Patent application title: TADG-15: an extracellular serine protease overexpossed in carcinomas
Inventors:
Timothy J. O'Brien (Little Rock, AR, US)
Hirotoshi Tanimoto (Kagawa, JP)
IPC8 Class: AA61K5110FI
USPC Class:
424 149
Class name: Drug, bio-affecting and body treating compositions radionuclide or intended radionuclide containing; adjuvant or carrier compositions; intermediate or preparatory compositions attached to antibody or antibody fragment or immunoglobulin; derivative
Publication date: 2011-09-01
Patent application number: 20110212023
Abstract:
The present invention provides DNA encoding a TADG-15 protein as well as
a TADG-15 protein. Also provided is a vector capable of expressing the
DNA of the present invention adapted for expression in a recombinant cell
and regulatory elements necessary for expression of the DNA in the cell.
The present invention further provides for methods of inhibiting TADG-15
expression and/or protease activity, methods of detecting TADG-15 mRNA
and/or protein and methods of screening for TADG-15 inhibitors.
Additionally, the present invention provides for cell-specific targeting
via TADG-15 and methods of vaccinating an individual against TADG-15. The
methods described are useful in the diagnosis, treatment and prevention
of cancer, particularly breast and ovarian cancer.Claims:
1. A vector comprising a DNA encoding a tumor antigen-derived gene
(TADG-15) protein and regulatory elements necessary for expression of the
DNA in a cell; wherein the DNA is positioned in reverse orientation
relative to the regulatory elements such that TADG-15 antisense mRNA is
produced, wherein the DNA is: (a) isolated DNA which encodes a TADG-15
protein; (b) isolated DNA which hybridizes under high stringency
conditions to the isolated DNA of (a) above and which encodes a TADG-15
protein; or (c) isolated DNA differing from the isolated DNAs of (a) and
(b) above in codon sequence due to the degeneracy of the genetic code,
and which encodes a TADG-15 protein.
2. The vector of claim 1, wherein the DNA has the sequence shown in SEQ ID NO: 1.
3. The vector of claim 1, wherein the TADG-15 protein has the amino acid sequence shown in SEQ ID NO: 2.
4. A host cell transfected with the vector of claim 1.
5. The host cell of claim 4, wherein the cell is a bacterial cell, a mammalian cell, a plant cell, or an insect cell.
6. The host cell of claim 5, wherein the bacterial cell is E. coli.
7. A method of inhibiting expression of TADG-15 in a cell, comprising: introducing the vector of claim 1 into a cell, wherein expression of the vector produces TADG-15 antisense mRNA in the cell, wherein the TADG-15 antisense mRNA hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
8. An oligonucleotide having the nucleotide sequence complementary to the DNA sequence of claim 1.
9. A method of treating a neoplastic state in an individual in need of such treatment, comprising: administering to the individual an effective dose of the oligonucleotide of claim 8.
10. The method of claim 9, wherein the neoplastic state is ovarian cancer, breast cancer, lung cancer, colon cancer, prostate cancer, or other cancers in which TADG-15 is overexpressed.
11. A method of inhibiting a TADG-15 protein in a cell, comprising: introducing an antibody into a cell, wherein the antibody is specific for a TADG-15 protein or a fragment thereof, wherein binding of the antibody to the TADG-15 protein inhibits the TADG-15 protein.
12. The method of claim 11, wherein the antibody is linked to a therapeutic moiety, said method further comprising targeting the therapeutic moiety to cells in an individual.
13. The method of claim 12, wherein the therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant, or a cytotoxic agent.
14. The method of claim 12, wherein the individual suffers from ovarian cancer, lung cancer, prostate cancer, colon cancer, or other cancers in which TADG-15 is overexpressed.
15. An immunogenic composition, comprising an immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
16. The immunogenic composition of claim 15, wherein the fragment is a 9-residue fragment up to a 20-residue fragment.
17. The immunogenic composition of claim 16, wherein the 9-residue fragment has a sequence shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89, or 90.
18. A method of vaccinating an individual against TADG-15, comprising: inoculating an individual with the TADG-15 protein or immunogenic fragment thereof of claim 15, wherein the TADG-15 protein or immunogenic fragment thereof lacks TADG-15 protease activity, wherein the inoculation with the TADG-15 protein or immunogenic fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-15.
19. The method of claim 18, wherein the individual has cancer, is suspected of having cancer or is at risk of getting cancer.
20. A method of screening for compounds that inhibit TADG-15, comprising: (a) contacting a sample with a compound, wherein the sample comprises TADG-15 protein; and (b) assaying for TADG-15 protease activity, wherein a decrease in the TADG-15 protease activity in the presence of the compound relative to TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of pending U.S. Ser. No. 11/978,259, filed Oct. 29, 2007, which is a divisional of U.S. Ser. No. 10/600,187, filed Jun. 20, 2003, now U.S. Pat. No. 7,291,462, which is a divisional of U.S. Ser. No. 09/654,600, filed Sep. 1, 2000, now U.S. Pat. No. 6,649,741, which is a divisional of U.S. Ser. No. 09/421,213, filed Oct. 20, 1999, now U.S. Pat. No. 7,022,821, which is a continuation-in-part of U.S. Ser. No. 09/027,337, filed Feb. 20, 1998, now U.S. Pat. No. 5,972,616 and thereby claim the benefit of priority under 35 USC §120 and the entirety of all of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of cellular biology and the diagnosis of neoplastic disease. More specifically, the present invention relates to an extracellular serine protease, termed tumor antigen-derived gene 15 (TADG-15), which is overexpressed in carcinomas.
[0004] 2. Description of the Related Art
[0005] Extracellular proteases have been directly associated with tumor growth, shedding of tumor cells and invasion of target organs. Individual classes of proteases are involved in, but not limited to, (a) digestion of stroma surrounding the initial tumor area, (b) digestion of the cellular adhesion molecules to allow dissociation of tumor cells; and (c) invasion of the basement membrane for metastatic growth and activation of both tumor growth factors and angiogenic factors.
[0006] In the process of cancer progression and invasion, proteases mediate specific proteolysis and contribute to the removal of extracellular matrix components surrounding tumor cells, the digestion of intercellular adhesion molecules to allow dissociation of malignant cells and the activation of many growth and angiogenic factors (1-3). Depending on the nature of their catalytic domain, proteases are classified into four families: serine proteases, metalloproteases, aspartic proteases and cysteine proteases (3). Among these proteases, the metalloproteases have been well studied in relation to tumor growth and progression, and they are known to be capable of degrading the extracellular matrix, thereby enhancing the invasive potential of malignant cells (1, 4-5). For serine proteases, previous studies have demonstrated an increased production of plasminogen activator in tumor cells and a positive correlation between plasminogen activator activity and aggressiveness of cancer (6-7). Prostate specific antigen (a serine protease) has also been widely used as an indicator of abnormal prostate growth (8). More recently, several other serine proteases have been reported, viz. hepsin and the stratum corneum chymotryptic enzyme (SCCE), which are overexpressed in ovarian cancer and which may contribute to malignant progression by increasing the extracellular lytic activity of these tumor cells (9).
[0007] The prior art is deficient in the lack of effective means of screening to identify proteases overexpressed in carcinoma. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
[0008] The present invention discloses a screening program to identify proteases overexpressed in carcinoma by examining PCR products amplified using differential display in early stage tumors and metastatic tumors compared to that of normal tissues. The approach herein to identify candidate genes overexpressed in tumor cells has been to utilize the well conserved domains surrounding the triad of amino acids (His-Asp-Ser) prototypical of the catalytic domain of serine proteases. Herein, evidence is presented for a unique form of serine protease not previously described in the literature which is highly expressed in ovarian carcinomas. Through the screening approach using differential PCR amplification of normal, low malignant potential and overt carcinomas, a PCR product present only in carcinoma was subcloned and sequenced, and was found to have a catalytic domain which was consistent with the serine protease family. Reported herein is the complete cloning and sequencing of this transcript and evidence for its expression in ovarian tumor cells.
[0009] In one embodiment of the present invention, there is provided a DNA encoding a tumor antigen-derived gene (TADG-15) protein, selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein. The embodiment further includes a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell. Additionally embodied is a vector in which the TADG-15 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-15 antisense mRNA is produced.
[0010] In another embodiment of the present invention, there is provided an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein.
[0011] In yet another embodiment of the present invention, there is provided a method for detecting TADG-15 mRNA in a sample, comprising the steps of: (a) contacting a sample with a probe which is specific for TADG-15; and (b) detecting binding of the probe to TADG-15 mRNA in the sample. In still yet another embodiment of the present invention, there is provided a kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe specific for TADG-15. A label for detection is further embodied in the kit.
[0012] The present invention additionally embodies a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of the antibody to TADG-15 protein in the sample. Similarly, the present invention also embodies a kit for detecting TADG-15 protein, comprising: an antibody specific for TADG-15 protein or a fragment thereof. Means for detection of the antibody is further embodied in the kit.
[0013] In another embodiment, the present invention provides an antibody specific for the TADG-15 protein or a fragment thereof.
[0014] In yet another embodiment, the present invention provides a method of screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample comprising TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity. Typically, a decrease in the TADG-15 protease activity in the presence of the compound relative to TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
[0015] In still yet another embodiment of the present invention, there is provided a method of inhibiting expression of TADG-15 in a cell, comprising the step of: (a) introducing a vector into a cell, whereupon expression of the vector produces TADG-15 antisense mRNA in the cell which hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
[0016] Further embodied by the present invention, there is provided a method of inhibiting a TADG-15 protein in a cell, comprising the step of: (a) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, whereupon binding of the antibody to the TADG-15 protein inhibits the TADG-15 protein.
[0017] In an embodiment of the present invention, there is provided a method of targeted therapy to an individual, comprising the step of: (a) administering a compound containing a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15.
[0018] In an embodiment of the present invention, there is provided a method of diagnosing cancer in an individual, comprising the steps of: (a) obtaining a biological sample from an individual; and (b) detecting TADG-15 in the sample, wherein the presence of TADG-15 in the sample is indicative of the presence of carcinoma in the individual and the absence of TADG-15 in the sample is indicative of the absence of carcinoma in the individual.
[0019] In another embodiment of the present invention, there is provided a method of vaccinating an individual against TADG-15, comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof that lacks TADG-15 protease activity, wherein the inoculation with the TADG-15 protein or fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-15.
[0020] In an embodiment of the present invention, there is provided a method of producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG-15 protein or fragment thereof that lacks TADG-15 protease activity, wherein the exposure to said TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune-activated cells directed toward TADG-15.
[0021] In another embodiment of the present invention, there is provided an immunogenic composition, comprising an immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
[0022] Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
[0024] FIGS. 1A-1B show a comparison of the serine protease catalytic domain of TADG-15 with Hepsin (Heps, SEQ ID NO: 3), SCCE (SEQ ID NO: 4), Trypsin (Try, SEQ ID NO: 5), Chymotrypsin (Chymb, SEQ ID NO: 6), Factor 7 (Fac7, SEQ ID NO: 7) and Tissue plasminogen activator (Tpa, SEQ ID NO: 8). The asterisks indicate conserved amino acids of catalytic triad.
[0025] FIGS. 2A-2D show the nucleotide sequence of the TADG-15 cDNA and the derived amino acid sequence of the TADG-15 protein. The putative start codon is located at nucleotides 23-25. The potential transmembrane sequence is underlined. Possible N-linked glycosylation sites are indicated by a broken line. The asterisks indicate conserved cysteine residues of CUB domain. The SDE-motifs of the LDL receptor ligand binding repeat-like domain are boxed. The arrow shows the arginine-valine bond cleaved upon activation. The conserved amino acids of the catalytic triad; histidine, aspartic acid and serine residues are circled.
[0026] FIG. 3 shows the amino acid sequence of the TADG-15 protease, including functional sites and domains.
[0027] FIG. 4 shows a diagram of the TADG-15 protein. 1; cytoplasmic domain, (aa #1-54), 2; transmembrane domain (aa #55-57), 3; extracellular domain (aa #78-213), 4-5; CUB repeat (aa #214-447), 6-9; LDL receptor ligand binding repeat (class A motif) like domain (aa #453-602), 10; serine protease (aa #615-855).
[0028] FIG. 5 shows Northern blot analysis of TADG-15 mRNA expression in normal ovary, ovarian carcinomas, carcinoma cell lines, normal fetal tissues and normal adult tissues. A single intense transcript of the TADG-15 was observed in every sub-type of carcinoma and the two ovarian carcinoma cell lines, SW626 and CAOV3, whereas no visible band was detected in normal ovary or the two breast cancer cell lines. In normal fetal tissues, fetal kidney showed increased transcript and faint expression was detected in fetal lung. In normal adult tissues, the TADG-15 was detected in colon with low expression in small intestine and prostate.
[0029] FIG. 6A shows quantitative PCR analysis of TADG-15 expression. Expression levels of TADG-15 relative to β-tubulin are significantly elevated in all LMP tumors and carcinomas compared to that of normal ovaries. m; mucinous, s; serous. FIG. 6B shows the ratio of TADG-15 expression to expression of β-tubulin in normal ovary, LMP tumor and ovarian carcinoma. TADG-15 mRNA expression levels were significantly elevated in both LMP tumor (*; p<0.001) and carcinoma (**; p<0.0001) compared to that in normal ovary. All 10 samples of normal ovary showed a low level of TADG-15 expression.
[0030] FIG. 7 shows the TADG-15 expression in tumor cell lines derived from both ovarian and breast carcinoma tissues.
[0031] FIG. 8 shows the overexpression of TADG-15 in other tumor tissues.
[0032] FIG. 9 shows SW626 and CAOV3 cell lysates that were separated by SDS-PAGE and immunoblotted. Lanes 1 and 2 were probed with rabbit pre-immune serum as a negative control. Lanes 3 and 4 were probed with polyclonal rabbit antibody generated to a carboxy terminal peptide from TADG-15 protein sequence.
[0033] FIG. 10 shows that immunohistochemical staining of normal ovarian epithelium (FIG. 10A) with a polyclonal antibody to a TADG-15 protease peptide shows no staining of the stroma or epithelium. However, antibody staining of carcinomas confirms the presence of TADG-15 expression in the cytoplasm of a serous low malignant potential tumor (FIG. 10B); a mucinous low malignant potential tumor (FIG. 10C); a serous carcinoma (FIG. 10D); and its presence in both the cytoplasm and cell surface of an endometrioid carcinoma (FIG. 10E).
[0034] FIGS. 11A-11B show an alignment of the human TADG15 protein sequence with that of mouse epithin which demonstrates that the proteins are 84% similar and 81% identical over 843 amino acids. Residues that are identical between the two proteins are indicated by a "--", while the "*" symbol represents the TADG15 translation termination. The most significant difference between these two proteins lies in the carboxy-termini, which for epithin, includes 47 amino acids that are not present in TADG15.
[0035] FIGS. 12A-12E show a nucleotide sequence comparison between TADG-15 and human SNC-19 (GeneBank Accession No. U20428).
DETAILED DESCRIPTION OF THE INVENTION
[0036] Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, redundant primers directed towards conserved amino acid domains surrounding the catalytic triad of His, Asp and Ser were utilized to amplify serine proteases differentially expressed in carcinomas. Using this method, a serine protease named TADG-15 (tumor antigen-derived gene 15) has been identified that is overexpressed in ovarian tumors. TADG-15 appears to be a transmembrane multidomain serine protease. TADG-15 is highly overexpressed in ovarian tumors based on PCR, Northern blot and immunolocalization.
[0037] The TADG-15 cDNA is 3147 base pairs long (SEQ ID NO: 1) encoding for a 855 amino acid protein (SEQ ID NO: 2). The availability of the TADG-15 gene provides numerous utilities. For example, the TADG-15 gene can be used as a diagnostic or therapeutic target in ovarian and other carcinomas, including breast, prostate, lung and colon.
[0038] The present invention is directed to DNA encoding a tumor antigen-derived gene (TADG-15) protein, selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein. It is preferred that the DNA has the sequence shown in SEQ ID NO: 1 and the TADG-15 protein has the amino acid sequence shown in SEQ ID NO: 2.
[0039] The present invention is directed toward a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell, or a vector in which the TADG-15 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-15 antisense mRNA is produced. Generally, the DNA encodes a TADG-15 protein having the amino acid sequence shown in SEQ ID NO: 2. The invention is also directed toward host cells transfected with either of the above-described vector(s). Representative host cells are bacterial cells, mammalian cells, plant cells and insect cells. Preferably, the bacterial cell is E. coli.
[0040] The present invention is directed toward an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein. Preferably, the protein has the amino acid sequence shown in SEQ ID NO: 2.
[0041] The present invention is directed toward a method for detecting TADG-15 mRNA in a sample, comprising the steps of: (a) contacting a sample with a probe which is specific for TADG-15; and (b) detecting binding of the probe to TADG-15 mRNA in the sample. The present invention is also directed toward a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of the antibody to TADG-15 protein in the sample. Generally, the sample is a biological sample; preferably, the biological sample is from an individual; and typically, the individual is suspected of having cancer.
[0042] The present invention is directed toward a kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe, wherein the probe is specific for TADG-15. The kit may further comprise: a label with which to label the probe; and means for detecting the label. The present invention is additionally directed toward a kit for detecting TADG-15 protein, comprising: an antibody which is specific for TADG-15 protein or a fragment thereof. The kit may further comprise: means to detect the antibody.
[0043] The present invention is directed toward a antibody which is specific for TADG-15 protein or a fragment thereof.
[0044] The present invention is directed toward a method of screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample containing TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity. Typically, a decrease in the TADG-15 protease activity in the presence of the compound relative to TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
[0045] The present invention is directed toward a method of inhibiting expression of TADG-15 in a cell, comprising the step of: (a) introducing a vector expressing TADG-15 antisense mRNA into a cell, which hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell. Generally, the inhibition of TADG-15 expression is for treating cancer.
[0046] The present invention is directed toward a method of inhibiting a TADG-15 protein in a cell, comprising the step of: (a) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, which inhibits the TADG-15 protein. Generally, the inhibition of the TADG-15 protein is for treating cancer.
[0047] The present invention is directed toward a method of targeted therapy to an individual, comprising the step of: (a) administering a compound having a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15. Representative targeting moiety are an antibody specific for TADG-15 and a ligand or ligand binding domain (e.g., CUB, LDLR, protease and extracellular) that binds TADG-15. Likewise, a representative therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent and immune stimulants. Typically, the above-described method is useful when the individual suffers from ovarian cancer, breast cancer or cancers of the prostate, lung, colon and cervix.
[0048] The present invention is directed toward a method of diagnosing cancer in an individual, comprising the steps of: (a) obtaining a biological sample from an individual; and (b) detecting TADG-15 in the sample. Generally, the presence of TADG-15 in the sample is indicative of the presence of carcinoma in the individual, and the absence of TADG-15 in the sample is indicative of the absence of carcinoma in the individual. Generally, the biological sample is blood, ascites fluid, urine, tears, saliva or interstitial fluid. Typical means of detecting TADG-15 are by Northern blot, Western blot, PCR, dot blot, ELIZA, radioimmunoassay, DNA chips or tumor cell labeling. This method may be useful in diagnosing cancers such as ovarian, breast and other cancers in which TADG-15 is overexpressed, such as lung, prostate and colon cancers.
[0049] The present invention is also directed to an antisense oligonucleotide having the nucleotide sequence complementary to a TADG-15 mRNA sequence. The present invention is also directed to a composition comprising such an antisense oligonucleotide according and a physiologically acceptable carrier therefore.
[0050] The present invention is also directed to a method of treating a neoplastic state in an individual syndrome in an individual in need of such treatment, comprising the step of administering to said individual an effective dose of an antisense oligonucleotide of. Preferably, the neoplastic state is selected from the group consisting of from ovarian cancer, breast cancer, lung cancer, prostate cancer, colon cancer and other cancers in which TADG-15 is overexpressed. For such therapy, the oligonucleotides alone or in combination with other anti-neoplastic agents can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The oligonucleotide active ingredient is generally combined with a pharamceutically acceptable carrier such as a diluent or excipient which can include fillers, extenders, binders, wetting agents, disintergrants, surface active agents or lubricants, depending on the nature of the mode of administration and dosage forms. Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions, and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
[0051] For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For injection, the oligonucleotides of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers. In addition, the oligonucleotides can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also incldued. Dosages that can be used for systemic administration preferably range from about 0.01 mg/kg to 50 mg/kg administered once or twice per day. However, different dosing schedules can be utilized depending on (1) the potency of an individual oligonucleotide at inhibiting the activity of its target DNA, (2) the severity or extent of the pathological disease state, or (3) the pharmacokinetic behavior of a given oligonucleotide.
[0052] The present invention is directed toward a method of vaccinating an individual against TADG-15 overexpression, comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof which lacks TADG-15 protease activity. The inoculation with the TADG-15 protein or fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-15. The vaccination with TADG-15 described herein is intended for an individual who has cancer, is suspected of having cancer or is at risk of getting cancer. Generally, the TADG-15 fragment useful for vaccinating an individual are 9-residue fragments up to 20-residue fragments, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
[0053] The present invention is directed toward a method of producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG-15 protein or fragment thereof that lacks TADG-15 protease activity, wherein exposure to the TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune-activated cells directed toward TADG-15. Representative immune-activated cells are B-cells, T-cells and dendrites. Generally, the TADG-15 fragment is a 9-residue fragment up to a 20-residue fragment, with preferable 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90. Preferably, the dendritic cells are isolated from an individual prior to exposure, and the activated dendritic cells reintroduced into the individual subsequent to exposure. Typically, the individual for which this method may apply has cancer, is suspected of having cancer or is at risk of getting cancer.
[0054] The present invention is directed toward an immunogenic composition, comprising an immunogenic fragment of a TADG-15 protein and an appropriate adjuvant. Generally, the fragment is a 9-residue fragment up to a 20-residue fragment, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
[0055] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. 1985); "Transcription and Translation" (B. D. Hames & S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I. Freshney, ed. 1986); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To MolecularCloning" (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.
[0056] As used herein, the term "cDNA" shall refer to the DNA copy of the mRNA transcript of a gene.
[0057] As used herein, the term "derived amino acid sequence" shall mean the amino acid sequence determined by reading the triplet sequence of nucleotide bases in the cDNA.
[0058] As used herein the term "screening a library" shall refer to the process of using a labeled probe to check whether, under the appropriate conditions, there is a sequence complementary to the probe present in a particular DNA library. In addition, "screening a library" could be performed by PCR.
[0059] As used herein, the term "PCR" refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, as well as other improvements now known in the art.
[0060] The amino acid described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are used as in customary in the art.
[0061] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
[0062] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
[0063] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "vector" may further be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid.
[0064] A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single-stranded form or as a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. The structure is discussed herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
[0065] An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See, for example, techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being "operably linked" if the transcription control sequences effectively control transcription of the gene. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses. In general, expression vectors contain promoter sequences which facilitate the efficient transcription of the inserted DNA fragment and are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
[0066] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.
[0067] A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
[0068] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
[0069] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters typically contain Shine-Dalgarno ribosome-binding sequences in addition to the -10 and -35 consensus sequences.
[0070] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
[0071] A "signal sequence" can be included near the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
[0072] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
[0073] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0074] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
[0075] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
[0076] The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, b-glucuronidase, b-D-glucosidase, b-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
[0077] A particular assay system developed and utilized in the art is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the label after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
[0078] An assay useful in the art is known as a "cis/trans" assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex. Thus, for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Pat. No. 4,981,784.
[0079] As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes a human TADG-15 protein of the present invention can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for the gene which encodes a human TADG-15 protein of the present invention for purposes of prokaryote transformation. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.
[0080] The invention includes a substantially pure DNA encoding a TADG-15 protein, a DNA strand which will hybridize at high stringency to a probe containing a sequence of at least 15 consecutive nucleotides of (SEQ ID NO: 1). The protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably 85%, more preferably 90%, and most preferably 95%) with the amino acids listed in FIGS. 3 and 4 (SEQ ID NO: 2). More preferably, the DNA includes the coding sequence of the nucleotides of FIG. 2 (SEQ ID NO: 1), or a degenerate variant of such a sequence. This invention also includes a substantially pure DNA containing a sequence of at least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region from nucleotides 1 to 3147 of the nucleotides shown in FIGS. 2A-2D (SEQ ID NO: 1).
[0081] By "substantially pure DNA" is meant DNA that is not part of a milieu in which the DNA naturally occurs, by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in FIGS. 2A-2D (SEQ ID NO: 1) and which encodes an alternative splice variant of TADG-15.
[0082] By a "substantially pure protein" is meant a protein which has been separated from at least some of those components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60% (by weight) free from the proteins and other naturally-occurring organic molecules with which it is naturally associated in vivo. Preferably, the purity of the preparation (by weight) is at least 75%, more preferably at least 90%, and most preferably at least 99%. A substantially pure TADG-15 protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a TADG-15 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, such as immunoaffinity chromatography using an antibody specific for TADG-15, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they do not naturally occur.
[0083] The term "oligonucleotide", as used herein, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer", as used herein, refers to an oligonucleotide, whether occurring naturally (as in a purified restriction digest) or produced synthetically, and which is capable of initiating synthesis of a strand complementary to a nucleic acid when placed under appropriate conditions, i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, sequence and/or homology of primer and the method used. For example, in diagnostic applications, the oligonucleotide primer typically contains 15-25 or more nucleotides, depending upon the complexity of the target sequence, although it may contain fewer nucleotides.
[0084] The primers herein are selected to be "substantially" complementary to particular target DNA sequences. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment (i.e., containing a restriction site) may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product.
[0085] The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the coding sequence of the nucleotides listed in FIGS. 2A-2D (SEQ ID NO: 1) or the complement thereof. Such a probe is useful for detecting expression of TADG-15 in a cell by a method including the steps of (a) contacting mRNA obtained from the cell with a labeled TADG-15 hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
[0086] By "high stringency" is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42° C. in the presence of about 50% formamide; a first wash at about 65° C. with about 2×SSC containing 1% SDS; followed by a second wash at about 65° C. with about 0.1×SSC.
[0087] The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in FIGS. 2A-2D (SEQ ID NO: 1), preferably at least 75% (e.g., at least 80%); and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching or identical positions. When a position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, VVI 53705).
[0088] The present invention comprises a vector comprising a DNA sequence which encodes a human TADG-15 protein, wherein said vector is capable of replication in a host, and comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said TADG-15 protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID NO: 1. Vectors may be used to amplify and/or express nucleic acid encoding a TADG-15 protein or fragment thereof.
[0089] In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) of the TADG-15 protein (SEQ ID NO: 2). As used herein, "fragment," as applied to a polypeptide, will ordinarily be at least 6 residues, more typically at least 9-12 residues, and preferably at least 13-20 residues in length, but less than the entire, intact sequence. Alternatively, a fragment may be an individual domain of 20-120 residues from SEQ ID NO: 2. Fragments of the TADG-15 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant TADG-15 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of TADG-15, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of TADG-15 (e.g., binding to an antibody specific for TADG-15) can be assessed by methods described herein. Purified TADG-15 or antigenic fragments of TADG-15 can be used to generate new antibodies or to test existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention is polyclonal antisera generated by using TADG-15 or a fragment of TADG-15 as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant TADG-15 cDNA clones, and to distinguish them from other cDNA clones.
[0090] Further included in this invention are TADG-15 proteins which are encoded, at least in part, by portions of SEQ ID NO: 2, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of TADG-15 sequence has been deleted. The fragment, or the intact TADG-15 polypeptide, may be covalently linked to another polypeptide, e.g., one which acts as a label, a ligand or a means to increase antigenicity.
[0091] The invention also includes a polyclonal or monoclonal antibody which specifically binds to TADG-15. The invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin.
[0092] In one embodiment, the antibody, or a fragment thereof, may be linked to a toxin or to a detectable label, e.g., a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or colorimetric label. Examples of suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc. Examples of suitable radioisotopic labels include 3H, 125I, 131P, 35S, 14C, etc.
[0093] Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. There are numerous examples of elements that are useful in magnetic resonance imaging. For discussions on in vivo nuclear magnetic resonance imaging, see, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986) Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys. Med. NMR 16, 93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR 16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examples of suitable fluorescent labels include a fluorescein label, an isothiocyalate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an ophthaldehyde label, a fluorescamine label, etc. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
[0094] Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known and used by those of ordinary skill in the art. Typical techniques are described by Kennedy et al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al., (1977) Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method. All of these methods are incorporated by reference herein.
[0095] Also within the invention is a method of detecting TADG-15 protein in a biological sample, which includes the steps of contacting the sample with the labeled antibody, e.g., radioactively tagged antibody specific for TADG-15, and determining whether the antibody binds to a component of the sample. Antibodies to the TADG-15 protein can be used in an immunoassay to detect increased levels of TADG-15 protein expression in tissues suspected of neoplastic transformation. These same uses can be achieved with Northern blot assays and analyses.
[0096] As described herein, the invention provides a number of diagnostic advantages and uses. For example, the TADG-15 protein is useful in diagnosing cancer in different tissues since this protein is highly overexpressed in tumor cells. Antibodies (or antigen-binding fragments thereof) which bind to an epitope specific for TADG-15, are useful in a method of detecting TADG-15 protein in a biological sample for diagnosis of cancerous or neoplastic transformation. This method includes the steps of obtaining a biological sample (e.g., cells, blood, plasma, tissue, etc.) from a patient suspected of having cancer, contacting the sample with a labeled antibody (e.g., radioactively tagged antibody) specific for TADG-15, and detecting the TADG-15 protein using standard immunoassay techniques such as an ELISA. Antibody binding to the biological sample indicates that the sample contains a component which specifically binds to an epitope within TADG-15.
[0097] Likewise, a standard Northern blot assay can be used to ascertain the relative amounts of TADG-15 mRNA in a cell or tissue obtained from a patient suspected of having cancer, in accordance with conventional Northern hybridization techniques known to those of ordinary skill in the art. This Northern assay uses a hybridization probe, e.g., radiolabelled TADG-15 cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID NO: 1 (FIG. 2), or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The DNA hybridization probe can be labeled by any of the many different methods known to those skilled in this art.
[0098] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
Example 1
Tissue Collection and Storage
[0099] Upon patient hysterectomy, bilateral salpingo-oophorectomy, or surgical removal of neoplastic tissue, the specimen is retrieved and placed on ice. The specimen was then taken to the resident pathologist for isolation and identification of specific tissue samples. Finally, the sample was frozen in liquid nitrogen, logged into the laboratory record and stored at -80° C.
[0100] Additional specimens were frequently obtained from the Cooperative Human Tissue Network (CHTN). These samples were prepared by the CHTN and shipped on dry ice. Upon arrival, these specimens (e.g., blood (serum), urine, saliva, tears and insterstitial fluid) were logged into the laboratory record and stored at -80° C. Participation of the following divisions of the Cooperative Human Tissue Network (CHTN) in providing tumor tissues is acknowledged: Western Division, Case Western Reserve University, (Cleveland, Ohio); Midwestern Division, Ohio state University, (Columbus, Ohio); Eastern Division, NDRI, (Philadelphia, Pa.); Pediatric Division, Children's Hospital, (Columbus, Ohio); Southern Division, University of Alabama at Birmingham, (Birmingham, Ala.).
Example 2
mRNA Isolation and cDNA Synthesis
[0101] Forty-one ovarian tumors (10 low malignant potential tumors and 31 carcinomas) and 10 normal ovaries were obtained from surgical specimens and frozen in liquid nitrogen. The human ovarian carcinoma cell lines SW626 and CAOV3, and the human breast carcinoma cell lines MDA-MB-231 and MDA-MB-435S, were purchased from the American Type Culture Collection (Rockville, Md.). Cells were cultured to sub-confluency in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and antibiotics.
[0102] Messenger RNA (mRNA) isolation was performed according to the manufacturer's instructions using the Mini RiboSep® Ultra mRNA Isolation Kit purchased from Becton Dickinson. In this procedure, polyA.sup.+ mRNA was isolated directly from the tissue lysate using the affinity chromatography media oligo(dT) cellulose. The amount of mRNA recovered was quantitated by UV spectrophotometry.
[0103] First-strand complementary DNA (cDNA) was synthesized using 5.0 μg of mRNA and either random hexamer or oligo(dT) primers according to the manufacturer's protocol utilizing a first strand synthesis kit obtained from CLONTECH (Palo Alto, Calif.). The purity of the cDNA was evaluated by PCR using primers specific for the p53 gene. These primers span an intron such that pure cDNA can be distinguished from cDNA that is contaminated with genomic DNA.
Example 3
PCR with Redundant Primers, Cloning of TADG-15 cDNA, T-Vector Ligation and Transformations and DNA Sequencing
[0104] Redundant primers, forward 5'-TGGGTIGTIACIGCIGCICA(C/T)TG-3' (SEQ ID NO: 11) and reverse 5'-A(A/G)IGGICCICCI(C/G)(T/A)(A/G)TCICC-3' (SEQ ID NO: 12), corresponding to the amino acids surrounding the catalytic triad for serine proteases, were used to compare the PCR products from normal and carcinoma cDNAs.
[0105] The purified PCR products were ligated into the Promega T-vector plasmid and the ligation products used to transform JM109 competent cells according to the manufacturer's instructions (Promega). Positive colonies were cultured for amplification, the plasmid DNA isolated using the Wizard® Minipreps DNA purification system (Promega), and the plasmids were digested with ApaI and SacI restriction enzymes to determine the size of the insert. Plasmids with inserts of the size(s) visualized by the previously described FCR product gel electrophoresis were sequenced.
[0106] Individual colonies were cultured and plasmid DNA was isolated using the Wizard Miniprep DNA purification system (Promega). Applied Biosystems Model 373A DNA sequencing system was used for direct cDNA sequence determination. Utilizing a plasmid-specific primer near the cloning site, sequencing reactions were carried out using PRISM' Ready Reaction Dye Deoxy® terminators (Applied Biosystems) according to the manufacturer's instructions. Residual dye terminators were removed from the completed sequencing reaction using a Centri-sep® spin column (Princeton Separation). Based upon the determined sequence, primers that specifically amplify the gene of interest were designed and synthesized.
[0107] The original TADG-15 subclone (436 bp) was randomly labeled and used as a probe to screen an ovarian tumor cDNA library by standard hybridization techniques (13). The library was constructed in 8ZAP using mRNA isolated from, the tumor cells of a stage III/grade III ovarian adenocarcinoma patient. Three overlapping clones were obtained which spanned 3147 nucleotides.
Example 4
Northern Blot Analysis
[0108] 10 mg mRNAs were size separated by electrophoresis through a 1% formaldehyde-agarose gel in 0.02 M MOPS, 0.05 M sodium acetate (pH 7.0), and 0.001 M EDTA. The mRNAs were then blotted to Hybond-N.sup.+ nylon membrane (Amersham) by capillary action in 20×SSPE. The RNAs are fixed to the membrane by baking for 2 hours at 80° C. 32P-labeled cDNA probes were made by Prime-a-Gene Labeling System (Promega). The PCR products amplified by the same primers described above were used for probes. The blots were prehybridized for 30 min and hybridized for 60 min at 68° C. with 32P-labeled cDNA probe in ExpressHyb Hybridization Solution (CLONTECH). Control hybridization to determine relative gel loading was performed with a β-tubulin probe.
[0109] Normal human tissues; spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte, and normal human fetal tissues; brain, lung, liver and kidney (Human Multiple Tissue Northern Blot; CLONTECH) were also examined by the same hybridization procedure. Additional multiple tissue northern (MTN) blots from CLONTECH include the Human MTN blot, the Human MTN II blot, the Human Fetal MTN II blot, and the Human Brain MTN III blot.
Example 5
Western Blot Analysis
[0110] Polyclonal rabbit antibody was generated by immunization with a poly-lysine linked multiple Ag peptide derived from the TADG-15 protein sequence `LFRDWIKENTGV` (SEQ ID NO: 13). Approximately 20 μg of cell lysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDF at 100 V for 40 min at 4° C. The proteins were fixed to the membrane by incubation in 50% MeOH for 10 min. The membrane was blocked overnight in TBS (pH 7.8) containing 0.2% non-fat milk. Primary antibody was added to the membrane at a dilution of 1:100 in 0.2% milk/TBS and incubated for 2 h at room temperature. The blot was washed and incubated with a 1:3000 dilution of alkaline-phosphatase conjugated goat anti-rabbit IgG (BioRad) for 1 h at room temperature. The blot was washed and incubated with a chemiluminescent substrate before a 10 sec exposure to X-ray film for visualization.
Example 6
Quantitative PCR
[0111] The mRNA overexpression of TADG-15 was determined using a quantitative PCR. Quantitative PCR was performed.11,12 Oligonucleotide primers were used for TADG-15: forward 5'-ATGACAGAGGATTCAGGTAC-3' (SEQ ID NO: 14) and reverse 5'-GAAGGTGAA GTCATTGAAGA-3' (SEQ ID NO: 15); and for β-tubulin: forward 5'-CGCATCAACGTGTACTAC AA-3' (SEQ ID NO: 16) and reverse 5'-TACGAGCTGGTGGACTGAGA-3' (SEQ ID NO: 17). β-tubulin was utilized as an internal control.
[0112] The PCR reaction mixture consists of cDNA derived from 50 ng of mRNA, 5 μmol of sense and antisense primers for both the TADG-15 gene and the β-tubulin gene, 200 μmol of dNTPs, 5 μCi of α-32PdCTP and 0.25 units of Taq DNA polymerase with reaction buffer (Promega) in a final volume of 25 μl. The target sequences were amplified in parallel with the β-tubulin gene. Thirty cycles of PCR were carried out in a Thermal Cycler (Perkin Elmer Gene Amp 2400; Perkin-Elmer Cetus). Each cycle of PCR included 30 sec of denaturation at 94° C., 30 sec of annealing at 60° C. and 30 sec of extension at 72° C. The annealing temperature varies according to the primers that are used in the PCR reaction. For the reactions involving degenerate primers, an annealing temperature of 48° C. was used. The appropriate annealing temperature for the TADG-15- and b-tubulin-specific primers is 62° C.
[0113] A portion of the PCR products were separated on 2% agarose gels and the radioactivity of each PCR product was determined by using a Phospholmager (Molecular Dynamics). In the present study, the expression ratio (TADG-15/13-tubulin) was used to evaluate gene expression and defined the value at mean±2SD of normal ovary as the cut-off value to determine overexpression. The student's t test was used for comparison of the mean values of normal ovary and tumors.
Example 7
Immunohistochemistry
[0114] Immunohistochemical staining was performed using a Vectastain Elite ABC Kit (Vector). Formalin-fixed and paraffin-embedded specimens were routinely deparaffinized and processed using microwave heat treatment in 0.01 M sodium citrate buffer (pH 6.0). The specimens were incubated with normal goat serum in a moist chamber for 30 min. After incubation with biotinylated anti-rabbit IgG for 30 min, the sections were then incubated with ABC reagent (Vector) for 30 min. The final products were visualized using the AEC substrate system (DAKO) and sections were counterstained with hematoxylin before mounting. Negative controls were performed using normal serum instead of the primary antibody.
Example 8
Antisense TADG-15
[0115] TADG-15 is cloned and expressed in the opposite orientation such that an antisense RNA molecule (SEQ ID NO: 18) is produced. For example, the antisense RNA is used to hybridize to the complementary RNA in the cell and thereby inhibit translation of TADG-15 RNA into protein.
Example 9
Peptide Ranking
[0116] For vaccine or immune stimulation, individual 9-mers to 11-mers were examined to rank the binding of individual peptides to the top 8 haplotypes in the general population (24). The computer program used for this analyses can be found at <www-bimas.dcrt.nih.gov/molbio/hla_ bind/>. Table 1 shows the peptide ranking based upon the predicted half-life of each peptide's binding to a particular HLA allele. A larger half-life indicates a stronger association with that peptide and the particular HLA molecule. The TADG-15 peptides that strongly bind to an HLA allele are putative immunogens, and are used to innoculate an individual against TADG-15.
TABLE-US-00001 TABLE 1 TADG-15 peptide ranking HLA Type Predicted SEQ & Ranking Start Peptide Dissociation1/2 ID No. HLA A0201 1 68 VLLGIGFLV 2537.396 19 2 126 LLYSGVPFL 1470.075 20 3 644 SLISPNWLV 521.640 21 4 379 KVSFKFFYL 396.525 22 5 386 YLLEPGVPA 346.677 23 6 257 SLTFRSFDL 123.902 24 7 762 ILQKGEIRV 118.238 25 8 841 RLPLFRDWI 106.842 26 9 64 GLLLVLLGI 88.783 27 10 57 VLAAVLIGL 83.527 28 HLA A0205 1 67 LVLLGIGFL 142.800 29 2 379 KVSFKFFYL 100.800 30 3 126 LLYSGVPFL 71.400 31 4 88 KVFNGYMRI 36.000 32 5 670 TQWTAFLGL 33.600 33 6 119 KVKDALKLL 25.200 34 7 60 AVLIGLLLV 24.000 35 8 62 LIGLLLVLL 23.800 36 9 57 VLAAVLIGL 23.800 37 10 61 VLIGLLLVL 23.800 38 HLA A1 1 146 FSEGSVIAY 337.500 39 2 658 YIDDRGFRY 125.000 40 3 449 SSDPCPGQF 75.000 41 4 401 YVEINGEKY 45.000 42 5 387 LLEPGVPAG 18.000 43 6 553 GSDEASCPK 15.000 44 7 97 TNENFVDAY 11.250 45 8 110 STEFVSLAS 11.250 46 9 811 SVEADGRIF 9.000 47 10 666 YSDPTQWTA 7.500 48 HLA A24 1 709 DYDIALLEL 220.000 49 2 408 KYCGERSQF 200.000 50 3 754 QYGGTGALI 50.000 51 4 153 AYYWSEFSI 50.000 52 5 722 EYSSMVRPI 50.000 53 6 326 GFEATFFQL 36.000 54 7 304 TFHSSQNVL 24.000 55 8 707 TFDYDIALL 20.000 56 9 21 KYNSRHEKV 16.500 57 10 665 RYSDPTQWT 14.400 58 HLA B7 1 686 APGVQERRL 240.000 59 2 12 GPKDFGAGL 80.000 60 3 668 DPTQWTAFL 80.000 61 4 461 TGRCIRKEL 60.000 62 5 59 AAVLIGLLL 36.000 63 6 379 KVSFKFFYL 20.000 64 7 119 KVKDALKLL 20.000 65 8 780 LPQQITPRM 20.000 66 9 67 LVLLGIGFL 20.000 67 10 283 SPMEPHALV 18.000 68 HLA B8 1 12 GPKDFGAGL 24.000 69 2 257 SLTFRSFDL 8.000 70 3 180 MLPPRARSL 8.000 71 4 217 GLHARGVEL 8.000 72 5 173 MAEERVVML 4.800 73 6 267 SCDERGSDL 4.800 74 7 567 CTKHTYRCL 4.000 75 8 724 SSMVRPICL 4.000 76 9 409 YCGERSQFV 3.600 77 10 495 TCKNKFCKP 3.200 78 HLA B2702 1 427 VRFHSDQSY 1000.000 79 2 695 KRIISHPFF 600.000 80 3 664 FRYSDPTQW 500.000 81 4 220 ARGVELMRF 200.000 82 5 492 HQFTCKNKF 100.000 83 6 53 GRWVVLAAV 100.000 84 7 248 LRGDADSVL 60.000 85 8 572 YRCLNGLCL 60.000 86 9 692 RRLKRIISH 60.000 87 10 24 SRHEKVNGL 60.000 88 HLA B4403 1 147 SEGSVIAYY 360.000 89 2 715 LELEKPAEY 360.000 90 3 105 YENSNSTEF 60.000 91 4 14 KDFGAGLKY 50.625 92 5 129 SGVPFLGPY 36.000 93 6 436 TDTGFLAEY 33.750 94 7 766 GEIRVINQT 30.000 95 8 402 VEINGEKYC 30.000 96 9 482 DELNCSCDA 24.000 97 10 82 RDVRVQKVF 22.500 98
Example 10
TADG-15 cDNA
[0117] A screening strategy to identify proteases which are overexpressed in human cancer has been developed in which RT-PCR products amplified specifically in tumors, as compared to normal tissue, are examined (9). During this effort, candidate genes were identified using redundant sense primers to the conserved amino acid histidine domain at the NH3 end of the catalytic domain and antisense primers to the downstream conserved amino acid serine domain. Subcloning and sequencing the appropriate 480 base pair band(s) amplified in such a PCR reaction provides the basis for identifying the gene(s) encoding proteases(s). Among these amplified catalytic domains, a new serine protease gene named TADG-15 (tumor antigen-derived gene 15) was identified. The catalytic domain of the newly identified TADG-15 protein is similar to other serine proteases and specifically contains conserved amino acids appropriate for the catalytic domain of the trypsin-like serine protease family.
[0118] A computerized search of GenEMBL databases using the FASTA program (Wisconsin Package Version 9.1, GCG, Madison, Wis.) for amino acid sequences homologous to the TADG-15 protease domain revealed that homologies with other known human proteases never exceeds 55%. FIGS. 1A-1B show the alignment of the protease domain of TADG-15 compared with other human serine proteases. Using the BESTFIT program available through GCG, the similarities between TADG-15 and trypsin, chymotrypsin, and tissue-type plasminogen activator are 51%, 46% and 52%, respectively.
[0119] From the sequence derived from the TADG-15 catalytic domain, specific primers were synthesized to amplify a TADG-15-specific probe for library screening. After screening an ovarian carcinoma library, one 1785 bp clone was obtained which included the 3' end of the TADG-15 transcript. Upon further screening using the 5' end of the newly detected clone, two additional clones were identified which provided another 1362 bp of the cDNA, including the 5' end of the TADG-15 transcript. The total length of the sequenced cDNA was approximately 3.15 kb. The total nucleotide sequence obtained includes a Kozak's consensus sequence preceding a single open reading frame encoding a predicted protein of 855 amino acids (FIG. 2).
[0120] The deduced open reading frame encoded by the TADG-15 nucleotide sequence (FIGS. 2A-2D, 3-4) contains several distinct domains as follows: an amino terminal cytoplasmic tail (amino acids (aa) #1-54), a potential transmembrane domain (aa #55-77), an extracellular membrane domain (aa #78-213), two complement subcomponents Clr/Cls, Uegf, and bone morphogenetic protein 1 (CUB) repeats (aa #214-447), four ligand binding repeats of the low density lipoprotein (LDL) receptor-like domain (aa #453-602) and a serine protease domain (aa #615-855). The TADG-15 protein also contains two potential N-linked glycosylation sites (aa #109 and 302) and a potential proteolytic cleavage site upstream from the protease domain (aa #614) which could release and/or activate the protease at the carboxy end of this protein. In addition, TADG-15 contains an RGD motif (aa #249-251) which is commonly found in proteins involved in cell-cell adhesion.
Example 11
TADG-15 Expression
[0121] To examine the size of the transcript for TADG-15 and its pattern of expression in various tissues, Northern blot hybridization was performed for representative histological types of carcinoma and in a series of cell lines, fetal tissues and normal adult tissues (FIG. 5). The transcript size for the TADG-15 message was determined to be approximately 3.2 kb and a single intense transcript appeared to be present in all of the carcinomas examined, whereas no visible band was detected in normal ovary (FIG. 5). This transcript size is also in good agreement with the sequence data predicting a transcript size of 3.15 kb. The ovarian tumor cell lines, SW626 and CAOV3, also showed an abundance of transcript, however little or no transcript was detectable in the breast carcinoma cell lines MDA-MB-231 and MDA-MB-4355. Among normal human fetal tissues, fetal kidney showed an abundance of the TADG-15 transcript and low expression was also detected in fetal lung. In normal adult tissues, TADG-15 was detected in colon with low levels of expression in small intestine and prostate (FIG. 5).
[0122] To evaluate mRNA transcript expression of TADG-15 in ovarian tumors and normal ovary, semi-quantitative PCR (FIG. 6) was performed. In a preliminary study, the linearity of this assay (10-11) was confirmed and its efficacy correlated with both Northern blots and immunohistochemistry. The data was quantified using a phosphoimager and compared as a ratio of expression (TADG-15/3-tubulin). Results herein indicate that TADG-15 transcript expression is elevated above the cut-off value (mean for normal ovary±2 SD) in all of the tumor cases examined and is either not detected or detected at extremely low levels in normal ovaries (FIGS. 6A-6B). Analysis of ovarian carcinoma subtypes, including early stage and late stage disease, confirms overexpression of TADG-15 in all carcinomas examined (Table 2). All of the carcinomas studied, which included 5 stage 1 and 3 stage 11 carcinomas, showed overexpression of the TADG-15 gene.
[0123] These data can also be examined with regard to tumor stage and histological sub-type, and results indicated that every carcinoma of every stage and histological sub-type overexpressed the TADG-15 gene. The expression ratio (mean value±SD) for normal ovary group was determined as 0.182±0.024, for LMP tumor group as 0.847±0.419 and for carcinoma group as 0.771±0.380 (Table 2). A comparison between the normal ovary group and tumor groups showed that overexpression of the TADG-15 gene is statistically significant in both the LMP tumor group and the carcinoma group (LMP tumor: p<0.001, carcinoma: p<0.0001).
[0124] As shown in FIG. 6, TADG-15 transcripts were noted in all ovarian carcinomas, but were not present at detectable levels in any of the following tissues: a) normal ovary, b) fetal liver and brain, c) adult spleen, thymus, testes, ovary and peripheral blood lymphocytes, d) skeletal muscle, liver, brain or heart. This evaluation was extended to a standard panel of about 40 tumors. Using TADG-15-specific primers, the expression was also examined in tumor cell lines derived from both ovarian and breast carcinoma tissues as shown in FIG. 7 and in other tumor tissues as shown in FIG. 8. Expression of TADG-15 was also observed in carcinomas of the breast, colon, prostate and lung.
[0125] Polyclonal antibodies developed to a synthetic peptide (a 12-mer) at the carboxy terminus of the protease domain were used to examine TADG-15 expression in cell lines by Western blot and by immunolocalization in normal ovary and ovarian tumors. Western blots of cell extracts from SW626 and CAOV3 cells were probed with both antibody and preimmune sera (FIG. 9). Several bands were detected with the antibody, including bands of approximately 100,000 daltons, approximately 60,000 daltons and 32,000 daltons. The anticipated molecular size of the complete TADG-15 molecule is estimated to be approximately 100,000 daltons, and the protease domain which may be released by proteolytic cleavage at aa #614 is estimated to be approximately 32,000 daltons. Some intermediate proteolytic product may be represented by the 60,000 dalton band.
[0126] Antibody staining of tumor cells confirms the presence of the TADG-15 protease in the cytoplasm of a serous LMP tumor, mucinous LMP tumor and serous carcinoma (FIGS. 10B-10D, respectively). This diffuse staining pattern may be due to detection of TADG15 within the cell as it is being packaged and transported to the cell surface. In endometrioid carcinoma, the antigen is clearly detectable on the surface of tumor cells (FIG. 10E). No staining was detected in normal ovarian epithelium or stromal cells (FIG. 10A). Immunohistochemical staining of a series of 27 tumors indicates the presence of the TADG-15 protein in all the carcinoma subtypes examined, including the low malignant potential group. Strong staining was noted in 7 of 9 low malignant potential tumors and 13 of 18 carcinomas (Table 3).
TABLE-US-00002 TABLE 2 Number of cases with overexpression of TADG-15 in normal ovaries and ovarian tumors overexpression expression N of TADG-15 ratioa Normal 10 0 (0%) 0.182 ± 0.024 LMP 10 10 (100%) 0.847 ± 0.419 serous 6 6 (100%) 0.862 ± 0.419 mucinous 4 4 (100%) 0.825 ± 0.483 Carcinoma 31 31 (100%) 0.771 ± 0.380 serous 18 18 (100%) 0.779 ± 0.332 mucinous 7 7 (100%) 0.907 ± 0.584 endometrioid 3 3 (100%) 0.502 ± 0.083 clear cell 3 3 (100%) 0.672 ± 0.077 aThe ratio of expression level of TADG-15 to β-tubulin (mean ± SD)
TABLE-US-00003 TABLE 3 Immunohistochemical staining using TADG-15 Lab No. Histology TADG-15 Surface epithelium of the ovary - H-3194 serous (LMP) ++ H-162 serous (LMP) ++ H-1182 serous (LMP) ++ H-4818 serous (LMP) ++ H-4881 serous (LMP) ++ H-675 mucinous (LMP) + H-2446 mucinous (LMP) + H-0707 mucinous (LMP) ++ H-2042 mucinous (LMP) ++ H-2555 serous carcinoma ++ H-1858 serous carcinoma ++ H-5266 serous carcinoma ++ H-5316 serous carcinoma + H-2597 serous carcinoma + H-4931 mucinous carcinoma ++ H-1867 mucinous carcinoma ++ H-5998 mucinous carcinoma ++ H-2679 endometrioid adenocarcinoma + H-5718 endometrioid adenocarcinoma ++ H-3993 endometrioid adenocarcinoma + H-2991 endometrioid adenocarcinoma ++ H-2489 endometrioid adenocarcinoma ++ H-5994 clear cell carcinoma ++ H-6718 clear cell carcinoma ++ H-1661 clear cell carcinoma ++ H-6201 clear cell carcinoma ++ H-5640 clear cell carcinoma + - Negative; + Weak Positive; ++ Strong Positive (more than 50% of cell staining)
Example 12
TADG-15 Homology
[0127] Recently, a mouse protein named epithin (GenBank Accession No. AF042822) has been described (13). Epithin is a 902 amino acid protein which contains a similar structure to TADG-15 in that it has a cytoplasmic domain, transmembrane domain, two CUB domains, four LDLR-like domains and a carboxy terminal serine protease domain. TADG-15 and epithin are 84% similar over 843 amino acids, suggesting that the proteins may be orthologous (FIGS. 11A-11B). The precise role of epithin remains to be elucidated.
[0128] A search of GeneBank for similar previously identified sequences yielded one such sequence with relatively high homology to a portion of the TADG-15 gene. The similarity between the portion of TADG-15 from nucleotide #182 to 3139 and SNC-19 GeneBank Accession No. #U20428) is approximately 97% (FIGS. 12A-12E). There are however significant differences between SNC-19 and TADG-15. For example, TADG-15 has an open reading frame of 855 amino acids whereas the longest open reading frame of SNC-19 is 173 amino acids. Additionally, SNC-19 does not include a proper start site for the initiation of translation, nor does it include the amino terminal portion of the protein encoded by TADG-15. Moreover, SNC-19 does not include an open reading frame for a functional serine protease because the His, Asp and Ser residues of the catalytic triad that are necessary for function are encoded in different reading frames.
Implications
[0129] The overall structure of the TADG-15 protein is relatively similar to the members of the tolloid/BMP-1 family and the complement subcomponents, Clr/Cls. These proteins contain both CUB and protease domains, and complex formation through the ligand binding domain is essential for their function. Activation of the serine protease domains of Clr and Cls requires proteolytic cleavage of Arg-Gly and Arg-Ile bonds, respectively (14). Similarly, it might be expected that the TADG-15 protein is synthesized as a zymogen, which is activated by cleavage between Arg614 and Val615 and analogous to the activation mechanism of other serine protease zymogens. Western blot analysis of cultured cell lysates confirmed both a 100 kDa and 32 kDa peptide, which correspond to the putative zymogen (whole molecule) and a cleaved protease product of TADG-15 (FIG. 9). These data support a model for proteolytic release and/or activation of TADG-15 as occurs for similar type II serine proteases.
[0130] CUB domains were first found in complement subcomponents C1r/C1s (15-17) and are known to be a widespread module in developmentally regulated proteins, such as the bone morphogenetic protein-1 (BMP-1) and the tolloid gene product (17-19). The role of these repeats remains largely unknown. However, some models suggest that the CUB domain may be involved in protein-protein interactions. The CUB domain of Clr and Cls participates in the assembly of the Cls-Clr-Clr-Cls tetrameric complex in the activation of the classical pathway of complement by providing protein-protein interaction domains (14). The Drosophila decapentaplegic (DPP) protein is essential for dorsal-ventral specification of the embryo, and the Drosophila tolloid (TLD) forms a complex with DPP to regulate its activity (18-19). Missense mutations in the CUB domain of the tolloid protein results in a phenotype that does not allow a protein interaction with the DPP complex (18).
[0131] The TADG-15 protein contains two tandem repeats of CUB-like domains between amino acid residues 214 and 447. Each of these is approximately 110 amino acids long and each has four conserved cysteine residues characteristic of other CUBs (amino acids 214, 244, 268, 294, 340, 366, 397, 410). By analogy, the CUB repeats of the TADG-15 protein may form an interactive domain capable of promoting multimeric complex formation and regulating the activity of the target protein or TADG-15 itself.
[0132] The TADG-15 protein also contains the LDL receptor ligand binding repeat (class A motif)-like domain, which consists of four contiguous cysteine-rich repeats (amino acid residues 453 to 602). Each cysteine-rich repeat is approximately 40 amino acids long and contains a conserved, negatively-charged sequence (Ser-Asp-Glu) with six cysteine residues. In the LDL receptor protein, this repeat is thought to function as a protein-binding domain which interacts with the lysine and arginine residues present in lipoproteins (20-21). In addition, the first repeat of the LDL receptor appears to bind Ca2+ and not the lipoproteins (22). By analogy, it is possible that the LDL receptor-like repeat in TADG-15 may act in a similar fashion, interacting with positively charged regions of other proteins and/or as a Ca2+ binding site. As a result of ligand binding and the formation of receptor-ligand complex, LDL receptor is internalized via clathrin-coated pits (23). These types of plasma membrane receptors contain a characteristic amino acid sequence in their cytoplasmic domain for binding to clathrin-coated pits (23) TADG-15 does not contain this motif in its cytosolic region, and furthermore, no similarities with other known protein sequences were found in the cytoplasmic domain of the TADG-15. This finding suggests that TADG-15 functions in a different manner from the endocytic receptors (such as the LDL receptor), although TADG-15 possesses similar ligand-binding repeats in the extracellular matrix.
[0133] Although the precise role of TADG-15 is unknown, this gene is clearly overexpressed in ovarian tumors. A variety of proteases, such as type IV collagenase and plasminogen activator, appear to be involved in the process of tumor invasion and are constituents of a protease cascade in malignant progression. TADG-15 may constitute such an activity and directly digest extracellular matrix components surrounding a tumor, or activate other proteases by cleavage of inactive precursors, indirectly enhancing tumor growth and invasion. It is also possible that TADG-15 may function like a member of the tolloid/BMP-1 family by forming complexes with other growth factors or signal transduction proteins to modulate their activities.
[0134] These data raise the possibility that the TADG-15 gene and its translated protein will be a useful marker for the early detection of ovarian carcinoma through release of the protease domain into the extracellular matrix and ultimately the circulation. These data also suggest the possibility of using TADG-15 as a target for therapeutic intervention through delivery systems directed at the CUB/LDLR ligand binding domains.
[0135] The following references were cited herein: [0136] 1. Liotta, L. A., et al. Cell, 64: 327-336, 1991. [0137] 2. Duffy, M. J. Clin. Exp. Metastasis, 10: 145-155, 1992. [0138] 3. Tryggvason, K., et al. Biochem. Biophys. Acta., 907: 191-217, 1987. [0139] 4. Levy, A. T., et al. Cancer Res., 51: 439-444, 1991. [0140] 5. Monsky, W. L. et al. Cancer Res., 53: 3159-3164, 1993. [0141] 6. Duffy, M. J. et al. Cancer, 62: 531-533, 1988. [0142] 7. Hackel, C., et al. Cancer, 79: 53-58, 1997. [0143] 8. Watt, K, et al. Proc. Natl. Acad. Sci. U.S.A., 83: 3166-3170, 1986. [0144] 9. Tanimoto, H. et al. Cancer Res., 57: 2884-2887, 1997. [0145] 10. Shigemasa, K. et al. J. Soc. Gynecol. Invest., 4: 95-102, 1997. [0146] 11. Tanimoto, H. et al. Gynecol. Oncol., 66: 308-312, 1997. [0147] 12. Maniatis et al. Molecular Cloning, p. 309-361 Cold Spring Harbor Laboratory, NY, 1982. [0148] 13. Kim, M. G., et al. Immunogenetics, 49(5): 420-428, 1999. [0149] 14. Arlaud et al. Method in Enzymology, 223: 61-82, 1993. [0150] 15. Joumet, A. & Tosi, M. Biochem. J., 240: 783-787, 1986. [0151] 16. Mackinnon, C. M., et al. Eur. J. Biochem., 169: 547-553, 1987. [0152] 17. Bork, P. & Beckmann, G. J. Mol. Biol., 231: 539-545, 1993. [0153] 18. Childs, S. R. & O'Connor, M. B. Dev. Biol., 162: 209-220, 1994. [0154] 19. Blader, P L, et al. Science, 278: 1937-1940, 1997. [0155] 20. Yamamoto, T. et al. Cell, 39: 27-38, 1984. [0156] 21. Daly, N. L., et al. Proc. Natl. Acad. Sci., 92: 6334-6338, 1995. [0157] 22. van Driel, I. R., et al. J. Biol. Chem., 262: 17443-17449, 1987. [0158] 23. Lodish, H. et al. Sorting of membrane proteins internalized from the cell surface. In: Molecular Cell Biology, 3rd ed., p. 722-733 Scientific American Books, Inc., New York, 1995. [0159] 24. Parker, K C et al. J. Immunol. 152:163, 1994.
[0160] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
Sequence CWU
1
9813147DNAHomo sapiensTADG-15 1tcaagagcgg cctcggggta ccatggggag cgatcgggcc
cgcaagggcg 50gagggggccc gaaggacttc ggcgcgggac tcaagtacaa
ctcccggcac 100gagaaagtga atggcttgga ggaaggcgtg gagttcctgc
cagtcaacaa 150cgtcaagaag gtggaaaagc atggcccggg gcgctgggtg
gtgctggcag 200ccgtgctgat cggcctcctc ttggtcttgc tggggatcgg
cttcctggtg 250tggcatttgc agtaccggga cgtgcgtgtc cagaaggtct
tcaatggcta 300catgaggatc acaaatgaga attttgtgga tgcctacgag
aactccaact 350ccactgagtt tgtaagcctg gccagcaagg tgaaggacgc
gctgaagctg 400ctgtacagcg gagtcccatt cctgggcccc taccacaagg
agtcggctgt 450gacggccttc agcgagggca gcgtcatcgc ctactactgg
tctgagttca 500gcatcccgca gcacctggtg gaggaggccg agcgcgtcat
ggccgaggag 550cgcgtagtca tgctgccccc gcgggcgcgc tccctgaagt
cctttgtggt 600cacctcagtg gtggctttcc ccacggactc caaaacagta
cagaggaccc 650aggacaacag ctgcagcttt ggcctgcacg cccgcggtgt
ggagctgatg 700cgcttcacca cgcccggctt ccctgacagc ccctaccccg
ctcatgcccg 750ctgccagtgg gccctgcggg gggacgccga ctcagtgctg
agcctcacct 800tccgcagctt tgaccttgcg tcctgcgacg agcgcggcag
cgacctggtg 850acggtgtaca acaccctgag ccccatggag ccccacgccc
tggtgcagtt 900gtgtggcacc taccctccct cctacaacct gaccttccac
tcctcccaga 950acgtcctgct catcacactg ataaccaaca ctgagcggcg
gcatcccggc 1000tttgaggcca ccttcttcca gctgcctagg atgagcagct
gtggaggccg 1050cttacgtaaa gcccagggga cattcaacag cccctactac
ccaggccact 1100acccacccaa cattgactgc acatggaaca ttgaggtgcc
caacaaccag 1150catgtgaagg tgagcttcaa attcttctac ctgctggagc
ccggcgtgcc 1200tgcgggcacc tgccccaagg actacgtgga gatcaatggg
gagaaatact 1250gcggagagag gtcccagttc gtcgtcacca gcaacagcaa
caagatcaca 1300gttcgcttcc actcagatca gtcctacacc gacaccggct
tcttagctga 1350atacctctcc tacgactcca gtgacccatg cccggggcag
ttcacgtgcc 1400gcacggggcg gtgtatccgg aaggagctgc gctgtgatgg
ctgggccgac 1450tgcaccgacc acagcgatga gctcaactgc agttgcgacg
ccggccacca 1500gttcacgtgc aagaacaagt tctgcaagcc cctcttctgg
gtctgcgaca 1550gtgtgaacga ctgcggagac aacagcgacg agcaggggtg
cagttgtccg 1600gcccagacct tcaggtgttc caatgggaag tgcctctcga
aaagccagca 1650gtgcaatggg aaggacgact gtggggacgg gtccgacgag
gcctcctgcc 1700ccaaggtgaa cgtcgtcact tgtaccaaac acacctaccg
ctgcctcaat 1750gggctctgct tgagcaaggg caaccctgag tgtgacggga
aggaggactg 1800tagcgacggc tcagatgaga aggactgcga ctgtgggctg
cggtcattca 1850cgagacaggc tcgtgttgtt gggggcacgg atgcggatga
gggcgagtgg 1900ccctggcagg taagcctgca tgctctgggc cagggccaca
tctgcggtgc 1950ttccctcatc tctcccaact ggctggtctc tgccgcacac
tgctacatcg 2000atgacagagg attcaggtac tcagacccca cgcagtggac
ggccttcctg 2050ggcttgcacg accagagcca gcgcagcgcc cctggggtgc
aggagcgcag 2100gctcaagcgc atcatctccc accccttctt caatgacttc
accttcgact 2150atgacatcgc gctgctggag ctggagaaac cggcagagta
cagctccatg 2200gtgcggccca tctgcctgcc ggacgcctcc catgtcttcc
ctgccggcaa 2250ggccatctgg gtcacgggct ggggacacac ccagtatgga
ggcactggcg 2300cgctgatcct gcaaaagggt gagatccgcg tcatcaacca
gaccacctgc 2350gagaacctcc tgccgcagca gatcacgccg cgcatgatgt
gcgtgggctt 2400cctcagcggc ggcgtggact cctgccaggg tgattccggg
ggacccctgt 2450ccagcgtgga ggcggatggg cggatcttcc aggccggtgt
ggtgagctgg 2500ggagacggct gcgctcagag gaacaagcca ggcgtgtaca
caaggctccc 2550tctgtttcgg gactggatca aagagaacac tggggtatag
gggccggggc 2600cacccaaatg tgtacacctg cggggccacc catcgtccac
cccagtgtgc 2650acgcctgcag gctggagact ggaccgctga ctgcaccagc
gcccccagaa 2700catacactgt gaactcaatc tccagggctc caaatctgcc
tagaaaacct 2750ctcgcttcct cagcctccaa agtggagctg ggaggtagaa
ggggaggaca 2800ctggtggttc tactgaccca actgggggca aaggtttgaa
gacacagcct 2850cccccgccag ccccaagctg ggccgaggcg cgtttgtgta
tatctgcctc 2900ccctgtctgt aaggagcagc gggaacggag cttcggagcc
tcctcagtga 2950aggtggtggg gctgccggat ctgggctgtg gggcccttgg
gccacgctct 3000tgaggaagcc caggctcgga ggaccctgga aaacagacgg
gtctgagact 3050gaaattgttt taccagctcc cagggtggac ttcagtgtgt
gtatttgtgt 3100aaatgggtaa aacaatttat ttctttttaa aaaaaaaaaa
aaaaaaa 31472855PRTHomo sapiensTADG-15 2Met Gly Ser Asp
Arg Ala Arg Lys Gly Gly Gly Gly Pro Lys Asp1 5
10 15Phe Gly Ala Gly Leu Lys Tyr Asn Ser Arg His
Glu Lys Val Asn 20 25
30Gly Leu Glu Glu Gly Val Glu Phe Leu Pro Val Asn Asn Val Lys
35 40 45Lys Val Glu Lys His Gly Pro
Gly Arg Trp Val Val Leu Ala Ala 50 55
60Val Leu Ile Gly Leu Leu Leu Val Leu Leu Gly Ile Gly Phe
Leu 65 70 75Val Trp His
Leu Gln Tyr Arg Asp Val Arg Val Gln Lys Val Phe 80
85 90Asn Gly Tyr Met Arg Ile Thr Asn Glu Asn
Phe Val Asp Ala Tyr 95 100
105Glu Asn Ser Asn Ser Thr Glu Phe Val Ser Leu Ala Ser Lys Val
110 115 120Lys Asp Ala Leu Lys Leu
Leu Tyr Ser Gly Val Pro Phe Leu Gly 125
130 135Pro Tyr His Lys Glu Ser Ala Val Thr Ala Phe Ser
Glu Gly Ser 140 145 150Val
Ile Ala Tyr Tyr Trp Ser Glu Phe Ser Ile Pro Gln His Leu
155 160 165Val Glu Glu Ala Glu Arg Val
Met Ala Glu Glu Arg Val Val Met 170 175
180Leu Pro Pro Arg Ala Arg Ser Leu Lys Ser Phe Val Val Thr
Ser 185 190 195Val Val Ala
Phe Pro Thr Asp Ser Lys Thr Val Gln Arg Thr Gln 200
205 210Asp Asn Ser Cys Ser Phe Gly Leu His Ala
Arg Gly Val Glu Leu 215 220
225Met Arg Phe Thr Thr Pro Gly Phe Pro Asp Ser Pro Tyr Pro Ala
230 235 240His Ala Arg Cys Gln Trp
Ala Leu Arg Gly Asp Ala Asp Ser Val 245
250 255Leu Ser Leu Thr Phe Arg Ser Phe Asp Leu Ala Ser
Cys Asp Glu 260 265 270Arg
Gly Ser Asp Leu Val Thr Val Tyr Asn Thr Leu Ser Pro Met
275 280 285Glu Pro His Ala Leu Val Gln
Leu Cys Gly Thr Tyr Pro Pro Ser 290 295
300Tyr Asn Leu Thr Phe His Ser Ser Gln Asn Val Leu Leu Ile
Thr 305 310 315Leu Ile Thr
Asn Thr Glu Arg Arg His Pro Gly Phe Glu Ala Thr 320
325 330Phe Phe Gln Leu Pro Arg Met Ser Ser Cys
Gly Gly Arg Leu Arg 335 340
345Lys Ala Gln Gly Thr Phe Asn Ser Pro Tyr Tyr Pro Gly His Tyr
350 355 360Pro Pro Asn Ile Asp Cys
Thr Trp Asn Ile Glu Val Pro Asn Asn 365
370 375Gln His Val Lys Val Ser Phe Lys Phe Phe Tyr Leu
Leu Glu Pro 380 385 390Gly
Val Pro Ala Gly Thr Cys Pro Lys Asp Tyr Val Glu Ile Asn
395 400 405Gly Glu Lys Tyr Cys Gly Glu
Arg Ser Gln Phe Val Val Thr Ser 410 415
420Asn Ser Asn Lys Ile Thr Val Arg Phe His Ser Asp Gln Ser
Tyr 425 430 435Thr Asp Thr
Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp Ser Ser 440
445 450Asp Pro Cys Pro Gly Gln Phe Thr Cys Arg
Thr Gly Arg Cys Ile 455 460
465Arg Lys Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Thr Asp His
470 475 480Ser Asp Glu Leu Asn Cys
Ser Cys Asp Ala Gly His Gln Phe Thr 485
490 495Cys Lys Asn Lys Phe Cys Lys Pro Leu Phe Trp Val
Cys Asp Ser 500 505 510Val
Asn Asp Cys Gly Asp Asn Ser Asp Glu Gln Gly Cys Ser Cys
515 520 525Pro Ala Gln Thr Phe Arg Cys
Ser Asn Gly Lys Cys Leu Ser Lys 530 535
540Ser Gln Gln Cys Asn Gly Lys Asp Asp Cys Gly Asp Gly Ser
Asp 545 550 555Glu Ala Ser
Cys Pro Lys Val Asn Val Val Thr Cys Thr Lys His 560
565 570Thr Tyr Arg Cys Leu Asn Gly Leu Cys Leu
Ser Lys Gly Asn Pro 575 580
585Glu Cys Asp Gly Lys Glu Asp Cys Ser Asp Gly Ser Asp Glu Lys
590 595 600Asp Cys Asp Cys Gly Leu
Arg Ser Phe Thr Arg Gln Ala Arg Val 605
610 615Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp Pro
Trp Gln Val 620 625 630Ser
Leu His Ala Leu Gly Gln Gly His Ile Cys Gly Ala Ser Leu
635 640 645Ile Ser Pro Asn Trp Leu Val
Ser Ala Ala His Cys Tyr Ile Asp 650 655
660Asp Arg Gly Phe Arg Tyr Ser Asp Pro Thr Gln Trp Thr Ala
Phe 665 670 675Leu Gly Leu
His Asp Gln Ser Gln Arg Ser Ala Pro Gly Val Gln 680
685 690Glu Arg Arg Leu Lys Arg Ile Ile Ser His
Pro Phe Phe Asn Asp 695 700
705Phe Thr Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro
710 715 720Ala Glu Tyr Ser Ser Met
Val Arg Pro Ile Cys Leu Pro Asp Ala 725
730 735Ser His Val Phe Pro Ala Gly Lys Ala Ile Trp Val
Thr Gly Trp 740 745 750Gly
His Thr Gln Tyr Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys
755 760 765Gly Glu Ile Arg Val Ile Asn
Gln Thr Thr Cys Glu Asn Leu Leu 770 775
780Pro Gln Gln Ile Thr Pro Arg Met Met Cys Val Gly Phe Leu
Ser 785 790 795Gly Gly Val
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser 800
805 810Ser Val Glu Ala Asp Gly Arg Ile Phe Gln
Ala Gly Val Val Ser 815 820
825Trp Gly Asp Gly Cys Ala Gln Arg Asn Lys Pro Gly Val Tyr Thr
830 835 840Arg Leu Pro Leu Phe Arg
Asp Trp Ile Lys Glu Asn Thr Gly Val 845
850 8553256PRTHomo sapiensHepsin 3Arg Ile Val Gly Gly Arg
Asp Thr Ser Leu Gly Arg Trp Pro Trp1 5 10
15Gln Val Ser Leu Arg Tyr Asp Gly Ala His Leu Cys Gly
Gly Ser 20 25 30Leu Leu
Ser Gly Asp Trp Val Leu Thr Ala Ala His Cys Phe Pro 35
40 45Glu Arg Asn Arg Val Leu Ser Arg Trp
Arg Val Phe Ala Gly Ala 50 55
60Val Ala Gln Ala Ser Pro His Gly Leu Gln Leu Gly Val Gln Ala
65 70 75Val Val Tyr His Gly Gly
Tyr Leu Pro Phe Arg Asp Pro Asn Ser 80 85
90Glu Glu Asn Ser Asn Asp Ile Ala Leu Val His Leu Ser
Ser Pro 95 100 105Leu Pro
Leu Thr Glu Tyr Ile Gln Pro Val Cys Leu Pro Ala Ala 110
115 120Gly Gln Ala Leu Val Asp Gly Lys Ile
Cys Thr Val Thr Gly Trp 125 130
135Gly Asn Thr Gln Tyr Tyr Gly Gln Gln Ala Gly Val Leu Gln Glu
140 145 150Ala Arg Val Pro Ile
Ile Ser Asn Asp Val Cys Asn Gly Ala Asp 155
160 165Phe Tyr Gly Asn Gln Ile Lys Pro Lys Met Phe Cys
Ala Gly Tyr 170 175 180Pro
Glu Gly Gly Ile Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro
185 190 195Phe Val Cys Glu Asp Ser Ile
Ser Arg Thr Pro Arg Trp Arg Leu 200 205
210Cys Gly Ile Val Ser Trp Gly Thr Gly Cys Ala Leu Ala Gln
Lys 215 220 225Pro Gly Val
Tyr Thr Lys Val Ser Asp Phe Arg Glu Trp Ile Phe 230
235 240Gln Ala Ile Lys Thr His Ser Glu Ala Ser
Gly Met Val Thr Gln 245 250
255Leu 4225PRTHomo sapiensSCCE 4Lys Ile Ile Asp Gly Ala Pro Cys Ala Arg
Gly Ser His Pro Trp1 5 10
15Gln Val Ala Leu Leu Ser Gly Asn Gln Leu His Cys Gly Gly Val
20 25 30Leu Val Asn Glu Arg Trp Val
Leu Thr Ala Ala His Cys Lys Met 35 40
45Asn Glu Tyr Thr Val His Leu Gly Ser Asp Thr Leu Gly Asp
Arg 50 55 60Arg Ala Gln
Arg Ile Lys Ala Ser Lys Ser Phe Arg His Pro Gly 65
70 75Tyr Ser Thr Gln Thr His Val Asn Asp Leu
Met Leu Val Lys Leu 80 85
90Asn Ser Gln Ala Arg Leu Ser Ser Met Val Lys Lys Val Arg Leu
95 100 105Pro Ser Arg Cys Glu Pro
Pro Gly Thr Thr Cys Thr Val Ser Gly 110
115 120Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro
Ser Asp Leu 125 130 135Met
Cys Val Asp Val Lys Leu Ile Ser Pro Gln Asp Cys Thr Lys
140 145 150Val Tyr Lys Asp Leu Leu Glu
Asn Ser Met Leu Cys Ala Gly Ile 155 160
165Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly
Pro 170 175 180Leu Val Cys
Arg Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr 185
190 195Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly
Val Tyr Thr Gln Val 200 205
210Cys Lys Phe Thr Lys Trp Ile Asn Asp Thr Met Lys Lys His Arg
215 220 2255225PRTHomo
sapiensTrypsin 5Lys Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro
Tyr1 5 10 15Gln Val Ser
Leu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu 20
25 30Ile Asn Glu Gln Trp Val Val Ser Ala Gly
His Cys Tyr Lys Ser 35 40
45Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Glu Val Leu Glu
50 55 60Gly Asn Glu Gln Phe Ile Asn
Ala Ala Lys Ile Ile Arg His Pro 65 70
75Gln Tyr Asp Arg Lys Thr Leu Asn Asn Asp Ile Met Leu Ile
Lys 80 85 90Leu Ser Ser
Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser 95
100 105Leu Pro Thr Ala Pro Pro Ala Thr Gly Thr
Lys Cys Leu Ile Ser 110 115
120Gly Trp Gly Asn Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu
125 130 135Leu Gln Cys Leu Asp Ala
Pro Val Leu Ser Gln Ala Lys Cys Glu 140
145 150Ala Ser Tyr Pro Gly Lys Ile Thr Ser Asn Met Phe
Cys Val Gly 155 160 165Phe
Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly
170 175 180Pro Val Val Cys Asn Gly Gln
Leu Gln Gly Val Val Ser Trp Gly 185 190
195Asp Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
Val 200 205 210Tyr Asn Tyr
Val Lys Trp Ile Lys Asn Thr Ile Ala Ala Asn Ser 215
220 2256231PRTHomo sapiensChymotrypsin 6Arg Ile
Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp1 5
10 15Gln Val Ser Leu Gln Asp Lys Thr Gly
Phe His Phe Cys Gly Gly 20 25
30Ser Leu Ile Ser Glu Asp Trp Val Val Thr Ala Ala His Cys Gly
35 40 45Val Arg Thr Ser Asp Val
Val Val Ala Gly Glu Phe Asp Gln Gly 50 55
60Ser Asp Glu Glu Asn Ile Gln Val Leu Lys Ile Ala Lys
Val Phe 65 70 75Lys Asn
Pro Lys Phe Ser Ile Leu Thr Val Asn Asn Asp Ile Thr 80
85 90Leu Leu Lys Leu Ala Thr Pro Ala Arg
Phe Ser Gln Thr Val Ser 95 100
105Ala Val Cys Leu Pro Ser Ala Asp Asp Asp Phe Pro Ala Gly Thr
110 115 120Leu Cys Ala Thr Thr
Gly Trp Gly Lys Thr Lys Tyr Asn Ala Asn 125
130 135Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala Leu Pro
Leu Leu Ser 140 145 150Asn
Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg Ile Thr Asp Val
155 160 165Met Ile Cys Ala Gly Ala Ser
Gly Val Ser Ser Cys Met Gly Asp 170 175
180Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp Thr
Leu 185 190 195Val Gly Ile
Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser Ser 200
205 210Pro Gly Val Tyr Ala Arg Val Thr Lys Leu
Ile Pro Trp Val Gln 215 220
225Lys Ile Leu Ala Ala Asn 2307255PRTHomo sapiensFactor 7
7Arg Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp1
5 10 15Gln Val Leu Leu Leu Val Asn
Gly Ala Gln Leu Cys Gly Gly Thr 20 25
30Leu Ile Asn Thr Ile Trp Val Val Ser Ala Ala His Cys Phe
Asp 35 40 45Lys Ile Lys
Asn Trp Arg Asn Leu Ile Ala Val Leu Gly Glu His 50
55 60Asp Leu Ser Glu His Asp Gly Asp Glu Gln
Ser Arg Arg Val Ala 65 70
75Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn His
80 85 90Asp Ile Ala Leu Leu Arg Leu
His Gln Pro Val Val Leu Thr Asp 95 100
105His Val Val Pro Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu
Arg 110 115 120Thr Leu Ala
Phe Val Arg Phe Ser Leu Val Ser Gly Trp Gly Gln 125
130 135Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu
Leu Met Val Leu Asn 140 145
150Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg Lys
155 160 165Val Gly Asp Ser Pro Asn
Ile Thr Glu Tyr Met Phe Cys Ala Gly 170
175 180Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp
Ser Gly Gly 185 190 195Pro
His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile
200 205 210Val Ser Trp Gly Gln Gly Cys
Ala Thr Val Gly His Phe Gly Val 215 220
225Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu
Met 230 235 240Arg Ser Glu
Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro 245
250 2558253PRTHomo sapiensTissue plasminogen
activator 8Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp1
5 10 15Gln Ala Ala Ile Phe
Ala Lys His Arg Arg Ser Pro Gly Glu Arg 20
25 30Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp
Ile Leu Ser 35 40 45Ala
Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr 50
55 60Val Ile Leu Gly Arg Thr Tyr Arg
Val Val Pro Gly Glu Glu Glu 65 70
75Gln Lys Phe Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp
80 85 90Asp Asp Thr Tyr Asp
Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser 95
100 105Asp Ser Ser Arg Cys Ala Gln Glu Ser Ser Val Val
Arg Thr Val 110 115 120Cys
Leu Pro Pro Ala Asp Leu Gln Leu Pro Asp Trp Thr Glu Cys
125 130 135Glu Leu Ser Gly Tyr Gly Lys
His Glu Ala Leu Ser Pro Phe Tyr 140 145
150Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu Tyr Pro Ser
Ser 155 160 165Arg Cys Thr
Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn 170
175 180Met Leu Cys Ala Gly Asp Thr Arg Ser Gly
Gly Pro Gln Ala Asn 185 190
195Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys
200 205 210Leu Asn Asp Gly Arg Met
Thr Leu Val Gly Ile Ile Ser Trp Gly 215
220 225Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr
Thr Lys Val 230 235 240Thr
Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro 245
25092900DNAHomo sapiensSNC-19; GeneBank Accession No. #U20428
9cgctgggtgg tgctggcagc cgtgctgatc ggcctcctct tggtcttgct
50ggggatcggc ttcctggtgt ggcatttgca gtaccgggac gtgcgtgtcc
100agaaggtctt caatggctac atgaggatca caaatgagaa ttttgtggat
150gcctacgaga actccaactc cactgagttt gtaagcctgg ccagcaaggt
200gaaggacgcg ctgaagctgc tgtacagcgg agtcccattc ctgggcccct
250accacaagga gtcggctgtg acggccttca gcgagggcag cgtcatcgcc
300tactactggt ctgagttcag catcccgcag cacctggttg aggaggccga
350gcgcgtcatg gccaggagcg cgtagtcatg ctgcccccgc gggcgcgctc
400cctgaagtcc tttgtggtca cctcagtggt ggctttcccc acggactcca
450aaacagtaca gaggacccag gacaacagct gcagctttgg cctgcacgcc
500gcggtgtgga gctgatgcgc ttcaccacgc cggcttccct gacagcccct
550accccgctca tgcccgctgc cagtgggctg cggggacgcg acgcagtgct
600gagctactcg agctgactcg cagcttgact gcgcctcgac gagcgcggca
650gcgacctggt gacgtgtaca acaccctgag ccccatggag ccccacgcct
700ggtgagtgtg tggcacctac cctccctcct acaacctgac cttccactcc
750ctcccacgaa cgtcctgctc atcacactga taaccaacac tgacgcggca
800tcccggcttt gaggccacct tcttccagct gcctaggatg agcagctgtg
850gaggccgctt acgtaaagcc caggggacat tcaacagccc ctactaccca
900ggccactacc cacccaacat tgactgcaca tggaaaattg aggtgcccaa
950caaccagcat gtgaaggtgc gcttcaaatt cttctacctg ctggagcccg
1000gcgtgcctgc gggcacctgc cccaaggact acgtggagat caatggggag
1050aaatactgcg gagagaggtc ccagttcgtc gtcaccagca acagcaacaa
1100gatcacagtt cgcttccact cagatcagtc ctacaccgac accggcttct
1150tagctgaata cctctcctac gactccagtg acccatgccc ggggcagttc
1200acgtgccgca cggggcggtg tatccggaag gagctgcgct gtgatggctg
1250ggcgactgca ccgaccacag cgatgagctc aactgcagtt gcgacgccgg
1300ccaccagttc acgtgcaaga gcaagttctg caagctcttc tgggtctgcg
1350acagtgtgaa cgagtgcgga gacaacagcg acgagcaggg ttgcatttgt
1400ccggacccag accttcaggt gttccaatgg gaagtgcctc tcgaaaagcc
1450agcagtgcaa tgggaaggac gactgtgggg acgggtccga cgaggcctcc
1500tgccccaagg tgaacgtcgt cacttgtacc aaacacacct accgctgcct
1550caatgggctc tgcttgagca agggcaaccc tgagtgtgac gggaaggagg
1600actgtagcga cggctcagat gagaaggact gcgactgtgg gctgcggtca
1650ttcacgagac aggctcgtgt tgttgggggc acggatgcgg atgagggcga
1700gtggccctgg caggtaagcc tgcatgctct gggccagggc cacatctgcg
1750gtgcttccct catctctccc aactggctgg tctctgccgc acactgctac
1800atcgatgaca gaggattcag gtactcagac cccacgcagg acggccttcc
1850tgggcttgca cgaccagagc cagcgcaggc cctggggtgc aggagcgcag
1900gctcaagcgc atcatctccc accccttctt caatgacttc accttcgact
1950atgacatcgc gctgctggag ctggagaaac cggcagagta cagctccatg
2000gtgcggccca tctgcctgcc ggacgcctgc catgtcttcc ctgccggcaa
2050ggccatctgg gtcacgggct ggggacacac ccagtatgga ggcactggcg
2100cgctgatcct gcaaaagggt gagatccgcg tcatcaacca gaccacctgc
2150gagaacctcc tgccgcagca gatcacgccg cgcatgatgt gcgtgggctt
2200cctcagcggc ggcgtggact cctgccaggg tgattccggg ggacccctgt
2250ccagcgtgga ggcggatggg cggatcttcc aggccggtgt ggtgagctgg
2300ggagacgctg cgctcagagg aacaagccag gcgtgtacac aaggctccct
2350ctgtttcggg aatggatcaa agagaacact ggggtatagg ggccggggcc
2400acccaaatgt gtacacctgc ggggccaccc atcgtccacc ccagtgtgca
2450cgcctgcagg ctggagactc gcgcaccgtg acctgcacca gcgccccaga
2500acatacactg tgaactcatc tccaggctca aatctgctag aaaacctctc
2550gcttcctcag cctccaaagt ggagctggga gggtagaagg ggaggaacac
2600tggtggttct actgacccaa ctggggcaag gtttgaagca cagctccggc
2650agcccaagtg ggcgaggacg cgtttgtgca tactgccctg ctctatacac
2700ggaagacctg gatctctagt gagtgtgact gccggatctg gctgtggtcc
2750ttggccacgc ttcttgagga agcccaggct cggaggaccc tggaaaacag
2800acgggtctga gactgaaaat ggtttaccag ctcccaggtg acttcagtgt
2850gtgtattgtg taaatgagta aaacatttta tttcttttta aaaaaaaaaa
290010902PRTMus musculusEpithin 10Met Gly Ser Asn Arg Gly Arg Lys Ala Gly
Gly Gly Ser Gln Asp1 5 10
15Phe Gly Ala Gly Leu Lys Tyr Asp Ser Arg Leu Glu Asn Met Asn
20 25 30Gly Phe Glu Glu Gly Val Glu
Phe Leu Pro Ala Asn Asn Ala Lys 35 40
45Lys Val Glu Lys Arg Gly Pro Arg Arg Trp Val Val Leu Val
Ala 50 55 60Val Leu Phe
Ser Phe Leu Leu Leu Ser Leu Met Ala Gly Leu Leu 65
70 75Val Trp His Phe His Tyr Arg Asn Val Arg
Val Gln Lys Val Phe 80 85
90Asn Gly His Leu Arg Ile Thr Asn Glu Ile Phe Leu Asp Ala Tyr
95 100 105Glu Asn Ser Thr Ser Thr
Glu Phe Ile Ser Leu Ala Ser Gln Val 110
115 120Lys Glu Ala Leu Lys Leu Leu Tyr Asn Glu Val Pro
Val Leu Gly 125 130 135Pro
Tyr His Lys Lys Ser Ala Val Thr Ala Phe Ser Glu Gly Ser
140 145 150Val Ile Ala Tyr Tyr Trp Ser
Glu Phe Ser Ile Pro Pro His Leu 155 160
165Ala Glu Glu Val Asp Arg Ala Met Ala Val Glu Arg Val Val
Thr 170 175 180Leu Pro Pro
Arg Ala Arg Ala Leu Lys Ser Phe Val Leu Thr Ser 185
190 195Val Val Ala Phe Pro Ile Asp Pro Arg Met
Leu Gln Arg Thr Gln 200 205
210Asp Asn Ser Cys Ser Phe Ala Leu His Ala His Gly Ala Ala Val
215 220 225Thr Arg Phe Thr Thr Pro
Gly Phe Pro Asn Ser Pro Tyr Pro Ala 230
235 240His Ala Arg Cys Gln Trp Val Leu Arg Gly Asp Ala
Asp Ser Val 245 250 255Leu
Ser Leu Thr Phe Arg Ser Phe Asp Val Ala Pro Cys Asp Glu
260 265 270His Gly Ser Asp Leu Val Thr
Val Tyr Asp Ser Leu Ser Pro Met 275 280
285Glu Pro His Ala Val Val Arg Leu Cys Gly Thr Phe Ser Pro
Ser 290 295 300Tyr Asn Leu
Thr Phe Leu Ser Ser Gln Asn Val Phe Leu Val Thr 305
310 315Leu Ile Thr Asn Thr Gly Arg Arg His Leu
Gly Phe Glu Ala Thr 320 325
330Phe Phe Gln Leu Pro Lys Met Ser Ser Cys Gly Gly Val Leu Ser
335 340 345Asp Thr Gln Gly Thr Phe
Ser Ser Pro Tyr Tyr Pro Gly His Tyr 350
355 360Pro Pro Asn Ile Asn Cys Thr Trp Asn Ile Lys Val
Pro Asn Asn 365 370 375Arg
Asn Val Lys Val Arg Phe Lys Leu Phe Tyr Leu Val Asp Pro
380 385 390Asn Val Pro Val Gly Ser Cys
Thr Lys Asp Tyr Val Glu Ile Asn 395 400
405Gly Glu Lys Gly Ser Gly Glu Arg Ser Gln Phe Val Val Ser
Ser 410 415 420Asn Ser Ser
Lys Ile Thr Val His Phe His Ser Asp His Ser Tyr 425
430 435Thr Asp Thr Gly Phe Leu Ala Glu Tyr Leu
Ser Tyr Asp Ser Asn 440 445
450Asp Pro Cys Pro Gly Met Phe Met Cys Lys Thr Gly Arg Cys Ile
455 460 465Arg Lys Glu Leu Arg Cys
Asp Gly Trp Ala Asp Cys Pro Asp Tyr 470
475 480Ser Asp Glu Arg Tyr Cys Arg Cys Asn Ala Thr His
Gln Phe Thr 485 490 495Cys
Lys Asn Gln Phe Cys Lys Pro Leu Phe Trp Val Cys Asp Ser
500 505 510Val Asn Asp Cys Gly Asp Gly
Ser Asp Glu Glu Gly Cys Ser Cys 515 520
525Pro Ala Gly Ser Phe Lys Cys Ser Asn Gly Lys Cys Leu Pro
Gln 530 535 540Ser Gln Lys
Cys Asn Gly Lys Asp Asn Cys Gly Asp Gly Ser Asp 545
550 555Glu Ala Ser Cys Asp Ser Val Asn Val Val
Ser Cys Thr Lys Tyr 560 565
570Thr Tyr Arg Cys Gln Asn Gly Leu Cys Leu Ser Lys Gly Asn Pro
575 580 585Glu Cys Asp Gly Lys Thr
Asp Cys Ser Asp Gly Ser Asp Glu Lys 590
595 600Asn Cys Asp Cys Gly Leu Arg Ser Phe Thr Lys Gln
Ala Arg Val 605 610 615Val
Gly Gly Thr Asn Ala Asp Glu Gly Glu Trp Pro Trp Gln Val
620 625 630Ser Leu His Ala Leu Gly Gln
Gly His Leu Cys Gly Ala Ser Leu 635 640
645Ile Ser Pro Asp Trp Leu Val Ser Ala Ala His Cys Phe Gln
Asp 650 655 660Asp Lys Asn
Phe Lys Tyr Ser Asp Tyr Thr Met Trp Thr Ala Phe 665
670 675Leu Gly Leu Leu Asp Gln Ser Lys Arg Ser
Ala Ser Gly Val Gln 680 685
690Glu Leu Lys Leu Lys Arg Ile Ile Thr His Pro Ser Phe Asn Asp
695 700 705Phe Thr Phe Asp Tyr Asp
Ile Ala Leu Leu Glu Leu Glu Lys Ser 710
715 720Val Glu Tyr Ser Thr Val Val Arg Pro Ile Cys Leu
Pro Asp Ala 725 730 735Thr
His Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp
740 745 750Gly His Thr Lys Glu Gly Gly
Thr Gly Ala Leu Ile Leu Gln Lys 755 760
765Gly Glu Ile Arg Val Ile Asn Gln Thr Thr Cys Glu Asp Leu
Met 770 775 780Pro Gln Gln
Ile Thr Pro Arg Met Met Cys Val Gly Phe Leu Ser 785
790 795Gly Gly Val Asp Ser Cys Gln Gly Asp Ser
Gly Gly Pro Leu Ser 800 805
810Ser Ala Glu Lys Asp Gly Arg Met Phe Gln Ala Gly Val Val Ser
815 820 825Trp Gly Glu Gly Cys Ala
Gln Arg Asn Lys Pro Gly Val Tyr Thr 830
835 840Arg Leu Pro Cys Ser Ser Gly Leu Asp Gln Arg Ala
His Trp Gly 845 850 855Ile
Ala Ala Trp Thr Asp Ser Arg Pro Gln Thr Pro Thr Gly Met
860 865 870Pro Asp Met His Thr Trp Ile
Gln Glu Arg Asn Thr Asp Asp Ile 875 880
885Tyr Ala Val Ala Ser Pro Pro Gln His Asn Pro Asp Cys Glu
Leu 890 895 900His
Pro1123DNAArtificial sequenceDegenerate oligonucleotide primer
11tgggtngtna cngcngcnca ytg
231220DNAArtificial sequenceDegenerate oligonucleotide primer
12arnggnccnc cnswrtcncc
201312PRTHomo sapiensFragment of TADG-15 13Leu Phe Arg Asp Trp Ile Lys
Glu Asn Thr Gly Val1 5
101420DNAArtificial sequenceTADG-15 forward oligonucleotide primer
14atgacagagg attcaggtac
201520DNAArtificial sequenceTADG-15 reverse oligonucleotide primer
15gaaggtgaag tcattgaaga
201620DNAArtificial sequenceb-tubulin forward oligonucleotide primer
16cgcatcaacg tgtactacaa
201720DNAArtificial sequenceb-tubulin reverse oligonucleotide primer
17tacgagctgg tggactgaga
20183147RNAArtificial sequenceAntisense of TADG-15 18uuuuuuuuuu
uuuuuuuuua aaaagaaaua aauuguuuua cccauuuaca 50caaauacaca
cacugaaguc cacccuggga gcugguaaaa caauuucagu 100cucagacccg
ucuguuuucc aggguccucc gagccugggc uuccucaaga 150gcguggccca
agggccccac agcccagauc cggcagcccc accaccuuca 200cugaggaggc
uccgaagcuc cguucccgcu gcuccuuaca gacaggggag 250gcagauauac
acaaacgcgc cucggcccag cuuggggcug gcgggggagg 300cugugucuuc
aaaccuuugc ccccaguugg gucaguagaa ccaccagugu 350ccuccccuuc
uaccucccag cuccacuuug gaggcugagg aagcgagagg 400uuuucuaggc
agauuuggag cccuggagau ugaguucaca guguauguuc 450ugggggcgcu
ggugcaguca gcgguccagu cuccagccug caggcgugca 500cacuggggug
gacgaugggu ggccccgcag guguacacau uuggguggcc 550ccggccccua
uaccccagug uucucuuuga uccagucccg aaacagaggg 600agccuugugu
acacgccugg cuuguuccuc ugagcgcagc cgucucccca 650gcucaccaca
ccggccugga agauccgccc auccgccucc acgcuggaca 700gggguccccc
ggaaucaccc uggcaggagu ccacgccgcc gcugaggaag 750cccacgcaca
ucaugcgcgg cgugaucugc ugcggcagga gguucucgca 800gguggucugg
uugaugacgc ggaucucacc cuuuugcagg aucagcgcgc 850cagugccucc
auacugggug uguccccagc ccgugaccca gauggccuug 900ccggcaggga
agacauggga ggcguccggc aggcagaugg gccgcaccau 950ggagcuguac
ucugccgguu ucuccagcuc cagcagcgcg augucauagu 1000cgaaggugaa
gucauugaag aagggguggg agaugaugcg cuugagccug 1050cgcuccugca
ccccaggggc gcugcgcugg cucuggucgu gcaagcccag 1100gaaggccguc
cacugcgugg ggucugagua ccugaauccu cugucaucga 1150uguagcagug
ugcggcagag accagccagu ugggagagau gagggaagca 1200ccgcagaugu
ggcccuggcc cagagcaugc aggcuuaccu gccagggcca 1250cucgcccuca
uccgcauccg ugcccccaac aacacgagcc ugucucguga 1300augaccgcag
cccacagucg caguccuucu caucugagcc gucgcuacag 1350uccuccuucc
cgucacacuc aggguugccc uugcucaagc agagcccauu 1400gaggcagcgg
uagguguguu ugguacaagu gacgacguuc accuuggggc 1450aggaggccuc
gucggacccg uccccacagu cguccuuccc auugcacugc 1500uggcuuuucg
agaggcacuu cccauuggaa caccugaagg ucugggccgg 1550acaacugcac
cccugcucgu cgcuguuguc uccgcagucg uucacacugu 1600cgcagaccca
gaagaggggc uugcagaacu uguucuugca cgugaacugg 1650uggccggcgu
cgcaacugca guugagcuca ucgcuguggu cggugcaguc 1700ggcccagcca
ucacagcgca gcuccuuccg gauacaccgc cccgugcggc 1750acgugaacug
ccccgggcau gggucacugg agucguagga gagguauuca 1800gcuaagaagc
cggugucggu guaggacuga ucugagugga agcgaacugu 1850gaucuuguug
cuguugcugg ugacgacgaa cugggaccuc ucuccgcagu 1900auuucucccc
auugaucucc acguaguccu uggggcaggu gcccgcaggc 1950acgccgggcu
ccagcaggua gaagaauuug aagcucaccu ucacaugcug 2000guuguugggc
accucaaugu uccaugugca gucaauguug gguggguagu 2050ggccugggua
guaggggcug uugaaugucc ccugggcuuu acguaagcgg 2100ccuccacagc
ugcucauccu aggcagcugg aagaaggugg ccucaaagcc 2150gggaugccgc
cgcucagugu ugguuaucag ugugaugagc aggacguucu 2200gggaggagug
gaaggucagg uuguaggagg gaggguaggu gccacacaac 2250ugcaccaggg
cguggggcuc cauggggcuc aggguguugu acaccgucac 2300caggucgcug
ccgcgcucgu cgcaggacgc aaggucaaag cugcggaagg 2350ugaggcucag
cacugagucg gcgucccccc gcagggccca cuggcagcgg 2400gcaugagcgg
gguaggggcu gucagggaag ccgggcgugg ugaagcgcau 2450cagcuccaca
ccgcgggcgu gcaggccaaa gcugcagcug uuguccuggg 2500uccucuguac
uguuuuggag uccgugggga aagccaccac ugaggugacc 2550acaaaggacu
ucagggagcg cgcccgcggg ggcagcauga cuacgcgcuc 2600cucggccaug
acgcgcucgg ccuccuccac caggugcugc gggaugcuga 2650acucagacca
guaguaggcg augacgcugc ccucgcugaa ggccgucaca 2700gccgacuccu
ugugguaggg gcccaggaau gggacuccgc uguacagcag 2750cuucagcgcg
uccuucaccu ugcuggccag gcuuacaaac ucaguggagu 2800uggaguucuc
guaggcaucc acaaaauucu cauuugugau ccucauguag 2850ccauugaaga
ccuucuggac acgcacgucc cgguacugca aaugccacac 2900caggaagccg
auccccagca agaccaagag gaggccgauc agcacggcug 2950ccagcaccac
ccagcgcccc gggccaugcu uuuccaccuu cuugacguug 3000uugacuggca
ggaacuccac gccuuccucc aagccauuca cuuucucgug 3050ccgggaguug
uacuugaguc ccgcgccgaa guccuucggg cccccuccgc 3100ccuugcgggc
ccgaucgcuc cccaugguac cccgaggccg cucuuga 3147199PRTHomo
sapiensResidues 68-76 of the TADG-15 protein 19Val Leu Leu Gly Ile Gly
Phe Leu Val1 5209PRTHomo sapiensResidues 126-134 of the
TADG-15 protein 20Leu Leu Tyr Ser Gly Val Pro Phe Leu1
5219PRTHomo sapiensResidues 644-652 of the TADG-15 protein 21Ser Leu Ile
Ser Pro Asn Trp Leu Val1 5229PRTHomo sapiensResidues
379-387 of the TADG-15 protein 22Lys Val Ser Phe Lys Phe Phe Tyr Leu1
5239PRTHomo sapiensResidues 386-394 of the TADG-15 protein
23Tyr Leu Leu Glu Pro Gly Val Pro Ala1 5249PRTHomo
sapiensResidues 257-265 of the TADG-15 protein 24Ser Leu Thr Phe Arg Ser
Phe Asp Leu1 5259PRTHomo sapiensResidues 762-770 of the
TADG-15 protein 25Ile Leu Gln Lys Gly Glu Ile Arg Val1
5269PRTHomo sapiensResidues 841-849 of the TADG-15 protein 26Arg Leu Pro
Leu Phe Arg Asp Trp Ile1 5279PRTHomo sapiensResidues 64-72
of the TADG-15 protein 27Gly Leu Leu Leu Val Leu Leu Gly Ile1
5289PRTHomo sapiensResidues 57-65 of the TADG-15 protein 28Val Leu Ala
Ala Val Leu Ile Gly Leu1 5299PRTHomo sapiensResidues 67-75
of the TADG-15 protein 29Leu Val Leu Leu Gly Ile Gly Phe Leu1
5309PRTHomo sapiensResidues 379-387 of the TADG-15 protein 30Lys Val
Ser Phe Lys Phe Phe Tyr Leu1 5319PRTHomo sapiensResidues
126-134 of the TADG-15 protein 31Leu Leu Tyr Ser Gly Val Pro Phe Leu1
5329PRTHomo sapiensResidues 88-96 of the TADG-15 protein 32Lys
Val Phe Asn Gly Tyr Met Arg Ile1 5339PRTHomo
sapiensResidues 670-678 of the TADG-15 protein 33Thr Gln Trp Thr Ala Phe
Leu Gly Leu1 5349PRTHomo sapiensResidues 119-127 of the
TADG-15 protein 34Lys Val Lys Asp Ala Leu Lys Leu Leu1
5359PRTHomo sapiensResidues 60-68 of the TADG-15 protein 35Ala Val Leu
Ile Gly Leu Leu Leu Val1 5369PRTHomo sapiensResidues 62-70
of the TADG-15 protein 36Leu Ile Gly Leu Leu Leu Val Leu Leu1
5379PRTHomo sapiensResidues 57-65 of the TADG-15 protein 37Val Leu Ala
Ala Val Leu Ile Gly Leu1 5389PRTHomo sapiensResidues 61-69
of the TADG-15 protein 38Val Leu Ile Gly Leu Leu Leu Val Leu1
5399PRTHomo sapiensResidues 146-154 of the TADG-15 protein 39Phe Ser
Glu Gly Ser Val Ile Ala Tyr1 5409PRTHomo sapiensResidues
658-666 of the TADG-15 protein 40Tyr Ile Asp Asp Arg Gly Phe Arg Tyr1
5419PRTHomo sapiensResidues 449-457 of the TADG-15 protein
41Ser Ser Asp Pro Cys Pro Gly Gln Phe1 5429PRTHomo
sapiensResidues 401-409 of the TADG-15 protein 42Tyr Val Glu Ile Asn Gly
Glu Lys Tyr1 5439PRTHomo sapiensResidues 387-395 of the
TADG-15 protein 43Leu Leu Glu Pro Gly Val Pro Ala Gly1
5449PRTHomo sapiensResidues 553-561 of the TADG-15 protein 44Gly Ser Asp
Glu Ala Ser Cys Pro Lys1 5459PRTHomo sapiensResidues 97-105
of the TADG-15 protein 45Thr Asn Glu Asn Phe Val Asp Ala Tyr
5469PRTHomo sapiensResidues 110-118 of the TADG-15 protein 46Ser Thr
Glu Phe Val Ser Leu Ala Ser1 5479PRTHomo sapiensResidues
811-819 of the TADG-15 protein 47Ser Val Glu Ala Asp Gly Arg Ile Phe1
5489PRTHomo sapiensResidues 666-674 of the TADG-15 protein
48Tyr Ser Asp Pro Thr Gln Trp Thr Ala1 5499PRTHomo
sapiensResidues 709-717 of the TADG-15 protein 49Asp Tyr Asp Ile Ala Leu
Leu Glu Leu1 5509PRTHomo sapiensResidues 408-416 of the
TADG-15 protein 50Lys Tyr Cys Gly Glu Arg Ser Gln Phe1
5519PRTHomo sapiensResidues 754-762 of the TADG-15 protein 51Gln Tyr Gly
Gly Thr Gly Ala Leu Ile1 5529PRTHomo sapiensResidues
153-161 of the TADG-15 protein 52Ala Tyr Tyr Trp Ser Glu Phe Ser Ile1
5539PRTHomo sapiensResidues 722-730 of the TADG-15 protein
53Glu Tyr Ser Ser Met Val Arg Pro Ile1 5549PRTHomo
sapiensResidues 326-334 of the TADG-15 protein 54Gly Phe Glu Ala Thr Phe
Phe Gln Leu1 5559PRTHomo sapiensResidues 304-312 of the
TADG-15 protein 55Thr Phe His Ser Ser Gln Asn Val Leu1
5569PRTHomo sapiensResidues 707-715 of the TADG-15 protein 56Thr Phe Asp
Tyr Asp Ile Ala Leu Leu1 5579PRTHomo sapiensResidues 21-29
of the TADG-15 protein 57Lys Tyr Asn Ser Arg His Glu Lys Val1
5589PRTHomo sapiensResidues 665-673 of the TADG-15 protein 58Arg Tyr
Ser Asp Pro Thr Gln Trp Thr1 5599PRTHomo sapiensResidues
686-694 of the TADG-15 protein 59Ala Pro Gly Val Gln Glu Arg Arg Leu1
5609PRTHomo sapiensResidues 12-20 of the TADG-15 protein 60Gly
Pro Lys Asp Phe Gly Ala Gly Leu1 5619PRTHomo
sapiensResidues 668-676 of the TADG-15 protein 61Asp Pro Thr Gln Trp Thr
Ala Phe Leu1 5629PRTHomo sapiensResidues 461-469 of the
TADG-15 protein 62Thr Gly Arg Cys Ile Arg Lys Glu Leu1
5639PRTHomo sapiensResidues 59-67 of the TADG-15 protein 63Ala Ala Val
Leu Ile Gly Leu Leu Leu1 5649PRTHomo sapiensResidues
379-387 of the TADG-15 protein 64Lys Val Ser Phe Lys Phe Phe Tyr Leu1
5659PRTHomo sapiensResidues 119-127 of the TADG-15 protein
65Lys Val Lys Asp Ala Leu Lys Leu Leu1 5669PRTHomo
sapiensResidues 780-788 of the TADG-15 protein 66Leu Pro Gln Gln Ile Thr
Pro Arg Met1 5679PRTHomo sapiensResidues 67-75 of the
TADG-15 protein 67Leu Val Leu Leu Gly Ile Gly Phe Leu1
5689PRTHomo sapiensResidues 283-291 of the TADG-15 protein 68Ser Pro Met
Glu Pro His Ala Leu Val1 5699PRTHomo sapiensResidues 12-20
of the TADG-15 protein 69Gly Pro Lys Asp Phe Gly Ala Gly Leu1
5709PRTHomo sapiensResidues 257-265 of the TADG-15 protein 70Ser Leu
Thr Phe Arg Ser Phe Asp Leu1 5719PRTHomo sapiensResidues
180-188 of the TADG-15 protein 71Met Leu Pro Pro Arg Ala Arg Ser Leu1
5729PRTHomo sapiensResidues 217-225 of the TADG-15 protein
72Gly Leu His Ala Arg Gly Val Glu Leu1 5739PRTHomo
sapiensResidues 173-181 of the TADG-15 protein 73Met Ala Glu Glu Arg Val
Val Met Leu 5749PRTHomo sapiensResidues 267-275 of the
TADG-15 protein 74Ser Cys Asp Glu Arg Gly Ser Asp Leu1
5759PRTHomo sapiensResidues 567-575 of the TADG-15 protein 75Cys Thr Lys
His Thr Tyr Arg Cys Leu1 5769PRTHomo sapiensResidues
724-732 of the TADG-15 protein 76Ser Ser Met Val Arg Pro Ile Cys Leu1
5779PRTHomo sapiensResidues 409-417 of the TADG-15 protein
77Tyr Cys Gly Glu Arg Ser Gln Phe Val1 5789PRTHomo
sapiensResidues 495-503 of the TADG-15 protein 78Thr Cys Lys Asn Lys Phe
Cys Lys Pro1 5799PRTHomo sapiensResidues 427-435 of the
TADG-15 protein 79Val Arg Phe His Ser Asp Gln Ser Tyr1
5809PRTHomo sapiensResidues 695-703 of the TADG-15 protein 80Lys Arg Ile
Ile Ser His Pro Phe Phe1 5819PRTHomo sapiensResidues
664-672 of the TADG-15 protein 81Phe Arg Tyr Ser Asp Pro Thr Gln Trp1
5829PRTHomo sapiensResidues 220-228 of the TADG-15 protein
82Ala Arg Gly Val Glu Leu Met Arg Phe1 5839PRTHomo
sapiensResidues 492-500 of the TADG-15 protein 83His Gln Phe Thr Cys Lys
Asn Lys Phe1 5849PRTHomo sapiensResidues 53-61 of the
TADG-15 protein 84Gly Arg Trp Val Val Leu Ala Ala Val1
5859PRTHomo sapiensResidues 248-256 of the TADG-15 protein 85Leu Arg Gly
Asp Ala Asp Ser Val Leu1 5869PRTHomo sapiensResidues
572-580 of the TADG-15 protein 86Tyr Arg Cys Leu Asn Gly Leu Cys Leu1
5879PRTHomo sapiensResidues 692-700 of the TADG-15 protein
87Arg Arg Leu Lys Arg Ile Ile Ser His1 5889PRTHomo
sapiensResidues 24-32 of the TADG-15 protein 88Ser Arg His Glu Lys Val
Asn Gly Leu1 5899PRTHomo sapiensResidues 147-155 of the
TADG-15 protein 89Ser Glu Gly Ser Val Ile Ala Tyr Tyr1
5909PRTHomo sapiensResidues 715-723 of the TADG-15 protein 90Leu Glu Leu
Glu Lys Pro Ala Glu Tyr1 5919PRTHomo sapiensResidues
105-113 of the TADG-15 protein 91Tyr Glu Asn Ser Asn Ser Thr Glu Phe1
5929PRTHomo sapiensResidues 14-22 of the TADG-15 protein 92Lys
Asp Phe Gly Ala Gly Leu Lys Tyr1 5939PRTHomo
sapiensResidues 129-137 of the TADG-15 protein 93Ser Gly Val Pro Phe Leu
Gly Pro Tyr1 5949PRTHomo sapiensResidues 436-444 of the
TADG-15 protein 94Thr Asp Thr Gly Phe Leu Ala Glu Tyr1
5959PRTHomo sapiensResidues 766-774 of the TADG-15 protein 95Gly Glu Ile
Arg Val Ile Asn Gln Thr1 5969PRTHomo sapiensResidues
402-410 of the TADG-15 protein 96Val Glu Ile Asn Gly Glu Lys Tyr Cys1
5979PRTHomo sapiensResidues 482-490 of the TADG-15 protein
97Asp Glu Leu Asn Cys Ser Cys Asp Ala1 5989PRTHomo
sapiensResidues 82-90 of the TADG-15 protein 98Arg Asp Val Arg Val Gln
Lys Val Phe1 5
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