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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• the invention relates to the early detection of colorectal adenoma and carcinoma. In particular it relates to the detection of secreted or cell surface markers in easily collectible bodily samples.
• Colorectal cancer is the second leading cause of cancer death in the United States, with ⁇ 130,000 patients diagnosed each year and -50,000 ultimately succumbing to the disease (1).
• Most colorectal cancers develop slowly, beginning as small benign colorectal adenomas which progress over several decades to larger and more dysplastic lesions which eventually become malignant. This gradual progression provides multiple opportunities for prevention and intervention.
• benign adenomas can be detected and removed by simple colonoscopy and polypectomy, precluding the need for radical surgical and adjuvant treatments. It is therefore believed that early detection and removal of these benign neoplasms provides the best hope for minimizing morbidity and mortality from colorectal cancer.
• Various screening methods for detecting early colorectal tumors are available, such as fecal occult blood testing, sigmoidoscopy, and colonoscopy (reviewed in 2). However, none of these methods are optimal, and new approaches are needed.
• a method for detection of colorectal adenoma and carcinoma.
• An mRNA sample is isolated from feces of a subject. Renal dipeptidase mRNA in said mRNA sample is detected. The amount of renal dipeptidase mRNA in said mRNA sample is compared to amounts of renal dipeptidase mRNA in normal subjects. An elevated amount of renal dipeptidase mRNA in said mRNA sample is an indicator of colorectal adenoma or carcinoma in the subject.
• a method for detection of colorectal adenoma or carcinoma is provided.
• Epithelial cells are isolated from blood of a subject.
• An mRNA sample is isolated from the epithelial cells. Renal dipeptidase mRNA in said mRNA sample is detected.
• the amount of renal dipeptidase mRNA in said mRNA sample is compared to amounts of renal dipeptidase mRNA in normal subjects.
• An elevated amount of renal dipeptidase mRNA in said mRNA sample is an indicator of colorectal adenoma or carcinoma in the subject.
• a third embodiment of the invention provides a method for detection of colorectal adenoma or carcinoma.
• Blood of a subject is contacted with a renal dipeptidase substrate.
• Activity of renal dipeptidase in said blood is determined by detection of increased reaction product or decreased renal dipeptidase substrate.
• the amount of activity of renal dipeptidase in blood of the subject is compared to that in normal subjects.
• An elevated amount of activity of renal dipeptidase in the blood of the subject is an indicator of colorectal adenoma or carcinoma in the subject.
• a method for detection of colorectal adenoma or carcinoma is provided.
• Feces of a subject is contacted with a renal dipeptidase substrate.
• Activity of renal dipeptidase in said feces is determined by detection of increased reaction product or decreased renal dipeptidase substrate.
• the amount of activity of renal dipeptidase in feces of the subject is compared to that in normal subjects, wherein an elevated amount of activity of renal dipeptidase in the feces of the subject is an indicator of colorectal adenoma or carcinoma in the subject.
• Another embodiment of the invention provides a method for detection of colorectal adenoma or carcinoma.
• An antibody is administered to a subject.
• the antibody specifically binds to renal dipeptidase and is labeled with a moiety which is detectable from outside of the subject.
• the moiety in the subject is detected from outside of the subject.
• An area of localization of the moiety within the subject but outside the proximal tubules of the kidney identifies colorectal adenoma or carcinoma.
• Another method is also provided for detection of colorectal adenoma or carcinoma.
• An inhibitor of renal dipeptidase is administered to a subject.
• the inhibitor is labeled with a moiety which is detectable from outside of the subject.
• the moiety in the subject is detected from outside of the subject.
• An area of localization of the moiety within the subject but outside the proximal tubules of the kidney identifies colorectal adenoma or carcinoma.
• a substrate for renal dipeptidase is administered to a subject.
• the substrate is labeled with a detectable moiety.
• Feces are isolated from the subject. Renal dipeptidase reaction product or renal dipeptidase substrate with the detecable moiety is detected in the feces. An increased reaction product or decreased reaction substrate in the feces indicates colorectal adenoma or carcinoma in the subject.
• Still another method for detection of colorectal adenoma or carcinoma is provided by the present invention.
• a substrate for renal dipeptidase is administered to a subject.
• the substrate is labeled with a detectable moiety.
• Blood from the subject is subsequently isolated. Renal dipeptidase reaction product or renal dipeptidase substrate with the detecable moiety is detected in the blood.
• An increased product or decreased substrate in the blood indicates colorectal adenoma or carcinoma in the subject.
• Still another embodiment of the invention is a method for detection of colorectal adenoma or carcinoma. Renal dipeptidase in blood of a subject is detected and compared to the amount of renal dipeptidase in normal subjects. An elevated amount of renal dipeptidase in the blood of the subject is an indicator of colorectal adenoma or carcinoma in the subject.
• Still another embodiment of the invention is a method for detection of colorectal adenoma or carcinoma. Renal dipeptidase in feces of a subject is detected and compared to the amount of renal dipeptidase in normal subjects. An elevated amount of renal dipeptidase in the feces of the subject is an indicator of colorectal adenoma or carcinoma in the subject.
• Yet another embodiment of the invention is a method for detection of colorectal adenoma or carcinoma.
• An mRNA sample is isolated from feces of a subject. Macrophage inhibitory cytokine mRNA is detected in the mRNA sample. The amount of macrophage inhibitory cytokine mRNA in said mRNA sample is compared to amounts of macrophage inhibitory cytokine mRNA in normal subjects. An elevated amount of macrophage inhibitory cytokine mRNA in said mRNA sample is an indicator of colorectal adenoma or carcinoma in the subject.
• Another embodiment of the invention is a method for detection of colorectal adenoma or carcinoma.
• Epithelial cells are isolated from blood of a subject.
• An mRNA sample is isolated from the epithelial cells.
• Macrophage inhibitory cytokine mRNA is detected in said mRNA sample.
• the amount of macrophage inhibitory cytokine mRNA in said mRNA sample is compared to amounts of macrophage inhibitory cytokine mRNA in normal subjects.
• An elevated amount of macrophage inhibitory cytokine mRNA in said mRNA sample is an indicator of colorectal adenoma or carcinoma in the subject.
• Fig. 1.A Distribution of the fold changes of differentially expressed transcript tags. Transcripts in which the significance criterion was met (p ⁇ 0.05, a total of 957 tags) in the comparisons between normal and adenoma or normal and cancer are plotted in the figure. The ratios of adenoma to normal and cancer to normal were plotted on a log scale. The shaded box in (Fig. 1.A) and enlarged in (Fig. 1.5) encloses the transcript tags detailed in Table 3. The two unlabeled dots correspond to tags whose differential expression could not be confirmed by quantitative PCR suggesting that the tags were derived from different transcripts than the ones indicated in Table 3.
• Fig. 2 Quantitative PCR analysis of genes elevated in both adenomas and cancers. Quantitation of expression of genes in tumors and matched normal tissues from five patients (Pt) are shown as fold elevation over that in matched normal colonic mucosa. Each bar represents the average of three independent measurements. TGFBI, LYS, RDP, MIC-1, REGA, and DEHL are as described in Table 3.
• Fig. 3 Quantitative PCR analysis of genes decreased in both adenomas and cancers. Quantitation of expression of genes in tumors and matched normal tissue from five patients (Pt) are shown as a fraction of matched normal. Each bar represents the average of three independent measurements.
• CA2 and DRA are described in Table 4. Dual Specificity Phosphatase (DUSP1), and Acid Sphingomylenase-like phosphodiesterase (ASML3a) represented transcripts that were repressed but did not meet the stringent criteria required for inclusion in Table 4. SAGE data indicated that DUSP1 was 5- and 76-fold repressed in adenomas and cancers, respectively. ASML3a was 15-fold repressed in both adenoma and cancer.
• DUSP1 Dual Specificity Phosphatase
• ASML3a Acid Sphingomylenase-like phosphodiesterase
• Fig. 4 Quantitative PCR analysis of mRNA from purified epithelial cells of genes elevated in both adenomas and cancers. Quantitation of expression of genes in the purified normal (N) or cancer (Ca) epithelial cells taken from two patients are shown as fold elevation over matched normal. Genes examined were the same as in Fig. 2.
• Fig. 5A - Fig. 5E In-situ hybridization analyses of elevated genes. Genes examined were REGA (Fig. 5A), TGFBI (Fig. 5B), LYS (Fig. 5C), RDP (Fig. 5D), and MIC- 1 (Fig. 5E). Positive cells appear red, arrows point to clusters of malignant epithelial cells, and arrow heads point to macrophages.
• FIG. 7 A comparison of the inhibitors shown in Fig. 6. compares the inhibition rate as a function of concentration of inhibitor.
• Fig. 8 Substrates of renal dipeptidase are shown.
• Fig. 9 shows the difference in activity of renal dipeptidase found in adenomas, cancer, and metastases compared to normal colonic tissue.
• Serum markers can be found and detected in whole blood, serum, plasma, or fractions thereof. These are collectively referred to as "blood” herein. Markers can also be found in stool. Samples for testing can be feces or processed or fractionated feces. All such samples are referred to herein as "feces.”
• Inhibitors of markers which are enzymes, such as Renal Dipeptidase can be used as affinity reagents for labeling the marker.
• the inhibitors are those which bind irreversibly.
• they are ones which bind and release, but release at a slow rate.
• Inhibitors with suitably slow release rates are those which have a binding half-life of greater than 30 minutes, or 1, 2, 3, 5, 8, or 10 hours.
• Many inhibitors of Renal Dipeptidase are known, including the commercially available Cilastatin, and phosphinic acid inhibitors. See Parsons et al., "A new class of potent, slowly reversibly dehydropeptidase inhibitors," Biochemistry International, vol. 23, pp. 1107-1115, 1991.
• Inhibitors which covalently bind to and/or modify Renal Dipeptidase are also known and can be used. See Wu and Mobashery, "Targeting renal dipeptidase (dehydropeptidase I) for inactivation by mechanism-based inactivators," J. Med. Chem., vol. 34, pp. 1914-1916, 1991. Some inhibitors mimic transition states between substrates and product. Some useful inhibitors are shown in Fig. 6. These include inhibitors having halogen substitutions. Such inhibitors can be readily made using radioactive halogens for ready labeling of renal dipeptidase and easy detection. Similar inhibitors of other enzymes are also known in the art and can be used. Inhibitors can be labeled using any detectable moiety known in the art, including but not limited to fluors and radioactive atoms.
• RNA for any of the markers can be detected using any of the known techniques in the art. Preferably an amplification step will be used, because the amount of RNA for the marker is expected to be very small from the sources contemplated. Suitable techniques include RT-PCR, hybridization of copy mRNA (cRNA) to an array of nucleic acid probes, and Northern blotting.
• cRNA copy mRNA
• Protein forms of the markers can be detected using any techniques known in the art. These include activity assays, immunological assays, binding to specific ligands, etc. Particularly suitable assays for Renal Dipeptidase include using L-L amino acid dipeptide substrates and L-D amino acid dipeptide substrates. Substrates which can be used for assaying renal dipeptidase are shown in Fig. 8, and include the generic structures for dipeptides and dehydrodipeptides. ⁇ (DNP)-L-Lysine-D -Amp can also be used as a substrate, yielding a colored product. Substrates for other enzymes can be used similarly to assess the presence of the tumor marker enzyme in the body or in a body sample.
• Such substrates can be labeled with detectable moieties, including but not limited to fluors and radioactive atoms.
• detectable moieties including but not limited to fluors and radioactive atoms.
• One particularly useful labeling scheme employs a substrate which is labeled with two moieties on opposite sides of the substrate cleavage site. One of the moieties is fluorescent and one of the moieties is a quencher. When the two moieties are close, as in an intact substrate, the fluorescence of the fluorescent moiety is quenched. Upon cleavage the quenching is released and an increase in fluorescence is observed.
• inhibitors can also be labeled and used for detecting suitable markers.
• antibodies can be used to label protein forms of the markers.
• the antibodies can be labeled as is known in the art.
• Suitable radioactive atoms for use in labeling inhibitors, substrates, and antibodies include In- 111, 1-123, Tc-99m, Re-186, Re-188, Ga-67, Ga-68, Tl-201, Fe-52, Pb-203, Co-58, Cu-64, 1-124, 1-125, 1-131, At-210, Br-76, Br-77, and F-18 and others known in the art for such purposes.
• Contrast enhancement agents can also be attached to the substrates, inhibitors, or antibodies. Such agents include gadolinium.
• imaging techniques can be used to detect such labels within the body.
• An example of an imaging technique which can be used is spiral computer tomography.
• the detecting agent such as inhibitor or antibody can be linked to a contrast enhancing agent.
• Other detection means include gamma cameras, magnetic resonance imaging, planar scintigraphic imaging, SPECT imaging, PET imaging, and ultrasound imaging.
• markers can be detected both in situ in the body or in vitro in an isolated body sample.
• Epithelial cells can be isolated from blood or other tissue samples to enrich for the markers or their mRNAs. Epithelial cells can be isolated, inter alia, by immunoaffinity techniques. Such a technique is described in more detail below.
• Substrates of enzymic markers can be administered to subjects and the reaction products measured in body samples, inhibitors can be administered to subjects and the subject can be imaged to detect the inhibitor bound to the marker.
• markers are preferably those which are not secreted proteins, but rather are those which are anchored to a tumor.
• Typical modes of administration of such agents can be any which is suitable, including but not limited to per os, intravenous, intramuscular, intraarterial, subdermal, transdermal, and rectal.
• a high background of certain markers may obscure detection of increased expression.
• Tumor-specific glycoforms of Renal Dipeptidase and MIC-1 bind to LPHA, an L lectin from Phaseolus vulgaris hemagglutinin, and thus can be distinguished on that basis.
• Other lectins such as with similar specificity for tumor-specific glycoforms, such as Sambucus Nigra Lectin isolated from Sambucus nigra (elderberry) bark can be used as well.
• the cDNA encoding this protein was isolated from a random screen of genes highly expressed in a regenerating-islet derived cDNA library (8) and subsequently shown to be elevated in colorectal cancers (9). More recently, REGA was isolated in a hybridization-based screen for genes elevated in colorectal cancers and shown to be elevated in many colorectal adenocarcinomas (10). Consistent with these published observations, we observed a strong elevation in expression of REGA in unpurified tumors, and a similar elevation in one purified tumor. In situ hybridization experiments demonstrated REGA to be strongly expressed in the epithelial cells of the tumors, with no expression evident in the stroma (Fig. 5A).
• TGFB-induced gene encodes a small polypeptide of unknown function initially isolated through a differential display screen for genes induced in response to treatment with TGF ⁇ (11).
• the protein is expressed in the keratinocytes of the cornea (12) and, interestingly, germline mutations of this gene cause familial corneal dystrophies (13).
• TGFBI was previously shown to be among the most significantly elevated genes in colorectal cancers (4), and our new data show that it is expressed at high levels in adenomas as well. Quantitative PCR results demonstrated strong elevation both in unpurified tumors and purified tumor epithelial cells. Accordingly, in situ hybridization experiments revealed TGFBI to be expressed in many cell types, in both the stromal and epithelial compartments (Fig. 5B).
• Lysozyme (LYS, 1 ,4- ⁇ -N-acetylmuramidase, EC 3.2.1.17) is an enzyme with bacteriolytic activity (14) capable of cleaving ⁇ -1,4 glycosidic bonds found in the cell walls of gram-positive bacteria. The enzyme is expressed in the secretory granules of monocytes, macrophages and leukocytes, as well as in the Paneth cells of the gastrointestinal tract. Fecal lysozyme levels are dramatically elevated in patients with inflammatory bowel disease (15, 16), and serum lysozyme activity is significantly elevated in patients with sarcoidosis (17), both of which are diseases characterized by aberrant chronic inflammation.
• LYS LYS in the macrophage compartment of colorectal tumors was also supported by its high representation in a SAGE library constructed from hematopoietic cells (CD45+, CD64+, CD 14+) purified from colorectal tumors (602 LYS tags/56,643 total tags) (6).
• RDP renal dipeptidase
• the enzyme has been extensively analyzed with respect to its catalytic mechanism and inhibition kinetics by a variety of synthetic inhibitors.
• RDP is unique among the dipeptidases in that it can cleave amide bonds in which the C-terminal partner is a D amino acid, providing excellent opportunity for the development of specific probes for its detection in vivo.
• Quantitative PCR revealed RDP to be markedly elevated in both unpurified and purified tumor epithelial cells, and in situ hybridization experiments showed that RDP was exclusively localized to epithelial cells of colorectal tumors (Fig. 5D).
• Macrophage Inhibitory Cytokine is a small polypeptide of 16 kDa first isolated from a differential screen for genes that were induced upon macrophage activation (20). Concurrently, it was identified in the IMAGE database by a search for molecules homologous to the Bone Morphogenic Protein/TGF ⁇ family of growth and differentiation factors (21). In addition to being highly expressed in activated macrophages, MIC-1 has been noted to be highly expressed in placenta and the epithelial cells of normal prostate. In the current study, we found MIC-1 expression to be elevated between 7 and 133 fold in the unpurified tumors.
• SAGE is a gene expression profiling method that associates individual mRNA transcripts with 15- base tags derived from specific positions near their 3' termini (3). The abundance of each tag provides a quantitative measure of the transcript level present within the mRNA population studied. SAGE is not dependent on pre-existing databases of expressed genes, and therefore provides an unbiased view of gene expression profiles.
• Elevated transcripts showed a significantly different (P ⁇ 0.05) tag count between normal and tum both tumor tissues analyzed, and had an expression level that was higher in the tumors than in the column one. For the purposes of calculation, 0.5 was substituted for the denominator when no ta samples.
• Repressed transcripts showed a significantly different (P ⁇ 0.05) tag count between normal and tu both normal tissues analyzed and had an expression level that was lower in the tumors than in the column one.
• RDP Renal Dipeptidase
• mRNA selection was performed from the purified total RNA using oligo(dT) cellulose (Life Technologies, Gaithersburg, MD). Two adenoma SAGE libraries were prepared as described (3, 4) and sequenced to a total depth of over 90,000 transcript tags. For SAGE of normal and malignant tissues, four previously described normal (NC-1 and NC-2) and primary cancer (Tu-98 and Tu-102) SAGE libraries were employed (4). In collaboration with the Cancer Genome Anatomy Project (CGAP) (5), the analyses of these libraries was extended from a total of 123,046 transcripts in the previously published work to 195,160 transcripts in the current work.
• CGAP Cancer Genome Anatomy Project
• Tags were extracted from the raw sequence data and, after excluding repeated ditags, linker sequences, and tags from the polymorphic Major Histocompatibility loci, the resulting tag libraries were compared and statistical analysis performed using SAGE software, version 4.0 .
• Data from the libraries are publicly available at the Uniform Resource Locator (URL) address for the http file type found on the www host server that has a domain name of ncbi.nlm.nih.gov, and a path to the directory SAGE, and detailed SAGE protocols are available at the Uniform Resource Locator (URL) address for the http file type found on the www host server that has a domain name ofleyet.org, and file name of sage_protocol.htm.
• URL Uniform Resource Locator
• the quantitative PCR experiments verified that five of the six selected genes (TGFBI, LYS, RDP, MIC-1, REGA) were expressed at significantly higher levels in every neoplastic sample analyzed compared to patient-matched normal mucosa (Fig. 2). Several tumors exhibited >20-fold higher levels of the studied transcripts compared to their patient-matched normal colonic mucosa, as predicted by SAGE. Another control was provided by the quantitative PCR analysis of four genes whose expression was observed to be reduced in the SAGE libraries prepared from adenomas and cancers compared to those from normal colonic mucosa. As shown in Fig. 3, the quantitative PCR confirmed the lower levels of expression of each of these genes, emphasizing that the dramatic elevations in expression observed in Fig. 2 represented gene-specific phenomena.
• Tumor epithelial cells were purified using a modification of the procedure previously developed for the isolation of tumor endothelial cells (6).
• fresh surgical specimens of tumor and matched normal tissue were obtained and digested with collagenase and the resulting material filtered through a nylon mesh to obtain single cell suspensions.
• the cells were then bound to a mixture of anti-CD14 and anti- CD45 immunomagnetic beads (Dynal, Oslo, Norway) to deplete the population of hematopoetic cells (negative selection).
• the remaining cell suspension was then incubated with anti-Ber-EP4 immunomagnetic beads to isolate epithelial cells (positive selection).
• Purified cells were lysed directly on the beads and mRNA purified using QuickPrep reagents (Amersham Pharmacia Biotech UK, Buckinghamshire, England).
• TAAMill GC 0 1 99 12 20 37273321 differentially expressed in hematopoietic lineages
• nucleophosmtn nucleolar phosphoprotein 823, numatrin
• Tag_SequenceNC1 t ⁇ C2 AD1 AD2 ( :AI ⁇ A2 UNI ID Description
• ATGTGGGCTC 7 2 0 0 0 0151641 glycoprotem A repetitions predominant
• ATTGGAGTGC 135 85 15 3S 37 19220529 carcinoembryonic antigen-related cell adhesion molecule 5
• CTCAGAACTT IS 3 1 0 0 0194710 glucosa iny! (N-acety!) tran ⁇ fcrasc 3, mucin type
• GCACCTGTCG 2 9 0 0 1 0 1 OS 059 mitochondrial ribosomal protein L1 alanyl (merr ⁇ rane) amincpeptidase (aminopcptidase N, amincpaptidase M,
• GCCAGGTTGC 14 5 1 1 1 55682 eukaryotic translation initiation factor 3, subunit 7 ( ⁇ eta, 66/67kD)
• GGAAGAGCAC 21 11 1 1 2 575268 sialyltransferase 4C (beta-gaiactosidase a!pha-2,3-sialytransferase)
• GTAGCAG6TG 24 27 11 7 7 7140452 cargo selection protein (mannose 6 phosphate receptor binding protein)
• TACTGTGGAT 4 11 0 2 2 121537 protein phosphatase 1 , catalytic subunit, beta isoform
• TGCTCCTACC 140 113 70 22 17 22111732 Fc fragment of IgG binding protein
• TGCTCCTACC 140 113 70 22 ⁇ -r 1 / 22301253 Homo sapiens chromosome 19, cosm.id R30SC9
• TTAACCCCTC 34 14 5 9 1 578224 rlbonuclease, RNase A family, 1 (pancreatic)

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