding of the polypeptide to the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of the bound polypeptide or molecule.
Examples of such molecules include antibodies, oligonucleotides, proteins (eg receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand (eg, fragment of the ligand) of the polypeptide or natural substrate, ligand, structural mimetic, or functional mimic (Coligan et al. , Current Prot
ocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, the molecule may be intimately associated with the natural receptor to which the polypeptide binds, or at least a fragment of the receptor (eg, the active site) that may be bound by the polypeptide. In any case, the molecule can be rationally designed using known techniques.

Preferably, screening for these molecules involves producing suitable cells that express the polypeptide. Preferred cells include mammalian, yeast, Drosophila, or E. cells derived from E. coli. The cells expressing the polypeptide (or cell membranes containing the expressed polypeptide) are then preferably observed for binding, stimulating, or inhibiting activity of either the polypeptide or the molecule. Are contacted with a test compound potentially containing the molecule of

The assay may simply test the binding of a candidate compound to the polypeptide, where binding is detected by the label or in an assay for competition with a labeled competitor. In addition, the assay can test whether a candidate compound produces a signal generated by binding to this polypeptide.

Alternatively, the assay can be performed with cell-free preparations, polypeptides / molecules attached to a solid support, chemical libraries, or a mixture of natural products. The assay also simplifies the steps of mixing the candidate compound with a solution containing the polypeptide, measuring the activity or binding of the polypeptide / molecule, and comparing the activity or binding of the polypeptide / molecule to a standard. May be included.

Preferably, the ELISA assay may measure the level or activity of a polypeptide in a sample (eg, biological sample) using monoclonal or polyclonal antibodies. Antibodies may be bound to the polypeptide either directly or indirectly, or by competition with the polypeptide for a substrate,
The level or activity of the polypeptide can be measured.

Furthermore, the receptors to which the polypeptides of the invention bind may be obtained by a number of methods known to those skilled in the art, such as ligand panning and FACS sorting (Coliga).
n et al., Current Protocols in Immun. , 1 (2),
Chapter 5 (1991)). For example, expression cloning is used when polyadenylated RNA is prepared from cells responsive to this polypeptide, eg, NIH3T3 cells, which are known to contain multiple receptors for FGF family proteins. , And SC-3 cells, and a cDNA library made from this RNA are pooled and used to transfect COS cells or other cells that are not responsive to the polypeptide. Transfected cells growing on glass slides are exposed to the polypeptides of the invention after labeling them. The polypeptide may be labeled by various means, including iodination or encapsulation of recognition sites for site-specific protein kinases.

After immobilization and incubation, slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an iterative subpooling and rescreening process to ultimately yield a single clone encoding the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptide may be photoaffinity linked to the cell membrane or a preparation expressing the receptor molecule may be extracted. The crosslinked material is separated by PAGE analysis and exposed to X-ray film. A labeled complex containing the receptor for the polypeptide can be excised, separated into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

Furthermore, the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”) may be used to modulate the activity of the polypeptides of the invention, thereby. Effectively produces agonists and antagonists of the polypeptides of the invention. Generally, US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,83.
7,458, and Patten, P .; A. Curr. Opinion
Biotechnol. 8: 724-33 (1997); Harayama,
S. Trends Biotechnol. 16 (2): 76-82 (1998)
); Hansson, L .; O. Et al., J. Mol. Biol. 287: 265-7
6 (1999); and Lorenzo, M .; M. And Blasco, R .;
See Biotechniques 24 (2): 308-13 (1998), each of which patents and publications are incorporated herein by reference. In one embodiment, alteration of the polynucleotide and corresponding polypeptide can be accomplished by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule of the invention by homologous or site-specific recombination. In another embodiment, the polynucleotides of the invention and corresponding polypeptides are subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Can be changed. In another embodiment, one or more components, motifs, fragments, portions, domains, fragments, etc. of the polypeptides of the invention are one or more components, motifs, fragments, portions, domains of one or more heterologous molecules. , Fragments, etc. In a preferred embodiment, the heterologous molecule is a member of the family. In a more preferred embodiment, the heterologous molecule is, for example, platelet derived growth factor (PDGF),
Insulin-like growth factor (IGF-I), transforming growth factor (TGF)
-Α, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-β,
Bone morphogenetic protein (BMP) -2, BMP-4, BMP-5, BMP-6, BM
P-7, activin A and activin B, decapentapresic (decap
entangled (dpp), 60A, OP-2, dosalin (dors)
alin), growth differentiation factor (GDF), nodule, MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3, TGF-β5, and glial-derived neurotrophic factor (GDNF). It is a growth factor.

Other preferred fragments are biologically active fragments of the polypeptides of the invention. A biologically active fragment is a fragment that exhibits an activity similar to, but not necessarily identical to, that of the polypeptide of the invention. The biological activity of this fragment may include an improved desired activity, or a decreased undesired activity.

The invention further provides methods of screening compounds to identify those that modulate the action of the polypeptides of the invention. An example of such an assay involves combining mammalian fibroblasts, a polypeptide of the invention, the compound to be screened and ³ [H] thymidine under cell culture conditions in which the fibroblasts normally grow. . Control assays are performed in the absence of the compound to be screened to obtain, and compared to the amount of fibroblast proliferation in the presence of the compound, ³ in each case [H] thymidine incorporation of The determination can determine whether the compound stimulates proliferation. The amount of fibroblast proliferation is ³ [H
] Measured by liquid scintillation chromatography, which measures thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

In another method, mammalian cells or membrane preparations that express the receptor for a polypeptide of the invention are incubated with a labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured. Alternatively, the response of a known second messenger system following interaction of the compound to be screened with the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured Determine whether a compound is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays can be used to treat or treat a disease or to bring a particular result to a patient (eg, blood vessel growth) by activating or inhibiting a polypeptide / molecule. . In addition, the assay can discover factors that can inhibit or enhance production of the polypeptides of the invention from appropriately engineered cells or tissues.

Accordingly, the invention includes a method of identifying a compound that binds to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and ( b) determining whether a bond has occurred. Further, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying biological activity, And (b) determining whether the biological activity of the polypeptide has been modified.

Targeted Delivery In another embodiment, the invention provides a composition comprising a targeted cell expressing a receptor for a polypeptide of the invention, or a cell-bound form of a polypeptide of the invention. Methods of delivering to expressing cells are provided.

As discussed herein, a polypeptide or antibody of the invention is a hydrophobic, hydrophilic, ionic and / or covalently linked heterologous polypeptide, heterologous nucleic acid, toxin or prodrug. May be associated through interaction. In one embodiment, the invention provides a polypeptide of the invention ((a) associated with a heterologous polypeptide or nucleic acid (
A method for specific delivery of a composition of the invention to cells by administering (including an antibody) is provided. In one example, the invention provides a method for delivering therapeutic proteins into targeted cells. In another example, the invention can be a single-stranded nucleic acid (eg, an antisense or ribozyme) or a double-stranded nucleic acid (eg, integrated into the genome of a cell, or can be episomally replicating, and transcribed). , DNA) for delivery to targeted cells.

In another embodiment, the invention provides cell specificity by administering a polypeptide of the invention (eg, a polypeptide of the invention or an antibody of the invention) associated with a toxin or a cytotoxic prodrug. Methods for selective destruction (eg, destruction of tumor cells).

A “toxin” is an endogenous cytotoxic effector system, radioisotope, holotoxin, modified toxin, catalytic subunit of a toxin, or in a cell that causes cell death under defined conditions. Alternatively, it means a compound that binds and activates any molecule or enzyme not normally present on the cell surface. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, compounds that bind an intrinsic or induced endogenous cytotoxic effector system. (For example, antibody (or a part including complement fixation thereof)), thymidine kinase, endonuclease, RNAse, α-toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin (gelonin). , Pokeweed antiviral protein, α-sarcin and cholera toxin.
By "cytotoxic prodrug" is meant a non-toxic compound that is converted into a cytotoxic compound by the enzymes normally present in cells. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. However, the present invention is not limited to these.

Drug Screening Further contemplated is the use of the polypeptides of the invention, or the polynucleotides encoding these polypeptides, to screen for molecules that alter the activity of the polypeptides of the invention. Such methods include the steps of contacting the polypeptides of the invention with selected compounds suspected of having antagonistic or agonistic activity, and assaying the activity of these polypeptides following binding. To do.

The present invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment used in such a test can be immobilized on a solid support, expressed on the cell surface, free in solution, or localized intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the polypeptide of the invention can be measured.

Accordingly, the present invention provides methods of screening for drugs or any other agents that affect the activity mediated by the polypeptides of the present invention. These methods involve contacting such agents with a polypeptide of the invention or fragment thereof by methods well known in the art, and the presence of a complex between the agent and the polypeptide or fragment thereof. The step of assaying is included. In such competitive binding assays, the drug to be screened is typically labeled. After incubation, free drug is separated from the drug present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular drug to bind to a polypeptide of the invention.

Another technique for drug screening provides high throughput screening for compounds with appropriate binding affinity for the polypeptides of the invention, and European Patent Application 84/03564 (published September 13, 1984). (Which is incorporated herein by reference) in great detail. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate (eg, plastic pins or some other surface). Peptide test compounds are reacted with the polypeptides of the invention and washed. Bound polypeptide is then detected by methods well known in the art. The purified polypeptide is
It is coded directly on the plate for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

The present invention also contemplates the use of competitive drug screening assays, wherein a neutralizing antibody capable of binding a polypeptide of the invention specifically competes with a test compound for binding to the polypeptide or fragment thereof. To do. In this style,
Antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with a polypeptide of the invention.

Antisense and Ribozymes (Antagonists) In certain embodiments, an antagonist according to the invention comprises a nucleic acid corresponding to the sequence contained in SEQ ID NO: X or a complementary strand thereof, and / or identified in Table 1c.
It is a nucleic acid corresponding to the nucleotide sequence contained in DNA plasmid Z. In one embodiment, the antisense sequence is produced internally by the organism, and in another embodiment the antisense sequence is administered separately (eg, O'Conn.
or. , Neurochem. 56: 560 (1991)).
Oligodexoxytides as Antisense I
nhibitors of Gene Expression, CRC Pre
ss, Boca Raton, FL (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA or through the formation of triple helices. Antisense technology is described in, for example, Okano, J. et al.
, Neurochem. 56: 560 (1991); Oligodeoxynu.
cleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Rato
n, FL (1988). Triple helix formation is described, for example, in Lee et al., N.
ucleic Acids Research 6: 3073 (1979); C
Ooney et al., Science, 241: 456 (1988); and Devv.
An, et al., Science, 251: 1300 (1991).
These methods are based on the binding of polynucleotides to complementary DNA or RNA.

For example, the use of the c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines has been previously described (Wickstrom et al. 1988); Anfossi et al.
9)). These experiments were performed in vitro by incubating cells with oligoribonucleotides. A similar procedure for in vivo use is
It is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is generated as follows: a sequence complementary to the first 15 bases of the open reading frame, an EcoRI site at the 5'end and a HindIII at the 3'end. Adjacent to the site. The oligonucleotide pairs are then heated at 90 ° C. for 1 minute and then 2 × ligation buffer (20 mM TRIS HC
pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DT
T) and 0.2 mM ATP) and then ligated into the EcoR1 / HindIII sites of the retroviral vector PMV7 (WO91).
/ 15580).

For example, the 5'coding portion of a polynucleotide encoding a polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby inhibiting transcription and production of receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a receptor polypeptide.

In one embodiment, the antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, the vector or a portion thereof is transcribed to produce the antisense nucleic acid (RNA) of the invention. Such a vector contains a sequence encoding the antisense nucleic acid of the present invention. Such a vector can be episomal or chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. The vector can be a plasmid, virus, etc. known in the art used for replication and expression in vertebrate cells. Expression of sequences encoding the polypeptides of the present invention or fragments thereof may be obtained by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include SV40 early promoter region (Bernist and Chamber, Nature, 29:
304-310 (1981)), a promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-797 (19).
80)), the herpes thymidine promoter (Wagner et al., Proc. Nat.
l. Acad. Sci. U. S. A. 78: 1441-1445 (1981)).
, Regulatory sequences of the metallothionein gene (Brinster et al., Nature, 2
96: 39-42 (1982)) and the like, but are not limited thereto.

The antisense nucleic acid of the present invention comprises at least a sequence complementary to a part of RNA transcript of the gene of the present invention. However, perfect complementarity, although preferred, is not necessary. A sequence "complementary to at least a portion of RNA" as referred to herein means a sequence that has sufficient complementarity to hybridize with RNA and forms a stable duplex; Therefore, in the case of the double-stranded antisense nucleic acid of the present invention,
Single strands of double-stranded DNA can be tested or triplex formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, longer hybridizing nucleic acids may contain more base mismatches with RNA, which may include stable duplexes (or triplexes) and still form. obtain. One of skill in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting temperature of the hybridizing complex.

Oligonucleotides that are complementary to the 5'end of the message (eg, 5'untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. is there. However, sequences complementary to the 3'untranslated sequences of mRNAs have likewise been shown to be effective in inhibiting translation of mRNAs. Generally, Wagner, R .; , Nature 372: 333-335 (199).
See 4). Therefore, 5 of the polynucleotide sequences described herein
Oligonucleotides complementary to either the'- or 3'-untranslated non-coding regions can be used in antisense approaches to inhibit translation of endogenous mRNAs. Oligonucleotides complementary to the 5'untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region, although less efficient inhibitors of translation, can be used in accordance with the present invention. Whether designed to hybridize to the 5'region, 3'region or coding region of the mRNA of the invention, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably 6 ~ About 5
An oligonucleotide with a length of 0 nucleotides. In particular aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the present invention may be DNA, or RNA, or chimeric mixtures, or derivative or modified versions thereof, single-stranded, or double-stranded. The oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone to improve, eg, molecular stability, hybridization, and the like.
The oligonucleotide may be attached to other additional groups such as peptides (eg, for targeting host cell receptors in vivo) or factors that facilitate transport across cell membranes (eg, Letsinger et al., Proc. Natl. Acad.
． Sci. U. S. A. 86: 6553-6556 (1989); Lemait.
Re et al., Proc. Natl. Acad. Sci. 84: 648-652 (19
87); PCT Publication No. WO88 / 09810 (Published December 15, 1988)
, Or the blood-brain barrier (eg PCT Publication No. WO89 / 101).
34 (published April 25, 1988)), hybridization-triggered cleavag.
e.g.) (eg Kroll et al., BioTechniques, 6: 9).
58-976 (1988)), or intercalating agents (see, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide is a different molecule (for example,
Peptides, hybridization-triggered cross-linking agents, transport agents, hybridization-triggered cleavage agents, etc.).

Antisense oligonucleotides can include at least one modified base moiety, which is selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5 -Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosyl cuocosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2
-Methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v
), Wybutoxosine, pseudouracil, queocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy. Acetic acid (v), 5-methyl-2-thiouracil,
3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)
w, and 2,6-diaminopurine.

Antisense oligonucleotides may also include at least one modified sugar moiety selected from the group including, but not limited to: arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0577] In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to: phosphorothioate, phosphorodithioate, phosphoro. Amidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof.

[0578] In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. The a-anomeric oligonucleotide has a complementary R
It forms a double-stranded hybrid specific to NA and, contrary to the normal b-unit,
The chains are parallel to each other (Gautier et al., Nucl. Acids Res
． 15: 6625-6641 (1987)). This oligonucleotide has 2
'-0-methyl ribonucleotide (Inoue et al., Nucl. Acid)
s Res. , 15: 6131-6148 (1987)), or chimeric RNA.
-A DNA analogue (Inoue et al., FEBS Lett. 215: 327).
-330 (1987)).

Polynucleotides of the invention may be prepared using standard methods known in the art (eg, automated D
By NA synthesizer (such equipment is Biosearch, Applied B
(commercially available from iosystems, etc.)). For example, phosphorothioate oligonucleotides are described by Stein et al. (Nuc
l. Acids Res. 16: 3209 (1988)),
Methylphosphonate oligonucleotides are used as controlled pore glass (con
polymerized polymer support (Sarin et al., Pr)
oc. Natl. Acad. Sci. U. S. A. , 85: 7448-7451
(1988)) and the like.

Although antisense nucleotides complementary to the coding region sequences may be used, antisense nucleotides complementary to the transcribed, untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, ie ribozymes (eg PCT International Publication WO 90/11364, October 4, 1990).
Published: Sarver et al., Science, 247: 1222-1225 (19).
90)). On the other hand, a ribozyme that cleaves an mRNA with a site-specific recognition sequence can be used to destroy the mRNA, but it is preferable to use a hammerhead ribozyme. Hammerhead ribozymes cleave mRNAs at positions determined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-
UG-3 '. Construction and production of hammerhead ribozymes are well known in the art, and Haseroff and Gerlach, Nature, 334:
585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within the nucleotide sequence of SEQ ID NO: X. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5'end of the mRNA; ie, to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts. It

For the antisense approach, the ribozymes of the invention may be composed of modified oligonucleotides (eg, to improve stability, targeting, etc.),
It should then be delivered in vivo to cells expressing the polypeptide of the invention. The DNA construct encoding the ribozyme can be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. Preferred methods of delivery include strong constitutive promoters (eg, pol III or pl
Under the control of a ribozyme (such as the II promoter), so that the transfected cells have a sufficient amount to disrupt the endogenous message and inhibit translation. Produces the ribozyme. Ribozymes, unlike antisense molecules, are catalytic, so lower intracellular concentrations are required for efficiency.

Antagonist / agonist compounds may be utilized to grow and proliferate the polypeptides of the invention against neoplastic cells and tissues.
feration) effect. That is, stimulating a tumor agonist,
It slows or prevents aberrant cell growth and proliferation (eg, in tumorigenesis or proliferation).

Antagonists / agonists may also be utilized to prevent diseases of hypervascularity and prevent proliferation of epithelial lens cells following extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the invention may also be required in cases such as restenosis after balloon angioplasty.

Antagonists / agonists may also be utilized to prevent scar tissue growth during wound healing.

Antagonists / agonists may also be utilized to treat the diseases described herein.

Accordingly, the present invention treats disorders or diseases including, but not limited to, those disorders or diseases listed throughout this application that are associated with overexpression of a polynucleotide of the present invention. A method is provided for treating a patient by administering (a) an antisense molecule relating to the polynucleotide of the present invention and / or (b) a ribozyme relating to the polynucleotide of the present invention to a patient. The invention, and / or (b) a ribozyme relating to the polynucleotide of the present invention.

Binding Peptides and Other Molecules The invention also provides screening methods for identifying non-polypeptides that bind polypeptides and steroid hormone receptor polypeptides, and steroid hormone receptor binding molecules identified thereby. Include. These binding molecules are useful, for example, as agonists and antagonists of steroid hormone receptor peptides. Such agonists and antagonists may be used in accordance with the present invention in the therapeutic embodiments described in detail below.

This method comprises the following steps: a. Contacting a steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide with a plurality of molecules; and b. Identifying a molecule that binds to a steroid hormone receptor polypeptide or a steroid hormone receptor-like polypeptide.

The step of contacting the steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide with multiple molecules can be provided in a number of ways. For example, one skilled in the art will appreciate that a steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide immobilized on a solid support, and a solution of a plurality of molecules immobilized steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide. It may be intended to come into contact with. Such a procedure is similar to the affinity chromatography process, where the affinity matrix is composed of immobilized steroid hormone receptor polypeptides or steroid hormone receptor-like polypeptides. Molecules having a selective affinity for steroid hormone receptor polypeptides or steroid hormone receptor-like polypeptides can then be purified by affinity selection. The nature of the solid support, the process for attachment of the steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide to the solid support, the solvent, and the conditions of affinity isolation or selection are very conventional, And it is well known to those skilled in the art.

Alternatively, one of skill in the art can also separate multiple polypeptides into substantial separation fractions that include individual polypeptides or subsets thereof. For example, multiple polypeptides can be separated by gel electrophoresis, column chromatography, or similar methods known to those of skill in the art for separating polypeptides. Individual polypeptides may also be produced by the transformed host cell so as to be expressed on or about its outer surface (eg, recombinant phage). The individual isolates are then "probed" by steroid hormone receptor polypeptides or steroid hormone receptor-like polypeptides, optionally in the presence of an inducer which should be required for expression. Thus, it may be determined whether any selective affinity interaction occurs between the steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide and the individual clone. Prior to contacting the steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide with each fraction containing the individual polypeptide, the polypeptide may first be transferred to a solid support for further convenience. . Such a solid support can be just a piece of filtration membrane, such as one made from nitrocellulose or nylon. In this way
Positive clones can be identified from a collection of transformed host cells of an expression library, which expression library encodes a polypeptide having a selective affinity for a steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide. Carrying a DNA construct that Furthermore, the amino acid sequence of a polypeptide having a selective affinity for a steroid hormone receptor polypeptide or a steroid hormone receptor-like polypeptide can be determined directly by conventional procedures, or the code for the DNA encoding this polypeptide. Sequences can often be more conveniently determined. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is determined from the polypeptide itself, one of skill in the art can use microsequencing techniques. Sequencing technology
Mass spectrometry may be included.

In certain circumstances, prior to attempting to determine or detect the presence of a selective affinity interaction, any unbound steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide, or unbound polypeptide To
It may be desirable to wash away a steroid hormone receptor polypeptide or a mixture of steroid hormone receptor-like polypeptides and multiple polypeptides. Such a washing step may be particularly desirable when the steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide, or multiple polypeptides are attached to a solid support.

Multiple molecules provided according to this method may be used in diverse libraries, such as random or recombinant peptide or non-peptide libraries, for molecules that specifically bind to steroid hormone receptor polypeptides. Can be screened))). Many libraries that can be used are known in the art and include, for example, chemically synthesized libraries, recombinant (eg, phage display libraries), and in vitro translocation-based libraries. Examples of chemically synthesized libraries are described below: Fodor et al., 1991, Science 251: 767-773; H.
Oughten et al., 1991, Nature 354: 84-86; Lam et al.
1991, Nature 354: 82-84; Medynski, 1994,
Bio / Technology 12: 709-710; Gallop et al., 19
94, J.M. Medicinal Chemistry 37 (9): 1233-
1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. S
ci. USA 90: 10922-10926; Erb et al., 1994, Proc.
． Natl. Acad. Sci. USA 91: 11422-11426; Ho.
ughten et al., 1992, Biotechniques 13: 412; Ja.
yawickreme et al., 1994, Proc. Natl. Acad. Sci.
USA 91: 1614-1618; Salmon et al., 1993, Proc. N
atl. Acad. Sci. USA 90: 11708-11712; PCT Application Publication No. WO 93/20242; and Brenner and Lerne.
r, 1992, Proc. Natl. Acad. Sci. USA 89: 538.
1-5383.

Examples of phage display libraries are described below: Scott and Smith, 1990, Science 249: 386-390; Dev.
Lin et al., 1990, Science, 249: 404-406; Christ.
ian, R.M. B. Et al., 1992, J. Am. Mol. Biol. 227: 711-71
8); Lenstra, 1992, J. Am. Immunol. Meth. 152: 1
49-157; Kay et al., 1993, Gene 128: 59-65; and PCT Application Publication No. WO 94/18318 (August 18, 1994).

In vitro transfer-based libraries include, but are not limited to, the libraries described below: PCT Application Publication No. WO 91/05058 (19).
91 April 18); and Mattheakis et al., 1994, Proc. N
atl. Acad. Sci. USA 91: 9022-9026.

For purposes of illustration of non-peptide libraries, benzodiazepine libraries (eg, Bunin et al., 1994, Proc. Natl. Acad. Sci. US).
A 91: 4708-4712) can be adapted for use.
Peptoid library (Simon et al., 1992, Proc. Natl. Ac
ad. Sci. USA 89: 9367-9371) is also used. Another example of a library that can be used, where the amide functionality in the peptide is permethylated to produce a chemically converted library, is described by Ostresh et al. (1994, Proc. Natl. . Ac
ad. Sci. USA 91: 11138-11142).

The variety of non-peptide libraries useful in the present invention is broad. For example, Ecker and Crooke, 1995, Bio / Technology.
13: 351-360, among the species that form the basis of various libraries, are benzodiazepines, hydantoins, piperazines.
dione), biphenyls, sugar analogs, β-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, amineimides, and oxazolones.

Non-peptide libraries can be broadly classified into two types: modified (de
correlated) Monomers and oligomers. The modified monomer library uses a relatively simple backbone structure as the various functional groups are added. Often, this scaffold is a molecule with known and useful pharmacological activity. For example, the backbone can be a benzodiazepine structure.

Non-peptidic oligomer libraries use a large number of monomers that are assembled together in a manner that produces new shapes depending on the order of the monomers. The monomer units used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers whose side chains are attached to α-amino groups rather than α-carbon, form the basis of another version of the non-peptide oligomer library. The first non-peptide oligomer library utilized one type of monomer and thus contained a repeating backbone. Recent libraries utilize more than one monomer, giving the library flexibility.

Screening the library can be accomplished by any of a variety of commonly known methods. See, for example, the following references, which disclose screening of peptide libraries: Parmley and Smith, 19
89, Adv. Exp. Med. Biol. 251: 215-218; Scot.
t and Smith, 1990, Science 249: 386-390; F.
owkes et al., 1992; BioTechniques 13: 422-42.
7; Oldenburg et al., 1992, Proc. Natl. Acad. Sci
． USA 89: 5393-5397; Yu et al., 1994, Cell 76: 9.
33-945; Staudt et al., 1988, Science 241: 577-.
580; Bock et al., 1992, Nature 355: 564-566; Tu.
erk et al., 1992, Proc. Natl. Acad. Sci. USA 89:
6988-6992; Ellington et al., 1992, Nature 355.
: 850-852; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223.
409, and US Pat. No. 5,198,346 (all Ladner et al.); R
ebar and Pabo, 1993, Science 263: 671-673.
And PCT Application Publication No. WO 94/18318.

In a specific embodiment, a screen to identify molecules that bind to a steroid hormone receptor polypeptide involves screening library members with a steroid hormone receptor polypeptide or steroid hormone receptor-like polypeptide bound on a solid phase. Can be performed by contacting with and collecting those library members that bind to steroid hormone receptor polypeptides or steroid hormone receptor-like polypeptides. An example of such a screening method (named the "panning" technique) is, by way of example, Palmley and Smith, 1988, Gene 73: 305-3.
18; Fowlkes et al., 1992, BioTechniques 13:42.
2-427; PCT Application Publication No. WO 94/18318; as well as the references described herein.

In another embodiment, a two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature.
e 340: 245-246; Chien et al., 1991, Proc. Natl.
Acad. Sci. USA 88: 9578-9582) can be used to identify molecules that specifically bind to steroid hormone receptor polypeptides or steroid hormone receptor-like polypeptides.

When the steroid hormone receptor binding molecule is a polypeptide, the polypeptide is conveniently selected from any peptide library (random peptide library, combinatorial peptide library, or biased peptide library). Can be done. The term “biased” means that in this case the method by which the library is generated governs the diversity of the resulting collection of molecules 1.
Used herein to mean manipulated to limit one or more parameters.

Thus, a truly random peptide library produces a collection of peptides in which the probability of finding a particular amino acid at a given position in the peptide is the same for all 20 amino acids. However, the bias specifies, for example, that lysine is present every fifth amino acid, or that positions 4, 8 and 9 of the decapeptide library are fixed to contain only arginine. Can be introduced into the library by. Obviously, many types of bias can be contemplated, and the invention is not limited to any particular bias. Further, the invention contemplates particular types of peptide libraries, such as those that utilize a DNA construct comprising a phage display peptide library and a lambda phage vector with a DNA insert.

As described above, in the case of a steroid hormone receptor binding molecule that is a polypeptide, the polypeptide will have from about 6 to less than about 60 amino acid residues, preferably from about 6 to about 10 amino acid residues, And most preferably about 6 to about 22
May have amino acid residues of In another embodiment, the steroid hormone receptor binding polypeptide has a range of 15-100 amino acids, or 20-50 amino acids.

The selected steroid hormone receptor binding polypeptide may be obtained by chemical synthesis or recombinant expression.

Other Activities The polypeptides, polynucleotides, agonists or antagonists of the invention result in a variety of disease states (eg, thrombosis, arteriosclerosis, and others) as a result of their ability to stimulate vascular endothelial cell proliferation. Cardiovascular conditions) to stimulate revascularization of ischemic tissue. The polypeptides, polynucleotides, agonists, or antagonists of the present invention can also be utilized to stimulate angiogenesis and limb regeneration as discussed above.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also be utilized in the treatment of wounds due to injury, burns, post-operative tissue repair, and ulcers. Because they are mitogenic for a variety of cells of different origin, such as fibroblasts and skeletal muscle cells, and therefore promote repair or replacement of damaged or diseased tissue.

The polypeptides, polynucleotides, agonists or antagonists of the invention also stimulate neuronal growth and impair the neuronal degeneration or conditions of certain neurons (eg Alzheimer's disease, Parkinson's disease, and AIDS-related complexes). A) to treat and prevent neuronal damage that occurs in The polypeptides, polynucleotides, agonists, or antagonists of the invention may have the ability to stimulate chondrocyte proliferation, thus enhancing bone and periodontal regeneration, and tissue grafts or bone grafts. It can be used to assist.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also be utilized to prevent aging of the skin due to sunburn by stimulating keratinocyte proliferation.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also be utilized to prevent hair loss. This is because members of the FGF family activate hair-forming cells and promote melanocyte proliferation. Following the same principles, the polypeptides, polynucleotides, agonists or antagonists of the invention may be utilized to stimulate hematopoietic and myeloid cell proliferation and differentiation when used in combination with other cytokines.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also be utilized to maintain pre-transplant organs or to support cell culture of primary tissues. The polypeptides, polynucleotides, agonists or antagonists of the invention can also be utilized to induce tissue of mesoderm origin for differentiation in early embryos.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also increase or decrease embryonic stem cell differentiation or proliferation in addition to hematopoietic lineages as discussed above.

Polypeptides, polynucleotides, agonists or antagonists of the invention may also have mammalian characteristics (eg height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size. , And shape) (eg, cosmetic surgery). Similarly, the polypeptides, polynucleotides, agonists, or antagonists of the invention can be used to regulate mammalian metabolism that affects catabolism, anabolic activity, processing, utilization, and energy storage.

Polypeptides, polynucleotides, agonists or antagonists of the invention can be used in biorhythms, caricadic rhythms, depression (
Depressive disorders included, tendency to violence, resistance to pain, fertility (preferably,
Activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities to alter the mental or physical state of a mammal Can be done.

The polypeptides, polynucleotides, agonists or antagonists of the invention may also increase or decrease, eg, storage capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional components. To let
It can be used as a food additive or preservative.

The above applications have use in a wide variety of hosts. Such hosts include humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, miniature pigs, chickens, goats, cows, sheep, dogs, cats, non-human primates, and humans. But are not limited to these. In a particular embodiment, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In the most preferred embodiment, the host is human. (Other Preferred Embodiments) Another preferred embodiment of the present invention is SEQ ID NO: X or its complementary strand, and / or
Or an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of cDNA plasmid Z.

Also preferred is a nucleic acid molecule wherein the above sequence of contiguous nucleotides is contained in the nucleotide sequence of SEQ ID NO: X in the range of positions identified relative to SEQ ID NO: X in Table 1.

An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 150 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: X or its complementary strand, and / or cDNA plasmid Z. Is also preferred.

An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X or its complementary strand, and / or cDNA plasmid Z. Is more preferable.

[0621] A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: X in the range of positions identified to SEQ ID NO: X in Table 1.

A further preferred embodiment is SEQ ID NO: X or its complement, and / or c
An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence of DNA plasmid Z.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising SEQ ID NO: X or its complementary strand and / or the nucleotide sequence of the cDNA plasmid Z, wherein Thus, this nucleic acid molecule does not hybridize to a nucleic acid molecule having a nucleotide sequence consisting of only A or T residues under stringent hybridization conditions.

[0624]   Also preferred is a composition comprising a DNA molecule containing the cDNA plasmid Z.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of cDNA plasmid Z.

Also preferred is an isolated nucleic acid molecule, wherein the sequence of at least 50 contiguous nucleotides described above is contained within the nucleotide sequence of the open reading frame sequence encoded by the cDNA plasmid Z.

At least 1 in the nucleotide sequences encoded by the cDNA plasmids
Also preferred is an isolated nucleic acid molecule that comprises a nucleotide sequence that is at least 95% identical to a sequence of 50 contiguous nucleotides.

A further preferred embodiment has at least 9 to at least 500 contiguous nucleotide sequences in the nucleotide sequence encoded by the cDNA plasmid.
An isolated nucleic acid molecule comprising a nucleotide sequence that is 5% identical.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence encoded by the cDNA plasmid.

A further preferred embodiment detects in a biological sample a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: A nucleotide sequence of SEQ ID NO: X or a strand complementary thereto, and a nucleotide sequence encoded by the cDNA plasmid Z; this method comprising converting the nucleotide sequence of at least one nucleic acid molecule in the sample as described above. Comparing with a sequence selected from the group and determining whether the sequence of the nucleic acid molecule in the sample is at least 95% identical to the selected sequence.

Comparing the sequences described above comprises determining the degree of nucleic acid hybridization between the nucleic acid molecule in the sample and a nucleic acid molecule comprising the sequence selected from the group above. The above method is also preferable. Similarly, the step of comparing the sequences described above, with a nucleotide sequence determined from the nucleic acid molecules in the sample,
Also preferred is the above method carried out by the step of comparing with a sequence selected from the above group. Nucleic acid molecules can include DNA or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample, the method comprising at least 50 contiguous sequences in a sequence selected from the group consisting of: Detecting a nucleic acid molecule (if any) in the sample that contains a nucleotide sequence that is at least 95% identical to the sequence of the selected nucleotides: the nucleotide sequence of SEQ ID NO: X or its complementary strand and cDNA.
Nucleotide sequence encoded by plasmid Z.

The method for identifying the species, tissue, or cell type of a biological sample can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein At least one sequence in the panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the sequence selected from the group above.

Also preferred is a method for diagnosing in a subject a pathological condition associated with aberrant structure or expression of the nucleotide sequence of SEQ ID NO: X or its complementary strand or cDNA plasmid Z encoding a protein, which method is also preferred. Is a biology obtained from said subject, a nucleic acid molecule (if any) comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: Detecting in a biological sample: the nucleotide sequence of SEQ ID NO: X or its complement and the nucleotide sequence of plasmid Z of the cDNA.

The method for diagnosing a pathological condition can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in the panel above. Is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the sequence selected from the above group.

Also preferred is a composition comprising an isolated nucleic acid molecule, wherein the nucleotide sequence of said nucleic acid molecule comprises a panel of at least two nucleotide sequences, wherein at least one sequence in the above panel is: At least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO: X or its complementary strand and the nucleotide sequence encoded by cDNA plasmid Z. The nucleic acid molecule can include a DNA molecule or an RNA molecule.

A polypeptide sequence of SEQ ID NO: Y; a sequence of at least about 10 contiguous amino acids in the polypeptide encoded by SEQ ID NO: X or its complement and / or the polypeptide encoded by cDNA plasmid Z, and at least 90. %
Also preferred are isolated polypeptides that include the same amino acid sequences.

Amino acid sequence of SEQ ID NO: Y; at least about 30 consecutive amino acids in the amino acid sequence of the polypeptide encoded by SEQ ID NO: X or its complement and / or the amino acid sequence of the polypeptide encoded by cDNA plasmid Z. Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of

[0639] The amino acid sequence of SEQ ID NO: Y; at least about 100 contiguous amino acids in the amino acid sequence of the polypeptide encoded by SEQ ID NO: X or its complement and / or the amino acid sequence of the polypeptide encoded by cDNA plasmid Z. Further preferred is an isolated polypeptide comprising an amino acid sequence which is at least 95% identical to the sequence of.

The complete amino acid sequence of SEQ ID NO: Y; at least 95% identical to the complete amino acid sequence of the polypeptide encoded by SEQ ID NO: X or its complement and / or the polypeptide encoded by cDNA plasmid Z. Further preferred is an isolated polypeptide comprising an amino acid sequence.

Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least about 10 consecutive amino acids in the complete amino acid sequence of the polypeptide encoded by cDNA plasmid Z.

The above contiguous amino acid sequence is a polypeptide encoded by the cDNA plasmid Z; an amino acid which is a part of the polypeptide encoded by SEQ ID NO: X or its complementary strand and / or the polypeptide sequence of SEQ ID NO: Y. Polypeptides included in the sequence are also preferred.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 30 consecutive amino acids in the amino acid sequence of the polypeptide encoded by cDNA plasmid Z.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the polypeptide encoded by cDNA plasmid Z.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the polypeptide encoded by cDNA plasmid Z.

An isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: , More preferred: the polypeptide of SEQ ID NO: Y; the polypeptide sequence encoded by SEQ ID NO: X or its complement and the polypeptide encoded by cDNA plasmid Z.

Following: at least 10 in the sequence selected from the group consisting of the polypeptide sequence of SEQ ID NO: Y; the polypeptide encoded by SEQ ID NO: X or its complement and the polypeptide encoded by cDNA plasmid Z. Further preferred is a method of detecting in a biological sample a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of contiguous amino acids; the method comprises determining the amino acid sequence of at least one polypeptide molecule in the sample. The step of comparing to a sequence selected from this group and the sequence of this polypeptide molecule in this sample is at least 90 with this sequence of at least 10 consecutive amino acids.
% Determining if they are the same.

This step of comparing the amino acid sequence of at least one polypeptide molecule in this sample with a sequence selected from this group is carried out by comparing the polypeptide in this sample with the polypeptide sequence of SEQ ID NO: Y; At least 90% identical to the sequence of at least 10 consecutive amino acids in the sequence selected from the group consisting of the polypeptide encoded by SEQ ID NO: X or its complementary strand and the polypeptide encoded by cDNA plasmid Z. Also preferred is the above method, which comprises determining the degree of specific binding to an antibody which specifically binds a polypeptide comprising an amino acid sequence.

Also preferred is the above method, wherein the step of comparing the sequences is performed by comparing the amino acid sequence determined from the polypeptide molecules in the sample with this sequence selected from this group.

Also preferred is a method of identifying the species, tissue or cell type of a biological sample, wherein the method, if present, is at least 10 consecutive amino acids in a sequence selected from the group consisting of: Detecting in this sample a polypeptide molecule comprising an amino acid sequence which is at least 90% identical to the sequence of: SEQ ID NO: Y, the polypeptide sequence; A polypeptide encoded by plasmid Z.

Also preferred is the above method of identifying a species, tissue or cell type of a biological sample, wherein the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences. , Wherein at least one sequence in this panel is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the above group.

Also preferred is a method of diagnosing in a subject a pathological condition identified in Table 1 associated with aberrant structure or expression of a nucleic acid sequence encoding a polypeptide, wherein the method comprises: Detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences in a biological sample obtained from a subject, wherein at least one sequence in this panel comprises the group At least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from: a polypeptide sequence of SEQ ID NO: Y; a polypeptide encoded by SEQ ID NO: X or its complement and a cDNA plasmid Z Polypeptide.

In any of these methods, the step of detecting the polypeptide molecule comprises the steps of:
Using an antibody is included.

A nucleotide that is at least 95% identical to a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Also preferred is an isolated nucleic acid molecule comprising a sequence: the polypeptide sequence of SEQ ID NO: Y; the polypeptide encoded by SEQ ID NO: X or its complement and the polypeptide encoded by cDNA plasmid Z.

Also preferred is an isolated nucleic acid molecule in which the nucleotide sequence encoding the above polypeptide has been optimized for expression of this polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: the polypeptide sequence of SEQ ID NO: Y; SEQ ID NO: X.
Alternatively, a polypeptide encoded by its complementary chain and a polypeptide encoded by cDNA plasmid Z.

Further preferred is a method of making a recombinant vector comprising the step of inserting any of the above isolated nucleic acid molecules into a vector. The recombinant vector produced by this method is also preferred. Also preferred is a method of making a recombinant host cell, which comprises the step of introducing the vector into a host cell, as well as a recombinant host cell produced by this method.

A method of making an isolated polypeptide, comprising culturing the recombinant host cell under conditions such that the polypeptide is expressed, and recovering the polypeptide. The method is also preferable. Also preferred is this method of producing an isolated polypeptide wherein the recombinant host cell is a eukaryotic cell and the polypeptide is a human protein comprising an amino acid sequence selected from the group consisting of: The polypeptide sequence of SEQ ID NO: Y; the polypeptide encoded by SEQ ID NO: X or its complement and the polypeptide encoded by cDNA plasmid Z. Also preferred are isolated polypeptides produced by this method.

Also preferred is a method of treating an individual in need of increased levels of protein activity, wherein the method is directed to such an individual in order to increase the level of activity of the protein in the individual. Administering an effective amount of a therapeutic agent comprising an isolated polypeptide, polynucleotide, immunogenic fragment or analog thereof, binding agent, antibody, or antigen fragment of the invention.

Also preferred is a method of treating an individual in need of a reduced level of protein activity, wherein the method is directed to such an individual in order to reduce the level of activity of the protein in the individual. Administering an effective amount of a therapeutic agent comprising an isolated polypeptide, polynucleotide, immunogenic fragment or analog thereof, binding agent, antibody, or antigen fragment of the invention.

In a particular embodiment of the present invention, for each “contig ID” listed in the fourth column of Table 2, preferably the nucleotide sequence referenced in the fifth column of Table 2 and the general formula comprises or consists of a nucleotide sequence described by ab, where a and b are uniquely determined for the corresponding SEQ ID NO: X referenced in column 3 of table 2. Excludes one or more polynucleotides. More particular embodiments relate to polynucleotide sequences that exclude 1, 2, 3, 4, or more of the particular polynucleotide sequences referenced in the fifth column of Table 2. This table is by no means meant to include all of the sequences that can be excluded by this general formula, this table is merely an illustrative example. All documents available through these registries are hereby incorporated by reference in their entirety.

[0662]

[Table 2]

[0663]

[Table 3]

[0664]

[Table 4] Although the invention has been generally described, the invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting.

EXAMPLES Example 1: Isolation of Selected cDNA Clones from Deposited Samples Each cDNA clone in the referenced ATCC deposit is contained in a plasmid vector. Table 1 identifies the vector used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a plasmid excised phage vector. The table immediately below correlates the relevant plasmids for each phage vector used in constructing the cDNA library. For example, if a particular clone is identified in Table 1 as isolated in the vector "Lambda Zap", the corresponding fiducial clone is "pBluescript".

[0666]

[Table 5] Vector Lambda Zap (US Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (US Pat. No. 5,128,25).
6 and 5,286,636), Zap Express (US Pat. Nos. 5,128,256 and 5,286,636), pBluescri.
pt (pBS) (Short et al., Nucleic Acids Res. 16:
7583-7600 (1988); Alting-Mees et al., Nucleic.
Acids Res. 17: 9494 (1989)) and pBK (Al
Ting-Mees et al., Strategies 5: 58-61 (1992)).
From Stratagene Cloning Systems, Inc. , 11
011 N.N. Torrey Pines Road, La Jolla, CA,
It is commercially available from 92037. pBS contains the ampicillin resistance gene and pBK contains the neomycin resistance gene. Both are E. coli strain XL-
1 Blue (also available from Stratagene). pBS arrives in four forms: SK +, SK-, KS + and KS. S and K are polylinker regions (“S” is SacI, and
"K" refers to KpnI, which refers to the orientation of the polylinker relative to the T7 and T3 primer sequences adjacent to the first site at each end of the linker). "+" Or "-" means f1 ori in a certain direction
Refers to the orientation of the f1 origin of replication (“ori”) such that a single-stranded rescue initiated from produces a sense-strand DNA and is antisense on the other.

Vectors pSport1, pCMVSport 2.0 and pCMVSpo
rt3.0 from Life Technologies, Inc. , P .; O. Box
6009, Gaithersburg, MD 20897. All Sport vectors contain the ampicillin resistance gene, and E. coli. coli strain D
H10B, which is also available from Life Technologies, can be transformed. (For example, Gruber, CE et al., Focus)
15:59 (1993)). Vector lafmid BA (Ben
to Soares, Columbia University, NY) contains the ampicillin resistance gene, and E. E. coli strain XL-1 Blue. Vector pCR® 2.1 (this is Invitroge
n, 1600 Faraway Avenue, Carlsbad, CA 92
(Available from E. 008) contains an ampicillin resistance gene and c
oli strain DH10B (available from Life Technologies) (eg, Clark, Nuc. Acids Res. 1).
6: 9677-9686 (1988) and Mead et al., Bio / Techno.
logy 9: (1991)). Preferably, the polynucleotides of the invention do not include the phage vector sequences identified for the particular clones in Table 1, as well as the corresponding plasmid vector sequences shown above.

The deposited material in the sample given the ATCC accession number for any given cDNA clone, referred to in Table 1, also contains one or more additional plasmids, each of which is Including different cDNA clones)
Can be included. Thus, deposits sharing the same ATCC Deposit Number include at least a plasmid for each cDNA clone identified in Table 1. Typically, the sample of each ATCC deposit referenced in Table 1 contains a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; , Such deposit samples have more or less than 50 c
Plasmids for DNA clones (up to about 500 cDNA clones) can be included.

Two approaches can be used to isolate a clone from the deposited sample of plasmid DNA cited for that particular clone in Table 1. First, the plasmid is isolated directly by screening the clones with the polynucleotide probe corresponding to SEQ ID NO: X.

In particular, certain polynucleotides having 30-40 nucleotides are synthesized according to the reported sequence using an Applied Biosystems DNA synthesizer. The oligonucleotide is labeled with, for example, ³² P-γ-ATP, T
Labeled with 4 polynucleotide kinase and purified according to conventional methods. (For example, Maniatis et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harb
or Press, Cold Spring, NY (1982)). Techniques known to those of skill in the art, such as those provided by vector suppliers or those provided in the relevant publications or patents cited above, are available for plasmid mixtures.
Using a suitable host as described above (eg XL-1 Blue (Strat.
a))). Transformants are plated on 1.5% agar plates (containing a suitable selection agent such as ampicillin) at a density of about 150 transformants (colony) per plate. These plates were subjected to conventional methods for bacterial colony screening (eg Sambrook et al., M.
olecular Cloning: A Laboratory Manual
, Second Edition, (1989), Cold Spring Harbor Labor.
Screening using nylon membranes according to the Atory Press, pages 1.93-1.104) or other techniques known to those of skill in the art.

Alternatively, both ends of SEQ ID NO: X (ie, the 5'N of the clone defined in Table 1)
17-2 from within the region of SEQ ID NO: X surrounded by T and 3′NT)
Two primers of 0 nucleotides are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under conventional conditions, for example in 25 μl of a reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl ₂ , 0.01% (w / v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 units of Taq polymerase. . 35 cycle PC
R (denaturation at 94 ° C. for 1 min; annealing at 55 ° C. for 1 min; extension at 72 ° C. for 1 min) is performed using a Perkin-Elmer Cetus automated thermal cycler. The amplification product is analyzed by agarose gel electrophoresis and the DNA band of the expected molecular weight is excised and purified. PCR product, D
Confirm the selected sequence by subcloning and sequencing the NA product.

Several methods are available for the identification of the 5'non-coding or 3'non-coding portions of genes that may not be present in the deposited clones. These methods include, but are not limited to: filter probe probing, clonal enrichment using specific probes, and 5'and 3 well known in the art.
'A protocol similar or identical to the "RACE" protocol. For example, 5 '
A method similar to RACE is available to generate the missing 5'end of the desired full length transcript (Frommont-Racine et al., Nucleic A.
cids Res. 21 (7): 1683-1684 (1993)).

Briefly, specific RNA oligonucleotides are ligated to the 5'end of a population of RNAs that probably contains full-length gene RNA transcripts. PCR of the 5'portion of the desired full-length gene is performed using a primer set containing a primer specific to the linked RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
Amplify. The amplified product can then be sequenced and used to generate the full length gene.

This above method starts with total RNA isolated from the desired source, but may also use poly A + RNA. The RNA preparation is then treated with phosphatase, if necessary, to degrade or damage the RN, which may interfere with subsequent RNA ligase steps.
The 5'phosphate group of A can be eliminated. The phosphatase should then be inactivated, and the RNA should be treated with tobacco acid pyrophosphatase to remove the cap structure present at the 5'end of the messenger RNA.
This reaction leaves a 5'phosphate group at the 5'end of the cap cleaved RNA that can then be ligated to the RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first-strand cDNA synthesis using gene-specific oligonucleotides. First strand synthesis reaction
It is used as a template for PCR amplification of the desired 5'end using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest. The resulting product is then sequenced and analyzed to confirm that the 5'end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to Polynucleotides Human Genome P1 Library (Genomic Systems, Inc.)
Are screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO: X according to the method described in Example 1 (Sa
See also mbrook).

[0677]   (Example 3: Tissue distribution of polypeptide)   The tissue distribution of mRNA expression of the polynucleotides of the invention can be determined, inter alia, by Samb
Use the protocol for Northern blot analysis described by look et al.
Decide. For example, the cDNA produced by the method described in Example 1.
Probe the rediprime^TM  DNA labeling system
(Amersham Life Science) and follow the manufacturer's instructions.
That's P³²Label with. After labeling, the probe was attached to CHROMA SPIN-100. ^TM Using a column (Clontech Laboratories, Inc.)
And purify according to manufacturer's protocol number PT1200-1. Then refined
Various human tissues are tested for mRNA expression using labeled probes
.

Multiple Tissue Northern (MTN) blots (Clontech) containing various human tissues (H) or human immune system tissues (IM) were analyzed using ExpressHyb ^™ Hybridization Solution (Clontech), manufacturer's protocol number P.
Test with labeled probe according to T1190-1. After hybridization and washing, the blots are mounted and exposed to film overnight at -70 ° C and the film developed according to standard procedures.

Example 4 Polynucleotide Chromosome Mapping A set of oligonucleotide primers is designed according to the sequence at the 5 ′ end of SEQ ID NO: X. This primer preferably spans about 100 nucleotides.
This primer set is then used for polymerase chain reaction under the following set of conditions: 95 ° C for 30 seconds; 56 ° C for 1 minute; 70 ° C for 1 minute. This cycle is repeated 32 times and then once at 70 ° C. for 5 minutes. Somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc)
In addition, human, mouse, and hamster DNA are used as templates. Reactions are analyzed on either an 8% polyacrylamide gel or a 3.5% agarose gel. Chromosome mapping is performed for about 100 in specific somatic cell hybrids.
Determined by the presence of the bp PCR fragment.

Example 5 Bacterial Expression of Polypeptides Polynucleotides encoding the polypeptides of the present invention are PCR oligos corresponding to the 5 ′ and 3 ′ ends of the DNA sequences, as outlined in Example 1. Amplification is performed using nucleotide primers to synthesize the insert fragment. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, and, if necessary, start / stop codons for cloning the amplification product into an expression vector. is there. For example, BamHI and XbaI are bacterial expression vectors pQE-9 (Qiagen, Inc., Ch.
atsworth, CA). This plasmid vector contains antibiotic resistance (Amp ^r ), bacterial origin of replication (ori), IPTG-regulatable promoter / operator (P / O), ribosome binding site (RBS),
It encodes a 6-histidine tag (6-His), and a restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated in bacterial RBS. The ligation mixture was then treated with the plasmid pR, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ).
E. coli, including multiple copies of EP4. E. coli strain M15 / rep4 (Qiagen,
Inc. ). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired construct were cloned into Amp (100 μg / ml) and Kan (100 μg / ml).
Liquid culture in LB medium supplemented with both (25 μg / ml) overnight (O / N)
Proliferate. The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells were placed at an optical density of 600 (OD) between 0.4 and 0.6.
． Grow to ⁶⁰⁰ ). Then IPTG (isopropyl-BD-thiogalactopyranoside) is added to a final concentration of 1 mM. IPTG is lacI
Induced by inactivation of the repressor, it removes P / O and leads to increased gene expression.

Cells are grown for an additional 3-4 hours. The cells are then centrifuged (6000 x
g for 20 minutes). The cell pellet is chaotropic agent 6
Solubilize by stirring in M guanidine HCl for 3-4 hours at 4 ° C. Cellular debris was removed by centrifugation and the polypeptide-containing supernatant was loaded onto a nickel-nitrilotriacetic acid (“Ni-NTA”) affinity resin column (QI).
AGEN, Inc. (Available from above). Proteins with a 6xHis tag bind Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (see, in particular, The QIA expressionlist (1
995) QIAGEN, Inc. , See above).

Briefly, the supernatant was loaded onto a column of 6M guanidine-HCl, pH8, which column was first washed with 10 volumes of 6M guanidine-HCl, pH8,
It is then washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

The purified protein was then loaded with phosphate buffered saline (PBS) or 5
Regenerate by dialysis against 0 mM sodium acetate, pH 6 buffer and 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. Recommended conditions are as follows: Regeneration using a linear gradient of 6M to 1M urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. Regeneration should take a minimum of 1.5 hours. After playing
The protein is eluted by the addition of 250 mM imidazole. Imidazole in PBS or 50 mM sodium acetate pH 6 buffer and 200 mM
It is removed by a final dialysis step against NaCl. 4 purified proteins
Store at ℃ or frozen at -80 ℃.

In addition to the expression vectors described above, the invention further includes an expression vector called pHE4a that includes a phage operator and promoter elements operably linked to the polynucleotide of the invention (ATCC Accession No. 209645).
, Deposited on February 25, 1998). This vector contains: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, and 6) lactose operon repressor gene (laqIq). The origin of replication (oriC) is pUC19 (LTI, Ga
Itersburg, MD). Promoter and operator sequences are made synthetically.

The vector was restricted with NdeI and XbaI, BamHI, XhoI, or Asp718, the restriction products were run on a gel, and the larger fragment (the stuffer fragment should be approximately 310 base pairs). DNA) can be inserted into pHEa by isolating D
The NA insert was labeled with NdeI (5 ′ primer) and XbaI, BamHI, Xh.
PCR with restriction sites for oI or Asp718 (3 'primer)
Primers are used and generated according to the PCR protocol described in Example 1.
The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

The engineered vector can be readily replaced in the above protocol to express the protein in bacterial systems.

Example 6 Purification of Polypeptides from Inclusion Bodies Another method described below is the procedure described in E. coli when the polypeptides are present in the form of inclusion bodies. co
It can be used to purify the polypeptide expressed in li. Unless otherwise specified, all the following steps are performed at 4-10 ° C.

E. After completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 ° C. and the cells are harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). Based on the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste (by weight) was added to 100 mM Tris, 50 mM EDTA, pH 7.
4. Suspend in buffer solution containing 4. The cells are dispersed in a homogenous suspension using a high shear mixer.

The cells were then transferred to a microfluidizer (
Microfluidics, Corp. Or APV Gaulin, Inc
． ) Is dissolved twice by passing the solution at 4000-6000 psi.
The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 minutes. The obtained pellet was treated with 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.
Wash again using 4.

The resulting washed inclusion bodies were washed with 1.5 M guanidine hydrochloride (GuHCl) to
Solubilize for 4 hours. After centrifugation at 7000 xg for 15 minutes, the pellet is discarded, and the polypeptide-containing supernatant is incubated at 4 ° C overnight to allow additional Gu.
Allows HCl extraction.

Following high speed centrifugation (30,000 × g) to remove insoluble particles, Gu
HCl solubilized protein was extracted with GuHCl extract, 50 mM sodium, pH
Refold by rapid mixing with 20 volumes of buffer containing 4.5, 150 mM NaCl, 2 mM EDTA with vigorous stirring. The refolded diluted protein solution is kept at 4 ° C. without mixing for 12 hours before further purification steps.

To clarify the refolded polypeptide solution, a pre-prepared tangential filtration unit with a 0.16 μm membrane filter with appropriate surface area, equilibrated with 40 mM sodium acetate, pH 6.0 (eg, Filtro
n) is used. The filtered sample is treated with a cation exchange resin (eg, Poros).
HS-50, Perseptive Biosystems). The column was washed with 40 mM sodium acetate, pH 6.0, and 250 mM, 500 mM, 1000 mM, and 1500 mM NaC in the same buffer.
With l, elute in a stepwise fashion. The absorbance of the eluate at 280 nm is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample was then added to a pre-prepared strong anion (Poros HQ
-50, Perseptive Biosystems exchange resin and weak anion (Poros CM-20, Perseptive Biosystems).
) Load into a set of in-line columns of exchange resin. The column is equilibrated with 40 mM sodium acetate, pH 6.0. Both columns were loaded with 40 mM sodium acetate,
Wash with 200 mM NaCl, pH 6.0. Next, the CM-20 column is replaced with 10
Linear gradient of column volume (0.2 M NaCl, 50 mM sodium acetate, pH
Elution with 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5). Fractions are collected under constant A ₂₈₀ monitoring of the eluate. Fractions containing the polypeptide (eg, found by 16% SDS-PAGE) are then pooled.

The resulting polypeptide should exhibit greater than 95% purity after the refolding and purification steps described above. When 5 μg of purified protein is loaded, no major contaminating band should be observed from Coomassie blue stained 16% SDS-PAGE gels. The purified protein is also endotoxin / LPS
It can be tested for contamination and typically the LPS content is less than 0.1 ng / ml according to the LAL assay.

Example 7: Cloning and Expression of Polypeptides in Baculovirus Expression System In this example, plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector is used for Autographa californica nuclear polyhedrosis virus (
AcMNPV) strong polyhedrin promoter followed by BamHI, Xba
I, and convenient restriction sites such as Asp718. Simian virus 40
The polyadenylation site of ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, this plasmid was engineered into E. coli under the control of the weak Drosophila promoter in the same orientation. It contains the β-galactosidase gene from E. coli, followed by the polyadenylation signal of the polyhedrin gene. The inserted gene is flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA, which produces a viable virus expressing the cloned polynucleotide.

Many other baculovirus vectors (eg pAc373, pVL941)
, And pAcIM1) are signals that the construct is properly positioned for transcription, translation, secretion, etc. (including signal peptide and in-frame AUG, if required), as will be readily appreciated by those of skill in the art. Can be used in place of the above vector as long as Such a vector is, for example, Lucco.
W et al., Virology 170: 31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone was amplified using the PCR protocol described in Example 1 using primers with appropriate restriction sites and start / stop codons. Let The pA2 vector does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Alternatively, Summers et al., “A Manual of Me.
hows for Baculovirus Vectors and In
sect Cell Culture Procedures ", Texas
Agricultural Experimental Station Bu
lletin NO. : 1555 (1987), this vector can be modified to include a baculovirus leader sequence using standard methods (
pA2GP).

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO
101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified on a 1% agarose gel.

This plasmid can be digested with the corresponding restriction enzymes and optionally dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. This DNA is then added to a commercially available kit (“Geneclean” BIO 10
1 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel.

This fragment and the dephosphorylated plasmid are ligated together using T4 DNA ligase. E. coli HB101 cells or XL-1 Blu
e (Stratagene Cloning Systems, La Joll)
a, CA) other suitable E. The E. coli host is transformed with the ligation mixture and spread on culture plates. Bacteria containing this plasmid are identified by digesting the DNA from individual colonies and analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of plasmid containing this polynucleotide was prepared as described in Felgner et al., Pr.
oc. Natl. Acad. Sci. USA 84: 7413-7417 (19).
87), using 1.0 μg of commercially available linearized baculovirus DNA ("BaculoGold ^™ baculovi").
rus DNA ", Pharmingen, San Diego, CA). 1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid were treated with 50 μl of serum-free Grace's medium (Life Tech).
Nologies Inc. , Gaithersburg, MD) in a sterile well of a microtiter plate. Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then seeded on a 35 mm tissue culture plate containing 1 ml of Grace's medium without serum (Sf9 insect cells (A
TCC CRL 1711). The plate is then incubated at 27 ° C for 5 hours. The transfection solution is then removed from the plate and 1 ml Grace's insect medium supplemented with 10% fetal bovine serum is added. The culture is then continued for 4 days at 27 ° C.

Supernatants are collected after 4 days and plaque assay performed as described by Summers and Smith (supra). "Blue Gal" (Life
Technologies Inc. , Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones (resulting in blue-stained plaques). (A detailed description of this type of "plaque assay" can also be found in Life Technologies Inc.
, Gaithersburg, pages 9-10, which can be found in the user guide for insect cell culture and baculovirology). After appropriate incubation, blue-stained plaques are picked up with a micropipette tip (eg Eppendorf). The agar containing the recombinant virus was then resuspended in a microcentrifuge tube containing 200 μl Grace's medium and the suspension containing the recombinant baculovirus was used to seed Sf9 cells seeded in a 35 mm dish. Infect. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C.

[0705]   In order to confirm the expression of the polypeptide, Glein supplemented with 10% heat-inactivated FBS was used.
Sf9 cells are grown in sucrose medium. The cells were infected with an infection efficiency of about 2 (“MO
I ") with a recombinant baculovirus containing this polynucleotide.
If radiolabeled protein is desired, remove the medium after 6 hours and
Methionine- and cysteine-free SF900 II medium (Life T
technologies Inc. Available from Rockville, MD
Noh). 42 hours later 5 μCi³⁵Of S-methionine and 5 μCi ³⁵ Add S-cysteine (available from Amersham). Cells further
Incubate for 16 hours, then harvest by centrifugation. Tampa in the supernatant
Cytoplasmic as well as intracellular proteins by SDS-PAGE followed by
Analyze by geography (if radiolabeled).

[0706] Minor sequencing of the amino-terminal amino acid sequence of the purified protein can be used to determine the amino-terminal sequence of the produced protein.

Example 8 Expression of Polypeptides in Mammalian Cells The polypeptides of the invention can be expressed in mammalian cells. A typical mammalian expression vector contains promoter elements that mediate the initiation of transcription of mRNA, protein coding sequences, and signals necessary for termination of transcription and polyadenylation of the transcript. Further elements are enhancers, Kozak (Koz
ak) sequences and intervening sequences flanking the donor and acceptor sites for RNA splicing. Very efficient transcription is achieved by the SV40-derived early and late promoters, retroviruses (eg RSV, HTLVI, H
IVI) -derived long terminal repeat (LTR), and cytomegalovirus (CMV)
Is achieved using the early promoter of. However, intracellular elements (eg,
The human actin promoter) can also be used.

Expression vectors suitable for use in practicing the present invention are, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVc.
at (ATCC 37152), pSV2dhfr (ATCC 37146),
Includes vectors such as pBC12MI (ATCC 67109), pCMVSport2.0, as well as pCMVSport3.0. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurka.
t cells, mouse NIH3T3 cells and C127 cells, Cos 1 cells, Co
s 7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide comprises a polynucleotide integrated into a chromosome,
It can be expressed in stable cell lines. Co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is
Useful for developing cell lines that carry hundreds or even thousands of copies of the gene of interest (eg, Alt, FW et al., J. Biol. Chem. 253: 1357).
-1370 (1978); Hamlin et al., Biochem. et Bioph
ys. Acta, 1097: 107-143 (1990); Page et al., Bio.
technology, 9: 64-68 (1991)). Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 277-279 (1991); Bebbing.
ton et al., Bio / Technology, 10: 169-175 (1992).
). These markers are used to grow mammalian cells in selective medium and select the cells with the highest resistance. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for protein production.

Expression vectors pC4 (ATCC Accession No. 209646) and pC6 (AT
CC Accession No. 209647) is for Rous sarcoma virus (Cullen et al., Mol.
electrical and Cellular Biology, 438-447 (
March 1985)), a strong promoter (LTR), and a fragment of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites with BamHI, XbaI, and Asp718 restriction enzyme cleavage sites facilitate cloning of the gene of interest. This vector also contains a 3'intron, polyadenylation and termination signals of the rat preproinsulin gene, and S
Contains the mouse DHFR gene under the control of the V40 early promoter.

Specifically, for example, plasmid pC6 is digested with an appropriate restriction enzyme,
Calf intestinal phosphatase (phosph) is then prepared by procedures known in the art.
ate) to dephosphorylate. The vector is then isolated from a 1% agarose gel.

The polynucleotides of the invention are amplified according to the protocol outlined in Example 1, using primers with appropriate restriction sites and start / stop codons, if necessary. If secretion is required, the vector can be modified to include a heterologous signal sequence (see, eg, WO96 / 34891).

The amplified fragment was transferred to a commercial kit (“Geneclean”, BIO 10).
1 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. Then E. E. coli HB101 cells or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC6 are identified using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of expression plasmid pC6 was cotransfected with 0.5 μg of plasmid pSVneo using lipofectin (
Felgner et al., Supra). Plasmid pSV2-neo contains the neo gene from Tn5 which encodes an enzyme that confers resistance to a group of antibiotics, including G418, which is the dominant and selectable marker. The cells were treated with 1 mg / ml G4
Seed in α minus MEM supplemented with 18. Two days later, the cells were trypsinized and treated with 10, 25, or 50 ng / ml methotrexate and 1 m.
Seed in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with g / ml G418. After about 10-14 days, single clones were trypsinized and then using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
Seed in 6-well Petri dishes or 10 ml flasks. The clones that grow at the highest concentration of methotrexate are then transferred to higher concentrations of methotrexate (
1 μM, 2 μM, 5 μM, 10 mM, 20 mM) in a new 6-well plate. The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

Example 9 Protein Fusions The polypeptides of the invention are preferably fused to other proteins. These fusion proteins can be used for various applications. For example, fusion of a polypeptide of the invention to a His tag, HA tag, protein A, IgG domain, and maltose binding protein facilitates purification (see Example 5; EP A 394,827). Traunecker et al., Nature 331;
: 84-86 (1988)). These polypeptides also
It may be fused to a heterologous polypeptide sequence to facilitate secretion or intracellular trafficking (eg, KDEL). Furthermore, IgG-1, Ig
Fusion to G-3 and albumin increases half-life in vivo. The nuclear localization signal fused to the polypeptide of the present invention allows the protein to bind to specific subcellular (
Subcellular localization can be targeted. On the other hand, covalent heterodimers or homodimers may increase or decrease the activity of the fusion protein. Fusion proteins can also create chimeric molecules that have more than one function. Finally, fusion proteins may increase the solubility and / or stability of fusion proteins as compared to non-fusion proteins. All forms of the above fusion proteins can be made by modifying the protocol below, which outlines the fusion of polypeptides to IgG molecules, or the protocol described in Example 5.

Briefly, the human Fc portion of an IgG molecule is the 5'and 3'of the sequence set forth below.
It can be PCR amplified using primers spanning the ends. These primers should also have convenient restriction enzyme sites and, if necessary, start / stop codons to facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be linked to the BamHI cloning site. Note that the 3'BamHI site should be destroyed. The vector containing the human Fc portion was then restricted again with BamHI to linearize the vector and Example 1
A polynucleotide of the invention isolated by the PCR protocol described in
It is ligated to this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise no fusion protein will be produced.

PC4 does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891).

[0721]

[Chemical 3] Example 10: Formulation of Polypeptides Polypeptide compositions are prepared according to the clinical condition of an individual patient (particularly the side effects of treatment with a secreted polypeptide alone), delivery site, mode of administration, dosing regimen and others known to those of skill in the art. Good medical practice taking factors into consideration
Prescription and dosing in a manner that complies with. Therefore, in the present specification, the target “
An "effective amount" is determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the polypeptide administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient body weight, This is at therapeutic discretion, as described above. More preferably, for the hormone, the dose is at least 0.01 mg / kg / day, and most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. When administered continuously, the polypeptide is typically injected one to four times daily or continuously subcutaneously (eg, using a minipump) at a dosage rate of about 1 μg / kg / hour to about 50 μg / kg / hour. It is administered by either. Intravenous bag solutions may also be used. The duration of treatment required to observe changes and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.

A pharmaceutical composition comprising the polypeptide of the invention may be administered orally, rectally, parenterally, intracistally, vaginally, intraperitoneally, topically (powder, ointment,
Given as a gel, drops, or transdermal patch), buccal or oral or nasal spray. "Pharmaceutically acceptable carrier" means a non-toxic solid,
Semi-solid or liquid filler, diluent, encapsulant or formulation auxiliary of any type. The term "parenteral" as used herein means intravenous, intramuscular, intraperitoneal, intrasternal,
Refers to modes of administration which include subcutaneous and intraarticular injection and infusion.

The polypeptide is also suitably administered by a sustained release system. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, eg films or microcapsules. Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Bio.
Polymers, 22: 547-556 (1983)), poly (2-hydroxyethylmethacrylate) (Langer et al., J. Biomed. Mater.
Res. 15: 167-277 (1981), and Langer, Chem.
Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R
． Langer et al.) Or poly-D-(−)-3-hydroxybutyric acid (EP133
, 988). Sustained-release compositions also include liposome-encapsulated polypeptides. Liposomes containing secreted polypeptides are prepared by methods known per se: DE 3,218,121; Epstein et al., P.
roc. Natl. Acad. Sci. USA 82: 3688-3692 (1
985); Hwang et al., Proc. Natl. Acad. Sci. USA 7
7: 4030-4034 (1980); EP52,322; EP36,676;
EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,54.
4,545; and EP 102,324. Usually, liposomes are small (about 200-800 angstroms) unilamellar form with a lipid content of about 30.
A selected percentage, greater than mol% cholesterol, is adjusted for optimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, the polypeptide generally will be administered to the recipient in the desired degree of purity, pharmaceutically acceptable carrier, ie, the dosage and concentration employed. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with a non-toxic compound compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be deleterious to polypeptides.

In general, the formulations are prepared by uniformly and intimately contacting the polypeptide with a liquid carrier or a finely divided solid carrier or both. Then, if necessary,
Mold the product into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, and include such substances as phosphate, citrate,
Buffering agents such as succinate, acetic acid and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg polyarginine or tripeptides); serum Proteins such as albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; cellulose or its derivatives, glucose, mannose or dextrin,
Monosaccharides, disaccharides, and other carbohydrates; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium;
And / or non-ionic surfactants such as polysorbates, poloxamers or PEG.

Polypeptides will typically be formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml, at a pH of about 3-8. . It is understood that the use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.

Any polypeptide used for therapeutic administration can be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (eg 0.2 micron membrane). Generally, the therapeutic polypeptide compositions are placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

The polypeptides are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, sterile filtered 1% (
w / v) Charge 5 ml of aqueous polypeptide solution and lyophilize the resulting mixture. The lyophilized polypeptide is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. A notice in the form of a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products may accompany such containers, which notice may include government notices regarding manufacture, use, or sale for human administration. Indicates approval by the institution. Furthermore, the polypeptides of the present invention may be used in combination with other therapeutic compounds.

Example 11 Methods of Treating Decreased Levels of Polypeptides Conditions caused by reduced normal or normal expression levels of a polypeptide in an individual are those in which the polypeptide of the invention is preferably secreted. It will be appreciated that treatment may be by and / or administration in a soluble form. Accordingly, the invention also provides a method of treating an individual in need of increased levels of this polypeptide. The method comprises the step of administering to such an individual a pharmaceutical composition comprising an amount of the polypeptide that increases the activity level of the polypeptide in such individual.

For example, a patient with reduced levels of a polypeptide receives the polypeptide at a daily dose of 0.1-100 μg / kg for 6 consecutive days. Preferably the polypeptide is in secreted form. The exact details of the dosing and formulation-based regimen are:
Provided in Example 10.

Example 12 Methods of Treating Elevated Levels of Polypeptides Antisense technology is used to inhibit production of the polypeptides of the invention. This technique uses polypeptides (preferably secretory forms) that result from various etiologies such as cancer.
Is one example of a method of reducing the level of.

For example, in patients diagnosed with abnormally elevated levels of polypeptide, antisense polynucleotides are administered at 0.5, 1.0, 1.5, 2.0 and 3.0 mg.
/ Kg / day for 21 days. If well tolerated, the procedure is repeated after a 7 day washout period. The formulation of this antisense polynucleotide is provided in Example 10.

Example 13 Treatment Method Using Gene Therapy—Ex Vivo One method of gene therapy involves transplanting fibroblasts capable of expressing a polypeptide into a patient. Fibroblasts are generally obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. A blob of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted and tightly closed, then left overnight at room temperature. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask, and fresh medium (eg,
Ham's F12 containing 10% FBS, penicillin and streptomycin
Medium) is added. The flask is then incubated at 37 ° C for approximately 1 week.

At this point, fresh medium is added and then replaced every few days. After culturing for another two weeks, a monolayer of fibroblasts appears. Trypsinizing this monolayer,
Scale up to a larger flask.

PMV-7 (Kirsc) flanked by long terminal repeats of Moloney murine sarcoma virus.
hmeier, P.M. T. Et al., DNA, 7: 219-25 (1988)).
After digestion with RI and HindIII, it is treated with calf intestinal phosphatase.
The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNAs encoding the polypeptides of the present invention were labeled with 5'terminal and 3'terminal sequences, respectively, as described in Example 1 using primers and having appropriate restriction sites and start / stop codons, respectively. Can be amplified using PCR primers corresponding to Preferably, the 5'primer contains an EcoRI site and the 3'primer contains a HindIII site. Equal amounts of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under suitable conditions for ligating the two fragments. The ligation mixture is then used to transform bacterial HB101. It is then plated on agar containing kanamycin to confirm that the vector has the gene of interest inserted correctly.

Amphotropic pA317 or GP + am12 packaging cells were
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle Medium (DMEM) with 0% Calf Serum (CS), Penicillin and Streptomycin. The MSV vector containing the gene of interest is then added to the medium and the packaging cells are transduced with this vector. At this time, the packaging cells produce infectious viral particles containing the gene (herein, the packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells, which is then harvested from 10 cm plates of confluent producer cells. The spent medium containing infectious viral particles is filtered through a Millipore filter to remove detached producer cells and then used to infect fibroblasts. Media is removed from the subconfluent plates of fibroblasts and quickly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts will be infected and no selection is required. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker such as neo or his. Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if the protein is being produced.

The engineered fibroblasts are then transplanted into the host either alone or after being grown to confluence on Cytodex 3 microcarrier beads.

Example 14 Gene Therapy Using an Endogenous Steroid Hormone Receptor Gene Another method of gene therapy according to the present invention is the use of an endogenous steroid hormone receptor gene sequence, eg, US Pat. No. 5,641,670. (June 24, 1997
International publication number WO 96/29411 (published September 26, 1996)
International publication number WO 94/12650 (published on August 4, 1994); Koll
er et al., Proc. Natl. Acad. Sci. USA, 86: 8932-8
935 (1989); and Zijlstra et al., Nature, 342: 43.
5-438 (1989), operably linked to the promoter via homologous recombination. The method involves activation of a gene that is present in the target cell but is not expressed in the cell or is expressed at a lower level than desired.

A polynucleotide construct containing a promoter and targeting sequence is made. This target sequence is homologous to the 5'non-coding sequence of the endogenous steroid hormone receptor gene, which is adjacent to the promoter. This targeting sequence is sufficiently close to the 5'end of the steroid hormone receptor gene so that the promoter is
It is operably linked to this endogenous sequence upon homologous recombination. The promoter and targeting sequence can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites on the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence is the 3'end of the amplified promoter. Contains the same restriction sites as.

The amplified promoter and amplified targeting sequence are digested with the appropriate restriction enzymes followed by treatment with calf intestinal phosphatase. The digested promoter and digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent such as liposomes, viral sequences, viral particles, precipitating agents and the like. Such methods of delivery are known in the art.

Once cells have been transfected, homologous recombination will occur and the promoter will be operably linked to the endogenous steroid hormone receptor gene sequence. This results in the expression of steroid hormone receptors in this cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the surface of the plastic with nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is mixed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM K).
Cl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. This final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting the steroid hormone receptor locus, plasmid pUC18 (MBI Fermentas, Amherst, NY) was transformed into H
Digest with indIII. The CMV promoter is amplified by PCR with an XbaI site at the 5'end and a BamHI site at the 3'end. Two steroid hormone receptor non-coding gene sequences are amplified via PCR: one steroid hormone receptor non-coding sequence (steroid hormone receptor fragment 1) with a HindIII site at the 5'end and an Xba site at the 3'end The other steroid hormone receptor non-coding sequence (steroid hormone receptor fragment 2) is amplified at the 5'end with a BamHI site and 3 '
Amplify with a HindIII site at the end. This CMV promoter and steroid hormone receptor fragment were prepared using the appropriate enzymes (CMV promoter-XbaI and BamHI; steroid hormone receptor fragment 1-
XbaI; steroid hormone receptor fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII and ligated with the HindIII digested pUC18 plasmid.

Plasmid DNA was added to a sterile cuvette (B
io-Rad). Final DNA concentration is generally at least 120μ
g / ml. Then 0.5 ml of the cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably integrate the introduced DNA into their genome. For these parameters, pulse time of about 14-20m
Sec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells were placed in a 10 cm dish containing pre-warmed nutrient medium (D containing 15% bovine serum).
MEM) 10 ml directly and incubate at 37 ° C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours.

The engineered fibroblasts are then injected into the host, either alone or after being grown to confluency on cytodex 3 microcarrier beads. To do. here,
The fibroblasts produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 15: Method of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. This gene therapy method involves the introduction of a naked nucleic acid (DNA, RNA, and antisense DNA or RNA) steroid hormone receptor sequence into an animal to increase or decrease the expression of a steroid hormone receptor polypeptide. The steroid hormone receptor polynucleotide may be operably linked to a promoter, or any other genetic element required for expression of the steroid hormone receptor polypeptide by the target tissue. Techniques and methods for such gene therapy and delivery are known in the art, eg, WO90 / 11092, WO98 / 11779; US Pat. No. 56.
93622, 5705151, 5580859; Tabata et al., Cardiovasc. Res. 35 (3): 470-479 (1997);
Chao J. Et al., Pharmacol. Res. 35 (6): 517-522.
(1997); Wolff, Neuromuscul. Disord. 7 (5)
: 314-318 (1997), Schwartz et al., Gene Ther.
3 (5): 405-411 (1996); Tsurumi Y et al., Circul.
ation 94 (12): 3281-3290 (1996), incorporated herein by reference.

The steroid hormone receptor polynucleotide construct is any method of delivering an injectable substance to cells of an animal (eg, injection into the interstitial space of a tissue (heart, muscle, skin, lung, liver, intestine, etc.)). Can be delivered by. The steroid hormone receptor polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle (viral sequence, viral particle, liposome formulation, lipofectin, or lipofectin that acts to assist, facilitate, or facilitate entry into cells. (Including a precipitating agent) is not included. However, steroid hormone receptor polynucleotides can also be prepared in liposome formulations (eg, Felgner et al., Ann. NY Acad. Sci. 772: 126-1) which can be prepared by methods well known to those of skill in the art.
39 (1995) and Abdallah et al., Biol. Cell 85 (1)
: 1-7 (1995)).

The steroid hormone receptor polynucleotide vector construct used in this gene therapy method is preferably one that does not integrate into the host genome and does not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies show that non-replicating DNA
It has been shown that the sequences can be introduced into cells to provide for production of the desired polypeptide for periods of up to 6 months.

This polynucleotide construct is used to determine the tissue (muscle, skin, brain, lung, liver,
(Including spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue) It can be delivered to the space. The interstitial spaces of this tissue are intercellular fluids, mucopolysaccharide substrates (between reticulum fibers of organ tissue, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue), or connective tissue sheath Includes the same matrix within muscle cells or in the hiatus of bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space is preferred for the reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression can be effected on undifferentiated cells or cells that are not fully differentiated (eg, blood stem cells). Or skin fibroblasts, etc.). In vivo, muscle cells
They are particularly qualified for their ability to incorporate and express polynucleotides.

For naked steroid hormone receptor polynucleotide infusions, effective dosages of DNA or RNA range from about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is from about 0.005 mg / kg to about 20.
mg / kg, and more preferably from about 0.05 mg / kg to about 5 mg
/ Kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, including, for example, inhalation of aerosol formulations, especially for delivery to the lungs or bronchial tissues, throat, or mucous membranes of the nose. In addition, the naked steroid hormone receptor polynucleotide construct can be delivered to the artery during the angioplasty procedure by the catheter used in this procedure.

The dose response effect of infused steroid hormone receptor polynucleotides in muscle in vivo is determined as follows. Suitable steroid hormone receptor template DNAs for the production of mRNAs encoding steroid hormone receptor polypeptides are prepared according to standard recombinant DNA methodologies. Either the template DNA, which can be either circular or linear, is used as naked DNA or is complexed with liposomes. Mice are then injected into the quadriceps with various amounts of template DNA.

To 5-6 week old female and male Balb / C mice, 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. Steroid hormone receptor template DNA was placed in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle for 1 minute into the knee about 0.5 cm from the distal insertion site of the muscle and a depth of about 0.2 cm. Inject. A suture is placed over the injection site for future localization and the skin is closed with a stainless steel clip.

After a suitable incubation time (eg 7 days), muscle extracts are prepared by cutting out whole quads. Every fifth 15 μm section of an individual quadriceps is histochemically stained for steroid hormone receptor protein expression. The time course for steroid hormone receptor protein expression can be done in a similar fashion, except that four animals from different mice are collected at different times.
Persistence of steroid hormone receptor DNA in muscle after injection can be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice can be used to extrapolate appropriate dosages and other treatment parameters in humans and other animals using naked DNA for steroid hormone receptors.

Example 16: Production of Antibodies a. Hybridoma Technology The antibodies of the present invention can be prepared by a variety of methods. (Current Pr
otocols, Chapter 2). As one example of such a method, cells expressing a steroid hormone receptor polypeptide are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of steroid hormone receptor polypeptide is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

Monoclonal antibodies specific for steroid hormone receptor polypeptides are prepared using hybridoma technology (Kohler et al., Natu.
re 256: 495 (1975); Kohler et al., Eur. J. Immun
ol. 6: 511 (1976); Kohler et al., Eur. J. Immunol
． 6: 292 (1976); Hammerling et al .: Monoclonal.
Antibodies and T-Cell Hybridomas, Els
evier, N.M. Y. , Pp. 563-681 (1981)). Generally, the animal, preferably a mouse, is immunized with a steroid hormone receptor polypeptide, or more preferably with secretory steroid hormone receptor polypeptide expressing cells. Such polypeptide-expressing cells can be prepared in any suitable tissue culture medium,
Preferably supplemented with 10% fetal bovine serum (inactivated at about 56 ° C) and about 1
0 g / l nonessential amino acid, about 1,000 U / ml penicillin, and about 10
It is cultured in Earle's modified Eagle medium supplemented with 0 μg / ml streptomycin.

Splenocytes from such mice are extracted and fused with the appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, AT
It is preferred to utilize the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232).
(1981)) and cloned by limiting dilution. The hybridoma cells obtained through such selection are then assayed to identify clones secreting antibodies capable of binding the steroid hormone receptor polypeptide.

Alternatively, additional antibodies capable of binding to the steroid hormone receptor polypeptide can be generated in a two step procedure using anti-idiotypic antibodies. Such a method takes advantage of the fact that the antibody itself is the antigen and therefore it is possible to obtain an antibody that binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones producing antibodies whose ability to bind the steroid hormone receptor protein-specific antibody can be blocked by the steroid hormone receptor polypeptide. Such antibodies include anti-idiotypic antibodies against steroid hormone receptor protein-specific antibodies and are used to immunize animals to induce the formation of additional steroid hormone receptor protein-specific antibodies.

For in vivo use of antibodies in humans, the antibody is "humanized." Such antibodies can be produced using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (for a review, Morrison, Science 229: 1202 (1985).
); Oi et al., BioTechniques 4: 214 (1986); Cabi.
Lly et al., US Pat. No. 4,816,567; Taniguchi et al., EP 1
71496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Bou.
Lianne et al., Nature 312: 643 (1984); Neuberg.
er et al., Nature 314: 268 (1985)).

B. Isolation of Antibody Fragments Directed against Steroid Hormone Receptor Polypeptides from a Library of scFvs The naturally occurring V gene isolated from human PBLs could be exposed to donors or Construction into a library of antibody fragments containing reactivity to steroid hormone receptor polypeptides that could not be exposed (eg US Pat.
885,793 (incorporated herein by reference in its entirety).

Library Rescue ScFvs libraries are constructed from human PBL RNA as described in PCT Publication WO 92/01047. To rescue the phage displaying the antibody fragment, about 10 ⁹ E. coli carrying the phagemid were rescued. 50 ml of 2xTY (containing 1% glucose and 100 μg / ml ampicillin) (2xTY-AMP-GLU) with E. coli and 0 with shaking.
． 8 of O. D. Grow to. Using 5 ml of this culture, 50 ml of 2xT
Y-AMP-GLU was inoculated and 2 × 10 8 TU of Δ gene 3 helper (M13Δ
Gene III, see PCT Publication WO92 / 01047) and the cultures are incubated at 37 ° C. for 45 minutes without shaking and then at 37 ° C. for 45 minutes with shaking. This culture was incubated for 10 minutes at 4000 rpm. p.
m. Centrifuge at 2 ° C. and pellet 2 liters of 2 × TY (100 μg / ml
Reconstitute in ampicillin and 50 μg / ml kanamycin) and grow overnight. Phages are prepared as described in PCT Publication WO 92/01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking, and then further incubated at 37 ° C with shaking. The cells were spun down (IEC-Centra 8, 400 r.p.
m. For 10 minutes) and 2 x TY broth (2 x TY-AMP-KAN) containing 100 μg / ml ampicillin and 25 μg / ml kanamycin 300 m.
Resuspend in 1 and grow overnight at 37 ° C with shaking. Phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and 0.45 μm filter (
Miniart NML; Sartorius) to give a final concentration of approximately 10 ¹³ transducing units / ml (ampicillin resistant clones).

(Paning of Library) Immunotubes (Nunc) were treated with 100 μg of the polypeptide of the present invention.
Coat overnight in PBS with 4 ml of either 10 μg / ml or 10 μg / ml. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C, then washed 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to the tubes and incubated for 30 minutes at room temperature with tumbling up and down on a turntable, then left to stand for a further 1.5 hours. Tube with PBS 0.1% T
Wash 10 times with ween-20 and 10 times with PBS. 1 ml of 100 mM
The phage were eluted by adding triethylamine and spinning up and down for 15 minutes on a rotating disc, then adding 0.5 ml of 1.0 M Tris-H to this solution.
Immediately neutralize with Cl, pH 7.4. Then, the eluted phage was incubated with the bacteria for 30 minutes at 37 ° C. to give 10 ml with the phage.
Mid-logarithmic growth of E. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Δ gene 3 helper phage as described above to prepare phage for the next round of selection. This process was then repeated for all 4 rounds of affinity purification, 3
For the second and fourth time, wash the tube with PBS, 0.1% Tween-20 for 2 times.
Increase to 0 times and 20 times with PBS.

Characterization of Binders Phage eluted from the third and fourth round of selection were used to transform E. coli. coli
Infect HB2151 and generate soluble scFv from a single colony for assay (Marks et al., 1991). 50 mM bicarbonate, pH 9.
ELISA is performed using microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in 6. Positive clones in the ELISA are further characterized by PCR fingerprinting (see, eg, PCT Publication WO92 / 01047) followed by sequencing. These EL
ISA positive clones can also be cloned using techniques known in the art (eg, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody / antigen binding, and competitive agonist activity or competition). Further antagonistic activity).

Example 17: Interaction of Steroid Hormone Receptor with Other Signal Molecules The purified steroid hormone receptor (SHR) polypeptide of the present invention is a protein that interacts with SHR, or a signal that SHR functions. It is a useful research tool for the identification, characterization and purification of other proteins in the transduction pathway (eg, receptor proteins or other signal transduction pathway proteins). Briefly, labeled steroid hormone receptor proteins are useful as reagents for the purification of interacting molecules. In one embodiment of affinity purification, the steroid hormone receptor protein is covalently attached to a chromatography column. Cell-free extracts from putative target cells such as hepatocytes or kidney cells were passed through the column. Molecules that bind to SHR remain on the column. The SHR protein complex is recovered from the column, dissociated, and the recovered SHR interacting protein subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify capture molecules or design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate DNA library.

Example 18: β-Gal of steroid hormone receptor interacting clones
Assays Among other assays known in the art, Seol et al. (Seol. W.,
Et al., Mol Endocrinology, 9: 72-84 (1995), incorporated herein by reference in its entirety), utilizing the yeast two-hybrid system. The interaction of the steroid hormone receptor (SHR) of the invention with members or other signaling molecules can be assayed. Briefly, one creates an expression vector for making two types of fusion proteins: one fusion protein
Le fused to a steroid hormone receptor or signal transduction molecule of interest
Another type of fusion protein contains the xA DNA binding domain, and the B42 transcriptional activation domain fused to the SHR of the invention. EGY48 [MATα,
leu2, trp1 ura3 his3 LEU2 :: pLexop6-LE
U2 (ΔUAS LEU2)] yeast strain (where the chromosome LEU2 gene is L
exH-A operator), 8H18-34lacZ reporter plasmid (where lacZ expression is under Lex-A operator control), Lex-A fusion protein expression vector and B-42 fusion protein expression. Successfully transform with vector. The LacZ vector contains the URA3 gene;
The Lex-A fusion protein vector contains the HIS3 gene and the B42 expression vector contains the TRP1 gene. At least two separate colonies from a plate containing glucose but lacking uracil, histidine and tryptophan were randomly selected for each co-expressing line and plated on liquid medium containing galactose but lacking uracil, histidine and tryptophan. To induce expression of the B42 fusion. Cultures are assayed for β-gal because β-gal expression is an indicator of the interaction between steroid hormone receptors or signaling molecules of interest and the SHRs of the invention.

Example 19: Gel Shift Assay To determine if the steroid hormone receptor (SHR) of the invention is involved in binding to DNA responsive elements, see Seol et al. (Seol. W., et al. (1995). ) Is used. Briefly, double-stranded oligonucleotides containing known or putative SHR binding sequences (eg, Drosophi containing the conserved heteromeric sequence AGGTCA).
The Ec-responsive element (EcRE) from the 1a heat shock protein (hsp) 27 promoter is end-labeled using [ ³² ATP] ATP and a kinase. In addition to naturally occurring responsive elements, the heteromeric sequence AGGTCA
It will be apparent to those skilled in the art to make synthetic oligonucleotides containing direct or converted repeats of (between 0 and 5 bp apart). 20μ of SHR protein
1 gel shift assay buffer (10 mM Tris (pH 8.0), 40 m
Either M or 150 mM KCl, 0.05% NP-40, 10% glycerol, 1 mM dithiothreitol, 2.5 mM MgCl ₂ , and 5n
g poly-dl-dC) pre-incubate on ice for 10 minutes. The mixture is then allowed to bind to the labeled labeled probe and incubated at room temperature for 20 minutes. The mixture is analyzed at 4 ° C. by 6% non-denaturing polyacrylamide gel electrophoresis using 0.5 × Tris-borate buffer, followed by autoradiography. The migration of probe incubated with SHR is compared to the migration of untreated probe. The slower migration of probe incubated with SHR is an indication that SHR binds to DNA responsive elements.

Example 20: Reporter Gene Assay for SHR For assaying the transcriptional activity of the reporter gene by the steroid hormone receptor (SHR) of the present invention, Kumar et al. (Kumer, MB.
Et al., J. Biol. Chem. 274: 22155-64 (1999): incorporated herein by reference in its entirety). Reporter constructs were prepared as described by Kumar et al. (Kumer,
M. B. , (1999)) and has an SHR binding site upstream of the luciferase reporter gene in addition to any other regulatory sequences of interest. Briefly, modified Eagle containing 10% fetal bovine serum (v / v)
Culture the cells in medium. For reporter gene assay, LipofectAMINE reagent (Life Technology) in 24-well plates.
es, Inc. ) Is used to transfect cells with a total of 750 ng of DNA. The transfected DNA mixture was mixed with various amounts of control plasmid and SHR expression plasmid with 100 ng of reporter plasmid and β-galatosidase internal control plasmid pCMV / La, respectively.
cZ (Invitrogen, San Diego, CA). The cells can then be treated with a substance known to activate SHR after transfection for a period of time. Cells are harvested and assayed for luciferase activity. Luciferase activity is averaged and β-galactosidase activity is observed. All transfections are performed 3 times.

It will be apparent to those skilled in the art that alternative reporter systems can be used to detect reporter gene activity by the SHRs of the present invention. For example, a GFP or GFP fusion protein whose expression is under the control of the SHR binding site can be used as a reporter gene. COS-1 cells seeded on 2 mm coverslips on a 6 mm dish are transfected with a total of 400 ng of GFP or GFP fusion plasmid containing GFP or GFP fusion along with SHR expression plasmid or control plasmid. The cells are then treated with a substance known or expected to activate SHR for a period of time after transfection. Wash these cells with phosphate buffered saline,
Then, 24 hours after transfection, the cells are visualized using a fluorescence microscope.

Certain steroid hormone receptor polynucleotides and polypeptides (including antibodies) of the invention and their uses are described in US Patent Application No. 60/1.
52,932 and 60 / 189,032, each of which is incorporated herein by reference in its entirety.

It will be appreciated that the present invention may be practiced other than as described in detail in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the appended claims.

The entire disclosure (including patents, patent applications, scholarly literature, abstracts, laboratory manuals, books or other disclosures) of each document cited in the background, detailed description and examples is included herein. Incorporated as a reference in. Further, both the hard copy of the sequence listing and the corresponding computer readout form submitted with this specification are hereby incorporated by reference in their entirety.

[0780]

[Table 6] ATCC Deposit No. PTA-629 (Canada) Applicant is nominated by the Commissioner of the Patent Office until a Canadian patent is issued under the application, or the application is refused or abandoned and cannot be recovered or withdrawn. of Patents), the applicant of the international application Before the regulatory preparations for publication are complete, the International Bureau must be notified in writing.

(Norway) Applicants hereby agree that the application of samples shall be made by the Norwegian Patent Office until the application has been published (by the Norwegian Patent Office) or has been decided by the Norwegian Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Australia) The Applicant hereby states that the donation of a sample of microorganisms is prior to the grant of the patent:
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

(Finland) The applicant hereby states that the application (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

(UK) Applicant hereby claims that a sample of microorganisms is only made available to the expert. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Deposit No. PTA-629 (Denmark) The applicant hereby approves the provision of samples until the application is either published (by the Danish Patent Office) or determined by the Danish Patent Office without publication. Claim that it is done only to technical experts. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Sweden) Applicants hereby give samples to the art until the application has been published (by the Swedish Patent Office) or determined by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be filed by the applicant with the International Bureau not later than 16 months from the priority date (preferably PCT Applicant's Gui).
Form PCT / RO / 13 described in Annex Z of Volume I of de
4). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts (lis) prepared by the Swedish Patent Office.
any of the persons listed in to of recognized experts),
Alternatively, it may be any person approved by the applicant in the individual case.

(Netherlands) Applicants hereby agree that until the issue date of the Dutch patent, or until the date when the application is rejected, withdrawn or abandoned (lapped), microorganisms are specialized under the provisions of Patent Law 31F (1). Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

[0787]

[Table 7] ATCC Deposit No. PTA-1477 (Canada) Applicant is nominated by the Commissioner of the Patent Office until a Canadian patent is issued under the application or the application is refused or abandoned and cannot be recovered or withdrawn. of Patents), the applicant is entitled to grant a sample of the deposited biological material referred to in the application only to an independent expert appointed by the Commissioner, Before the regulatory preparations for publication are complete, the International Bureau must be notified in writing.

(Norway) Applicants hereby agree that the application of samples is not subject to specialization in the art until the application is published (by the Norwegian Patent Office) or determined by the Norwegian Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Australia) The Applicant hereby states that the donation of a sample of microorganisms is prior to the granting of a patent.
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

(Finland) The applicant hereby states that the application is (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

(UK) Applicants hereby claim that samples of microorganisms are only made available to professionals. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Deposit No. PTA-1477 (Denmark) Applicants hereby give samples until the application has been published (by the Danish Patent Office) or has not been published and has been decided by the Danish Patent Office. Claim that it is done only to technical experts. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Sweden) Applicants hereby note that the provision of samples is not subject to technical expertise until the application has been published (by the Swedish Patent Office) or determined by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be filed by the applicant with the International Bureau not later than 16 months from the priority date (preferably PCT Applicant's Gui).
Form PCT / RO / 13 described in Annex Z of Volume I of de
4). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts (lis) prepared by the Swedish Patent Office.
any of the persons listed in to of recognized experts),
Alternatively, it may be any person approved by the applicant in the individual case.

(Netherlands) Applicants hereby agree that until the issue date of the Dutch patent, or until the date when the application is rejected, withdrawn or lapped, the microorganism is specialized under the provisions of Patent Act 31F (1). Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/16 A61P 5/14 4C084 3/10 7/00 4C085 5/14 7/04 4C086 7/00 7 / 06 4H045 7/04 13/12 7/06 15/08 13/12 21/00 15/08 25/28 21/00 29/00 25/28 101 29/00 31/18 101 37/02 31/18 37/08 37/02 43/00 111 37/08 C07K 14/705 43/00 111 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21 / 02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/566 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A 33/566 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH , GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ni, Gian United States Maryland 20853, Rock Bill, Manorfield Road 5502 (72) Inventor Shi, Young United States Maryland 20878, Gaithersburg, Apartment 102, West Side Drive 437 (72) Inventor Reuben, Stephen M. United States Maryland 20832, Ornay, Heritage Hills Drive 18528 F Term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 AA12 BA61 BA63 CA02 CA04 CA09 GA11 HA01 HA14 HA17 4B063 QA19 QQ03 QQ08 QQ43 QR55 CC20 DA14 CA05 DA20 DA14 CA0 DAQDA DAB26 CA0 4B065 AB01 BA02 CA24 CA44 CA46 4C084 AA02 AA06 AA07 BA03 CA53 DB63 NA14 ZA152 ZA162 ZA532 ZA552 ZA592 ZA812 ZA892 ZB052 ZB072 ZB112 ZB132 ZB152 ZB352 ZC022 ZC062 ZC352 4C085 AA13 AA14 BB11 CC02 CC04 CC05 DD22 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 ZA15 ZA16 ZA53 ZA55 ZA59 ZA81 ZA89 ZB05 ZB07 ZB11 ZB13 ZB15 ZB35 ZC02 ZC06 ZC35 4H045 AA10 AA11 AA20 AA30 CA40 DA50 DA75 EA21 EA28 EA50 EA51 FA74

Claims (21)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) a polynucleotide set forth in SEQ ID NO: X, or a polynucleotide encoded by the cDNA contained in ATCC Deposit No. Z; (b) SEQ ID NO: Biologically active polypeptide fragment of Y, or AT
A polynucleotide encoding a biologically active polypeptide fragment encoded by the cDNA sequence contained in CC Accession No. Z; (c) the polypeptide epitope of SEQ ID NO: Y, or the cDNA sequence contained in ATCC Accession No. Z A polynucleotide encoding a polypeptide epitope encoded by: (d) a polynucleotide capable of hybridizing to any one of the polynucleotides specified in (a) to (c) under stringent conditions. Wherein the polynucleotide comprises a polynucleotide selected from the group consisting of: a polynucleotide that does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence with only A residues or only T residues. , An isolated nucleic acid molecule. 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide comprises a nucleotide sequence that encodes a soluble polypeptide. 3. The polynucleotide, wherein the polynucleotide encodes a sequence identified as SEQ ID NO: Y, or a cDNA contained in ATCC Accession No.Z.
A nucleotide sequence encoding a polypeptide encoded by the sequence,
An isolated nucleic acid molecule according to claim 1. 4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide comprises the entire nucleotide sequence of SEQ ID NO: X, or the cDNA contained in ATCC Deposit No.Z. 5. The isolated nucleic acid molecule of claim 2, wherein the polynucleotide is DNA. 6. The isolated nucleic acid molecule of claim 3, wherein the polynucleotide is RNA. 7. A vector comprising the isolated nucleic acid molecule of claim 1. 8. A host cell containing the vector of claim 7. 9. A recombinant host cell comprising the nucleic acid molecule of claim 1 operably linked to a heterologous regulatory element that controls gene expression. 10. A method for producing a polypeptide, which comprises a step of expressing the encoded polypeptide from the host cell according to claim 9 and a step of recovering the polypeptide. 11. An isolated polypeptide, which is: (a) a polypeptide represented by SEQ ID NO: Y, or a polypeptide encoded by a cDNA; (b) a polypeptide fragment of SEQ ID NO :, or at least 95 in the sequence selected from the group consisting of: (c) a polypeptide epitope of SEQ ID NO: or a polypeptide encoded by cDNA; and (d) a variant of SEQ ID NO: % Containing amino acid sequences,
Isolated polypeptide. 12. The isolated polypeptide of claim 11, which comprises a polypeptide having SEQ ID NO: Y. 13. An isolated antibody that specifically binds to the isolated polypeptide of claim 11. 14. Expressing the isolated polypeptide of claim 11.
Recombinant host cell. 15. A method for producing an isolated polypeptide, comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that the polypeptide is expressed. A step; and (b) recovering the polypeptide. 16. A polypeptide produced by the method of claim 15. 17. A method of preventing, treating or alleviating a medical condition, comprising:
13. A method comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 11 or the polynucleotide of claim 1. 18. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1. And (b) diagnosing a pathological condition, or susceptibility to a pathological condition, based on the presence or absence of the mutation. 19. A method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) the presence of expression of the polypeptide of claim 11 in a biological sample. Or a step of determining the amount; and (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or amount of expression of the polypeptide. 20. A method of identifying a binding partner for the polypeptide of claim 11, comprising: (a) contacting the polypeptide of claim 11 with a binding partner;
And (b) determining whether the binding partner results in the activity of the polypeptide. 21. A method of screening for a molecule that modifies the activity of the polypeptide of claim 11, comprising: (a) contacting the polypeptide with a compound suspected of having agonistic or antagonistic activity. And (a) assaying the activity of the polypeptide.