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EP1711525A2 - Polypeptides specifiques aux tumeurs et leurs utilisations - Google Patents

Polypeptides specifiques aux tumeurs et leurs utilisations

Info

Publication number
EP1711525A2
EP1711525A2 EP05780003A EP05780003A EP1711525A2 EP 1711525 A2 EP1711525 A2 EP 1711525A2 EP 05780003 A EP05780003 A EP 05780003A EP 05780003 A EP05780003 A EP 05780003A EP 1711525 A2 EP1711525 A2 EP 1711525A2
Authority
EP
European Patent Office
Prior art keywords
cell
seq
polypeptide
antibody
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05780003A
Other languages
German (de)
English (en)
Inventor
Heinz Peter Vollmers
Hans Konrad MÜLLER-HERMELINK
Frank Hensel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Debiovision Inc
Original Assignee
Debiovision Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/764,730 external-priority patent/US7378247B2/en
Application filed by Debiovision Inc filed Critical Debiovision Inc
Priority to EP10011483A priority Critical patent/EP2361929A1/fr
Publication of EP1711525A2 publication Critical patent/EP1711525A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention is related to the field of cancer diagnosis and treatment and, more specifically, to polypeptides and methods of using these polypeptides in the diagnosis, detection, monitoring, and treatment of neoplasms in a mammal, e.g., a human.
  • a mammal e.g., a human.
  • the present invention is related to the field of cancer diagnosis and treatment and, more specifically, to polypeptides and methods of using these polypeptides in the diagnosis, detection, monitoring, and treatment of neoplasms in a mammal, e.g., a human.
  • a mammal e.g., a human.
  • neoplasms e.g., stomach adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, adenocarcinoma of the pancreas
  • human monoclonal antibodies produced from B-cell hybridomas may be used in the treatment of tumors, viral and microbial infections, B-cell immunodeficiencies with reduced antibody production, and other impairments of the immune system.
  • Gastric carcinoma is one of the most frequently occurring types of cancer worldwide and is histologically divided into diffuse adenocarcinoma and intestinal adenocarcinoma. Intestinal gastric carcinomas are often accompanied by chronic type B gastritis and particularly by intestinal metaplasias, which are considered to be precursors of dysplastic changes and of gastric carcinomas. Differences between these two types of gastric carcinomas are also evident in that patients having carcinomas of the diffuse type often belong to blood group A, from which the influence of genetic factors on the cancer risk may be concluded, while environmental factors, e.g., a Helicobacter pylori infection, is possibly significant for the occurrence of carcinomas of the intestinal type.
  • environmental factors e.g., a Helicobacter pylori infection
  • stomach cancer is a multi-step and multi-factor process (Correa, Cancer Res. 52:6735-6740 (1992)). Although little is known about molecular mechanisms, factors such as high salt intake, alcohol, nitrosamines, and infection with the bacterium Helicobacter pylori (H. pylori) are clearly proven to be involved in the initiation of stomach carcinogenesis. Due to a strong correlation between H. pylori infection and the occurrence of gastritis, dysplasia, and development of gastric cancer, the bacterium has been classified as a class I carcinogen by the WHO. H.
  • Gastroenterol. 29:961-965 (1994)) and Lewis blood group antigens (Appelmelk et al, Trends. Microbiol. 5:70-73 (1997)) are frequently found in stomach mucosa or stomach cancer.
  • Therapeutic methods for treating stomach cancer are currently restricted to gastrectomy and lymphadenectomy. Due to the poor prognosis associated with these methods, there is a need for alternative or additional therapeutic and diagnostic methods.
  • the present invention features polypeptides and polypeptide fragments that specifically bind a novel isoform of CFR-1 that is expressed on neoplastic cells as well as on cells of pre-cancerous lesions, but not on normal cells. These polypeptides may be used in the therapy and diagnosis not only of tumors, but also of pre-cancerous structures both in vitro and in vivo.
  • the novel isoform of CFR-1, fragments of this novel isoform, and nucleic acids encoding this novel isoform may be used in methods of inducing an immune response against a neoplastic cell, in methods of generating antibodies that specifically bind this novel isoform or fragments of this isoform, and in screening methods to identify additional therapeutic compounds.
  • the first aspect of the invention features an isolated polypeptide that specifically binds to a neoplastic cell or a cell of a pre-cancerous lesion, but does not specifically bind to a normal cell, where the normal cell is not a cell of the glomerular, fascicular zone of the adrenal gland or an epithelial cell of the collection tubes of the kidney.
  • This isolated polypeptide may include amino acids 28-32, 51-53, and/or 90-100 of the sequence of SEQ ID NO:27.
  • the isolated polypeptide also includes amino acids 11-18, 36-43, and/or 82-104 of the sequence of SEQ ID NO:26.
  • the invention features an isolated polypeptide that includes amino acids 11-15, 30-46, and/or 79-88 of the sequence of SEQ ID NO:2 and/or amino acids 17-32, 48-54, and/or 87-95 of the sequence of SEQ ID NO:4, but does not include the full-length sequence of SEQ ID NO:2 or SEQ ID NO:4 and that that specifically binds to a neoplastic cell or a cell of a pre-cancerous lesion, but does not specifically bind to a normal cell, where the normal cell is not a cell of the glomerular, fascicular zone of the adrenal gland or an epithelial cell of the collection tubes of the kidney.
  • the polypeptide includes amino acids 11-18, 36-43, and/or 82-104 of SEQ ID NO:26 or amino acids 28-32, 51-53, and/or 90-100 of SEQ ID NO:27, but does not include the full-length amino acid sequence of SEQ ID NO:26 or SEQ ID NO:27.
  • the polypeptide is capable of inducing apoptosis of the neoplastic cell or the cell of the pre-cancerous lesion, but does not induce apoptosis of the normal cell
  • the neoplastic may be a Barrett's tumor cell or a cell of a tumor of the esophagus, stomach, intestine, rectum, liver, gallbladder, pancreas, lungs, bronchi, breast, cervix, prostate, heart, ovary, or uterus.
  • the pre-cancerous lesion may be a dysplasia of the gastric mucosa, interstitial metaplasia of the stomach, inflammation of the gastric mucosa which is associated with the bacteria Helicobacter pylori, tubular and tubulovillous adenomas of the stomach, tubular adenoma of the colon, villous adenoma of the colon, dysplasia in ulcerative colitis, Barrett's dysplasia, Barrett's metaplasia of the esophagus, cervical intraepithelial neoplasia I, cervical intraepithelial neoplasia TJ, cervical intraepithelial neoplasia DJ, squamous epithelial metaplasia, squamous epithelial dysplasia of the bronchus, low grade and high grade prostate intraepithelial neoplasia (PIN) > breast ductal carcinoma in situ (D-CIS) or breast lobal
  • the polypeptide is a functional fragment of an antibody, e.g., a V L , V H , F V , F , Fab, Fab', or F(ab') 2 antibody fragment, h addition, the polypeptide may specifically bind to a polypeptide comprising SEQ ID NO: 6 or a fragment thereof.
  • the invention features an isolated nucleic acid molecule containing nucleic acids 31-54, 106-129, and/or 244-312 of the sequence of SEQ ID NO:28, and/or 82-96, 151-159, and/or 268-300 of the sequence of SEQ ID NO:29.
  • the isolated nucleic acid molecule does not include the full-length sequence or SEQ ID NO:28 and or SEQ ID NO:29.
  • the invention features an isolated nucleic acid molecule containing nucleic acids 31-45, 88-138, and/or 235-264 of SEQ ID NO:l. Desirably, this nucleic acid molecule does not include the full-length sequence of SEQ ID NO: 1.
  • the invention features an isolated nucleic acid molecule containing nucleic acids 49-96, 142-162, and/or 259-285 of SEQ ID NO:3.
  • the nucleic acid molecule does not include the full- length sequence of SEQ ID NO:3.
  • the invention features an isolated nucleic acid molecule including the sequence of SEQ ID NO: 5 and in the fifth aspect, the invention features a vector containing the nucleic acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:28, and/or SEQ ID NO:29.
  • the invention features an isolated cell, e.g., a mammalian cell, containing a vector that includes the nucleic acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:28, and/or SEQ ID NO:29.
  • the invention features an isolated cell, e.g., a mammalian cell, that expresses the polypeptide of the first aspect of the invention.
  • the cell of the seventh aspect of the invention is a human cell.
  • the invention features a method of producing the purified polypeptide of the first aspects of the invention. This method involves contacting a cell with a vector that includes SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:28, and/or SEQ ID NO:29 and isolating the polypeptide expressed by the vector.
  • the ninth aspect of the invention features a method of diagnosing a neoplasm or a pre-cancerous lesion in a mammal, e.g., a human.
  • This method involves the steps of (a) contacting a cell or tissue sample derived from the mammal with a purified polypeptide of the first aspects of the invention, and (b) detecting whether the purified polypeptide specifically binds to the cell or tissue sample, where specific binding of the purified polypeptide to the cell or tissue sample is indicative of the mammal having a neoplasm or pre-cancerous lesion.
  • the cell or tissue sample maybe Barrett's tumors, tumors of the esophagus, stomach, intestine, rectum, liver, gallbladder, pancreas, lungs, bronchi, breast, cervix, prostate, heart, ovary, and uterus, dysplasia of the gastric mucosa, interstitial metaplasia of the stomach, inflammation of the gastric mucosa which is associated with the bacteria Helicobacter pylori, tubular and tubulovillous adenomas of the stomach, tubular adenoma of the colon, villous adenoma of the colon, dysplasia in ulcerative colitis, Barrett's dysplasia, Barrett's metaplasia of the esophagus, cervical intraepithelial neoplasia I, cervical intraepithelial neoplasia ⁇ , cervical intraepithelial neoplasia HI, squa
  • the polypeptide is an antibody, such as murine antibody 58/47-69.
  • the polypeptide is conjugated to a detectable agent.
  • This detectable agent may be a radionuclide, a fluorescent marker, an enzyme, a cytotoxin, a cytokine, or a growth inhibitor and the detectable agent may also be capable of inducing apoptosis of the cell.
  • the polypeptide of the ninth aspect may be conjugated to a protein purification tag, e.g., a cleavable protein purification tag.
  • the tenth aspect of the invention features a method of treating a proliferative disorder in a mammal, for example, a human.
  • This method involves the step of contacting a cell with the purified polypeptide of the first aspect, where binding of the purified polypeptide to the cell results in the induction of apoptosis of the cell.
  • the polypeptide is an antibody, e.g., a humanized antibody, a chimeric antibody (i.e., one that comprises amino acid sequence derived from more than one species) or murine antibody 58/47-69.
  • the polypeptide of the tenth aspect of the invention may be conjugated to a detectable agent.
  • This detectable agent may be a radionuclide, a fluorescent marker, an enzyme, a cytotoxin, a cytokine, or a growth inhibitor.
  • the polypeptide may also be conjugated to a protein purification tag, such as a cleavable protein purification tag.
  • the invention features a pharmaceutical composition that contains the isolated polypeptide of the first aspect in a pharmaceutically acceptable carrier and in the twelfth aspect, the invention features a diagnostic agent containing the isolated polypeptide of the first aspect of the invention.
  • the invention features an isolated polypeptide, for example, one that is 90%, 95%, or 99% pure, that includes amino acids 469-518 of SEQ ID NO:6 and/or amino acids 739-748 of SEQ ID NO:6.
  • the polypeptide does not include the full-length sequence of SEQ ID NO:6.
  • the polypeptide of the thirteenth aspect is encoded by the nucleic acid sequence of SEQ ID NO:5.
  • the polypeptide is specifically bound by murine antibody 58/47-69 and/or includes a tumor-specific glycostructure.
  • the polypeptide may have an apparent molecular weight of approximately 130 IcD on a polyacrylamide gel and may be a polypeptide expressed by adenocarcinoma cell line 23132. In other desirable embodiments of the thirteenth aspect of the invention, the polypeptide is expressed by a pre-cancerous lesion and not by normal cells of the same tissue type.
  • This pre-cancerous lesion may be dysplasia of the gastric mucosa, interstitial metaplasia of the stomach, inflammation of the gastric mucosa which is associated with the bacteria Helicobacter pylori, tubular and rubulovillous adenomas of the stomach, tubular adenoma of the colon, villous adenoma of the colon, dysplasia in ulcerative colitis, Barrett's dysplasia, Barrett's metaplasia of the esophagus, cervical intraepithelial neoplasia I, cervical intraepithelial neoplasia IL cervical intraepithelial neoplasia HI, squamous epithelial metaplasia, squamous epithelial dysplasia of the bronchus, low grade and high grade prostate intraepithelial neoplasia (PIN), breast ductal carcinoma in situ (D-CIS) or breast lobular carcinoma in situ
  • the polypeptide of the thirteenth aspect of the invention may be expressed by a tumor such as Barrett's tumor, or tumors of the esophagus, stomach, intestine, rectum, liver, gallbladder, pancreas, lungs, bronchi, breast, cervix, prostate, heart, ovary, or uterus, and not by a normal cell of the same tissue type.
  • the fourteenth aspect of the invention features a pharmaceutical composition that contains the polypeptide of the thirteenth aspect in a pharmaceutically acceptable carrier and the fifteenth aspect features a diagnostic agent including the isolated polypeptide of the thirteenth aspect.
  • the sixteenth aspect of the invention features a method of inducing a tumor- specific immune response in a mammal.
  • This method includes the step of contacting the mammal, e.g., a human, with an isolated polypeptide containing the sequence of SEQ ID NO:6 or an isolated polypeptide that comprises amino acids 469-518 of SEQ ID NO:6 or amino acids 739-748 of SEQ ID NO:6 and does not comprise the full- length sequence of SEQ ID NO: 6, where the contacting induces a tumor-specific immune response in the mammal.
  • the tumor-specific immune response involves the production of an antibody that induces apoptosis of a cell which is specifically bound by the antibody.
  • the invention features a DNA vaccine comprising a plasmid vector or a viral vector which includes a nucleotide sequence encoding SEQ ID NO:6, or a fragment thereof, where, upon administration into a patient, a tumor-specific immune response is induced in the patient, e.g., a human patient.
  • This immune response results in the formation of antibodies that specifically bind to a neoplasm or a pre-cancerous lesion.
  • the invention features a method of producing an isolated polypeptide containing the sequence of SEQ ID NO: 6 or a fragment thereof that comprises amino acids 469-518 of SEQ ID NO:6 or amino acids 739-748 of SEQ ID NO:6 and does not comprise the full-length sequence of SEQ ID NO:6.
  • This method involves the steps of (a) contacting a cell with a vector containing a nucleic acid sequence that is substantially identical or identical to SEQ TD NO: 5 or a fragment thereof and (b) isolating the polypeptide expressed by the cell.
  • the fragment comprises amino acids 469-518 of SEQ ID NO:6 and amino acids 739-748 of SEQ ID NO:6 and does not comprise the full-length sequence of SEQ ID NO:6.
  • the eighteenth aspect of the invention features a method of identifying a candidate therapeutic compound. This method involves the steps of (a) contacting a cell expressing a polypeptide containing the amino acid sequence of SEQ ID NO:6, e.g., adenocarcinoma cell line 23132 (DSMZ Accession No.
  • DSM ACC 201 DSM ACC 201
  • test compound determining whether the test compound induces apoptosis of the cell and not of a control cell contacted with the test compound, where a test compound that induces apoptosis of the cell and not of the control cell is a candidate therapeutic compound.
  • fragments of SEQ ID NO:6, for example, ones that comprise amino acids 469-518 of SEQ ID NO:6 or amino acids 739-748 of SEQ ID NO: 6 and do not comprise the full-length sequence of SEQ ID NO:6, may be used.
  • the invention features a method of producing an antibody that specifically binds to a neoplastic cell.
  • This method involves (a) administering a purified polypeptide having amino acids 469-518 of SEQ ID NO: 6 and/or amino acids 739-748 of SEQ ID NO:6 and not including the full length sequence of SEQ ID NO:6 to a mammal and (b) isolating from the mammal, an antibody that specifically binds to the polypeptide having amino acids 469-518 of SEQ ID NO:6 and/or amino acids
  • the purified polypeptide administered to the mammal may comprise the full-length sequence of SEQ ID NO:6.
  • the polypeptide is purified from adenocarcinoma cell line 23132 (DSMZ Accession No. DSM ACC 201).
  • the polypeptide having amino acids 469-518 of SEQ ID NO:6 and/or amino acids 739-748 of SEQ ID NO:6 includes a tumor-specific glycostructure.
  • the method also involves isolating a cell expressing the antibody from the mammal.
  • PAM-1 antibody is meant an isoform of CFR-1 that includes amino acids 469-518 of SEQ ID NO:6 and/or amino acids 739-748 of SEQ ID NO:6 and that is expressed by neoplastic cells and cells of a pre-cancerous lesion and not by a normal cell, where cells of the glomerular, fascicular zone of the adrenal gland or an epithelial cell of the collection tubes of the kidney are excluded from the group of normal cells.
  • the novel CFR-1 isoform does not comprise the full-length amino acid sequence of SEQ ID NO:6.
  • the novel CFR-1 isoform is specifically-bound by human monoclonal antibody 103/51 and/or murine antibody 58/47-69 and has a molecular mass of approximately 130 kD.
  • the use of the term "approximately” reflects that one skilled in the art would recognize that these types of size determinations are affected by changes or variations of the methods ofthe molecular size determination, e.g., gel electrophoresis conditions.
  • the novel CFR-1 isoform is a polypeptide present on adenocarcinoma cell line 23132 (DSMZ Accession No. DSM ACC 201).
  • the novel CFR-1 isoform is expressed by the cells of the following types of pre-cancerous lesion, but not normal cells: H. pylori induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis- related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung, cervical intraepithelial neoplasia, low grade and high grade prostate intraepithelial neoplasia (PIN), breast ductal carcinoma in situ (D-CIS) and breast lobular carcinoma in situ (L-CIS).
  • H. pylori induced gastritis induced gastritis
  • intestinal metaplasia and dysplasia of the stomach ulcerative colitis- related dysplasia and adenomas of the colon
  • Barrett metaplasia and dysplasia of the esophagus s
  • the novel CFR-1 isoform is expressed by carcinomas of the following tissues, but not normal cells of the same tissues: esophagus, stomach, colon, liver, pancreas, lung, breast, ovary, uterus, cervix, and prostate.
  • a "PAM-1 antibody” is meant a polypeptide that specifically binds to the isoform of CFR-1 that comprises the amino acid sequence of SEQ ID NO: 6 and that is expressed by 23132 cells.
  • a PAM-1 antibody binds a tumor-specific glycostructure of the CFR-1 isoform having the amino acid sequence of SEQ ID NO:6.
  • a PAM-1 antibody may be human monoclonal antibody 103/51, murine antibody 58-49/69, or a humanized or chimeric antibody containing all or part of the sequence of SEQ ID NO:2 and/or 4.
  • a PAM-1 antibody can induce apoptosis or alter proliferation, or both, in a neoplastic cell or a cell of a pre-cancerous lesion, but not a normal cell.
  • a PAM-1 antibody comprises the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO:4 or is encoded, in part, by the nucleic acid sequence of SEQ ID NO: 1 and/or SEQ ID NO:3.
  • a PAM-1 antibody may comprise amino acids 11-18, 36-43, and/or 82- 104 of SEQ ID NO:26 and/or amino acids 28-32, 51-53, and/or 90-100 of SEQ ID NO:27.
  • a “functional fragment” or a "fragment of a PAM-1 antibody” as used herein in reference to polypeptide is meant a fragment that retains at least one biological activity of the full-length polypeptide. Examples of such a biological activity are the ability to specifically bind an antigen, induce apoptosis, and/or inhibit cell proliferation. The biological activities of a functional fragment may be determined, for example, using any one of the assays described herein.
  • a "functional fragment” has an amino acid sequence that is substantially identical to a fragment, e.g., 3, 4, 5, 10, 15, 20, 15, 30, 50, 75, or 100 contiguous amino acids, of the amino acid sequence of SEQ ID NO:2 , SEQ ID NO:4, SEQ ID NO:26, or SEQ ID NO:27.
  • a "functional fragment” is identical to a fragment of the sequence of SEQ ID NO:2 , SEQ ID NO:4, SEQ ID NO:26, or SEQ ID NO:27.
  • Such a "functional fragment” may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 15, 30, 50, 75, or 100 contiguous amino acids of SEQ ID NO:2 , SEQ ID NO:4, SEQ ID NO:26, or SEQ ID NO:27, or maybe the entire amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:26, or SEQ ID NO:27.
  • such a fragment includes one or more of the Complement Determining Regions (CDR) of the V H or the V regions of the murine PAM-1 antibody 58-49/69 or human PAM-1 antibody 103/51.
  • CDR Complement Determining Regions
  • a functional fragment may include amino acids 11-15, 30-46, and/or 79-88 of SEQ ID NO:2; and/or amino acids 17-32, 48-54, and/or 87-95 of SEQ ID NO:4.
  • Other examples of functional fragments include polypeptides having amino acids 11-18, 36-43, and/or 82-104 of SEQ ID NO:26 and/or amino acids 28-32, 51-53, and/or 90-100 of SEQ ID NO:27.
  • detecttable agent is meant a compound that is linked to a diagnostic agent to facilitate detection. Such a “detectable agent” may be covalently or non-covalently linked to a diagnostic agent, hi addition, the linkage may be direct or indirect. Examples of “detectable agents” include, protein purification tags, cytotoxins, enzymes, paramagnetic labels, enzyme substrates, co-factors, enzymatic inhibitors, dyes, radionuclides, chemiluminescent labels, fluorescent markers, growth inhibitors, cytokines, antibodies, and biotin.
  • a diagnostic agent is meant a compound that may be used to detect a neoplastic cell by employing any one of the assays described herein as well as any other method that is standard in the art.
  • a diagnostic agent may include, for example, an antibody which specifically binds to cell line 23132 (DSMZ Accession No. ACC 201), but not to normal cells, h addition, a diagnostic agent may specifically bind to cells of pre-cancerous lesions such as those exhibiting H.
  • pylori induced gastritis intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung, cervical intraepithelial neoplasia, low grade and high grade prostate intraepithelial neoplasia (PIN), breast ductal carcinoma in situ (D-CIS) and breast lobular carcinoma in situ (L-CIS).
  • PIN prostate intraepithelial neoplasia
  • D-CIS breast ductal carcinoma in situ
  • L-CIS breast lobular carcinoma in situ
  • a diagnostic agent may also specifically bind carcinomas of the esophagus, stomach, colon, liver, pancreas, lung, breast, ovary, uterus, cervix, and prostate, but not non-neoplastic cell of the same tissue type.
  • a "diagnostic agent” may alter cell proliferation, induce apoptosis, or both only when it is bound to a neoplastic cell or a cell of a pre- cancerous lesion, but not a normal cell.
  • a “diagnostic agent” may include, for example, peptides, polypeptides, synthetic organic molecules, naturally-occurring organic molecules, nucleic acid molecules, and components thereof, as well as one or more detectable agent covalently or non-covalently linked to the diagnostic agent.
  • high stringency hybridization conditions is meant, for example, hybridization at approximately 42°C in about 50% formamide, 0.1 mg/ml sheared salmon sperm DNA, 1% SDS (Sodium Dodecyl Sulfate), 2X SSC (Sodium Citrate Buffer), 10% Dextran Sulfate, a first wash at approximately 65°C in about 2X SSC, 1% SDS, followed by a second wash at approximately 65°C in about 0.1X SSC.
  • high stringency hybridization conditions may include hybridization at approximately 42°C in about 50% formamide, 0.1 mg/ml sheared salmon sperm DNA, 0.5% SDS, 5X SSPE, IX Denhardt's, followed by two washes at room temperature in 2X SSC, 0.1% SDS, and two washes at between 55-60°C in 0.2X SSC, 0.1% SDS.
  • Altering cell proliferation refers to a reduction or an increase in the rate of cell division of a cell in comparison with the normal rate of cell division of that type of cell under the same conditions.
  • Cell proliferation may be assayed using a number of methods standard in the art, for example, the MTT cell proliferation assay described herein, BrdU incorporation, and 3 H thymidine uptake.
  • MTT cell proliferation assay described herein, BrdU incorporation, and 3 H thymidine uptake.
  • assays are described, for example, in Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, N.Y., 2001.
  • the increase or decrease of cell proliferation is 20%, 40%, 50%, or 75%.
  • the increase or decrease of cell proliferation is 80%, 90%, 95%).
  • cell proliferation is completely inhibited.
  • “Inducing apoptosis,” as used herein, refers to the appearance of characteristics in a cell that are well defined in the art (see, e.g., Wyllie et al., Br. J. Cancer 80 Suppl. 1:34-37, 1999; Kerr et al, Br. J. Cancer 26:239-257, 1972). These characteristics include morphological characteristics, such as membrane blebbing, DNA condensation, as well as changes in F-actin content, mitochondrial mass, and membrane potential.
  • the induction of apoptosis may be assayed using a number of methods standard in the art, for example, a cell death ELISA, TUNEL staining, DNA stains, e.g., Hoechst 33258, and staining with various vital dyes such as acridine orange, Mito Tracker Red ® staining (Molecular Probes, Eugene, OR), and Annexin V ® staining (Becton Dickinson, NJ).
  • a cell death ELISA TUNEL staining
  • DNA stains e.g., Hoechst 33258
  • staining with various vital dyes such as acridine orange, Mito Tracker Red ® staining (Molecular Probes, Eugene, OR), and Annexin V ® staining (Becton Dickinson, NJ).
  • inducing apoptosis refers to an increase in the number of cells undergoing apoptosis when compared with a control cell population under
  • the increase of apoptosis may be 10%, 20%o, 40%), 50%, or 75%.
  • the induction of apoptosis results in an increase of apoptosis that is 2-fold, 3-fold, 10-fold, or even 100-fold over that seen in a control cell population.
  • a “humanized antibody” as used herein is a genetically engineered antibody in which a minimum of a non-human, e.g., a murine, antibody sequence is combined with human antibody sequence and still maintains the binding specificity of the original non-human antibody, h desirable embodiments, a humanized antibody contains 15%, 20%, 25%, 30%, or 40% non-human sequence, h more desirable embodiments, a humanized antibody contains 5% or 10% non-human sequence, h addition, a humanized antibody desirably induces no or only a minimal human immune response.
  • a "neoplastic cell,” as used herein, refers to a cell which is undergoing cell division, not undergoing apoptosis, or both, under inappropriate conditions.
  • a "neoplastic cell” may undergo cell division when a corresponding normal cell does not undergo cell division, or, alternatively, a “neoplastic cell” may not respond to normal cell-cycle checkpoint controls.
  • a "cell of a pre-cancerous lesion” is meant cells that are undergoing cell division, not undergoing apoptosis, or both, under inappropriate conditions, but that have not developed into a cancerous tumor.
  • cells in a pre-cancerous lesion may undergo cell division when a corresponding normal cell does not undergo cell division, or, alternatively, cells of a pre-cancerous lesion may not respond to normal cell-cycle checkpoint controls.
  • pre-cancerous lesions include H.
  • a "normal cell” as used herein is meant a cell that is neither a neoplastic cell nor a cell of a pre-cancerous lesion.
  • proliferative disease refers to any disorder that results in the abnormal proliferation of a cell.
  • proliferative diseases are various types of neoplasms, such as stomach adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, and adenocarcinoma of the pancreas.
  • a "protein purification tag,” as used herein, is a peptide, e.g., an epitope tag, that is covalently or non-covalently added to a protein to aid in the purification of the protein. Desirably such peptides bind with high affinity to an antibody or to another peptide such as biotin or avidin.
  • epitope tags include His-tags, HA-tags, FLAG ® -tags, and c-Myc-tags.
  • any epitope that is recognized by an antibody also may be used as a protein purification tag.
  • Protein purification tags may be cleaved from a protein, for example, by using an enzyme, e.g., thrombin, or a chemical, e.g., cyanogen bromide.
  • an antibody e.g., an antibody
  • an antibody e.g., the human or murine PAM-1 antibody
  • an affinity for its antigen that is least 2-fold, 5-fold, 10-fold, 30-fold, or 100-fold greater than for an equal amount of any other antigen, including related antigens.
  • Binding of a polypeptide to another polypeptide may be determined as described herein, and by any number of standard methods in the art, e.g., Western analysis, ELISA, or co-immunoprecipitation.
  • substantially identical is meant a polypeptide or nucleic acid exl ibiting at least 80%), 85%, 90%, or 95% identity to a reference amino acid (e.g., the sequence of SEQ ID NO:2, 4, 6, 26, or 27) or nucleic acid sequence (e.g., the sequence of SEQ ID NO:l, 3, 5, 28, or 29), or a fragment thereof, hi desirable embodiments, the , polypeptide or nucleic acid sequence is at least 98%, 99%, 99.4%, 99.5%, 99.6 %, 99.7%, 99.8%, 99.9%, or even 100% identical to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 3, 4, 5, 6, 8, 10, or 15 amino acids and desirably at least 20 or 25 contiguous amino acids. In more desirable embodiments, the length of comparison sequences is at least 30, 50, 75, 90, or 95 contiguous amino acids, or even the full- length amino acid sequence.
  • the length of comparison sequences will generally be at least 9, 10, 12, 15, 18, 20, 24, or 25 contiguous nucleotides, and desirably at least 30 contiguous nucleotides. In more desirable embodiments, the length of comparison sequences is at least 50, 75, 150, 225, 270, 280, 285, or 290 contiguous nucleotides, or even the full-length nucleotide sequence.
  • Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Multiple sequences may also be aligned using the Clustal W(1.4) program (produced by Julie D.
  • the pairwise alignment parameters to include an open gap penalty of 10.0 and an extend gap penalty of 0.1, as well as setting the similarity matrix to "blosum.”
  • the multiple alignment parameters may include an open gap penalty of 10.0, an extend gap penalty of 0.1, as well as setting the similarity matrix to "blosum,” the delay divergent to 40%o, and the gap distance to 8.
  • a factor is "purified” or “isolated” when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated, or in reference to a nucleic acid molecule, is free from the nucleic acid sequences that naturally flank the sequence of the nucleic acid molecule in the genome of an organism.
  • the factor is at least 75%>, more desirably, at least 90% or 95% and most desirably, at least 99%, by weight, pure.
  • a substantially pure factor may be obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor.
  • Proteins, vesicles, and organelles may be purified by one skilled in the art using standard techniques, such as those described by Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001).
  • the factor is desirably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or Western analysis (Ausubel et al, Current Protocols in Molecular Biology, Wiley hiterscience, New York, 2001).
  • Desirable methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography and nickel affinity columns, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody.
  • a “tumor-specific glycostructure” is meant a glycostructure on a polypeptide which is present on a neoplastic cell or a pre-cancerous cell, but not on a normal cell of the same cell type.
  • vector or "expression vector” is meant an expression system, a viral vector, a nucleic acid-based shuttle vehicle, a nucleic acid molecule adapted for nucleic acid delivery, or an autonomous self-replicating circular DNA (e.g., a plasmid).
  • the vector can either be stably replicated by the cell during mitosis as an autonomous structure, incorporated into the genome of the host cell, or maintained in the host cell's nucleus or cytoplasm.
  • proliferation markers e.g., Ki67
  • the polypeptides of the invention can be used to identify cells that are likely to give rise to a tumor, such as cells of a pre-cancerous lesion.
  • Membrane fractions from stomach carcinoma cell line 23132 were purified using chromatographic procedures and whole membrane fraction (lane 2), or purified proteins (lane 3) were stained with Coomassie (lane 1: 10 kDa ladder).
  • Western blot analysis with antibody 103/51 on membrane fractions of cell line 23132 showed one reaction with a protein with a molecular mass of approximately 130 kD (lane 4).
  • Specificity of processed membrane extracts was controlled by Western blotting with 103/51 (lane 5).
  • the protein band indicated by the arrow was excised from a preparative gel and used for MALDI mass mapping and immunization of mice.
  • Fig IB is a spectrograph of the identification of the 130 kDa gel-separated protein by high resolution MALDI peptide mass mapping.
  • Figs. 2 A - 2F are a series of images showing the effect of CFR-1 antisense transfection on antibody 103/51 staining and live cell staining (Magnification 200x). Fig.
  • FIG. 2 A shows cell line 23132 transiently transfected with a control vector and, after acetone fixation, shows intensive staining with antibody 103/51.
  • Fig. 2B shows reduced staining in cells transiently transfected with a CFR-1 antisense vector. To reduce background staining in immunohistochemical staining, live cell staining was performed with cell line 23132. A clear membrane staining is visible (Fig. 2C). Fig.
  • FIG. 2D shows control live cell staining, using only secondary antibody, on cell line 23132.
  • Fig. 2E shows a lack of live cell staining on cell line Colo-699 with antibody 103/51, which indicates that this cell line is negative for expression of CFR-1.
  • Fig. 2F shows control live cell staining, using only secondary antibody, on cell line Colo-699.
  • Fig. 2G is a graph of flow cytometry results of cell line 23132 with antibodies Chromopure human IgM (grey) and 103/51.
  • Fig. 2H is a graph of the analysis of cells transfected with control vector pHOOK-2 with flow cytometry 48 hours after transfection.
  • FIG. 21 is a graph of cells transfected with CFR-1 antisense vector, which shows a clear decrease in binding of antibody 103/51.
  • Figs. 3 A and 3B are images showing the effect of deglycosylation on staining with antibody 103/51.
  • Fig. 3 A shows 23132 cells incubated with deglycosylation buffer and acetone-fixed show intense staining with antibody 103/51 and
  • Fig. 3B shows 23132 cells treated with N-glycosidase followed by acetone fixation. A clear reduction in staining is evident as a result of the N-glycosidase treatment.
  • Fig. 3C is a Western blot showing the effect of deglycosylation of membrane extracts of cell line 23132 on the reaction with antibody 103/51.
  • Figs. 4A-4D are a series of images showing immunohistochemical staining with murine antibody 58/47-69 and 103/51 on stomach adenocarcinoma cells. To show identical specificity of antibody 103/51 and murine antibody 58/47-69, diffuse- type stomach adenocarcinoma was stained with haematoxilin-eosin (Fig. 4A), antibodies 103/51 (Fig.
  • Figs. 5A-5D are a series of images showing immunohistochemical staining of antibody 103/51 on different gastric tissues. Cryo-sections of gastric tissues were stained with haematoxilin-eosin ("HE"), antibody Ki67 (to indicate proliferating cells) and antibody 103/51. The magnification is lOOx. Fig. 5A shows gastric tissue with inflammation. Fig.
  • Fig. 5B shows H pylori induced gastritis (inserts show a higher magnification of marked glands).
  • Fig. 5C shows dysplasia and
  • Fig. 5D shows gastric adenocarcinoma cells.
  • Figs. 6A-6F are a series of images of immunohistochemical staining with antibody 103/51 on different cancerous and normal tissues. The staining of antibody 103/51 on the following tissues is shown: Carcinoma of the ampulla of Vater (Fig. 6A), mamma carcinoma invasive lobular (Fig. 6B), adenocarcinoma of the colon and no staining of normal goblet-cell epithelium of the colon (Fig. 6C), hepatocellular carcinoma (Fig.
  • Figs. 6D glomerular and fascicular zones of the adrenal gland (Fig. 6E), collection tubes of the kidney-specific staining of the Golgi apparatus (arrow) (Fig. 6F).
  • Arrows in Figs. 6A-6D indicate tumor cells
  • the arrow in Fig. 6C points to goblet cells
  • the arrow in (Fig. 6F) indicates Golgi apparatus (Magnification 400x (Figs. 6A-6E) and 200x (Fig. 6F)).
  • Figs. 7A-7D are a series of graphs showing stimulation of cell lines with antibodies 103/51 and 58/47-69 as determined by colorimetric MTT-assays.
  • Fig. 6D are a series of graphs showing stimulation of cell lines with antibodies 103/51 and 58/47-69 as determined by colorimetric MTT-assays.
  • Fig. 7B is a graph of the results of an MTT-assay with equal concentrations (4 ⁇ g/ml) of purified antibodies 103/51 and 58/47-69 and shows comparable stimulation of
  • FIG. 7C is a graph of the results of an experiment in which cell line 23132 was transiently transfected with control vector pHOOK-2 or CFR-1 antisense vector, incubated for 24 hours, and tested in the MTT assay for stimulation with 4 ⁇ g/ml purified antibody 103/51 after 24 hours. Untransfected cells were also incubated as control (Control, uncorrelated human IgM).
  • Fig. 7D is a graph of the results of an MTT-assay with equal concentrations (4 ⁇ g/ml) of antibody 103/51 on different epithelial tumor cell lines. This graph shows stimulation only on the cell line 23132 24 hours after addition of antibody.
  • Figs. 8A and 8B are the nucleic acid sequence (SEQ ID NO:l) and the amino acid sequence (SEQ ID NO:2) of the variable region of the heavy chain of murine antibody NM58-49/69.
  • the J-gene and D-gene, as well as Complement Determining Regions (CDR)1 to 3 are indicated in Fig. 8B.
  • Figs. 9 and 9B are the nucleic acid sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO:4) of the variable region of the light chain of murine antibody NM58-49/69.
  • Figs. 10-1 to 10-5 are the nucleic acid (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO:6) of the novel isoform of CFR-1 expressed by cell line 23132.
  • Figs. 11 A and 1 IB are a series of Coomassie blue stained SDS gels and Western blots showing fragments of antibody PAM-1. Intact PAM-1 antibody was digested with pepsin to yield antibody fragments. PAM-1 antibody was digested with Pepsin to yield antibody fragments.
  • Fig. 10-1 to 10-5 are the nucleic acid (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO:6) of the novel isoform of CFR-1 expressed by cell line 23132.
  • Figs. 11 A and 1 IB are a series of Coomassie blue stained SDS gels and Western blots showing fragments of antibody PAM-1. Intact PAM-1 antibody was digested with pepsin to yield antibody fragments. PAM-1 antibody was digest
  • FIG. 11A shows a Coomassie blue stained SDS gel of intact PAM-1 antibody (lane 1) and the fragmented antibody after 10 min (lane 2) and 15 min (lane 3) of pepsin treatment.
  • Fig. 11B shows the specific protein bands of intact (lane 1) and fragmented (lane 2 and 3) PAM-1 antibody.
  • Figs. 12A and 12B are a series of images showing immunohistochemical staining of intact and fragmented PAM-1 antibody on pre-malignant and malignant tissue. Paraffin sections were stained with positive control (anti-cytokeratin 8), unrelated fragmented IgM as a negative control, intact PAM-1 antibody or fragmented PAM-1 antibody.
  • FIG. 12A shows vilous adenoma of the colon (precursor lesion of coloncarcinoma); and Fig 12 B shows gastric adenocarcinoma.
  • the original magnification was lOOx.
  • FIGs. 13A and 13B are graphs showing functional analyses of fragmented PAM-1 antibody in vitro. The consequences of fragmented PAM-1 antibody treatment on the proliferation of stomach cancer cell line 23132/87 was measured by MTT proliferation assay (Fig 13A) and Cell Death Detection ELISA PLUS apoptosis assay (Fig 13B). Fig 13 A shows concentration dependent inhibition of cell proliferation with fragmented PAM-1 antibody.
  • Fig. 13A shows concentration dependent inhibition of cell proliferation with fragmented PAM-1 antibody.
  • FIG. 13B shows antibody induced apoptosis with the fragmented form of PAM-1 after 24 hours of treatment (control 1 : no antibody was added; control 2: unrelated fragmented IgM was added at a similar concentration).
  • Figs. 14A-14C are a series of graphs showing the results of in vivo experiments with human carcinoma cells treated with fragmented PAM-1 antibody in NMRI nu/nu mice.
  • Fig. 14A shows tumor size development of mice treated with fragmented form of PAM-1 antibody or unrelated fragmented IgM (control group) over a period of 23 days.
  • Figs. 14B and 14C show that antibody-reduced tumor mass is measurable in mice treated with fragmented PAM-1 antibody after 23 days.
  • FIGs. 15A-15F are a series of images showing immunoperoxidase staining of paraffin-embedded tumor sections with Klenow FragEL DNA fragmentation Kit for apoptosis. Sections were obtained from mice treated with either unrelated fragmented IgM (Figs. 15A-15C) or fragmented PAM-1 antibody (Figs. 15D-15F). Figs. 15A and 15D show HE staining; Figs. 15B and 15E show immunoperoxidase staining with apoptosis positive control, all cell nuclei are stained; and Figs.
  • Fig. 16 is a comparison of the amino acid sequence of the CFR-1 obtained from cell line 23132 (SEQ ID NO:6) to the published sequences of CFR-1 and MG160 (SEQ ID NOS:7 and 8). These experimental comparisons primarily show that the CFR-1 protein obtained from cell line 23132 is not identical to the CFR-1 sequences previously known, but represents a novel isoform thereof. The differences between the sequences are underlined. Fig.
  • Fig. 17 is the nucleic acid sequence (SEQ ID NO:26) and the amino acid sequence (SEQ ID NO:28) of the variable region of the heavy chain of the human PAM-1 antibody (clone 103/51).
  • the Complement Determining Regions (CDR)1 to 3 are indicated.
  • Fig. 18 is the nucleic acid sequence (SEQ ID NO:27) and the amino acid sequence (SEQ ID NO:29) of the variable region of the light chain of the human PAM-1 antibody (clone 103/51).
  • the Complement Determining Regions (CDR)1 to 3 are indicated.
  • Figs. 19A and 19B are a series of graphs showing that a PAM-1 recombinant
  • IgG antibody induces both a reduction of tumor volume (Fig. 19A) and tumor weight (Fig. 19B) of stomach cancer in vivo using a mouse model.
  • Mice receiving the control IgG antibody Chrompure human IgG; "CP IgG" are indicated by shaded circles and mice receiving the PAM-1 Ab are indicated by open circles.
  • Figs. 20A and 20B are a series of graphs showing that a PAM-1 recombinant
  • IgG antibody induces both a reduction of tumor volume (Fig. 20 A) and tumor weight (Fig. 20B) of pancreatic cancer in vivo using a mouse model.
  • Mice receiving the control IgG antibody Chrompure human IgG; "CP IgG" are indicated by shaded circles and mice receiving the PAM-1 Ab are indicated by open circles.
  • Figs. 21 A - 21D are a series of images showing keratin staining (Figs. 21 A and 21C) and haematoxilin-eosin ("H & E") staining (Figs. 21B and 21D) of pancreatic cancer in mice injected either with a control IgG antibody (Figs. 21 A and 21B) or PAM-1 IgG (Figs. 21C and 21D).
  • a reduction of keratin staining in Fig. 21 C indicates tumor cell death and apoptotic cells are indicated by arrows in Fig. 21D.
  • the present invention features novel polypeptides and methods of using these polypeptides in the diagnosis, detection, monitoring, and treatment of neoplasms in a mammal, e.g., a human.
  • a mammal e.g., a human.
  • the isoform of CFR-1 described herein and polypeptides that bind this antigen such as human monoclonal antibody 103/51 and murine antibody 58-49/69, enable the therapy and diagnosis not only of tumors, but also of pre-cancerous structures both in vitro and in vivo.
  • the novel isoform of CFR-1 maybe used in screening methods to identify additional therapeutic compounds.
  • PAM-1 antibody (58/47-69).
  • the isoform of CFR-1 described herein is specific for tumor cells, particularly for gastric carcinoma cells and is not expressed by normal stomach tissue.
  • the specificity for the antigen of human PAM-1 antibody 103/51 was confirmed by producing murine antibodies from purified molecules having identical reactions and functions, through immunohistochemical staining, and an MTT assay of two cell lines that do not express the CFR-1 isoform recognized by the PAM-1 antibody.
  • the isoform of the CFR-1 molecule which was detected by both the human and the murine antibodies, is localized in the cell membranes of the epithelial cells, and has an expression pattern which differs from that previously described for wild- type CFR-1 (Burrus et al, 1992, Mol. Cell. Biol. 12:5600-5609).
  • CFR-1 Wild-type CFR- 1 which was isolated as a high-affinity FGF-binding protein from chicken fibroblasts (Burrus et al, 1992, Mol. Cell. Biol. 12:5600-5609), binds to a number of FGFs and may have a role in the regulation of cellular proliferation.
  • CFR-1 was found to be expressed only in the Golgi apparatus (Burrus et al, 1992, Mol. Cell. Biol. 12:5600-5609), but it can also be secreted in a mutant form (Zuber et al, 1997, J. Cell Physiol. 170:217-227).
  • MG-160 which is a medial Golgi sialoglycoprotein and was purified from rat brains, plays a role in intracellular FGF trafficking (Zuber et al, 1997, J. Cell Physiol. 170:217-227). Recent findings have shown that the localization of this protein is not restricted to the Golgi apparatus. However, if truncated at the c-terminus, the protein can be localized to the plasma membrane and filopodia (Gonatas et al, 1998, J. Cell Sci. Ill :249-260).
  • ESL- 1 which was isolated from mouse neutrophilic progenitor cells (32Dcl3), is located in the Golgi apparatus as well on the cell surface of the microvilli (Steegmaier et al,
  • ESL-1 was identified as ligand for E-selectin in neutrophils with an approximate molecular mass of 150 kD. Immunoprecipitation with anti ESL-1 antibodies showed that a non- defined isoform of this protein could be precipitated from various cells, including some cancerous cell lines (Steegmaier et al, 1995, Nature 373:615-620).
  • the tissue distribution shows that the tumor- and pre-cancerous lesion-specific isoform of CFR-1 described herein is correlated with cellular activation and proliferation demonstrated by staining with antibody Ki67 (Ramires et al, 1997, J. Pathol. 182:62-67).
  • Ki67 Ki67
  • Variable cellular distribution of a protein is a known phenomenon (Smalheiser, 1996, Mol. Biol. Cell 7:1003-1014).
  • An altered distribution might be caused by a different glycosylation pattern in malignant cells, which may lead to a transport to the plasma membrane.
  • Normal stomach mucosa does not express this receptor in a measurable amount, but H pylori infiltrated epithelia and dysplastic epithelia express this antigen.
  • CFR-1 both tissues proliferate and may be precursors for gastric carcinoma.
  • the isoform of CFR-1 described herein is not found on normal cells, but exclusively on proliferating cells associated with malignancies, such as the tumor cells found in the growth and corresponding precursor stages.
  • the CFR-1 isoform described herein may be used not only in the diagnosis and treatment of gastric carcinomas, but rather also for all epithelial tumors.
  • An antibody e.g., a PAM-1 antibody, which binds to the isoform of CFR-1 described herein therefore has a targeted activity on the cancerous or pre-cancerous (and not normal) cells.
  • Antibodies and Polypeptides play an essential role in maintaining the health of an individual, particular, antibodies are present in serum and bind to and help eliminate diverse pathogens such as bacteria, viruses, and toxins.
  • Antibodies consist of Y-shaped protein structures built from two heavy chains and two light chains. Each chain has a modular construction: each light chain consists of two domains, and each heavy chain has at least four domains.
  • the antigen binding site is fashioned by one domain from the heavy chain (VH domain) and one domain from the light chain (V L domain). Indeed, small antigen binding fragments can be prepared by linking these two domains, either associated non-covalently, or covalently via disulphide bonds or a peptide linker.
  • the antigen binding domains are more variable in amino acid sequence than the other domains of the antibody, and are therefore termed variable (V) domains, in contrast to the constant (C) domains.
  • the constant domains of the antibody are responsible for triggering antibody effector mechanisms, such as complement lysis and cell-mediated killing.
  • Antibodies are made by B-lymphocytes in a process involving gene rearrangement. During the development of these cells, the genes encoding the variable domains are assembled from genetic elements. In the case of the V H domains there are three elements, the un-rearranged V H gene, D segment, and J H segment.
  • V L domains there are two elements, the un-rearranged V L (V Lambda or V Kappa) gene and the J L (J Lambda or J Kappa) segment. Random combination of these gene segments and random combination of the rearranged V H and V L domains generate a large repertoire of antibodies, capable of binding to a large number of equally diverse antigens. Further, the V H and V L regions each have three Complement Determining Regions (CDR) and four framework regions (FR). The FRs are the backbone of the antibody and the CDRs are the parts of the antibody that bind the antigen.
  • CDR Complement Determining Regions
  • FR framework regions
  • One skilled in the art can determine the FR and CDR regions of an antibody by comparing the amino acid sequence of a number of antibodies raised in the same species (see, e.g., Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; and Kabat et al., Sequences of Proteins of Immunological Interest, 5 th edition, NTH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991).
  • Neoplasm-Specific Polypeptides A PAM-1 antibody or a fragment thereof may be produced by expression in a hybridoma or recombinantely in a host cell such as E. coli or yeast, e.g., S. cerevisiae, or a mammalian cell line. Functional fragments of polypeptides may also be generated, for example, by direct synthesis using recombinant methods. These methods are standard in the art. For example, a nucleic acid sequence may be amplified using the polymerase chain reaction (PCR). The PCR technique is known in the art and is described, for example in U.S. Patent No. 4,683,195.
  • PCR polymerase chain reaction
  • the sequence of a monoclonal antibody expressed by a hybridoma or trioma may be obtained and functional fragments of the antibody may be amplified.
  • whole RNA may be isolated from a hybridoma expressing a tumor-specific monoclonal antibody.
  • cDNA may then be generated from the RNA using reverse transcriptase and the cDNAs which contain the functional fragments of the variable regions of the heavy and light chains may be amplified using PCR.
  • the PCR products may then be purified and cloned into expression vectors, e.g., plasmid or viral vectors.
  • nucleic acid molecules identified using the methods of the invention may be expressed in a variety of standard vectors and host cells. Any promoter that is active in the host cell may be used to express a nucleic acid molecule. Nonetheless, for expression of an antibody or a fragment of an antibody in a mammalian cell, use of an immunoglobulin gene promoter is desirable.
  • Methods of introducing a vector into a host cell include, electroporation, use of synthetic lipid polymers, e.g., LipofectinTM, use of calcium chloride, and use of DEAE Dextran.
  • synthetic lipid polymers e.g., LipofectinTM
  • Such methods are also described in, for example, Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, N. Y., 2001.
  • the purified antigen recognized by an antibody may be used to generate additional antibodies that specifically recognize the same antigen.
  • Such methods are standard in the art and generally involve immunizing a mammal, such as a mouse, rat, rabbit, goat, or horse, with the purified antigen to illicit an immune response against the antigen in the mammal.
  • Antibodies produced by the mammal in response to this immunization may either be purified from blood obtained from the mammal and characterized for binding specificity and function, or antibody producing lymphocytes or splenocytes may be obtained from the mammal and used to generate antibody-producing hybridoma cell lines. The antibodies produced by these hybridoma cell lines are then characterized for binding specificity and function using standard assays as described herein.
  • Amino acid sequence variants of a PAM-1 antibody can be prepared by introducing appropriate nucleotide changes into the DNA encoding the antibody, or by in vitro synthesis of the desired polypeptide.
  • Such variants include, for example, deletion, insertion, or substitution of, residues within the amino acid sequence of a PAM-1 antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., the ability to induce apoptosis of a neoplastic cell, but not a non-neoplastic cell, or the ability to inhibit the proliferation of a cell.
  • the amino acid changes also may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites, altering the membrane anchoring characteristics, or modifying its susceptibility to proteolytic cleavage.
  • an antibody such as changing the number or position of glycosylation sites, altering the membrane anchoring characteristics, or modifying its susceptibility to proteolytic cleavage.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, or deleting the target residue.
  • a useful method for identification of specific residues or regions for mutagenesis in a polypeptide is called "alanine scanning mutagenesis" and is described, for example, by Cunningham and Wells (Science 244:1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most desirably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • the domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed variants are screened for, e.g., the ability to induce apoptosis of a neoplastic cell and not a non-neoplastic cell, or to inhibit the proliferation of a neoplastic cell and not a non-neoplastic cell.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as affecting the biological activity of a polypeptide.
  • ala may be substituted with val, leu, or ile; arg may be substituted with lys, gin, or asn; asn may be substituted with gin, his, lys, or arg; asp may be substituted with glu; cys may be substituted with ser; gin may be substituted with asn; glu may be substituted with asp; gly may be substituted with pro; his may be substituted with asn, gin, lys, or arg; ile may be substituted with leu, val, met, ala, or phe; leu may be substituted with ile, val, met, ala, or phe; lys may be substituted with arg, gin, or asn; met may be substituted with leu, phe, or ile; phe may be substituted with arg, gin, or asn; met may be substituted with leu, phe, or ile; phe may be substitute
  • a PAM-1 antibody, a fragment thereof, or the novel CFR-1 isoform described herein may be linked to a detectable agent to facilitate the purification of the polypeptide as well as the diagnosis, monitoring, or treatment of a neoplasm or a pre- cancerous lesion in a mammal in need thereof.
  • a detectable agent to facilitate the purification of the polypeptide as well as the diagnosis, monitoring, or treatment of a neoplasm or a pre- cancerous lesion in a mammal in need thereof.
  • suitable detectable agent will depend on the intended use of the polypeptide and will be apparent to those of ordinary skill in the art.
  • Detectable agents include, for example, protein purification tags, cytotoxins, enzymes, paramagnetic labels, enzyme substrates, co-factors, enzyme inhibitors, dyes, radionuclides, chemiluminescent labels, fluorescent markers, growth inhibitors, and biotin.
  • a protein purification tag may be conjugated to a PAM-1 antibody, fragment thereof, or the novel CFR-1 isoform described herein to facilitate isolation of the polypeptide.
  • tags that can be used include His-tags, HA-tags, FLAG ® - tags, and c-Myc tags.
  • An enzymatic or a chemical cleavage site maybe engineered between the polypeptide and the tag moiety so that the tag can be removed following purification.
  • Suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin.
  • suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.
  • suitable radioisotopic labels include 3 H, 125 1, 131 1, 32 P, 35 S, and 14 C.
  • the radioisotope will emit in the 10-5,000 kev range, more desirably 100- 500 kev.
  • Paramagnetic isotopes may also be conjugated to the polypeptide and used in vivo for the diagnosis and treatment of cancer.
  • the use of such conjugated polypeptides may be for in vivo nuclear magnetic resonance imaging.
  • Such methods are known in the art (see, for example, Schaefer et al., JACC 14:472-480, 1989;
  • a radiolabeled polypeptide may also be used in radioimmunoguided surgery (RIGS), which involves the surgical removal of any tissue the labeled antibody binds to.
  • RIGS radioimmunoguided surgery
  • Radiolabels useful for tumor imaging are preferably short-lived radioisotopes.
  • Various radioactive metals with half-lives ranging from 1 hour to 11.4 days are available for conjugation to antibodies, such as scandium-47 (3.4 days), gallium-67 (2.8 days), gallium-68 (68 minutes), technetium-99m (6 hours), indium-Il l (3.2 days), and radium-223 (11.4 days), of which gallium-67, technetium- 99m, and indium- 111 are preferable for gamma camera imaging, gallium-68 is preferable for positron emission tomography, and scandium-47 and radium-223 (and other alpha-emitting radionuclides) are preferable for tumor therapy.
  • fluorescent markers examples include fluorescein, isothiocyalate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde, and fluorescamine.
  • chemiluminescent markers include a luminal label, isoluminal label, aromatic acridinium ester label, imidazole label, acridinium salt label, oxalate ester label, luciferin label, luciferase label, and aequorin label.
  • Conjugation of these detectable agents to a polypeptide described herein, such as PAM-1 antibodies, fragments thereof, and the novel isoform of CFR-1 can be accomplished using standard techniques known in the art.
  • Typical antibody conjugation techniques are described by Kennedy et al. (Clin. Chim. Acta 70, 1-31, 1976) and Schurs et al. (Clin. Chim. Acta 81, 1-40 , 1977) and include, for example, the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method.
  • Antibodies may be radiolabeled by any of several techniques known to the art, described, for example, in U.S.
  • polypeptides Conjugated to Anti-Tumor Agents Although a PAM-1 antibody or fragment thereof can induce apoptosis of neoplastic cells, inhibit cellular proliferation of neoplastic cells, or both, the polypeptide may in addition be conjugated to an agent that kills neoplastic cells or that inhibits their proliferation.
  • the targeting ability of the PAM-1 antibody or fragment thereof results in the delivery of the cytotoxic or anti-proliferative agent to the tumor to enhance the destruction of the tumor.
  • the polypeptide therefore may be used for the treatment and prevention of a neoplasm or pre-cancerous lesion in a mammal, such as a human patient.
  • the cytotoxic agent linked to the polypeptide may be any agent that destroys or damages a tumor cell or tumor to which the polypeptide has bound.
  • agents include chemotherapeutic agents or radioisotopes, enzymes which activate a pro-drug, or a cytokine.
  • chemotherapeutic agents include, for example, taxol, mithramycin, deoxyco-formycin, mitomycin-C, L- asparaginase, interferons (especially IFN-alpha), etoposide, teniposide, anthracyclines (e.g., daunomycin and doxorubicin), methotrexate, vindesine, neocarzinostatin, cis- platinum, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan, ricin, and calicheamicin.
  • the chemotherapeutic agents may be conjugated to the antibody using conventional methods known in the art.
  • Suitable radioisotopes for use as cytotoxic agents are also known to those skilled in the art and include, for example, 131 I, or an astatine such as 211 At. These isotopes may be attached to the polypeptide, either covalently or non-covalently, using conventional techniques known in the art.
  • the cytotoxic agent may also be an enzyme, which activates a pro-drug. This allows the conversion of an inactive pro-drug to its active, cytotoxic form at the tumor site and is called "antibody-directed enzyme pro-drug therapy" (ADEPT).
  • ADEPT antibody-directed enzyme pro-drug therapy
  • the polypeptide-enzyme conjugate may be administered to the patient and allowed to localize in the region of the tumor to be treated.
  • the pro-drug is then administered to the patient such that conversion to the cytotoxic drug is localized in the region of the tumor to be treated under the influence of the localized enzyme.
  • An exemplary enzyme is bacterial carboxypeptidase G2 (CPG2) the use of which is described in, for example, WO 88/07378.
  • CPG2 carboxypeptidase G2
  • the polypeptide-enzyme conjugate may, if desired, be modified in accordance with the teaching of WO 89/00427, such as to accelerate its clearance from areas of the body that are not in the vicinity of a neoplasm.
  • the polypeptide-enzyme conjugate may also be used in accordance with WO 89/00427, for example, by providing an additional component, which inactivates the enzyme in areas of the body that are not in the vicinity of the tumor.
  • the cytotoxic agent conjugated to a PAM-1 antibody or fragment thereof may also be a cytokine such as interleukin-2 (IL-2), interleukin-4 (EL-4), or tumor necrosis factor alpha (TNF-alpha).
  • IL-2 interleukin-2
  • EL-4 interleukin-4
  • TNF-alpha tumor necrosis factor alpha
  • the polypeptide targets the cytokine to the tumor so that the cytokine mediates damage to or destruction of the tumor without affecting other tissues.
  • the cytokine may be fused to the polypeptide at the DNA level using conventional recombinant DNA techniques.
  • any inhibitor of cell proliferation e.g., genistein, tamoxifen, or cyclophospham
  • a polypeptide of the invention to a patient be limited to a particular mode of administration, dosage, or frequency of dosing; the present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intravesicular, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to decrease the number of neoplastic cells by inducing apoptosis of neoplastic cells, by inhibiting proliferation of tumor cells, or both.
  • the compound(s) may be administered to the patient in a single dose or in multiple doses.
  • the doses may be separated from one another by, for example, one day, two days, one week, two weeks, or one month.
  • the polypeptide e.g., a PAM-1 antibody, fragment thereof, the novel CFR-1 isoform described herein
  • a vector including a nucleic acid molecule that encodes this novel isoform of CFR-1 maybe administered once a week for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • the precise dose will vary dependent on the polypeptide or vector used, the density, on the tumor surface, of the ligand to which the polypeptide binds, and the rate of clearance of the polypeptide.
  • the dosage of a PAM-1 antibody or novel isoform of CFR- 1 described herein can be increased if the lower dose does not provide sufficient anti- neoplastic activity.
  • the dosage a PAM-1 antibody or novel isoform of CFR-1 described herein can be decreased if the neoplasm is cleared from the patient.
  • a therapeutically effective amount of a polypeptide such as a monoclonal antibody or a fragment thereof, may be, for example, in the range of about 0.1 mg to 50 mg/kg body weight/day or 0.70 mg to 350 mg/kg body weight/week.
  • a therapeutically effective amount is in the range of about 0.50 mg to 20.0 mg/kg, and more desirably in the range of about 0.50 mg to 15.0 mg/kg, for example, about 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 8.5, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, or 15.0 mg/kg body weight administered daily, every other day, or twice a week.
  • a suitable dose is an amount of the polypeptide that, when administered as described above, is capable of inducing apoptosis, and is at least 20% above the basal (i.e., untreated) level, hi general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non- treated patients.
  • the administration of the polypeptide can induce neoplastic cell apoptosis by at least 20%, 40%>, 50%, or 75% > above that of an untreated control as measured by any standard assay known in the art. More desirably, apoptosis is induced by 80%, 90%, 95%), or even 100% above that of an untreated control.
  • the administration of the polypeptide can inhibit neoplastic cell proliferation by at least 20%, 40%, 50%>, or 75%> below that of an untreated control as measured by any standard assay known in the art. More desirably, proliferation is inhibited by 80%, 90%, 95%, or even 100%> below that of an untreated control.
  • the polypeptide can simultaneously inhibit proliferation and induce apoptosis of neoplastic cells relative to untreated control cells.
  • Such responses can be monitored by any standard technique known in the art, including those described herein, h general, for pharmaceutical compositions, the amount of antibody present in a dose ranges from about 25 ⁇ g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • a DNA vaccine including a plasmid vector or a viral vector which includes a nucleotide sequence encoding SEQ ID NO:6, or a fragment thereof, may be used to induce a tumor-specific immune response in a patient.
  • an effective dose range of about 1 ng to 5 mg, 100 ng to 2.5 mg, 1 ⁇ g to 750 ⁇ g, and preferably about 10 ⁇ g to 300 ⁇ g of DNA is administered directly into muscle tissue.
  • Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also suitable.
  • booster vaccinations may be provided.
  • a PAM-1 antibody, fragment thereof, or the novel CFR-1 isoform described therein may be administered by any suitable means that results in a concentration having anti-neoplastic properties upon reaching the target region.
  • the polypeptide may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route.
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R.
  • the pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • the neoplastic cells are in direct contact with the blood (e.g., leukemias), or if the tumor is only accessible by the bloodstream then the intravenous (LV.) route may be used, h cases in which tumors grow in confined spaces such as the pleural cavity or the peritoneal cavity, the polypeptide may be directly administered into the cavity rather than into the blood stream.
  • LV. intravenous
  • the formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found, for example, in Remington (The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott, Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
  • aspects of the present invention are directed to methods of detecting or diagnosing a neoplasm in a mammal, preferably a human patient.
  • a neoplasm which is specifically bound by a PAM-1 antibody, or a fragment thereof, may be detected either in vitro or in vivo according to the methods of the present invention.
  • a neoplasm may be one that expressed the isoform of CFR-1 that is bound by a PAM-1 antibody.
  • a cell which expressed the isoform of CFR-1 described herein is likely to be a neoplastic cell or a cell in a pre- cancerous lesion.
  • the isoform of CFR-1 recognized by a PAM-1 antibody may be used as a marker to detect whether a patient has neoplasm or is likely to develop a neoplasm.
  • PAM-1 antibodies and fragments thereof are particularly useful because they specifically bind neoplastic, cells in pre-cancerous lesions, as well as proliferating malignant cells, and not normal cells and normal proliferating cells or tissue. Accordingly, such polypeptides can bind to neoplastic cells within the tumor, but not the normal surrounding tissue, thus allowing the detection, the treatment, or both, of a neoplasm in a mammal.
  • PAM-1 antibodies and fragments thereof may be used to determine if a biopsy removed the entire tumor by verifying that no cells bound by the polypeptide remain in the patient or, by verifying that tumor removed from the patient is entirely surrounded by cells that are not bound by the polypeptide. It is understood that to improve the sensitivity of detection, multiple neoplastic markers may be assayed within a given sample or individual. Thus, polypeptides such as antibodies or functional fragments specific for different antigens may be combined within a single assay, or in multiple assays. Further, multiple primers or probes specific to neoplasms may be used concurrently. The selection of markers may be based on routine experiments to determine combinations that results in optimal sensitivity.
  • the diagnosis of a neoplasm in a mammal involves obtaining a biological sample from the mammal (e.g., human patient), contacting such sample with a PAM-1 antibody or fragment thereof, detecting, in the test sample, the level of reactivity or binding of the polypeptide to neoplastic cells relative to a control sample, which corresponds to non-neoplastic cells derived from healthy tissue from the mammal in which the cancer is being diagnosed or from another patient known not to have a neoplasm.
  • the novel isoform of CFR-1 described herein, as well as the PAM-1 antibodies of the invention are particularly useful for the detection of early stage tumors or metastases, which are otherwise undetectable. Accordingly, in addition to diagnosing a neoplasm in a patient, the methods of this invention may also be used to monitor progression of a neoplasm in a mammal.
  • the polypeptides described herein therefore may be used as markers for the progression of a neoplasm.
  • the assays described below, which are used for the diagnosis of a neoplasm may be performed over time, and the change in the level of reactive polypeptide(s) evaluated.
  • the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed, hi general, a neoplasm is progressing in those patients in whom the level of bound PAM- 1 antibody detected increases over time or where the expression of the isoform of CFR-1 described herein increases over time.
  • the neoplasm is not progressing when the level of bound PAM-1 antibody either remains constant or decreases with time or where the expression of the isoform of CFR-1 described herein descreases over time.
  • the CFR-1 isoform described herein and the PAM-1 antibodies may be used to determine the presence of tumor cells in the mammal following tumor resection by surgical intervention to determine whether the tumor has been completely removed from the mammal.
  • the polypeptide is linked to a detectable agent, which facilitates detection, or measurement of polypeptide reactivity.
  • the biological sample is any biological material which may contain neoplastic or proliferating cells and includes, for example, blood, saliva, tissue, serum, mucus, sputum, urine, or tears.
  • the biological sample may also be a tissue section, which may be fixed tissue, fresh tissue, or frozen tissue.
  • a neoplasm is detected or diagnosed in the mammal from wliich the sample was obtained if there is an increase in the level of reactivity of a PAM-1 antibody or fragment thereof with the biological sample, or increased expression of the isoform of CFR-1 described herein over the control sample. Such increase is at least 10%, 20%), 30%, 40%, 50%, or more than 50% over control levels.
  • the level of binding or reactivity can be determined by any method known in the art and is described in further detail below.
  • the diagnosis of neoplasms or a pre-cancerous lesion using a polypeptide of the invention may be performed by any method known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers, e.g., the isoform of CFR-1 recognized by a PAM-1 antibody, in a sample. See, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y., 1999.
  • the polypeptide may be used for enzyme-linked immunosorbent assay (ELISA), Western blotting, or in situ detection of tumor cells in a tissue sample.
  • the ELISA assay typically involves the use of a polypeptide, such as an antibody, immobilized on a solid support to bind to the tumor cells in the biological sample.
  • the bound tumor cell may then be detected using a detection reagent that contains a reporter group and that specifically binds to the antibody/rumor cell complex.
  • detection reagents include, for example, any binding agent that specifically binds to a PAM-1 antibody or fragment thereof, such as an anti- immunoglobulin, protein G, protein A, or a lectin.
  • a competitive assay may be utilized, in which the polypeptide is a PAM-1 antibody and in which the antigens, to which the antibody is specific to is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the biological sample.
  • the extent to which components of the sample inhibit the binding of the labeled antigens to the antibody is indicative of the reactivity of the sample with the immobilized antibody.
  • Diagnosis of a neoplasm in a patient, or the identification of a pre-cancerous lesion in a patient may also be determined by a two-antibody sandwich assay.
  • This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
  • the cut-off value for the detection of a neoplasm is the average mean signal obtained when the antibody is incubated with samples from patients without a neoplasm.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods may be used. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups.
  • Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a defined period of time), followed by spectroscopic or other analysis of the reaction products. PAM-1 antibodies and fragments thereof may also be employed histologically for in situ detection or quantitative determination of tumor cells, for example, by immunofluorescence or immunoelectron microscopy. In situ detection or determination may be accomplished by removing a tissue specimen from a patient and allowing a labeled antibody to bind to any tumor cell in the specimen.
  • the biological sample can be a smear of biological material containing neoplastic cells on a slide, and the detection of neoplastic cells in the biological material is achieved by examining the smear with a microscope or by fluocytometry.
  • a PAM-1 antibody may also be used in vivo for detecting and localizing a neoplasm or pre-cancerous lesion.
  • Such a method may involve injecting a mammal, desirably a human subject, parenterally with a PAM-1 antibody or fragment thereof, which has been labeled with a detectable agent, and is described, for instance, in U.S. Patent No. 4,444,744.
  • the antibody or antibody fragment can be radiolabeled with a pharmacologically inert radioisotope and administered to the patient.
  • the activity of the radioisotope can be detected in the mammal using a photoscanning device, and an increase in activity relative to a control reflects the detection and localization of a neoplasm.
  • the present invention also features methods for treating neoplasms in a mammal, desirably a human patient.
  • the method generally involves the administration of a biologically effective amount of a polypetide, e.g., the isoform of CFR-1 described herein, or a PAM-1 antibody or fragment thereof.
  • the polypeptide is typically administered to the mammal by means of injection using any routes of administration such as by intrathecal, subcutaneous, submucosal, or intracavitary injection as well as for intravenous or intraarterial injection.
  • the polypeptide may be injected systemically, for example, by the intravenous injection of the polypeptide into the patient's bloodstream or alternatively, the polypeptide can be directly injected at the site of the neoplasm or at a location in proximity to the neoplastic cells.
  • purified polypeptides corresponding to all or a part of the CFR-1 isoform described herein may be used to reinforce and amplify antibody formation and, therefore, maybe used to induce elevated apoptosis of the tumor cells or for a complement-mediated lysis.
  • CFR-1 given CFR-1 's likely function in nutrient uptake, the cells expressing this rumor-specific isoform of CFR-1 would "starve," since blocking of the receptor leads to growth arrest.
  • the isoform of CRF- 1 described herein is particularly suitable for treating the following tumors and pre-cancerous lesions: dysplasia of the gastric mucosa, interstitial metaplasia of the stomach, inflammation of the gastric mucosa which is associated with the bacteria Helicobacter pylori, tubular and tubulovillous adenomas of the stomach, tubular adenoma of the colon, villous adenoma of the colon, dysplasia in ulcerative colitis, Barrett's dysplasia, Barrett's metaplasia of the esophagus, cervical intraepithelial neoplasia I, cervical intraepithelial neoplasia U, cervical intraepithelial neoplasia m, squamous epithelial metaplasia, squamous epithelial dysplasia of the bronchus, low grade and high grade prostate intraepithelial neop
  • PAM-1 antibodies and fragments thereof may also be used in methods to treat neoplasms or kill cells of a pre-cancerous lesion.
  • binding of a PAM-1 antibody or fragment thereof to a neoplastic or a cell of a pre-cancerous lesion results in an induction in apoptosis, a reduction in cellular proliferation, or both relative to the control sample.
  • the antibodies may also activate the complement pathway, which ultimately causes holes to be punctured into the cellular membrane, resulting in cell death.
  • the PAM-1 antibody or fragment thereof may also be conjugated to drugs or toxins as described above.
  • the conjugate may be engulfed into the cell cytoplasm where cell enzymes cleave, and, thus, activate or free the drugs or toxins from the conjugate. Once released, the drugs or toxins damage the cell and irreversibly induce cell death.
  • binding to neoplastic cells or cells of a pre-cancerous lesion and the resulting emission of radiation, at a short distance from the cell DNA produces damage to the latter thus inducing cell death in the next replication round.
  • a higher dose of labeled antibody is injected.
  • Injection may be intravenous, intraarterial, intralymphatic, intrathecal, or intracavitary, and may be repeated more than once.
  • Radiolabeled polypeptides or polypeptide mixtures may be advantageous for some therapies to administer multiple, divided doses of radiolabeled polypeptides or polypeptide mixtures, e.g., in the range of 20-120 mCi (70 kg patient), thus providing higher cell-killing doses to the neoplasm usually without effecting a proportional increase in radiation of normal tissues
  • Therapy using labeled polypeptides is advantageously used as a primary therapeutic treatment, but may also be used in combination with other anti-neoplastic therapies, e.g., radiation and chemotherapy, and as an adjunct to surgery.
  • the administration of such conjugated polypeptides is particularly useful in the case where small metastases cannot be surgically removed.
  • Chemotherapeutic agents and/or radiation and/or surgical removal of the neoplasm can optionally be combined with any of the methods of the present invention.
  • Classes of compounds that can be used as the chemotherapeutic agent include: alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), and synthetics.
  • alkylating agents e.g., nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes
  • alkylating agents include Uracil mustard, Chlormethine, Cyclophosphamide (Cytoxan ® ), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene- melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, dacarbazine, and Temozolomide.
  • Antimetabolites may include, for example, Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6- Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.
  • Natural products and their derivatives may also be used and include, for example, Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, paclitaxel (paclitaxel is commercially available as Taxol ® ), Mithramycin, Deoxyco-formycin, Mitomycin-C, L-Asparaginase, Interferons (especially IFN-alpha), Etoposide, and Teniposide.
  • Vinblastine Vincristine
  • Vindesine Bleomycin
  • Dactinomycin Daunorubicin
  • Doxorubicin Doxorubicin
  • Epirubicin Idarubicin
  • paclitaxel paclitaxel is commercially available as Taxol ®
  • Mithramycin Deoxyco-formycin
  • Mitomycin-C L-Asparaginase
  • Interferons especially IFN-alpha
  • Hormones and steroids include, for example, 17-alpha-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Tamoxifen, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, or Zoladex.
  • Exemplary synthetics include Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, and Hexamethylmelamine.
  • Methods and dosages for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. h addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the "Physicians' Desk Reference” (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ. 07645-1742, USA), the disclosure of which is incorporated herein by reference.
  • PDR Physicalians' Desk Reference
  • the novel isoform of CFR-1 described herein, or fragments thereof maybe used to produce an anti-neoplastic or anti-proliferative agent, in which compounds that are potentially effective against tumors are assayed for their ability to specifically bind to the novel CFR-1 isoform described herein.
  • this compound Upon a positive result, i.e., upon the occurrence of binding, this compound maybe assayed for its ability to induce apoptosis in neoplastic cells or cells of a pre-cancerous lesion or its ability to alter the rate of proliferation of such cells using the methods described herein.
  • a pharmaceutical composition for the treatment of a neoplastic disease.
  • Test extracts and compounds In general, compounds that alter a biological activity of the novel isoform of CFR-1 described herein are identified from large libraries of both natural products, synthetic (or semi-synthetic) extracts or chemical libraries, according to methods known in the art.
  • such compounds may be human antibodies, murine antibodies, humanized antibodies of any arbitrary species, as well as antibody fragments such as Fab and F(ab) 2 and/or Fab' fragments obtained by proteolytic cleavage of antibodies. These also include single strand antibodies and/or tetrameric and/or dimeric antibody forms and/or bispecific antibodies.
  • Fab and F(ab) 2 fragment antigen binding
  • Fab' fragments obtained by proteolytic cleavage of antibodies.
  • These also include single strand antibodies and/or tetrameric and/or dimeric antibody forms and/or bispecific antibodies.
  • the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention.
  • any number of chemical extracts or compounds can be screened using the exemplary methods described herein.
  • examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds.
  • Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WJ).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including, but not limited to, Biotics (Sussex, UK), Xenova (Slough, UK), Harbor
  • the antibody was eluted with 20 mM sodium phosphate, pH 7.5 in 30% isopropanol. Buffer exchange with PBS was perfo ⁇ ned using NAPTM- 10 columns (Amersham Pharmacia Biotech). Purity was determined by SDS gel electrophoresis and Western blotting.
  • the supernatant was centrifuged for 30 min at 100,000g in a swing-out rotor to pellet membranes. After washing the pellet with hypotonic buffer, it was resuspended in membrane lysis buffer (50 mM HEPES pH 7.4, 0.1 mM EDTA, 10% glycerol, and 1% TRITON X-100). A protease inhibitor (Boehringer, Mannheim, Germany) was added to all solutions.
  • the secondary antibody (peroxidase-coupled rabbit anti-human IgM antibody (Dianova, Hamburg, Germany)) was detected with the SUPERSIGNAL chemiluminescence kit from Pierce (KMF, St. Augustin, Germany). After three washes with PBS + 0.05%o Tween-20, the blots were incubated with the secondary antibody (peroxidase-coupled rabbit antihuman IgM antibody (Dianova, Hamburg, Germany)). The reaction was detected with the aid of the SUPERSIGNAL chemiluminescence kit from Pierce (KMF, St. Augustin, Germany).
  • FPLC unit Pharmacia (Freiburg, Germany) FPLC unit.
  • a Pharmacia SUPERDEX 200 column XK16/60
  • buffer A 100 mM Tris/Cl, pH 7.5, 2 mM EDTA, 40 mM NaCl, 1% Triton X-100.
  • the eluate was fractionated and examined in Western blot analysis for reaction with antibody 103/51. Positive fractions were loaded on a MONOQ (5/5) purification column using buffer A.
  • the bound proteins were eluted with a linear gradient using buffer B (100 mM Tris/Cl, pH 7.5, 1 M NaCl, 2 mM EDTA, 1 M NaCl, 1% Triton X-100), fractionated and examined in Coomassie- stained SDS-PAGE and Western blot analysis. Positive bands were cut out from gel and sequenced or used for immunization of mice.
  • buffer B 100 mM Tris/Cl, pH 7.5, 1 M NaCl, 2 mM EDTA, 1 M NaCl, 1% Triton X-100
  • Amplification was performed using the following cycle profile: 95°C, 2 minutes, followed by 35 cycles of 94°C, 30 seconds; 60°C, 30 seconds; 72°C, 60 seconds, and a final extension of 72°C for 4 minutes.
  • Cloning into the pCR-Script Amp SK (+) vector and DNA sequencing were performed as described, e.g., in Hensel et al. (Int. J. Cancer 81:229- 235 (1999)). The insert was subcloned into the pHook-2 vector (h vitrogen, Leek, Netherlands), and the accuracy of the cloning was verified by sequencing.
  • Transfection of cell line 23132 with pHOOK2-antiCFR-l was accomplished with PRIMEFECTOR reagent (PQLab, Er Weg, Germany) according to supplier's manual, h short, plasmid DNA was diluted to 10 ⁇ g/ml and the PRIMEFECTOR reagent was added in a 1 : 10 ratio to a serum-free growth medium. Diluted plasmid DNA (450 ⁇ l), diluted PRIMEFECTOR reagent (90 ⁇ l), and serumfree medium (460 ⁇ l) were mixed and incubated at room temprature ("RT"). 60-milliliter cell culture plates (70% confluent) were washed two times with serumfree medium, and then the PRIMEFECTOR/DNA mixture was added dropwise.
  • RT room temprature
  • Glycosidase assays Detached and washed cells were resuspended in RPMI-1640 containing 10% FCS and incubated for 1 hour on ice, then counted, and cytospins were prepared. After air-drying, cytospin preparations were acetone-fixed (10 min), washed, and incubated with 20 ⁇ U/ml O-glycosidase or 5 mU/ml N-glycosidase (Boehringer) for 4 hours at 37°C. Slides then were washed and immunohistochemically stained.
  • membrane extracts were incubated for 16 hours at 37°C with 1 mU/ml N-glycosidase diluted in deglycosylation buffer (50 mM PO 4 -Buffer, pH 7.4). As a control, extracts were incubated with deglycosylation buffer alone. Extracts then were separated by SDS- PAGE and Western blots were performed as described above.
  • BALB/c mice were immunized two times within 17 days with 5 ⁇ g purified antigen of antibody 103/51, and killed 4 days after the second immunization. Spleens were disrupted mechanically and fused with 1 x 10 NSO cells as described, e.g., m Vollmers et al. (Cell 40:547-557 (1985)). Antibody-producing hybridomas were tested through immunohistochemical staining and reaction in Western blot analysis. Clone 58/47-69 with positive reactivity was used for further experiments.
  • cytospins were prepared, air-dried at RT and fixed in acetone as described above. Cytospins then were blocked for 15 minutes with PBS/BSA (0.1 %) and incubated for 30 minutes with 10 ⁇ g/ml primary antibodies followed by three washings. Incubation with secondary antibody and staining was performed as described above.
  • MTT-proliferation assay The MTT-assay with the established cell line 23132 was performed as described, e.g., in Vollmers et al. (Cancer 74:1525-1532 (1994)). In short, trypsinized cells were diluted to lxl 0 6 cells/ml in complete growth medium, and 50 ⁇ l of cell suspension was added to each well of a 96-well plate. 50 ⁇ l of the antibody, diluted to the indicated concentrations with complete growth medium, were added to the wells, and plates were incubated for one or two days at 37°C in a humidified incubator.
  • MTT (3(4,5 dimethylthiazol)-2,5 diphenyltetrazolium bromide) solution (5 mg/ml) were added to each well, and plates were incubated for 30 minutes. After incubation, plates were centrifuged at 800g for 5 minutes, MTT solution was removed, the stained cell pellet was dissolved in 150 ⁇ l dimethylsulphoxide, and absorption was measured at wavelengths of 540 nm and 690 nm.
  • the cells were centrifuged for 10 minutes at 200 g, the supernatants were removed followed by an incubation with lysis-buffer for 30 minutes at RT. After centrifugation, the supernatants were transferred into a streptavidin-coated microtitre plate (MTP), immunoreagent added (mixture of 10% Anti-Histone-Biotin, 10% Anti-DNA-peroxidase (Anti-DNA POD) and 80% incubation buffer) and incubated for 2 hours at RT on a MTP shaker at 250 rpm. Following incubation, unbound components were removed by washing with incubation buffer.
  • MTP streptavidin-coated microtitre plate
  • immunoreagent added mixture of 10% Anti-Histone-Biotin, 10% Anti-DNA-peroxidase (Anti-DNA POD) and 80% incubation buffer
  • Peroxidase is determined photometrically with an ABTSTM as a substrate (1 ABTSTM (2,2'-Azino-di[3-ethyl-benz-thiazolin-sufonat) tablet in 5 ml substrate buffer).
  • the antibody induced apoptosis was measured at 405 nm against ABTS solution as a blank (reference wavelength approx. 490 nm).
  • mice grown in PAM-1 treated mice and in the control group were fixed in 3%> formaldehyde and embedded in paraffin. Sections were then processed using the apoptosis-specific FragEL-Klenow DNA Fragmentation Kit (Calbiochem-
  • RNA was prepared for the cDNA synthesis with the aid of the RNEASY kit from Quiagen.
  • lxl 0 6 cells were washed twice using ice cold PBS and pelletized at 1000 x g for 5 minutes and the RNA was prepared in accordance with the manufacturer description.
  • 5 ⁇ g RNA (1-5 ⁇ l solution) was mixed with 1 ⁇ l oligo-dTis (1 ⁇ g/ ⁇ l) and 2 ⁇ l random primer (40 ⁇ M) and brought up to a total volume of 8 ⁇ l using H 2 O.
  • the RNA was denatured for 10 minutes at 65°C and the sample was subsequently cooled on ice.
  • the PCR Mastermix consisted of 2.5 ⁇ l lOx Taq- polymerase buffer, 0.5 ⁇ l 10 mM NTPs, 1.5-2 ⁇ l 25 mM MgCl 2 , 0.5 ⁇ l each 20 pM 3' and 5' primer, and 0.2 ⁇ l Taq polymerase (1 U).
  • the amplification conditions for the various PCR products are shown in the following overview of the PCR program used for amplifying the various cDNAs.
  • 3 ⁇ l plasmid DNA was mixed with 1 ⁇ l primer (3.2 pM), 11 ⁇ l H 2 O, and 5 ⁇ l reaction mixture of the ABIPRISM Sequencing Kit and incubated in the thermocycler for 25 cycles using the following parameters:
  • the reaction mixture was purified via a Sephadex G-50 filled purification column.
  • a 100 ⁇ l pipette tip was loaded up to the upper edge with column material and centrifuged for 3 minutes at 2000 x g. Subsequently the sample was applied and the small column was centrifuged again.
  • the DNA was then precipitated by 2 ⁇ l Na acetate (pH 5.2) and 50 ⁇ l 100% ethanol and pelletized by centrifuging at 13,000 x g for 15 minutes. After drying, the DNA was received in 3 ⁇ l formamide/25 mM EDTA (5:1) and analyzed in the sequencer.
  • RNA-Isolation RNA from nonnal and cancerous gastric tissue of the stomach was isolated using the phenol-guanidine-isothiocyanate method with TRIZOL ® Reagent (hivitrogen).
  • TRIZOL ® Reagent hivitrogen
  • frozen normal and tumor tissues were cut in serial 5 ⁇ m sections on a freezing microtome.
  • 1 ml TRIZOL ® Reagent was added to the tissue samples and the solutions were homogenized subsequently.
  • the insoluble material was removed from the homogenate by centrifugation at 12,000 x g for 10 minutes at 4°C. 200 ⁇ l chloroform was added to the RNA containing supernatant, and, after mixing the solution was incubated for 3 minutes at RT.
  • RNA pellet was washed with 1ml of 75% ethanol and centrifuged for 5 minutes at 7,500 x g at 4°C.
  • the RNA pellet was air-dried and re-suspended in 80 ⁇ l DEPC-treated water. The integrity and quality of purified total RNA were controlled by 1% agarose gel electrophoresis and the concentrations were evaluated by spectrophotometry.
  • RT-PCR Semi-quantitative Reverse Transcription-PCR mRNA levels were examined using semi-quantitative Reverse Transcription- PCR (RT-PCR) method. Synthesis of first-strand cDNA from normal and cancerous gastric tissue was performed with 5 ⁇ g of total RNA using Moloney murine leukemia virus reverse transcriptase (M-MLV RT, hivitrogen GmbH, Düsseldorf, Germany) and oligo-dT primer according to the supplier's manual. The PCR method was used to detect CFR-1 mRNA. PCR reactions were carried out in a 25 ⁇ l volume with 2 nM MgCl 2 , 0,4 pM primer, 200 ⁇ m each dNTP and 1 unit of Taq polymerase (MBI).
  • M-MLV RT Moloney murine leukemia virus reverse transcriptase
  • CFR-1 mRNA was normalized to GAPDH mRNA levels.
  • the primers specific for CFR-1 and GAPDH were designed on their reported sequences and commercially synthesized by MWG-BIOTECH AG (Ebersberg, Germany).
  • the sequences of these oligonucleotides are 5' CAAGAGCAGACAG-GTCAGGTGG 3' (SEQ ID NO:22) and 5 ' CCGGAAGTTCTGTTG-GTATGAG 3 ' (SEQ ID NO:23) for CFR-1 and 5' GTGGAAGGACTCATGACCACAGTC 3' (SEQ ID NO:24) and 5' CATGTGGGCCATGAGGTCCACCAC 3' (SEQ LD NO:25) for GAPDH.
  • Pepsin cleavage For pepsin digestion of the PAM-1 antibody a buffer exchange with lOOmM sodium citrate (pH 3.5) using NAPTM-10 columns (Amersham Pharmacia Biotech) was used. Additionally pepsin digestion was done with an unrelated human IgM antibody (Chrompure IgM, Dianova, Hamburg, Germany) to obtain a suitable negative control. For each milligram of antibody, 5 ⁇ g pepsin (Sigma Aldrich, Taufkirchen, Germany) was added, followed by incubation for 10-15 minutes in a 37 °C water bath. The reaction was stopped by adding 1/10 volume of 3.0 M Tris (pH 8.8) followed by centrifuging at 10,000 g for 30 minutes.
  • cytospin preparations were prepared and stained with antibodies 103/51 and anti-cytokeratin 18 as a control (data not shown).
  • the immunohistochemistry showed a clear reduction of staining in cells transfected with the pHOOK-CFR antisense vector when compared to mock- transfected cells (Figs. 2A and 2B). This confirmed the binding of antibody 103/51 to CFR-1.
  • the slight cytoplasmic staining visible in both stainings might be due to nonspecific binding often observed in staining with human IgM antibodies on acetone- fixed cells.
  • Membrane expression and the effect of transfection were also tested by flow cytometry (Figs. 2G-2I).
  • Glycosidase assay CFR-1 is a sialoglycoprotein with 5 possible N-glycosylation sites and it has been shown by treatment with glycosidase F that the molecule is glycosylated at these sites (Steegmaier et al, Nature 373:615-620 (1995)). Since tumor-reactive antibodies often react with carbohydrate residues, we investigated whether this is the case for the antibody 103/51. Cytospin preparations of cell line 23132 were incubated for 4 hours with O- and N-glycosidases, and then subjected to immunohistochemical staining with antibody 103/51. Treatment of cells with N-glycosidase led to a dramatic decrease in 103/51 staining (Fig.
  • the nucleic acid sequence (SEQ ID NO:l) and the amino acid sequence (SEQ LD NO:2) of the variable region of the heavy chain of murine antibody 58-49/69 are shown in Figures 8A and 8B.
  • CDR1 of the 58-49/69 variable region heavy chain spans nucleotides 31-45 which encode amino acids 11-15
  • CDR2 spans nucleotides 88-138 which encode amino acids 30-46
  • CDR3 spans nucleotides 235-264 which encode amino acids 79-88.
  • the D-gene spans nucleotides 235-243 and the J-gene spans nucleotides 243-288.
  • variable region of the light chain of murine antibody 58-49/69 are shown in Figures 9A and 9B.
  • CDR1 of the 58-49/69 variable region light chain spans nucleotides 49-96 which encode amino acids 17-32
  • CDR2 spans nucleotides 142-162 which encode amino acids 48-54
  • CDR3 spans nucleotides 259-285 which encode amino acids 87-95.
  • the nucleic acid sequence (SEQ ID NO:28) and the amino acid sequence (SEQ ID NO:26) of the variable region of the heavy chain of human antibody 103/51 are shown in Figure 17.
  • CDR1 of the 103/51 variable region heavy chain spans nucleotides 31-54 which encode amino acids 11-18
  • CDR2 spans nucleotides 106-129 which encode amino acids 36-43
  • CDR3 spans nucleotides 244-312 which encode amino acids 82-104.
  • the nucleic acid sequence (SEQ ID NO: 29) and the amino acid sequence (SEQ ID NO:27) of the variable region of the light chain of human antibody 103/51 are shown in Figure 18.
  • CDR1 of the 103/51 variable region light chain spans nucleotides 82-96 which encode amino acids 28-32
  • CDR2 spans nucleotides 151-159 which encode amino acids 51-53
  • CDR3 spans nucleotides 268-300 which encode amino acids 90-100.
  • the staining pattern of antibody 103/51 shows a strong co ⁇ elation with the activation pattern shown by Ki67 staining (Ramires et al, 1997, J. Pathol. 182:62-67).
  • Ki67 staining A more intensive staining of antibody 103/51 was seen in the proliferation zone of gastric dysplasia also correlating with Ki67 staining. The strongest staining was found in the proliferating zone of gastric adenocarcinoma.
  • Table IB Reaction pattern of antibody 103/51 with different normal tissues and pre- cancerous lesions.
  • HCC hepatocellular carcinoma, l Proliferation zone, Glandular foveola, 2 Glomerular, fascicular zone (membranous staining), 3 Collection tubes of the endoplasmic reticulum.
  • Antibody 103/51 leads to the stimulation of cell line 23132 in vitro.
  • MTT mitochondrial hydroxylase assay
  • MTT mitochondrial hydroxylase assay
  • Example 3 The antigen recognized by the PAM-1 antibody is specifically expressed on cancerous and pre-cancerous cells
  • Table 2 Expression of the isoform of CFR-1 recognized by PAM-1 on tumor tissues.
  • Esophagus Squamous cell 4 1 50-70 4/1 5/0 Adeno (Barrett) 12 1 48-85 11/2 11/2
  • HCC Liver Adeno
  • the CFR-1 isoform recognized by the PAM-1 antibody is expressed in stomach cancer precursor lesions like H. pylori- ⁇ nducQd gastritis and gastric dysplasia and the level of expression likely increases with the escalation of malignancy.
  • stomach cancer precursor lesions like H. pylori- ⁇ nducQd gastritis and gastric dysplasia and the level of expression likely increases with the escalation of malignancy.
  • Ki67 proliferation marker
  • Esophagus Barrett metaplasia 9 0 42-69 8/1 9/0 Barrett dysplasia 4 2 62-86 3/3 6/0
  • Colon Dysplasia (ulcerative colitis) 4 1 42-57 4/1 5/0 Tubular adenoma 5 2 54-85 5/2 6/1 Villous adenoma 8 2 45-85 9/1 8/2
  • Bronchus Squamous metaplasia 5 0 61-72 3/2 5/0
  • Epithelial dysplasia 3 0 64-75 3/0 3/0
  • Prostate PIN 15 0 55-76 nd 14/1 Adenoma-carcinoma sequence Malignant changes and the resulting carcinomas of the colon belong to the frequently occurring neoplasia and are often associated with high mortality. The formation of colon carcinomas is a multi-step process which could be retraced to the so-called adenoma-carcinoma sequence. All, adenomatous lesions arise as a result of epithelial proliferative changes and there is strong evidence that adenomas are a precursor lesion for invasive colorectal adenocarcinoma (Cummings, Semin. Gastrointest. Dis. 11:229-237 (2000); Scheiden et al., hit. J.
  • colorectal carcinogenesis provides the. ideal opportunity to investigate the reactive pattern of the PAM-1 antibody in precancerous lesions in more detail. Additional immunohistochemical stainings were therefore performed on different types of mucosa and epithelia.
  • PAM-1 antibody reacts with H y/orz ' -associated chronic active gastritis, high-grade dysplasia and gastric adenocarcinoma.
  • Ulcerative colitis- related dysplasia which consists of atypical changes in epithelial cells, is also recognized to be involved in the development of colorectal adenocarcinoma (Wong et al., Histopathology 37:108-114 (2000)).
  • colorectal adenocarcinoma On this high grade dysplasia, clear staining by the PAM-1 antibody, especially of these atypical epithelial cells, was observed. The most intense staining was found in colorectal adenocarcinoma, following the obtained results in case of gastric mucosa.
  • the expression of the isoform of CFR-1 recognized by the PAM-1 antibody c ⁇ elates with the pattern of Ki67.
  • Barrett carcinogenesis Barrett esophagus is a complication of long-standing gastroesophageal reflux.
  • the distal squamous mucosa is replaced by metaplastic columnar epithelium, as a response to prolonged injury.
  • the carcinogenesis of esophagial adenocarcinoma takes 5 place from Barrett metaplasia to Barrett dysplasia (Spechler, Seudin. Gastrointest. Dis. 7:51-60 (1996); Haggitt, Hum. Pathol. 25:982-933 (1994); Devesa et al, Cancer 83:2049-2053 (1998); and Spechler, Am. J. Med. I l l Suppl 8A:130-136 (2001)).
  • Cervical neoplasia Increased expression of the isoform of CFR-1 recognized by the PAM-1 antibody on cervical epithelium with architectural abnormalities was also observed.
  • the precursors of the cervical squamous cell carcinoma are classified as cervical ; • intraepithelial neoplasia grade I, ⁇ and HJ. Mild dysplasiae are termed CIN I up to ' 25 carcinoma in situ lesions CTN HJ (Arends et al., J. Clin. Pathol. 51:96-103 (1998)).
  • the precurs ⁇ rlesions (CLN I-HI) and invasive malignancy of the cervix, the squamous cell carcinoma were included in this study.
  • Bronchial carcinogenesis ' ⁇ Carcinomas of the lung are one of the most frequently occurring carcinomas world-wide. The most common type is the squamous cell carcinoma, which correlates closely with a history of smoking. In the airways of smokers, squamous metaplasia and dysplasia are usually present. In squamous metaplasia the normal bronchial ciliated epithelium is replaced by squamous epithelium. With occurrence of cyto logical disturbance and severe atypia, the lesion becomes known as squamous dysplasia (Colby et al, Adv. Anat. Pathol. 5:205-212 (1998); and Franklin, J. Thorac. Imaging 15:3-12 (2000)).
  • Normal ciliated epithelium shows no expression of the isoform of CFR-1 recognized by the PAM-1 antibody, while the PAM-1 antibody reacted with metaplasia and dysplasia of bronchus epithelium.
  • Squamous cell metaplasia of the bronchus represents the initial stages of carcinogenesis and shows a lower intensity of staining compared to dysplasia.
  • For dysplasia the preliminary stage of cancer, a more intensive staining was observed. The most intense staining was again observed in the squamous cell carcinoma, hi each of the three cases in this study, the staining of PAM-1 correlated with the reaction pattern of Ki67.
  • PAM-1 antibody is specific for malignant proliferation and not involved in normal proliferation processes (e.g., regeneration of tissue), we stained different proliferative regions of healthy and pre-malignant tissue with PAM-1 and Ki67 antibodies. We observed that the proliferation zone of normal colon mucosa is positive for Ki67, but negative for expression of the isoform of CFR-1 recognized by the PAM-1 antibody.
  • Ki67 shows a positive staining of the proliferation zone while the isoform of CFR-1 recognized by the PAM-1 antibody is not expressed.
  • non-dysplastic intestinal-type Barrett metaplasia which defines Ba ⁇ ett's esophagus the pre-malignant lesion for adenocarcinoma of the esophagus, shows a positive expression of Ki67 and the isoform of CFR-1 recognized by the PAM-1 antibody. This clearly shows that the isoform of CFR-1 recognized by the PAM-1 antibody is not expressed in healthy proliferating cells.
  • Apoptosis is the programmed cell death, suicide of cells, through fragmentation of the DNA, cell shrinkage, and dilatation of the endoplasmic reticulum, followed by cell fragmentation and the fo ⁇ nation of membrane-bound vesicles, or apoptotic bodies.
  • Apoptosis the physiological form of cell death, guarantees rapid and clean removal of unnecessary cells, without triggering inflammation processes or tissue trauma, as in the case of necrosis. Under pathological conditions, it is also used for removing malignant cells, such as cancer precursor cells.
  • cytotoxic T-lymphocytes or cytokines such as tumor necrosis factor, glucocorticoids, and antibodies. It is the most frequent cause of death of eukaryotic cells and occurs in embryogenesis, metamorphosis, and tissue atrophy. Apoptotic receptors on the cell surface, such as those of the NGF/TNF family, are predominantly expressed on lymphocytes, but are also found on various other cell types, wherefore they are not suitable for cancer therapy, h particular, ligands and antibodies for these receptors have led to liver damage in in vivo tests. Therefore, tumor-specific receptors having apoptotic function are especially important.
  • PAM-1 cleavage Pepsin digestion was used to cleave the intact pentameric PAM-1 antibody into IgM antibody fragments. Following cleavage the resulting fragments were analyzed by SDS-PAGE and Western blotting under non-reducing conditions. After blotting, the intact antibody showed the characteristic bands corresponding to intact antibody, monomeric forms and light chains. By SDS-PAGE, the intact pentameric IgM of about 900 kDa was, unable to migrate into the stacking gel. Following 10-15 minutes of treatment with pepsin the pentameric form was completely digested into monomeric, F(ab) , Fab, and light chain fragments which could be clearly identified by molecular weight (Figs. 11 A and 1 IB).
  • PAM-1 in vitro activity To define the in vitro activity of the fragmented PAM-1 we used the colorimetric mitochondrial hydroxylase assay (MTT). We incubated cell line 23132/87 with various concentrations of the fragmented PAM- 1 and found that the fragmented PAM-1 antibody inhibited cell proliferation in a concentration dependent manner (Fig. 13 A). In contrast the cell growth of cells treated with different concentrations of unrelated fragmented IgM is not affected. To further investigate the inhibitory effect of fragmented PAM-1 antibody on tumor cell growth, the apoptosis-specific Cell Death Detection ELISA PLUS Kit was used. The experiment illustrated in Fig. 13B clearly showed that fragmented PAM-1 antibody inhibits cell growth by inducing apoptosis in stomach carcinoma cells in vitro.
  • MTT colorimetric mitochondrial hydroxylase assay
  • Fragmented PAM-1 in vivo activity To determine the effects of fragmented PAM-1 on tumor cell growth in vivo, a nude mouse-human stomach carcinoma cell system was used. A concentration of 2 x 10 6 cells derived from the human stomach carcinoma cell line 23132 were injected intraperitoneal (i.p.) into NMRI nu/nu mice. Four days after the inoculation of tumor cells, 200 ⁇ g of fragmented PAM-1 antibody, diluted in PBS, was injected i.p. Control mice received the same quantity of unrelated fragmented human IgM. Throughout the duration of the study, tumor growth was controlled macroscopically. After 23 days the mice were sacrificed. The control mice developed measurable tumors i.p. from day 5 onwards.
  • mice treated with fragmented PAM-1 antibody did not develop detectable tumors before day 10. Moreover, the tumors which developed during the course of the experiment showed a significant reduction in growth and size. (Fig. 14A).
  • the study was also extended to include an inspection of organs and tissues of the mice for hidden spread of tumors and other alterations, hi two additional experiments mice were inoculated i.p. with gastric cancer received fragmented PAM-1 or control antibody. After 23 days mice were inspected for tumor-growth. The control group showed expanded tumor spreading into the peritoneum, diaphragm, kidney, stomach, intestine, liver and spleen (the spleen was enlarged in all cases).
  • mice treated with fragmented PAM-1 antibody showed a reduced spread of tumors and in addition there were no enlarged spleens observable.
  • the overall weight of tumor mass was significantly reduced by the PAM- 1 antibody (Figs. 14B and 14C).
  • Morphological analysis of the tumors revealed that tumors from mice treated with fragmented PAM-1 antibody exhibited not only a reduced size, but also regressive changes in growth pattern like tumor-regression, infiltration and a high number of pyknotic cells (Figs. 15A and 15D).
  • Figs. 15C and 15F show immunoperoxidase staining with an apoptosis-positive control in which all cell nucleic are stained.
  • mice in the respective test and control groups were injected with 200 ⁇ g of the PAM-1 IgG antibody or the control IgG antibody (Chrompure human IgG) at day 1, 3, 5, 7, and 9.
  • the mice were sacrificed at day 14 and the tumor weight and volume were compared between the control and test groups.
  • mice were sacrificed at day 27 and the tumor weight and volume were compared between the control and test groups.
  • PAM-1 -induced tumor regression was also verified by staining tissue obtained from the mice injected with pancreatic cancer cells and either the PAM-1, or the control IgG antibody, for keratin or with haematoxilin-eosin. The reduction in keratin staining seen in Fig.
  • FIG. 21C when compared with the control (Fig. 21 A) indicates tumor cell death in the mice receiving the PAM-1 antibody, as does the existence of apoptotic cells in tumor tissue obtained from mice receiving the PAM-1 IgG antibody (indicated by arrows in Fig. 2 ID) and not in the control tissue (Fig. 21B).
  • a patient suspected of having a neoplasm, such as stomach cancer may be given a dose of radioiodmated PAM-1 antibody or fragment thereof, or another tumor- specific polypeptide, and radiolabeled unspecific antibody using the methods described herein. Localization of the tumor for imaging may be effected according to the procedure of Goldenberg et al. (N. Engl. J. Med., 298:1384, 1978). By LV. an 1 1 infusion of equal volumes of solutions of I-PAM-1 antibody and Tc-99m-labeled unspecific antibody may be administered to a patient.
  • the patient Prior to administration of the reagents I.V., the patient is typically pre-tested for hypersensitivity to the antibody preparation (unlabeled) or to antibody of the same species as the antibody preparation.
  • I ⁇ To block thyroid uptake of I, Lugol's solution is administered orally, beginning one or more days before injection of the radioiodmated antibody, at a dose of 5 drops twice or three-times daily. Images of various body regions and views may be taken at 4, 8, and 24 hours after injection of the labeled preparations.
  • the neoplasm e.g., a stomach adenocarcinoma
  • the neoplasm is detected by gamma camera imaging with subtraction of the Tc-99m counts from those of I, as described for I -labeled anti- CEA antibody and Tc- 99m-labeled human serum albumin by DeLand et al. (Cancer Res. 40:3046, 1980).
  • imaging is usually clear and improves with time up to the 24 hour scans.
  • a patient diagnosed with a neoplasm may be treated with PAM-1 antibodies or fragments thereof as follows.
  • Lugol's solution may be administered, e.g., 7 drops 3 times daily, to the patient.
  • a therapeutic dose of 131 I-PAM-1 antibody may be administered to the patient.
  • a 131 I dose of 50 mCi maybe given weekly for 3 weeks, and then repeated at intervals adjusted on an individual basis, e.g., every three months, until hematological toxicity interrupts the therapy.
  • the exact treatment regimen is generally determined by the attending physician or person supervising the treatment.
  • the radioiodmated antibodies may be administered as slow LV. infusions in 50 ml of sterile physiological saline.
  • a reduction in the size of the primary tumor and metastases may be noted, particularly after the second therapy cycle, or 10 weeks after onset of therapy.
  • Example 7 Treatment Using Conjugated Antibodies
  • a patient diagnosed with a neoplasm for example, a patient with stomach cancer that has metastasized, may be treated with solutions of 131 I-PAM-1, 10 B-PAM-
  • I-labeled PAM-1 antibody in 50 ml of sterile physiological saline
  • An amount of I-labeled PAM-1 antibody sufficient to provide 100 mCi of 131 I activity based on a 70 kg patient weight may be administered to the patient.
  • This dosage is equal to 3.3 mg of an antibody having 40-80 Boron atoms and 8-16 Boron- 10 atoms per antibody molecule.
  • the neoplasm is first precisely localized using the procedure of Example 5.
  • Lugol's solution should be continuously administered to the patient, as in the previous example.
  • a well-collimated beam of thermal neutrons may then be focused on the defined tumor locations.
  • Irradiation with an external neutron beam dose of 400-800 rads, delivered in a period of from 8-20 min, is effected for each tumor locus, and is optionally repeated with administration of the tumor-locating antibody, with or without the radiolabel, at intervals adjusted on an individual basis, but usually not exceeding a total dose of 3200 rads unless simultaneous external irradiation therapy is indicated.
  • an anti-tumor agent such as a chemotherapeutic agent, may also be administered to the patient.

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Abstract

L'invention concerne des nouveaux polypeptides et des procédés pour utiliser lesdits polypeptides dans le diagnostic, la détection, la surveillance et le traitement de tumeurs chez les mammifères, par exemple chez un humain.
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EP2361929A1 (fr) 2011-08-31

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