Patent Application
20120039811
The present invention relates to methods for detecting, prognosing and staging cancers, in particular cancers of the gastrointestinal tract. The methods of the invention comprise detecting specific protein markers in a tissue of interest, wherein the detected levels thereof may be indicative of pre-cancerous or cancerous tissue, or the stage or prognosis of a cancer. Further provided are methods of using the markers for treating cancer, and cancer detection kits.
Colorectal cancer, also referred to as colon cancer or large bowel cancer, is a malignant neoplastic disease associated with tumors in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world.
Colorectal cancers originate in the colorectal epithelium and typically are not extensively vascularized (and therefore not invasive) during the early stages of development. The transition to a highly vascularized, invasive and ultimately metastatic cancer, which spreads throughout the body, commonly takes ten years or longer. If the cancer is detected prior to invasion, surgical removal of the cancerous tissue is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, often after metastasis has occurred, and the prognosis for the patient is poor, even after surgical resection of the cancerous tissue. For example, patients diagnosed with early colon cancer generally have a much greater, five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized colon cancer. Accordingly, early detection of colorectal cancer is of critical importance for reducing its morbidity.
Diagnostic methods for colon cancer most frequently depend on direct visual inspection of the gastrointestinal (GI) tract. Endoscopy involves inspection with a miniaturized light source at a probe end of a coherent bundle fiber optic cable. Reflected light beam images are returned through the fiber optic cable for detection by an external digital camera and display on an external monitor or for recording on an external video recorder or both. While this technique allows for identification, removal, and biopsy of potentially cancerous growths such as polyps, its use is associated with certain disadvantages, such as being expensive, uncomfortable, inherently risky due to its invasive nature, and the inability to access some portions of the large intestine and most of the small intestine.
Swallowable endoscopy capsules containing miniaturized optical, digital camera and radio transmission systems have been subsequently developed along with complementary external monitoring systems for inspecting the GI tract. For example, the capsule marketed under the trade name PillCam® SB was initially approved by the U.S. Food and Drug Administration in 2001 for detection and diagnosis of disorders of the small intestine. U.S. Pat. No. 5,604,531 discloses an in vivo video camera system comprising a swallowable capsule. The transit of endoscopy capsules through the small intestine is dependent on peristalsis, meaning that some areas with lesions may be missed if the capsule is not retained in that area for a sufficient amount of time. Further, the endoscopy capsules in current use are not capable of identifying molecular markers, which may be early indicators of colorectal cancer, even prior to the development of pre-cancerous polyps.
U.S. Pat. No. 7,468,044 discloses a system and method for in vivo and in situ detection of body lumen conditions, such as in the GI tract. The system comprises an interaction chamber comprising an indicator; a light source for illuminating the interaction chamber; and an optical detector for detecting in vivo optical changes occurring in the interaction chamber upon reaction of the indicator with an endo-luminal sample.
U.S. Pat. No. 7,515,953 discloses a method for detecting fluorescence emitted by cells in a wall of a body lumen, such as an intestinal wall, the method comprising use of a swallowable capsule, the capsule comprising a light source and a fluorescent-labeled probe, which is released from a reservoir in the capsule. According to the disclosure, an electric field generated from an electrode in the capsule enhances uptake of the probe, and a detector in the capsule detects the fluorescent signal emitted. By determining the intensity and/or position in the lumen wall of the fluorescent signal, a drug for killing abnormal cells is released from a second reservoir in the capsule. According to the disclosure, the abnormal cells may be cancer cells, colon polyps or precancerous cells.
U.S. Patent Application Publication No. 2008/0146896 discloses a device, such as an autonomous capsule, for in vivo analysis which includes a reaction chamber to store a detecting reagent able to react with a sample collected in vivo; and optionally a labeled-substance chamber to store a labeled substance able to bind to a compound resulting from a reaction of the detecting reagent and the sample. According to the disclosure, the detecting reagent may be an antibody.
U.S. Patent Application Publication No. 2009/0216082 discloses a device system for in vivo detection of target molecules in an endo-luminal sample, and a method for in vivo magnetic immunoassay, which may be used for the detection of cancer in the gastrointestinal tract.
Yet other methods of colon cancer detection are based on detection of particular proteins or genes which are considered to be specifically or differentially expressed in colon cancer.
U.S. Pat. No. 7,507,541 discloses a method of detecting the presence of inter alia colon cancer that is based on determining the level of 36P6D5 protein expressed by cells in a test tissue sample from an individual, and comparing the level to that expressed in a corresponding normal tissue sample.
U.S. Pat. No. 7,501,242 discloses a method of detecting colon cancer that is based on detecting levels of expression of tyrosine threonine kinase (TTK) in a test sample, such as a colon sample, that are increased by at least two fold relative to the level of expression in a normal non-cancer sample of the same type.
U.S. Pat. No. 7,452,727 discloses a automatable method for identifying cancer cells and their precursor cells that is based on detecting at least two molecular markers, wherein the detection of each marker alone is not a reliable indicator of the presence of cancer cells and their precursor cells. According to the disclosure, the molecular markers may be selected from her2/neu, Ki67, p53, her2/neu, bcl-2, MN, mdm-2, EGF receptor, bcl-2 and p16.
U.S. Pat. No. 7,402,403 discloses a method for the detection of cancer or early neoplastic change that is based on detecting autoantibodies directed to tumor marker antigens in a sample of bodily fluids, wherein the tumor marker antigens are selected from MUC1, p53, c-erbB2, Ras, c-myc, BRCA1, BRCA2, PSA, APC and CA125.
U.S. Pat. No. 7,129,043 discloses a method of identifying a human subject having an increased risk of developing colon cancer that is based on detecting upregulation of the CLN3 gene.
U.S. Pat. No. 7,115,368 relates to a method of detecting epithelial cancer cells inter alia colon cancer that is based on detection of expression in a biological sample of pellino proteins.
U.S. Pat. No. 7,098,008 relates to a method for detection of cancer inter alia colon cancer that is based on detecting expression of melanoma antigen gene (MAGE).
U.S. Pat. No. 7,078,180 relates to a method of diagnosing a cancer inter alia colon cancer that is based on detection of a ZEB (zfh-1/delta EF1) polypeptide.
U.S. Pat. No. 6,949,339 relates to methods for detecting, diagnosing, monitoring, staging, and prognosticating colon cancers, based on detection of Colon Specific Genes.
U.S. Pat. No. 6,919,176 discloses a method of detecting cancer inter alia colon cancer that is based on detection of expression of specific G-protein coupled receptors.
There remains an unmet need for methods of early detection, prognosis and treatment of colon cancer.
The present invention provides methods of detecting cancer that are based on the qualitative or quantitative identification of particular proteins, also referred to herein as molecular markers. Further provided are methods of cancer prevention, prognosis and treatment.
Disclosed herein for the first time is a specific group of protein markers which are indicative of both pre-cancerous and cancerous lesions. Further disclosed herein for the first time is an additional specific group of protein markers which are primarily indicative of pre-cancerous lesions.
The invention is based in part, on the unexpected discovery that the level of expression within the gastrointestinal tract of certain proteins is significantly increased in both pre-cancerous and cancerous tissue relative to the level of expression of the same proteins in healthy tissue of the same type Surprisingly, the expression of these markers is elevated, even compared to healthy tissue bordering tumor growth. It has also been surprisingly found that the expression of yet other markers is significantly increased in early stage cancer, and significantly decreased in later stages of cancer
Without wishing to be bound by any particular theory or mechanism of action, the invention enables identification of individuals at risk of developing cancer, in particular colorectal cancer, even prior to observable histological changes in affected tissue. Since the methods of the invention are based on changes in protein expression patterns in cells, rather than later occurring pathological changes in tissue, a level of sensitivity is obtained that is greater by many orders of magnitude than current techniques of cancer detection. Thus, in the case of colorectal cancer, the invention provides a means of predicting the disease well in advance of the possibility of detecting potentially cancerous polyps by conventional endoscopic examination, the latter being the current yet inadequate standard for early detection.
The principles of the current invention are exemplified herein by quantitative mass spectroscopy analysis of healthy, pre-cancerous and cancerous tissue obtained from the gastrointestinal tract of patients during surgical excision of early stage (e.g. polyps) or more advanced stage tumors, which has resulted in the identification of a specific group of proteins, the expression of each of which is significantly increased in diseased colon tissue, as compared to healthy colon tissue. This group of proteins, which includes KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), has not been previously disclosed, either in part or as a whole, to be useful for detection of colorectal cancer at any stage of the disease.
It is to be specifically understood however, that the current method of the invention need not be limited to examination of colon tissue obtained by surgical means, nor should it be limited to detection and quantification of the subject molecular markers using mass spectrometry techniques. Rather, the invention may be advantageously practiced using for example immunological techniques and reagents for detection of the subject molecular markers, either in vivo or ex vivo. For example, labeled monoclonal antibodies can be used for in vivo detection and quantitation of such proteins in different tissue compartments and regions. The detection may be accomplished for example, using pharmaceutical compositions or endoscopy probes which incorporate specifically designed chemical, immunological or nucleic acid reagents. Advantageously, labeled reagents such as antibodies, which specifically interact with the subject molecular markers may be prepared as injectable or ingestible pharmaceutical compositions and following administration the interaction with their molecular targets may be externally monitored. Alternately or in addition, the invention may be practiced by analysis of biological samples obtained from a subject, such as biopsy tissue.
In a first aspect, the invention provides a method of detecting cancer in a subject, the method comprising: (i) detecting in a biological sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein, wherein detection of a level of said at least one protein in the biological sample which is significantly different from the reference level, is indicative of cancer in the subject.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In another aspect, the invention provides a method for determining the stage of a cancerous or pre-cancerous growth in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the stage of the growth.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In another aspect, the invention provides a method for determining the prognosis of a cancerous disease in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the prognosis of the cancerous disease.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the biological sample is selected from the group consisting of blood, serum, nipple aspirate fluid, lymph node aspirate, a biopsy sample, a tumor sample, a tissue sample, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. Each of the aforementioned materials represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In currently preferred embodiments, the biological sample comprises a tissue sample. In currently preferred embodiments, the tissue sample comprises gastrointestinal tissue, particularly colorectal tissue or pre-cancerous tissue such as polyps. In currently preferred embodiments, the biological sample is selected from a tumor within the gastrointestinal tract, particularly a colorectal tumor. In particular embodiments, the gastrointestinal tissue is from an area or organ selected from the group consisting of the esophagus, the stomach, the small intestine, the large intestine (colon), the rectum, the appendix and a combination thereof.
In particular embodiments, the cancer being detected is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gastric cancer, head and neck cancer, hepatic cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid and parathyroid cancer, renal cancer, sarcoma, thymoma, hematological malignancies and germ cell tumors. In a currently preferred embodiment, the cancer is colorectal cancer. Each of the aforementioned cancers represents a separate embodiment of the invention and may be used independently from any of the others.
In a particular embodiment, the at least one protein detected is indicative of a disorder selected from the group consisting of pre-cancerous polyps, early stage colorectal cancer and advanced stage colorectal cancer. In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20).
In particular embodiments, said reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the biological sample or test sample is obtained from the subject by a procedure selected from the group consisting of biopsy, flexible endoscopy, double balloon endoscopy and surgical colorectal re-sectioning.
In particular embodiments, the biological sample or test sample is assessed in vivo in the subject. In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one of a pharmaceutical composition and an endoscopy apparatus.
In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20)4. In particular embodiments, the body tissue, cavity or fluid is contacted with a reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the method further comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the method comprises administering a diagnostic pharmaceutical composition. In particular embodiments, the administering of the pharmaceutical composition is by a route selected from the group consisting of oral, parenteral, subcutaneous, intramuscular, intrathoracic and intraarticular.
In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of the aforementioned proteins. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2).
In particular embodiments, the reagent specifically interacts with at least one of the aforementioned proteins or with nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprises a multiplicity of reagents, wherein each reagent of the multiplicity specifically interacts with one distinct protein or with nucleic acid encoding one distinct protein or a fragment thereof. In particular embodiments, the reagent is selected from an antibody, an antibody mimetic and a nucleic acid. In particular embodiments, the antibody is selected from a monoclonal antibody, a humanized antibody, a single chain antibody, an antibody fragment and combinations thereof. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise a multiplicity of antibody mimetics, wherein each antibody mimetic of the multiplicity specifically interacts with one of the aforementioned proteins.
In particular embodiments, the reagent comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.
In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one least one nucleic acid, wherein the at least one nucleic acid is complementary to a nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the at least one nucleic acid comprises a multiplicity of nucleic acids, wherein each nucleic acid of the multiplicity is complementary to a nucleic acid encoding one distinct protein of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the nucleic acid comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.
In particular embodiments, the detecting in step (i) comprises use of an assay system. In particular embodiments, the assay system comprises an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library or combinations thereof. In particular embodiments, the array is a protein array, or a phage display library array. In particular embodiments, the assay system comprises at least one reagent suitable for detecting the level of at least one of the aforementioned proteins, as described herein. In particular embodiments, the assay system comprises a multiplicity of such reagents, for example antibodies, wherein each reagent in the multiplicity specifically interacts with one of the aforementioned proteins.
In particular embodiments, the detecting comprises use of an external monitoring system. In particular embodiments, the external monitoring system is configured to display the level of the at least one protein detected.
In particular embodiments, a method of the invention further comprises detecting in the sample at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR;C1QTNF3 (SEQ ID NO:67), ARID1A (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR;C1QTNF3 (SEQ ID NO:67), ARID (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the cancer is a cancer other than colorectal cancer and the at least one protein includes at least one of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), TNC(SEQ ID NO:57), RBM39 (SEQ ID NO:58), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), NAMPT (SEQ ID NO:2), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64).
In particular embodiments, the level of the at least one protein in the biological or test sample is increased by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, relative to the reference level. In particular embodiments, the reference level is representative of the level of the same protein in non-diseased tissue. In particular embodiments, the reference level is representative of the level of the same protein in a particular stage or form of cancer. In particular embodiments, the reference level is representative of the level of the same protein in a cancer having a known prognosis.
In another aspect, the invention provides a method of treating cancer, the method comprising administering to a subject in need thereof at least one pharmaceutical agent, wherein the pharmaceutical agent specifically interacts with at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). Each of the aforementioned agents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the at least one protein is selected from the group consisting of KIAA0152 (SEQ ID NO:1), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). In particular embodiments, the pharmaceutical agent specifically interacts with KIAA0152 (SEQ ID NO:1). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the pharmaceutical agent is selected from an antibody and an antibody mimetic. In particular embodiments, the pharmaceutical agent further comprises a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive moiety or a chemotherapeutic agent. In particular embodiments, the pharmaceutical agent is selected from a chemical conjugate and fusion protein.
In another aspect, the invention provides an antigen composition comprising at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93), or an immunogenic fragment of said at least one protein. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the composition comprises at least one of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50), or an immunogenic fragment thereof. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the composition comprises KIAA0152 (SEQ ID NO:1) or an immunogenic fragment thereof. In a particular embodiment, a method of preventing or treating cancer in a subject in need thereof comprises administering to the subject an effective amount of said antigen composition. Further provided is an antigen composition as previously specified; for use in preventing or treating cancer.
In another aspect, the invention provides a kit for detecting, staging or prognosing cancer, the kit comprising at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20); CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the kit is for detecting pre-cancerous polyps or early stage colorectal cancer and comprises at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). In particular embodiments, the kit comprises a reagent suitable for detecting the level of KIAA0152 (SEQ ID NO:1).
According to some embodiments the present invention excludes proteins known to be associated with colorectal cancers. According to other embodiments the methods of the present invention exclude proteins that were known to be associated with other types of cancers.
Other objects, features and advantages of the present invention will become clear from the following description and examples.
The present invention provides methods of identification of cancerous cells by detection of levels of particular proteins, also referred to herein as “cancer-associated proteins or “molecular markers”, which have been found to be differentially expressed in cancer tissue, in particular, colorectal cancer tissue. Details of the subject cancer-associated proteins disclosed herein are provided in Tables 1 and 3-7. Some of the identified proteins for which significantly elevated levels were detected in colorectal cancer tissue as compared to normal colorectal tissue have not been previously disclosed to be associated with cancerous disease. Examples of such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Newly identified as cancer-associated” and include the proteins FAM62B, SLC1A5. RSL1D1, LYZ, TNC, RBM39, ILVBL, ERO1L, LOC442497, TCOF1, NAMPT, SERPINB9 and HSDL2. Yet other proteins, in particular THBS1 and LMO7, have been disclosed in the prior art to be down-regulated in cancerous conditions, whereas the inventors of the present invention now disclose significantly increased levels of these proteins in cancer tissue, indicative of up-regulation. These proteins are indicated in Table 1 as denoted by the symbol “UR” in the column labeled “Newly identified as cancer-associated”. Yet other proteins were detected at significant levels in colorectal cancer tissue but were completely absent from healthy colorectal tissue in the same patients. Such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Detected in cancerous but not healthy” and include the proteins AMACR, AMACR;C1 QTNF3, COL5A1, FKBP10, FKBP9, GPRC5A, POLR1c, SPARC and UBAP2.
Table 3 list proteins which are highly expressed in colorectal cancer, both in pre-cancerous polyps and in more advanced stages of the disease. Accordingly, any of the proteins listed in Table 3 represent candidates for use as diagnostic markers of colorectal cancer, which can be indicative of any of pre-cancerous lesions, early stage or late stage forms of the disease. Two currently preferred protein markers are KIAA0152 and NAMPT.
A quantitative analysis of some of the proteins listed in Table 3 is shown in
Table 4 lists proteins which appear to be highly expressed in pre-cancerous polyps, yet their expression tends to be diminished in more advanced stages of the disease. Accordingly, proteins listed in Table 4 represent promising candidates for use as very early diagnostic markers to identify individuals at risk of developing colorectal cancer.
Identification and quantification of a panel of tumor-associated proteins such as those listed in Tables 1 and 3-6, provides a specific means of detecting colorectal cancer in a subject, as well as means of prognosing and staging previously diagnosed colorectal cancers. Such methods can conveniently be carried out using detectably-labeled reagents which specifically bind the selected target proteins. Typically such reagents comprise monoclonal antibodies or small chemical mimetics thereof. In some cases however, it may be advantageous to employ nucleic acids complementary to mRNA species encoding the target proteins so as to quantitate expression activity which may be correlated with the corresponding protein levels. Furthermore, therapeutic pharmaceutical agents directed against tumor-associated proteins, for example, those disclosed in Table 7 herein, may be prepared, for example monoclonal antibodies conjugated to cytotoxic moieties, for use in targeted therapy of colorectal cancer.
The terms “subject” and “patient” as used herein refer to any single subject for which cancer detection, prognosis, staging or therapy is desired, including humans and non-human mammals, such as primate, bovine, ovine, canine, feline and rodent mammals. Also included are subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
The terms “cancer detection” and “cancer diagnosis” and related grammatical terms, such as “detecting cancer” and “diagnosing cancer”, respectively, are used herein interchangeably to refer to any of determination of a subject's susceptibility to a malignant cancer disease; determination as to whether a subject is presently affected by a malignant cancer disease; determination of a subject's stage of cancer, determination of and monitoring the effect on the cancer in response to anti-cancer therapy.
The term “characteristics of a cancerous or pre-cancerous growth” as used herein refers to one or more molecular, physiological, histological, clinical or other properties which may be used to define the nature and behavior of the growth.
The terms “cancer”, “neoplasm”, “tumor”, “growth” and the like are used interchangeably herein to refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include pre-malignant (e.g., benign hyperplasic), malignant, metastatic, and non-metastatic cells.
The term “prognosis” as used herein refers to the expected or predicted outcome of a disease, such as a cancer, in a patient following diagnosis. A prognosis may predict the relative chance of disease progression, arrest or cure. A prognosis may be established on the basis of prognostic indicators specific for a particular disease. Prognostic indicators in cancer may include for example, the grade and stage of cancer at initial diagnosis, the genetic make-up of the patient, the presence and level of cancer-associated antigens in the tumor, and patient responsiveness to a particular therapy.
The terms “biological sample” and “test sample” as used herein encompass a variety of types of biological materials that can be used in the methods of the invention. The sample may be procured from the body of an individual or investigated without removal from the body of an individual. The term encompasses solid tissue samples, such as from biopsy specimens, tumors or tumor metastases, or tissue cultures or cells derived there from and the progeny thereof. The terms also encompass blood and other liquid samples of biological origin, such as nipple aspirate fluid, lymph node aspirate, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. The terms encompass samples that have been manipulated in any way after their procurement, such as by lysis, treatment with reagents, solubilization, or enrichment for certain components. Also included are clinical samples, cells in cell culture, cell supernatants and cell lysates. It is to be explicitly understood that in accordance with the invention, a biological or test sample may be obtained i.e. removed, from the body of a subject, or accessed in vivo, for example by contacting with a specific reagent or apparatus.
As used herein, a biological or test sample that is “obtained from a subject”, means that the sample is removed from the body of the subject, and any subsequent analysis thereof may be performed outside the body for example under in vitro or ex vivo conditions. When however, a biological or test sample is “assessed in vivo” or “accessed in vivo” it means that the sample is maintained within the body of the subject and direct or indirect contact is established using any suitable means, such as via a reagent, composition, device or apparatus. Subsequent analysis of the accessed sample is performed under in vivo conditions, which can include conditions of local or general anaesthesia. Means of accessing and assessing a sample in vivo include for example, contacting the tissue or organ or region thereof of interest with a pharmaceutical composition, a swallowable endoscopy capsule or an endoscopy probe.
The term “a normal biological sample of the same type” as used herein refers to a non-diseased sample consisting of the same biological material or type of tissue and/or cells e.g. blood, colorectal tissue, as that of the test sample. The normal biological sample may be that from a single individual, including the subject in which cancer detection, prognosing or characterizing is performed, or from a group of individuals of known healthy status. Accordingly, the level of a protein in a normal biological sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.
The term “a control sample” as used herein refers to the standard provided by either a normal i.e. non-diseased sample or group of samples, or a sample or group of samples corresponding to an established form, type, stage or grade of a disease, in particular a cancer disease. Accordingly, the level of a protein in a control sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, for example from a group of healthy individuals, or from a group of diseased individuals established to have the same form, type, stage or grade of a cancer disease.
The term “non-diseased tissue” as used herein refers to tissue which is determined to be free of a cancer disease, on the basis of any technique known in the art, for example, histological and immuno-histochemical investigation. It is to be understood that diseased and non-diseased tissue of the same tissue type may be present in close proximity in a individual patient.
The term “immunogenic fragment” as used herein in reference to a protein refers to a portion of the protein which is capable of inducing an immune response, such as antibody production, following administration to an individual.
As used herein, the terms “a protein associated with cancer”, “tumor associated protein”, “molecular marker” and the like, interchangeably refer to a protein that is present at relatively higher or lower levels in a cancer cell relative to a normal cell of the same type (e.g., as in protein associated with colon cancer).
As used herein the terms “nucleic acid encoding a protein,” encompass polynucleotides and polypeptides respectively having sequence similarity or sequence identity to the respective gene and gene products having the accession numbers of the particular protein referred to, of at least about 65%, preferably at least about 80%, more preferably at least about 85%, and can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence similarity and sequence identity are calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. In general, percent sequence identity is calculated by counting the number of residue matches (e.g., nucleotide residue or amino acid residue) between the query and test sequence and dividing total number of matches by the number of residues of the individual sequences found in the region of strongest alignment, as is known in the art. Algorithms for computer-based sequence analysis are known in the art, such as BLAST (see, e.g., Altschul et al., J. Mol. Biol., 215:403-10 (1990)), particularly the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular).
The terms “protein” and “polypeptide” are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
As used herein, a “fusion protein” or “chimeric peptide” refers to a protein or polypeptide which comprises at least a portion of a first naturally occurring protein or polypeptide fused to least a portion of a second protein or polypeptide. For example, a fusion protein for targeting FAM62B may include a portion or all of an anti-FAM62B antibody fused with another peptide or polypeptide such as a protein label moiety or a cytotoxic protein.
The term “antibody” as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, humanized antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies and antibody fragments (e.g., F(ab′)2, Fab′, Fab, Fv) so long as they bind specifically to a target antigen or epitope of interest.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries, as is known in the art, for example using techniques such as those described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).
Furthermore, monoclonal antibodies specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see for example U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term “reagent suitable for detecting the level of a protein” as used herein refers to a reagent which either specifically binds the protein itself or is complementary to a nucleic acid molecule that is involved in expression of the protein. Particularly suitable examples of reagents which specifically bind to proteins are antibodies and chemical mimetics thereof having similar level of binding affinity and/or avidity. Nucleic acid reagents which are complementary to mRNA encoding a protein of interest are suitable for determining the level of the protein, typically by correlating the amount of mRNA detected with the corresponding level of protein product produced in a suitable translation system.
The terms “specifically interacts” and “specifically binds” are used herein interchangeably to refer to high avidity and/or high affinity binding of a reagent, such as an antibody to a specific polypeptide or epitope thereof, such as for example, an epitope of any of the proteins FAM62B, SLC1A5, RSL1D1, LYZ, RBM39 and TCOF1. Antibody binding to its epitope is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls.
The term “primary antibody” as used herein refers to an antibody which binds specifically to the target protein antigen in a biological or test sample. A primary antibody is generally the first antibody used in an immunoassay procedure. In some embodiments, the primary antibody is the only antibody used in an immunoassay procedure.
The term “secondary antibody” as used herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is typically directed against the Fc portion of the immunoglobulin type of the primary antibody (e.g., anti-mouse Fc antibody).
The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides, including but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Further include are mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component, capping, substitution of one or more of naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
The term “complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules or a DNA/RNA hybrid. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
The term “hybridization” as used herein encompasses any process by which a strand of nucleic acid joins with a complementary strand through base pairing (see for example, Coombs, Dictionary of Biotechnology, Stockton Press, New York N.Y. (1994)). Accordingly, a hybridization assay is a quantitative means of determining the extent of hybridization between a nucleic acid in a test sample, such as a tissue sample, and a nucleic acid probe corresponding to at least a fragment of a gene (DNA or RNA) encoding a protein of interest, such as a cancer associated protein. “Stringency” typically occurs in a range from about Tm −5° C. (5° C. below the Tm of the nucleic acid probe) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a stringency hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach et al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. (1995), and may be performed prior to or as part of a hybridization assay.
As used herein, the term “differentially expressed” generally refers to a polynucleotide and/or the corresponding protein that is expressed at levels in a test cell that differ significantly from levels in a reference or control cell, e.g., a cancer-associated protein as disclosed herein is found at levels at least about 50% to about 100% increased, generally at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold or more increased in a cancerous cell when compared with a cell of the same type that is not cancerous. The comparison can be made between two tissues, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also be made between cells removed from their tissue source, or cells maintained in their native state in vivo. “Differential expression” refers to both quantitative, as well as qualitative, differences in the genes' temporal and/or cellular expression patterns among, for example, normal and neoplastic tumor cells, and/or among tumor cells which have undergone different tumor progression events.
The terms “correspond to” or “represents” as used in, for example, the phrase “polynucleotide corresponds to a differentially expressed gene” are used to refer to the relationship between a given polynucleotide and the gene from which the polynucleotide sequence is derived (e.g., a polynucleotide that is derived from a coding region of the gene, a splice variant of the gene, an exon, and the like) or to which the polynucleotide hybridizes to under stringent conditions.
The term “label” as used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as an antibody, a nucleic acid probe or a chemical agent and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
The term “mimetic” as used herein refers to any entity, including natural and synthesized inorganic or organic molecules, including recombinant molecules, that mimic the properties of the molecule of which it is a mimetic. Accordingly, a mimetic of a particular antibody has the same, similar or enhanced epitope binding properties of that antibody.
The term “gene” as used herein refers to any nucleic acid sequence or portion thereof with a functional role in encoding or transcribing a protein or regulating other gene expression. The gene may consist of all the nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein. The nucleic acid sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences or unique adjacent regions to the gene.
The singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the reagent” includes reference to one or more reagents and equivalents thereof known to those skilled in the art, and so forth.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
The present invention is based on the discovery that specific proteins, including for example, KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and MCM3 are present at significantly higher levels in cancerous or pre-cancerous colorectal tissue as compared to normal tissue of the same cell type, even in the same individual.
This discovery serves as the basis for identification of cancerous tissue, as well as for staging and prognosing tumors, and development of therapeutic agents which target the particular cancer-associated markers. Detection of the markers disclosed herein and/or the genes encoding them, enables early diagnosis based on molecular changes leading to carcinogenesis and/or decision making in disease management. For example, a relatively increased level of a particular protein, such as FAM62B, compared to normal cells or tissues of the same type can be indicative of a poorer prognosis, and therefore warrant more aggressive therapy (e.g., chemotherapy following surgery). The correlation of tumor specific markers with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting, antagonists (e.g., small molecules), and gene therapy. Determining colon cancer-specific protein levels and comparison of a patient's profile with known levels in normal tissue and variants of the disease allows a determination of optimal treatment strategies. The marker expression pattern can also be used to better classify (i.e. stage and grade), and thus diagnose and treat different forms of cancer. Furthermore, a protein identified as being differentially or specifically expressed in, one type of cancer may also have implications for development or risk of development of other types of cancer.
Colorectal cancer is one of the most common neoplasms in humans and perhaps the most frequent form of hereditary neoplasia. Prevention and early detection are key factors in controlling and curing colorectal cancer. Colorectal cancer begins as polyps, which are small, benign growths of cells that form on the inner lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. Multiple familial colorectal cancer disorders have been identified, as follows: 1) Familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3) Hereditary nonpolyposis colon cancer (HNPCC); and 4) Familial colorectal cancer in Ashkenazi Jews.
Staging is a process used in the medical arts to describe how advanced the cancerous state is in a patient. While staging systems vary with the types of cancer, they generally involve the “TNM” system: “T” indicates the type of tumor, “N” indicates whether the cancer has metastasized to nearby lymph nodes; and “M” indicates whether the cancer has metastasized to other parts of the body. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.
The grade of a cancer describes how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well differentiated or low-grade tumors. The following guidelines are generally used for grading tumors: GX, Grade cannot be assessed; G1, Well differentiated; G2, Moderately well differentiated; G3, Poorly differentiated; G4, Undifferentiated.
Methods of detection, prognosis, and characterization as disclosed herein are based on levels of at least one, and preferably a panel or multiplicity of cancer-associated proteins, in comparison to levels of the same protein(s) in suitable non-cancerous or cancerous control samples. For example, a detection of cancer may be enabled by the detection of a level of one or more proteins of interest in a sample, such as FAM62b, that is increased at least by 50% or 100% or greater or, alternatively by 2-fold, 3-fold 5-fold, 10-fold, 30-fold, or greater, relative to a normal non-cancerous sample of the same tissue type. The normal non-cancerous sample may be from the same individual as the test sample or from one or more different individuals. Preferably, the level in the normal non-cancerous sample is a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.
Similarly, prognosis and characterization of the cancer may be established on the levels of proteins of interest relative to reference standards established for particular grades, stages and forms of cancer. For example, detection of a level of a particular protein which is 1.5-fold compared to that of the control may be taken to indicate a relatively positive prognosis and/or relatively non-aggressive type of cancer, whereas detection of a level of the same protein of 10-fold or more compared to that of the control may be taken to indicate a poorer prognosis and/or a substantially aggressive type of cancer.
The methods of the invention may be carried out using various assay systems and methods for detection of the level of the protein of interest in a test biological sample. Suitable systems include those employing an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library, or a combination thereof.
Immunoassays
Immunoassays for detecting levels of specific binding between an antibody and its target antigen are known in the art and include for example, radioimmunoassay, (RIA), fluorescent immunoassay, (FIA) enzyme-linked immunosorbant assay (ELISA), immunohistochemistry (IHC) and fluorescent activated cell sorting (FACS) (see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999)). In general, an immunoassay may be either direct or indirect. In a direct assay, the binding of antibody to the target antigen is determined directly using a labeled reagent, such as a fluorescent labeled or an enzyme-labeled primary antibody, which can be detected without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. Alternately, both the primary and secondary antibodies may be unlabeled and labeled tertiary antibody is employed. Where the antibody (primary, secondary or tertiary) is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide detection of the antigen.
The primary antibody used for detection of the protein(s) of interest is stably associated with e.g., directly or indirectly bound to, the surface of a solid support, e.g. column, microtiter plate, beads, membrane, typically made of glass, plastic, polysaccharides, nylon or nitrocellulose. A multiplicity of antibody specificities for detecting a panel of tumor-associated proteins may be simultaneously bound to the same support, such as in an array.
The test sample is allowed to contact the support during a period of incubation, generally following blocking of non-specific binding sites with non-interfering proteins such as bovine serum albumin. After incubation with each reagent e.g. blocking agent, primary antibody, secondary antibody, the support is washed to remove non-bound components. Determination of suitable reagents, conditions for washing, incubation etc. is within the ability of one of average skill in the art.
Immunoassays can also be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of tumor associated antigen. In situ detection can be accomplished by removing a histological sample from a subject, and contacting the sample with a labeled antibody. The antibody is typically contacted with the sample by overlaying the labeled antibody onto the sample. Through the use of such a procedure, the presence of the tumor associated antigen can be determined and/or the distribution of the antigen in the histological sample can be examined. Those of ordinary skill in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Detectable labels suitable for conjugation to antibodies and other binding reagents include radioisotopes, fluorescent labels, enzyme-substrate labels, chromogenic labels, chemiluminescent labels and colloidal gold particles.
Radioisotopes include for example, 35S, 14C, 125I, 32P and 131I. Fluorescent labels include for example, fluorescent molecules fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a (PerCP), Cy3 (indocarbocyanine), Cy5 (indodicarbocyanine), lanthanide phosphors, and the like.
Enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of enzyme-substrate combinations include, for example: horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and β-D-galactosidase β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase).
A label may be indirectly conjugated with an antibody or other reagent, as is known in the art. For example, an antibody can be conjugated with biotin and any of the types of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and thus, the label can be conjugated with the antibody in an indirect manner. In some cases, detectable labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
Detection of bound, labeled antibody can be carried out by standard colorimetric, radioactive, photometric and/or fluorescent detection means. For fluorescent labels, signal can be detected by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by, for example, U.S. Pat. Nos. 5,578,832 and 5,631,734. For antibodies labeled with biotin, the reaction can be treated with the appropriate streptavidin-conjugate (e.g., streptavidin-horseradish peroxidase, streptavidin-alkaline phosphatase, streptavidin-luciferase, and the like) and then treated with the appropriate reagents for calorimetric or photometric detection. For radiolabeled antibody, signal can be detected using a scintillation counter, phosphoimager or similar device.
Arrays
Cancer associated proteins in a sample may be detected using an array-based binding assay system. Such an array-based system may incorporate an immunoassay as described above, or incorporate small molecule chemical entities which specifically interact with particular cancer associated proteins. In either case, the solid substrate used for the array comprises a plurality of binding reagents attached to the substrate, wherein each binding reagent has specificity for a different cancer associated protein. The protein panel or “set” for which the array is predetermined to specifically bind and detect is characteristic of a particular disease, or stage of form of a disease. The binding reagents, are immobilized onto the substrate surface, preferably in a spatially addressable manner. The binding reagents may be antibodies, antibody fragments or small molecule chemical entities.
The nature and geometry of the solid substrate will depend upon a variety of factors, including, among others, the type of array (e.g., one-dimensional, two-dimensional or three-dimensional). Generally, the surface can be composed of any material which will permit immobilization of the binding reagents and which will not substantially degrade under the conditions used in the applications of the array.
The solid substrate used for the array may be in the form of beads, particles or sheets, and may be permeable or impermeable, depending on the type of array, wherein the surface is coated with a suitable material enabling binding of the binding reagents at high affinity. For example, for linear or three-dimensional arrays the surface may be in the form of beads or particles, fibers (such as glass wool or other glass or plastic fibers) or glass or plastic capillary tubes. For two-dimensional arrays, the solid surface may be in the form of plastic, micromachined chips, membranes, slides, plates or sheets in which at least one surface is substantially flat, wherein these surfaces may comprise glass, plastic, silicon, low cross-linked and high cross-linked polystyrene, silica gel, polyamide, and the like.
Fluorescence tagged beads are also an addressable (liquid) array in which each bead is tagged with a different set of fluorescent colors and bound with an antibody; specific to an array is detected with devices such as fluorescence scanners for arrays or FACS for beads.
The arrays used for the present invention may be of any desired size. The upper and lower limits on the size of the array are determined solely by the practical considerations of resolution, size of molecules expressed at each address and the like.
Either a population of discrete proteins is employed to form the array, such that each address presents a different molecule, or a single or a few addresses are employed with a similar protein. In many applications, redundancies in the spots are desirable for the purposes of acting as internal controls.
Technologies for the deposition of droplets containing protein binding reagents onto a suitable solid surface are known in the art. An ink-jet printing technology for deposition of small droplets while avoiding overlap or splatter is disclosed in U.S. Pat. No. 5,449,754.
In order to conduct array-based binding assays, the test sample is allowed to contact the array comprising a coated surface containing the anchored binding reagents. Following contact, the array is optionally washed, typically under conditions such that any complexes formed will remain immobilized on the solid surface and unbound material will be removed.
The detection of complexes anchored on the solid surface can be accomplished in a number of ways. In some embodiments, the non-immobilized sample is pre-labeled, and the detection is directed to label immobilized on the surface indicating that complexes were formed. In other embodiments, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized sample (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). In another embodiment, the immobilized molecules of the microarray are labeled, the array can be scanned or otherwise analyzed for detectable assay signal, and the signal from each labeled spot, or alternatively from all spots, quantified.
An important consideration is the presence of an amount of a label at each position within the array that is proportional to the amount of molecule immobilized at that particular spot. Thus, it is important that the efficiencies of the coupling reactions which are used to immobilize the labeled molecules are substantially similar.
Virtually any label that produces a detectable, quantifiable signal and that is capable of being attached to an immobilized binding reagent on a substrate can be used in conjunction with the array of the invention. Suitable labels include: radioisotopes, fluorophores, chromophores, chemiluminescent moieties, as described above.
Preferably, the position of the label will not interfere with interaction between a desired sample and the immobilized molecules and with the detection in case of an interaction between the desired sample and an immobilized molecule of the array. Suitable methods of making labeled molecules are well known in the art.
In the case where each spot in the array contains an amount of a label or “tracer” proportional to the amount of molecules immobilized at the particular spot, the signals obtained from the arrays of the invention can be normalized. As a consequence, signal intensities from spots within a single array, or across multiple arrays, can be directly compared. A normalized signal of a particular spot may be defined by (It−I0)/Io, where It is the intensity of the signal of the spot after contacting with a sample of interest and Io is the intensity of the background signal of the spot before contacting with a sample of interest.
Various methods and devices for detection and analysis of the array are known in the art. Practically, any imaging system that is capable of detecting with a resolution appropriate to the size of the array features can be utilized. For example, a method for screening an array of proteins for interactions with a fluid sample is disclosed in U.S. Pat. No. 6,475,809. Imaging apparatus may be selected, for example, from ScanArray 4000 (General Scanning), Biochip Imager (Hewlett Packard), GMS 418 Array Scanner (Genetic Microsystems), GeneTAC 1000 (Genomic Solutions), Chip Reader (Virtek). Phosphorimager systems are available for detecting radiolabels, e.g. Cyclone (Packard Instrument Co.) and BAS-5000 (Fujifilm).
Hybridization Assays
Hybridization assays generally comprise contacting a sample containing nucleic acids (target nucleic acids) with a nucleic acid probe capable of hybridizing to tumor associated antigen nucleic acids, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
Suitable hybridization assays include, for example, Northern blots, dot blots, RT-PCR, and quantitative PCR. Such procedures can be performed in situ directly for example, in tissue sections (e.g., fixed and/or frozen) of subject tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Tumor associated antigen nucleic acids can be used as probes and/or primers for such procedures (see e.g., Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, NY (1992)).
Detection of tumor associated antigen nucleic acids typically involves contacting and incubating nucleic acids from a test sample with one or more labeled nucleic acids, (i.e. “probes”) under conditions favorable for the specific annealing of the nucleic acids to their complementary sequences. Typically, the lengths of the nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed. The presence of bound i.e. hybridized, nucleic acids from the sample, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the tissue or cell type of interest can be immobilized, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
Nucleic acid arrays can be used to monitor the expression of tumor associated genes, such as, for example, those corresponding to KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and/or MCM3. For detection of a multiplicity of genes encoding distinct cancer associated proteins, an array of polynucleotide probes may be contacted with a sample of target nucleic acids to produce a hybridization pattern. The binding of the target nucleic acids to one or more probes of the array is then detected to obtain a qualitative and/or quantitative profile of expression of the tumor associated antigen gene.
A variety of different arrays can be used and are known in the art. The polymeric or probe molecules of the arrays can be polynucleotides or hybridizing derivatives or analogs thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methyl-phosphonate, phosphoramidate, guanidine, and the like; nucleic acids in which the ribose subunit has been substituted, for example, hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from about 10 to about 1000 nucleotides, typically, from about 15 to about 150 nucleotides in length, but also possibly from about 150 to about 1000 nucleotides in length. The probes can be single or double stranded, usually single stranded, and can be PCR fragments amplified from cDNA. The probe molecules on the surface of the substrates will typically correspond to at least one of the tumor associated antigen genes and be positioned on the array at known locations so that positive hybridization events can be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived. Because of the manner in which the target nucleic acid sample is generated, the arrays of probes will generally have sequences that are complementary to the non-template strands of the gene to which they correspond.
The substrate for the array can be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like. The arrays can be produced according to any convenient methodology, such as pre-forming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.
The target nucleic acid is typically contacted with the array under conditions sufficient for hybridization of target nucleic acid to probe to occur. Suitable hybridization conditions are well known to those of skill in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor, Cold Spring Harbor, N.Y. (2001)).
The amount of tumor associated antigen nucleic acids in the sample can be quantitated (see, e.g., U.S. Pat. No. 6,004,755). For example, the target nucleic acids in the sample can be end-labeled in a manner such that each of the target nucleic acids in the sample produces a signal of the same specific activity. By generating the same specific activity is meant that each individual target polynucleotide in the sample being assayed is labeled in a manner such that the molecule is capable of providing the same signal (e.g., the same intensity of signal) as every other labeled target in the sample. Each of the target nucleic acids generates a signal of the same specific activity because the number of labeled nucleotide bases in each of the target molecules is either identical or substantially the same.
The label is capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Suitable detectable labels include radioactive, fluorescent and enzymatic labels as described above.
In some applications, it is desired to analyze populations of target nucleic acids from two or more samples. Such samples can be differentially labeled. Alternatively, target nucleic acids from different samples are separately contacted to identical probe arrays under conditions of hybridization, typically stringent hybridization conditions, such that labeled nucleic acids hybridize to their complementary probes on the substrate surface, and the target nucleic acids bound to the array separately detected. A set of standard nucleic acid molecules can optionally be used. For example, the standard nucleic acids can be provided by reverse transcribing standard RNA.
Following hybridization, a washing step is usually employed to remove non-specifically bound nucleic acid from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface. Various wash solutions and protocols for their use are known to those of skill in the art.
Where the label on the target nucleic acid is not directly detectable, the array can be contacted with the other member(s) of the signal producing system that is being employed. For example, where the label on the target is biotin, the array can be contacted with streptavidin-fluorophore conjugate under conditions sufficient for binding between the specific binding member pairs to occur. Following contact, any unbound members of the signal producing system will then be removed (e.g., by washing).
The resultant hybridization pattern(s) of target nucleic acids bound to the array can be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid. For example, detection means can include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement, and the like.
Prior to detection or visualization, the array of hybridized target/probe complexes can be optionally treated with an endonuclease, for example, mung bean nuclease, S1 nuclease, and the like. The endonuclease degrades single stranded, but not double stranded DNA.
Following detection or visualization, the hybridization pattern can be used to determine qualitative and/or quantitative information about the expression of tumor associated antigen genes. The hybridization patterns of different samples can be compared with each other, or with a control sample, to identify differences between the patterns. The hybridization arrays can also be used to identify differential gene expression, in the analysis of diseased and normal tissue.
Antibodies directed against cancer associated proteins include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv, or hypervariable regions), bi-specific antibodies and humanized antibodies, methods of production of which are known in the art.
For the production of polyclonal antibodies, a host animal (e.g., rabbits, mice, rats, sheep, goats, and the like) can be immunized by injection with a tumor associated antigen, fragment, derivative or analog. Various adjuvants can be used to increase the immunological response, depending on the host species. Such adjuvants include, for example, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and other adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Techniques for preparation of monoclonal antibodies include the original Kohler and Milstein hybridoma technique (see, e.g., Nature 256:495 97 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15 19 (1993); Hering et al., Biomed. Biochim. Acta 47:211 16 (1988)), the human B-cell hybridoma technique (see, e.g., Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique (see, e.g., Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96 (1985)). Human antibodies can be obtained using human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA 80:2026 30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g., Cole et al., supra).
Chimeric antibodies are typically prepared by splicing the genes (of one species) for an antibody molecule specific for tumor associated antigen together with genes from another species of antibody molecule of appropriate biological activity. It can be desirable to transfer the antigen binding regions (e.g., Fab′, F(ab′)2, Fab, Fv, or hypervariable regions) of antibodies from one species into the framework of an antibody from another species by recombinant DNA techniques to produce a chimeric molecule. Methods for producing such molecules are described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397; 5,693,762, and 5,712,120. A human monoclonal antibody or portion(s) thereof can be identified by screening a human B-cell cDNA library for nucleic acid molecules that encode antibodies that specifically bind to a tumor associated antigen according to the method generally set forth by Huse et al. (Science 246:1275 81 (1989)). The nucleic acid molecule can then be cloned and amplified to obtain sequences that encode the antibody (or antigen-binding domain) of the desired specificity. Phage display technology offers another technique for selecting antibodies that bind to tumor associated antigens, fragments, derivatives or analogs thereof (see, e.g., International Patent Publications WO 91/17271 and WO 92/01047; Huse et al., supra.)
Techniques for the production of single chain antibodies are described for example in U.S. Pat. Nos. 4,946,778 and 5,969,108. A Fab expression library (see, e.g., Huse et al., supra) allows rapid and easy identification of monoclonal Fab fragments with the desired specificity for tumor associated antigens, fragments, derivatives, or analogs thereof.
F(ab′)2 antibody fragments can be produced by pepsin digestion of an antibody molecule. Fab′ fragments can be generated by reducing the disulfide bridges of a F(ab′)2 fragment, Fab and Fv fragments can be generated by treating an antibody molecule with papain and a reducing agent. Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, as described in U.S. Pat. No. 5,965,405.
Bi-specific antibodies can be monoclonal antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities can be for a tumor associated antigen and the other one is for any other antigen. Alternatively, one specificity is for a first tumor associated antigen, while the other specificity is for a second, different tumor associated antigen. Methods for making bi-specific antibodies may be based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature 305:537 39 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas produce a potential mixture of different antibody molecules, some of which have the desired bi-specific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion typically is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. The first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is usually present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bi-specific antibodies see, for example, Suresh et al (Methods in Enzymology 121:210 (1986)).
To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which expresses the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al., supra.
A pharmaceutical or antigen composition according to the invention comprises at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1, or an immunogenic fragment thereof.
The composition may be used in a therapeutic method for preventing or treating cancer, wherein an effective amount of the composition is administered to a subject in need thereof. A subject in need thereof may be for example, an individual in which colorectal polyps have been identified, and/or has been determined to have other risk factors for development of colorectal cancer. Accordingly, a method of preventing colorectal cancer may comprise administering an effective amount of a pharmaceutical composition comprising at least one protein highly expressed in polyps, for example any of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1.
Further provided are pharmaceutical compositions comprising one or more reagents suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, UGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1.
As hereinbefore described, such reagents may comprise labeled antibodies, antibody mimetics or nucleic acids as the active ingredients. Advantageously such reagents are provided as a “cocktail” tailored to detect in vivo a particular panel of cancer associated proteins, or genes expressing same, Following administration, the localization of such reagents to specific body and organ regions may be detected by means of an external monitoring apparatus or system appropriate for detection and quantitation of the specific label incorporated into the reagents.
In yet other embodiments, pharmaceutical agents, in particular antibodies and an antibody mimetics, are provided, which may be used for targeted therapy of cancers expressing particular cancer associated proteins. Such, agents may advantageously incorporate a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive atom or a chemotherapeutic agent. Further, the pharmaceutical agent may be in the form of a chemical conjugate or a fusion protein and provided in a suitably formulated pharmaceutical composition.
Plant toxins include for example, ricin, abrin, pokeweed antiviral protein, saporin, gelonin, and derivatives thereof. Bacterial toxins include for example, Pseudomonas exotoxin, diphtheria toxin and derivatives thereof, iodine-125 and radon. Radioactive atoms include for example, radium, cesium-137, iridium-192, americium-241 and gold-198.
Chemotherapeutic agents include, without limitation, alkylating agents, for example cyclophosphamide; nitrosoureas, for example carmustine and lomustine; antimetabolites, for example 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine and pemetrexed; anthracyclines, for example daunorubicin, doxorubicin respinomycin D and idarubicin; topoisomerase inhibitors, for example topotecan, irinotecan, etoposide and teniposide; mitotic inhibitors, for example paclitaxel, docetaxel, etoposide, vinblastine and vincristine; platinum based drugs, for example cisplatin, carboplatin and oxaliplatin; steroids for example hydrocortisone, dexamethasone, methylprednisolone and prednisolone; and anti-angiogenic agents, for example bevacizumab, thalidomide, dopamine and tetrathiomolybdate.
The pharmaceutical composition can be used to administer diagnostic reagents designed to detect levels of proteins disclosed herein, in particular KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), including antibodies, antibody mimetics or nucleic acids, or therapeutic reagents designed to exert a cytotoxic affect on their cancer targets. Therapeutic compositions for targeting tumors may include a reagent which specifically interacts with a protein selected from NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, KIAA0152, UGCGL1, FAM3D, CCT4, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP, KIAA0152 and CLIC1.
The pharmaceutical composition may be prepared by suspending the desired reagent in any pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
The pharmaceutical compositions may be prepared in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents. Sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art.
Kits
The invention also includes kits for detecting, diagnosing, prognosing or staging a cancer or a tumor in a mammal. The cancer or tumor can be of any of the types described herein, and is preferably colorectal cancer. The kit comprises a container or a sample tube, or the like, for storing a sample of a cell, a population of cells, a tissue or a body fluid obtained from the mammal.
The kit also comprises one or more detection reagents selected from: an antibody or antibody mimetic which specifically bind with a cancer associated protein disclosed herein; a nucleic acid such as an oligonucleotide which specifically binds a nucleic acid (such as mRNA) encoding said cancer associated protein, and a PCR primer pair specific for a nucleic acid encoding said cancer associated protein. These detection reagents are as described herein. The kit comprises the one or more detection reagents in an amount effective to permit detection of the protein(s) of interest or a corresponding nucleic acid in the sample. Detection of the proteins or the nucleic acids is accomplished using any of the methods described herein or known to a skilled artisan for detecting a specific protein or specific nucleic acid molecule within a biological sample.
Reagents in the kit may be directed to a protein selected from KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. The kit may be intended for detecting pre-cancerous or early stage colorectal cancer and include reagents detecting any protein selected from the group consisting of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. In particular embodiments, the kit comprises a reagent suitable for detecting KIAA0152.
The kit also comprises at least one control biological sample, such as from non-diseased tissue, for comparison to a biological sample obtained from a subject under investigation. Control biological samples which correspond to samples from a cancer of a known stage or having a known prognosis, may also be included.
The kit also comprises an instructional material which directs the use of the reagents and the samples for the determining the amount and the location of the proteins or the nucleic acids in one or more cells of the sample. The instructional material also directs the correlation of the amount and the location of the protein or the nucleic acid in the cells of the sample with the diagnosis, prognosis and/or stage of a cancer or a tumor in the mammal.
As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which directs or dictates the use of the components of a kit for performing the function of a method of the invention described herein. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition.
The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
The following methods were used in the Examples.
Protein Extraction from Frozen Tissues
Surplus snap-frozen tumor and margin non-tumoral tissue were collected from patients undergoing surgery for colorectal cancer at either of two university teaching hospitals in Israel. Around 30 mg of frozen tissue were extracted from every tissue sample by mixing the tissue sample with in 0.5 ml of 8 M urea, 400 mM ammonium bicarbonate, and homogenized by high speed tissue homogenizer (Omni-TH) for 1 min, activated at full speed.
Identification of Proteins
1) Gel-slicing method. About 50 μg of the proteins extracted from the tumor tissues or the healthy tissues were resolved by 10% of SDS-PAGE. Each gel lane was subsequently stained with Coomassie blue. The stained gel lanes were cut into 12 slices. The gel pieces were de-stained by extensive washing with acetonitrile and ammonium bicarbonate. The proteins in each gel slice were proteolyzed inside the stained gel: stained slices were reduced with 10 mM DTT, incubated at 60° C. for 30 min, alkylated with 10 mM iodoacetamide, at room temperature for 30 min and digested with trypsin overnight at 37° C., using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The tryptic peptides were analyzed by AC-MS/MS using the OrbitrapXL mass spectrometer (Thermo-Fisher) fitted with a capillary HPLC (Eksigent). The peptides were resolved on homemade capillary columns (75 micron ID) packed with reversed phase 3.5 micron beads Reprosil C18-Aqua, using a method described by (Ishihama, Rappsilber et al. 2002). The HPLC separations of the peptides were at flow rates of about 250 nanoliters per minute during 2 hrs and with 7-40% gradients of acetonitrile in the presence of 0.1% formic acid. The capillary columns were connected on line to the Orbitrap mass spectrometer through an electrospray interface. The mass spectrometer was operated in a data-dependent mode where the masses of the eluting peptides were measured at high accuracy in the Orbitrap part of the machine and the seven most intense masses, detected at each full MS spectra whose charge states were determined to be double and triple, were selected for fragmentation by CID in the linear trap at the subsequent seven CID fragmentations.
2) Multidimensional chromatography coupled with mass spectrometry. 50 μg of total protein extracts mixed with 100 μg in 8 M urea, 100 mM ammonium bicarbonate were treated for blocking all the sulfhydryls by first reducing disulfides by addition of 10 mM DTT, incubation at 60° C. for 30 min. The free disulfides were next blocked by carboxymethylation using 10 mM iodoacetamide and incubation at room temperature for 30 min. The denatured and carboxymethylated protein mixtures were diluted three-fold with water to reduce urea concentration to about 2M followed by digestion in solution, overnight at 37° C. using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The resulting peptides from the trypsinized proteins were desalted with reversed-phase with a C18 tip disposable micro-columns (Harvard), eluted with 90% acetonitrile, dried and dissolved in 0.1% formic acid. The resulting peptides were resolved by multi-dimensional chromatography with on-line first dimension of strong cation exchange (SCX) chromatography 0.3×5 mm columns (LC Packings) using ten salt steps of 20, 40, 60, 80, 100, 120, 160, 200, 300 and 500 mM ammonium acetate in 5% acetonitrile with 0.1% acetic acid. The peptides from each increased salt elution from the SCX columns were transferred on-line to a C18 trap column (0.3×5 mm, LC-Packings), which was connected on-line to a Reprosil C18 homemade capillary columns (75 micron ID), resolved by 7-40% acetonitrile gradients, during 2 hrs, in the presence of 0.1% formic acid as described before for the gel-slicing method.
Resolving peptides by two-dimensional capillary chromatography has been described (Link, Eng et al. 1999), reviewed in (Link 2002). The mass spectrometry analysis was performed on-line as described above using data-dependent LC-MS/MS analysis with full MS in the Orbitrap and subsequent seven dependent ion-trap CID spectra of the most abundant doubly and triply charged peptides, detected in the full MS.
3) Isotope labeling peptides to enable quantitative analysis. Labeling tryptic peptides with light or heavy stable isotope reagents may rely on commercial reagents, reviewed in (Ong and Mann 2005; Regnier and Julka 2006). To facilitate the accurate comparison of the relative amounts of each of the proteins in the different samples, the mixture of peptides produced by the trypsinization of the entire in-solution protein proteolysis were covalently modified with light and heavy stable-isotope reagents. Reductive dimethylation labeling was done with heavy and light formaldehyde as described by (Hsu, Huang et al. 2003).
50 ug of proteolytic peptides were covalently modified with stable-isotope labeled (heavy and light formaldehyde) reagents (reductive dimethylation). The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.
Stable isotope labeling was also performed by iTRAQ (Ross, Huang et al. 2004) using a labeling kit that was purchased from Applied Biosystems and labeling was done according to the manufacturer's protocol. The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.
Bioinformatics
The mass spectrometry data of both the tryptic peptides obtained from proteolysis in the gel slices mentioned above and the tryptic peptides resolved by multidimensional chromatography were clustered and analyzed using the Pep-Miner software tool (Beer, Barnea et al. 2004). The search against the human part of the IPI database was done by using multiple search engines: Pep-Miner (Beer, Barnea et al. 2004), Mascot (Perkins, Pappin et al. 1999) and Sequest (Eng, McCormack et al. 1994). Both Mascot and Sequest were run together using the Protein Discoverer software tool (Thermo-Fisher).
Peptides were selected according to the following critera: 1) Mascot: ionScore>identityHigh and expValue<0.05 and deltaScore=0; 2) Sequest: ((xCorr>2 and chg</=2) or (xCorr>2.5 and chg>/=3)) and probability>15 and deltaScore=0 A peptide was used for the analysis if it was identified with the above criteria at least once for Sequest and once for Mascot, although not necessarily both in the same scan or run or patient.
The entire protein repertoires of healthy and diseased gastrointestinal tissues were analyzed from samples obtained from greater than 50 patients undergoing surgery for colorectal cancer.
The clinical characteristics of the patients studied are provided in Table 2.
Table 3 lists proteins that were observed to be highly expressed in polyps, as well as in early and advanced stages of various colorectal cancers. As shown in Table 3 and in
Table 4 lists proteins that were observed to be highly expressed in polyps, whereas in more advanced stages of colorectal cancer these proteins tended to have decreased levels of expression. Accordingly, a diagnostic array of reagents directed to detection of at least some of the proteins in this group may be used as a screening test for very early detection of colorectal cancer. Such a screening test could identify susceptible at-risk individuals, even prior to the stage at which polyp visualization is possible by endoscopic techniques.
The proteins identified by isotopic labeling included those listed in Table 5. All of the listed proteins were found to be present in colon cancer tissue at levels that were at least 2.5-fold greater than the level of the same protein in healthy colon tissue from the same subject, as indicated by the median ratios of the protein levels (i.e. cancerous tissue:healthy tissue) listed in the column denoted “MED”.
The proteins identified on the basis of the peptides detected by the multidimensional chromatography technique include those listed in Table 6, and reflects the abundance of these proteins in cancerous tissues. None of the proteins listed in Table 6 were identified by the isotopic labeling technique. The proteins listed in Tables 3 and 6 appear to be specifically expressed in colorectal cancer tissue, as they were substantially undetectable in all healthy colorectal tissues obtained from the colorectal cancer patients.
Table 7 lists proteins that are considered potential targets for development of cytotoxic reagents specifically directed to these proteins, for example, specific antibody or antibody fragments conjugated to toxic moieties for targeted elimination of cancer cells. As well as being highly expressed in early stage polyps and in tumors, these proteins are generally exposed, and considered vulnerable to attack by targeted cytotoxic reagents.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the brand concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL10/00342 | 4/27/2010 | WO | 00 | 10/27/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61172800 | Apr 2009 | US |
