WO1997025431A1

WO1997025431A1 - Compositions and methods for the treatment and diagnosis of cancer

Info

Publication number: WO1997025431A1
Application number: PCT/US1997/000398
Authority: WO
Inventors: Tony N. Frudakis; John M. Smith
Original assignee: Corixa Corporation
Priority date: 1996-01-10
Filing date: 1997-01-10
Publication date: 1997-07-17
Also published as: AU1575697A

Abstract

Compositions and methods for the detection and therapy of cancer are disclosed. The compounds provided include human endogenous retroviral sequences that are preferentially expressed in tumor tissue, as well as polypeptides encoded by such nucleotide sequences. Vaccines and pharmaceutical compositions comprising such compounds are also provided and may be used, for example, for the prevention and treatment of cancer. The polypeptides may also be used for the production of antibodies, which are useful for diagnosing and monitoring the progression of cancer in a patient.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF CANCER

Technical Field

The present invention relates generally to the detection and therapy of cancer. The invention is more specifically related to nucleotide sequences that are preferentially expressed in a tumor tissue and to polypeptides encoded by such nucleotide sequences. The invention is more particularly related to nucleotide sequences comprising at least a portion of a human endogenous retroviral sequence that is preferentially expressed in a tumor tissue, and to polypeptides encoded by such nucleotide sequences. The nucleotide sequences and polypeptides may be used in vaccines and pharmaceutical compositions for the prevention and treatment of cancer. The polypeptides may also be used for the production of compounds, such as antibodies, useful for diagnosing and monitoring the progression of cancer in a patient.

Background ofthe Invention

In recent years, considerable research has been directed to the identification of tumor markers, which may be useful for the diagnosis of particular cancers, for predicting the outcome of the disease or for developing a therapy in a patient-specific manner. Such research has generally focused on oncogenes, which are normal cellular genes whose expression has been altered (e.g., by gene amplification, increased transcription, alteration of mRNA splicing or mutation within the coding region) such that otherwise normal cells assume neoplastic growth behavior. To date, however, the established markers have had a limited utility, and their use often leads to a result that is difficult to interpret.

Management of cancer currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. However, current diagnostic methods often fail to detect a cancer until the disease has progressed to a state that is difficult to treat, and existing treatments often have serious side effects. The high mortality observed among cancer patients indicates that improvements are needed in the diagnosis and treatment ofthe disease. Accordingly, there is a need in the art for improved tumor markers, and methods for therapy and diagnosis of cancer. The present invention fulfills these needs and further provides other related advantages. Summary of the Invention

Briefly stated, this invention provides compositions and methods for the diagnosis and therapy of cancer. In one aspect, isolated DNA molecules are provided, comprising: (a) a human endogenous retroviral sequence, wherein the retroviral sequence is preferentially expressed in a tumor tissue; (b) a variant of the human endogenous retroviral sequence that contains one or more nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% (preferably no more than 5%) of the nucleotide positions, such that the antigenic and/or immunogenic properties of the polypeptide encoded by the human endogenous retroviral sequence are retained; or (c) a nucleotide sequence encoding an epitope of a polypeptide encoded by at least one of the above sequences. Isolated DNA and RNA molecules comprising a nucleotide sequence complementary to a DNA molecule as described above are also provided. In another aspect, the present invention provides an isolated DNA molecule encoding an epitope of a polypeptide, the polypeptide being encoded by: (a) a nucleotide sequence transcribed from the sequence of SEQ ID NO:l l ; or (b) a variant of the nucleotide sequence that contains one or more nucleotide substitutions, deletions, insertions and/or modifications at not more than 20% of the nucleotide positions, such that the antigenic and/or immunogenic properties of the polypeptide encoded by the nucleotide sequence are retained. Isolated DNA and RNA molecules comprising a nucleotide sequence complementary to a DNA molecule as described above are also provided.

In related aspects, the present invention provides recombinant expression vectors comprising a DNA molecule as described above and host cells transformed or transfected with such expression vectors.

In further aspects, polypeptides, comprising an amino acid sequence encoded by a DNA molecule as described above, and monoclonal antibodies that bind to such polypeptides are provided. In another aspect, methods are provided for determining the presence of a cancer in a patient. In one embodiment, the method comprises detecting, within a biological sample obtained from a patient, a polypeptide as described above. In another embodiment, the method comprises detecting, within a biological sample, an RNA molecule encoding a polypeptide as described above. In yet another embodiment, the method comprises (a) intradermally injecting a patient with a polypeptide as described above; and (b) detecting an immune response on the patient's skin and therefrom detecting the presence of a cancer in the patient. In a related aspect, diagnostic kits useful in the determination of breast cancer are provided. The diagnostic kits generally comprise one or more monoclonal antibodies as described above, and a detection reagent. Within another related aspect, the diagnostic kit comprises a first polymerase chain reaction primer and a second polymerase chain reaction primer, the first and second primers each comprising at least about 10 contiguous nucleotides of an RNA molecule encoding a polypeptide as described above. Within yet another related aspect, the diagnostic kit comprises at least one oligonucleotide probe, the probe comprising at least about 15 contiguous nucleotides of a DNA molecule as described above. In another aspect, the present invention provides methods for monitoring the progression of a cancer in a patient. In one embodiment, the method comprises: (a) detecting an amount, in a biological sample, of a polypeptide as described above; (b) subsequently repeating step (a); and (c) comparing the amounts of polypeptide detected in steps (a) and (b), and therefrom monitoring the progression of cancer in the patient. In another embodiment, the method comprises (a) detecting an amount, within a biological sample, of an RNA molecule encoding a polypeptide as described above; (b) subsequently repeating step (a); and (c) comparing the amounts of RNA molecules detected in steps (a) and (b), and therefrom monitoring the progression of cancer in the patient.

In other aspects, pharmaceutical compositions, which comprise a polypeptide as described above and a physiologically acceptable carrier, and vaccines, which comprise a polypeptide as described above and an immune response enhancer are provided.

In related aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as described above.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

Brief Description ofthe Drawings

Figure 1 shows the differential display PCR products, separated by gel electrophoresis. obtained from cDNA prepared from normal breast tissue (lanes 1 and 2) and from cDNA prepared from breast tumor tissue from the same patient (lanes 3 and 4). The arrow indicates the band corresponding to B 18 Ag 1.

Figure 2 is a northern blot comparing the level of B18Agl mRNA in breast tumor tissue (lane 1) with the level in normal breast tissue. Figure 3 shows the level of B18Agl mRNA in breast tumor tissue compared to that in various normal and non-breast tumor tissues as determined by RNase protection assays.

Figure 4 is a genomic clone map showing the location of additional retroviral sequences (provided in SEQ ID NO:3 - SEQ ID NO: 10) relative to B18Agl.

Figures 5A and 5B show the sequencing strategy, genomic organization, and predicted open reading frame for the retroviral element containing B18Agl .

Figure 6 shows the nucleotide sequence of the representative human endogenous retroviral element BlδAgl.

Detailed Description ofthe Invention

As noted above, the present invention is generally directed to compositions and methods for the diagnosis, monitoring and therapy of cancer. The compositions described herein include polypeptides, nucleic acid sequences and antibodies. Polypeptides of the present invention generally comprise at least a portion of a protein that is encoded by a human endogenous retroviral sequence, wherein the human endogenous retroviral sequence is expressed at substantially greater levels in a human tumor tissue than in normal tissue (i.e., the level of RNA encoding the polypeptide is at least two fold higher, and preferably at least five fold higher, in a tumor tissue than in normal tissue). Such sequences are said to be "preferentially expressed" in a tumor tissue. Any cancer characterized by increased expression of a human endogenous retroviral sequence within a tumor may be detected and/or treated according to the present invention. Representative cancers include breast cancer, prostate cancer, leukemia, lymphoma and Kaposi's sarcoma. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins (and epitopes thereof) encoded by a human endogenous retroviral sequence.

Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes a polypeptide as described above, or that is complementary to such a sequence. Antibodies are generally immune system proteins, or fragments thereof, that are capable of binding to a portion of a polypeptide as described above. Antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. Polypeptides within the scope of this invention include, but are not limited to, polypeptides (and epitopes thereof) encoded by the human endogenous retroviral sequences described herein. Such sequences include the sequence designated B18Agl (SEQ ID NO:l) as well as other sequences such as those recited in SEQ ID NO:3-SEQ ID NO: 10, found within the retroviral genome containing B 18Agl (SEQ ID NO: 1 1). B18Agl has homology to the P30 gene of the endogenous human retroviral element S71, as described in Werner et al., Virology 174:225-22 (1990). As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins encoded by a human endogenous retroviral element. A polypeptide comprising an epitope of a human endogenous retroviral element may consist entirely of the epitope, or may contain additional sequences. The additional sequences may be derived from the native protein or may be heterologous, and such sequences may (but need not) possess immunogenic or antigenic properties.

An "epitope," as used herein is a portion of a polypeptide that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Epitopes may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An epitope of a polypeptide is a portion that reacts with such antisera and/or T-cells at a level that is similar to the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis. Polypeptides comprising an epitope of a polypeptide that is preferentially expressed in a tumor tissue (with or without additional amino acid sequence) are within the scope ofthe present invention.

The compositions and methods of the present invention also encompass variants of the above polypeptides and nucleic acid sequences encoding such polypeptides. A polypeptide "variant," as used herein, is a polypeptide that differs from the native polypeptide in substitutions and/or modifications such that the antigenic and/or immunogenic properties of the polypeptide are retained. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antisera and/or T-cells as described above. Nucleic acid variants may contain one or more substitutions, deletions, insertions and/or modifications such that the antigenic and/or immunogenic properties of the encoded polypeptide are retained. One preferred variant of a human endogenous retroviral sequence, or an epitope thereof, is a variant that contains nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% of the nucleotide positions within the native polypeptide sequence.

Preferably, a variant contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (l) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenic or antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N- terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region. Human endogenous retroviral sequences that are expressed at substantially greater levels in a human tumor tissue than in normal tissue may be prepared using any of several techniques. For example, the human endogenous retroviral sequence designated B18Agl (Figure 6 and SEQ ID NO:l) may be cloned on the basis of its breast tumor specific expression, using differential display PCR. This technique compares the amplified products from poly A+ or total RNA template prepared from normal and breast tumor tissue. cDNA may be prepared by reverse transcription of RNA using a (dT)₁₂AG primer. Following amplification using the primer CCTCAACCTC (SEQ ID NO: 13), a band corresponding to an amplified product specific to the tumor RNA may be cut out from a silver stained gel and subcloned into a suitable vector (e.g., the T-vector, Novagen, Madison, WI).

Alternatively, the B18Agl gene (or a portion thereof) may be amplified from human genomic DNA, or from breast tumor cDNA, via polymerase chain reaction. For this approach. B18Agl sequence-specific primers may be designed based on the sequence provided in SEQ ID NOT, and may be purchased or synthesized. One suitable primer pair for amplification from breast tumor cDNA is (5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO:14) and (5'CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 15). An amplified portion of B18Agl may then be used to isolate the full length gene from a human genomic DNA library or from a breast tumor cDNA library, using well known techniques such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1989). Other sequences within the retroviral genome containing B18Agl, such as those recited in SEQ ID NO:3 - SEQ ID NO:10, may be similarly prepared by screening human genomic libraries using BlδAgl -specific sequences as probes.

Other human endogenous retroviral sequences that are expressed at substantially greater levels in a human tumor tissue than in normal tissue may be prepared using methods known to those of ordinary skill in the art. For example, such sequences may be identified using low stringency hybridization, followed by PCR to identify conserved motifs. The level of expression in tumor tissue may generally be evaluated using the methods described herein, such as PCR and Northern blot analysis.

Recombinant polypeptides encoded by the DNA sequences described above may be readily prepared from the DNA sequences. For example, supernatants from suitable host/vector systems which secrete recombinant polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.

Such techniques may also be used to prepare polypeptides comprising epitopes or variants of the native polypeptides. For example, variants of a native polypeptide may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides. Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc., Foster City, CA, and may be operated according to the manufacturer's instructions. In specific embodiments, polypeptides of the present invention encompass polypeptides encoded by a human endogenous retroviral sequence that is expressed at substantially greater levels in a human tumor tissue than in normal tissue (such as the sequence recited in SEQ ID NO:l), variants of such polypeptides that are encoded by DNA molecules containing one or more nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% of the nucleotide positions, and epitopes of the above polypeptides. Polypeptides within the scope of the present invention also include polypeptides (and epitopes thereof) encoded by DNA sequences that hybridize to the above sequences under stringent conditions, wherein the DNA sequences are at least 80% identical in overall sequence to the sequence recited in SEQ ID NO: 1 , and wherein RNA corresponding to said nucleotide sequence is expressed at a greater level in human tumor tissue than in the corresponding normal tissue. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing overnight at 65°C in 6X SSC, 0.2% SDS; followed by washing twice at 65° C for 30 minutes each with IX SSC, 0.1% SDS, and then washing twice at 65°C for 30- 60 minutes each with 0.1X SSC, 0.1% SDS. DNA molecules according to the present invention include molecules that encode any ofthe above polypeptides.

In another aspect of the present invention, antibodies are provided. Such antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. (5:51 1-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred. Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection ofthe hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

Antibodies may be used, for example, in methods for detecting a cancer (such as breast cancer, prostate cancer, leukemia, lymphoma or Kaposi's sarcoma) in a patient. Such methods involve using one or more antibodies to detect the presence or absence of a polypeptide as described herein in a suitable biological sample. As used herein, suitable biological samples include tumor or normal tissue biopsy, mastectomy, blood, lymph node, serum and urine samples or other tissue, homogenate or extract thereof, obtained from a patient. It will be evident to those of ordinary skill in the art that, following detection of a polypeptide within a non-biopsy sample, additional tumor markers may be employed to identify the particular type of cancer.

There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect polypeptide markers in a sample. See, e.g., Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react with antibody. The presence of antibody on the membrane may then be detected using a suitable detection reagent, as described below.

In another embodiment, the assay involves the use of an antibody immobilized on a solid support to bind to the polypeptide and remove it from the remainder of the sample. The bound polypeptide may then be detected using a second antibody that binds to the binding partner/polypeptide complex and contains a reporter group. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding ofthe labeled polypeptide to the antibody is indicative ofthe reactivity of the sample with the immobilized antibody, and as a result is indicative of the concentration of polypeptide in the sample.

The solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose filter or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681. The antibody may be immobilized on the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the antibody, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of antibody ranging from about 10 ng to about 1 μg, and preferably about 100-200 ng, is sufficient to immobilize an adequate amount of polypeptide.

Covalent attachment of antibody to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the antibody. For example, the antibody may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g. , Pierce Immunotechnology Catalog and Handbook (1991) at A12-A13).

In certain embodiments for detection of polypeptide in a sample, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the biological sample, such that the polypeptide within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a second antibody (containing a reporter group) capable of binding to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is that period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with breast cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and bio tin. The conjugation of antibody to reporter group may be achieved using standard methods known to those of ordinary skill in the art.

The second antibody is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound second antibody is then removed and bound second antibody is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. To determine the presence or absence of a cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value may be considered positive for a cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic- Science for Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer. In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose. In the flow-through test, the polypeptide within the sample binds to the immobilized antibody as the sample passes through the membrane. A second, labeled antibody then binds to the antibody-polypeptide complex as a solution containing the second antibody flows through the membrane. The detection of bound second antibody may then be performed as described above. In the strip test format, one end of the membrane to which antibody is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second antibody and to the area of immobilized antibody. Concentration of second antibody at the area of immobilized antibody indicates the presence of breast cancer. Typically, the concentration of second antibody at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about lμg, and more preferably from about 50 ng to about lμg. Such tests can typically be performed with a very small amount of biological sample.

The presence or absence of a cancer in a patient may also be determined by evaluating the level of mRNA encoding a polypeptide of the present invention within the biological sample (e.g., a biopsy, mastectomy and/or blood sample from a patient) relative to a predetermined cut-off value. Such an evaluation may be achieved using any of a variety of methods known to those of ordinary skill in the art such as, for example, in situ hybridization and amplification by polymerase chain reaction. For example, polymerase chain reaction may be used to amplify sequences from cDNA prepared from RNA that is isolated from one of the above biological samples. Sequence-specific primers for use in such amplification may be designed based on a cDNA or genomic sequence, such as a sequence provided in SEQ ID NO: 1 or SEQ ID NO:3 - SEQ ID NO: 10, and may be purchased or synthesized. In the case of B18Agl, as noted herein, one suitable primer pair is (5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 14) and (5'CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 15). The PCR reaction products may then be separated and visualized using gel electrophoresis, according to methods well known to those of ordinary skill in the art. Amplification is typically performed on samples obtained from matched pairs of tissue (tumor and non-tumor tissue from the same individual) or from unmatched pairs of tissue (tumor and non-tumor tissue from different individuals). The amplification reaction is preferably performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the tumor sample as compared to the same dilution of the non-tumor sample is considered positive. Conventional RT-PCR protocols using agarose and ethidium bromide staining, while important in defining gene specificity do not lend themselves to diagnostic kit development because of the time and effort required in making them quantitative (i.e., construction of saturation and/or titration curves), and their sample throughput. This problem is overcome by the development of procedures such as real time RT-PCR which allows for assays to be performed in single tubes, and in turn can be modified for use in 96 well plate formats. Instrumentation to perform such methodologies are available from ABI/Perkin Elmer. Alternatively, other high throughput assays using labelled probes (e.g., digoxygenin) in combination with labelled (e.g., enzyme fluorescent, radioactive) antibodies to such probes can also be used in the development of 96 well plate assays.

In yet another method for determining the presence or absence of a cancer in a patient, one or more of the polypeptides described above may be used in a skin test. As used herein, a "skin test" is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above. Such injection may be achieved using any suitable device sufficient to contact the polypeptide or polypeptides with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.

The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to a test antigen (i.e., an immunogenic portion of a polypeptide employed, or a variant thereof). The response may measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of a cancer. As noted above, additional tumor markers may be employed, using methods known to those of ordinary skill in the art, to identify the type of cancer present.

The polypeptides of this invention are preferably formulated, for use in a skin test, as pharmaceutical compositions containing at least one polypeptide and a physiologically acceptable carrier, such as water, saline, alcohol, or a buffer. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 μg to 100 μg, preferably from about 10 μg to 50 μg in a volume of 0.1 mL. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80™.

In other aspects of the present invention, the progression and/or response to treatment of a cancer may be monitored by performing any of the above assays over a period of time, and evaluating the change in the level ofthe response (i.e., the amount of polypeptide or mRNA detected or, in the case of a skin test, the extent of the immune response detected). For example, the assays may be performed every 1-2 months for a period of 1-2 years. In general, a cancer is progressing in those patients in whom the level of the response increases over time. In contrast, a cancer is not progressing when the signal detected either remains constant or decreases with time.

In further aspects of the present invention, the compounds described herein may be used for the immunotherapy of a cancer. In these aspects, the compounds (which may be polypeptides, antibodies or nucleic acid molecules) are preferably incorporated into pharmaceutical compositions or vaccines. Pharmaceutical compositions comprise one or more such compounds and a physiologically acceptable carrier. Vaccines may comprise one or more polypeptides and an immune response enhancer, such as an adjuvant or a liposome (into which the compound is incorporated). Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, including one or more separate polypeptides.

Alternatively, a vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retro virus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259: 1745-1749 (1993) and reviewed by Cohen, Science 25P: 1691 -1692 (1993). The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. Any of a variety of adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune responses, such as lipid A, Bordella pertussis or Mycobacterium tuberculosis-deήved proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), alum, biodegradable microspheres, monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants. The above pharmaceutical compositions and vaccines may be used, for example, for the therapy of cancer in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with a cancer. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. To prevent the development of a cancer, a pharmaceutical composition or vaccine comprising one or more polypeptides as described herein (or naked, plasmid or viral vector DNA encoding such a polypeptide) may be administered to a patient. For treating a patient with a cancer, the pharmaceutical composition or vaccine may comprise one or more polypeptides, antibodies or nucleic acid molecules complementary to DNA encoding a polypeptide as described herein (e.g., antisense RNA or antisense deoxyribonucleotide oligonucleotides).

For example, tumor cells that express a polypeptide as described herein may be preferentially killed by administering to a patient a conjugate in which a cytotoxic agent or "prodrug" is linked to antisense RNA, an antisense deoxyribonucleotide oligonucleotide or an antibody that binds to such a polypeptide. As used herein, the term "prodrug" refers to a group that is not itself toxic to the cell, but that can be rendered toxic after the conjugate is directed to the target cell by the addition of a second activating compound, such as an enzyme that can convert the prodrug into an active drug. Any suitable cytotoxic agent (including radionuclides) or prodrug known to those of ordinary skill in the art may be employed in such methods. Suitable prodrugs include boron, doxifluridine, or the prodrug precursor of palytoxin. Routes and frequency of administration, as well as dosage, will vary from individual to individual. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 10 doses may be administered for a 52 week period. Preferably, 6 doses are administered, at intervals of one month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response. Such a response can be monitored by measuring the level of anti-tumor antibodies in a patient or by vaccine- dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. A suitable dose should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non- vaccinated patients.. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 μg to about 5 mg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1 Preparation of B18Agl cDNA and Genomic Clones Using Differential Display RT- PCR

This Example illustrates the preparation of cDNA and genomic DNA molecules encoding B18Agl using a differential display screen.

Tissue samples were prepared from breast tumor and normal tissue of a patient with breast cancer that was confirmed by pathology after removal from the patient. Normal RNA and tumor RNA was extracted from the samples and mRNA was isolated and converted into cDNA using a (dT)_]2AG anchored 3' primer. Differential display PCR was then executed using a randomly chosen primer (CTTCAACCTC) (SEQ ID NO: 16). Amplification conditions were standard buffer containing 1.5 mM MgCl₂, 20 pmol of primer, 500 pmol dNTP, and 1 unit of Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ). Forty cycles of amplification were performed using 94°C denaturation for 30 seconds, 42°C annealing for 1 minute, and 72°C extension for 30 seconds. An RNA fingerprint containing 76 amplified products was obtained. Although the RNA fingerprint of breast tumor tissue was over 98% identical to that of the normal breast tissue, a band was repeatedly observed to be specific to the RNA fingerprint pattern of the tumor. This band was cut out of a silver stained gel and subcloned into the T-vector (Novagen, Madison, WI) and sequenced.

The sequence of the cDNA, referred to as B18Agl , is provided in SEQ ID NO:l . A database search of GENBANK and EMBL revealed that the B18Agl fragment initially cloned is 77% identical to the endogenous human retroviral element S71, which is a truncated retroviral element homologous to the Simian Sarcoma Virus (SSV). S71 contains a complete gag gene, a portion of the pol gene and an LTR-like structure at the 3' terminus (see Werner et al., Virology 174:225-238 (1990)). B18Agl is also 64% identical to SSV in the region corresponding to the P30 (gag) locus. B18Agl contains three separate and incomplete reading frames covering a region which shares considerable homology to a wide variety of gag proteins of retroviruses which infect mammals. In addition, the homology to S71 is not just within the gag gene, but spans several kb of sequence including an LTR.

B18Agl -specific PCR primers were synthesized using computer analysis guidelines. RT-PCR amplification (94°C, 30 seconds; 60°C → 42°C, 30 seconds; 72°C, 30 seconds, for 40 cycles) confirmed that B18Agl represents an actual mRNA sequence present at relatively high levels in the patient's breast tumor tissue. The primers used in amplification were B18Agl-l (CTG CCT GAG CCA CAA ATG) (SEQ ID NO: 17) and B18Agl-4 (CCG GAG GAG GAA GCT AGA GGA ATA) (SEQ ID NO: 18) at a 3.5 mM magnesium concentration and a pH of 8.5, and B18Agl-2 (ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 14) and B18Agl-3 (CCG GTA TCT CCT CGT GGG TATT) (SEQ ID NO: 15) at 2 mM magnesium at pH 9.5. The same experiments showed exceedingly low to nonexistent levels of expression in this patient's normal breast tissue (see Figure 1). RT-PCR experiments were then used to show that B18Agl mRNA is present in nine other breast tumor samples (from Brazilian and American patients) but absent in, or at exceedingly low levels in, the normal breast tissue corresponding to each cancer patient. RT-PCR analysis has also shown that the B18Agl transcript is not present in various normal tissues (including lymph node, myocardium and liver) and present at relatively low levels in PBMC and lung tissue. The presence of B18Agl mRNA in breast tumor samples, and its absence from normal breast tissue, has been confirmed by Northern blot analysis, as shown in Figure 2.

The differential expression of BlδAgl in breast tumor tissue was also confirmed by RNase protection assays. Figure 3 shows the level of B18Agl mRNA in various tissue types as determined in four different RNase protection assays. Lanes 1 - 12 represent various normal breast tissue samples, lanes 13-25 represent various breast tumor samples; lanes 26-27 represent normal prostate samples; lanes 28-29 represent prostate tumor samples; lanes 30-32 represent colon tumor samples; lane 33 represents normal aorta; lane 34 represents normal small intestine; lane 35 represents normal skin, lane 36 represents normal lymph node; lane 37 represents normal ovary; lane 38 represents normal liver; lane 39 represents normal skeletal muscle; lane 40 represents a first normal stomach sample, lane 41 represents a second normal stomach sample; lane 42 represents a normal lung; lane 43 represents normal kidney; and lane 44 represents normal pancreas. Interexperimental comparison was facilitated by including a positive control RNA of known B-actin message abundance in each assay and normalizing the results ofthe different assays with respect to this positive control. RT-PCR and Southern blot analysis has shown the B18Agl locus to be present in human genomic DNA as a single copy endogenous retroviral element. A genomic clone of approximately 12-18 kb was isolated using the initial B18Agl sequence as a probe. Four additional subclones were also isolated by Xbal digestion. Additional retroviral sequences obtained from these clones (located as shown in Figure 4) are shown as SEQ ID NO:3 - SEQ ID NO: 10, where SEQ ID NO:3 shows the location ofthe sequence labeled 10 in Figure 4, SEQ ID NO:4 shows the location of the sequence labeled 1 1-29, SEQ ID NO:5 shows the location of the sequence labeled 3, SEQ ID NO:6 shows the location of the sequence labeled 6, SEQ ID NO:7 shows the location of the sequence labeled 12, SEQ ID NO: 8 shows the location of the sequence labeled 13, SEQ ID NO:9 shows the location of the sequence labeled 14 and SEQ ID NO: 10 shows the location ofthe sequence labeled 11-22. Subsequent studies demonstrated that the 12-18 kb genomic clone contains a retroviral element of about 7.75 kb, as shown in Figures 5A and 5B. The sequence of this retroviral element is shown in SEQ ID NO: 11. The numbered line at the top of Figure 5A represents the sense strand sequence of the retroviral genomic clone. The box below this line shows the position of selected restriction sites. The arrows depict the different overlapping clones used to sequence the retroviral element. The direction of the arrow shows whether the single-pass subclone sequence corresponded to the sense or anti-sense strand. Figure 5B is a schematic diagram ofthe retroviral element containing B18Agl depicting the organization of viral genes within the element. The open boxes correspond to predicted reading frames, starting with a methionine, found throughout the element. Each of the six likely reading frames is shown, as indicated to the left of the boxes, with frames 1-3 corresponding to those found on the sense strand.

Using the cDNA of SEQ ID NO:l as a probe, a longer cDNA was obtained (SEQ ID NO: 12) which contains minor nucleotide differences (less than 1%) compared to the genomic sequence shown in SEQ ID NOT 1.

Example 2 Preparation of B18Agl DNA from Human Genomic DNA

This example illustrates the preparation of B18Agl DNA by amplification from human genomic DNA.

B18Agl DNA may be prepared from 250 ng human genomic DNA using 20 pmol of B18Agl specific primers, 500 pmol dNTPS and 1 unit of Taq DNA polymerase (Perkin Elmer, Branchburg, NJ) using the following amplification parameters: 94°C denaturing for 30 seconds, 30 second 60°C to 42°C touchdown annealing in 2°C increments every two cycles and 72°C extension for 30 seconds. The last increment (a 42°C annealing temperature) should cycle 25 times. Primers (B18Agl-l, B18Agl-2, B18Agl-3 and B18Agl-4) were selected using computer analysis. Primers synthesized were. Primer pairs that may be used are 1+3, 1+4, 2+3, and 2+4.

Following gel electrophoresis, the band corresponding to B18Agl DNA may be excised and cloned into a suitable vector. Example 3 Preparation of B18Agl DNA from Breast Tumor cDNA

This example illustrates the preparation of B18Agl DNA by amplification from human breast tumor cDNA.

First strand cDNA is synthesized from RNA prepared from human breast tumor tissue in a reaction mixture containing 500 ng poly A+ RNA, 200 pmol of the primer (T)12AG (i.e., TTT TTT TTT TTT AG) (SEQ ID NO: 19), IX first strand reverse transcriptase buffer, 6.7 mM DTT, 500 mmol dNTPs, and 1 unit AMV or MMLV reverse transcriptase (from any supplier, such as Gibco-BRL (Grand Island, NY)) in a final volume of 30 μl. After first strand synthesis, the cDNA is diluted approximately 25 fold and 1 μl is used for amplification as described in Example 2. While some primer pairs can result in a heterogeneous population of transcripts, the primers B18Agl-2 (5 'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 14) and B18Agl-3 (5'CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 15) yield a single 151 bp amplification product.

Example 4 Identification of B-cell and T-cell Epitopes of B18Agl

This Example illustrates the identification of B18Agl epitopes. The B18Agl sequence can be screened using a variety of computer algorithms. To determine B-cell epitopes, the sequence can be screened for hydrophobicity and hydrophilicity values using the method of Hopp, Prog. Clin. Biol. Res. 7725:367-77 (1985) or, alternatively, Cease et al., 164 J. Exp. Med. 1779-84 (1986) or Spouge et al., J. Immunol. 138:204-12 (1987). Additional Class II MHC (antibody or B-cell) epitopes can be predicted using programs such as AMPHI (e.g., Margalit et al., J Immunol. 735:2213 (1987)) or the methods of Rothbard and Taylor (e.g., EMBOJ. 7:93 (1988)).

Once peptides (15-20 amino acids long) are identified using these techniques, individual peptides can be synthesized using automated peptide synthesis equipment (available from manufacturers such as Applied BioSystems, Inc., Foster City, CA) and techniques such as Merrifield synthesis. Following synthesis, the peptides can used to screen sera harvested from either normal or breast cancer patients to determine whether patients with breast cancer possess antibodies reactive with the peptides. Presence of such antibodies in breast cancer patient would confirm the immunogenicity of the specific B-cell epitope in question. The peptides can also be tested for their ability to generate a serologic or humoral immune in animals (mice, rats, rabbits, chimps etc.) following immunization in vivo. Generation of a peptide- specific antiserum following such immunization further confirms the immunogenicity of the specific B-cell epitope in question.

To identify T-cell epitopes, the B18Agl sequence can be screened using different computer algorithms which are useful in identifying 8-10 amino acid motifs within the B18Agl sequence which are capable of binding to HLA Class I MHC molecules, (see, e.g., Rammensee et al., Immunogenetics ¥7:178-228 (1995)). Following synthesis such peptides can be tested for their ability to bind to class I MHC using standard binding assays (e.g., Sette et al., J. Immunol. 753:5586-92 (1994)) and more importantly can be tested for their ability to generate antigen reactive cytotoxic T- cells following in vitro stimulation of patient or normal peripheral mononuclear cells using, for example, the methods of Bakker et al., Cancer Res. 55:5330-34 (1995); Visseren et al., J. Immunol. 75 :3991-98 (1995); Kawakami et al., J. Immunol. 754:3961-68 (1995); and Kast et al., J. Immunol. 752:3904-12 (1994). Successful in vitro generation of T-cells capable of killing autologous (bearing the same class I MHC molecules) tumor cells following in vitro peptide stimulation further confirms the immunogenicity of the B18Agl antigen. Furthermore, such peptides may be used to generate murine peptide and B18Agl reactive cytotoxic T-cells following in vivo immunization in mice rendered transgenic for expression of a particular human MHC Class I haplotype (Vitiello et al., J. Exp. Med. 773:1007-15 (1991).

A representative a list of predicted B18Agl B-cell and T-cell epitopes, broken down according to predicted HLA Class I MHC binding antigen, is shown below:

Predicted Th Motifs (B-cell epitopes)

SSGGRTFDDFHRYLLVGI (SEQ ID NO:20) QGAAQKPINLSKXIEVVQGHDE (SEQ ID NO:21) SPGVFLEHLQEAYRIYTPFDLSA (SEQ ID NO:22)

Predicted HLA A2.1 Motifs (T-cell epitopes) YLLVGIQGA (SEQ ID NO:23) GAAQKPINL (SEQ ID NO:24) NLSKXIEVV (SEQ ID NO:25)

EVVQGHDES (SEQ ID NO:26) HLQEAYRIY (SEQ ID NO:27) NLAFVAQAA (SEQ ID NO:28) FVAQAAPDS (SEQ ID NO:29)

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope ofthe invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT Coπxa Corporation

(π) TITLE OF INVENTION COMPOUNDS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF CANCER

(in) NUMBER OF SEQUENCES 29

(iv) CORRESPONDENCE ADDRESS

(A) ADDRESSEE SEED and BERRY LLP

(B) STREET 6300 Columbia Center 701 Fifth Avenue

(C) CITY Seattle

(D) STATE Washington

(E) COUNTRY USA

(F) ZIP 98104-7092

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE Floppy disk

(B) COMPUTER IBM PC compatible

(C) OPERATING SYSTEM PC-DOS/MS-DOS

(D) SOFTWARE Patentin Release #10 Version #130

(vi) CURRENT APPLICATION DATA

(A) APPLICATION NUMBER

(B) FILING DATE 10-JAN-1997

(C) CLASSIFICATION

(viπ) ATTORNEY/AGENT INFORMATION

(A) NAME Maki, David J

(B) REGISTRATION NUMBER 31.392

(C) REFERENCE/DOCKET NUMBER 210121418PC (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 622-4900

(B) TELEFAX: (206) 682-6031

⁽2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 415 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

πGANTGTCA AAAACCTTNT AGGCTATCTC TAAAAGCTGA CTGGTATTCA TTCCAGCAAA 60

ATCCCTCTAG TTTTTGGAGT TTCCTTTTAC TATCTGGGGC TGCCTGAGCC ACAAATGCCA 120

AATTAAGAGC ATGGCTATTT TCGGGGGCTG ACAGGTCAAA AGGGGTGTAA ATCCGATAAG 180

CCTCCTGGAG GTGCTCTAAA AACACTCCTG GTGACTCATC ATGCCCCTGG ACGACTTCAA 240

TCGNCTTAGA CAAGTTTATA GGTTTCTGGG CAGTCCCTGA ATACCCACGA GGAGATACCG 300

GTGGAAATCG TCAAAAGTTC TCCCTCCACT TGAGAAATTT GGGTCCCAAT TAGGTCCCAA 360

TTGGGTCTCT AATCACTATT CCTCTAGCTT CCTCCTCCGG NCTATTGGTT GATGT 415

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 96 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Trp Asp Pro Asn Phe Ser Ser Gly Gly Arg Thr Phe Asp Asp Phe His 1 5 10 15

Arg Tyr Leu Leu Val Gly He Gin Gly Ala Ala Gin Lys Pro He Asn 20 25 30

Leu Ser Lys Xaa He Glu Val Val Gin Gly His Asp Glu Ser Pro Gly 35 40 45

Val Phe Leu Glu His Leu Gin Glu Ala Tyr Arg He Tyr Thr Pro Phe 50 55 60

Asp Lys Ser Ala Pro Glu Asn Ser His Ala Leu Asn Leu Ala Phe Val 65 70 75 80

Ala Gin Ala Ala Pro Asp Ser Lys Arg Lys Leu Gin Lys Leu Glu Gly 85 90 95

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1180 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY- linear

(xi ) SEQUENCE DESCRI PTION : SEQ ID N0 : 3 :

NCNNNNNTTA TGATTACGCC AAGCGNGCAA TTAACCCTCA CTAAAGGGAA CAAMGCTGG 60

AGCTCCACCG CGGTGGCGGC CGCTAGAATC TTCATACCCC GAACTCTTGG GAAAACTTTA 120

ATCAGTCACC TACAGTCTAC CACCCATTTA GGAGGAGCAA AGCTACCTCA GCTCCTCCGG 180

AGCCGTTTTA AGATCCCCCA TCTTCAAAGC CTAACAGATC AAGCAGCTCT CCGGTGCACA 240

ACCTGCGCCC AGGTAAATGC CAAAAAAGGT CCTAAACCCA GCCCAGGCCA CCGTCTCCAA 300

GAAAACTCAC CAGGAGAAAA GTGGGAAATT GACTTTACAG AAGTAAAACC ACACCGGGCT 360

GGGTACAAAT ACCTTCTAGT ACTGGTAGAC ACCTTCTCTG GATGGACTGA AGCATTTGCT 420

ACCAAAAACG AAACTGTCAA TATGGTAGTT AAGTTTTTAC TCAATGAAAT CATCCCTCGA 480

CGTGGGCTGC CTGTTGCCAT AGGGTCTGAT AATGGAACGG CCTTCGCCTT GTCTATAGTT 540

TAATCAGTCA GTAAGGCGTT AAACATTCAA TGGAAGCTCC ATTGTGCCTA TCGACCCAGA 600

GCTCTGGGAA GTAGAACGCA TGAACTGCAC CCTAAAAAAA CACTCTTACA AAATTAATCT 660

TAAAAACCGG TGπAAπGT GπAGTCTCC TTCCCTTAGC CCTACπAGA GTTAAGGTGC 720

ACCCCπACT GGGCTGGGTT CπTACCTTT TGAAATCATN TΓTNGGAAGG GGCTGCCTAT 780

CTTTNCTTAA CTAAAAAANG CCCATTTGGC AAAAATTTCN CAACTAATTT NTACGTNCCT 840

ACGTCTCCCC AACAGGTANA AAAATCTNCT GCCCTTTTCA AGGAACCATC CCATCCATTC 900 CTNAACAAAA GGCCTGCCNT TCTTCCCCCA GTTAACTNTT TπTNπAAA AHCCCAAAA 960

AANGAACCNC CTGCTGGAAA AACNCCCCCC TCCAANCCCC GGCCNAAGNG GAAGGTTCCC 1020

πGAATCCCN CCCCCNCNAA NGGCCCGGAA CCNπAAANT NGπCCNGGG GGTNNGGCCT 1080

AAAAGNCCNA πTGGTAAAC CTANAAATπ πTCπTTNT AAAAACCACN NTπNNππ 1140

TCπAAACAA AACCCTNTTT NTAGNANCNT ATπCCCNCC 1180

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1163 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

TNCTπGATA CCCNAGCGπ CAAπAACCC TCACTAAAGG GAACAAAAGC TGGAGCTCCA 60

CCGCGGTGGC GGCCGCTCTA GAGCTGCGCC TGGATCCCGC CACAGTGAGG AGACCTGAAG 120

ACCAGAGAAA ACACAGCAAG TAGGCCCTπ AAACTACTCA CCTGTGπGT CπCTAATπ 180

AπCTGTlTT AππGπTC CATCATπTA AGGGGπAAA ATCATCπGT TCAGACCTCA 240

GCATATAAAA TGACCCATCT GTAGACCTCA GGCTCCAACC ATACCCCAAG AGπGTCTGG 300

ππGTπAA AπACTGCCA GGTπCAGCT GCAGATATCC CTGGAAGGAA TAπCCAGAT 360 JPθUiL λ9010d01 (0)

ΘLSULS ^• SS3Nα3αNVyiS (3)

PL09 OLθLonu :3 λl (9) s^jLPd aseq ZZU ^:H19N31 (V)

^:S3IiSiy313VyVH3 33N3D03S X)

^:5^:0N 01 03S rJOd N0I1VWH0JNI (Z)

C9II INI NVN01V1WV NN133MM3

OHΪ 1V1331NWV V3333N LL31111113133 33333331311111W333319NV03333N

080T 33199933NV V33311199999NW1N1N1 N33333133N 1JJL1NV33333NNNNMMI

0201 U111VN33V WUJLL33311119993NN1 W91131NV991N13ULL1V WN11V3313

096 3111119NN9 N33333333V 11399NV1NN 99N11N1WN 313339N313 33331N31VN

006 1N3333V913 1NNV333991 V99VH9V111191331119993VWVW19V11111911

Ot78 MlFll I IWi 3993333333 333N3333399VNV11W99133V11W99 NN3333V331

08 33V3331111133N33V11V WV3331N3913N1WNW3 91133N1393 3131W3399

QZL 1LLLN1LLLV V999139NV3 3191193313 91131NV993 WWIIIII I NN131N1N11

099 11NN1133N3 N3131311W V1V9VN31V3 33LLLLV1V1 JJL1919W9V V9 L31191V

009 VL 31131V V9V1199199111191VW3 31LW1333V 119V3111V3 3913WV11V

0179 99V99V3113 311WW11V W9V919V1L 913V19VW11139V13333 V131LLLVW

0817 WWV1V139 3VW919113 91W1113W MM IV9911 LV91V3WV W9V9V3191

02fr 33119V9W9 9V9111191311311399V1 V1331WW1199V331119 V19V913331

63 6e00/Z.6Sfl/13d IZVSVL^β OΛV

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

NNGGTCCNNC TCAAAGTCAN TATAGGGCGA AπGGGTACC GGGCCCCCCC TCGAGGTCGA 60

CGGTATCGAT AAGCπGATA TCGAAπCCT GCAGCCCGGG GGATCCACTA GπCTAGACC 120

AAGAAATGGA GGATTTTAGA GTGACTGATG ATπCTCTAT CATCTGCAGT TAGTAAACAT 180

TCTCCACAGT πATGCAAAA AGTAACAAAA CCACTGCAGA TGACAAACAC TAGGTAACAC 240

ACATACTATC TCCCAAATAC CTACCCACAA GCTCAACAAT ITTAAACTGT TAGGATCACT 300

GGCTCTAATC ACCATGACAT GAGGTCACCA CCAAACCATC AAGCGCTAAA CAGACAGAAT 360

GTπCCACTC CTGATCCACT GTGTGGGAAG AAGCACCGAA CπACCCACT GGGGGGCCTG 420

CNTCANAANA AAAGCCCATG CCCCCGGGTN TNCCTTTNAA CCGGAACGAA TNAACCCACC 480

ATCCCCACAN CTCCTCTGπ CNTGGGCCCT GCATCπGTG GCCTCNTNTN CπTNGGGGA 540

NACNTGGGGA AGGTACCCCA πTCNπGAC CCCNCNANAA AACCCCNGTG GCCCTTTGCC 600

CTGAπcNCN TGGGCcππ CTCΠTTCCC ππGGGπG πTAAAπcc CAATGTCCCC 660

NGAACCCTCT CCNTNCTGCC CAAAACCTAC CTAMπNCT CNCTANGNNT TπcπGGTG 720

πNCTTπCA AAGGTNACCT TNCCTGπCA NNCCCNACNA AAAπTNπC CNTATNNTGG 780

NCCCNNAAAA ANNNATCNNC CCNAAπGCC CGAAπGGπ NGGππTCC TNCTGGGGGA 840

AACCCTFTAA AπTCCCCCT TGGCCGGCCC CCCM M MC CCCCCπTNG AAGGCAGGNR 900 02t? 333133NNNN NNN933NN1V 999913913V NN313NV9NN 333N3333331V919NN313

09C 3V913333W N3131991VN NV3399LL911333LLL199991V9V9V1V VULLLLV191

OOC 111W1399333V3V33V3V V3919399V3 V11V999139 V19W3311339V3133913

QPZ 31311V9391 V31999933333913131W 3913V3139V 3131V9313V 93193999V1

081 V13V313V93 V1W13139V 9393399393139V9V13119V13V331V999993339V3

OZl 913311W931V1V91139V V1V931V1993V93199V931333333399933V199911

09 W93999V1V 13V313V93V 1W19393939V919V3N933V9WVN191 HN33NNN3N

^•9-ON Ql D3S N0IldIαOS3(] 33N3D03S (LX)

eeuLL A9010d01 (0)

ΘLBULS SS3Nα30NVdlS (3)

PLOB 0L3[3nU 3 λl (8) s^jied aseq I60I H19N31 (V)

^■S3USIb313VdVH3 33N3R03S ( L)

^:9^:0N αi 03S yθd NOIIVWαOdNI (Z)

ZZU 3311313119N113313V33N111NW1V1W 99WWWN3

0801 111133WN1 WVW99N3333313V333319N333339V 11391I99W VWN3333V1

0201 1331NVW9N 9NN999N99N 3313333111313399NN99 NN119933NV IWWWLLV

096 WV1331111333VW91N911V33391N339V3W3N11 W33113W93331131199

ιε 6€00/Z,6Sfl/XDd 1&SVL6 OW JP9U L L : λθOIOdOl (0 )

Θ L5U L S - S 3Nα3αNVyiS ( 3 ) piDB θ Lθ [onu : 3 λl (8) s L9d oseq ggπ -- H19N31 (V)

: S3USIb31DVaVH3 33N3DQ3S X )

^: X ON 01 03S bOJ NOI1VWHOJNI ( Z)

1601 3 31133JJL31N

0801 3313N13N1N 33V1NN13N1 3N3N1NN133 3N1N333NN3 133NN1NNNN N13N13131N

0201 31LLN31N33 NN3NNN13N1 NN33N3NN1N 1N1N1NN333 N33N311N31 3N133N3133

096 3311331N1N 133131N13N 11N331N33N 31N1N3N31N 3N31NNN131 N1N1N33N13

006 N3N1NN3NN3 NN31N13N11 N3N1NN31N3 11N3N31N13 N331N3NNNN 1N1NNN1NNN

0fr8 333N313131 N133N33333 3N331NNN33 3N311NN313 NN3331NNN1 3313N111N3

08 1NN31N3N3N NN31N1NNNN 1N3N3N1133 11NN33N333 11N1LN3NNN 3N31N3NN3N

02Z N1NN331NNN 311N33N1N3 31NNN3NN3N NN111NN133 NN3N3133N3 333N3N33NN

099 1NN3133N1N 3NN3N31N31 31NNNNN331 3NNNNNN1NN 3133131N11 N3N3N13N31

009 NN3NN3133N N1N131N331 NNN3NNN1N3 1N3V1NN33N N1N3NNNN3N NN3NN31NNN

0175 N3NN331N1N N3N1N3N311 NNN3N333N1 N3N33NNN3N N33NN3NN31 N11N1NNN33

0817 N313113NN3 31NN31NN3N NNNNNNN313 N11331NN1N 331NN3N3NN NNNNN3N3NN

Z£

86eθO/L6Sfl/13d \ttSVL6 OΛV 0201 VNV9N33311 33333N3331 1N311331N9 9333191N3V V333313311 1NN3399913

096 33193333W 3311133331 9911933311 11N3WW33 1W999V93N 1VW1N3119

006 1VNWW919 9NWV33331 33331V3339 1339911V13 3NV11V1333 3313133W9

0178 3913311113 N31191113V WW3VNV91 V I I I I I 0W1 1331113333 NV33999V33

08Z 1V33911W1 N1911191V9 V93V391W1 JJL313913V1 V399399133 3V3V9V9199

02Z V9913999V1 1331V3WW 9V99V1W9V 913VJLL3V13 1191V191W 9333WVLL9

099 199113191V VW1W3999 1333193139 1331319131 399W9W9V V19W91LV3

009 911131399V V99V9WW1 NV11V9W99 V913919131 999WW933 3V3V93339V

0f9 333331V3V9 3339V33333 1V3V93339V 333331913V 9133V9W19 99W9991V3

08fr 133991V1V9 V91339V1V9 191V333313 111V9W331 911V199V33 9VW99V131

0217 913133V999 V393399W9 93913V3V1V V3V3V999V3 33V19W313 1W131311V

098 33V31V319V VW1L91139 1V1W1V9W 9113919VW VW1191113 91V13999V1

OOC 11V991W31 199W313V9 1191913919 1V3V9V9V3V 3139191333 W33339V13

0t?2 33W19131V V1191391V9 V91L339131 1311WWV9 W13V19131 191111V331

081 1V9V9W1V3 V9V19W9W V9V191V131 91913V1L91 3V9V31V931 3V9319V999

021 VW13V3133 3W11W313 9V93933993 93139V9V13 1393399399 1993933V33

09 139V99139V VW3W999V W13V3133V V11W3NN93 VN3393V11V 91V11NNN3N

X ^:ON Ql 03S ^; NOUdIb3S3α 33N3HQ3S ( LX )

εε £00/L6Sfl/I3d ItPSZIL^β OΛV 08^ 9V99193V191V391913V1391113393V 199V39V3V913V0W1193 W11V1V999

0217 W199991V993131399V313V31333931V911W91399WNN3N3N V3133V3VW

098 3NV33V111399V31399919119191VN13V9V99W33 V11913NV39199339999V

008 93V13V999V V9V931331133N33V19V3 V9V3V31191933V3391V99199VW99V

017 133339191V WV31131V9133V9339913VW913V13 WV9911913 W99V31913

081 V91V311913 311V13V3313VW313V9133113V13V3 V139V331W V1LL91991V

021 3V33313333113V9111V31339V31V9V 1L913V1119 V9V31V19V991331V39W

09 V1V913V339 WWW9139 V399V3139N 39W3VW1N NW3V91191 V9V111N33N

^•8^:0N Ql 03S -NOIldiyDSH^Q 33N3RQ3S X*)

^jeauLL :λ9010d01 (0)

ΘL6ULS :sS30 αNVyiS (3) pLoe DiθLonu :]dλl (8) s^jLed θseq ZTI :H19N3^~I (V)

X3USiy313VyVH333N3H03S X)

^'8^:0N 0103S dOJ NOIlVWdOJNI (2)

S9II 33331 NN333911V111111119N3

OHI 0NW99H1V V3333V3N1333333199V91133991399391993311N 911199199V

0801 VN31199NNV W3WVN3311133W1V11 WV31333339N99NV91N193333VWW

C00/ .6Sf-/X3d ie*SZ/£6 OΛV CCATCTACCA GGAGCGTGGG CTACTCACTC GGCAGGTGGC TGTNATCCAC TGTAAANGGA 540

CATCAAAAGG AAAACNNGGC TGπGCCCGT GGTAACCANA AANCTGATCN NCAGCTCNAA 600

GATGCTGTGT TGACTπCAC TCNCNCCTCT TAAACπGCT GCCCACANTC TCCTTTCCCA 660

ACCAGATCTG CCTGACAATC CCCATACTCA AAAAAAAAAN AANACTGGCC CCGAACCCNA 720

ACCAATAAAA ACGGGGANGG TNGGTNGANC NNCCTGACCC AAAAATAATG GATCCCCCGG 780

GCTGCAGGAA πCAAπCAN CCπATCNAT ACCCCCAACN NGGNGGGGGG GGCCNGTNCC 840

CAπNCCCCT NTAπNAπc πTNNCCCCC CCCCCGGCNT CCTTπTNAA CTCGTGAAAG 900

GGAAAACCTG NCπACCAAN πATCNCCTG GACCNTCCCC πCCNCGGTN GNπANAAAA 960

AAAAGCCCNC ANTCCCNTCC NAAATπGCA CNGAAAGGNA AGGAATπAA CCTπAππ 1020

πNNTCCTπ ANπTGTNNN CCCCCTπTA CCCAGGCGAA CNGCCATCNT πAANAAAAA 1080

AAANAGAANG πTAππTC CπNGAACCA TCCCAATANA AANCACCCGC NGGGGAACGG 1140

GGNGGNAGGC CNCTCACCCC CiπNTGTNG GNGGGNC 1177

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1146 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear 0201 3333331119 NN1V9N13N9 9N33111W3 333339WN1 V9V9V999V1 1W1111311

096 N1333N33U 333HN1133 3N333WWV 3WWW33V 1133311WN 3VWW331L

006 1V99399999 9N33N333W WWVW333 3333N311W N133W911V WJJLN111N3

0fr8 99W911111 l l l l l l l i n 3N1LL1WV9 W991399V3 3V11333NV3 3399WW99

08Z 1311V133N3 9131VWWV WN3VWWN 13331NV1N3 31W93399V V I M M 1 331

0 13VWV3311 1VWN39111 3V131LV339 91113W331 3V3313V339 999W31133

099 3V919V3119 9V9191LV3V V91139V3V3 V339333W3 W11V3LLLV V999339W9

009 99V31V913V W999V1191 W31V911V3 39V313VW3 3V91331391 9999W991V

0179 339V991W9 1V391991W 91V11VWV1 9W991133V 9W99V3139 9131993V3V

08 VW9399V93 199W91399 33W9139V9 3999V3V19V 39W9V9V9V 3K3113133

0217 1313VW999 V9199913V9 99199399W LLLL991399 V19V999W1 333V91V999

098 1V3V9V99V9 33NV1V3V99 99V3V1339V 91V919991V V399V9W33 W3911993V

008 33V3331331 13V139999V 9V19V19919 V313131339 19113V999V 9V333V31V3

0 2 13V39V9919 9999W99V3 3VW1V3V99 9W9WV9V1 9W9V319W 3V1V11W33

081 11119991V1 1V99V999W 9V9V311LL3 99991339W V3199V919V 31V3313V9V

021 3V19V9V331 9191W3399 9199V3331V 319V3V9119 139V99WW 31999WW9

09 199V9V3113 1313331113 V1V991LL31 3133991111 13191191V9 1NN11NN33N

^: 6 ^: ON Ql 03S ^: NOIld iy3S3Q 30N3flθ3S ( LX )

9 eOO/ .6Sn/13d lεpSVLό OAX 08* 1391911113339V311W31V93999V919V91339V9V 9931V3333V 11331V1W1

02* 193W11919 V319139133 V19399VW39V19V3V391 V3V193V3313991V9V199

098 1331393V3339V19V9199 V9139133V339V3V11V9919V3V11133191V911113

008 31L1L919339V3W39993 V33V11991311139V31W 9139139V91131V3913V3

0*2 V319VW913 V919399V9V 11L9W39V39991913V9V 99VW99191399131V9V3

081 99V31911V999391V19V91191311311311119V3399V9131L9V91313991LV1

021 1111V91331133W33V33 W9W99V319V9V131L9V 13V331V999993339V391

09 3311W931V 1V91139W1 V931V1993V 93133V931333339993V199911V3113

^:0HON Ql 03S ^:NOIldIαOS3Q 33N3H03S X*)

9ΘULL :λ9010d01 (G)

Θ[6ULS :sS3Nα3αNVHlS (3) ppe DLθLonu :3 λl (8)

S L9d θseq g^g :H19N31 (V)

^•S3USiy313VdVH333N3I103S ( )

ΌHON αi DB ^yoj NouvwaodNi (z)

9*11 NV9V1V

0*11 1111NN133V NVWW999N 99N1N9111N VWWNN331 1111V33WV 331133W3N

0801 VN11V9N319 9N1111L113 33N33NW99 VN3NV33311 1113WVN33 1L11V33333

zε C00/ .6Sn/lDd l£ϊ$ZIL6 OM. 009 131191V1911V9333WW 19199113191W9V1W3999133319313913313191

0*5 31V399W99 V9W199V9119V39111313399W99V9 WW19V11V 99V99V9139

08* 19131999W W9333V3V93339V333331V3V93339V 333331V3V93339V33333

02* 1913V9133V 9W1999W9991V3133991V1V9V91339V1V9191V3 33313111V9

098 W331911V199V339VW99V131913133V999V393399W993913 V3V1W3V3V

008 999V333V19 W3131W131311V33V31 V319WWLL 911391V1W 1V9W91139

0*2 19VWWV119111391V13999V111V991W31199W 313V911919139191V3V9

081 V9V3V3139191333W33339V1333W19131W1191391V9V9113 39131131LV

021 WW9W13V 19131191111V3311V9V9 W1V3V9V19 W9WV9V191V13191913

09 V1L913V9V31V9313V9319V999VW13 V31333W11 W3139V939 D399393139

XHON αi 03S ^:N0IldIbOS3α 33N3FI03S ( P

JBΘULL :λ9010d01 (0)

9LDULS :SS3N03αNVyiS (3) p p9 O L9 [onu : 3 λl (8 ) s^j Led 9S q 8886 ^: H19N31 (V)

: S3USIfJ313VyVH3 33N3HQ3S X )

^■ U ^'-OH 01 Q3S rJOd NOUVWrJOJNI (2)

5*5 9933V

0*5 39139V3W1 991V331319 131V3V3W3 V333W9133 39W119913 91119199V9

C00/_,6SfV.L3d IWSZIL6 OM 0891 1393999193 13V9933V33 19331V3131 1193399333 33391399V3 13V99913W

0291 9993993V33 911V339911 WV9991999 3V33193911 1133991331 9V991939V3

0951 9V31999V99 1393V9991V 3V99339133 9193V33993 11V1399911 319V313319

0051 V91919W99 919WV3919 9V33133991 99V3913199 1L99W3939 91339W993

0**1 31LLV33313 3393339W9 9331119339 33991LV333 U193V31V9 131V3W19V

08CT 1339131913 V993WV991 3391191913 1131113311 93939V913V 91399V9111

0281 V131933131 VW913991L V331V3V391 33W999V99 191W13991 1VJJ.DJJ.3VD

0921 9W333399V 199V399V19 1139191391 1LL39WV99 V39391WW V3939V3311

002T V339V199V3 9939399111 V9UJ1VW1 911V91999V V33V39V3W V99V3919W

0*11 V9W33131V W39199133 939V331319 99V999199V 339991V399 V399199933

0801 V31L919911 319991199V V9V3919919 913V9W3W 3V91393119 1339V13V11

0201 1W1W31L3 333V3399V3 9999V99191 99V3V333V1 VW9V93V91 33V3311931

096 31319V3131 1113111313 1919131319 1L13V1V131 3JJJ31JJ13 V313999113

006 3319933939 V913919193 3133199V19 13331919V3 3V9V9V313V V999V913V1

0*8 WV1W31V9 1W1V9V9V1 991V9V9331 31333331V3 V339133991 1V1333V1LV

08Z 13V3303131 333V913913 3JJJ193V31 1911133V9V 9V3V3V91V1 1LV113VW1

02Z 1331L133V3 9V3V399W3 1V3V3919W 1V1911191V 9V933V391V V1LL313913

099 V1W399399 1393V3V9V9 199V991399 9V11331V3V WV9V99V1V 9V913V113V

eOO/ .6Sn/X3d IZ SZIL6 OΛV 09Z2 9333999339 V9V9399933 V3193VW9V 99933V3193 VW9V939V3 33399V9911

00 2 1V3V93V199 V3139313W 1313111193 991919VW3 1319111W1 11L3V991W

0*92 V1VW139V3 3993991131 31V339VJ1V 39V9111LW 9V9V9939V9 V31L311333

0852 39V131V9W 9VWV1V19V W33WV131 1133331333 3133V9V13V V1UJ911W

0252 W19131 V 1113V11391 I I I I I 3W1L 1L331V1LLV 33W191331 JJDWJJVID

09*2 W99133V39 133339WV1 131W1W19 1133V31V13 193933339V VW33391V1

00*2 1V91LV9331 V311W99V9 331L9V3933 1V1V139W3 991913V1V9 WJ1913W1

0*82 1W391W9V V331V3V39V 33313V9139 W39191L3V V113V99W3 1331133339

0822 V39913V311 1313991131 V3313V991V 9V33939V9V V139V11333 1313V911W

0222 9133V33JJJ 91L339V133 V31VW1133 1113331313 V9V M I 1 191 333W13V11

0912 11199133W 33391W191 9V19V1131V 1V91L11991 33119W19V V99WVJJJ1

0012 911W99111 9V119V939V 3V399W191 91311LVW9 9999113111 V1V33WW1

0*02 V1L1399V31 3113W3W3 9V1W13319 1119199111 19V I I I I I W 33WV11W1

0861 3911911911 11W991WV V119V339V3 V99W1V913 131913V991 9V1LLV11V1

026 T 91L9V33911 11L3V3131V VW3V1LL33 W3333V39V WlWlVlll 91V1L91V11

0981 19913311V3 VlWWllVl 1L99V91L99 V39333V919 V999V99913 393V391191

0081 11L9199V39 9V131L1V91 1113393VW V1999313V3 339V3111L1 399993WW

0* I V199VW11V 9V33131911 V19333V913 133V913311 19V191V913 939913V393

0* εoo/ .6sn/x3d iersz/z,6 OΛV 0*88 9911V91339311V3WWV W1919V91V V9993V119119199V319V V39W9V339

08Z8 13111131913319V3V33V 33991399V11LV31W31V 313JJJVW1199V99V3V3

02Z8 191LLL11V913919VJJ33199W3913113W9W199913193V9W 3339JJV1V9

0998 3193V99V3191V391LL199W3191L9919VJJ191131911V131919931191311

0098 9JJVJJ191V 11119111913W31999V133391V3V111913W1V31913VW9W9

0*58 VWWWVW WV13191L19113333133333V9993V31V3331V913 V9131V3999

08*8 399V339939 V99999W1333319W3339V99V9W1391W3191339933V1V111

02*8 99999339V9331V93139V 1V399331V39V399WJJ399W1V3133 V939919331

0988 19939133V3133399919V V3119V993V 139VW93993V33139V1911L9V33133

0088 19113939113999W3V99 V999991W91W91991W 919191313V W1V11913V

0*28 VWV9VWW V9VW99913131W9V99113JJ199V3119113311131W13131V1

081ε 311111313131313131319913V9V399131119913319V913V999991391V99V

02iε 3331V3V3319V319V9113331V339999 V3V913339919W999V999131913131

090ε 9191313393 V9VJJ91311 JJ9913339V 9V1999V9V1391V99V1313111V33391

000ε 313V9111131333191999 V91V91333V 939W999V9 V399131199 MI3V9IMV

0*62 9V3199V199 V3199993333V913913W 313319V913133V3311W W33V33391

0882 999391V33V 13V931V9939V3V99V99331L31933V91999VW333 3199V33JJJ

0282 VWWWW9133V33919V 999999V191139V339V3V 99W993333 3139191913

I* 00/t6Sn/X3d l£PSZIL6 OΛV 026* 139V31199339V31333V9313393111191933V9V339V9133133199W33133V

098* 11LLW1V311V33V391V313V3313991 V331L3333V 39V99V3199 U19V91399

008* 1W31V91LV 3V13331L19 V31V913331339933313V W913VJJ911999399191

0*Z* 9139W31L911W3V3133 W913V31999W91L33339919V91L9V 99J119VW3

089* 3991W9V19 VW391313V 3V9913139V V399WWW 113393319V 991VUV99V

029* 9V3131L199 D13131LLLL W9V391391 V1V19V331313339V991L 999W191L3

095* 39V333133113V3VW999 WVWWWV VWVW31133911WV11V 1131WV31L

005* 91V131V1V911199913919131913131119V919V999V3333339V 3313V1V931

0*** 1131191311 V91191V3199131113139131 I II 19919119139V111V19919W3

088* 99191V919V 3V139VW1L 31W3133313313319V1313399139993131V9V99V

028* 333V913W3 31V9V133V3 W9V9V9V9V V9W9V131V 9V3V1999V111V199WW

092* 3V3V9V991V 319V9111339JJJV139V9111999139V VWV9WW11333W9113

002* 313V313139131399113V 99V33131939V3119W39 V3V93919V33111193131

0*1* V3V3V3333V 1333339133313313311113319919W 331V93WW V1131V3331

080* 313V139919131331191313V1131LV9 V111931L3V JJJ3913V1931333V133V

020* W999V191913V3V39V1V W1L3339191331LLL3W 191311V31991LL3133V3

096ε 1391133111 V9V391333V 339V3WW31199919919913VW13W V13133133V

0068 991LV91L99 V331V3V3333V1V3191LV 9V11LV1911991V91V33V 3991311LV9

Z 00/-.6Sfl/X3d l£f9Z/L6 OΛV 0009 WD1LD1V91 33V9339913 VW913V13V W9911913V V99V31913V 91V31L9133

0*65 11V13V3313 VW313V913 31L3V13V3V 139V331WV 1LL91991V3 V333133331

0885 13V9JJ1V31 339V31V9V1 1913V1119V 9V31V19V99 1331V39VW 1V913V339V

0285 VWW9139V 399V3139V3 9W3WWV3 VWW91199 V9W319V3V 91111199W

09 5 W1339V1V9 V9V11L139V 319V33V1W 91W991391 JJJV999V9V 13VWW331

00Z5 3WV99VWV 19V1V9V333 39V399V313 9919111V39 91HW1131 391V339V1V

0*95 VW9333339 V399133V91 1113333V3V 1L1V9931V1 1399V99V33 133V39V9V1

0855 1L11919V99 V33V319V91 V91V39999V 3319319W9 13V9399W1 319113VW1

0255 V133VW9V3 139139V999 V311V19991 93133131V1 9933V331L1 V93V9ULLL3

09*5 W9V999V99 19W313111 VW333V999 1LW133V99 91LW333V9 V9V11V919V

00*5 1W99V9V13 9W99V99V9 93399V1W3 3W31V3V31 33W3V19W VW3399V9V

0*85 9WV331V19 VW999V9V9 W913133V3 111L333V91 11133139V3 W31913V91

0825 V999133V33 39911V3V3V 991313V191 319V991V91 3V3133V911 1399V33339

0225 VWV913131 13331331W 1V333WW9 911W1V191 13V913113V 1311131133

09T5 3191V11199 1331133333 V3313919V9 1V91999131 33319W3V3 99V9V9V913

00T5 V33V13V131 33339V19W 99V9991991 933W39JJ9 9113133911 V333V31V31

0*05 399V191333 3191V13993 13313191V3 331V31999V 1133313V13 39V33WW1

086* 133933V333 19V333V313 331319V9V9 9V9W9W91 3131313391 3V19133393

ε* €00/t.6Sn/XDd l£tSZIL6 OΛV 080 L 3913W91V393W9V19W 399913139V 9V3333V931 V1339191LV 33139W991

020Z W311V3VW 119399W19 V319V319V1119V1V131911339311339933V991W

0969 1V9131999V 1V3391L9133913999193 V9313331V31VW91W313VM II I9W

0069 119V1991V1 W31913WV 93WWV33V 1391LLV39V V913V991V9913131L33V

0*89 3V9V19913V 19V131L33V 1VW3V19991399933V3V 33WW19W 9V3V1113V9

08Z9 11VW99919 WW9V99V33V313WW9 W33131933 V3399V3339 V333VW133

02Z9 199WVWV3391WV199V 333939133V V3V391993313139V39W 31V9V3W13

0999 39WV31131 V333331V9V V1L119339V 9933133139 V3133V139V W39V99V99

0099 V111V333V33V1319V3V133V319V31V V1JJL3WW9991L313W93333V1V311

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0989 3JJV9139W V9V33W19919333911913993V3WW 99WW31V3 V99VW1913

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08T9 3131399V313V31333931 V91LW91399WW3V39V 3133V3VW39V33V11139

0219 9V313999191L9191V9V3 V9V991V33V 1L9139V391993399VW93V13V999W

0909 9V931331139V39V19V3V 9V3V31191933V3391V99199VW99V133339191W

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0*08 V31V131939 33339WW3 33V3V1LV91 1W331931L W99V333JJ 9V39331V1V

086Z 139W39919 13V1V9W1V 913W11W3 91W9W331 V3V39V3331 3V9139W39

026Z 19113W1L3 V99W31331 139339V399 13V3JJ1313 99JJ31V331 3V991V9V33

098Z 939V9W139 V113331313 V911W9133 V3311191L3 39V313V31V VUJ331113

008Z 331313V9V1 U1191333V V13VUJ119 9133W3319 19V191W99 9919W3V3V

0*ZZ WW333311 V91LLV91W 31W99V913 W1V1191L3 3333W1V1V V313V9W11

089Z 31V191V1L3 VW31913JJ 39V1V391W 3VWV19JJLL 1W1131JLLL 13HWJ1V9

029Z V11V339W9 19991L9W1 33V3913VW V11333399V 3199913999 V333319991

09SZ V3VW9V13V V3339V993V V3399VWW 31V3933313 V31V311V99 193913311V

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0*IZ 31319W191 91LWV1919 933VW9V11 31W1LWW 3VJJ313V3V VWW1333V

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0216 19131319113V3339V1999W3V3999V 3V9V3111V99V3199V31991V3131LV9

0906 9191V191V11V1111V31V V3VW3139919V1HWW 33V3V1LW99V3191V19V

0006 11V13V3V9V 3V1L91399199W119191 WJ13V11911V139113V139J13V91V9

0*68 19V333113V 3W1WV3311W3V9W333JJ3913V3191L991V311339V1VW33

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0228 93JJJW9V9 V93399V31131133339V131VW39WV V1V19VW33 VW1311133

9v 00/Z.6Sα/XDd ICrSZ/16 OM 009 V3313919V91V9199913133319W3V399V9V9V913 V33V13V13133339V19W

0*5 99V9991991933W39JJ99113133911 V333V31V31399V1913333191V13993

08* 13313191V3331V31999V 1133313V1339V33WW1133933V33319V333V313

02* 331319V9V99V9W9W9131313133913V19133393139931199339V31333V9

098 313393111191933V9V339V9133133199W33133V JJJJW1VD11V33V391V3

008 13V3313991933113333V 39V99V31991119V913991W31V911V 3V13331119

0*2 V31V913331339933313V W913VJJ9119993991919139W31191LW3V3133

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021 3V9913139V V399WWW 1L3393319V 991V11V99V 9V3131119931313ULUJ

09 W9V391391 V1V19V331313339V991L 999W191L339V333133113V3VW999

^:2I^:ON OI 03S ^:N0Udiy3S3α 33N3HD3S (PO

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: S3USiy313VyVH3 33N3D03S ( !■ )

: ZT ^: 0N 01 03S dOd NOUVWdOdNI (2)

8886 31V99V33 3V331VW39 V3V39V199V

0986 91VJJ11319 999V91391V 9V3999V331 3999V3V399 V99V1131LV V9V319V199

0086 9V31339V39 91339V3V11 331L3V9113 3991V1V331 1L919V191V 1V133919V9

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0951 V9V991V33V 1L9139V391 993399VW9 3V13V999W 9V93133113 9V39V19V3V

0051 9V3V31191V 33V3391V99 199VW99V1 33339191W W3U31V91 33V9339913

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0881 3113V13V3V 139V331VW 11191991V3 V333133331 13V91L1V31 339V31V9V1

0281 1913V1L19V 9V31V19V99 1331V39VW 1V913V339V WWV9139V 399V3139V3

0921 9W3WVW3 WWV91L99 V9W319V3V 9U11L99W W1339V1V9 V9V11L139V

0021 319V33V1W 91W991391 11LV999V9V 13VWW331 3VW99WW 19V1V9V333

0*11 39V399V313 9919111V39 91LLW1L31 391V339V1V VW9333339 V399133V91

0801 1113333V3V ULLV9V31V1 1399V99V33 133V39V9V1 JJJJ919V99 V33V319V91

0201 V91V39999V 3319319W9 13V9399W1 319JJ3WV1 V133WV9V3 139139V999

096 V3JJV19991 93133131V1 9933V331LL V93V911H3 W9V999V99 19W313111

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0*8 93399V1W3 3W31V3V31 33W3V19W WV3399V9V 9WV331V19 VW999V9V9

08Z W913133V3 JJ 1333V91 11133139V3 W31913V91 V999133V33 39911V3V3V

02Z 991313V191 319V991V91 3V3133V911 1399V33339 WW913131 13331331W

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8* C00/-.6Sfl/XDd 1 SZIL6 OΛV 09Z2 1319V3V133 V19V3VJJV1 11W13WV3 V31V1VWW 3991LW333 91V9V9W13

00Z2 9W13391L3 1V13391391 999V9991V1 91V31VW91 11133V1131 1999139991

0*92 3V1L3333V3 9199W19V9 V1L3V13339 VJJ1331L33 1319W1919 11WV19199

0852 33VW9V1L3 1W11WW3 V11313V3W WW1333V3 913W91V39 3W9V19W3

0252 99913139V9 V3333V931V 1339191LV3 3139W991V V31LV3VW1 19399W19V

09*2 319V319V11 19V1V13191 1339311339 933V991W1 V9131999V1 V3391LL913

00*2 39139991V3 V9313331V3 1VW91W31 3VI I I I I 9W 119V1991V1 W31913WV

0*82 93VWW33V 139JJJV39V V913V991V9 913131133V 3V9V19913V 19V131L33V

0822 1VW3V1999 1399933V3V 33WW19W 9V3VJJ13V9 11VW99919 WW9V99V3

0222 3V313WW9 W33131933 V3399V3339 V333WV133 199WWW3 391VW199V

0912 333939133V V3V3919933 13139V39W 31V9V3W13 39VW31L31 V333331V9V

0012 VJ1119339V 9933133139 V3133V139V W39V99V99 V11LV333V3 3V1319V3V1

0*02 33V319V31V V1113WW9 991L313W9 3333V1V31L 31W9V1313 V913311311

086 T V99199JJ99 W99V31VW W1W339V9 V313W9V31 339913WW 9W9W9V3V

0261 V313V1V393 331W3V913 39131V9V33 9V3V33J113 31319V3V33 391391L3W

0981 V1313393V3 19V311L3V9 1919V391V9 W3139V39V 311V9139W V9V33W199

0081 1933391191 3993V3WW 99WW31V3 V99VW1913 V331W1913 99199V39V3

0*ZT 133V313V13 9991939V99 V33V131V33 9V99193V19 1V391913V1 39ULL3393V

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0*58 W399V933V 39V31V339V 39V319V31V 91333V33V3 3W39V3113 33V1313333

08*8 9V3V31V91L W03193LLV V99V333119 V39331V1V1 39W399191 3V1V9W1V9

02*8 13W11W39 1W9W331V 3V39V33313 V9139W391 9JJ3W113V 99W313311

0988 39339V3991 3V31113139 91L31V3313 V991V9V339 39V9W139V 1L3331313V

0088 91LW9133V 3311191139 V313V31W1 1133111333 1313V9VJJLL 1191333W1

0*28 3V1LL11991 33W331919 V191W9999 19W3V3VW W3333311V 91JLLV91W3

0818 1W99V913V V1V1L91133 333W1V1W 313V9W113 1V191V113V W31913113

021 ε 9V1V391W3 WW19JJJ1 WI 131 I l I 311W1LV9V JJV339W91 999119W13

0908 3V3913WW 11333399V3 199913999V 333319991V 3VW9V13W 3339V9V3W

0008 3399WVW3 1V3933313V 31V31LV991 93913311V3 991V99199V V91313993V

0*62 V3391V93V3 1V31933V3V 3133V9V9W 9911391331 33313V99W 9V9V9V33JJ

0882 9VWW1191 119119133V 9199V33933 31LV31LV33 91333999V3 V9V3W9133

0282 11W331W3 331V333W9 9V931191L3 V3391331V3 1V1V9W3V1 99V3W3333

e00/i.6Sr-/X3d I€frSZ/ .6 OΛV (D) TOPOLOGY linear

(xi ) SEQUENCE DESCRIPTION SEQ ID NO 13

CCTCAACCTC 10

(2) INFORMATION FOR SEQ ID NO 14

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 23 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO 14

ATGGCTATπ TCGGGGGCTG ACA 23

(2) INFORMATION FOR SEQ ID NO 15

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 22 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CCGGTATCTC CTCGTGGGTA π 22

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CπCAACCTC 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17 CTGCCTGAGC CACAAATG 18

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

CCGGAGGAGG AAGCTAGAGG AATA 24

(2) INFORMATION FOR SEQ ID NO:19:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

I I I I M I ! I I πAG 14

(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Ser Ser Gly Gly Arg Thr Phe Asp Asp Phe His Arg Tyr Leu Leu Val 1 5 10 15

Gly He

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Gin Gly Ala Ala Gin Lys Pro He Asn Leu Ser Lys Xaa He Glu Val 1 5 10 15

Val Gin Gly His Asp Glu 20

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Ser Pro Gly Val Phe Leu Glu His Leu Gin Glu Ala Tyr Arg He Tyr 1 5 10 15

Thr Pro Phe Asp Leu Ser Ala 20

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Tyr Leu Leu Val Gly He Gin Gly Ala 1 5

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

Gly Ala Ala Gin Lys Pro He Asn Leu 1 5

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

Asn Leu Ser Lys Xaa He Glu Val Val 1 5 (2) INFORMATION FOR SEQ ID NO 26

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 9 amino acids

(B) TYPE amino acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO 26

Glu Val Val Gin Gly His Asp Glu Ser 1 5

(2) INFORMATION FOR SEQ ID NO 27

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 9 amino acids

(B) TYPE amino acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(xi) SEQUENCE DESCRIPTION SEQ ID NO 27

His Leu Gin Glu Ala Tyr Arg He Tyr 1 5

(2) INFORMATION FOR SEQ ID NO 28 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

Asn Leu Ala Phe Val Ala Gin Ala Ala 1 5

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

Phe Val Ala Gin Ala Ala Pro Asp Ser 1 5

Claims

1. An isolated DNA molecule, comprising:

(a) a human endogenous retroviral sequence, wherein said retroviral sequence is preferentially expressed in a tumor tissue;

(b) a variant of said human endogenous retroviral sequence that contains one or more nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% of the nucleotide positions, such that the antigenic and/or immunogenic properties ofthe polypeptide encoded by the human endogenous retroviral sequence are retained; or

(c) a nucleotide sequence encoding an epitope of a polypeptide encoded by at least one ofthe above sequences.

2. An isolated DNA molecule encoding an epitope of a polypeptide, wherein said polypeptide is encoded by:

(a) a nucleotide sequence transcribed from the sequence of SEQ ID NO:l l ; or

(b) a variant of said nucleotide sequence that contains one or more nucleotide substitutions, deletions, insertions and/or modifications at no more than 20% of the nucleotide positions, such that the antigenic and/or immunogenic properties of the polypeptide encoded by the nucleotide sequence are retained.

3. A recombinant expression vector comprising a DNA molecule according to claim 1 or claim 2.

4. A host cell transformed or transfected with an expression vector according to claim 3.

5. A polypeptide comprising an amino acid sequence encoded by a DNA molecule according to claim 1 or claim 2.

6. A monoclonal antibody that binds to a polypeptide according to claim 5.

7. A method for determining the presence of a cancer in a patient comprising detecting, within a biological sample obtained from a patient, a polypeptide according to claim 5, and therefrom determining the presence of cancer in the patient.

8. The method of claim 7 wherein the biological sample is a tumor sample.

9. The method of claim 7 wherein the step of detecting comprises contacting the biological sample with a monoclonal antibody according to claim 6.

10. The method of claim 7 wherein the polypeptide comprises an amino acid sequence encoded by a human endogenous retroviral sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3 - SEQ ID NO: 10 and SEQ ID NO: 12.

1 1. A method for determining the presence of a cancer in a patient comprising detecting, within a biological sample obtained from a patient, an RNA molecule encoding a polypeptide according to claim 5, and therefrom determining the presence of cancer in the patient.

12. The method of claim 1 1 wherein the biological sample is a tumor sample.

13. The method of claim 1 1 wherein the step of detecting comprises:

(a) preparing cDNA from RNA molecules within the biological sample; and

(b) specifically amplifying cDNA molecules that are capable of encoding at least a portion of a polypeptide according to claim 5.

14. The method of claim 1 1 wherein the polypeptide comprises an amino acid sequence encoded by a human endogenous retroviral sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3 - SEQ ID NO: 10 and SEQ ID NO: 12.

15. A polypeptide according to claim 5 for use within a method for detecting the presence of a cancer in a patient.

16. The polypeptide of claim 15 wherein the polypeptide comprises an amino acid sequence encoded by a human endogenous retroviral sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3 - SEQ ID NO:10 and SEQ ID NO: 12.

17. A method for monitoring the progression of a cancer in a patient, comprising: (a) detecting an amount, in a biological sample obtained from a patient, of a polypeptide according to claim 5;

(b) subsequently repeating step (a); and

(c) comparing the amounts of polypeptide detected in steps (a) and (b), and therefrom monitoring the progression of cancer in the patient.

18. The method of claim 17 wherein the biological sample is a tumor sample.

19. The method of claim 17 wherein the step of detecting comprises contacting a portion of the biological sample with a monoclonal antibody according to claim 6.

20. The method of claim 17 wherein the polypeptide comprises an amino acid sequence encoded by a human endogenous retroviral sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3 - SEQ ID NO: 10 and SEQ ID NO: 12.

21. A method for monitoring the progression of a cancer in a patient, comprising:

(a) detecting an amount, within a biological sample obtained from a patient, of an RNA molecule encoding a polypeptide according to claim 5;

(b) subsequently repeating step (a); and

(c) comparing the amounts of RNA molecules detected in steps (a) and (b), and therefrom monitoring the progression of cancer in the patient.

22. The method of claim 21 wherein the step of detecting comprises:

(a) preparing cDNA from RNA molecules within the biological sample; and

23. The method of claim 21 wherein the polypeptide comprises an amino acid sequence encoded by a human endogenous retroviral sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3 - SEQ ID NO: 10 and SEQ ID NO: 12.

24. A pharmaceutical composition, comprising: (a) a polypeptide according to claim 5; and (b) a physiologically acceptable carrier.

25. A vaccine, comprising:

(a) a polypeptide according to claim 5; and

(b) an immune response enhancer.

26. A diagnostic kit comprising:

(a) one or more monoclonal antibodies according to claim 6; and

(b) a detection reagent.

27. The kit of claim 26 wherein the monoclonal antibody(s) are immobilized on a solid support.

28. A diagnostic kit comprising a first polymerase chain reaction primer and a second polymerase chain reaction primer, the first and second primers each comprising at least about 10 contiguous nucleotides of an RNA molecule encoding a polypeptide according to claim 5.

29. A diagnostic kit comprising at least one oligonucleotide probe, the oligonucleotide probe comprising at least about 15 contiguous nucleotides of a DNA molecule according to claim 1 or claim 2.