WO2013192242A2 - Cell surface marker and methods of use thereof - Google Patents

Cell surface marker and methods of use thereof Download PDF

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Publication number
WO2013192242A2
WO2013192242A2 PCT/US2013/046436 US2013046436W WO2013192242A2 WO 2013192242 A2 WO2013192242 A2 WO 2013192242A2 US 2013046436 W US2013046436 W US 2013046436W WO 2013192242 A2 WO2013192242 A2 WO 2013192242A2
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gctm
cells
antibody
analysis
epitope
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French (fr)
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WO2013192242A3 (en
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Martin Pera
Jun Wu
David BRAXTON
Kouichi Hasegawa
Veronika AKOPIAN
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University Of Southern California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention generally relates to novel bio markers for detection and screening of cancer, and more particularly, to biomarkers comprising GCTM-5 antigen, the distinct population of cells identified by the expression pattern of GCTM-5, diagnostic methods for utilizing the biomarkers and reagents for detecting the biomarkers.
  • the liver is capable of regeneration not only through hepatocyte proliferation but also via a biliary ductular reaction, wherein bipotential cells give rise to bile duct and hepatic parenchyma. These facultative bipotential progenitor cells reside in the Canals of Hering (14-17). Although there is significant heterogeneity in this population of ductular progenitors, epithelial cell adhesion molecule (EpCAM) and neural cell adhesion molecule (NCAM) provide useful markers for these cells (9, 17).
  • EpCAM epithelial cell adhesion molecule
  • NCAM neural cell adhesion molecule
  • pancreas Regenerative mechanisms in the pancreas are not as well established as those in the liver.
  • a candidate population of progenitors found in chronic pancreatitis resides in a poorly defined structure termed the tubular complex, that has been proposed to originate in various compartments including acinar cells, centroacinar cells, and terminal ductules (19- 21).
  • these structures have been identified as a susceptible population for neoplastic transformation, and they may represent the cell of origin of pancreatic intraepithelial neoplasia (PanIN), the precursor of pancreatic ductal adenocarcinoma (22-26).
  • PanIN pancreatic intraepithelial neoplasia
  • pancreatic ductal adenocarcinoma An analogous sequence of premalignant changes has been proposed for intrahepatic cholangiocarcinoma, but the distinct cell of origin is largely unknown for this highly malignant tumor (27, 28).
  • one aspect of the present invention is the discovery that expression of the GCTM-5 antigen marks putative progenitor compartments in gastrointestinal and hepatopancreatic tissues.
  • cells expressing the GCTM-5 epitope participate in the process of regeneration, metaplasia, and neoplasia in these tissues.
  • the GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a useful cell surface marker for the study of nonnal differentiation lineages and regeneration in tissues of endodermal origin.
  • GCTM-5 antigen is a serum-based biomarker for diagnostic or screening tests for pancreatic adenocarcinoma.
  • pancreatic tissues maybe biopsied first and analyzed for their GCTM-5 expression, wherein a higher level of GCTM-5 expression compared to a reference indicates the likelihood of pancreatic adenocarcinoma.
  • the invention provides a method for detecting and characterizing a subpopulation of cells in a sample.
  • the method includes: a) detecting expression or presence of a biomarker of one or more cells in the sample, wherein the biomarker is a GCTM-5 epitope; and b) analyzing the results of (a) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as a subpopulation of progenitor cells.
  • the invention provides a kit.
  • the kit includes an antibody that specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP, reagents for conducting a diagnostic test; and instructions for conducting and analyzing results of the diagnostic test.
  • Another aspect of the present invention is directed to an antibody, or fragment thereof which specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP.
  • the antibody may be an immunoconjugate.
  • Another aspect of the present invention is directed to a pharmaceutical composition including the antibody of the invention and a pharmaceutically acceptable carrier.
  • Another aspect of the present invention is directed to an isolated nucleic acid molecule encoding the antibody of the invention. Another aspect of the present invention is directed to a method for treating cancer.
  • the method includes administering to a subject the antibody or pharmaceutical composition of the invention to the subject.
  • Cirrhotic liver demonstrates overlapping coexpression (yellow) of EpCAM (red) and GCTM-5 (green) on a subpopulation of biliary ducts by immunofluorescence; Note that not all of the GCTM-5 expressing cells are also expressing EpCAM.
  • D Immunofluorescence showing GCTM-5 (red) and Albumin (green) coexpression (yellow) at the periphery of a regenerating nodule.
  • E Intrahepatic .
  • Cholangiocarcinoma variably express the GCTM-5 antigen (red).
  • FIG. 1 Distribution of GCTM-5 staining in the biliary tract. Serial sections of normal pediatric liver stained with GCTM-5 (top, pink) and CK-19 (bottom, brown) antibodies. Each section is Sum thick. Scale bar, lOOum. Figure 3. GCTM-5 antigen expression in normal and pathologic pancreatic tissues.
  • A An exocrine lobe shows a single focus of GCTM-5 staining (red) in a small ductule in cross section amongst a larger field of acini.
  • B In an area of mild fibrosis due to chronic pancreatitis the expression of GCTM-5 antigen in ductules is expanded.
  • C In severe chronic pancreatitis GCTM-5 expression is detected a subpopulation of cells in an expansion of ductules also known as a tubular complex formation or acinar-ductal metaplasia.
  • Cardia-type metaplasia expresses the antigen in a subpopulation of the epithelia.
  • Intestinal Metaplasia expresses the GCTM-5 antigen in the apical membrane mainly in the deep pits and glands.
  • Dysplasia (E) and Adenocarcinoma (F) react more extensively with GCTM-5.
  • Figure 6 Indirect immunofluorescence microscopy of hepatic marker expression in cell colonies derived from GCTM-5 positive cells in the non-parenchymal fraction of adult liver. Fixed colonies were stained with GCTM-5 and various cell surface and intracellular markers, then counterstained with DAPI.
  • FIG. 7 Biochemical characterization of the GCTM-5 antigen.
  • A Immunoblot of CFPAC-1 cell lysate with GCTM-5. Positions of 200 and 55 kDa markers are shown.
  • B GCTM-5 immunoblot of CFPAC-1 conditioned media treated with no enzyme control (Lane 1), N-Glycanase (Lane 2), sialidase A (Lane 3), O-Glycanase (Lane 4), all three enzymes (Lane 5). Position of 200 kDa marker is shown.
  • C Immunoprecipitates of concentrated CFPA-1 conditioned medium blotted with GCTM-5. Lane 1, GCTM-5 immunoprecipitate; Lane 2, control IgGl.
  • Position of 250 kDa marker is shown. Lower bands are immunoglobulins reacting with the secondary antibody.
  • D Immunoblot of affinity purified GCTM-5 antigen (Lanes 1 and 3) or CFPAC-1 concentrated conditioned medium (Lanes 2 and 4) with antisera to C-terminal (Lanes 1 and 2) or N-terminal (Lanes 3 and 4) peptide sequences of FCGBP. Positions of 250, 105 and 50 kDa markers are shown.
  • E Immunoblot of GCTM-5 immunoprecipitate of CFPAC-1 conditioned medium probed with antiserum against C-terminal peptide sequence of FCGBP. Position of 105 kDa marker is shown. Lower bands are immunoglobulins reacting with secondary antibody.
  • GCTM-5 expression shown in pink, in the crypts of the human fetal gut at 13 weeks gestation, and (B) an antibody isotype negative control.
  • GCTM-5 expression in (C) 16 week and (D) 18.5 week human fetal pancreas. Cytokeratin 19 staining of (E) 16 week and (F) 18.5 week fetal pancreas; detecting a larger number of ductal structures.
  • Figure 9 Depicts distribution of the GCTM-5 antigen in the gastrointestinal tract
  • Normal gastric epithelial does not react with GCTM-5, however, gastric intestinal metaplasia (B) and gastric carcinoma (C) express the GCTM-5 antigen.
  • B gastric intestinal metaplasia
  • C gastric carcinoma
  • D The epithelium of the gail blader stains positive.
  • the normal colonic crypts express GCTM-5 with less intense reactivity in dysplastic colonic lesions (F).
  • Non-gastrointestinal definitive endoderm derived tissues express GCTM-
  • FIG. 11 Shows that some GCTM-5+ cells in biliary atresia livers are also positive for Ki-67. Scale bar, lOOum.
  • Figure 12. GCTM-5 expression in biliary atresia livers.
  • B GCTM-5 stained positive on enlarged bile duct epithelium, majority of these cells are CK19+, N-CAM- & EpCAM+(very weak).
  • C GCTM-5 was also found positive on malformed bile duct epithelia, majority of the cells are CK-19+/-, N-CAM-, EpCAM+
  • D GCTM-5 staining was also found on newly formed bile ducts, majority of these cells are CK19+/-, N-CAM- (positive on surrounding cells) & EpCAM+. Scale bar, lOOum.
  • FIG. 13 GCTM-5 antigen expression in normal and pathologic pancreatic tissues.
  • A Adult pancreas shows an islet stained with c-peptide (red) that is exclusive of GCTM-5 staining (green).
  • B Cytokeratin 19
  • C Cytokeratin 19
  • GCTM-5 antigen or its epitopes are useful as biomarkers to identify endodermal cellular origin and other related cellular states and conditions.
  • the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP.
  • the tissue specificity of the GCTM-5 epitope distinguishes it from that of known mucins.
  • the only mucin expressed by both bile ducts and pancreas is Muc-1 (49, 50), and Muc-1 is far more broadly expressed in epithelia and in carcinomas than the GCTM-5 epitope. It is however possible that Muc-1, the GCTM-5 epitope, and FCGBP are present in some form of complex in bile duct and liver.
  • Mucins are known to exert cytoprotective effects, and in the context of tissue regeneration in the face of microenvironmental damage (gastroinitestinal reflux, pancreatitis, and biliary obstruction), progenitor cells may upregulate their expression of surface mucins to enhance their capacity for survival and repair.
  • progenitor cells in liver express the GCTM-5 antigen.
  • the GCTM-5 monoclonal antibody was used to isolate the biliary ductular cells that express the antigen on the membrane surface. Gene expression showed that these cells express transcription factors characteristic of endodermal progenitors, including GATA-6, HNFlbeta, HNF6, HNF4, HNF3a, and PDX1. Some of these transcription factors are found in progenitor populations in the developing liver, in the ductal plate, the cell type thought to correspond to adult liver stem cells (9, 15). When these GCTM-5-positive cells were cultured in vitro under conditions that promote growth of hepatocyte progenitors, they maintained a pattern of protein markers consistent with hepatic stem cells.
  • Intrahepatic cholangiocarcinoma is a highly aggressive malignancy of the biliary epithelia with a poor prognosis.
  • the GCTM-5 antigen is expressed variably and heterogeneously in these tumors. If in fact GCTM-5 marks a ductal progenitor population, this may indicate that the cell of origin of a subset of these tumors (51) is GCTM-5 positive.
  • GCTM-5 is expressed on sialomucin containing epithelia that are proliferating.
  • the multilayered epithelium is the candidate precursor of intestinal metaplasia of the esophagus.
  • the GCTM-5 antigen is variably expressed in the multilayered epithelium (ME), but its patterns of expression suggest it marks tissue that is progressing toward intestinal metaplasia. Those foci that express abundant GCTM-5 antigen are typically adjacent to or contiguous with metaplastic columnar epithelia that are also strongly immunoreactive. Further studies confirmed that when this occurs, these epithelia share a common profile of mucins and differentiation markers (not shown). This indicates a direct transition from multilayered epithelium into metaplastic columnar epithelia that is marked by GCTM-5 expression.
  • the GCTM-5 antigen is a novel marker for a subpopulation of epithelial cells in normal pancreatic ducts, PanIN, and pancreatic ductal adenocarcinoma.
  • the GCTM-5 antigen is expressed on the surface membrane of a subpopulation of epithelia in the developing and adult pancreas.
  • the normal pancreas demonstrates GCTM- 5 expression restricted to a minor proportion of the large ducts and terminal ductules. As in the liver, this population in the pancreas overlaps with those cells expressing Sox-9, thought to be a marker for pancreatic progenitor cells (S. Bonner- Weir, personal communication).
  • the proportion of positive cells increases, with severe disease demonstrating GCTM-5 expression in a subpopulation of the massive collection of ductules, often referred to as the tubular complex.
  • GCTM-5 was strongly reactive in a high percentage of tumor cells and mucinous material from ail cases of pancreatic adenocarcinoma that we examined.
  • CFPAC-1 pancreatic adenocarcinoma cells secrete or shed GCTM-5 antigen into the culture medium. Therefore, GCTM-5 antigen is a potential serum-based biomarker for diagnostic or screening tests for pancreatic adenocarcinoma.
  • FCGBP maps to chromosome 19ql3, a region of the genome that is often overrepresented in a subset of patients with pancreatic adenocarcinoma (53).
  • FCGBP locus lies within a 650 kbp minimal amplicon on 19ql3 defined on a panel of pancreatic cancer cell lines and patient tumors, and two-third cell lines that harbored the amplicon expressed FCGBP at levels >20-fold above levels in controls that did not contain the amplicon (49), Although these limited data do not of course implicate FCGBP in tumor progression, it is possible that as in other types of cancer, mucin overexpression is characteristic of the disease state.
  • a biomarker is indicative of certain biological state, such as the presence of cancerous tissue.
  • different forms of biomarkers can be indicative of certain disease states.
  • the presence of the biomarker may be used to distinguish and isolate certain population of cell types from others.
  • GCTM-5 epitope is a serum-based biomarker for diagnostic or screening tests for neoplasia and cancer.
  • tissue may be biopsied first and analyzed for GCTM-5 expression or presence, wherein a higher level of GCTM-5 expression or presence compared to a reference indicates the likelihood of cancer, such as pancreatic adenocarcinoma.
  • the invention provides a method for detecting cancer in a sample.
  • the method includes: a) obtaining the sample from the subject; b) detecting expression or presence of a biomarker in the sample, wherein the biomarker is a GCTM-5 epitope; and c) analyzing the results of (b) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as cancer.
  • Another aspect of the present invention is directed to biomarkers for colorectal carcinoma which comprises measuring the expression of both GCTM-5 and FCGBP in tissues from apical surface of intestine. Reduced expression of both GCTM-5 and FCBGP as compared with a reference level in the intestinal tissues indicates a likelihood of colorectal carcinoma.
  • the invention provides a method for detecting and characterizing a subpopulation of cells in a sample.
  • the method includes: a) detecting expression or presence of a biomarker of one or more cells in the sample, wherein the biomarker is a GCTM-5 epitope; and b) analyzing the results of (a) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as a subpopulation of progenitor cells.
  • the invention provides a method of isolating a progenitor subpopulation of cells from a sample, The method includes a) contacting the sample with an agent that specifically binds a biomarker present on a subpopulation of progenitor cells present in the sample to form a plurality of cellular complexes, wherein the biomarker is native GCTM-5 epitope of FCGBP; and b) isolating the cellular complexes from the sample, thereby isolating the progenitor subpopulation of cells.
  • Another aspect of the present invention is directed to diagnostic systems and reagent kits for diagnosing and screening cancers that may characterized by the expression pattern of GCTM-5 antigen, including but not limited to pancreatic adenocarcinoma and colorectal carcinoma.
  • the invention provides a kit.
  • the kit includes an antibody that specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP, reagents for conducting a diagnostic test; and instructions for conducting and analyzing results of the diagnostic test.
  • sample refers to any sample suitable for the methods provided by the present invention.
  • the sample may be any sample that includes native GCTM-5 epitope for detection.
  • Sources of samples include whole blood, pleural fluid, peritoneal fluid, central spinal fluid, urine, saliva bronchial washes, and tissue.
  • the sample is a tissue sample, including, for example, pancreatic, esojphageal, gastrointestinal, colon and liver tissue or fraction or component thereof.
  • GCTM-5 epitope positive cells are detected and analyzed to derive clinically significant data via antibodies of the present invention.
  • Analysis of GCTM-5 epitope positive cells may be performed by a variety of methods depending of the type of data desired. For example, in various aspects, subsequent to detecting, GCTM-5 epitope positive cells may be analyzed by detecting and characterizing the cells via assays utilizing recognition and/or binding of cellular components, such as biomarkers including cell surface markers.
  • GCTM-5 epitope positive cells are captured by techniques commonly used to enrich a sample for a particular cell type, for example those involving immunospecific interactions, such as immunomagnetic capture.
  • techniques commonly used to enrich a sample for a particular cell type for example those involving immunospecific interactions, such as immunomagnetic capture.
  • immunocapture methods are known, including immunocapture with beads or posts.
  • a magnetic field or solid supports may aid the immunocapture.
  • Immunomagnetic capture also known as immunomagnetic cell separation typically involves attaching antibodies directed to proteins found on a particular cell type to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, they attach to and surround the particular cell. The sample is then placed in a strong magnetic field, causing the beads to pellet to one side. After removing the blood, captured cells are retained with the beads. Many variations of this general method are well known in the art and suitable for use with the methods of the present invention.
  • GCTM-5 epitope positive cells are further processed prior to an enrichment step using filtration.
  • the GCTM-5 epitope positive cells are further processed via cell separation by density gradient sedimentation. Typically, the process relies on a gross physical distinction, such as cellular density. Many variations of this general method are well known in the art and suitable for use with the methods of the present invention.
  • the revealed cells are enriched by a technique called "panning".
  • panning a technique called "panning”.
  • such processes utilize an antibody specific to the cell type in question in which the antibody is adhered to a solid surface.
  • the cell mixture is layered on top of the antibody-coated surface, the targeted cells tightly adhere to the solid surface due to the immunospecific interaction involving antibody-antigen binding.
  • Non-adherent cells are rinsed off the surface, thereby effecting a cell separation and enrichment.
  • Cells that express a cell surface protein recognized by the antibody are retained on the solid surface whereas other cell types are not.
  • Detection and characterization of GCTM-5 epitope positive cells using the methods of the invention is useful in disease detection and diagnosis as well as assessing cancer prognosi s and in monitoring therapeutic efficacy for early detection of treatment failure that may lead to disease relapse.
  • cellular analysis according to the invention enables the detection of early relapse in presymptomatic patients who have completed a course of therapy. This is possible because the presence of GCTM-5 epitope positive cells is likely associated and/or correlated with tumor progression and spread, poor response to therapy, relapse of disease, and/or decreased survival over a period of time.
  • enumeration and characterization of GCTM-5 epitope positive cells provides methods to stratify patients for baseline characteristics that predict initial risk and subsequent risk based upon response to therapy.
  • subject refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
  • rodents including mice, rats, hamsters and guinea pigs
  • cats dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc.
  • primates including monkeys, chimpanzees, orangutans and gorillas
  • analysis of a subject's GCTM-5 epitope positive cell number and characterization may be made over a particular time course in various intervals to assess a subject's progression and pathology. For example, analysis may be performed at regular intervals such as one day, two days, three days, one week, two weeks, one month, two months, three months, six months, or one year, in order to track level and
  • characterization of GCTM-5 epitope positive cells as a function of time In the case of existing cancer patients, this provides a useful indication of the progression of the disease and assists medical practitioners in making appropriate therapeutic choices based on the increase, decrease, or lack of change in circulating epithelial cells, such as the presence of GCTM-5 epitope positive cells in the patient's bloodstream. Any increase, be it 2-fold, 5- fold, 10-fold or higher, in the GCTM-5 epitope positive cells over time decreases the patient's prognosis and is an early indicator that the patient should change therapy.
  • additional analysis may also be performed to characterize GCTM-5 epitope positive cells to provide additional clinical assessment.
  • PCR techniques may be employed, such as multiplexing with primers specific for particular cancer markers to obtain information.
  • cell size, DNA or RNA analysis, proteome analysis, or metabolome analysis may be performed as a means of assessing additional information regarding characterization of the patient's cancer.
  • the additional analysis may provide data sufficient to make determinations of responsiveness of a subject to a particular therapeutic regime, or for determining the effectiveness of a candidate agent in the treatment of cancer.
  • the present invention provides a method of determining responsiveness of a subject to a particular therapeutic regime or determining the effectiveness of a candidate agent in the treatment of cancer by detecting GCTM-5 epitope positive cells of the subject as described herein and analyzing the GCTM-5 epitope positive cells.
  • a drug treatment is administered to a patient, it is possible to determine the efficacy of the drug treatment using the methods of the invention.
  • a sample taken from the patient before the drug treatment, as well as one or more cellular samples taken from the patient concurrently with or subsequent to the drug treatment may be processed using the methods of the invention.
  • By comparing the results of the analysis of each processed sample one may determine the efficacy of the drug treatment or the responsiveness of the patient to the agent. In this manner, early identification may be made of failed compounds or early validation may be made of promising compounds.
  • Antibodies may be made of failed compounds or early validation may be made of promising compounds.
  • Certain embodiments of the present invention include immunopeptides directed against native GCTM-5 epitope of mucin-like glycoprotein FCGBP.
  • the immunoglobulin peptides, or antibodies, described herein are shown to bind to the native GCTM-5 epitope.
  • the GCTM-5 epitope binding activity is specific.
  • These GCTM-5 epitope specific antibodies can be used to differentiate between subpopulations of cells to identify progenitor cells, or discriminate between diseased cells and normal cells.
  • the GCTM-5 epitope specific antibodies can also be used in immunotherapy against various cancers, to determine the response after therapy for cancer and to inhibit metastasis.
  • immunopeptides can be raised in a variety of means known to the art.
  • the term antibody encompasses all types of antibodies and antibody fragments, e.g., polyclonal, monoclonal, and those produced by the phage display methodology.
  • Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for GCTM-5 epitope. In certain embodiments, the antibodies exhibit an affinity for GCTM-5 epitope of about d ⁇ 10 "8 M.
  • Substantially purified generally refers to a composition which is essentially free of other cellular components with which the antibodies are associated in a non-purified, e.g., native state or environment. Purified antibody is generally in a homogeneous state, although it can be in either in a dry state or in an aqueous solution. Purity and
  • homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • Substantially purified GCTM-5 epitope specific antibody will usually comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the antibody with a pharmaceutical carrier, excipient, adjuvant, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient. More typically, the antibody is purified to represent greater than 90% of all proteins present in a purified preparation. In specific embodiments, the antibody is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.
  • Immunoglobulin peptides include, for example, polyclonal antibodies, monoclonal antibodies, and antibody fragments. The following describes generation of
  • immunoglobulin peptides specifically GCTM-5 epitope antibodies, via methods that can be used by those skilled in the art to make other suitable immunoglobulin peptides having similar affinity and specificity which are functionally equivalent to those used in the examples.
  • Monoclonal antibody (mAb) technology can be used to obtain mAbs to GCTM-5 epitope.
  • hybridomas are produced using spleen cells from mice immunized with GCTM-5 antigen.
  • the spleen cells of each immunized mouse are fused with mouse myeloma Sp 2/0 cells, for example using the polyethylene glycol fusion method of Galfre, G. and Milstein, C, Methods Enzymol., 73:3-46 (1981).
  • Growth of hybridomas, selection in HAT medium, cloning and screening of clones against antigens are carried out using standard methodology (Galfre, G. and Milstein, C, Methods Enzymol, 73:3-46 (1981)).
  • HAT-selected clones are injected into mice to produce large quantities of mAb in ascites as described by Galfre, G. and Milstein, C, Methods Enzymol., 73:3-46 (1981), which can be purified using protein A column chromatography (BioRad, Hercules, Calif.).
  • mAbs are selected on the basis of their (a) specificity for GCTM-5 antigen, (b) high binding affinity, (c) isotype, and (d) stability. mAbs can be screened or tested for GCTM-5 epitope specificity using any of a variety of standard techniques, including Western Blotting (Koren, E. et al, Biochim. Biophys. Acta 876:91-100 (1986)) and enzyme-linked immunosorbent assay (ELISA) (Koren, E. et al, Biochim. Biophys. Acta 876:91-100 (1986)).
  • Humanized Antibodies Humanized Antibodies
  • the well-known basic structure of a typical IgG molecule is a symmetrical tetrameric Y-shaped molecule of approximately 150,000 to 200,000 daltons consisting of two identical light polypeptide chains (containing about 220 amino acids) and two identical heavy polypeptide chains (containing about 440 amino acids). Heavy chains are linked to one another through at least one disulfide bond. Each light chain is linked to a contiguous heavy chain by a disulfide linkage. An antigen-binding site or domain is located in each arm of the Y-shaped antibody molecule and is formed between the amino terminal regions of each pair of disulfide linked light and heavy chains. These amino terminal regions of the light and heavy chains consist of approximately their first 110 amino terminal amino acids and are known as the variable regions of the light and heavy chains.
  • variable regions of the light and heavy chains there are hypervariable regions which contain stretches of amino acid sequences, known as complementarity determining regions (CDRs). CDRs are responsible for the antibody's specificity for one particular site on an antigen molecule called an epitope.
  • CDRs complementarity determining regions
  • the carboxy terminal regions of light and heavy chains are similar or identical to those of other antibody molecules and are called constant regions.
  • the amino acid sequence of the constant region of the heavy chains of a particular antibody defines what class of antibody it is, for example, IgG, IgD, IgE, IgA or IgM.
  • Some classes of antibodies contain two or more identical antibodies associated with each other in multivalent antigen-binding arrangements.
  • Fab and F(ab') 2 fragments of mAbs that bind GCTM-5 epitope can be used in place of whole mAbs. Because Fab and F(ab') 2 fragments are smaller than intact antibody molecules, more antigen-binding domains are available than when whole antibody molecules are used. Proteolytic cleavage of a typical IgG molecule with papain is known to produce two separate antigen binding fragments called Fab fragments which contain an intact light chain linked to an amino terminal portion of the contiguous heavy chain via by disulfide linkage. The remaining portion of the papain-digested immunoglobin molecule is known as the Fc fragment and consists of the carboxy terminal portions of the antibody left intact and linked together via disulfide bonds.
  • an antibody is digested with pepsin, a fragment known as an F(ab3 ⁇ 4 fragment is produced which lacks the Fc region but contains both antigen-binding domains held together by disulfide bonds between contiguous light and heavy chains (as Fab fragments) and also disulfide linkages between the remaining portions of the contiguous heavy chains (Handbook of Experimental Immunology. Vol 1 : Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications, Oxford (1986)).
  • ScFvs single chain antigen-binding polypeptides known as single chain Fv fragments (ScFvs or ScFv antibodies). Further, ScFvs can be dimerized to produce a diabody. ScFvs bind a specific epitope of interest and can be produced using any of a variety of recombinant bacterial phage-based methods, for example as described in Lowman et al. (1991 ) Biochemistry, 30, 10832-10838;
  • RNA isolated from a hybridoma that produces an mAb for GCTM- 5 antigen.
  • the cDNA molecules encoding the variable regions of the heavy and light chains of the mAb can then be amplified by standard polymerase chain reaction (PCR) methodology using a set of primers for mouse immunoglobulin heavy and light variable regions (Clackson (1991) Nature, 352, 624-628).
  • PCR polymerase chain reaction
  • the amplified cDNAs encoding mAb heavy and light chain variable regions are then linked together with a linker
  • the ScFv DNA is Hgated into a filamentous phage plasmid designed to fuse the amplified cDNA sequences into the 5' region of the phage gene encoding the minor coat protein called g3p.
  • Escherichia coli bacterial cells are than transformed with the recombinant phage plasmids, and filamentous phage grown and harvested.
  • the desired recombinant phages display antigen-binding domains fused to the amino terminal region of the minor coat protein.
  • Such “display phages” can then be passed over immobilized antigen, for example, using the method known as "pasng”, see Parmley and Smith (1989) Adv. Exp. Med. Biol. 251, 215-218; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382, to adsorb those phage particles containing ScFv antibody proteins that are capable of binding antigen.
  • the antigen-binding phage particles can then be amplified by standard phage infection methods, and the amplified recombinant phage population again selected for antigen- binding ability.
  • Another method to select for enhanced antigen-binding activity is to alter nucleotide sequences within the cDNA encoding the binding domain of the ScFv and subject recombinant phage populations to successive rounds of selection for antigen- binding activity and amplification (see Lowman et al. (1991) Biochemistry 30, 10832- 10838; and Cwirla et al. ( 990) Proc. Natl. Acad. Sci. USA 87, 6378-6382).
  • the recombinant GCTM-5 antigen antibody can be produced in a free form using an appropriate vector in conjunction with E. coli strain HB2151. These bacteria actually secrete ScFv in a soluble form, free of phage components (Hoogenboom et al. (1991) Nucl. Acids Res. 19, 4133-4137).
  • the purification of soluble ScFv from the HB2151 bacteria culture medium can be accomplished by affinity chromatography using antigen molecules immobilized on a solid support such as
  • AFFIGELTM BioRad, Hercules, Calif.
  • ScFvs are even smaller molecules than Fab or F(ab') 2 fragments, they can be used to attain even higher densities of antigen binding sites per unit of surface area when immobilized on a solid support material than possible using whole antibodies, F(ab') 2 , or Fab fragments. Furthermore, recombinant antibody technology offers a more stable genetic source of antibodies, as compared with hybridomas. Recombinant antibodies can also be produced more quickly and economically using standard bacterial phage production methods.
  • Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab ! and F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies .
  • Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CHS domains.
  • antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI , CH2, and CH3 domains.
  • Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region may be condricthoid in origin (e.g., from sharks).
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous
  • immunoglobulins as described infra and, for example in, U.S. Pat. No. 5,939,598 by ucherlapati et al.
  • GCTM-5 epitope antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be "multispecific,” e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time.
  • an GCTM-5 epitope antibody is "monospecific” or “multispecific,” e.g., "bispecific,” refers to the number of different epitopes with which a binding polypeptide reacts.
  • Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • valency refers to the number of potential binding domains, e.g., antigen binding domains, present in an GCTM-5 epitope antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope.
  • binding polypeptide or antibody comprises more than one binding domain
  • each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed “bivalent monospecific,” or to different epitopes, for an antibody with two binding domains, termed “bivalent bispecific.”
  • An antibody may also be bispecific and bivalent for each specificity (termed “bispecific tetravalent antibodies”).
  • tetr tetr
  • Bispecific bivalent antibodies, and methods of making them are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein.
  • Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCX publications WO
  • the present invention includes multispecific GCTM-5 epitope antibodies.
  • a bispecific antibody comprised of two scFv antibody fragments, both of which bind GCTM-5 epitope.
  • the scFv antibody fragments may bind the same or different epitopes on GCTM-5,
  • Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • GCTM-5 epitope specific antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
  • Radiolabled GCTM-5 epitope antibodies of the invention will be particularly useful, while antibody drug conjugates (ADCs) remain to be developed.
  • ADCs antibody drug conjugates
  • GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding GCTM-5 antigen.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • GCTM-5 epitope specific antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in the GCTM-5 epitope specific antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given GCTM-5 epitope specific antibody.
  • the present invention also provides for fusion proteins comprising an GCTM-5 epitope antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide.
  • the heterologous polypeptide to which the antibody is fused may be useful for function or is useful to target the GCTM-5 epitope expressing cells.
  • a fusion protein of the invention comprises a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence.
  • a fusion protein for use in the methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three of the VH- CDRs of an GCTM-5 epitope specific antibody, or fragments,, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an GCTM-5 epitope specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence.
  • the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an GCTM-5 epitope specific antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of GCTM-5.
  • a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an GCTM-5 epitope specific antibody of the invention and the amino acid sequence of at least one VL region of an GCTM-5 epitope specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence.
  • the VH and VL regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds at least one epitope of GCTM-5.
  • a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an GCTM-5 epitope specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an GCTM-5 epitope specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence.
  • VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention.
  • Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. Fusion proteins can be prepared using methods that are well known in the art (see for example U.S. Pat. Nos. 5,1 16,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
  • the invention provides for a particularly preferred anti-GCTM-5 epitope antibody; i.e., an isolated anti-GCTM-5 epitope antibody which specifically bind native GCTM-5 epitope.
  • native GCTM-5 epitope refers to GCTM-5 epitope produced from CFPAC-1 cells in its native state, e.g., unaltered by enzyme or chemical treatment, such as treatment with acid, sialydase A, N-glycosidase or O-glycosidase.
  • a pharmaceutical formulation comprising the antibody against GCTM-5 epitope and a pharmaceutically acceptable carrier.
  • the invention provides an isolated nucleic acid encoding the antibody against GCTM-5 epitope, In another embodiment, the invention provides for an expression vector comprising the nucleic acid according to nucleic acid encoding an antibody against GCTM-5 epitope. In an additional embodiment, the invention provides for a host cell comprising the nucleic acid encoding an antibody against GCTM-5 epitope. In a further embodiment, the invention provides for a method of producing an anti-GCTM-5 epitope antibody comprising culturing the host cells under conditions to produce the antibody. In one aspect, the method of producing an antibody further comprises recovering the antibody.
  • the anti-GCTM-5 epitope antibodies and GCTM-5 epitope binding peptides described herein can be used for the treatment or prevention of a GCTM-5 epitope cancer, such as pancreatic carcinoma or to inhibit metastasis of a GCTM-5 epitope cancer cell in a subject.
  • treat or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues.
  • Cancer includes, but is not limited to neoplasms, whether benign or malignant, located in the: colon, digestive system, gastrointestinal tract, liver, pancreas, peritoneum and esophagus. Such neoplasms, in certain embodiments, express, over-express, or abnormally express GCTM-5 epitope.
  • the selected antibody will typically be an anti-
  • GCTM-5 epitope antibody which may be administered alone, or in combination with, or conjugated to, one or more combinatorial therapeutic agents.
  • the antibodies described herein may exert a beneficial effect in the subject by a variety of mechanisms.
  • monoclonal antibodies that specifically bind GCTM-5 epitope are purified and administered to a patient.
  • the immunotherapeutic reagents of the invention may include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as anti-inflammatory ant anti-fibrinolytic drugs.
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as anti-inflammatory ant anti-fibrinolytic drugs.
  • therapeutic antibodies described herein are coordinately administered with, co-formulated with, or coupled to (e.g., covalently bonded) a combinatorial therapeutic agent, for example a radionuclide, a differentiation inducer, a drug, or a toxin.
  • a combinatorial therapeutic agent for example a radionuclide, a differentiation inducer, a drug, or a toxin.
  • a radionuclide for example a radionuclide, a differentiation inducer, a drug, or a toxin.
  • a combinatorial therapeutic agent for example a radionuclide, a differentiation inducer, a drug, or a toxin.
  • a combinatorial therapeutic agent for example a radionuclide, a differentiation inducer, a drug, or a toxin.
  • Various known radionuclides can be employed, including 90 Y, ,23 1, 12S I, i3l I, I86 Re, 188 Re, and 21 I
  • Suitable differentiation inducers include phorbol esters and butyric acid.
  • Suitable toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,
  • GCTM-5 antigen antibody can be coupled to an GCTM-5 antigen antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency.
  • a variety of bifunctional or polyfunctional reagents both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, 111.), may be employed as a linker group. Coupling may be affected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.
  • immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used. A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration is intravenous, intramuscular, or subcutaneous.
  • a safe and effective amount of a GCTM-5 antigen agent is, for example, that amount that would cause the desired therapeutic effect in a patient while minimizing undesired side effects.
  • a therapeutically effective amount is that sufficient to promote production of one or more cytokines and/or to cause complement- mediated or antibody-dependent cellular cytotoxicity.
  • the dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, and so on.
  • the dosage of administered GCTM-5 epitope antibodies, GCTM-5 epitope antibody components, immunoconjugates thereof and fusion proteins will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history.
  • a dosage of antibody component, vaccine, immunoconjugate or fusion protein which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of antibodies, antibody components, vaccines, immunoconjugates or fusion proteins to a patient can be intravenous, intraarterial,, intraperitoneal,
  • intramuscular, subcutaneous, intrapleural, intrathecal by perfusion through a regional catheter, or by direct intralesional injection.
  • administration may be by continuous infusion or by single or multiple boluses.
  • Intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies.
  • Intravenous administration is subject to limitation by a vascular barrier comprising endothelial cells of the vasculature and the subendothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors. Lymphomas have relatively high blood flow rates, contributing to effective antibody delivery. Intraiymphatic routes of administration, such as subcutaneous or intramuscular injection, or by catherization of lymphatic vessels, also provide a useful means of treating lymphomas.
  • GCTM-5 epitope antibodies, binding peptides, immunoconjugates thereof and fusion proteins are administered at low protein doses, such as 20 to 100 milligrams protein per dose, given once, or repeatedly, parenterally.
  • administration is in doses of 30 to 90 milligrams protein per dose, or 40 to 80 milligrams protein per dose, or 50 to 70 milligrams protein per dose.
  • the present invention also contemplates therapeutic methods in which GCTM-5 epitope antibody components are radiolabeled or supplemented with radiolabeled immunoconjugate or fusion protein administration.
  • GCTM-5 epitope antibodies are administered as or with low-dose radiolabeled GCTM-5 epitope antibodies or fragments.
  • GCTM-5 epitope antibodies may be administered with low-dose radiolabeled GCTM-5 epitope-cytokine immunoconjugates.
  • Those of ordinary skill in Hie art will be familiar with pharmaceutically acceptable radiolabelling molecules and their appropriate dosing levels.
  • a preferable dosage is in the range of 15 to 40 mCi, while the most preferable range is 20 to 30 mCi.
  • a preferred dosage of Y-labeled immunoconjugates is in the range from 10 to 30 mCi, while the most preferable range is 10 to 20 mCi.
  • IgGl isotype negative control (DakoCytomation, Carpenteria, CA), anti-Ki- 67 (DakoCytomation), Rabbit anti c-peptide (Millipore Corporation, Billerica, MA), mouse anti-cyto keratin 19 (DakoCytomation), mouse anti-Cytokeratin 7 (OV-TL 12/30; DakoCytomation), EpCAM (Ber Ep4, DakoCytomation). Appropriate isotype controls were used for all experiments and were shown to be negative on the relevant tissue.
  • esophagectomy cases were evaluated for foci of GCTM-5 reactivity.
  • the GCTM-5 stained esophageal specimens were evaluated by two expert gastrointestinal surgical pathologists (L.M.P. and P.T.C.). Isolation of GCTM-5 positive cells from adult human liver.
  • liver non-parenchymal cells were provided by CellzDirect (Life Technologies, Grand island, NY). The supernatant post isolation of hepatocytes was rinsed with ice-cold 0.1% bovine serum albumin/ 2mM EDTA in phosphate buffered saline several times and processed further as liver non-parenchymal cells. The liver non-parenchymal cells were then loaded onto a 20-40% percoll- gradient column which was centrifuged at lOOOg for 10 minutes. The cell fraction banding 20% percoll included majority of GCTM-5 positive cells, and this fraction was collected and rinsed.
  • GCTM-positive cells were isolated by magnetic positive isolation with purified GCTM-5 antibody and Dynabeads Rat anti- Mouse IgGl (DYNAL, Life Technologies) according to manufacture's protocol.
  • the GCTM-5 positive cells were directly lysed for RNA purification, or seeded on collagen IV-coated plates and then cultured in Kubota's medium supplemented with 2% fetal calf serum, lOng/ml HGF and EGF. The cultured cells were gently dissociated with TripLETM (Invitrogen, Life Technologies) and passaged every week. Quantitative PCR analysis.
  • RNeasyTM mini kit with DNase I (Qiagen, Valencia, CA). Reverse transcription was performed using the OmniscriptTM kit (Qiagen) and random hexamer primers. Quantitative PCR was performed using gene-specific primer/probe mixtures (TaqMan Gene Expression Assays, Life Technologies), TaqManTM 2x Master Mix, and the ABI PRISMTM 7900 Sequence Detection system (Applied Biosystems, Life Technologies) according to the manufacturer's protocols. The PCR data was analyzed by the delta/delta CT method and normalized to PPIA expression with RQ Manager software (Applied Biosystems). The fold expression was calculated relative to human embryonic stem cells. Human dermal fibroblast and HepG2 cells were used as negative and positive control respectively.
  • the isolated GCTM-5 positive cells ware passaged to remove magnetic beads after 5 days culture, and fixed with 4% paraformaldehyde in phosphate buffered saline two days after passage. All mouse IgGl antibodies were labeled with a fluorescent-conjugated F(ab') 2 fragment (Zenon Mouse IgG Labeling kit (Invitrogen), and staining was performed according to manufacture's protocol.
  • the other antibodies used were detected indirectly with fluorescent-conjugated secondary antibodies *
  • CFPAC-1 pancreatic adenocarcinoma cells were grown in TI75 flasks to 75% confluence in Iscove's Modified Dulbecco's Medium containing 10% fetal calf serum. Serum containing medium was then removed and the cells washed 3 times with phosphate buffered saline. Serum-free/antibiotic free medium was then added to the CFPAC-1 cells which were subsequently cultured in a humidified environment at 37°C S 5% C02 for a further three days.
  • the conditioned medium was collected, passed through a 0.22uM filter and stored at 4°C, CFPAC-1 concentrated conditioned medium was added to 2x Laemmli sample buffer and proteins separated on either 10% or 5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose or polyvinylideneflouride membranes, and immunoblotted with GCTM-5 antibody as previously described (18).
  • SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • CFPAC-1 conditioned medium was concentrated down to ⁇ 15ml under nitrogen gas pressure ( ⁇ 20 psi). Concentrated conditioned medium was then filtered through a 0.22 ⁇ filter. Immunoprecipitation (with Protein A or Protein G beads) or immunoblotting with GCTM-5 or with commercial antibodies against peptides from the C- or N- terminus of FCGBP (Sigma Aldrich, St. Louis MO) were carried out on concentrated conditioned medium.
  • Conditioned medium or immunoprecipitates were separated on SDS PAGE on precast 4-20% gradient polyacrylamide gels or 8% gels in Tris-HEPES-SDS running buffer. Detection was carried out either with anti-mouse or anti-rabbit Ig or with or the corresponding F(Ab') 2 conjugates.
  • GCTM-5 antibody purified by affinity chromatography on Protein G SepharoseTM, was covalently coupled to Protein G SepharoseTM using dimethylpimelimidate in 0.2 M sodium borate buffer.
  • the column was washed with 10mm sodium phosphate buffer at pH 6.8 and eluted with 100mm glycine at pH 2.5. Immunob lotting was carried out with GCTM-5 or with commercial antisera to FCGBP as described above.
  • the column eluate was sequenced at the USC Proteomics Core facility on a Thermo LTQ-ETD mass spectrometer.
  • GCTM-5 immunoreactivity is highly specific for tissues of endodermal origin (Table 1, Table 2 and Figures 8-10).
  • the GCTM-5 antigen was detected in normal biliary ducts and pancreatic ducts, with expansion of the population in diseased states in these tissue and in metaplastic esophagus (Table 1).
  • Table 1 A survey of normal and diseased tissues representative of the three embryonic germ layers indicated that GCTM-5 immunoreactivity is highly specific for tissues of endodermal origin (Table 1, Table 2 and Figures 8-10).
  • the GCTM-5 antigen was detected in normal biliary ducts and pancreatic ducts, with expansion of the population in diseased states in these tissue and in metaplastic esophagus (Table 1).
  • Table 1 A survey of normal and diseased tissues representative of the three embryonic germ layers indicated that GCTM-5 immunoreactivity is highly specific for tissues of endodermal origin (Table 1, Table 2 and Figures 8-10).
  • GCTM-5 staining was associated with mature cells, such as mucin-secreting cells in the adult colon. However, in many tissues, the antibody stained progenitor compartments.
  • Putative biliary progenitors and cholangiocarcinoma express the GCTM-5 antigen.
  • GCTM-5 colocalized with EpCAM ( Figure 1C) and demonstrated co-expression with albumin where GCTM-5 positive cells merge at the periphery of regenerative nodules of parenchymal hepatocytes ( Figure ID).
  • the malignant cells of intrahepatic cholangiocarcinoma reacted variably and heterogeneously with GCTM-5 ( Figure IE; Table 1).
  • GCTM-5 positive cells were more widely distributed in bile ducts from pediatric liver. Staining of serial sections showed that immunoreactive cells could be found in the distal portions of the duct extending well into the hepatic parenchyma ( Figure 2), consistent with localization in the Canals of Hering.
  • GCTM-5 positive cells in adult liver have a progenitor cell phenotype
  • GCTM-5 To characterize further the GCTM-5 subpopulation in adult liver, antigen-positive cells were isolated from the non-parenchymal fraction of dissociated liver tissue using GCTM-5 coupled to immunomagnetic beads, then analyzed the cells by QRT-PCR for expression of liver progenitor and hepatocyte markers (Figure 5). Relative to whole liver, total non-parenchymal cells, HepG2 hepatocellular carcinoma, or human dermal fibroblasts, the GCTM-5 positive fraction expressed high levels of HNFlbeta, HNF6, HHex, GATA-6, and Pdx-1. GCTM-5 cells expressed GATA-4, HNF3a, HNF4, Sox-17, Sox-7, albumin and alpha I-antitrypsin at similar levels to other hepatic cell types, but did not express AFP.
  • GCTM-5 positive cells from adult liver could be serially cultivated in vitro for several passages in media previously reported to support propagation of human liver progenitor cells.
  • the isolated cells formed small epithelial colonies throughout which most cells retained GCTM-5 expression (Figure 6). Most cells in the colonies were positive for Ep-CAM, E-cadherin, and albumin. A subpopulation of cells in growing colonies expressed CD 133, and cytokerations 8 and 19. Most cells were negative for N- CAM and I-CAM, and a subset expressed CD133 (not shown).
  • GCTM-5 antigen is expressed in a subpopulation of normal pancreatic ducts with expansion of expression in ductal metaplasia, Pancreatic intraepithelial neoplasia
  • FIG. 3C Poorly defined structures known as transforming tubular complexes, which show morphological overlap with PanIN but occur in the acinar compartment, variably expressed the GCTM-5 antigen (Figure 3D) (22, 40). Both low grade and high grade PanIN lesions showed intense membrane reactivity ( Figure 3E). Primary and metastatic pancreatic ductal adenocarcinoma strongly and consistently expressed the GCTM-5 antigen in a high percentage of tumour cells, irrespective of the degree of differentiation of the tumor ( Figure 3F).
  • GCTM-5 antigen is expressed in the reflux to adenocarcinoma sequence of Barrett's esophagus in proliferating cells.
  • the GCTM-5 antigen was characterized as an epitope on a -50 kDA protein present in cell lysates of primary biliary epithelial cells and differentiating populations of human embryonic stem cells (J 8).
  • the antigen is also expressed on the cell surface of cultured pancreatic adenocarcinoma cell line CFPAC-1 (live cell staining analyzed by flow cytometry; immunoblotting of whole cell lysates of CFPAC-1 cells lysed into SDS-PAGE gel reducing sample buffer showed a strong band of ⁇ 55kDa and diffuse high molecular weight bands ( Figure 7 A).
  • CFPAC-1 cells secreted or shed the antigen into the cell culture medium, where the high molecular weight species represented the predominant form ( Figure 7B, lane 1).
  • stamp L Crosby HA. Hawes SM, Strain AJ, Pera MF. A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells. Stem Cells 2005;23:103-112.
  • Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia.
  • Zen Y Adsay NV, Bardadin K, Colombari R, Ferrell L, Haga H, Hong SM, et al.
  • Biliary intraepithelial neoplasia an international interobserver agreement study and proposal for diagnostic criteria.
  • FYPAGDVLR (SEQ ID NO: 1) 1038.18 0.33 184-192 0.30
  • YDLAFVVASQATK (SEQ ID 1413.60 0.44 263-275 0.43 NO: 3)
  • AISGLTIDGHAVGAK SEQ ID 1410.60 0.44 366-380 0.50 NO: 4

Abstract

The present invention provides biomarkers comprising GCTM-5 antigen for detection and screening of cancer, as well as a distinct population of cells identified by the expression pattern of GCTM-5, diagnostic methods for utilizing the biomarkers and reagents for detecting the biomarkers.

Description

CELL SURFACE MARKER AND METHODS OF USE THEREOF
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
This invention was made with government support under Grant No. P30-DK04852 by the National Institute for Health, and Grant No. CIRM TG2-01168 by the California Institute of Regenerative Medicine. The government has certain rights in the invention.
FIELD OF THE INVENTION
This invention generally relates to novel bio markers for detection and screening of cancer, and more particularly, to biomarkers comprising GCTM-5 antigen, the distinct population of cells identified by the expression pattern of GCTM-5, diagnostic methods for utilizing the biomarkers and reagents for detecting the biomarkers. BACKGROUND OF THE INVENTION
Cell markers are biochemical or genetic characteristics that distinguish between different cell types. They are important tools for understanding mechanisms of cellular differentiation and regulation. They are particularly useful in aiding understanding of regenerative mechanisms at the organ or tissue level. Longstanding evidence suggests that the molecular factors regulating embryonic morphogenesis and lineage determination also govern tissue regeneration and repair (1, 2). The gastrointestinal tract, liver, and pancreas are formed from the definitive endoderm. These tissues share a close ontogeny and common developmental regulation of many critical transcriptional and growth factors (3-5). For example, cell lineage tracing experiments in mice show that Sox-9 expression characterizes progenitors in the liver and pancreatic ducts, and small intestinal crypts (6). In these adult tissues, resident stem cells or facultative progenitors may mediate regeneration through recapitulation of the developmental program (7-9). Recent experiments have demonstrated interconversion of mature pancreatic and hepatic lineages through the introduction of transcriptional regulators or growth factors, demonstrating extraordinary plasticity in hepatopancreatic ontogeny (10-13). Furthermore, these reprogramming studies suggest a key role for stem cells and progenitors in the pathological processes of metaplasia and neoplasia that occur in these organs.
The liver is capable of regeneration not only through hepatocyte proliferation but also via a biliary ductular reaction, wherein bipotential cells give rise to bile duct and hepatic parenchyma. These facultative bipotential progenitor cells reside in the Canals of Hering (14-17). Although there is significant heterogeneity in this population of ductular progenitors, epithelial cell adhesion molecule (EpCAM) and neural cell adhesion molecule (NCAM) provide useful markers for these cells (9, 17). The GCTM-5 monoclonal antibody epitope is a candidate marker for this putative progenitor cell marker, since GCTM-5 reacts with a subpopulation of biliary cells in normal liver and demonstrates co- localization with NCAM in the expanded biliary population found in cirrhotic liver (18).
Regenerative mechanisms in the pancreas are not as well established as those in the liver. A candidate population of progenitors found in chronic pancreatitis, resides in a poorly defined structure termed the tubular complex, that has been proposed to originate in various compartments including acinar cells, centroacinar cells, and terminal ductules (19- 21). In both animal and human studies, these structures have been identified as a susceptible population for neoplastic transformation, and they may represent the cell of origin of pancreatic intraepithelial neoplasia (PanIN), the precursor of pancreatic ductal adenocarcinoma (22-26). An analogous sequence of premalignant changes has been proposed for intrahepatic cholangiocarcinoma, but the distinct cell of origin is largely unknown for this highly malignant tumor (27, 28).
Columnar metaplasia of the esophagus is a preneoplastic lesion that occurs secondary to gastroesophageal reflux disease (29-32). For many years the incomplete intestinal metaplasia found in this condition has been thought to originate from
transdifferentiation that occurs during regeneration following reflux-induced injury.
However, more recently, the ductal epithelium of the esophageal submucosal glands has been implicated as the source of a multipotent stem or progenitor cell that might give rise to the pathogenesis of neoplasia (33-38). Currently, there is a paucity of cell surface markers for the identification and prospective isolation of progenitor cell populations in all of these tissues.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that the GCTM-5 epitope is specifically expressed on tissues derived from the definitive endoderm, more particularly in the fetal gut, liver and pancreas tissues. GCTM-5 antibodies distinctively identified subpopulations of normal adult biliary and pancreatic duct cells. These cells are also found to expand in disease states such as biliary atresia, cirrhosis and pancreatitis. Neoplasms originating in these tissues express the GCTM-5 antigen, with pancreatic adenocarcinoma in particular. The GCTM-5 epitope is also found to express strongly on cells undergoing intestinal metaplasia in Barrett's esophagus. Moreover, GCTM-5 epitope positive cells isolated from the non-parenchymal fraction of adult liver are found to express markers of progenitor cells. The GCTM-5 epitope is found to be associated with the mucin-like glycoprotein FCGBP, which may play a cytoprotective role on progenitor cells during tissue repair.
Accordingly, one aspect of the present invention is the discovery that expression of the GCTM-5 antigen marks putative progenitor compartments in gastrointestinal and hepatopancreatic tissues. As will be described more fully below, cells expressing the GCTM-5 epitope participate in the process of regeneration, metaplasia, and neoplasia in these tissues. Hence, the GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a useful cell surface marker for the study of nonnal differentiation lineages and regeneration in tissues of endodermal origin.
Another aspect of the present invention is the first-time definition and
characterization of the distribution of the GCTM-5 antigen in regenerative, metaplastic, and neoplastic states in the gastrointestinal tract and hepatopancreatic ducts. As described herein, the population of GCTM-5 expressing epithelia in the biliary ductular reaction of liver, cirrhosis is characterized. Also characterized herein is the antigen biochemically. Another aspect of the present invention is directed to the use of GCTM-5 as a biomarker for disease states in the gastrointestinal tract and the hepatopancreatic ducts. In particular, GCTM-5 antigen is a serum-based biomarker for diagnostic or screening tests for pancreatic adenocarcinoma. For example, pancreatic tissues maybe biopsied first and analyzed for their GCTM-5 expression, wherein a higher level of GCTM-5 expression compared to a reference indicates the likelihood of pancreatic adenocarcinoma.
Accordingly, in one embodiment, the invention provides a method for detecting cancer in a sample. The method includes: a) obtaining the sample from the subject; b) detecting expression or presence of a biomarker in the sample,, wherein the biomarker is a GCTM-5 epitope; and c) analyzing the results of (b) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as cancer.
Another aspect of the present invention is directed to biomarkers for colorectal carcinoma which comprises measuring the expression of both GCTM-5 and FCGBP in tissues from apical surface of intestine. Reduced expression of both GCTM-5 and FCBGP as compared with a reference level in the intestinal tissues indicates a likelihood of colorectal carcinoma.
Another aspect of the present invention is directed to a method of isolating a distinct progenitor cell population characterized by GCTM-5 antigen expression and the cells isolated therefrom.
Accordingly, in one embodiment, the invention provides a method for detecting and characterizing a subpopulation of cells in a sample. The method includes: a) detecting expression or presence of a biomarker of one or more cells in the sample, wherein the biomarker is a GCTM-5 epitope; and b) analyzing the results of (a) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as a subpopulation of progenitor cells.
In another embodiment, the invention provides a method of isolating a progenitor opulation of cells from a sample. The method includes a) contacting the sample with an agent that specifically binds a biomarker present on a subpopulation of progenitor cells present in the sample to form a plurality of cellular complexes, wherein the biomarker is native GCTM-5 epitope of FCGBP; and b) isolating the cellular complexes from the sample, thereby isolating the progenitor subpopulation of cells. Another aspect of the present invention is directed to diagnostic systems and reagent kits for diagnosing and screening cancers that may characterized by the expression pattern of GCTM-5 antigen, including but not limited to pancreatic adenocarcinoma and colorectal carcinoma.
Accordingly, in one embodiment, the invention provides a kit. The kit includes an antibody that specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP, reagents for conducting a diagnostic test; and instructions for conducting and analyzing results of the diagnostic test.
Another aspect of the present invention is directed to an antibody, or fragment thereof which specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP. In some embodiments, the antibody may be an immunoconjugate.
Another aspect of the present invention is directed to a pharmaceutical composition including the antibody of the invention and a pharmaceutically acceptable carrier.
Another aspect of the present invention is directed to an isolated nucleic acid molecule encoding the antibody of the invention. Another aspect of the present invention is directed to a method for treating cancer.
The method includes administering to a subject the antibody or pharmaceutical composition of the invention to the subject.
Other aspects and advantages of the present invention will be further illustrated by the following figures and detailed descriptions. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. GCTM-5 niarlts putative progenitors of liver regeneration and neoplasia of biliary origin. (A) Normal liver shows GCTM-5 reactive biliary ducts (red; arrows) confined to the small areas of the portal tracts. (B) Cirrhotic liver demonstrates a regenerative hepatocyte nodule (center) surrounded by an expanded population of GCTM- 5 expressing biliary ducts. (C) Cirrhotic liver demonstrates overlapping coexpression (yellow) of EpCAM (red) and GCTM-5 (green) on a subpopulation of biliary ducts by immunofluorescence; Note that not all of the GCTM-5 expressing cells are also expressing EpCAM. (D) Immunofluorescence showing GCTM-5 (red) and Albumin (green) coexpression (yellow) at the periphery of a regenerating nodule. (E) Intrahepatic .
Cholangiocarcinoma variably express the GCTM-5 antigen (red).
Figure 2. Distribution of GCTM-5 staining in the biliary tract. Serial sections of normal pediatric liver stained with GCTM-5 (top, pink) and CK-19 (bottom, brown) antibodies. Each section is Sum thick. Scale bar, lOOum. Figure 3. GCTM-5 antigen expression in normal and pathologic pancreatic tissues.
(A) An exocrine lobe shows a single focus of GCTM-5 staining (red) in a small ductule in cross section amongst a larger field of acini. (B) In an area of mild fibrosis due to chronic pancreatitis the expression of GCTM-5 antigen in ductules is expanded. (C) In severe chronic pancreatitis GCTM-5 expression is detected a subpopulation of cells in an expansion of ductules also known as a tubular complex formation or acinar-ductal metaplasia. (D), An area of acinar lobe replaced by a range of irregular duct like structures that express the GCTM-5 antigen (E) PanlN lesions consistently express the GCTM-5 antigen. (F) Adenocarcinoma strongly expresses GCTM-5 antigen in the cytoplasm, membrane, and mucinous material. Figure 4. Distribution of the GCTM-5 antigen in metaplastic columnar epithelia of the esophagus. (A) Squamous epithelium does not express the antigen. (B) The multilayered epithelium shows surface mucous cells with abundant GCTM-5 antigen (red).
(C) Cardia-type metaplasia expresses the antigen in a subpopulation of the epithelia.
(D) Intestinal Metaplasia expresses the GCTM-5 antigen in the apical membrane mainly in the deep pits and glands. Dysplasia (E) and Adenocarcinoma (F) react more extensively with GCTM-5.
Figure 5. QRT-PCR analysis of gene expression in GCTM-5 positive cells from nonparenchymal fraction of adult liver. Non-parenchyma cells were isolated by immunomagnetic affinity, and QRTJPCR was carried out on positive cells, the total nonparenchymal cell fraction, and whole liver. HepG2 hepatocellular carcinoma cells and human dermal fibroblasts were included as controls. Graphs display log of relative expression.
Figure 6. Indirect immunofluorescence microscopy of hepatic marker expression in cell colonies derived from GCTM-5 positive cells in the non-parenchymal fraction of adult liver. Fixed colonies were stained with GCTM-5 and various cell surface and intracellular markers, then counterstained with DAPI.
Figure 7. Biochemical characterization of the GCTM-5 antigen. (A) Immunoblot of CFPAC-1 cell lysate with GCTM-5. Positions of 200 and 55 kDa markers are shown. (B) GCTM-5 immunoblot of CFPAC-1 conditioned media treated with no enzyme control (Lane 1), N-Glycanase (Lane 2), sialidase A (Lane 3), O-Glycanase (Lane 4), all three enzymes (Lane 5). Position of 200 kDa marker is shown. (C) Immunoprecipitates of concentrated CFPA-1 conditioned medium blotted with GCTM-5. Lane 1, GCTM-5 immunoprecipitate; Lane 2, control IgGl. Position of 250 kDa marker is shown. Lower bands are immunoglobulins reacting with the secondary antibody. (D) Immunoblot of affinity purified GCTM-5 antigen (Lanes 1 and 3) or CFPAC-1 concentrated conditioned medium (Lanes 2 and 4) with antisera to C-terminal (Lanes 1 and 2) or N-terminal (Lanes 3 and 4) peptide sequences of FCGBP. Positions of 250, 105 and 50 kDa markers are shown. (E) Immunoblot of GCTM-5 immunoprecipitate of CFPAC-1 conditioned medium probed with antiserum against C-terminal peptide sequence of FCGBP. Position of 105 kDa marker is shown. Lower bands are immunoglobulins reacting with secondary antibody. (F) Immunoblots of affinity purified GCTM-5 antigen (Lanes 1 3 5 and 6) or concentrated CFPAC-1 antigen (Lanes 2 and 4) probed with C-terminal peptide sequence of FCGBP (Lanes 1 and 2 detected with anti-rabbit Ig, lane 5 with anti-rabbit Ig F(ab')2 precleared with Protein A) or anti-rabbit Ig alone (lanes 3 and 4) or anti-rabbit Ig F(ab')2 precleared with Protein A alone (Lane 6). Positions of 250 and 105 kDa markers are shown. Figure 8. Fetal tissues express the GCTM-5 antigen. (A) GCTM-5 expression, shown in pink, in the crypts of the human fetal gut at 13 weeks gestation, and (B) an antibody isotype negative control. GCTM-5 expression in (C) 16 week and (D) 18.5 week human fetal pancreas. Cytokeratin 19 staining of (E) 16 week and (F) 18.5 week fetal pancreas; detecting a larger number of ductal structures.
Figure 9. Depicts distribution of the GCTM-5 antigen in the gastrointestinal tract
Normal gastric epithelial (A) does not react with GCTM-5, however, gastric intestinal metaplasia (B) and gastric carcinoma (C) express the GCTM-5 antigen. (D) The epithelium of the gail blader stains positive. The normal colonic crypts (E) express GCTM-5 with less intense reactivity in dysplastic colonic lesions (F).
Figure 10. Non-gastrointestinal definitive endoderm derived tissues express GCTM-
5. (A) The ducts of the salivary glands react with GCTM-5 (B) Human paediatric thymus stained with pan-Keratin (red), GCTM-5 (green) reveal GCTM-5/pan-keratin colcalisation in the Hassall's Corpuscles (C) The GCTM-5 antigen is expressed in the endocervix.
Figure 11. Shows that some GCTM-5+ cells in biliary atresia livers are also positive for Ki-67. Scale bar, lOOum. Figure 12. GCTM-5 expression in biliary atresia livers. (A) GCTM-5 expression on small reactive bile ductules, majority of these cells are CK19+, N-CAM+ &
EpCAM+(weak). (B) GCTM-5 stained positive on enlarged bile duct epithelium, majority of these cells are CK19+, N-CAM- & EpCAM+(very weak). (C) GCTM-5 was also found positive on malformed bile duct epithelia, majority of the cells are CK-19+/-, N-CAM-, EpCAM+ (D) GCTM-5 staining was also found on newly formed bile ducts, majority of these cells are CK19+/-, N-CAM- (positive on surrounding cells) & EpCAM+. Scale bar, lOOum.
Figure 13. GCTM-5 antigen expression in normal and pathologic pancreatic tissues. (A) Adult pancreas shows an islet stained with c-peptide (red) that is exclusive of GCTM-5 staining (green). Comparison of Cytokeratin 19 (B) and GCTM-5 (C)
immunofluorescence staining of normal human pancreatic ducts reveal that GCTM-5 reacts with a subpopulation of the ductal epithelium. Figure 14. Detection of transcripts for FCGBP in non-parenchymaS cells from normal adult human liver. Non-parenchymal cells were isolated from normal adult liver and fractionated into GCTM-5 positive and negative populations. QRT-PCR was carried out and transcript levels estimated by the delta-delta Ct method relative to housekeeping controls (GAPDA and PPIA) and normal cultured human fibroblast RNA. Means and range of data shown.
DETAILED DESCRIPTION OF THE INVENTION
As disclosed herein, it has surprisingly been found that expression of the GCTM-5 antigen in cells marks putative progenitor compartments in gastrointestinal and
hepatopancreatic tissues. As such, GCTM-5 antigen or its epitopes are useful as biomarkers to identify endodermal cellular origin and other related cellular states and conditions.
Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described.
To facilitate a full and complete understanding of the present invention, the following discussion of specific findings regarding the properties of GCTM-5 antigen and epitopes are provided.
1. The GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP.
Mass spectrometry sequencing data along with immunoprecipitation and immunoblotting indicate that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. GCTM-5 bound peptides of -55 and 100 kDa in conditioned medium from CFPAC-1 pancreatic adenocarcinoma cells (ATCC CRL- 1 18) that were cross-reactive with antisera to the N and C termini of FCGBP, respectively; these fragment sizes are consistent with previously described autocatalytic degradation fragments of FCGBP (41, 44). FCGBP was originally isolated from the small intestine as a protein that bound the Fc portion of immunoglobulins (42, 43). In our study, secondary antisera against mouse or rabbit Ig showed a degree of reactivity with purified GCTM-5 antigen, and predictably this reactivity was greatly decreased using F(ab')2 conjugates as probes instead of intact Ig. The anti-mouse or anti-rabbit Ig secondary antibodies reacted with the high molecular weight form of FCGBP isolated by acid elution from affinity columns, but not the native antigen in concentrated conditioned medium, or the 100 or 55 kDa bands.
Neither commercial antisera to FCGBP could immunoprecipitate the native GCTM-5 antigen and neither reagent decorated bile ducts in liver sections. These antisera did not recognize native high molecular weight GCTM-5 antigen in conditioned medium. However, both antisera recognized fragments of FCGBP in conditioned medium and in immunoaffinity purified material, and both reacted with high molecular weight bands in immunopurified GCTM-5 eluted by acid from a GCTM-5 affinity column. Therefore, it appears likely that the peptide epitopes recognized by the commercial antisera are not exposed on the native intact form of FCGBP expressed in CFPAC-1 cells or in liver.
Instead, the relevant epitopes become exposed in degraded fragments of the intact native protein. As noted above, secondary antisera containing intact immunoglobulins bind the GCTM-5 antigen eluted by acid from immunoaffinity columns but not the native antigen or the putative degradation products. It has been proposed that FCGBP undergoes autocleavage at specific sites at acidic pH to generate these fragments (41, 44), and it is thus likely that the acidic buffer (glycine, pH 2.5) used to elute the antigen from the affinity column exposes these sites.
FCGBP is encoded by a 16 kbp transcript that is transcribed to yield a 300 kDa protein with multiple Von Wille-brand factor and mucin-like repeats (43). FCGBP^ like GCTM-5, is found on the apical surface of intestine (45), and expression of both FCGBP and GCTM-5 is downregulated in colorectal carcinoma (46). FCGBP protein has also been identified in bile (47) and salivary glands (48). In the intestine, FCGBP exists in a large complex with Muc2 and Trefoil peptide (41, 44). Like FCGBP, the expression pattern of the GCTM-5 epitope resembles that of mucins. However, the tissue specificity of the GCTM-5 epitope distinguishes it from that of known mucins. The only mucin expressed by both bile ducts and pancreas is Muc-1 (49, 50), and Muc-1 is far more broadly expressed in epithelia and in carcinomas than the GCTM-5 epitope. It is however possible that Muc-1, the GCTM-5 epitope, and FCGBP are present in some form of complex in bile duct and liver.
The GCTM-5 epitope clearly contains sialic acid, and since FCGBP likely exists in a supermolecular complex in its secreted form, it is only possible to conclude that the epitope is associated with FCGBP. Further studies are required to define the exact location of the epitope on the FCGBP polypeptide, which contains multiple repeats and multiple island O-glycosylation sites. Our results are consistent with a model in which FCGBP is secreted from CFPAC-1 cells in a form that cannot bind immunoglobulin and in which the C- and N- terminal epitopes recognized by commercial antisera against the peptide sequence are concealed. Mucins are known to exert cytoprotective effects, and in the context of tissue regeneration in the face of microenvironmental damage (gastroinitestinal reflux, pancreatitis, and biliary obstruction), progenitor cells may upregulate their expression of surface mucins to enhance their capacity for survival and repair. 2. Progenitor cells in liver express the GCTM-5 antigen.
Histological studies here have confirmed previous work showing consistent GCTM-5 antigen expression in a subpopulation of cells in the normal biliary ducts and in an expanded population of biliary ductular reaction cells of cirrhotic liver, in this ductular reaction, we demonstrated coexpression of GCTM-5 with EpCAM, and with albumin at the periphery of regenerative nodules. These studies suggest that the GCTM-5 antigen marks a subset of cells in the heterogeneous population of facultative progenitors of the biliary ductular reaction. Although the finding of coexpression of albumin and GCTM-5 at the margin of regenerating foci of hepatocytes suggests that GCTM-5 cells can give rise to hepatocytes, the exact lineages that the GCTM-5-expressing ductular cells contribute to are unknown at this time. The lack of complete overlap of expression between GCTM-5, EpCAM, and NCAM in normal or regenerating liver suggests that this progenitor cell compartment may in fact be quite heterogeneous.
The GCTM-5 monoclonal antibody was used to isolate the biliary ductular cells that express the antigen on the membrane surface. Gene expression showed that these cells express transcription factors characteristic of endodermal progenitors, including GATA-6, HNFlbeta, HNF6, HNF4, HNF3a, and PDX1. Some of these transcription factors are found in progenitor populations in the developing liver, in the ductal plate, the cell type thought to correspond to adult liver stem cells (9, 15). When these GCTM-5-positive cells were cultured in vitro under conditions that promote growth of hepatocyte progenitors, they maintained a pattern of protein markers consistent with hepatic stem cells.
Further studies using multiple markers including GCTM-5 to isolate
subpopulations of cells within this compartment will aid in defining those cells with the greatest capacity for repopulation. These studies will also assist in the isolation of progenitor cells from embryonic stem cells or induced pluripotent stem cells undergoing differentiation into the hepatic lineage.
Intrahepatic cholangiocarcinoma is a highly aggressive malignancy of the biliary epithelia with a poor prognosis. The GCTM-5 antigen is expressed variably and heterogeneously in these tumors. If in fact GCTM-5 marks a ductal progenitor population, this may indicate that the cell of origin of a subset of these tumors (51) is GCTM-5 positive.
3. The GCTM-5 Antigen Is Expressed by the putative precursors of Barrett's Esophagus.
We have detected expression of GCTM-5 antigen in the metaplasia to
adenocarcinoma sequence of the columnar lined esophagus and characterized the immunoreactive populations. In general, GCTM-5 is expressed on sialomucin containing epithelia that are proliferating.
The multilayered epithelium is the candidate precursor of intestinal metaplasia of the esophagus. The GCTM-5 antigen is variably expressed in the multilayered epithelium (ME), but its patterns of expression suggest it marks tissue that is progressing toward intestinal metaplasia. Those foci that express abundant GCTM-5 antigen are typically adjacent to or contiguous with metaplastic columnar epithelia that are also strongly immunoreactive. Further studies confirmed that when this occurs, these epithelia share a common profile of mucins and differentiation markers (not shown). This indicates a direct transition from multilayered epithelium into metaplastic columnar epithelia that is marked by GCTM-5 expression.
4. The GCTM-5 antigen is a novel marker for a subpopulation of epithelial cells in normal pancreatic ducts, PanIN, and pancreatic ductal adenocarcinoma. The GCTM-5 antigen is expressed on the surface membrane of a subpopulation of epithelia in the developing and adult pancreas. The normal pancreas demonstrates GCTM- 5 expression restricted to a minor proportion of the large ducts and terminal ductules. As in the liver, this population in the pancreas overlaps with those cells expressing Sox-9, thought to be a marker for pancreatic progenitor cells (S. Bonner- Weir, personal communication). In chronic pancreatitis, the proportion of positive cells increases, with severe disease demonstrating GCTM-5 expression in a subpopulation of the massive collection of ductules, often referred to as the tubular complex.
The tubular complex is thought to represent a progenitor population with experimental and circumstantial evidence suggesting that these structures may originate from a variety of sources including centroacinar and the terminal ductule cells (19). Our data not only demonstrate GCTM-5 expression in the terminal ductules but also
demonstrate GCTM-5 expression in the tubular complex. This suggests that GCTM-5 may mark the progenitors in the normal terminal ductules that give rise to the tubular complex found in chronic pancreatitis. Additionally, the PanlN lesions and pancreatic adenocarci- noma express the GCTM-5 antigen strongly and consistently. As in the liver, a progenitor cell population in the termini of the pancreatic ducts marked by GCTM-5 may be the cell of origin for pancreatic adenocarcinoma. Identification and isolation of GCTM-5-positive cells will help dissect out relationships between normal, preneoplastic, and cancer progenitors in the pancreas (52). GCTM-5 was strongly reactive in a high percentage of tumor cells and mucinous material from ail cases of pancreatic adenocarcinoma that we examined. CFPAC-1 pancreatic adenocarcinoma cells secrete or shed GCTM-5 antigen into the culture medium. Therefore, GCTM-5 antigen is a potential serum-based biomarker for diagnostic or screening tests for pancreatic adenocarcinoma. FCGBP maps to chromosome 19ql3, a region of the genome that is often overrepresented in a subset of patients with pancreatic adenocarcinoma (53). The FCGBP locus lies within a 650 kbp minimal amplicon on 19ql3 defined on a panel of pancreatic cancer cell lines and patient tumors, and two-third cell lines that harbored the amplicon expressed FCGBP at levels >20-fold above levels in controls that did not contain the amplicon (49), Although these limited data do not of course implicate FCGBP in tumor progression, it is possible that as in other types of cancer, mucin overexpression is characteristic of the disease state.
5. Summary.
The GCTM-5 monoclonal antibody is specific for tissue derivatives of the definitive endoderm in the fetus and adult. In particular, the GCTM-5 antigen is expressed in regenerating and pathological states of gastrointestinal and hepatopancreatic ductal epithelia in fetal and normal adult. The distribution of GCTM-5 antigen localizes to many known or putative progenitor compartments such as the biliary ductular reaction, colonic crypts, pancreatic tubular complex, and the esophageal multilayered epithelium.
Prospective isolation of these progenitors in liver, pancreas, and esophagus will enable elucidation of their role in tissue regeneration and neoplasia. Expression of FCGBP on the cell surface may protect progenitor cells from adverse environmental conditions during tissue repair. Antibodies to the GCTM-5 epitope finds application in the diagnosis or treatment of pancreatic cancer. As discussed above, herein is described a monoclonal antibody GCTM-5 that is reactive with a large glycoprotein complex present on the surface of stem and progenitor cells of endodermal origin. There are a limited number of specific cell surface markers for endodermal progenitor cells. Therefore, in this study we sought to characterize further the expression of the GCTM-5 epitope in normal and diseased tissues. The antigen is strongly expressed on hepatoblasts at early stages (7.5 weeks) of human liver development. Later in gestation, the marker was found on periportal hepatocytes and the ductal plate, a structure that contains a bipotential stem cell population that persists in the Canal of Hering in the adult liver. Cells with the features of stem or progenitor cells in the ducts of the normal adult liver, pancreas, and esophagus all stained strongly, as did precancerous cells in the esophagus (intestinal metaplasia) and pancreas (pancreatic intraepithelial neoplasia). Submucosal gland cells of the esophagus, now regarded as a putative precursor of intestinal neoplasia, were also reactive with GCTM-5. Five out of seven cultured cell lines from pancreatic ductal carcinoma tested showed cell surface staining by indirect immunofluorescence and flow cytometry. The antigen secreted or shed into the culture medium by the antigen positive pancreatic carcinoma cell lines was not well resolved in immunoblots of conventional SDS-polyacrylamide gels, but migrated as a single diffuse band with an apparent MR of -850 kDa in native Coomassie polyacrylamide gel electrophoresis. The large mucin-like glycoprotein. FCGBP represented a major component of this multiprotein complex. Immunohistochemistry on tissue microarrays showed strong eel! surface staining on 100/116 cases of pancreatic ductal carcinoma. The GCTM-5 antigen identifies progenitor cells in ducts within tissues of endodermal origin that are implicated in repair and neoplasia.
As used herein, the term "biomarker" means a distinctive biological or
biologically-derived indicator of a process, event or condition. In other words, a biomarker is indicative of certain biological state, such as the presence of cancerous tissue. In some cases, different forms of biomarkers can be indicative of certain disease states. In other cases, the presence of the biomarker may be used to distinguish and isolate certain population of cell types from others.
One aspect of the present invention is directed to the use of GCTM-5 epitope as a biomarker for disease states. In particular GCTM-5 antigen is a serum-based biomarker for diagnostic or screening tests for neoplasia and cancer. For example, tissue may be biopsied first and analyzed for GCTM-5 expression or presence, wherein a higher level of GCTM-5 expression or presence compared to a reference indicates the likelihood of cancer, such as pancreatic adenocarcinoma. In one embodiment, the invention provides a method for detecting cancer in a sample. The method includes: a) obtaining the sample from the subject; b) detecting expression or presence of a biomarker in the sample, wherein the biomarker is a GCTM-5 epitope; and c) analyzing the results of (b) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as cancer.
Another aspect of the present invention is directed to biomarkers for colorectal carcinoma which comprises measuring the expression of both GCTM-5 and FCGBP in tissues from apical surface of intestine. Reduced expression of both GCTM-5 and FCBGP as compared with a reference level in the intestinal tissues indicates a likelihood of colorectal carcinoma.
In embodiments, the invention provides a method for detecting and characterizing a subpopulation of cells in a sample. The method includes: a) detecting expression or presence of a biomarker of one or more cells in the sample, wherein the biomarker is a GCTM-5 epitope; and b) analyzing the results of (a) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as a subpopulation of progenitor cells.
In embodiments, the invention provides a method of isolating a progenitor subpopulation of cells from a sample, The method includes a) contacting the sample with an agent that specifically binds a biomarker present on a subpopulation of progenitor cells present in the sample to form a plurality of cellular complexes, wherein the biomarker is native GCTM-5 epitope of FCGBP; and b) isolating the cellular complexes from the sample, thereby isolating the progenitor subpopulation of cells. Another aspect of the present invention is directed to diagnostic systems and reagent kits for diagnosing and screening cancers that may characterized by the expression pattern of GCTM-5 antigen, including but not limited to pancreatic adenocarcinoma and colorectal carcinoma.
Accordingly, in one embodiment, the invention provides a kit. The kit includes an antibody that specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP, reagents for conducting a diagnostic test; and instructions for conducting and analyzing results of the diagnostic test.
The methods of the present disclosure may be performed using any suitable sample type. As used herein, the term "sample" refers to any sample suitable for the methods provided by the present invention. The sample may be any sample that includes native GCTM-5 epitope for detection. Sources of samples include whole blood, pleural fluid, peritoneal fluid, central spinal fluid, urine, saliva bronchial washes, and tissue. In one embodiment, the sample is a tissue sample, including, for example, pancreatic, esojphageal, gastrointestinal, colon and liver tissue or fraction or component thereof. In one
embodiment, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. A blood sample, suitable for use with the present invention may be extracted from any source known that includes blood cells or components thereof, such as veinous, arterial, peripheral, tissue, cord, and the like, For example, a sample may be obtained and processed using well known and routine clinical methods {e.g., procedures for drawing and processing whole blood). In one aspect, an exemplary sample may be peripheral blood drawn from a subject with cancer.
As used herein, a cellular component is intended to include any component of a cell that may be at least partially isolated upon lysis of the cell. Cellular components may be organelles, such as nuclei, perinuclear compartments, nuclear membranes, mitochondria, chloroplasts, or cell membranes; polymers or molecular complexes, such as lipids, polysaccharides, proteins (membrane, trans-membrane, or cytosoHc); nucleic acids, viral particles, or ribosomes; or other molecules, such as hormones, ions, cofactors, or drugs.
In various embodiments of the present invention, GCTM-5 epitope positive cells are detected and analyzed to derive clinically significant data via antibodies of the present invention. Analysis of GCTM-5 epitope positive cells may be performed by a variety of methods depending of the type of data desired. For example, in various aspects, subsequent to detecting, GCTM-5 epitope positive cells may be analyzed by detecting and characterizing the cells via assays utilizing recognition and/or binding of cellular components, such as biomarkers including cell surface markers. A variety of
detection/immobilization assays are contemplated for use with the present invention from which useful data may be derived. Additional analysis methods may include image analysis.
As used herein, image analysis includes any method which allows direct or indirect visualization of GCTM-5 epitope positive cells and may be in vivo or ex vivo. For example, image analysis may include, but not limited to, ex vivo microscopic or cytometric detection and visualization of cells bound to a solid substrate, flow cytometry, fluorescent imaging, and the like. In an exemplary aspect, GCTM-5 epitope positive cells are detected using antibodies of the present invention and may be further analyzed using antibodies directed to additional biomarkers and cell surface markers via being bound to a solid substrate and visualized using microscopic or cytometric detection.
In various embodiments, a variety of cell surface markers or biomarkers may be used to analyze GCTM-5 epitope positive cells. As used herein, cell surface markers include any cellular component that may be detected within or on the surface of a cell, or a macromolecuie bound or aggregated to the surface of the cell. As such, cell surface markers are not limited to markers physically on the surface of a cell. For example, cell surface markers may include, but are not limited to surface antigens, transmembrane receptors or coreceptors, macromolecules bound to the surface, such as bound or aggregated proteins or carbohydrates, internal cellular components, and the like.
In various embodiment, GCTM-5 epitope positive cells are captured by techniques commonly used to enrich a sample for a particular cell type, for example those involving immunospecific interactions, such as immunomagnetic capture. A variety of
immunocapture methods are known, including immunocapture with beads or posts. A magnetic field or solid supports may aid the immunocapture.
Immunomagnetic capture, also known as immunomagnetic cell separation typically involves attaching antibodies directed to proteins found on a particular cell type to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, they attach to and surround the particular cell. The sample is then placed in a strong magnetic field, causing the beads to pellet to one side. After removing the blood, captured cells are retained with the beads. Many variations of this general method are well known in the art and suitable for use with the methods of the present invention.
In another embodiment, GCTM-5 epitope positive cells are further processed prior to an enrichment step using filtration. In another embodiment, the GCTM-5 epitope positive cells are further processed via cell separation by density gradient sedimentation. Typically, the process relies on a gross physical distinction, such as cellular density. Many variations of this general method are well known in the art and suitable for use with the methods of the present invention.
In another embodiment, the revealed cells are enriched by a technique called "panning". Typically, such processes utilize an antibody specific to the cell type in question in which the antibody is adhered to a solid surface. The cell mixture is layered on top of the antibody-coated surface, the targeted cells tightly adhere to the solid surface due to the immunospecific interaction involving antibody-antigen binding. Non-adherent cells are rinsed off the surface, thereby effecting a cell separation and enrichment. Cells that express a cell surface protein recognized by the antibody are retained on the solid surface whereas other cell types are not.
Detection and characterization of GCTM-5 epitope positive cells using the methods of the invention, is useful in disease detection and diagnosis as well as assessing cancer prognosi s and in monitoring therapeutic efficacy for early detection of treatment failure that may lead to disease relapse. In addition, cellular analysis according to the invention enables the detection of early relapse in presymptomatic patients who have completed a course of therapy. This is possible because the presence of GCTM-5 epitope positive cells is likely associated and/or correlated with tumor progression and spread, poor response to therapy, relapse of disease, and/or decreased survival over a period of time. Thus, enumeration and characterization of GCTM-5 epitope positive cells provides methods to stratify patients for baseline characteristics that predict initial risk and subsequent risk based upon response to therapy. The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
In various aspects, analysis of a subject's GCTM-5 epitope positive cell number and characterization may be made over a particular time course in various intervals to assess a subject's progression and pathology. For example, analysis may be performed at regular intervals such as one day, two days, three days, one week, two weeks, one month, two months, three months, six months, or one year, in order to track level and
characterization of GCTM-5 epitope positive cells as a function of time. In the case of existing cancer patients, this provides a useful indication of the progression of the disease and assists medical practitioners in making appropriate therapeutic choices based on the increase, decrease, or lack of change in circulating epithelial cells, such as the presence of GCTM-5 epitope positive cells in the patient's bloodstream. Any increase, be it 2-fold, 5- fold, 10-fold or higher, in the GCTM-5 epitope positive cells over time decreases the patient's prognosis and is an early indicator that the patient should change therapy.
Similarly, any increase, be it 2-fold, 5-fold, 10-fold or higher, indicates that a patient should undergo further testing such as imaging to further assess prognosis and response to therapy. Any decrease, be it 2-fold, 5-fold, 10-fold or higher, in the GCTM-5 epitope positive cells over time shows disease stabilization and a patient's response to therapy, and is an indicator to not change therapy. For those at risk of cancer, a sudden increase in the number of circulating epithelial cells detected may provide an early warning that the patient has developed a tumor thus providing an early diagnosis. In one embodiment, the detection of GCTM-5 epitope positive cells increases the staging of the cancer.
In any of the methods provided herein, additional analysis may also be performed to characterize GCTM-5 epitope positive cells to provide additional clinical assessment. For example, in addition to image analysis and bulk number measurements, PCR techniques may be employed, such as multiplexing with primers specific for particular cancer markers to obtain information. Additionally, cell size, DNA or RNA analysis, proteome analysis, or metabolome analysis may be performed as a means of assessing additional information regarding characterization of the patient's cancer. For example, the additional analysis may provide data sufficient to make determinations of responsiveness of a subject to a particular therapeutic regime, or for determining the effectiveness of a candidate agent in the treatment of cancer. Accordingly, the present invention provides a method of determining responsiveness of a subject to a particular therapeutic regime or determining the effectiveness of a candidate agent in the treatment of cancer by detecting GCTM-5 epitope positive cells of the subject as described herein and analyzing the GCTM-5 epitope positive cells. For example, once a drug treatment is administered to a patient, it is possible to determine the efficacy of the drug treatment using the methods of the invention. For example, a sample taken from the patient before the drug treatment, as well as one or more cellular samples taken from the patient concurrently with or subsequent to the drug treatment, may be processed using the methods of the invention. By comparing the results of the analysis of each processed sample, one may determine the efficacy of the drug treatment or the responsiveness of the patient to the agent. In this manner, early identification may be made of failed compounds or early validation may be made of promising compounds. Antibodies
Certain embodiments of the present invention include immunopeptides directed against native GCTM-5 epitope of mucin-like glycoprotein FCGBP. The immunoglobulin peptides, or antibodies, described herein are shown to bind to the native GCTM-5 epitope. The GCTM-5 epitope binding activity is specific. These GCTM-5 epitope specific antibodies can be used to differentiate between subpopulations of cells to identify progenitor cells, or discriminate between diseased cells and normal cells. The GCTM-5 epitope specific antibodies can also be used in immunotherapy against various cancers, to determine the response after therapy for cancer and to inhibit metastasis. Such
immunopeptides can be raised in a variety of means known to the art. As used herein, the term antibody encompasses all types of antibodies and antibody fragments, e.g., polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for GCTM-5 epitope. In certain embodiments, the antibodies exhibit an affinity for GCTM-5 epitope of about d<10"8 M. Substantially purified generally refers to a composition which is essentially free of other cellular components with which the antibodies are associated in a non-purified, e.g., native state or environment. Purified antibody is generally in a homogeneous state, although it can be in either in a dry state or in an aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
Substantially purified GCTM-5 epitope specific antibody will usually comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the antibody with a pharmaceutical carrier, excipient, adjuvant, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient. More typically, the antibody is purified to represent greater than 90% of all proteins present in a purified preparation. In specific embodiments, the antibody is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.
Immunoglobulin peptides include, for example, polyclonal antibodies, monoclonal antibodies, and antibody fragments. The following describes generation of
immunoglobulin peptides, specifically GCTM-5 epitope antibodies, via methods that can be used by those skilled in the art to make other suitable immunoglobulin peptides having similar affinity and specificity which are functionally equivalent to those used in the examples.
Monoclonal Antibodies
Monoclonal antibody (mAb) technology can be used to obtain mAbs to GCTM-5 epitope. Briefly, hybridomas are produced using spleen cells from mice immunized with GCTM-5 antigen. The spleen cells of each immunized mouse are fused with mouse myeloma Sp 2/0 cells, for example using the polyethylene glycol fusion method of Galfre, G. and Milstein, C, Methods Enzymol., 73:3-46 (1981). Growth of hybridomas, selection in HAT medium, cloning and screening of clones against antigens are carried out using standard methodology (Galfre, G. and Milstein, C, Methods Enzymol, 73:3-46 (1981)). HAT-selected clones are injected into mice to produce large quantities of mAb in ascites as described by Galfre, G. and Milstein, C, Methods Enzymol., 73:3-46 (1981), which can be purified using protein A column chromatography (BioRad, Hercules, Calif.). mAbs are selected on the basis of their (a) specificity for GCTM-5 antigen, (b) high binding affinity, (c) isotype, and (d) stability. mAbs can be screened or tested for GCTM-5 epitope specificity using any of a variety of standard techniques, including Western Blotting (Koren, E. et al, Biochim. Biophys. Acta 876:91-100 (1986)) and enzyme-linked immunosorbent assay (ELISA) (Koren, E. et al, Biochim. Biophys. Acta 876:91-100 (1986)). Humanized Antibodies
Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (see, e.g., Queen et al.f Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989 and WO 90/07861 , each incorporated by reference). Human antibodies can be obtained using phage-display methods (see, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047). In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity may be selected by affinity enrichment.
Antibody Fragments
It may be desirable to produce and use functional fragments of a mAb for a particular application. The well-known basic structure of a typical IgG molecule is a symmetrical tetrameric Y-shaped molecule of approximately 150,000 to 200,000 daltons consisting of two identical light polypeptide chains (containing about 220 amino acids) and two identical heavy polypeptide chains (containing about 440 amino acids). Heavy chains are linked to one another through at least one disulfide bond. Each light chain is linked to a contiguous heavy chain by a disulfide linkage. An antigen-binding site or domain is located in each arm of the Y-shaped antibody molecule and is formed between the amino terminal regions of each pair of disulfide linked light and heavy chains. These amino terminal regions of the light and heavy chains consist of approximately their first 110 amino terminal amino acids and are known as the variable regions of the light and heavy chains.
In addition, within the variable regions of the light and heavy chains there are hypervariable regions which contain stretches of amino acid sequences, known as complementarity determining regions (CDRs). CDRs are responsible for the antibody's specificity for one particular site on an antigen molecule called an epitope. Thus, the typical IgG molecule is divalent in that it can bind two antigen molecules because each antigen-binding site is able to bind the specific epitope of each antigen molecule. The carboxy terminal regions of light and heavy chains are similar or identical to those of other antibody molecules and are called constant regions. The amino acid sequence of the constant region of the heavy chains of a particular antibody defines what class of antibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes of antibodies contain two or more identical antibodies associated with each other in multivalent antigen-binding arrangements.
Fab and F(ab')2 fragments of mAbs that bind GCTM-5 epitope can be used in place of whole mAbs. Because Fab and F(ab')2 fragments are smaller than intact antibody molecules, more antigen-binding domains are available than when whole antibody molecules are used. Proteolytic cleavage of a typical IgG molecule with papain is known to produce two separate antigen binding fragments called Fab fragments which contain an intact light chain linked to an amino terminal portion of the contiguous heavy chain via by disulfide linkage. The remaining portion of the papain-digested immunoglobin molecule is known as the Fc fragment and consists of the carboxy terminal portions of the antibody left intact and linked together via disulfide bonds. If an antibody is digested with pepsin, a fragment known as an F(ab¾ fragment is produced which lacks the Fc region but contains both antigen-binding domains held together by disulfide bonds between contiguous light and heavy chains (as Fab fragments) and also disulfide linkages between the remaining portions of the contiguous heavy chains (Handbook of Experimental Immunology. Vol 1 : Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications, Oxford (1986)).
Recombinant DNA methods have been developed which permit the production and selection of recombinant immunoglobulin peptides which are single chain antigen-binding polypeptides known as single chain Fv fragments (ScFvs or ScFv antibodies). Further, ScFvs can be dimerized to produce a diabody. ScFvs bind a specific epitope of interest and can be produced using any of a variety of recombinant bacterial phage-based methods, for example as described in Lowman et al. (1991 ) Biochemistry, 30, 10832-10838;
Clackson et al. (1991) Nature 352, 624-628; and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382. These methods are usually based on producing genetically altered filamentous phage, such as recombinant M13 or fd phages, which display on the surface of the phage particle a recombinant fusion protein containing the antigen-binding ScFv antibody as the amino terminal region of the fusion protein and the minor phage coat protein g3p as the carboxy terminal region of the fusion protein. Such recombinant phages can be readily grown and isolated using well-known phage methods. Furthermore, the intact phage particles can usually be screened directly for the presence (display) of an antigen-binding ScFv on their surface without the necessity of isolating the ScFv away from the phage particle.
To produce an ScFv, standard reverse transcriptase protocols are used to first produce cDNA from mRNA isolated from a hybridoma that produces an mAb for GCTM- 5 antigen. The cDNA molecules encoding the variable regions of the heavy and light chains of the mAb can then be amplified by standard polymerase chain reaction (PCR) methodology using a set of primers for mouse immunoglobulin heavy and light variable regions (Clackson (1991) Nature, 352, 624-628). The amplified cDNAs encoding mAb heavy and light chain variable regions are then linked together with a linker
oligonucleotide in order to generate a recombinant ScFv DNA molecule. The ScFv DNA is Hgated into a filamentous phage plasmid designed to fuse the amplified cDNA sequences into the 5' region of the phage gene encoding the minor coat protein called g3p. Escherichia coli bacterial cells are than transformed with the recombinant phage plasmids, and filamentous phage grown and harvested. The desired recombinant phages display antigen-binding domains fused to the amino terminal region of the minor coat protein. Such "display phages" can then be passed over immobilized antigen, for example, using the method known as "parming", see Parmley and Smith (1989) Adv. Exp. Med. Biol. 251, 215-218; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382, to adsorb those phage particles containing ScFv antibody proteins that are capable of binding antigen. The antigen-binding phage particles can then be amplified by standard phage infection methods, and the amplified recombinant phage population again selected for antigen- binding ability. Such successive rounds of selection for antigen-binding ability, followed by amplification, select for enhanced antigen-binding ability in the ScFvs displayed on recombinant phages. Selection for increased antigen-binding ability may be made by adjusting the conditions under which binding takes place to require a tighter binding activity. Another method to select for enhanced antigen-binding activity is to alter nucleotide sequences within the cDNA encoding the binding domain of the ScFv and subject recombinant phage populations to successive rounds of selection for antigen- binding activity and amplification (see Lowman et al. (1991) Biochemistry 30, 10832- 10838; and Cwirla et al. ( 990) Proc. Natl. Acad. Sci. USA 87, 6378-6382).
Once an ScFv is selected, the recombinant GCTM-5 antigen antibody can be produced in a free form using an appropriate vector in conjunction with E. coli strain HB2151. These bacteria actually secrete ScFv in a soluble form, free of phage components (Hoogenboom et al. (1991) Nucl. Acids Res. 19, 4133-4137). The purification of soluble ScFv from the HB2151 bacteria culture medium can be accomplished by affinity chromatography using antigen molecules immobilized on a solid support such as
AFFIGEL™ (BioRad, Hercules, Calif.).
Other developments in the recombinant antibody technology demonstrate possibilities for further improvements such as increased avidity of binding by
polymerization of ScFvs into dimers and tetramers {see Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6444-6448).
Because ScFvs are even smaller molecules than Fab or F(ab')2 fragments, they can be used to attain even higher densities of antigen binding sites per unit of surface area when immobilized on a solid support material than possible using whole antibodies, F(ab')2, or Fab fragments. Furthermore, recombinant antibody technology offers a more stable genetic source of antibodies, as compared with hybridomas. Recombinant antibodies can also be produced more quickly and economically using standard bacterial phage production methods.
Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab! and F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies . ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CHS domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI , CH2, and CH3 domains. Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous
immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by ucherlapati et al.
Multiple specific antibodies, antibody immunoconjugates and fusion molecules.
GCTM-5 epitope antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be "multispecific," e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time. Thus, whether an GCTM-5 epitope antibody is "monospecific" or "multispecific," e.g., "bispecific," refers to the number of different epitopes with which a binding polypeptide reacts. Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. As used herein the term "valency" refers to the number of potential binding domains, e.g., antigen binding domains, present in an GCTM-5 epitope antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope. When an GCTM-5 epitope antibody, binding polypeptide or antibody comprises more than one binding domain, each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed "bivalent monospecific," or to different epitopes, for an antibody with two binding domains, termed "bivalent bispecific." An antibody may also be bispecific and bivalent for each specificity (termed "bispecific tetravalent antibodies"). In another embodiment, tetravaient minibodies or domain deleted antibodies can be made.
Bispecific bivalent antibodies, and methods of making them, are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein. Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCX publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al, J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681 ; 4,925,648; 5,573,920; 5,601,819; Kostelny et al, J. Immunol. 148: 1547-1553 (1992). The present invention includes multispecific GCTM-5 epitope antibodies. For example, a bispecific antibody comprised of two scFv antibody fragments, both of which bind GCTM-5 epitope. The scFv antibody fragments may bind the same or different epitopes on GCTM-5,
The invention further extends to fusion proteins. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, GCTM-5 epitope specific antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. Radiolabled GCTM-5 epitope antibodies of the invention will be particularly useful, while antibody drug conjugates (ADCs) remain to be developed.
GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding GCTM-5 antigen. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. GCTM-5 epitope antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. GCTM-5 epitope specific antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the GCTM-5 epitope specific antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given GCTM-5 epitope specific antibody.
The present invention also provides for fusion proteins comprising an GCTM-5 epitope antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused may be useful for function or is useful to target the GCTM-5 epitope expressing cells. In one embodiment, a fusion protein of the invention comprises a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence.
In another embodiment, a fusion protein for use in the methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three of the VH- CDRs of an GCTM-5 epitope specific antibody, or fragments,, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an GCTM-5 epitope specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an GCTM-5 epitope specific antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of GCTM-5. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an GCTM-5 epitope specific antibody of the invention and the amino acid sequence of at least one VL region of an GCTM-5 epitope specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds at least one epitope of GCTM-5. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an GCTM-5 epitope specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an GCTM-5 epitope specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. Fusion proteins can be prepared using methods that are well known in the art (see for example U.S. Pat. Nos. 5,1 16,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression. The invention provides for a particularly preferred anti-GCTM-5 epitope antibody; i.e., an isolated anti-GCTM-5 epitope antibody which specifically bind native GCTM-5 epitope. As used herein, native GCTM-5 epitope refers to GCTM-5 epitope produced from CFPAC-1 cells in its native state, e.g., unaltered by enzyme or chemical treatment, such as treatment with acid, sialydase A, N-glycosidase or O-glycosidase. One embodiment of the invention provides for a pharmaceutical formulation comprising the antibody against GCTM-5 epitope and a pharmaceutically acceptable carrier. In an additional embodiment, the invention provides an isolated nucleic acid encoding the antibody against GCTM-5 epitope, In another embodiment, the invention provides for an expression vector comprising the nucleic acid according to nucleic acid encoding an antibody against GCTM-5 epitope. In an additional embodiment, the invention provides for a host cell comprising the nucleic acid encoding an antibody against GCTM-5 epitope. In a further embodiment, the invention provides for a method of producing an anti-GCTM-5 epitope antibody comprising culturing the host cells under conditions to produce the antibody. In one aspect, the method of producing an antibody further comprises recovering the antibody.
The anti-GCTM-5 epitope antibodies and GCTM-5 epitope binding peptides described herein can be used for the treatment or prevention of a GCTM-5 epitope cancer, such as pancreatic carcinoma or to inhibit metastasis of a GCTM-5 epitope cancer cell in a subject.
As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
As used herein, the term "cancer" or "cancer cell" or "GCTM-5 epitope expressing cancer" or "GCTM-5 epitope expressing cancer cell" refers to all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues. Cancer includes, but is not limited to neoplasms, whether benign or malignant, located in the: colon, digestive system, gastrointestinal tract, liver, pancreas, peritoneum and esophagus. Such neoplasms, in certain embodiments, express, over-express, or abnormally express GCTM-5 epitope. In certain therapeutic embodiments, the selected antibody will typically be an anti-
GCTM-5 epitope antibody, which may be administered alone, or in combination with, or conjugated to, one or more combinatorial therapeutic agents. When the antibodies described herein are administered alone as therapeutic agents, they may exert a beneficial effect in the subject by a variety of mechanisms. In certain embodiments, monoclonal antibodies that specifically bind GCTM-5 epitope are purified and administered to a patient.
The immunotherapeutic reagents of the invention may include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as anti-inflammatory ant anti-fibrinolytic drugs.
In other embodiments, therapeutic antibodies described herein are coordinately administered with, co-formulated with, or coupled to (e.g., covalently bonded) a combinatorial therapeutic agent, for example a radionuclide, a differentiation inducer, a drug, or a toxin. Various known radionuclides can be employed, including 90Y, ,231, 12SI, i3lI, I86Re, 188Re, and 21 IAt. Useful drugs for use in such combinatorial treatment formulations and methods include methotrexate, and pyrimidine and purine analogs.
Suitable differentiation inducers include phorbol esters and butyric acid. Suitable toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,
Shigella toxin, and poke weed antiviral protein. These combinatorial therapeutic agents can be coupled to an GCTM-5 antigen antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other. Alternatively, it may be desirable to couple a combinatorial therapeutic agent and an antibody via a linker group as a spacer to distance an antibody from the combinatorial therapeutic agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. It will be further evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, 111.), may be employed as a linker group. Coupling may be affected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.
It may also be desirable to couple more than one agent to an anti- GCTM-5 epitope antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used. A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration is intravenous, intramuscular, or subcutaneous.
It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon such factors as the antibody used, the antigen density, and the rate of clearance of the antibody. A safe and effective amount of a GCTM-5 antigen agent is, for example, that amount that would cause the desired therapeutic effect in a patient while minimizing undesired side effects. Generally, a therapeutically effective amount is that sufficient to promote production of one or more cytokines and/or to cause complement- mediated or antibody-dependent cellular cytotoxicity. The dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, and so on.
In general, the dosage of administered GCTM-5 epitope antibodies, GCTM-5 epitope antibody components, immunoconjugates thereof and fusion proteins will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibody component, vaccine, immunoconjugate or fusion protein which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate. Administration of antibodies, antibody components, vaccines, immunoconjugates or fusion proteins to a patient can be intravenous, intraarterial,, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins, peptides or conjugates by injection, the administration may be by continuous infusion or by single or multiple boluses.
Those of skill in the art are aware that intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. Intravenous administration, however, is subject to limitation by a vascular barrier comprising endothelial cells of the vasculature and the subendothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors. Lymphomas have relatively high blood flow rates, contributing to effective antibody delivery. Intraiymphatic routes of administration, such as subcutaneous or intramuscular injection, or by catherization of lymphatic vessels, also provide a useful means of treating lymphomas.
Preferably, GCTM-5 epitope antibodies, binding peptides, immunoconjugates thereof and fusion proteins are administered at low protein doses, such as 20 to 100 milligrams protein per dose, given once, or repeatedly, parenterally. Alternatively, administration is in doses of 30 to 90 milligrams protein per dose, or 40 to 80 milligrams protein per dose, or 50 to 70 milligrams protein per dose.
The present invention also contemplates therapeutic methods in which GCTM-5 epitope antibody components are radiolabeled or supplemented with radiolabeled immunoconjugate or fusion protein administration. In one variation, GCTM-5 epitope antibodies are administered as or with low-dose radiolabeled GCTM-5 epitope antibodies or fragments. As an alternative, GCTM-5 epitope antibodies may be administered with low-dose radiolabeled GCTM-5 epitope-cytokine immunoconjugates. Those of ordinary skill in Hie art will be familiar with pharmaceutically acceptable radiolabelling molecules and their appropriate dosing levels. For reference, consider "low doses" of 131I-labeled immunoconjugates, wherein a preferable dosage is in the range of 15 to 40 mCi, while the most preferable range is 20 to 30 mCi. In contrast, a preferred dosage of Y-labeled immunoconjugates is in the range from 10 to 30 mCi, while the most preferable range is 10 to 20 mCi.
The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
Example I
Experimental materials and methods.
The following experimental materials and methods were utilized throughout the
Examples.
Human fetal, paediatric and adult tissue samples.
All human tissue specimens were obtained following appropriate ethical consent or institutional review board approval at the respective institutions. Details on tissue types and acquisitions can be found in Supplementary Material.
Immunohistochemistry, histochemistry, and histopathological analysis.
Immunohistochemistry was performed using standard protocols. Primary antibodies used in this study were: GCTM-5 (neat hybridoma cell line culture
supernatant), IgGl isotype negative control (DakoCytomation, Carpenteria, CA), anti-Ki- 67 (DakoCytomation), Rabbit anti c-peptide (Millipore Corporation, Billerica, MA), mouse anti-cyto keratin 19 (DakoCytomation), mouse anti-Cytokeratin 7 (OV-TL 12/30; DakoCytomation), EpCAM (Ber Ep4, DakoCytomation). Appropriate isotype controls were used for all experiments and were shown to be negative on the relevant tissue.
A total of 21 esophageal biopsy cases and 12 paraffin blocks from 6
esophagectomy cases were evaluated for foci of GCTM-5 reactivity. The GCTM-5 stained esophageal specimens were evaluated by two expert gastrointestinal surgical pathologists (L.M.P. and P.T.C.). Isolation of GCTM-5 positive cells from adult human liver.
Adult human liver cells were provided by CellzDirect (Life Technologies, Grand island, NY). The supernatant post isolation of hepatocytes was rinsed with ice-cold 0.1% bovine serum albumin/ 2mM EDTA in phosphate buffered saline several times and processed further as liver non-parenchymal cells. The liver non-parenchymal cells were then loaded onto a 20-40% percoll- gradient column which was centrifuged at lOOOg for 10 minutes. The cell fraction banding 20% percoll included majority of GCTM-5 positive cells, and this fraction was collected and rinsed. Then GCTM-positive cells were isolated by magnetic positive isolation with purified GCTM-5 antibody and Dynabeads Rat anti- Mouse IgGl (DYNAL, Life Technologies) according to manufacture's protocol. The GCTM-5 positive cells were directly lysed for RNA purification, or seeded on collagen IV-coated plates and then cultured in Kubota's medium supplemented with 2% fetal calf serum, lOng/ml HGF and EGF. The cultured cells were gently dissociated with TripLE™ (Invitrogen, Life Technologies) and passaged every week. Quantitative PCR analysis.
Total RNA was purified with Trizol (Invitrogen), and further purified with
RNeasy™ mini kit with DNase I (Qiagen, Valencia, CA). Reverse transcription was performed using the Omniscript™ kit (Qiagen) and random hexamer primers. Quantitative PCR was performed using gene-specific primer/probe mixtures (TaqMan Gene Expression Assays, Life Technologies), TaqMan™ 2x Master Mix, and the ABI PRISM™ 7900 Sequence Detection system (Applied Biosystems, Life Technologies) according to the manufacturer's protocols. The PCR data was analyzed by the delta/delta CT method and normalized to PPIA expression with RQ Manager software (Applied Biosystems). The fold expression was calculated relative to human embryonic stem cells. Human dermal fibroblast and HepG2 cells were used as negative and positive control respectively.
Immunostammg.
The isolated GCTM-5 positive cells ware passaged to remove magnetic beads after 5 days culture, and fixed with 4% paraformaldehyde in phosphate buffered saline two days after passage. All mouse IgGl antibodies were labeled with a fluorescent-conjugated F(ab')2 fragment (Zenon Mouse IgG Labeling kit (Invitrogen), and staining was performed according to manufacture's protocol. The other antibodies used were detected indirectly with fluorescent-conjugated secondary antibodies* The purified GCTM-5, anti-N-CAM (CD54) (clone HA58, BD Pharmingen, BD Biosciences, San Diego, CA), anti-CD133 (clone EMK08, eBioscience, San Diego, CA), anti-Cytokeratin 8 (C51, Santa Cruz
Biotechnology, Santa Cruz, CA), anti-cytokeratin 19 (DakoCytomation), anti-E-cadherin antibody (HECD-1 , Invitrogen, Life Technologies), anti-epithelial antigen (Ep-CAM) (clone Ber-EP4, DakoCytomation), anti-I-CAM antibody (BD Pharmingen) and anti- Albumin (DakoCytomation) were used as primary antibodies, and Alexa Fluor 488- or 594-conjugated antibodies (Molecular Probes, Life Technologies) were used as secondary antibodies.
Immunochemical and Biochemical Characterization of the GCTM-5 antigen.
CFPAC-1 pancreatic adenocarcinoma cells were grown in TI75 flasks to 75% confluence in Iscove's Modified Dulbecco's Medium containing 10% fetal calf serum. Serum containing medium was then removed and the cells washed 3 times with phosphate buffered saline. Serum-free/antibiotic free medium was then added to the CFPAC-1 cells which were subsequently cultured in a humidified environment at 37°CS 5% C02 for a further three days. The conditioned medium was collected, passed through a 0.22uM filter and stored at 4°C, CFPAC-1 concentrated conditioned medium was added to 2x Laemmli sample buffer and proteins separated on either 10% or 5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose or polyvinylideneflouride membranes, and immunoblotted with GCTM-5 antibody as previously described (18).
Using a Stirred Ultracentrifuge Cell (Millipore) and a 30kDa molecular weight cutoff filter, 500ml of CFPAC-1 conditioned medium was concentrated down to~15ml under nitrogen gas pressure (~20 psi). Concentrated conditioned medium was then filtered through a 0.22μΜ filter. Immunoprecipitation (with Protein A or Protein G beads) or immunoblotting with GCTM-5 or with commercial antibodies against peptides from the C- or N- terminus of FCGBP (Sigma Aldrich, St. Louis MO) were carried out on concentrated conditioned medium. Conditioned medium or immunoprecipitates were separated on SDS PAGE on precast 4-20% gradient polyacrylamide gels or 8% gels in Tris-HEPES-SDS running buffer. Detection was carried out either with anti-mouse or anti-rabbit Ig or with or the corresponding F(Ab')2 conjugates.
For carbohydrate analysis, 200μΤ of concentrated condition medium was added to ΙΟΟμΙ, of 3x Laemmli sample buffer containing dithiothreitol then treated with
deglycosylating enzymes according to manufacturers' instructions (GLYKO, Prozyme, Hayward, CA). Denatured concentrated conditioned medium in sample buffer was treated with N-Glycanase, O-Glycanase, Sialidase A, or all three enzymes, and the samples were run on SDS PASGE gels and blotted with GCTM-5 antibody as described above to assess the effect of enzyme treatment on antibody reactivity. GCTM-5 antibody, purified by affinity chromatography on Protein G Sepharose™, was covalently coupled to Protein G Sepharose™ using dimethylpimelimidate in 0.2 M sodium borate buffer. Concentrated conditioned medium from CFPAC-1 cells, prepared as described above, was applied to a column of Protein G Sepharose™ coupled to GCTM-5. The column was washed with 10mm sodium phosphate buffer at pH 6.8 and eluted with 100mm glycine at pH 2.5. Immunob lotting was carried out with GCTM-5 or with commercial antisera to FCGBP as described above. The column eluate was sequenced at the USC Proteomics Core facility on a Thermo LTQ-ETD mass spectrometer.
Supplementary Material and Methods
Human fetal, paediatric and adult tissue samples.
All human tissue specimens were obtained following appropriate ethical consent or institutional review board approval at the respective institutions. Fetal gut tissue was obtained from Monash Medical Centre, fetal pancreas was obtained from the Diabetes Transplant Unit, Prince of Wales Hospital, Sydney Australia, and human paediatric thymus tissue was obtained from Monash University. Additional normal and diseased adult tissues were obtained from archival tissue of the Alfred Hospital. Esophageal biopsies, pancreatic adenocarcinoma, and cholangiocarcinoma was retrieved from the routine formalin fixed, paraffin embedded archives of the Los Angeles County-University of Southern California (LAC+USC) medical center. Esophagectomy resection specimens were obtained from the USC University Hospital. Biopsy specimens were selected from patients who had undergone endoscopic evaluation for gastroesophageal reflux disease.
Example II
Expression of the epitope detected by the GCTM-5 monoclonal antibody is specific for endodermal tissues of the fetus and adult and is modulated in disease states.
A survey of normal and diseased tissues representative of the three embryonic germ layers indicated that GCTM-5 immunoreactivity is highly specific for tissues of endodermal origin (Table 1, Table 2 and Figures 8-10). In particular, the GCTM-5 antigen was detected in normal biliary ducts and pancreatic ducts, with expansion of the population in diseased states in these tissue and in metaplastic esophagus (Table 1). In some tissues,
GCTM-5 staining was associated with mature cells, such as mucin-secreting cells in the adult colon. However, in many tissues, the antibody stained progenitor compartments.
In normal tissues, GCTM-5 immuonoreactivity was found at the apical surface of epithelia, in a mucin-like pattern of distribution. In cancerous tissue, where epithelial polarity is disrupted, staining was distributed throughout the surface of the cell.
Example III
Putative biliary progenitors and cholangiocarcinoma express the GCTM-5 antigen.
Previous studies indicated that the GCTM-5 monoclonal antibody is a novel cell surface marker of putative biliary progenitors in normal liver that demonstrates colocaliz ation with NCAM and C 19 in cirrhotic tissues (18). Further immunohistochemical investigation confirmed that GCTM-5 is expressed in a subpopulation of biliary ductal cells in the portal tracts of normal adult liver (Figure 1A). In regenerating cirrhotic livers, GCTM-5 marked an expanded population of ductal cells with the appearance of biliary ductular reaction (Figure IB). Co-expression of the GCTM-5 antigen with EpCAM and albumin, markers of hepatic stem cells was studied (15). GCTM-5 colocalized with EpCAM (Figure 1C) and demonstrated co-expression with albumin where GCTM-5 positive cells merge at the periphery of regenerative nodules of parenchymal hepatocytes (Figure ID). The malignant cells of intrahepatic cholangiocarcinoma reacted variably and heterogeneously with GCTM-5 (Figure IE; Table 1). GCTM-5 positive cells were more widely distributed in bile ducts from pediatric liver. Staining of serial sections showed that immunoreactive cells could be found in the distal portions of the duct extending well into the hepatic parenchyma (Figure 2), consistent with localization in the Canals of Hering. GCTM-5 reacted with a subset of C 19 positive cells in the distal portions of the bile ducts. In livers undergoing repair after biliary atresia, GCTM-5 reactive cells were positive for Ki-67 (Figure 1 1). In these specimens there was some overlap with N-CAM and EpCAM, but as in the adult, the two markers were not always coincident (Figure 12). An antibody to the transcription factor Sox-9 decorated a subset of GCTM-5-positive cells within the hepatic ducts (data not shown).
Example IV
GCTM-5 positive cells in adult liver have a progenitor cell phenotype
To characterize further the GCTM-5 subpopulation in adult liver, antigen-positive cells were isolated from the non-parenchymal fraction of dissociated liver tissue using GCTM-5 coupled to immunomagnetic beads, then analyzed the cells by QRT-PCR for expression of liver progenitor and hepatocyte markers (Figure 5). Relative to whole liver, total non-parenchymal cells, HepG2 hepatocellular carcinoma, or human dermal fibroblasts, the GCTM-5 positive fraction expressed high levels of HNFlbeta, HNF6, HHex, GATA-6, and Pdx-1. GCTM-5 cells expressed GATA-4, HNF3a, HNF4, Sox-17, Sox-7, albumin and alpha I-antitrypsin at similar levels to other hepatic cell types, but did not express AFP.
GCTM-5 positive cells from adult liver could be serially cultivated in vitro for several passages in media previously reported to support propagation of human liver progenitor cells. The isolated cells formed small epithelial colonies throughout which most cells retained GCTM-5 expression (Figure 6). Most cells in the colonies were positive for Ep-CAM, E-cadherin, and albumin. A subpopulation of cells in growing colonies expressed CD 133, and cytokerations 8 and 19. Most cells were negative for N- CAM and I-CAM, and a subset expressed CD133 (not shown). Example V
GCTM-5 antigen is expressed in a subpopulation of normal pancreatic ducts with expansion of expression in ductal metaplasia, Pancreatic intraepithelial neoplasia
(PanIN), and adenocarcinoma.
In the adult pancreas, infrequent and scattered GCTM-5 reactivity in small ductules and in larger ducts was observed (Figure 3 A), Expansion of the GCTM-5 expressing ductules was observed in areas of tissue, affected by mild chronic pancreatitis (Figure 3B).
In cases of severe chronic pancreatitis, a subpopulation of ductules in the tubular complex formation, also known as acinar-ductal metaplasia, express the GCTM-5 antigen
(Figure 3C). Poorly defined structures known as transforming tubular complexes, which show morphological overlap with PanIN but occur in the acinar compartment, variably expressed the GCTM-5 antigen (Figure 3D) (22, 40). Both low grade and high grade PanIN lesions showed intense membrane reactivity (Figure 3E). Primary and metastatic pancreatic ductal adenocarcinoma strongly and consistently expressed the GCTM-5 antigen in a high percentage of tumour cells, irrespective of the degree of differentiation of the tumor (Figure 3F).
Example VI
GCTM-5 antigen is expressed in the reflux to adenocarcinoma sequence of Barrett's esophagus in proliferating cells.
Further study of the reflux to adenocarcinoma sequence of Barrett's esophagus was performed to evaluate the significance of GCTM-5 antigen expression in this condition. The normal squamous epithelium of the esophagus did not express the GCTM-5 antigen (Figure 4A). However, GCTM-5 immunoreacted with the majority of cases containing metaplastic epithelia, dysplasia, and adenocarcinoma in the esophagus (Table 1 and Figure 4B-F). This indicates that the GCTM-5 antigen is expressed in the metaplasia to adenocarcinoma sequence of Barrett's esophagus. A lesion known as the multilayered epithelium, thought to be a precursor to intestinal metaplasia, was strongly decorated with GCTM-5 (Figure 4B)> as were cardiac mucosa type metaplasia (Figure 4C) and intestinal metaplasia (Figure 4D) itself. The majority of dysplastic lesions (Figure 4E) expressed the antigen, as did adenocarcinoma (Figure 4F), although only portions of these tissues were reactive with the antibody. Example VII
Biochemical Characterization of the GCTM-5 antigen.
Previously the GCTM-5 antigen was characterized as an epitope on a -50 kDA protein present in cell lysates of primary biliary epithelial cells and differentiating populations of human embryonic stem cells (J 8). The antigen is also expressed on the cell surface of cultured pancreatic adenocarcinoma cell line CFPAC-1 (live cell staining analyzed by flow cytometry; immunoblotting of whole cell lysates of CFPAC-1 cells lysed into SDS-PAGE gel reducing sample buffer showed a strong band of ~55kDa and diffuse high molecular weight bands (Figure 7 A). CFPAC-1 cells secreted or shed the antigen into the cell culture medium, where the high molecular weight species represented the predominant form (Figure 7B, lane 1).
It was desired to characterize the GCTM-5 antigen further. Treatment of CFPAC-1 lysates with deglycosylating enzymes demonstrated significant sensitivity of the epitope to sialidase A treatment (Figure 7B, lanes 3 and 5) but not to N- or O-glycosidases
(Figure 7B, lanes 2 and 4). The antigen was difficult to extract from CFPAC-1 cells using non-ionic detergents. However, the secreted form could be recovered from concentrated conditioned medium by immunoprecipitation (Figure 7C, lane 1) or affinity
chromatography. Affinity purified antigen from CFPAC-1 supernatants often contained only the high molecular weight form. The eluate from GCTM-5 affinity columns was subjected to mass spectrometry. The only protein represented by multiple peptide bands in the sample was the mucin-like protein FCGBP (Table 3). Commercially available antibodies against protein epitopes from either the C-terminal or N-terminal of FCGBP reacted with a high molecular weight band in the eluate of GCTM-5 affinity columns (Figure 7D, lanes 1 and 3). Immunoblotting of concentrated condition medium from CFPAC-1 cells showed that the antibody against the C-terminal epitope reacted with a protein band of approximately 100 kDa (Figure 7D, lane 2) and the antibody directed against the N-terminal epitope reacted with a band of approximately 55 kDa (Figure 7D, lane 4). The 55 kDa and 100 kDa bands approximate roughly to the sizes of fragments of FCGBP protein isolated from intestinal mucous that are thought to be generated by a process of proteolytic degradation of the intact FCGBP molecule (41). Neither commercially available anti-FCGBP antisera recognized the native form of the GCTM-5 antigen in concentrated conditioned medium from CFPAC-l cells, and neither reagent immunoprecipitated the high molecular weight form of the GCTM-5 antigen from CFPAC-l cells (not shown). Neither reagent reacted in bile duct cells in liver tissue sections, though transcripts for FCGBP were readily detected in adult liver non- parenchymal fractions. (Figure 14). However, anti-FCGBP C-terminal antisera reacted with the 100 kDa band seen in conditioned medium in GCTM-5 immunoprecipitates of CFPAC1 concentrated conditioned medium (Figure 7e)
FCGBP was originally isolated from the small intestine as a protein that bound the Fc portion of immunoglobulins (42, 43). In the study secondary antisera against mouse or rabbit Ig showed a degree of reactivity with the high molecular weight form of purified GCTM-5 antigen (Figure 7F, compare lanes 1 and 3). Secondary antisera did not react with the lOOkDa or 50kDa fragments recognized by the C- and N-terminal anti-FCGBP antibody in concentrated conditioned medium (C-terminal antiserum, Figure 7F, compare tracks 2 and 4), or the native high molecular weight form of the antigen. This reactivity of secondary antibodies was greatly decreased by using F(ab')2 conjugates instead of intact immunoglobulins to probe the immunoblots (Figure 7F, lanes 5 and 6).
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
Those skilled in the art will recognize that it is common within the art to describe devices and/or processes in the fashion set forth herein, and thereafter use engineering practices to integrate such described devices and/or processes into data processing systems. That is, at least a portion of the devices and/or processes described herein can be integrated into a data processing system via a reasonable amount of experimentation. Those having skill in the art will recognize that a typical data processing system generally includes one or more of a system unit housing, a video display device, a memory such as volatile and non-volatile memory, processors such as microprocessors and digital signal processors, computational entities such as operating systems, drivers, graphical user interfaces, and applications programs, one or more interaction devices, such as a touch pad or screen, and/or control systems including feedback loops and control motors (e.g., feedback for sensing position and/or velocity; control motors for moving and/or adjusting components and/or quantities). A typical data processing system may be implemented utilizing any suitable commercially available components, such as those typically found in data computing/communication and/or network computing/communication systems.
References:
The following references are incorporated herein in their entireties:
1. Slack JM. Origin of stem cells in organogenesis. Science 2008;322:1498-1501.
2. Zaret KS, Grompe M. Generation and regeneration of cells of the liver and
pancreas. Science 2008;322:1490-1494.
Zaret KS. Regulatory phases of early liver development: paradigms of
organogenesis. Nat Rev Genet 2002;3 :499-512.
Oikawa T. Kamiya A, Kakinuma S, Zeniya M, Nishinakamura R, Tajiri H,
Nakauchi H. Sall4 regulates cell fate decision in fetal hepatic stem/progenitor cells. Gastroenterology 2009;136:1000-1011.
Sherwood RI, Chen TY, Melton DA. Transcriptional dynamics of endodermal organ formation. Dev Dyn 2009;238:29-42.
Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S, Kuhara T} Hosokawa S, et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat Genet 201 1 ;43:34-41.
Jensen JN, Cameron E, Garay MV, Starkey TW, Gianani R, Jensen J.
Recapitulation of elements of embryonic development in adult mouse pancreatic regeneration. Gastroenterology 2005;128:728-741.
Limaye PB, Alarcon G, Walls AL, Nalesnik MA, Michalopoulos GK, Demetris AJ, Ochoa ER. Expression of specific hepatocyte and cholangiocyte transcription factors in human liver disease and embryonic development. Lab Invest
2008;88:865-872. . Zhang L, Theise N, Chua M, Reid LM. The stem cell niche of human livers:
symmetry between development and regeneration. Hepatology 2008;48: 1598-
1607. 0. Krakowski ML, Kritzik MR, Jones EM, Krahl T, Lee J, Arnush M, Gu D, et al. Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells. Am J Pathol 1999; 154:683-691.
11. Xu X, D'Hoker J, Stange G, Bonne S, De Leu N, Xiao X, Van de Casteele M, et al.
Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 2008;132: 197-207.
12. Yechoor V, Liu V, Espiritu C, Paul A, Oka K, Kojima H, Chan L. Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes. Dev Cell 2009;16:358-373. 13. Zaret KS. Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation. Nat Rev Genet 2008;9:329-340.
14. Riehle KJ, Dan YY, Campbell JS, Fausto N. New concepts in liver regeneration. J Gastroenterol Hepatol 201 1;26 Suppl 1 :203-212.
15. Turner R, Lozoya O, Wang Y, Cardinale V, Gaudio E, Alpini G, Mendel G, et al.
Human hepatic stem cell and maturational liver lineage biology. Hepatology 2011 ;53:1035-1045. 16. Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, Kumar A, et al. The canals of Hering and hepatic stem cells in humans. Hepatology
1999;30: 1425-1433.
17. Zhou H, Rogler LE, Teperman L, Morgan G, Rogler CE. Identification of
hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver. Hepatology 2007;45:716-724.
18. Stamp L, Crosby HA. Hawes SM, Strain AJ, Pera MF. A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells. Stem Cells 2005;23:103-112.
19. Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, Leach SD. Isolation and
characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci U S A; 107:75-80. Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JR, Marino CR, et al. Expansion of Pdxl-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor alpha. Gastroenterology 199 ;1 17: 1416-1426. Strobel O, Dor Y, Alsina J, Stirman A, Lauwers G, Trainor A, Castillo CF, et al. In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastroenterology 2007; 133: 1999-2009. Esposito I, Seiler C, Bergmann F, Kleeff J, Friess H, Schirmacher P. Hypothetical progression model of pancreatic cancer with origin in the centroacinar-acinar compartment. Pancreas 2007;35:212-217. Fendrich V, Esni F, Garay MV, Feldmann G, Habbe N, Jensen JN, Dor Y, et al. Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology 2008; 135:621-631. Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, lacobuzio-Donahue CA, Sriuranpong V, et al. Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565-576. Stanger BZ, Stiles B, Lauwers GY, Bardeesy N, Mendoza M, Wang Y, Greenwood A, et al. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer Cell 2005;8:185-195. Zhu L, Shi G} Schmidt CM, Hruban RH, Konieczny SF. Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia. Am J Pathol 2007;171 :263-273. Komuta M, Spee B, Vander Borght S, De Vos R, Verslype C, Aerts R, Yano H, et al. Clinicopathological study on cholangiolocellular carcinoma suggesting hepatic progenitor cell origin. Hepatology 2008;47:1544-1556. Zen Y, Adsay NV, Bardadin K, Colombari R, Ferrell L, Haga H, Hong SM, et al. Biliary intraepithelial neoplasia: an international interobserver agreement study and proposal for diagnostic criteria. Mod Pathol 2007;20:701-709. Chandrasoma PT, Der R, Dalton P, Kobayashi G, Ma Y, Peters J, Demeester T. Distribution and significance of epithelial types in columnar-lined esophagus. Am J Surg Pathol 2001 ;25:1 188-1 193. Chandrasoma PT, Lokuhetty DM, Demeester TR, Bremmer CG, Peters JH, Oberg S, Groshen S. Definition of histopathologic changes in gastroesophageal reflux disease. Am J Surg Pathol 2000;24:344-351. 31. Chaves P, Crespo M, Ribeiro C, Laranjeira C, Pereira AD, Suspiro A, Cardoso P, et al. Chromosomal analysis of Barrett's cells: demonstration of instability and detection of the metaplastic lineage involved. Mod Pathol 2007;20:788-796. 32. Liu W, Hahn H, Odze RDf Goyal RK. Metaplastic esophageal columnar
epithelium without goblet cells shows DNA content abnormalities similar to goblet cell-containing epithelium. Am J Gastroenterol 2009;104:816-824,
33. Coad RA, Woodman AC, Warner PJ, Barr H, Wright NA, Shepherd NA. On the histogenesis of Barrett's oesophagus and its associated squamous islands: a three- dimensional study of their morphological relationship with native oesophageal gland ducts. J Pathol 2005;206:388-394.
34. Glickman JN, Chen YY, Wang HH, Antonioli DA, Odze RD. Phenotypic
characteristics of a distinctive multilayered epithelium suggests that it is a precursor in the development of Barrett's esophagus. Am J Surg Pathol 2001 ;25:569-578.
35. Glickman JN, Spechler SJ, Souza RF, Lunsford T, Lee E, Odze RD. Multilayered epithelium in mucosal biopsy specimens from the gastroesophageal junction region is a histologic marker of gastroesophageal reflux disease. Am J Surg Pathol
2009;33:818-825.
36. Leedham SJ, Preston SL, McDonald SA, Elia G, Bhandari P, Poller D, Harrison R, et al, Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett's oesophagus. Gut 2008;57:1041-1048.
37. Shi L, Der R, Ma Y, Peters J, Demeester T, Chandrasoma P. Gland ducts and
multilayered epithelium in mucosal biopsies from gastroesophageal-junction region are useful in characterizing esophageal location. Dis Esophagus 2005;18:87-92.
38. Shields HM, Rosenberg SJ, Zwas FR, Ransil BJ, Lembo AJ, Odze R. Prospective evaluation of multilayered epithelium in Barrett's esophagus. Am J Gastroenterol 2001 ;96:3268-3273. 39. Schmelzer E, Zhang L, Bruce A et al. Human hepatic stem cells from fetal and postnatal donors. J Exp Med 2007;204:1973-1987.
40. Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, Boivin GP, Furth EE, et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res 2006;66:95-
106.
41. Albert TK, Laubinger W, Muller S, Hanisch FG, alinski T, Meyer F, Hoffmann W. Human intestinal TFF3 forms disulfide-linked heteromers with the mucus- associated FCGBP protein and is released by hydrogen sulfide. J Proteome Res
2010;9:3108-3117. 42. Kobayashi K, Blaser MJ, Brown WR. Identification of a unique IgG Fc binding site in human intestinal epithelium. J Immunol 1989;143:2567-2574. 43. Harada N, Iijima S, Kobayashi K, Yoshida T, Brown WR, Hibi T, Oshima A, et al.
Human IgGFc binding protein (FcgammaBP) in colonic epithelial cells exhibits mucin-like structure. J Biol Chem 1997;272: 15232- 15241.
44. Johansson ME, Thomsson KA, Hansson GC. Proteomic analyses of the two mucus layers of the colon barrier reveal that their main component, the Muc2 mucin, is strongly bound to the Fcgbp protein. J Proteome Res 2009;8:3549-3557.
45. Kobayashi K, Hamada Y, Blaser MJ, Brown WR. The molecular configuration and ultrastructural locations of an IgG Fc binding site in human colonic epithelium. J Immunol 1991 ;146:68-74.
46. Yasui Y, Tanaka T. Protein expression analysis of inflammation-related colon carcinogenesis. J Carcinog 2009;8:10. 47. Kristiansen TZ, Bunkenborg J, Gronborg M, Molina H, Thuluvath PJ, Argani P, Goggins MG, et al. A proteomic analysis of human bile. Mol Cell Proteomics 2004;3 :715-728.
48. Kouznetsova I, Gerlach KL, Zahl C, Hoffmann W. Expression analysis of human salivary glands by laser microdissection: differences between submandibular and labial glands. Cell Physiol Biochem 2010;26:375-382.
49. Sasaki M, Ikeda H, Nakanuma Y. Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: Physiological distribution and pathological significance. Prog Histochem Cytochem 2007;42:61 -
110.
50. Yonezawa S, Higashi M, Yamada N et al. Significance of mucin expression in pancreatobiliary neoplasms. J Hepatobiliary Pancreat Sci 2010;17:108-124.
51. Roskams T. Liver stem cells and their implication in hepatocellular and
cholangiocarcinoma. Oncogene 2006;25:3818-3822.
52. Rooman I, Real FX. Pancreatic ductal adenocarcinoma and acinar cells: A matter of differentiation and development? Gut 2012;61 : 449-458.
53. Kuuselo R, Savinainen K, Azorsa DO et al. Intersex-like (IXL) is a cell survival regulator in pancreatic cancer with 19ql 3 amplification. Cancer Res 2007;67: 1943- 1949. Table 1. Distribution of the GCTM-5 antigen in gastrointestinal and hepatopancreatic epithelia.
Tissue Positive/ Grade Pattern & Localization total cases
Metaplastic Esophagus
Cardia-type Metaplasia 1 1/17 (65%) Columnar mucous cells
(a, c)
Intestinal Metaplasia 11/12 (92%) Columnar and goblet cells (a,c)
Dysplasia 5/7 (74%) (a, c)
Adenocarcinoma 6/8 (75%) +++ (a, c)
Gastric Mucosa
Normal gastric mucosa 0/2
Gastric Polyp 1/1 + Mucous cells
Intestinal Metaplasia 1/1 (a, c)
Carcinoma 1/1 (a, c)
Liver
Normal Liver 4/4 ++ Biliary ducts (a, c) Cirrhotic Liver 3/3 Biliary ductular reaction
(a, c)
Bening Hepatic Adenoma 1/1 + Tumour cells (c)
Gallbladder Adenoma 1/1 ++ (a)
Hepatocellular Carcinoma 3/5 + Tumour cells (c)
Intrahepatic 3/5 +++ Tumour cells (a, c) Cholangiocarcinoma
Pancreas
Normal Pancreas 33 Ducts (a)
Chronic Pancreatitis 2/2 Ducts (a)
Pancreatic Adenocarcinoma 9/9 Tumour cells (a,
Bowels
Duodenum 1/1 Ducts of brunners glands (a)
Small Bowel 1/3 Crypts (a, c)
Ampulla of Vater Adeoma 1/1 (a) Colon 3/3 +++ Crypts (a, c)
Colonic Adenocarcinoma 0/5
- = no staining + = Scant, ++= moderate, +++ = extensive (a) apical membrane, (c) cytoplasmic
Table 2. Histological snrvey of GCTM-5 reactivity.
Tissue Positive/ Grade Pattern & Localization total cases
Non-Gastrointestinal Endoderm
Salivary Gland 1/1 Salivary ducts (a)
Lung 1/1 Respiratory epithelium mucous cells (a)
Normal Endocervix 1/1 Endocervical epithelium
(a)
Endocervical Polyp 1/1 Mucin secreting glands
(a, c)
Benign prostatic Hypertrophy 1 / 1 Prostatic ducts (a)
Ectodermal & Mesodermal
Cerebral Cortex 0/2
Keratinizing epithelia 0/4
Dermis 0/4
Burns and Normal Skin 2/2 Adenexal structures (a)
Kidney ½ Proximal tubule (c)
Blood many
Lymph Node 0/4+
Vasculature 0/30+
Smooth Muscle 0/5+
Cardiac Muscle 0/2
- = no staining + = Scant, ++= moderate, +++ = extensive (a) apical membrane, (c) cytoplasmic Table 3. Mass spectrometry identification of peptide sequences corresponding to FCGBP in GCTM-5 affinity column eluate.
Sequence MH+ %Mass AA %AA
FYPAGDVLR (SEQ ID NO: 1) 1038.18 0.33 184-192 0.30
VTLQPYNVAQLQS SVDLSGSK 2235.48 0.70 193-212 0.70
(SEQ ID NO: 2)
YDLAFVVASQATK (SEQ ID 1413.60 0.44 263-275 0.43 NO: 3)
AISGLTIDGHAVGAK (SEQ ID 1410.60 0.44 366-380 0.50 NO: 4)
LPVVLANGQIR (SEQ ID NO: 1 180.43 0.37 1338-1348 0.37 5)
YLPVNSSLLTSDCSER (SEQ ID 1784.97 0.56 5182-5197 0.53 NO: 6)
TOTALS 6183.04 1.94 60 2.00 Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:
1. A method for detecting cancer in a sample obtained from a subject, comprising:
a) obtaining the sample from the subject;
b) detecting expression or presence of a biomarker in the sample, wherein the biomarker is a GCTM-5 epitope; and
c) analyzing the results of (b) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as cancer, thereby detecting cancer.
2. The method of claim 1, wherein the cancer is liver, pancreatic, colorectal or esophagus cancer.
3. The method of claim 2, wherein the cancer is pancreatic adenocarcinoma.
4. The method of claim 1, wherein the sample comprises cells from liver, colorectal, pancreas or gastrointestinal tissue.
5. The method of claim 1, further comprising contacting the sample with an agent that specifically binds the biomarker.
6. The method of claim 5, wherein the agent is an antibody or antibody fragment which specifically binds the native form of the GCTM-5 epitope.
7. The method of claim 6, wherein the agent is fiuorescently labeled.
8. The method of claim 1, wherein analysis further comprises image analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay analysis, or nuclear exclusion analysis.
9. The method of claim 8, wherein the image analysis comprises detection of a plurality of biomarkers.
10. The method of claim 9, wherein the plurality of biomarkers are detected via antibodies.
1 1. The method of claim 10, wherein the antibodies are fluorescently labeled.
12. The method of claim 1 1 wherein the image analysis is performed by
immunomicroscopy or flow cytometry.
13. The method of claim 8, wherein the PCR analysis comprises multiplexing using primers specific for a plurality of genes.
14. The method of claim 1, further comprising providing a diagnosis or a prognosis to the subject.
15. The method of claim 1, wherein the subject is known to have cancer and is undergoing cancer therapy.
1 . The method of claim 15, wherein the therapy is chemotherapy.
17. The method of claim 17, wherein the subject is being administered a candidate agent.
18. The method of claim 17, further comprising determining the responsiveness of the subject to the cancer therapy.
19. The method of claim 15, wherein a decrease in the presence of GCTM-5 epitope is indicative of remission or survival.
20. A method for detecting and characterizing a subpopulation of cells in a sample, comprising:
a) detecting expression or presence of a biomarker of one or more cells in the sample, wherein the biomarker is a GCTM-5 epitope; and
b) analyzing the results of (a) via a computer generated algorithm to characterize the one or more cells of the sample which are positive for the biomarker as a subpopulation of progenitor cells, thereby detecting and characterizing the subpopulation of cells in the sample.
21. The method of claim 20, further comprising isolating the subpopulation from the sample.
22. The method of claim 20, wherein the sample comprises cells of liver, pancreatic, colorectal or esophagus tissue.
23. The method of claim 20, wherein the subpopulation is a progenitor population of liver, pancreatic, or esophagus cells.
24. The method of claim 20, further comprising contacting the sample with an agent that specifically binds the biomarker.
25. The method of claim 24, wherein the agent is an antibody or antibody fragment which specifically binds the native form of the GCTM-5 epitope.
26. The method of claim 25, wherein the agent is fluorescently labeled.
27. The method of claim 20, wherein analysis further comprises image analysis, cell morphology analysis, polymerase chain reaction (PCR) analysis, sequence analysis, DNA analysis, RNA analysis, gene expression profiling, proteome analysis, metabolome analysis, immunoassay analysis, or nuclear exclusion analysis.
28. The method of claim 27, wherein the image analysis comprises detection of a plurality of biomarkers.
29. The method of claim 28, wherein the plurality of biomarkers are detected via antibodies.
30. The method of claim 29, wherein the antibodies are fluorescently labeled.
31. The method of claim 30, wherein the image analysis is performed by
immunomicroscopy or flow cytometry.
32. The method of claim 27, wherein the PCR analysis comprises multiplexing using primers specific for a plurality of genes.
33. An isolated antibody which specifically binds native GCTM-5 epitope of mucin-line glycoprotein FCGBP.
34. The antibody of claim 33, wherein the antibody specifically binds native GCTM-5 epitope having a molecular weight of about 100 kDA.
35. The antibody of claim 33, wherein the antibody is an immunoconjugate.
36. The antibody of claim 35, wherein the antibody is conjugated to a reporter molecule.
37. The antibody of claim 36, wherein the reporter is a fluorophore, chromophore or radioisotope.
38. The antibody of claim 35, wherein the antibody is conjugated to an enzyme, chemotherapeutic agent or toxin.
39. A pharmaceutical composition comprising:
a) the antibody of claim 33; and
b) a pharmaceutically acceptable carrier.
40. A kit comprising:
a) the antibody of claim 33; and
b) reagents for conducting a diagnostic test; and
c) instructions for conducting and analyzing results of the diagnostic test,
41. The kit of claim 40, wherein the reagents comprise one or more additional antibodies.
42. The kit of claim 40, wherein the instructions provide a computer algorithm for determining the presence of cancer in a sample from a subject.
43. An isolated nucleic acid molecule encoding the antibody of claim 33.
44. A method for treating cancer comprising administering to a subject the antibody of claim 33, thereby treating cancer in the subject.
45. The method of claim 44, wherein the subject is coadministered a therapeutic agent.
46. A method of isolating a progenitor subpopulation of cells from a sample, comprising:
a) contacting the sample with an agent that specifically binds a biomarker present on a subpopulation of progenitor cells present in the sample to form a plurality of cellular complexes, wherein the biomarker is native GCTM-5 epitope of FCGBP; and b) isolating the cellular complexes from the sample, thereby isolating the progenitor subpopulation of cells.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016077881A1 (en) * 2014-11-17 2016-05-26 The University Of Queensland Glycoprotein biomarkers for esophageal adenocarcinoma and barrett's esophagus and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040009492A1 (en) * 2002-07-10 2004-01-15 Mi-Kyung Kim Multiplex PCR primer set for human glucokinase gene amplification
WO2004099432A2 (en) * 2003-05-02 2004-11-18 The Johns Hopkins University Identification of biomarkers for detecting pancreatic cancer
US20070218059A1 (en) * 2003-10-09 2007-09-20 Martin Pera Cell Surface Marker
US20080286284A1 (en) * 2001-10-23 2008-11-20 Psma Development Company, Llc Compositions of PSMA antibodies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080286284A1 (en) * 2001-10-23 2008-11-20 Psma Development Company, Llc Compositions of PSMA antibodies
US20040009492A1 (en) * 2002-07-10 2004-01-15 Mi-Kyung Kim Multiplex PCR primer set for human glucokinase gene amplification
WO2004099432A2 (en) * 2003-05-02 2004-11-18 The Johns Hopkins University Identification of biomarkers for detecting pancreatic cancer
US20070218059A1 (en) * 2003-10-09 2007-09-20 Martin Pera Cell Surface Marker

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016077881A1 (en) * 2014-11-17 2016-05-26 The University Of Queensland Glycoprotein biomarkers for esophageal adenocarcinoma and barrett's esophagus and uses thereof
CN107430126A (en) * 2014-11-17 2017-12-01 昆士兰大学 Glycoprotein biomarker of adenocarcinoma of esophagus and Barrett esophagus and application thereof
AU2015349613B2 (en) * 2014-11-17 2022-01-13 The Council Of The Queensland Institute Of Medical Research Glycoprotein biomarkers for esophageal adenocarcinoma and barrett's esophagus and uses thereof

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