Patent application title: Transmembrane serine protease overexpressed in ovarian carcinoma and uses thereof
Timothy J. O'Brien (Little Rock, AR, US)
Lowell J. Underwood (Little Rock, AR, US)
IPC8 Class: AA61K39395FI
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds expression product or fragment thereof of cancer-related gene (e.g., oncogene, proto-oncogene, etc.)
Publication date: 2010-05-13
Patent application number: 20100119513
The present invention provides a TADG-12 protein and a DNA fragment
encoding such protein. Also provided is a vector/host cell capable of
expressing the DNA. The present invention further provides various
methods of early detection of associated ovarian and other malignancies,
and of interactive therapies for cancer treatment by utilizing the DNA
and/or protein disclosed herein.
1. An antibody directed against a Tumor Associated Differentially
Expressed Gene-12 (TADG-12) protein, a variant Tumor Associated
Differentially Expressed Gene-12 (TADG-12V) protein or a protein fragment
2. The antibody of claim 1, wherein the TADG-12 protein sequence is shown in SEQ ID NO: 2 and is encoded by (a) isolated DNA comprising the polynucleotide sequence of SEQ ID NO: 1 or (b) isolated DNA differing from the isolated DNA of (a) above in codon sequence due to the degeneracy of the genetic code.
3. The antibody of claim 1, wherein the TADG-12V protein sequence is shown in SEQ ID NO: 4 and is encoded by (a) isolated DNA comprising the polynucleotide sequence of SEQ ID NO: 2 or (b) isolated DNA differing from the isolated DNA of (a) above in codon sequence due to the degeneracy of the genetic code.
4. The antibody of claim 1, wherein the TADG-12V protein fragment sequence is shown in SEQ ID NO: 8 and is encoded by (a) isolated DNA comprising the polynucleotide sequence of SEQ ID NO: 7 or (b) isolated DNA differing from the isolated DNA of (a) above in codon sequence due to the degeneracy of the genetic code.
5. The antibody of claim 1, wherein the TADG-12 protein amino acid sequence is shown in SEQ ID NO: 2.
6. The antibody of claim 1, wherein the TADG-12V protein amino acid sequence is shown in SEQ ID NO: 4.
7. The antibody of claim 1, wherein the TADG-12V protein fragment amino acid sequence is shown in SEQ ID NO: 8.
8. A method of inhibiting expression of a Tumor Associated Differentially Expressed Gene-12 (TADG-12) protein or variant protein (TADG-12V) in a cell, comprising:introducing the antibody of claim 1 into a cell, wherein binding of the antibody to the TADG-12 protein or TADG-12V variant protein or fragment thereof inhibits expression of the same.
9. The method of claim 8, wherein the antibody binds to the TADG-12 protein sequence shown in SEQ ID NO: 2.
10. The method of claim 8, wherein the antibody binds to the TADG-12V protein sequence shown in SEQ ID NO: 4.
11. The method of claim 8, wherein the antibody binds to the TADG-12V protein fragment sequence shown in SEQ ID NO: 8.
12. A method of targeted therapy to an individual having a cancer, comprising:administering to the individual a compound comprising the antibody of claim 1 and a therapeutic moiety.
13. The method of claim 12, wherein the therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant and a cytotoxic agent.
14. The method of claim 12, wherein the cancer is ovarian cancer, lung cancer, prostate cancer, colon cancer, or other cancer in which a TADG-12 protein is overexpressed.
15. An antibody directed against a Tumor Associated Differentially Expressed Gene-12 (TADG-12) protein with a sequence shown in SEQ ID NO: 2.
16. An antibody directed against a variant Tumor Associated Differentially Expressed Gene-12 (TADG-12) protein with a sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. Ser. No. 11/400,825, filed Apr. 7, 2006, which is a divisional of U.S. Ser. No. 10/455,720, filed Jun. 5, 2003, now issued as U.S. Pat. No. 7,067,630, which is a divisional of U.S. Ser. No. 09/650,371, filed on Aug. 28, 2000, now issued as U.S. Pat. No. 6,942,978 which is a divisional of U.S. Ser. No. 09/518,046, filed Mar. 2, 2000, now issued as U.S. Pat. No. 6,294,663, which is a continuation-in-part of U.S. Ser. No. 09/261,416, filed Mar. 3, 1999, now issued as U.S. Pat. No. 6,291,663.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of cellular biology and diagnosis of neoplastic disease. More specifically, the present invention relates to a transmembrane serine protease termed Tumor Associated Differentially-Expressed Gene-12 (TADG-12), which is overexpressed in ovarian carcinoma.
2. Description of the Related Art
Tumor cells-rely on the expression of a concert of proteases to be released from their primary sites and move to distant sites to inflict lethality. This metastatic nature is the result of an aberrant expression pattern of proteases by tumor cells and also by stromal cells surrounding the tumors (1-3). For most tumors to become metastatic, they must degrade their surrounding extracellular matrix components, degrade basement membranes to gain access to the bloodstream or lymph system, and repeat this process in reverse fashion to settle in a secondary host site (3-6). All of these processes rely upon what now appears to be a synchronized protease cascade. In addition, tumor cells use the power of proteases to activate growth and angiogenic factors that allow the tumor to grow progressively (1). Therefore, much research has been aimed at the identification of tumor-associated proteases and the inhibition of these enzymes for therapeutic means. More importantly, the secreted nature and/or high level expression of many of these proteases allows for their detection at aberrant levels in patient serum, e.g. the prostate-specific antigen (PSA), which allows for early diagnosis of prostate cancer (7).
Proteases have been associated directly with tumor growth, shedding of tumor cells and invasion of target organs. Individual classes of proteases are involved in, but not limited to 1) the digestion of stroma surrounding the initial tumor area, 2) the digestion of the cellular adhesion molecules to allow dissociation of tumor cells; and 3) the invasion of the basement membrane for metastatic growth and the activation of both tumor growth factors and angiogenic factors.
For many forms of cancer, diagnosis and treatment has improved dramatically in the last 10 years. However, the five year survival rate for ovarian cancer remains below 50% due in large part to the vague symptoms which allow for progression of the disease to an advanced stage prior to diagnosis (8). Although the exploitation of the CA125 antigen has been useful as a marker for monitoring recurrence of ovarian cancer, it has not proven to be an ideal marker for early diagnosis. Therefore, new markers that may be secreted or released from cells and which are highly expressed by ovarian tumors could provide a useful tool for the early diagnosis and for therapeutic intervention in patients with ovarian carcinoma.
The prior art is deficient in the lack of identification of the proteases overexpressed in carcinoma, therefore, deficient in the lack of a tumor marker useful as an indicator of early disease, particularly for ovarian cancers. Specifically, TADG-12, a transmembrane serine protease, has not been previously identified in either nucleic acid or protein form. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention discloses TADG-12, a new member of the Tumor Associated Differentially-Expressed Gene (TADG) family, and a variant splicing form of TADG-12 (TADG-12V) that could lead to a truncated protein product. TADG-12 is a transmembrane serine protease overexpressed in ovarian carcinoma. The entire cDNA of TADG-12 has been identified (SEQ ID NO: 1). This sequence encodes a putative protein of 454 amino acids (SEQ ID NO: 2) which includes a potential transmembrane domain, an LDL receptor like domain, a scavenger receptor cysteine rich domain, and a serine protease domain. These features imply that TADG-12 is expressed at the cell surface, and it may be used as a molecular target for therapy or a diagnostic marker.
In one embodiment of the present invention, there is provided a DNA fragment encoding a TADG-12 protein selected from the group consisting of: (a) an isolated DNA fragment which encodes a TADG-12 protein; (b) an isolated DNA fragment which hybridizes to isolated DNA fragment of (a) above and which encodes a TADG-12 protein; and (c) an isolated DNA fragment differing from the isolated DNA fragments of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-12 protein. Specifically, the DNA fragment has a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.
In another embodiment of the present invention, there is provided a vector/host cell capable of expressing the DNA of the present invention.
In yet another embodiment of the present invention, there is provided an isolated and purified TADG-12 protein encoded by DNA selected from the group consisting of: (a) isolated DNA which encodes a TADG-12 protein; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a TADG-12 protein; and (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-12 protein. Specifically, the TADG-12 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
In still yet another embodiment of the present invention, there is provided a method for detecting expression of a TADG-12 protein, comprising the steps of: (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
The present invention further provides methods for diagnosing a cancer or other malignant hyperplasia by detecting the TADG-12 protein or mRNA disclosed herein.
In still another embodiment of the present invention, there is provided a method of inhibiting expression of endogenous TADG-12 mRNA in a cell by introducing a vector into the cell, wherein the vector comprises a DNA fragment of TADG-12 in opposite orientation operably linked to elements necessary for expression. In still yet another embodiment of the present invention, there is provided a method of inhibiting expression of a TADG-12 protein in a cell by introducing an antibody directed against a TADG-12 protein or fragment thereof. In still yet another embodiment of the present invention, there is provided a method of targeted therapy by administering a compound having a targeting moiety specific for a TADG-12 protein and a therapeutic moiety. Specifically, the TADG-12 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
The present invention still further provides a method of vaccinating an individual against TADG-12 by inoculating the individual with a TADG-12 protein or fragment thereof. Specifically, the TADG-12 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The TADG-12 fragment includes the truncated form of TADG-12V peptide having a sequence shown in SEQ ID NO: 8, and a 9-residue up to 12-residue fragment of TADG-12 protein.
In yet another embodiment of the present invention, there is provided an immunogenic composition, comprising an immunogenic fragment of a TADG-12 protein and an appropriate adjuvant. The TADG-12 fragment includes the truncated form of TADG-12V peptide having a sequence shown in SEQ ID NO: 8, and a 9-residue up to 12-residue fragment of TADG-12 protein.
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
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.
FIG. 1A shows that the expected PCR product of approximately 180 bp and the unexpected PCR product of approximately 300 bp using the redundant serine protease primers were not amplified from normal ovary cDNA (Lane 1) but were found in abundance from ovarian tumor cDNA (Lane 2). The primer sequences for the PCR reactions are indicated by horizontal arrows. FIG. 1B shows that TADG-12 was subcloned from the 180 bp band while the larger 300 bp band was designated TADG-12V. The sequences were found to overlap for 180 bp (SEQ ID NO: 5 for nucleotide sequence, SEQ ID NO: 6 for deduced amino acid sequence) with the 300 bp TADG-12V (SEQ ID NO: 7 for nucleotide sequence, SEQ ID NO: 8 for deduced amino acid sequence) having an additional insert of 133 bases. This insertion (vertical arrow) leads to a frame shift, which causes the TADG-12V transcript to potentially produce a truncated form of TADG-12 with a variant amino acid sequence.
FIG. 2 shows that Northern blot analysis for TADG-12 revealed three transcripts of 2.4, 1.6 and 0.7 kilobases. These transcripts were found at significant levels in ovarian tumors and cancer cell lines, but the transcripts were found only at low levels in normal ovary.
FIG. 3 shows an RNA dot blot (CLONTECH) probed for TADG-12. The transcript was detectable (at background levels) in all 50 of the human tissues represented with the greatest abundance of transcript in the heart. Putamen, amygdala, kidney, liver, small intestine, skeletal muscle, and adrenal gland were also found to have intermediate levels of TADG-12 transcript.
FIGS. 4A-4C show the entire cDNA sequence for TADG-12 (SEQ ID NO: 1) with its predicted open reading frame of 454 amino acids (SEQ ID NO: 2). Within the nucleotide sequence, the Kozak's consensus sequence for the initiation of translation and the poly-adenylation signal are underlined. In the protein sequence, a potential transmembrane domain is boxed. The LDLR-A domain is underlined with a solid line. The SRCR domain is underlined with a broken line. The residues of the catalytic triad of the serine protease domain are circled, and the beginning of the catalytic domain is marked with an arrow designated as a potential proteolytic cleavage site. The * represents the stop codon that terminates translation.
FIG. 5A shows the 35 amino acid LDLR-A domain of TADG-12 (SEQ ID NO: 13) aligned with other LDLR-A motifs from the serine protease TMPRSS2 (U75329, SEQ ID NO: 14), the complement subunit C8 (P07358, SEQ ID NO: 9), two LDLR-A domains of the glycoprotein GP300 (P98164, SEQ ID Nos. 11-12), and the serine protease matriptase (AF118224, SEQ ID NO: 10). TADG-12 has its highest similarity with the other serine proteases for which it is 54% similar to TMPRSS2 and 53% similar to matriptase. The highly conserved cysteine residues are shown in bold type. FIG. 5B shows the SRCR domain of TADG-12 (SEQ ID NO: 17) aligned with other domain family members including the human macrophage scavenger receptor (P21757, SEQ ID NO: 16), human enterokinase (P98073, SEQ ID NO: 19), bovine enterokinase (P21758, SEQ ID NO: 15), and the serine protease TMPRSS2 (SEQ ID NO: 18). Again, TADG-12 shows its highest similarity within this region to the protease TMPRSS2 at 43%. FIG. 5C shows the protease domain of TADG-12 (SEQ ID NO: 23) in alignment with other human serine proteases including protease M (U62801, SEQ ID NO: 20), trypsinogen I (P07477, SEQ ID NO: 21), plasma kallikrein (P03952, SEQ ID NO: 22), hepsin (P05981, SEQ ID NO: 25), and TMPRSS2 (SEQ ID NO: 24). Cons represents the consensus sequence for each alignment.
FIG. 6 shows semi-quantitative PCR analysis that was performed for TADG-12 (upper panel) and TADG-12V (lower panel). The amplification of TADG-12 or TADG-12V was performed in parallel with PCR amplification of β-tubulin product as an internal control. The TADG-12 transcript was found to be overexpressed in 41 of 55 carcinomas. The TADG-12V transcript was found to be overexpressed in 8 of 22 carcinomas examined. Note that the samples in the upper panel are not necessarily the same as the samples in the lower panel.
FIGS. 7A-7C show immunohistochemical staining of normal ovary and ovarian tumors which were performed using a polyclonal rabbit antibody developed to a TADG-12 specific peptide. No significant staining was detected in normal ovary (FIG. 7A). Strong positive staining was observed in 22 of 29 carcinomas examined. FIGS. 7B and 7C represent a serous and mucinous carcinoma, respectively. Both show diffuse staining throughout the cytoplasm of tumor cells while stromal cells remain relatively unstained.
FIG. 8 is a model to demonstrate the progression of TADG-12 within a cellular context. In normal circumstances, the TADG-12 transcript is appropriately spliced and the resulting protein is capable of being expressed at the cell surface where the protease may be cleaved to an active form. The role of the remaining ligand binding domains has not yet been determined, but one can envision their potential to bind other molecules for activation, internalization or both. The TADG-12V transcript, which occurs in some tumors, may be the result of mutation and/or poor mRNA processing may be capable of producing a truncated form of TADG-12 that does not have a functional protease domain. In addition, this truncated product may present a novel epitope at the surface of tumor cells.
DETAILED DESCRIPTION OF THE INVENTION
To examine the serine proteases expressed by ovarian cancers, a PCR based differential display technique was employed utilizing redundant PCR primers designed to the most highly conserved amino acids in these proteins . As a result, a novel cell-surface, multi-domain serine protease, named Tumor Associated Differentially-expressed Gene-12 (TADG-12) was identified. TADG-12 appears to be overexpressed in many ovarian tumors. The extracellular nature of TADG-12 may render tumors susceptible to detection via a TADG-12 specific assay. In addition, a splicing variant of TADG-12, named TADG-12V, was detected at elevated levels in 35% of the tumors that were examined. TADG-12V encodes a truncated form of TADG-12 with an altered amino acid sequence that may be a unique tumor specific target for future therapeutic approaches.
The TADG-12 cDNA is 2413 base pairs long (SEQ ID NO: 1) encoding a 454 amino acid protein (SEQ ID NO: 2). A variant form, TADG-12V (SEQ ID NO: 3), encodes a 294 amino acid protein (SEQ ID NO: 4). The availability of the TADG-12 and/or TADG-12V gene opens the way for a number studies that can lead to various applications. For example, the TADG-12 and/or TADG-12V gene can be used as a diagnostic or therapeutic target in ovarian carcinoma and other carcinomas including breast, prostate, lung and colon.
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 Molecular Cloning" (1984).
Therefore, if appearing herein, the following terms shall have the definitions set out below.
As used herein, the term "cDNA" shall refer to the DNA copy of the mRNA transcript of a gene. 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. 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. 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.
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. COON refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (J Biol. Chem., (1969) 243:3552-59), abbreviations for amino acid residues are known in the art. 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.
A "replicon" is any genetic element, e.g., plasmid, chromosome, or virus, that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. 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 "DNA molecule" refers to the polymeric form of deoxyribonucleotides, i.e., adenine, guanine, thymine, or cytosine, in its either single stranded form, or 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. In discussing the structure 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.
An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. 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. 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.
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream, or 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, or 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 or consensus sequences responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences. 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.
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. The term "oligonucleotide", as used herein in referring to the probe of the present invention, 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, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, 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, source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. 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 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 or hybridize therewith and thereby form the template for the synthesis of the extension product.
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.
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.
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.
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.
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.
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. 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.
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-12 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-12 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. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted DNA fragment 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.
The invention includes a substantially pure DNA encoding a TADG-12 protein, a strand of which DNA will hybridize at high stringency to a probe containing a sequence of at least 15 consecutive nucleotides of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. 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 SEQ ID NO: 2 or SEQ ID NO: 4. More preferably, the DNA includes the coding sequence of the nucleotides of FIGS. 4A-4C (SEQ ID NO: 1), or a degenerate variant of such a sequence.
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. 4A-4C (SEQ ID NO: 1) or the complement thereof. Such a probe is useful for detecting expression of TADG-12 in a human cell by a method including the steps of (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
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 2413 of the nucleotides listed in SEQ ID NO: 1, or of the region from nucleotides 1 to 2544 of the nucleotides listed in SEQ ID NO: 3. The present invention also comprises antisense oligonucleotides directed against this novel DNA. Given the teachings of the present invention, a person having ordinary skill in this art would readily be able to develop antisense oligonucleotides directed against this DNA.
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.
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 shown in SEQ ID NO: 3 which encodes an alternative splice variant of TADG-12 (TADG-12V).
The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in SEQ ID NO: 1 or SEQ ID NO: 3, 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 subunit 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 (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
The present invention comprises a vector comprising a DNA sequence which encodes a human TADG-12 protein and the vector is capable of replication in a host which comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. A "vector" may be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. Vectors may be used to amplify and/or express nucleic acid encoding a TADG-12 protein. 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, the 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 the 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.
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 is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure TADG-12 protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an TADG-12 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-12, 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.
In addition to substantially full-length proteins, the invention also includes fragments, e.g., antigenic fragments, of the TADG-12 protein. As used herein, "fragment," as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30, e.g., 50, residues in length, but less than the entire, intact sequence. Fragments of the TADG-12 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant TADG-12 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of TADG-12, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of TADG-12, e.g., binding to an antibody specific for TADG-12, can be assessed by methods described herein. Purified TADG-12 or antigenic fragments of TADG-12 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 are polyclonal antisera generated by using TADG-12 or a fragment of TADG-12 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-12 cDNA clones, and to distinguish them from known cDNA clones.
Further included in this invention are TADG-12 proteins which are encoded at least in part by portions of SEQ ID NO: 1 or SEQ ID NO: 3, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of TADG-12 sequence has been deleted. The fragment, or the intact TADG-12 polypeptide, may be covalently linked to another polypeptide, e.g. which acts as a label, a ligand or a means to increase antigenicity.
The invention also includes a polyclonal or monoclonal antibody which specifically binds to TADG-12. 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.
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, 13II, 32P, 35S, 14C, etc.
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.
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 to 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.
Also within the invention is a method of detecting TADG-12 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-12, and determining whether the antibody binds to a component of the sample.
As described herein, the invention provides a number of diagnostic advantages and uses. For example, the TADG-12 protein disclosed in the present invention 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-12, are useful in a method of detecting TADG-12 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-12, and detecting the TADG-12 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-12.
Likewise, a standard Northern blot assay can be used to ascertain the relative amounts of TADG-12 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-12 cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID NO: 1 or SEQ ID NO: 3, 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.
Antibodies to the TADG-12 protein can be used in an immunoassay to detect increased levels of TADG-12 protein expression in tissues suspected of neoplastic transformation. These same uses can be achieved with Northern blot assays and analyses.
The present invention is directed to DNA fragment encoding a TADG-12 protein selected from the group consisting of: (a) an isolated DNA fragment which encodes a TADG-12 protein; (b) an isolated DNA fragment which hybridizes to isolated DNA fragment of (a) above and which encodes a TADG-12 protein; and (c) an isolated DNA fragment differing from the isolated DNA fragments of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-12 protein. Preferably, the DNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. More preferably, the DNA encodes a TADG-12 protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The present invention is also directed to a vector and/or a host cell capable of expressing the DNA of the present invention. Preferably, the vector contains DNA encoding a TADG-12 protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Representative host cells include bacterial cells, yeast cells, mammalian cells and insect cells.
The present invention is also directed to an isolated and purified TADG-12 protein coded for by DNA selected from the group consisting of: (a) isolated DNA which encodes a TADG-12 protein; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a TADG-12 protein; and (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-12 protein. Preferably, the isolated and purified TADG-12 protein has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
The present invention is also directed to a method of detecting expression of the TADG-12 protein described herein, comprising the steps of: (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
A number of potential applications are possible for the TADG-12 gene and gene product including the truncated product TADG-12V. In one embodiment of the present invention, there is provided a method for diagnosing a cancer by detecting a TADG-12 protein in a biological sample, wherein the presence or absence of a TADG-12 protein indicates the presence or absence of a cancer. Preferably, the biological sample is selected from the group consisting of blood, urine, saliva, tears, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells. Still preferably, the detection of TADG-12 protein is by means selected from the group consisting of Northern blot, Western blot, PCR, dot blot, ELIZA sandwich assay, radioimmunoassay, DNA array chips and flow cytometry. Such method is used for detecting an ovarian cancer, breast cancer, lung cancer, colon cancer, prostate cancer and other cancers in which TADG-12 is overexpressed.
In another embodiment of the present invention, there is provided a method for detecting malignant hyperplasia by detecting a TADG-12 protein or TADG-12 mRNA in a biological sample. Further by comprising the TADG-12 protein or TADG-12 mRNA to reference information, a diagnosis or a treatment can be provided. Preferably, PCR amplification is used for detecting TADG-12 mRNA, wherein the primers utilized are selected from the group consisting of SEQ ID Nos. 28-31. Still preferably, detection of a TADG-12 protein is by immunoaffinity to an antibody directed against a TADG-12 protein.
In still another embodiment of the present invention, there is provided a method of inhibiting expression of endogenous TADG-12 mRNA in a cell by introducing a vector comprising a DNA fragment of TADG-12 in opposite orientation operably linked to elements necessary for expression. As a result, the vector produces TADG-12 antisense mRNA in the cell, which hybridizes to endogenous TADG-12 mRNA, thereby inhibiting expression of endogenous TADG-12 mRNA.
In still yet another embodiment of the present invention, there is provided a method of inhibiting expression of a TADG-12 protein by introducing an antibody directed against a TADG-12 protein or fragment thereof. As a result, the binding of the antibody to the TADG-12 protein or fragment thereof inhibits the expression of the TADG-12 protein.
TADG-12 gene products including the truncated form can be used for targeted therapy. Specifically, a compound having a targeting moiety specific for a TADG-12 protein and a therapeutic moiety is administered to an individual in need of such treatment. Preferably, the targeting moiety is selected from the group consisting of an antibody directed against a TADG-12 protein and a ligand or ligand binding domain that binds a TADG-12 protein. The TADG-12 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Still preferably, the therapeutic moiety is selected from the group consisting of a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant and a cytotoxic agent. Such method can be used for treating an individual having a disease selected from the group consisting of ovarian cancer, lung cancer, prostate cancer, colon cancer and other cancers in which TADG-12 is overexpressed.
In yet another embodiment of the present invention, there is provided a method of vaccinating, or producing an immune response in, an individual against TADG-12 by inoculating the individual with a TADG-12 protein or fragment thereof. Specifically, the TADG-12 protein or fragment thereof lacks TADG-12 activity, and the inoculation elicits an immune response in the individual, thereby vaccinating the individual against TADG-12. Preferably, the individual has a cancer, is suspected of having a cancer or is at risk of getting a cancer. Still preferably, TADG-12 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, while TADG-12 fragment has a sequence shown in SEQ ID NO: 8, or is a 9-residue fragment up to a 20-residue fragment. Examples of 9-residue fragment are shown in SEQ ID Nos. 35, 36, 55, 56, 83, 84, 97, 98, 119, 120, 122, 123 and 136.
In still yet another embodiment of the present invention, there is provided an immunogenic composition, comprising an immunogenic fragment of a TADG-12 protein and an appropriate adjuvant. Preferably, the immunogenic fragment of the TADG-12 protein has a sequence shown in SEQ ID NO: 8, or is a 9-residue fragment up to a 20-residue fragment. Examples of 9-residue fragment are shown in SEQ ID Nos. 35, 36, 55, 56, 83, 84, 97, 98, 119, 120, 122, 123 and 136.
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.
Tissue Collection and Storage
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. 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 were logged into the laboratory record and stored at -80° C.
mRNA Extraction and cDNA Synthesis
Sixty-nine ovarian tumors (4 benign tumors, 10 low malignant potential tumors and 55 carcinomas) and 10 normal ovaries were obtained from surgical specimens and frozen in liquid nitrogen. The human ovarian carcinoma cell lines SW 626 and Caov 3, 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.
Extraction of mRNA and cDNA synthesis were carried out by the methods described previously (14-16). mRNA was isolated by using a RiboSep mRNA isolation kit (Becton Dickinson Labware). In this procedure, poly A+ mRNA was isolated directly from the tissue lysate using the affinity chromatography media oligo(dT) cellulose. cDNA was synthesized with 5.0 μg of mRNA by random hexamer priming using 1st strand cDNA synthesis kit (CLONTECH).
PCR with Redundant Primers and Cloning of TADG-12 cDNA
Redundant primers, forward 5'-TGGGTIGTIACIGCIGCICA(CT)TG-3' (SEQ ID NO: 26) and reverse 5'-A(AG)IA(AG)IGCIATITCITTICC-3' (SEQ ID NO: 27), for the consensus sequences of amino acids surrounding the catalytic triad for serine proteases were used to compare the PCR products from normal and carcinoma cDNAs. The appropriate bands were ligated into Promega T-vector plasmid and the ligation product was used to transform JM109 cells (Promega) grown on selection media. After selection of individual colonies, they were cultured and plasmid DNA was isolated by means of the Wizard miniprep DNA purification system (Promega). Nucleotide sequencing was performed using PRISM Ready Reaction Dye Deoxy terminator cycle sequencing kit (Applied Biosystems). Applied Biosystems Model 373A DNA sequencing system was used for direct cDNA sequence determination.
The original TADG-12 subclone was randomly labeled and used as a probe to screen an ovarian tumor cDNA library by standard hybridization techniques (11, 15). The library was constructed in λZAP using mRNA isolated from the tumor cells of a stage III/grade III ovarian adenocarcinoma patient. Three overlapping clones were obtained which spanned 2315 nucleotides. The final 99 nucleotides encoding the most 3' sequence including the poly A tail was identified by homology with clones available in the GenBank EST database.
The mRNA overexpression of TADG-12 was determined using a quantitative PCR. Quantitative PCR was performed according to the procedure as previously reported (16). Oligonucleotide primers were used for: TADG-12, forward 5'-GAAACATGTCCTTGCTCTCG-3' (SEQ ID NO: 28) and reverse 5'-ACTAACTTCCACAGCCTCCT-3' (SEQ ID NO: 29); the variant TADG-12, forward 5'-TCCAGGTGGGTCTAGTTTCC-3' (SEQ ID NO: 30), reverse 5'-CTCTTTGGCTTGTACTTGCT-3' (SEQ ID NO: 31); β-tubulin, forward 5'-CGCATCAACGTGTACTACAA-3' (SEQ ID NO: 32) and reverse 5'-TACGAGCTGGTGGACTGAGA-3' (SEQ ID NO: 33). β-tubulin was utilized as an internal control. The PCR reaction mixture consists of cDNA derived from 50 ng of mRNA, 5 pmol of sense and antisense primers for both the TADG-12 gene and the β-tubulin gene, 200 μmol of dNTPs, 5 μCi of α-32PdCTP and 0.25 unit 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 Cetus). Each cycle of PCR included 30 seconds of denaturation at 94% C, 30 seconds of annealing at 60% C and 30 seconds of extension at 72% C. The PCR products were separated on 2% agarose gels and the radioactivity of each PCR product was determined by using a Phospho Imager (Molecular Dynamics). The present study used the expression ratio (TADG-12/β-tubulin) as measured by phosphoimager 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.
Sequencing of TADG-12/TADG-12V
Utilizing a plasmid specific primer near the cloning site, sequencing reactions were carried out using PRISM® Ready Reaction Dye Deoxy® terminators (Applied Biosystems NO: 401384) according to the manufacturer's instructions. Residual dye terminators were removed from the completed sequencing reaction using a Centri-sep® spin column (Princeton Separation NO: CS-901). An Applied Biosystems Model 373A DNA Sequencing System was available and was used for sequence analysis.
Polyclonal rabbit antibodies were generated by immunization of white New Zealand rabbits with a poly-lysine linked multiple antigen peptide derived from the TADG-12 carboxy-terminal protein sequence NH2-WIHEQMERDLKT-COOH (WIHEQMERDLKT, SEQ ID NO: 34). This peptide is present in full length TADG-12, but not TADG-12V. Rabbits were immunized with approximately 100 μg of peptide emulsified in Ribi adjuvant. Subsequent boost immunizations were carried out at 3 and 6 weeks, and rabbit serum was isolated 10 days after the boost inoculations. Sera were tested by dot blot analysis to determine affinity for the TADG-12 specific peptide. Rabbit pre-immune serum was used as a negative control.
Northern Blot Analysis
10 μg of mRNA were loaded onto a 1% formaldehyde-agarose gel, electrophoresed and blotted on a Hybond-N+nylon membrane (Amersham). 32P-labeled cDNA probes were made by Prime-a-Gene Labeling System (Promega). The PCR products amplified by the same primers as 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 the β-tubulin probe.
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 same hybridization procedure.
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 minutes. TADG-12 peptide antibody was allowed to incubate with the specimens in a moisture chamber for 1 hour. Excess antibody was washed away with phosphate buffered saline. After incubation with biotinylated anti-rabbit IgG for 30 minutes, the sections were then incubated with ABC reagent (Vector) for 30 minutes. The final products were visualized using the AEC substrate system (DAKO) and sections were counterstained with hematoxylin before mounting. Negative controls were performed by using normal serum instead of the primary antibody.
Isolation of Catalytic Domain Subclones of TADG-12 and TADG-12 Variant
To identify serine proteases that are expressed in ovarian tumors, redundant PCR primers designed to the conserved regions of the catalytic triad of these enzymes were employed. A sense primer designed to the region surrounding the conserved histidine and an anti-sense primer designed to the region surrounding the conserved aspartate were used in PCR reactions with either normal ovary or ovarian tumor cDNA as template. In the reaction with ovarian tumor cDNA, a strong product band of the expected size of approximately 180 bp was observed as well as an unexpected PCR product of approximately 300 bp which showed strong expression in some ovarian tumor cDNA's (FIG. 1A). Both of these PCR products were subcloned and sequenced. The sequence of the subclones from the 180 bp band (SEQ ID NO: 5) was found to be homologous to the sequence identified in the larger, unexpected band (SEQ ID NO: 7) except that the larger band had an additional insert of 133 nucleotides (FIG. 1B). The smaller product of the appropriate size encoded for a protein sequence (SEQ ID NO: 6) homologous to other known proteases while the sequence with the insertion (SEQ ID NO: 8) encoded for a frame shift from the serine protease catalytic domain and a subsequent premature translational stop codon. TADG-12 variants from four individual tumors were also subcloned and sequenced. It was found that the sequence and insert to be identical. The genomic sequences for these cDNA derived clones were amplified by PCR, examined and found to contain potential AG/GT splice sites that would allow for the variant transcript production (data not shown).
Northern Blot Analysis of TADG-12 Expression
To examine transcript size and tissue distribution, the catalytic domain subclone was randomly labeled and used to probe Northern blots representing normal ovarian tissue, ovarian tumors and the cancer cell lines SW626, CAOV3, HeLa, MD-MBA-435S and MD-MBA-231 (FIG. 2). Three transcripts of 2.4, 1.6 and 0.7 kilobases were observed. In blots of normal and ovary tumor the smallest transcript size 0.7 kb was lowly expressed in normal ovary while all transcripts (2.4, 1.6 and 0.7 kb) were abundantly present in serous carcinoma. In addition, Northern blots representing the normal human tissues spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte, and normal human fetal tissues of brain, lung, liver and kidney were examined. The same three transcripts were found to be expressed weakly in all of these tissues (data not shown). A human β-tubulin specific probe was utilized as a control for relative sample loading. In addition, an RNA dot blot was probed representing 50 human tissues and determined that this clone is weakly expressed in all tissues represented (FIG. 3). It was found most prominently in heart, with intermediate levels in putamen, amygdala, kidney, liver, small intestine, skeletal muscle, and adrenal gland.
Sequencing and Characterization of TADG-12
An ovarian tumor cDNA library constructed in λZAP was screened by standard hybridization techniques using the catalytic domain subclone as a probe. Two clones that overlapped with the probe were identified and sequenced and found to represent 2316 nucleotides. The 97 nucleotides at the 3' end of the transcript including the poly-adenylation signal and the poly (A) tail were identified by homology with clones available in GenBank's EST database. This brought the total size of the transcript to 2413 bases (SEQ ID NO: 1, FIGS. 4A-4C). Subsequent screening of GenBank's Genomic Database revealed that TADG-12 is homologous to a cosmid from chromosome 17. This cosmid has the accession number AC015555.
The identified cDNA includes an open reading frame that would produce a predicted protein of 454 amino acids (SEQ ID NO: 2), named Tumor Associated Differentially-Expressed Gene 12 (TADG-12). The sequence has been submitted to the GenBank database and granted the accession no. AF201380. Using homology alignment programs, this protein contains several domains including an amino-terminal cytoplasmic domain, a potential Type II transmembrane domain followed by a low-density lipoprotein receptor-like class A domain (LDLR-A), a scavenger receptor cysteine rich domain (SRCR), and an extracellular serine protease domain.
As predicted by the ®Pred program, TADG-12 contains a highly hydrophobic stretch of amino acids that could serve as a potential transmembrane domain, which would retain the amino terminus of the protein within the cytoplasm and expose the ligand binding domains and protease domain to the extracellular space. This general structure is consistent with other known transmembrane proteases including hepsin (17), and TMPRSS2 (18), and TADG-12 is particularly similar in structure to the TMPRSS2 protease.
The LDLR-A domain of TADG-12 is represented by the sequence from amino acid 74 to 108 (SEQ ID NO: 13). The LDLR-A domain was originally identified within the LDL Receptor (19) as a series of repeated sequences of approximately 40 amino acids, which contained 6 invariant cysteine residues and highly conserved aspartate and glutamate residues. Since that initial identification, a host of other genes have been identified which contain motifs homologous to this domain (20). Several proteases have been identified which contain LDLR-A motifs including matriptase, TMPRSS2 and several complement components. A comparison of TADG-12 with other known LDLR-A domains is shown in FIG. 5A. The similarity of these sequences range from 44 to 54% of similar or identical amino acids.
In addition to the LDLR-A domain, TADG-12 contains another extracellular ligand binding domain with homology to the group A SRCR family. This family of protein domains typically is defined by the conservation of 6 cysteine resides within a sequence of approximately 100 amino acids (23). The SRCR domain of TADG-12 is encoded by amino acids 109 to 206 (SEQ ID NO: 17), and this domain was aligned with other SRCR domains and found to have between 36 and 43% similarity (FIG. 5B). However, TADG-12 only has 4 of the 6 conserved cysteine residues. This is similar to the SRCR domain found in the protease TMPRSS2.
The TADG-12 protein also includes a serine protease domain of the trypsin family of proteases. An alignment of the catalytic domain of TADG-12 with other known proteases is shown in FIG. 5C. The similarity among these sequence ranges from 48 to 55%, and TADG-12 is most similar to the serine protease TMPRSS2 which also contains a transmembrane domain, LDLR-A domain and an SRCR domain. There is a conserved amino acid motif (RIVGG; SEQ ID NO. 154) downstream from the SRCR domain that is a potential cleavage/activation site common to many serine proteases of this family (25). This suggests that TADG-12 is trafficked to the cell surface where the ligand binding domains are capable of interacting with extracellular molecules and the protease domain is potentially activated. TADG-12 also contains conserved cysteine residues (amino acids 208 and 243) which in other proteases form a disulfide bond capable of linking the activated protease to the other extracellular domains.
Quantitative PCR Characterization of the Alternative Transcript
The original TADG-12 subclone was identified as highly expressed in the initial redundant-primer PCR experiment. The TADG-12 variant form (TADG-12V) with the insertion of 133 bp was also easily detected in the initial experiment. To identify the frequency of this expression and whether or not the expression level between normal ovary and ovarian tumors was different, a previously authenticated semi-quantitative PCR technique was employed (16). The PCR analysis co-amplified a product for β-tubulin with either a product specific to TADG-12 or TADG-12V in the presence of a radiolabelled nucleotide. The products were separated by agarose gel electrophoresis and a phosphoimager was used to quantitate the relative abundance of each PCR product. Examples of these PCR amplification products are shown for both TADG-12 and TADG-12V in FIG. 6. Normal expression was defined as the mean ratio of TADG-12 (or TADG-12V) to β-tubulin +/-2SD as examined in normal ovarian samples. For tumor samples, overexpression was defined as >2SD from the normal TADG-12/β-tubulin or TADG-12V/β-tubulin ratio. The results are summarized in Table 1 and Table 2. TADG-12 was found to be overexpressed in 41 of 55 carcinomas examined while the variant form was present at aberrantly high levels in 8 of 22 carcinomas. As determined by the student's t test, these differences were statistically significant (p<0.05).
TABLE-US-00001 TABLE 1 Frequency of Overexpression of TADG-12 in Ovarian Carcinoma Histology Type TADG-12 (%) Normal 0/16 (0%) LMP-Serous 3/6 (50%) LMP-Mucinous 0/4 (0%) Serous Carcinoma 23/29 (79%) Mucinous Carcinoma 7/12 (58%) Endometrioid Carcinoma 8/8 (100%) Clear Cell Carcinoma 3/6 (50%) Benign Tumors 3/4 (75%)
TABLE-US-00002 TABLE 2 Frequency of Overexpression of TADG-12V in Ovarian Carcinoma Histology Type TADG-12V (%) Normal 0/10 (0%) LMP-Serous 0/5 (0%) LMP-Mucinous 0/3 (0%) Serous Carcinoma 4/14 (29%) Mucinous Carcinoma 3/5 (60%) Endometrioid Carcinoma 1/3 (33%) Clear Cell Carcinoma N/D Overexpression = more than two standard deviations above the mean for normal ovary; LMP = low malignant potential tumor
Immunohistochemical Analysis of TADG-12 in Ovarian Tumor Cells
In order to examine the TADG-12 protein, polyclonal rabbit anti-sera to a peptide located in the carboxy-terminal amino acid sequence was developed. These antibodies were used to examine the expression level of the TADG-12 protein and its localization within normal ovary and ovarian tumor cells by immuno-localization. No staining was observed in normal ovarian tissues (FIG. 7A) while significant staining was observed in 22 of 29 tumors studied. Representative tumor samples are shown in FIGS. 7B and 7C. It should be noted that TADG-12 is found in a diffuse pattern throughout the cytoplasm indicative of a protein in a trafficking pathway. TADG-12 is also found at the cell surface in these tumor samples as expected. It should be noted that the antibody developed and used for immunohistochemical analysis would not detect the TADG-12V truncated protein.
The results of the immunohistochemical staining are summarized in Table 3. 22 of 29 ovarian tumors showed positive staining of TADG-12, whereas normal ovarian surface epithelium showed no expression of the TADG-12 antigen. 8 of 10 serous adenocarcinomas, 8 of 8 mucinous adenocarcinomas, 1 of 2 clear cell carcinomas, and 4 of 6 endometrioid carcinomas showed positive staining.
TABLE-US-00003 TABLE 3 Case Stage Histology Grade LN* TADG12 Prognosis 1 Normal ovary 0- 2 Normal ovary 0- 3 Normal ovary 0- 4 Mucinous B ND 0- Alive 5 Mucinous B ND 1+ Alive 6 1a Serous LMP G1 ND 1+ Alive 7 1a Mucinous LMP G1 ND 1+ Alive 8 1a Mucinous CA G1 ND 1+ Alive 9 1a Mucinous CA G2 ND 1+ Alive 10 1a Endometrioid CA G1 ND 0- Alive 11 1c Serous CA G1 N 1+ Alive 12 1c Mucinous CA G1 N 1+ Alive 13 1c Mucinous CA G1 N 2+ Alive 14 1c Clear cell CA G2 N 0- Alive 15 1c Clear cell CA G2 N 0- Alive 16 2c Serous CA G3 N 2+ Alive 17 3a Mucinous CA G2 N 2+ Alive 18 3b Serous CA G1 ND 1+ Alive 19 3c Serous CA G1 N 0- Dead 20 3c Serous CA G3 P 1+ Alive 21 3c Serous CA G2 P 2+ Alive 22 3c Serous CA G1 P 2+ Unknown 23 3c Serous CA G3 ND 2+ Alive 24 3c Serous CA G2 N 0- Dead 25 3c Mucinous CA G1 P 2+ Dead 26 3c Mucinous CA G2 ND 1+ Unknown 27 3c Mucinous CA G2 N 1+ Alive 28 3c Endometrioid CA G1 P 1+ Dead 29 3c Endometrioid CA G2 N 0- Alive 30 3c Endometrioid CA G2 P 1+ Dead 31 3c Endometrioid CA G3 P 1+ Alive 32 3c Clear Cell CA G3 P 2+ Dead LN* = Lymph Node: B = Benign; N = Negative; P = Positive; ND = Not Done
For vaccine or immune stimulation, individual 9-mers to 11-mers of the TADG-12 protein were examined to rank the binding of individual peptides to the top 8 haplotypes in the general population (27). The computer program used for this analysis can be found at www.bimas.dcrt.nih.gov/molbio/hla_bind/. Table 4 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-12 peptides that strongly bind to an HLA allele are putative immunogens, and are used to innoculate an individual against TADG-12.
TABLE-US-00004 TABLE 4 TADG-12 peptide ranking HLA Type Predicted SEQ & Ranking Start Peptide Dissociation1/2 ID NO: HLA A0201 1 40 ILSLLPFEV 685.783 35 2 144 AQLGFPSYV 545.316 36 3 225 LLSQWPWQA 63.342 37 4 252 WIITAAHCV 43.992 38 5 356 VLNHAAVPL 36.316 39 6 176 LLPDDKVTA 34.627 40 7 13 FSFRSLFGL 31.661 41 8 151 YVSSDNLRV 27.995 42 9 436 RVTSFLDWI 21.502 43 10 234 SLQFQGYHL 21.362 44 11 181 KVTALHHSV 21.300 45 12 183 TALHHSVYV 19.658 46 13 411 RLWKLVGAT 18.494 47 14 60 LILALAIGL 18.476 48 15 227 SQWPWQASL 17.977 49 16 301 RLGNDIALM 11.426 50 17 307 ALMKLAGPL 10.275 51 18 262 DLYLPKSWT 9.837 52 19 416 LVGATSFGI 9.001 53 20 54 SLGIIALIL 8.759 54 HLA A0205 1 218 IVGGNMSLL 47.600 55 2 60 LILALAIGL 35.700 48 3 35 AVAAQILSL 28.000 56 4 307 ALMKLAGPL 21.000 51 5 271 IQVGLVSLL 19.040 57 6 397 CQGDSGGPL 16.800 58 7 227 SQWPWQASL 16.800 49 8 270 TIQVGLVSL 14.000 59 9 56 GIIALILAL 14.000 60 10 110 RVGGQNAVL 14.000 61 11 181 KVTALHHSV 12.000 45 12 151 YVSSDNLRV 12.000 42 13 356 VLNHAAVPL 11.900 39 14 144 AQLGFPSYV 9.600 36 15 13 FSFRSLFGL 7.560 41 16 54 SLGIIALIL 7.000 54 17 234 SLQFQGYHL 7.000 44 18 217 RIVGGNMSL 7.000 62 19 411 RLWKLVGAT 6.000 47 20 252 WIITAAHCV 6.000 38 HLA A1 1 130 CSDDWKGHY 37.500 63 2 8 AVEAPFSFR 9.000 64 3 328 NSEENFPDG 2.700 65 4 3 ENDPPAVEA 2.500 66 5 98 DCKDGEDEY 2.500 67 6 346 ATEDGGDAS 2.250 68 7 360 AAVPLISNK 2.000 69 8 153 SSDNLRVSS 1.500 70 9 182 VTALHHSVY 1.250 71 10 143 CAQLGFPSY 1.000 72 11 259 CVYDLYLPK 1.000 73 12 369 ICNHRDVYG 1.000 74 13 278 LLDNPAPSH 1.000 75 14 426 CAEVNKPGV 1.000 76 15 32 DADAVAAQI 1.000 77 16 406 VCQERRLWK 1.000 78 17 329 SEENFPDGK 0.900 79 18 303 GNDIALMKL 0.625 80 19 127 KTMCSDDWK 0.500 81 20 440 FLDWIHEQM 0.500 82 HLA A24 1 433 VYTRVTSFL 280.000 83 2 263 LYLPKSWTI 90.000 84 3 169 EFVSIDHLL 42.000 85 4 217 RIVGGNMSL 12.000 62 5 296 KYKPKRLGN 12.000 86 6 16 RSLFGLDDL 12.000 87 7 267 KSWTIQVGL 11.200 88 8 81 RSSFKCIEL 8.800 89 9 375 VYGGIISPS 8.000 90 10 110 RVGGQNAVL 8.000 91 11 189 VYVREGCAS 7.500 92 12 60 LILALAIGL 7.200 48 13 165 QFREEFVSI 7.200 93 14 271 IQVGLVSLL 7.200 57 15 56 GIIALILAL 7.200 60 16 10 EAPFSFRSL 7.200 94 17 307 ALMKLAGPL 7.200 51 18 407 CQERRLWKL 6.600 95 19 356 VLNHAAVPL 6.000 39 20 381 SPSMLCAGY 6.000 96 HLA B7 1 375 VYGGIISPS 200.000 97 2 381 SPSMLCAGY 80.000 98 3 362 VPLISNKIC 80.000 99 4 35 AVAAQILSL 60.000 56 5 373 RDVYGGIIS 40.000 100 6 307 ALMKLAGPL 36.000 51 7 283 APSHLVEKI 24.000 101 8 177 LPDDKVTAL 24.000 102 9 47 EVFSQSSSL 20.000 103 10 110 RVGGQNAVL 20.000 91 11 218 IVGGNMSLL 20.000 55 12 36 VAAQILSLL 12.000 104 13 255 TAAHCVYDL 12.000 105 14 10 EAPFSFRSL 12.000 94 15 138 YANVACAQL 12.000 106 16 195 CASGHVVTL 12.000 107 17 215 SSRIVGGNM 10.000 108 18 298 KPKRLGNDI 8.000 109 19 313 GPLTFNEMI 8.000 110 20 108 CVRVGGQNA 5.000 111 HLA B8 1 294 HSKYKPKRL 80.000 112 2 373 RDVYGGIIS 16.000 100 3 177 LPDDKVTAL 4.800 102 4 265 LPKSWTIQV 2.400 113 5 88 ELITRCDGV 2.400 114 6 298 KPKRLGNDI 2.000 109 7 81 RSSFKCIEL 2.000 89 8 375 VYGGIISPS 2.000 97 9 79 RCRSSFKCI 2.000 115 10 10 EAPFSFRSL 1.600 94 11 215 SSRIVGGNM 1.000 108 12 36 VAAQILSLL 0.800 104 13 255 TAAHCVYDL 0.800 116 14 381 SPSMLCAGY 0.800 98 15 195 CASGHVVTL 0.800 107 16 362 VPLISNKIC 0.800 99 17 138 YANVACAQL 0.800 106 18 207 ACGHRRGYS 0.400 117 19 154 SDNLRVSSL 0.400 118
20 47 EVFSQSSSL 0.400 103 HLA B2702 1 300 KRLGNDIAL 180.000 119 2 435 TRVTSFLDW 100.000 120 3 376 YGGIISPSM 100.000 121 4 410 RRLWKLVGA 60.000 122 5 210 HRRGYSSRI 60.000 123 6 227 SQWPWQASL 30.000 49 7 109 VRVGGQNAV 20.000 124 8 191 VREGCASGH 20.000 125 9 78 YRCRSSFKC 20.000 126 10 113 GQNAVLQVF 20.000 127 11 91 TRCDGVSDC 20.000 128 12 38 AQILSLLPF 20.000 129 13 211 RRGYSSRIV 18.000 130 14 216 SRIVGGNMS 10.000 131 15 118 LQVFTAASW 10.000 132 16 370 CNHRDVYGG 10.000 133 17 393 GVDSCQGDS 10.000 134 18 235 LQFQGYHLC 10.000 135 19 271 IQVGLVSLL 6.000 57 20 408 CQERRLWKL 6.000 95 HLA B4403 1 427 AEVNKPGVY 90.000 136 2 162 LEGQFREEF 40.000 137 3 9 VEAPFSFRS 24.000 138 4 318 NEMIQPVCL 12.000 139 5 256 AAHCVYDLY 9.000 140 6 98 DCKDGEDEY 9.000 67 7 46 FEVFSQSSS 8.000 141 8 38 AQILSLLPF 7.500 129 9 64 LAIGLGIHF 7.500 142 10 192 REGCASGHV 6.000 143 11 330 EENFPDGKV 6.000 144 12 182 VTALHHSVY 6.000 145 13 408 QERRLWKLV 6.000 146 14 206 TACGHRRGY 4.500 147 15 5 DPPAVEAPF 4.500 148 16 261 YDLYLPKSW 4.500 149 17 33 ADAVAAQIL 4.500 150 18 168 EEFVSIDHL 4.000 151 19 304 NDIALMKLA 3.750 152 20 104 DEYRCVRVG 3.600 153
In this study, a serine protease was identified by means of a PCR based strategy. By Northern blot, the largest transcript for this gene is approximately 2.4 kb, and it is found to be expressed at high levels in ovarian tumors while found at minimal levels in all other tissues examined. The full-length cDNA encoding a novel multi-domain, cell-surface serine protease was cloned, named TADG-12. The 454 amino acid protein contains a cytoplasmic domain, a type II transmembrane domain, an LDLR-A domain, an SRCR domain and a serine protease domain. Using a semi-quantitative PCR analysis, it was shown that TADG-12 is overexpressed in a majority of tumors studied. Immunohistochemical staining corroborates that in some cases this protein is localized to the cell-surface of tumor cells and this suggests that TADG-12 has some extracellular proteolytic functions. Interestingly, TADG-12 also has a variant splicing form that is present in 35% of the tumors studied. This variant mRNA would lead to a truncated protein that may provide a unique peptide sequence on the surface of tumor cells.
This protein contains two extracellular domains which might confer unusual properties to this multidomain molecule. Although the precise role of LDLR-A function with regard to proteases remains unclear, this domain certainly has the capacity to bind calcium and other positively charged ligands (21-22). This may play an important role in the regulation of the protease or subsequent internalization of the molecule. The SRCR domain was originally identified within the macrophage scavenger receptor and functionally described to bind lipoproteins. Not only are SRCR domains capable of binding lipoproteins, but they may also bind to molecules as diverse as polynucleotides (23). More recent studies have identified members of this domain family in proteins with functions that vary from proteases to cell adhesion molecules involved in maturation of the immune system (24). In addition, TADG-12, like TMPRSS2 has only four of six cysteine residues conserved within its SRCR domain. This difference may allow for different structural features of these domains that confer unusual ligand binding properties. At this time, only the function of the CD6 encoded SRCR is well documented. In the case of CD6, the SRCR domain binds to the cell adhesion molecule ALCAM (23). This mediation of cell adhesion is a useful starting point for future research on newly identified SRCR domains, however, the possibility of multiple functions for this domain can not be overlooked. SRCR domains are certainly capable of cell adhesion type interactions, but their capacity to bind other types of ligands should be considered.
At this time, the precise role of TADG-12 remains unclear. Substrates have not been identified for the protease domain, nor have ligands been identified for the extracellular LDLR-A and SRCR domains. FIG. 8 presents a working model of TADG-12 with the information disclosed in the present invention. Two transcripts are produced which lead to the production of either TADG-12 or the truncated TADG-12V proteins. Either of these proteins is potentially targeted to the cell surface. TADG-12 is capable of becoming an activated serine protease while TADG-12V is a truncated protein product that if at the cell surface may represent a tumor specific epitope.
The problem with treatment of ovarian cancer today remains the inability to diagnose the disease at an early stage. Identifying genes that are expressed early in the disease process such as proteases that are essential for tumor cell growth (26) is an important step toward improving treatment. With this knowledge, it may be possible to design assays to detect the highly expressed genes such as the TADG-12 protease described here or previously described proteases to diagnose these cancers at an earlier stage. Panels of markers may also provide prognostic information and could lead to therapeutic strategies for individual patients. Alternatively, inhibition of enzymes such as proteases may be an effective means for slowing progression of ovarian cancer and improving the quality of patient life. Other features of TADG-12 and TADG-12V must be considered important to future research too. The extracellular ligand binding domains are natural targets for drug delivery systems. The aberrant peptide associated with the TADG-12V protein may provide an excellent target drug delivery or for immune stimulation.
The following references are cited herein. 1. Duffy, M. J., Clin. Exp. Metastasis, 10: 145-155, 1992. 2. Monsky et al., Cancer Res., 53: 3159-3164, 1993. 3. Powell et al., Cancer Res., 53: 417-422, 1993. 4. Neurath, H., The Diversity of Proteolytic Enzymes. In: R. J. Beynon and J. S. Bond (eds.), pp. 1-13, Proteolytic enzymes, Oxford: IRL Press, 1989. 5. Liotta et al., Cell, 64: 327-336, 1991. 6. Tryggvason et al., Biochem. Biophys. Acta., 907: 191-217, 1987. 7. McCormack et al., Urology, 45:729-744, 1995. 8. Landis et al., CA Cancer J. Clin., 48: 6-29, 1998. 9. Tanimoto et al., Cancer Res., 57: 2884-2887, 1997. 10. Tanimoto et al., Cancer, 86: 2074-2082, 1999. 11. Underwood et al., Cancer Res., 59:4435-4439, 1999. 12. Tanimoto et al. Tumor Biology, 22: 104-114, 2000. 13. Tanimoto et al., Proc. of the Amer. Assoc. for Canc. Research 39:648, 1998. 14. Tanimoto et al., Tumor Biology, 20: 88-98, 1999. 15. Maniatis et al., J. Molecular Cloning, p. 309-361 1982. 16. Shigemasa et al., J. Soc. Gynecol. Invest., 4:95-102, 1997. 17. Leytus et al., Biochemistry, 27: 1067-1074, 1988. 18. Paoloni-Giacobino et al., Genomics, 44: 309-320, 1997. 19. Sudhof et al., Science, 228: 815-822, 1985. 20. Daly et al., Proc. Natl. Acad. Sci. USA 92: 6334-6338, 1995. 21. Mahley, R. W., Science 240: 622-630, 1988. 22. Van Driel et al., J. Biol. Chem. 262: 17443-17449, 1987. 23. Freeman et al., Proc. Natl. Acad. Sci. USA 87: 8810-8814, 1990. 24. Aruffo et al., Immunology Today 18(10): 498-504, 1997. 25. Rawlings, N. D., and Barrett, A. J., Methods Enzymology 244: 19-61, 1994. 26. Torres-Rosado et al., Proc. Natl. Acad. Sci. USA, 90: 7181-7185, 1993. 27. J. R. Parker. Practical Computer Vision Using C. John Wiley & Sons inc., New York, 1994.
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 indicated to be 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.
15412413DNAHomo sapiensCDSentire cDNA sequence of TADG-12 gene 1cgggaaaggg ctgtgtttat gggaagccag taacactgtg gcctactatc 50tcttccgtgg tgccatctac atttttggga ctcgggaatt atgaggtaga 100ggtggaggcg gagccggatg tcagaggtcc tgaaatagtc accatggggg 150aaaatgatcc gcctgctgtt gaagccccct tctcattccg atcgcttttt 200ggccttgatg atttgaaaat aagtcctgtt gcaccagatg cagatgctgt 250tgctgcacag atcctgtcac tgctgccatt tgaagttttt tcccaatcat 300cgtcattggg gatcattgca ttgatattag cactggccat tggtctgggc 350atccacttcg actgctcagg gaagtacaga tgtcgctcat cctttaagtg 400tatcgagctg ataactcgat gtgacggagt ctcggattgc aaagacgggg 450aggacgagta ccgctgtgtc cgggtgggtg gtcagaatgc cgtgctccag 500gtgttcacag ctgcttcgtg gaagaccatg tgctccgatg actggaaggg 550tcactacgca aatgttgcct gtgcccaact gggtttccca agctatgtga 600gttcagataa cctcagagtg agctcgctgg aggggcagtt ccgggaggag 650tttgtgtcca tcgatcacct cttgccagat gacaaggtga ctgcattaca 700ccactcagta tatgtgaggg agggatgtgc ctctggccac gtggttacct 750tgcagtgcac agcctgtggt catagaaggg gctacagctc acgcatcgtg 800ggtggaaaca tgtccttgct ctcgcagtgg ccctggcagg ccagccttca 850gttccagggc taccacctgt gcgggggctc tgtcatcacg cccctgtgga 900tcatcactgc tgcacactgt gtttatgact tgtacctccc caagtcatgg 950accatccagg tgggtctagt ttccctgttg gacaatccag ccccatccca 1000cttggtggag aagattgtct accacagcaa gtacaagcca aagaggctgg 1050gcaatgacat cgcccttatg aagctggccg ggccactcac gttcaatgaa 1100atgatccagc ctgtgtgcct gcccaactct gaagagaact tccccgatgg 1150aaaagtgtgc tggacgtcag gatggggggc cacagaggat ggaggtgacg 1200cctcccctgt cctgaaccac gcggccgtcc ctttgatttc caacaagatc 1250tgcaaccaca gggacgtgta cggtggcatc atctccccct ccatgctctg 1300cgcgggctac ctgacgggtg gcgtgaacag ctgccagggg gacagcgggg 1350ggcccctggt gtgtcaagag aggaggctgt ggaagttagt gggagcgacc 1400agctttggca tcggctgcgc agaggtgaac aagcctgggg tgtacacccg 1450tgtcacctcc ttcctggact ggatccacga gcagatggag agagacctaa 1500aaacctgaag aggaagggga caagtagcca cctgagttcc tgaggtgatg 1550aagacagccc gatcctcccc tggactcccg tgtaggaacc tgcacacgag 1600cagacaccct tggagctctg agttccggca ccagtagcgg gcccgaaaga 1650ggcacccttc catctgattc cagcacaacc ttcaagctgc tttttgtttt 1700ttgttttttt gaggtggagt ctcgctctgt tgcccaggct ggagtgcagt 1750ggcgaaatac cctgctcact gcagcctccg cttccctggt tcaagcgatt 1800ctcttgcctc agcttcccca gtagctggga ccacaggtgc ccgccaccac 1850acccaactaa tttttgtatt tttagtagag acagggtttc accatgttgg 1900ccaggctgct ctcaaacccc tgacctcaaa tgatgtgcct gcttcagcct 1950cccacagtgc tgggattaca ggcatgggcc accacgccta gcctcacgct 2000cctttctgat cttcactaag aacaaaagaa gcagcaactt gcaagggcgg 2050cctttcccac tggtccatct ggttttctct ccagggtctt gcaaaattcc 2100tgacgagata agcagttatg tgacctcacg tgcaaagcca ccaacagcca 2150ctcagaaaag acgcaccagc ccagaagtgc agaactgcag tcactgcacg 2200ttttcatctt tagggaccag aaccaaaccc accctttcta cttccaagac 2250ttattttcac atgtggggag gttaatctag gaatgactcg tttaaggcct 2300attttcatga tttctttgta gcatttggtg cttgacgtat tattgtcctt 2350tgattccaaa taatatgttt ccttccctca aaaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaa 24132454PRTHomo sapienscomplete amino acid sequence of TADG-12 protein 2Met Gly Glu Asn Asp Pro Pro Ala Val Glu Ala Pro Phe Ser Phe1 5 10 15Arg Ser Leu Phe Gly Leu Asp Asp Leu Lys Ile Ser Pro Val Ala20 25 30Pro Asp Ala Asp Ala Val Ala Ala Gln Ile Leu Ser Leu Leu Pro35 40 45Phe Glu Val Phe Ser Gln Ser Ser Ser Leu Gly Ile Ile Ala Leu50 55 60Ile Leu Ala Leu Ala Ile Gly Leu Gly Ile His Phe Asp Cys Ser65 70 75Gly Lys Tyr Arg Cys Arg Ser Ser Phe Lys Cys Ile Glu Leu Ile80 85 90Thr Arg Cys Asp Gly Val Ser Asp Cys Lys Asp Gly Glu Asp Glu95 100 105Tyr Arg Cys Val Arg Val Gly Gly Gln Asn Ala Val Leu Gln Val110 115 120Phe Thr Ala Ala Ser Trp Lys Thr Met Cys Ser Asp Asp Trp Lys125 130 135Gly His Tyr Ala Asn Val Ala Cys Ala Gln Leu Gly Phe Pro Ser140 145 150Tyr Val Ser Ser Asp Asn Leu Arg Val Ser Ser Leu Glu Gly Gln155 160 165Phe Arg Glu Glu Phe Val Ser Ile Asp His Leu Leu Pro Asp Asp170 175 180Lys Val Thr Ala Leu His His Ser Val Tyr Val Arg Glu Gly Cys185 190 195Ala Ser Gly His Val Val Thr Leu Gln Cys Thr Ala Cys Gly His200 205 210Arg Arg Gly Tyr Ser Ser Arg Ile Val Gly Gly Asn Met Ser Leu215 220 225Leu Ser Gln Trp Pro Trp Gln Ala Ser Leu Gln Phe Gln Gly Tyr230 235 240His Leu Cys Gly Gly Ser Val Ile Thr Pro Leu Trp Ile Ile Thr245 250 255Ala Ala His Cys Val Tyr Asp Leu Tyr Leu Pro Lys Ser Trp Thr260 265 270Ile Gln Val Gly Leu Val Ser Leu Leu Asp Asn Pro Ala Pro Ser275 280 285His Leu Val Glu Lys Ile Val Tyr His Ser Lys Tyr Lys Pro Lys290 295 300Arg Leu Gly Asn Asp Ile Ala Leu Met Lys Leu Ala Gly Pro Leu305 310 315Thr Phe Asn Glu Met Ile Gln Pro Val Cys Leu Pro Asn Ser Glu320 325 330Glu Asn Phe Pro Asp Gly Lys Val Cys Trp Thr Ser Gly Trp Gly335 340 345Ala Thr Glu Asp Gly Gly Asp Ala Ser Pro Val Leu Asn His Ala350 355 360Ala Val Pro Leu Ile Ser Asn Lys Ile Cys Asn His Arg Asp Val365 370 375Tyr Gly Gly Ile Ile Ser Pro Ser Met Leu Cys Ala Gly Tyr Leu380 385 390Thr Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu395 400 405Val Cys Gln Glu Arg Arg Leu Trp Lys Leu Val Gly Ala Thr Ser410 415 420Phe Gly Ile Gly Cys Ala Glu Val Asn Lys Pro Gly Val Tyr Thr425 430 435Arg Val Thr Ser Phe Leu Asp Trp Ile His Glu Gln Met Glu Arg440 445 450Asp Leu Lys Thr32544DNAHomo sapiensCDSentire cDNA sequence of TADG-12 variant gene 3cgggaaaggg ctgtgtttat gggaagccag taacactgtg gcctactatc 50tcttccgtgg tgccatctac atttttggga ctcgggaatt atgaggtaga 100ggtggaggcg gagccggatg tcagaggtcc tgaaatagtc accatggggg 150aaaatgatcc gcctgctgtt gaagccccct tctcattccg atcgcttttt 200ggccttgatg atttgaaaat aagtcctgtt gcaccagatg cagatgctgt 250tgctgcacag atcctgtcac tgctgccatt tgaagttttt tcccaatcat 300cgtcattggg gatcattgca ttgatattag cactggccat tggtctgggc 350atccacttcg actgctcagg gaagtacaga tgtcgctcat cctttaagtg 400tatcgagctg ataactcgat gtgacggagt ctcggattgc aaagacgggg 450aggacgagta ccgctgtgtc cgggtgggtg gtcagaatgc cgtgctccag 500gtgttcacag ctgcttcgtg gaagaccatg tgctccgatg actggaaggg 550tcactacgca aatgttgcct gtgcccaact gggtttccca agctatgtaa 600gttcagataa cctcagagtg agctcgctgg aggggcagtt ccgggaggag 650tttgtgtcca tcgatcacct cttgccagat gacaaggtga ctgcattaca 700ccactcagta tatgtgaggg agggatgtgc ctctggccac gtggttacct 750tgcagtgcac agcctgtggt catagaaggg gctacagctc acgcatcgtg 800ggtggaaaca tgtccttgct ctcgcagtgg ccctggcagg ccagccttca 850gttccagggc taccacctgt gcgggggctc tgtcatcacg cccctgtgga 900tcatcactgc tgcacactgt gtttatgaga ttgtagctcc tagagaaagg 950gcagacagaa gaggaaggaa gctcctgtgc tggaggaaac ccacaaaaat 1000gaaaggacct agaccttccc atagctaatt ccagtggacc atgttatggc 1050agatacaggc ttgtacctcc ccaagtcatg gaccatccag gtgggtctag 1100tttccctgtt ggacaatcca gccccatccc acttggtgga gaagattgtc 1150taccacagca agtacaagcc aaagaggctg ggcaatgaca tcgcccttat 1200gaagctggcc gggccactca cgttcaatga aatgatccag cctgtgtgcc 1250tgcccaactc tgaagagaac ttccccgatg gaaaagtgtg ctggacgtca 1300ggatgggggg ccacagagga tggaggtgac gcctcccctg tcctgaacca 1350cgcggccgtc cctttgattt ccaacaagat ctgcaaccac agggacgtgt 1400acggtggcat catctccccc tccatgctct gcgcgggcta cctgacgggt 1450ggcgtggaca gctgccaggg ggacagcggg gggcccctgg tgtgtcaaga 1500gaggaggctg tggaagttag tgggagcgac cagctttggc atcggctgcg 1550cagaggtgaa caagcctggg gtgtacaccc gtgtcacctc cttcctggac 1600tggatccacg agcagatgga gagagaccta aaaacctgaa gaggaagggg 1650acaagtagcc acctgagttc ctgaggtgat gaagacagcc cgatcctccc 1700ctggactccc gtgtaggaac ctgcacacga gcagacaccc ttggagctct 1750gagttccggc accagtagcg ggcccgaaag aggcaccctt ccatctgatt 1800ccagcacaac cttcaagctg ctttttgttt tttgtttttt tgaggtggag 1850tctcgctctg ttgcccaggc tggagtgcag tggcgaaata ccctgctcac 1900tgcagcctcc gcttccctgg ttcaagcgat tctcttgcct cagcttcccc 1950agtagctggg accacaggtg cccgccacca cacccaacta atttttgtat 2000ttttagtaga gacagggttt caccatgttg gccaggctgc tctcaaaccc 2050ctgacctcaa atgatgtgcc tgcttcagcc tcccacagtg ctgggattac 2100aggcatgggc caccacgcct agcctcacgc tcctttctga tcttcactaa 2150gaacaaaaga agcagcaact tgcaagggcg gcctttccca ctggtccatc 2200tggttttctc tccagggtct tgcaaaattc ctgacgagat aagcagttat 2250gtgacctcac gtgcaaagcc accaacagcc actcagaaaa gacgcaccag 2300cccagaagtg cagaactgca gtcactgcac gttttcatct ttagggacca 2350gaaccaaacc caccctttct acttccaaga cttattttca catgtgggga 2400ggttaatcta ggaatgactc gtttaaggcc tattttcatg atttctttgt 2450agcatttggt gcttgacgta ttattgtcct ttgattccaa ataatatgtt 2500tccttccctc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 25444294PRTHomo sapienscomplete amino acid sequence of TADG-12 variant protein 4Met Gly Glu Asn Asp Pro Pro Ala Val Glu Ala Pro Phe Ser Phe1 5 10 15Arg Ser Leu Phe Gly Leu Asp Asp Leu Lys Ile Ser Pro Val Ala20 25 30Pro Asp Ala Asp Ala Val Ala Ala Gln Ile Leu Ser Leu Leu Pro35 40 45Phe Glu Val Phe Ser Gln Ser Ser Ser Leu Gly Ile Ile Ala Leu50 55 60Ile Leu Ala Leu Ala Ile Gly Leu Gly Ile His Phe Asp Cys Ser65 70 75Gly Lys Tyr Arg Cys Arg Ser Ser Phe Lys Cys Ile Glu Leu Ile80 85 90Thr Arg Cys Asp Gly Val Ser Asp Cys Lys Asp Gly Glu Asp Glu95 100 105Tyr Arg Cys Val Arg Val Gly Gly Gln Asn Ala Val Leu Gln Val110 115 120Phe Thr Ala Ala Ser Trp Lys Thr Met Cys Ser Asp Asp Trp Lys125 130 135Gly His Tyr Ala Asn Val Ala Cys Ala Gln Leu Gly Phe Pro Ser140 145 150Tyr Val Ser Ser Asp Asn Leu Arg Val Ser Ser Leu Glu Gly Gln155 160 165Phe Arg Glu Glu Phe Val Ser Ile Asp His Leu Leu Pro Asp Asp170 175 180Lys Val Thr Ala Leu His His Ser Val Tyr Val Arg Glu Gly Cys185 190 195Ala Ser Gly His Val Val Thr Leu Gln Cys Thr Ala Cys Gly His200 205 210Arg Arg Gly Tyr Ser Ser Arg Ile Val Gly Gly Asn Met Ser Leu215 220 225Leu Ser Gln Trp Pro Trp Gln Ala Ser Leu Gln Phe Gln Gly Tyr230 235 240His Leu Cys Gly Gly Ser Val Ile Thr Pro Leu Trp Ile Ile Thr245 250 255Ala Ala His Cys Val Tyr Glu Ile Val Ala Pro Arg Glu Arg Ala260 265 270Asp Arg Arg Gly Arg Lys Leu Leu Cys Trp Arg Lys Pro Thr Lys275 280 285Met Lys Gly Pro Arg Pro Ser His Ser2905174DNAArtificial sequencenucleotide sequence of the subclone containing the 180 bp band from the PCR product for TADG-12 5tgggtggtga cggcggcgca ctgtgtttat gacttgtacc tccccaagtc 50atggaccatc caggtgggtc tagtttccct gttggacaat ccagccccat 100cccacttggt ggagaagatt gtctaccaca gcaagtacaa gccaaagagg 150ctgggcaacg acatcgccct ccta 174658PRTArtificial sequencededuced amino acid sequence of the 180 bp band from the PCR product for TADG-12 6Trp Val Val Thr Ala Ala His Cys Val Tyr Asp Leu Tyr Leu Pro1 5 10 15Lys Ser Trp Thr Ile Gln Val Gly Leu Val Ser Leu Leu Asp Asn20 25 30Pro Ala Pro Ser His Leu Val Glu Lys Ile Val Tyr His Ser Lys35 40 45Tyr Lys Pro Lys Arg Leu Gly Asn Asp Ile Ala Leu Leu50 557328DNAArtificial sequencenucleotide sequence of the subclone containing the 300 bp band from the PCR product for TADG-12 variant, which contains an additional insert of 133 bases 7gggtggtgac ggcggcgcac tgtgtttatg agattgtagc tcctagagaa 50agggcagaca gaagaggaag gaagctcctg tgctggagga aacccacaaa 100aatgaaagga cctagacctt cccatagcta attccagtgg accatgttat 150ggcagataca ggcttgtacc tccccaagtc atggaccatc caggtgggtc 200tagtttccct gttggacaat ccagccccat cccacttggt ggagaagatt 250gtctaccaca gcaagtacaa gccaaagagg ctgggcaacg acatcgccct 300cctaatcact agtgcggccg cctgcagg 328842PRTArtificial sequencededuced amino acid sequence of the 300 bp band from the PCR product for TADG-12 variant, which is a truncated form of TADG-12 8Val Val Thr Ala Ala His Cys Val Tyr Glu Ile Val Ala Pro Arg1 5 10 15Glu Arg Ala Asp Arg Arg Gly Arg Lys Leu Leu Cys Trp Arg Lys20 25 30Pro Thr Lys Met Lys Gly Pro Arg Pro Ser His Ser35 40934PRTHomo sapiensDOMAINLDLR-A domain of the complement subunit C8(Compc8) 9Cys Glu Gly Phe Val Cys Ala Gln Thr Gly Arg Cys Val Asn Arg1 5 10 15Arg Leu Leu Cys Asn Gly Asp Asn Asp Cys Gly Asp Gln Ser Asp20 25 30Glu Ala Asn Cys1034PRTHomo sapiensDOMAINLDLR-A domain of the serine protease matriptase (Matr) 10Cys Pro Gly Gln Phe Thr Cys Arg Thr Gly Arg Cys Ile Arg Lys1 5 10 15Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Thr Asp His Ser Asp20 25 30Glu Leu Asn Cys1137PRTHomo sapiensDOMAINLDLR-A domain of the glycoprotein GP300 (Gp300-1) 11Cys Gln Gln Gly Tyr Phe Lys Cys Gln Ser Glu Gly Gln Cys Ile1 5 10 15Pro Ser Ser Trp Val Cys Asp Gln Asp Gln Asp Cys Asp Asp Gly20 25 30Ser Asp Glu Arg Gln Asp Cys351235PRTHomo sapiensDOMAINLDLR-A domain of the glycoprotein GP300 (Gp300-2) 12Cys Ser Ser His Gln Ile Thr Cys Ser Asn Gly Gln Cys Ile Pro1 5 10 15Ser Glu Tyr Arg Cys Asp His Val Arg Asp Cys Pro Asp Gly Ala20 25 30Asp Glu Asn Asp Cys351335PRTHomo sapiensDOMAINLDLR-A domain of TADG-12 13Cys Ser Gly Lys Tyr Arg Cys Arg Ser Ser Phe Lys Cys Ile Glu1 5 10 15Leu Ile Thr Arg Cys Asp Gly Val Ser Asp Cys Lys Asp Gly Glu20 25 30Asp Glu Tyr Arg Cys351436PRTHomo sapiensDOMAINLDLR-A domain of the serine protease TMPRSS2 Tmprss2 14Cys Ser Asn Ser Gly Ile Glu Cys Asp Ser Ser Gly Thr Cys Ile1 5 10 15Asn Pro Ser Asn Trp Cys Asp Gly Val Ser His Cys Pro Gly Gly20 25 30Glu Asp Glu Asn Arg Cys3515101PRTBos taurusDOMAINSRCR domain of bovine enterokinase (BovEntk) 15Val Arg Leu Val Gly Gly Ser Gly Pro His Glu Gly Arg Val Glu1 5 10 15Ile Phe His Glu Gly Gln Trp Gly Thr Val Cys Asp Asp Arg Trp20 25 30Glu Leu Arg Gly Gly Leu Val Val Cys Arg Ser Leu Gly Tyr Lys35 40 45Gly Val Gln Ser Val His Lys Arg Ala Tyr Phe Gly Lys Gly Thr50 55 60Gly Pro Ile Trp Leu Asn Glu Val Phe Cys Phe Gly Lys Glu Ser65 70 75Ser Ile Glu Glu Cys Arg Ile Arg Gln Trp Gly Val Arg Ala Cys80 85 90Ser His Asp Glu Asp Ala Gly Val Thr Cys Thr95 10016101PRTHomo sapiensDOMAINSRCR domain of human macrophage scavenger receptor (MacSR) 16Val Arg Leu Val Gly Gly Ser Gly Pro His Glu Gly Arg Val Glu1 5 10 15Ile Leu His Ser Gly Gln Trp Gly Thr Ile Cys Asp Asp Arg Trp20 25 30Glu Val Arg Val Gly Gln Val Val Cys Arg Ser Leu Gly Tyr Pro35 40 45Gly Val Gln Ala Val His Lys Ala Ala His Phe Gly Gln Gly Thr50 55 60Gly Pro Ile Trp Leu Asn Glu Val Phe Cys Phe Gly Arg Glu Ser65 70 75Ser Ile Glu Glu Cys Lys Ile Arg Gln Trp Gly Thr Arg Ala Cys80 85 90Ser His Ser Glu Asp Ala Gly Val Thr Cys Thr95 1001798PRTHomo sapiensDOMAINSRCR domain of TADG-12 (TADG12) 17Val Arg
Val Gly Gly Gln Asn Ala Val Leu Gln Val Phe Thr Ala1 5 10 15Ala Ser Trp Lys Thr Met Cys Ser Asp Asp Trp Lys Gly His Tyr20 25 30Ala Asn Val Ala Cys Ala Gln Leu Gly Phe Pro Ser Tyr Val Ser35 40 45Ser Asp Asn Leu Arg Val Ser Ser Leu Glu Gly Gln Phe Arg Glu50 55 60Glu Phe Val Ser Ile Asp His Leu Leu Pro Asp Asp Lys Val Thr65 70 75Ala Leu His His Ser Val Tyr Val Arg Glu Gly Cys Ala Ser Gly80 85 90His Val Val Thr Leu Gln Cys Thr951894PRTHomo sapiensDOMAINSRCR domain of the serine protease TMPRSS2 (Tmprss2) 18Val Arg Leu Tyr Gly Pro Asn Phe Ile Leu Gln Met Tyr Ser Ser1 5 10 15Gln Arg Lys Ser Trp His Pro Val Cys Gln Asp Asp Trp Asn Glu20 25 30Asn Tyr Gly Arg Ala Ala Cys Arg Asp Met Gly Tyr Lys Asn Asn35 40 45Phe Tyr Ser Ser Gln Gly Ile Val Asp Asp Ser Gly Ser Thr Ser50 55 60Phe Met Lys Leu Asn Thr Ser Ala Gly Asn Val Asp Ile Tyr Lys65 70 75Lys Leu Tyr His Ser Asp Ala Cys Ser Ser Lys Ala Val Val Ser80 85 90Leu Arg Cys Leu1990PRTHomo sapiensDOMAINSRCR domain of human enterokinase (HumEntk) 19Val Arg Phe Phe Asn Gly Thr Thr Asn Asn Asn Gly Leu Val Arg1 5 10 15Phe Arg Ile Gln Ser Ile Trp His Thr Ala Cys Ala Glu Asn Trp20 25 30Thr Thr Gln Ile Ser Asn Asp Val Cys Gln Leu Leu Gly Leu Gly35 40 45Ser Gly Asn Ser Ser Lys Pro Ile Phe Ser Thr Asp Gly Gly Pro50 55 60Phe Val Lys Leu Asn Thr Ala Pro Asp Gly His Leu Ile Leu Thr65 70 75Pro Ser Gln Gln Cys Leu Gln Asp Ser Leu Ile Arg Leu Gln Cys80 85 9020149PRTHomo sapiensDOMAINprotease domain of protease M (ProM) 20Leu Trp Val Leu Thr Ala Ala His Cys Lys Lys Pro Asn Leu Gln1 5 10 15Val Phe Leu Gly Lys His Asn Leu Arg Gln Arg Glu Ser Ser Gln20 25 30Glu Gln Ser Ser Val Val Arg Ala Val Ile His Pro Asp Tyr Asp35 40 45Ala Ala Ser His Asp Gln Asp Ile Met Leu Leu Arg Leu Ala Arg50 55 60Pro Ala Lys Leu Ser Glu Leu Ile Gln Pro Leu Pro Leu Glu Arg65 70 75Asp Cys Ser Ala Asn Thr Thr Ser Cys His Ile Leu Gly Trp Gly80 85 90Lys Thr Ala Asp Gly Asp Phe Pro Asp Thr Ile Gln Cys Ala Tyr95 100 105Ile His Leu Val Ser Arg Glu Glu Cys Glu His Ala Tyr Pro Gly110 115 120Gln Ile Thr Gln Asn Met Leu Cys Ala Gly Asp Glu Lys Tyr Gly125 130 135Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys140 14521151PRTHomo sapiensDOMAINprotease domain of trypsinogen I (Try1) 21Gln Trp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Gln1 5 10 15Val Arg Leu Gly Glu His Asn Ile Glu Val Leu Glu Gly Asn Glu20 25 30Gln Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro Gln Tyr Asp35 40 45Arg Lys Thr Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Ser Ser50 55 60Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro Thr65 70 75Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly80 85 90Asn Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln Cys95 100 105Leu Asp Ala Pro Val Leu Ser Gln Ala Lys Cys Glu Ala Ser Tyr110 115 120Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe Leu Glu125 130 135Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val140 145 150Cys22158PRTHomo sapiensDOMAINprotease domain of plasma kallikrein (Kal) 22Gln Trp Val Leu Thr Ala Ala His Cys Phe Asp Gly Leu Pro Leu1 5 10 15Gln Asp Val Trp Arg Ile Tyr Ser Gly Ile Leu Asn Leu Ser Asp20 25 30Ile Thr Lys Asp Thr Pro Phe Ser Gln Ile Lys Glu Ile Ile Ile35 40 45His Gln Asn Tyr Lys Val Ser Glu Gly Asn His Asp Ile Ala Leu50 55 60Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr Glu Phe Gln Lys Pro65 70 75Ile Cys Leu Pro Ser Lys Gly Asp Thr Ser Thr Ile Tyr Thr Asn80 85 90Cys Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys Gly Glu Ile95 100 105Gln Asn Ile Leu Gln Lys Val Asn Ile Pro Leu Val Thr Asn Glu110 115 120Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg Met125 130 135Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly140 145 150Asp Ser Gly Gly Pro Leu Val Cys15523157PRTHomo sapiensDOMAINprotease domain of TADG-12 (TADG12) 23Leu Trp Ile Ile Thr Ala Ala His Cys Val Tyr Asp Leu Tyr Leu1 5 10 15Pro Lys Ser Trp Thr Ile Gln Val Gly Leu Val Ser Leu Leu Asp20 25 30Asn Pro Ala Pro Ser His Leu Val Glu Lys Ile Val Tyr His Ser35 40 45Lys Tyr Lys Pro Lys Arg Leu Gly Asn Asp Ile Ala Leu Met Lys50 55 60Leu Ala Gly Pro Leu Thr Phe Asn Glu Met Ile Gln Pro Val Cys65 70 75Leu Pro Asn Ser Glu Glu Asn Phe Pro Asp Gly Lys Val Cys Trp80 85 90Thr Ser Gly Trp Gly Ala Thr Glu Asp Gly Gly Asp Ala Ser Pro95 100 105Val Leu Asn His Ala Ala Val Pro Leu Ile Ser Asn Lys Ile Cys110 115 120Asn His Arg Asp Val Tyr Gly Gly Ile Ile Ser Pro Ser Met Leu125 130 135Cys Ala Gly Tyr Leu Thr Gly Gly Val Asp Ser Cys Gln Gly Asp140 145 150Ser Gly Gly Pro Leu Val Cys15524159PRTHomo sapiensDOMAINprotease domain of TMPRSS2 (Tmprss2) 24Glu Trp Ile Val Thr Ala Ala His Cys Val Glu Lys Pro Leu Asn1 5 10 15Asn Pro Trp His Trp Thr Ala Phe Ala Gly Ile Leu Arg Gln Ser20 25 30Phe Met Phe Tyr Gly Ala Gly Tyr Gln Val Gln Lys Val Ile Ser35 40 45His Pro Asn Tyr Asp Ser Lys Thr Lys Asn Asn Asp Ile Ala Leu50 55 60Met Lys Leu Gln Lys Pro Leu Thr Phe Asn Asp Leu Val Lys Pro65 70 75Val Cys Leu Pro Asn Pro Gly Met Met Leu Gln Pro Glu Gln Leu80 85 90Cys Trp Ile Ser Gly Trp Gly Ala Thr Glu Glu Lys Gly Lys Thr95 100 105Ser Glu Val Leu Asn Ala Ala Lys Val Leu Leu Ile Glu Thr Gln110 115 120Arg Cys Asn Ser Arg Tyr Val Tyr Asp Asn Leu Ile Thr Pro Ala125 130 135Met Ile Cys Ala Gly Phe Leu Gln Gly Asn Val Asp Ser Cys Gln140 145 150Gly Asp Ser Gly Gly Pro Leu Val Thr15525164PRTHomo sapiensDOMAINprotease domain of Hepsin (Heps) 25Asp Trp Val Leu Thr Ala Ala His Cys Phe Pro Glu Arg Asn Arg1 5 10 15Val Leu Ser Arg Trp Arg Val Phe Ala Gly Ala Val Ala Gln Ala20 25 30Ser Pro His Gly Leu Gln Leu Gly Val Gln Ala Val Val Tyr His35 40 45Gly Gly Tyr Leu Pro Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser50 55 60Asn Asp Ile Ala Leu Val His Leu Ser Ser Pro Leu Pro Leu Thr65 70 75Glu Tyr Ile Gln Pro Val Cys Leu Pro Ala Ala Gly Gln Ala Leu80 85 90Val Asp Gly Lys Ile Cys Thr Val Thr Gly Trp Gly Asn Thr Gln95 100 105Tyr Tyr Gly Gln Gln Ala Gly Val Leu Gln Glu Ala Arg Val Pro110 115 120Ile Ile Ser Asn Asp Val Cys Asn Gly Ala Asp Phe Tyr Gly Asn125 130 135Gln Ile Lys Pro Lys Met Phe Cys Ala Gly Tyr Pro Glu Gly Gly140 145 150Ile Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Phe Val Cys155 1602623DNAArtificial sequenceforward redundant primer for the consensus sequences of amino acids surrounding the catalytic triad for serine proteases 26tgggtngtna cngcngcnca ytg 232720DNAArtificial sequencereverse redundant primer for the consensus sequences of amino acids surrounding the catalytic triad for serine proteases 27arnarngcna tntcnttncc 202820DNAArtificial sequenceforward oligonucleotide primer for TADG-12 used for quantitative PCR 28gaaacatgtc cttgctctcg 202920DNAArtificial sequencereverse oligonucleotide primer for TADG-12 used for quantitative PCR 29actaacttcc acagcctcct 203020DNAArtificial sequenceforward oligonucleotide primer for TADG-12 variant (TADG-12V) used for quantitative PCR 30tccaggtggg tctagtttcc 203120DNAArtificial sequencereverse oligonucleotide primer for TADG-12 variant (TADG-12V) used for quantitative PCR 31ctctttggct tgtacttgct 203220DNAArtificial sequenceforward oligonucleotide primer for beta-tubulin used as an internal control for quantitative PCR 32cgcatcaacg tgtactacaa 203320DNAArtificial sequencereverse oligonucleotide primer for beta-tubulin used as an internal control for quantitative PCR 33tacgagctgg tggactgaga 203412PRTArtificial sequencea poly-lysine linked multiple antigen peptide derived from the TADG-12 carboxy-terminal protein sequence, present in full length TADG-12, but not in TADG-12V 34Trp Ile His Glu Gln Met Glu Arg Asp Leu Lys Thr1 5 10359PRTHomo sapiens40..48TADG-12 peptide 35Ile Leu Ser Leu Leu Pro Phe Glu Val1 5369PRTHomo sapiens144..152TADG-12 peptide 36Ala Gln Leu Gly Phe Pro Ser Tyr Val1 5379PRTHomo sapiens225..233TADG-12 peptide 37Leu Leu Ser Gln Trp Pro Trp Gln Ala1 5389PRTHomo sapiens252..260TADG-12 peptide 38Trp Ile Ile Thr Ala Ala His Cys Val1 5399PRTHomo sapiens356..364TADG-12 peptide 39Val Leu Asn His Ala Ala Val Pro Leu1 5409PRTHomo sapiens176..184TADG-12 peptide 40Leu Leu Pro Asp Asp Lys Val Thr Ala1 5419PRTHomo sapiens13...21TADG-12 peptide 41Phe Ser Phe Arg Ser Leu Phe Gly Leu1 5429PRTHomo sapiens151...159TADG-12 peptide 42Tyr Val Ser Ser Asp Asn Leu Arg Val1 5439PRTHomo sapiens436..444TADG-12 peptide 43Arg Val Thr Ser Phe Leu Asp Trp Ile1 5449PRTHomo sapiens234..242TADG-12 peptide 44Ser Leu Gln Phe Gln Gly Tyr His Leu1 5459PRTHomo sapiens181..189TADG-12 peptide 45Lys Val Thr Ala Leu His His Ser Val1 5469PRTHomo sapiens183..191TADG-12 peptide 46Thr Ala Leu His His Ser Val Tyr Val1 5479PRTHomo sapiens411..419TADG-12 peptide 47Arg Leu Trp Lys Leu Val Gly Ala Thr1 5489PRTHomo sapiens60..68TADG-12 peptide 48Leu Ile Leu Ala Leu Ala Ile Gly Leu1 5499PRTHomo sapiens227..235TADG-12 peptide 49Ser Gln Trp Pro Trp Gln Ala Ser Leu1 5509PRTHomo sapiens301..309TADG-12 peptide 50Arg Leu Gly Asn Asp Ile Ala Leu Met1 5519PRTHomo sapiens307..315TADG-12 peptide 51Ala Leu Met Lys Leu Ala Gly Pro Leu1 5529PRTHomo sapiens262..270TADG-12 peptide 52Asp Leu Tyr Leu Pro Lys Ser Trp Thr1 5539PRTHomo sapiens416..424TADG-12 peptide 53Leu Val Gly Ala Thr Ser Phe Gly Ile1 5549PRTHomo sapiens54..62TADG-12 peptide 54Ser Leu Gly Ile Ile Ala Leu Ile Leu1 5559PRTHomo sapiens218..226TADG-12 peptide 55Ile Val Gly Gly Asn Met Ser Leu Leu1 5569PRTHomo sapiens35..43TADG-12 peptide 56Ala Val Ala Ala Gln Ile Leu Ser Leu1 5579PRTHomo sapiens271..279TADG-12 peptide 57Ile Gln Val Gly Leu Val Ser Leu Leu1 5589PRTHomo sapiens397..405TADG-12 peptide 58Cys Gln Gly Asp Ser Gly Gly Pro Leu1 5599PRTHomo sapiens270..278TADG-12 peptide 59Thr Ile Gln Val Gly Leu Val Ser Leu1 5609PRTHomo sapiens56..64TADG-12 peptide 60Gly Ile Ile Ala Leu Ile Leu Ala Leu1 5619PRTHomo sapiens110..118TADG-12 peptide 61Arg Val Gly Gly Gln Asn Ala Val Leu1 5629PRTHomo sapiens217..225TADG-12 peptide 62Arg Ile Val Gly Gly Asn Met Ser Leu1 5639PRTHomo sapiens130..138TADG-12 peptide 63Cys Ser Asp Asp Trp Lys Gly His Tyr1 5649PRTHomo sapiens8..16TADG-12 peptide 64Ala Val Glu Ala Pro Phe Ser Phe Arg1 5659PRTHomo sapiens328..336TADG-12 peptide 65Asn Ser Glu Glu Asn Phe Pro Asp Gly1 5669PRTHomo sapiens3..11TADG-12 peptide 66Glu Asn Asp Pro Pro Ala Val Glu Ala1 5679PRTHomo sapiens98..106TADG-12 peptide 67Asp Cys Lys Asp Gly Glu Asp Glu Tyr1 5689PRTHomo sapiens346..354TADG-12 peptide 68Ala Thr Glu Asp Gly Gly Asp Ala Ser1 5699PRTHomo sapiens360..368TADG-12 peptide 69Ala Ala Val Pro Leu Ile Ser Asn Lys1 5709PRTHomo sapiens153..161TADG-12 peptide 70Ser Ser Asp Asn Leu Arg Val Ser Ser1 5719PRTHomo sapiens182..190TADG-12 peptide 71Val Thr Ala Leu His His Ser Val Tyr1 5729PRTHomo sapiens143..151TADG-12 peptide 72Cys Ala Gln Leu Gly Phe Pro Ser Tyr1 5739PRTHomo sapiens259..267TADG-12 peptide 73Cys Val Tyr Asp Leu Tyr Leu Pro Lys1 5749PRTHomo sapiens369..377TADG-12 peptide 74Ile Cys Asn His Arg Asp Val Tyr Gly1 5759PRTHomo sapiens278..286TADG-12 peptide 75Leu Leu Asp Asn Pro Ala Pro Ser His1 5769PRTHomo sapiens426..434TADG-12 peptide 76Cys Ala Glu Val Asn Lys Pro Gly Val1 5779PRTHomo sapiens32..40TADG-12 peptide 77Asp Ala Asp Ala Val Ala Ala Gln Ile1 5789PRTHomo sapiens406..414TADG-12 peptide 78Val Cys Gln Glu Arg Arg Leu Trp Lys1 5799PRTHomo sapiens329..337TADG-12 peptide 79Ser Glu Glu Asn Phe Pro Asp Gly Lys1 5809PRTHomo sapiens303..311TADG-12 peptide 80Gly Asn Asp Ile Ala Leu Met Lys Leu1 5819PRTHomo sapiens127..135TADG-12 peptide 81Lys Thr Met Cys Ser Asp Asp Trp Lys1 5829PRTHomo sapiens440..448TADG-12 peptide 82Phe Leu Asp Trp Ile His Glu Gln Met1 5839PRTHomo sapiens433..441TADG-12 peptide 83Val Tyr Thr Arg Val Thr Ser Phe Leu1 5849PRTHomo sapiens263..271TADG-12 peptide 84Leu Tyr Leu Pro Lys Ser Trp Thr Ile1 5859PRTHomo sapiens169..177TADG-12 peptide 85Glu Phe Val Ser Ile Asp His Leu Leu1 5869PRTHomo sapiens296..304TADG-12 peptide 86Lys Tyr Lys Pro Lys Arg Leu Gly Asn1 5879PRTHomo sapiens16..24TADG-12 peptide 87Arg Ser Leu Phe Gly Leu Asp Asp Leu1 5889PRTHomo sapiens267..275TADG-12 peptide 88Lys Ser Trp Thr Ile Gln Val Gly Leu1 5899PRTHomo sapiens81..89TADG-12 peptide 89Arg Ser Ser Phe Lys Cys Ile Glu Leu1 5909PRTHomo sapiens375..383TADG-12 peptide 90Val Tyr Gly Gly Ile Ile Ser Pro Ser1 5919PRTHomo sapiens110..118TADG-12 peptide 91Arg Val Gly Gly Gln Asn Ala Val Leu1 5929PRTHomo sapiens189..197TADG-12 peptide 92Val Tyr Val Arg Glu Gly Cys Ala Ser1 5939PRTHomo sapiens165..173TADG-12 peptide 93Gln Phe Arg Glu Glu Phe Val Ser Ile1 5949PRTHomo sapiens10..18TADG-12 peptide 94Glu Ala Pro Phe Ser Phe Arg Ser Leu1 5959PRTHomo sapiens407..415TADG-12 peptide 95Cys Gln Glu Arg Arg Leu Trp Lys Leu1 5969PRTHomo sapiens381..389TADG-12 peptide 96Ser Pro Ser Met Leu Cys Ala Gly Tyr1 5979PRTHomo sapiens375..383TADG-12 peptide 97Val Tyr Gly Gly Ile Ile Ser Pro Ser1 5989PRTHomo sapiens381..389TADG-12 peptide 98Ser Pro Ser Met Leu Cys Ala Gly Tyr1 5999PRTHomo sapiens362..370TADG-12 peptide 99Val Pro Leu Ile Ser Asn Lys Ile Cys1 51009PRTHomo sapiens373..381TADG-12 peptide 100Arg Asp Val Tyr Gly Gly Ile Ile Ser1 51019PRTHomo sapiens283..291TADG-12 peptide 101Ala Pro Ser His Leu Val Glu Lys Ile1 51029PRTHomo sapiens177..185TADG-12 peptide 102Leu Pro Asp Asp Lys Val Thr Ala Leu1 51039PRTHomo sapiens47..55TADG-12 peptide 103Glu Val Phe Ser Gln Ser Ser Ser Leu1 51049PRTHomo sapiens36..44TADG-12 peptide 104Val Ala Ala Gln Ile Leu Ser Leu Leu1 51059PRTHomo sapiens255..263TADG-12 peptide 105Thr Ala Ala His Cys Val Tyr Asp Leu1 51069PRTHomo sapiens138..146TADG-12 peptide 106Tyr Ala Asn Val Ala Cys Ala Gln Leu1 51079PRTHomo sapiens195..203TADG-12 peptide 107Cys Ala Ser Gly His Val Val Thr Leu1 51089PRTHomo sapiens215..223TADG-12 peptide 108Ser Ser Arg Ile Val Gly Gly Asn Met1 51099PRTHomo sapiens298..306TADG-12 peptide 109Lys Pro Lys Arg Leu Gly Asn Asp Ile1 51109PRTHomo sapiens313..321TADG-12 peptide 110Gly Pro Leu Thr Phe Asn Glu Met Ile1 51119PRTHomo sapiens108..116TADG-12 peptide 111Cys Val Arg Val
Gly Gly Gln Asn Ala1 51129PRTHomo sapiens294..302TADG-12 peptide 112His Ser Lys Tyr Lys Pro Lys Arg Leu1 51139PRTHomo sapiens265..273TADG-12 peptide 113Leu Pro Lys Ser Trp Thr Ile Gln Val1 51149PRTHomo sapiens88..96TADG-12 peptide 114Glu Leu Ile Thr Arg Cys Asp Gly Val1 51159PRTHomo sapiens79..87TADG-12 peptide 115Arg Cys Arg Ser Ser Phe Lys Cys Ile1 51169PRTHomo sapiens255..263TADG-12 peptide 116Thr Ala Ala His Cys Val Tyr Asp Leu1 51179PRTHomo sapiens207..215TADG-12 peptide 117Ala Cys Gly His Arg Arg Gly Tyr Ser1 51189PRTHomo sapiens154..162TADG-12 peptide 118Ser Asp Asn Leu Arg Val Ser Ser Leu1 51199PRTHomo sapiens300..308TADG-12 peptide 119Lys Arg Leu Gly Asn Asp Ile Ala Leu1 51209PRTHomo sapiens435..443TADG-12 peptide 120Thr Arg Val Thr Ser Phe Leu Asp Trp1 51219PRTHomo sapiens376..384TADG-12 peptide 121Tyr Gly Gly Ile Ile Ser Pro Ser Met1 51229PRTHomo sapiens410..418TADG-12 peptide 122Arg Arg Leu Trp Lys Leu Val Gly Ala1 51239PRTHomo sapiens210..218TADG-12 peptide 123His Arg Arg Gly Tyr Ser Ser Arg Ile1 51249PRTHomo sapiens109..117TADG-12 peptide 124Val Arg Val Gly Gly Gln Asn Ala Val1 51259PRTHomo sapiens191..199TADG-12 peptide 125Val Arg Glu Gly Cys Ala Ser Gly His1 51269PRTHomo sapiens78..86TADG-12 peptide 126Tyr Arg Cys Arg Ser Ser Phe Lys Cys1 51279PRTHomo sapiens113..121TADG-12 peptide 127Gly Gln Asn Ala Val Leu Gln Val Phe1 51289PRTHomo sapiens91..99TADG-12 peptide 128Thr Arg Cys Asp Gly Val Ser Asp Cys1 51299PRTHomo sapiens38..46TADG-12 peptide 129Ala Gln Ile Leu Ser Leu Leu Pro Phe1 51309PRTHomo sapiens211..219TADG-12 peptide 130Arg Arg Gly Tyr Ser Ser Arg Ile Val1 51319PRTHomo sapiens216..224TADG-12 peptide 131Ser Arg Ile Val Gly Gly Asn Met Ser1 51329PRTHomo sapiens118..126TADG-12 peptide 132Leu Gln Val Phe Thr Ala Ala Ser Trp1 51339PRTHomo sapiens370..378TADG-12 peptide 133Cys Asn His Arg Asp Val Tyr Gly Gly1 51349PRTHomo sapiens393..401TADG-12 peptide 134Gly Val Asp Ser Cys Gln Gly Asp Ser1 51359PRTHomo sapiens235..243TADG-12 peptide 135Leu Gln Phe Gln Gly Tyr His Leu Cys1 51369PRTHomo sapiens427..435TADG-12 peptide 136Ala Glu Val Asn Lys Pro Gly Val Tyr1 51379PRTHomo sapiens162..170TADG-12 peptide 137Leu Glu Gly Gln Phe Arg Glu Glu Phe1 51389PRTHomo sapiens9..17TADG-12 peptide 138Val Glu Ala Pro Phe Ser Phe Arg Ser1 51399PRTHomo sapiens318..326TADG-12 peptide 139Asn Glu Met Ile Gln Pro Val Cys Leu1 51409PRTHomo sapiens256..264TADG-12 peptide 140Ala Ala His Cys Val Tyr Asp Leu Tyr1 51419PRTHomo sapiens46..54TADG-12 peptide 141Phe Glu Val Phe Ser Gln Ser Ser Ser1 51429PRTHomo sapiens64..72TADG-12 peptide 142Leu Ala Ile Gly Leu Gly Ile His Phe1 51439PRTHomo sapiens192..200TADG-12 peptide 143Arg Glu Gly Cys Ala Ser Gly His Val1 51449PRTHomo sapiens330..338TADG-12 peptide 144Glu Glu Asn Phe Pro Asp Gly Lys Val1 51459PRTHomo sapiens182..190TADG-12 peptide 145Val Thr Ala Leu His His Ser Val Tyr1 51469PRTHomo sapiens408..416TADG-12 peptide 146Gln Glu Arg Arg Leu Trp Lys Leu Val1 51479PRTHomo sapiens206..214TADG-12 peptide 147Thr Ala Cys Gly His Arg Arg Gly Tyr1 51489PRTHomo sapiens5..13TADG-12 peptide 148Asp Pro Pro Ala Val Glu Ala Pro Phe1 51499PRTHomo sapiens261..269TADG-12 peptide 149Tyr Asp Leu Tyr Leu Pro Lys Ser Trp1 51509PRTHomo sapiens33..41TADG-12 peptide 150Ala Asp Ala Val Ala Ala Gln Ile Leu1 51519PRTHomo sapiens168..176TADG-12 peptide 151Glu Glu Phe Val Ser Ile Asp His Leu1 51529PRTHomo sapiens304..312TADG-12 peptide 152Asn Asp Ile Ala Leu Met Lys Leu Ala1 51539PRTHomo sapiens104..112TADG-12 peptide 153Asp Glu Tyr Arg Cys Val Arg Val Gly1 51545PRTHomo sapiensconserved motif downstream SRCR domaain of TADG-12 154Arg Ile Val Gly Gly1 5
Patent applications by Lowell J. Underwood, Little Rock, AR US
Patent applications by Timothy J. O'Brien, Little Rock, AR US
Patent applications in class Binds expression product or fragment thereof of cancer-related gene (e.g., oncogene, proto-oncogene, etc.)
Patent applications in all subclasses Binds expression product or fragment thereof of cancer-related gene (e.g., oncogene, proto-oncogene, etc.)