JP2003528584A - DNA encoding PROST07 polypeptide - Google Patents

Abstract

(57) Abstract: The present invention provides a novel serine protease polypeptide designated PROST 07, a polynucleotide encoding the polypeptide, a method for producing the polypeptide, an expression vector, and a genetic construct for expression of the polypeptide. Host cells that have been The invention further relates to the use of said polynucleotides and polypeptides in research, diagnostic and therapeutic applications.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to newly identified polynucleotides and polypeptides; variants and derivatives of said polynucleotides and polypeptides; methods for producing said polynucleotides and polypeptides, and variants and derivatives thereof. Antibodies directed against said polypeptides, variants and derivatives thereof; and said polynucleotides,
It relates to the use of polypeptides, variants and antibodies. In particular and with respect to those and others, the present invention provides novel PROST 07 polypeptides, polynucleotides encoding those polypeptides, antibodies directed against those polypeptides, and PROST 07
To an antisense polynucleotide that blocks the expression of

BACKGROUND OF THE INVENTION Prostate cancer is a frequently occurring disease in men, in that it is found in about one-third of men over the age of 45. There is evidence of genetic and environmental causes, and their major cause is probably the result of a combination of the above factors. Studies of familial cancers suggest that genetic predisposition plays a role in about 5-10% of all prostate cancers and about 45% of young men younger than 55 years.

There is evidence that prostate cancer progresses as a multi-stage disease, and one of the precursor diseases is prostate intraepithelial neoplasia (PIN). The early stages of the disease are androgen dependent, and the late stages are hormone independent. The proliferative disease of the prostate, known as benign prostatic hyperplasia, is often detected clinically, but is probably not the stage in the progression of cancer. However, it is often associated with prostate cancer. Cancers in the prostate are often multifocal, generally slow growing, and heterogeneous. Late stage cancers often metastasize to lymph nodes and bone.

Prostate cancer is usually diagnosed by physical examination and by serum levels of prostate specific antigen (PSA). Basic prostatectomy is a selective treatment for localized disease. Advanced metastatic disease is currently treated by androgen degeneration induced by orchidectomy or treatment with GnRH (gonadotropin releasing hormone), and by anti-androgen therapy. However, advanced disease becomes almost constant, becoming hormone resistant, and treatment for advanced disease is needed. In addition, there are severe side effects associated with both basic prostatectomy and androgen degeneration treatment. They include incontinence and impotence associated with basic prostatectomy and high risk of bone damage and osteoporosis associated with androgen degeneration treatment.

Therefore, there is a considerable need for new therapeutic approaches for both early and late stage prostate cancer. This has a significant impact on the treatment, so a new diagnostic agent,
In particular, there is a significant need for agents that can distinguish stages of disease. For example, if the disease progresses beyond the prostate and is transferred to the lymph nodes, basic prostatectomy is not warranted because it has no effect on progression and has significant unwanted side effects. Any agent that detects metastasis in vivo would be of considerable value.

[0006] Altered expression of specific proteins may result in abnormal p53 expression in prostate cancer, eg, late stage prostate cancer, reduced levels of TGF-β receptor, reduced levels of E-cadhen, C-CAM (cell Adhesion molecules) and some integrins have been shown. Expression of the oncogene bcl-2 is significantly elevated in late-stage androgen-dependent tumors, and the prognosis for patients who express bcl-2 at elevated levels is relatively poor. The changes in gene expression referred to above are well documented, but the changes in expression that have been shown to be responsible for the disease have not been identified. Therefore, it would be useful to identify novel proteins whose expression is linked to the presence or progression of prostate tumors, which act as molecular targets for the diagnosis and treatment of prostate cancer.

The present invention discloses novel human serine proteases that are homologous to the human kallikrein gene family. This homologue, designated PPOST 07, is specifically expressed in prostate tissue and may be overexpressed in prostate tumors as compared to other tissues.

Kallikreins are a group of serine proteases involved in post-translational processing of certain polypeptide precursors (Clements. Endocr. Rev. 10: 393-4.
19, 1989; Young et al., J. Androl. 16: 97-99, 1995). The human genome is
Species kallikrein members include: pancreas / saliva / kidney kallikrein (hK1), glandular kallikrein-2 (hK2) and prostate specific antigen (PSA or hK3) (Young et al., J.
Androl. 16, 97-99, 1995; Carbini et al., J. Hypertens. 11: 893-898, 1993).
. All three kallikrein genes are located as clusters and spans 80 kb on chromosome 19q132-q13.4 (Riegman et al., Genomics 14.6-11, 1992). DNA (65-80%) and amino acids (57-78) of the three gene and protein products.
) Sequence homologues indicate that they are generated by gene duplication from the same ancestral gene (Schedlich et al., DNA 6: 429-437, 1987; Morris, Physiol. 16: 345.
-351, 1989).

Recently, a novel serine protease with homology to kallikrein and trypsin has been isolated and characterized from growing enamel matrix in pig teeth (Simmer et al., J. Dent. Res. 77, 377-387, 1998). A serine protease, called enamel matrix serine protease 1 (EMSP1), appears to be a porcine tooth-specific protease, and may play a key role in enamel and dentin formation. EMSP1 has been shown to be activated by matrix metalloproteases in the outer enamel layer (Tanabe et al., Arch. Oral. Biol. 39: 277-281, 1996).

Two major types of serine proteases are involved in a wide range of proteolytic reactions. The first group of trypsin-like enzymes includes digestive (chymotrypsin, trypsin), lysosomal (cathepsin G) and fibrinolytic (plasminogen activator) enzymes. A second main group of serine proteases is homologous to the bacterial protease subtilisin. They act within the secretory pathway and cleave polypeptide precursors at specific base sites to produce their biologically active forms. Serine protease inhibitors (serpins) make up a protein family that includes at least α1-antitrypsin or α1-antichymotrypsin.

The contribution of serine proteases in tumor invasion and metastasis appeared to be primarily extracellular matrix destruction. However, recent evidence suggests that serine proteases primarily affect tumor growth rather than invasion (Noel et al.
., Invasion Metastasis 17: 221-239, 1997). Proteases have been shown to affect growth factor release, activation and bioavailability (Whitelock et al.
, J. Biol. Chem. 71: 10079-10086, 1996), and consequently tumor cell proliferation,
Regulates apoptosis and angiogenesis.

[0012] These data indicate that serine proteases are good candidates for use in the diagnosis and possible treatment of cancer. Based on the present inventors and other protein expression data, the present inventors are the subject of the present invention, namely a novel serine protease.
Predict that PROST 07 will be used primarily in the treatment and diagnosis of prostate, ovarian and uterine cancers.

SUMMARY OF THE INVENTION The present invention provides a polynucleotide sequence that specifically encodes a novel serine protease designated herein as PROST 07. The PROST 07 polypeptide is characterized by a catalytic trimer composed of serine, histidine and aspartic acid, as described for other serine proteases. It shows homology to the kallikrein family of serine proteases and shows 82.9% similarity to the newly described serine protease EMSP1. p
The polynucleotide sequence designated herein as rost 07 and set forth herein in Figure 1 (SEQ ID NO: 1) encodes the amino acid sequence for PROST 07 shown in Figure 2 (SEQ ID NO: 2). To do.

Towards their ends and towards the other, due to the homology between the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and the known amino acid sequences of other serine protease polypeptides, it is considered as a novel serine protease. It is an object of the invention to provide the identified polypeptides. In addition, polynucleotides encoding such polypeptides, especially PROST
It is another object of the invention to provide a polynucleotide encoding the polypeptide designated herein as 07.

According to this aspect of the invention, isolated polynucleotides encoding PROST 07, such as mRNA, cDNA, genomic DNA and, in a further embodiment of the aspects herein, biological, diagnostic Also provided are clinically or therapeutically useful variants, analogs, or derivatives thereof, or fragments thereof, such as fragments of said variants, analogs and derivatives. In a particularly preferred embodiment of this aspect of the invention there is a naturally occurring allelic variant of the polynucleotide encoding the variant of the polypeptide designated herein as PROST 07.

According to this aspect of the invention, a novel polypeptide of human origin, referred to herein as PROST 07, and biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, Variants and derivatives of the above fragments, and analogs of the above are provided. In a particularly preferred embodiment of this aspect of the invention there are provided variants of PROST 07 encoded by naturally occurring allelic variants of PROST 07 polynucleotides.

It is another object of the invention to provide a method for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of said variants and derivatives, and analogues of the foregoing. In a preferred embodiment of this aspect of the invention there is provided a method of producing said PROST 07 polypeptide, wherein said method expressably incorporates an exogenously derived PROST 07 encoded polypeptide therein. Culturing the host cell under conditions for expression in a host of human PROST 07 and recovering the expressed polypeptide. According to another object of the invention, there are provided products, compositions and methods, which use said polypeptides and polynucleotides, inter alia for research, biology, clinical and therapeutic purposes.

According to a preferred embodiment of this aspect of the invention the PROST 07 polypeptide or PROST 07
Products, compositions and methods are provided for assessing the expression of PROST 07 in cells by determining the 07-encoding mRNA and for assaying genetic variations and abnormalities in the prost 07 gene, such as defects. According to a preferred embodiment of this and other aspects of the invention there is provided a probe which hybridizes to the prost 07 sequence.

Antibodies or antibodies that are highly selective for PROST 07 polypeptides and that can be used in methods for the diagnosis and / or detection of PROST 07 expression associated with various cancers, namely prostate and ovarian cancer. It is a further object of the invention to provide the fragments. In a preferred embodiment of this aspect of the invention, the antibody is labeled with a means that produces a detectable signal. Antibodies labeled with radioactive labels, enzymes, chromophores and fluorescent agents are especially preferred.

In a further aspect of the invention there is provided an antibody conjugated to a therapeutic agent for administration to in vitro cells, ex vivo cells and cells or multicellular organisms. In this regard, therapeutic agents that are cytotoxic are particularly preferred. In a preferred embodiment in this regard, such conjugated antibodies are administered to human patients for the treatment of disease states characterized by PROST 07 activity or expression, eg prostate or ovarian cancer. In a further aspect of the invention there are provided peptides and anti-idiotypic antibodies that can be used to stimulate an immune response.

In a further aspect of the invention, ribozymes and polynucleotides complementary to prost 07 polynucleotides (ie, antisense polynucleotides) for administration to cells in vitro, cell ex vivo and cells in vivo or to multicellular organisms. Nucleotides are provided. In this regard, it is warmed by lowering the level of PROST 07 activity. Particularly preferred is the administration of antisense molecules to human patients for the treatment of disease states such as prostate cancer or benign prostatic hyperplasia.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. However, it should be utilized that the following description and specific examples, which illustrate preferred embodiments of the invention, are provided for illustrative purposes only. Various changes and modifications within the scope of the disclosed invention will be readily apparent to those skilled in the art from the following description and other parts of the disclosed invention.

Specific Description of the Invention: Definitions: As used in the specification, examples and claims, unless otherwise stated, the following terms have the meanings indicated. "PROST 07" refers to a polypeptide having an amino acid sequence as shown in Figure 2 (SEQ ID NO: 2), variants, analogs, derivatives and fragments thereof, and fragments of said variants, analogs and derivatives. . The terms "fragment" and "
“Analog” when referring to the polypeptide of FIG. 2 (SEQ ID NO: 2) is a polypeptide that retains substantially the same biological and / or immunological activity as the polypeptide of FIG. 2 (SEQ ID NO: 2). Means peptide.

“Prost 07” refers to a polynucleotide having the sequence shown in FIG. 1 (SEQ ID NO: 1) and a polypeptide encoding the polypeptide having the amino acid sequence of PROST 07 shown in FIG. 2 (SEQ ID NO: 2). Nucleotides; and PROST 07 variants, analogs, derivatives and fragments, and polynucleotides encoding fragments of said variants, analogs and derivatives. Prost 07 also refers to such polynucleotides composed of RNA, and polynucleotides that are the complement of the polynucleotides encoding the polypeptide sequences shown in Figure 2 (SEQ ID NO: 2).

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, a polynucleotide, as used herein, includes, among other things, single-stranded and double-stranded DNA, DNA that is a mixture of 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; including DNA and RNA that can be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions A hybrid molecule consisting of

Furthermore, polynucleotides as used herein are RNA or DNA.
, Or a triple-stranded region comprising both RNA and DNA. The chains in such regions can be from the same or different molecules. The region includes all molecules of one or more, but more typically only some regions of the molecule. One of the molecules of the triple-stranded helix is often an oligonucleotide.

As used herein, the term “polynucleotide” includes DNA or RNA, as described above, containing one or more modified bases. Thus, a DNA or RNA that has a backbone that has been modified for stability or for other reasons is a "polynucleotide" as intended herein. Furthermore, DNA or RNA comprising unusual bases, such as inosine, or modified bases, such as tritium-labeled bases, are polynucleotides as used herein.

It will be appreciated that numerous modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide", as used herein, refers to such chemically, enzymatically, or metabolically modified forms of polynucleotides, and viral and cellular characteristics, such as simple and complex cells. Includes chemical forms of DNA and RNA.

“Polypeptide”, as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in countless textbooks and other publications in the art. In such a context, the term refers to any peptide or protein comprising a plurality of amino acids linked to each other in a straight chain by peptide bonds. As used herein, the term also generally refers to
It refers to both the short chains that are referred to in the art as peptides, oligopeptides and oligomers, and the long chains that are commonly referred to in the art as proteins of which many types exist.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and many amino acids, including the terminal amino acids, may be subjected to natural processes such as glycosylation and It will be appreciated that it may be modified in a given polypeptide either by other post-translational modifications or by chemical modification techniques well known in the art. Even the usual naturally occurring modifications in a polypeptide are too numerous to list exhaustively herein, but are well documented in the basic text and more detailed monographs, and in numerous research references. And they are well known to those skilled in the art.

Known modifications that can be present in the polypeptides of the invention include: acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme components, Polynucleotide or polynucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol covalent bond, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate Formation, formylation, γ-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, methylation, myristoylation, oxidation, proteolytic process, phosphorylation, prenylation, ceramilation, selenium oxidation, sulfuric acid , RNA-mediated addition of amino acids to proteins, eg alkyneation And ubiquitination.

Such modifications are well known to the person skilled in the art and are described in more detail in the chemical literature. Some particularly common modifications, namely glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamate residues, hydroxylation and ADP-ribosylation, are found in most basic texts such as IE Creighton,
Proteins-Structure and Molecular Properties, 2 nd Ed., WH Freeman and
Company New York, 1993. Many detailed reconsideration on this is
Available from those supplied by: Wala. F. Posttransla
tional Covalent Modidication of Proteins, BC Johnson, Ed., Academic P
ress, New York, pp. 1-12, 1983; Seifter et al., Meth. Enzymol. 182: 626-6.
46, 1990 and Rattan et al., Protein Synthesis: Posttranslational Modificati.
ons and Aging, Ann. N. Y Acad. Sci. 663: 48-62, 1992.

It will be appreciated that the polypeptide is not necessarily perfectly linear, as is well known and as indicated above. For example, polypeptides may be branched as a result of ubiquitylation, and they may or may not have branching as a result of post-translational events, such as natural processing events and phenomena resulting from non-natural human manipulation. It may be a ring that does not. Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translated natural and fully synthetic methods.

Modifications can occur at any position in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Indeed, blocking amino or carboxyl groups in a polypeptide, or both, by covalent modifications is common in naturally occurring and synthetic polypeptides, and such modifications are also present in polypeptides of the invention. be able to. For example, the amino terminal residue of a polypeptide produced in E. coli prior to the proteolytic step would be almost unchanged, N-formylmethionine.

The modifications that occur in a polypeptide often will be a function of how it is made. For example, for a polypeptide produced by expression of a cloned gene in a host, the nature and extent of modification will be determined, in large part, by the host cell's posttranslational modification capacity and modification signals present in the polypeptide amino acid sequence. Will. For example, as is well known, glycosylation often is not present in bacterial hosts such as E. coli. Thus, if glycosylation is desired, the polypeptide should be expressed in a glycosylation host, generally a eukaryotic cell. Insect cells often undergo the same post-translational glycosylation as mammalian cells, and for this reason insect cell expression systems, among other things, elicit efficient expression of mammalian proteins with an inherent pattern of glycosylation. Has been done. Similar considerations apply to other modifications.

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 have many types of modifications. In general, the term polypeptide, as used herein, includes all modifications thereof, particularly those modifications present in a polypeptide synthesized by expressing the polynucleotide in a host cell.

“Polynucleotide encoding a polypeptide”, as used herein, encodes a polypeptide of the present invention, particularly a PROST 07 polypeptide having the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2). To include the sequence. The terms are
Included are polynucleotides that include a single contiguous or discontinuous region (eg, interrupted by an intron) encoding the polypeptide, along with additional regions.

“Biological” refers to the structural, regulatory or biochemical function of a naturally occurring PRUST 07 polypeptide. "Immune activity" refers to eliciting a specific immune response in a suitable animal or cell,
It then refers to the ability of native, recombinant or synthetic PROST 07, or any fragment thereof, to bind a particular antibody.

“Oligonucleotide” refers to a relatively short polynucleotide. Often, the term refers to single-stranded deoxyribonucleotides, but it includes single- or double-stranded ribonucleotides, RNA: DNA hybrids and double-stranded, among others.
Mention may be made of DNA. Oligonucleotides, such as single stranded DNA probe oligonucleotides, are often synthesized by chemical methods, such as those performed on an automated oligonucleotide synthesizer.

However, oligonucleotides can be produced by various other methods, such as in vitro recombinant DNA-mediated techniques, and expression of DNA in cells and organisms. "Oligonucleotide" or "oligomer" or oligonucleotide "fragment", "portion" or "segment" means at least about 10 nucleotides, and about 60 nucleotides, preferably about 15-30 nucleotides. , And more preferably about 20-25 nucleotide sequences.

“Naturally occurring PROST 07” refers to PROST 07 produced by human cells that have not been genetically constructed, and specifically refers to polypeptide post-translational modifications such as acetylation, carboxylation. , A variety of PROST 07 forms from, but not limited to, glycosylation, phosphorylation, lipidation, acylation and degradation. A “variant” of a polynucleotide or polypeptide, as used herein, is a polynucleotide or polypeptide that differs from the polynucleotide or polypeptide of interest, respectively. This variant is described in more detail below and elsewhere in the present disclosure.

(1) A polynucleotide that differs in polynucleotide sequence from another, reference polynucleotide. In general, the differences are limited so that the control and variant polynucleotide sequences are closely similar overall and are identical in many regions.

As shown below, changes in the polynucleotide sequence of the variant may be silent. That is, they cannot change the amino acids encoded by the polynucleotide. If the alteration is restricted to this type of silent alteration, the variant will encode a polypeptide having the same amino acid sequence as the control. As shown below, changes in the polynucleotide sequence of the variant can alter the amino acid sequence of the polypeptide encoded by the control polynucleotide. Such polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the control sequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another, reference polypeptide. In general, the differences are limited so that the control and variant sequences are closely similar overall and are identical in many regions. Variants and control polypeptides can differ in amino acid sequence by one or more substitutions, deletions, fusions and truncations, which can occur in any combination. Recombinant variants encoding those same or similar polypeptides can be synthesized or selected by the use of "redundancy" in the genetic code. Various codon substitutions, such as silent changes that create various restriction sites, can be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations can also be introduced to modify the properties of a polypeptide, ie alter ligand binding affinity, interchain affinity or polypeptide degradation or turnover rate.

“Allelic variant” refers to other forms of prost 07 polynucleotides.
Alleles result from mutations, i.e. changes in the polynucleotide sequence, and are generally altered mRs that may or may not have altered structure or function.
Produces NA or polypeptide. Either given gene has nothing,
It can have one or many allelic forms. The usual mutational changes that give rise to alleles are generally ascribed to natural deletions, additions or substitutions of nucleotides. Each of these types of variations can occur in a given arrangement, alone or in combination with one another, or one or more times.

“Derivative” refers to an amino acid that is not normally present in a chemical modification, such as ubiquitination, labeling (eg, by radionuclide, various enzymatic modifications), pegylated (derivatized with polyethylene glycol) or human protein. Refers to a polynucleotide or polypeptide derived from naturally occurring prost 07 or PROST 07, respectively, for example by inserting or substituting ornithine (or substituting, for example, a nucleotide encoding an amino acid).

“Deletion” is defined as a change in a polynucleotide or amino acid sequence that is absent of one or more polynucleotide or amino acid residues, respectively. An "insertion" or "addition" is a change in a polynucleotide or acid sequence resulting from the addition of one or more polynucleotide or amino acid residues, respectively, as compared to a naturally occurring polynucleotide or amino acid sequence. . "Substitution" results from the replacement of one or more polynucleotides or amino acids by different polynucleotides or amino acids, respectively.

Preferably, the amino acid substitution is the substitution of one amino acid with another amino acid having similar structural and / or chemical properties, for example leucine with isoleucine or valine, asparagine with glutamic acid or threonine with serine, That is, it is due to a conservative amino acid substitution. Insertions or deletions typically range from about 1 to 5 amino acids. The variation is determined experimentally by systematically producing amino acid insertions, deletions or substitutions in the polypeptide using recombinant DNA techniques, and assaying the resulting recombinant variants for activity. obtain.

A “fragment” is a polypeptide having an amino acid sequence which is exactly the same as part, but not all, of the amino acid sequence of the PROST 07 polypeptide and variants or derivatives thereof. A polypeptide "fragment", "portion" or "segment" is at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9-13 amino acids and in various embodiments at least It is an amino acid residue with a length of 17 or more amino acids.

“Recombinant” or “recombinant DNA molecule” refers to a polynucleotide sequence that does not occur in nature or is produced by the artificial combination of two separate segments of sequence. By "recombinantly produced" is meant an artificial combination accomplished by chemical synthetic means, or by artificial manipulation of isolated segments of polynucleotides, such as genetic construction techniques. Such manipulations are usually performed to replace one codon with a redundant codon that encodes the same or a conservative amino acid, and is typically performed by introducing or removing a sequence recognition site. Be seen.

On the other hand, it is for ligation with a polynucleotide segment having the desired function to produce a single gene entity comprising the desired combination of functions not found in the usual natural forms. Done. Restriction enzyme recognition sites, regulatory sequences, control sequences or other useful features may be incorporated by design. "Recombinant DNA molecule" includes cloning and expression vectors. "Recombinant" can also refer to a polynucleotide that encodes a polypeptide and is prepared using recombinant DNA techniques.

“Isolated” means altered “by the hand of man” from its natural state, ie, if it is present in nature, it has been altered or removed from its original environment. ing. For example, a polynucleotide or polypeptide naturally occurring in a living animal in its natural state is not "isolated", but the same polynucleotide or polypeptide isolated from its naturally occurring coexisting material is , As the term is used herein, is "isolated".

For example, with regard to a polynucleotide, the term “isolated” means that it is separated from its naturally occurring chromosome and cell. Polynucleotides and polypeptides are compositions, such as media formulations or solutions for the introduction of the polynucleotides or polypeptides into cells, not naturally occurring compositions, compositions for chemical or enzymatic reactions, or It can be present in solution and can remain as an isolated polynucleotide or polypeptide within the meaning of the term as used herein.

“Substantially simple” and “substantially homologous” are used interchangeably and describe a PROST 07 polypeptide or fragment thereof, or a polynucleotide segment that encodes it, where Such a polypeptide or polynucleotide is separated from the components that naturally accompany it .. A PROST 07 polypeptide or fragment thereof, or a DNA segment that encodes it, is a natural segment with which it is naturally associated. When separated from contaminants, it is substantially free of naturally relevant components.

Thus, a polypeptide that is chemically synthesized, or synthesized in a cellular system different from the cell from which it naturally originates, has substantially no naturally associated components. Let's do it. Similarly, a polynucleotide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will have substantially no naturally associated components.

“Homologous” when used to describe a polynucleotide, means that at least 70% of the two polynucleotides, or planned sequences thereof, are optionally aligned and compared. Nucleotides, usually about 75-99% and more preferably at least about 99% nucleotides, are shown to be identical, with appropriate nucleotide insertions or deletions.

“Polymerase chain reaction” or “PCR” means that a particular piece of DNA is
Reference is made to a method of amplification as described in U.S. Pat. No. 4,683,195 issued date. In general, sequence information from the ends of the polypeptide fragment of interest or otherwise must be available in order for the oligonucleotide primers to be designed; the primers are oriented towards each other and amplified It will be identical or similar in sequence to the opposite strand of the template. Two
The nucleotides at the 5'end of the seed primer are co-located with the ends of the amplified material. PCR is a specific DNA sequence from total genomic DNA, cD transcribed from total cellular RNA
It may be used to amplify NA, plasmid sequences, etc. (generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51, 263, 1987, Erich, ed. PC.
R Technology, Stackton Press, NY, 1989).

“Stringency” is typically in the range of about Tm (melting temperature) −5 ° C. (65 ° C. below the Tm of the probe) to about 20 ° C.-25 ° C. (25 ° C. below Tm). To do. As will be appreciated by those in the art, stringent hybridization is used to identify or detect identical polynucleotide sequences, or to identify or detect similar or related polynucleotide sequences. obtain. As used herein, the term "stringent conditions" allows hybridization to occur at least between sequences.
It is meant to be present only if 95% and preferably at least 97% identity is present.

“Hybridization” as used herein will include “any method by which a polynucleotide strand is linked to a complementary strand through base pairing” (Coombs, J., Dictionary of Biotechnology, Stockton Press, New
York, NY, 1994).

“Therapeutically effective dose” refers to an amount of a polypeptide or its antibody, antagonist, or inhibitor, such as an antisense molecule and a ribozyme, that ameliorates the symptoms or condition of a disease state. The therapeutic efficacy and toxicity of such compounds, such as ED ₅₀ (the dose therapeutically effective in 50% of the population) and LD ₅₀ (the dose that is lethal in 50% of the population) are standard in cell cultures or experimental animals. Can be determined by the pharmaceutical method of The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio ED ₅₀ / LD ₅₀ . “Treat” or “treatment”, as used herein, includes disease states in which the disease state is associated with prostate tumor growth and the patient has a reduced need for PROST 07. , Treatment of disease states in human patients.

Specific Description of the Invention: The present invention relates to novel PROST 07 polypeptides, prost 07 polynucleotides and antibodies directed against PROST 07 polypeptides, among others, as described in more detail below. In particular, the present invention relates to novel PROST 07 polypeptides and their PROS.
With respect to the polynucleotide encoding the T 07 polypeptide, and particularly, FIG.
PROST 07 having the amino acid sequence shown in SEQ ID NO: 2 and FIG. 1 (SEQ ID NO: 1)
), Prost 07 having the polynucleotide sequence shown in FIG. The present invention also provides
Includes PROST 07 variants.

Preferred PROST 07 variants have at least 70% similarity (preferably at least 70% identity) and more preferably at least 90% to the polypeptide sequence shown in Figure 2 (SEQ ID NO: 2). % Variants (more preferably at least 90% identity), and even more preferably at least 95% similarity (even more preferably at least 95% identity), and also Also included is a portion of such a polypeptide having such a portion of the polypeptide generally comprising at least 30 amino acids and more preferably at least 50 amino acids.

The coding sequence for the predicted active form of the PROST 07 polypeptide begins 31 base pairs from the 5'end of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1).
The first 30 base pairs encode a predicted pro-sequence and part of the signal sequence.

The present invention is based, in part, on the identification of catalytic trimers with serine protease characteristics that can be identified in the PROST 07 polypeptide sequence. This ternary is composed of histidine, aspartic acid and serine residues (positions 51, 96 and 187, respectively in Figure 2 (SEQ ID NO: 2)). The amino acids surrounding those residues (underlined in Figure 2) are sometimes conserved in serine proteases. The present invention is also based in part on the structural homology shown in Figure 3 between PROST 07 and another member of the serine protease family, EMPS 1. PROST
The amino acid sequence of 07 is approximately 77.8% identical to EMSP1.

The present invention also relates to the expression of its PROST 07 in a prostate tissue library and the overexpression of PROST 07 in a prostate tumor library, as shown by its PR.
It is based in part on the expression profile of OST 07. It is also found in the analysis of mRNA expression in tissue samples from normal and tumor tissues by Northern blot and PCR-based Taqman analysis. Both analysis methods code PROST 07
It has been shown that mRNA is expressed in prostate tissue but not in other tumor tissue samples or other normal tissue samples. However, recent studies have shown the presence of PROST 07 in tissue samples from patients with late stage ovarian cancer (Obiezu, et al., Clin. Biochem. 33: 234, 2000), which indicates that this type of cancer It also suggests that it can be treated with PROST 07-based therapeutic agents.

The present invention is also based, in part, on functional studies demonstrating the effect of reduced levels of PROST 07-encoding mRNA on prostate tumor cell growth in culture. This was shown in a prostate tumor cell line, LNCaP, using antisense oligonucleotides to reduce prost 07 mRNA expression. Two antisense oligonucleotide reagents were selected that reduced mRNA levels for prost 07 when administered to cells in the presence of lipids.

Administration of those antisense oligonucleotide reagents through LNCaP cells inhibited proliferation of cells in a dose-dependent manner. At the same dose range, a control oligonucleotide mix composed of a set of random 23-mer oligonucleotides had no effect on cell proliferation. Next, those experiments showed that inhibition of PROST 07 expression was
It was shown that OST 07 affected the proliferation of normally expressed tumor cell lines. The present invention is further based on studies demonstrating the ability of antibodies directed against PROST 07 to neutralize the activity of proteins and inhibit tumor cell migration and invasion in vivo.

Polynucleotides : One aspect of the invention provides an isolated polynucleotide encoding a PROST 07 polypeptide having the deduced amino acid sequence of Figure 2 (SEQ ID NO: 2).

Using the information provided herein, eg, the polynucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), the polynucleotide of the invention encoding a PROST 07 polypeptide can be labeled using standard cloning and screening methods. , Using, for example, those methods for the cloning of cDNA using mRNA from cells of human tissue as starting material. The polynucleotide sequence in Figure 1 (SEQ ID NO: 1) is
It was found in a cDNA cloning obtained from human prostate tissue. prost 07 is Incy
It was identified as a gene expressed in the prostate by searching the te's LifeSeq gene expression database.

The mRNA encoding PROST 07 is a cDNA of Incyte's prostate tumor cells with LifeSeq “transcriptor imaging” features and multiple copies of more than one per library.
It was expressed using the study parameters that allowed the selection of new genes expressed in the library. This set of genes was evaluated by electronic Northern analysis to determine their distribution in other prostate tissue libraries and all other tissue libraries in the database. Clone 1651585 was identified in the library PROST 12 of LIFESEQ version 4.1 SEP96 (Beta) and was shown by electronic northern analysis to cluster with other clones containing prost 07 polynucleotides in a prostate specific manner.

This prostate-specific expression has been consistently found in recent versions of the LifeSeg database. All clones encoding fragments of PROST 07 were assembled into contiguous sequences and the assembled mRNA sequences were evaluated using the Genetics Computer Group (GCG Wisconsin Package) gene assembly program. An open reading frame encoding the protein was identified, and the predicted protein was shown to be homologous to a family of serine proteases. An additional clone encoding a fragment of PROST 07 is LifeSeq
Incyte containing the most complete 5'sequence in the database, eg clone 3357511
Acquired from.

In order to obtain additional sequences at the 5'end of the prost 07 polynucleotide sequence, five RACE PCR methods were utilized (using an amplification kit obtained from Clontech) to
An additional cDNA product encoding 07 was generated. Samples of cDNA generated from prostate tissue RNA from Clontech Laboratories were used as templates for their synthesis. The PCR products were cloned and the selected clones were sequenced. The sequences include their 5'terminal sequences to further include the predicted full-length coding sequence for the active form of PROST 07, and part of the putative pro-sequence and putative signal sequence. Extended. Additional sequence information is PROST 0
It was obtained from a genomic clone encoding 7 and confirmed the sequence determined by the RACE PCR method. Sequences encompassing the three regions of the transcript were obtained by sequencing Incyte clone 181800 encompassing the complete three regions of the mRNA.

The polynucleotides of the present invention may be obtained in the form of RNA, such as mRNA, or in the form of DNA, such as by cloning, or by chemical synthesis techniques, or combinations thereof, or described herein. It can exist in the form of cDNA and genomic DNA produced by the method. The DNA may be double-stranded or single-stranded.
Single-stranded DNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the antisense strand. The sequence encoding the polypeptide may be identical to the coding sequence of the polynucleotide shown in Figure 1 (SEQ ID NO: 1). It can also be a polynucleotide having a different sequence, which codes for the polypeptide of Figure 2 (SEQ ID NO: 2) as a result of the redundancy (degeneracy) of the genetic code.

Polynucleotides of the invention encoding the polypeptide of FIG. 2 (SEQ ID NO: 2) include, but are not limited to, the coding sequences for the following polypeptides themselves: Coding sequences for and additional coding sequences, such as those sequences encoding leader or secretory sequences, such as pre- or pro- or prepro-protein sequences; additional non-coding sequences, such as introns and non-coding 5'and 3 '. Sequences, eg transcribed, non-translated sequences that play a role in transcription, mRNA processing (eg splicing and polyadenylation signals)
, Or an additional coding sequence that encodes additional amino acids, eg, a coding sequence for a polypeptide with or without the additional coding sequences described above, along with those sequences that provide additional functionality.

Thus, for example, the polypeptide may be fused to a marker sequence, eg a peptide, which facilitates purification of the fused polypeptide. In a preferred embodiment of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as
Labels supplied to the pTrchisB vector (Invitrogen, Carisbad, CA), many of which are commercially available. For example, Geniz. (Proc. Natl. Acad. Sci.
USA 86: 821-824, 1989), hexa-histidine provides convenient purification of the fusion protein. HA labeling is performed by Wilson et al. (Cell 37: 767, 1984.
) Corresponds to an epitope derived from the influenza hemagglutinin protein described by

A polynucleotide may be one polypeptide which is said polypeptide, and additional amino or carboxyl terminal amino acids, or the internal amino acids of the polypeptide (
Active form having more than one polypeptide chain). Such sequences may play a role in the processing of the polypeptide from the precursor to its final form, among others, may facilitate polypeptide trafficking, and extend or shorten the half-life of the polypeptide. Or can facilitate manipulation of the polypeptide for assay or production. Generally, as is often the case, additional amino acids can be removed from the polypeptide by cellular enzymes.

A precursor polypeptide having the final form of the polypeptide fused to one or more prosequences can be an inactive form of the polypeptide. If the pro sequence is removed,
Such inactive precursors are generally activated. Some or all of the prosequences may be removed prior to activation. Generally, such precursors are called propolypeptides.

The present invention further relates to variants of the polynucleotides described above which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 2 (SEQ ID NO: 2). The variant of the polynucleotide may be a naturally occurring variant, eg a naturally occurring allelic variant, or it may be a variant known to occur in nature. Such non-naturally occurring variants of the polynucleotide may be produced by mutagenesis techniques such as those applied to the polynucleotide, cell or organism.

Variants in this regard are variants that differ from the aforementioned polynucleotides by virtue of polynucleotide substitutions, deletions or additions. Substitutions, deletions or additions can include one or more polynucleotides. Variants can be altered in coding or non-coding regions, or in both regions. Alteration of the coding region can produce conservative or non-conservative amino acid substitutions, deletions or additions. Among them, a particularly preferred embodiment of the present invention in this regard is a polynucleotide encoding a polypeptide having the amino acid sequence of PROST 07 shown in FIG. 2 (SEQ ID NO: 2), variants, analogs, derivatives and fragments thereof, and Fragments of the variants, analogs and derivatives.

Even more particularly preferred in this regard are several, several, 5-10, 1
~ 5, 1-3, 2, 1 amino acid residues are substituted, deleted or added in any combination, or any amino acid is also substituted, deleted or added 2 is a PROST 07 variant, analog, derivative and fragment having the amino acid sequence of the PROST 07 polypeptide of FIG. 2 (SEQ ID NO: 2) and a polynucleotide encoding the variant, analog and derivative of said fragment. .. Especially preferred among them are silent substitutions, additions and deletions, which do not alter the properties and activities of the PROST 07 polypeptide. Also particularly preferred in this regard are conservative substitutions. Most preferred is a polynucleotide encoding a polypeptide having the amino acid sequence of Figure 2 (SEQ ID NO: 2) without substitutions.

A further preferred embodiment of the present invention is at least 70 relative to a polypeptide encoding a PROST 07 polypeptide having the amino acid sequence shown in Figure 2 (SEQ ID NO: 2).
% Polynucleotides that are% identical, and polynucleotides that are complementary to such polynucleotides. On the other hand, most preferred are polynucleotides that comprise a region that is at least 80% identical to a polypeptide encoding a PROST 07 polypeptide, and polynucleotides that are complementary thereto.

In this regard, polynucleotides with at least 90% identity are particularly preferred, and among those particularly preferred polynucleotides, those with at least 95% identity are particularly preferred. Furthermore, those with at least 97% identity are highly preferred, and among them those with at least 98% and at least 99% identity are very particularly preferred, and at least 99%. Those having the same identity are more preferred.

A particularly preferred embodiment in this respect is a polynucleotide encoding a polypeptide which retains substantially the same biological activity as the polypeptide encoded by the polynucleotide sequence of Figure 1 (SEQ ID NO: 1). . The invention further relates to polynucleotides that hybridize to the above sequences.
In this regard, the invention particularly relates to polynucleotides that hybridize to the above polynucleotides under stringent conditions.

With respect to the polynucleotide assays of the present invention, as discussed further herein, the polynucleotides of the present invention, as discussed above, encode full-length cDNA and genomic clones encoding PROST 07. To isolate, and pros
It can be used as a hybridization probe for cDNA and genomic DNA to isolate cDNA and genomic clones of other genes with high sequence similarity to the T07 gene. Such a probe will generally comprise at least 15 bases. Preferably, such a probe will have at least 30 bases, and can have at least 50 bases.

For example, the coding region of the PROST 07 gene can be isolated by screening with known DNA sequences to synthesize an oligonucleotide probe. Next, a labeled oligonucleotide having a sequence complementary to the sequence of the polynucleotide of the present invention is a library of human cDNA, genomic DNA or mRNA screened and the members of the library to which the probe hybridizes. Used to determine.

In summary, the polynucleotides of the present invention include polypeptides, polypeptides and leader sequences (which may be referred to as prepolypeptides), precursors of polypeptides having one or more prosequences that are not leader sequences of prepolypeptides. Generally removed during the processing step of producing the active form of the body or polypeptide,
A prepropeptide that is a precursor to a propolypeptide can be encoded that has a leader sequence and one or more prosequences.

The invention also includes, among other things, polynucleotides that encode polypeptide fragments, polypeptide fragments, particularly polynucleotides that hybridize to polynucleotides that encode those fragments that hybridize under stringent conditions, and polypeptide fragments. To a polynucleotide for amplifying a polynucleotide encoding, for example PCR primers. For those, preferred polynucleotides are those polynucleotides that correspond to preferred polypeptide fragments, as discussed below.

Polypeptides : The present invention further provides PROS having the deduced amino acid sequence of Figure 2 (SEQ ID NO: 2).
T07 polypeptide. The invention also relates to fragments, analogs and derivatives of those polypeptides. The terms, fragments, derivatives and analogs when referring to the polypeptide of Figure 2 (SEQ ID NO: 2) mean a polypeptide that retains substantially the same biological activity as such polypeptide. Thus, analogs include propolypeptides that can be activated by cleavage of the propolypeptide moiety to produce an active polypeptide of the invention. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In a preferred embodiment it is a recombinant polypeptide.

Fragments, derivatives or analogs of the polypeptide of FIG. 2 (SEQ ID NO: 2) are (
i) one or more amino acid residues are replaced by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such replaced amino acid residues are in the genetic code Or not, or (ii) one or more amino acid residues containing a substituent, or (iii) the polypeptide is another compound, for example Those fused with a compound to increase the half-life of the peptide (eg polyethylene glycol), or (iV) an additional amino acid polypeptide, eg a leader or secretory sequence, or a sequence used for purification of the polypeptide Can be fused to. Such fragments, derivatives and analogs are believed to be within the skill of those in the art from the techniques herein.

A particularly preferred embodiment of the invention in this regard is shown in Figure 2 (SEQ ID NO: 2).
A polypeptide having the amino acid sequence of PROST 02, its variants, derivatives and fragments, and variants, analogs and derivatives of said fragments. Preferred variants are those that differ from the control by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide with another amino acid of similar characteristics. Typically, the following conservative substitutions are found: Substitutions between the aliphatic amino acids Ala, Val, Leu and Ile,
Exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and aromatic residues Phe and T
Substitution between yr.

Some, a few, 5-10, 1-5, 1-3, 2, 1, amino acid residues are substituted, deleted or added in any combination. Has been done,
Or variants, analogs, derivatives and fragments having the amino acid residues of the PROST 07 polypeptide of FIG. Particularly preferred in this regard. Especially preferred among them are silent substitutions, additions and deletions, which do not alter the properties and activities of the PROST 07 polypeptide. Particularly preferred in this regard are conservative substitutions. Most preferred is a polypeptide having the amino acid sequence of Figure 2 (SEQ ID NO: 2) without substitutions.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form and are preferably purified to homogeneity. The polypeptide of the present invention also includes the polypeptide of FIG. 2 (rupture number 2), and FIG.
At least 70% similarity (preferably at least 70% identity) and more preferably at least 90% analog (more preferably at least 90% identity) to the polypeptide of (SEQ ID NO: 2), and Even more preferably it includes polypeptides having at least 95% similarity (even more preferably at least 95% identity), and also generally at least 30 amino acids and more preferably at least 50 amino acids. Includes portions of such polypeptides having such portions of the polypeptide including.

As known in the art, “similarity” between two polypeptides refers to
It is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. A fragment or portion of a polypeptide of the invention may be used to produce its corresponding full-length polypeptide by peptide synthesis; thus, a fragment may be used to produce a full-length polypeptide. Can be used as an intermediate.

Fragments : Also preferred embodiments of this aspect of the invention are fragments of PROST 07, most particularly the fragment of PROST 07 of FIG. 2 (SEQ ID NO: 2) and FIG. 2 (SEQ ID NO: 2).
A) PROST 07 variants and derivatives thereof. In this regard, a fragment is a polypeptide having an amino acid sequence which is exactly the same as part, but not all, of the amino acid sequence of said PROST 07 polypeptide and variants or derivatives thereof.

Such fragments may be “free-standing,” ie, they may be a part of, or not fused to, another amino acid or polypeptide, or they may have a part or region. It can be included within the large polypeptide that forms it. When included within a large polypeptide, the currently discussed fragments most preferably form a single contiguous region. However, some fragments may be contained within a single large polypeptide. For example, certain preferred embodiments have a heterologous pre- and propolypeptide region designed for expression in a host and fused to the amino terminus of the PROST 07 fragment, and an additional region fused to the carboxyl terminus of the fragment. It relates to fragments of the PROST 07 polypeptides of the invention which are included within the precursor polypeptide. Thus, a fragment in one aspect of the meaning contemplated by the invention refers to a portion of a fusion polypeptide or fusion protein derived from PROST 07.

As representative examples of polypeptide fragments of the present invention, those fragments having about 25 to about 145 amino acids can be mentioned. As used herein, the term "about" refers to a range that is specifically referred to, and some, a few, a few, 5,4,3,2 or 1 amino acid at any or both limits, more or less. Including a small range. For example, about 145 amino acids means 25 ± some, a few, 5,4,3,2 or 1 amino acid to 145 ± some, a few, 5 amino acids.
, 4, 3, 2 or 1 amino acid residue polypeptide fragment, ie 25-some amino acids as a wide range ~ 145 + some amino acids ~ 25 + several amino acids as a narrow range ~ 145- It is a range of some amino acids.

In this regard, the recited range ± 5 amino acids in either or both extremes is highly preferred. Very particular preference is given to ranges of amino acids as long as the limits quoted ± 3 in any or both extremes. Particularly preferred are the recited ranges (without additions or deletions) ± 1 amino acid at either or both limits. In this regard, about 25 to about 145 amino acids are most preferred.

A particularly preferred fragment of the present invention is a truncation mutant of PROST 07. Cleavage variants include PROST 07 of Figure 2 (SEQ ID NO: 2), or variants or derivatives thereof, except for the following: the amino terminus of the sequence shown in Figure 2 (SEQ ID NO: 2). A contiguous series of residue deletions (ie, contiguous regions, parts or portions),
Or at the carboxyl terminus, or as in the double truncation variant, a deletion of two consecutive series of residues, a deletion involving the amino terminus and a series of consecutive residues including the deletion involving the carboxyl terminus . Fragments having the size ranges indicated above are also preferred embodiments of cleavage fragments which are particularly preferred between fragments in general.

Particularly preferred in this aspect of the invention are fragments characterized by the biological and / or immunological properties of PROST 07. A fragment containing the predicted active domain of PROST 07, which includes amino acids 11-234, or amino acids 4-10
Most preferred are fragments containing the putative proprotein sequence of PROST 07, including

Certain preferred regions for them are shown in Figure 2 (SEQ ID NO: 2) and
It includes, but is not limited to, regions of the above type identified by analysis of the amino acid sequence set forth in SEQ ID NO: 2). As shown in FIG. 2 (SEQ ID NO: 2), such preferred regions are amino acids 47-52, 96-99 of PROST 07 and
Includes the catalytic ternary found in serine proteases, including 181-192.

Highly preferred fragments in this regard are those fragments which comprise a region of PROST 07 which combine several structural features, such as those set out above. In this regard, the active domain defined by the 11-234 amino acid residues of Figure 2 (SEQ ID NO: 2), which is characteristic of the serine protease family of proteins, is a particularly highly preferred region. Such regions may be contained within large polypeptides, as discussed above, or may themselves be preferred fragments of the invention. The term "about" as used in this text generally has the meaning given above for fragments.

Further preferred regions are those regions which mediate the activity of PROST 07. Most preferred in this regard are fragments of PROST 07 having a chemical, biological or other activity, such as those having a similar activity, or an improved activity, or a reduced undesired activity. In this regard, it includes regions that are homologous in sequence, or in position, or in both sequence and position, to the active region of other proteins of the serine protease family, including related polypeptides, such as PROST 07. Fragments are highly preferred.

Vectors, Host Cells and Expression Systems : The present invention also includes vectors comprising the polynucleotides of the invention, host cells genetically constructed with the vectors of the invention, and polypeptides of the invention by recombinant techniques. Regarding generation. Such techniques are described in Sambrook et al., Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY 1989, and Ausubel, FM, etc., Current Protocols in Molecular Biology, John Wil
ey & New York, N, Y., 1989.

Host cells can be genetically constructed to incorporate the polynucleotides of the invention and express the polypeptide. For example, polynucleotides can be introduced into host cells using the well known techniques of infection, transduction, transfection, transvection and transformation. The polynucleotide may be introduced alone or in combination with other polynucleotides. Such other polynucleotides may be independently introduced, co-introduced, or introduced ligated to the polynucleotides of the invention.

Thus, for example, a polynucleotide of the invention is transduced into a host cell with another polynucleotide that encodes a selectable marker, eg, using standard techniques for co-transfection and selection in mammalian cells. Can be perfected. In this case, the polynucleotide will generally be stably integrated into the host cell genome.

On the other hand, the polynucleotide may be linked to a vector containing a selectable marker for growth in host cells. The vector construct can be introduced into host cells by the techniques described above. Generally, plasmid vectors are introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or a complex with charged lipids. Electroporation can also be used to introduce a polynucleotide into a host. If the vector is a virus, it can be packaged in vitro or introduced into a packaging cell and the packaged virus transduced into the cell.

In this respect of the invention, a wide variety of techniques suitable for making polynucleotides and introducing polynucleotides into cells are well known and routine to those of skill in the art. is there. Such techniques are reviewed in Sanbrook et al., Cited above, which exemplifies the numerous laboratory manuals that describe them in detail. According to this aspect of the invention, the vector may be, for example, a plasmid vector, a single-stranded or double-stranded phage vector, a single-stranded or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably RNA, by well known techniques for introducing DNA and RNA into cells.

Vectors are introduced into cells as packaged or encapsulated virus, in the case of phage and viral vectors, and preferably by well known techniques for infection and transduction. Viral vectors can be replication competent or replication defective. In the latter case, viral propagation generally will only exist in complementing the host cell.

In one aspect, preferred vectors are those for expression of the polynucleotides and polypeptides of the invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operably linked to the polynucleotide to be expressed. Appropriate trans-acting factors are supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon introduction into the host.

In a preferred embodiment in this regard, the vector provides for specific expression.
Such specific expression may be inducible expression or expression only in certain types of cells, or inducible and cell specific expression. Particularly preferred among inducible vectors are those that are inducible for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable for this aspect of the invention, such as constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and can be routinely used by those of skill in the art.

The constructed host cells can be cultured in conventional nutrient media which can be suitably modified to, among other things, activate the promoter, select for transformants, or amplify the gene. Culture conditions traditionally used by the host cell selected for expression, such as temperature, pH and the like, are generally for expression of the polypeptides of the invention, as will be apparent to those of skill in the art. Would be appropriate.

A wide variety of expression vectors can be used to express the polypeptides of the invention. Suitable vectors include vectors derived from chromosomes, episomes and viruses such as bacterial plasmids, bacteriophage, yeast episomes, enzyme chromosomal elements, viruses such as baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, pox pox virus, pseudo- Includes vectors derived from sexual rabies virus and retroviruses, and vectors derived from combinations thereof, such as plasmids and bacteriophage genetic elements such as those vectors derived from cosmids and phagemids, all of which It can be used for expression according to this aspect. Generally, any vector suitable for maintaining, propagating or expressing a polynucleotide for expressing a polypeptide in a host can be used for expression in this regard. A preferred vector is a baculovirus-derived vector.

The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and conventional techniques. Generally, the DNA sequence for expression is cleaved with the DNA sequence and the expression vector by one or more restriction endonucleases, and the T4 DN
It is ligated into the expression vector by ligating together the restriction fragments with A ligase. Restrictions and methods for ligation that can be used for this purpose are well known and routine to those of skill in the art. In this regard, and suitable methods for constructing expression vectors using other techniques well known and routine to those of skill in the art are detailed in Sambrook et al., Which is incorporated herein by reference. ing.

The DNA sequence in the expression vector is operably linked to appropriate expression control sequences, such as a promoter to direct mRNA transcription. Representative of such promoters are the phage random P _L promoter, E. Coli lac, trp and tac promoter, SV40 early and late promoters, and may for naming few promoters known promoter retroviral LTR Include. Many promoters not mentioned are suitable, well known for use in this aspect of the invention, and can be readily used by those skilled in the art in the manner illustrated by the discussion and examples herein. It will be understood.

In general, the expression construct will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct will include a translation initiation AUG at the start, and a stop codon approximately located at the end of the polypeptide to be translated. In addition, the construct can include control regions that regulate and effect expression. In general, according to many commonly practiced methods, such regions, such as repressor binding sites and enhancers, among others, will operate by controlling transcription.

Vectors for propagation and expression will generally include selectable markers. Such markers are also suitable for amplification, or the vector can include additional markers for this purpose. In this regard, expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers are dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and E. It includes the tetracycline, theomycin, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.

Vectors containing suitable DNA sequences, as described elsewhere in this specification, and suitable polo motors, as well as other suitable control sequences, are provided for expression at the bottom of the desired polypeptide. It may be introduced into a suitable host using a variety of suitable and well known techniques. Representative examples of suitable hosts are bacterial cells such as E. E. coli, Streptomyces and Salmonella
typhimurium) cells, fungal cells, eg yeast cells; insect cells, eg Drosophila
52 and Spodoptera Sf9 cells, animal cells such as CHO, COS and Bowes melanoma cells; and plant cells, preferably insect cells BT1-TN-5B1-4. Hosts for a wide variety of expression constructs are well known, and those of skill in the art are, according to this aspect of the invention, able to readily select a host for expressing a polypeptide.

A variety of mammalian cell culture systems can be used for expression. Examples of mammalian expression systems include the COS-7 system of monkey kidney fibroblasts (Gluzman et al.,
Cell 23: 175, 1991). Other cell lines capable of expressing compatible vectors include, for example, C127, 3T3, CHO, Hela, human kidney 293 and BHK cell lines. Many viral-based expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the polynucleotide sequence encoding PROST07 can be ligated into the adenovirus transcription / translation complex consisting of the late promoter and ternary leader sequence. Insertions in the nonessential E1 or E3 regions of the viral genome will result in a viable virus allowing expression of PROST 07 in infected host cells (Logon and Shenk, Proc. Natl. Acad. Sci. USA 81
: 3656-59, 1984). In addition, transcription enhancers such as Rous sarcoma virus (R
VS) enhancers can be used to increase expression in mammalian host cells.

More specifically, the present invention also includes a recombinant construct, eg an expression construct, comprising one or more sequences as described above. The construct comprises a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted. The sequences can be inserted in the forward or reverse orientation. In a preferred embodiment in this regard, the construct further comprises regulatory sequences, such as a promoter operably linked to said sequences. Many suitable vectors and promoters include
There are many commercially available vectors known to those of skill in the art and suitable for use in the present invention.

The following vectors, which are commercially available, are provided by way of example. Vectors suitable for use in bacteria include: pQE70, pQE60 and pQE-9, available from Qiagen USA (Valencia, CA), pQE70; Stratagene (La Jolla, CA), p
BS vector, Phagescript (trademark) vector, Bluescript (trademark) vector, pNH
8A, pNH16a, pNH18A, pNH46A; and ptrc99a, pk223-3, pkk233-3, pDR540, pRIT5 available from Pharmacia Biotech (Piscataway, NJ). Most preferred is the pTrcHisB vector available from Introgen. A preferred eukaryotic vector is Strategene
PWLNEO, pSV2CAT, pOG44, PXTI and pSG available from Pharmacia Biotech
PSVK3, pBPV, pMSG and pSVL available from

Most preferred is the pCineo vector available from Promega. Those vectors are listed by a number of well known vectors available in the art for use in accordance with this aspect of the invention. For example, it is understood that any other plasmid or vector suitable for introducing, maintaining, propagating or expressing the polynucleotide or polypeptide of the invention in the host may be used in this aspect of the invention. Let's do it.

The promoter region is a chloramphenicol acetyl transferase (“cat ") Can be selected from any desired gene using a vector containing the transcription unit. As is well known, the introduction of a promoter-comprising fragment into the vector at a restriction site upstream of the cat gene is standard.
It results in the production of CAT activity that can be detected by the CAT assay. Vectors that are suitable for this purpose are well known and readily available. Two such vectors are pKK232-8 and pCM7. Therefore, the promoter for expressing the polynucleotide of the present invention includes not only well-known and easily available promoters but also promoters easily obtained by the above-mentioned technique using a reporter gene.

Known cell promoters suitable for expression of the polynucleotides and polypeptides of the present invention are E. coli. E. coli lact and lacZ promoters, T3 and T7 promoters, T
5 tac promoter, lambda PR, PL promoter, trp promoter and trc hybrid promoter (due to trp and lac promoter). Suitable known eukaryotic promoters in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs such as the Teus sarcoma virus (“RSV”) and the metallothionein promoters such as the mouse metallothionein. -1 promoter.

Selection of the appropriate vector and promoter for expression in a host cell is a well known method and necessary techniques for expression vector construction, the introduction of the vector into a host, and the host Expression is within the normal range of one of ordinary skill in the art. In general, recombinant expression vectors allow the isolation of vector-containing cells after exposure to an origin of replication, a promoter derived from a highly expressed gene to direct the transcription of downstream structural sequences, and the vector. Will include a selectable marker.

The present invention also relates to host cells containing the above structures. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. The polypeptide may be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. A cell-free translation system is also of the present invention.
RNA derived from DNA constructs can also be used to produce such proteins. Suitable cloning and expression vectors for use with eukaryotes and prokaryotes are described by Sambrook et al., Cited herein.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are elements of DNA, usually about 10-300 bp, that act to enhance the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the replication origin at bp 100-270, the cytomegalovirus early promoter enhancer, the polymer enhancer on the late side of the replication origin, and the adenovirus enhancer.

A polynucleotide of the invention encoding a heterologous structural sequence of a polypeptide of the invention may generally be inserted into the vector using standard techniques so that it is operably linked to a promoter for expression. Connected to. Transcription of a polynucleotide
It will be located on the 5'side of the ribosome binding site. The ribosome binding site will be located on the 5'AUG side that initiates translation of the polypeptide to be expressed. Generally, there will be no other open reading frames starting with the initiation codon, usually the AUG, and being between the ribosome binding site and the initiation AUG. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be polyadenylation and transcription termination signals that are located approximately at the 3'end of the transcribed region.

Appropriate secretory signals may be incorporated into the expressed polypeptide for insertion of the translated protein into the lumen, periplasmic space or extracellular environment of the endoplasmic reticulum. The signals can be endogenous to the polypeptide or they can be heterologous signals. The polypeptide may be expressed in a modified form, for example in the form of a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. Thus, for example, a region of additional amino acids, particularly charged amino acids, is added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or subsequent handling and storage. Can be done.

In addition, specific regions may also be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Among others, the addition of peptide moieties to polypeptides to produce secretion or excretion, improve stability, and facilitate purification is a common technique well known in the art. For example, if large amounts of PROST 07 are required for the production of antibodies, a vector that directs high level expression of the easily purified fusion protein is desired.

Such vectors include, but are not limited to, the following: polyfunctional E. Coli cloning and expression vectors, such as Bluescript ™ (Stratagene), where the sequence encoding prost 07 reads with the amino-terminal Met and the next 7 residues of β-galactosidase to produce a hybrid protein. Can be ligated into the vector in line with the frame); pIN vector (Van H
eede and Shuster, J. Biol. Chem. 264: 5503-5509, 1989) and the like. P
The trcHis vector (Invitrogene, Carisbad, CA) can be used to express foreign polypeptides as a fusion protein containing a polyhistidine (6xHis) tag for rapid purification. Proteins produced by such systems are designed to contain a cleavage site, such as an enterokinase cleavage site, so that
The cloned polypeptide of interest may be deliberately released from the fusion peptide component.

Following transformation of a suitable host strain and growth of the host strain to a suitable cell density, if the selected promoter is inducible, it is suitable means (eg, temperature shift, or to a chemical inducer). Exposure) and the cells are cultured for an additional period of time. The cells are then typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for additional purification. The microbial cells used to express the protein may be any conventional method such as freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents (such methods being well known to those skilled in the art. Can be destroyed by.

PROST 07 polypeptides can be recovered and purified from recombinant cell culture by the following well known methods: ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography. Chromatography, hydrophobic interaction chromatography affinity chromatography, hydroxyapatite chromatography, and cyretin chromatography. Most preferably high performance liquid chromatography ("HPLC") is used for purification.

Well-known techniques for folding proteins can be used to regenerate the active conformation when the polypeptide is denatured during isolation and / or purification. Various other methods of protein purification well known in the art are available from Deutsc
her, M., Methods in Enzymology, Vol. 182, Academic Press, San Diego, 198
2; and Scopes, R., Protein Purification: Principles and Practice Springe
and those methods described in r-Veriag, New York, 1982.

On the other hand, the polypeptides of the present invention can be produced by direct peptide synthesis using solid phase techniques (Stewart et al., Solid-Phase Peptide Synthesis, WH.
Freeman Co., San Francisco, 1969; Merrifield, j., J. Am. Chem. Soc. 85:
2149-2154, 1963). In vitro protein synthesis can be performed using manual techniques or by automated equipment. Applied Biosystems 431A Peptide Synt
It can be achieved using a hesizer (Perkin Elmer, Faster City, Calif) according to the instructions provided by its manufacturer. The various fragments of PROST 07 can be chemically synthesized separately and combined using chemical methods to produce molecules of full length.

The polypeptides of the present invention are produced by recombinant techniques from naturally purified products, products of chemical synthetic methods and from prokaryotic or eukaryotic hosts such as bacteria, yeast, higher plants, insects and mammalian cells. Including living creatures. Depending on the host used in a recombinant production method, the polypeptides of the invention may or may not be glycosylated. In addition, the polypeptides of the present invention may also include methionine residues that are initially modified, in some cases as a result of host-mediated methods.

Uses of PROST 07 Polypeptides and Polynucleotides Encoding them : The prost 07 polynucleotides and PROST 07 polypeptides have various uses, eg
It can be used according to their application using the chemical and biological properties of PROST 07. Additional uses relate to the diagnosis and treatment of cell proliferative disorders, such as prostate cancer.
Those aspects of the invention are further illustrated by the following discussion and further described herein.

The basic principles for use of the polynucleotide and polypeptide sequences of the invention are:
Based in part on the chemical and structural homology between PROST 07 and other serine molecules disclosed herein, and the selective expression of PROST 07 in prostate tissue as compared to other tissues. There is. PROST 07 can be used in the diagnosis and treatment of conditions, disorders or diseases associated with inappropriate growth of prostate cancer. They include but are not limited to cancer and metastatic tumor growth.

The prost 07 polynucleotide sequence can be used as a DNA probe and as a target for antisense and ribozyme therapy or as a template for the production of antisense polynucleotides. PROST 07 polypeptides are used to generate antibodies to PROST 07, which are useful in detecting levels of PROST 07 polypeptides in cells and tissues and in targeting agents to primary and metastatic tumors. Can be done. PROST 07 polypeptides can be used to stimulate an immune response against cells containing PROST 07.

Polynucleotides encoding PROST 07 are useful in diagnostic assays for detecting the level of polynucleotides encoding PROST 07 in cells and tissues. In conditions associated with PROST 07 expression, such as prostate cancer, it is advantageous to control PROST 07 expression or activity. Expression of PROST 07 can be suppressed by administration of antisense oligonucleotides or ribozymes. On the other hand, an antibody that specifically recognizes a region of PROST 07 polypeptide that is responsible for its activity is
It can be administered to treat diseases or conditions associated with 07 activity.

Polynucleotide Assays : The present invention also relates to the use of prost 07-related polynucleotides to detect complementary polynucleotides, eg as diagnostic reagents. Related to disease state
Detection of prost 07 polynucleotides will provide a means for the development of in vitro and in vivo diagnostics that can define the diagnosis of disease or susceptibility to disease resulting from tissue-specific expression of PROST 07.

Individuals carrying mutations in the gene encoding PROST 07 have been identified by various techniques.
It can be detected at the DNA level. Polynucleotide samples for diagnosis are obtained from patient cells, such as blood, urine, saliva, tissue biopsies and necropsy material. Genomic DNA
Can be used directly for detection or can be enzymatically amplified by using PCR prior to analysis (Saiki et al., Nature, 324: 163-166, 1986). RNA
Alternatively, the cDNA can also be used by the same means.

As an example, a PCR complementary to the polynucleotide sequence encoding PROST 07
Primers can be used to identify and analyze prost 07 expression and mutations. For example, deletions and insertions can be detected by changes in the size of the amplified product in comparison to the normal genotype. Point mutations are either radiolabeled prost 07 RNA or, on the other hand, radiolabeled prost 07 antisense DNA.
It can be identified by hybridizing amplified DNA to the sequence. Perfectly matched sequences can be distinguished from mismatched duplicates by differences in RNase A digestion or melting temperatures.

Sequence differences between control genes and genes with mutations also result in direct DN
A sequencing can show. Further, the cloned DNA segment can be used as a probe to detect a particular DNA segment. The sensitivity of such methods can be greatly enhanced by the appropriate use of PCR or another amplification method. For example, sequencing primers are used with double-stranded PCR products, or single-stranded template molecules generated by improved PCR. Sequencing is performed by conventional methods with radiolabeled polynucleotides or by automated sequencing methods with fluorescent labels.

Genetic testing based on DNA sequence differences can be accomplished by detection of alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can even be distinguished on denaturing formamide gradient gels, in which the mobility of different DNA fragments is delayed in the gel at different positions according to their particular melting or partial melting temperatures (eg Myers et al. .
, Science, 230: 1242, 1985).

Sequence changes at specific positions can also be represented by nuclease protection assays, such as RN-ase and S1 protection, or chemical cleavage methods (eg, Catton et al.,.
Proc. Natl. Acad. Sci., USA, 85: 4397-4401, 1985). Thus, the detection of a particular DNA sequence may be accomplished by a variety of methods, such as hybridization, RNase protected chemical cleavage, direct DNA sequencing or the use of restriction (eg,
Restriction fragment length polymorphism in genomic DNA (“RFLP”) and Southern blotting)
Can be achieved by In addition to conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Polypeptide Assays : The invention also includes diagnostic assays, such as determining normal and abnormal levels.
It also relates to quantitative and diagnostic assays for determining the level of PROST 07 polypeptides in cells and tissues, and body fluids. Thus, for example, a diagnostic assay of the invention for detecting overexpression of PROST 07 polypeptide as compared to a normal control tissue sample can be used to detect the presence of hyperplasia, eg prostate cancer. Such diagnostic tests can be used to detect metastatic tumor growth. Assay techniques that can be used to determine the level of a polypeptide, eg, a PROST 07 polypeptide of the invention, in a sample derived from a host are well known to those of skill in the art.

Such assay methods include radioimmunoassay (RIA), competitive binding assay, Western blot analysis and enzyme-linked immunosorbent assay (ELISA).
And fluorescence activated cell sorting (FACS). Of those, ELISA is preferred. The ELISA assay first comprises preparing an antibody specific for PRCST 07, preferably a monoclonal antibody. In addition, reporters that bind monoclonal antibodies are generally prepared. The reporter antibody is linked to a detectable reagent, such as a radioactive, fluorescent or enzymatic reagent. In this example, the horseradish peroxidase enzyme is used.

To perform an ELISA, the sample is removed from the host and incubated on a solid support, such as a polystyrene dish that binds the polypeptide in the sample. Next, any free polypeptide binding sites on the dish are coated by incubating with a non-specific protein such as bovine serum albumin. The monoclonal antibody then binds to any PROST 07 polypeptide that is bound to the polystyrene dish during which the monoclonal antibody is incubated in the dish.

Unbound monoclonal antibody is washed out with buffer. A reporter antibody bound to horseradish peroxidase is placed in the dish and PROST 07
Binding of the reporter antibody to any monoclonal antibody that is bound to. The unbound reporter antibody is then washed. Next, reagents for peroxidase activity, including a colorimetric substrate, are added to the dish. The immobilized peroxidase, which is bound to PROST 07 through the first and the first antibody, produces a colored reaction product. The amount of color progressing over time indicates the PROST 07 polypeptide present in the sample. Quantitative results are typically obtained with a standard curve.

A competition assay can be used in which an antibody specific for PROST 07 is bound to the solid support and labeled PROST 07 and a sample from the host is passed over the solid support, and The amount of label detected that is bound to the solid support correlates to the amount of PROST 07 in the sample. These and other assays are described, inter alia, in the following references: Hampton et al. (Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.
, 1990) and Maddox et al. (J. Exp. Med. 158: 1211, 1983).

Antibodies : The present invention is further referred to herein as PROST 07 antibodies, PROST 07
To an antibody that specifically binds to. The prostate specificity of PROST 07 and its cell surface location characterize outstanding markers for screening, prognosis, follow-up assays and imaging methods. In addition, their features are PROST
07 can be an excellent target for therapeutic methods, such as targeted antibody therapy, immunotherapy and gene therapy. As used herein, the term "specifically binds" refers to the interaction of an antibody and a polypeptide, wherein said interaction is a particular structure on the polypeptide (i.e., Depending on the presence of antigenic determinants or epitopes, in other words, antibodies generally recognize and bind to particular polypeptide structures rather than proteins.

PROST 07 polypeptides, their fragments or derivatives, or analogs thereof,
Or its analogs, or cells expressing them can be used as an immunogen to generate antibodies against it (Harlow, Antibodies, Cold Spring Harbor Pre.
ss NY (1989)). The antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric, single chain and human antibodies, and Fab fragments, or the products of Fab expression libraries. Various methods known in the art can be used for the production of such antibodies and fragments.

Antibodies generated against the peptides corresponding to the sequences of the present invention are obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a non-human. . The antibody so obtained will then bind the polypeptide itself. In this embodiment, even fragments of the polypeptide, even sequences, can be used to generate antibodies that bind the complete native polypeptide. Such antibodies can be used to isolate the polypeptide from tissues expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. For example, the hybridoma technique (
Kohler and Milstein, Nature 256: 495-497, 1975), human B-cell hybridoma technique (Kozbor et al., Immunology Today 4, 72 1983) and EBV-hybridoma technique (Cole et al., In Monoclonal) for producing human monoclonal antibodies. Antibo
ies and Cancer, Alan R. Liss. Inc., 77-96, 1985).

In addition, techniques for the production of "chimeric antibodies", ie splicing of the mouse antibody gene against the human antibody gene, may be used to obtain molecules with the appropriate antigen specificity and biological activity (Morrison. Etc., Proc. Natl. Acad. Sci
USA 81: 6851, 6855, 1984; Neuberger et al., Nature 312: 604-608, 1984; T.
Akeda et al., Nature 314: 452-454, 1985). On the other hand, the techniques described for the production of single chain antibodies (US Pat. No. 4,846,778) can be adapted to produce PROST 07-specific single chains.

Furthermore, "human" antibodies are described in US Pat.
It can be produced using the methods described in 877,397 and 5,569,825. Antibodies can also be generated by in vivo production in a lymphocyte population, or by Oriandi et al. (Proc. Natl. Acad. Sci. USA 86: 3833-3937, 1989) and Wint.
er and Milstein (Nature 349: 293-299, 1991) can be produced by screening a panel of highly specific binding reagents or a recombinant immunoglobulin library.

Antibody fragments which contain a specific binding site for PROST 07 can also be generated. For example, such fragments include F (ab ′) ₂ fragments that can be generated by pepsin digestion of antibody molecules and Fab fragments that can be generated by reducing the disulfide bridges of F (ab ′) ₂ fragments, However, it is not limited thereto. On the other hand, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 256: 1270-1281, 1989).

The amino acid sequence of PROST 07 provided herein can be used to select a specific region of a PROST 07 polypeptide to generate an antibody. As will be appreciated by those in the art, the region or epitope of the PROST 07 polypeptide to which the antibody is directed may vary depending on its intended use. For example, an antibody intended for use in an immunoassay for the detection of membrane-bound PROST 07 on prostate cells should be directed against accessible epitopes on the PROST 07 polypeptide.

Regions of the PROST 07 polypeptide exhibiting immunogenic structure and other regions and domains may be identified by a variety of other methods known in the art, eg Chou-Fasman, Garni.
It can be easily identified using er-Robson or Jameson-Wolf analysis. Fragments containing those residues are particularly suitable in generating anti-PROST 07 antibodies. Particularly useful fragments include, but are not limited to, the sequence SQPWQAALMENELF (SEQ ID NO: 10); the sequence EASLSVRHPEYNRP (SEQ ID NO: 11);
Sequence SKLYDPLYHPSMF (SEQ ID NO: 12); and sequence CKFTEWIEKTVQAS (SEQ ID NO: 13).
The production of polyclonal antibodies against those regions is described in Example 5.

The PROST 07 antibodies of the invention may be particularly useful in diagnostic assays, imaging methods and therapeutic methods for the management of prostate cancer. The present invention provides various immunological assays useful for detecting PROST 07 polypeptides and diagnosing prostate cancer. Such an assay generally comprises one or more PROST 07 antibodies capable of recognizing and binding to the PROST 07 polypeptide. Most preferred antibodies will selectively bind PROST 07 and will not bind (or bind weakly) to non-PROST 07 polypeptides.

The assays include, but are not limited to, various immunological assay types well known in the art: various types of radioimmunoassays, enzyme-linked immunosorbent assays and the like. Things. In addition, immunological imaging methods capable of detecting prostate cancer, eg radioscintigraphic imaging methods using labeled PROST 07 antibody, are also provided by the present invention. Such assays are clinically useful in the detection, monitoring and prognosis of prostate cancer.

The above antibodies may be used to isolate or identify clones expressing the polypeptide,
Alternatively, it can be used to purify a polypeptide of the invention by binding the antibody to a solid support for isolation and / or purification by affinity chromatography. In addition, PROST 07 antibodies can be used to isolate PROST 07 positive cells using cell sorting and purification techniques. In particular, the PROST 07 antibody can be used to isolate prostate cancer cells from xenograft tumor tissue, cultured cells, etc. using antibody-based cell sorting or affinity purification techniques. Another use of the PROST 07 antibody of the invention is PROST 07
The production of anti-idiotypic antibodies that mimic a polypeptide is included.

The presence of such PROST 07-comprising cells in various biological samples such as serum, prostate and other tissue biopsy specimens can be detected by PROST 07 antibodies. further
PROST 07 antibodies can be used in various imaging methods, including Tc-99m (or other isotope).
It can be used for immunoscintigraphy with conjugated antibodies. For example, an imaging protocol similar to the recently described protocol using an In-111 conjugated anti-PSMA antibody can be used to detect recurrent and metastatic prostate cancer (Sodee et al., Clin. Nuc. Med. 21: 759-766, 1997).

The PROST 07 antibody of the invention may be labeled with a detectable marker or conjugated to a second molecule, eg cytotoxicity, and to PROST 07 positive cells the second antibody.
Can be used to target molecules (Vitetla, ES et al., Immunotoxin The
rapy, in Darita. Jr. VT et al., eds, Cancer: Principles and Practice o
f Oncology 4 ^th ed., JB Lippincott Co., Philadelphia, 2624-2636, 1993
). Examples of cytotoxic agents include, but are not limited to, lysine, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinbrathine, colchicine, dihydroxyanthracin, Diones, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, glucocorticoids and other chemotherapeutic agents, and radioisotopes.

Suitable detection markers include, but are not limited to: radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators or enzymes. Suitable radioisotopes include the following: antimony-12
4, antimony-125, arsenic-74, barium-103, barium-140, beryllium-
206, bismuth-207, cadmium-109, cadmium-115m, calcium-45,
Cesium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51,
Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60, Cobalt-64, Erbium-169, Europium-152, Gadolinium-153, All-195, All-199,
Hafnium-175, hafnium-175-182, indium-11, iridium-192,
Iron-55, Iron-59, Krypton-85, Lead-210, Manganese-54, Mercury-197, Mercury-
203,

Molybdenum-99, Neodymium-147, Neptium-237, Nickel-63, Niobium-
95, Osmium-185 + 191, Palladium-103, Platinum-195m, Praseodymium-1
43, promethium-147, protactinium-233, radium-2226, rhenium-186, rubidium-86, ruthenium-103, ruthenium-106, scandium-4
4, scandium-46, selenium-75, silver-110m, silver-11, sodium-22,
Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125, Tellurium-132, Thallium-204
, Thorium-228, Thorium-232, Thallium-170, Tin-113, Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169, Yttrium-88, Yttrium-90, Yttrium-91, Zinc. -65 and zirconium-95.

Immunotherapy for Prostate Cancer : The present invention provides various immunotherapies for treating prostate cancer, including antibody therapy, in vivo vaccines and ex vivo immunotherapy approaches. In one approach, the invention can be used systemically to treat prostate cancer PROST
07 Providing antibodies. For example, unconjugated PROST 07 antibody is introduced into a patient such that the antibody binds to PROST 07 on or in prostate cancer cells and complement-mediated cytolysis, antibody-dependent cell Toxicity, alteration of the physiological function of PROST 07 and
/ Or Mediates cell and tumor destruction by mechanisms involving ligand binding or inhibition of signal transduction pathways. PROST 07 antibodies conjugated to toxic agents, such as ricin or radioisotopes, also directly deliver toxicity to PROST 07-bearing prostate tumor cells, and thereby therapeutically to destroy the tumor cells. Can also be used.

Prostate immunotherapy with the PROST 07 antibody can follow the teachings generated from the various approaches that have been successfully used for the following other types of cancer:
Colon cancer (Arlen et al., Crit. Rev. Immunol. 18: 133-138, 1998), multiple myeloma (Ozaki et al., Blood 90: 3179-3185, 1997; Tsunenari et al., Blood 90: 243)
7-2444, 1997), gastric cancer (Kasprzyk et al., Cancer Res. 52: 2771-2776, 1992),
B-cell lymphoma (Funakoshi et al., Immunther. Emphosis Tumer Immunol. 19:93
-101, 1996), leukemia (Zhong et al., Leuk. Res. 20: 581-589, 1996), colorectal cancer (Moun et al., Cancer Res. 54: 6160-6166, 1994; Velders et al., Cancer R.
es. 55: 4398-4403, 1995) and breast cancer (Shepard et al., J. Clin. Immunol. 11: 1.
17-127, 1991).

The invention further provides a vaccine formulated to include a PROST 07 polypeptide or fragment thereof. The use of tumor antigens in vaccines to generate humoral and cell-mediated immunity for use in anti-cancer therapy is well known in the art, and human RSMA and rodent PAP immunity. Hara has been used for prostate cancer (Hodge et al., Int. J. Cancer 63: 231-237, 1995; Fung et al., J.
Immunol. 159: 3113-3117, 1997). Such a method is facilitated by using a PROST 07 polypeptide or fragment thereof, or a PROST 07-encoding nucleic acid molecule, and a recombinant vector capable of expressing and appropriately representing the PROST 07 immunogen. Can be done.

For example, a viral gene delivery system can be used to deliver a PROST 07-encoding nucleic acid molecule. Various viral gene delivery systems that may be used in practicing this aspect of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, Adeno-associated virus, lentivirus and Sindbis virus (Re
stifo, in Curr. Opin. Immunol. 8: 658-663, 1996). Non-viral delivery systems can also be used by using naked DNA encoding a PROST 07 polypeptide or fragment thereof introduced into a patient (ie, intramuscularly) to elicit an anti-tumor response.

In one embodiment, full length human PROST 07 cDNA can be used. In another embodiment, human PROST 07 cDNA fragment can be used. In another embodiment, pros encoding a specific T lymphocyte (CTL) epitope
t 07 nucleic acid molecules can be used. CTL epitopes can be determined using specific algorithms (eg, Epimer, Brown University) to identify peptides within the PROST 07 polypeptide that can optimally bind to the identified HLA allele.

Various ex vivo methods may also be used. One approach involves dendritic cells to provide the PROST 07 polypeptide as an antigen to the patient's immune system. Dendritic cells express MHC class I and II, B7 costimulators and IL-12, and are therefore highly specialized in antigen-presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of prostate-specific membrane antigen (PSMA) are used in Phase I clinical trials to stimulate the immune system of patients with prostate cancer (Tjoa etc.,
Prostate 28: 65-69, 1996; Murphy et al., Prostate 29: 371-380, 1996). Dendritic cells can be used to provide PROST 07 polypeptides to T cells of MHC class I and II molecules. In one embodiment, the autologous dendritic cells are MH
Pulsed by a PROST 07 polypeptide capable of binding to the C molecule.

In another embodiment, dendritic cells are pulsed with intact PROST 07 polypeptide. In yet another embodiment, various implementation vectors known in the art, such as adenovirus (Arthur et al., Cancer Gene Ther. 4:
17-25, 1997), retrovirus (Henderson et al., Cancer Res. 56: 3763-3770).
, 1996), lentivirus, adeno-associated virus, DNA transfection (R
ibas et al., Cancer Res. 50: 2865-2869, 1997) and tumor-derived RNA transfection (Ashley et al., J. Exp. Med. 186: 1177-1182, 1997) in dendritic cells. Includes the construction of overexpression of the prost 07 gene.

Anti-idiotypic anti-PROST 07 antibodies can also be used in anti-cancer therapy as a vaccine to elicit an immune response against cells expressing PROST 07 polypeptides. In particular, generation of anti-idiotype antibodies is well known in the art and is readily adapted to generate anti-idiotype anti-PROST 07 antibodies that mimic an epitope on a PROST 07 polypeptide. Get (eg Wagner etc.,
Hybridoma 16: 33-40, 1997; Foon et al., J. Clin. Invest. 96: 334-342, 199.
5; Hertyn et al., Cancer Immunol. Immunother. 43: 65-76, 1996). Such anti-idiotypic antibodies can be used for anti-idiotypic therapy, as currently practiced by other anti-idiotypic antibodies directed against tumor antigens.

Gene immunization methods can be used to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing PROST 07. Using the PROST 07-encoding DNA molecules described herein, a structure comprising DNA encoding a PROST 07 polypeptide / immunogen and appropriate regulatory sequences can be directly incorporated into muscle or skin of an individual. Injected, resulting in muscle or skin cells taking up the construct and expressing the encoded PROST 07 polypeptide / immunogen.

The PROST 07 polypeptide / immunogen can be expressed as a cell surface polypeptide to be secreted. Expression of the PROST 07 polypeptide / immunogen also results in the generation of prophylactic or therapeutic humoral and cellular immunity against prostate cancer. Various prophylactic and therapeutic gene immunization techniques known in the art may be used (for review, see information and published literature at internet address www.genweb.com).

Antisense Oligonucleotides, Antisense Vectors and Ribozymes : Antisense polynucleotides complementary to prost 07 can be prepared synthetically. Such oligonucleotides, with or without lipids that can aid in uptake of antisense oligonucleotides into cells,
It can be provided in cells. On the other hand, expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or various cellular plasmids also contain antisense pr.
It can also be used for the construction and supply of recombinant vectors that will express ost 07. See, for example, the techniques described in Sambrook et al. (Supra) and Ausubel et al. (Supra).

A polynucleotide comprising a full-length cDNA sequence and / or its regulatory elements may be used in the sense strand (Youssoufian and Lodish, Mol. Cel.
l. Biol. 13: 98-104, 1993) or antisense strand (Eguchi. et al., Annu. Rev.
Biochem. 60: 631-652, 1991) allowing the use of prost 07 polynucleotides by researchers as a research tool. Such techniques are now well known in the art, and sense or antisense oligomers, or larger fragments, can be engineered from various positions along the control region code.

The gene encoding PROST 07 can be stopped by transfecting cells or tissues with an expression vector that expresses high levels of the engineered prost 07 polynucleotide fragment. Such constructs fill cells with untranslated sense or antisense sequences. Particularly preferred antisense sequences include, but are not limited to, CAACGGGUACUUGCAGGGCCUUG.
(SEQ ID NO: 17), ACCUCUGCAAAUUCACUGAGUGG (SEQ ID NO: 18), and UACUUGCAGGGCC
UUGUGUCUUU (SEQ ID NO: 19). Even in the presence of integration into the DNA, such a vector will not contain RNA until all copies have been disabled by endogenous nucleotides.
The molecule can continue to be transcribed. Transient expression can last for a month or more with a non-replicating vector, and even longer if the appropriate replication elements are part of the vector system.

As mentioned above, modification of gene expression may include modification of the antisense molecule, DN, to control regions of prost 07, namely promoters, enhancers and introns.
Obtained by planning A or RNA. Oligonucleotides derived from the transcription initiation site, eg, the −10 to +10 region of the leader sequence, are preferred. Antisense molecules can also be designed to block translation of mRNA by interfering with the binding of the transcript to the ribosome. Similarly, inhibition may be achieved using the "triple helix" base pairing method. Triple-stranded helix pairing loses sufficient ability to cleave double-stranded helices for the binding of polymerases, transcription factors, or regulatory molecules. Triple-stranded DNA
Recent advances in treatment using Gee, JE et al. (In Huber and Car. Molecula
r and Immunologic Approaches. Futura Publishing Co., Mt. Kisco, NY, 19
Revised by 94.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (US Pat. No. 4,987,071; WO93 / 23057). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are constructed hammerbed motif ribozyme molecules that specifically and effectively catalyze endonucleolytic cleavage of RNA encoding PROST 07.

Specific ribozyme cleavage sites within any potential RNA target were first identified by scanning the target molecule for ribozyme cleavage sites including the following sequences, GUA, GUU and GUC. It Once identified, a short RNA sequence of 15-20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, can be evaluated for secondary structural features that can render the oligonucleotide operable. The suitability of candidate targets can also be assessed by testing accessibility to hybridization with complementary oligonucleotides using a ribonuclease protection assay (Irie et al., Advace. Pharmacol. 40: 207-257. , 1997)
.

The antisense molecules and ribozymes of the invention can be prepared by any method known in the art for the synthesis of RNA molecules. They include techniques for chemically synthesizing oligonucleotides, such as solid phase phosphoramidide chemical synthesis. RNA molecules, on the other hand, can be expressed by in vitro and in vivo transcription, or
It can be produced by a DNA sequence encoding ROST 07. Some DNA sequences may be incorporated into a wide variety of vectors with a suitable RNA polymerase promoter, such as T7 or SP6. On the other hand, antisense cRNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include the addition of flanking sequences at the 5'and / or 3'ends of the molecule, or the use of phosphothioates or 2'O-methyl rather than phosphodiesterase chains within the backbone of the molecule, provided that It is not limited to that. Increased stability is also attributed to non-conventional bases such as inosine and cueosin, and acetyl-, thio-.
, And likewise modified forms of adenine, cytidine, guanine, thymine and uridine which are not readily recognized by endogenous endonucleases.

Methods for introducing antisense vectors into cells or tissues are discussed below and include those methods that are equally suitable for in vivo, in vitro and ex vivo therapies. For ex vivo therapy, an antisense vector is introduced into cells harvested from a patient and clonally propagated for autologous grafts, U.S. Pat.
Returned in the same patient as provided in 493 and 5,437,994. Transfection and liposome delivery, or other lipid-based or non-lipid-based agents are well known in the art.

Assays for Identifying Agents that Bind PROST 07 : The present invention also relates to assays and methods that can be used to identify agents that bind PROST 07. In particular, agents that bind to PROST 07 will bind to PROST 07, PR
It may be identified by the ability of the OST 07 ligand or other agent or constituent organism and / or the ability to inhibit / stimulate PROST 07 activity. PROST 0 with PROST 07 polypeptide
Assays for 7 activity (eg, binding) are suitable for use in high throughput screening methods.

In one embodiment, the assay comprises mixing PROST 07 with a test agent or cell extract. After mixing under conditions that allow the association of PROST 07 with components of the agent or extract, the mixture is analyzed to determine if the agent / component was bound to PROST 07. Binders / components are identified as those capable of binding PROST 07. Alternatively, or sequentially, PROST 07 activity can be assessed directly as a means to identify agonists and antagonists of PROST 07 activity using a chromogenic assay, such as the assay described in Example 4. .

On the other hand, agents that bind PROST 07 polypeptides can be identified using the yeast two-hybrid system or binding capture assay. In the yeast two-hybrid system, an expression unit encoding a fusion protein composed of one subunit of the two-subunit transcription factor and a PROST07 polypeptide is introduced into yeast cells and expressed. The cell further comprises (1) an expression unit whose expression encodes a detectable marker that requires two subunit transcription factors for expression, and (2) a cloning of a second subunit of the transcription factor and DNA. Modified to include an expression unit encoding a fusion protein made from the segment.

When the cloned segment of DNA encodes a protein that binds a PROST 07 polypeptide, expression results in an interaction between PROST 07 and the encoded protein. This closes binding and crosslinks the two subunits of the transcription factor, allowing reconstitution of the transcription factor. This results in the expression of the detectable marker. The yeast two-hybrid system is particularly useful in screening libraries of cDNA encoding segments for the cell binding partners of PROST07.

PROST 07 polypeptides that can be used in the above assays include, but are not limited to, the following: isolated PROST 07 polypeptide, PROST
A fragment of a 07 polypeptide, a cell modified to express a PROST 07 polypeptide, or a fraction of cells modified to express a PROST 07 polypeptide. Furthermore, the PROST 07 polypeptide can be the complete polypeptide or a defined fragment of the PROST 07 polypeptide. It will be apparent to those skilled in the art that this assay can be used so long as the PROST 07 polypeptide is assayed for a binding agent by a shift in molecular weight or activity.

The method used to identify whether an agent / cellular component binds to a PROST 07 polypeptide will be based primarily on the nature of the PROST 07 polypeptide used. For example, a gel retardation assay can be used to determine if an agent binds PROST 07 or a fragment thereof. On the other hand, immunodetection and biochip techniques can be employed for use with PROST 07 polypeptides. One of ordinary skill in the art can readily use many techniques known in the art to determine whether a particular agent binds to a PROST 07 polypeptide.

Agents and cellular components can be further tested for their ability to modulate the activity of PROST 07 polypeptides using cell free or cell assay systems. PROS
As the activity of the T07 polypeptide becomes more defined, functional assays based on the identified activity can be used. One such assay would be the assay for serine protease activity described in Example 4 using synthetic peptide substrates with known serine protease cleavage sites within their sequence.

As used herein, an agent is said to antagonize PROST 07 activity if the agent reduces PROST 07 activity. Preferred antagonists are
It would selectively antagonize PROST 07 without affecting any other cellular proteins. Furthermore, preferred antagonists will reduce PROST 07 activity by 50% or more, more preferably 90% or more, most preferably all.

The agents assayed in the above methods can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected if the agent is randomly selected without considering the particular sequence of the PROST 07 polypeptide. Examples of randomly selected agents are the use of chemical or peptide binding libraries, or growth broth of biological or plant extracts.

As used herein, an agent is rationally selected if the agent is selected on a non-random basis that takes into account the sequence of the target site and / or its conformation regarding the action of the agent. It is said to be selected or planned. Agent is PROS
It can be rationally selected or rationally designed by using a peptide sequence which produces a T 07 polypeptide. For example, a rationally selected peptide agent is
It may be a peptide whose amino acid sequence is identical to a fragment of the PROST 07 polypeptide.

Agents tested by the methods of the invention can be, for example, peptides, antibodies, oligonucleotides, small molecules and butamine derivatives, and carbohydrates. One of ordinary skill in the art can readily recognize that there are no restrictions regarding the structural properties of the agents used in the screening methods of the present invention. One type of agent of the present invention is a peptide agent, the amino acid sequence of which is PROST
The selection is based on the amino acid sequence of the 07 polypeptide.

Peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as known in the art. DNA encoding those peptides
Can be synthesized using commercially available oligonucleotide synthesizers and can be recombinantly produced using standard recombinant production systems. Generation using solid phase peptide synthesis is required if the amino acids of the genetic code are not included.

Another type of agent of the invention is an antibody that is immunoreactive with a determinant position of a PROST 07 polypeptide. As was done above, the antibody is the PR targeted by the antibody.
It is obtained by immunizing a suitable mammalian subject with the peptides containing those parts of the OST 07 polypeptide as antigenic regions. Such agents can be used in competitive binding studies to identify second generation inhibitors and block PROST 07 activity. The cell extract tested by the method of the invention may be, for example, an aqueous extract of cells or tissues, an organic extract of cells or tissues, or a partially purified cell fraction.
Those skilled in the art can easily understand that there is no limitation as to the source of the cell extract used in the screening method of the present invention.

Agents that bind PROST 07 polypeptides, such as PROST 07 antibodies, can be used to identify agents that modulate PROST 07, target anti-cancer agents to appropriate mammalian cells, or block interaction with PROST 07. Can be used. Cells expressing PROST 07 can be targeted or identified using agents that bind PROST 07. How the PROST 07 binder is used depends on the nature of the PROST 07 binder.
For example, PROST 07 binding provides conjugated toxins such as diphtheria toxin, cholera toxin, wasin or Pseudomonas exotoxin to cells expressing PROST 07; PROS
T07 activity can be prepared; killing PROST07-expressing cells directly; or used to identify competitive binders in the screen. For example, PROST 07 inhibitor
Can be used to directly inhibit the growth of PROST 07 expressing cells, and PROST 07
Binders can be used as diagnostic agents.

Pharmaceutical Compositions and Administration : The present invention also includes a prost 07 polynucleotide, a PROST 07 polypeptide, an antibody, an agonist, or an inhibitor, alone or in at least one other agent, such as any sterile, It also relates to a pharmaceutical composition comprising a biocompatible carrier, such as a salt solution, a buffer solution, dextrose and a stable compound that can be administered in a water including but not limited to water, in combination with the stable compound. Any of those molecules can be administered to a patient alone or in combination with other agents, drugs or hormones as a pharmaceutical composition admixed with an excipient or pharmaceutically acceptable carrier. In one aspect of the invention, the pharmaceutically acceptable carrier is pharmaceutically inactive.

The present invention also relates to the administration of pharmaceutical compositions. Such administration is oral or parenteral. Parenteral delivery methods include local, intraarterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration. In addition to the active organism, those pharmaceutical compositions comprise suitable pharmaceutically acceptable carriers which comprise excipients and auxiliaries which facilitate the processing of the active compounds into pharmaceutically usable formulations. Can be included. Additional details regarding techniques for formulation and administration may be found in Remington's Pharmaceutical Science (Ed. Maack Publishing Co.
Easton, Pa.).

Pharmaceutical compositions for oral administration may be formulated using pharmaceutically acceptable carriers well known in the art as appropriate doses for oral administration. Such carriers enable the formulation of pharmaceutical compositions into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like for ingestion by a patient.

Pharmaceutical preparations for oral use may be obtained by combining the active compounds with solid excipients, optionally by grinding the resulting mixture and adding suitable auxiliaries, and then, if desired, tablets. Alternatively, it is obtained by processing a mixture of the above granules to obtain a dragee core. Suitable excipients are carbohydrate or protein fillers such as sugars such as lactose, sucrose, mannitol or sorbitol; starches from corn, wheat, rice, potato or other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose or sodium. Carboxymethyl cellulose and gums such as gum arabic and gum tragacanth; and proteins,
Examples are gelatin and collagen. If desired, disintegrating or dissolving agents such as cross-linked polyvinylpyrrolidone, agar, alginsan or salts thereof such as sodium alkyneate may be added.

Dragee cores may also contain suitable coatings, eg, acacia, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. , Supplied by a concentrated sugar solution. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity or volume of active compound.

Pharmaceutical agents that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with fillers or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and optionally stabilizers.
In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical composition of the invention may be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiological buffer solution.
Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions.

Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or intranasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Kits : The present invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more ingredients in the aforementioned compositions of the invention. Such containers exist in a form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, and authorizes the manufacture, use or sale of products for human administration. be able to.

Manufacture and Storage : The pharmaceutical compositions of the present invention may be formulated in any manner known in the art, eg, by conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping or entrapping. It can be manufactured by a freeze-drying process. The pharmaceutical composition may be supplied as a salt and formed with acids such as hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in their corresponding free base form, aqueous or other protic solvents. In other cases, the preferred formulations are pH 4.5-5 combined with a buffer prior to use.
It can be a lyophilized powder in 1 mM to 50 mM histidine at 0.5, 0.1 to 2% sucrose, 2 to 7% mannitol.

After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier are prepared, they are placed in a suitable container and labeled for treatment of the indicated pathology PROST 07. With regard to the administration of, such labeling includes the amount, frequency and method of administration.

Therapeutically Effective Amount : A pharmaceutical composition suitable for use in the present invention is one in which the active ingredient is to achieve its intended purpose, ie, treatment of a particular disease state characterized by PROST 07 expression. The composition is included in an effective amount. Determination of the effective amount is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, such as neoplastic cells or animal models, usually mice, rabbits, dogs or pigs. Animal models are also used to obtain the desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective amount is referred to as an amount of the protein or its antibody, agonist or inhibitor that ameliorates the symptoms or condition. The therapeutic efficacy and toxicity of such compounds, such as the ED ₅₀ (therapeutically effective dose in 50% of the population) and LD ₅₀ (lethal dose to 50% of the population) are determined by cell culture or experimental It can be determined by standard pharmaceutical methods in animals. The dose of therapeutic effect and toxicity is the therapeutic index, and it can be expressed as the ratio, ED ₅₀ / LD ₅₀ . Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose will vary within this range depending upon the dosage form used, the sensitivity of the patient and the route of administration.

The exact dosage will be chosen according to the individual medicine in view of the patient to be treated. Dosage and administration are adjusted to provide a sufficient level of active ingredient or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state, such as tumor size and location; age, weight and sex of the patient; diet, time and frequency of administration, drug combination, response sensitivity and resistance / response to treatment. To do. Long-acting pharmaceutical compositions have doses of 3-4 depending on the half-life and clearance rate of the particular formulation.
Dosed daily, weekly, or biweekly.

The usual dose can vary from 0.1 to 100,000 μg to a total dose of 1 g, depending on the route of administration. Guidance on specific doses and delivery methods is provided in the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344 or 5,225,212. Those skilled in the art will use different formulations for the polynucleotide rather than for the proteins or their inhibitors. Similarly, the supply of polynucleotides or polypeptides will be specific to particular cells, pathologies, locations, etc.

The invention is further described by the following examples. The examples are provided only to illustrate the invention according to particular embodiments. The examples are illustrative of particular aspects of the invention and do not limit the scope of the disclosed invention. All examples were made using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques in the following examples can be found in standard laboratory manuals such as Sambrook et al., Molecular Cloning AL.
^{aboratory Manual, 2 nd Ed. Cold} Spring Harbor Laboratory Press, Cold Spri
ng Harbor, NY, 1989.

[0216] Example Example 1. Identification of human prost 07 polynucleotides prost 07 was identified as a gene expressed in the prostate by searching the Incyte's LifeSeq gene expression database. MRNAs encoding PROST 07 polypeptides enable LifeSeq "transcriptor imaging" features and selection of novel genes expressed in Incyte's prostate tumor cell cDNA library in multiple copies of more than one per library Discovered using survey parameters. This set of genes was evaluated by electronic Northern analysis to determine their analysis in other prostate tissue libraries and all other tissue libraries in the database. Clone 1651585 was identified in the library PROSTUT 12 of LIFESEQ Version 4.1 SEP96 (Beta) and was shown by electronic Northern analysis to cluster with other clones containing the prost 07 polynucleotide in a prostate specific manner.

This prostate-specific expression was found to be consistent in the latest version of the LifeSeq database. All clones encoding fragments of PROST 07 were assembled into a contiguous sequence using the Genetics Computer Group (GCG, Wisconsin package) gene assembly program and the sequence of the assembled mRNA was evaluated. An open reading frame encoding the protein was identified and shown that the predicted protein is homologous to a family of serine proteases. An additional clone encoding a fragment of PROST 07,
Clone 3357511 containing most complete 5'sequences in the LifeSeq database
Acquired from Incyte which embraces.

To obtain additional sequences at the 5'end, primers were selected 3'to the predicted stop codon and Clo to generate a cDNA product encoding PROST07.
Used with primers and reagents in a 5 RACE PCR amplification kit obtained from ntech. A sample of cDNA generated from prostate tissue RNA from Clontech Laboratories was used as a template for this synthesis. The PCR products were
It was cloned into a TA vector obtained from itrogen and the selected clones were sequenced. This sequence corresponds to the nucleotide sequence of prost 07 predicted by assembly of individual clone pairs in Life Seq, and PROST 07
Of the active form of Escherichia coli was included, and additional sequences were included, including part of the putative pro-sequence and putative signal sequence.

Additional confirmatory sequence information was obtained from the genomic clone encoding PROST 07 (P1 clone onc77). The sequence containing the 3'region of the transcript should be changed to Incyt containing the complete 3'region.
E-clone # 1818001 was obtained by sequencing.

A. 5'RACE (5'rapid amplification of cDNA ends). 5'RACE-RCR, Clontech Marathon c
Performed on prost 07 using the DNA amplification kit. Normal human prostate mRNA (Clo
ntech) was used as the template. First-strand cDNA synthesis by Marat for prost 07
Sensitization was performed with either the hon cDNA synthesis primer or the gene-specific primer. After second strand synthesis and adapter ligation, PCR amplification was performed with either Advantage Klen Taq polymerase mix, AmpliTaq or AmpliTaq-Gold. A panel of PCR reactions was then performed and the PCR products were gel isolated and TA cloned into pCR2.1 using the TA cloning kit from Invitrogen.

Plasmid DNA was prepared from individual transformant colonies and prost 07-
Confirmed by Southern blot using a specific oligonucleotide probe.
The confirmed clones were then sequenced to obtain an additional 5'sequence for prost 07.

Sequence of PCR primer and oligonucleotide probe: Prost 07 RACE # 1 set: Pr7GSP1: 5'-TCCACCATCTGGCTCCCTGGCTC-3 '(SEQ ID NO: 3) Pr7NGSP1: 5'-AGGCCCAGCCCGATGGTGTAGGAG-3' (SEQ ID NO: 4) Pr7Probe1: 5 '-GCACCAGGACGCCCGAGCAGAACAATTCGTTTTCCATGACCAGTGCCGCC-3' (SEQ ID NO: 7) Pr7Probe2: 5'-TCTGGAAACAGTGTGCGGCTGACAGCACCCACTGCGGATGCACCAGGACG-3 '(SEQ ID NO: 8)

[0223] Prost 07 RACE # 2:   Pr7GSP2:     5'-CTGACAGCACCCACTGCGGATGCAC-3 '(SEQ ID NO: 5)   Pr7NGSP2:     5'-GAACAATTCGTTTTCCATGACCAGTGCC-3 '(SEQ ID NO: 6)   Pr7Probe3:     5'-TGCCAGGGCTGCGAGTGCGGGCTGCAGTCC-3 '(SEQ ID NO: 9)

B. DNA and protein sequence analysis. The protein sequence predicted by the cDNA and genomic sequences evaluated was that of the activated form of serine protease (
(Fig. 2). Comparison of the nucleotide sequence of Prost 07 with the sequence of the known serine protease-comprising EMSP1 showed that all the sequences required to encode the active enzyme were present. The additional 5'sequence encoded the predicted pro-sequence and some signal peptide sequences.

Example 2 prost 07 mRNA expression prost 07 mRN in various samples of normal and tumor tissues and in cell lines
Expression of A was determined by two methods, Northern blot analysis, and semi-quantitative PCR using the Taqman assay (Perkin-Elmer). Prostate normal, benign and tumor tissue samples graded according to the improved Gleason classification system,
Obtained from Urology Department at Stanford University School of Medicine. R
NA was isolated from them by standard methods. RNA from other tumors and normal tissues was purchased from commercial sources such as Clonetech and Biochain. Prostate tumor cell line (PC-
3, LNCaP and DU145) were obtained from the American Type Culture Collection and grown in culture by standard methods with serum-containing medium. Xenograft tumors derived from those cell lines were established in nude mice and approximately 4 of transplants.
Harvested from mice after ~ 6 weeks. RNA was isolated from the tumor by standard methods.

A. Northern blot analysis. Northern blots were performed with 2 μg of total RNA (
See FIG. 4). RNA was loaded on a 1% agarose gel, tested at 60 volts for 4 hours and subsequently transferred to Hybond membranes. A radiolabeled probe for Prost 07 mRNA was generated using a prost 07-containing plasmid (pINCY-containing Incyte clone 3357511), followed by isolation of the 3357511 insert from the vector. Randomly sensitized, radiolabeled probe was applied to the Pharmacia Biotech oligo-
It was generated using a labeling kit and used to probe Prost 07 mRNA on the blot. An mRNA species with a size of 1.6 Kb was detected. It was specifically expressed in prostate tissue and the LNCaP prostate cell line, but not in any of the other tissues evaluated. Expression was generally higher in prostate tumor tissue than in normal tissue from the prostate.

B. Taqman analysis. Taqman-based PCR analysis with the following primers: GGG AAC TCT TG
C CTC GTT TCT (SEQ ID NO: 14) and GAC ACC ACC GAC ACG TTC AC (SEQ ID NO: 15)
, And Taqman probe: FAM-TGCTGGCGAACGGCAGAATGC-TAMRA (SEQ ID NO: 16). The primers and probes were designed using Perkin Elmer's Primer Express and synthesized by Synthetic Genetics. PCR reactions were performed for 30-40 cycles and quantified with prostate RNA to generate a standard curve for relative comparison. This analysis showed that prost 07 mRNA was most exclusively detected in the prostate, confirming the results obtained by Northern analysis (see Figure 5).

Example 3 Histochemical Detection of PROST 07 The presence of PROST 07 in various human tissues, eg prostate tissue, was tested using histochemical methods. Histochemical studies were performed with polyclonal rabbit (Prost7.4) or monoclonal mouse (4B8) antibodies followed by alkaline phosphatase-conjugated antibodies as the first antibody (see Example 5 below). As a negative control, staining was performed in the absence of the first antibody or with a non-specific IgG antibody as the first antibody. Both PROST 07 antibodies showed the same staining pattern when used to determine PROST 07 expression in prostate tissue.

The normal human tissues used in the study included 11 organs, such as kidney, liver, lung and prostate. Most prostates and ducts were strongly positive for staining (see Figure 8A). Basal cells were negative for staining. Weak granular deposits were found in the region of fibromuscular stroma. The seminal vesicle epithelium showed weak cytoplasmic staining in most cells, but a few lesions showed strong staining in the middle. In addition to strong-to-moderate expression of PROST 07 in the prostate, reddening of the cytoplasm results in cells of other normal tissues, such as liver (hepatocytes), pancreas (Islets of Langerhans) or podocytes (kidneys) in the glomerulus. Occasionally found on the skin. Most cells in normal tissue
It was negative for PROST 07 expression.

A detailed analysis of PROST 07 expression in prostate tumor tissue was also performed (see Figures 8A and 8B). The expression of PROST 07 in prostate cancer (n = 2), lymph nodes (n = 3), bone and spinal cord metastases from prostate tumors (n = 6) was determined. The cancer used in this study was found to express the PROST 07 protein. Within cancer, most tumor cells were positive for PROST 07 expression, although a wide range of weak to strong staining varied between cells and regions of the tumor. Prostate intraepithelial neoplasia (PIN
The glands exhibiting changes in) showed moderate to strong staining, similar to hyperplastic regions. Weak granular deposits were found on most of the fibromuscular stroma. Endoluminal secretion was also strongly positive.

All cases of prostate cancer metastases to lymph nodes showed positive staining for PROST 07, as did cases of metastases to bone marrow. Two out of three cases of prostate cancer metastases to bone showed expression of the PROST 07 protein. The surrounding normal tissue was not stained.

Example 4 PROST 07 Expression, purification and assay for biological activity 1. GST-PROST 07 structure: A. Expression of GST-PROST 07 (Glutathione-S-transferase-PROST 07
Fusion protein). Correctly folded, and thus virtually active PROST
To generate 07, a cDNA fragment encoding amino acid residues 11-234, containing the putative mature PROST 07 protease domain, was amplified using the following primers: M7 5'-CGC GGA TCC GAG AAT. CTT TAT TTT CAG ATC ATC AAC GGC GAG GAC TGC
-3 '(SEQ ID NO: 20); M9 5'-CCG GAA TTC TTA ACT GGC CTG GAC GGT TTT C-3' (SEQ ID NO: 21).

The resulting fragment was ligated into the Bac HI / EcoRI restricted pAcSecG2T plasmid (Pharmingen). Stock solutions of recombinant baculovirus were produced as well known to those skilled in the art. The recombinant fusion protein GST-PROST 07 was secreted from insect cells infected with recombinant baculovirus (BT1-TN-5B1-4) and purified from the supernatant by glutathione chromatography.

B. Purification of GST-PROST 07. Insect cell culture supernatant was centrifuged (Sorvall H-50
Clarified by 0 rotor for 30 minutes at 4500 rpm). The resulting supernatant is filtered (0.22 μm filter) and then using a P-1 pump at a rate of 4 ml / min at 4 ° C., a layer volume of 5 ml glutathione sepharose 4B (Pharmacia cat. # 17). -0756-
Loaded onto a FPLC column packed according to 01). Next, attach the column to Pharmacia FP.
It was connected to an LC machine and washed with 15 ml PBS (phosphate buffer solution) at a flow rate of 10 ml / min.

The bound GST-PROST 07 fusion protein was loaded with 15 ml of elution buffer (10 mM reduced glutathione, 50 mlM Tris, 100 mM NaCl, pH 8.0) at a rate of 10 ml / min.
Was eluted from the column by. Fractions (1 or 2 ml / fraction) were collected and the elution profile was monitored by _A280 . The eluted fractions were analyzed by PAGE (polyacrylamide gel electrophoresis), and the protein concentration was determined by Bio-Rad Protein Assay (cat.
# 500-006), determined by Western blot with anti-GST and / or anti-PROST 07 antibodies, and enzyme activity monitored by zymogram gel assay.

C. Enzymatic activity of GST-PROST 07: 1. Zymography. To determine if the fusion protein exhibits enzymatic activity, a sample of glutathione eluate was subjected to zymography using a 10% gelatin gel (Figure 7). 5 × sample buffer lacking β-mercaptoethanol, DTT or any other reducing agent was appropriately mixed with the sample. Samples were preheated to gelatin zymogram gel (Novex cat. #EC
61752) Loaded directly on. Zymograms were performed with SDS running buffer according to standard PAGE protocols. After running the gel, it was removed from the plastic plate and soaked in 50 ml of regeneration solution (2.5% Triton X-100) at 21 ° C for 50 minutes.

The gel is then loaded with 100 ml of regeneration buffer (50 mM Tris, 40 mM HCl, 200 mM NaCl).
, CaCl ₂ of 5 mM, the 0.2% Brij; Novex-Cat. (Available from # LC2671) at 37 ° C. for 4 hours without shaking. After regeneration, the gel was first stained with Coomassie blue for 30 minutes and then decontaminated in standard PAGE destaining buffer for 30 minutes. As shown in FIG. 7, the GST-PROST07 fusion protein showed enzymatic activity to digest gelatin incorporated into the gel. PROST 07
The second band migrating at an apparent molecular weight corresponding to was also active in digesting the gelatin matrix. This band was due to autocatalytic degradation at the assembled thrombin site, as confirmed by N-terminal sequencing (data not shown). This indicated that PROST 07 was an active serine protease.

2. Assays with peptide substrates. PROST 07 activity was also measured by HPLC analysis following hydrolysis of the synthetic peptide substrate. In a reaction volume of 15 μl,
1-5 μM PROST 07 or activated PROST 07 was added to 1 mM peptide substrate (SDLVPR
MSENLYFQ (SEQ ID NO: 24) or SDLVPRGSENLYFQ (SEQ ID NO: 25)), and
Incubated at 37 ° C for various times. The buffer for this assay is 25 mM
It is HEPES (pH 7.4). After incubation, 5 μl of 10% TFA was added to stop the reaction, and then 10 μl of sample was loaded onto an analytical C18RP-HPLC column (Phen).
umenex jupiter 5 μ300A 150 × 4.5 mm Patent No. 00F-4053-E0).
The column was first equilibrated with 5% solvent B, where solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in acetonitrile. Peptide fragments were eluted with a 5-35% acetonitrile gradient for 30 minutes at a flow rate of 1 ml / min. The eluate was monitored at 214 nm.

3. Assay of PROST 07 with a chromogenic substrate. PROST 07 activity can be monitored according to the release of the chromophore from the chromogenic substrate. 96 in a final 200 μl reaction volume
-Using a well plate, 1-5 μM Prost 7 or activated Prost 7
1 mM commercial chromogenic trobin substrate (American Diagnostica, Greenwith, (T; c
at. No. 238L TH). The reaction rate was monitored at 405 nm for 30 minutes using a plate reader. The buffer for this assay is 25 mM HEPES (pH 7.4)
Is.

II. 6X-HIS PROST 07 Structure: A. Expression of 6X-HIS PROST 07 (polyhistidine PROST 07 fusion protein). Activity
Putative maturation to improve expression and secretion levels of PROST 07 protease
C encodes amino acid residues 11-234, including the PROST 07 protease domain c
DNA was amplified using the following primers: M8 5'-CGC GGA TCC ATC ATC AAC GGC GAG GAC TGC-3 '(SEQ ID NO: 22); M29 5'-CCG GAA TTC TTA ATG ATG ATG ATG ATG ATG GCT GCT GCC GCC ACT GG
C CTG GAC GGT TTT-3 '(SEQ ID NO: 23).

The resulting fragment was ligated into the Bam HI / EcoRI restricted pAcGP67-8 plasmid (Pharmingen). Stock solutions of recombinant baculovirus were produced as well known to those skilled in the art. Recombinant fusion protein 6X-His PROST 07
Was secreted from insect cells (BT1-TN-5B1-4) infected with recombinant baculovirus and purified from the supernatant by glutathione chromatography.

B. Purification of 6X-HIS PROST 07. 6X-HIS PROST 07 with 10k MWCO tangential flow membrane
Ten liters of conditioned medium from insect cells expressing L. was concentrated to approximately 200 ml. Using a 14 k dialysis tube, concentrate the concentrate into 2 x 4.0 L of buffer A (50 mM Na ₂ HPO ₄ , pH 8.0, 300 mM NaCl).
). The dialysate was loaded onto a 20 ml Qiagen Ni-NTA column at a flow rate of 5 ml / min.
Loaded on rmacia XK26 / 20. After loading, the column was loaded with 10 ml buffer A, followed by 20 m.
100 ml of M imidazole or 8% buffer B (50 mM Na ₂ HPO ₄ , pH 8.0, 300 mM NaCl,
Washed with 150 mM imidazole). The enzyme was eluted with an imidazole gradient of 20 mM-250 mM (8-100% buffer B). Fractions containing PROST 07 were pooled based on SDS-PAGE, Western blot and activity assay.

Using a 14 K dialysis tube, the Ni-NTA pool was filled with 2 x 1.0 L of buffer C (25 mM HEPES,
It was dialyzed against pH 7.4). Apply 1 ml of dialysate onto a Pharmacia Mono-Q HR5 / 5 column.
Loaded at a flow rate of / min. After loading, the column was washed with 10 ml of buffer B and the enzyme was loaded with 0-500 mM NaCl or 0-50% buffer D (25 mM HEPES, pH 7.4, 500 m).
Elution with a salt gradient (M NaCl). Fractions containing PROST 07 were pooled based on SDS-PAGE, Western blot and activity assay.

Two porcine pancreatic elastases (Sigma E-0258) were used to activate PROST 07.
: 1 ratio, ie 1 mg PROST 07: 0.5 mg elastase ratio, was added to the Mono-Q pool and incubated for 60 minutes at 37 ° C. Using the same conditions mentioned above, elastase-treated PROST 07 was loaded onto a Mono-Q column to remove elastase. Elastase flows through this column. SD
Fractions containing elastase-treated PROST 07 were pooled based on S-PAGE, Western blot activity assay. The protein concentration of PROST 07 and elastase-treated PROST 07 was determined using the Pierce BCA Protein Assay Kit (Pierce 23225
). For Western blots, either rabbit anti-6X-HIS or mouse anti-PROST 07 was used.

Example 5 Antibody-producing rabbit polyclonal antisera were raised against four synthetic peptide sequences derived from the PROST 07 polypeptide sequence. The sequences were chosen for their predicted position on the surface of the protein in order to generate antisera that likely recognize surface epitopes. The cysteine residue was replaced by aminobutyric acid (Abu) to aid synthesis. Listed below are specific amino sequences, positions on PROST 07 polypeptides and names for the three peptides.

Name Position Amino acid sequence Pab1 21-36 SQPWQAALMENELF (SEQ ID NO: 10) Pab2 78-91 EASLSVRHPEYNRP (SEQ ID NO: 11) Pab4 159-172 SKLYDPLYHPSMF (SEQ ID NO: 12) Pab5 221-234 CKFTEWIEKTVQAS (SEQ ID NO: 13)

The peptide was covalently attached to limpet hemocyanin (KLH) through an additional amino-terminal cysteine for use as an immunogen. Similarly, bovine serum albumin (BSA) conjugate was prepared for analysis of antiserum titer through ELISA. Two animals were immunized with the individual peptides. Initial immunizations were performed in Freund's Complete Adjuvant (0.5 mg / animal), followed by booster immunizations with 0.25 mg / animal in Freund's Incomplete Adjuvant at regular intervals of 3 weeks by intramuscular administration. Periodic test bleeds were taken and antibody titers to specific BSA-peptide conjugates were measured by ELISA and compared to preimmune sera.

Antisera were tested by well turn blot for binding to purified PROST07 protein expressed as a 6His-PROST07 fusion protein in E. coli. Antisera that recognized the PROST 07 fusion protein were further tested for their ability to recognize naturally occurring proteins in LNCAP tumors, LNCAP cells, PC3 tumors and lysates prepared from PC3 cells. Binding specificity was determined by antibody binding in the presence and absence of homologous and heterologous peptides.

Rabbit polyclonal antisera and murine monoclonal antibodies were also raised against the 6His-PROST 07 fusion protein purified from E. coli. Immunogen,
Following extraction with 8M urea, metal chelating affinity resin (Ni-NTA-Sepharose) was used to e. Purified from E. coli cell lysate. Ten murine hybridoma clones that recognize the PROST 07 fusion protein in the denatured state were obtained. The monoclonal antibodies were tested for recognition of baculovirus expressing the PROST 07 polypeptide using an ELISA assay. Six of those clones were native (catalytically active) PROST 07 (monclonal antibodies 5F4, 4A12, 8D11, 8H5, 2F).
9, 3F4 and 438) were found.

Example 6 Antibody neutralization of PROST 07 activity E. A murine monoclonal antibody raised against PROST 07 expressed in E. coli (see Example 5 above) was used in American Diagnostica Greenwich, CT.
(Cat. # 238L TH) using commercially available dye-forming thrombin substrate, PROST
07 Tested for their ability to neutralize enzyme activity. Finally, the chromogen p-nitro-anilide was coupled through the peptide HDHHTAR-pNA-2AcOH12, the carboxyl terminus.

An assay for the ability of the antibody to neutralize PROST 07 activity was performed using 0.1% PEG-6000.
In 50 mM Hepes buffer (pH 7.4) containing 96 well plates. Pr
Following purification on otein Sepharose, the monoclonal antibody was incubated overnight with benzamidine sepharose to remove contaminating proteases.
Sepharose beads were removed by a brief centrifugation and 1.6 x 10 ^-9 mol (7
Individual mabs (in 5 μl) were preincubated on ice with 1.6 × 10 ⁻¹⁰ moles (in 25 μl) of purified PROST 07 (see Example 4). After pre-incubation, 100 μl of a 1:10 diluted solution of chromogenic substrate was added, immediately followed by a change in absorbance at 405 nm for 15 minutes at 2 minute intervals using an automated microtiter plate reader. It was measured. Controls included substrate only, monoclonal antibody + substrate, or reaction mixture + buffer, normal mouse IgG, or 10 mM benzamidine.

Example 7 Inhibition of Tumor Cell Migration and Invasion by Anti- PROST 07 Antibodies The ability of PROST 07 neutralizing monoclonal antibody 4B8 to inhibit tumor cell migration and / or in vitro was tested using a Boyden chamber (Pathology O
ncology Research, Vol. 4, 1998). PROST 07-positive CAOV-3 cells (ATCC)
Incubated with either ST07-specific monoclonal antibody 4B8 or non-specific IgG mouse antibody. For migration assays, cells were seeded in transwells with a low profile coated with fibronectin.

For the invasion assay, cells were seeded on transwells coated on the upper side with Matrigel. PROST 07 neutralizing monoclonal antibody 4B8 partially inhibits the ability of CAOV-3 cells to migrate through fibronectin coated wells when compared to the effects of non-specific IgG mouse antibodies or untreated cells ( Figure 10A
), And partially inhibited the entry capacity of CAOV-3 cells through Matrigel-coated wells (see FIG. 10B).

Example 8 Specific shrinking effect of PROST 07 mRNA on prostate tumor cells Prost tumor cell lines PC-3 and LNCaP using antisense oligonucleotides to reduce mRNA expression in selected cell lines
Selected for evaluation of 07 roles. PC-3 cell line shows very low levels of prost 07
mRNA is expressed, and LNCaP cells express significant levels of prost 07 mRNA.
The antisense oligonucleotide used has the sequence CAACGGGUACUUGCAGGGCCUUG
(SEQ ID NO: 17), ACCUCUGCAAAUUCACUGAGUGG (SEQ ID NO: 18) and UACUUGCAGGGCCUU
It has GUGUCUUU (SEQ ID NO: 19) and is referred to as 13577, 13581 and 13598, respectively. The reagents are RNA / DNA hybrid molecules consisting of 23 nucleotides in length. The reagent of choice is the mRNA for PROST 07 when administered to cells in the presence of lipids (NC388 lipid mixture supplied by Atugen Corp.).
Lower the level.

The reagents include an oligonucleotide region that is complementary to prost 07 mRNA, along with the specific recruitment regions for each of them shown above. LNCaP
Administration of those sequences to, when given as a complex with lipid NC388
It inhibited the proliferation of cells in a dose-dependent manner in the range of 120 nM. At the same dose range, a control oligonucleotide consisting of a set of random 23-mer oligonucleotides had no effect on cell proliferation. Antisense oligonucleotides directed against the enzyme inosyl monophosphate dehydrogenase (IMPDH II) and the enzyme required for nucleic acid synthesis also caused growth inhibition in LNCaP cells (see Figure 6). ).

All publications and patents mentioned in this specification are herein incorporated by reference. Although the present invention has been described in terms of particular embodiments thereof, it should be understood by those skilled in the art that various modifications can be made within the scope of the invention. further,
Many modifications may be made to adapt a particular situation, material, composition of matter, process, process step, objective, within the scope of the invention. All modifications are intended within the scope of the invention.

[Brief description of drawings]

FIG. 1: Polynucleotide sequence of prost 07 (SEQ ID NO: 1), which encodes the biologically or immunologically active form of PROST 07.

FIG. 2. Deduced amino acid sequence of PROST 07 in active form (SEQ ID NO: 2), with the catalytic ternary underlined.

[Fig. 3] Sequence of enamel matrix serine protease (EMSP1) with sufficient length and PRO
A sequence of amino acids with the sequence of ST 07. PROST 07 is at the bottom.

FIG. 4: Expression of prost 07 mRNA in human tissues and cell lines by Northern blot analysis. RNA from human tissues (tumors and normals) and human cell lines was
The rost 07 mRNA was tested for hybridization. 1.6 kb size mRN
Type A human prostate (tumor and normal), human prostate tumor cell line LNCaP, and LNCaP
It was detected in tumor tissue derived from cells. Expression was not detected in tissues other than prostate tissue.

FIG. 5. Expression of prost 07 mRNA in human tissues by Taqman-based PCR analysis. RNA from human tissues (tumor and normal) was isolated by standard methods. Primers and probes for detecting prost 07 mRNA expression were designed using Perkin Elmer's Espress software and synthesized by Synthetic Genetics. pro
st 07 mRNA was detected in human prostate tissue (tumor and normal) but not in other normal and tumor tissues.

FIG. 6: Effect of specific reduction of prost 07 mRNA expression on prostate tumor cell proliferation. prost 0
Three antisense oligonucleotides targeted to the 7 sequence (see Example 8) were used to determine the effect of specific reduction of prost 07 mRNA in the PROST 07-expressing human prostate cell line LNCaP. Was used.

FIG. 7: Measurement of PROST 07 activity. FIG. 7A shows a zymography of purified GST-PROST 07. A sample of glutathione eluate was fractionated on an SDS gel containing 10% gelatin under non-reducing conditions. To regenerate the separated protein, SDS
It was sequestered using Triton X-100 and the gel was stained with Coomassie. Zymograms are due to auto-catalysis, GST-fusion proteins and PRO
Two types of enzyme activity showing the apparent molecular weight of ST07 were identified. Lanes 1 and 3, GST-
Different preparations of PROST 07; lane 5, molecular weight marker. Samples were unloaded in lanes 2 and 4. FIG. 7B shows an enzymatic assay of GST-PROST 07 activity. PROST 07 protease activity was determined using an enzymatic assay according to the rate of hydrolysis of the peptide substrate (see Example 4). The proteolytic activity is
Shown as% material hydrolyzed over time.

FIG. 8: Immunohistochemical staining of PROST 07. Polyclonal and monoclonal antibodies specific for PROST 07 have been identified in PROS 07 in various human tissues as described in Example 3.
It was used to test T07 expression. FIG. 8A shows staining of normal and prostate tumor tissue. FIG. 8B shows staining of bone marrow containing prostate tumor metastases.

FIG. 9: Neutralization of PROST 07 activity by a monoclonal antibody. A mouse monoclonal antibody specific for PROST 07 was prepared using the chromogenic assay described in Example 4 for PROS
They were tested for their ability to neutralize T 07 activity (see Example 6). The release rate of the chromogenic substrate was followed over time. Three monoclonal antibodies, 5F4, 4B8 and 4A12, were found to significantly inhibit PROST 07 activity.

FIG. 10. Inhibition of tumor migration / invasion by monoclonal antibodies. A mouse monoclonal antibody specific for PROST 07 (4B8) was used to block migration (FIG. 10A) or invasion (FIG. 10B) of PROST 07 positive CAOV-3 cells (see Example 7). It was Control cells are untreated or treated with a non-specific IgG mouse antibody.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/475 A61K 31/573 4C076 31/573 31/704 4C084 31/704 31/7048 4C085 31/7048 31 / 7105 4C086 31/7105 39/395 E 4C206 38/00 T 4H045 39/395 45/00 47/48 45/00 48/00 47/48 A61P 13/08 48/00 35/00 51/00 43/00 105 A61P 13/08 C07K 16/40 35/00 C12N 1/15 43/00 105 1/19 C07K 16/40 1/21 C12N 1/15 9/48 1/19 C12Q 1/68 A 1/21 G01N 33 / 53 D 5/10 33/566 9/48 33/574 C12Q 1/68 C12P 21/08 G01N 33/53 C12N 15/00 ZNAA 33/566 5/00 A 33/574 A61K 37/02 // C12P 21 / 08 43/00 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, S, 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 Harkins, Richard United States, California 94502, Alameda, Seaview Parkway 2819 (72) Inventor Wright, David Earl. United States, California 94402, San Mateo, South Fremont Street 614 (72) Inventor Lin, Richard Jay. United States, California 94526, Danville, Hill Meadow Place 1026 (72) Inventor Parks, Deborah United States, California 94541, Hayward, Hampton Place 19050 (72) Inventor Parry, Gordon United States, California 94596, Walnut Creek, Hackamore Coat 2255 (72) Inventor Schneider, Douglas W. United States, California 94549, Lafayette, Walnut Lane 3329 (72) Inventor Steinblecher, Renate United States, California 94596, Walnut Creek, Hackamore Coat 2255 (72) Inventor Van Hewitt, Pamela Toy United States, California 94556, Moraga , Camporindo Drive 3965 (72) Inventor Suan, Gian-i USA 94591, Castro Valley, Kit Lane 6729 F term (reference) 4B024 AA01 AA12 BA14 CA03 CA04 CA09 CA11 HA01 HA14 HA17 4B050 CC04 DD11 LL01 LL03 4B063 QA19 QQ44 QR32 QR55 QR62 QS25 QS34 4B064 AG27 DA05 4B065 AB01 BA02 CA33 CA44 CA46 4C076 AA95 CC27 CC42 EE41 EE59 4C084 AA02 AA07 AA12 AA13 AA17 BA01 BA08 BA16 BA17 BA18 BA22 BA01 BA02 BA01 BA01 BA01 NA01 NA13 NA01 NA13 NA14 NA03 NA05 NA11 CC21 DD62 EE01 4C086 AA01 AA02 BA02 BC30 CB03 CB05 DA10 EA10 EA11 EA16 MA01 MA04 NA05 NA13 NA14 NA15 ZA81 ZB26 4C206 AA01 AA02 AA03 GA02 GA30 MA01 MA04 NA05 NA13 NA14 NA15 ZA81 ZB26 4H045 AA11 AA30 CA40 DA40 DA40 CA76 DA40

Claims (45)

[Claims] 1. (a) A polynucleotide encoding a polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a biologically or immunologically active fragment thereof; (b) A polynucleotide encoding a polypeptide comprising amino acid 11 to amino acid 234 as shown in FIG. 2 (SEQ ID NO: 2); (c) amino acid 4 to amino acid 234 as shown in FIG. 2 (SEQ ID NO: 2) A polynucleotide encoding a polypeptide comprising; and (d) a polynucleotide which is complementary to the polynucleotide of (a), (b) or (c) above; for a member selected from the group consisting of: An isolated polynucleotide comprising a polynucleotide that is 70% identical. 2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA. 3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA. 4. The polynucleotide according to claim 1, wherein the polynucleotide is genomic DNA. 5. The polynucleotide encodes a polypeptide comprising amino acids 1 to 234 as shown in FIG. 2 (SEQ ID NO: 2).
The described polynucleotide. 6. The polynucleotide encodes a polypeptide comprising amino acids 11 to 234 as shown in FIG. 2 (SEQ ID NO: 2).
The described polynucleotide. 7. The polynucleotide encodes a polypeptide comprising amino acid 4 to amino acid 234 as shown in FIG. 2 (SEQ ID NO: 2).
The described polynucleotide. 8. The polynucleotide of claim 1, wherein the polynucleotide comprises the sequence of nucleotide 1 to nucleotide 1354 as shown in FIG. 1 (SEQ ID NO: 1). 9. The polynucleotide of claim 1, wherein the polynucleotide comprises the sequence from nucleotide 31 to nucleotide 1354 as shown in FIG. 1 (SEQ ID NO: 1). 10. The polynucleotide of claim 1, wherein the polynucleotide comprises the sequence from nucleotide 10 to nucleotide 1354 as shown in FIG. 1 (SEQ ID NO: 1). 11. A vector comprising the polynucleotide of claim 2. 12. A host cell comprising the vector of claim 11. 13. A method for producing a polypeptide comprising expressing a polypeptide encoded by said polynucleotide from the host cell of claim 12. 14. The method of claim 13, wherein said polypeptide comprises amino acids 1 to 234 as shown in Figure 2 (SEQ ID NO: 2). 15. The method of claim 13, wherein said polypeptide comprises amino acids 11 to 234 as shown in Figure 2 (SEQ ID NO: 2). 16. The method of claim 13, wherein the polypeptide comprises amino acids 4 to 234 as shown in Figure 2 (SEQ ID NO: 2). 17. A method for producing a polypeptide comprising the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), comprising: (a) expressing the host cell of claim 12 in which the polypeptide is expressed. Culturing under the following conditions: and (b) recovering the polypeptide from the culture. 18. A method of producing a cell expressing a polypeptide, which comprises:
A method comprising genetically engineering said cell with the vector of claim 11. 19. (a) A polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment of biological or immunological activity thereof; (b) FIG. 2 (SEQ ID NO: 2). 2) A polypeptide comprising amino acids 11 to 234 as shown in FIG. 2); (c) A polypeptide comprising amino acids 4 to 234 as shown in FIG. 2 (SEQ ID NO: 2); (d) Diagram 2 (SEQ ID NO: 2) as shown in amino acid 21 to amino acid 36; (e) Figure 2 (SEQ ID NO: 2) as shown in amino acid 78 to amino acid 91. (F) a polypeptide comprising amino acids 159 to 172 as shown in Figure 2 (SEQ ID NO: 2); (g) comprising amino acids 221 to 234 as shown in Figure 2 (SEQ ID NO: 2) Comprised of Polypepchi And (h) a polypeptide which is at least 70% identical to the polypeptide of (a), (b), (c), (d), (e), (f) or (g) above. A polypeptide comprising a member selected from the group. 20. The polypeptide of claim 19, wherein the polypeptide comprises amino acids 1 to 234 as shown in Figure 2 (SEQ ID NO: 2). 21. The polypeptide of claim 19, wherein said polypeptide comprises amino acids 11 to 234 as shown in Figure 2 (SEQ ID NO: 2). 22. The polypeptide of claim 19, wherein said polypeptide comprises amino acid 4 to amino acid 234 as shown in Figure 2 (SEQ ID NO: 2). 23. (a) A polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment of biological or immunological activity thereof; (b) FIG. 2 (SEQ ID NO :). 2) A polypeptide comprising amino acids 11 to 234 as shown in FIG. 2); (c) A polypeptide comprising amino acids 21 to 36 as shown in FIG. 2 (SEQ ID NO: 2); (d) Diagram 2 (SEQ ID NO: 2) comprising amino acids 78 to 91, (e) a polypeptide comprising amino acids 159 to 172 as shown in FIG. 2 (SEQ ID NO: 2); (F) a polypeptide comprising amino acids 221 to 234 as shown in FIG. 2 (SEQ ID NO: 2); and (g) above (a), (b), (c), (d), (e). ) Or (f) polypeptide Isolated antibody or antibody fragment that specifically binds to a polypeptide comprising a member selected from the group consisting of; polypeptide is 70% identical even without. 24. The antibody has the amino acid sequence SQPWQAALMENELF (SEQ ID NO: 10).
24. The antibody of claim 23, which specifically binds to. 25. The antibody has the amino acid sequence EASLSVRHPEYNRP (SEQ ID NO: 11).
24. The antibody of claim 23, which specifically binds to. 26. The antibody has the amino acid sequence SKLYDPLYHPSMF (SEQ ID NO: 12).
24. The antibody of claim 23, which specifically binds to. 27. The antibody has the amino acid sequence CKFTEWIEKTVQAS (SEQ ID NO: 13).
24. The antibody of claim 23, which specifically binds to. 28. The antibody according to claim 23, wherein the antibody is a polyclonal antibody. 29. The antibody according to claim 23, wherein the antibody is a monoclonal antibody. 30. (a) A polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment of biological or immunological activity thereof; (b) FIG. 2 (SEQ ID NO :). 2) A polypeptide comprising amino acids 11 to 234 as shown in FIG. 2); (c) A polypeptide comprising amino acids 21 to 36 as shown in FIG. 2 (SEQ ID NO: 2); (d) Diagram 2 (SEQ ID NO: 2) comprising amino acids 78 to 91, (e) a polypeptide comprising amino acids 159 to 172 as shown in FIG. 2 (SEQ ID NO: 2); (F) a polypeptide comprising amino acids 221 to 234 as shown in FIG. 2 (SEQ ID NO: 2); and (g) above (a), (b), (c), (d), (e). ) Or (f) polypeptide A polypeptide that is at least 70% identical; comprising an isolated antibody or antibody fragment that specifically binds to a polypeptide conjugated to a therapeutic agent, comprising a member selected from the group consisting of: Consisting of immunoconjugates. 31. The immunoconjugate of claim 30, wherein the therapeutic agent is a cytotoxic agent. 32. The cytotoxic agent is lysine, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinbrastin, colchicine, dihydroxyanthracin,
Dione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A,
32. The immunoconjugate of claim 31, selected from the group consisting of PE40, abrin, glucocorticoids and radioisotopes. 33. The immunoconjugate of claim 30, wherein the antibody fragment is selected from the group consisting of Fv, F (ab ') 2 and F (ab') ₂ fragments. 34. A method for selectively destroying cells expressing the polypeptide of FIG. 2 (SEQ ID NO: 2), which comprises the immunoconjugate according to claim 30 and the cells.
A method comprising reacting a therapeutic agent of the immunoconjugate so as to destroy the cell. 35. A method of treating a disease state associated with the expression of PROST 07 in a human patient, comprising administering to said patient a therapeutically effective amount of the immunoconjugate of claim 30. 36. (a) A polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment of biological or immunological activity thereof; (b) FIG. 2 (SEQ ID NO :). 2) a polypeptide comprising amino acids 11 to 234 as shown in FIG. 2); (c) a polypeptide comprising amino acids 4 to 234 as shown in FIG. 2 (SEQ ID NO: 2); and (d) At least 70% relative to the polypeptide of (a), (b) or (c) above
A method of treating a disease state associated with inappropriate expression of PROST 07 in a patient in need of reduced levels of a polypeptide comprising a member selected from the group consisting of: Specifically decomposes RNA encoding the polypeptide,
A method comprising administering to said patient a therapeutically effective amount of a ribozyme. 37. Treatment of a disease state associated with inappropriate expression of PROST 07 in a patient in need of reduced levels of a polypeptide having an amino acid sequence as shown in Figure 2 (SEQ ID NO: 2). A method comprising administering to said patient a therapeutically effective amount of a polynucleotide that is complementary to a polynucleotide encoding said polypeptide or a portion thereof. 38. The administered polynucleotide has the sequence CAACGGGUACUUGC.
38. The method of claim 37 having AGGGCCUUG (SEQ ID NO: 17). 39. The administered polynucleotide has the sequence ACCUCUGCAAAUUC.
38. The method of claim 37 having ACUGAGUGG (SEQ ID NO: 18). 40. The administered polynucleotide has the sequence UACUUGCAGGGCCU
38. The method of claim 37 having UGUGUCUUU (SEQ ID NO: 19). 41. (a) A polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment thereof of biological or immunological activity; (b) FIG. 2 (SEQ ID NO: 2). 2) A polypeptide comprising amino acids 11 to 234 as shown in FIG. 2); (c) A polypeptide comprising amino acids 21 to 36 as shown in FIG. 2 (SEQ ID NO: 2); (d) Diagram 2 (SEQ ID NO: 2) comprising amino acids 78 to 91, (e) a polypeptide comprising amino acids 159 to 172 as shown in FIG. 2 (SEQ ID NO: 2); (F) a polypeptide comprising amino acids 221 to 234 as shown in FIG. 2 (SEQ ID NO: 2); and (g) above (a), (b), (c), (d), (e). ) Or (f) polypeptide Without even the polypeptide is 70% identical; for the presence of a polypeptide comprising a member selected from the group consisting of, a diagnostic method comprising analyzing a sample derived from a host. 42. Contacting the sample with an antibody or antibody fragment of claim 23, wherein the assay specifically binds to the polypeptide, and detects binding of the antibody to the polypeptide in the sample. 42. The method of claim 41, comprising: 43. (a) A polynucleotide encoding a polypeptide comprising an amino acid sequence as shown in FIG. 2 (SEQ ID NO: 2), or a fragment thereof having biological or immunological activity; (b) A polynucleotide encoding a polypeptide comprising amino acid 11 to amino acid 234 as shown in FIG. 2 (SEQ ID NO: 2); (c) amino acid 4 to amino acid 234 as shown in FIG. 2 (SEQ ID NO: 2) A polynucleotide encoding a polypeptide comprising; and (d) a polynucleotide which is complementary to the polynucleotide of (a), (b) or (c) above; for a member selected from the group consisting of: A polynucleotide that is 70% identical; and a diagnostic method comprising: analyzing for the presence of a polynucleotide comprising the polynucleotide. 44. A method for diagnosing metastasis associated with the polypeptide of Figure 2 (SEQ ID NO: 2) in a patient, comprising: (a) obtaining a tissue and / or fluid sample from the patient; 24. Contacting the sample with the antibody of claim 23; and (c) detecting binding of the polypeptide to the antibody in the sample. 45. The antibody is labeled with a compound selected from the group consisting of a radioactive label, an enzyme, a chromophore and a fluorescent agent to directly or indirectly produce a detectable signal. The method described.