[go: up one dir, main page]

WO2003020115A2 - Methodes et compositions de detection du cancer - Google Patents

Methodes et compositions de detection du cancer Download PDF

Info

Publication number
WO2003020115A2
WO2003020115A2 PCT/US2002/027747 US0227747W WO03020115A2 WO 2003020115 A2 WO2003020115 A2 WO 2003020115A2 US 0227747 W US0227747 W US 0227747W WO 03020115 A2 WO03020115 A2 WO 03020115A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibodies
cancer
polypeptide
biological sample
antibody
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.)
Ceased
Application number
PCT/US2002/027747
Other languages
English (en)
Other versions
WO2003020115A9 (fr
Inventor
George Doellgast
Jennifer Freeman
Deyrick O. Dean
James W. Geyer
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.)
AMPLISTAR Inc
Original Assignee
AMPLISTAR 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
Application filed by AMPLISTAR Inc filed Critical AMPLISTAR Inc
Publication of WO2003020115A2 publication Critical patent/WO2003020115A2/fr
Anticipated expiration legal-status Critical
Publication of WO2003020115A9 publication Critical patent/WO2003020115A9/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the detection of cancer by detection and/or measurement of autoantibodies.
  • the invention also relates to compositions and kits adapted to provide an assay for such autoantibody cancer markers.
  • the invention also relates to polypeptides and antibodies useful in the detection and treatment of cancer.
  • SEREX in a manner that demonstrates at least some reactivity of the marker with antibodies from cancer patient serum.
  • the observed serological reactivity of the markers has been characterized as a factor establishing the significance of the antigen marker. Little consideration has been afforded the concept of anti-cancer antibodies as markers themselves.
  • Current tests have focused on the detection of the protein marker, e.g. , the prostate-specific antigen test ("PSA" test) for prostate cancer, and CA-125 test for ovarian cancer.
  • PSA prostate-specific antigen test
  • Blood tests have many advantages for screening and/or diagnosis of cancer. Such tests are convenient and minimally objectionable in many respects.
  • protein markers have several drawbacks as cancer screening/diagnostic markers. Important markers may reside in disease tissue and fail to circulate sufficiently to provide the basis for a reliable blood test. Further, tests for circulating markers may not provide an adequate basis for detecting disease in early stages, possibly because circulating markers may only appear after significant cellular breakdown has begun during an advanced disease state. For at least these reasons, current tests have significant drawbacks.
  • nucleic acid probes used as cancer gene tests have proved to be far from simple. Recently, researchers completed the first sequence map of the human genome, and can now identify virtually all of the nearly 100,000 human genes. As the function of each gene become known, mutations that either directly cause disease or are associated with a predisposition to disease are likely to become valuable markers for diagnosis, prognosis, and risk assessment. However, gene discovery and mutation analyses represent only the initial step.
  • Gene probe tests for cancer have the following disadvantages: it is problematic to identify every mutation for a "diseased" gene and, because every mutation/cancer gene is not known, a "negative” test has limited clinical utility; genes may not be cancer-specific; by their nature, such tests only indicate that a subject is at risk (no indication of the presence of the actual disease); and most probe tests require tissue samples that are difficult and inconvenient to obtain.
  • a need exists for an effective method of detecting cancer that may be carried out based on a circulating marker detectable and/or measurable in a sample that is convenient to obtain in a manner acceptable to test subjects.
  • the present invention relates to a method of detecting cancer in a mammalian subject comprising contacting a biological sample from the mammalian subject with at least one Al 14 polypeptide; and detecting antibodies in the biological sample that react with at least one Al 14 polypeptide.
  • the invention provides a method of screening for mammalian subjects having an increased risk for the presence of cancer, comprising contacting a biological sample from the mammalian subject sample with Al 14; measuring a level of antibodies in the biological sample bound to Al 14; and comparing the level of patient antibodies to a predetermined level of Al 14 antibodies present in one or more samples from one or more non-cancerous subjects.
  • An elevated level of the antibodies in the biological sample is indicative of an increased risk for the presence of cancer in the mammalian subject.
  • the invention provides a method of determining the status of a cancerous condition in a mammalian subject, comprising contacting a biological sample from the mammalian subject sample with Al 14; measuring a level of antibodies in the biological sample bound to Al 14; and comparing the level to a level determined previously, wherein any difference between the levels indicates a change in the status of the cancerous condition.
  • the invention provides polypeptides and antibodies useful in vaccination and other immunotherapy protocols effective against cancer or cancerous conditions.
  • the methods of the invention can be used for screening, diagnosis, prognosis or other detection or evaluation of cancer or cancer-related disease, in particular, but not limited to, the breast, colon and ovarian forms of the disease.
  • the methods and compositions can also be used directly or indirectly to provide therapy to subjects suffering from cancer or a cancerous condition.
  • Figure 1 illustrates a nucleotide sequence (SEQ ID NO: 1) including a sequence encoding the Al 14 marker protein (shaded portion of the sequence).
  • Figure 2 illustrates the amino acid sequence (SEQ ID NO: 2) of the Al 14 marker protein.
  • the C-terminal LEQ is a product of the expression vector sequence.
  • Figure 3 illustrates the primer sequences (Forward, SEQ ID NO 3; and Reverse, SEQ ID NO: 4) used to amplify the coding sequence as shown in Figure 1, and to introduce a 5' Notl and a 3' Xbal restriction endonuclease site into the resultant nucleic acid molecule to facilitate expression cloning of the Al 14-encoding sequence.
  • Al 14 means a polypeptide having the amino acid sequence of SEQ ED NO: 2 or conservative amino acids substitutions thereof, or an immunoreactive fragment or epitope of a polypeptide having such an amino acid sequence.
  • amino acid or “amino acid residue” mean naturally occurring L amino acids or D amino acids.
  • amino acids The commonly used one- and three-letter abbreviations for amino acids are used herein (see, e.g., Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)).
  • antibody means a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that specifically binds and recognizes an analyte (antigen).
  • Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain has a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms “variable light chain” (or “V ”) and “variable heavy chain” (or “V H ”) refer to these light and heavy chains, respectively.
  • Antibodies exist, for example, as intact immunoglobulins or as a number of well- characterized antigen-binding fragments produced by digestion with various peptidases. For example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce an F(ab') 2 fragment, a dimer of Fab which itself is a light chain joined to V H - CHI by a disulfide bond. The F(ab') fragment can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab') 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology, Third Edition, W.E.
  • antibody also includes antibody fragments, such as a single chain antibody, an antigen binding F(ab') 2 fragment, an antigen binding Fab 1 fragment, an antigen binding Fab fragment, an antigen binding Fv fragment, a single heavy chain or a chimeric antibody.
  • antibody fragments such as a single chain antibody, an antigen binding F(ab') 2 fragment, an antigen binding Fab 1 fragment, an antigen binding Fab fragment, an antigen binding Fv fragment, a single heavy chain or a chimeric antibody.
  • Such antibodies can be produced by modifying whole antibodies, or synthesizing the antibodies de novo using recombinant DNA methodologies. (See, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999), the disclosure of which is inco ⁇ orated by reference herein).
  • autoantibody means an antibody produced by a subject in response to an antigen produced within the subject, e.g. by a tumor, cancer, or cancerous condition with the subject.
  • autoantibodies although produced by the subject in response to an endogenous antigen, can be characterized (detected or measured) by their reaction with a test antigen produced or obtained from a variety of sources including by recombinant techniques.
  • a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not substantially alter activity.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. See, e.g., Creighton, Proteins, W. H. Freeman and Company (1984), inco ⁇ orated herein by reference. For example, the following six groups each contain amino acids that are conservative substitutions for one another:
  • A alanine
  • S serine
  • T threonine
  • D aspartic acid
  • E glutamic acid
  • heterologous refers to a nucleic acid or polypeptide from a different source, such as a tissue, organism or species, as compared with another nucleic acid or polypeptide.
  • immunoreactive means that such a substance has an antibody as a specific binding partner. Immunoreactive substances may or may not be immunogenic (antigenic). Accordingly, immunoreactive substances include peptide epitopes which can bind specifically to particular antibody, but not exhibit immunogenicity (antigenicity).
  • isolated refers to a nucleic acid, polypeptide or antibody that has been removed from its natural cellular environment.
  • an isolated nucleic acid is typically at least partially purified from other cellular nucleic acids, polypeptides and other constituents.
  • the term “marker” means any detectable or measurable factor or substance that can be correlated with the presence or progression of cancer or a cancerous disease condition.
  • the specific autoantibodies of the invention are “markers,” as are antigens or other substances characterized by their association with a cancer disease state.
  • the terms “polynucleotide” and “nucleic acid” mean a polymer comprising multiple nucleotide units, including ribo- or deoxyribonucleotides or related structural variants.
  • a polynucleotide or nucleic acid can be of any length, and include RNA, genomic and cDNA, sense and antisense strands, single-strands and double strands, synthetic forms, and any mixtures thereof. Nonnatural or synthetic polynucleotide units can be included.
  • polypeptide means a polymer of amino acids and its equivalent and does not refer to a specific length of the polymer. Peptides, oligopeptides and proteins are included. A “fragment” refers to a portion of a polypeptide having at least 6 contiguous amino acids, typically 8-10 contiguous amino acids, more typically at least 20 contiguous amino acids, still more typically at least 50 contiguous amino acids of the polypeptide of interest.
  • polypeptide are polypeptides containing one or more analogs of an amino acid (e.g., nonnatural amino acids, and the like), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
  • primer means a polynucleotide, typically an oligonucleotide, that acts as a point of initiation of polynucleotide synthesis used under conditions that lead to the synthesis of a primer extension product.
  • oligonucleotide means a polynucleotide of from about six (6) to about one hundred (100) nucleotides in length. Oligonucleotides can be synthesized on an automated synthesizer.
  • reactive when used to describe the relationship between a substance and an antibody, means that the antibody has an affinity for the substance, which results in the formation of a non-covalent complex between the antibody and the substance.
  • the present invention can be practiced using conventional molecular biological, microbiological, and recombinant nucleic acid techniques unless otherwise indicated. Such techniques are well known to those skilled in the art and ample guidance is presented in the current literature, e.g. as in Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001); and "Current Protocols in Molecular Biology,” Ausebel, et al, eds., Current Protocols, (1994).
  • the present invention provides a method of detecting cancer by detecting and/or measuring anti-cancer antibodies that bind specifically to Al 14.
  • the detection methods of the present invention can be used in screening, diagnosis, and prognosis relating to detection and monitoring of cancer and cancer-related disease.
  • Figure 1 illustrates SEQ ID NO: 1, showing the portion encoding Al 14 in gray shading.
  • Figure 2 illustrates the amino acid sequence of the protein encoded by the nucleotide sequence from position 128 to position 1816 of SEQ ID NO: 1.
  • Figure 3 illustrates the primers which are used in a polymerase chain reaction (PCR) in order to amplify the coding region of SEQ ID NO: 1 and to generate a 5' Notl restriction endonuclease site, as well as a 3' Xbal site to facilitate insertion into an expression vector.
  • PCR polymerase chain reaction
  • a partial nucleotide sequence with homo logy to Al 14 has been reported to show colon-specific expression (see WO 00/07632 (PCT/US99/16357) - sequence designated "Clnl 17").
  • WO 00/07632 suggests that mRNA corresponding to the Clnl 17 sequence is expressed at elevated levels in the colon, when compared with a variety of other non- colon associated tissues. Over-expression of Clnl 17 was also shown in colon cancer tissue, when compared with the respective, adjacent non-cancerous tissue for each sample. Such findings suggest that a protein marker encoded by a sequence related to Clnl 17 may be tissue- and or cancer-specific. (See also GenBank Accession No. XM_015636; GI: 14745930). The present inventors have demonstrated that autoantibodies directed against Al 14 become elevated in a subpopulation of cancer patient's sera during the course of this disease (see Examples). The present invention provides methods using Al 14 to detect specific autoantibodies in order to determine the presence or increased risk of cancer or a cancerous condition.
  • autoantibodies specific for Al 14 are useful as a cancer-specific marker. Detection and/or measurement of such autoantibodies allows detection of "positive" cancer patients in several cancers, including, but not limited to, breast, colon and ovarian (see Examples).
  • Al 14 autoantibodies can be detected in samples such as, but not limited to, biological fluids and lavages or washes of various natures. Biological fluid samples can be blood, serum, ascites fluid, saliva, urine, sweat, or any other fluid obtained from a subject, which is found to contain a measurable amount of autoantibody specific for Al 14. Lavages and washes can also be analyzed for the presence of specific autoantibodies.
  • the autoantibodies relevant to the present invention include IgA, IgD, IgE, and IgM, in addition to IgG.
  • the invention provides isolated nucleic acids which encode a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, and conservative substitutions thereof.
  • the invention also provides nucleic acids encoding antibody-binding fragments or epitopes of SEQ ID NO: 2.
  • Polypeptides are also provided which have the amino acid sequence as shown in SEQ ID NO: 2 or conservative substitutions thereof.
  • Antibody- binding fragments are also provided.
  • autoantibodies are detected using a polypeptide, fragment or epitope to which they specifically bind.
  • Immobilized or labeled antigens can be used.
  • the antigens useful in the methods of the invention are those peptides, polypeptides and/or proteins comprising all or a portion of the sequence of Al 14 (SEQ ID NO:2), as well as conservative, immunoreactive substitutions thereof.
  • the present invention relates to a method of detecting cancer in a mammalian subject comprising contacting a biological sample from the mammalian subject with at least one Al 14 polypeptide; and detecting antibodies in the biological sample bound to the at least one Al 14 polypeptide. Detection of antibodies bound to the at least one Al 14 polypeptide is indicative of the presence of cancer in the mammalian subject. Alternatively, the detection of antibodies binding to Al 14 can indicate an increased risk of cancer in the mammalian subject providing the biological sample.
  • the invention provides a method of screening for mammalian subjects having an increased risk for the presence of cancer, comprising contacting a biological sample from the mammalian subject sample with Al 14; measuring a level of antibodies in the biological sample bound to Al 14; and comparing the level of patient antibodies to a predetermined level of Al 14 antibodies present in one or more samples from one or more non-cancerous subjects.
  • An elevated level of the antibodies in the biological sample is indicative of an increased risk for the presence of cancer in the mammalian subject.
  • the invention provides a method of determining the status of a cancerous condition in a mammalian subject.
  • the method involves contacting a biological sample from the mammalian subject sample with Al 14; measuring a level of antibodies in the biological sample bound to Al 14; and comparing the level to a level determined previously. Any difference between the levels indicates a change in the status of the cancerous condition.
  • the methods of the invention can be used for screening, diagnosis, prognosis or other detection or evaluation of cancer, including, but not limited to, the breast, colon and ovarian forms of the disease.
  • the Al 14 polypeptide can be a polypeptide containing the amino acid sequence of SEQ ID NO: 2 or conservative amino acid substitutions thereof.
  • the polypeptide can also be an immunoreactive fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a sequence having conservative amino acid substitutions thereof.
  • polypeptides corresponding to portions of the Al 14 marker are particularly useful according to the methods of the invention.
  • Polypeptides corresponding to at least about nine consecutive amino acids of any of the following regions of SEQ ID NO: 2 can be used: from about position 1 to about position 31; from about position 70 to about position 85; from about position 215 to about 223; from about position 311 to about position 350; from about 401 to about 409; and from about position 551 to about position 563.
  • Polypeptides can be used corresponding to at least about 9 consecutive amino acids of one of the following regions of SEQ ID NO: 2: from about position 5 to about position 15; from about position 71 to about position 82; from about position 315 to about position 325; from about 338 to about position 348; and from about position 552 to about 565.
  • a polypeptide can also be used corresponding to at least about 9 consecutive amino acids of a region of SEQ ID NO: 2 from about position 71 to about position 82.
  • a polypeptide can be used corresponding to at least about 9 consecutive amino acids of SEQ ID NO: 2 from about position 552 to about 565.
  • the present invention also provides antibodies that bind specifically to at least one Al 14 polypeptide.
  • These antibodies are recoverable from biological samples according to the present invention, but can be subsequently analyzed to determine the antibody primary structure. This information allows the antibodies to be produced recombinantly and such recombinantly produced antibodies are within the scope of the present invention.
  • the antibody-binding polypeptides can be used to produce antibodies having a high affinity for the same or similar polypeptides in vivo. As discussed in greater detail below, the polypeptides can be used to generate specific antibodies using a variety of well-known techniques.
  • Such antibodies are useful in preparation of therapeutic agents where such polypeptides are associated with cancer cells or are functionally associated with cancer or its progression.
  • Antibodies specific for the polypeptides of the invention can be coupled to cytotoxic or other chemotherapeutic agents.
  • agents can include radioactive substances, ricin toxin (or ricin A chain), diptheria toxin, Pseudomonas toxin, or adriamycin.
  • Fy proteins derived from monoclonal antibodies can be used for immunotoxin therapy. The small size of these antibody proteins allows increased penetration of tumor tissue. Further guidance regarding targeted immunotoxin therapy can by found, e.g., in Thrush, G.R., et al, Ann. Rev. Immunol, 14:49-11 (1996); as well as U.S. Patent Nos. 5,686,072, 4,792,447, and 4,664911 (the disclosures of which are inco ⁇ orated herein by reference).
  • polypeptides are immunogenic, they can also be used to prepare vaccines. As will be recognized by the skilled person, some polypeptides should be administered with an adjuvant in order to produce an immune response.
  • Useful antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, antigen binding antibody fragments (e.g., Fab, Fab', F(ab') 2 , F v , or hypervariable regions), bi-specific antibodies, and an Fab expression library.
  • polyclonal and/or monoclonal antibodies to a cancer-specific antigen marker are produced.
  • antibodies to a domain of the antigen are produced.
  • fragments of the antigen that are identified as immunogenic are used as immunogens for antibody production. Procedures known in the art can be used to produce polyclonal antibodies.
  • Various host animals including, but not limited to, rabbits, mice, rats, sheep, goats, camels, and the like
  • Various adjuvants can be used to increase the immunological response, depending on the host species.
  • Such adjuvants include, for example, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, GM-CSF (granulocyte macrophage colony stimulating factor), and other adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium paryum. Any technique that produces antibody molecules by continuous cell lines in culture can be used.
  • Such techniques include, for example, the hybridoma technique originally developed by Kohler and Milstein (see, e.g., Nature 256:495-91 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15-19 (1993); Hering et al, Biomed. Biochim. Acta 47:211-16 (1988)), the human B-cell hybridoma technique (see, e.g., Kozbor et al, Immunology Today 4:12 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (see, e.g., Cole et al, In: Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be used and can be obtained by using human hybridomas (see, e.g. Cote et al, Proc. Natl Acad. Sci. USA 50:2026-30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g. , Cole et al. , supra).
  • Chimeric antibodies see, e.g., Momson et al, Proc. Natl. Acad. Sci. USA 57:6851-55 (1984); Neuberger et al, Nature 312:604-08 (1984); Takeda et al, Nature 314:452-54 (1985)) can be prepared.
  • Such chimeric antibodies are typically prepared by splicing the genes (of one species) for an antibody molecule specific for tumor-associated antigen together with genes from another species of antibody molecule of appropriate biological activity.
  • the antigen binding regions e.g.
  • Fab', F(ab') 2 , Fab, Fv, or hypervariable regions) of antibodies from one species can be transferred into the framework of an antibody from another species by recombinant DNA techniques to produce a chimeric molecule.
  • Methods for producing such "chimeric" molecules are well known and described in, e.g., U.S. Patent Nos. 4,816,397; 4,816,567; 5,693,762; and 5,712,120; PCT Patent Publications WO 87/02671 and WO 90/00616; and European Patent Publication EP 239 400, inco ⁇ orated herein by reference.
  • a human monoclonal antibody or portions thereof can be identified by first screening a human B-cell cDNA library for nucleic acid molecules that encode antibodies that specifically bind to a cancer- specific antigen according to the method generally set forth by Huse et al, Science 246:1275-1281 (1989). The nucleic acid molecule can then be cloned and amplified to obtain sequences that encode the antibody (or antigen-binding domain) of the desired specificity. Phage display technology can also be used for selecting antibodies that bind to tumor associated antigens, fragments, derivatives or analogs thereof. See, e.g., PCT Patent Publications WO 91/17271 and WO 92/01047; as well as Huse et al, supra.
  • techniques described for producing single chain antibodies can be used.
  • An additional aspect of the invention uses the techniques described for constructing an Fab expression library (see, e.g., Huse et al, supra) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for cancer-specific antigens, fragments, derivatives, or analogs thereof.
  • Antibody fragments that contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include, but are not limited to, the
  • F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, the methods described in U.S. Patent No. 5,965,405 (the disclosure of which is inco ⁇ orated by reference herein).
  • bi-specific antibodies are provided.
  • Bi-specific antibodies can be monoclonal antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities can be for a cancer-specific antigen and the other one can be for any other antigen.
  • one specificity is for a first cancer-specific antigen, while the other specificity can be for a second, different antigen.
  • bi-specific antibodies can be based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature 305:531-39 (1983), inco ⁇ orated herein by reference). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of different antibody molecules, of which only one has the correct bi-specific structure. The correct molecule is usually purified by affinity chromatography. Similar procedures are disclosed in PCT Patent Publication WO 93/08829, and in Trauneeker et al, EMBO J. 70:3655-59 (1991), inco ⁇ orated herein by reference.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion typically is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding is usually present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co- transfected into a suitable host organism. Further details of generating bi-specific antibodies can be found, e.g., in Suresh et al, Methods in Enzymology 727:210 (1986), inco ⁇ orated herein by reference.
  • polypeptides can be used in connection with techniques and methods employing dentritic cells. Such techniques are discussed, for example, in
  • the antibodies detected or measured according to the methods of the invention can be IgG antibodies.
  • the antibodies can also be IgA, IgD, IgE or IgM antibodies.
  • the antibodies can be detected as free antibodies, or as part of an immune complex (e.g., as discussed in Freeman, J.L. et al, Amer. J. Reprod. Immunol, 42(2): ⁇ 49 (2001), inco ⁇ orated herein by reference).
  • the biological sample can be blood, serum, ascites fluid, urine, sweat, saliva, sputum, stool, mucus, fluid, or milk.
  • the biological sample can also be fluid obtained by wash or lavage.
  • the methods of the invention can further comprise detecting at least one additional cancer marker other than antibodies bound to Al 14 polypeptide.
  • the additional cancer marker and the antibodies bound to the at least one Al 14 polypeptide can be detected using a variety of corresponding specific binding partners.
  • the at least one additional cancer marker can also be an antibody.
  • Al l 4 polypeptide is one of a panel of polypeptides to cancer-associated genes and this panel is used to measure antibodies to Al 14 and the additional polypeptides for screening, diagnosis, prognosis or other detection or evaluation of cancer.
  • Antibodies can be detected or measured using, for example, an ELIS A assay, an ELISA/ELCA assay, a microbead-flow cytometry-based assay, such as LUMINEX®, a Western blot assay, or an immunoprecipitation assay. These methods can be adapted to simultaneously provide for detecting or measuring more than one antibody/marker in a single assay, e.g. as when a panel of binding partners is presented in order to assess the presence or levels of multiple antibodies/markers. According to the present invention, at least one of the antibodies/markers analyzed is an antibody that binds specifically to at least one Al 14 polypeptide.
  • the method of detection also includes one or more steps to account for, or remove, "nonspecific" binding of immunoglobulins that do not specifically recognize the polypeptide. (Total immunoglobulin levels are present in serum at concentrations up to a million times higher than that of the polypeptide-specific antibody).
  • Al 14 autoantibodies can be detected in a biological sample, especially in a blood or serum sample, using Al 14 polypeptides in immunoassays.
  • the polypeptides can be used in liquid phase or bound to a solid phase carrier.
  • the Al 14 polypeptides can be detectably labeled in various ways for use in immunoassays for Al 14 autoantibodies.
  • Immunoassays for detecting Al 14 antibodies using the methods of this invention include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), an amplified ELISA assay called ELISA-enzyme-linked coagulation assay (ELISA-ELCA) or other assays known in the art, such as immunofluorescent assays, chemiluminescent assays, or bioluminescent assays.
  • Al 14 polypeptides or antibodies can also be labeled using fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, using techniques known to the art.
  • Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, dichlorotriazinylamino fluorescein, Texas Red, XRITC, mo ⁇ holinorhodamine isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine.
  • Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and the oxalate esters.
  • Typical bioluminescent compounds include luciferin, luciferase, and aequorin.
  • Typical enzymes include alkaline phosphatase, ⁇ -galactosidase, glucose-6- phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, urease and peroxidase.
  • enzyme assays Two general types of enzyme assays are the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT) (Syva Co ⁇ ., Palo Alta, California) and enzyme-linked immunosorbent assay (ELISA).
  • EMIT enzyme-multiplied immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.
  • the ELISA assay is ELISA-ELCA, because this modification of ELISA possesses enhanced sensitivity over traditional ELISA assays for antibody detection in particular embodiments of the invention (ELISA-ELCA is described in Doellgast, G.J., Anal. Biochem., 167:91- 105 (1987); Beard, G. A.
  • Latex agglutination assays have been described in Beltz, G. A. et al, in Molecular Probes: Techniques and Medical Applications, A. Albertini et al, eds., Raven Press, New York, 1989, inco ⁇ orated herein by reference.
  • a polypeptide is immobilized on latex particles. A drop of the latex particles is added to an appropriate dilution of the serum to be tested and mixed by gentle rocking of the mixture, e.g. as applied to a test card.
  • the latex particles With samples lacking antibodies to the polypeptide, the latex particles remain in suspension and retain a smooth, milky appearance. However, if antibodies reactive with the polypeptide are present, the latex particles clump into visibly detectable aggregates.
  • the latex agglutination assay is especially suitable for small volume users, emergency situations, and areas lacking the sophisticated laboratory equipment and supplies needed for immunornetric assays.
  • An agglutination assay can also be used to detect antibodies where the desired immunoreactive polypeptide is immobilized on a suitable particle other than latex beads, for example, on gelatin, red blood cells, nylon, liposomes, gold particles, etc.
  • a suitable particle other than latex beads for example, on gelatin, red blood cells, nylon, liposomes, gold particles, etc.
  • the presence of antibodies causes agglutination, similar to that of a precipitation reaction, which can then be detected, for example, using nephelometry, turbidity, infrared spectrometry, visual inspection, colorimetry, and the like.
  • Immunornetric assays include forward sandwich immunoassays, reverse sandwich immunoassays and simultaneous assays. Each of these terms is well understood by those skilled in the art.
  • the polypeptide is bound to a solid-phase carrier and antibodies, generally anti-IgG antibodies, are detectably labeled.
  • a sample suspected of containing antibodies against the immunoreactive polypeptide is first incubated with a solid-phase immunoadsorbent to which at least one appropriate polypeptide has been bound. Incubation is continued for a period of time sufficient to allow the antibodies in the sample to bind to the immobilized polypeptide(s). After the first incubation, the solid- phase immunoadsorbent is separated from the incubation mixture and washed to remove interfering substances, which also may be present in the sample. Solid-phase immunoadsorbent-containing antibodies bound to the immobilized polypeptide are subsequently incubated for a second time with soluble labeled antibody cross-reactive with a different domain on the antibody to be detected.
  • a reverse sandwich assay the sample suspected of containing test antibodies against an immunoreactive polypeptide of interest is initially incubated with labeled anti- antibody (e.g. goat anti-human IgG), after which the solid-phase immunoadsorbent containing immobilized polypeptide cross-reactive with a different domain on the antibody (the polypeptide of interest) is added thereto, and a second incubation is carried out.
  • labeled anti- antibody e.g. goat anti-human IgG
  • the initial washing step required by a forward sandwich assay is not required, although a wash is performed after the second incubation.
  • Reverse sandwich assays have been described, for example, in U.S. Pat. Nos. 4,098,876 and 4,376,110.
  • a simultaneous sandwich assay the sample, the immunoadsorbent having immobilized polypeptide thereon and labeled soluble antibody specific to a different domain of the test antibody are incubated simultaneously in one incubation step.
  • the simultaneous assay requires only a single incubation and does not require washing steps.
  • the use of a simultaneous assay is a very helpful technique, providing ease of handling, homogeneity, reproducibility, linearity of the assays, and high precision. See U.S. Pat. No. 4,376,110 to David et al, inco ⁇ orated by reference herein.
  • delayed immunornetric assays can also be used. These assays are described, for example, in Chu, U.S. Pat. No. 4,289,747, and Wolters, U.S. Pat. No. 4,343,896, inco ⁇ orated herein by reference.
  • the antibody-containing sample, solid-phase immunoadsorbent with immobilized polypeptide, and labeled soluble antibody are incubated under conditions and for a period of time sufficient to allow the test antibodies to bind to the immobilized polypeptide and to the labeled, soluble antibodies.
  • the specific concentrations of labeled antibodies and immobilized fragments, the temperature and time of incubation, as well as other such assay conditions can be varied, depending upon various factors including the concentration of antibody in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • solid-phase immunoadsorbents there are many solid-phase immunoadsorbents that have been employed and that can be used in the present invention.
  • Well-known immunoadsorbents include beads formed from glass, polystyrene, paper, polypropylene, dextran, nylon, and other material; tubes formed from or coated with such materials, and the like.
  • the immobilized polypeptides can be covalently or physically bound to the solid-phase immunoadsorbent, by techniques such as covalent bonding via an amide or ester linkage or by adso ⁇ tion.
  • Those skilled in the art will know many other suitable carriers for binding polypeptides, or will be able to ascertain such, using routine experimentation.
  • the soluble antibody can be labeled with any detectable label, such as a radiolabel, a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label. Most commonly, the label is a radiolabel or an enzyme label.
  • the presence of autoantibodies to immunoreactive polypeptides can be detected in biological fluids and tissues. Any sample containing an unknown amount of specific autoantibodies can be analyzed. Normally, a sample is a liquid such as, for example, urine, saliva, tear drops, cerebrospinal fluid, blood, serum and the like, or a solid or semi- solid such as, for example, tissues, feces, and the like. Relative concentration of the antibody in a sample, as well as various other conditions, can be evaluated to determine the most appropriate assay for a particular type of sample or set of assay conditions. Sensitivity varies considerably depending on the type of assay.
  • Immunogenicity of protein cancer markers/antigens can be evaluated rapidly and efficiently using microsphere-based, flow cytometric assays.
  • One method of simultaneously analyzing multiple reactivities in a single sample is represented by the LabMAP (laboratory multiple analyte pro filing) system of Luminex Co ⁇ oration, Austin, Texas. See, e.g., Iannonne, M.A., et al, Cytometry 39:131-140 (2000); Chen, J-W., et al, Genome Research 70:549-557 (2000); and, especially, Vignali, D.A.A., J. Immun. Meth. 243:243-255 (2000)(review), and Carson, R.T. and Vignali, D.A.A., J. Immun. Meth. 227:41-52 (1999).
  • This system makes use of beads that inco ⁇ orate dyes, allowing their detection via lasers as a feature of a flow cytometric process.
  • the specific polypeptide/antibody binding event is measured using a second reporter molecule.
  • the reporter molecule e.g. phycoerythrin-labeled goat anti-human IgG, signals the extent of the reaction by attaching to the molecules on the microspheres.
  • a colorimetric reporter two sources of color will be present: the color-coded dye inside each microsphere and the reporter color on the surface of the microsphere.
  • color-coded microspheres having the antigen of interest attached to their surface, anti-human IgG reporter molecules, and sample, e.g. human sera or a dilution thereof, are combined.
  • This mixture is injected into an instrument such as the LUMINEX ® 100.
  • the LUMINEX ® 100 aligns the microspheres in single file where lasers illuminate the colors inside and on the surface of each microsphere.
  • Advanced optics integrated into the LUMINEX ® 100 capture the color signals, and digital signal processing translates the signals into quantitative data for each reaction.
  • the particular spectral address of a particular color-coded microsphere allows multiple analytes to be evaluated in a single experiment. Accordingly, multiple antigen or marker analytes, and appropriate controls can be tested simultaneously against samples, e.g. patient sera, to determine the reactivity of a number of potentially imrnunogenic markers in a single experiment.
  • Proteins can be produced by recombinant methods that introduce one or more tags (or "molecular handles") onto the protein antigen in the form of a N-terminal or C-terminal sequence having a particular functionality, facilitating purification or other manipulations.
  • tags or "molecular handles"
  • polyhistidine sequence which is made up of five or six histidine resides in a row.
  • poly-His is a commonly used tag
  • different expression vector systems use a variety of different molecular "handles.”
  • the pBAD/glll vectors available from Invitrogen Co ⁇ oration (Carlsbad, CA) include a c-myc epitope, as well as the poly-His tag (Information regarding the pBAD expression systems offered by Invitrogen can be found at www.invitrogen.com and in "pBAD Prokaryotic Expression Vectors," the disclosure of which is inco ⁇ orated by reference herein).
  • the pET family of vectors available from Novagen, Inc, Madison, WI use either the CBD tag, the GST tag, the polyHis tag, the HSV tag, the Nus tag, the S-tag, the T7 tag, the Trx tag, or some combination of those listed herein (for more info, see www.novagen.com and Novagen's newletter, inNovations vol. 1 p.1-3 ).
  • the first mentioned expression system utilizes the pBAD vectors. This system provides a series of expression vectors subject to very finely controlled, arabinose- dependent expression. The system also facilitates cloning of proteins of interest in conjunction with a variety of features advantageous for further purification and/or manipulation of the expressed protein.
  • vector features useful according to the methods of the present invention include: a multiple cloning site, an N-terminal polyhistidine tag, ANTI-EXPRESS epitope, and an enterokinase cleavage site for removal of the fusion partner (pBAD/His); and a C-terminal polyhistidine tag, a 10 amino acid epitope for the cellular homolog of the myc oncogene (c-myc epitope) (pBAD/MycHis or pBAD/glll). See Example 1 below.
  • tags e.g., N-terminal or C-terminal (e.g., polyHis, c-myc epitope) such that the appropriate end of a protein of interest can be tagged in order to minimize interference with native protein structure and function.
  • C-terminal e.g., polyHis, c-myc epitope
  • Methods using a poly-His tag can exploit the high affinity for metals exhibited by the tag.
  • a chromatographic medium containing immobilized nickel, or cobalt can be used in a one-step purification protocol for many proteins comprising the poly-His tag.
  • One example of a useful system for nickel-based purification of protein antigens is provided in the QIAEXPRESS Protein Purification System by Qiagen, Inc., Valencia, California (Ni- NTA (nickel-nitrilotriacetic acid) magnetic beads, spin columns, agarose beads).
  • Cobalt- based purification of proteins antigens can be performed using the TALONTM system of reagents supplied by BD Sciences Clontech, Palo Alto, CA (see their website at www.clontech.com for further details). Proteins tagged with poly-His (e.g. 6X-His) can also be purified immobilized using methods inco ⁇ orating a highly specific anti-6X-His antibody. Such antibodies are available from a number of suppliers, e.g. R&D Systems, Inc., Minneapolis, Minnesota (mouse IgG monoclonal anti-6X histidine antibody, Cat. No. MAB050).
  • antibodies specific for a variety of epitope tags can be used for affinity purification/immobilization.
  • Such antibodies can be attached to a solid matrix, e.g. beads, and used to accomplish an initial purification of the protein antigen.
  • affinity constructs can also be used directly to detect corresponding antibodies in patient samples. Further, such constructs can also be adapted for use within the context of the various assay techniques described herein.
  • Another exemplary expression system involves the pET family of vectors.
  • the vectors in this family are all subject to very finely controlled, IPTG-dependent expression.
  • the system also facilitates cloning of proteins of interest in conjunction with a variety of features advantageous for further purification and/or manipulation of the expressed protein.
  • the pET30 series has the following features useful according to the methods of the present invention: a multiple cloning site, a stop codon in each of the three open reading frames, an N-terminal polyhistidine tag, a C-terminal polyhistidine tag, an S-tag, a thrombin cleavage site for removal of N-terminal portion of the fusion protein containing the N-terminal polyhistidine tag and an enterokinase cleavage site for removal of the N-terminal portion of the fusion protein containing the S tag and the N-terminal polyhistidine tag.
  • a very useful tag in many of the pET vectors, including the pET30 series, is the S- tag.
  • a fusion protein carrying this tag is able to reconstitute a fully functional ribonuclease enzyme, in the presence of wild- type S-protein, which can cleave a fluorescent substrate (see inN ovations Vol. 10. pi 0 for more details).
  • the activity of the ribonuclease can be used to optimize expression conditions and to locate and quantitate fractions of the fusion protein during the course of a purification protocol. Assay kits for this pu ⁇ ose are available from Novagen Inc. (Cat. Nos. 70724-3 and 70724-4).
  • recombinant methods and expression systems can be used to prepare polypeptides.
  • methods to produce recombinant proteins as required herein include, but are not limited to in vitro expression, insect cell expression mammalian cell expression and yeast cell expression.
  • a nucleic acid encoding a polypeptide can be transcribed and translated using a vector with a T7 promoter (e.g., pCRT7/VP22-l-TOPO from Invitrogen Co ⁇ oration, Carlsbad, California; see www.invitrogen.com) in an in vitro transcription/translation system such as those offered by Promega Co ⁇ oration, Madison, WI (e.g., TNT T7 Quick Coupled Transcription Translation Systems, TNT Coupled Wheat Germ Extract Systems, or E. coli S30 Extract Systems). Information regarding these methods can be found on the internet at www.promega.com.
  • Polypeptides can also be expressed in insect cells using a vector with a 6X-His epitope tag (e.g., pMIB/V5-His). Information regarding this vector and insect cell expression systems can be found at www.invitrogen.com.
  • yeast as an expression system is quite common, as will be recognized by those skilled in the art. Multiple references are available, such as "Current Protocols in Molecular Biology,” Ausebel, et al, eds., Current Protocols, (1994).
  • Immunoprecipitation techniques are known in the art and can be used to detect and/or measure anti-cancer antibodies according to the present invention. Immunoprecipitation can be performed as described by Harlow, E. and D. Lane, in
  • Al 14 polypeptides are produced as fusion constructs comprising a 6X-His tag and a cellular myc 10 amino acid epitope.
  • Al 14 polypeptides are produced as fusion constructs comprising a N-terminal His tag, a C-terminal His tag and an S tag.
  • the fusion constructs are purified in conjunction with various beads linked to either specific antibodies for the fusion partners, or nickel-containing moieties having affinity for the 6X-His tag. Further, the bound protein marker can then be used directly in immunogenicity testing or in a serum assay protocol.
  • Marker proteins can be used in an ELISA format assay in order to detect and/or measure anti-cancer antibodies according to the present invention.
  • ELISA assays can be performed as described by Harlow, E. and D. Lane, in Chapter 14 of "Antibodies: A laboratory manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988), the disclosure of which in fully inco ⁇ orated herein by reference.
  • ELISA-ELCA ELISA-ELCA
  • Marker proteins can be used in an ELISA-ELCA format assay in order to detect and/or measure trace levels of anti-cancer antibodies according to the present invention.
  • ELISA-ELCA assays can be performed as described by the references cited previously in the document, or as disclosed in U.S. Patent Nos. 4,668,621 and 5,071,745, which are inco ⁇ orated herein by reference.
  • Marker proteins can be used in a Western blotting protocol in order to detect and/or measure anti-cancer antibodies according to the present invention. Generally,
  • individual assays of the present invention can detect and/or measure the serological reactivity of multiple marker proteins. It will be recognized that all of the immunoassay techniques can be adapted from their basic format to allow for the simultaneous testing of the immunoreactivity of more than one marker.
  • the Al 14 polypeptides can be provided in conjunction with other immunogenic cancer markers of particular specificity, to generate or optimize a test panel useful for detection of cancer or a cancerous condition.
  • One or more Al 14 polypeptides can be included as members of a test panel of immunoreactive substances.
  • the members of such panels can be selected in order to obtain a particular data set relevant to the detection of a particular disease state or physical condition of the test subject, including the presence or progress of a disease state relating to colon cancer.
  • panel immunoassays include e.g., panel ELISA assays, microplate immunoassays, and microbead systems (see, e.g., the Luminex system discussed herein).
  • the assay can also comprise a test card system, which can be automated (see, e.g., U.S. Patent No. 5,075,077, inco ⁇ orate herein by reference). Protein chips can also be used.
  • cDNA (SEQ ID NO: 1) comprising the shaded sequence as shown in Fig. 1, encoding Al 14 (Fig. 2; SEQ ID NO: 2), was inserted into the pBAD/glll expression vector (Invitrogen Co ⁇ oration, Carlsbad, California).
  • the cDNA for Al 14 was cloned, by polymerase chain reaction (PCR), from a human colon cDNA library prepared from normal colon tissue (Human Colon 5 '-STRETCH PLUS cDNA library (Clontech, Palo Alto, CA).
  • Primers were prepared to facilitate cloning of the Al 14 coding region.
  • the forward and reverse primers were ordered from Integrated DNA Technologies, Coralville, IA and were designed to contain the first and last 20-25 base pairs of the Al 14 coding sequence, respectively.
  • Forward primer 5'- GCCGCCGCGGCCGCTATGTCAAGGCCCAGCAGCAGAGCC-3' (SEQ ID NO: 3) and reverse primer, 5'- TTTTGTTCTAGAGGGCTTATCCCCAGCATCCTTCT -3', (SEQ ID NO: 4).
  • the primers were also designed to introduce unique restriction endonuclease cleavage sites at the 5'(7VotI) and 3' (Xbal) ends of the amplified sequence.
  • PCR polymerase chain reaction
  • the PCR product was examined on a 1.0% TAE agarose gel. Bands of the predicted sizes were excised from the gel and purified using the QIAX II DNA purification kit (Qiagen, Valencia, CA.).
  • both insert and vector DNA were digested with the restriction endonucleases, TVotl and Xbal.
  • the sequence of the expression vector can be found on the Invitrogen website at www.invitrogen.com (Cat. Number V450-01).
  • both DNA fragments were purified on a 0.7% TAE agarose gel.
  • Vector and insert DNA were excised and purified using the QIAX II kit, as noted above.
  • T4 DNA ligase (Promega Co ⁇ oration, Madison, WI) was used to ligate the Al 14 cDNA into pBAD/glll. The ligation mixture was then transformed into One Shot ® competent E. coli Top 10 cells purchased from Invitrogen. The transformation mixture was transferred to LB media plates containing antibiotic (ampicillin) and the bacteria grown on these plates to select for E. coli colonies containing pBAD/III and a DNA insert.
  • Plasmid DNA from twenty colonies was isolated using a Qiagen Mini-Prep Kit and tested for the presence of the Al 14 cDNA. Plasmid DNA was cleaved using the restriction endonucleases, Notl and Xbal to excise the Al 14 DNA fragment from the pBAD/glll vector. The size of the DNA fragments were checked on a 0.8% TAE agarose gel. DNA clones that yielded DNA fragments of the expected size were considered for DNA sequencing. Plasmid DNA from six bacterial colonies was sequenced at the Wake Forest
  • Bacteria containing pBAD/gll/Al 14 were grown in TB under the optimal induction conditions just described and the cells were harvested by centrifugation.
  • the bacterial pellet was resuspended in ice-cold 0.1 M Tris-HCl, pH 8.0, (5 ml per 2 g wet weight of bacterial pellet) and the cellular extract solubilized by addition of 20 ml of 8 M urea and 2.7 ml of 10% (w/v) sodium N-lauroyl-sarcosinate.
  • the mixture was then incubated at RT for 30 minutes, with shaking, on a rotary shaker (200 rpm) and sonicated to break-up any particulate matter in the sample.
  • the volume of the bacterial extract was brought up to 50 ml through the addition of 22.3 ml of equilibration buffer (20 mM Tris-HCl pH8.0, 0.2 M NaCl, 10% (v/v) glycerol and 0.1% (w/v) sodium N-lauroyl- sarcosinate), the sample was sonicated again to reduce the viscosity of the mixture and the cellular debris was pelleted by centrifugation. The supernantant was removed. Three ml of Ni-NTA SUPERFLOW (Qiagen) was equilibrated in equilibration buffer and then mixed with 50 ml of supernantant from the bacterial extract.
  • equilibration buffer 20 mM Tris-HCl pH8.0, 0.2 M NaCl, 10% (v/v) glycerol and 0.1% (w/v) sodium N-lauroyl- sarcosinate
  • the sample was incubated at RT for 1-2 hrs, with shaking, on a rotary shaker (200 ⁇ m), after which the mixture was centrifuged and the liquid was replaced with 50 ml of renaturation buffer (this buffer is identical to equilibration buffer, except that the sodium N-lauroyl- sarcosinate has been exchanged for 0.1% (v/v) Triton X-100).
  • the Ni-NTA SUPERFLOW was incubated with two changes of renaturation buffer at RT for 1-2 hrs, with shaking and then the Ni-NTA SUPERFLOW was transferred to a chromatography column.
  • the column was washed with 30 ml (5x 6 ml aliquots) of renaturation buffer, plus 10 mM, 20 mM, 50 mM, 0.1 M and 0.5 M imidazole, respectively (total 150 ml). The fractions were collected for gel analysis. The purified Al 14 recombinant protein eluted usually in renaturation buffer, plus 0.1 M imidazole.
  • Example 4 Total Protein Stain ofSDS-PAGE Gels and Western Blotting.
  • Electrophoresis was performed according to the manufacturers instructions until the bromophenol blue dye front ran off the bottom of the gel.
  • Gels to be stained for total protein were placed in 7.5% (v/v) acetic acid containing a 1:5,000 dilution of SYPRO Tangerine stain (Amersham Biosciences, Piscataway, NJ) and maintained in the dark, with gentle agitation, for approximately 1 hr.
  • the gels were rinsed in 7.5% (v/v) acetic acid for 1 min with gentle agitation.
  • Total protein was visualized with a standard UV transilluminator (300 nm) on an imaging system from Alpha Innotech Co ⁇ oration, San Leandro, CA.
  • the sensitivity of detection with Sypro Tangerine staining is 4-8 ng/well, which is comparable with Silver staining, traditionally the most sensitive method of detecting total protein (Amersham Biosciences product bulletin).
  • Proteins were transferred from the gel to the PVDF membrane using 22 volts for 45 min.
  • the membrane was removed from the sandwich and marked for orientation pu ⁇ oses.
  • the PVDF filter was blocked by incubation in T-TBS (20 mM Tris-HCl, 500 mM NaCl, 0.1% (v/v) Tween-20, ⁇ H7.4) with 5% (w/v) non-fat dry skimmed milk (Carnation, Nestle USA Inc., Ohio) and gentle agitation at RT for 1 hr, or 4°C overnight. Following the blocking step, the membrane was washed 3x 5 min in T-TBS at RT with gentle agitation.
  • HRP horseradish peroxidase
  • Qiagen horseradish peroxidase-labeled anti-poly-His
  • HRP chemiluminescent substrate Supersignal ® West Femto Maximum Sensitivity Substrate, Pierce, Rockford, IL
  • His-tagged proteins were visualized using an imaging system from Alpha Innotech Co ⁇ oration. Because recombinant protein from pBAD/III also posseses a myc tag, HRP-anti-myc (Qiagen) can also be used to detect recombinant Al 14 protein.
  • Example 6 Microbead-based Flow Cytometry A key feature of the LUMINEX ® assay (developed by Luminex Inc., Austin,
  • Texas is the coupling of protein to microbeads.
  • this coupling reaction can be completed via direct coupling of the protein to the bead, or indirect coupling of the protein to the bead through an antibody intermediate specific for the poly-His tag, or myc epitope of the purified protein.
  • an antibody to the poly-His tag is coupled to the bead and then the protein is attached to the bead through the interaction of the antibody and the poly-His tag at the C-terminal end of the protein.
  • Mouse anti-poly-His antibodies (Qiagen) were attached to different 'colored' microbeads according to the LUMINEX ® 100 User's Manual, version 1.7.
  • the coupling reaction featured carbodiimide coupling of the antibody to LabMAPTM carboxylated microbeads (Luminex). 500,000 microbeads per 'color' set were coupled in order to perform 96 assays in a 96-well microtiter plate.
  • a specific bead set was incubated with 30 ⁇ g Al 14 protein overnight at 4°C.
  • a control bead set was also included at this point.
  • the control set was treated in the same manner as the Al 14 protein bead set, except that no protein was added to the beads.
  • the control beads were intended to measure background serum reactivity in the assay.
  • microspheres were washed twice in PBS-T (PBS, plus 0.05% (v/v) Tween-20, pH7.4) and resuspended in PBS-BSA (1% (w/v) albumin-free bovine serum albumin, PBS, 0.05% (v/v) NaN 3 ) containing 10% (v/v) normal mouse serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
  • PBS-BSA 1% (w/v) albumin-free bovine serum albumin, PBS, 0.05% (v/v) NaN 3 ) containing 10% (v/v) normal mouse serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
  • the number of beads in each 'colored' set was counted using a haemacytometer, and the bead sets were mixed in the same buffer in such a way that the concentration of each bead set was 100,000 microspheres per ml.
  • the liquid was removed from the plate using a vacuum manifold, which draws the buffer through the filter whilst keeping the microspheres trapped in the wells of the plate.
  • 100 ⁇ l R-Phycoerythrin (PE) conjugated donkey anti-human IgG (Jackson ImmunoResearch Laboratories, Inc.)) was diluted 1 :200 in PBS-BSA and added to the appropriate wells of the plate.
  • the samples were mixed and allowed to incubate at room temperature for 45 min with agitation.
  • the microbeads in the wells of the microtiter plate were washed three times with PBS-BSA, as described previously and resuspended in 100 ⁇ l PBS-BSA.
  • the Luminex ® 100 system counts 100 beads per set and quantifies the amount of reporter (PE-labeled-IgG) and thus, antigen-specific antibody bound to each set as median fluorescence intensity (MFI).
  • MFI median fluorescence intensity
  • stage III/iV cancer patients 90 serum samples from late stage, or stage III/iV cancer patients and 23 samples from normal healthy individuals.
  • the cancer patients were comprised of 10 lung, 6 colon, 33 breast and 41 ovarian patients. Within the breast and ovarian groups, it should be noted that there were 5 sets of 4 serial samples. In other words, 20 breast and 20 ovarian samples came from 5 breast and 5 ovarian patients, respectively.
  • Dr. Taylor supplied 105 serum samples consisting of the following: 5 serum samples from patients diagnosed with benign adenoma, or benign fibrioma; 6 serum samples from patients diagnosed with adenocarcinoma of the endometrium; 59 serum samples from patients diagnosed with adenocarcinoma of the ovary; 3 serum samples from patients diagnosed with endometriod cancer of the ovary; 30 serum samples from individuals at high risk of developing ovarian cancer, who have been referred to the University of Louisville for testing; and 2 serum samples from patients diagnosed with squamous carcinoma of the cervix.
  • Bacterial or mammalian cells are washed several times in PBS or TBS to remove proteins from the supernatant.
  • Mammalian cells are resuspended in as small volume of ice cold NP-40 lysis buffer (10 7 mammalian cells in 1 ml lysis buffer), and incubated for 30 minutes with gentle mixing.
  • Bacterial cells are resuspended in 10 volumes of ice cold RIP A lysis buffer, sonicated four times with 10-30 second bursts (maximum intensity), with cooling on ice between bursts. Preparations are centrifuged at 10,000 g for 10 min. The supernatant is precleared by adding 100 ul of al0% protein- A/G slurry per 1 ml of supernatant, mixing for 1 hr at 4°C, then centrifuging at 10,000g for 10 min.
  • purified proteins can be used at 10-50 ng per reaction.
  • a polypeptide-only (antigen-only) control is included.
  • NP-40 lysis buffer 500ml
  • RJPA lysis buffer 500ml
  • 150mM NaCl 15 ml of 5M NaCl
  • 150mM NaCl 15 ml of 5M NaCl
  • mice Laboratories, Chantilly, VA are coated with 100 ul of mouse anti-His or mouse anti-myc antibodies in coating buffer (0.2M sodium bicarbonate, pH [9.2]), and incubated for 2 hours to overnight (ON) at room temperature (RT).
  • coating buffer 0.2M sodium bicarbonate, pH [9.2]
  • antibody is prepared at 1-5 ug/ml. Plates are washed with PBS and the wells are blocked with 200ul of casein buffer or 5% BSA/PBS, then incubated 2 hours to overnight at RT. Plates are washed three times with PBS. A polypeptide source containing His- or myc-tagged polypeptides is added to each well. The amount of polypeptide added is pre-determined. Typically, the amounts are in the 1-5 ug/ml range in 5% BSA/PBS. Plates are incubated 1 hour at RT and washed three times with PBS.
  • Human serum (lOOul), diluted in 5% BSA/PBS with mouse serum (5%) to reduce anti-mouse activity, is added per well. Plates are incubated for 1 hour at RT and washed three times with PBS. lOOul of alkaline phosphatase labeled anti-human IgG (Jackson Immunoresearch, Inc., West Grove, PA) is added at a pre-determined dilution, typically
  • alkaline phosphatase substrate e.g., PNPP, Sigma Inc.
  • substrate absorbance e.g., 405nm
  • Example 10 Western Blotting For Western blotting, proteins (bacterial lysate, mammalian cell lysate, or pure protein) are prepared by boiling for 5 min in SDS-PAGE loading buffer (50 ug of total protein per each SDS-PAGE well). The protein sample is centrifuged, and the supernatant is loaded onto an SDS-PAGE gel (e.g., BIO-RAD pre-made gels, (Bio-Rad Laboratories, Inc., Hercules, CA)). Gels are run at 200 constant volts until the samples are l A inch from the bottom of the gel. The gel is removed and soaked in Western transfer buffer for 30 min prior to blotting.
  • SDS-PAGE loading buffer 50 ug of total protein per each SDS-PAGE well.
  • SDS-PAGE gel e.g., BIO-RAD pre-made gels, (Bio-Rad Laboratories, Inc., Hercules, CA)
  • Filter paper soaked in Western buffer is placed on the surface of a semi-dry transfer blotter (Bio-Rad). After pre-wetting a PVDF membrane with methanol, the membrane is soaked in Western transfer buffer for 5 min and placed on top of the filter paper, removing all trapped air. The gel is placed on top of the membrane and trapped air is removed. The sandwich is completed by placing a Western buffer-soaked piece of filter paper on top of the gel and removing any trapped air.
  • Proteins are transferred to the membrane using 22 volts for 45 min.
  • the membrane is removed from the sandwich, and marked for orientation pu ⁇ oses.
  • the membrane is blocked by incubating in TTBS with 5% milk 2 hours to overnight.
  • HRP horseradish peroxidase
  • the blocked gel is incubated with a dilution of serum for 1 hour at room temperature (RT). Then blots are washed four times in TTBS; HRP-labeled anti-human IgG is added and incubated for 1 hour at RT, then rinsed four times with TTBS. Chemilummescence substrate is added as above. Buffer compositions are as follows- in Table 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des méthodes et des compositions de détection du cancer par détection et/ou mesure des autoanticorps. L'invention concerne également des compositions et des kits adaptés pour l'analyse des marqueurs cancéreux d'autoanticorps, ainsi que des polypeptides et des anticorps utilisés dans la détection et le traitement du cancer.
PCT/US2002/027747 2001-08-31 2002-08-30 Methodes et compositions de detection du cancer Ceased WO2003020115A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31669501P 2001-08-31 2001-08-31
US60/316,695 2001-08-31

Publications (2)

Publication Number Publication Date
WO2003020115A2 true WO2003020115A2 (fr) 2003-03-13
WO2003020115A9 WO2003020115A9 (fr) 2013-10-10

Family

ID=23230233

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/027747 Ceased WO2003020115A2 (fr) 2001-08-31 2002-08-30 Methodes et compositions de detection du cancer

Country Status (1)

Country Link
WO (1) WO2003020115A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2299275A2 (fr) 2004-07-30 2011-03-23 Adeza Biomedical Corporation Classification de la teneur en fibronectine oncofoetale pour l'indication des maladies et autres conditions
CN110208543A (zh) * 2019-06-06 2019-09-06 威海威高生物科技有限公司 血管内皮生长因子检测试剂盒及其使用方法和应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2299275A2 (fr) 2004-07-30 2011-03-23 Adeza Biomedical Corporation Classification de la teneur en fibronectine oncofoetale pour l'indication des maladies et autres conditions
US9513298B2 (en) 2004-07-30 2016-12-06 Hologic, Inc. Methods for detecting oncofetal fibronectin
CN110208543A (zh) * 2019-06-06 2019-09-06 威海威高生物科技有限公司 血管内皮生长因子检测试剂盒及其使用方法和应用

Also Published As

Publication number Publication date
WO2003020115A9 (fr) 2013-10-10

Similar Documents

Publication Publication Date Title
JP5711196B2 (ja) 卵巣癌を判定するためのｈｅ４及び他の生化学マーカーの使用
CA3088235C (fr) Utilisation de la proteine rep bmmf1 en tant que biomarqueur pour le cancer colorectal
US8440415B2 (en) BORIS isoforms and methods of detecting and treating disease
US8642347B2 (en) Urinary CA125 peptides as biomarkers of ovarian cancer
US8658769B2 (en) Diagnostic marker for hepatocellular carcinoma comprising anti-FASN autoantibodies and a diagnostic composition for hepatocellular carcinoma comprising antigens thereof
KR101777259B1 (ko) En2 단백질을 특이적으로 인식하는 단클론 항체 또는 이를 함유하는 전립선암 진단용 조성물
JP4515099B2 (ja) Ｌａｓｐ−１免疫反応性の決定によって炎症性疾患および感染を診断するための方法
CN103429617B (zh) 包含抗-ck8/18复合物自身抗体的标记物及其诊断癌症的用途
KR101777254B1 (ko) En2 단백질을 특이적으로 인식하는 특정 항원으로부터 얻어진 단클론 항체 또는 이를 함유하는 전립선암 진단용 조성물
JP6122779B2 (ja) 抗ｗｔ１抗体の測定方法
KR20150129932A (ko) 보체인자 b 단백질에 특이적으로 결합하는 항체를 포함하는 췌장암 진단용 키트
EP3699594A1 (fr) Utilisation de protéine rep bmmf1 en tant que biomarqueur pour le cancer de la prostate
WO2003020115A2 (fr) Methodes et compositions de detection du cancer
EP3928095B1 (fr) Utilisation de protéines rep bmmf1 en tant que biomarqueur pour le cancer du sein
CN115698718A (zh) 胰腺癌的检测方法和检测试剂
JP5358808B2 (ja) 腫瘍マーカー、腫瘍診断キット、腫瘍マーカーの測定方法および腫瘍診断方法
JP3888695B2 (ja) ヒトｌｅｃｔ２に対する抗体、それを産生する細胞、その測定法及び測定用キット
EP1796723B1 (fr) Méthode de diagnose du cancer en détectant l'expression de spag9 ou des anticorps contre le spag9
AU2020223867B2 (en) Use of BMMF1 Rep protein as a biomarker for prostate cancer
US7306919B1 (en) Antigen-antibody cancer recognition system
WO2005094422A2 (fr) Fragments d'angiocidine et leurs utilisations dans des analyses cliniques relatives au cancer et a d'autres maladies
US20050233390A1 (en) Device including a proteinaceous factor, a recombinant proteinaceous factor, and a nucleotide sequence encoding the proteinaceous factor
JP2018062466A (ja) 新規甲状腺癌関連抗原に結合する抗体および甲状腺癌診断剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VN YU ZA ZM

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 69(1) EPC DATED 21/05/04

122 Ep: pct application non-entry in european phase
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)