EP1238281A2 - Antigenes associes au cancer et utilisations correspondantes - Google Patents

Antigenes associes au cancer et utilisations correspondantes

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Publication number
EP1238281A2
EP1238281A2 EP00982348A EP00982348A EP1238281A2 EP 1238281 A2 EP1238281 A2 EP 1238281A2 EP 00982348 A EP00982348 A EP 00982348A EP 00982348 A EP00982348 A EP 00982348A EP 1238281 A2 EP1238281 A2 EP 1238281A2
Authority
EP
European Patent Office
Prior art keywords
group
nucleic acid
molecule
cancer associated
associated antigen
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Withdrawn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
EP00982348A
Other languages
German (de)
English (en)
Inventor
Toshiro Okayama University Medical School ONO
Eiichi Okayama University Medical School NAKAYAMA
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.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
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 Ludwig Institute for Cancer Research Ltd, Ludwig Institute for Cancer Research New York filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP1238281A2 publication Critical patent/EP1238281A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention relates to nucleic acids and encoded polypeptides which are cancer associated antigens expressed in methylcholanthrene-induced fibrosarcomas.
  • the invention also relates to agents which bind the nucleic acids or polypeptides.
  • the nucleic acid molecules, polypeptides coded for by such molecules and peptides derived therefrom, as well as related antibodies and cytolytic T lymphocytes, are useful, ter alia, in diagnostic and therapeutic contexts.
  • T cells recognize foreign materials has been implicated in cancer.
  • CTL cytolytic T lymphocyte
  • autologous CTLs for identifying tumor antigens requires that the target cells which express the antigens can be cultured in vitro and that stable lines of autologous CTL clones which recognize the antigen-expressing cells can be isolated and propagated. While this approach has worked well for melanoma antigens, other tumor types, such as epithelial cancers including breast and colon cancer, have proved refractory to the approach.
  • the invention provides, inter alia, isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules.
  • the invention also provides isolated proteins and peptides, antibodies to those proteins and peptides and CTLs which recognize the proteins and peptides. Fragments including functional fragments and variants of the foregoing also are provided. Kits containing the foregoing molecules additionally are provided.
  • the foregoing can be used in the diagnosis, monitoring, research, or treatment of conditions characterized by the expression of one or more cancer associated antigens.
  • the invention involves the surprising discovery of several genes, some previously known and some previously unknown, which are expressed in mice bearing methylcholanthrene-induced fibrosarcomas. These individuals all have serum antibodies against the proteins (or fragments thereof) encoded by these genes. Thus, abnormally expressed genes are recognized by the host's immune system and therefore can form a basis for diagnosis, monitoring and therapy.
  • the human equivalents (i.e., homologs) of the nucleic acids have in some cases also been identified.
  • the invention involves the use of a single material, a plurality of different materials and even large panels and combinations of materials.
  • a single gene, a single protein encoded by a gene, a single functional fragment thereof, a single antibody thereto, etc. can be used in methods and products of the invention.
  • pairs, groups and even panels of these materials and optionally other cancer associated antigen genes and/or gene products can be used for diagnosis, monitoring and therapy.
  • the pairs, groups or panels can involve 2, 3, 4, 5 or more genes, gene products, fragments thereof or agents that recognize such materials.
  • a plurality of such materials are not only useful in monitoring, typing, characterizing and diagnosing cells abnormally expressing such genes, but a plurality of such materials can be used therapeutically.
  • the invention has in vivo and in vitro uses, including for therapeutic, diagnostic, monitoring and research purposes.
  • One aspect of the invention is the ability to fingerprint a cell expressing a number of the genes identified according to the invention by, for example, quantifying the expression of such gene products. Such fingerprints will be characteristic, for example, of the stage of the cancer, the type of the cancer, or even the effect in animal models of a therapy on a cancer. Cells also can be screened to determine whether such cells abnormally express the genes identified according to the invention.
  • the invention in one aspect, is a method of diagnosing a disorder characterized by expression of a cancer associated antigen precursor coded for by a nucleic acid molecule.
  • the method involves the steps of contacting a biological sample isolated from a subject with an agent that specifically binds to the nucleic acid molecule, an expression product thereof, or a fragment of an expression product thereof complexed with a MHC, preferably an HLA, molecule, wherein the nucleic acid molecule is a NA Group 1 nucleic acid molecule, and determining the interaction between the agent and the nucleic acid molecule, the expression product or fragment of the expression product as a determination of the disorder.
  • the agent is selected from the group consisting of (a) a nucleic acid molecule comprising NA Group 1 nucleic acid molecules or a fragment thereof, (b) a nucleic acid molecule comprising NA Group 3 nucleic acid molecules or a fragment thereof, (c) a nucleic acid molecule comprising NA Group 5 nucleic acid molecules or a fragment thereof, (d) an antibody that binds to an expression product, or a fragment thereof, of NA group 1 nucleic acids, (e) an antibody that binds to an expression product, or a fragment thereof, of NA group 3 nucleic acids, (f) an antibody that binds to an expression product, or a fragment thereof, of NA group 5 nucleic acids, (g) and agent that binds to a complex of an MHC, preferably HLA, molecule and a fragment of an expression product of a NA Group 1 nucleic acid, (h) an agent that binds to a complex of an MHC, preferably HLA, molecule and a fragment of an expression
  • the disorder may be characterized by expression of a plurality of cancer associated antigen precursors.
  • the methods of diagnosis may include use of a plurality of agents, each of which is specific for a different cancer associated antigen precursor (including at least one of the cancer associated antigen precursors disclosed herein), and wherein said plurality of agents is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 such agents.
  • the agent may be specific for a cancer associated antigen precursor, including the cancer associated antigen precursors disclosed herein, and preferably human homologs thereof.
  • the invention is a method for determining regression, progression or onset of a condition characterized by expression of abnormal levels of a protein encoded by a nucleic acid molecule that is a NA Group 1 molecule.
  • the method involves the steps of monitoring a sample, from a subject who has or is suspected of having the condition, for a parameter selected from the group consisting of (i) the protein, (ii) a peptide derived from the protein, (iii) an antibody which selectively binds the protein or peptide, and (iv) cytolytic T cells specific for a complex of the peptide derived from the protein and a MHC molecule, as a determination of regression, progression or onset of said condition.
  • the sample is a body fluid, a body effusion or a tissue.
  • the step of monitoring comprises contacting the sample with a detectable agent selected from the group consisting of (a) an antibody which selectively binds the protein of (i), or the peptide of (ii), (b) a protein or peptide which binds the antibody of (iii), and (c) a cell which presents the complex of the peptide and MHC molecule of (iv).
  • a detectable agent selected from the group consisting of (a) an antibody which selectively binds the protein of (i), or the peptide of (ii), (b) a protein or peptide which binds the antibody of (iii), and (c) a cell which presents the complex of the peptide and MHC molecule of (iv).
  • the antibody, the protein, the peptide or the cell is labeled with a radioactive label or an enzyme.
  • the sample in a preferred embodiment is assayed for the peptide.
  • the nucleic acid molecule is one of the following: a NA Group 3 molecule or a NA Group 5 molecule.
  • the protein is a plurality of proteins
  • the parameter is a plurality of parameters, each of the plurality of parameters being specific for a different of the plurality of proteins.
  • the invention in another aspect is a pharmaceutical preparation, preferably for a human subject.
  • the pharmaceutical preparation includes an agent which when administered to the subject enriches selectively the presence of complexes of a MHC molecule, preferably a HLA molecule, and a cancer associated antigen, and a pharmaceutically acceptable carrier, wherein the cancer associated antigen is a fragment of a cancer associated antigen precursor encoded by a nucleic acid molecule which comprises a NA Group 1 molecule, or a human homolog thereof.
  • the nucleic acid molecule is a NA Group 3 nucleic acid molecule.
  • the agent in one embodiment comprises a plurality of agents, each of which enriches selectively in the subject complexes of a MHC molecule and a different cancer associated antigen.
  • the plurality is at least two, at least three, at least four or at least 5 different such agents.
  • the agent is selected from the group consisting of (1) an isolated polypeptide comprising the cancer associated antigen, a human homolog thereof, or a functional variant thereof, (2) an isolated nucleic acid operably linked to a promoter for expressing the isolated polypeptide, homolog or functional variant thereof, (3) a host cell expressing the isolated polypeptide, homolog or functional variant thereof, and (4) isolated complexes of the polypeptide, homolog or functional variants thereof, and an HLA molecule.
  • the agent may be a cell expressing an isolated polypeptide.
  • the agent is a cell expressing an isolated polypeptide comprising the cancer associated antigen, a human homolog thereof or a functional variant thereof.
  • the agent is a cell expressing an isolated polypeptide comprising the cancer associated antigen, a homolog or a functional variant thereof, and wherein the cell expresses an HLA molecule that binds the polypeptide.
  • the cell can express one or both of the polypeptide and HLA molecule recombinantly.
  • the cell is nonproliferative.
  • the agent is at least two, at least three, at least four or at least five different polypeptides, each representing a different cancer associated antigen, homolog or functional variant thereof.
  • the agent in one embodiment is a PP Group 2 polypeptide. In other embodiments the agent is a PP Group 3 polypeptide or a PP Group 4 polypeptide.
  • each of the pharmaceutical preparations described herein also includes an adjuvant.
  • a composition which includes an isolated agent that binds selectively a PP Group 1 polypeptide.
  • the agent binds selectively to a polypeptide selected from the following: a PP Group 2 polypeptide, a PP Group 3 polypeptide, a PP Group 4 polypeptide, and a PP Group 5 polypeptide.
  • the agent is a plurality of different agents that bind selectively at least two, at least three, at least four, or at least five different such polypeptides.
  • the agent may be an antibody.
  • the invention is a composition of matter composed of a conjugate of the agent of the above-described compositions of the invention and a therapeutic or diagnostic agent.
  • the conjugate is of the agent and a therapeutic or diagnostic that is an antineoplastic.
  • the invention in another aspect is a pharmaceutical composition which includes an isolated nucleic acid molecule selected from the group consisting of: (1) NA Group 1 molecules, and (2) NA Group 2 molecules, and a pharmaceutically acceptable carrier.
  • the isolated nucleic acid molecule comprises a NA Group 3 or NA Group 4 molecule.
  • the isolated nucleic acid molecule comprises at least two isolated nucleic acid molecules coding for two different polypeptides, each polypeptide comprising a different cancer associated antigen.
  • the pharmaceutical composition also includes an expression vector with a promoter operably linked to the isolated nucleic acid molecule.
  • the pharmaceutical composition also includes a host cell recombinantly expressing the isolated nucleic acid molecule.
  • a pharmaceutical composition includes an isolated polypeptide comprising a PP Group 1 or a PP Group 2 polypeptide, and a pharmaceutically acceptable carrier.
  • the isolated polypeptide comprises a PP Group 3 or a PP Group 4 polypeptide.
  • the isolated polypeptide comprises at least two different polypep tides, each comprising a different cancer associated antigen at least one of which is encoded by a NA group 1 molecule as disclosed herein.
  • the isolated polypeptides are selected from the following: PP Group 3 polypeptides or HLA binding fragments thereof and PP Group 5 polypeptides or HLA binding fragments thereof.
  • each of the pharmaceutical compositions described herein also includes an adjuvant.
  • Another aspect the invention is an isolated nucleic acid molecule comprising a NA Group 3 molecule. Another aspect the invention is an isolated nucleic acid molecule comprising a NA Group 4 molecule.
  • the invention in another aspect is an isolated nucleic acid molecule selected from the group consisting of (a) a fragment of a nucleic acid selected from the group of nucleic acid molecules consisting of SEQ ID NOs numbered below and comprising all nucleic acid sequences among SEQ ID Nos: 9, 13, 15, 17, 19 and 23, of sufficient length to represent a sequence unique within the mouse or human genomes, and identifying a nucleic acid encoding a cancer associated antigen precursor, (b) complements of (a), provided that the fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from the sequence group consisting of (1) sequences having the GenBank accession numbers of Table 11, (2) complements of (1), and (3) fragments of (1) and (2).
  • sequence of contiguous nucleotides is selected from the, group consisting of: (1) at least two contiguous nucleotides nonidentical to the sequences in Table 11 , (2) at least three contiguous nucleotides nonidentical to the sequences in Table 11 , (3) at least four contiguous nucleotides nonidentical to the sequences in Table 11 , (4) at least five contiguous nucleotides nonidentical to the sequences in Table 11, (5) at least six contiguous nucleotides nonidentical to the sequences in Table 11, or (6) at least seven contiguous nucleotides nonidentical to the sequences in Table 11.
  • the fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and every integer length therebetween.
  • the molecule encodes a polypeptide which, or a fragment of which, binds a MHC molecule, preferably a human HLA receptor, or an antibody, preferably one having human amino acid sequences.
  • Another aspect of the invention is an expression vector comprising an isolated nucleic acid molecule of the invention described above operably linked to a promoter.
  • the invention is an expression vector comprising a nucleic acid operably linked to a promoter, wherein the nucleic acid is a NA Group 1 or Group 2 molecule.
  • the invention is an expression vector comprising a NA Group 1 or Group 2 molecule and a nucleic acid encoding an MHC, preferably HLA, molecule.
  • the invention is a host cell transformed or transfected with an expression vector of the invention described above.
  • the invention is a host cell transformed or transfected with an expression vector comprising an isolated nucleic acid molecule of the invention described above operably linked to a promoter, or an expression vector comprising a nucleic acid operably linked to a promoter, wherein the nucleic acid is a NA Group 1 or 2 molecule and further comprising a nucleic acid encoding a MHC molecule.
  • an isolated polypeptide encoded by the isolated nucleic acid molecules the invention, described above, is provided.
  • These include PP Group 1-5 polypeptides, including SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 24.
  • the invention also includes a fragment of the polypeptide which is immunogenic. In one embodiment the fragment, or a portion of the fragment, binds a MHC molecule, preferably HLA ,or an antibody, preferably one having human amino acid sequences.
  • the invention includes in another aspect an isolated fragment of a cancer associated antigen precursor which, or portion of which, binds a MHC molecule, preferably HLA, or a human antibody, or an antibody, preferably one having human amino acid sequences, wherein the precursor is encoded by a nucleic acid molecule that is a NA Group 1 molecule.
  • the fragment is part of a complex with MHC/HLA.
  • the fragment is between 8 and 12 amino acids in length.
  • the invention includes an isolated polypeptide comprising a fragment of the polypeptide of sufficient length to represent a sequence unique within the mouse or human genomes and identifying a polypeptide that is a cancer associated antigen precursor.
  • a kit for detecting the presence of the expression of a cancer associated antigen precursor includes a pair of isolated nucleic acid molecules each of which consists essentially of a molecule selected from the group consisting of (a) a 12-32 nucleotide contiguous segment of the nucleotide sequence of any of the NA Group 1 molecules and (b) complements of ("a"), wherein the contiguous segments are nonoverlapping.
  • the pair of isolated nucleic acid molecules is constructed and arranged to selectively amplify an isolated nucleic acid molecule that is a NA Group 3 molecule.
  • the pair amplifies a human NA Group 3 molecule.
  • a method for treating a subject with a disorder characterized by expression of a cancer associated antigen precursor includes the step of administering to the subject an amount of an agent, which enriches selectively in the subject the presence of complexes of a MHC/HLA molecule and a cancer associated antigen, effective to ameliorate the disorder, wherein the cancer associated antigen is a fragment of a cancer associated antigen precursor encoded by a nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule comprising NA group 1 nucleic acid molecules, (b) a nucleic acid molecule comprising NA group 3 nucleic acid molecules, (c) a nucleic acid molecule comprising NA group 5 nucleic acid molecules.
  • the disorder is characterized by expression of a plurality of cancer associated antigen precursors and wherein the agent is a plurality of agents, each of which enriches selectively in the subject the presence of complexes of a MHC/HLA molecule and a different cancer associated antigen.
  • the plurality is at least 2, at least 3, at least 4, or at least 5 such agents.
  • the agent is an isolated polypeptide selected from the group consisting of PP Group 1, PP Group 2, PP Group 3, PP Group 4, and PP group 5 polypeptides.
  • the disorder is cancer.
  • the invention is a method for treating a subject having a condition characterized by expression of a cancer associated antigen precursor in cells of the subject.
  • the method includes the steps of (i) removing an immunoreactive cell containing sample from the subject, (ii) contacting the immunoreactive cell containing sample to the host cell under conditions favoring production of cytolytic T cells against a cancer associated antigen which is a fragment of the precursor, (iii) introducing the cytolytic T cells to the subject in an amount effective to lyse cells which express the cancer associated antigen, wherein the host cell is transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operably linked to a promoter, the isolated nucleic acid molecule being selected from the group of nucleic acid molecules consisting of NA Group 1, NA Group 2, NA Group 3, NA Group 4, NA Group 5.
  • the host cell recombinantly expresses an HLA molecule which binds the cancer associated antigen. In another embodiment the host cell endogenously expresses a MHC/HLA molecule which binds the cancer associated antigen.
  • the invention includes in another aspect a method for treating a subject having a condition characterized by expression of a cancer associated antigen precursor in cells of the subject.
  • the method includes the steps of (i) identifying a nucleic acid molecule expressed by the cells associated with said condition, wherein said nucleic acid molecule is a NA Group 1 molecule (ii) transfecting a host cell with a nucleic acid selected from the group consisting of (a) the nucleic acid molecule identified, (b) a fragment of the nucleic acid identified which includes a segment coding for a cancer associated antigen, (c) deletions, substitutions or additions to (a) or (b), and (d) degenerates of (a), (b), or (c); (iii) culturing said transfected host cells to express the transfected nucleic acid molecule, and; (iv) introducing an amount of said host cells or an extract thereof to the subject effective to increase an immune response against the cells of the subject associated with the condition.
  • the method also includes the step of (a) identifying a MHC molecule which presents a portion of an expression product of the nucleic acid molecule, wherein the host cell expresses the same MHC molecule as identified in (a) and wherein the host cell presents a MHC binding portion of the expression product of the nucleic acid molecule.
  • the method also includes the step of treating the host cells to render them non-proliferative.
  • the immune response comprises a B-cell response or a T cell response.
  • the response is a T-cell response which comprises generation of cytolytic T-cells specific for the host cells presenting the portion of the expression product of the nucleic acid molecule or cells of the subject expressing the cancer associated antigen.
  • the nucleic acid molecule is a NA Group 3 molecule.
  • Another aspect of the invention is a method for treating or diagnosing or monitoring a subject having a condition characterized by expression of an abnormal amount of a protein encoded by a nucleic acid molecule that is a NA Group 1 molecule. The method includes the step of administering to the subject an antibody which specifically binds to the protein or a peptide derived therefrom, the antibody being coupled to a therapeutically useful agent, in an amount effective to treat the condition.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody is a chimeric antibody or a humanized antibody.
  • the invention is a method for treating a condition characterized by expression in a subject of abnormal amounts of a protein encoded by a nucleic acid molecule that is a NA Group 1 nucleic acid molecule.
  • the method involves the step of administering to a subject at least one of the pharmaceutical compositions of the invention described above in an amount effective to prevent, delay the onset of, or inhibit the condition in the subject.
  • the condition is cancer.
  • the method includes the step of first identifying that the subject expresses in a tissue abnormal amounts of the protein.
  • the invention in another aspect is a method for treating a subject having a condition characterized by expression of abnormal amounts of a protein encoded by a nucleic acid molecule that is a NA Group 1 nucleic acid molecule.
  • the method includes the steps of (i) identifying cells from the subject which express abnormal amounts of the protein; (ii) isolating a sample of the cells; (iii) cultivating the cells, and (iv) introducing the cells to the subject in an amount effective to provoke an immune response against the cells.
  • the method includes the step of rendering the cells non- proliferative, prior to introducing them to the subject.
  • the invention is a method for treating a pathological cell condition characterized by abnormal expression of a protein encoded by a nucleic acid molecule that is a NA Group 1 nucleic acid molecule.
  • the method includes the step of administering to a subject in need thereof an effective amount of an agent which inhibits the expression or activity of the protein.
  • the agent is an inhibiting antibody which selectively binds to the protein and wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fragment thereof.
  • the agent is an antisense nucleic acid molecule which selectively binds to the nucleic acid molecule which encodes the protein.
  • the nucleic acid molecule is a NA Group 3 nucleic acid molecule.
  • the invention includes in another aspect a composition of matter useful in stimulating an immune response to a plurality of proteins encoded by nucleic acid molecules that are NA Group 1 molecules.
  • the composition is a plurality of peptides derived from the amino acid sequences of the proteins, wherein the peptides bind to one or more MHC molecules presented on the surface of the cells which express an abnormal amount of the protein. In one embodiment at least a portion of the plurality of peptides bind to MHC molecules and elicit a cytolytic response thereto.
  • composition of matter includes an adjuvant.
  • adjuvant is a saponin, GM-CSF, or an interleukin.
  • the compositions also includes at least one peptide useful in stimulating an immune response to at least one protein which is not encoded by nucleic acid molecules that are NA Group 1 molecules, wherein the at least one peptide binds to one or more MHC molecules.
  • the invention is an isolated antibody which selectively binds to a complex of: (i) a peptide derived from a protein encoded by a nucleic acid molecule that is a NA Group 1 molecule and (ii) and an MHC molecule to which binds the peptide to form the complex, wherein the isolated antibody does not bind to (i) or (ii) alone.
  • the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fragment thereof.
  • the invention also involves the use of the genes, gene products, fragments thereof, agents which bind thereto, and so on in the preparation of medicaments.
  • a particular medicament is for treating cancer, preferably bladder cancer, colon cancer, lung cancer, breast cancer or hepatoma.
  • nucleic acid molecule include the nucleotide sequence of SEQ ID NO:23 or fragments thereof, and polypeptides comprise SEQ ID NO:24 or fragments thereof, or are encoded by SEQ ID NO:23 or fragments thereof.
  • nucleic acid microarrays that include a NA Group
  • nucleic acid molecule comprises SEQ ID NO:23, or a fragment thereof.
  • Protein microarrays that include a PP Group 1 through 5 polypeptide also are provided.
  • the polypeptide comprises SEQ ID NO:24, or a fragment thereof, preferably an immunogenic fragment. The use of such microarrays in diagnostic applications, particularly for diagnosing cancer, also is provided.
  • FIG. 1 shows an EST cluster for OY-TES-1 cDNA. Each arrow represents an EST sequence in the cluster.
  • Fig. 2 shows nucleotide (SEQ ID NO: 23) and deduced amino acid (SEQ ID NO: 24) sequences of OY-TES-1. Primers used for PCR are indicated by arrows. Polyadenylation signal consensus sequence is underlined. The sequence has been deposited in the GenBank under accession number .
  • Fig. 3 shows sequence alignment of guinea pig (GP; SEQ ID NO:37 ), porcine (Pig; SEQ ID NO: 38), mouse (SEQ ID NO: 39), and human (OY-TES-1, SEQ ID NO: 23) sp32 precursor protein.
  • the conserved residues are indicated by asterisks.
  • the dots indicate the identical residues with those in the guinea pig.
  • Twenty cysteine residues are totally conserved (highlighted in black).
  • a glutamic acid- and glutamine rich-domain (EQ-rich domain) is boxed.
  • An acidic amino acid region is underlined.
  • Identity guinea pig/human, 77.2%; porcine/human, 81.9%; mouse/human, 75.2%.
  • Fig. 4 shows Southern blot analysis of the OY-TES-1 gene. Genomic DNA from normal testis was digested with EcoRI, HindHI, and BamHI, and analyzed with an OY-TES-1 probe. EcoRI and BamHI digests showed a strong and a weak band, suggesting that there are two homologue genes in the human genome.
  • Fig. 5 is a photomicrograph showing chromosome localization of OY-TES-1 gene by fluorescence in situ hybridization. Metaphases showed twin signals with brightly red fluorescence on 12pl2-pl3 (arrows). The chromosome 12-specif ⁇ c ⁇ satellite probe at the centromere was also shown in green fluorescence.
  • Fig. 6 is a diagram shows genomic structure of OY-TES-1.
  • the open reading frame is shown in shaded boxes and spliced introns as lines.
  • the exon/intron structure is determined based on the PAC clone RP4-761 J14 and BAC clone RP11-433J6.
  • Fig. 7 shows RT-PCR analysis of OY-TES-1 expression in normal tissues.
  • PCR primers were: ht-5, 5'-AAGGACAGGGGACTAAGGAG-3' (SEQ ID NO: 27) and ht-3, 5'-CCGTACAAATCCAGCCCGTA-3' (SEQ ID NO: 28).
  • the same cDNA samples were tested for ⁇ -actin as an internal control. OY-TES-1 restriction was restricted to testis.
  • Fig. 8 shows RT-PCR analysis of OY-TES-1 expression in tumors using primers ht-5 and ht-3. The same cDNA samples were tested for ⁇ -actin as an internal control.
  • Fig. 9 shows representative results of ELISA reactivity with sera from bladder and prostate cancer patients.
  • antigen precursors and antigens are substantially the full-length protein encoded by the coding region of the isolated DNA and the antigen is a peptide which complexes with MHC, preferably HLA, and which participates in the immune response as part of that complex.
  • antigens are typically 9 amino acids long, although this may vary slightly.
  • a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • human cancer associated antigens which have substantial nucleotide and/or amino acid sequence identity to the presently identified cancer associated antigens, and human subjects are preferred.
  • the present invention in one aspect involves the cloning of cDNAs encoding cancer associated antigen precursors using autologous antisera of mice having methyl cholanthrene- induced fibrosarcomas.
  • the sequences of the clones representing genes identified according to the methods described herein are presented in the attached Sequence Listing. Of the foregoing, it can be seen that some of the clones are considered completely novel as no nucleotide or amino acid homologies to coding regions were found in the databases searched. Other clones are novel but have some homology to sequences deposited in databases (mainly EST sequences). Nevertheless, the entire gene sequence was not previously known.
  • the invention thus involves in one aspect cancer associated antigen polypeptides, genes encoding those polypeptides, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as diagnostics and therapeutics relating thereto.
  • Homologs and alleles of the cancer associated antigen nucleic acids of the invention can be identified by conventional techniques.
  • an aspect of the invention is those nucleic acid sequences which code for cancer associated antigen precursors. The following chart is provided to identify the various groups of sequences discussed in the claims and in the summary:
  • NA Group 1 nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid sequence selected from the group consisting of nucleic acid sequences among SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 23 and which code for a cancer associated antigen precursor,
  • nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code
  • NA Group 2 Fragments of NA Group 1, which codes for a polypeptide which, or a portion of which, binds an MHC molecule to form a complex recognized by a an autologous antibody or lymphocyte.
  • NA Group 3 The subset of NA Group 1 where the nucleotide sequence is selected from the group consisting of:
  • nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code
  • NA Group 4 Fragments of NA Group 3, which code for a polypeptide which, or a portion of which, binds to an MHC molecule to form a complex recognized by an autologous antibody or lymphocyte.
  • NA Group 5 A subset of NA Group 1, comprising cancer associated antigens that react with allogeneic cancer antisera.
  • Example 8 Identification of human homologs of cancer associated antigens will be familiar to those of skill in the art. In particular, the methods described in Example 8 were effective in identifying the human homolog of OY-MC-4, OY-TES-1, as well as establishing it as a cancer-testis antigen useful in therapeutic and diagnostic applications for treatment and diagnosis of cancer.
  • nucleic acid hybridization is a suitable method for identification of homologous sequences of another species (e.g., human) which correspond to a known sequence (e.g., mouse cancer associated sequences presented herein).
  • Standard nucleic acid hybridization procedures can be used to identify related nucleic acid sequences of selected precent identity. For example, one can construct a library of cDNAs reverse transcribed from the mRNA of a selected tissue (e.g., testis) and use the cancer associated antigen nucleic acids identified herein to screen the library for related nucleotide sequences. The screening can be performed at various stringencies to identify those sequences whieh are closely related by sequence identity, and more distantly related. Nucleic acids so identified ca be translated into polypeptides and the polypeptides can be tested for activity.
  • PCR primers are selected to amplify portions of a nucleic acid sequence believed to be conserved (e.g., a catalytic domain, a DNA-binding domain, etc.).
  • nucleic acids are preferably amplified from a tissue-specific library (e.g., such as testis to find sequences related to OY-MC-4).
  • tissue-specific library e.g., such as testis to find sequences related to OY-MC-4.
  • stringent conditions refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • stringent conditions refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
  • SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.
  • the membrane upon which the DNA is transferred is washed, for example, in 2 x SSC at room temperature and then at 0.1 - 0.5 x SSC/0.1 x SDS at temperatures up to 68°C.
  • homologs and alleles typically will share at least 75% nucleotide identity and/or at least 90% amino acid identity to the sequences of cancer associated antigen nucleic acid and polypeptides, respectively, in some instances will share at least 90% nucleotide identity and/or at least 95% amino acid identity and in still other instances will share at least 95% nucleotide identity and/or at least 99% amino acid identity.
  • the homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the internet (ftp:/ncbi.nlm.nih.gov/pub/).
  • Exemplary tools include the BLAST system available at http://www.ncbi.nlm.nih.gov, using default settings. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte- Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
  • a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film to detect the radioactive signal.
  • Northern blot hybridizations using the foregoing conditions can be performed on samples taken from cancer patients or subjects suspected of having a condition characterized by expression of cancer associated antigen genes.
  • Amplification protocols such as polymerase chain reaction using primers which hybridize to the sequences presented also can be used for detection of the cancer associated antigen genes or expression thereof.
  • isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • PCR polymerase chain reaction
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • An isolated nucleic acid as used herein is not a naturally occurring chromosome.
  • isolated means separated from its native environment and present in sufficient quantity to permit its identification or use.
  • Isolated when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis.
  • Isolated proteins or polypeptides may, but need not be, substantially pure.
  • the term "substantially pure” means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be produced by techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e. isolated from other proteins.
  • the cancer associated genes correspond to SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 23.
  • the preferred cancer associated antigens for the methods of diagnosis disclosed herein are those which are found to react with allogeneic cancer antisera (i.e. NA Group 5).
  • Encoded polypeptides e.g., proteins
  • peptides and antisera thereto are also preferred for diagnosis.
  • the nucleic acid is SEQ ID NO:23 and the polypeptide is SEQ ID NO:24.
  • the invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.
  • Each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating cancer associated antigen polypeptide.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the invention also provides modified nucleic acid molecules which include additions, substitutions and deletions of one or more nucleotides.
  • these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or the polypeptides, such as antigenicity, enzymatic activity, receptor binding, formation of complexes by binding of peptides by MHC class I and class II molecules, etc.
  • the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein.
  • the modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of skill in the art.
  • modified nucleic acid molecules which encode polypeptides having single amino acid changes can be prepared. Each of these nucleic acid molecules can have one, two or three nucleotide substitutions exclusive of nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Likewise, modified nucleic acid molecules which encode polypeptides having two amino acid changes can be prepared which have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid molecules like these will be readily envisioned by one of skill in the art, including for example, substitutions of nucleotides in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and so on.
  • each combination of two amino acids is included in the set of modified nucleic acid molecules, as well as all nucleotide substitutions which code for the amino acid substitutions.
  • Additional nucleic acid molecules that encode polypeptides having additional substitutions (i.e., 3 or more), additions or deletions (e.g., by introduction of a stop codon or a splice site(s)) also can be prepared and are embraced by the invention as readily envisioned by one of ordinary skill in the art. Any of the foregoing nucleic acids or polypeptides can be tested by routine experimentation for retention of structural relation or activity to the nucleic acids and/or polypeptides disclosed herein.
  • the invention also provides isolated fragments of cancer associated antigen nucleic acid sequences or complements thereof, preferably unique fragments.
  • a unique fragment is one that is a 'signature' for the larger nucleic acid. It, for example, is long enough to assure that its precise sequence is not found in molecules within the human genome outside of the cancer associated antigen nucleic acids defined above (and human alleles). Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human genome.
  • a fragment which is completely composed of the sequence described in the foregoing GenBank deposits is one which does not include any of the nucleotides unique to the sequences of the invention.
  • a unique fragment must contain a nucleotide sequence other than the exact sequence of those in GenBank or fragments thereof. The difference may be an addition, deletion or substitution with respect to the GenBank sequence or it may be a sequence wholly separate from the GenBank sequence.
  • Fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acids, or can be used in amplification assays such as those employing PCR. As known to those skilled in the art, large probes such as 200, 250, 300 or more nucleotides are preferred for certain uses such as Southern and Northern blots, while smaller fragments will be preferred for uses such as PCR. Fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments, or for generating immunoassay components. Likewise, fragments can be employed to produce nonfused fragments of the cancer associated antigen polypeptides, useful, for example, in the preparation of antibodies, and in immunoassays. Fragments further can be used as antisense molecules to inhibit the expression of cancer associated antigen nucleic acids and polypeptides, particularly for therapeutic purposes as described in greater detail below.
  • the size of the unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of cancer associated antigen sequences and complements thereof will require longer segments to be unique while others will require only short segments, typically between 12 and 32 nucleotides (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 or more bases long, up to the entire length of the disclosed sequence.
  • this disclosure intends to embrace each and every fragment of each sequence, beginning at the first nucleotide, the second nucleotide and so on, up to 8 nucleotides short of the end, and ending anywhere from nucleotide number 8, 9, 10 and so on for each sequence, up to the very last nucleotide (provided the sequence is unique as described above).
  • polytopes Especially preferred include nucleic acids encoding a series of epitopes, known as "polytopes".
  • the epitopes can be arranged in sequential or overlapping fashion (see, e.g., Thomson et al., Proc. Natl. Acad. Sci. USA 92:5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15:1280-1284, 1997), with or without the natural flanking sequences, and can be separated by unrelated linker sequences if desired.
  • the polytope is processed to generated individual epitopes which are recognized by the immune system for generation of immune responses.
  • peptides derived from a polypeptide having an amino acid sequence encoded by one of the nucleic acid disclosed herein, and which are presented by MHC molecules and recognized by CTL or T helper lymphocytes can be combined with peptides from one or more other cancer associated antigens (e.g. by preparation of hybrid nucleic acids or polypeptides) to form "polytopes".
  • the two or more peptides (or nucleic acids encoding the peptides) can be selected from those described herein, or they can include one or more peptides of previously known cancer associated antigens.
  • Exemplary cancer associated peptide antigens that can be administered to induce or enhance an immune response are derived from tumor associated genes and encoded proteins including MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-B2, MAGE-B3, MAGE-B4, tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-1 and CT-7.
  • polypeptides comprising one or more peptides and one or more of the foregoing cancer associated peptides, or nucleic acids encoding such polypeptides, according to standard procedures of molecular biology.
  • polytopes are groups of two or more potentially immunogenic or immune response stimulating peptides which can be joined together in various arrangements (e.g. concatenated, overlapping).
  • the polytope (or nucleic acid encoding the polytope) can be administered in a standard immunization protocol, e.g. to animals, to test the effectiveness of the polytope in stimulating, enhancing and/or provoking an immune response.
  • the peptides can be joined together directly or via the use of flanking sequences to form polytopes, and the use of polytopes as vaccines is well known in the art (see, e.g., Thomson et al., Proc. Acad. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J Immunol. 157(2):822-826, 1996; Tarn et al., J Exp. Med. 171(l):299-306, 1990).
  • Tarn showed that polytopes consisting of both MHC class I and class II binding epitopes successfully generated antibody and protective immunity in a mouse model. Tarn also demonstrated that polytopes comprising "strings" of epitopes are processed to yield individual epitopes which are presented by MHC molecules and recognized by CTLs. Thus polytopes containing various numbers and combinations of epitopes can be prepared and tested for recognition by CTLs and for efficacy in increasing an immune response.
  • tumors express a set of tumor antigens, of which only certain subsets may be expressed in the tumor of any given patient.
  • Polytopes can be prepared which correspond to the different combination of epitopes representing the subset of tumor rejection antigens expressed in a particular patient. Polytopes also can be prepared to reflect a broader spectrum of tumor rejection antigens known to be expressed by a tumor type. Polytopes can be introduced to a patient in need of such treatment as polypeptide structures, or via the use of nucleic acid delivery systems known in the art (see, e.g., Allsopp et al., Eur. J. Immunol. 26(8): 1951-1959, 1996).
  • Adenovirus, pox virus, Ty- virus like particles, adeno-associated virus, plasmids, bacteria, etc. can be used in such delivery.
  • the expression vector may also include a nucleic acid sequence coding for the MHC molecule that presents any particular tumor rejection antigen derived from these nucleic acids and polypeptides.
  • the nucleic acid sequence coding for such a MHC molecule can be contained within a separate expression vector.
  • the single vector can be used to transfect a cell which does not normally express either one.
  • the expression vectors can be cotransfected.
  • the cancer associated antigen precursor coding sequence may be used alone, when, e.g. the host cell already expresses a MHC molecule which presents a cancer associated antigen derived from precursor molecules.
  • the host cell already expresses a MHC molecule which presents a cancer associated antigen derived from precursor molecules.
  • the vectors which contain the two coding sequences may be used in any antigen-presenting cells if desired, and the gene for cancer associated antigen precursor can be used in host cells which do not express a MHC molecule which presents a cancer associated antigen.
  • cell-free transcription systems may be used in lieu of cells.
  • the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a cancer associated antigen polypeptide, to reduce the expression of cancer associated antigens. This is desirable in virtually any medical condition wherein a reduction of expression of cancer associated antigens is desirable, e.g., in the treatment of cancer. This is also useful for in vitro or in vivo testing of the effects of a reduction of expression of one or more cancer associated antigens.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the sequences of nucleic acids encoding cancer associated antigens, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
  • the listed sequences are cDNA sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of a cancer associated antigen.
  • the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to nucleic acids encoding cancer associated antigens.
  • antisense to allelic cDNAs, homologous cDNAs (e.g., human) and genomic DNAs are enabled without undue experimentation.
  • the antisense oligonucleotides of the invention maybe composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage.
  • These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
  • the antisense oligonucleotides of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic intemucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • Preferred synthetic intemucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O- alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • the present invention contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding cancer associated antigen polypeptides, together with pharmaceutically acceptable carriers.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • a cloning vector is one which is able to replicate autonomously or integrated in the genone in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
  • Preferred systems for mRNA expression in mammalian cells are those such as pcDNA3.1 or pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • CMV cytomegalovirus
  • suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr Virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element.
  • EBV Epstein Barr Virus
  • Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor l ⁇ , which stimulates efficiently transcription in vitro.
  • the plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996).
  • Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992).
  • adenovirus as an Adeno.Pl A recombinant for the expression of an antigen is disclosed by Warnier et al., in intradermal injection in mice for immunization against PI A (Int. J. Cancer, 67:303-310, 1996). Additional vectors for delivery of nucleic acid are provided below.
  • the invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors.
  • Such expression kits include at least separate portions of a vector and one or more of the previously discussed cancer associated antigen nucleic acid molecules. Other components may be added, as desired, as long as the previously mentioned nucleic acid molecules, which are required, are included.
  • kits for amplification of a cancer associated antigen nucleic acid including at least one pair of amplification primers which hybridize to a cancer associated antigen nucleic acid.
  • the primers preferably are 12-32 nucleotides in length and are non- overlapping to prevent formation of "primer-dimers".
  • One of the primers will hybridize to one strand of the cancer associated antigen nucleic acid and the second primer will hybridize to the complementary strand of the cancer associated antigen nucleic acid, in an arrangement which permits amplification of the cancer associated antigen nucleic acid.
  • Selection of appropriate primer pairs is standard in the art. For example, the selection can be made with assistance of a computer program designed for such a purpose, optionally followed by testing the primers for amplification specificity and efficiency.
  • the invention further includes nucleic acid or protein microarrays which include nucleic acid molecules selected from NA Groups 1 through 5 or polypeptides selected from PP Groups 1 through 5.
  • the microarrays contain SEQ ID NO:23 or SEQ ID NO:24, respectively.
  • nucleic acid microarray technology which is also known by other names including: DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an array of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization.
  • reporter molecules e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP
  • a probe with a nucleic acid sequence that perfectly matches the target sequence will, in general, result in detection of a stronger reporter- molecule signal than will probes with less perfect matches.
  • Many components and techniques utilized in nucleic acid microarray technology are presented in "The Chipping Forecast", Nature Genetics, Vol.21, Jan 1999, the entire contents of which is incorporated by reference herein.
  • standard techniques of protein microarray technology are utilized to assess expression of the polypeptides of the invention, and fragments thereof (including HLA binding peptides , and/or identify biological constituents that bind such proteins and peptides.
  • Protein microarray technology which is also known by other names including: protein chip technology and solid-phase protein array technology, is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., G. MacBeath and S.L. Schreiber, "Printing Proteins as Microarrays for High-Throughput Function Determination," Science 289(5485):1760-1763, 2000.
  • the constituents of biological samples that can be analyzed by protein microarrays include polypeptides, antibodies, HLA molecules, lymphocytes (particularly T lymphocytes), and the like.
  • microarray substrates may include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. In all embodiments a glass substrate is preferred.
  • probes are selected from the group of nucleic acids including, but not limited to: DNA, genomic DNA, cDNA, and oligonucleotides; and may be natural or synthetic. Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides and DNA/cDNA probes preferably are 500 to 5000 bases in length, although other lengths may be used.
  • preferred nucleic acid probes are sets of two or more of the OY-TES-1 nucleic acid molecules set forth herein.
  • Preferred protein microarray probes include OY-TES-1 protein (SEQ ID NO:24) and fragments thereof. Probes may be purified to remove contaminants using standard methods known to those of ordinary skill in the art such as gel filtration or precipitation.
  • the microarray substrate may be coated with a compound to enhance synthesis of the probe on the substrate.
  • a compound to enhance synthesis of the probe on the substrate include, but are not limited to, oligoethylene glycols.
  • coupling agents or groups on the substrate can be used to covalently link the first nucleotide or oligonucleotide to the substrate. These agents or groups may include, for example, amino, hydroxy, bromo, and carboxy groups.
  • These reactive groups are preferably attached to the substrate through a hydrocarbyl radical such as an alkylene or phenylene divalent radical, one valence position occupied by the chain bonding and the remaining attached to the reactive groups.
  • These hydrocarbyl groups may contain up to about ten carbon atoms, preferably up to about six carbon atoms.
  • Alkylene radicals are usually preferred containing two to four carbon atoms in the principal chain.
  • probes are synthesized directly on the substrate in a predetermined grid pattern using methods such as light-directed chemical synthesis, photochemical deprotection, or delivery of nucleotide precursors to the substrate and subsequent probe production.
  • the substrate may be coated with a compound to enhance binding of the probe to the substrate.
  • a compound to enhance binding of the probe to the substrate include, but are not limited to: polylysine, amino silanes, amino-reactive silanes (Chipping Forecast, 1999) or chromium.
  • presynthesized probes are applied to the substrate in a precise, predetermined volume and grid pattern, utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery.
  • Probes may be covalently linked to the substrate with methods that include, but are not limited to, UV-irradiation.
  • probes are linked to the substrate with heat.
  • Targets are nucleic acids, including but not limited to: DNA, genomic DNA, cDNA,
  • RNA, mRNA and may be natural or synthetic.
  • nucleic acid target molecules from human tissue are preferred.
  • the tissue may be obtained from a subject or may be grown in culture (e.g. from a cancer cell line).
  • control polypeptide or nucleic acid molecules are attached to the substrate.
  • control polypeptide or nucleic acid molecules allow determination of factors such as polypeptide or nucleic acid quality and binding characteristics, reagent quality and effectiveness, binding success, and analysis thresholds and success.
  • Control polypeptide or nucleic acids may include but are not limited to expression products of genes such as housekeeping genes or fragments thereof.
  • the invention also permits the construction of cancer associated antigen gene "knockouts" and transgenics in cells and in animals, providing materials for studying certain aspects of cancer and immune system responses to cancer.
  • the invention also provides isolated polypeptides (including whole proteins and partial proteins) encoded by the foregoing cancer associated antigen nucleic acids. Such polypeptides are useful, for example, alone or as fusion proteins to generate antibodies, as components of an immunoassay or diagnostic assay or as therapeutics.
  • Cancer associated antigen polypeptides can be isolated from biological samples including tissue or cell homogenates, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Short polypeptides, including protein fragments and antigenic peptides (such as are presented by MHC molecules on the surface of a cell for immune recognition) also can be synthesized chemically using well-established methods of peptide synthesis.
  • a unique fragment of a cancer associated antigen polypeptide in general, has the features and characteristics of unique fragments as discussed above in connection with nucleic acids. As will be recognized by those skilled in the art, the size of the unique fragment will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of cancer associated antigens will require longer segments to be unique while others will require only short segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acids including each integer up to the full length).
  • Human homologs of cancer associated antigen polypeptides are related in sequence to the cancer associated antigens described herein, and preferably are also related in function. Preferably the homologs are 90% or more identical to one or more portions of the amino acid sequence of the cancer associated antigens, more preferably are 95% or more identical, and still more preferably are 99% or more identical. Most preferably, the homologs contains at least one fragment of 10 or more amino acids that are identical to the corresponding amino acids of the cancer associated antigens. In some embodiments a human homolog has the same or similar activity or function as a cancer associated antigen. Activities and functions include, but are not limited to, enzymatic activity, recognition by antibodies, DNA binding activity, transcriptional activity, binding to MHC molecules, and the like.
  • Unique fragments and human homologs of a polypeptide preferably are those fragments and homologs which retain a distinct functional capability of the polypeptide.
  • Functional capabilities which can be retained in a fragment, preferably an unique fragment, of a polypeptide include interaction with antibodies, interaction with other polypeptides or fragments thereof, selective binding of nucleic acids or proteins, and enzymatic activity.
  • One important activity is the ability to act as a signature for identifying the polypeptide.
  • Another is the ability to complex with MHC and to provoke in a mammal, preferably a human, an immune response.
  • Those skilled in the art are well versed in methods for selecting unique amino acid sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from non-family members. A comparison of the sequence of the fragment to those on known databases typically is all that is necessary.
  • a "variant" of a cancer associated antigen polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a cancer associated antigen polypeptide.
  • Modifications which create a cancer associated antigen variant can be made to a cancer associated antigen polypeptide 1) to reduce or eliminate an activity of a cancer associated antigen polypeptide; 2) to enhance a property of a cancer associated antigen polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; 3) to provide a novel activity or property to a cancer associated antigen polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety; or 4) to provide equivalent or better binding to a MHC molecule.
  • Modifications to a cancer associated antigen polypeptide are typically made to the nucleic acid which encodes the cancer associated antigen polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non- amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the cancer associated antigen amino acid sequence.
  • variants include cancer associated antigen polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its desired physiological activity.
  • cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages.
  • certain amino acids can be changed to enhance expression of a cancer associated antigen polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
  • Mutations of a nucleic acid which encode a cancer associated antigen polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.
  • Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant cancer associated antigen polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art.
  • variants of cancer associated antigen polypeptides can be tested by cloning the gene encoding the variant cancer associated antigen polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant cancer associated antigen polypeptide, and testing for a functional capability of the cancer associated antigen polypeptides as disclosed herein.
  • the variant cancer associated antigen polypeptide can be tested for reaction with autologous or allogeneic sera as described in the Examples.
  • variants may favor testing of other activities, as will be known to one of ordinary skill in the art.
  • conservative amino acid substitutions may be made in cancer associated antigen polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, the variants retain the functional capabilities of the cancer associated antigen polypeptides.
  • a "conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
  • Exemplary functionally equivalent variants of the cancer associated antigen polypeptides include conservative amino acid substitutions of in the amino acid sequences of proteins disclosed herein.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • a peptide derived from a cancer associated antigen polypeptide is presented by an MHC molecule and recognized by antibodies or CTLs, one can make conservative amino acid substitutions to the amino acid sequence of the peptide, particularly at residues which are thought not to be direct contact points with the MHC molecule.
  • conservative amino acid substitutions to the amino acid sequence of the peptide, particularly at residues which are thought not to be direct contact points with the MHC molecule.
  • methods for identifying functional variants of HLA class II binding peptides are provided in a published PCT application of Strominger and Wucherpfennig (PCT/US96/03182).
  • Peptides bearing one or more amino acid substitutions also can be tested for concordance with known HLA/MHC motifs prior to synthesis using, e.g.
  • the substituted peptides can then be tested for binding to the MHC molecule and recognition by antibodies or CTLs when bound to MHC. These variants can be tested for improved stability and are useful, inter alia, in vaccine compositions.
  • amino acid sequence of cancer associated antigen polypeptides to produce functionally equivalent variants of cancer associated antigen polypeptides typically are made by alteration of a nucleic acid encoding a cancer associated antigen polypeptide.
  • substitutions can be made by a variety of methods known to one of ordinary skill in the art.
  • amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene encoding a cancer associated antigen polypeptide.
  • substitutions are made to a small unique fragment of a cancer associated antigen polypeptide, such as an antigenic epitope recognized by autologous or allogeneic sera or cytolytic T lymphocytes
  • the substitutions can be made by directly synthesizing the peptide.
  • the activity of functionally equivalent fragments of cancer associated antigen polypeptides can be tested by cloning the gene encoding the altered cancer associated antigen polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered cancer associated antigen polypeptide, and testing for a functional capability of the cancer associated antigen polypeptides as disclosed herein.
  • Peptides which are chemically synthesized can be tested directly for function, e.g., for binding to antisera recognizing associated antigens.
  • the invention permits isolation of the cancer associated antigen protein molecules.
  • a variety of methodologies well-known to the skilled practitioner can be utilized to obtain isolated cancer associated antigen molecules.
  • the polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition.
  • an expression vector may be introduced into cells to cause production of the polypeptide.
  • mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide.
  • cancer associated antigen polypeptides include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
  • Identification of the cancer associated antigens also permits the identification and isolation of related human sequences (e.g. human homologs such as SEQ ID NOs:23 and 24), including immuno genie fragments of polypeptides and nucleic acids which encode such fragments.
  • human homologs such as SEQ ID NOs:23 and 24
  • immuno genie fragments of polypeptides and nucleic acids which encode such fragments.
  • the isolation of homologs of different species is described elsewhere herein, and exemplified in Example 8.
  • cancer associated antigen genes also makes it possible for the artisan to diagnose a disorder characterized by expression of cancer associated antigens.
  • These methods involve determining expression of one or more cancer associated antigen nucleic acids, and/or encoded cancer associated antigen polypeptides and/or peptides derived therefrom. In the former situation, such determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labeled hybridization probes. In the latter situation, such determinations can be carried out by screening patient antisera for recognition of the polypeptide.
  • the invention also makes it possible isolate proteins which bind to cancer associated antigens as disclosed herein, including antibodies and cellular binding partners of the cancer associated antigens. Additional uses are described further herein.
  • Expression of cancer associated nucleic acid molecules can also be determined using protein measurement methods to determine expression of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 23, e.g., by determining the expression of polypeptides encoded by these nucleic acid molecules (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24).
  • Preferred methods of specifically and quantitatively measuring proteins include, but are not limited to: protein microarrays, mass spectroscopy-based methods such as surface enhanced laser desorption ionization (SELDI; e.g., Ciphergen ProteinChip System), non-mass spectroscopy- based methods, and immunohistochemistry-based methods such as 2-dimensional gel electrophoresis.
  • SELDI surface enhanced laser desorption ionization
  • non-mass spectroscopy-based methods such as 2-dimensional gel electrophoresis.
  • Protein microarrays are similar in concept to the nucleic acid microarrays described above. Proteins are spotter or printed on a substrate and exposed to a sample to determine interactions of molecules with the proteins fixed on the substrate. For example, MacBeath and Schrieiber described a protein microarray system and several of its uses. See, Science 289:1760-1763, 2000. In one embodiment of the invention, cancer-associated proteins are spotted on a substrate in a microarray and the microarray is used to determine the presence of proteins in a biological sample that bind to the cancer associated proteins. This type of assay permits rapid determination of the amount and kind of binding proteins expressed, such as antibodies present in a cancer patient's blood.
  • SELDI methodology may, through procedures known to those of ordinary skill in the art, be used to vaporize microscopic amounts of tumor tissue and to create a "fingerprint" of individual proteins, thereby allowing simultaneous measurement of the abundance of many proteins in a single sample.
  • SELDI-based assays may be utilized to identify and/or classify tumors. Such assays preferably include, but are not limited to the following examples. Gene products that are encoded by nucleic acids analyzed by nucleic acid microarrays may be selectively measured by specific (antibody mediated) capture to the SELDI protein disc (e.g., selective SELDI).
  • total protein SELDI optimized to visualize the expression of those genes of interest from among the nucleic acid molecules described herein.
  • SELDI measurement of multiple markers from among the genes disclosed herein may be utilized for tumor diagnosis and/or classification.
  • SELDI also can be used diagnostically or prognostically for analysis of precancerous tissues to determine the risk of cancer based on SELDI results.
  • the protein analysis methods also can be used as a prognostic method for selecting treatment strategies for cancer patients, based on the particular pattern and amounts of gene expression in a patient's tumor.
  • the invention also provides, in certain embodiments, "dominant negative" polypeptides derived from cancer associated antigen polypeptides.
  • a dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein.
  • a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand.
  • a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal.
  • a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.
  • the end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins.
  • One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides.
  • cancer associated antigens especially those which are similar to known proteins which have known activities (e.g., OY-MC-4 (pern)
  • one of ordinary skill in the art can modify the sequence of the cancer associated antigens by site- specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then can test the population of mutagenized polypeptides for diminution in a selected and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art.
  • the invention also involves agents such as polypeptides which bind to cancer associated antigen polypeptides.
  • agents such as polypeptides which bind to cancer associated antigen polypeptides.
  • binding agents can be used, for example, in screening assays to detect the presence or absence of cancer associated antigen polypeptides and complexes of cancer associated antigen polypeptides and their binding partners and in purification protocols to isolated cancer associated antigen polypeptides and complexes of cancer associated antigen polypeptides and their binding partners.
  • agents also can be used to inhibit the native activity of the cancer associated antigen polypeptides, for example, by binding to such polypeptides.
  • the invention therefore, embraces peptide binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to cancer associated antigen polypeptides.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology. 7th Ed., Blackwell Scientific Publications, Oxford).
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin.
  • Such antibodies including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and or CDR2 and or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • polypeptides of numerous size and type that bind specifically to cancer associated antigen polypeptides, and complexes of both cancer associated antigen polypeptides and their binding partners.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties. Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g.
  • inserts from 4 to about 80 amino acid residues using conventional procedures.
  • the inserts may represent, for example, a completely degenerate or biased array.
  • One then can select phage-bearing inserts which bind to the cancer associated antigen polypeptide. This process can be repeated through several cycles of reselection of phage that bind to the cancer associated antigen polypeptide. Repeated rounds lead to enrichment of phage bearing particular sequences.
  • DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that binds to the cancer associated antigen polypeptide can be determined.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the cancer associated antigen polypeptides.
  • the cancer associated antigen polypeptides of the invention, or a fragment thereof can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the cancer associated antigen polypeptides of the invention.
  • Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of cancer associated antigen and for other purposes that will be apparent to those of ordinary skill in the art.
  • antibodies and other binding molecules may be used for example to identify tissues expressing protein or to purify protein.
  • Antibodies also may be coupled to specific diagnostic labeling agents for imaging of cells and tissues that express cancer associated antigens or to therapeutically useful agents according to standard coupling procedures.
  • Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium, diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate sodium and radiodiagnostics including positron emitters such as fluorine-18 and carbon- 11, gamma emitters such as iodine- 123, technitium-99m, iodine-131 and indium- 111, nuclides for nuclear magnetic resonance such as fluorine and gadolinium.
  • Other diagnostic agents useful in the invention will be apparent to one of ordinary skill in the art.
  • therapeutically useful agents include any therapeutic molecule which desirably is targeted selectively to a cell expressing one of the cancer antigens disclosed herein, including antineoplastic agents, radioiodinated compounds, toxins, other cytostatic or cytolytic drugs, and so forth.
  • Antineoplastic therapeutics include: aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon- ⁇ , lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCI, thioguanine, vinblastine sulfate and vincristine sulfate.
  • Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
  • Toxins can be proteins such as, for example, pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin.
  • Toxin moieties can also be high energy-emitting radionuclides such as cobalt-60.
  • antibodies prepared according to the invention also preferably are specific for the cancer associated antigen/MHC complexes described herein.
  • disorder refers to any pathological condition where the cancer associated antigens are expressed.
  • An example of such a disorder is cancer, with fibrosarcoma as a particular example.
  • additional particular examples include bladder cancer, breast cancer, lung cancer, colon cancer, and hepatoma.
  • Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods such as tissue biopsy, including punch biopsy and cell scraping, and collection of blood or other bodily fluids by aspiration or other methods.
  • an immunoreactive cell sample is removed from a subject.
  • immunoreactive cell is meant a cell which can mature into an immune cell (such as a B cell, a helper T cell, or a cytolytic T cell) upon appropriate stimulation.
  • immunoreactive cells include CD34 + hematopoietic stem cells, immature T cells and immature B cells.
  • the immunoreactive cell When it is desired to produce cytolytic T cells which recognize a cancer associated antigen, the immunoreactive cell is contacted with a cell which expresses a cancer associated antigen under conditions favoring production, differentiation and/or selection of cytolytic T cells; the differentiation of the T cell precursor into a cytolytic T cell upon exposure to antigen is similar to clonal selection of the immune system.
  • Some therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, leading to lysis of antigen presenting cells, such as cancer cells which present one or more cancer associated antigens.
  • One such approach is the administration of autologous CTLs specific to a cancer associated antigen/MHC complex to a subject with abnormal cells of the phenotype at issue. It is within the ability of one of ordinary skill in the art to develop such CTLs in vitro.
  • An example of a method for T cell differentiation is presented in International Application number PCT/US96/05607.
  • a sample of cells taken from a subject such as blood cells, are contacted with a cell presenting the complex and capable of provoking CTLs to proliferate.
  • the target cell can be a transfectant, such as a COS cell.
  • transfectants present the desired complex of their surface and, when combined with a CTL of interest, stimulate its proliferation.
  • COS cells are widely available, as are other suitable host cells. Specific production of CTL clones is well known in the art. The clonally expanded autologous CTLs then are administered to the subject.
  • Tetramers are formed by mixing the biotinylated peptide-MHC complex with labeled avidin (e.g. phycoerythrin) at a molar ratio or 4: 1. Tetramers are then contacted with a source of CTLs such as peripheral blood or lymph node. The tetramers bind CTLs which recognize the peptide antigen/MHC class I complex. Cells bound by the tetramers can be sorted by fluorescence activated cell sorting to isolate the reactive CTLs. The isolated CTLs then can be expanded in vitro for use as described herein.
  • avidin e.g. phycoerythrin
  • the foregoing therapy assumes that at least some of the subject's abnormal cells present the relevant HLA/cancer associated antigen complex. This can be determined very easily, as the art is very familiar with methods for identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA of the pertinent sequences, in this case a cancer associated antigen sequence.
  • Once cells presenting the relevant complex are identified via the foregoing screening methodology, they can be combined with a sample from a patient, where the sample contains CTLs. If the complex presenting cells are lysed by the mixed CTL sample, then it can be assumed that a cancer associated antigen is being presented, and the subject is an appropriate candidate for the therapeutic approaches set forth herein.
  • Adoptive transfer is not the only form of therapy that is available in accordance with the invention.
  • CTLs can also be provoked in vivo, using a number of approaches.
  • One approach is the use of non-proliferative cells expressing the complex.
  • the cells used in this approach may be those that normally express the complex, such as irradiated tumor cells or cells transfected with one or both of the genes necessary for presentation of the complex (i.e. the antigenic peptide and the presenting MHC molecule).
  • Chen et al. Proc. Natl. Acad. Sci. USA 88:110-114, 1991
  • exemplifies this approach showing the use of transfected cells expressing HPV E7 peptides in a therapeutic regime.
  • Various cell types may be used.
  • vectors carrying one or both of the genes of interest may be used.
  • Viral or bacterial vectors are especially preferred.
  • nucleic acids which encode a cancer associated antigen polypeptide or peptide may be operably linked to promoter and enhancer sequences which direct expression of the cancer associated antigen polypeptide or peptide in certain tissues or cell types.
  • the nucleic acid may be incorporated into an expression vector.
  • Expression vectors may be unmodified extrachromosomal nucleic acids, plasmids or viral genomes constructed or modified to enable insertion of exogenous nucleic acids, such as those encoding cancer associated antigen, as described elsewhere herein.
  • Nucleic acids encoding a cancer associated antigen also may be inserted into a retroviral genome, thereby facilitating integration of the nucleic acid into the genome of the target tissue or cell type.
  • the gene of interest is carried by a microorganism, e.g., a vaccinia virus, pox virus, herpes simplex vims, retrovims or adenovirus, and the materials de facto "infect" host cells.
  • a similar effect can be achieved by combining the cancer associated antigen or a stimulatory fragment thereof with an adjuvant to facilitate incorporation into antigen presenting cells in vivo.
  • the cancer associated antigen polypeptide is processed to yield the peptide partner of the MHC molecule while a cancer associated antigen peptide may be presented without the need for further processing.
  • subjects can receive an intradermal injection of an effective amount of the cancer associated antigen. Initial doses can be followed by booster doses, following immunization protocols standard in the art.
  • Preferred cancer associated antigens include those found to react with allogeneic cancer antisera, shown in the examples below.
  • the invention involves the use of various materials disclosed herein to "immunize” subjects or as “vaccines".
  • "immunization” or “vaccination” means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition but rather contemplates the clinically favorable enhancement of an immune response toward an antigen.
  • Generally accepted animal models including the MethA fibroasrcoma model used herein, can be used for testing of immunization against cancer using a cancer associated antigen nucleic acid.
  • human cancer cells can be introduced into a mouse to create a tumor, and one or more cancer associated antigen nucleic acids can be delivered by the methods described herein.
  • the effect on the cancer cells can be assessed as a measure of the effectiveness of the cancer associated antigen nucleic acid immunization.
  • testing of the foregoing animal model using more conventional methods for immunization include the administration of one or more cancer associated antigen polypeptides or peptides derived therefrom, optionally combined with one or more adjuvants and/or cytokines to boost the immune response.
  • Methods for immunization including formulation of a vaccine composition and selection of doses, route of administration and the schedule of administration (e.g. primary and one or more booster doses), are well known in the art.
  • the tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating CTLs against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth.
  • one or more cancer associated antigens or stimulatory fragments thereof are administered with one or more adjuvants to induce an immune response or to increase an immune response.
  • An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response.
  • Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes. Adjuvants of many kinds are well known in the art.
  • adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract; DQS21, described in PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So et al., Mol.
  • MPL monophosphoryl lipid A
  • SPL SmithKline Beecham
  • saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract
  • DQS21 described in PCT application WO96/33739 (SmithKline Beecham)
  • QS-7, QS-17, QS-18, and QS-L1 So et al., Mol.
  • the peptides are administered mixed with a combination of DQS21/MPL.
  • the ratio of DQS21 to MPL typically will be about 1 : 10 to 10:1, preferably about 1:5 to 5:1 and more preferably about 1 :1.
  • DQS21 and MPL will be present in a vaccine formulation in the range of about 1 ⁇ g to about 100 ⁇ g.
  • Other adjuvants are known in the art and can be used in the invention (see, e.g. Goding, Monoclonal Antibodies: Principles and Practice, 2nd Ed., 1986). Methods for the preparation of mixtures or emulsions of peptide and adjuvant are well known to those of skill in the art of vaccination.
  • Other agents which stimulate the immune response of the subject can also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties.
  • cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-12 (IL-12) which has been shown to enhance the protective effects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSF and IL-18.
  • IL-12 interleukin-12
  • GM-CSF GM-CSF
  • IL-18 interleukin-18
  • costimulatory molecules provided in either protein or nucleic acid form.
  • costimulatory molecules include the B7-1 and B7-2 (CD80 and CD86 respectively) molecules which are expressed on dendritic cells (DC) and interact with the CD28 molecule expressed on the T cell.
  • DC dendritic cells
  • This interaction provides costimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) T cell, increasing T cell proliferation and effector function.
  • B7 also interacts with CTLA4 (CD 152) on T cells and studies involving CTLA4 and B7 ligands indicate that the B7-CTLA4 interaction can enhance antitumor immunity and CTL proliferation (Zheng P., et al. Proc. Natl. Acad. Sci. USA 95 (11):6284- 6289 (1998)).
  • B7 typically is not expressed on tumor cells so they are not efficient antigen presenting cells (APCs) for T cells. Induction of B7 expression would enable the tumor cells to stimulate more efficiently CTL proliferation and effector function.
  • a combination of B7/IL-6/EL-12 costimulation has been shown to induce IFN-gamma and a Thl cytokine profile in the T cell population leading to further enhanced T cell activity (Gajewski et al., J Immunol, 154:5637-5648 (1995)).
  • Tumor cell transfection with B7 has ben discussed in relation to in vitro CTL expansion for adoptive transfer immunotherapy by Wang et al., (J. Immunol., 19:1-8 (1986)).
  • B7 molecule delivery mechanisms for the B7 molecule would include nucleic acid (naked DNA) immunization (Kim J., et al. Nat. Biotechnol., 15:7:641-646 (1997)) and recombinant viruses such as adeno and pox (Wendtner et al., Gene Ther., 4:7:726-735 (1997)).
  • nucleic acid naked DNA
  • recombinant viruses such as adeno and pox
  • adeno and pox Wang et al., Gene Ther., 4:7:726-735 (1997).
  • These systems are all amenable to the construction and use of expression cassettes for the coexpression of B7 with other molecules of choice such as the antigens or fragment(s) of antigens discussed herein (including polytopes) or cytokines.
  • These delivery systems can be used for induction of the appropriate molecules in vitro and for in vivo vaccination situations.
  • anti-CD28 antibodies to directly stimulate T cells in vitro and in vivo could also be considered.
  • the inducible co-stimulatory molecule ICOS which induces T cell responses to foreign antigen could be modulated, for example, by use of anti-ICOS antibodies (Hutloff et al., N ⁇ twre 397:263-266, 1999).
  • Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCs and some tumor cells and interacts with CD2 expressed on T cells. This interaction induces T cell IL-2 and IF ⁇ -gamma production and can thus complement but not substitute, the B7/CD28 costimulatory interaction (Parra et al., J. Immunol., 158:637-642 (1997), Fenton et al., J Immunother., 21:2:95-108 (1998)).
  • Lymphocyte function associated antigen- 1 (LFA-1) is expressed on leukocytes and interacts with ICAM-1 expressed on APCs and some tumor cells. This interaction induces T cell IL-2 and IF ⁇ -gamma production and can thus complement but not substitute, the B7/CD28 costimulatory interaction (Fenton et al., J. Immunother., 21 :2:95-108 (1998)). LFA-1 is thus a further example of a costimulatory molecule that could be provided in a vaccination protocol in the various ways discussed above for B7.
  • Th cell help through the interaction between the Th cell CD40L (CD40 ligand) molecule and the CD40 molecule expressed by DCs (Ridge et al., Nature, 393:474 (1998), Bennett et al., Nature, 393:478 (1998), Schoenberger et al., Nature, 393:480 (1998)).
  • This mechanism of this costimulatory signal is likely to involve upregulation of B7 and associated IL-6/IL-12 production by the DC (APC).
  • the CD40-CD40L interaction thus complements the signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.
  • anti-CD40 antibodies to stimulate DC cells directly, would be expected to enhance a response to tumor antigens which are normally encountered outside of a inflammatory context or are presented by non-professional APCs (tumor cells). In these situations Th help and B7 costimulation signals are not provided. This mechanism might be used in the context of antigen pulsed DC based therapies or in situations where Th epitopes have not been defined within known TRA precursors.
  • a cancer associated antigen polypeptide, or a fragment thereof also can be used to isolate their native binding partners. Isolation of such binding partners may be performed according to well-known methods. For example, isolated cancer associated antigen polypeptides can be attached to a substrate (e.g., chromatographic media, such as polystyrene beads, or a filter), and then a solution suspected of containing the binding partner may be applied to the substrate. If a binding partner which can interact with cancer associated antigen polypeptides is present in the solution, then it will bind to the substrate-bound cancer associated antigen polypeptide. The binding partner then may be isolated.
  • a substrate e.g., chromatographic media, such as polystyrene beads, or a filter
  • the invention embraces the use of the cancer associated antigen cDNA sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., dendritic cells, B cells, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells).
  • prokaryotic e.g., E. coli
  • eukaryotic e.g., dendritic cells, B cells, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells.
  • mammalian cells such as human, mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. Specific examples include dendritic cells, peripheral blood leukocytes, bone marrow stem cells and embryonic stem cells.
  • the expression vectors require that the pertinent sequence, i.
  • the invention also contemplates delivery of nucleic acids, polypeptides or peptides for vaccination. Delivery of polypeptides and peptides can be accomplished according to standard vaccination protocols which are well known in the art. In another embodiment, the delivery of nucleic acid is accomplished by ex vivo methods, i.e. by removing a cell from a subject, genetically engineering the cell to include a cancer associated antigen, and reintroducing the engineered cell into the subject.
  • ex vivo methods i.e. by removing a cell from a subject, genetically engineering the cell to include a cancer associated antigen, and reintroducing the engineered cell into the subject.
  • U.S. Patent 5,399,346 One example of such a procedure is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents.
  • a vims vector for delivering a nucleic acid encoding a cancer associated antigen is selected from the group consisting of adenovimses, adeno- associated viruses, poxviruses including vaccinia vimses and attenuated poxvimses, Semliki Forest vims, Venezuelan equine encephalitis virus, retroviruses, Sindbis vims, and Ty vims- like particle.
  • vimses and vims- like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenovimses (e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovims (Townsend et al., J. Virol. 71 :3365-3374, 1997), a nonreplicating retrovims (Irwin et al., J. Virol.
  • replication-defective adenovimses e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:
  • the vims vector is an adenovims.
  • the adeno-associated vims is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated vims can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression.
  • adeno-associated vims infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated vims genomic integration is a relatively stable event.
  • the adeno-associated vims can also function in an extrachromosomal fashion.
  • other preferred viral vectors are based on non-cytopathic eukaryotic vimses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic vimses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Adenovimses and retrovimses have been approved for human gene therapy trials.
  • the retrovimses are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • Standard protocols for producing replication-deficient retrovimses including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retrovimses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry, E.J. Ed.
  • nucleic acid delivery vectors (1) contain exogenous genetic material that can be transcribed and translated in a mammalian cell and that can induce an immune response in a host, and (2) contain on a surface a ligand that selectively binds to a receptor on the surface of a target cell, such as a mammalian cell, and thereby gains entry to the target cell.
  • a target cell such as a mammalian cell
  • Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the foregoing vimses including the nucleic acid of interest, liposome mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell e.g., a retrovims, or other vims; a liposome
  • a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.
  • Preferred antibodies include antibodies which selectively bind a cancer associated antigen, alone or as a complex with a MHC molecule.
  • monoclonal antibodies are employed.
  • proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.
  • compositions of the present invention can be administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • a preferred route of administration is by pulmonary aerosol.
  • Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences. 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations of the invention, slow intravenous administration is preferred.
  • the compositions of the invention are administered in effective amounts.
  • an “effective amount” is that amount of a cancer associated antigen composition that alone, or together with further doses, produces the desired response, e.g. increases an immune response to the cancer associated antigen.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • Monitoring the progression, regression or onset of a condition can include, for example, obtaining samples from a patient or a person suspected of having the condition at sequential times and assaying such samples for the presence and/or amount of protein or nucleic acid markers of the condition.
  • markers include the nucleic acids and polypeptides of the invention, antibodies that bind to the polypeptides, nucleic acids that hybridize to the nucleic acids, and other molecules known to be useful in diagnostic applications.
  • Onset of a cancerous condition is indicated by the appearance of marker(s) in a subject's samples where there were no such marker(s) previously.
  • Progression and regression of a cancerous condition are generally indicated by the increase or decrease, respectively, of marker(s) in a subject's samples over time.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of cancer associated antigen or nucleic acid encoding cancer associated antigen for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by determining the immune response following administration of the cancer associated antigen composition via a reporter system by measuring downstream effects such as gene expression, or by measuring the physiological effects of the cancer associated antigen composition, such as regression of a tumor or decrease of disease symptoms.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • cancer associated antigen compositions e.g., polypeptide, peptide, antibody, cell or nucleic acid
  • doses of cancer associated antigen compositions can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • cancer associated antigen are formulated and administered in doses between 1 ng and 1 mg, and preferably between 10 ng and 100 ⁇ g, according to any standard procedure in the art.
  • doses of between 1 ng and 0.1 mg generally will be formulated and administered according to standard procedures.
  • Other protocols for the administration of cancer associated antigen compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration (e.g., intra-tumoral) and the like vary from the foregoing.
  • cancer associated antigen peptides are used for vaccination
  • modes of administration which effectively deliver the cancer associated antigen and adjuvant, such that an immune response to the antigen is increased, can be used.
  • preferred methods include intradermal, intravenous, intramuscular and subcutaneous administration. Although these are preferred embodiments, the invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remington 's Pharmaceutical Sciences, 18th edition, 1990) provide modes of administration and formulations for delivery of immunogens with adjuvant or in a non-adjuvant carrier.
  • the pharmaceutical preparations of the invention When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • a cancer associated antigen composition may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • the pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a symp, elixir or an emulsion.
  • Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of cancer associated antigen polypeptides or nucleic acids, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
  • a non-toxic parenterally-acceptable diluent or solvent for example, as a solution in 1,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • Meth A is a prototype tumor for analysis of tumor rejection antigens (Gross, L., Cancer Res. 3:326-333, 1943; Foley, Cancer Res. 13:835-842, 1953; Prehn et al., J. Natl. Cancer Inst. 18:769-778, 1957; and Old et al., Ann. NY. Acad. Sci. 101 :80-106, 1962).
  • mice BALB/c mice were purchased from Japan SLC (Shizuoka, Japan).
  • Meth A, CMS5a, CMS5j, CMS8, CMS13 and CMS17 are methylcholanthrene- induced fibrosarcomas in BALB/c mice (DeLeo et al., J. Exp. Med. 146:720-734, 1977; Palladino et al., Cancer Res. 47:5074-5079, 1987).
  • RL 1 1 is a radiation-induced leukemia in a BALB/c mouse (Nakayama et al., Proc. Natl. Acad. Sci USA 76:3486-3490, 1979).
  • RVA, RVC and RVD are leukemias induced by an injection of radiation leukemia vims into neonatal BALB/c mice (Stockert et al., J.
  • RV2 is leukemia induced by an injection of radiation leukemia vims into a neonatal C57BL/6 mouse (Nakayama et al., Cancer Res. 44:5138-5144, 1984).
  • A20.2J is a BALB/c B cell lymphoma (Kim et al., J. Immunol. 122:549-554, 1979).
  • MOPC-70A is a mineral oil-induced myeloma in a BALB/c mouse (Potter, M. In: Methods in Cancer Research, 1967, pp. 106-157).
  • P815 is a methylcholanthrene-induced mastocytoma in a DBA/2 mouse (Dunn et al., J. Natl. Cancer Inst. 18:587-601, 1957).
  • EL4 is an dimethylbenzanthracene-induced leukemia in a C57BL mouse (Gorer et al., Br. J. Cancer 4:372-379, 1950).
  • Meth A (K ⁇ ) cells used for constmction of cDNA expression library were Meth A cells which were retrovirally introduced with murine IFN- ⁇ cDNA (Watanabe et al., Eur. J. Immunol. 18:1627-1630, 1988).
  • Meth A (K ⁇ ) cells were highly antigenic and regressed in CB6F 1 mice. Concomitant inoculation of the cells caused rejection of the parental Meth A cells (Hamtsumi et al., Int. J. Oncol. 7:233-238, 1995).
  • Sera were obtained from BALB/c mice at various days after 5 x 10 5 Meth A inoculation (i.d. in the back). Sera were obtained from 3-5 mice each at 16, 23, 31 and 45 days after MethA inoculation.
  • RNA was purified from Meth A (K ⁇ ) cells and a cDNA expression library was constmcted in a ⁇ ZAP Express vector using a cDNA library kit (Stratagene, La Jolla, CA).
  • cDNA libraries was screened with semm from BALB/c mice obtained on day 45 after inoculation of Meth A.
  • the nitrocellulose membranes containing the phages were incubated overnight at room temperature with the semm diluted to 1 :200, which has been preabsorbed with lysate from E. coli coupled to Sepharose 4B (5 Prime — • 3 Prime, Boulder, CO).
  • Semm antibodies binding to recombinant proteins expressed in lytic phages were detected by incubation with peroxidase-conjugated goat anti -mouse IgG (Jackson Immuno Research, West Grove, PA) and visualized by staining with 3-3'-diaminobenzidine (Sigma, St. Louis, MO).
  • CMV plasmid forms (Stratagene, La Jolla, CA).
  • the nucleotide sequence ofcDNA inserts was determined by using ABI PRISM automated sequencers (PE Applied Biosystems, Foster City, CA). Sequence alignments were performed with BLAST software on GenBank.
  • the mRNA was reverse transcribed into single strand cDNA using Moloney murine leukemia vims reverse transcriptase and oligo(DT) ⁇ 5 as primer (Amersham Pharmacia, Piscataway, NJ). cDNAs were tested for integrity by amplification of ⁇ -actin transcripts in a 30 cycle reaction. Gene specific oligonucleotide primers were designed to amplify cDNA segments for 300-600 bp in length.
  • RT-PCR was performed by using 30 amplification cycles at an annealing temperature of 58°C using the following primers: MC4A 5'-GTGGACAAGAGGAAGCACAA-3' (SEQ ID NO:21) and MC4B 5'-TGAAAAGTAAGGGCTGTCAT-3' (SEQ ID NO:22).
  • cDNA expression libraries were prepared from Meth A (K ⁇ ) cells, which were Meth A cells retrovirally introduced with IFN- ⁇ in order to augment antigenicity. Immunoscreening of 80,000 clones with semm from BALB/c mice obtained at day 45 after inoculation of parental Meth A yielded a total of 35 positive clones. The nucleotide sequences of the cDNA inserts were determined. As shown in Table 1, ten different genes were isolated (OY-MC-1 through OY-MC-10), five of which were identical to or had homology to genes in the DNA database.
  • OY-MC-1 was represented by 13 overlapping clones, OY-MC-2 by 10 overlapping clones, OY-MC-3 by 3 overlapping clones, OY-MC-4 by 2 overlapping clones and OY-MC-6 by a single clone.
  • OY-MC-1 was homologous to rat ribosomal protein LI 1.
  • OY-MC-3 was homologous to bovine NAD + dependent isocitrate dehydrogenase subunits 3 and 4.
  • OY-MC-2 was homologous to mouse glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).
  • OY-MC-4 was homologous to mouse placenta and embryonic expression gene (pern).
  • OY-MC-6 was identical to a C4 and cytochrome P450 hydroxylase A gene. The remaining five genes (OY-MC-5, 7, 8, 9, 10) were unknown genes which had no homology to any published genes.
  • OY-MC-1, OY-MC-2 and OY-MC-9 showed ubiquitous mRNA expression in normal tissues, other genes showed a variety of expression patterns. Of those, OY-MC-4 showed a restricted expression in normal tissues as it was expressed only in testis and placenta, and to a lesser extent in ovary (Table 2).
  • Adrenal gland Bone marrow - Weak expression
  • OY-MC-4 showed ubiquitous expression in 15 mouse tumors tested. Expression of OY-MC-4 was rather restricted and found in 10 of 15 mouse tumors tested (Table 3). Of 6 methylcholanthrene-induced fibrosarcomas examined, Meth A,
  • CMS5j, CMS8 and CMS13 showed OY-MC-4 expression, CMS17showed a faint expression, and CMS5a was negative.
  • Expression of OY-MC-4 was much less frequent in 6 leukemias examined. Strong expression of OY-MC-4 was only found in EL4. RVD and RV2 showed a faint expression, and RLd" 1, RVA, RVC were negative (Table 3).
  • the nucleotide sequence of OY-MC-4 revealed that the cDNA was 845 bp in length (SEQ ID NO:7) and nearly identical to a mouse homeobox gene, placenta and embryonic expression gene (pern).
  • the pern gene was identified from an AKR T-cell lymphoma and shown to be coded for by the X chromosome (Wilkinson et al., Dev. Biol. 141 :451-455, 1990; Lin et al., Dev. Biol. 166:170-179, 1994).
  • C to A One nucleotide difference at position 619 was observed in OY-MC-4 compared with the published pern sequence.
  • This nucleotide difference results in a Thr— >Asp change at amino acid residue 174 (SEQ ID NO: 8) as compared with the published pern sequence.
  • the sequences of OY-MC-4 PCR products from Meth A, CMS5j, CMS8, CMS 13 and testis that were subcloned into pCR2.1 showed A (adenine) at position 619, indicating that A at position 619 of SEQ ID NO: 7 is a normal pern nucleotide sequence in BALB/c mouse.
  • mice were injected s.c. with 3-methylcholanthrene dissolved in peanut oil at a concentration of 125 ⁇ g/ml in a volume of 0.2 ml. Semm was obtained 42 weeks after injection.
  • OY-MC-4 was nearly identical to mouse placenta and embryonic expression gene (pern), which was previously shown to be expressed during embryogenesis (Wilkinson et al., Dev. Biol. 141 :451-455, 1990). Pern gene was distantly related to Xheprd/pax homeobox gene family (Lin et al., Dev. Biol. 166:170-179, 1994; Sasaki et al., Mech. Dev. 34:155-164, 1991) and was shown to be coded for by the X chromosome (Lin et al., Dev. Biol.
  • OY-MC-4 nucleotide sequence differed from the published pern cDNA sequence, which was isolated from a T-lymphoma of AKR mouse, in having an A instead of a C at position 619.
  • the corresponding predicted amino acid sequence of OY-MC- 4 included asparagine instead of threonine at position 174. Threonine at position 174 was thought to be a phosphorylation site for protein kinase C (Wilkinson et al., 1990. Dev. Biol. 141:451-455).
  • CT cancer/testis
  • NY-ESO-1 was isolated from an esophageal squamous cell carcinoma.
  • NY-ESO- 1 has been shown to be recognized by CTL, in the context of HLA-A2 and HLA-A31.
  • SCPl which was isolated from a cDNA library enriched for testis specific clones by using sera from patients with renal cancer, is presently the only CT antigen with known function.
  • the SCPl protein is involved in the meiotic chromosome synapses of sperm cells.
  • OY-MC-4 described here, is a new member of SEREX-defined CT antigens with known function.
  • Example 2 Identification of acrosomal protein, sp32, as a new human cancer/testis antigen.
  • Poly (A) + RNA was isolated from tumor tissue using the QuickPrep Micro mRNA Purification kit (Amersham Pharmacia, Piscataway, NJ).
  • the mRNA was reverse transcribed into single strand cDNA using the Moloney murine leukemia vims reverse transcriptase and oligo(dT) ⁇ 5 as a primer (Amersham Pharmacia, Piscataway, NJ).
  • GSP1-T1 (SEQ LD NO:30) 5'-TTCCTGGGCTGATCGAATGAG-3'
  • GSP2-T1 (SEQ ED NO:31) 5'-GCAAAAGAGGAAGGGTTAGAAG-3'
  • Tes-Nl (SEQ ED NO:32) 5'-CCGTGGTTTTCATATTGGTC-3' (sequence primer)
  • human testis cDNA was supplemented with 5 ⁇ l of lOx PCR buffer (100 mM Tris-HCl, 500 mM KC1, 0.01% (w/v) gelatin), 3 ⁇ l of 25 mM MgCl 2 , 2 ⁇ l of 10 mM dNTPs, 5 ⁇ l of 5 ⁇ M each primer (pem5 and EST-2), 1.25 units of AmpliTaq DNA Polymerase (Perkin Elmer) and water to a final volume of 50 ⁇ l.
  • PCR cycle conditions were 1 min at 94°C, 1 min at 54°C and 1.5 min at 72°C for 35 cycles.
  • PCR product of 1.1 kb was obtained and sequenced using an ABI PRISM automated sequencer. Homology walking was performed using the human dbEST database maintained by the National Center for Biotechnology Information (http:/www.ncbi.nlm.nih.gov/dbEST). Several ESTs corresponded to the PCR product. Most of the ESTs were cDNA clones derived from human testis. The EST clusters are shown in Fig. 1. The 3' end of the gene was extended by the sequence information of more than 11 ESTs which exhibited homology to the PCR product.
  • Nucleotide sequence (nucleotides 261- 1796 of SEQ LD NO:23) of the PCR product of 1.5 kb which was obtained using only anti- sense primer ht-1 (SEQ ED NO:29) was identical to the deduced sequence.
  • 5' RACE was performed to identify the cDNA 5' end sequence using the 5' RACE System for Rapid Amplification kit following the manufacturer's protocols (Life Technologies, Inc., Rockville, MD). Total RNA from human testis was used as a template and first-strand cDNA was synthesized using specific primer, GSP1-T1 (SEQ ED NO:30). The RACE product was sequenced using an ABI PRISM automated sequencer with sequence primer, Tes-Nl (SEQ ED NO:32).
  • a homology search of the cDNA sequence through the GenBank database revealed that the full length cDNA is the guinea pig and porcine acrosomal protein sp32 genes.
  • Other homologous nucleic acid sequences are set forth in Table 11 below.
  • the amino-terminal region composed of 25 residues in theprotein was highly hydrophobic, suggesting that it was a signal sequence.
  • the amino-terminal region composed of 24 residues in mature human sp32 corresponding to the same region composed of 25 residues in the porcine, 22 residues in the guinea pig, and 23 residues in the mouse are abundant in acidic amino acids.
  • ht-5 SEQ ID NO:27
  • ht-3 SEQ ED NO:28
  • RT-PCR was performed as described above for 30 amplification cycles with an annealing temperature of 62°C. Expression of the mRNA was observed with only testis in normal adult tissues and with various malignant tumors (Table 7).
  • Table 7 OY-TES-1 mRNA expression in human tumors Tumor type mRNA, positive/total
  • the cDNA identified is the human homologue of acrosomal protein sp32 and has the expression characteristics of a cancer/testis antigen. Therefore, the cDNA was named OY-TES-1 antigen.
  • PCR was applied to the Stanford G3 Radiation Hybrid Panel (purchased from Research Genetics, USA). PCR primers designed from the sequence of the 3'UTR of OY- TES-1 were as follows: sense, 5'-CTGGCGTCTATTCTGCCCA-3' (SEQ ED NO: 33); antisense, 5'-TGTAAAGTCATCTTTTAAGGAGG-3' (SEQ ED NO: 34).
  • PCR was performed under conditions of 33 cycles at 94°C for 30 sec for denaturation at 57°C for 30 sec for annealing, and at 72°C for 30 sec for extension in a mixture containing 250mM of each dNTP, lOmM of each primer, PCR buffer (lOmM Tris-HCI, 50mM KCI, 1.5mM MgCl 2 , pH 8.3), 0.5 unit oiTaq polymerase (Takara Shuzo, Japan) and 25ng of template DNA.
  • the PCR products were loaded onto 6% polyacrylamide gels. The presence of specific PCR products was scored, and screening results were submitted to Online RH-server at Stanford Human Genome Center.
  • PACs PI -derived artificial chromosomes
  • Genomic DNA was digested with 100 units of EcoRI, Hindlfl and BamHI at 37°C overnight. The DNA was separated on an 0.8% agarose gel, blotted onto nylon transfer membrane (Hybond-N+, Amersham Pharmacia Biotech, Amersham Place, UK). The blot was hybridized to the 602-bp cDNA probe (nt 749-1351 of SEQ ED NO:23) directly labeled with alkaline phosphatase (AlkPhos Direct, Amersham. Pharmacia Biotech, Amersham Place, UK), washed and processed for chemiluminescence according to the manufacturer's instmctions.
  • OY-TES-1 was expressed in E. coli using histidine-tag-containing vector pQE32
  • cDNA amplification primers were designed to encompass entire coding sequences of the gene, corresponding to amino acid positions from 1 to 543.
  • the induction of recombinant protein synthesis and subsequent purification by Ni 2+ -NTA column were performed according to the manufacturer's instmctions.
  • secondary antibody HRP- conjugated goat-anti human IgG, Medical Biological Lab., Tokyo, Japan
  • OY-TES-1 5'- CCCCATCTCACCCCAC-3' (SEQ ED NO: 35) from EST-2 corresponding to nt 501-516 of SEQ ED NO:23 with one nucleotide (underlined) difference and 3 '-CTTCGTGTT-5 ' (SEQ ED NO: 36) from pem5 corresponding to nt 1633-1625 of SEQ ED NO:23.
  • OY-TES-1 contains a single long open reading frame that extends from bp 49 to 1677 and predicts a protein containing 543 amino acids (SEQ ED NO:24; Fig. 2).
  • a homology search of the gene through the GenBank database revealed that it is a human homologue of the gene coding for porcine, guinea pig and mouse proacrosin binding protein sp32 (Baba, T., et al., J. Biochem.
  • the scores obtained with the radiation hybrid panel were as follows: 00100 - 01001 - 01000 - 01001 - 01000 - 10000 - 00000 - 00010 - 00110 - 01100 - 00011 - 00001 - 01000 - 00110 - 00000 - 01000 - 000.
  • the result was matched to the cell line SHGC- 12737 that was mapped to chromosome 12 and a reference interval estimated was 13.9 cM between two markers, D12S99-DI2S358.
  • More fine chromosomal localization was defined by the FISH analysis with a PAC probe (145N7): of the 20 metaphase cells analyzed, 18 showed brightly fluorescent twin signals 12pl2-pl3 (Fig. 5). Genomic structure of OY-TES-1.
  • OY-TES-1 Genomic sequence of OY-TES-1 was searched in the GenBank hgts database by mnning the publicly available BLASTN software.
  • OY-TES-1 cDNA sequence was found in the sequences of PAC clone RP4-761 J14 and BAC clone RP11-433J6. Those two genes are located on chromosome 12pl3, confirming the FISH analysis.
  • the OY-TES-1 gene is composed of 10 exons, spanning a distance of 9339 bp.
  • OY-TES-1 mRNA expression was examined by RT-PCR using a panel of normal and malignant tissues. As shown in Fig. 7, in adult tissues, OY-TES-1 expression was observed only in testis. In tumors, OY-TES-1 was expressed at various frequencies in different origin. As shown in Fig. 8 and Table 7, 28% (11/39) of bladder cancer tumor samples, 36% (4/11) of lung cancer tumor samples, 40% (2/5) of breast cancer tumor samples, 40% (2/5) of liver cancer tumor samples and 15% (2/13) of colon cancer tumor samples showed detectable OY- TES-1 mRNA. However, no expression of OY-TES-1 mRNA was observed in renal or stomach cancer tumor samples.
  • Table 8 ELISA reactivity of sera from normal blood donors and cancer patients with OY-TES-1 Sera ELISA, positive/total
  • Example 3 Preparation of recombinant cancer associated antigens.
  • recombinant proteins are prepared according to standard procedures.
  • the clones encoding cancer associated antigens are subcloned into a baculovims expression vector, and the recombinant expression vectors are introduced into appropriate insect cells.
  • Baculovims/insect cloning systems are preferred because post-translational modifications are carried out in the insect cells.
  • Another preferred eukaryotic system is the Drosophila Expression System from Invitrogen. Clones which express high amounts of the recombinant protein are selected and used to produce the recombinant proteins.
  • the recombinant proteins are tested for antibody recognition using semm from the mouse or patient which was used to isolated the particular clone, or in the case of cancer associated antigens recognized by allogeneic sera, by the sera from any of the mouse strains or patients used to isolate the clones or sera which recognize the clones' gene products.
  • cancer associated antigen clones are inserted into a prokaryotic expression vector for production of recombinant proteins in bacteria.
  • Other systems including yeast expression systems and mammalian cell culture systems also can be used.
  • Example 4 Preparation of antibodies to cancer associated antigens.
  • the recombinant cancer associated antigens produced as in Example 2 above are used to generate polyclonal antisera and monoclonal antibodies according to standard procedures.
  • the antisera and antibodies so produced are tested for correct recognition of the cancer associated antigens by using the antisera/antibodies in assays of cell extracts of mice or patients known to express the particular cancer associated antigen (e.g. an ELISA assay).
  • These antibodies can be used for experimental purposes (e.g. localization of the cancer associated antigens, immunoprecipitations, Western blots, etc.) as well as diagnostic purposes (e.g., testing extracts of tissue biopsies, testing for the presence of cancer associated antigens).
  • Example 5 Expression of cancer associated antigens in cancers of similar and different origin.
  • the expression of one or more of the cancer associated antigens is tested in a range of tumor samples to determine which, if any, other malignancies should be diagnosed and/or treated by the methods described herein.
  • Tumor cell lines and tumor samples are tested for cancer associated antigen expression, preferably by RT-PCR according to standard procedures.
  • Northern blots also are used to test the expression of the cancer associated antigens.
  • Antibody based assays such as ELISA and western blot, also can be used to determine protein expression.
  • a preferred method of testing expression of cancer associated antigens is allogeneic serotyping using a modified SEREX protocol (as described above).
  • extracts from the tumors of mice or patients who provided sera for the initial isolation of the cancer associated antigens are used as positive controls.
  • the cells containing recombinant expression vectors described in the Examples above also can be used as positive controls.
  • results generated from the foregoing experiments provide panels of multiple cancer associated nucleic acids and/or polypeptides for use in diagnostic (e.g. determining the existence of cancer, determining the prognosis of a patient undergoing therapy, etc.) and therapeutic methods (e.g., vaccine composition, etc.).
  • diagnostic e.g. determining the existence of cancer, determining the prognosis of a patient undergoing therapy, etc.
  • therapeutic methods e.g., vaccine composition, etc.
  • Example 6 HLA typing of patients positive for cancer associated antigen.
  • cells of the patients which express the cancer associated antigens are HLA typed (mice likewise can be typed for relevant MHC molecules).
  • Peripheral blood lymphocytes are taken from the patient and typed for HLA class I or class II, as well as for the particular subtype of class I or class II.
  • Tumor biopsy samples also can be used for typing.
  • HLA typing can be carried out by any of the standard methods in the art of clinical immunology, such as by recognition by specific monoclonal antibodies, or by HLA allele- specific PCR (e.g. as described in WO97/31126).
  • Example 7 Characterization of cancer associated antigen peptides presented by MHC class I and class II molecules.
  • Antigens which provoke an antibody response in a subject may also provoke a cell- mediated immune response.
  • Cells process proteins into peptides for presentation on MHC class I or class II molecules on the cell surface for immune surveillance.
  • Peptides presented by certain MHC/HLA molecules generally conform to motifs. These motifs are known in some cases, and can be used to screen the cancer associated antigens for the presence of potential class I and/or class II peptides. Summaries of class I and class II motifs have been published (e.g., Rammensee et al., Immunogenetics 41 :178-228, 1995). Based on the results of experiments such as those described above, the HLA types which present the individual cancer associated antigens are known. Motifs of peptides presented by these HLA molecules thus are preferentially searched.
  • HLA binding peptides for the OY-TES-1 polypeptide are listed in Table 10 below. These peptides were predicted using the algorithm of Parker et al (J. Immunol. 152:163, 1994). The binding peptides having the top scores for several HLA molecules are reported. The score for each peptide corresponds to an estimated half time of dissociation of a peptide from an HLA molecule.
  • Example 8 Identification of the portion of a cancer associated polypeptide encoding an antigen.
  • CTL clones are generated by stimulating the peripheral blood lymphocytes (PBLs) of a patient (or mouse) with autologous normal cells transfected with one of the clones encoding a cancer associated antigen polypeptide or with irradiated PBLs loaded with synthetic peptides corresponding to the putative protein and matching the consensus for the appropriate HLA class I molecule (as described above) to localize an antigenic peptide within the cancer associated antigen clone (see, e.g., Knuth et al., Proc. Natl. Acad. Sci.
  • PBLs peripheral blood lymphocytes
  • CTL clones are screened for specificity against COS cells transfected with the cancer associated antigen clone and autologous HLA alleles as described by Brichard et al. (Eur. J. Immunol. 26:224-230, 1996).
  • CTL recognition of a cancer associated antigen is determined by measuring release of TNF from the cytolytic T lymphocyte or by 51 Cr release assay (Herin et al., Int. J. Cancer 39:390-396, 1987).
  • a CTL clone specifically recognizes a transfected COS cell, then shorter fragments of the cancer associated antigen clone transfected in that COS cell are tested to identify the region of the gene that encodes the peptide. Fragments of the cancer associated antigen clone are prepared by exonuclease III digestion or other standard molecular biology methods. Synthetic peptides are prepared to confirm the exact sequence of the antigen. Optionally, shorter fragments of cancer associated antigen cDNAs are generated by
  • Synthetic peptides corresponding to portions of the shortest fragment of the cancer associated antigen clone which provokes TNF release are prepared. Progressively shorter peptides are synthesized to determine the optimal cancer associated antigen tumor rejection antigen peptides for a given HLA molecule.
  • a similar method is performed to determine if the cancer associated antigen contains one or more HLA class II peptides recognized by T cells.
  • class II peptides are presented by a limited number of cell types.
  • dendritic cells or B cell clones which express HLA class IE molecules preferably are used.
  • AI598144 1, AIO 95590, AI017929, AA310175, AA873566, AA868795, AA868719,
  • AI863340 1, AI520938, AA829435, AA508601, AI815674.1, AI720708.1, AI161342,
  • AI469742 AI281705, AA885866, A 139387.1, AA152181, AA729741, N35532,
  • AI131223 1, AI091519 1, AI122673 1, AA709069.1, AA443593.1, AI147148.1,
  • AI574731 1, R07740.1, AA992230.1 AI326707.1, AA154158.1, AI125674.1,

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Abstract

L'invention concerne des antigènes associés au cancer identifiés par criblage d'anticorps autologue de librairies d'acides nucléiques exprimés dans les cellules cancéreuses de fibrosarcome induit par le méthylcholanthrène, en utilisant des antisérums de souris atteintes par ces tumeurs. L'invention concerne en outre des acides nucléiques et des polypeptides codés qui sont des antigènes associés au cancer exprimés chez des souris atteintes par des fibrosarcomes induits par le méthylcholanthrène, ainsi que leurs homologues et notamment leurs homologues humains. L'invention concerne, entre autres, des molécules d'acides nucléiques isolés, des vecteurs d'expression renfermant ces molécules et des cellules hôtes transfectées avec les molécules en question. L'invention concerne par ailleurs des protéines et des peptides isolés, des anticorps vis-à-vis de ces protéines et peptides, et des lymphocytes T cytotoxiques reconnaissant les protéines et peptides en question. L'invention concerne aussi des fragments de ce type de molécule, qui renferment des fragments et des variants fonctionnels. L'invention concerne enfin des kits contenant les molécules considérées. Les molécules décrites peuvent être utilisées dans les applications suivantes: diagnostic, surveillance, recherche, ou traitement pour des états caractérisés par l'expression d'un ou plusieurs antigènes associés au cancer.
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