Cancer associated antigens and uses therefor

Abstract

Cancer associated antigens have been identified by autologous antibody screening of libraries of nucleic acids expressed in testis cells using antisera from seminoma patients. The invention relates to nucleic acids and encoded polypeptides which are cancer associated antigens expressed in patients afflicted with a variety of cancers. 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 cytotoxic T lymphocytes which recognize the proteins and peptides. Fragments of the foregoing including functional fragments and variants also are provided. Kits containing the foregoing molecules additionally are provided. The molecules provided by the invention can be used in the diagnosis, monitoring, research, or treatment of conditions characterized by the expression of one or more cancer associated antigens.

Description

FIELD OF THE INVENTION

The invention relates to nucleic acids and encoded polypeptides which are cancer associated antigens. 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, inter alia, in diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The mechanism by which T cells recognize foreign materials has been implicated in cancer. A number of cytolytic T lymphocyte (CTL) clones directed against autologous melanoma antigens, testicular antigens, and melanocyte differentiation antigens have been described. In many instances, the antigens recognized by these clones have been characterized.

The use of 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.

Recent progress in the identification of human tumor antigens has generated a repertoire of target molecules for use in antigen-specific tumor vaccines. The continued expansion of this repertoire allows for the potential development of polyvalent cancer vaccines that can be applied to several tumor types and may help circumvent some of the difficulties associated with active immunotherapy, such as tumor heterogeneity and antigen loss. Human tumor antigens fall into several categories, including autoimmunogenic differentiation antigens (e.g., tyrosinase), mutated gene products (e.g., beta catenin), oncogenes (e.g., Her2/neu), and a collection of immunogenic proteins that have an expression pattern restricted to testis, as well as a proportion of various cancers. These proteins, referred to as cancer-testis antigens (CT antigens, CTAs), were initially identified in cancer patients by expression cloning using either the autologous cellular or humoral immune response as the election criteria. The first members of this group were those molecules defined by cytotoxic T-lymphocyte (CTL) recognition such as the MAGE, BAGE, and GAGE antigens. Subsequently, by employing autologous antibody recognition, the SEREX (serological analysis of recombinant cDNA expression libraries) method uncovered other members of this group including the HOM-MEL-40/SSX2, NY-ESO-1, SCP1 and CT7 antigens.

The SEREX procedure was described by Sahin et al. ( Proc. Natl. Acad. Sci. USA 92:11810-11813, 1995). Also, see U.S. Pat. No. 5,698,396, incorporated herein by reference. According to this approach, autologous antisera are used to identify immunogenic protein antigens expressed in cancer cells by screening expression libraries constructed from tumor cell cDNA. Antigen-encoding clones so identified have been found to have elicited an high-titer humoral immune response in the patients from which the antisera were obtained. Such a high-titer IgG response implies helper T cell recognition of the detected antigen. These tumor antigens can then be screened for the presence of MHC/HLA class I and class II motifs and reactivity with CTLs.

More recently, non-immunological techniques such as representational difference analysis (RDA) have led to the identification of molecules such as MAGE-C1 that has an expression profile similar to CT antigens. A standardized nomenclature has been proposed for organizing the CT antigens into families. Due to a general lack of functional information, it is based on their order of discovery, i.e., sequentially, MAGE, BAGE, GAGE, HOM-MEL-40/SSX2, NY-ESO-1, SCP-1, CT7 and CT8.

Since individual CTAs are expressed only in a variable proportion of tumors of a given entity, the availability of additional CTAs would significantly enlarge the proportion of patients who are potentially eligible for therapeutic interventions. Despite the fact that the pool of available tumor antigens has grown since the introduction of SEREX, the proportion of antigen-negative tumors is still high. Thus there presently is a need for additional cancer antigens for development of therapeutics and diagnostics applicable to a greater number of cancer patients having various cancers.

SUMMARY OF THE INVENTION

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 BRDT/CT9 and optionally one or more additional cancer associated antigens.

The invention involves the surprising discovery that a previously known gene, BRDT, is expressed in individuals who have cancer. In addition, the invention also includes the characterization of expression of CT antigens in individuals with lung cancer. These individuals 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 invention involves the use of a single material, a plurality of different materials and even large panels and combinations of materials. For example, 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. Likewise, 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. An example of this is the use of a plurality of such materials prophylactically or acutely for the prevention, delay of onset, amelioration, etc. of cancer in cells which express or will express such genes. Any and all combinations of the genes, gene products, and materials which recognize the genes and gene products can be tested and identified for use according to the invention. It would be far too lengthy to recite all such combinations; those skilled in the art, particularly in view of the teaching contained herein, will readily be able to determine which combinations are most appropriate for which circumstances.

As will be clear from the following discussion, 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 part, relates to compositions and methods of use thereof of nucleic acid molecules and polypeptides. For simplicity, these nucleic acid molecules and polypeptides have been classified into different groups. NA Group 1 molecules represent (a) nucleic acid molecules coding for a cancer associated antigen precursor and which hybridize under stringent conditions to a molecule consisting of the nucleotide sequence of SEQ ID NO:4, (b) deletions, additions and substitutions which code for a respective cancer associated antigen precursor, and (c) complements of (a) or (b). NA Group 2 molecules represent fragments of NA Group 1 molecules which code 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 molecules represent (a) nucleic acid molecules which code for a cancer associated antigen precursor and 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:13-19, (b) deletions, additions and substitutions which code for a respective cancer associated antigen precursor, (c) complements of (a) or (b). NA Group 4 molecules represent fragments of NA Group 3 molecules 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. PP Group 1 polypeptides are polypeptides encoded by NA Group 1 molecules. PP Group 2 polypeptides are polypeptides encoded by NA Group 2 molecules. PP Group 3 polypeptides are polypeptides encoded by NA Group 3 molecules. PP Group 4 polypeptides are polypeptides encoded by NA Group 4 molecules. As used herein, nucleic acid molecules and polypeptides will be referred to by group. Where appropriate and stated, degenerate sequences of nucleic acid molecules are also embraced by the invention. Degenerate sequences or degenerates refer to nucleic acid molecules that differ from a nucleic acid molecule in codon sequence due to the degeneracy of the genetic code.

The invention, in one aspect, is a method of diagnosing a disorder characterized by expression of a preferably human cancer associated antigen precursor coded for by at least one nucleic acid molecule. The method involves the steps of contacting a biological sample isolated from a subject with an agent that specifically binds to a first nucleic acid molecule, a fragment of a first nucleic acid molecule, an expression product of a first nucleic acid molecule, or a fragment of an expression product of a first nucleic acid molecule complexed with an 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 first nucleic acid molecule, the fragment of the first nucleic acid molecule, the expression product or fragment of the expression product of the first nucleic acid molecule as a determination of the disorder.

In one embodiment, the agent is selected from the group consisting of (a) a nucleic acid molecule comprising a complement of the first nucleic acid molecule or a fragment thereof, (b) an antibody that binds to an expression product, or a fragment thereof, of the first nucleic acid molecule, and (c) an agent that binds to a complex of an MHC, preferably HLA, molecule and a fragment of an expression product of the first nucleic acid.

In another embodiment, the method further comprises the use of a second agent that specifically binds to a second nucleic acid molecule, a fragment of the second nucleic acid molecule, an expression product of the second nucleic acid molecule, or a fragment of the expression product of the second nucleic acid molecule complexed with an HLA molecule, wherein the second nucleic acid molecule is a NA Group 3 molecule. The method further involves determining the interaction between the second agent and the second nucleic acid molecule, the fragment of the second nucleic acid molecule, the expression product or fragment of the expression product of the second nucleic acid molecule as a determination of the disorder.

In a further embodiment, the second agent is selected from the group consisting of (a) a nucleic acid molecule comprising a complement of the second nucleic acid molecule or a fragment thereof, (b) an antibody that binds to an expression product, or a fragment thereof, of the second nucleic acid molecule, and (c) agent that binds to a complex of an MHC, preferably HLA, molecule and a fragment of an expression product of the second nucleic acid.

The disorder may be characterized by expression of a plurality of cancer associated antigen precursors. Thus the methods of diagnosis may include use of a plurality of agents, each of which is specific for a human cancer associated antigen precursor (including at least one of the cancer associated antigen precursors disclosed herein). The second nucleic acid molecule may be a plurality of different second nucleic acid molecules and the second agent may be a plurality of second agents each of which bind to a respective second nucleic acid molecule, a fragment of a respective second nucleic acid molecule, an expression product of a respective second nucleic acid molecule or a fragment of an expression product of a respective second nucleic acid molecule complexed with an HLA molecule. The plurality of agents may be at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7.

In another aspect the invention is a method for determining regression, progression or onset of a condition characterized by expression of abnormal levels of at least one protein. 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) a first protein encoded by a nucleic acid molecule which comprises a NA Group 1 molecule, (ii) a first peptide derived from the first protein, (iii) an antibody which selectively binds the first protein or the first peptide, and (iv) cytolytic T cells specific for a complex of the first peptide derived from the first protein and an MHC molecule, as a determination of regression, progression or onset of said condition. In one embodiment the sample is a body fluid, a body effusion or a tissue.

In another embodiment 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 first protein of (i), or the first peptide of (ii), (b) a protein or a peptide which binds the antibody of (iii), and (c) a cell which presents the complex of the first peptide and MHC molecule of (iv). In a preferred embodiment the antibody, the first protein, the first peptide or the cell is labeled with a detectable molecule, such as a radioactive label or an enzyme. The sample in a preferred embodiment is assayed for the first peptide.

According to another embodiment the protein is a plurality of proteins, at least one of the plurality being encoded by a second nucleic acid molecule comprising a NA Group 3 molecule and the at least one of the plurality is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7. In yet another embodiment, the parameter is a plurality of parameters, each of the plurality of parameters respective for one plurality of proteins.

The invention in another aspect is a pharmaceutical composition for a human subject. The pharmaceutical preparation includes an effective amount of an agent which when administered to the subject enriches selectively the presence of complexes of an HLA molecule and a human cancer associated antigen, and a pharmaceutically acceptable carrier, wherein the human cancer associated antigen is a fragment of a human cancer associated antigen precursor encoded by a nucleic acid molecule which comprises a NA Group 1 molecule. In one embodiment the nucleic acid molecule is a NA Group 3 molecule.

The agent in another embodiment comprises a plurality of agents, each of which enriches selectively in the subject complexes of an HLA molecule and a different human cancer associated antigen. Preferably the plurality is at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 different such agents, and at least one of the different human cancer associated antigens is encoded by a NA Group 3 molecule.

In another embodiment the agent is selected from the group consisting of (1) an isolated polypeptide comprising the human cancer associated antigen, or a functional variant thereof, (2) an isolated nucleic acid operably linked to a promoter for expressing the isolated polypeptide, or functional variant thereof, (3) a host cell expressing the isolated polypeptide, or functional variant thereof, and (4) isolated complexes of the polypeptide, or functional variants thereof, and an HLA molecule.

The agent may be a cell expressing an isolated polypeptide. In one embodiment the agent is a cell expressing an isolated polypeptide comprising the human cancer associated antigen or a functional variant thereof. In another embodiment the agent is a cell expressing an isolated polypeptide comprising the human cancer associated antigen 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. In preferred embodiments the cell is nonproliferative. In yet another embodiment the agent is a plurality of agents, and the plurality is at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 different polypeptides, each representing a different human cancer associated antigen or functional variant thereof.

The agent in one embodiment is a PP Group 1 polypeptide. In another embodiment, the agent is a PP Group 2 polypeptide. In yet other embodiments, the agent is a PP Group 3 polypeptide or a PP Group 4 polypeptide.

In an embodiment each of the pharmaceutical compositions described herein also includes an adjuvant.

According to another aspect the invention, a composition of matter is provided which includes an isolated agent that binds selectively to a PP Group 1 polypeptide. In separate embodiments the agent binds selectively to a polypeptide selected from the following: a PP Group 2 polypeptide, a PP Group 3 polypeptide and a PP Group 4 polypeptide. In other embodiments, the agent is a plurality of different agents that bind selectively at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 different such polypeptides. In each of the above described embodiments the agent may be an antibody. In a further embodiment, the agent is a therapeutic or a diagnostic agent.

In another aspect the invention is a composition of matter comprising a conjugate of the agent of the above-described compositions of the invention and a therapeutic or diagnostic agent. Preferably the conjugate is of the agent and a therapeutic or diagnostic agent that is a toxin.

In another aspect of the invention, pharmaceutical compositions are provided each comprising an effective amount of any one of the compositions of matter described above and a pharmaceutically acceptable carrier.

The invention in another aspect is a pharmaceutical composition comprising an effective amount of an isolated nucleic acid molecule selected from the group consisting of: (1) NA Group 1 molecules, (2) NA Group 2 molecules, and (3) degenerates of NA Group 1 and NA Group 2 molecules, and a pharmaceutically acceptable carrier. In one embodiment the composition further comprises at least two isolated nucleic acid molecules coding for two different polypeptides, each polypeptide comprising a different human cancer associated antigen, wherein at least one of the nucleic acid molecules is selected from the group consisting of a NA Group 3 molecule and a NA Group 4 molecule.

Preferably the pharmaceutical composition also includes an expression vector with a promoter operably linked to the isolated nucleic acid molecule. In another embodiment the pharmaceutical composition also includes a host cell recombinantly expressing the isolated nucleic acid molecule.

According to another aspect of the invention a pharmaceutical composition is provided that includes an effective amount of an isolated polypeptide comprising a PP Group 1 or a PP Group 2 polypeptide, and a pharmaceutically acceptable carrier. In one embodiment the isolated polypeptide further comprises a PP Group 3 or a PP Group 4 polypeptide.

In another embodiment the isolated polypeptide comprises at least two different polypeptides, each comprising a different preferably human cancer associated antigen. In separate embodiments the isolated polypeptides are selected from PP Group 3 polypeptides or HLA binding fragments thereof.

In an embodiment each of the pharmaceutical compositions described herein also includes an adjuvant.

Another aspect of 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.

According to another aspect of the invention isolated polypeptides encoded by the isolated nucleic acid molecules of the invention, described above, are provided. These include PP Group 1-4 polypeptides. In a preferred embodiment, a fragment of a PP Group 1 polypeptide is provided. The invention also includes a fragment of the polypeptide which is immunogenic. In one embodiment the fragment, or a portion of the fragment, binds HLA molecule or a human antibody.

The invention includes in another aspect an isolated fragment of a human cancer associated antigen precursor which, or portion of which, binds an HLA molecule or a human antibody, wherein the precursor is encoded by a nucleic acid molecule comprising a NA Group 1 molecule. In one embodiment the fragment is part of a complex with an HLA molecule. In another embodiment the fragment is between 8 and 12 amino acids in length. In another embodiment the invention includes an isolated polypeptide comprising a fragment of the polypeptide of sufficient length to represent a sequence unique within the human genome and identifying a polypeptide that is a human cancer associated antigen precursor.

According to another aspect of the invention a method for treating a subject with a disorder characterized by expression of a human cancer associated antigen precursor is provided. The method includes the step of administering to the subject an amount of an agent, which enriches selectively in the subject the presence of complexes of an HLA molecule and a human cancer associated antigen, effective to ameliorate the disorder, wherein the human cancer associated antigen is a fragment of a human cancer associated antigen precursor encoded by a nucleic acid molecule comprising a NA group 1 nucleic acid molecule.

In one embodiment the disorder is characterized by expression of a plurality of human cancer associated antigen precursors and the agent is a plurality of agents, each of which enriches selectively in the subject the presence of complexes of an HLA molecule and a different human cancer associated antigen wherein at least one of the human cancer associated antigens is encoded by a NA Group 3 molecule. Preferably the plurality is at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 such agents.

In another embodiment the agent is an isolated polypeptide selected from the group consisting of PP Group 1, PP Group 2, PP Group 3, and PP Group 4 polypeptides.

In yet another embodiment the disorder is cancer. In preferred embodiments, the disorder is lung cancer.

According to another aspect the invention is a method for treating a subject having a condition characterized by expression of at least one human 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 a host cell under conditions favoring production of cytolytic T cells against a human 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 human cancer associated antigen, wherein the host cell is transformed or transfected with an expression vector comprising an isolated nucleic acid molecule, or human cancer associated antiagen encoding fragment thereof, operably linked to a promoter. The isolated nucleic acid molecule is selected from the group of nucleic acid molecules consisting of NA Group 1, NA Group 2, NA Group 3, and NA Group 4 nucleic acid molecules. In preferred embodiments, the isolated nucleic acid molecule is a NA Group 1 nucleic acid molecule.

In one embodiment the host cell recombinantly expresses an HLA molecule which binds the human cancer associated antigen. In another embodiment the host cell endogenously expresses an HLA molecule which binds the human cancer associated antigen.

The invention includes in another aspect a method for treating a subject having a condition characterized by expression of at least one 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. Preferably, the antigen is a human antigen and the subject is a human.

In one embodiment the method also includes the step of (a) identifying an 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 an MHC binding portion of the expression product of the nucleic acid molecule.

In another embodiment the method also includes the step of treating the host cells to render them non-proliferative.

In yet another embodiment the immune response comprises a B-cell response or a T cell response. Preferably 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 human cancer associated antigen.

In another embodiment the method comprises identifying a second nucleic acid molecule expressed by the cells associated with the condition, wherein the second nucleic acid molecule comprises 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 or an agent which specifically binds to the protein or a peptide derived therefrom, the antibody optimally being coupled to a therapeutically useful agent, in an amount effective to treat the condition.

In one embodiment the antibody is a monoclonal antibody. Preferably the monoclonal antibody is a chimeric antibody or a humanized antibody.

In another aspect 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 comprising a NA Group 1 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. In one embodiment the condition is cancer. In another embodiment 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.

In one embodiment the method includes the step of rendering the cells non-proliferative, prior to introducing them to the subject.

In another aspect the invention is a method for treating a pathological condition characterized by aberrant expression of a protein encoded by a nucleic acid molecule comprising a NA Group 1 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.

In one embodiment 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. In another embodiment the agent is an antisense nucleic acid molecule which selectively binds to the nucleic acid molecule which encodes the protein. In yet another important embodiment the nucleic acid molecule further comprises a NA Group 3 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 and optionally NA Group 3 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 or antibody response thereto. In another embodiment the composition of matter includes an adjuvant and/or a costimulatory molecule. In another embodiment the adjuvant is a saponin, GM-CSF, or an interleukin. In still another embodiment, 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.

According to another aspect 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 which binds the peptide to form the complex, wherein the isolated antibody does not bind to (i) or (ii) alone.

In one embodiment 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 and a more particular medicament is for treating seminomas, melanomas, teratomas, gliomas, lung cancer, ovarian cancer and/or colorectal cancer.

Sequence Listing

SEQ ID NO:1 is the nucleotide sequence of human RING3 cDNA.

SEQ ID NO:2 is the nucleotide sequence of human KIAA0043 cDNA.

SEQ ID NO:3 is the nucleotide sequence of human HUNK1 cDNA.

SEQ ID NO:4 is the nucleotide sequence of human BRDT cDNA.

SEQ ID NO:5 is the nucleotide sequence of the forward primer for BRDT.

SEQ ID NO:6 is the nucleotide sequence of the reverse primer for BRDT.

SEQ ID NO:7 is the nucleotide sequence of the forward primer for RING3.

SEQ ID NO:8 is the nucleotide sequence of the reverse primer for RING3.

SEQ ID NO:9 is the nucleotide sequence of the forward primer for KIAA0043.

SEQ ID NO:10 is the nucleotide sequence of the reverse primer for KIAA0043.

SEQ ID NO:11 is the nucleotide sequence of the forward primer for HUNK1.

SEQ ID NO:12 is the nucleotide sequence of the reverse primer for HUNK1.

SEQ ID NO:13 is the nucleotide sequence of human SCP-1 cDNA.

SEQ ID NO:14 is the nucleotide sequence of human NY-ESO-1 cDNA.

SEQ ID NO:15 is the nucleotide sequence of human CT7 cDNA.

SEQ ID NO:16 is the nucleotide sequence of human SSX2 cDNA.

SEQ ID NO:17 is the nucleotide sequence of human SSX4 cDNA.

SEQ ID NO:18 is the nucleotide sequence of human MAGEA1 cDNA.

SEQ ID NO:19 is the nucleotide sequence of human MAGEA3 cDNA.

SEQ ID NO:20 is the amino acid sequence of human RING3 protein.

SEQ ID NO:21 is the amino acid sequence of human KIAA0043 protein.

SEQ ID NO:22 is the amino acid sequence of human HUNK1 protein.

SEQ ID NO:23 is the amino acid sequence of human BRDT protein.

SEQ ID NO:24 is the amino acid sequence of human SCP-1 protein.

SEQ ID NO:25 is the amino acid sequence of human NY-ESO-1 protein.

SEQ ID NO:26 is the amino acid sequence of human CT7 protein.

SEQ ID NO:27 is the amino acid sequence of human SSX2 protein.

SEQ ID NO:28 is the amino acid sequence of human SSX4 protein.

SEQ ID NO:29 is the amino acid sequence of human MAGE-A1 protein.

SEQ ID NO:30 is the amino acid sequence of human MAGE-A3 protein.

Genbank accession numbers for cDNA sequences listing above are as follows: RING3 is X96670, KIAA0043 is D26362, HUNK1 is Y12059, BRDT is AF019085, SCP-1 is D67035, NY-ESO-1 is U87459, CT7 is AF056334, SSX2 is X86175, SSX4 is U90841, MAGE-A1 is NM—004988, MAGE-3A is NM — 005362.

DETAILED DESCRIPTION OF THE INVENTION

A previously identified gene, bromo domain testis-specific gene product (BRDT), has been cloned and molecularly characterized as a cancer-testis antigen (CT9). This cancer associated antigen was identified by its homology to another gene, RING3, isolated by SEREX analysis of the serum of a breast cancer patient. The potential oncogenic function of BRDT, and/or its function as a marker of cancer cells, and the function of the other cancer associated antigens can be inhibited by specific immunological and genetic therapeutic interventions.

In the above summary and in the ensuing description, lists of sequences are provided. The lists are meant to embrace each single sequence separately, two or more sequences together where they form a part of the same gene, any combination of two or more sequences which relate to different genes, including and up to the total number on the list, as if each and every combination were separately and specifically enumerated. Likewise, when mentioning fragment size, it is intended that a range embrace the smallest fragment mentioned to the full-length of the sequence (less one nucleotide or amino acid so that it is a fragment), each and every fragment length intended as if specifically enumerated. Thus, if a fragment could be between 10 and 15 in length, it is explicitly meant to mean 10, 11, 12, 13, 14, or 15 in length.

The summary and the claims mention antigen precursors and antigens. As used in the summary and in the claims, a precursor is 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. Such antigens are typically 9 amino acids long (for HLA class II molecules), although this may vary slightly.

The nucleic acid molecules described herein preferably are isolated. As used herein with respect to nucleic acids, the term “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. Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, 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.

The polypeptides described herein also preferably are isolated. As used herein with respect to polypeptides, “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.

As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, rodent or other verterinary animal. In all embodiments human cancer antigens and human subjects are preferred.

The present invention in one aspect involves the identification of a cDNA encoding RING3 as a human cancer associated antigen precursor using autologous antisera of subjects having breast cancer. In another aspect, the invention involves the identification of a RING3 homolog, BRDT, as a cancer-testis antigen. The nature of the BRDT gene as encoding a CT antigen is, of course, unexpected.

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. Preferably the cancer associated antigens are cancer-testis antigens as described herein.

Homologs and alleles of the cancer associated antigen nucleic acids of the invention can be identified by conventional techniques. Thus, an aspect of the invention is those nucleic acid sequences which code for cancer associated antigen precursors. Because this application contains several nucleotide and amino acid sequences, the following chart is provided to identify the various groups of sequences discussed in the claims and in the summary:

Nucleic Acid Sequences

NA Group 1. (a) nucleic acid molecules coding for a cancer associated antigen precursor and which hybridize under stringent conditions to a molecule consisting of the nucleotide sequence of SEQ ID NO:4,

(b) deletions, additions and substitutions which code for a respective cancer associated antigen precursor, and

(c) complements of (a) or (b).

NA Group 2. Fragments of NA Group 1, which code 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. (a) nucleic acid molecules which code for a cancer associated antigen precursor and 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:13-19,

(b) deletions, additions and substitutions which code for a respective cancer associated antigen precursor,

(c) complements of (a) or (b).

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.

Polypeptide Sequences

PP Group 1. Polypeptides encoded by NA Group 1.

PP Group 2. Polypeptides encoded by NA Group 2

PP Group 3. Polypeptides encoded by NA Group 3.

PP Group 4. Polypeptides encoded by NA Group 4

The term “stringent conditions” as used herein 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, N.Y., 1989, or Current Protocols in Molecular Biology , F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH 2 PO 4 (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, the membrane upon which the DNA is transferred is washed, for example, in 2×SSC at room temperature and then at 0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of cancer associated antigen nucleic acids of the invention (e.g., by using lower stringency conditions). The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to the sequences of cancer associated antigen nucleic acid and polypeptides, respectively, in some instances will share at least 50% nucleotide identity and/or at least 65% amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75% amino acid identity. Preferably the homologs and alleles will share at least 80% nucleotide identity and/or at least 90% amino acid identity, and more preferably will share at least 90% nucleotide identity and/or at least 95% 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, used with default settings. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained, for example, using the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.

In screening for cancer associated antigen genes, 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. In screening for the expression of cancer associated antigen nucleic acids, Northern blot hybridizations using the foregoing conditions (see also the Examples) 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.

The cancer associated genes correspond to NA Groups 1 and 3 The preferred cancer associated antigens for the methods of diagnosis disclosed herein are BRDT (SEQ ID NO:4) and previously identified cancer-testis antigens (SEQ ID NOs:13-19), each of which may be used alone or in combination. Encoded polypeptides (e.g., proteins), peptides and antisera thereto are also preferred for diagnosis.

The invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, 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. Thus, it will be apparent to one of ordinary skill in the art that 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. Similarly, 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. Thus, 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. In preferred embodiments, 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. In certain embodiments, 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.

For example, 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. In the foregoing example, 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.

Fragments of the cancer-testis antigen nucleic acids 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 polypeptide fragments, or for generating immunoassay components. Likewise, fragments can be employed to produce nonfused fragments of the cancer-testis 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-testis nucleic acids and polypeptides, particularly for therapeutic purposes.

Especially preferred nucleic acid molecules 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.

Thus, for example, peptides derived from a polypeptide having an amino acid sequence encoded by one of the nucleic acid molecules 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 (which are presented by MHC class I or II) that can be administered to induce or enhance an immune response are derived from tumor associated genes and encoded proteins including MAGE-A2, 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, LAGE-1, SSX-1, and SSX-5. See, for example, PCT application publication no. WO96/10577. Other examples will be known to one of ordinary skill in the art (for example, see Coulie, Stem Cells 13:393-403, 1995), and can be used in the invention in a like manner as those disclosed herein. One of ordinary skill in the art can prepare polypeptides comprising one or more peptides of the invention and one or more of the foregoing known cancer associated peptides, or nucleic acids encoding such polypeptides, according to standard procedures of molecular biology.

Thus 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 Biolechnol . 15(12):1280-1284, 1997; Thomson et al., J. Immunol . 157(2):822-826, 1996; Tam et al., J. Exp. Med . 171(1):299-306, 1990). For example, Tam showed that polytopes consisting of both MHC class I and class II binding epitopes successfully generated antibody and protective immunity in a mouse model. Tam 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.

It is known that 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, for example, as described in the Examples below. 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. One can test the polytope delivery systems in mouse models to determine efficacy of the delivery system. The systems also can be tested in human clinical trials.

In instances in which a human HLA molecule presents tumor rejection antigens derived from cancer associated nucleic acids, the expression vector may also include a nucleic acid sequence coding for the HLA molecule that presents any particular tumor rejection antigen derived from these nucleic acids and polypeptides. Alternatively, the nucleic acid sequence coding for such a HLA molecule can be contained within a separate expression vector. In a situation where the vector contains both coding sequences, the single vector can be used to transfect a cell which does not normally express either one. Where the coding sequences for a cancer associated antigen precursor and the HLA molecule which presents it are contained on separate expression vectors, 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 HLA molecule which presents a cancer associated antigen derived from precursor molecules. Of course, there is no limit on the particular host cell which can be used. As 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 HLA molecule which presents a cancer associated antigen. Further, cell-free transcription systems may be used in lieu of cells.

As used herein, 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. In the case of plasmids, 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., β-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.

As used herein, 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. If it is desired that the coding sequences be translated into a functional protein, 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. Thus, 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.

The precise nature of the 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. Especially, such 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.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a cancer associated antigen polypeptide or fragment or variant thereof. 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 pRc/CMV (available from Invitrogen, Carlsbad, Calif.) 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. Additionally, 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. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor 1α, 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 E1 and E3 proteins ( J. Clin. Invest . 90:626-630, 1992). The use of the adenovirus as an Adeno.P1A recombinant for the expression of an antigen is disclosed by Warnier et al., in intradermal injection in mice for immunization against P1A ( 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. The invention also includes 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 also permits the construction of cancer associated antigen gene “knock-outs” and stable or transient transgenic expression in cells and in animals, providing materials for studying certain aspects of cancer and immune system responses to cancer.

As mentioned above, 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, preferably cancer-testis 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.

As used herein, the term “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. Those skilled in the art will recognize that 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 antigen, 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. In order to be sufficiently selective and potent for inhibition, such 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.

Although 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 . 14(5):439-457, 1994) and at which proteins are not expected to bind. Finally, although 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. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to nucleic acids encoding cancer associated antigens. Similarly, antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of the invention may be 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 intemucleoside 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.

In preferred embodiments, however, 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.

The term “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. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, 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. Thus modified oligonucleotides may include a 2′-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, 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.

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art, as further described below.

The invention also provides isolated polypeptides (including whole proteins and partial proteins, i.e., fragments) 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 preferably are 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 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.

Fragments of a polypeptide preferably are those fragments which retain a distinct functional capability of the polypeptide. Functional capabilities which can be retained in a fragment of a polypeptide include interaction with antibodies, interaction with other polypeptides or fragments thereof, selective binding of nucleic acids or proteins, and enzymatic actyivity. One important activity is the ability to complex with HLA and to provoke in a human an immune response.

The invention embraces variants of the cancer associated antigen polypeptides described above. As used herein, 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 an HLA 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. One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus “design” a variant cancer associated antigen polypeptide according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary a only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a cancer associated antigen polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.

In general, variants include cancer associated antigen polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its desired physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, 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. Still other mutations can be made to the noncoding sequences of a cancer associated antigen gene or cDNA clone to enhance expression of the polypeptide. The activity of 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. For example, the variant cancer associated antigen polypeptide can be tested for reaction with autologous or allogeneic sera as disclosed in the Examples. Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acid substitutions may be made in cancer associated antigen polypeptides to provide functional variants of the foregoing polypeptides, i.e., variants that retain the functional capabilities of the cancer associated antigen polypeptides such as stimulation of an immune response, binding to HLA molecules, etc. As used herein, 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. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology , F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functional 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.

For example, upon determining that a peptide derived from a cancer associated antigen polypeptide is presented by an MHC molecule and recognized by CTLs (e.g., in a subject or tumor type, for making polyvalent vaccines, as described in the Examples), 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. For example, 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 computer program described by D'Amaro and Drijfhout (D'Amaro et al., Human Immunol . 43:13-18, 1995; Drijfhout et al., Human Immunol . 43:1-12, 1995). The substituted peptides can then be tested for binding to the MHC molecule and recognition by CTLs when bound to MHC. These variants can be tested for improved stability and are useful, inter alia, in vaccine compositions.

Conservative amino-acid substitutions in the amino acid sequence of cancer associated antigen polypeptides to produce functional variants of cancer associated antigen polypeptides typically are made by alteration of a nucleic acid encoding a cancer associated antigen polypeptide. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, 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. Where amino acid 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 as described herein has a number of uses, some of which are described elsewhere herein. First, the invention permits treatment of disorders characterized by expression of the cancer associated antigen nucleic acid or protein molecules, preferably by stimulation of an immune response which recognizes fragments of the polypeptides presented by HLA molecules. A variety of methodologies well-known to the skilled practitioner can be utilized to stimulate our immune response against the cancer associated antigen molecules. Immunological recognition can be stimulated by administering to a subject the cancer associated antigen polypeptides or fragments thereof. Alternatively, an expression vector may be introduced into cells to cause production of the polypeptide. Those skilled in the art also can readily follow known methods for isolating cancer associated antigen polypeptides. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.

The isolation and identification of 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 involves agents such as polypeptides which bind to cancer associated antigen polypeptides. Such 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. Such 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 Modern Immunology Wiley & Sons, Inc., New York; Roitt, 1. (1991) Essential Immunology , 7th Ed., Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, designated an F(ab′) 2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, 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. Proceeding further, 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 Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the 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. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

Thus, for example, 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.

Thus, as will be apparent to one of ordinary skill in the art, 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 CDR1 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 CDR1 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 CDR1 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 CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

Thus, the invention involves 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. These polypeptides may be derived also from sources other than antibody technology. For example, such 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. ml3, fd, or lambda phage), displaying 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. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the cancer associated antigen polypeptides. Thus, 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.

As detailed herein, the foregoing 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. As used herein, “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 are well known and include: aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl, 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.

In the foregoing methods, antibodies prepared according to the invention also preferably are specific for the cancer associated antigen/MHC complexes described herein.

When “disorder” is used herein, it refers to any pathological condition where the cancer associated antigens are expressed. An example of such a disorder is cancer, with lung cancers as particular examples.

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.

In certain embodiments of the invention, an immunoreactive cell sample is removed from a subject. By “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. Thus immunoreactive cells include CD34 + hematopoietic stem cells, immature T cells and immature B cells. 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. Generally, sample of cells taken from a subject, such as blood cells, are contacted with a cell presenting he complex and capable of provoking CTLs to proliferate (e.g., dendritic cells). The target ell, such as a dendritic cell, can be a transfectant. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation. Other suitable host cells, such as COS cells, are widely available. Specific production of CTL clones is well known in the art. The clonally expanded autologous CTLs then are administered to the subject.

Another method for selecting antigen-specific CTL clones has recently been described (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol . 8:413-416, 1998), in which fluorogenic tetramers of MHC class I molecule/peptide complexes are used to detect specific CTL clones. Briefly, soluble MHC class I molecules are folded in vitro in the presence of β 2 -microglobulin and a peptide antigen which binds the class I molecule. After purification, the MHC/peptide complex is purified and labeled with biotin. 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.

To detail a therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol . 136(5): 1917, 1986; Riddel et al., Science 257: 238, 1992; Lynch et al, Eur. J Immunol . 21: 1403-1410,1991; Kast et al., Cell 59: 603-614, 1989), cells presenting the desired complex (e.g., dendritic cells) are combined with CTLs leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs then lyse the abnormal cells, thereby achieving the desired therapeutic goal.

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, particularly the CT antigens described herein. 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 supra.

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 HLA molecule). Chen et al. ( Proc. Natl. Acad Sci. USA 88: 110-114,1991) exemplifies this approach, showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime. Various cell types may be used. Similarly, vectors carrying one or both of the genes of interest may be used. Viral or bacterial vectors are especially preferred. For example, 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. In these systems, the gene of interest is carried by a microorganism, e.g., a Vaccinia virus, pox virus, herpes simplex virus, retrovirus or adenovirus, and the materials de facto “infect” host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate.

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 HLA molecule while a cancer associated antigen peptide may be presented without the need for further processing. Generally, 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”. As used herein, “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 can be used for testing of immunization against cancer using a cancer associated antigen nucleic acid. For example, 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 (e.g., reduction of tumor size) can be assessed as a measure of the effectiveness of the cancer associated antigen nucleic acid immunization. Of course, 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.

As part of the immunization compositions, 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. Specific examples of 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. Cells 7:178-186, 1997); incomplete Freund's adjuvant; complete Freund's adjuvant; montanide; and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. Preferably, the peptides are administered and 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. Typically for human administration, DQS21 and MPL will be present in a vaccine formulation in the range of about 1 μg to about 100 μg. 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. Many other 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. Thus cytokines can be administered in conjunction with antigens and adjuvants to increase the immune response to the antigens.

There are a number of immune response potentiating compounds that can be used in vaccination protocols. These include costimulatory molecules provided in either protein or nucleic acid form. Such 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. 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 (CD152) 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/IL-12 costimulation has been shown to induce IFN-gamma and a Th1 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 been discussed in relation to in vitro CTL expansion for adoptive transfer immunotherapy by Wang et al., ( J. Immunol ., 19:1-8 (1986)). Other 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)). 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. The use of anti-CD28 antibodies to directly stimulate T cells in vitro and in vivo could also be considered. Similarly, the inducible co-stimulatory molecule ICOS which induces T cell responses to foreign antigen could be modulated, for example, by use of anti-1COS antibodies (Hutloff et al., Nature 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 IFN-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 IFN-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.

Complete CTL activation and effector function requires 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.

The use of 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. Other methods for inducing maturation of dendritic cells, e.g., by increasing CD40-CD40L interaction, or by contacting DCs with CpG-containing oligodeoxynucleotides or stimulatory sugar moieties from extracellular matrix, are known in the art.

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.

It will also be recognized that 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). Especially useful are 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, keratinocytes, peripheral blood leukocytes, bone marrow stem cells and embryonic stem cells. The expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.

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. One example of such a procedure is outlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo nucleic acid delivery using vectors such as viruses and targeted liposomes also is contemplated according to the invention.

In preferred embodiments, a virus vector for delivering a nucleic acid encoding a cancer associated antigen is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle. Examples of viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenoviruses (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 retrovirus (Townsend et al., J. Virol . 71:3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J. Virol . 68:5036-5044, 1994), a replication defective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995), canarypox virus and highly attenuated vaccinia virus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol. Stand . 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis et al., J. Virol . 70:3781-3787, 1996), Sindbis virus (Pugachev et al., Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al., Eur. J. Immunol 26:1951-1959, 1996). In preferred embodiments, the virus vector is an adenovirus.

Another preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus 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 virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Adenoviruses and retroviruses have been approved for human gene therapy trials. In general, the retroviruses are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses 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. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

Preferably the foregoing 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.

Various techniques may be employed for introducing nucleic acids of the invention into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host.

Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid of interest, liposome mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid to particular cells. In such instances, a vehicle used for delivering a nucleic acid of the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, 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. Especially preferred are monoclonal antibodies. Where liposomes are employed to deliver the nucleic acids of the invention, 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 internalization 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.

When administered, the therapeutic 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. When antibodies are used therapeutically, 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. In the case of treating a particular disease or condition characterized by expression of one or more cancer associated antigens, such as seminoma, 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.

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 practioner. 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.

The pharmaceutical 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.

The doses of cancer associated antigen compositions (e.g., polypeptide, peptide, antibody, cell or nucleic acid) administered to a subject 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.

In general, for treatments for eliciting or increasing an immune response, doses of cancer associated antigen are formulated and administered in doses between 1 ng and 1 mg, and preferably between 10 ng and 100 μg, according to any standard procedure in the art. Where nucleic acids encoding cancer associated antigen of variants thereof are employed, 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. Administration of cancer associated antigen compositions to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.

Where 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. For administration of a cancer associated antigen peptide in adjuvant, 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.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term “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. 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. Also, 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. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “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 (not preferred for pediatric applications).

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 syrup, 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, 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.

EXAMPLES

Materials and Methods

Sera, tissues and cell lines. Sera were obtained during routine diagnostic or therapeutic procedures. Sera were stored at −80° C. until use. Tumor specimens were obtained from the Departments of Pathology at the Memorial Sloan-Kettering Cancer Center and New York Hospital-Comell Medical Center. The lung cancer cell lines used in this study were from the cell bank of the Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center.

Reverse transcribed PCR. Total RNA from tumor specimens and cell lines was prepared by the guanidinium thiocyanate method. Total RNA from various normal tissues was obtained commercially (Clontech Laboratories, Inc. Palo Alto, Calif.).

The mRNA expression patterns of RING3 (SEQ ID NO:1), KIAA0043 (SEQ ID NO:2), HUNK1 (SEQ ID NO:3) and BRDT (SEQ ID NO:4) were determined by RT-PCR using a panel of normal tissue RNA. The initial test panel consisted of RNA from lung, testis, small intestine, breast, liver, brain and placenta. In the case of BRDT expression, RNA samples from various tumor specimens, tumor cell lines and additional normal tissues (spleen, kidney, skeletal muscle pancreas, adrenal gland, heart, thymus, uterus and prostate) were also analyzed by RT-PCR. The cDNA preparations used in the RT-PCR reactions were synthesized by incubating total RNA template (2 μg), random hexamers (1.66 μg, Boehringer Mannheim, Indianapolis, Ind.) and MuLV reverse transcriptase (200U, Gibco BRL), in a total reaction volume of 25 μl, at 42° C. for 1 hour. MuLV synthesized cDNA (2.5 μl/PCR reaction) was them amplified using gene specific primers (0.2 μM and AmpliTaq Gold DNA polymersase (2.5 U, PE Applied Biosystems, Branchburg, N.J.) in 25 μl PCR reactions consisting of 30 cycles at a denaturation temperature of 94° C. (1 min/cycle); an annealing temperature of 60° C. (1 min/cycle); and an extension temperature of 72° C. (2 min/cycle). As a control for genomic DNA contamination in the RT-PCR reactions, duplicate cDNA templates were prepared as above in the absence of MuLV reverse transcriptase and used in equivalent PCR reactions. In addition to RT-PCR product identification on the basis of size, selected products were subcloned into pCR2.1 (Invitrogen, San Diego, Calif.) and multiple clones were subjected to automated DNA sequencing (Cornell University DNA services, Ithaca, N.Y.). The gene specific amplification primers used in RT-PCR reactions of RING3 gene family members are as follows:

BDRT Forward 5′ TCAAGAAAGGCACTCAACAG (bp 544; SEQ ID NO:5);

BRDT Reverse 5′ TTCACTACTTGCTTTAACTGC (bp 797; SEQ ID NO:6);

RING3 Forward 5′ GGGAAGGGAATGCAGGGTTG (bp 1074; SEQ ID NO:7);

RING3 Reverse 5′ TGGCTGTTCTTAGGGATGGTC (bp 1623; SEQ ID NO:8);

KIAA0043 Forward 5′ TAATGGCCCAAGCTTTAGAG (bp 510; SEQ ID NO:9);

KIAA0043 Reverse 5′ GTTTGCAGTGATGGTTGG TAC (bp 760; SEQ ID NO:10);

HUNK1 Forward 5′AGCACCCAGCACCAGAGAAG (bp 1241; SEQ ID NO:11); and

HUNK1 Reverse 5′ TGCTGCTGTCGCTGGATGAG (bp 1690; SEQ ID NO:12). Gene specific primers for a panel of CT antigens including SCP-1 (SEQ ID NO:13), NY-ESO-1(SEQ ID NO:14), CT7 (SEQ ID NO:15), HOM-MEL-40/SSX2 (SEQ ID NO:16), SSX4 (SEQ ID NO:17), MAGE-A1 (SEQ ID NO:18) and MAGE-A3 (SEQ ID NO:19), were prepared according to published sequences.

Nuclease protection assays. A 32 P labeled singled-stranded antisense DNA probe corresponding to nucleotides 544-797 of BRDT was prepared using the Prime-A-Probe kit as per the manufacturer's protocol (Ambion, Austin Tex.). Total RNA (20 μg) preparations from various normal tissues (testis, liver, prostate, placenta, spleen, kidney, mammary gland), 2 lung cancer specimens, normal lung tissue from the same patients, and the SK-LC-5 lung cancer cell line were hybridized with 2.0×10 5 cpm BRDT probe (specific activity=5×10 9 cpm/pg) at 44° C. for 16 hours. Samples were then digested with 10 U of S1 nuclease at 37° C. for 1 hour and electrophoresed on a 8M urea/5%polyacrylamide/TBE gel. The protected fragments were visualized by autoradiography.

Immunoscreening of transfectants. Sera (diluted 1:500) from cancer patients were used to immunoscreen for clones reactive with IgG antibodies as described in detail previously (Sahin, et al., Proc. Natl. Acad. Sci. USA , 92:11810-11813, 1995; Türeci et al., Cancer Res . 56:4766-4772, 1996).

Example 1

Identification of BRDT as a Cancer-testis Antigen

SEREX analysis of human breast cancer identified the RING3 gene product as an antigen that reacts with autologous antibody. RING3 was originally defined as the human homologue to the Drosophila female sterile homeotic (fsh) protein. It is a nuclear serine-threonine kinase containing 2 copies of the bromo domain, a motif of approximately 100 amino acids found in several mammalian, invertebrate and yeast transcriptional regulators. Preliminary expression analysis based on the tissue distribution of identical expressed sequence tags (ESTs) indicates that RING3 is expressed in a wide range of normal tissues. A database search of proteins displaying a high degree of similarity to RING3 revealed a testis specific transcript, termed the bromo domain testis-specific gene product (BRDT), which shares 52% amino acid identity with RING3. BRDT is a putative nuclear protein of 947 amino acids (108 kDa), also containing two copies of the bromo domain. The region in BRDT with similarity to serine/threonine kinase domains, is lacking in essential subdomains required for catalytic activity indicating that it does not function as a kinase. Initial characterization studies reported RNA expression of BRDT to be restricted to testis. In addition to BRDT, two uncharacterized members of the human RING3 gene family have also been identified, KIAA0043 and HUNKI (unpublished GenBank submission Y12059). An analysis of identical EST sequences showed that expression of KIAA0043 and HUNKI transcripts is not tissue restricted.

The current study examined the mRNA expression pattern of BRDT in normal and malignant tissues to determine if it has an expression profile similar to CT antigens. In addition, the co-expression patterns of various CT antigens and BRDT were evaluated in a series of lung cancer specimens.

As shown in Table 1, seventeen different normal tissue RNA samples were analyzed for BRDT expression by RT-PCR. A BRDT/CT9 specific fragment of 250 bp was obtained using BRDT/CT9 gene specific primers. Only testis and placenta were positive for BRDT mRNA expression. Subsequent RT-PCR expression analysis of BRDT in tumor tissue (Table 1) revealed expression in 12 of 47 (25%) non-small cell lung cancer specimens (NSCLC), 1 of 12 (8%) esophageal cancer specimens and in 1 of 12 (8%) squamous cell carcinomas of the head and neck. BRDT mRNA was not detected in melanoma (12 cases), colon cancer (6 cases), breast cancer (18 cases), renal cancer (6 cases), and bladder cancer (12 cases). Also, it was not detected in available normal lung tissue (3 cases) from BRDT positive lung cancer patients. The PCR products obtained from testis and lung cancer were subcloned and subjected to DNA sequencing. The resultant DNA sequences were identical to BRDT. Thus, this cancer/testis (placenta) expression profile of BRDT was similar to the expression profile of CT antigens and accordingly, it was given the CT designation CT9. BRDT transcript expression was also analyzed in fifteen lung cancer cell lines (Table 1). Only the SK-LC-5 cell line demonstrated BRDT expression by RT-PCR. Expression of RING3, KIAA0043 and HUNKI mRNA was also determined by RT-PCR and was found to be universal in a panel consisting of normal lung, testis, small intestine, breast, liver, brain and placenta RNA.

Table 1. Expression BRDT/CT9 mRNA in various normal human tissues, tumor tissue and lung cancer cell lines as determined by RT-PCR.

Normal Tissue     RT-PCR
Testis            +
Breast            Neg.
Liver             Neg.
Sm. Intest.       Neg.
Brain             Neg.
Lung              Neg.
Fetal Brain       Neg.
Placenta          +
Spleen            Neg.
Kidney            Neg.
Skel. Muscle      Neg.
Pancreas          Neg.
Adrenal           Neg.
Heart             Neg.
Thymus            Neg.
Uterus            Neg.
Prostate          Neg.
Tumor Tissue
Lung Cancer       12/47
Colon Cancer      0/6
Melanoma          0/12
Breast Cancer     0/18
SQCC of Head      1/12
and Neck
Bladder Cancer    0/13
Esophageal Cancer 1/12
Renal Cancer      0/6
Lung Tumor Cell Lines
SK-LC-1           Neg.
SK-LC-2           Neg.
SK-LC-3           Neg.
SK-LC-5           +
SK-LC-6           Neg.
SK-LC-7           Neg.
SK-LC-8           Neg.
SK-LC-14          Neg.
SK-LC-17          Neg.
SW1271            Neg.
CaLu-1            Neg.
CaLu-3            Neg.
KNS-62            Neg.
NCI-H740          Neg.

Example 2

Analysis of BRDT Expression by S1 Nuclease Assay

S1 nuclease protection assays were performed to confirm the RT-PCR results of BRDT mRNA expression. A BRDT protected fragment of 250 bases is present only in normal testis, 2 lung cancer specimens and the SK-LC-5 cell line. S1 nuclease failed to detect BRDT expression in normal liver, prostate, placenta, spleen, kidney, mammary gland and normal lung tissue from lung cancer patients whose tumors were BRDT positive. The ability to detect weak BRDT expression in placenta by RT-PCR, but not by S1 nuclease protection, probably reflects the ability of RT-PCR to detect trace levels of mRNA. In this regard, the restricted tissue distribution of BRDT/CT-9 mRNA is not absolute. Under exhaustive RT-PCR conditions of 35 amplification cycles, weak expression, as determined by presence of faint signals of appropriate size fragments, were obtained in some normal issues and other tumor types. Normal tissues that showed weak BRDT mRNA expression at 35 PCR cycles failed to yield corresponding BRDT protected fragments in S1 nucleus protection assays. Thus, it was concluded that weak BRDT expression at 35 PCR cycles likely represents leaky expression of questionable biological significance.

Example 3

Expression of BRDT in Lung Cancer

Table 2 shows BRDT mRNA expression as a function of the histological type of lung cancer. Of the twelve BRDT positive lung cancers, the majority were moderately differentiated adenocarcinomas (42%) and undifferentiated large cell carcinomas (33%). Among the eleven undifferentiated large cell carcinomas tested, 36% (4/11) were positive for expression of BRDT transcripts. Of the 17 moderately differentiated adenocarcinomas, 29% (5/17) showed expression of this transcript. Single cases of squamous cell carcinoma (1/6), poorly differentiated adenocarcinoma (1/8) and adenocarcinoma of the bronchioloalveolar type (1/2) were positive for BRDT transcript expression. SK-LC-5, the only lung cancer cell line that tested positive for BRDT expression (Table 1), was derived from a large cell carcinoma.

Table 2. BRDT/CT9 mRNA expression in lung cancer as a function of the histological type

Lung Cancer Type                 RT-PCR Positive Tumors
Undifferentiated Large Cell Carcinoma 4/11
Squamous Cell Carcinoma          1/6
Poorly Differentiated Adenocarcinoma 1/8
Moderately Differentiated Adenocarcinoma 5/17
Well Differentiated Adenocarcinoma 0/1
Adenocarcinoma (Papilary)        0/1
Adenocarcinoma (Bronchioloalveolar) 1/2
Poorly Differentiated Carcinosarcoma 0/1

Example 4

Expression of Multiple CT Antigens in Lung Cancer

In order to place BRDT mRNA expression in the context of general CT antigen expression, a series of 33 lung cancer specimens were co-typed for expression of MAGE-A1, MAGE-A3, HOM-MEL-40/SSX-2, SSX-4, NY-ESO-1, SCP-1, CT7 and BRDT/CT9 transcripts by RT-PCR. As shown in Table 3, twenty-two of thirty-three lung cancer specimens (67%) showed expression of at least 1 of the transcripts in this panel. MAGE-3 transcripts were detected most often (60% of the specimens), followed by MAGE-1 (36% of the specimens), CT7 (30%), SSX4 (23%), BRDT (21%), NY-ESO-1 (21%), HOM-MEL-40/SSX2 (15%) and SCP-1 (3%). Of the twenty-two lung cancers that tested positive for transcripts encoding CT antigens, twenty were positive for MAGE-A3. The two exceptions were a specimen that expressed only MAGE-A1 transcripts and another that expressed both BRDT and SSX-4 transcripts. Of the eleven lung cancer specimens that did not express transcripts encoding any of the nine CT antigens analyzed, nine were adenocarcinomas of various states of differentiation, one was a squamous cell carcinoma and one was a carcinosarcoma. The staining intensities of the resultant PCR products varied from weak to intense (+ to +++, Table 3), likely reflecting the mRNA abundance level. In general, strong signals were associated with MAGE-A3, MAGE-A1, NY-ESO-1 and BRDT transcripts. The very faint RT-PCR signals (Table 3, designated Neg.*) obtained from certain tumor samples are of questionable biological significance. These samples were included among the eleven negative cases.

TABLE 3
CT antigens in non-small cell lung cancer by RT-PCR
									Total Number
Lung									Positive CT
Cancer	MAGE 1	MAGE 3	SSX2	SSX4	NY-ESO-1	SCP1	CT7	BRDT/CT9	Angtigens
Lu-2	Neg	+	Neg	Neg	Neg	Neg	Neg*	+++	2
Lu-4	+++	+++	+	++	+++	Neg	++	+++	7
Lu-5	Neg	+	Neg	++	Neg	Neg	+	Neg	3
Lu-6	++	+++	Neg	Neg	Neg	Neg	Neg	Neg	2
Lu-7	Neg	+++	+	Neg*	+++	Neg	++	Neg	4
Lu-9	Neg	Neg*	Neg	Neg	Neg	Neg	Neg*	Neg	0
Lu-10	Neg	+	Neg	Neg*	Neg	Neg	Neg	Neg	1
Lu-14	Neg	Neg	Neg	Neg*	Neg	Neg	Neg	Neg	0
Lu-16	++	++=	Neg	Neg	Neg	Neg	+	Neg	3
Lu-17	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-18	Neg	Neg*	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-19	++	+++	Neg*	Neg	+++	Neg	Neg	Neg	3
Lu-21	+++	+++	+	+	+++	Neg	++	Neg	6
Lu-22	+++	Neg*	Neg	Neg	Neg	Neg	Neg*	Neg	1
Lu-23	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-24	Neg	+	Neg	Neg*	Neg	Neg	Neg	+++	2
Lu-28	Neg	+++	Neg	Neg	Neg	Neg	Neg	Neg	1
Lu-29	Neg	Neg	Neg	+	Neg	Neg	Neg	++	2
Lu-38	+	+++	Neg	+	Neg	Neg	+++	Neg	4
Lu-39	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-40	+++	+++	Neg	Neg	Neg	Neg	Neg*	++	3
Lu-43	Neg	+	Neg	Neg	Neg	Neg	Neg*	+	2
Lu-44	++	+++	Neg	Neg	Neg	Neg	Neg*	+++	3
Lu-46	+++	+++	+	+++	+	Neg	+	Neg	6
Lu-47	Neg	+++	Neg	Neg	Neg	+	Neg*	Neg	2
Lu-51	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-53	Neg*	Neg	Neg	Neg*	Neg	Neg	Neg*	Neg	0
Lu-54	+++	+++	+	+	+++	Neg	+	Neg	6
Lu-55	Neg	Neg	Neg	Neg*	Neg	Neg	Neg	Neg	0
Lu-57	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-58	+++	+++	Neg	+	+	Neg	+	Neg	5
Lu-63	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	0
Lu-65	Neg	++	Neg	Neg	Neg	Neg	+	Neg	2
Totals	12/33	20/33	5/33	8/33	7/33	1/33	10/33	7/33	Frequency of CT
	(36%)	(60%)	(15%)	(24%)	(21%)	(3%)	(30%)	(21%)	expression = 22/33
									or 67%

Expression of CT antigen transcripts in lung cancer appears to be clustered. In these studies, 11 of 33 lung tumors (36%) lacked reliable expression of any of the eight CT antigens tested and 3 of 33 lung tumors (9%) express only one of the antigens tested (2 cases of MAGE-A3 expression and 1 case of MAGE-A1 expression). On the other hand, 19 of 33 patients (57%) express 2 or more antigens and 12 of 33 (36%) express 3 or more of these antigens. A summary of the co-typing data is shown in Table 4. In terms of BRDT mRNA expression, it did not consistently co-type with 5 of the 6 relevant CT antigens, the exception being MAGE-A3. Of the seven BRDT positive lung tumors, 43% were positive for MAGE-A1; 86% were positive for MAGE-A3; 28% were positive for SSX-4 and 14% was positive for HOM-MEL-40/SSX-2, NY-ESO-1 and CT-7. No lung cancer specimen was exclusively BRDT positive. In contrast, six of the remaining seven CT antigens (SCP-1 is the exception) had a higher incidence of coexpression. For example, all of the five HOM-Mel-40/SSX2 positive tumors express MAGE-A3, NY-ESO-1 and CT7 and 80% express MAGE-A1 and SSX-4. Only 20% of the HOM-MEL-40/SSX-2 positive tumors express BRDT/CT-9. Furthermore, of the seven NY-ESO-1 positive lung tumors, all express MAGE A3, 86% express MAGE-A1 and CT7, 71% express HOM-MEL-40/SSX-2 and SSX-4 and 14% express BRDT/CT-9.

TABLE 4
The incidence of CT antigen coexpression in non-small lung cancer.
	Co-typing CT Antigens
Reference	MAGE-	MAGE-
CT Antigen	A1	A3	SSX2	SSX4	NY-ESO-1	SCP1	CT7	BRDT/CT9
MAGE-A1	X	11/12	4/12	6/12	6/12	0/12	7/12	3/12
MAGE-A3	11/20	X	5/20	7/20	7/20	1/20	10/20	6/20
SSX2	4/5	5/5	X	4/5	5/5	0/5	5/5	1/5
SSX4	6/5	7/8	4/8	X	5/8	0/8	7/8	2/8
NY-ESO-1	6/7	7/7	5/7	5/7	X	0/7	6/7	1/7
SCP1	1/1	0/1	0/1	0/1	0/1	X	0/1	0/1
CT7	7/10	10/10	5/10	7/10	6/10	0/10	X	1/10
BRDT/CT9	3/7	6/7	1/7	2/7	1/7	0/7	1/7	X

Data were derived from Table 3.

The expression profile of CT9/BRDT in cancer and its potential transcriptional regulatory activity make it an interesting molecule for future immunological and functional investigation. Immunologically, the ability of BRDT/CT9 or peptide derivatives to stimulate T lymphocyte reactivity is imperative for it to be considered a candidate molecule for tumor vaccines. In this regard, the current study has assembled a group of CT antigens for further studies aimed at generating a polyvalent lung cancer vaccine(s). A minimal panel, covering all CT-antigen positive tumors (67% of all NSCLC specimens tested) and all but 1 case of multi-CT antigen expression, would consist of MAGE-A3, MAGE-A1, NY-ESO-1, BRDT/CT9, CT7 and SSX-4. With such a panel, the frequency of CT antigen coexpression within the subgroup CT antigen positive tumors examined in the current study is 82%, 59%, 45%, 27% and 23% for expression of two, three, four, five and six of these CT antigens, respectively. Thus, such a panel represents a promising repertoire of target antigens for a polyvalent cancer vaccine to be used in the treatment of lung cancer.

Example 5

Identification of the Portion of a Cancer Associated Polypeptide Encoding an Antigen

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.

One also can search for class I and class II motifs using computer algorithms. For example, computer programs for predicting potential CTL epitopes based on known class I motifs has been described (see, e.g., Parker et al, J. Immunol . 152:163, 1994; D'Amaro et al., Human Immunol . 43:13-18, 1995; Drijfhout et al., Human Immunol . 43:1-12, 1995). HLA binding predictions can conveniently be made using an algorithm available via the Internet on the National Institutes of Health World Wide Web site at URL http://bimas.dcrt.nih.gov, for example, using default settings.

To determine if the cancer associated antigens isolated as described above can provoke a cytolytic T lymphocyte response, the following method is performed for generation of CD8 + T cells using the predicted HLA peptides described above.

Briefly, peripheral blood mononuclear cells (PBMCs) are isolated using Ficoll-Hypaque (Pharmacia) from blood of HLA-A201 + healthy donors (or donors positive for whatever HLA binding peptide is being investigated). Adherent monocytes are separated from non-adherent peripheral blood lymphocytes (PBLs) by plastic adherence via overnight incubation at 37° C. on plastic. The non-adherent PBLs are cryopreserved until needed, while adherent cells are stimulated to differentiate into dendritic cells (DCs) by incubation in AIMV-medium (Gibco), containing 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days.

On day 7, 8×10 5 DCs are loaded with 50 mg/ml exogenously added peptide for 2 hours at 37° C. in medium containing 1000 U/ml TNFα and 10,000 U/ml IL-1β. The peptide pulsed DCs are then washed twice in excess medium containing no peptide. Autologous PBLs are thawed and 4×10 7 PBLs are coincubated with 8×10 5 peptide loaded DCs (ratio 50:1), in medium containing 5 ng/ml IL-7 and 20 U/ml IL-2. These cultures are then incubated at 37° C.

On days 14, 21 and 28 the lymphocyte cultures are further stimulated with autologous peptide pulsed DC cells as for day 7. Functional assays such as Elispot and cytotoxicity assays are done on days 14, 21 and/or 28. The Elispot assays are performed using commercially available Elispot-system for IFN-γ; NC plates from Millipore; antibodies from Holzel, following manufacturers instructions. The cytotoxicity assays are performed using a Europium labelling based assay which is analogous to chromium release type assay (Blomberg et al., J. Immunol Methods 114:191-195, 1988).

A similar method is performed to determine if the cancer associated antigen contains one or more HLA class II peptides recognized by T cells. One can search the sequence of the cancer associated antigen polypeptides for HLA class II motifs as described above. In contrast to class I peptides, class II peptides are presented by a limited number of cell types. Thus for these experiments, dendritic cells or B cell clones which express HLA class II molecules preferably are used.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in their entirety.

30 1 14561 DNA Homo sapiens 1tccacacccc tggcggttcc aggcaggcta cgccacgcga cccctcccgt ttccctgctt 60tggccaatgg aggagctacg aatggcacga cctgctcgag cttggcagtc tccagttggg 120ctgtgcatgg aagcttggga agactttgtt ggaaggggag gcggggagag agtgctggag 180gctctggggc gatggcttcc gcacctcttc caaccaccct ctttccctgg agtcggcgga 240ccacagctca gccaattggc ttggagatgt ggcgggttgc cacttccctg tgggtctctg 300cggcactctt ctgcctggtg actgacacct tggaaatgaa gtttatgacg tcatcgttgc 360ggctggccaa tagaaaaagc tcccgcggag aggtgttcct tccccttcga ctcagcttct 420tcacccgcgt gagcgagcgc gcgcgcgcgg agggggtggg gaaaatctca agcagggtgg 480cgcgcatgag cggcgaagct cctcctcccc gcctatatat aaagggctgg cgcggggctc 540ggcggcgcca tttcgtgctg gagtggagca gcctctagaa cgagctggag gattctgcct 600accgatacag agccttcgag tcgtccgggg ccgccattac aatccacctc catccgcttg 660gaaatggcct tcgtcccggc ctatgactgg tcccagcggg cagtacagac cccctagaag 720cccctggagc tccccttttt cgggccccgc ccaatcctcg gagtctgtcc accccctcta 780ctccgccctc aagaggattt caaagatgga ggcggcggct ccctaaacca cttttcgtgt 840tcatccgcct ccatccgaga tcgaaacggg acctcgtcgg ccccgtaggg gcccgacaag 900aagagggaat ccctgcagac caacagcggg ctatattgac gacggtgtct gagatcgggg 960accgtctttt gaagagtcag tccctcctta gttgcccgcc tcagctgagg ccgccgccat 1020tttcttgctg tccgccgtct gcagagcgcg ccaagctgcc cggagctctc cgagaggccc 1080caaagagact gctttcgtgc cggccaggca gggggtttgt cgcctggagg cccaagagga 1140acggcctccc cccaacttag cgggttatgc tggaccgggc ggtgagggga accgaggcca 1200cccggacttt ccgcggctga gggcagcgcc ggttccttgc ggtcaagatg ctgcaaaacg 1260tgactcccca caataagtac gtttccgcga gccgcgtgtg ggaaggggat gttgcagggc 1320ggcggcacag gggtgtgggg cgccgtgttg ggagtactga gcggccccgg cgcgctgctg 1380ttgcggcgca gctgtcgact cggtcgcgcg gagggaattg agcgacggtt ttggaacggt 1440ggtggcggct cggctactgc tcgtggaggg gaatacaggt tgtcaattta tacgctatta 1500atgccgccgt ggcccagtct taaccgagtc aggcagagct agtttgacgg tggagtggag 1560tgaggttgaa cagcaggttt ggcgtttggt gggtctggta tctagcggcg gtctgttagc 1620cttttagggg ggattcacgg acacctctag cgccctgtag ggttgccatg gtgacggagc 1680gcttaaggga ctggcaacgg ggattcccag agaagggtaa agggatcact ctcccgtgtg 1740tgcaggttcc taatgcccag ggcatgtcat taaatctttt gctttctttg ggtgggtggg 1800ttgtgtgtgg tgtttgttgg tgcagggatt gttttttcct aacattaaaa gtttgattca 1860gggcaggagg gtagagctaa ggttcctagt tcagctctgc gatgtaaaca atgagattcc 1920catatgatgt tttaattctt aggtggtagg aaagactgat cggaggagca ccagagggac 1980tgtaaatgaa ccactgttag cgtttggtgt ccggagttgg tgctacaggg ggaactggta 2040gtggaatcgt gttgtgtagt gggtgggtgg aagggggcta tcacttggtg accttgactg 2100ttttgtacgg ctttttgact tccttggagt gaggagactc tgatttggtg cgaataattt 2160tgagggcctg gaagttacgg gctgtgaagt ctgacaaatt cttccttgtc tgaatttgtt 2220tttaagttga tatggttctt cctctgggtt tctagtctat gttctgttgt ggcgtgaact 2280acccagacct tgtggaagat ggtgctctct cttctatcta ggtggattat tctgtgtctt 2340atcagcattt tatggaattt tttatagcca taatttgttc ttttcctcct taccggcgct 2400caaccaccat ggcaaccacc aaacccctag tgaggaggaa gcttggggtt tgagtttctt 2460aactccaccc attttgctta atccccatcc ccatagggct gtagttctga gatgtcgtgc 2520cttgtcagaa acaatttggg agttttttaa aatatgaaaa agaacagata gagcctatca 2580gacttaagaa ggtgggatct agatagtata ctaaaaatat taataaaagg aaggcggggc 2640cagcaataaa agctccacag attgtttgga tattgtttct gcttaagaag cacttggcat 2700aagcttaacc acctcactag ggccagcacc tggattcatc agactattgt gcagatgcac 2760tttttcctca tttggacgat attgccctaa ttttgttccc atctttacag gctccctggg 2820gaagggaatg cagggttgct ggggctgggc ccagaagcag cagcaccagg gaaaaggatt 2880cgaaaaccct ctctcttgta tgagggcttt gagagcccca caatggcttc ggtgcctgct 2940ttgcaactta cccctgccaa cccaccaccc ccggaggtgt ccaatcccaa aaagccagga 3000cgagttacca accagctgca atacctacac aaggtagtga tgaaggctct gtggaaacat 3060cagttcgcat ggccattccg gcagcctgtg gatgctgtca aactgggtct accggtgagt 3120agagacattg gagccgggga ggtgtgggat gagcaagaat gcgtgtgaat gggggtggtc 3180tgcctagtgt agatgctgcg gcccctaggg agttcccatt tctcccctgt agggcagtta 3240gctaccagat ttctgggtat cttggtcctt tgtgattgat ccgaccgctt gctgtaacta 3300tcttggcatc tttccttgtg ccctccatgt gtccttcctt aacttttgtg ccctggctcc 3360attttacaga ttcccacctc gggttgggag aggaccacgg tggccaaaat tcttagcttc 3420ttcctttccc tcatgcagcc catggatagc cagccccaga ggtaatgtca caggatggga 3480agtttccaga gtgggtggga ggtgggtggt tagagaaagg cagcaggggc ctccctgtgg 3540atgtcaagaa tcttttttat ttatttattt attttgtccc acagtttaat tggggccgca 3600gtttaactgt tcctttgatg catagggggt gtgtgtgtgt gtgtgtgtgt gtgtgagagt 3660cggggatcgg tagtctccct ataagcattt atttttctgt ggttctgacc taacatttct 3720ttatttagga ttatcacaaa attataaaac agcctatgga catgggtact attaagagga 3780gacttgaaaa caattattat tgggctgctt cagagtgtat gcaagatttt aataccatgt 3840tcaccaactg ttacatttac aacaaggtga gtttttctgt gtgttcattt agtaggtggg 3900gagaaacagt aatttctatt attgctggat atgttgtcta cataaagttt aaatcctttg 3960ctactgaagg tgttatccag gtagggtagt cggagtctta aaaacctgac tctagatggt 4020actattgaac acagtgatgt gacttcagag ctctagttga aggttattta gaacacttca 4080tacttggggg tggtggtcct gtttcttaga aatcaccaga gacctgagta gaccagggat 4140ctgttttctt gtcagctctc aagttttttc ttctttcgaa ttttgggaga cagttaggag 4200aaagtggaaa ttagtagtgg cctggagtag aaattttctt taagatttga tgacaagatg 4260actggtgggg gtatggtaat ggcctagggc ctgaatgcct ctgagaaaga tggtgtgtat 4320ctatcttctg ttggcatttt ttaactttct ttattgctgt ctgtgttctc atagcccact 4380gatgatattg tcctaatggc acaaacgctg gaaaagatat tcctacagaa ggttgcatca 4440atgccacaag aagaacaaga gctggtagtg accatcccta agaacagcca caagaagggg 4500gccaagttgg caggtaggaa gagtgggagt tttgcaaatg gacaacttaa agatggggaa 4560gagaatcaaa ctacactttt ttcctttttt ctagcgctcc agggcagtgt taccagtgcc 4620catcaggtgc ctgccgtctc ttctgtgtca cacacagccc tgtatactcc tccacctgag 4680atacctacca ctgtcttcaa cattccccac ccatcagtca tttcctctcc acttctcaag 4740tccttgcact ctgctggacc cccgctcctt gctgttactg cagctcctcc agcccagccc 4800cttgccaagg tatgatctgt ggatttcctc tgggcagcag ggaggcaagg gtcttaagta 4860aagtgggctt ggagtgacag gttccctatc ttgtttcttt ctgcagaaaa aaggcgtaaa 4920gcggaaagca gatactacca cccctacacc tacagccatc ttggctcctg gttctccagc 4980tagccctcct gggagtcttg agcctaaggc agcacggctt ccccctatgc gtagagagag 5040tggtcgcccc atcaagcccc cacgcaaaga cttgcctgac tctcagcaac aacaccagag 5100ctctaagaaa ggaaagcttt cagaacagtt aaaacattgc aatggcattt tgaaggagtt 5160actctctaag aagcatgctg cctatgcttg gcctttctat aaaccagtgg atgcttctgc 5220acttggcctg catgactacc atgacatcat taagcacccc atggacctca gcactgtcaa 5280ggtacccact gcatggggca gatgggatgc tcaagcagtg atgggagcct aggtgcaaaa 5340caataagtct ccttatgtgg gcacacagca gtctttggtt cttggcattt tacttttata 5400aaataatagt ggaacagaag gtctggtgtt ttgagaattt gtatttcttg gagtttgaaa 5460cagtagggtg gggtttcttt gtcttgagaa aaatactgtc tataattaag tactaatgtg 5520gcagtgttgg gttaaggaag ttatagggtg gaaagacagg cataggccac ctctctgtca 5580cttagaaatg atttcttttt ctagacataa atatttcttc aacccaccca aattcctttg 5640acttcaaact tgaaccccag ggcacagatc cttaaggtca tccccactgt gctctcaaga 5700gagggctctt cttgtggtgt ctggggttgg cagggaaagg tgagtcttcc tgcctgtgca 5760gcttctgatg ctgcctcctt ctgcagcgga agatggagaa ccgtgattac cgggatgcac 5820aggagtttgc tgctgatgta cggcttatgt tctccaactg ctataagtac aatcccccag 5880atcacgatgt tgtggcaatg gcacgaaagc tacaggtgag tggaaaggtt ggagtttgaa 5940aaataaatgg tatggggagt tattttgtca tgtgtgctgc atagcctcaa cgtgagggtc 6000tcactgttct gtacagttgt aaattggagc tatatcactt ggtggctggg tatgtagggc 6060actgtttatc agcatagttt tgagtttgtg cctctttcta ggatgtattt gagttccgtt 6120atgccaagat gccagatgaa ccactagaac cagggccttt accagtctct actgccatgc 6180cccctggctt ggccaaatcg tcttcagagt cctccagtga ggaaagtagc agtgagagct 6240cctctgagga agaggaggag gaagatgagg aggacgagga ggaagaagag agtgaaagct 6300cagactcaga ggaagaaagg gctcatcgct tagcagaact acaggaacag gtattttgtc 6360actcttgaaa gtttttattg ggtaagaggt tcatgccctt tgtcctcatt ttttcttctt 6420gttattttat ctttatttac tttttccact tcatgttttt tttcctttag cttcgggcag 6480tacatgaaca actggctgct ctgtcccagg gtccaatatc caagcccaag aggaaaagag 6540agaaaaaaga gaaaaagaag aaacggaagg cagagaagca tcgaggccga gctggggccg 6600atgaagatga caaggggcct agggcacccc gcccacctca acctaagaag tccaagaaag 6660caagtggcag tgggggtggc agtgctgctt taggcccttc cggctttgga ccttctggag 6720gaagtggcac caagtgagtt agagtaggaa gcagagacta gtttggctat ttctgtctct 6780ctgggggatg ccatctctct ttgcaaagat aattctaaat ggccagttaa cagatacaat 6840aggctttgag cagtggtccc caaccttttt ggcaccaggg accagtttcg tggaacacag 6900attttaccac agacagggtt tgaggggatg gtttttggga tgaaactgtt ccacctcaga 6960tcattgggcc attggattcc cataaggagc atgcagcctg gatatgtacc atgcgcactt 7020cacagtaggg ttcatgcttc tatgagaatc taatgcttct gctgatgtga caggcagtga 7080tgcccacatg ccggctgttc acctcctgcg tagcccagta acaggccacg gactggtact 7140ggtctggggg ttgggacccc tggctttggg agtcagggtg tttcacagct actctgacag 7200tgaactcaaa gtagccataa actagaaaca tgaagatggc tgtgttccaa aaagacttta 7260tttgcaaaga cacgtggcga tcagatttgt tctctgggcc atatagtttg cctgttgctc 7320taaatcaatg agtctagact tgtttttcat ggcgtagtag ttttgggttt tttggtgtgg 7380ttttgtgttt ttttttgttt gttttttttt tttttttttt ttttttaaag actccaggct 7440ggagtgcagt ggcgtgatct cggcttactg caacctccac ttctcgggtt caagcgattc 7500tcctgcctca gcctcccaag tagccagaat tacaggcatg cgccaccacg cccagctaat 7560ttttgtattt ttagtgcgca gctagtttat gtagttttag tggagacggg gtttcgccat 7620gttgggcagg ctggtcttga actcctgacc tcaagtgatc tgcccgcctt ggcctcccaa 7680agtgctggga ttacaaatct gagccactgc agctggccca tggtgtagtt tggtagtgtt 7740taagggagca gaaagaccca tgtcagtata cctaaacagg tataccttgt tttattgtgc 7800ttcactttac ggagtttttt ttagatacta cttttttttt tagttgaaga tttgtgacaa 7860ccctgtgtgg agcaagtctt tcaacagttt ttccaacatg tttgtgtgtc acatttttag 7920taatattttt tcattaaggt atgtacgtac attgtctttt taaagacatg ttattgccta 7980cttacagtca agagcaaaat gctctgtttc actatacagt gtcccagtag cccacctctt 8040acttggccat tgaatggaaa aacagaagct ccactctggg caggaaatag gatcactgaa 8100ttataacagt gggaacatac tggaagaggt taatgaagct tcttttgctg acaactcttt 8160ttgcccttag gctccccaaa aaggccacaa agacagcccc acctgccctg cctacaggtt 8220atgattcaga ggaggaggaa gagagcaggc ccatgagtta cgatgagaag cggcagctga 8280gcctggacat caacaaatta cctggggaga agctgggccg agttgtgcat ataatccaag 8340ccagggagcc ctctttacgt gattcaaacc cagaagagat tgagattgat tttgaaacac 8400tcaagccatc cacacttaga gagcttgagc gctatgtcct ttcctgccta cgtaagaaac 8460cccggaagcc ctacagtacg tatgaaatga ggttcatctc atggttctga ggacagttga 8520ggaaagatgg tggggtctgt ttgcattcag gattgtcagc tcccaggata atgggatgtg 8580ttggttggca gctgacgttc aagaagggaa cttgggaacc ttaggggccc ataataagat 8640gcttggggca atcttaatgt atcctgataa atttctttca ttagccatta agaagcctgt 8700gggaaagaca aaggaggaac tggctttgga gaaaaagcgg gaattagaaa agcggttaca 8760agatgtcagc ggacagctca attctactaa aaagcccccc aagaaaggtg agtatatact 8820ttcatgccac tacagattga ctccatcctg ccttcttgac tgtcttttat tgacaaatga 8880agattcagac ttgaacgtct ttaactttcg aatttgttct gcagcgaatg agaaaacaga 8940gtcatcctct gcacagcaag tagcagtgtc acgccttagc gcttccagct ccagctcaga 9000ttccagctcc tcctcttcct cgtcgtcgtc ttcagacacc agtgattcag actcaggcta 9060aggggtcagg ccagatgggg caggaaggct ccgcaggacc ggacccctag accaccctgc 9120cccacctgcc ccttccccct ttgctgtgac acttcttcat ctcacccccc cctgcccccc 9180tctaggagag ctggctctgc agtgggggag ggatgcaggg acatttactg aaggagggac 9240atggacaaaa caacattgaa ttcccagccc cattggggag tgatctcttg gacacagagc 9300ccccattcaa aatggggcag ggcaagggtg ggagtgtgca aagccctgat ctggagttac 9360ctgaggccat agctgcccta ttcacttcta agggccctgt tttgagattg tttgttctaa 9420tttattttaa gctaggtaag gctgggggga gggtggggcc gtggtcccct cagcctccat 9480ggggagggaa gaagggggag ctcttttttt acgttgattt ttttttttct actctgtttt 9540ccctttttcc ttccgctcca tttggggccc tgggggtttc agtcatctcc ccatttggtc 9600ccctggactg tctttgttga ttctaacttg taaataaaga aaatattatt caagttttga 9660gttaccttaa tatttgcttt tgtagtgttt caaaaggaac atcataagaa ttgtcttgat 9720aattttgagg gaaatattac tgcagtgaga aaaggcaata gctaacctat aattggattg 9780tcttaatttt taaaccagta ggcttttgct gtgtttttaa taaagtaaat atgacttttg 9840taaattgagt ccttagaagt aatctttagg tctacaattt gctcttgttt aaatgaaaaa 9900tagtactgtg gctcattcat gctttaacca agaactcaaa attttgaggt aggctttagg 9960tttttccctg tggcactgga tgtgtgaatt ttctcctgag cagacttaaa atatgagaaa 10020agggtgggag gtagccgaac ataagtactt tatgcattga gtttattgcc ttttaaaagg 10080aaattggcct gtaatcccag cactttggga ggccgaggcg ggcagatcac gaggtcagga 10140gatcgagacc atggtgaaac cctgtctact aaaaaaaaat tagctgggcg aggtggcggg 10200tacctgtagt cccagctact cgggaggctg aggcagcaga atggcgtgaa ctcgggaggc 10260ggggttcagt gagccgagat cgcgccactg cactccagcc tgggtggtag acactccgtc 10320tcaaaaaaaa aagtaattgg gcctactaca ttgttaaaca ttgttaaatt ttgctgccat 10380ggtcacacac aaatttacag atagtttatt agtagaatac taaagagtat tccaacgatt 10440aaatcacaaa actgtgcttt ctgcataccc ccttgtcttg ctaaggggag agaagggttg 10500tataaaaagt ttagggggtt gggatgtttg cattctggaa tttggggctt taatactgga 10560aaagtgagac atttgcttag tatagtgtac catagtagga aacctggata gagacatgga 10620aattagaatc aggaatgtag taaagcaaat ggtttatttt gctgtaaatg acaccacaaa 10680ctaagtgtag ggcaacacca caaactaaat gtaggaagca ataaatttta ctagtgatgc 10740tcagccctct ttaggaattc cggctaaact ggggcttgag caacaatttt caaaagctcg 10800ggagatggta ataaaaaatt aggtttgtga accacctgct actgtttgcc aagcacttag 10860agggaaacaa acccttgttt gggctttctt gctaacttgt gtgcaccagt gaaagctctt 10920gagctccctt tgagctctgg ttcccttttg agaataacag atgttgagga ttcgtaagta 10980cttaatagag acgcgttggg caataggtga tgagatacaa attaaagttc tgaaaatcgg 11040agtaaataga tttaagctaa gtgcatgtct atgtcaagga ttacatctca tttcagaggg 11100aattgaagga tttagttgga ttagttttgg gacaaaatat agaatatttt gactgcagcc 11160ccttctgctc atgtactttt aaggtttgtt ttctgtagtt cggaaaaata aaagtttcaa 11220cctgacattg gaggcccctg agtacttaat tccctgtaaa tggaacccag accgccctaa 11280atgctttaag agagagaagg gctggctgac acaggggctt cagacctgcc ttaaaccaat 11340tggactagtc tcttaattga ctttagtttg aacttatttc aagcctgtct cacttaggga 11400ttgtaattgt ttcaggagtt tggttgagtt ccatcttggt tgccaaagga ctttattcca 11460aaatagcagt ctccagcaca actcaaagga ctagtggagt cctgtgggca ttatttcccc 11520ctatgctcct ctcagctctt ggaatcatgg gttctattgc tgctgctttt tccctctccc 11580ctcatgctgc catttactgc cttttattgc gtccatagga agccttttgt tgggttgtgg 11640gggaaggtga gagtcggtct tatttactca gtcacacatt tattgagttc ccttgattgc 11700cttttcagca aactgttagg cctgtagacc tggacgttgc caagccagag ggtataaggt 11760gaagataaga caaggtctta tcatggagtt tgcttagcac gaatagggtg caattattaa 11820ccatcatggt gtggtaatta tactgattga atccaagata taggcatgac ttggtcttca 11880cagaccatcc ttattgtgca cccatatgtg gaccccagtt ccagcctgcc tgtaaccttc 11940cccaaagtcc tgctcttagg ttcactcggg actaccttga ttggaaggcc ctgcttccat 12000gagtgaccag catgaaggac tcccaggaca aggaccagag gtgactggtg ttactctggg 12060gcttagtgca agactggagg aagattttcc tgagcaatta gagagtggct gtaatggaga 12120gctgggaagg agatgtggga atggtagcat ggaactaatg tgttgtcacc atgatttcat 12180tttttcctgg gtcatcaccc taaagatact cacaaaatcc cactagctgg tctccagcac 12240tgaatgagac gacagcttct tgctctcaat ttatattcta ggatcgggga aaggggcagc 12300attagacaac tagtcacacg atttttaaca aaaataagta attctaacag gacaatgtgc 12360agaggtctag caagaattta gaaaagaggg ttctctggtc taagctgaga gctattcaga 12420tatctcagaa cgatgagttc cttcctgttt gtaaggggtg gatgggtggg agcagggagt 12480agtgaacatt gccagcagaa gaaacagcaa ctgagaaggt aactaggtga tgctgaagca 12540gcaaggatga aggtgagtgc tttgagatgc tttgaattta catctcaaag actaatagct 12600aacatttatt gagcacttac tgtgtcccat gcactgtgct aaataaaaac ttaccctgta 12660aactcatttg gtcctcacta taatcctgtg aggtacatct tgtctgcatt taacagataa 12720agaaatgagg cacagagaga ttagtcaatt tgcccaacat caccacattg tcagtgagca 12780ctggaggtgg gttttgaaac caggcgatct ggcttcaggg tccacattta taactactgc 12840actagacttc cagtgctgtg ggccagtagg gagctaggag atagacatgc tttaaggaaa 12900ttgcactagg gacaatcatg ttagggttgg tggggtgttg agagtggata gtttagaaag 12960caattgcagc agttaatccc agccagtgag aatagtggtg tagaagagaa aggcagcaat 13020gtagatgaag aaatgtagat agatttgaga gctatgtagg agttaaaata gatgggactt 13080attctcaaaa ggttcttgtc tccacacatt aaaggaacag cagagcacac gtgcacttgc 13140agacatacag ctctcagagt ccctagcaca gagctttcca atagtggaag cttgatattt 13200tgttgactaa aggagtgcca cctggctgac tggcagattt gaatggagct ccctccagca 13260tggcttgcca aaggggcagg ggttctgagg ctcttgtaga ctgccactga aggtatttgg 13320cgccacctgt tgggcgtgtt acccacgatt gcctcctgaa tctattgtca tttttgtgtc 13380ctgcccccga ggttaggtgg ttcttccctt cttactttcc taaacttcaa ctcttaaaat 13440gtgagccttc atttttatga cccagagggt cacaaaagaa ggagattagg cctttttagt 13500ccttatctcc ctttacctct gaagcatccc atggggcttc cctagcattt tattttatat 13560tgatttattt atttattttt aagaaagagg atttctgtca cccaggctgg agtgcagtgg 13620tgtgatcata gctcactgta gcttcgacct tctgggctca agtaatgctc ttgcctcagc 13680ctcctgagta gctgggatta caggcatgag ccaccgcacc ctgcatctcc tgacttcttt 13740gacttgacac cacttgttcc tagctctcat acttttcaga cagccatttc ttagtctgct 13800tctttaaagt ctctgcctct gcctcccata atatattgtc ccttatgaca cctttctttg 13860tcctcttttt atttgcacag tatttttaag caagtttacc cattcctctg gaggcacctc 13920ccacccataa aatctgtgtc tctgtttaga ccttgtatta gtccattctc acatggctat 13980gaggaaatac ctgagactag gtgatttata aaggaaagag gtttaattga ctcacagttc 14040tgcatggctg gggaggcctc aggaaactta caatcatggc ggaagacacc tcttcacagg 14100gtggcaggag aatgagtgcc aaagtgaagt ggggaagccc cttataaaac cataagatct 14160cgtgagaact cactatcacg agaacagcat gggggaaact gcccccatat caaaccttga 14220gtcaaagttc caagtgccta atgcaatgcc taatggcaag gtcccataag ctcgtgcaca 14280catttttaga aaagattcta cctttttccc caaacatgtc cctcgcagtg agttctctaa 14340gtcagcagtc cccagctttt ttggcaccaa ggactggttt tgtggaagac aacttttcta 14400tggatggagg cggggagaat ggtttccaga tcaaactgtt ctacttcaga tcaccaggca 14460ttagattctc aaaaggagtt tgcaaccttc cctcgcatgc tcagttcaca atagggtttg 14520cagtcctgtg agaatctaat gccgctgctg atcccgtcga c 14561 2 3028 DNA Homo sapiens 2gacggccgcc ggagccgcga ggccaactgt cgcctggttg ggcccggaaa tgggacgtcg 60cgctttctca gggagcgtag aagcagccag ggcctctcca agccgctgct gtgacagaaa 120gtgagtgagc tgccggagga tgtccaccgc cacgacagtc gcccccgcgg ggatcccggc 180gaccccgggc cctgtgaacc cacccccccc ggaggtctcc aaccccagca agcccggccg 240caagaccaac cagctgcagt acatgcagaa tgtggtggtg aagacgctct ggaaacacca 300gttcgcctgg cccttctacc agcccgtgga cgcaatcaaa ttgaacctgc cggattatca 360taaaataatt aaaaacccaa tggatatggg gactattaag aagagactag aaaataatta 420ttattggagt gcaagcgaat gtatgcagga cttcaacacc atgtttacaa attgttacat 480ttataacaag cccacagatg acatagtgct aatggcccaa gctttagaga aaatttttct 540acaaaaagtg gcccagatgc cccaagagga agttgaatta ttaccccctg ctccaaaggg 600caaaggtcgg aagccggctg cgggagccca gagcgcaggt acacagcaag tggcggccgt 660gtcctctgtc tccccagcga ccccctttca gagcgtgccc cccaccgtct cccagacgcc 720cgtcatcgct gccacccctg taccaaccat cactgcaaac gtcacgtcgg tcccagtccc 780cccagctgcc gccccacctc ctcctgccac acccatcgtc cccgtggtcc ctcctacgcc 840gcctgtcgtc aagaaaaagg gcgtgaagcg gaaagcagac acaaccactc ccacgacgtc 900ggccatcact gccagccgga gtgagtcgcc cccgccgttg tcagacccca agcaggccaa 960agtggtggcc cggcgggaga gtggtggccg ccccatcaag cctcccaaga aggacctgga 1020ggacggcgag gtgccccagc acgcaggcaa gaagggcaag ctgtcggagc acctgcgcta 1080ctgcgacagc atcctcaggg agatgctatc caagaagcac gcggcctacg cctggccctt 1140ctacaagcca gtggatgccg aggccctgga gctgcacgac taccacgaca tcatcaagca 1200cccgatggac ctcagcaccg tgaaaaggaa gatggatggc cgagagtacc cagacgcaca 1260gggctttgct gctgatgtcc ggctgatgtt ctcgaattgc tacaaataca atcccccaga 1320ccacgaggtt gtggccatgg cccggaagct ccaggacgtg tttgagatga ggtttgccaa 1380gatgccagat gagcccgtgg aggcaccggc gctgcctgcc cccgcggccc ccatggtgag 1440caagggcgct gagagcagcc gtagcagtga ggagagctct tcggactcag gcagctcgga 1500ctcggaggag gagcgggcca ccaggctggc ggagctgcag gagcagctga aggccgtgca 1560cgagcagctg gccgccctgt ctcaggcccc agtaaacaaa ccaaagaaga agaaggagaa 1620gaaggagaag gagaagaaga agaaggacaa ggagaaggag aaggagaagc acaaagtgaa 1680ggccgaggaa gagaagaagg ccaaggtggc tccgcctgcc aagcaggctc agcagaagaa 1740ggctcctgcc aagaaggcca acagcacgac cacggccggc agacagctga agaaaggcgg 1800caagcaggca tctgcctcct acgactcaga ggaagaggag gagggcctgc ccatgagcta 1860cgatgaaaag cgccagctta gcctggacat caaccggctg cccggggaga agctgggccg 1920ggtagtgcac atcatccaat ctcgggagcc ctcgctcagg gactccaacc ccgacgagat 1980agaaattgac tttgagactc tgaaacccac cactttgcgg gaactggaga gatatgtcaa 2040gtcttgttta cagaaaaagc aaaggaaacc gttctcagca agcgggaaga aacaggcagc 2100caagtcgaaa gaggagctag ctcaggaaaa gaagaaggag ctggaaaagc gtctgcagga 2160tgtcagcggg cagctgagca gcagcaagaa gcccgcccgg aaagagaagc ccggctcagc 2220accctcaggg ggcccgtcca ggctcagcag cagcagctcc tccgagtctg ggagcagcag 2280ctccagcggg tccagctctg acagcagtga ctcagaatga actggcttcg gacagaacag 2340gacagatgga tgtcgcacac gccgagactc tgccgtaccc ctctgtggtt catattacta 2400cttctgttcc atggtgtgca ggtctgcttc ttaattcagt gttatgatat cttccagttt 2460ttgctttcat aggtcagaga tctatcttgt gtgtgcgtta gacttgatga gaaggtgtga 2520actctgcaga aagtctcttc ttcatcactg aattcagtca cttggagatg acaacttcaa 2580atgctaaccc gatgacccca gaaaaccgtg tgagattcgt accgaagaac cttgtggaat 2640ccctttgctt aggcccaacc tggtcgatag ctcgagaaag aattttttcc aaggaaatgt 2700ctcggatatg ggtactgtat ttgaaagctg ttagctttgt caacacgcat tgtccttgtc 2760atttgggccc cgagctctga ccctcgtgtc tgacgcggcc acctctttct ggaggggctg 2820aggacagatg tgcctgcttg tggagaccag gctgggccta agcgaagggt catcgcagcc 2880ccagcccgga gcgtggagcc cttggggggt ggtcgggtgg gatgtgcgtt ctccgctcgt 2940ggtgatgtca ggagctcctc ggagggaaca gagcggctgt gtatgcagcc tgcaggtttc 3000catacactga agcttttacc tcaacttt 3028 3 3149 DNA Homo sapiens 3attctttgga atactactgc tagaagtctg acttaagacc cagcttatgg gccacatggc 60acccagctgc ttctgcagag aaggcaggcc actgatgggt acagcaaagt gtggtgctgc 120tggccaagcc aaagacccgt gtaggatgac tgggcctctg ccccttgtgg gtgttgccac 180tgtgcttgag tgcctggtga agaatgtgat gggatcacta gcatgtctgc ggagagcggc 240cctgggacga gattgagaaa tctgccagta atgggggatg gactagaaac ttcccaaatg 300tctacaacac aggcccaggc ccaaccccag ccagccaacg cagccagcac caaccccccg 360cccccagaga cctccaaccc taacaagccc aagaggcaga ccaaccaact gcaatacctg 420ctcagagtgg tgctcaagac actatggaaa caccagtttg catggccttt ccagcagcct 480gtggatgccg tcaagctgaa cctccctgat tactataaga tcattaaaac gcctatggat 540atgggaacaa taaagaagcg cttggaaaac aactattact ggaatgctca ggaatgtatc 600caggacttca acactatgtt tacaaattgt tacatctaca acaagcctgg agatgacata 660gtcttaatgg cagaagctct ggaaaagctc ttcttgcaaa aaataaatga gctacccaca 720gaagaaaccg agatcatgat agtccaggca aaaggaagag gacgtgggag gaaagaaaca 780gggacagcaa aacctggcgt ttccacggta ccaaacacaa ctcaagcatc gactcctccg 840cagacccaga cccctcagcc gaatcctcct cctgtgcagg ccacgcctca ccccttccct 900gccgtcaccc cggacctcat cgtccagacc cctgtcatga cagtggtgcc tccccagcca 960ctgcagacgc ccccgccagt gcccccccag ccacaacccc cacccgctcc agctccccag 1020cccgtacaga gccacccacc catcatcgcg gccaccccac agcctgtgaa gacaaagaag 1080ggagtgaaga ggaaagcaga caccaccacc cccaccacca ttgaccccat tcacgagcca 1140ccctcgctgc ccccggagcc caagaccacc aagctgggcc agcggcggga gagcagccgg 1200cctgtgaaac ctccaaagaa ggacgtgccc gactctcagc agcacccagc accagagaag 1260agcagcaagg tctcggagca gctcaagtgc tgcagcggca tcctcaagga gatgtttgcc 1320aagaagcacg ccgcctacgc ctggcccttc tacaagcctg tggacgtgga ggcactgggc 1380ctacacgact actgtgacat catcaagcac cccatggaca tgagcacaat caagtctaaa 1440ctggaggccc gtgagtaccg tgatgctcag gagtttggtg ctgacgtccg attgatgttc 1500tccaactgct ataagtacaa ccctcctgac catgaggtgg tggccatggc ccgcaagctc 1560caggatgtgt tcgaaatgcg ctttgccaag atgccggacg agcctgagga gccagtggtg 1620gccgtgtcct ccccggcagt gccccctccc accaaggttg tggccccgcc ctcatccagc 1680gacagcagca gcgatagctc ctcggacagt gacagttcga ctgatgactc tgaggaggag 1740cgagcccagc ggctggctga gctccaggag cagctcaaag ccgtgcacga gcagcttgca 1800gccctctctc agccccagca gaacaaacca aagaaaaagg agaaagacaa gaaggaaaag 1860aaaaaagaaa agcacaaaag aaaagaggaa gtggaagaga ataaaaaaag caaagccaag 1920gaacctcctc ctaaaaagac gaagaaaaat aatagcagca acagcaatgt gagcaagaag 1980gagccagcgc ccatgaagag caagccccct cccacgtatg agtcggagga agaggacaag 2040tgcaagccta tgtcctatga ggagaagcgg cagctcagct tggacatcaa caagctcccc 2100ggcgagaagc tgggccgcgt ggtgcacatc atccagtcac gggagccctc cctgaagaat 2160tccaaccccg acgagattga aatcgacttt gagaccctga agccgtccac actgcgtgag 2220ctggagcgct atgtcacctc ctgtttgcgg aagaaaagga aacctcaagc tgagaaagtt 2280gatgtgattg ccggctcctc caagatgaag ggcttctcgt cctcagagtc ggagagctcc 2340agtgagtcca gctcctctga cagcgaagac tccgaaacag gtcctgccta atcattggac 2400acggactctt aataaaacgg tcttcagttc cagattcctt cccagcaagc tatagcttaa 2460gtccattttc ttccgtgaaa gggacaggac tccatcaagt tatggaattc ctcagagccc 2520tgggcctgtc ccccggggtg gattagtcat gtccagcagc acacgcctag tcccgccttc 2580gggaaggctg cctgcctggc cagccgccca ggcctctctg tgtaaagact gcctggctgt 2640cctgcccagc cttcctggtt ctctggggtc ctctgggtgg gtggcatctc ctggagggtg 2700atgacaatcc ccaacacatg cattcatgtg gtgctactct gtgtgcaaag ccagacccca 2760agtatgtttt ctctctttgt cccatccctc tttttctggg actttggacc ctaactactt 2820ccctcctgaa ccttgcagtg acatcagtcc aggagagctc tcgttcagtg tgcggaagaa 2880cactctgacc tctagagctg tcctagataa ggagtgggag ctttagaggc aaggcctcta 2940gaccctggaa ggctcagtga ggctcttccc acagcatgct tctcactggt gccctgtaag 3000ctcgagccac cgctgactct gagccttttt ggagtctttc ctccttcgtc tccattgttc 3060cgtgcatttc caaagcttaa gttgctggtg ggcatttccc cagtttctat gggctccgtc 3120ttctcaagtc acatagggaa agtaccttc 3149 4 3106 DNA Homo sapiens 4gtggcaagat gttcctggga ggtcaagtta agagtcaaaa ataattcatt agatttaaca 60atttagcatg gacatgtact tgtagacagg attcaaagca gttaagaatg tctctgccaa 120gtcgacaaac agctattatt gttaaccctc ctccaccaga atatataaat actaagaaaa 180atgggcgatt gacaaatcaa cttcagtatc tacaaaaagt tgtcctaaag gatttatgga 240agcatagttt ttcatggccc tttcaacgtc ctgtggatgc tgtgaaacta aagttgcctg 300attattatac cattataaaa aacccaatgg atttaaatac aattaagaag cgcttggaga 360ataaatatta tgcgaaggct tcagaatgta tagaagactt caatacaatg ttctcaaatt 420gttatttata taacaagcct ggagatgaca ttgttcttat ggcacaagct ctagagaagc 480tgtttatgca gaaattatct cagatgccac aagaagagca agttgtgggt gttaaggaaa 540gaatcaagaa aggcactcaa cagaatatag ctgtttcttc tgctaaagaa aaatcatcac 600ccagcgcaac agaaaaagta tttaagcagc aagaaattcc ttctgtattt cctaagacat 660ctatttctcc cttgaacgtg gtacagggag cttcagtcaa ctccagttca caaactgcgg 720cccaagttac aaaaggtgtg aagaggaaag cagatacaac aactcctgca acttcagcag 780ttaaagcaag tagtgaattt tctccaacat tcacagaaaa atcagtggca ctgccaccta 840taaaagaaaa tatgccaaag aatgttttgc cagattctca gcaacaatat aatgttgtgg 900agactgttaa agtaactgaa caattaaggc actgtagtga gattcttaaa gaaatgcttg 960caaagaaaca tttttcatat gcatggccct tttataatcc tgttgacgtt aatgctttgg 1020gactccataa ctactatgac gttgtcaaaa atccgatgga tcttggaact attaaggaga 1080aaatggataa ccaagaatat aaggatgcat actcatttgc ggcagatgtt agattaatgt 1140tcatgaattg ctacaagtac aatcctccag atcacgaagt tgtgacaatg gcaagaatgc 1200ttcaggatgt tttcgaaacg catttttcaa agatcccgat tgaacctgtt gagagtatgc 1260ctttatgtta catcaaaaca gatatcacag aaaccactgg tagagagaac actaatgaag 1320cctcctctga agggaactct tctgatgatt ctgaagatga gcgagttaag cgtcttgcaa 1380agcttcagga gcagcttaaa gctgtacatc aacagctcca ggttttgtcc caagtacctt 1440tccgtaagct aaataaaaag aaagagaagt ctaaaaagga aaagaaaaaa gaaaaggtta 1500ataacagcaa tgaaaatcca agaaaaatgt gtgagcaaat gaggctaaag gaaaagtcca 1560agagaaatca gccaaagaaa aggaaacaac agttcattgg tctaaaatct gaagatgaag 1620ataatgctaa acctatgaac tatgatgaga aaaggcagtt aagtctgaat ataaacaaac 1680tccctggaga taaacttggg cgagtagttc acataataca atcaagagag ccttctctga 1740gcaattccaa tcctgatgag atagagatag actttgaaac actgaaagca tcaacactaa 1800gagaattaga aaaatatgtt tcggcatgtc taagaaagag accattaaaa cctcctgcta 1860agaaaataat gatgtccaaa gaagaacttc actcacagaa aaaacaggaa ttggaaaagc 1920ggttactgga tgttaataat cagttaaatt ctagaaaacg tcaaacaaaa tctgataaaa 1980cgcaaccatc caaagctgtt gaaaatgttt cccgactgag tgagagcagc agcagcagca 2040gcagctcatc agagtctgaa agtagcagca gtgacttaag ctcttcagac agcagtgatt 2100ctgaatcaga aatgttccct aagtttacag aagtaaaacc aaatgattct ccttctaaag 2160agcatgtaaa gaaaatgaag aatgaatgca tactgcctga aggaagaaca ggcgtcacac 2220agataggata ttgtgtgcaa gacacaacct ctgccaatac tacccttgtt catcagacca 2280caccttcaca tgtaatgcca ccaaatcacc accaattagc atttaattat caagaattag 2340aacatttaca gactgtgaaa aacatttcac ctttacaaat tctgcctccc tcaggtgatt 2400ctgaacagct ctcaaatggc ataactgtga tgcatccatc tggtgatagt gacacaacga 2460tgttagaatc tgaatgtcaa gctcctgtac agaaggatat aaagattaag aatgcagatt 2520catggaaaag tttaggcaaa ccagtgaaac catcaggtgt aatgaaatcc tcagatgagc 2580tcttcaacca atttagaaaa gcagccatag aaaaggaagt aaaagctcgg acacaggaac 2640tcatacggaa gcatttggaa caaaatacaa aggaactaaa agcatctcaa gaaaatcaga 2700gggatcttgg gaatggattg actgtagaat ctttttcaaa taaaatacaa aacaagtgct 2760ctggagaaga gcagaaagaa catccgcagt catcagaagc tcaagataaa tccaaactct 2820ggcttctcaa agaccgtgat ttagccaggc cgaaagaaca agagaggagg aggagagaag 2880ccatggtggg taccattgat atgacccttc aaagtgacat tatgacaatg tttgaaaaca 2940actttgatta aaactcagtt tttaaattaa ccatccactt aaaatgaatg gtaaaagatc 3000aaaatgcata tggtaaaatg attgctttca gataacaaga taccaatctt atattgtatt 3060ttgactgctc taaaatgatt aaacagtttt cacttacaaa aaaaaa 3106 5 20 DNA Homo sapiens 5tcaagaaagg cactcaacag 20 6 21 DNA Homo sapiens 6ttcactactt gctttaactg c 21 7 20 DNA Homo sapiens 7gggaagggaa tgcagggttg 20 8 21 DNA Homo sapiens 8tggctgttct tagggatggt c 21 9 20 DNA Homo sapiens 9taatggccca agctttagag 20 10 21 DNA Homo sapiens 10gtttgcagtg atggttggta c 21 11 20 DNA Homo sapiens 11agcacccagc accagagaag 20 12 20 DNA Homo sapiens 12tgctgctgtc gctggatgag 20 13 3446 DNA Homo sapiens 13gcgcaggaac ttaagacagt tcctcctggc gatgtgatgg aatttaatgg gacaggagaa 60gggaacgggc tttcttttca ggccagcgtg gcagcgggcg gtagggcgaa agggagaagg 120aaacgagggt ttattccgtt gcccactccg cgatatttac aaccgtaaca gagaaaatgg 180aaaagcaaaa gccctttgca ttgttcgtac caccgagatc aagcagcagt caggtgtctg 240cggtgaaacc tcagaccctg ggaggcgatt ccactttctt caagagtttc aacaaatgta 300ctgaagatga ttttgagttt ccatttgcaa agactaatct ctccaaaaat ggggaaaaca 360ttgattcaga tcctgcttta caaaaagtta atttcttgcc cgtgcttgag caggttggta 420attctgactg tcactatcag gaaggactaa aagactctga tttggagaat tcagagggat 480tgagcagagt gtattcaaaa ctgtataagg aggctgaaaa gataaaaaaa tggaaagtaa 540gtacagaagc tgaactgaga cagaaagaaa gtaagttgca agaaaacaga aagataattg 600aagcacagcg aaaagccatt caggaactgc aatgtggaaa tgaaaaagta agtttgacat 660tagaagaagg aatacaagac aataaagatt taataaaaga gaataatgcc acaaggcatt 720tatgtaatct actcaaagaa acctgtgcta gatctgcaga aaagacaaag aaatatgaat 780atgaacggga agaaaccagg caagtttata tggatctaaa tagtaacatt gagaaaatga 840taacagcttt tgaggaactt cgtgtgcaag ctgagaattc cagactggaa atgcatttta 900agttaaagga agattatgaa aaaatccaac accttgaaca agaatacaag aaggaaataa 960atgacaagga aaagcaggta tcactactat tgatccaaat cactgagaaa gaaaataaaa 1020tgaaagattt aacatttctg ctagaggaat ccagagataa agttaatcaa ttagaggaaa 1080agacaaaatt acagagtgaa aacttaaaac aatcaattga gaaacagcat catttgacta 1140aagaactaga agatattaaa gtgtcattac aaagaagtgt gagtactcaa aaggctttag 1200aggaagattt acagatagca acaaacacaa tttgtcagct aactgaagaa aaagacactc 1260aaatggaaga atctaataaa gctagagctg ctcattcgtt tgtggttact gaatttgaaa 1320ctactgtctg cagcttggaa gaattattga gaacagaaca gcaaagattg gaaaattatg 1380aagatcaatt gataatactt accatggagc ttcaaaagac atcaagtgag ctggaagaga 1440tgactaagct tacaaataac aaagaagtag aacttgaaga attgaaaaaa gtcttgggag 1500aaaaggaaac acttttatat gacaataaac aatttgagaa gattgctgaa gaattaaaag 1560gaacagaaca agaactaatt ggtcttctcc aagccagaga gaaagaagta catgatttgg 1620aatacagtta ctgccattac cacaagtgga cagtattacc caaaagaggt caaagaccaa 1680aactgagctc gaaacgagaa ctcaagaata ctgaatactt cacactgcaa caagcttcac 1740ccccccccaa cgagctcaca caggaaacaa gtgatatgac cctagaactc aagaatcagc 1800aagaagatat aattaataac aaaaagcaag aagaaaggat gttgacacaa atagaaaatc 1860ttcaagaaac agaaacccaa ttaagaaatg aactagaata tgtgagagaa gagctaaaac 1920agaaaagaga tgaagttaaa tgtaaattgg acaagagtga agaaaattgt aacaatttaa 1980ggaaacaagt tgaaaataaa aacaagtata ttgaagaact tcagcaggag aataaggcct 2040tgaaaaaaaa aggtacagca gaaagcaagc aactgaatgt ttatgagata aaggtcaata 2100aattagagtt agaactagaa agtgccaaac agaaatttgg agaaatcaca gacacctatc 2160agaaagaaat tgaggacaaa aagatatcag aagaaaatct tttggaagag gttgagaaag 2220caaaagtaat agctgatgaa gcagtaaaat tacagaaaga aattgataag cgatgtcaac 2280ataaaatagc tgaaatggta gcacttatgg aaaaacataa gcaccaatat gataagatca 2340ttgaagaaag agactcagaa ttaggacttt ataagagcaa agaacaagaa cagtcatcac 2400tgagagcatc tttggagatt gaactatcca atctcaaagc tgaacttttg tctgttaaga 2460agcaacttga aatagaaaga gaagagaagg aaaaactcaa aagagaggca aaagaaaaca 2520cagctactct taaagaaaaa aaagacaaga aaacacaaac atttttattg gaaacacctg 2580acatttattg gaaattggat tctaaagcag ttccttcaca aactgtatct cgaaatttca 2640catcagttga tcatggcata tccaaagata aaagagacta tctgtggaca tctgccaaaa 2700atactttatc tacaccattg ccaaaggcat atacagtgaa gacaccaaca aaaccaaaac 2760tacagcaaag agaaaacttg aatataccca ttgaagaaag taaaaaaaag agaaaaatgg 2820cctttgaatt tgatattaat tcagatagtt cagaaactac tgatcttttg agcatggttt 2880cagaagaaga gacattgaaa acactgtata ggaacaataa tccaccagct tctcatcttt 2940gtgtcaaaac accaaaaaag gccccttcat ctctaacaac ccctggatct acactgaagt 3000ttggagctat aagaaaaatg cgggaggacc gttgggctgt aattgctaaa atggatagaa 3060aaaaaaaact aaaagaagct gaaaagttat ttgtttaatt tcagagaatc agtgtagtta 3120aggagcctaa taacgtgaaa cttatagtta atattttgtt cttatttgcc agagccaaat 3180tttatctgga agttgagact taaaaaatac ttgcatgaat gatttgtgtt tctttatatt 3240tttagcctaa atgttaacta catattgtct ggaaacctgt cattgtattc agataattag 3300atgattatat attgttgtta ctttttcttg tattcatgaa aactgttttt actaagtttt 3360caaatttgta aagttagcct ttgaatgcta agaatgcatt attgagggtc attctttatt 3420ctttactatt aaaatatttt ggatgc 3446 14 752 DNA Homo sapiens 14atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gccatgcagg 60ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga ggccctggca 120ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca 180gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg 240gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc 300cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag 360agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc 420tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc 480gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca 540cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaagccc 600agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc agcacgagtg 660gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt acatgtttgt 720ttctgtagaa aataaaactg agctacgaaa aa 752 15 4265 DNA Homo sapiens 15gtctgaagga cctgaggcat tttgtgacga ggatcgtctc aggtcagcgg agggaggaga 60cttatagacc tatccagtct tcaaggtgct ccagaaagca ggagttgaag acctgggtgt 120gagggacaca tacatcctaa aagcaccaca gcagaggagg cccaggcagt gccaggagtc 180aaggttccca gaagacaaac cccctaggaa gacaggcgac ctgtgaggcc ctagagcacc 240accttaagag aagaagagct gtaagccggc ctttgtcaga gccatcatgg gggacaagga 300tatgcctact gctgggatgc cgagtcttct ccagagttcc tctgagagtc ctcagagttg 360tcctgagggg gaggactccc agtctcctct ccagattccc cagagttctc ctgagagcga 420cgacaccctg tatcctctcc agagtcctca gagtcgttct gagggggagg actcctcgga 480tcctctccag agacctcctg aggggaagga ctcccagtct cctctccaga ttccccagag 540ttctcctgag ggcgacgaca cccagtctcc tctccagaat tctcagagtt ctcctgaggg 600gaaggactcc ctgtctcctc tagagatttc tcagagccct cctgagggtg aggatgtcca 660gtctcctctg cagaatcctg cgagttcctt cttctcctct gctttattga gtattttcca 720gagttcccct gagagtattc aaagtccttt tgagggtttt ccccagtctg ttctccagat 780tcctgtgagc gccgcctcct cctccacttt agtgagtatt ttccagagtt cccctgagag 840tactcaaagt ccttttgagg gttttcccca gtctccactc cagattcctg tgagccgctc 900cttctcctcc actttattga gtattttcca gagttcccct gagagaagtc agagaacttc 960tgagggtttt gcacagtctc ctctccagat tcctgtgagc tcctcctcgt cctccacttt 1020actgagtctt ttccagagtt cccctgagag aactcagagt acttttgagg gttttcccca 1080gtctccactc cagattcctg tgagccgctc cttctcctcc actttattga gtattttcca 1140gagttcccct gagagaactc agagtacttt tgagggtttt gcccagtctc ctctccagat 1200tcctgtgagc ccctccttct cctccacttt agtgagtatt ttccagagtt cccctgagag 1260aactcagagt acttttgagg gttttcccca gtctcctctc cagattcctg tgagctcctc 1320cttctcctcc actttattga gtcttttcca gagttcccct gagagaactc agagtacttt 1380tgagggtttt ccccagtctc ctctccagat tcctggaagc ccctccttct cctccacttt 1440actgagtctt ttccagagtt cccctgagag aactcacagt acttttgagg gttttcccca 1500gtctcctctc cagattccta tgacctcctc cttctcctct actttattga gtattttaca 1560gagttctcct gagagtgctc aaagtgcttt tgagggtttt ccccagtctc ctctccagat 1620tcctgtgagc tcctctttct cctacacttt attgagtctt ttccagagtt cccctgagag 1680aactcacagt acttttgagg gttttcccca gtctcctctc cagattcctg tgagctcctc 1740ctcctcctcc tccactttat tgagtctttt ccagagttcc cctgagtgta ctcaaagtac 1800ttttgagggt tttccccagt ctcctctcca gattcctcag agtcctcctg aaggggagaa 1860tacccattct cctctccaga ttgttccaag tcttcctgag tgggaggact ccctgtctcc 1920tcactacttt cctcagagcc ctcctcaggg ggaggactcc ctatctcctc actactttcc 1980tcagagccct cctcaggggg aggactccct gtctcctcac tactttcctc agagccctca 2040gggggaggac tccctgtctc ctcactactt tcctcagagc cctcctcagg gggaggactc 2100catgtctcct ctctactttc ctcagagtcc tcttcagggg gaggaattcc agtcttctct 2160ccagagccct gtgagcatct gctcctcctc cactccatcc agtcttcccc agagtttccc 2220tgagagttct cagagtcctc ctgaggggcc tgtccagtct cctctccata gtcctcagag 2280ccctcctgag gggatgcact cccaatctcc tctccagagt cctgagagtg ctcctgaggg 2340ggaggattcc ctgtctcctc tccaaattcc tcagagtcct cttgagggag aggactccct 2400gtcttctctc cattttcctc agagtcctcc tgagtgggag gactccctct ctcctctcca 2460ctttcctcag tttcctcctc agggggagga cttccagtct tctctccaga gtcctgtgag 2520tatctgctcc tcctccactt ctttgagtct tccccagagt ttccctgaga gtcctcagag 2580tcctcctgag gggcctgctc agtctcctct ccagagacct gtcagctcct tcttctccta 2640cactttagcg agtcttctcc aaagttccca tgagagtcct cagagtcctc ctgaggggcc 2700tgcccagtct cctctccaga gtcctgtgag ctccttcccc tcctccactt catcgagtct 2760ttcccagagt tctcctgtga gctccttccc ctcctccact tcatcgagtc tttccaagag 2820ttcccctgag agtcctctcc agagtcctgt gatctccttc tcctcctcca cttcattgag 2880cccattcagt gaagagtcca gcagcccagt agatgaatat acaagttcct cagacacctt 2940gctagagagt gattccttga cagacagcga gtccttgata gagagcgagc ccttgttcac 3000ttatacactg gatgaaaagg tggacgagtt ggcgcggttt cttctcctca aatatcaagt 3060gaagcagcct atcacaaagg cagagatgct gacgaatgtc atcagcaggt acacgggcta 3120ctttcctgtg atcttcagga aagcccgtga gttcatagag atactttttg gcatttccct 3180gagagaagtg gaccctgatg actcctatgt ctttgtaaac acattagacc tcacctctga 3240ggggtgtctg agtgatgagc agggcatgtc ccagaaccgc ctcctgattc ttattctgag 3300tatcatcttc ataaagggca cctatgcctc tgaggaggtc atctgggatg tgctgagtgg 3360aataggggtg cgtgctggga gggagcactt tgcctttggg gagcccaggg agctcctcac 3420taaagtttgg gtgcaggaac attacctaga gtaccgggag gtgcccaact cttctcctcc 3480tcgttacgaa ttcctgtggg gtccaagagc tcattcagaa gtcattaaga ggaaagtagt 3540agagtttttg gccatgctaa agaataccgt ccctattacc tttccatcct cttacaagga 3600tgctttgaaa gatgtggaag agagagccca ggccataatt gacaccacag atgattcgac 3660tgccacagaa agtgcaagct ccagtgtcat gtcccccagc ttctcttctg agtgaagtct 3720agggcagatt cttccctctg agtttgaagg gggcagtcga gtttctacgt ggtggagggc 3780ctggttgagg ctggagagaa cacagtgcta tttgcatttc tgttccatat gggtagttat 3840ggggtttacc tgttttactt ttgggtattt ttcaaatgct tttcctatta ataacaggtt 3900taaatagctt cagaatccta gtttatgcac atgagtcgca catgtattgc tgtttttctg 3960gtttaagagt aacagtttga tattttgtaa aaacaaaaac acacccaaac acaccacatt 4020gggaaaacct tctgcctcat tttgtgatgt gtcacaggtt aatgtggtgt tactgtagga 4080attttcttga aactgtgaag gaactctgca gttaaatagt ggaataaagt aaaggattgt 4140taatgtttgc atttcctcag gtcctttagt ctgttgttct tgaaaactaa agatacatac 4200ctggtttgct tggcttacgt aagaaagtcg aagaaagtaa actgtaataa ataaaagtgt 4260cagtg 4265 16 766 DNA Homo sapiens 16ctctctttcg attcttccat actcagagta cgcacggtct gattttctct ttggattctt 60ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcct ttgcaaggag 120acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatg atattgccaa 180atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatct tctatgtgta 240tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccc tcccaccttt 300catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatg accctaaccg 360tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaa tctccccgaa 420gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgc cagaagcatc 480tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaacta cctctgagaa 540gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccaca gactgcgtga 600gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatg acgagtaact 660cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgac ctttcacgaa 720catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg 766 17 576 DNA Homo sapiens 17atgaacggag acgacgcctt tgcaaggaga cccagggatg atgctcaaat atcagagaag 60ttacgaaagg ccttcgatga tattgccaaa tacttctcta agaaagagtg ggaaaagatg 120aaatcctcgg agaaaatcgt ctatgtgtat atgaagctaa actatgaggt catgactaaa 180ctaggtttca aggtcaccct cccacctttc atgcgtagta aacgggctgc agacttccac 240gggaatgatt ttggtaacga tcgaaaccac aggaatcagg ttgaacgtcc tcagatgact 300ttcggcagcc tccagagaat cttcccgaag atcatgccca agaagccagc agaggaagaa 360aatggtttga aggaagtgcc agaggcatct ggcccacaaa atgatgggaa acagctgtgc 420cccccgggaa atccaagtac cttggagaag attaacaaga catctggacc caaaaggggg 480aaacatgcct ggacccacag actgcgtgag agaaagcagc tggtggttta tgaagagatc 540agcgaccctg aggaagatga cgagtaactc ccctcg 576 18 1624 DNA Homo sapiens 18gttttcaggg gacaggccaa cccagaggac aggattccct ggaggccaca gaggagcacc 60aaggagaaga tctgcctgtg ggtcttcatt gcccagctcc tgcccacact cctgcctgct 120gccctgacga gagtcatcat gtctcttgag cagaggagtc tgcactgcaa gcctgaggaa 180gcccttgagg cccaacaaga ggccctgggc ctggtgtgtg tgcaggctgc cacctcctcc 240tcctctcctc tggtcctggg caccctggag gaggtgccca ctgctgggtc aacagatcct 300ccccagagtc ctcagggagc ctccgccttt cccactacca tcaacttcac tcgacagagg 360caacccagtg agggttccag cagccgtgaa gaggaggggc caagcacctc ttgtatcctg 420gagtccttgt tccgagcagt aatcactaag aaggtggctg atttggttgg ttttctgctc 480ctcaaatatc gagccaggga gccagtcaca aaggcagaaa tgctggagag tgtcatcaaa 540aattacaagc actgttttcc tgagatcttc ggcaaagcct ctgagtcctt gcagctggtc 600tttggcattg acgtgaagga agcagacccc accggccact cctatgtcct tgtcacctgc 660ctaggtctct cctatgatgg cctgctgggt gataatcaga tcatgcccaa gacaggcttc 720ctgataattg tcctggtcat gattgcaatg gagggcggcc atgctcctga ggaggaaatc 780tgggaggagc tgagtgtgat ggaggtgtat gatgggaggg agcacagtgc ctatggggag 840cccaggaagc tgctcaccca agatttggtg caggaaaagt acctggagta ccggcaggtg 900ccggacagtg atcccgcacg ctatgagttc ctgtggggtc caagggccct cgctgaaacc 960agctatgtga aagtccttga gtatgtgatc aaggtcagtg caagagttcg ctttttcttc 1020ccatccctgc gtgaagcagc tttgagagag gaggaagagg gagtctgagc atgagttgca 1080gccaaggcca gtgggagggg gactgggcca gtgcaccttc cagggccgcg tccagcagct 1140tcccctgcct cgtgtgacat gaggcccatt cttcactctg aagagagcgg tcagtgttct 1200cagtagtagg tttctgttct attgggtgac ttggagattt atctttgttc tcttttggaa 1260ttgttcaaat gttttttttt aagggatggt tgaatgaact tcagcatcca agtttatgaa 1320tgacagcagt cacacagttc tgtgtatata gtttaagggt aagagtcttg tgttttattc 1380agattgggaa atccattcta ttttgtgaat tgggataata acagcagtgg aataagtact 1440tagaaatgtg aaaaatgagc agtaaaatag atgagataaa gaactaaaga aattaagaga 1500tagtcaattc ttgccttata cctcagtcta ttctgtaaaa tttttaaaga tatatgcata 1560cctggatttc cttggcttct ttgagaatgt aagagaaatt aaatctgaat aaagaattct 1620tcct 1624 19 945 DNA Homo sapiens 19atgcctcttg agcagaggag tcagcactgc aagcctgaag aaggccttga ggcccgagga 60gaggccctgg gcctggtggg tgcgcaggct cctgctactg aggagcagga ggctgcctcc 120tcctcttcta ctctagttga agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180cctccccaga gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg 240agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac cttccctgac 300ctggagtccg agttccaagc agcactcagt aggaaggtgg ccgagttggt tcattttctg 360ctcctcaagt atcgagccag ggagccggtc acaaaggcag aaatgctggg gagtgtcgtc 420ggaaattggc agtatttctt tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480gtctttggca tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc 540tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc caaggcaggc 600ctcctgataa tcgtcctggc cataatcgca agagagggcg actgtgcccc tgaggagaaa 660atctgggagg agctgagtgt gttagaggtg tttgagggga gggaagacag tatcttgggg 720gatcccaaga agctgctcac ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780gtccccggca gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa 840accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc tcacatttcc 900tacccacccc tgcatgagtg ggttttgaga gagggggaag agtga 945 20 754 PRT Homo sapiens 20Met Ala Ser Val Pro Ala Leu Gln Leu Thr Pro Ala Asn Pro Pro Pro 1 5 10 15Pro Glu Val Ser Asn Pro Lys Lys Pro Gly Arg Val Thr Asn Gln Leu 20 25 30Gln Tyr Leu His Lys Val Val Met Lys Ala Leu Trp Lys His Gln Phe 35 40 45Ala Trp Pro Phe Arg Gln Pro Val Asp Ala Val Lys Leu Gly Leu Pro 50 55 60Asp Tyr His Lys Ile Ile Lys Gln Pro Met Asp Met Gly Thr Ile Lys65 70 75 80Arg Arg Leu Glu Asn Asn Tyr Tyr Trp Ala Ala Ser Glu Cys Met Gln 85 90 95Asp Phe Asn Thr Met Phe Thr Asn Cys Tyr Ile Tyr Asn Lys Pro Thr 100 105 110Asp Asp Ile Val Leu Met Ala Gln Thr Leu Glu Lys Ile Phe Leu Gln 115 120 125Lys Val Ala Ser Met Pro Gln Glu Glu Gln Glu Leu Val Val Thr Ile 130 135 140Pro Lys Asn Ser His Lys Lys Gly Ala Lys Leu Ala Ala Leu Gln Gly145 150 155 160Ser Val Thr Ser Ala His Gln Val Pro Ala Val Ser Ser Val Ser His 165 170 175Thr Ala Leu Tyr Thr Pro Pro Pro Glu Ile Pro Thr Thr Val Phe Asn 180 185 190Ile Pro His Pro Ser Val Ile Ser Ser Pro Leu Leu Lys Ser Leu His 195 200 205Ser Ala Gly Pro Pro Leu Leu Ala Val Thr Ala Ala Pro Pro Ala Gln 210 215 220Pro Leu Ala Lys Lys Lys Gly Val Lys Arg Lys Ala Asp Thr Thr Thr225 230 235 240Pro Thr Pro Thr Ala Ile Leu Ala Pro Gly Ser Pro Ala Ser Pro Pro 245 250 255Gly Ser Leu Glu Pro Lys Ala Ala Arg Leu Pro Pro Met Arg Arg Glu 260 265 270Ser Gly Arg Pro Ile Lys Pro Pro Arg Lys Asp Leu Pro Asp Ser Gln 275 280 285Gln Gln His Gln Ser Ser Lys Lys Gly Lys Leu Ser Glu Gln Leu Lys 290 295 300His Cys Asn Gly Ile Leu Lys Glu Leu Leu Ser Lys Lys His Ala Ala305 310 315 320Tyr Ala Trp Pro Phe Tyr Lys Pro Val Asp Ala Ser Ala Leu Gly Leu 325 330 335His Asp Tyr His Asp Ile Ile Lys His Pro Met Asp Leu Ser Thr Val 340 345 350Lys Arg Lys Met Glu Asn Arg Asp Tyr Arg Asp Ala Gln Glu Phe Ala 355 360 365Ala Asp Val Arg Leu Met Phe Ser Asn Cys Tyr Lys Tyr Asn Pro Pro 370 375 380Asp His Asp Val Val Ala Met Ala Arg Lys Leu Gln Asp Val Phe Glu385 390 395 400Phe Arg Tyr Ala Lys Met Pro Asp Glu Pro Leu Glu Pro Gly Pro Leu 405 410 415Pro Val Ser Thr Ala Met Pro Pro Gly Leu Ala Lys Ser Ser Ser Glu 420 425 430Ser Ser Ser Glu Glu Ser Ser Ser Glu Ser Ser Ser Glu Glu Glu Glu 435 440 445Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Ser Glu Ser Ser Asp 450 455 460Ser Glu Glu Glu Arg Ala His Arg Leu Ala Glu Leu Gln Glu Gln Leu465 470 475 480Arg Ala Val His Glu Gln Leu Ala Ala Leu Ser Gln Gly Pro Ile Ser 485 490 495Lys Pro Lys Arg Lys Arg Glu Lys Lys Glu Lys Lys Lys Lys Arg Lys 500 505 510Ala Glu Lys His Arg Gly Arg Ala Gly Ala Asp Glu Asp Asp Lys Gly 515 520 525Pro Arg Ala Pro Arg Pro Pro Gln Pro Lys Lys Ser Lys Lys Ala Ser 530 535 540Gly Ser Gly Gly Gly Ser Ala Ala Leu Gly Pro Ser Gly Phe Gly Pro545 550 555 560Ser Gly Gly Ser Gly Thr Lys Leu Pro Lys Lys Ala Thr Lys Thr Ala 565 570 575Pro Pro Ala Leu Pro Thr Gly Tyr Asp Ser Glu Glu Glu Glu Glu Ser 580 585 590Arg Pro Met Ser Tyr Asp Glu Lys Arg Gln Leu Ser Leu Asp Ile Asn 595 600 605Lys Leu Pro Gly Glu Lys Leu Gly Arg Val Val His Ile Ile Gln Ala 610 615 620Arg Glu Pro Ser Leu Arg Asp Ser Asn Pro Glu Glu Ile Glu Ile Asp625 630 635 640Phe Glu Thr Leu Lys Pro Ser Thr Leu Arg Glu Leu Glu Arg Tyr Val 645 650 655Leu Ser Cys Leu Arg Lys Lys Pro Arg Lys Pro Tyr Thr Ile Lys Lys 660 665 670Pro Val Gly Lys Thr Lys Glu Glu Leu Ala Leu Glu Lys Lys Arg Glu 675 680 685Leu Glu Lys Arg Leu Gln Asp Val Ser Gly Gln Leu Asn Ser Thr Lys 690 695 700Lys Pro Pro Lys Lys Ala Asn Glu Lys Thr Glu Ser Ser Ser Ala Gln705 710 715 720Gln Val Ala Val Ser Arg Leu Ser Ala Ser Ser Ser Ser Ser Asp Ser 725 730 735Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Asp Thr Ser Asp Ser Asp 740 745 750Ser Gly 21 726 PRT Homo sapiens 21Met Ser Thr Ala Thr Thr Val Ala Pro Ala Gly Ile Pro Ala Thr Pro 1 5 10 15Gly Pro Val Asn Pro Pro Pro Pro Glu Val Ser Asn Pro Ser Lys Pro 20 25 30Gly Arg Lys Thr Asn Gln Leu Gln Tyr Met Gln Asn Val Val Val Lys 35 40 45Thr Leu Trp Lys His Gln Phe Ala Trp Pro Phe Tyr Gln Pro Val Asp 50 55 60Ala Ile Lys Leu Asn Leu Pro Asp Tyr His Lys Ile Ile Lys Asn Pro65 70 75 80Met Asp Met Gly Thr Ile Lys Lys Arg Leu Glu Asn Asn Tyr Tyr Trp 85 90 95Ser Ala Ser Glu Cys Met Gln Asp Phe Asn Thr Met Phe Thr Asn Cys 100 105 110Tyr Ile Tyr Asn Lys Pro Thr Asp Asp Ile Val Leu Met Ala Gln Ala 115 120 125Leu Glu Lys Ile Phe Leu Gln Lys Val Ala Gln Met Pro Gln Glu Glu 130 135 140Val Glu Leu Leu Pro Pro Ala Pro Lys Gly Lys Gly Arg Lys Pro Ala145 150 155 160Ala Gly Ala Gln Ser Ala Gly Thr Gln Gln Val Ala Ala Val Ser Ser 165 170 175Val Ser Pro Ala Thr Pro Phe Gln Ser Val Pro Pro Thr Val Ser Gln 180 185 190Thr Pro Val Ile Ala Ala Thr Pro Val Pro Thr Ile Thr Ala Asn Val 195 200 205Thr Ser Val Pro Val Pro Pro Ala Ala Ala Pro Pro Pro Pro Ala Thr 210 215 220Pro Ile Val Pro Val Val Pro Pro Thr Pro Pro Val Val Lys Lys Lys225 230 235 240Gly Val Lys Arg Lys Ala Asp Thr Thr Thr Pro Thr Thr Ser Ala Ile 245 250 255Thr Ala Ser Arg Ser Glu Ser Pro Pro Pro Leu Ser Asp Pro Lys Gln 260 265 270Ala Lys Val Val Ala Arg Arg Glu Ser Gly Gly Arg Pro Ile Lys Pro 275 280 285Pro Lys Lys Asp Leu Glu Asp Gly Glu Val Pro Gln His Ala Gly Lys 290 295 300Lys Gly Lys Leu Ser Glu His Leu Arg Tyr Cys Asp Ser Ile Leu Arg305 310 315 320Glu Met Leu Ser Lys Lys His Ala Ala Tyr Ala Trp Pro Phe Tyr Lys 325 330 335Pro Val Asp Ala Glu Ala Leu Glu Leu His Asp Tyr His Asp Ile Ile 340 345 350Lys His Pro Met Asp Leu Ser Thr Val Lys Arg Lys Met Asp Gly Arg 355 360 365Glu Tyr Pro Asp Ala Gln Gly Phe Ala Ala Asp Val Arg Leu Met Phe 370 375 380Ser Asn Cys Tyr Lys Tyr Asn Pro Pro Asp His Glu Val Val Ala Met385 390 395 400Ala Arg Lys Leu Gln Asp Val Phe Glu Met Arg Phe Ala Lys Met Pro 405 410 415Asp Glu Pro Val Glu Ala Pro Ala Leu Pro Ala Pro Ala Ala Pro Met 420 425 430Val Ser Lys Gly Ala Glu Ser Ser Arg Ser Ser Glu Glu Ser Ser Ser 435 440 445Asp Ser Gly Ser Ser Asp Ser Glu Glu Glu Arg Ala Thr Arg Leu Ala 450 455 460Glu Leu Gln Glu Gln Leu Lys Ala Val His Glu Gln Leu Ala Ala Leu465 470 475 480Ser Gln Ala Pro Val Asn Lys Pro Lys Lys Lys Lys Glu Lys Lys Glu 485 490 495Lys Glu Lys Lys Lys Lys Asp Lys Glu Lys Glu Lys Glu Lys His Lys 500 505 510Val Lys Ala Glu Glu Glu Lys Lys Ala Lys Val Ala Pro Pro Ala Lys 515 520 525Gln Ala Gln Gln Lys Lys Ala Pro Ala Lys Lys Ala Asn Ser Thr Thr 530 535 540Thr Ala Gly Arg Gln Leu Lys Lys Gly Gly Lys Gln Ala Ser Ala Ser545 550 555 560Tyr Asp Ser Glu Glu Glu Glu Glu Gly Leu Pro Met Ser Tyr Asp Glu 565 570 575Lys Arg Gln Leu Ser Leu Asp Ile Asn Arg Leu Pro Gly Glu Lys Leu 580 585 590Gly Arg Val Val His Ile Ile Gln Ser Arg Glu Pro Ser Leu Arg Asp 595 600 605Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe Glu Thr Leu Lys Pro Thr 610 615 620Thr Leu Arg Glu Leu Glu Arg Tyr Val Lys Ser Cys Leu Gln Lys Lys625 630 635 640Gln Arg Lys Pro Phe Ser Ala Ser Gly Lys Lys Gln Ala Ala Lys Ser 645 650 655Lys Glu Glu Leu Ala Gln Glu Lys Lys Lys Glu Leu Glu Lys Arg Leu 660 665 670Gln Asp Val Ser Gly Gln Leu Ser Ser Ser Lys Lys Pro Ala Arg Lys 675 680 685Glu Lys Pro Gly Ser Ala Pro Ser Gly Gly Pro Ser Arg Leu Ser Ser 690 695 700Ser Ser Ser Ser Glu Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Ser705 710 715 720Asp Ser Ser Asp Ser Glu 725 22 722 PRT Homo sapiens 22Met Ser Ala Glu Ser Gly Pro Gly Thr Arg Leu Arg Asn Leu Pro Val 1 5 10 15Met Gly Asp Gly Leu Glu Thr Ser Gln Met Ser Thr Thr Gln Ala Gln 20 25 30Ala Gln Pro Gln Pro Ala Asn Ala Ala Ser Thr Asn Pro Pro Pro Pro 35 40 45Glu Thr Ser Asn Pro Asn Lys Pro Lys Arg Gln Thr Asn Gln Leu Gln 50 55 60Tyr Leu Leu Arg Val Val Leu Lys Thr Leu Trp Lys His Gln Phe Ala65 70 75 80Trp Pro Phe Gln Gln Pro Val Asp Ala Val Lys Leu Asn Leu Pro Asp 85 90 95Tyr Tyr Lys Ile Ile Lys Thr Pro Met Asp Met Gly Thr Ile Lys Lys 100 105 110Arg Leu Glu Asn Asn Tyr Tyr Trp Asn Ala Gln Glu Cys Ile Gln Asp 115 120 125Phe Asn Thr Met Phe Thr Asn Cys Tyr Ile Tyr Asn Lys Pro Gly Asp 130 135 140Asp Ile Val Leu Met Ala Glu Ala Leu Glu Lys Leu Phe Leu Gln Lys145 150 155 160Ile Asn Glu Leu Pro Thr Glu Glu Thr Glu Ile Met Ile Val Gln Ala 165 170 175Lys Gly Arg Gly Arg Gly Arg Lys Glu Thr Gly Thr Ala Lys Pro Gly 180 185 190Val Ser Thr Val Pro Asn Thr Thr Gln Ala Ser Thr Pro Pro Gln Thr 195 200 205Gln Thr Pro Gln Pro Asn Pro Pro Pro Val Gln Ala Thr Pro His Pro 210 215 220Phe Pro Ala Val Thr Pro Asp Leu Ile Val Gln Thr Pro Val Met Thr225 230 235 240Val Val Pro Pro Gln Pro Leu Gln Thr Pro Pro Pro Val Pro Pro Gln 245 250 255Pro Gln Pro Pro Pro Ala Pro Ala Pro Gln Pro Val Gln Ser His Pro 260 265 270Pro Ile Ile Ala Ala Thr Pro Gln Pro Val Lys Thr Lys Lys Gly Val 275 280 285Lys Arg Lys Ala Asp Thr Thr Thr Pro Thr Thr Ile Asp Pro Ile His 290 295 300Glu Pro Pro Ser Leu Pro Pro Glu Pro Lys Thr Thr Lys Leu Gly Gln305 310 315 320Arg Arg Glu Ser Ser Arg Pro Val Lys Pro Pro Lys Lys Asp Val Pro 325 330 335Asp Ser Gln Gln His Pro Ala Pro Glu Lys Ser Ser Lys Val Ser Glu 340 345 350Gln Leu Lys Cys Cys Ser Gly Ile Leu Lys Glu Met Phe Ala Lys Lys 355 360 365His Ala Ala Tyr Ala Trp Pro Phe Tyr Lys Pro Val Asp Val Glu Ala 370 375 380Leu Gly Leu His Asp Tyr Cys Asp Ile Ile Lys His Pro Met Asp Met385 390 395 400Ser Thr Ile Lys Ser Lys Leu Glu Ala Arg Glu Tyr Arg Asp Ala Gln 405 410 415Glu Phe Gly Ala Asp Val Arg Leu Met Phe Ser Asn Cys Tyr Lys Tyr 420 425 430Asn Pro Pro Asp His Glu Val Val Ala Met Ala Arg Lys Leu Gln Asp 435 440 445Val Phe Glu Met Arg Phe Ala Lys Met Pro Asp Glu Pro Glu Glu Pro 450 455 460Val Val Ala Val Ser Ser Pro Ala Val Pro Pro Pro Thr Lys Val Val465 470 475 480Ala Pro Pro Ser Ser Ser Asp Ser Ser Ser Asp Ser Ser Ser Asp Ser 485 490 495Asp Ser Ser Thr Asp Asp Ser Glu Glu Glu Arg Ala Gln Arg Leu Ala 500 505 510Glu Leu Gln Glu Gln Leu Lys Ala Val His Glu Gln Leu Ala Ala Leu 515 520 525Ser Gln Pro Gln Gln Asn Lys Pro Lys Lys Lys Glu Lys Asp Lys Lys 530 535 540Glu Lys Lys Lys Glu Lys His Lys Arg Lys Glu Glu Val Glu Glu Asn545 550 555 560Lys Lys Ser Lys Ala Lys Glu Pro Pro Pro Lys Lys Thr Lys Lys Asn 565 570 575Asn Ser Ser Asn Ser Asn Val Ser Lys Lys Glu Pro Ala Pro Met Lys 580 585 590Ser Lys Pro Pro Pro Thr Tyr Glu Ser Glu Glu Glu Asp Lys Cys Lys 595 600 605Pro Met Ser Tyr Glu Glu Lys Arg Gln Leu Ser Leu Asp Ile Asn Lys 610 615 620Leu Pro Gly Glu Lys Leu Gly Arg Val Val His Ile Ile Gln Ser Arg625 630 635 640Glu Pro Ser Leu Lys Asn Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe 645 650 655Glu Thr Leu Lys Pro Ser Thr Leu Arg Glu Leu Glu Arg Tyr Val Thr 660 665 670Ser Cys Leu Arg Lys Lys Arg Lys Pro Gln Ala Glu Lys Val Asp Val 675 680 685Ile Ala Gly Ser Ser Lys Met Lys Gly Phe Ser Ser Ser Glu Ser Glu 690 695 700Ser Ser Ser Glu Ser Ser Ser Ser Asp Ser Glu Asp Ser Glu Thr Gly705 710 715 720Pro Ala 23 947 PRT Homo sapiens 23Met Ser Leu Pro Ser Arg Gln Thr Ala Ile Ile Val Asn Pro Pro Pro 1 5 10 15Pro Glu Tyr Ile Asn Thr Lys Lys Asn Gly Arg Leu Thr Asn Gln Leu 20 25 30Gln Tyr Leu Gln Lys Val Val Leu Lys Asp Leu Trp Lys His Ser Phe 35 40 45Ser Trp Pro Phe Gln Arg Pro Val Asp Ala Val Lys Leu Lys Leu Pro 50 55 60Asp Tyr Tyr Thr Ile Ile Lys Asn Pro Met Asp Leu Asn Thr Ile Lys65 70 75 80Lys Arg Leu Glu Asn Lys Tyr Tyr Ala Lys Ala Ser Glu Cys Ile Glu 85 90 95Asp Phe Asn Thr Met Phe Ser Asn Cys Tyr Leu Tyr Asn Lys Pro Gly 100 105 110Asp Asp Ile Val Leu Met Ala Gln Ala Leu Glu Lys Leu Phe Met Gln 115 120 125Lys Leu Ser Gln Met Pro Gln Glu Glu Gln Val Val Gly Val Lys Glu 130 135 140Arg Ile Lys Lys Gly Thr Gln Gln Asn Ile Ala Val Ser Ser Ala Lys145 150 155 160Glu Lys Ser Ser Pro Ser Ala Thr Glu Lys Val Phe Lys Gln Gln Glu 165 170 175Ile Pro Ser Val Phe Pro Lys Thr Ser Ile Ser Pro Leu Asn Val Val 180 185 190Gln Gly Ala Ser Val Asn Ser Ser Ser Gln Thr Ala Ala Gln Val Thr 195 200 205Lys Gly Val Lys Arg Lys Ala Asp Thr Thr Thr Pro Ala Thr Ser Ala 210 215 220Val Lys Ala Ser Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Val225 230 235 240Ala Leu Pro Pro Ile Lys Glu Asn Met Pro Lys Asn Val Leu Pro Asp 245 250 255Ser Gln Gln Gln Tyr Asn Val Val Glu Thr Val Lys Val Thr Glu Gln 260 265 270Leu Arg His Cys Ser Glu Ile Leu Lys Glu Met Leu Ala Lys Lys His 275 280 285Phe Ser Tyr Ala Trp Pro Phe Tyr Asn Pro Val Asp Val Asn Ala Leu 290 295 300Gly Leu His Asn Tyr Tyr Asp Val Val Lys Asn Pro Met Asp Leu Gly305 310 315 320Thr Ile Lys Glu Lys Met Asp Asn Gln Glu Tyr Lys Asp Ala Tyr Ser 325 330 335Phe Ala Ala Asp Val Arg Leu Met Phe Met Asn Cys Tyr Lys Tyr Asn 340 345 350Pro Pro Asp His Glu Val Val Thr Met Ala Arg Met Leu Gln Asp Val 355 360 365Phe Glu Thr His Phe Ser Lys Ile Pro Ile Glu Pro Val Glu Ser Met 370 375 380Pro Leu Cys Tyr Ile Lys Thr Asp Ile Thr Glu Thr Thr Gly Arg Glu385 390 395 400Asn Thr Asn Glu Ala Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu 405 410 415Asp Glu Arg Val Lys Arg Leu Ala Lys Leu Gln Glu Gln Leu Lys Ala 420 425 430Val His Gln Gln Leu Gln Val Leu Ser Gln Val Pro Phe Arg Lys Leu 435 440 445Asn Lys Lys Lys Glu Lys Ser Lys Lys Glu Lys Lys Lys Glu Lys Val 450 455 460Asn Asn Ser Asn Glu Asn Pro Arg Lys Met Cys Glu Gln Met Arg Leu465 470 475 480Lys Glu Lys Ser Lys Arg Asn Gln Pro Lys Lys Arg Lys Gln Gln Phe 485 490 495Ile Gly Leu Lys Ser Glu Asp Glu Asp Asn Ala Lys Pro Met Asn Tyr 500 505 510Asp Glu Lys Arg Gln Leu Ser Leu Asn Ile Asn Lys Leu Pro Gly Asp 515 520 525Lys Leu Gly Arg Val Val His Ile Ile Gln Ser Arg Glu Pro Ser Leu 530 535 540Ser Asn Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe Glu Thr Leu Lys545 550 555 560Ala Ser Thr Leu Arg Glu Leu Glu Lys Tyr Val Ser Ala Cys Leu Arg 565 570 575Lys Arg Pro Leu Lys Pro Pro Ala Lys Lys Ile Met Met Ser Lys Glu 580 585 590Glu Leu His Ser Gln Lys Lys Gln Glu Leu Glu Lys Arg Leu Leu Asp 595 600 605Val Asn Asn Gln Leu Asn Ser Arg Lys Arg Gln Thr Lys Ser Asp Lys 610 615 620Thr Gln Pro Ser Lys Ala Val Glu Asn Val Ser Arg Leu Ser Glu Ser625 630 635 640Ser Ser Ser Ser Ser Ser Ser Ser Glu Ser Glu Ser Ser Ser Ser Asp 645 650 655Leu Ser Ser Ser Asp Ser Ser Asp Ser Glu Ser Glu Met Phe Pro Lys 660 665 670Phe Thr Glu Val Lys Pro Asn Asp Ser Pro Ser Lys Glu His Val Lys 675 680 685Lys Met Lys Asn Glu Cys Ile Leu Pro Glu Gly Arg Thr Gly Val Thr 690 695 700Gln Ile Gly Tyr Cys Val Gln Asp Thr Thr Ser Ala Asn Thr Thr Leu705 710 715 720Val His Gln Thr Thr Pro Ser His Val Met Pro Pro Asn His His Gln 725 730 735Leu Ala Phe Asn Tyr Gln Glu Leu Glu His Leu Gln Thr Val Lys Asn 740 745 750Ile Ser Pro Leu Gln Ile Leu Pro Pro Ser Gly Asp Ser Glu Gln Leu 755 760 765Ser Asn Gly Ile Thr Val Met His Pro Ser Gly Asp Ser Asp Thr Thr 770 775 780Met Leu Glu Ser Glu Cys Gln Ala Pro Val Gln Lys Asp Ile Lys Ile785 790 795 800Lys Asn Ala Asp Ser Trp Lys Ser Leu Gly Lys Pro Val Lys Pro Ser 805 810 815Gly Val Met Lys Ser Ser Asp Glu Leu Phe Asn Gln Phe Arg Lys Ala 820 825 830Ala Ile Glu Lys Glu Val Lys Ala Arg Thr Gln Glu Leu Ile Arg Lys 835 840 845His Leu Glu Gln Asn Thr Lys Glu Leu Lys Ala Ser Gln Glu Asn Gln 850 855 860Arg Asp Leu Gly Asn Gly Leu Thr Val Glu Ser Phe Ser Asn Lys Ile865 870 875 880Gln Asn Lys Cys Ser Gly Glu Glu Gln Lys Glu His Pro Gln Ser Ser 885 890 895Glu Ala Gln Asp Lys Ser Lys Leu Trp Leu Leu Lys Asp Arg Asp Leu 900 905 910Ala Arg Pro Lys Glu Gln Glu Arg Arg Arg Arg Glu Ala Met Val Gly 915 920 925Thr Ile Asp Met Thr Leu Gln Ser Asp Ile Met Thr Met Phe Glu Asn 930 935 940Asn Phe Asp945 24 973 PRT Homo sapiens 24Met Glu Lys Gln Lys Pro Phe Ala Leu Phe Val Pro Pro Arg Ser Ser 1 5 10 15Ser Ser Gln Val Ser Ala Val Lys Pro Gln Thr Leu Gly Gly Asp Ser 20 25 30Thr Phe Phe Lys Ser Phe Asn Lys Cys Thr Glu Asp Asp Phe Glu Phe 35 40 45Pro Phe Ala Lys Thr Asn Leu Ser Lys Asn Gly Glu Asn Ile Asp Ser 50 55 60Asp Pro Ala Leu Gln Lys Val Asn Phe Leu Pro Val Leu Glu Gln Val65 70 75 80Gly Asn Ser Asp Cys His Tyr Gln Glu Gly Leu Lys Asp Ser Asp Leu 85 90 95Glu Asn Ser Glu Gly Leu Ser Arg Val Tyr Ser Lys Leu Tyr Lys Glu 100 105 110Ala Glu Lys Ile Lys Lys Trp Lys Val Ser Thr Glu Ala Glu Leu Arg 115 120 125Gln Lys Glu Ser Lys Leu Gln Glu Asn Arg Lys Ile Ile Glu Ala Gln 130 135 140Arg Lys Ala Ile Gln Glu Leu Gln Cys Gly Asn Glu Lys Val Ser Leu145 150 155 160Thr Leu Glu Glu Gly Ile Gln Asp Asn Lys Asp Leu Ile Lys Glu Asn 165 170 175Asn Ala Thr Arg His Leu Cys Asn Leu Leu Lys Glu Thr Cys Ala Arg 180 185 190Ser Ala Glu Lys Thr Lys Lys Tyr Glu Tyr Glu Arg Glu Glu Thr Arg 195 200 205Gln Val Tyr Met Asp Leu Asn Ser Asn Ile Glu Lys Met Ile Thr Ala 210 215 220Phe Glu Glu Leu Arg Val Gln Ala Glu Asn Ser Arg Leu Glu Met His225 230 235 240Phe Lys Leu Lys Glu Asp Tyr Glu Lys Ile Gln His Leu Glu Gln Glu 245 250 255Tyr Lys Lys Glu Ile Asn Asp Lys Glu Lys Gln Val Ser Leu Leu Leu 260 265 270Ile Gln Ile Thr Glu Lys Glu Asn Lys Met Lys Asp Leu Thr Phe Leu 275 280 285Leu Glu Glu Ser Arg Asp Lys Val Asn Gln Leu Glu Glu Lys Thr Lys 290 295 300Leu Gln Ser Glu Asn Leu Lys Gln Ser Ile Glu Lys Gln His His Leu305 310 315 320Thr Lys Glu Leu Glu Asp Ile Lys Val Ser Leu Gln Arg Ser Val Ser 325 330 335Thr Gln Lys Ala Leu Glu Glu Asp Leu Gln Ile Ala Thr Asn Thr Ile 340 345 350Cys Gln Leu Thr Glu Glu Lys Asp Thr Gln Met Glu Glu Ser Asn Lys 355 360 365Ala Arg Ala Ala His Ser Phe Val Val Thr Glu Phe Glu Thr Thr Val 370 375 380Cys Ser Leu Glu Glu Leu Leu Arg Thr Glu Gln Gln Arg Leu Glu Asn385 390 395 400Tyr Glu Asp Gln Leu Ile Ile Leu Thr Met Glu Leu Gln Lys Thr Ser 405 410 415Ser Glu Leu Glu Glu Met Thr Lys Leu Thr Asn Asn Lys Glu Val Glu 420 425 430Leu Glu Glu Leu Lys Lys Val Leu Gly Glu Lys Glu Thr Leu Leu Tyr 435 440 445Asp Asn Lys Gln Phe Glu Lys Ile Ala Glu Glu Leu Lys Gly Thr Glu 450 455 460Gln Glu Leu Ile Gly Leu Leu Gln Ala Arg Glu Lys Glu Val His Asp465 470 475 480Leu Glu Tyr Ser Tyr Cys His Tyr His Lys Trp Thr Val Leu Pro Lys 485 490 495Arg Gly Gln Arg Pro Lys Leu Ser Ser Lys Arg Glu Leu Lys Asn Thr 500 505 510Glu Tyr Phe Thr Leu Gln Gln Ala Ser Pro Pro Pro Asn Glu Leu Thr 515 520 525Gln Glu Thr Ser Asp Met Thr Leu Glu Leu Lys Asn Gln Gln Glu Asp 530 535 540Ile Ile Asn Asn Lys Lys Gln Glu Glu Arg Met Leu Thr Gln Ile Glu545 550 555 560Asn Leu Gln Glu Thr Glu Thr Gln Leu Arg Asn Glu Leu Glu Tyr Val 565 570 575Arg Glu Glu Leu Lys Gln Lys Arg Asp Glu Val Lys Cys Lys Leu Asp 580 585 590Lys Ser Glu Glu Asn Cys Asn Asn Leu Arg Lys Gln Val Glu Asn Lys 595 600 605Asn Lys Tyr Ile Glu Glu Leu Gln Gln Glu Asn Lys Ala Leu Lys Lys 610 615 620Lys Gly Thr Ala Glu Ser Lys Gln Leu Asn Val Tyr Glu Ile Lys Val625 630 635 640Asn Lys Leu Glu Leu Glu Leu Glu Ser Ala Lys Gln Lys Phe Gly Glu 645 650 655Ile Thr Asp Thr Tyr Gln Lys Glu Ile Glu Asp Lys Lys Ile Ser Glu 660 665 670Glu Asn Leu Leu Glu Glu Val Glu Lys Ala Lys Val Ile Ala Asp Glu 675 680 685Ala Val Lys Leu Gln Lys Glu Ile Asp Lys Arg Cys Gln His Lys Ile 690 695 700Ala Glu Met Val Ala Leu Met Glu Lys His Lys His Gln Tyr Asp Lys705 710 715 720Ile Ile Glu Glu Arg Asp Ser Glu Leu Gly Leu Tyr Lys Ser Lys Glu 725 730 735Gln Glu Gln Ser Ser Leu Arg Ala Ser Leu Glu Ile Glu Leu Ser Asn 740 745 750Leu Lys Ala Glu Leu Leu Ser Val Lys Lys Gln Leu Glu Ile Glu Arg 755 760 765Glu Glu Lys Glu Lys Leu Lys Arg Glu Ala Lys Glu Asn Thr Ala Thr 770 775 780Leu Lys Glu Lys Lys Asp Lys Lys Thr Gln Thr Phe Leu Leu Glu Thr785 790 795 800Pro Asp Ile Tyr Trp Lys Leu Asp Ser Lys Ala Val Pro Ser Gln Thr 805 810 815Val Ser Arg Asn Phe Thr Ser Val Asp His Gly Ile Ser Lys Asp Lys 820 825 830Arg Asp Tyr Leu Trp Thr Ser Ala Lys Asn Thr Leu Ser Thr Pro Leu 835 840 845Pro Lys Ala Tyr Thr Val Lys Thr Pro Thr Lys Pro Lys Leu Gln Gln 850 855 860Arg Glu Asn Leu Asn Ile Pro Ile Glu Glu Ser Lys Lys Lys Arg Lys865 870 875 880Met Ala Phe Glu Phe Asp Ile Asn Ser Asp Ser Ser Glu Thr Thr Asp 885 890 895Leu Leu Ser Met Val Ser Glu Glu Glu Thr Leu Lys Thr Leu Tyr Arg 900 905 910Asn Asn Asn Pro Pro Ala Ser His Leu Cys Val Lys Thr Pro Lys Lys 915 920 925Ala Pro Ser Ser Leu Thr Thr Pro Gly Ser Thr Leu Lys Phe Gly Ala 930 935 940Ile Arg Lys Met Arg Glu Asp Arg Trp Ala Val Ile Ala Lys Met Asp945 950 955 960Arg Lys Lys Lys Leu Lys Glu Ala Glu Lys Leu Phe Val 965 970 25 180 PRT Homo sapiens 25Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala65 70 75 80Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met145 150 155 160Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175Gly Gln Arg Arg 180 26 1142 PRT Homo sapiens 26Met Gly Asp Lys Asp Met Pro Thr Ala Gly Met Pro Ser Leu Leu Gln 1 5 10 15Ser Ser Ser Glu Ser Pro Gln Ser Cys Pro Glu Gly Glu Asp Ser Gln 20 25 30Ser Pro Leu Gln Ile Pro Gln Ser Ser Pro Glu Ser Asp Asp Thr Leu 35 40 45Tyr Pro Leu Gln Ser Pro Gln Ser Arg Ser Glu Gly Glu Asp Ser Ser 50 55 60Asp Pro Leu Gln Arg Pro Pro Glu Gly Lys Asp Ser Gln Ser Pro Leu65 70 75 80Gln Ile Pro Gln Ser Ser Pro Glu Gly Asp Asp Thr Gln Ser Pro Leu 85 90 95Gln Asn Ser Gln Ser Ser Pro Glu Gly Lys Asp Ser Leu Ser Pro Leu 100 105 110Glu Ile Ser Gln Ser Pro Pro Glu Gly Glu Asp Val Gln Ser Pro Leu 115 120 125Gln Asn Pro Ala Ser Ser Phe Phe Ser Ser Ala Leu Leu Ser Ile Phe 130 135 140Gln Ser Ser Pro Glu Ser Ile Gln Ser Pro Phe Glu Gly Phe Pro Gln145 150 155 160Ser Val Leu Gln Ile Pro Val Ser Ala Ala Ser Ser Ser Thr Leu Val 165 170 175Ser Ile Phe Gln Ser Ser Pro Glu Ser Thr Gln Ser Pro Phe Glu Gly 180 185 190Phe Pro Gln Ser Pro Leu Gln Ile Pro Val Ser Arg Ser Phe Ser Ser 195 200 205Thr Leu Leu Ser Ile Phe Gln Ser Ser Pro Glu Arg Ser Gln Arg Thr 210 215 220Ser Glu Gly Phe Ala Gln Ser Pro Leu Gln Ile Pro Val Ser Ser Ser225 230 235 240Ser Ser Ser Thr Leu Leu Ser Leu Phe Gln Ser Ser Pro Glu Arg Thr 245 250 255Gln Ser Thr Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile Pro Val 260 265 270Ser Arg Ser Phe Ser Ser Thr Leu Leu Ser Ile Phe Gln Ser Ser Pro 275 280 285Glu Arg Thr Gln Ser Thr Phe Glu Gly Phe Ala Gln Ser Pro Leu Gln 290 295 300Ile Pro Val Ser Pro Ser Phe Ser Ser Thr Leu Val Ser Ile Phe Gln305 310 315 320Ser Ser Pro Glu Arg Thr Gln Ser Thr Phe Glu Gly Phe Pro Gln Ser 325 330 335Pro Leu Gln Ile Pro Val Ser Ser Ser Phe Ser Ser Thr Leu Leu Ser 340 345 350Leu Phe Gln Ser Ser Pro Glu Arg Thr Gln Ser Thr Phe Glu Gly Phe 355 360 365Pro Gln Ser Pro Leu Gln Ile Pro Gly Ser Pro Ser Phe Ser Ser Thr 370 375 380Leu Leu Ser Leu Phe Gln Ser Ser Pro Glu Arg Thr His Ser Thr Phe385 390 395 400Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile Pro Met Thr Ser Ser Phe 405 410 415Ser Ser Thr Leu Leu Ser Ile Leu Gln Ser Ser Pro Glu Ser Ala Gln 420 425 430Ser Ala Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile Pro Val Ser 435 440 445Ser Ser Phe Ser Tyr Thr Leu Leu Ser Leu Phe Gln Ser Ser Pro Glu 450 455 460Arg Thr His Ser Thr Phe Glu Gly Phe Pro Gln Ser Pro Leu Gln Ile465 470 475 480Pro Val Ser Ser Ser Ser Ser Ser Ser Thr Leu Leu Ser Leu Phe Gln 485 490 495Ser Ser Pro Glu Cys Thr Gln Ser Thr Phe Glu Gly Phe Pro Gln Ser 500 505 510Pro Leu Gln Ile Pro Gln Ser Pro Pro Glu Gly Glu Asn Thr His Ser 515 520 525Pro Leu Gln Ile Val Pro Ser Leu Pro Glu Trp Glu Asp Ser Leu Ser 530 535 540Pro His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Leu Ser545 550 555 560Pro His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Leu Ser 565 570 575Pro His Tyr Phe Pro Gln Ser Pro Gln Gly Glu Asp Ser Leu Ser Pro 580 585 590His Tyr Phe Pro Gln Ser Pro Pro Gln Gly Glu Asp Ser Met Ser Pro 595 600 605Leu Tyr Phe Pro Gln Ser Pro Leu Gln Gly Glu Glu Phe Gln Ser Ser 610 615 620Leu Gln Ser Pro Val Ser Ile Cys Ser Ser Ser Thr Pro Ser Ser Leu625 630 635 640Pro Gln Ser Phe Pro Glu Ser Ser Gln Ser Pro Pro Glu Gly Pro Val 645 650 655Gln Ser Pro Leu His Ser Pro Gln Ser Pro Pro Glu Gly Met His Ser 660 665 670Gln Ser Pro Leu Gln Ser Pro Glu Ser Ala Pro Glu Gly Glu Asp Ser 675 680 685Leu Ser Pro Leu Gln Ile Pro Gln Ser Pro Leu Glu Gly Glu Asp Ser 690 695 700Leu Ser Ser Leu His Phe Pro Gln Ser Pro Pro Glu Trp Glu Asp Ser705 710 715 720Leu Ser Pro Leu His Phe Pro Gln Phe Pro Pro Gln Gly Glu Asp Phe 725 730 735Gln Ser Ser Leu Gln Ser Pro Val Ser Ile Cys Ser Ser Ser Thr Ser 740 745 750Leu Ser Leu Pro Gln Ser Phe Pro Glu Ser Pro Gln Ser Pro Pro Glu 755 760 765Gly Pro Ala Gln Ser Pro Leu Gln Arg Pro Val Ser Ser Phe Phe Ser 770 775 780Tyr Thr Leu Ala Ser Leu Leu Gln Ser Ser His Glu Ser Pro Gln Ser785 790 795 800Pro Pro Glu Gly Pro Ala Gln Ser Pro Leu Gln Ser Pro Val Ser Ser 805 810 815Phe Pro Ser Ser Thr Ser Ser Ser Leu Ser Gln Ser Ser Pro Val Ser 820 825 830Ser Phe Pro Ser Ser Thr Ser Ser Ser Leu Ser Lys Ser Ser Pro Glu 835 840 845Ser Pro Leu Gln Ser Pro Val Ile Ser Phe Ser Ser Ser Thr Ser Leu 850 855 860Ser Pro Phe Ser Glu Glu Ser Ser Ser Pro Val Asp Glu Tyr Thr Ser865 870 875 880Ser Ser Asp Thr Leu Leu Glu Ser Asp Ser Leu Thr Asp Ser Glu Ser 885 890 895Leu Ile Glu Ser Glu Pro Leu Phe Thr Tyr Thr Leu Asp Glu Lys Val 900 905 910Asp Glu Leu Ala Arg Phe Leu Leu Leu Lys Tyr Gln Val Lys Gln Pro 915 920 925Ile Thr Lys Ala Glu Met Leu Thr Asn Val Ile Ser Arg Tyr Thr Gly 930 935 940Tyr Phe Pro Val Ile Phe Arg Lys Ala Arg Glu Phe Ile Glu Ile Leu945 950 955 960Phe Gly Ile Ser Leu Arg Glu Val Asp Pro Asp Asp Ser Tyr Val Phe 965 970 975Val Asn Thr Leu Asp Leu Thr Ser Glu Gly Cys Leu Ser Asp Glu Gln 980 985 990Gly Met Ser Gln Asn Arg Leu Leu Ile Leu Ile Leu Ser Ile Ile Phe 995 1000 1005Ile Lys Gly Thr Tyr Ala Ser Glu Glu Val Ile Trp Asp Val Leu Ser 1010 1015 1020Gly Ile Gly Val Arg Ala Gly Arg Glu His Phe Ala Phe Gly Glu Pro1025 1030 1035 1040Arg Glu Leu Leu Thr Lys Val Trp Val Gln Glu His Tyr Leu Glu Tyr 1045 1050 1055Arg Glu Val Pro Asn Ser Ser Pro Pro Arg Tyr Glu Phe Leu Trp Gly 1060 1065 1070Pro Arg Ala His Ser Glu Val Ile Lys Arg Lys Val Val Glu Phe Leu 1075 1080 1085Ala Met Leu Lys Asn Thr Val Pro Ile Thr Phe Pro Ser Ser Tyr Lys 1090 1095 1100Asp Ala Leu Lys Asp Val Glu Glu Arg Ala Gln Ala Ile Ile Asp Thr1105 1110 1115 1120Thr Asp Asp Ser Thr Ala Thr Glu Ser Ala Ser Ser Ser Val Met Ser 1125 1130 1135Pro Ser Phe Ser Ser Glu 1140 27 188 PRT Homo sapiens 27Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala Gln 1 5 10 15Ile Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30Ser Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr 35 40 45Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys 50 55 60Ala Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe Gln65 70 75 80Gly Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu Arg 85 90 95Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met 100 105 110Pro Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro Glu 115 120 125Ala Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly Lys 130 135 140Pro Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Pro Lys Arg Gly145 150 155 160Glu His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Ile 165 170 175Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu 180 185 28 188 PRT Homo sapiens 28Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Arg Asp Asp Ala Gln 1 5 10 15Ile Ser Glu Lys Leu Arg Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe 20 25 30Ser Lys Lys Glu Trp Glu Lys Met Lys Ser Ser Glu Lys Ile Val Tyr 35 40 45Val Tyr Met Lys Leu Asn Tyr Glu Val Met Thr Lys Leu Gly Phe Lys 50 55 60Val Thr Leu Pro Pro Phe Met Arg Ser Lys Arg Ala Ala Asp Phe His65 70 75 80Gly Asn Asp Phe Gly Asn Asp Arg Asn His Arg Asn Gln Val Glu Arg 85 90 95Pro Gln Met Thr Phe Gly Ser Leu Gln Arg Ile Phe Pro Lys Ile Met 100 105 110Pro Lys Lys Pro Ala Glu Glu Glu Asn Gly Leu Lys Glu Val Pro Glu 115 120 125Ala Ser Gly Pro Gln Asn Asp Gly Lys Gln Leu Cys Pro Pro Gly Asn 130 135 140Pro Ser Thr Leu Glu Lys Ile Asn Lys Thr Ser Gly Pro Lys Arg Gly145 150 155 160Lys His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Val 165 170 175Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu 180 185 29 309 PRT Homo sapiens 29Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu 1 5 10 15Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Thr 20 25 30Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr 35 40 45Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe 50 55 60Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser65 70 75 80Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser 85 90 95Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe 100 105 110Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met 115 120 125Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe 130 135 140Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys145 150 155 160Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly 165 170 175Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr 180 185 190Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His 195 200 205Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr 210 215 220Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr225 230 235 240Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp 245 250 255Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala 260 265 270Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala 275 280 285Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu 290 295 300Glu Glu Glu Gly Val305 30 314 PRT Homo sapiens 30Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5 10 15Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp65 70 75 80Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135 140Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu145 150 155 160Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly225 230 235 240Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 245 250 255Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260 265 270Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300His Glu Trp Val Leu Arg Glu Gly Glu Glu305 310

Claims

1. A method of diagnosing lung, cancer, comprising:contacting a biological sample comprising lung, cells from a subject with a probe that hybridizes under high-stringency hybridization conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:4, or a full-length complement thereof, wherein the high-stringency hybridization conditions are hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4(pH7), 0.5% SDS, 2 mM EDTA) and wherein SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid, and determining expression of the nucleic acid molecule or its full-length complement in the sample by measuring hybridization of the probe to the nucleic acid molecule or its full-length complement, wherein the expression of the nucleic acid molecule or its full-length complement is diagnostic for lung cancer in the sample.

2. The method of claim 1, wherein the probe is a second nucleic acid molecule comprising the nucleic acid molecule that selectively binds to a full-length complement of the nucleic acid molecule.

3. The method of claim 1, wherein the expression of the nucleic acid molecule in the sample is determined by determining the binding between the probe and the nucleic acid molecule, or the fragment of the nucleic acid molecule produced by nucleic acid amplification of the nucleic acid set forth as SEQ ID NO:4.

4. The method of claim 1, further comprising a reverse transcription polymerase chain reaction (RT-PCR) amplification.