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WO2003097666A2 - Utilisation du peptide liberateur de gastrine (grp) et de son recepteur dans le depistage du cancer - Google Patents

Utilisation du peptide liberateur de gastrine (grp) et de son recepteur dans le depistage du cancer Download PDF

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WO2003097666A2
WO2003097666A2 PCT/US2003/015466 US0315466W WO03097666A2 WO 2003097666 A2 WO2003097666 A2 WO 2003097666A2 US 0315466 W US0315466 W US 0315466W WO 03097666 A2 WO03097666 A2 WO 03097666A2
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grp
level
cancer
receptor
asymptomatic subject
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PCT/US2003/015466
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WO2003097666A3 (fr
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Bijan Nejadnik
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Euro-Celtique, S.A.
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Priority to AU2003234623A priority Critical patent/AU2003234623A1/en
Publication of WO2003097666A2 publication Critical patent/WO2003097666A2/fr
Publication of WO2003097666A3 publication Critical patent/WO2003097666A3/fr

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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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the use of GRP and GRP-R nucleic acids, proteins, derivatives, and analogs thereof for screening for multiple malignancies in asymptomatic subjects.
  • the invention provides screening methods for cancers of the gastrointestinal tract (colon, pancreas, stomach) and prostate.
  • CRC Colorectal cancer
  • CRC ulcerative colitis .
  • Colonoscopy is currently the "gold standard" for detection of small and pre- malignant lesions, although it is not cost-effective for screening an average-risk population.
  • FOBT has a poor sensitivity and specificity.
  • Sigmoidoscopy may be sensitive and specific for lesions within the reach of the instrument, but misses the roughly 50% of neoplasms that are proximal or right-sided (reviewed by Ransohoff et al, 2002, New England Journal of Medicine, 346(l):40-44).
  • Prostate cancer kills several hundred thousand patients worldwide each year. Prostate cancer tumors most commonly occur in older men, with the median age of 70 years at diagnosis. Treatment choices for prostate cancer are influenced by age and coexisting medical problems. Given the late age at which most prostate cancer tumors emerge, the practice of "watchful waiting" or palliative hormonal treatment is often sufficient to prevent the cancer, or its treatment, from affecting the patient's life. However, the likelihood for rapidly growing and/or metastasizing tumors is markedly higher in younger men. When the cancer is confined to the prostate gland, survival of 5 years or more is common. However, poorly differentiated tumors often have metastasized when diagnosed, with the bone being the most common site of secondary tumor growth.
  • treatment regimes include radical prostatectomy, radiation therapy with curative intent and/or hormonal (androgen deprivation) therapy.
  • none of these therapies is effective in prolonging longevity relative to untreated patients.
  • the main hope for more effective therapy lies in earlier and more accurate diagnosis of this disease.
  • Prostate specific antigen a 32 kilodalton (kd) serine protease synthesized by prostate epithelial cells, is normally present at less than about 4 ng/ml in serum.
  • PSA Prostate specific antigen
  • Intermediate PSA levels of about 4-10 ng/ml are seen with inflammation (i.e., prostatism and benign prostatic hypertrophy), as well as with early stages of prostate cancer.
  • High serum PSA levels i.e., greater than about 10 ng/ml suggest that prostate cancer is likely to be present.
  • Stomach cancer or gastric adenocarcinoma
  • Men are affected twice as often as women.
  • Symptoms may not occur until local disease is advanced (reviewed in Bowles et ⁇ , 2001, BMJ, 323: 1413-6).
  • Endoscopy and barium meal are complementary diagnostic methods for detecting and staging this type of cancer.
  • Endoscopy allows direct visualization and biopsy of the carcinoma.
  • Barium meals are done to visualize the anatomy of the tumor and the degree of obstruction as visualized by a CT scan. Differentiation between benign and malignant gastric ulcers by endoscopy can be difficult, and several biopsies are therefore taken (i.e up to six, typically) from all parts of the ulcer.
  • a barium study gives a better impression of the anatomy of the tumor and the degree of the obstruction. Once the diagnosis is made, the difficulty lies in deciding which patients need urgent investigation. A good initial symptomatic response to acid suppression does not exclude malignancy.
  • the disease is incurable in about half of the patients at presentation. With regional lymph node metastases, five year survival after gastrectomy is about 10%. In those with only perigastric lymph node involvement, survival rises to 30%, and in those with gastric carcinoma confined to the stomach five year survival is about 70%.
  • pancreatic cancer rises with age, and the disease is slightly more common in men than women.
  • pancreatic neoplasm is ductal adenocarcinoma.
  • Most cancers arise in the head, neck, or uncinate process of the pancreas and may compress the common bile duct (reviewed in Bowles et al, 2001, BMJ, 323:1413-6).
  • the classic presentation is painless, progressive, obstructive jaundice.
  • Serum biochemistry will confirm jaundice and also give information about its cause.
  • Alkaline phosphatase and g-glutamyltransferase tend to be raised in obstructive jaundice.
  • Tumor markers may also be of value in diagnosis.
  • Carcinoembryonic antigen is elevated in up to 85% of the cases.
  • Ultrasonography and endoscopic retrograde cholangiopancreatography are used in diagnosis.
  • Neoplastic proliferation of neuroendocrine cells (NE) of the gastrointestinal (GI) tract are commonly called "carcinoids". They can occur in all age groups and even in infants. The incidence of carcinoids has been reported to be higher than 1%. Most GI NE tumors are found in the appendix (50%) and the ileum (30%). Practically all appendiceal NE tumors are benign. Among the ileal NE tumors, a large number can metastasize and result in a fatal outcome. The ability to metastasize is related to size and the multiplicity of the primary tumors. Exceptionally highly malignant NE neoplasms can arise from the colon/rectum as well as from the esophagus.
  • Carcinoids rarely present with symptoms or signs of disseminated disease (Chejfec G. et al, 1988, Path. Res. Pract. 183:143-54; Lyss A. P., 1988, Seminars in Oncology, 15(2):129-37; Falkmer S., 1993, Gastrointestinal Hormones in Medicine, 22(4):731-52).
  • Gastrin-releasing peptide is the primary member of the bombesin family of neuropeptides. Bombesin was originally isolated from the skin of the amphibian Bombina bombina. The first "mammalian bombesin" was isolated from porcine non-antral stomach. The carboxy terminal region of this peptide possesses striking sequence homology to bombesin and stimulates the release of gastrin in vivo. Gastrin is a hormone secreted by the glands in the mucous membrane of the stomach and stimulates the production of gastric juice in the stomach, which is an acidic digestive fluid consisting essentially of hydrochloric acids, pepsin, rennin, and mucin.
  • GRP G protein-coupled receptor
  • GRP has been implicated in the proliferation of human cancers including those arising in the lung, breast, stomach, pancreas, prostate, and colon (reviewed by Jehnsen et al, 2001, Peptides 22, 689-699; Sunday et al, 1988, Laboratory Investigation, 59, 5-24).
  • GRP and GRP-R are frequently expressed by cancers of the GI tract. GRP and GRP-R are expressed in 70% and 76% of colon cancer tissues, respectively (84% of these tissues expressed one of them and 62% expressed both of them) (Carroll et al, 1999, Am. J Physiology, 276 (3 Pt 1): G655-65). All cell lines derived from human colon cancers (and 93% of resected colon cancer tissues) have been found to express mRNA for both GRP and GRP-R.
  • GRP-R mRNA can be detected in most colorectal tumor specimens, suggesting a link between high GRP-R mRNA levels and both tumor de-differentiation and lymph vessel invasion (Saurin et al, 1999, Eur. J. Cancer, 35(l):125-32; Carroll et al, 1999, Am. J. Physiology, 276 (3 Pt 1): G655-65).
  • the tumors express GRP-R mRNA, functional protein has only been detected in 24-40% of colon cancers (Carroll et al, 2000, Molecular Pharmacology, 58, Issue 3, 601-607).
  • GRP and GRP-R are not believed to function primarily as mitogens when aberrantly expressed in cancer. Rather, they appear to regulate tumor morphology and/or differentiation and thus, any mitogenic properties appear subordinated to their action as morphogens (Carroll et al, 2000, Cell Growth Differ., 11(7):385-93).
  • the present invention relates to the use of GRP and GRP-R encoding nucleic acids, proteins, derivatives, and analogs thereof and antibodies thereto to screen for one or more multiple malignancies in asymptomatic subjects.
  • the invention provides screening methods for cancers of the gastrointestinal tract (e.g., colon, pancreas, stomach) and prostate.
  • the invention provides a method of screening for cancer (e.g., colon, prostate, stomach, pancreatic cancer or carcinoid tumors of the GI tract) in an asymptomatic subject comprising: (a) measuring a level of GRP, GRP-R, GRP mRNA, or GRP-R mRNA in a sample obtained from the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP and/or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for cancer.
  • a particular sample can be either stool or blood.
  • the sample can be urine, blood, or seminal fluid.
  • the present invention provides methods to screen for cancers of the gastrointestinal tract (e.g., colon, pancreas, stomach, esophogeal), prostate and carcinoid tumors.
  • the invention provides a method of screening for colon cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for colon cancer.
  • the subject is defined to be at increased risk for colon cancer and further referred to tests known in the art for the screening and diagnosis of colon cancer including but not limited to, colonoscopy, sigmoidoscopy, fecal occult-blood testing.
  • the invention provides a method of screening for prostate cancer in an asymptomatic subject comprising:(a) measuring a level of GRP or GRP-R in urine or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for prostate cancer, hi a further specific embodiment, should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for prostate cancer and further referred to tests known in the art for the screening and diagnosis of prostate cancer.
  • the invention provides a method of screening for stomach cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for stomach cancer.
  • the asymptomatic subject should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for stomach cancer and further referred to tests known in the art for the screening and diagnosis of stomach cancer.
  • the invention further provides a method of screening for pancreatic cancer in an asymptomatic subject comprising :(a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for pancreatic cancer, hi a further specific embodiment, should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for pancreatic cancer and further referred to tests known in the art for the screening and diagnosis of pancreatic cancer.
  • the invention provides a method of screening for a carcinoid tumor of the gastrointestinal (GI) tract in an asymptomatic subject comprising :
  • step (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for development of a carcinoid tumor of the GI tract.
  • the subject is defined to be at increased risk for carcinoid tumor of the GI tract and further referred to tests known in the art for the screening and diagnosis of carcinoid tumors of the GI tract.
  • At least one standard sample can be obtained from at least one subject having no prior history or current indications of cancer.
  • the cancer history is selected from the group consisting of colon cancer, prostate cancer, stomach cancer, pancreatic cancer, and lung cancer.
  • the invention in another specific embodiment, relates to a method of screening for cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP receptor in a stool or blood sample of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP receptor, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for cancer.
  • measuring at least one GRP or GRP-R level for any of the above-mentioned cancer screens can be carried out using a GRP or GRP-R probe, respectively.
  • the probe is selected from the group consisting of an antibody or a fragment thereof, an antigen, a nucleic acid, a protein, and a small molecule.
  • the protein is GRP or a fragment thereof, hi another specific embodiment, the protein is GRP-R or a fragment thereof.
  • the probe is an antibody, wherein the antibody is a monoclonal antibody.
  • the probe is an antibody which immunospecifically binds GRP or a fragment or derivative thereof.
  • the antibody is a monoclonal antibody of GRP or a fragment or derivative thereof.
  • the GRP fragment comprises residues 1-27 or residues 31-98.
  • the probe is an antibody which immunospecifically binds GRP-R or a fragment or derivative thereof.
  • the probe is a nucleic acid which is an mRNA.
  • the invention further relates to a kit comprising: (a) at least one reagent selected from the group consisting of an anti-GRP antibody or a fragment thereof, anti-GRP receptor antibody or a fragment thereof, a nucleic acid probe capable of hybridizing with a GRP mRNA, a nucleic acid probe capable of hybridizing with a GRP receptor mRNA, and a pair of nucleic acid primers capable of priming amplification of at least a portion of a GRP or GRP receptor nucleic acid; and (b) instructions for use in cancer screening in an asymptomatic subject, hi a particular embodiment, the kit further comprises a predetermined amount of a purified GRP or GRP-R protein or nucleic acid for use as a standard or control.
  • the reagent in the kit is labeled with a detectable marker.
  • the detectable marker can be a chemiluminescent, enzymatic, fluorescent, or radioactive label.
  • the step of measuring a level of GRP or GRP receptor comprises testing at least one aliquot from the sample, said step of testing comprising: (a) contacting the aliquot with an antibody or a fragment thereof that is immunospecific for GRP or GRP receptor; and (b) detecting whether binding has occurred between the antibody or a fragment thereof and at least one species in the aliquot.
  • the step of measuring a level of GRP or GRP receptor comprises testing at least one aliquot from the sample, said step of testing comprising: (a) contacting the aliquot with an isolated nucleic acid that is hybridizable to GRP or GRP receptor mRNA; and (b) detecting whether hybridization has occurred between the nucleic acid probe and at least one mRNA species in the aliquot.
  • FIGURE 1 Schematic of Cancer Screening Methods using GRP and its Receptor.
  • a flow chart is presented summarizing a preferred embodiment of the invention.
  • FIGURE 2 Nucleotide and Amino acid Sequence for Human GRP.
  • the nucleotide sequence for GRP is presented. This sequence was obtained from GENBANK with accession number XM-008719 (SEQ ID NO: 1). In the lower panel, the corresponding single-letter-code amino acid sequence is presented (SEQ ID NO: 2).
  • FIGURE 3 Nucleotide and Amino acid Sequence for Human GRP-R.
  • the nucleotide sequence for GRP-R is presented (SEQ ID NO: 3). This sequence was obtained from GENBANK with accession number M73481 In the lower panel, the corresponding single-letter-code amino acid sequence is presented (SEQ ID NO: 4).
  • the present invention provides methods to screen for cancers of the gastrointestinal tract (e.g., colon, pancreas, stomach, esophogeal), prostate and carcinoid tumors.
  • the invention provides a method of screening for colon cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for colon cancer.
  • the subject is defined to be at increased risk for colon cancer and further referred to tests known in the art for the screening and diagnosis of colon cancer including but not limited to, colonoscopy, sigmoidoscopy, fecal occult-blood testing.
  • the invention provides a method of screening for prostate cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP-R in urine or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for prostate cancer.
  • the asymptomatic subject should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for prostate cancer and further referred to tests known in the art for the screening and diagnosis of prostate cancer (e.g., rectal ultrasound with or without biopsy).
  • the invention provides a method of screening for stomach cancer in an asymptomatic subject comprising: (a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for stomach cancer, fn a further specific embodiment, should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for stomach cancer and further referred to tests known in the art for the screening and diagnosis of stomach cancer (e.g., endoscopy with or without biopsy).
  • the invention further provides a method of screening for pancreatic cancer in an asymptomatic subject comprising :(a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for pancreatic cancer.
  • the asymptomatic subject should the asymptomatic subject have an increased level of GRP or GRP-R, the subject is defined to be at increased risk for pancreatic cancer and further referred to tests known in the art for the screening and diagnosis of pancreatic cancer (e.g., MRI or CAT scan).
  • the invention provides a method of screening for a carcinoid tumor of the gastrointestinal (GI) tract in an asymptomatic subject comprising : (a) measuring a level of GRP or GRP-R in stool or blood of the asymptomatic subject; and (b) comparing the level measured in step (a) to a standard level of GRP or GRP-R, wherein elevation of the measured level relative to the standard level indicates that the asymptomatic subject is at increased risk for development of a carcinoid tumor of the GI tract.
  • GI gastrointestinal
  • the subject is defined to be at increased risk for carcinoid tumor of the GI tract and further referred for tests known in the art for the screening and diagnosis of carcinoid tumors of the GI tract (e.g., MRI or CAT scan).
  • asymptomatic subject refers to any subject (e.g., animals including humans), to which the test is applicable having no symptoms associated with a malignant disease (for example: an abnormal mass) to which a method of the invention is applied.
  • measurement of a GRP or GRP-R level may be carried out by the use of a GRP or GRP-R specific probe.
  • elevation of a measured level of GRP or GRP-R relative to a standard level means that the amount or concentration of GRP or GRP-R in a sample is sufficiently greater in an asymptomatic subject relative to the standard to be detected by any method now known in the art or to be developed in the future for measuring GRP or GRP-R levels.
  • elevation of the measured level relative to a standard level may be any statistically significant elevation detectable. Such an elevation may include, but is not limited to about a 1%, about a 10%, about a 20%, about a 40%, about an 80%, about a 2- fold, about a 4-fold, about an 8-fold, about a 20-fold, or about a 100-fold elevation, relative to the standard.
  • the term "about” as used herein, refers to levels of elevation of the standard numerical value plus or minus 10% of the numerical value.
  • a level of GRP or GRP-R in a method of the invention relates to any proxy for GRP or GRP-R levels.
  • levels may include, but are not limited to, the abundance of GRP or GRP-R nucleic acid or amino acid sequences in a sample from a subject.
  • a level of GRP or GRP-R may correspond to the abundance of full-length GRP or GRP-R protein.
  • a level of GRP or GRP-R may correspond to abundance of a fragment of GRP or GRP-R protein.
  • a level of GRP or GRP-R can be determined by measuring the abundance of nucleic acids (or sequences complementary thereto) that corresponds to all or fragments of GRP or GRP-R. In a preferred embodiment, the abundance of mRNA encoding GRP or GRP-R is measured.
  • a probe with which the amount or concentration of either GRP or GRP-R can be determined includes but is not limited to an antibody, an antigen, a nucleic acid, a protein, or a small molecule, hi a specific embodiment, the probe is the GRP protein or a fragment thereof. In a further specific embodiment, the probe is amino acids 1-27 of GRP protein, which can bind GRP-R. In another embodiment, the probe is an antibody that immunospecifically binds to GRP or GRP-R, such as e.g., a monoclonal antibody or a binding fragment thereof.
  • measuring a level of GRP or GRP-R comprises testing at least one aliquot of the sample, said step of testing comprising: (a) contacting the aliquot with an antibody or a fragment thereof that is i munospecific for GRP or GRP-R, and (b) detecting whether and how much binding has occurred between the antibody or a fragment thereof and at least one species of GRP or GRP-R in the aliquot.
  • measuring a level of GRP or GRP-R comprises testing at least one aliquot, said step of testing comprising: (a) contacting the aliquot with a nucleic acid probe that is hybridizable to GRP or GRP-R mRNA, and (b) detecting whether and how much hybridization has occurred between the nucleic acid probe and at least one species of GRP or GRP-R mRNA in the aliquot, hi both embodiments measuring a level of GRP or GRP-R involves quantitating the amount of complex formation. For example the amount of complex formation between the antibody or a fragment thereof and at least one species of GRP or GRP-R in the aliquot would correlate with the amount of at least one species of GRP or GRP-R in the aliquot of the sample.
  • the antibody or other probe is labeled with a detectable marker.
  • the detectable marker is a chemiluminescent, enzymatic, fluorescent, or radioactive label.
  • the invention provides a kit comprising : (a) at least one reagent capable of detecting GRP or GRP-R or mRNA encoding GRP or GRP-R, such as e.g., a reagent selected from the group consisting of an anti-GRP antibody, anti-GRP-R antibody, a nucleic acid probe capable of hybridizing with a GRP mRNA, a nucleic acid probe capable of hybridizing with a GRP-R mRNA, and a pair of nucleic acid primers capable of priming amplification of at least a portion of a GRP or GRP-R nucleic acid; and (b) printed instructions for use in cancer screening in an asymptomatic subject.
  • the kit further comprises a predetermined amount of a purified GRP or GRP-R protein or nucleic acid encoding GRP or GRP-R or a fragment thereof sufficient as a standard, for use as a standard or control, h a further specific embodiment, the reagent in the kit is labeled with a detectable marker, hi yet another specific embodiment, the detectable marker is a chemiluminescent, enzymatic, fluorescent, or radioactive label.
  • GRP or GRP-R is defined as a compound having GRP or GRP-R functional activity as determined by any standard assay or from which antibodies can be raised. Such a GRP or GRP-R analog does not necessarily share any structural relationship with GRP or GRP-R.
  • a GRP or GRP-R analog can be modified by the substitution of one or more amino acids with a different amino acid or amino acid analog (for example, a d- amino acid, or an amino acid mimic).
  • a “derivative" of GRP or GRP-R is defined as a variant obtained from or based on GRP or GRP-R, which derivative contains essential elements of the parent GRP or GRP-R (e.g., a fragment of GRP or GRP-R or a fusion protein containing all or fragments of GRP or GRP-R) sufficient to raise anti-GRP or anti- GRP-R antibodies useful in any assay of the present invention.
  • the GRP and GRP-R proteins and nucleic acids, derivatives and analogs thereof useful in the methods and kits of the invention can be produced by any method known in the art.
  • nucleotide sequences encoding human GRP and human GRP-R and the corresponding amino acid sequences of human GRP and human GRP-R are provided in Figures 2 and 3, respectively.
  • Isolated nucleic acids encoding GRP and GRP-R can be obtained by any method known in the art, e.g., from a deposited plasmid, by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, and/or by cloning from a cDNA or genomic library using an oligonucleotide probe specific for the gene sequence.
  • nucleic acids comprising all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the encoded protein by the desired sequences.
  • Promoters which may be used include, but are not limited to the SV40 early promoter (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22: 787-797); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl Acad. Sci.
  • promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase He ⁇ era-Estrella et al, 1984, Nature 310: 115-120
  • promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38: 639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald 1987, Hepatology
  • insulin gene control region which is active in pancreatic beta cells (Hanahan et al, 1985, Nature 315: 115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38: 647-658; Adams et al, 1985, Nature 318: 533-538; Alexander et al, 1987, Mol Cell Biol. 1: 1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45: 485-495), albumin gene control region which is active in liver (Pinckert et al, 1987, Genes andDevel.
  • alpha- fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol Cell. Biol. 5: 1639-1648; Hammer et al, 1987, Science 235: 53-58), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al, 1987, Genes andDevel.
  • beta globin gene control region which is active in myeloid cells (Mogra et al, 1985, Nature 315: 338-340; Kollias et al, 1986, Cell 46: 89-94), myelin basic protein gene control region which is active in ohgodendrocyte cells of the brain (Readhead et al, 1987, Cell 48: 703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani 1985, Nature 314: 283-286), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al, 1986, Science 234: 1372-1378).
  • a variety of host- vector systems may be utilized to express the protein-coding sequence for GRP or GRP-R. These include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • GRP or GRP-R protein, derivative or analog may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • GRP and GRP-R proteins may also be purified from natural sources by any standard purification method.
  • a GRP or GRP-R protein, analog or derivative can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al, 1984, Nature 310:105-111).
  • proteins are provided consisting of or comprising a fragment of GRP or GRP-R protein consisting of at least ten contiguous amino acids.
  • the fragment consists of at least 20, 30, 40, or 50 contiguous amino acids of GRP or GRP-R for use, for example, in raising antibodies, hi a specific embodiment the fragment of GRP comprises the N-terminal portion of the protein, comprising e.g., amino acids 1 through 27.
  • the fragment of GRP comprises amino acids 31 through 98.
  • Such fragments can also be useful, for example, as standards or controls in the methods and kits of the invention.
  • GRP or GRP-R derivatives and analogs can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the cloned gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the modified coding region remains within the same translational reading frame as that encoding native GRP or GRP-R, m interrupted by translational stop signals, in the coding region where the desired activity is encoded (e.g. the "desired activity" of GRP can be the binding capacity to GRP-R and the “desired activity” of GRP-R can be the binding capacity for GRP).
  • a GRP or GRP-R-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or delete translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), etc.
  • GRP or GRP-R sequences may also be made at the protein level. Included within the scope of the invention are GRP or GRP-R protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • GRP or GRP-R can be chemically synthesized.
  • a peptide corresponding to a portion of a GRP protein which comprises a desired region (e.g., amino acids 1-27), or which mediates the desired functional activity (i.e. binding to GRP-R) in vitro can be synthesized by use of a peptide synthesizer.
  • one or more non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the GRP or GRP-R sequence.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, 7-Abu, 6-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • the GRP or GRP-R derivative is a chimeric, or fusion, protein comprising a GRP or GRP-R protein or fragment thereof preferably consisting of at least a domain or motif of the GRP or GRP-R protein, or at least e.g., 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 160, and preferably at least about 20, amino acids of a GRP or GRP-R protein joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a GRP or GRP-R-coding sequence joined in-frame to a coding sequence for a different protein).
  • a nucleic acid encoding the protein comprising a GRP or GRP-R-coding sequence joined in-frame to a coding sequence for a different protein.
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • Chimeric genes comprising portions of GRP or GRP-R fused to any heterologous protein-encoding sequences may be constructed.
  • GRP, or GRP-R or derivatives or analogs thereof can be fused to various epitope tags known in the art. Epitope tagging is a valuable tool for quick detection, isolation, and analysis of protein-protein interactions, without any prior knowledge of the target proteins.
  • the FLAG epitope tag an eight amino acid peptide (DYKDDDDK) (SEQ ID NO; 5) that can be detected by anti-FLAG monoclonal antibody can be used (Zhang et al , 2001 , Mol Biotechnol, 19(3):313-21) for the construction of chimeric proteins of the invention.
  • DYKDDDDK eight amino acid peptide
  • Preferred polynucleotides and polypeptides of GRP or GRP-R for use in the invention have at least a portion of the sequences presented in Figures 2 and 3, respectively.
  • Other useful polynucleotides and polypeptides of the invention are ones that are substantially identical (e.g., degenerate variants or those that have at least about 45%, preferably about 55%, about 65%, about 75%, about 85%, about 95%, about 98%, or about 99% sequence identity) to any of such polypeptides or polynucleotides of the invention and which retain a functional and/or structural activity of the corresponding naturally-occurring protein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990 Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993 Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, 1990 J.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997 Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted. Additional standard methods for sequence comparisons are available in the art.
  • nucleotide and amino acid sequences of GRP and GRP-R are provided in Figures 2 and 3 (GENBANK accession numbers for human GRP cDNA and protein are XM-008719 and XP-008719, respectively; GENBANK accession numbers for human GRP-R cDNA and protein are M73481 and P30550, respectively). Nucleotide sequences and corresponding amino acid sequences for GRP and GRP-R having similarity (as described by the definitions and methods above) to the ones presented in Figures 2 and 3 are also provided for use in the methods and kits of the invention.
  • GRP-R nucleotide sequences and corresponding amino acid sequences for GRP-R from mouse and rat are provided (GENBANK nucleotide accession numbers M57922 , U84263, NM-00817, and X56661, with corresponding GENBANK protein accession numbers P21729, AAD00557, NP-032203, P52500, respectively; see also Giladi et al. 1993, J. of Molecular Neuroscience, 4(l):41-54; Battey et al, 1991, Proc. Natl. Acad. Sci.
  • the screening methods and kits of the invention may include anti-GRP or anti-GRP-R antibodies. Accordingly, any of GRP or GRP-R fragments, derivatives, or analogs thereof, may be used as immunogens to generate antibodies which immunospecifically bind to GRP or GRP-R. Such antibodies may include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a specific embodiment, antibodies to human GRP protein or human GRP-R protein are produced. In another embodiment, antibodies to a region of GRP (e.g. residues 1-27, or residues 31-98) or GRP-R protein are produced.
  • GRP e.g. residues 1-27, or residues 31-98
  • fragments of GRP or GRP-R that have been identified as hydrophilic are used as immunogens for antibody production.
  • design of antibodies that can recognize and be used to quantify GRP-R may optionally take into account the possibly dispersed nature of the GRP binding site on GRP-R.
  • polyclonal antibodies to GRP or GRP-R proteins may be obtained.
  • various host animals including but not limited to rabbits, mice, rats, etc. can be immunized by injection with the native GRP or GRP-R protein, or synthetic version, or derivatives (e.g., fragments) thereof.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) .
  • BCG Bacille Calmette-Guerin
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), or the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), or the EBV-hybridoma technique to produce human monoclonal antibodies can be used.
  • Monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
  • human antibodies may be used and can be obtained using human hybridomas (Cole et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
  • Techniques developed for the production of "chimeric antibodies” can also be used (see, e.g., Morrison et al, 1984, Proc. Natl. Acad. Sci. U.S.A.
  • humanized monoclonal antibodies may be used in the methods and kits of the invention.
  • humanized antibodies are antibody molecules from non-human species having one or more hypervariable or complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs hypervariable or complementarity determining regions
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three CDRs. The extent of the framework region and CDRs have been precisely defined (see, Kabat et al, 1983, "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services). While such humanized antibodies are commonly developed with therapeutics in mind, such antibodies may also be useful for quantitating GRP or its receptor in the methods and kits of the invention.
  • the monoclonal antibody is a murine monoclonal antibody, designated as "2All"or an antibody having equivalent properties.
  • This antibody is an anti- GRP antibody described by Kelley et ⁇ /., 1997 Chest, 112(1):256-261.
  • Antibody fragments which contain the GRP or GRP-R binding region of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment, which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, the Fab fragments, which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • Such fragments may be used for quantitating GRP or GRP-R according to any available method known in the art.
  • Antibodies to a peptide portion of a GRP or GRP-R can be utilized to generate anti-idiotype antibodies that "mimic" the peptide, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J1(5), 437-444; and Nissinoff, 1991, J. Immunol. 147(8), 2429-2438).
  • antibodies which bind to GRP and competitively inhibit the binding of GRP to GRP-R can be used to generate anti-idiotypes that "mimic" the peptide receptor and, therefore, bind the peptide.
  • Such antibodies may be used for quantitating GRP or GRP-R according to any available method known in the art.
  • a molecular clone of an antibody to an antigen of interest can be prepared by well known techniques. Recombinant DNA methodology (see e.g., Maniatis et al, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
  • Antibody molecules may be purified by well known techniques, e.g. , immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • the foregoing antibodies can be used according to known methods to localize and/or quantify the GRP or GRP-R protein, e.g., to measure levels thereof in appropriate physiological samples, in diagnostic methods, etc.
  • a sample for screening and/or diagnostic purposes may include but is not limited to stool, urine, seminal fluid, or blood obtained from an asymptomatic subject. Such samples may be obtained by any method known in the art (e.g., a finger-stick blood sample).
  • Diagnosis is defined as the act or process of determining the nature or cause of a disease through examination of a patient. Screening is defined as testing a sample taken from the patient for the presence of a disease or infection which does not necessarily require physical examination of the patient and, which involves any of various biochemical assays.
  • GRP and GRP-R proteins, derivatives, analogs and sub-sequences thereof, GRP and GRP-R nucleic acids (and sequences complementary thereto), and anti-GRP and anti-GRP-R antibodies are suitable for use in diagnostics and screening for cancers according to the invention.
  • Such molecules can be used in assays, such as immunoassays, hybridization assays, or PCR assays to detect or monitor aberrant levels of GRP and/or GRP-R.
  • an immunoassay may be carried out by a method comprising contacting a sample derived from an asymptomatic subject with an anti-GRP or anti-GRP-R antibody under conditions such that immunospecific binding to form a complex can occur, and detecting or measuring the amount of the complex, hi a specific embodiment, an antibody to GRP or GRP-R can be used to assay a sample (i.e stool, urine, and blood) for the presence of GRP or GRP-R, respectively, or a metabolite or derivative thereof, wherein an increased level of either compared to a standard sample indicates the presence of a cancerous state in the asymptomatic subject.
  • a sample i.e stool, urine, and blood
  • the presence of GRP in a sample obtained from an asymptomatic subject can be detected by a solid phase GRP radioimmunoassay (RIA) using 125 I-labeled antibodies (Yang et al, 1998, Lung Cancer, 21(3): 165-75.).
  • the 125 I-labeled-antibody may be an 125 I-labeled monoclonal antibody having the binding characteristics of monoclonal antibody 2A11 as described by Cuttitta et al. (1985, Nature, 316:823-826).
  • antibodies to a region of GRP for e.g., GRP(31-98) in human gastrin releasing peptide precursor can be used in blood samples isolated from asymptomatic subject(s).
  • This GRP precursor is extremely stable in blood and is extracellularly secreted through the rough endoplasmic reticulum without being affected by any of the proteolytic enzymes, rather than passing through the regulated pathway involving secretory particles.
  • This method of detection of GRP(31 -98) may be advantageous to detecting GRP or other fragments of GRP such as GRP(l-27) (see EP 0 811 683 Al; October 12, 1997), as conventionally done, since this latter fragment of GRP may not be stable in blood.
  • the immunoassays that can be used include but are not limited to competitive and non- competitive assay systems using techniques such as Western blots, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, imtnunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, chemiluminescent immunoassays, and protein A immunoassays.
  • Western blots such as Western blots, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, imtnunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays
  • Nucleic acids encoding GRP or GRP-R proteins, and related nucleotide sequences and sub- sequences, including complementary sequences, can be used in hybridization assays to detect and quantify mRNA.
  • Oligonucleotide molecules comprising or consisting of the GRP or GRP-R nucleotide sequences, or sub-sequences thereof comprising at least about 8, 10, 12, 14, 16, 18, or 20 sequence-specific nucleotides, can be used as hybridization probes. Longer probes can also be used.
  • Such assays can be used to detect or screen for the various cancers associated with increased levels of GRP or GRP-R.
  • such a hybridization assay may be carried out by a method comprising contacting a sample containing mRNA from a subject with a nucleic acid probe capable of hybridizing to GRP or GRP-R mRNA, under conditions conducive to the formation of a hybridization complex, and detecting or measuring the amount of the resulting hybridization complex by any method known to those skilled in the art.
  • cancers involving or characterized by increased levels of GRP or GRP-R can be screened for or diagnosed or their presence can be detected by detecting an increased level of GRP or GRP-R protein or nucleic acid, or by detecting increased GRP or GRP-R-functional activity.
  • GRP-R pharmacology differs from what is observed for GRP-R that is expressed by non-malignant tissues, h non-malignant cells, human GRP-R, for example, binds agonists in the nanomolar range.
  • GRP-R' s that are aberrantly expressed by human malignant cells have been shown to bind agonists with altered affinities relative to the non-malignant GRP-R' s.
  • accumulating evidence indicates that GRP-R is frequently mutated and rendered nonfunctional (i.e.
  • a sample from the asymptomatic subject is analyzed by Southern blotting or by RFLP analysis using nucleic acid probes in order to detect mutations in GRP-R.
  • a sample from the asymptomatic subject is subjected to DNA sequencing of GRP-R and compared to the wild type sequence of GRP-R in order to detect any mutations or deviations from the wild type sequence.
  • GRP or GRP-R nucleic acids in a sample from an asymptomatic subject may be amplified and/or quantitated by PCR using well known methods (Innis et al, 1990, PCR Protocols, Academic Press, Inc., San Diego, CA; Erlich, ed., 1989, PCR Technology, Principles and Applications for DNA Amplification, Stockton Press, New York; Erlich et al, 1991, Recent Advances in the Polymerase Chain Reaction, Science 252, 1643-1651). DNA may also be cloned inpCR2.1, a vector commonly used for GRP-R (Carroll et al. , 2000, Molecular Pharmacology, 58(3):601-607).
  • GRP-R may also be subjected to conformational fragment length polymorphism (RFLP) and dideoxy sequencing, methods known to those skilled in the art.
  • RFLP conformational fragment length polymorphism
  • methods incorporated herein by reference and known to those skilled in the art Carroll et al, 2000, Molecular Pharmacology, 58(3):601-607; Brow et al, 1996, Focus, 18:2-5; Carroll et al, 1999, Peptides, 20:229-37; Lyamichev et al, 1993, Science, 260:778-83
  • mutations in GRP-R can be rapidly determined, h a specific embodiment, mutation resulting in substitution of prolinel45 to tyrosine, and also mutation leading to substitution of valine317 to glutamic acid in GRP-R can be screened in the methods and kits of the invention.
  • the PPEM method may be used for detection of mutations in GRP-R together with the methods and kits of theinvention.
  • the PPEM (PNA-directed PCR, primer extension, MALDI-TOF) method has been described and is known to those skilled in the art (Sun et al, 2002, Nature Biotechnology, 20(2): 186-9). Briefly, this method is highly sensitive and specific for detecting mutations in samples that contain a small percentage of mutated cells in a large background of normal cells.
  • DNA samples obtained from the asymptomatic subject can be amplified using PNA-directed PCR clamping reactions in which the mutated DNA can be enriched. The PCR-amplified DNA fragments are then sequenced through primer extension.
  • mutations are identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI- TOF) (Sun et al, 2002, Nature Biotechnology, 20(2): 186-9).
  • MALDI- TOF matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • the PPEM method can be used to detect GRP in the samples obtained from the asymptomatic subject. Even though GRP mutations have not been reported in malignancies, this method can be used to rapidly screen for the presence of GRP.
  • a sample from an asymptomatic subject may be subjected to reverse transcriptase PCR (RT-PCR) and the presence of GRP or GRP-R mRNA can be detected from hybridization assays, by conventional methods (Bold et al, 1998, Cancer Investigation,16(l):12-ll; Aprikian et al, 1996, J. Mol Endocrinologyl6(3):291 -306).
  • RT-PCR reverse transcriptase PCR
  • sample(s) from an asymptomatic subject can be analyzed using HPLC chromatographic methods for detection of GRP and/or GRP derivatives (i.e. fragments) thereof, by conventional methods known to those skilled in the art (Kim et al, 2001, Am J. Physiology Regul Integr. Comp. Physiol 281(2):R902-8).
  • the HPLC fractions can be assayed for the presence of GRP and/or derivatives thereof by immunodetection.
  • the fractions can be subjected to MALDI mass spectrometric analysis for identification of GRP and/or derivatives thereof.
  • a nucleic acid for use in the methods and kits of the invention which is hybridizable to a GRP or GRP-R encoding nucleic acid (e.g., having a sequence as set forth in Figure 2 and 3) or to its complement, or to a nucleic acid encoding a GRP or GRP-R derivative, or to its complement, in order to, for example, quantitate mRNA levels of GRP or GRP-R via hybridization.
  • Methods for quantitation of mRNA are well known in the art (see e.g., Jensen LE et al, 1998, J. Immunol.
  • the hybridization can be performed under various conditions of stringency.
  • procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792).
  • Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used.
  • Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and re-exposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • a nucleic acid is provided which is hybridizable to a GRP or GRP-R encoding nucleic acid, or its complement, under conditions of high stringency.
  • procedures using such conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured sahnon sperm DNA.
  • Filters are hybridized for 48 h at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe. Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.
  • a nucleic acid is provided which is hybridizable to a GRP or GRP-R nucleic acid, or its reverse complement, under conditions of moderate stringency. Selection of appropriate conditions for such stringencies is well known in the art (see e.g., Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
  • Nucleic acids encoding derivatives and analogs of GRP or GRP-R proteins, and GRP and GRP-R antisense nucleic acids are additionally provided.
  • stringency conditions for washing are as follows. Each membrane is washed two times each for 30 minutes each at 45 °C in 40 mM sodium phosphate, pH 7.2, 5% SDS, 1 mM EDTA, 0.5% bovine serum albumin, followed by four washes each for 30 minutes in sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA, and subsequently each membrane is treated differently as described below for low, medium, or high stringency hybridization conditions. For low stringency hybridization, membranes are not washed further.
  • membranes are additionally subjected to four washes each for 30 minutes in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 55°C.
  • membranes are additionally subjected to four washes each for 30 minutes in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 55°C, followed by four washes each for 30 minutes in sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 65°C.
  • a nucleic acid is provided which is hybridizable to a GRP or GRP-R nucleic acid having a sequence as set forth in Figure 2 or 3, or to a complement thereof, under conditions of low, medium or high stringency.
  • Hybridization protocols of differing stringencies are well known in the art. The temperature and salt concentrations at which hybridizations are performed have a direct effect on the results that are obtained.
  • a denaturing agent such as formamide is typically used during hybridization.
  • Formamide is typically used at 25% to 50% (v/v) in a buffered diluent comprising IX to 6X SSC (IX SSC is 150 mM NaCl and 15mM sodium citrate; SSPE may be substituted for SSC, where IX SSPE is 150mM NaCl, 10 mM NaH 2 P0 4 , and 1.25 mM EDTA, ⁇ H7.4).
  • the hybridization temperature is typically about 42°C.
  • High stringency conditions also employ a wash buffer with low ionic strength, such as from about 0.1X to about 0.5X SSC, at relatively high temperature, typically greater than about 55°C up to about 70°C.
  • Moderately stringent conditions typically use 0% to 25% formamide in IX to 6X SSC, and use reduced hybridization temperatures, usually in the range of about 27°C to about 40°C.
  • the wash buffer can have increased ionic strength, e.g., from about 0.6X to about 2X SSC, and is used at reduced temperatures, usually from about 45°C to about 55°C.
  • the hybridization buffer is the same as that used for moderately stringent or high stringency, but does not contain a denaturing agent.
  • a reduced hybridization temperature is used, typically in the range of from about 25°C to about 30°C.
  • the wash buffer has increased ionic strength, usually from about 2X to about 6X SSC, and the wash temperature is in the range of from about 35°C to about 47°C.
  • GRP-R agonists and antagonists can be used to quantify the number of receptors in a sample from an asymptomatic subject, using well known methods (see Goodman et al , eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, New York, McGraw Hill, 1996).
  • Such agonists and antagonists are also well known in the art (Spindel E.R. et al, 1993, Recent Progress in Hormone Research, 48:365- 391; Schutte J. et al, 1993;129:115-29; Viallet J, and Minna JD., 1989, Prog. Growth Factor Res.;l(2):89-91; Sunday ME.
  • a sample from an asymptomatic subject can be subjected to an affinity assay, whereby affinity for agonists of GRP-R, e.g., bombesin, GRP, GRP(18-27), or neuromedin B, is determined based on the ability of any one of the peptide agonists known in the art to inhibit the binding of 50pM 125 I[Tyr 4 ]Bn, an antagonist of GRP-R (see Jensen RT. and Coy DH., 1991, Trends Pharmacol Sci; 12(1): 13-9). The binding of GRP-R and the agonist is proportional to the number of receptors occupied.
  • GRP-R e.g., bombesin, GRP, GRP(18-27), or neuromedin B
  • Kits for diagnostic and screening use comprise in one or more sterile containers anti-GRP or anti-GRP-R antibodies, and optionally, a labeled binding partner to the antibody.
  • the anti-GRP or anti-GRP-R antibodies can be labeled (with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety).
  • a kit is also provided that comprises in one or more sterile containers a nucleic acid probe or probes capable of hybridizing to GRP or GRP-R encoding mRNA.
  • a kit can comprise in one or more containers a pair of primers (e.g., each in the size range of 6-30 nucleotides) that are capable of priming amplification (e.g., by PCR (see, e.g., Innis et al, 1990, PCR Protocols, Academic Press, Inc., San Diego, CA; Erlich, ed., 1989, PCR Technology, Principles and Applications for DNA Amplification, Stockton Press, New York; Erlich et al, 1991, Recent Advances in the Polymerase Chain Reaction, Science 252, 1643-1651), ligase chain reaction (EP 320,308), use of b-replicase, cyclic probe reaction, or other methods known in the art), under appropriate conditions of at least a portion of GRP or GRP-R encoding nucleic acids.
  • primers e.g., each in the size range of 6-30 nucleotides
  • kits can optionally further comprise in a sterile container a predetermined amount of a purified GRP or GRP-R protein or nucleic acids thereof for use as a standard or control.
  • the kits may also include printed instructions and/or a printed label describing the practicing of the invention in accordance with one or more of the embodiments described herein.

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  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
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  • Physics & Mathematics (AREA)
  • Oncology (AREA)
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Abstract

La présente invention concerne l'utilisation d'acides nucléiques GRP et GRP-R, de protéines, de dérivés et d'analogues correspondants pour le dépistage de tumeurs malignes chez des sujets asymptomatiques. Plus particulièrement, l'invention concerne des méthodes de dépistage du cancer du tractus gastro-intestinal (côlon, pancréas, estomac) et de la prostate.
PCT/US2003/015466 2002-05-16 2003-05-16 Utilisation du peptide liberateur de gastrine (grp) et de son recepteur dans le depistage du cancer WO2003097666A2 (fr)

Priority Applications (1)

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AU2003234623A AU2003234623A1 (en) 2002-05-16 2003-05-16 Use of gastrin releasing peptide (grp) and its receptor in cancer screening

Applications Claiming Priority (2)

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US38115302P 2002-05-16 2002-05-16
US60/381,153 2002-05-16

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WO2003097666A2 true WO2003097666A2 (fr) 2003-11-27
WO2003097666A3 WO2003097666A3 (fr) 2004-07-01

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008507261A (ja) * 2004-01-27 2008-03-13 コンピュゲン ユーエスエイ，インク． 肺癌診断のための新規のヌクレオチド配列およびアミノ酸配列、ならびにそのアッセイおよび使用方法
US7927814B2 (en) * 2005-04-13 2011-04-19 Advanced Life Science Institute, Inc. Antibody directed against gastrin-releasing peptide precursor and use thereof
WO2015143525A1 (fr) * 2014-03-27 2015-10-01 Ziel Biosciências Pesquisa, Desenvolvimento E Diagnóstico Ltda. Anticorps anti-grpr, procédé d'obtention, procédé de détection, utilisation d'anticorps, trousse et construction génétique
WO2016011068A1 (fr) 2014-07-14 2016-01-21 Allegro Diagnostics Corp. Procédés pour évaluer le stade d'un cancer du poumon
US11639527B2 (en) 2014-11-05 2023-05-02 Veracyte, Inc. Methods for nucleic acid sequencing
US11976329B2 (en) 2013-03-15 2024-05-07 Veracyte, Inc. Methods and systems for detecting usual interstitial pneumonia
US12110554B2 (en) 2009-05-07 2024-10-08 Veracyte, Inc. Methods for classification of tissue samples as positive or negative for cancer
US12297505B2 (en) 2014-07-14 2025-05-13 Veracyte, Inc. Algorithms for disease diagnostics
US12305238B2 (en) 2024-09-23 2025-05-20 Veracyte, Inc. Methods for treatment of thyroid cancer

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* Cited by examiner, † Cited by third party
Title
BAI G. ET AL.: 'Development and application of a sandwich enzyme immunoassay for glycyrrhizae radix protein (GRP) using monoclonal antibodies' BIOL. PHARM. BULL. vol. 20, no. 12, December 1997, pages 1224 - 1228, XP002976914 *
CARROLL R.E. ET AL.: 'Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans' AM. J. PHYSIOL. vol. 276, 1999, pages G655 - G665, XP002976919 *
CARROLL R.E. ET AL.: 'Characterization of gastrin-releasing peptide and its receptor aberrantly expressed by human colon cancer cell lines' MOLECULAR PHARMACOLOGY vol. 58, no. 3, 2000, pages 601 - 607, XP002976918 *
CARROLL R.E. ET AL.: 'Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer' CELL GROWTH AND DIFFERENTIATION vol. 11, July 2000, pages 385 - 393, XP002976916 *
CHAVE H.S. ET AL.: 'Bombesin family receptor and ligand gene expression in human colorectal cancer and normal mucosa' BRITISH JOURNAL OF CANCER vol. 82, no. 1, 2000, pages 124 - 130, XP002976915 *
JENSEN J.A.G. ET AL.: 'The case for gastrin-releasing peptide acting as a morphogen when it and its receptor are aberrantly expressed in cancer' PEPTIDES vol. 22, 2001, pages 689 - 699, XP002976912 *
MATKOWSKYJ K.A. ET AL.: 'Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407' JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY vol. 51, no. 8, 2003, pages 1041 - 1048, XP002976920 *
MENORET A. ET AL.: 'Expression of the 100-kda glucose-regulated protein (GRP100/endoplasmin) is associated with tumorigenicity in a model of rat colon adenocarcinoma' INT. J. CANCER vol. 56, no. 3, 01 February 1994, pages 400 - 405, XP002976913 *
SAURIN J-C. ET AL.: 'High gastrin releasing peptide receptor mRNA level is related to tumour dedifferentiation and lymphatic vessel invasion in human colon cancer' EUROPEAN JOURNAL OF CANCER vol. 35, no. 1, 1999, pages 125 - 132, XP002976917 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008507261A (ja) * 2004-01-27 2008-03-13 コンピュゲン ユーエスエイ，インク． 肺癌診断のための新規のヌクレオチド配列およびアミノ酸配列、ならびにそのアッセイおよび使用方法
US7927814B2 (en) * 2005-04-13 2011-04-19 Advanced Life Science Institute, Inc. Antibody directed against gastrin-releasing peptide precursor and use thereof
US12110554B2 (en) 2009-05-07 2024-10-08 Veracyte, Inc. Methods for classification of tissue samples as positive or negative for cancer
US12297503B2 (en) 2009-05-07 2025-05-13 Veracyte, Inc. Methods for classification of tissue samples as positive or negative for cancer
US11976329B2 (en) 2013-03-15 2024-05-07 Veracyte, Inc. Methods and systems for detecting usual interstitial pneumonia
WO2015143525A1 (fr) * 2014-03-27 2015-10-01 Ziel Biosciências Pesquisa, Desenvolvimento E Diagnóstico Ltda. Anticorps anti-grpr, procédé d'obtention, procédé de détection, utilisation d'anticorps, trousse et construction génétique
WO2016011068A1 (fr) 2014-07-14 2016-01-21 Allegro Diagnostics Corp. Procédés pour évaluer le stade d'un cancer du poumon
EP3169814B1 (fr) * 2014-07-14 2022-05-04 Veracyte, Inc. Procédés pour évaluer le stade d'un cancer du poumon
US12297505B2 (en) 2014-07-14 2025-05-13 Veracyte, Inc. Algorithms for disease diagnostics
US11639527B2 (en) 2014-11-05 2023-05-02 Veracyte, Inc. Methods for nucleic acid sequencing
US12305238B2 (en) 2024-09-23 2025-05-20 Veracyte, Inc. Methods for treatment of thyroid cancer

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AU2003234623A1 (en) 2003-12-02
WO2003097666A3 (fr) 2004-07-01

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