IE84173B1 - Tumor rejection antigen precursors, tumor rejection antigens and uses thereof - Google Patents

Description

TUMOR REJECTION ANTIGEN PRECURSORS, TUMOR REJECTION ANTIGENS AND USES THEREOF This invention relates in general to the field of immunogenetics as applied to the study of oncology. More specifically, it relates to the study and analysis of mechanisms by which tumors are recognized by the organism's immune system such as through the presentation of so- called tumor rejection antigens, and the expression of what will be referred to herein as "tumor rejection antigen precursors".

The study of the recognition or lack of recognition of cancer cells by a host organism has proceeded. in many different directions. Understanding of the field presumes some understanding of both basic immunology and oncology.

Early research on mouse tumors revealed that these displayed molecules which led to rejection of tumor cells animals. These when transplanted into syngeneic molecules are "recognized" by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced in vitro by chemical carcinogens, such as methylcholanthrene. The antigens expressed by the tumors and which elicited the T-cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30: 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as "tumor specific transplantation antigens" or "TSTAS".

Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types of tumor described su ra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. see Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum" antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp.

Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum" antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., "tum*" cells). when these tum+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "tum'"). See Boon et al., Proc. Natl.

Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum7 variants fail to form progressive tumors because they elicit an immune rejection process.

The evidence in favor of this hypothesis includes the ability of "tum'" variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum" cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tum" variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., gupra; Uyttenhove et al., supra).

Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp.

Med. 157: 1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called "tumor rejection antigen" in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.

A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytotoxic T cells, leading to lysis. This class of antigens will be referred to as "tumor rejection antigens" or "TRAs" hereafter. TRAS may or may not elicit antibody responses. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cell ("CTL" hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed. Characteriza- tion studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysis is required for other types of antigens recognized by CTLs, including minor histocompatibility antigens, the male specific H-Y antigens, and a class of antigens, referred to as "tum-" antigens, and discussed herein.

A tumor exemplary of the subject. matter described ggpra is known as P815. See DePlaen et al., Proc. Natl.

Acad. Sci. USA 85: 2274-2278 (1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. Med. 172: 35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methyl- cholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum’ variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, ggpga), and P198 (Sibille, ggpga). In contrast to tumor rejection antigens - and this is a key distinction — the tum" antigens are only present after the tumor cells are mutagenized. Tumor rejection antigens are present on cells of a given tumor without mutagenesis. Hence, with reference to the literature, a cell line can be tum+, such as the line referred to as "P1", and can be provoked to produce tum’ variants. Since the tum" phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum" cell lines as compared to their tum* parental lines, and this difference can be exploited to locate the gene of interest in tum" cells. As a result, it was found that genes of tum’ variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, ggpgg, and Lurquin et al., Cell 58: 293-303 (1989). This has proved pg; to be the case with the TRAS of this invention. These papers also demonstrated that peptides derived from the tum" antigen are presented by the La molecule for recognition by CTLs. P91A is presented by La, P35 by Dd and P198 by Kd.

It has now been found that the genes which code for the molecules which are processed to form the presentation tumor rejection antigens (referred to as "tumor rejection antigen precursors", "precursor molecules" or "TRAPS" hereafter), are not expressed in most normal adult tissues but are expressed in tumor cells. Genes which code for the TRAPS have now been isolated and cloned, and represent a portion of the invention disclosed herein.

The gene is useful as a source for the isolated and purified tumor rejection antigen precursor and the TRA themselves, either of which can be used as an agent for treating the cancer for which the antigen is a "marker", as well as in various diagnostic and surveillance approaches to oncology, discussed infra. It is known, for example, that tum" cells can be used to generate CTLs which lyse cells presenting different tum" antigens as well as tum+ cells. See, e.g., Maryanski et al., Eur. J. Immunol 12: 401 (1982); and Van den Eynde et al., Modern Trends in Leukemia IX (June 1990), the disclosures of which are incorporated by reference. The tumor rejection antigen precursor may be expressed in cells transfected by the gene, and then used to generate an immune response against a tumor of interest.

In the parallel case of human neoplasms, it has been observed that autologous mixed lymphocyte-tumor cell cultures ("MLTC" hereafter) frequently generate responder lymphocytes which lyse autologous tumor cells and do not lyse natural killer targets, autologous EBV-transformed B cells, or autologous fibroblasts (see Anichini et al., Immunol. Today 8: 385-389 (1987)). This response has been particularly well studied for melanomas, and MLTC have been carried out either with peripheral blood cells or with tumor infiltrating lymphocytes. Examples of the literature in this area including Knuth et al., Proc. Natl. Acad. Sci.

USA 86: 2804-2802 (1984); Mukherji et al., J. Exp. Med. :’ 240 (1983); Hérin et all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol 6: 839-853 (1988).

Stable cytotoxic T cell clones ("CTLs" hereafter) have been derived from MLTC responder cells, and these clones are specific for the tumor cells. See Mukherji et al., supra, Bérin et all, su ra, Knuth et al., supra. The antigens recognized on tumor cells by these autologous CTLS do not appear to represent a cultural artifact, since they are found on fresh tumor cells. it Topalian et al., su ra; Degiovanni et al., Eur. J. Immunol. 20: 1865-1868 (1990).

These observations, coupled with the techniques used herein to isolate the genes for specific murine tumor rejection antigen precursors, have led to the isolation of nucleic acid sequences coding for tumor rejection antigen precursors of TRAS presented on human tumors. It is now possible to isolate the nucleic acid sequences which code for tumor rejection antigen precursors, including, but not being limited to those most characteristic of a particular tumor, with ramifications that are described infra. These isolated nucleic acid sequences for human tumor rejection antigen precursors and applications thereof, as described infra, are also the subject of this invention.

Accordingly, the present invention provides, in a first aspect, isolated nucleic molecules. An isolated nucleic acid molecule in accordance with this aspect of the invention is:- (a) a.DN1\ molecule comprising a nucleotide sequence selected from SEQ.ID.NOS. 7, 8, 9, 11, 12, 13, 15, 16, 18 and 19, equal length complementary sequences and portions thereof; or, (b) hybridizable under stringent conditions to a DNA molecule having a nucleotide sequence selected from SEQ.ID.NOS. 7, 8, 9, 11, 12, 13,15, 16, 18 and 19 and equal length complementary sequences; and codes for, or is an equal length complement of anucleic acid molecule which codes for a polypeptide or proteincapable, when presented on a cell surface, of eliciting a cytolytic response from human T-lymphocytes, or a precursor for such a polypeptide or protein.

An isolated nucleic acid molecule accordance with the invention can be a cDNA, genomic DNA, or RNA molecule. It can also be an RNA transcript of a DNA molecule in accordance with the invention.

An isolated nucleic acid molecule in accordance with the invention can comprise a nucleic acid sequence coding for a polypeptide or protein capable, when presented on a cell surface, of eliciting a cytolytic response from human T-lymphocytes, or a precursor for such a polypeptide or protein, that is coded for by a nucleic acid molecule in accordance with the first aspect of the invention, or an equal length complementary nucleic acid sequence.

Preferably, an isolated nucleic acid molecule in accordance with the invention codes for, or is an equal length complement of a nucleic acid molecule which codes for a polypeptide or protein comprising the amino acid sequence set out in SEQ.lD.NO. 26.

Preferably, an isolated nucleic acid molecule in accordance with the invention is hybridizable under stringent conditions to a nucleic acid molecule having a nucleotide sequence as set out in any one ofSEQ.ID.NOs. 7, 8, 9 and 11, or to an equal length complementary nucleotide sequence, and more preferably has a nucleotide sequence as set out in figure 9, or in any one of SEQ.ID.NOs. 7, 8, 9 and 11, or an equal length complementary nucleotide sequence.

In a second aspect, the present invention provides expression vectors. An expression vector in accordance with this aspect of the invention comprises a nucleic acid molecule in accordance with the first aspect of the invention. The nucleic acid molecule can be operably linked to a promoter and an expression vector in accordance with the invention can further comprise a nucleic acid sequence coding for a major histocompatability antigen (MHC) or a human leukocyte antigen (HLA), a cytokine, or a bacterial or viral genome or a portion thereof. The cytol-tine can be an interleukin, preferably IL-2 or IL-4.

In a third aspect, the present invention provides transfected cells. A cell in accordance with this aspect of the invention is transfected with a nucleic acid molecule in accordance with the first aspect of the invention, or an expression vector in accordance with the second aspect of the invention. Such a cell can be transfected i with a nucleic acid molecule coding for an MHC or l-ILA, or a cytokine.

Preferably, a cell in accordance with the invention is capable of expressing an MHC or HLA, or a cytokine, wherein the cytoltine is preferably an interleukin, more preferably IL-2 or IL-4. Preferred cells in accordance with the invention are non- proliferative.

In a fourth aspect, the present invention provides polypeptides and proteins. A polypeptide or protein in accordance with this aspect of the invention is capable of eliciting a cytolytic response from human lymphocytes, or is a precursor to such a polypeptide or protein, and is coded for by a nucleic acid molecule i.n accordance with the first aspect of the invention. A preferred polypeptide or protein in accordance with this aspect of the invention has an amino acid sequence as set out in SEQ.lD.NO. 26.

In a fifth aspect, the present invention relates to viruses. A virus in accordance with this aspect of the invention contains a nucleic acid molecule in accordance with the first aspect of the invention. A virus in accordance with this aspect of the invention can be mutated or etiolated.

In a sixth aspect, the present invention relates to antibodies. An antibody in accordance with this as ect of the invention s ecificall binds a e tide or P Y P P protein in accordance with the fourth aspect of the invention, or a complex of such a polypeptide or protein and an MI-1C or I-ILA, but does not bind to the MHC or HLA alone.

Preferred antibodies in accordance with the invention include monoclonal antibodies.

An isolated nucleic acid molecule, expression vector, cell, polypeptide, protein, virus, or antibody in accordance with the invention can be for use in the therapy, prophylaxis or diagnosis of tumors.

In a Further aspect, the present invention provides pharmaceudcal compositions for the prophylaxis, therapy or diagnosis of tumors. A pharmaceutical composition in accordance with this aspect of the invention comprises a nucleic acid molecule, expression vector, cell, polypeptide, protein, virus, or antibody in accordance with the invention, optionally in admixture with a pharmaceutically acceptable carrier and optionally further comprising an MHC or HLA.

In an additional aspect, the present invention provides a method of producing a cytolytic T-cell culture. reactive against autologous tumor cells of an individual, comprising the step of culturing a sample of lymphocytes from the individual with cells in accordance with the third aspect of the invention. ln a yet further aspect, the present invention provides the use of an isolated nucleic acid molecule, expression vector, cell, polypeptide, protein, virus, antibody, or pharmaceutical composition in accordance with the invention, for use in the preparation of a medicament for the prophylaxis, therapy or diagnosis of tumors.

Preferred such tumors include melanoma, sarcoma, and carcinoma such as a small cell lung, non-small cell lung, squamous cell, thyroid, colon, pancreatic, prostate, breast or larynx carcinoma.

In another aspect, the invention provides a method for determining the regression, progress or onset of a tumor in an individual comprising the steps oE- A (at) determining the amount of polypeptide or protein in accordance with the invention present in a sample of body fluid, tissue, or tumor from said individual, optionally by employing an immunoassay involving an antibody in accordance with the invention, (b) determining the number oficytolytic T-cells responsive to a cell in accordance with the invention, or a polypeptide or protein in accordance with the invention, present in a sample of body fluid, tissue, or tumor from said individual, or (c) determiningthe expression of a polypeptide in accordance with the ‘ invention, optionally by nucleic acid hybridization employing a nucleic acid molecule in accordance with the invention as a probe, present inasample of.bodyflui_d, tissue, or tumor from said individual.

These and other aspects of the invention are elaborated upon in the disclosure which . follows; BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts detection of transfectants expressing antigen P815A.

Figure 2 shows the sensitivity of clones P1.HTR, PO.HTR, genomic transfectant P1A.T2 and cosmid transfectant P1A.TC3.1 to lysis by various CTLs, as determined by chromium release assays.

Figure 3 is a restriction map of cosmid C1A.3.1.

Figure 4 shows Northern Blot analysis of expression of gene PlAI Figure 5 sets forth the structure of gene P1A with its restriction sites.

Figure 6 shows the results obtained when cells were transfected with the gene from P1A, either isolated from P815 or normal cells and then tested with CTL lysis.

Figure 7 shows lytic studies using mast cell line L138. 8A.

Figure 8 is a map of subfragments of the 2.4 kb antigen E fragment sequence which also express the antigen.

Figure 9 shows homology of sections of exon 3 from genes mage 1, 2 and 3.

Figure 10 shows the result of Northern blots for MAGE genes on various tissues.

Figure 11 presents the data of Figure 13 in table form.

Figure 12 shows Southern Blot experiments using the various human melanoma cell lines employed in this application.

Figure 13 is a generalized schematic of the expression of MAGE 1, 2 and 3 genes by tumor and normal tissues.

BRIEF DESCRIPTION OF SEQUENCE8 SEQ ID NO: 1 is cDNA for part of gene PIA.

SEQ ID NO: 2 presents coding region of CDNA for gene PIA.

SEQ ID NO: 3 shows non coding DNA for PIA CDNA which is 3’ to the coding region of SEQ ID NO: 2.

SEQ ID NO: 4 is the entire sequence of CDNA for P1A.

SEQ ID NO: 5 is the genomic DNA sequence for P1A.

SEQ ID NO: 6 shows the amino acid sequence for the antigenic peptides for P1A TRA. The sequence is for cells which are A* B+, i.e., express both the A and B antigens.

SEQ ID NO: 7 is a nucleic acid sequence coding for antigen SEQ ID NO: 8 is a nucleic acid sequence coding for MAGE- SEQ ID NO: 9 is the gene for MAGE-2.

SEQ ID NO: 10 is the gene for MAGE-21.

SEQ ID NO: 11 is CDNA for MAGE-3.

SEQ ID NO: 12 is the gene for MAGE-31.

SEQ ID NO: 13 is the gene for MAGE-4.

SEQ ID NO: 14 is the gene for MACE-41.

SEQ ID NO: 15 is CDNA for MAGE-4.

SEQ ID NO: 16 is CDNA for MAGE-5.

SEQ ID NO: 17 is genomic DNA for MAGE—51.

SEQ ID NO: 18 is CDNA for MAGE-6.

SEQ ID NO: 19 is genomic DNA for MAGE-7.

SEQ ID NO: 20 is genomic DNA for MAGE-8.

SEQ ID NO: 21 is genomic DNA for MAGE-9.

SEQ ID NO: 22 is genomic DNA for MAGE-10.

SEQ ID NO: 23 is genomic DNA for MAGE-11.

SEQ ID NO: 24 is genomic DNA for smage-I.

SEQ ID NO: 25 is genomic DNA for smage—II.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Many different "MAGE" genes have been identified, as will be seen from the sequences which follow the application. The protocols described in the following examples were used to isolate these genes and cDNA sequences.

"MAGE" as used herein refers to a nucleic acid sequence isolated from human cells. The acronym "smage" is used to describe sequences of murine origin. when "TRAP" or "TRAS" are discussed herein as being specific to a tumor type, this means that the molecule under consideration is associated with that type of tumor, although not necessarily to the exclusion of other tumor types.

Example 1 In order to identify and isolate the gene coding for antigen P815A, gene transfection was used. This approach requires both a source of the gene, and a recipient cell line. Highly transfectable cell line Pl.HTR was the starting material for the recipient, but it could not be used without further treatment, as it presents "antigen A", one of four recognized P815 tumor antigens. See Van Pel et al., Molecular Genetics 11: 467-475 (1985). Thus, screening experiments were carried out to isolate cell lines which did not express the antigen and which nonetheless possessed P1.HTR's desirable qualities.

To do this, P1.HTR was screened with CTLs which were specific for each of tumor antigens A, B, C and D. Such CTLs are described by Uyttenhove et al., J. Exp. Med. 157: -1052 (1983).

To carry out the selection, 106 cells of P1.HTR were mixed with 2-4x106 cells of the CTL clone in a round bottom tube in 2 ul of medium, and centrifuged for three minutes at 150xg. After four hours at 37°C, the cells were washed and resuspended in 10 ul of medium, following Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982). Additional information on the CTL assay and screening protocol, in general may be found in Boon et al., J. Exp. Med. 152: 1184-1193 (1980), and Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982), the disclosure of which are incorporated by reference.

When these selections were carried out, a cell line variant was found which expressed neither antigen A or B.

Additional selections with CTLs specific for antigen E then yielded a variant which also lacked antigen C. Please see figure 2 for a summary of the results of these screenings.

The variant PO.HTR is negative for antigens A, B and C, and was therefore chosen for the transfection experiments.

The cell line PO.HTR has been deposited in accordance with the Budapest Treaty at the Institute Pasteur Collection Nationale De Cultures De Microorganismes, 28, Rue de Docteur Roux, 75724 Paris France, and has accession number I-1117.

This methodology is adaptable to secure other cell lines which are variants of a cell type which normally presents at least one of the four recognized P815 tumor antigens, i.e., antigens A, B, C and D, where the variants present none of antigens A, B and C. P1.HTR is a mastocytoma cell line, so it will be seen that the protocol enables the isolation of biologically pure mastocytoma cell lines which express none of P815 antigens A, B and C, but which are highly transfectable. Other tumor types may also be screened in this fashion to secure desired, biologically pure cell lines. The resulting cell lines should be at least as transfectable with foreign DNA as is P1.HTR, and should be selected so as to Q9; express a specific antigen.

Example 2 Previous work reported by DePlaen et al., Proc. Natl.

Acad. Sci. USA 85: 2274-2278 (1988) the disclosure of which is incorporated by reference herein had shown the efficacy of using cosmid library transfection to recover genes coding for tum" antigens. selective plasmid and genomic DNA of P1.HTR were prepared, following Wdlfel et al., Immunogenetics ggz 178- 187 (1987). The transfection procedure followed Corsaro et al., Somatic Cell Molec. Genet 7: 603-616 (1981), with some modification. Briefly, 60 pg of cellular DNA and 3 pg of DNA of plasmid pHMR272, described by Bernard et al., Exp.

Cell. Biol. 158: 237-243 (1985) were mixed. This plasmid confers hygromycin resistance upon recipient cells, and therefore provides a convenient way to screen for transfectants.

The mixed DNA was combined with 940 ul of mM Tris-HCl (pH 7.5), 0.1 mM EDTA; and 310 ul 1M CaCl2.

The solution was added slowly, and under constant agitation to 1.25 ml of 50 mM Hepes, 280 mM NaCl, 1.5 mM Na2HPO4, adjusted to pH 7.1 with NaOH. Calcium phosphate - DNA precipitates were allowed to form for 30-45 minutes at room temperature. Following this; fifteen groups of P0.HTR cells (5x10°) per group were centrifuged for 10 minutes at 400 g. Supernatants were removed, and pellets were resuspended directly into the medium containing the DNA precipitates. This mixture was incubated for 20 minutes at °C, after which it was added to an 80 cm2 tissue culture flask containing 22.5 ml DMEM, supplemented with 10% fetal calf serum. After 24 hours, medium was replaced. Forty- eight hours after transfection, cells were collected and counted. Transfected cells were selected in mass culture using culture medium supplemented with hygromycin B (350 ug/ml). This treatment selected cells for hygromycin resistance.

For each group, two flasks were prepared, each containing 8x106 cells in 40 ml of medium. In order to estimate the number of transfectants, 1x106 cells from each group were plated in 5 ml DMEM with 10% fetal calf serum (FCS), 0.4% bactoagar, and 300 ug/ml hygromycin B. The colonies were then counted 12 days later. Two independent determinations were carried out and the average taken.

This was multiplied by 5 to estimate the number of transfectants in the corresponding group. Correction had to be made for the cloning efficiency of P815 cells, known to be about 0.3.

Example 3 Eight days after transfection as described in example 2, su ra, antibiotic resistant transfectants were separated from dead cells, using density centrifugation with Ficoll- Paque. These cells were maintained in non-selective medium for 1 or 2 days. The cells were plated in 96 well microplates (round bottom), at 30 cells/microwell in 200 ul of culture medium. Anywhere from 100-400 microwells were prepared, depending on the number of transfectants prepared. Agar colony tests gave estimates of 500-3000.

After 5 days, the wells contained about 6x104 cells and replicate plates were prepared by transferring 1/10 of the wells to microplates which were then incubated at 30°C.

One day later, master plates were centrifuged, medium removed, and 750 CTLs against P815 antigen A (CTL-P1:5) were added to each well together with 106 irradiated syngeneic feeder spleen cells in CTL culture medium containing 40 U/ml recombinant human IL-2, and HAT medium to kill stimulator cells. Six days later, plates were examined visually to identify wells where CTLs had proliferated. Where plates showed proliferating microcultures, aliquots of 100 ul of the wells were transferred to another plate containing 51Cr labeled P1.HTR target cells (2x103 - 4x103 per well), and chromium release was measured after 4 hours. Replicate microcultures corresponding to those showing high CTL activity were expanded and cloned by limited dilution in DMEM with 10% FCS. Five days later, about 200 clones were collected and screened with the CTL.P1:5 cell line, described su ra, in a visual lysis assay. See figure 1A for these results.

In these experiments, three of the fifteen groups of transfectants yielded a few positive microcultures. These microcultures were tested for lytic activity against P1.HTR, as described supra. Most of the microcultures where proliferation was observed showed lytic activity.

This activity was well above background, as shown in figure 1B. This figure summarizes data wherein two groups of cells (groups "5" and "14"), 400 and 300 microwells were seeded with 30 hygromycin resistant transfected cells.

Amplification and duplication of the microcultures was followed by addition of anti-A CTL P1:5. Six days later, lytic activity against P1.HTR was tested. In the figure, each point represents lytic activity of a single microculture.

Duplicate microcultures corresponding to several positive wells were subcloned, and more than 1% of the subclones were found to be lysed by anti—A CTL. Thus, three independent transfectants expressing P815A were obtained from 33,000 hygromycin resistant transfectants. one of these lines, referred to hereafter as P1A.T2 was tested further.

The relevant antigen profile of P1A.T2 is shown in figure 2, this being obtained via anti-CTL assays of the type described supr .

Example 4 The CTL assays carried out for PlA.T2 demonstrated that it presented antigen A ("P815A"), and therefore had received the gene from P1.HTR. To that end, this cell line was used as a source for the gene for the antigen precursor in the following experiments.

Prior work had shown that genes coding for tum" antigens could be recovered directly from transfectants obtained with a cosmid library. See DePlaen et al., Proc.

Natl. Acad. Sci. USA 85: 2274-2278 (1988). This procedure was followed for recovery of the P815 gene.

Total genomic DNA of P1A.T2 was partially digested with restriction endonuclease Sau 3A1, and fractionated by Nacl density gradient ultracentrifugation to enrich for 35- 50 kb DNA fragments, following Grosveld et al., Gene 19: 6715-6732 (1982). These fragments were ligated to cosmid arms of C2RB, described by Bates et al., Gene 26: 137-146 (1983), the disclosure of which is incorporated by reference. These cosmid arms had been obtained by cleavage with SmaI and treatment with calf intestinal phosphatase, followed by digestion with BamHI. Ligated DNA was packaged into lambda phage components, and titrated on E. coli ED , following Grosveld et al., supr . Approximately 9x1 ampicillin resistant colonies were obtained per microgram of DNA insert.

The cosmid groups were amplified by mixing 30,000 independent cosmids with 2 ml of ED 8767 in 10 mM MgCl2, incubated 20 minutes at 37°C, diluted with 20 ml of Luria Bertani ("LB") medium, followed by incubation for one hour.

This suspension was titrated and used to inoculate 1 liter of LB medium in the presence of ampicillin (50 ug/ml). At a bacterial concentration of 2x108 cells/ml (OD6oo=0.8), a ml aliquot was frozen, and 200 ug/ml chloramphenicol was added to the culture for overnight incubation. Total cosmid DNA was isolated by alkaline lysis procedure, and purified on Cscl gradient.

In these experiments, a library of 650,000 cosmids was prepared. The amplification protocol involved the use of groups of approximately 30,000 cosmids.

Example 5 Using the twenty—one groups of cosmids alluded to ggpgg, (60 ug) and 4 ug of pHMR272, described su ra, groups of 5x106 P0.HTR cells were used as transfectant hosts.

Transfection was carried out in the same manner as described in the preceding experiments. An average of 3000 transfectants per group were tested for antigen presentation, again using CTL assays as described. One group of cosmids repeatedly yielded positive transfectants, at a frequency of about 1/5,000 drug resistant transfectants. The transfectants, as with PlA.T2, also showed expression of both antigen A and B. The pattern of expression of transfectant P1A.TC3.1 is shown in figure 2.

Example 6 As indicated in Example 5, supra, three independent cosmid transfected cells presenting P815A antigen were isolated. The DNA of these transfectants was isolated and packaged directly with lambda phage extracts, following DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988). The resulting product was titrated on E. coli ED 8767 with ampicillin selection, as in Example 5.

Similarly, amplification of the cosmids and transfection followed Example 5, again using P0.HTR.

High frequencies of transfection were observed, as described in Table 1, which follows: »Tabk I 1Wanshr ofihc cxpnshon of anngcn PSISA by rosnnds obzancd by dncu packaging - Transfcclant obxantd vilh No. of cosmids obxaincd N0. of 1T2??Sf=C130l5 by dimct packaging of m cmnw hbmn’ Q5ugofDNA M HmB’numhcnnu TCSJ 32 87/192 'rc3.2 32000 W35‘ /72 cxpnsnng PSISA / no.

The cosmids were analyzed with restriction enzymes and it was found that directly packaged transfectant P1A.TC3.1 contained 32 cosmids, 7 of which were different. Each of these 7 cosmids was transfected into PO.HTR, in the manner described supra, and again,_ following the protocols described above, transfectants were studied for presentation of P815A. Four of the cosmid transfectants showed P815A presentation and, as with all experiments described herein, P815B was co-expressed. of the four cosmids showing presentation of the two antigens, cosmid C1A.3.1 was only 16.7 kilobases long, and was selected for further analysis as described igfrg.

The cosmid C1A.3.1 was subjected to restriction endonuclease analysis, yielding the map shown in Figure 3.

All EcoRI fragments were transfected, again using the above described protocols, and only the 7.4 kilobase fragment produced a transfectant that anti-A CTLs could lyse. similar experiments were carried out on the PstI fragments, and only a 4.1 kb fragment fully contained within the 7.4 kb EcoRI fragment produced lysable transfectants.

This fragment (i.e., the 4.1 kb PstI fragment), was digested with Smal, giving a 2.3 kb fragment which also yielded host cells presenting antigens A and B after transfection. Finally, a fragment 900 bases long, secured with SmaI/XbaI, also transferred expression of the precursors of these two antigens, i.e., the transfected host cell presented both antigen A and antigen B.

Example 7 The 900 base fragment described above was used as a probe to detect the expression of the P815A gene in parent cell line P1.HTR. To accomplish this, total cellular RNA was first isolated using the guanidine-isothiocyanate procedure of Davis et al., Basic Methods In Molecular Biology (Elseview Science Publishing Co, New York) (1986).

The same reference was the source of the method used to isolate and purify polyA* mRNA using oligodT cellulose column chromatography.

Samples were then subjected to Northern Blot analysis.

RNA samples were fractionated on 1% agarose gels containing 0.66 M formaldehyde. The gels were treated with 10xsSC (SSC: 0.15 M Nacl; 0.015 M sodium citrate, pH 7.0) for 30 minutes before overnight blotting on nitrocellulose membranes. These were baked for two hours at 80°C, after which the membranes were prehybridized for 15 minutes at 60°C in a solution containing 10% dextran sulfate, 1% SDS and 1M NaCl. Hybridization was then carried out using denatured probe (the 900 base fragment), together with 100 ug/ml salmon sperm DNA.

When this protocol was carried out using P1.HTR poly A7 RNA, a band of 1.2 kb and two fainter bands were identified, as shown in Figure 4, lane 1 (6 ug of the RNA).

The same probe was used to screen a cDNA library, prepared from poly-A+ RNA from the cell line. This yielded a clone with a lkb insert, suggesting a missing 5' end.

The Northern blots for the cDNA are not shown.

Hybridization experiments in each case were carried out overnight at 60°C. The blots were washed twice at room temperature with 2xSsC and twice at 60°C with 2xSSC supplemented with 1% SDS.

The foregoing experiments delineated the DNA expressing the P815A antigen precursor sufficiently to allow sequencing, using the well known Sanger dideoxy chain termination method. This was carried out on clones generated using a variety of restriction endonucleases and by specific priming with synthetic oligonucleotide primers.

The results for exons of the gene are set forth in sequence id no: 4.

Example 8 The Northern analysis described spppg suggested that the 5' end of the CDNA was missing. To obtain this sequence, cDNA was prepared from P1.HTR RNA using a primer corresponding to positions 320-303. The sequence was then amplified using the polymerase chain reaction using a 3' primer corresponding to positions 286-266 and a 5' primer described by Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002 (1988). A band of the expected size (270 bases) was found, which hybridized to the 900 bp SmaI/XbaI fragment described spppg on a Southern blot. Following cloning into m13tg 130 X tg 131, the small, 270 bp fragment was sequenced. The sequence is shown in sequence id no: 1.

Example 9 Following the procurement of the sequences described in Examples 7 and 8 and depicted in seq id no: 4, a 5.7 kb region of cosmid C1A.3.1 was sequenced. This fragment was known to contain the 900 base fragment which expressed P815A in transfectants. This experiment permitted delineation of introns and exons, since the cosmid is genomic in origin.

The delineated structure of the gene is shown in figure 5. Together with seq id no: 4, these data show that the gene for the antigen precursor, referred to as "P1A" hereafter, is approximately 5 kilobases long and contains 3 exons. An ORF for a protein of 224 amino acids starts in exon 1, ending in exon 2. The 900 base pair fragment which transfers expression of precursors for antigens A and B only contains exon 1. The promoter region contains a CAAT box, as indicated in seq. id no: 1, and an enhancer sequence. This latter feature has been observed in promoters of most MHC class I genes, as observed by Geraghty et al., J. Exp. Med 171: 1-18 (1990); Kimura et al., Cell 44: 261-272 (1986).

A computer homology search was carried out, using program FASTA with K-triple parameters of 3 and 6, as suggested by Lipman et al., Science 227: 1435-1441 (1985), and using Genbank database release 65 (October 1990). No homology was found except for a stretch of 95 bases corresponding to part of an acid region coded by exon 1 (positions 524-618), which is similar to sequences coding for acidic regions in mouse nucleolar protein N038/B23, as described by Bourbon et al., Mol. Biol. 200: 627-638 (1988), and Schmidt-Zachmann et al., Chromosoma 96: 417- 426 (1988). Fifty six of 95 bases were identical. In order to test whether these homologies were the reason for cross hybridizing, experiments were carried out using a mouse spleen CDNA library screened with the 900 base fragment. cDNA clones corresponding closely to the sizes of the cross hybridizing bands were obtained. These were partially sequenced, and the 2.6 kb cDNA was found to correspond exactly to reported cDNA sequence of mouse nucleolin, while the 1.5 kb CDNA corresponded to mouse nucleolar protein N038/B23.

Analysis of the nucleotide sequence of the gene, referred to as "P1A" hereafter, suggests that its coded product has a molecular mass of 25 kd. Analysis of the sequence id no: 4 shows a potential nuclear targeting signal at residues 5-9 (Dingwall et al., Ann. Rev. Cell Biol. 2: 367-390 (1986)), as well as a large acidic domain at positions 83-118. As indicated su ra, this contains the region of homology between P1A and the two nucleolar proteins. A putative phosphorylation site can be found at position 125 (serine). Also, a second acidic domain is found close to the C-terminus as an uninterrupted stretch of 14 glutamate residues. A similar C-terminal structure has been found by Kessel et al. Proc. Natl. Acad. Sci. USA : 5306-5310 (1987), in a murine homeodomain protein having nuclear localization.

In studies comparing the sequence of gene P1A to the sequences for P91A, 35B and P198, no similarities were found, showing that P1A is indicative of a different class of genes and antigens.

Example 10 With the P1A probe and sequence in hand, investigations were carried out to determine whether the gene present in normal tissue was identical to that expressed by the tumor. To do this, phage libraries were prepared, using lambda zapII 10 and genomic DNA of DBA2 murine kidney cells. P1A was used as a probe.

Hybridization conditions were as described su ra, and a hybridizing clone was found. The clone contained exons one and two of the P1A gene, and corresponded to positions — 0.7 to 3.8 of figure 5. Following localization of this sequence, PCR amplification was carried out to obtain the sequence corresponding to 3.8 to 4.5 of figure 5.

Sequence analysis was carried out, and no differences were found between the gene from normal kidneys and the PIA gene as obtained from the P815 tumor cells.

In further experiments, the gene as found in DBA/2 kidney’ cells was transfected into PO.HTR, as described ggpgg. These experiments, presented pictorially in figure 7, showed that antigens A and B were expressed as efficiently by the kidney gene isolated from normal kidney cells as with the P11A gene isolated from normal kidney cells from the P815 tumor cells.

These experiments lead to the conclusion that the gene coding for the tumor rejection antigen precursor is a gene that does not result from a nmtation; rather, it would appear that the gene is the same as one present in normal cells, but is not expressed therein. The ramifications of this finding are important, and are discussed infra.

In studies not elaborated upon herein, it was found that variants of the gene were available. Some cells were "P1A'B+", rather than the normal "P1A". The only difference between these is a point mutation in exon 1, with the 18th triplet coding for Ala in the variant instead of Val.

Example 11 Additional experiments were carried out with other cell types. Following the protocols described for Northern blot hybridizations sgpra, RNA of normal liver and spleen cells was tested to determine if a transcript of the P1A gene could be found. The Northern blot data are presented in figure 4 and, as can be seen, there is no evidence of expression.

The murine P815 cell line from which P1A was isolated is a mastocytoma. Therefore, mast cell lines were studied to determine if they expressed the gene. Mast cell line MC/9, described by Nabel et al., Cell 23: 19-28 (1981), and short term cultures of bone marrow derived mast cells were tested in the manner described guprg (Northern blotting), but no transcript was found.

In contrast when a Balb/C derived IL-3 dependent cell line L138.8A (Hfiltner et al., J. Immunol. 142: 3440-3446 (1989)) was tested, a strong signal was found. The mast cell work is shown in figure 4.

Further tests were carried out on other murine tumor cell lines, i.e., teratocarcinoma cell line PCC4 (Boon et al., Proc. Natl. Acad. Sci. USA 74: 272-275 (1977), and leukemias LEC and WEH1—3B.

Expression could not be detected in any of these samples.

Example 12 The actual presentation of the P1A antigen by MHC molecules was of interest. To test this, cosmid C1A.3.1 was transfected into fibroblast cell line DAP, which shows phenotype H-2*. The cell lines were transfected with genes expressing one of the Kd, Dd , and La antigen. Following transfection with both the cosmid and the MHC gene, lysis with CTLs was studied, again as described supr . These studies, summarized in Table 2, show that L" is required for presentation of the P1A antigens A and B.

Tab}: 2. H»?-xesmcxion of znzigcns PEISA and P8158 Retipéent eel)’ No of clones lysed by the CTL/ no. of P55’ clones‘ CT]. nu"-A CTL mi-B DA? (H.210 0/202 0/194 DA?-+Kd 0/165 0/162 D;_p.,Dd 0/157 0/129 DAp+1_d 25/33 15/20 ‘Cosmid C1A.3.) conxaininp the entire PM gene was uzzzsfecxed in DA? cells prex-ious1_v transfecxed wimh H-2‘ class 1 genes as indicaled.

‘Independent drug-resisxam colonies were tested for lysis by anti-A ox ami-B CTL in a \'is'.=al assay.

The observation that one may associate presentation of a tumor rejection antigen with a particular MHC molecule was confirmed in experiments with human cells and HLA molecules, as elaborated upon infra.

Example 13 Using the sequence of the P1A gene as well as the amino acid sequence derivable therefrom, antigenic peptides which were A+ B+ (i.e., characteristic of cells which express both the A and B antigens), and those which are A" B+ were identified. The peptide is presented in Figure 10.

This peptide when administered to samples of PO.HTR cells in the presence of CTL cell lines specific to cells presenting it, led to lysis of the P0.HTR cells, lending support to the view that peptides based on the product expressed by the gene can be used as vaccines.

Example 14 The human melanoma cell line referred to hereafter as MZ2-MEL is not a clonal cell line. It expresses four stable antigens recognized by autologous CTLs, known as antigens "D, E, F, and A". In addition, two other antigens "B" and "C" are expressed by some sublines of the tumor.

CTL clones specific for these six antigens are described by Van den Eynde et al., Int. J. Canc. 44: 634-640 (1989).

Among the recognized subclones of MZ2-MEL are MEL.43, MEL3.0 and MEL3.l. (Van den Eynde et al., ggprg). Cell line MEL3.1 expresses antigen E, as determined by CTL studies as described for P815 variants, ggpra, so it was chosen as a source for the nucleic acid sequence expressing the antigen precursor.

In isolating the pertinent nucleic acid sequence for a tumor rejection the antigen precursor, techniques developed sgprg, showed that a recipient cell is needed which fulfills two criteria: (i) the recipient cell must not express the TRAP of interest under normal conditions, and (ii) it must express the relevant class I HLA molecule.

Also, the recipient cell must have a high transfection frequency, i.e., it must be a "good" recipient.

In order to secure such a cell line, the clonal subline ME3.1 was subjected to repeated selection with anti-E CTL 82/30 as described by Van den Eynde, ggpga. The repeated cycles of selection led to isolation of subclone MZ2-MEL-2 .2 isc E‘.

This subclone is also HPRT', (i.e., sensitive to HAT medium: 10" M hypoxanthine, 3.8 x 10'7 aminopterine, 1.6 x 10'5 M 2—deoxythymidine). The subclone is referred to as "MEL-2.2" for simplicity hereafter.

Example 15 The genomic DNA of MEL3.0 was prepared following Wblfel et al., Immunogenetics 26: 178-187 (1987), the disclosure of which is incorporated by reference. The plasmid pSVtkneoB, as described by Nicolas et al., Cold Spring Harb., Conf. Cell Prolif. 10: 469-485 (1983) confers geneticin resistance, so it can be used as a marker for cotransfection, as it was in this experiment.

Following a procedure similar but not identical to that of Corsao et al., Somatic Cell Molec. Genet 7: 603- 616 (1981), total genomic DNA and the plasmid were cotransfected. The genomic DNA (60 pg) and plasmid DNA (6 pg) were mixed in 940 pl of 1 mM Trisfflfl. (pH 7.5), 0.1 mM EDTA, after which 310 pl of 1M CaCl2 was added. This solution was slowly added, under constant agitation, to 1.25 ml of ZXHBS (50 mM HEPES, 280 mM NaCl 1.5 mM Na2HPO4, adjusted to pH 7.1 with NaOH). The calcium phosphate DNA precipitates were allowed to form for 30-45 minutes at room temperature, after which they were applied to 80 cm: tissue culture flasks which had been seeded 24 hours previously with 3x106 MEL2.2 cells, in 22.5 ml of melanoma culture medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum. After 24 hours, the medium was replaced. Forty eight hours after transfection, the cells were harvested and seeded at 4x106 cells per 80 cm2 flask in melanoma culture medium supplemented with 2 mg/ml of geneticin. The geneticin serves as a selection marker.

Example 16 Thirteen days after transfection, geneticin-resistant colonies were counted, cultured in harvested, and nonselective medium for 2 or 3 days. Transfected cells were then plated in 96-well microplates at 200 cells/well in 200 ul of culture medium with 20% fetal calf serum (FCS) in order to obtain approximately 30 growing colonies per well. The number of microcultures was aimed at achieving redundancy, i.e., such that every independent transfectant should be represented at least four times.

After 10 days, wells contained approximately 6x104 cells. These cells were detached, and 1/3 of each microculture was transferred to a duplicate plate. After 6 hours, i.e., after readherence, medium was removed and 1500 anti—E CTL (CTL 82/30), were added to each well in 100 pl of CTL culture medium with 35 U/ml of IL-2. one day later, the supernatant (50 pl) was harvested and examined for TNF concentration, for reasons set forth in the following example.

Example 17 The size of the mammalian genome is 6x106 kb. As the average amount of DNA integrated in each drug-resistant transfectant was expected to be about 200 kb, a minimum of ,000 transfectants would need to be examined to ascertain whether antigen E had been transfected. Prior work with murine cells had shown that when a CTL stimulation assay was used, groups containing only 3% of cells expressing the antigen of interested could be identified. This should reduce the number of assays by a factor of 30. While an anti-E CTL assay, as described su ra, in mixed E+/E’ cells was helpful, it was not sufficient in that consistent results could not be obtained.

As a result, an alternative test was devised.

Stimulation of CTLs was studied by release of tumor necrosis factor ("TNF") using well known methodologies which need not be repeated here. As described in Example , 1500 CTL 82/30 cells had been added per well of transfectants. These CTLs were collected 6 days after stimulation. As indicated su ra, after 1/3 of the cells in each well had been removed and the remaining 2/3 (4x104) had readhered, the CTLs and IL-2 were added thereto. The 50 pl of supernatant was removed 24 hours later and transferred to a microplate containing 3x10‘ W13 (WEHI-164 clone 13; Espevik et al., J. Immunol. Meth. 95: 99-105 (1986)) cells in 50 ul of W13 culture medium (RPMI-1640, supplemented with L-arginine (116 mg/1), L-asparagine (36 mg/1), L- glutamine (216 mg/l), and 10% FCS supplemented with 2 pg of actinomycin D at 37% in an 8% CO2 atmosphere. The cell line W13 is a mouse fibrosarcoma cell line sensitive to TNF.

Dilutions of recombinant TNF-B in RPMI 1640 were added to target cell controls.

The W13 cultures were evaluated after 20 hours of incubation, and dead cell percentage was measured using an adaptation of the colorimetric assay of Hansen et al., J.

Immunol. Meth. 119: 203-210 (1989). This involved adding 50 ml of (3-(4,5-dimethylthiazolyl)-2,5—dipheny1 tetrazolium bromide at 2.5 mg/ml in PBS, followed by two hours of incubation at 37°C. Dark blue formazan crystals were dissolved by adding 100 pl of lysis solution (1 volume N,N dimethyl formamide mixed at 37°C with two volumes of water containing 30% (w/v) sodium dodecyl sulphate, at pH 4.7 from 1.6% acetic acid and 2.5% 1N Hcl). Plates were incubated at 37°C overnight, and 0Ds were taken at 570 nm using 650 nm as control.

Dead cell percentage was determined via the formula: -(0D57° sample well) X 1 - OD57o well + medium following Espevik et al., J. Immunol. Meth. 95: 99-105 (1986). The results showed that even when the ratio of E+/E" cells was as low as 1/45, significant production of TNF was observed, thus showing active CTLs. This led to the decision to test the drug resistant transfectants in groups of 30.

Example 18 Cells were tested for TNF production as discussed in Example 17, supra. A total of 100 groups of B" cells (4x106 cells/group) were tested following transfection, and 7x10‘ independent resistant geneticin transfectants were obtained, for an average of 700 per group. only one group of transfected cells led to a microculture which caused anti-E antigen CTL clone 82/30 to produce TNF. Of 300 clones tested, 8 were positive. These clones were then tested for lysis by anti-E CTL, using the standard 51Cr release assay, and were found to be lysed as efficiently as the original E+ cell line. The transfectant E.T1, discussed herein, had the same lysis pattern as did MEL2.2 for CTLs against antigens B,C,D and F.

The fact that only one transfectant. presented the antigen out of 70,000 geneticin resistance transfectants may at first seem very low, but it is not. The work described supra for P815 showed an average frequency of 1/13,000. Human DNA recipient MEL2.2 appears to integrate times less DNA than P1.HTR.

Example 19 once transfectant E.T1 was found, analysis had to address * several questions including whether an E contaminant of the cell population was the cause. The analysis of antigen presentation, described su ra, shows that E.T1 is B" and C", just like the recipient cell MEL2.2.

It was also found to be HPRT', using standard selection procedures. All E+ cells used in the work described herein, however, were HPRT+.

It was also possible that an E+revertant.of MEL2.2 was the source for E.T1. To test this, the observation by Perucho et al., Cell 22: 309-317 (1980), that cotransfected sequences usually integrate together at a single location of recipient genome was employed. If antigen E in a transfectant results from cotransfec-tion with pSVtkneoB, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoB sequences.

Wolfel et al., su ra, has shown this to be true. If a normally B’ cell is transfected with pSVtkneoB, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoB sequences. If a normally E+ cell transfected with psVtkneoB is E.T1, however, "co-deletion" should not take place. To test this, the transfectant E.T1 was subjected to immunoselection with 82/30, as described supra. Two antigen loss variants were obtained, which resisted lysis by this CTL. Neither of these had lost geneticin resistance; however, Southern blot analysis showed loss of several neo" sequences in the variants, showing close linkage between the E gene and neo’ gene in E.T1, leading to the conclusion that E.T1 was a transfectant.

Example 20 The E* subclone MZ2-MEL 4B was used as a source of DNA for preparation of a cosmid library. This library of nearly 700,000 cosmids was transfected into MZ2-MEL 2.2 cells, following the cosmid transfection By packaging the DNA of cosmid transfectants directly into lambda phase components, it is sometimes possible to retrieve cosmids that contain the sequences of interest.

This procedure was unsuccessful here, so we rescued the transfected sequence by ligating DNA of the transfectant to appropriate restriction fragments of cosmid vector pTL6.

This was tried with two transfectants and was successful with one of them. One cosmid, referred to as B3, was recovered from this experiment, and subjected to restriction endonuclease digestion via Xmal, or by BamHI digestion of a large, 12 kb XmaI transfected fragment. The fragments were cloned into vector pTZ 18R, and then transfected into MEL2.2. Again, TNF production was the measure by which successful transfection was determined.

The experiments led to the determination of a gene sequence capable of transfecting antigen E on the 12 kb Xmal fragment, and then on the 2.4 kb fragment of BamHI digestion of the 12 kb segment.

The 2.4 kb fragment hybridizes with a 2.4 kb fragment from MZ2-MEL and with a T cell clone of patient MZ-2, as determined by Southern Blots (BamHI/SmaI digested DNA).

The band is absent from E" antigen loss variants of M22- MEL, as seen in Figure 12.

C1 111 101 241 101 361 421 451 541 601 661 721 761 I41 01 $61 1021 1062 1141 1201 1261 1121 1361 1441 1501 1561 1621 1661 1741 1001 1061 1021 1021 2041 3101 2161 2221 2201 3141 34 The sequence for the E antigen precursor gene has been determined, and is presented herein: I 10 I 20 I cmvrccxss: cc'rc.:c.\.c.:A MMTAZLLG ' a.:':~c;:z.::;Ac .\c:c;:.s:.uc ‘runner: caocrscrcra crrococrcr acxcccrsxc aaucaxooc AGTGAGGCCT 1-osrctsxsa cAcAss:::=': amounts nrcmocc as-gum: Assxcrccxc master:-5: csxccrcrsc roeccaocrc D.‘:CC7GAG? csaxuosx ucwsmsc Aasaurcc mtcrczms no5:c':::G TIAGAGTCTC uucrccc: crcrccccuc BCCTGTGC‘-5‘? occtscrocc c':a.Ac:.>.c.A5 !‘CJ.!CJ.?G‘rC ‘rams;-.2»:-cc C'.':‘GAC-.'5-CCC Mcnrm-3: TCCTCCTCCT G$?CCTCCCC CAGAGGCKAC A?C3TG$AG7 C?GC?CC?CA ITCLAAAATT CTGG7C?T! ACCTGCCZAG G5CTTCC7GA GAA1?CTG3G GGGGAGCCCA AGGT "CGGA AAACCAGCTA TCTTCCCATC ?1GCAGC$A1 CAGCTTCCCC GTTCTCAGTA 16611316?! Affifikffikfih CTCCTCTGGT AGAGTCCTCA CCA5?5A5GG CCTTGTTCCG AASAECGAGC ACAAGZACTG GCA??GACGT G?C?CfCC?A IAATTGTCCT AGGAGCTGAE GGAAGCTGC? CAG7Gk?CCC TGTGALAGTC CC?G¢G?GLA GGCCAGTGGG 103326076! GTAGGSESCT CAAATG7?71 ccras....>....."- ~~ oosmc:-rcc ncaoas: :.s:a.:-rm-c usssxsccx m-rcc-ms auosucm rc.a.::.scc:a caraxrom taraaroaas uccumsx: acxcszurc CTTGAGIASG next-c‘.'7‘:cA Aoaesaxnc mnrmaoc cncrmoc 2222:2155: GCA3?CA£AC A&T?C?G?G? o cacccraca: cA:.c.:cA:cc c:.:cccrc..'..A oxsmrccrc crsurszac cmxcctccc ACLCTCICAC c-.-mac-....a*" cauxcvmus ‘CTTCATTGCC c::*s>.s:us ca-.5.-.c.c:::-s crssxamoc 4'-CC'T‘."‘.‘CC.'.'.A CGTGAAGAGG xstumss G1'C).:A.AAfrG arcrrcssu mccccxscs C7GGGTGATA acm.c.a.- -- 3TGIA‘!‘3P.'.'G 2:05:14."-~ ~- M:-rrccrcrc tcoxcnscr GAGAGGAGGA cc-c:.>.a¢oc1 curtcmzr.

UTGAC?TGGA ur~".'1~eu.: an-rm-m I so mmams tcczosnsc '::c::c::cc Ac...-toxucx scans-:—s:c txcrcruse _?1‘C-CTCC77 cAr.As:.:.:;:.x !"1"C‘?CAG::1‘G uscrcmsc ' '4 60 GGGG?:A?CC ACYGLSKASC 1531532 GCAGAGGA?G CCACCTGCCA CCTGIASAA? AGG??22CAG CCA153AGLA AGGCCSCTCA CCACACTCCT A5GAGTC?GC AC?GCAAGCC IGTGTSTGSA ?GCCCAC1GC CTACCATCXA AGGSSCCKAG T50: T33 CAGAAATGCT A$GC2fCTGA GCCACTCC1A A?:AGA?CAT GCGGCC£?GC GGAGGGAGCA AAAAGZACCT GGETCCAAGG CAGTGCLAGA AGAGGSAGTC CCTTGCAGGG CTCTGAAEAG GKTTTATCTT GAACTTCAGC 8566151535 GGCTGCCACC TGGG?CLL£A CTTCACTCGA CACCSCTTGI 5577553333 GGAGAGTG?C O7CC?1GCAG 1GICCT7G?C OCCCLAGALA ?CCTGAGGA5 CAGSGCCSA7 GGAG7ACGGC GCCCTCSCTG STTCGCTTSS TGAGCATGAG CCGCGTCCA5 AGCSSTCAGT TGTTCTCSTS ATCCAAGTT1 TCTTGTGTTT IATTCAGATT OGGLLLSCCA T7C7A?tTTG 02AC?7AGLA 1?G2GAAAAA TQAGCAGSLA LSLEAIAETC LA3TC7TGCC TTATLSCTCA GCATACC?G& 13??CCTTGG CTTCTTTGAG 12?C?:CCTG TTCACTGGC1 C?f?TC??CT CCCTGGGTIA GTAGTGGAGA QGCIALGGTA lGA3?C21GG A5C7GCAG?C hCC1AA?CG% IAAAGTGAGA GAGGGG?6LG GGTGTGGGGC CTGA5C?GGG GCA??TTGGG CIIEGGGLLA 1TGATC??GG CTGGLTCC I 10 I _2o ! an ?GAAT?G5GA IAATLACAGC AGTGGLASAA IAGATGAG AILAAGAAC? AALGAAATTA GTC1A?1CTG IAAAATTTST AAAGAZAZAT l1?G€AAGAG 1Al?7AAATC TGAATAAAGA C£ATGCAS?G A5CATCTGC1~?f?TGGAAGG lGCCLGL2TC.hIACCCA£CC AIAGGGTCGT GGTGGCLAGA EGTCCTCIAA IGATGTAGGG ICCGGGTGAG 8573335515 ?GTCAA?GCC C?GCAUT$CC ?TC?GGGGGA OC?GATTGTA I40 Isa I. 150 240 300 360 (20 460 540 600 660 720 750 840 900 960 1020 1060 1140 1200 1260 1320 1350 1440 1500 1560 1620 1690 1740 1600 1060 1920 1900 2040 2100 2160 2220 2250 2340 1400 24 Example 21 After the 2.4 kb genomic segment had been identified, studies were carried out to determine if an "E*" subline expressed any homologous DNA. Cell line MZ2-MEL 3.0 was used as a source, and a CDNA library was prepared from its mRNA, using art known techniques. The 2.4 kb segment was used as a probe, and mRNA of about 1.8 kb was identified as homologous, using Northern blot analysis. When CDNA was screened, clones were obtained showing almost complete identity to parts of the 2.4 kb fragment. Two exons were thus identified. An additional exon was located upstream of these, via sequencing segments of cosmid B3 located in front of the 2.4 kb BamHI fragment. The gene extends over about 4.5 kb, as shown in Figure 8. The starting point of the transcribed region was confirmed using PCR for the 5’ end of the CDNA. The three exons comprise 65, 73, and 1551 base pairs. An ATG is located at position 66 of exon 3, followed by an 828 base pair reading frame.

Example 22 To determine if smaller segments of the 2.4 kb fragment could transfer the expression of antigen E, smaller pieces corresponding to the larger gene were prepared, using art recognized techniques, and transferred into E" cells. Figure 8 shows the boundaries of the three segments. rather, MAGE refers to a family of tumor rejection antigen precursors and the nucleic acid sequences coding therefore.

The antigens resulting therefrom are referred to herein as "MAGE TRAS" or "melanoma antigen tumor rejection antigens" Example 24 Experiments with mouse tumors have demonstrated that new antigens recognized by T cells can result from point mutations that modify active genes in a region that codes for the new antigenic peptide. New antigens can also arise from the activation of genes that are not expressed in most normal cells. To clarify this issue for antigen MZ2-E, the mage-1 gene present in the melanoma cells was compared to that present in normal cells of patient MZ2.

Amplification by polymerase chain reaction (PCR) of DNA of phytohemagglutinin—activated blood lymphocytes using primers surrounding a 1300 bp stretch covering the first half of the 2.4 kb fragment was carried out. As expected, a PCR product was obtained whereas none was obtained with the DNA of the E" variant. The sequence of this PCR product proved identical to the corresponding sequence of the gene carried by the E+ melanoma cells. Moreover, it was found that antigen MZ2-E was expressed by cells transfected with the cloned PCR product. This result suggests that the activation of a gene normally silent is responsible for the appearance of tumor rejection antigen MZ2-E.

Example 25 In order to evaluate the expression of gene mage-1 by various normal and tumor cells, Northern blots were hybridized with a probe covering most of the third exon.

In contrast with the result observed with human tumor cell line MZ2-MEL 3.0, no band was observed with RNA isolated from a CTL clone of patient M22 and phytohemagglutinin- activated blood lymphocytes of the same patient. Also negative were several normal tissues of other individuals (Figure 10 and Figure 11). Fourteen melanoma cell lines of other patients were tested. Eleven were positive with bands of varying intensities. In addition to these culture cell lines, four samples of melanoma tumor tissue were analyzed. Two samples, including a metastasis of patient MZ2 proved positive, excluding the possibility that expression of the gene represented a tissue culture artefact. A few tumors of other histological types, including lung tumors were tested. Most of these tumors were positive (Figures 10 and 11). These results indicated that the MAGE gene family is expressed by many melanomas and also by other tumors. However, they provided no clear indication as to which of genes mage-1, 2 or 3 were expressed by these cells, because the DNA probes corresponding to the ‘three genes cross-hybridized to a considerable extent. To render this analysis more specific, PCR amplification and hybridization with highly specific o1igo- nucleotide probes were used. cDNAs were obtained and amplified by PCR using oligonucleotide primers corresponding to sequences of exon 3 that were identical for the three MAGE genes discussed herein. The PCR products were then tested for their ability to hybridize to three other oligonucleotides that showed complete specificity for one of the three genes (Figure 9). Control experiments carried out by diluting RNA of melanoma M22- MEL 3.0 in RNA from negative cells indicated that under the conditions used herein the intensity of the signal decreased proportionally to the dilution and that positive signals could still be detected at a dilution of 1/300.

The normal cells (lymphocytes) that were tested by PCR were confirmed to be negative for the expression of the three MAGE genes, suggesting therefore a level of expression of less than 1/300th that of the MZ2 melanoma cell line (Figure ). For the panel of melanoma cell lines, the results clearly showed that some melanomas expressed MAGE genes mage 1, 2 and 3 whereas other expressed only mage-2 and 3 (Figures 11 and 10). Some of the other tumors also expressed all three genes whereas others expressed only mage-2 and 3 or only mage-3. It is impossible to exclude formally that some positive PCR results do not reflect the expression of one of the three characterized MAGE genes but that of yet another closely related gene that would share the sequence of the priming and hybridizing oligo- nucleotides. It can be concluded that the MAGE gene family is expressed by a large array of different tumors and that these genes are silent in the normal cells tested to this point.

Exammple 26 The availability of a sequence that transfects at high efficiency and efficiently expresses a TRAP made it possible to search for the associated major histo- compatibility complex (MHC) class I molecule. The class I specificities of patient MZ2 are HLA-A1, A29, B37, B44 and C6. Four other melanomas of patients that had A1 in common with MZ2 were cotransfected with the 2.4 kb fragment and pSVtkneoB. Three of them yielded neo’ transfectants that stimulated TNF release by anti-E CTL clone 82/30, which is CD8+ (Figure 10). No E- transfectant was obtained with four other melanomas, some of which shared A29, B44 or C6 with MZ2. This suggests that the presenting molecule for antigen MZ2-E is HLA-A1. In confirmation, it was found that, out of 6 melanoma cell lines derived from tumors of HLA-A1 patients, two stimulated TNF release by anti-E CTL clone 82/30 of patient MZ2. One of these tumor cell lines, MI13443—MEL also showed high sensitivity to lysis by these anti-E CTL. These two melanomas were those that expressed mage-1 gene (Figure 13). Eight melanomas of patients with HLA haplotypes that did not include A1 were examined for their sensitivity to lysis and for their ability to stimulate TNF release by the CTL. None was found to be positive. The ability of some human anti-tumor CTL to lyse allogeneic tumors sharing an appropriate HLA specificity with the original tumor has been reported previously (Darrow, et al., J. Immunol. 142: 3329 (1989)). It is quite possible that antigenic peptides encoded by genes mage 2 and 3 can also be presented to autologous CTL by HLA-A1 or other class I molecules, especially in view of the similar results found with murine tumors, as elaborated upon supr .

Example 27 As indicated su ra, melanoma MZ2 expressed antigens F, D and A’, in addition to antigen E. Following the isolation of the nucleic acid sequence coding for antigen E, similar experiments were carried out to isolate the nucleic acid sequence coding for antigen F.

To do this, cultures of cell line MZ2-MBL2.2, an B cell line described su ra, were treated with anti—F CTL clone 76/6, in the same manner described for treatment with anti-E CTL clones. This resulted in the isolation of an F antigen loss variant, which was then subjected to several rounds of selection. The resulting cell line, "M22- MEL2.2.5" was completely resistant to lysis by anti-F CTLs, yet proved to be lysed by anti-D CTLs.

Again, following the protocols set forth for isolation of antigen -E precursor DNA, the F’ variant was transfected with genomic DNA from F+ cell line MZ2-MEL3.0. The experiments yielded 90,000 drug resistant transfectants.

These were tested for MZ2—F expression by using pools of 30 cells in the TNF detection assay elaborated upon gupgg.

One pool stimulated TNF release by anti-F CTLs, and was cloned. Five of 145 clones were found to stimulate anti- F CTLs. Lysis assays, also following protocols described supra, confirmed (i) expression of the gene coding for antigen F, and (ii) presentation of antigen F itself.

Example 28 Following identification of F+ cell lines, the DNA therefrom was used to transfect cells. To do this, a cosmid library of F+ cell line MZ2-MEL.43 was prepared, again using the protocols described ggpra. The library was divided into 14 groups of about 50,000 cosmids, and DNA from each group was transfected into MZ2-MEL2.2.S.

Transfectants were then tested for their ability to stimulate TNF release from anti-F CTL clone 76/6. Of 14 groups of cosmids, one produced two independent transfectants expressing antigen F; a yield of two positives out of 17,500 geniticin resistant transfectants.

Example 29 The existence of a gene family was suggested by the pattern observed on the Southern blot (Figure 12). To do this, the 2.4 kb BamHI fragment, which transferred the expression of antigen M22-E, was labelled with 32p and used as a probe on a Southern Blot of BamHI digested DNA of E + cloned subclone M22—MEL2.2. Hybridization conditions included 50 pl/cm: of 3.5xSSC, 1xDenhardt's solution; 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, 2mM EDTA, where herein thus refers to the foregoing conditions; subject to routine, art recognized modification.

Example 30 The CDNA coding for mage 4 was identified from a sample of the human sarcoma cell line LB23-SAR. This cell line was found to not express mage 1, 2 or 3, but the mRNA of the cell line did hybridize to the 2.4 kb sequence for mage 1. To study this further, a CDNA library was prepared from total LB23-SAR mRNA, and was then hybridized to the 2.4 kb fragment. A CDNA sequence was identified as hybridizing to this probe, and is identified hereafter as mage 4.

Example 31 Experiments were carried out using PHA-activated lymphocytes from patient "MZ2", the source of the "MZ" cells discussed supra. An oligonucleotide probe which showed homology to mage 1 but not mage 2 or 3 was hybridized. with a cosmid library derived from the PHA activated cells. The size of the hybridizing BamHI cosmid fragment, however, was 4.5 kb, thus indicating that the material was not mage 1; however, on the basis of homology to mage 1-4, the fragment can be referred to as "mage 5".

The sequence of MAGE 5 is presented in SEQ ID NO: 16.

Example 32 Melanoma cell line LBMEL was tested. Total mRNA from the cell line was used to prepare cDNA, which was then amplified. with oligos CHO9: (ACTCAGCTCCTCCCAGATTT), and CHO10: (GAAGAGGAGGGGCCAAG). These oligos correspond to regions of exon 3 that are common to previously described mage 1, 2 and 3.

To do this, 1 pg of RNA was diluted to a total volume of 20 pl, using 2 pl of 10x PCR buffer, 2 pl of each of 10 mM dNTP, 1.2 pl of 25 mM MgCl2, 1 pl of an 80 mM solution of CHO9, described su ra, 20 units of RNAsin, and 200 units of M-MLV reverse transcriptase. This was followed by incubation for 40 minutes at 42°C. PCR amplification followed, using 8 pl of 10x PCR buffer, 4.8 pl of 25 mM MgCl2, 1 pl of CHO10, 2.5 units of Thermus acquaticus ("Taq") polymerase, and water to a total volume of 100 pl.

Amplification was then carried out for 30 cycles (1 minute 94°C; 2 minutes at 52°C, 3 minutes at 72°C). Ten pl of each reaction were then size fractionated on agarose gel, followed by nitrocellulose blotting. The product was found to hybridize with oligonucleotide probe CH018 (TCTTGTATCCTGGAGTCC). This probe identified mage 1 but not mage 2 or 3. However, the product did not hybridize to probe SEQ 4 (TTGCCAAGATCTCAGGAA). This probe also binds mage 1 but not 2 and 3. This indicated that the PCR product contained a sequence that differed from mage 1, 2 and 3. Sequencing of this fragment also indicated differences with respect to mage 4 and 5. These results indicate a sequence differing from previously identified mage 1, 2, 3, 4 and 5, and is named mage 6.

Example 33 In additional experiments using cosmid libraries from PHA—activated lymphocytes of M22, the 2.4 kb mage 1 fragment was used as a probe and isolated a complementary fragment. This clone, however, did not bind to oligo- nucleotides specific for mage 1, 2, 3 or 4. The sequence obtained shows some homology to exon 3 of mage 1, and differs from mages 1-6.

It is referred to as mage 7 hereafter. Additional screenings yielded mage 8-11.

Example 34 The usefulness of the TRAPs, as well as TRAs derived therefrom, was exemplified by the following.

Exon 3 of mage 1 was shown to transfer expression of antigen E. As a result, it was decided to test whether WO 921203 PCWTUS9bm4Ifi synthetic peptides derived from this eicon‘ 3 could be used to confer sensitivity to anti-E CTL.

To do this, and using standard protocols, ' cells normally insensitive to anti-‘E/c'I‘Ls were incubated with the synthetic peptides derived from Exon 3.1. Using the CTL lytic ‘assays described gum on P8153," and a peptide concentration of 3 mx, the peptide G1u-Ala-Asp-Pro-Thr- .G1y-His-Ser-‘ryr was ‘shown to be ‘ibest; The assay showed \ 1ysis._ofv'_3.0%’, indicating conferringviof sensitivity to the anti-E A Example 35 T A _ p Further tissue samples were tested for the presence of IMAGE genes, using the protocols discussed supra. some of these results" follow.

There‘ was no expression of the MACE genes in brain or kidney tumor tissue. Colon tumor tissue showed expression r of MACE 1, 2, 3 and 4, although not "all tumors tested showed [expression of all MACE genes. This is also true for pancreatic tumor (MAGE 1); non—small cell lung (MAGE 1, 2, 3 and 4), prostate (MAGE 1), sarcomas (MAGE 1, 2, 3 and 4), breast (MAGE 1, 2 and 3), and larynx (MAGE 1 and 4).

Glu-Ala-Asp—Pro-Thr-Gly—His-Ser-Tyr.

The foregoing disclosure, including the examples, places many tools of extreme value in the hands of the skilled artisan. To begin, the examples identify and provide a methodology for isolating nucleic acid molecules which code for tumor rejection antigen precursors as well as the nucleic acid molecules complementary thereto. It is known that DNA exists in double stranded form, and that each of the two strands is complementary to the other.

Nucleic acid hybridization technology has developed to the point where, given a strand of DNA, the skilled artisan can isolate its complement, or synthesize it.

"Nucleic acid molecule" as used herein refers to all species of DNA and RNA which possess the properties discussed ggpra. Genomic and complementary DNA, or "CDNA" both code for particular proteins, and as the examples directed to isolation of MAGE coding sequences show, this disclosure teaches the artisan how to secure both of these.

Similarly, RNA molecules, such as mRNA can be secured.

Again, with reference to the skilled artisan, once one has a coding sequence in hand, mRNA can be isolated or synthesized.

Complementary sequences which do not code for TRAP, such as "antisense DNA" or mRNA are useful, e.g., in probing for the coding sequence as well as in methodologies for blocking its expression.

It will also be clear that the examples show ‘the manufacture of biologically pure cultures of cell lines which have been transfected with nucleic acid sequences which code for or express the TRAP molecules. Such cultures can be used as a source for tumor rejection antigens, e.g., or as therapeutics. This aspect of the invention is discussed infra.

Cells transfected with the TRAP coding sequences may also be transfected with other coding sequences. Examples of other coding sequences include cytokine genes, such as interleukins (e.g., IL-2 or IL-4), or major histo- compatibility complex (MHC) or human leukocyte antigen (HLA) molecules. Cytokine gene transfection is of value because expression of these is expected to enhance the therapeutic efficacy of the biologically pure culture of the cells i_ yiyg. The art is well aware of therapies where interleukin transfectants have been administered to subjects for treating cancerous conditions. In a particularly preferred embodiment, cells are transfected with sequences coding for each of (i) a TRAP molecule, (ii) an HLA/MHC molecule, and (iii) a cytokine.

Transfection with an MHC/HLA coding sequence is desirable because certain of the TRAs may be preferentially or specifically presented only by particular MMC/HLA molecules. Thus, where a recipient cell already expresses the MHC/HLA molecule associated with presentation of a TRA, additional transfection may not be necessary although further transformation could be used to cause over- expression of the antigen. on the other hand, it may be desirable to transfect with a second sequence when the recipient cell does not normally express the relevant MHC/HLA molecule. It is to be understood, of course, that transfection with one additional sequence does not preclude further transfection with other sequences.

The term "biologically pure" as used in connection with the cell line described herein simply means that these are essentially free of other cells. Strictly speaking, a "cell line" by definition is "biologically pure", but the recitation will establish this fully.

Transfection of cells requires that an appropriate vector be used. Thus, the invention encompasses expression vectors where a coding sequence for the TRAP of interest is operably linked to a promoter. The promoter may be a strong promoter, such as those well known to the art, or a differential promoter, i.e., one which is operative only in specific cell types. The expression vectors may also contain all or a part of a viral or bacterial genome, such as vaccinia virus or BCG. Such vectors are especially useful in preparing vaccines.

The expression vectors may incorporate several coding sequences, as long as the TRAP sequence is contained therein. The cytokine and/or MHC/HLA genes discussed gupra may be included in a single vector with the TRAP sequence.

Where this is not desired, then an expression system may be provided, where two or more separate vectors are used where each coding sequence is operably linkad to a promoter.

Again, the promoter may be a strong or differential promoter. Co-transfection is a well known technique, and the artisan in this field is expected to have this technology available for utilization. The vectors may be constructed so that they code for the TRA molecule directly, rather than the TRAP molecule. This eliminates the need for post—translational processing.

As the foregoing discussion makes clear, the sequences code for "tumor rejection antigen precursors" ("TRAPS") which, in turn, are processed into tumor rejection antigens ("TRAS"). Isolated forms of both of these categories are described herein, including specific examples of each.

Perhaps their most noteworthy aspect is as vaccines for treating various cancerous conditions. The evidence points to presentation of TRAs on tumor cells, followed by the development of an immune response and deletion of the cells. The examples show that when various TRAs are administered to cells, a CTL response is mounted and presenting cells are deleted. This is behavior characteristic of vaccines, and hence TRAPs, which are processed into TRAS, and the TRAs themselves may be used, either alone or in pharmaceutically appropriate compositions, as vaccines. Similarly, presenting cells may be used in the same manner, either alone or as combined with ingredients to yield pharmaceutical compositions.

Additional materials which may be used as vaccines include The generation of an immune response, be it T-cell or B-cell related, is characteristic of the effect of the presented tumor rejection antigen. With respect to the B- cell response, this involves, inter alga, the generation of antibodies to the TRA, i.e., which specifically bind thereto. In addition, the TRAP molecules are of sufficient size to render them immunogenic, and antibodies which specifically bind thereto are a part of this invention.

These antibodies may be polyclonal or monoclonal, the latter being prepared by any of the well recognized methodologies‘ for their preparation which need not be repeated here. For example, mAbs may be prepared using an animal model, e.g., a Balb/C mouse or in a test tube, using, e.g., EBV transformants. In addition, antiserum may be isolated from a subject afflicted with a cancerous condition where certain cells present a TRA. Such antibodies may also be generated to epitopes defined by the interaction of TRA and HLA/MHC molecules.

Review of the foregoing disclosure will show that there are a number of facets to the system which may be referred to as "tumor rejection antigen presentation and recognition".

Recognition of these phenomena has diagnostic consequences. For example, the existence of specific CTL clones, or antibodies to the TRA makes it possible to diagnose or monitor cancerous conditions (explained infra), by monitoring the CTLs in a sample from a subject, binding of antibodies to TRAs, or the activity of anti-TRA CTLs in connection with subject samples.

Similarly, the expression of nucleic acid molecules for TRAPS can be monitored via amplification (e.g., "polymerase chain reaction"), anti-sense hybridization, probe technologies, and so forth. Various subject samples, including body fluids (blood, serum, and other exudates, e.g.), tissues and tumors may be so assayed.

A particular manner of diagnosis is to use an adaptation of the standard "tuberculin test" currently used for diagnosis of tuberculosis. This standard skin test administers a stable form of "purified protein derivative" or "PPD" as a diagnostic aid. In a parallel fashion, TRAS in accordance with this invention may be used in such a skin test as a diagnostic aid or monitoring method.

The term "cancerous condition" is used herein to embrace all physiological events that commence with the initiation of the cancer and result in final clinical manifestation. Tumors do not spring up "ab initio" as visible tumors; rather there are various events associated with the transformation of a normal cell to malignancy, followed by development of a growth of biomass, such as a tumor, metastasis, etc. In addition, remission may be conceived of as part of "a cancerous condition" as tumors seldom spontaneously disappear. The diagnostic aspects of this invention involved in include all events carcinogenesis, from the first transformation to malignancy of a single cell, through tumor development and metastasis, as well as remission. All are embraced herein.

Where "subject" is used, the term embraces any species which can be afflicted with a cancerous condition. This includes humans and non—humans, such as domesticated animals, breeding stock, and so forth. vitro and then administer these to the subject. Antibodies may be administered, either polyclonal or monoclonal, which specifically bind to cells presenting the TRA of interest.

These antibodies may be coupled to specific antitumor agents, including, but not being limited to, methotrexate radio-iodinated compounds, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth. Thus, "targeted" antibody therapy is included herein, as is the application of deletion of the cancerous cells by the use of CTLs.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

SEQUENCE LISTING INFORMATION FOR SEQUENCE ID NO: 1: (i) SEQUENCE CHARACTERISTICS: 462 base pairs (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: linear nucleic acid genomic DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACCACAGGAG ATCACTCATT CTCGTGGGGG CTTGTGAATT CCCCCTCCCA AGAACTCTTC GTCAACGCCA TATCTTAACT AATGAAAAGA GGGTGTCTGA GTTTGTGAGC TGTACCCTTT CCTCGTGCTG CGGAGGAAGG TTGCACTGAG TAGCTCGGCT ACCCGGGACT GTTCTGCGAT CTTGGGTAGG CACGTAAAAA TGCTGAGTTT AGGGAGGACC CTGGTCGAAG TCCTGCTGGT CCCAAAGACG ATTCATCCCT AAGTTTTGCA AGTAGTCCAG AGAAGTCTTC CCCCCCCTTT AAGTAAGCCG ACCCTTTGTG CTAGATGTGT CAGCCAATGA AGTTCCGCCT AGTTTACTAC CTTATAGAAG GCTCTCCCAG CTAGCTTGCG CC GAAGATCCTG GCTTACTGTT ACAGCTCTAG ACCCTCCCTC TCTTCCGTAT CATGCATTGT ACTCTACTCT 180 240 300 360 420 462 ATG Met GAC Asp GAA Glu AGT Ser TAT Tyr TCT Ser GAC Asp GAG Glu GCC Ala GGT Gly 145 AAT Asn CTG Leu GAA Glu GAG Glu 220 INFORMATION FOR SEQUENCE ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: TCT Ser GGT Gly ATT Ile TTT Phe GAA Glu GTC Val GAC Asp GAA Glu GAA Glu 130 GCT Ala GAA Glu GTG Val AAT Asn GAG Glu GAT Asp GAT Asp CTG Leu CTG Leu AGG Arg GAT Asp GAG Glu GAA Glu 115 GAA Glu GGC Gly GTG Val TCT Ser GCT Ala 195 GAG Glu AAC Asn GGG Gly CCT Pro GCG Ala GAT Asp GAG Glu GAC Asp 100 GAA Glu GAG Glu GCT Ala AAG Lys ATA Ile 180 GAT Asp GAG Glu AAG Lys AAT Asn TAT Tyr CTC Leu GTG Val GAT Asp GAC Asp TTG Leu ATG Met AAC Asn TGT Cys 165 CCA Pro GAA Glu GAA Glu nucleic acid linear genomic DNA (Xi) SEQUENCE DESCRIPTION: AAA Lys AGG Arg CTA Leu CAG Gln GCC Ala GAA Glu GAC Asp GAG Glu AGC Ser TGT Cys 150 AGG Arg GTG Val GAG Glu GAG Glu 225 CCA Pro TGC Cys GGG Gly ATG Met TGG Trp GAC Asp GAC Asp AAC Asn GTG Val 135 GCC Ala ATG Met AAC Asn GTT Val GAA Glu GAC Asp AAT Asn TGG Trp TTC Phe ATA Ile GAT Asp GAT Asp CTG Leu 120 GAA Glu TGT Cys ATT Ile CCT Pro GCA Ala 200 ATG Met base pairs SEQ ID AAA Lys TTA Leu CTG Leu ATA Ile GCC Ala GAG Glu GCC Ala 105 ATG Met ATG Met GTT Val TAT Tyr AAG Lys 185 ATG Met GGA Gly GCC Ala TTG Leu GTC Val GAC Asp AGG Arg GAT Asp TTC Phe GAT Asp GGT Gly CCT Pro TTC Phe 170 GAA Glu GAA Glu AAC Asn CAC" CAC His TTC Phe GCC Ala CAA Gln GAT Asp TAT Tyr GAT Asp GCC Ala GGC Gly 155 TTC Phe CAA Gln GAG Glu CCG Pro 230 AGT Ser cos Arg GCT Ala CTT Leu AGC Ser GAG Glu GAT Asp GAA Glu GGA Gly 140 CAT His CAC His ATG Met GAA Glu 210 GAT Asp GGC Gly TAC Tyr GTT Val TAT Tyr AAG Lys GAT Asp GAT Asp TCA Ser 125 GCT Ala CAT His GAC Asp GAG Glu GAA Glu GGC Gly TCA Ser TCC Ser GTC Val GAG Glu CGC Arg GAC Asp GAG Glu 110 GAA Glu GAG Glu TTA Leu CCT Pro TGT Cys 190 GAA Glu TTC Phe GGT Gly CTG Leu ACA Thr GAG Glu ATG Met TAC Tyr GAT Asp GAT Asp GAA Glu AGG Arg AAT Asn 175 AGG Arg GAA Glu TCA Ser GGT Gly GAA Glu ACA Thr CAG Gln TCC Ser TAC Tyr GAT Asp GAG Glu ATG Met AAG Lys 160 TTC Phe TGT Cys GAG Glu CCT Pro 2 96 (2) INFORMATION FOR SEQUENCE ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 228 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG GAAGAAGTGG TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA ATTTGATTTT GTTCTAAAGT TCATTATGCA AAGATGTCAC CAACAGACTT CTGACTGCAT GGTGAACTTT CATATGATAC ATAGGATTAC ACTTGTACCT GTTAAAAATA AAAGTTTGAC TTGCATAC 180 228 INFORMATION FOR SEQUENCE ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: base linear (Xi) SEQUENCE DESCRIPTION: ACCACAGGAG GAAGATCCTG CAGCCAATGA AAGTTTTGCA CACGTAAAAA CCTCGTGCTG AGAACTCTTC CATGCATTGT CTAGCTTGCG ACCCTTTGTG AATGAAAAGA ATCACTCATT GCTTACTGTT AGTTCCGCCT AGTAGTCCAG TGCTGAGTTT CGGAGGAAGG GTCAACGCCA ACTCTACTCT CC ACCCGGGACT GGGTGTCTGA CTCGTGGGGG ACAGCTCTAG AGTTTACTAC AGAAGTCTTC AGGGAGGACC TTGCACTGAG TATCTTAACT pairs nucleic acid genomic DNA SEQ ID NO: CCCAAAGACG GTTCTGCGAT GTTTGTGAGC CTTGTGAATT ACCCTCCCTC CTTATAGAAG CCCCCCCTTT CTGGTCGAAG TAGCTCGGCT CTAGATGTGT ATTCATCCCT CTTGGGTAGG TGTACCCTTT CCCCCTCCCA TCTTCCGTAT GCTCTCCCAG AAGTAAGCCG TCCTGCTGGT ATG GGT TAC TTC ATA TGG GAT GAG GAG GAT GCT GGC TAT AAC GAA GAG TAG TCT GGT TCC GCT GAC ATA GAA GAC GAA GAG GAG CAT TTC CCT GAG GAG GAT GAC CTG GTT GCC GCC GAC GAC GAA GCC GAA CAT TTC AAG GTT GAA AAC GGT GAA GTC CTT AGG GAT GAC GAA GAA ATG TTA CAC GAA GCA GAG AAG GAT GAA ACA TAT CAA GAG GAC TTG GAA GGT AGG GAC CAA ATG GAA AAA GGG ATT ACA GAG AGC GAT GAT GAG GAG GCT AAG CCT ATG GAA ATG CCA AAT CTG AGT GAG AAG GAT GCC AAC ATG GGC AAT AAT GAG GAG GGA GAC AGG CCT TTT CAG CGC GAG TTC CTG AGC GCT GAA TTC TGT GAA AAC AAA TGC TAT CTG TAT ATG GAT TAT ATG GTG AAC GTG CTG AGG GAA CCG GCC AAT CTA GCG GAA TCC GAC GAT GAT GAA TGT AAG GTG TGT GAA GAT CAC TTA GGG CTC AGG TCT TAC GAT GAT ATG GCC TGT TCT GAA GAA GGC AGT TTG TGG CAG GAT GTC TAC GAG GAA GGT TGT AGG ATA AAT GAG TTC GGC CAC CTG ATG GTG GAT GAC GAT TCA GCC GTT ATG CCA GCT GAG TCA TCA CGG GTC TTC GCC GAG GAC GAT GAA GGA CCT ATT GTG GAT GAG CCT GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTTTTTAGT ATTTGATTTT GTTCTAAAGT TCATTATGCA AAGATGTCAC CTGACTGCAT GGTGAACTTT CATATGATAC ATAGGATTAC GTTAAAAATA AAAGTTTGAC TTGCATAC GAAGAAGTGG AATCCAGAAA CAACAGACTT ACTTGTACCT 150 200 250 300 350 400 450 462 504 546 588 630 672 714 756 798 840 882 924 966 1092 1134 1137 1187 1237 1287 1337 1365 INFORMATION FOR SEQUENCE ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: base linear pairs nucleic acid genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACCACAGGAG GAAGATCCTG CAGCCAATGA AAGTTTTGCA CACGTAAAAA CCTCGTGCTG AGAACTCTTC CATGCATTGT CTAGCTTGCG ACCCTTTGTG ATG TCT AATGAAAAGA ATCACTCATT GCTTACTGTT AGTTCCGCCT AGTAGTCCAG TGCTGAGTTT CGGAGGAAGG GTCAACGCCA ACTCTACTCT CC ACCCGGGACT GGGTGTCTGA CTCGTGGGGG ACAGCTCTAG AGTTTACTAC AGAAGTCTTC AGGGAGGACC TTGCACTGAG TATCTTAACT CCCAAAGACG GTTCTGCGAT GTTTGTGAGC CTTGTGAATT ACCCTCCCTC CTTATAGAAG CCCCCCCTTT CTGGTCGAAG TAGCTCGGCT CTAGATGTGT ATTCATCCCT CTTGGGTAGG TGTACCCTTT CCCCCTCCCA TCTTCCGTAT GCTCTCCCAG AAGTAAGCCG TCCTGCTGGT GGT TAC TTC ATA TGG GAT GAG GAG GAT GCT GGT TCC GCT GAC ATA GAA GAC GAA GAG GAG GAT GAC CTG GTT GCC GCC GAC GAC GAA GCC GAA AAC GGT GAA GTC CTT AGG GAT GAC GAA GAA GAA ATG GGT AAG GAT GAA ACA TAT CAA GAG GAC TTG AAA GGG ATT ACA GAG AGC GAT GAT GAG GAG GCT CCA AAT CTG AGT GAG AAG GAT GCC AAC ATG GAC AGG CCT TTT CAG CGC GAG TTC CTG AGC AAA TGC TAT CTG TAT ATG GAT TAT ATG GTG GCC AAT CTA GCG GAA TCC GAC GAT GAT GAA CAC TTA GGG CTC AGG TCT TAC GAT GAT ATG AGT TTG TGG CAG GAT GTC TAC GAG GAA GGT GGC CAC CTG ATG GTG GAT GAC GAT TCA GCC TCA CGG GTC TTC GCC GAG GAC GAT GAA GGA GTGAGTAACC CTCTTGCCCA TGGAGCCATT CCCCACTCCT TTCAGTCCAT TCCCCCTCGG TTCAGGCTTC CCTTTTCGCG TCACCAGCTT TCCTGCTCCC CTACCTGCTT TGCTCCTCCC CCTCCCTCCC TTGGTTTTTC TCACTCTGTA CCTCCCAAAT GCCTTTCTTT AACTCCCCTT TTCCCTTCCG CCTCCCCCTC GCCCCGTTCC CGTGGTCTTT CATCTGTAGT CCTGGCTCTC TGCTCCGCTC CCTGCTCTGC CTCAACTTTT CCCATTTGCT CCTTTTCTTT TGCTCTCCCT CTCCCCCTCC CCCTCCCCCT TCCCCCTCCC CCTCCCCAGG GAGACAGGGT GACCAGGCTG GCTGGGATTA TTTCTCCTCT TTGGCACCTT GCACCCTTCC TTTGCTCGAC CCTTTTTTGT GGC GCT ACTCTAGATT AAAGACCACA CTGTCCACGC TCTTTCCTTT TCCCTTTCCC CGTGCCTTCT CCTCTCCCGA CCTGCTCCCC GCTCCCCTCC CCTCCCTGTT TGCTGCTCCC CCTCCCTCCC CCTTTTTTTT TTCTCTTTGT GCCTCAAACT AAGGCTTGCA CTGGTCTCCC TCCTTTACAG TAGCCCTGCT TTTTAGCAGC GCCTTTCCTC TGT GCC CAGGTGGGGT TTTTGGTTGG CTATCCCCGC TCCCACCTTG CTTTGCTCTC GCTCTCTGAT AACCCTCCCC TCCCCCTCCC CCCTTTTGCA TACCCTTCAC TCCCTATTTG TATTTGCATT TTTTTTTTTT ATCCCTGGCT CAGAAATCTG CCAGGACTGC TAATCCCTTT GACCCCCTCC CTGTTCCCTC CTTACCTCTC CTGGCTCCCC GCATTCTTTA GGGTCATTGC TCCTCCCATC CCTCTGGAGC CTTGCTCCCC CCCCACCCTC TTCCTGTTCC TATTTACCTT CCTTTTCTTT CGCTTTTCCT CATTTTCGGG TTCGGGTGCT TTTTTTTTTT GTCCTGGCAC CCTGCCTCTG CCCAGTGCAG TCTGCATGTT CCCTCCCTGT TCCCTGCTCC CCTGCTTTCT TCCACCTTCC 150 200 250 300 350 400 450 462 504 546 588 630 672 714 756 798 840 882 916 966 1116 1166 1216 1266 1316 1366 1416 1466 1516 1566 1616 1666 1716 1766 1816 1866 1916 1966 AGCTCACCTT TTTTTTTTTT CCTCTGTGTG TCTGCCTTTC CTTTTCTAGA CCTGACCCTG CCTTTCTCCA TCCTGCTTCC GACTTCCTCT CTCTCTGTCC ATGTGTCTCT CCATCACCTC CCTGCTTCTT TCCATGTCCC ATTTCCCTCT TTCCCTTTGC TACTTGATCT CTTTGTCCCC ATCAACAACA AAGGCTGGAT AAGTGGCTCC CTTGATCCTT CAGGCCATGC GAATCTGAAA TAGTGATATT TCCTTCTACA GGCTAAAGAT TTGCTAAAAT TTTGTTTGTT GCACCTTGTT CCTTTCCTGT CTGTCCCTGC CTCCCCCCTC CTCCCCTTCC GCCTGTCACC TTTACCCCTT CCAGCCGCCC ATCACTTCCC CTTCCTATCT TCTCCTCCCT TACCCTGCCT CTCTCAATTC TTCTCCCTTA TTCTCCCTCC TCTCTCCTCT AGACCCTACA AGGAGGCAAG GAAAATAAGG TATAACCCTA GCTGCTTCTT TCCATGCTTG ACTAGGGGCC TCCCCCTAAA GGTGAGAAGT ACTTGGAACC ATTCTTTCTC TGGTTGTTTG TTCCAAGATC TCCCTCCCCC TCCCTTCTCT CAGGCTTGCT CCTCCCAGCT CCTCCTTCTC CCCTCTCCCT AGTTCCCTGC CCTAGTTTCA ATCCCTTCCT TCCCTTTCCT CTCCCATTGC CCTGTCCCAT GCCTCTTCTT TCCTTTCCCC CCACATACCC GTATCCTGTG AAACAGAGCA CCAGGTTCTG AGTACCAAGG TTACATATGT GCGCTTGCTC AGTGGTTTGT AATTATAACA GGAAAAATTG ATAGAAGCGT ACATATTCAT GTTGTTTGGT CCCCTCCCCC TCGCTGGCTC GCTAACCTTT GTTTGCTTCT CCCCCCTCTT TCCTCTCTGT ACTCTCCTCC AGTCCTGGAG CTTCCCTTTC TTCTGTCCCC CTCTCTTCCA CCTCTTACCT TGTGCTCCCT CCTCTTCTCT TTCCCCTATG TTTTTCCTTT CACAGGAAGT AAATCCCAAA AGGACAGCTG GAGAAAGTGA TGGCACATCT AGCGTGGTTA TTTGGGGACA AACAGATTCA TCACTATGAA TGTTAAAATA ATTCTCCAG TTGCTTTTTT TCCGGCTTCC CCCCTCCTT TAATGCCTTT GTGCACTTTT TTCCCACCTC TTCTCCCACT CTGCCTGCTG TCTTTCCTGC ACTCTCCCCT TCTCCTCTGT TTTTCTTCCA TTATGCCCAT CACATCTTCC TGTATCTCCC CCCTCTACTC CCACCCTGCC GGGAGGTGCC ATCAGCAGGA GAATCTAGCC TGGTGAAGTT TTCTCAAATG AGTAATGGGA AATTAGCACG TGATTTGAGA GTTCTTTTTA CTGCTTTCTT GT GTT CCT GGC CAT CAT TTA AGG AAG AAT GAA AGG ATG ATT TAT TTC TTC CAC GAC CCT AAT TTC ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAG GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC GTG AAG TGT CTG GTG TCT AGG TGT GAA GAA GAA GAA CCG GAT GGC TTC TCA CCT TAG GCATGCAGGT GCTAAGAGCA TCTTTTTACA CCCTAAGTTA GTAGTGAGAC GACCAGTAAA TTCTTATAGT TTCAAGAAAG TTCTGATTTT CTTAAAATTT TAGAATTCAA AATGTTTTTT GTAACTGGGG GTTCTGGTCT CAGTAGGTTA ATAAATACTC ATTTTAGTTT ACTGGCTTCA TCTTTTTAAA TTAATAAGTA AACAGAAGTC TACTTACTAC AGATCATGCA ACCTTTGAGA ATCACACGCC TTTCATTTCT CCTTCATCTT TTCAAATTCT AAAAAAAATG GGCTTAGGGA CTGAGAAGCA GTGAGGTTGA TAACAGCTAA CTCCTTGAGA CTAACCAACC AAATATTATT TTAAATTAAT AATGATGTCT AGATGAGAAG GTGAAATGTG CAGCTGATAA ATGGTTCACA AGACCTGTGG TAATTTTCCT TAATTCAATC CAAATCTCAT ATCTGTAGGG GTCAGAGAGA TATGATCAGA GGATCTCTGA AACAATGACA ATTCCTAACA GGTAAACTAA CCAGTATACA AGATGCCTGT TTGTTAGACT GCCATGGAAA CAGCTGACAA TGCAAATTAT TTTTAAAGAG TAACTTTAGT TTAATTTTTA TTTTAAGAGA TTGCGGTATA ATGGAAAACC TTATGGACAC GGGAAACACA AGACATAAAA TATGCCTGTA ACAATTGTTA GTTTTAAGAA TCTTTAGATT CGGGAGTAGA TCGCATATTG AAATAAGTGT TATTTTGTCG ATGAAAATCT TTTTTTCACT GATTTCTTAA TGAAAGCAGA GCAATAGGGA AGGCCCTTGC TCTCCAAATC ACAGGGAAAT TTGGCAAGAA 2116 2166 2216 2266 2316 2366 2416 2466 2516 2566 2616 2666 2716 2766 2816 2866 2916 2966 3016 3066 3116 3166 3216 3266 3316 3355 3396 3438 3480 3522 3564 3576 3626 3676 3726 3776 3826 3876 3926 3976 4026 4076 4126 4176 4226 4276 4326 4376 44 AGTCAGGAGT GTATTCTAAT AAGTGTTGCT TATCTCTTAT TTTCTTCTAC AGTTGCAAAG CCCAGAAGAA AGAAATGGAC AGCGGAAGAA GTGGTTGTTT TTTTTTCCCC TTCATTAATT TTCTAGTTTT TAGTAATCCA GAAAATTTGA TTTTGTTCTA AAGTTCATTA TGCAAAGATG TCACCAACAG ACTTCTGACT GCATGGTGAA CTTTCATATG ATACATAGGA TTACACTTGT ACCTGTTAAA AATAAAAGTT TGACTTGCAT AC 4576 4626 4676 46 (2) INFORMATION FOR SEQUENCE ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Leu Pro Tyr Leu Gly Trp Leu Val Phe (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: INFORMATION FOR SEQUENCE ID NO: 7: (i) SEQUENCE CHARACTERISTICS: base pairs linear genomic nucleic acid (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGATCCAGGC GGGGTCATCC TCCTGGTAGC GGCCCGTGGA TGGTCTGAGA GCCAGCAGTG CAGGACACAT CCTGTAGAAT TTCCTCCTTC CTGGAGGCCA TTAGAGTCTC CTCTCCCCAG GCCTGCTGCC ACTGCAAGCC GTGTGTGTGC CCTGGAGGAG AGGGAGCCTC CCCAGTGAGG TATCCTGGAG TGGTTGGTTT GCAGAAATGC GATCTTCGGC TGAAGGAAGC GGTCTCTCCT AGGCTTCCTG CTCCTGAGGA GGGAGGGAGC TTTGGTGCAG CGCACGCTAT ATGTGAAAGT TTCTTCCCAT CTGAGCATGA ACCTTCCAGG CCCATTCTTC TGTTCTATTG TCAAATGTTT TATGAATGAC GTCTTGTGTT ATAATAACAG AAATAGATGA CTTATACCTC CCTGCCAGGA ACTGCATGAG ACTGAGAAGC TTCCTCTTCC CAGTATCCTC AATGTTTGCC AGGACTCCAC CGACCTCTGC AGGTTTTCAG CAGAGGAGCA CAAGGTTCAG GCCTGTGGGT CTGACGAGAG TGAGGAAGCC AGGCTGCCAC GTGCCCACTG CGCCTTTCCC GTTCCAGCAG TCCTTGTTCC TCTGCTCCTC TGGAGAGTGT AAAGCCTCTG AGACCCCACC ATGATGGCCT ATAATTGTCC GGAAATCTGG ACAGTGCCTA GAAAAGTACC GAGTTCCTGT CCTTGAGTAT CCCTGCGTGA GTTGCAGCCA GCCGCGTCCA ACTCTGAAGA GGTGACTTGG TTTTTTAAGG AGCAGTCACA TTATTCAGAT CAGTGGAATA GATAAAGAAC AGTCTATTCT AAAATATAAG AGTGGGGATG CAGGGCTGTG TGGAGCTCCA AGGTCACAGA CTGAATGCAC AGAGTCTGGC TGGCCGGCTG GGGACAGGCC CCAAGGAGAA TTCTCAGCTG CTTCATTGCC TCATCATGTC CTTGAGGCCC CTCCTCCTCC CTGGGTCAAC ACTACCATCA CCGTGAAGAG GAGCAGTAAT AAATATCGAG CATCAAAAAT AGTCCTTGCA GGCCACTCCT GCTGGGTGAT TGGTCATGAT GAGGAGCTGA TGGGGAGCCC TGGAGTACGG GGGGTCCAAG GTGATCAAGG AGCAGCTTTG AGGCCAGTGG GCAGCTTCCC GAGCGGTCAG AGATTTATCT GATGGTTGAA CAGTTCTGTG TGGGAAATCC AGTACTTAGA TAAAGAAATT GTAAAATTTT GGCCCTGCGT TCACAGAGTC CTTGCGGTCT GGAACCAGGC GCAGAGGATG ACCAAGGGCC CTCACCTCCC TACCCTGAGT AACCCAGAGG GATCTGTAAG AGGCCTCTCA CAGCTCCTGC TCTTGAGCAG AACAAGAGGC TCTCCTCTGG AGATCCTCCC ACTTCACTCG GAGGGGCCAA CACTAAGAAG CCAGGGAGCC TACAAGCACT GCTGGTCTTT ATGTCCTTGT AATCAGATCA TGCAATGGAG GTGTGATGGA AGGAAGCTGC CAGGTGCCGG GGCCCTCGCT TCAGTGCAAG AGAGAGGAGG GAGGGGGACT CTGCCTCGTG TGTTCTCAGT TTGTTCTCTT TGAACTTCAG TATATAGTTT ATTCTATTTT AATGTGAAAA AAGAGATAGT TAAAGATATA GAGAACAGAG CAGCCCACCC GCACCCTGAG AGTGAGGCCT CACAGGGTGT CCACCTGCCA TACTGTCAGT ACCCTCTCAC ACAGGATTCC TAGGCCTTTG CACACTCCCT CCACACTCCT AGGAGTCTGC CCTGGGCCTG TCCTGGGCAC CAGAGTCCTC ACAGAGGCAA GCACCTCTTG GTGGCTGATT AGTCACAAAG GTTTTCCTGA GGCATTGACG CACCTGCCTA TGCCCAAGAC GGCGGCCATG GGTGTATGAT TCACCCAAGA ACAGTGATCC GAAACCAGCT AGTTCGCTTT AAGAGGGAGT GGGCCAGTGC TGACATGAGG AGTAGGTTTC TTGGAATTGT CATCCAAGTT AAGGGTAAGA GTGAATTGGG ATGAGCAGTA CAATTCTTGC TGCATACCTG 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 GATTTCCTTG AATTCTTCCT TTTTTGGAAG CATACCCACC AGGTGGCAAG GGGTGTGGGG GGCATTTTGG AATGATCTTG GCTTCTTTGA GTTCACTGGC GCCCTGGGTT CATAGGGTCG ATGTCCTCTA CTCCGGGTGA GCTTTGGGAA GGTGGATCC GAATGTAAGA TCTTTTCTTC AGTAGTGGAG TAGAGTCTAG AAGATGTAGG GAGTGGTGGA ACTGCAGTTC GAAATTAAAT TCCATGCACT ATGCTAAGGT GAGCTGCAGT GAAAAGTGAG GTGTCAATGC CTTCTGGGGG CTGAATAAAG GAGCATCTGC AAGCCAGACT CACGTAATCG AGAGGGGTGA CCTGAGCTGG AGCTGATTGT 2200 2250 2300 2350 2400 24 (2) INFORMATION FOR SEQUENCE ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: CCCGGGGCAC TACGCCACCC AGAATCCGGT ATGTGACGCC CGGTCTGAGG TAAGGAGGCA AGATAGAGGA GGTGGACTTC CTGGGGACTC AGAGGGCAGC AGGGCTGAGG ATGCTCACTC CCCCACATCC ATTCCACCCT CAGGCAGGAT GCCACTGACT AGGGACGGCG AGGCAAGGTG GAGAGCCCCA CGCGGGAAGA CCTTGAGAGA TGTGACCAGG GGCATCAAGA ACTCCAATCC CCCATCTCCT CCTGACCACC TCACCCTCAC CCCATCGCCT CCAGGGAAGC AGATCTGAGA ATCCACTGAG CTGAGGGAGG ATCCAGTACC GTCTCAGCTG GGCAATCTGT GGCAGGGCCC ACCCTGGGAG CACCGCCACC TCTTGTCAGA CAGGCACTCG GCTTGAACAG CACTGGCATC ATCCAAACAT TCCACCCCTG ACTGACTTGA GGCGGCTTGA AGGTGACATG CCCCAAATAA TCAGGCTGGG GAAGTCAGAG GTCCAGGCTC GTCCCTAAGA CCGTGACCCA CCCACCCCAT CACCCCCACC CCGGTTCCCG TGCGCATTGT TAGAGTTCGG AGAGGCTGAG AATATTCCAG CGTCTCAGCC CACCAGGTTC GCAGGACTGG TCAGCACCCA CCACTCCCAC CAGCTACACC ACCCTCCAGC TGCCCCCAAC CCCCCATTCT CCTGGTAGGC GAAGCCAGGT GGGAGTGGTT ACTGAGGAGG ACCCCTGCTG GACCACCCCC AGTCATAGCT AGGCATCAAG GGAACTGAGG CCACTCACAT ATCCCTGCTG GATCTTGACG GGCCTCAGGG CCTCCCCCTA CTTCACGCTC CTCTCAACCC GCATTAGTGG GATCGGTGGA CTGAGGGAGG TCCCTTCATG CCACCCCCAG CTCCGTGTGA TGCCAGACAT CCCCACTCCC ACCCCCTCTT CCCTCAACCC CCCACCCCCA CCAGGAAACA GGGGCAGAGA CCGAAGGAAC GGAGGACTGA CCCCGCCCTT TGGGCTGCCC TTCTCCCCAA TTAGGAGAGG AGAGGGAGGG CCCATTCGCA TCCACCCCCA CCCAGCACCA CCCACCCTCA GGCAGAATCC CCGATGTGAA TCATTTAATG TTAGGCTCTG CACACACCCC CCAGCCCTGG CGTCCCGTCC TATGTGACCG GTCCAGCATC GTTCCCCACC TCCCATACCT TCAACCCACG TCCCCATCCA GAGCAGAGGG genomic DNA MAGE-1 gene SEQ ID NO: CCACCCCCAA ACCCCCAGCC AGGGAAGCCC TTAGAGAGAA GGGAAGCGGG ACTGAGGACC CCAGTCCTGG CCCCCTTGCT TCAGGGAAGG CATGCTCAGG GTGACCCAAC CATTGTCATT TGATGCCCAT CGCCCACTCC TCCGGGTGCC GAAGCGAGGT CTGACCCAGG GGACCCCGCC GCTGCCAGCC CCAGACCCCT GCTCTGGAAT GCAGGGCACA CTGTGGGCCC TTCCCATTCC TCCCTACTCC GCCCCAACCC TCTCTCTCAT GGTTTGCCCC ACCACTGACT GTTCTGAGGG TGAGGAGGCA AGGTAGATGG ACCACCCGGC CACTGCCACT GGGCAGGGTT CGCCCGGCAT CACACCTGTC ACCCCCTACC GAAGCCACGG GGGTCTGATG AGGGCCCTAC TCCCTCCCTT CAAGCCAGGC AGGTGCCCAG GCGAGGTTTT CCCAGCTCTG CACTTACCCC ACCATCTGGT GCTTAAACCA GCTGCTTAGG ATTCTCAAGG CCCCACTCCA CCAACCCCCA CCGCCCAGCC CACCCCCACC CGGATGTGAC TTCCATTCTG CTCTGTGAGG ACTCCAAATA CTGGCCCACC GCTCCAAAAG CAGAGGTTGC GGCTCTGCCA CCAAGACTGC CCACCCAACC TACTCCGTCA TTCTGCCACC GTGCCCCACT TGCTCTCAAC TGAACCTCAC GCGGCTTGAG AGGTGAGATG CCCCAAAATG CAGGACAGAT TAACCCACAG GGTCAGGAGA TAGGGTCAGG TCCTCATCTC CCCAACCTCA GAATGGCGGC GAGGGAAGGG TGCGAGATGA 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 GGGAGGCCTC ACTGAGGCTG TTGCATGGGG GGGAGGACTC GAGAGGTCCA TGAGGTGACA AGAGGGAGGA AGGACTGGGG CTGTCCCCTT TTCCATTCTC ATGGGGTCTT GGTTGAGGAA AGGCTATTGG TCACCCAGGA GTGAGGACCT TCTGGTCTAA GAACATGAGG CTGCACAGAA GGGCCGTCTG ACGGGGAGGC GTCCCAGGCC CAGGACACAT AAGGACCTAG TCCTTATCAT GGGCAGGATC CAGAGGGGGT CACCCTCCTG CTGAGGGCCC GGCCTTGGTC GGTGTGCCAG TGCCACAGGA TCAGTCCTGT CTCACTTCCT ATTCCCTGGA CTTTGTTAGA TCCCTCTCTC CTCCTGCCTG AGAGGACCCA CCACTTCTGG GTGGGACCCA AGGGGACCTT GGGCACGGTG GGACAGAGCT GTTCCAGGAT ATATCCCCGG TTAGTAGCTC ACTTGTACCA GGGGTAAAGG GCACAGGCGC AATCCACACC TGTGGCTTCT CATTCTCAGA AGACAGAGCG GAGGACTGAG ATCAGCCCTG CCGAGGTCCT CTTGGTCTGA CTGCCAGGAG TAATTCCAAT GCACGTGTGG GGATGTGAAC CAGGCCCTGC CATCCACTGC GTAGCACTGA GTGGATTCCT TGAGACAGTA CAGTGAATGT CACATAGGAC AGAATCGACC CCTTCAGGTT GGCCACAGAG GTCTCCAAGG CCCAGGCCTG CTGCCCTGAC GCACCCTAGG CCTCAAGAAT GGCCTGCAAG GGAATCCAGA GCCACATATG GTGGTCTGAG CCATATGGCC CTCAGAAAGA TAGGGGGACC CAGGCAGGAA GGGGATGTCT TGGCAGGAAT CCAGAACCAA TTTTCACTCC GGGTGACTCA GTCCCAGGAT GGTACCCCAG CCCCTGCTGT TCCGTTATCC GAAGGCTGCG TCAAGGTGAG GAATTTTGAT CCAGATGTTT TCTTGATTTG CAGGAAAAAT ATGAGAGTGG GAAGCCAGGG CTTCCTGGAG TCCTCAGGTC TTGCCCTGAA TCCACAGAGT TCTGCTGGCC TTCAGGGGAC GAGCACCAAG TTCAGTTCTC TGGGTCTTCA GAGAGTCATC ACACCGCACC CAGAACGATG GCTTACGCGG TCAGTGTGGA GCCCATATTT AAGTGGGGCC CAAGATGTGC AGGGACTCCA AGATCAGGGA GTTGGGGGGC ACTCATGTCA AAAGATGAGT AGGGGTCAGC TGTTTCCAGA GGTCAACGTA CTGCCATGCG GACCAGAACA CACCCCAGAG TGGGATCATT CTCAGGTCAG GACCAAGCGG ATCTCTTGCT GTCCCCTCCT GATTTCTCAG ATAAGGGCCC GGATGTCACA CTGTGCTTGC CTCCAGGAAC ACAGAGCAGA TGCACACCAA CTGGCCTCAC GGCTGTACCC AGGCCAACCC GAGAAGATCT AGCTGAGGCC TTGCCCAGCT CCTGTCTGAG GGGACTCAGA AGGAAGAGGA CCTCGGCCCT CCTGCATCTT TCAGGTCAAC CCCCTTCATG CACAGTCTGG TGGCGGTATG CCTCAGGGAG GGGAATTGGG GAGACAGACA CCTGGACACC TCTGGGGCAG GGGACCCCCA TTCGGGTGAG CTGAGGGAGA AGCATGGGCT GATGTCAGGG TAGAGGGAGC GCACCTCACC GCCCTTCCCC GTCCTTCCAT ACCAGCAAAA TGCGTGAGAA GAGTCCAGCC GGTCTGCACC CAGGCAGTGA GGATGCACAG GGGCCCCACC CTCCCTACTG TGAGTACCCT AGAGGACAGG GTAAGTAGGC TCTCACACAC CCTGCCCACA ATG TCT CTT GAG CAG AGG GCC CTT GAG GCC CAA CAA CAG GCT GCC ACC TCC TCC CTG GAG GAG GTG CCC ACT AGT CCT CAG GGA GCC TCC ACT CGA CAG AGG CAA CCC GAG GAG GGG CCA AGC ACC CGA GCA GTA ATC ACT AAG CTG CTC CTC AAA TAT CGA GAA ATG CTG GAG AGT GTC CCT GAG ATC TTC GGC AAA TTT GGC ATT GAC GTG AAG TAT GTC CTT GTC ACC TGC CTG GGT GAT AAT CAG ATC ATT GTC CTG GTC ATG ATT GAG GAG GAA ATC TGG GAG GAT GGG AGG GAG CAC AGT AGT GAG TCC GCT GCC AGT TCT AAG GCC ATC GCC GAA CTA ATG GCA GAG GCC CTG GCC TCT GGG TTT GAG TGT GTG AGG AAA TCT GCA GGT CCC ATG CTG TAT CAC CTG CCT TCA CCC GGT ATC GCT GAG AAT GAG GAC CTC AAG GAG AGT GGG TGC GGC CTG ACA ACT TCC CTG GAT CCA TAC TCC CCC TCC ACA GGC GTG GAG AAG CTG GTC GAT ACC AGC GAG TTG GTC AAG TTG ACC TAT GGC GGC ATG CCC CCT GTG CTG CCT ATC AGC TCC GTT ACA CAC CAG GGC GAT TTC CAT GAG AGG GAG TGT GGC CCC AAC CGT TTG GGT AAG TGT CTG CAC GGC CTG GCT GTG AAG GAA GTG ACC CAG TTC GAA TTC TTT GCA TTT GTC TCC CTG ATA CCT TAT CTG 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3930 3972 4014 4056 4098 4140 4182 4224 4266 4308 4350 4392 4434 4476 4518 4560 4602 46 CTC ACC CAA GAT TTG GTG CAG GAA AAG TAC CTG GAG TAC GGC AGG TGC CGG ACA GTG ATC CCG CAC GCT ATG AGT TCC TGT GGG 4728 GTC CAA GGG CCC TCG CTG AAA CCA GCT ATG TGA 4761 AAGTCCTTGA GTATGTGATC AAGGTCAGTG CAAGAGTTC 4800 GCTTTTTCTT CCCATCCCTG CGTGAAGCAG CTTTGAGAGA GGAGGAAGAG 4850 GGAGTCTGAG CATGAGTTGC AGCCAAGGCC AGTGGGAGGG GGACTGGGCC 4900 AGTGCACCTT CCAGGGCCGC GTCCAGCAGC TTCCCCTGCC TCGTGTGACA 4950 TGAGGCCCAT TCTTCACTCT GAAGAGAGCG GTCAGTGTTC TCAGTAGTAG 5000 GTTTCTGTTC TATTGGGTGA CTTGGAGATT TATCTTTGTT CTCTTTTGGA 5050 ATTGTTCAAA TGTTTTTTTT TAAGGGATGG TTGAATGAAC TTCAGCATCC 5100 AAGTTTATGA ATGACAGCAG TCACACAGTT CTGTGTATAT AGTTTAAGGG 5150 TAAGAGTCTT GTGTTTTATT CAGATTGGGA AATCCATTCT ATTTTGTGAA 5200 TTGGGATAAT AACAGCAGTG GAATAAGTAC TTAGAAATGT GAAAAATGAG 5250 CAGTAAAATA GATGAGATAA AGAACTAAAG AAATTAAGAG ATAGTCAATT 5300 CTTGCCTTAT ACCTCAGTCT ATTCTGTAAA ATTTTTAAAG ATATATGCAT 5350 ACCTGGATTT CCTTGGCTTC TTTGAGAATG TAAGAGAAAT TAAATCTGAA 5400 TAAAGAATTC TTCCTGTTCA CTGGCTCTTT TCTTCTCCAT GCACTGAGCA 5450 TCTGCTTTTT GGAAGGCCCT GGGTTAGTAG TGGAGATGCT AAGGTAAGCC 5500 AGACTCATAC CCACCCATAG GGTCGTAGAG TCTAGGAGCT GCAGTCACGT 5550 AATCGAGGTG GCAAGATGTC CTCTAAAGAT GTAGGGAAAA GTGAGAGAGG 5600 GGTGAGGGTG TGGGGCTCCG GGTGAGAGTG GTGGAGTGTC AATGCCCTGA 5650 GCTGGGGCAT TTTGGGCTTT GGGAAACTGC AGTTCCTTCT GGGGGAGCTG 5700 ATTGTAATGA TCTTGGGTGG ATCC 57 (2) INFORMATION FOR SEQUENCE ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4157 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: (xi) SEQUENCE DESCRIPTION: CCCATCCAGA CCCAGGGAAG GGTCAGAGGA GAGGGAAGCA AGGACTGAGG GCTGCCTCTG GCTCAGTCGC GGAACTCTGG TGCTCAGGGC GACTGAGGGC ACCAACCCCA TCCCATCTCC CAATCAACCC ACGTTCACAT GGCCTTTGGG TCCTTAGGGG TCAAACTGAG CCCACTTCAG AAGAGGGAGG GCTGGGGGAT CTTCAGGGTG GGTCAGCAGA CTTCATGAGG AGTCTGGAAG CCCTAAGTGA CAGGGAGATA GGTTCCCCCT CCACAGGAGG GGACAACGCA CAGATCTCAG ACAGGGGCCC ATCCAGGTGG GCAGCAAGGG AGACCCTGGG TGATGTCAGG GTAGAGGGAG GACTCGTCAC GTCCTTCGCG TCTCCTTCTG CAAGCCAGCA CCTGAGTGAG TCCCCATCCG TCACGGGCCC CAGCGAGATT GGCGCAGGCT CGGGCCTCAC CTGCCGGGCC CACCACCTCA CGTAAGAGCT CCAGACTCAG AACCCACCCC CCCCCATCCC TCCCCCACCA ACGGAAGCTC GTACGGCTAA ATGCAGAGGA ACCCAGCATG CCACCTTTTC GGGGTTGGGG ACTGAGGGGA CCTGGGCACA ACAGAGAGTT GGGAGGAATC ACTCCCCATA TAAATTGTTC CAATCTCATT AGGTGTTGGT TGAGAAAGGG CCATCATAAC CGTGGGGTAA GGAGTTGATG CTCTGGTCGA AGAGCCTGAG GGCCCCATAG CAGGGCTGTC GAAGGGGAGG GGTCTCAGGC CCAGGACACC GAGGACCTGG TACCATATCA AAAGGGTGGG CACAGAGGGG GGCAGAATCC GGATGTGACG CTCGCCCTGA CCGTGAGGAG CCCAGACAGA TGGACCACCC CCCCGCCACC TTGTGTGACC CCAGGAATCA CTACCCTCAC TCAAACACCA CCATCCTGGC CGGGAATGGC GGGAGGGAAG AGGGCCCAGG CCAGGACAGG ATTCAGCCGA CCCAGCCTGC CCTTGGAGTC GTGGCCGAAT GAGGGCTGTG CCAGGATCTG CCCCCGGCCC TTAGCTCTGG TGTACCACAG GTAAAGAGGA CAGTCCCTGG GTTCACCCTA CAGGATGTGG ACCTTGTTTT CAGATGCAGT GTAGGATTGA AAATCTGCCC AGCTGAAGTC CCTTGGTCTG CCTGCCAGGA TGGACTCCAA TCACGTATGG GGGATGTGAG ATTAGGCCCT ACCCTCCACC genomic DNA MAGE-2 gene SEQ ID Nozu GGTTCCACCC CCACTGACTT GCAACGGCCT GCAAGGTAAG GGGCCCCCAA TGCAGGGGAA CCCCGCCGCT AGGGCAGGGC AGGTCAGGAC TACCAATCCC ACCCCACCCC AGAATCCGGC GGCCAAGCAC GGGTTGGGTC CCTCCTGGAA GGGCCCACTG GGGAATCCTA GAGGAGTCAA CAGATCAGTG GTGCCCCGTG GTCTGAGGGC CCGGACCCAA AGAAAGAAGG GGGAACCTGA GCAGGAGGTT GCTGTCTGCT CAGGAGTAAA GAACCAAAGG CCCCTCCTCA CAGAAGGTGA GGTTCTAGGA GGGTACCCCT TGCCCCTGCG CCTCCATTAT AAGGGGCTGG GTGGACGTGA TGAATTTGAC CCAGATGTGG TTCTTGACAT ACAAGGAGAA CAAGTAGAGT TTGCCGTGAA GCACATTGGA GACGTCGGCG ACGCCGAGGG TTAATCCAGC GACTTCTCAG TTAACCGCAG TGGTTAGAAG CCCAAGAGGG ATCCCCCAAC CAAACCCCAT TTTGCCCCTG GCGGATCCTG TCGTGAGTAT GACAGTGGAG TACCCCTGTC GGGATGCAGA GGGGAGGAAG GCAACCTTGG CTCATTGCAC TGGGACTTCA GGTGTGCCCC GATGCCACAG TCAGGGATGG GGGGAACCCT CATTTCAGGG GATGAGTAAC GGTCAGCCCT CTTGTCTTTC CTCAGTCAAC TCTGCCAAGC GGGCCAGAAT GTTACTTCAG CTGGGATCTT AGTCAGGTCA GGACCAAGCG ATCTCTCGTT GTCCCCTCTA GAGAGATTCT AGGTGAGGGC GGGGACCTCA 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 CGGAGTCTGG GCAGTCTGCA TCCAGGAACC AGAGCAGAGG CACACCAAGG GCCTCACCCT CTGTACCCTG AGGCTGACAA CTGTAAGTAA TAAGGCCTCA CCCAGCTCCT CCAACCCTGC CACTGAAGGC AGGCAGTGAG GGACGCAGAC GCCCCACCCG CCCTATTCTC AGGTGCCCTC GTAGGACCCG GCCTTTGTCA CACACGCTCC GCCCGCACTC TGAGACTTCT CCGTGCATTC GCCTTGGTCT AGTGCCAACA CCCAGAACAA AGTCCTGCAG CCACTTCCTC AGGCACTGGA GAGCCTCCAA TTCTCTCCCC CTGCCTGCTG GGGAATCCGT CTCTCCCAGG GAGTCAGTGC CTGAAGGTTT ATGGGACTCC CCTGAGCATG CTTCAGGTTC GGAGCATTGA GGTTCAGTTC AGGCCTGTGG CCCTGACCAG GGCTGTGCTT AATCAGGAGC CTCAGGTCAC GCCTGGAATG AGAGGGCCTG TGCTGGCCGG TGAGGGGGAC AGGAGAAGAT AGTTCTCACC GTCTTCATTG AGTCATC ATG GGC CAG TCT GAC TTC GAG CCC ATG AGG AGA TCC GTG GGC CCC ATA CTG TTC GAA GCA ACC GGA TTG CCT CTT GCT ACT TCA TCG GGC GAC GTT GAG AAT GAG GTC CTC AAG GAG AGT GCA AAC TGC AGC GAA AGA CTT GAG CCT CTA CCG ACT TCC CTG GAG CCG TGC TAC CCC TCC ACA GGC ATG CAT TAC TAC TAT CCT GAG GAG GCC GCT GTG AGT ACC AGC GAG TTG GTC CAG TTG ATC TAC GGC GAC TTG CCC CTG GAG GTG CAC GGA CAG CGA ACT GAA CCT ATC AAC TCC GTT ACA GAC CAG AGC GAT CTC TGT GAG AGG GAG TTC AAA ATT GAA AGG GGA GAG GTT CCC AAC CAA GAG CAT AAG TTC CTG CAC GGC CTG GCC GTG AAG TAC CTG GTC TCC GAG AGT GAG GAG ACC CAC TAC GAA TTC TTT GCA TTT GTC TTG CTG ATA CCT TTT CTG CGG TGG CTG TAC TGA CAG GCC CAG CTG AGT ACT GAG CAA CTG GAA CCC TTT TAC CTG ATC GAG GAG CTC CAG GGT CAC CCA CAC CTG CAG GGG CCT CTT GAG GCA CTC ATG GTG GGC ATC GGC GTC GAG GGG ATG GTG CCA CAT CCC TGC GGC ACC GAG CAG TGG GGG GCA CTC CTG ATC ATC CTT GAC CTG AAA AGG CAA CCC AGG ACA CTG AAG CTG GCT GTG GGA AGA CCA ATC AAG GAG TTC GAG GTC AAT GCC ATC GAG GAT GGC GCC CTA CAT CCT GTG TCT CCT GCC CAA AGA AGT TAT AGT AGC GTG ACC CAG ATA TGG GAC CTG AGT CTC AAG GAA GAA GGT TCC GCT TCC TCC ATG AGG CGA GTC AAA GTG TGC GTC ATC GAG AGT GTG GAT ATT ATC CGG GAA GCG TCT GCC AGC GAT TTT AAG GCC CTC GCC GAA CTG ATG GCA GAG GTC CAG CCT GAA GGT GCT GTCTCAGCAC GCACCTTCCA GGCCCATTCC TTTCTGTTCT TTGTTCAAAT GTTTATGAAT TAAGAGTCCT TTGTCACATA AATTAGCAGT TGCCTTATAC TGCTTCTTTG TCACTGGCTC CCTGGTAGTA ATGTTGCAGC GGGCCCCATC TGCCTCTTTG GTTGGATGAC GTTCCTTTTA GACAGTAGTC GTTTTTTATT ATAACAGCAG AAAATACATG CTCAGTCTAT AGAATGCAAA ATTTCTTTAC GTGGG CAGGGCCAGT CATTAGCTTC AAGAGAGCAG TTTGAGATTT ACAAATGGTT ACACATAGTG CAGATTGGGA TGGAATATGT ATACAAGGAA TATGTAAAAT AGAAATTAAA CATTCACTCA GGGAGGGGGT CACTGCCTCG TCAGCATTCT ATCTTTCTTT GGATGAACTT CTGTTTATAT AATCCATTCC ATTTGCCTAT CTCAAAAGAT TAAAAATATG TCTGAATAAA GCATCTGCTC CTGGGCCAGT TGTGATATGA TAGCAGTGAG CCTGTTGGAA CAGCATCCAA AGTTTAGGGG ATTTTGTGAG ATTGTGAACG AGTTAATTCT TGTATGTTTT TTCTTCCTGT TGTGGAAGGC 2200 2250 2300 2350 2400 2450 2500 2550 2597 2639 2681 2723 2765 2807 2849 2891 2933 2975 3017 3059 3101 3143 3185 3227 3269 3311 3353 3395 3437 3479 3521 3542 3592 3642 3692 3742 3792 3842 3892 3942 3992 4042 4092 4142 41 (2) INFORMATION FOR SEQUENCE ID NO: 10: (i) SEQUENCE CHARACTERISTICS: 662 base pairs nucleic acid (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: linear MAGE-21 (xi) SEQUENCE DESCRIPTION: GGATCCCCAT AGGGAAGTCA CAGAGAACAG GGAAGCAGGC ACTGAGGCGG CCTCTGCTGC AGTCGCCACC CTCTGGTGTA CAGGGCCCAG GAGGGTAACC CCCCACCCCC ACGGCACCCC GAGCTTTGCC GCACGCGGAT GGATCCAGGA CGGGGCCGGA CGAGATTCTC GCAGGCTCCG GCCTCACCCC CAGGCCTGGA ACCTCACCCC AGAGCTTTGT ACTCAGCCAG CCCCCGCACC ATCCCCCAAC CAAACCCCGA CCTGCAATCA CC AGAATCCAGT TGTGACGCCA GCCCTGAGCA TGAGGAGGCA AGACAGAGGG CCACCCTGCA GCCACCCCCC GTGACCAGGG GAATCAAGGT CCCACCACCA ACCAAACCCA TTCCCATCCC ACCCACGGAA genomic DNA gene SEQ ID NO: TCCACCCCTG CTGACTTGCG ACGGCCTGAC AGGTAAGATG CCCCCAATAA GGGGAAGACT GCCGCTTTAA CAGGGCTGGT CAGGACCCCA TTCCCATCCC CCACCATCGC CACCCATCCT GCTCCGGGAA CTGTGAACCC CGTTGGAGGT GTCGGCGGAG CCGAGGGAGG TCCAGCGCTG TCTCAGGCTC CCGCAGGGAA TAGAAGTGCT AGAGGGGACT CCAACACCAA TCAAACATCA GGCAGAATCG TGGCGGCCAA 150 200 250 300 350 400 450 500 550 600 650 6 (2) INFORMATION FOR SEQUENCE ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (Xi) SEQUENCE DESCRIPTION: CDNA to mRNA CDNA MAGE-3 SEQ ID NO: GCCGCGAGGG AAGCCGGCCC AGGCTCGGTG AGGAGGCAAG GACAGGCTGA CCTGGAGGAC CAGAGGCCCC CGGAGGAGCA AGATCTGCCA GTGGGTCTCC ATTGCCCAGC TCCTGCCCAC GTTGCCCTGA CCAGAGTCAT C GAG CAG AGG AGT CAG CAC GCC CGA GGA GAG GCC CTG GCT ACT GAG GAG CAG GAG GTT GAA GTC ACC CTG GGG GAG TCA CCA GAT CCT CCC CAG AGT CCT ACC ATG AAC TAC CCT CTC AGC AAC CAA GAA GAG GAG GAG TCC GAG TTC CAA GCA TTG GTT CAT TTT CTG CTC GTC ACA AAG GCA GAA ATG CAG TAT TTC TTT CCT GTG TTG CAG CTG GTC TTT GGC ATC GGC CAC TTG TAC ATC TAC GAT GGC CTG CTG GGT CCC AAG GCA GGC CTC CTG ATA ATC GTC GAC TGT GCC CCT GAG GAG TTA GAG GTG TTT GAG GGG CCC AAG AAG CTG CTC ACC CTG GAG TAC CGG CAG GTC GCA TGT TAT GAA TTC CTG TGG GGT CCA GTG AAA GTC CTG CAC CAT CAC ATT TCC TAC CCA CCC TTG AGA GAG GGG GAA GAG TGA ATG CCT CTT GGC CTT GAG CAG GCT CCT TCT ACT CTA CTC CCC ACT GAG GAC TCC CCT GAC CTG GTG GCC GAG AGG GAG CCG GGA AAT TGG TCC AGT TCC GTG GAC CCC GGC CTC TCC AGA GAG GGC CTG AGT GTG TTG GGG GAT GAA AAC TAC ACC AGC TAT GGA GGA CCT GTCTGAGCAC GCACCTTCCG GGCCCATTCT TTTCTGTTCT TTGTTCAAAT GTTTATGAAT TAAGAGTCTT TTGTGACATA GAATTAGCAA ATTCTTGCCT ACCAGGATTT GAGTTGCAGC GGGCCGCATC TCACTCTTTG GTTGGATGAC GTTCCTTTTA GACAGTAGTC GttTTTTACT ATAATAGCAG TAACATACAT TGTACCTCAA CCTTGACTTC GTTCTGAGGG CTGAAGGAGA ACTCCCGCCT TGC GGC GCT GAG CAG TGG GGG GCA CTC CTG ATC ATC TTT GAC CTG AAA AGG CAA CCC AGG ATG CTG AAG CTG GCC GTG GGA AGC CCA CTC AAG GGG TTC GAG GCC AAT GCC ATC GAA CAT GGC GCC GTA CAT CCT GTG TCC CCT GCC CAA AGC AGT TAT AGT AGC CTG ACC CAG ATA TGG GAC TTC AGT CTC AAG GAG GAA GGT TCC GCT TCC TCC ACC AGG CGA GTC AAA ATG TGC ATC ATC GAG AGT GTG GAT GTT ATC TGG GAA GCG TCT GCC AGC TAT TTC AAG GCC GTC GCT GAA CTG ATG GCA GAG ATG CAG CCT GAA AGT GTT CAGGGCCAGT CCTTAGTTTC AAGCGAGCAG TTTGAGATTA ACGGATGGTT ACACATAGTG CAAATTgGGA TGGTAAAAGT GAGATAACTC TCTATTCTGT TTTG GGGAGGGGGT CACTGCCTCC TCAGCATTCT TTCTTTGTTT GAATGAGCGT CTGTTTATAT AATCCATTCC ATTTGCTTAA AAGAAATCAA AAAATTAAAC CTGGGCCAGT TGTGACGTGA TAGTAGTGGG CCTGTTGGAG CAGCATCCAG AGTTTAGGAG ATTTTGTGAA AATTGTGAGC AAGATAGTTG AAATATGCAA 150 171 213 255 297 339 381 423 465 507 549 591 633 675 717 759 801 843 885 927 969 1095 1116 1166 1216 1266 1316 1366 1416 1466 1516 1566 1616 1640 INFORMATION FOR SEQUENCE ID NO: 12: (i) SEQUENCE CHARACTERISTICS: 943 base pairs (A) LENGTH: (ii) MOLECULE TYPE: (B) TYPE: (D) TOPOLOGY: (ix) FEATURE: (A) NAME/KEY: nucleic acid linear genomic DNA MAGE-31 gene (xi) SEQUENCE DESCRIPTION: GGATCCTCCA CCTGACAGTT GCCCGTGGAT AGGACTTGGT GATAGTGCCA CTGCCCCAGA TTCAGTCCTG CTCTCACTTC AGAGGCCCCC TTAGAGCCTC TCCCTCTCTC CTCCCGCCTG ATG CCT CCCCAGTAGA CTGGGAATCC TCCTCTCCCA CTGAGGCAGT ACGGTGAAGG ACACATGGAC CAGCCTCAGC CTCCTTCAGG GGAGGAGCAC CAAGGTTCCA CCCAGGCCAG TTGCCCTGAC GTGGGGACCT GTGGCTGCGT GGAATCAGGA GTCCTCAGGT TTTGCCTTGG TCCAGAGCGC ATGCGCTGGC TTCTGAGGGG TGAAGGAGAA TTCAGTACTC TGGGTCTCCA CAGAGTCATC SEQ ID NO: CACAGAGTCT TTGCTGTCTG GCTCCAGGAA CACAGAGTAG ATTCAAACCA CTGGCCTCAC CGGATGTACC ACAGGCTGAC GATCTGTAAG AGCTGAGGTC TTGCCCAGCT GGCCAACCCT CACATTGGGG CAAGGCAGTG AGGGGgCTCA AGGGCCCCAC CCTCAATACT CTGAGGTGCC CTGGAGGACC TAAGCCTTTG TCTCACATGC CCTGCCCACA GGC CAG TCT GAG CTC GAG CCT GTG CTT GCT AGT TCA CCC GAC GAC GCC GAG CCT GTA CCA ACT TCC CTG AAG GCC GCT GTT GAT ACC AGC GAG TTG CAG CGA ACT GAA CCT ATG AAC TCT GTT AGG GGA GAG GTC CCC AAC CAA GAG CAT AGT GAg GAG ACC CAG TAC GAA TTC TTT CAG GCC CAG CTG AGT CCT GAG CAA CTG CAC CTG GAG GGG CCT CTC GAG GCA CTC TGC GGC GCT GAG CAG TGG GGG GCA AAG CTG GCC GTG GGA AGC CCA CTC CCT GTG TCC CCT GCC CAA AGC AGT GAA GGT TCC GCT TCC TCC ACC AGG GAA GCG TCT GCC AGC TAT TTC AAG 150 200 250 300 350 400 450 500 550 580 622 A 790 832 874 916 943 INFORMATION FOR SEQUENCE ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: GGATCCAGGC GGGATCATCC TCTTGATGGC GGCCCATGGA TGGTCTGAGA GCCAGCAGTG CAAGACACAT CCTGCAGAAT CTTCCTCCTT TGGAGGCCAC TAGAGCCTCT TCTCCGTAGG CCTGCTGCCC CCTGCCTGGA ACTCCATGAG ACTGAGGGAC TTCCTCTCCT CAGTGTCCTC AATGTTTGCC AGGACTCCAA CGACCTCTGC CAGGTTCTGA AGAGGAGCAC AAGATTTGGT CCTGTGGGTC TGACCAGAGT GAAATGTGAG AGTGGGGACC CGGGGCTGTG AGGAGCTCCA AGGTTACAGA CTGAATGCAC AGAGTCTGGC TGGCCGGCTA GCAGACAGGC CAAGGAGAAG TCTCAGCTGA CCCATTGCCC CATC genomic DNA MAGE-4 gene SEQ ID NO: GGCCCTGAGT TCACAGAGTC CTTACAGTCT GGAACAAGGC GCAGAGGATG ACCAAGGGCC CTCACCTCCC TACCCTGAGG CAACCGGAGA ATCTGTAAGT GGTCTCTCAC AGCTTTTGCC GAACACAGTG CAGCCTACCC GCACCCTAAG AGTGAGGCCT CACAGGCTGT CCACCTGCCA TACCATCAAT TGCTCTCTCA CAGGATTCCC AAGCCTTTGT ATGCTCCCTC TGCACTCTTG ATG GGC CAG TCC GCT GCC AAT TCG AAG GCC ATC GCC GAA CTG TTT GCA GAG GTC CAG CCT GAA AAT GCT TCT GTT GCT TCT GAG TTA GAG CCT GTG AAG AAA TCC GTG GGC CCC ATG CTG TAT GAA GCG ACC GCA TTG TCT GAG CCT CCT TCA CCC GGT GAC GAT GAG AAT GAG GAC CTT AAG GAG GGT GGG AAC CGC AGC AGA TTA GAG GCC ACT CTG GCA ACT TCC GCA GAG CTG TAC TCC CCC TCC ACA GGC GTG GAG TAC TAT TAT GTT GAG CAG CAA ACT GTC GGT ACC AGC GAG TTG GTC AAG CTG GCC TAT GGC GAC ATG CCC CTG GAG GTG CGC GAG AAG GAA GAG CCT CCT ATC AGC TCC GCT ACA CGC AAG AGC GAT CTT AGC GGG AGG GAG TTC AAA ATT GAA AGT GAG GAG GGC CCC AGC CAA TTG CAT AAG TGC ATG AAC GGC CTG GCC GTG AAA TAC CTG GTC GCC GAG CAG GCC CAG ACC CAG TTC GAA TTC TTT GCA TTT ATC ACC CTG ATA TCT TAT CTG CGG TGG CTG TAC GGA CAC CTG GAG CTG AGT ACT GAG CGA CTG GAA CCT TTT TAC CTG ATC GAG GAT CTC CAG GGT GAG CCA GTC TGC GGC GCT GAG CCT TGC GAG GAA CTC ATG GTG GGC ACC GGT GTC GAG GGG ACC GTA CCA CAT TCC TGA AAG CTG GCT GAA CAG TGG GGG GCA CGC CTG ATC ATT CTT AAT CTG GAA AGG CAA CCC AGG GTG CTG CCT GTG GTC GTG GGA AGG CCA CTC AAG GAG TTC GAC GTC AAT GGC ATC GAG GAT GGC GCT GTC CGT GAG GGT TCC CCT GCC CAA AGC AGT TAT AGA GGC GTG ACC CAG ACA TGG CAC TGG AGT CTG AGG GAA GAA GCA TCC GCT TCT CCC ACC AAC CGA GTC AAA AAG TGC ATC ATT GAG ACT GTG AAT GCT GTC GCA GCATGAGTTG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT ATCTAACAGC CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTA TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA GGGCCAGTGC ACATGAGGCC GTAGTGGGTT TTTTACAATT GCATCCAAGT 150 200 250 300 350 400 450 500 550 600 624 666 708 750 792 834 876 918 960 1086 1128 1170 1212 1254 1296 1338 1380 1422 1464 1506 1548 1578 1628 1678 1728 1778 1828 TTATGAATCG AGTCTTGTTT GGACATAATA GAAATAGGTG GTCTATTCTG CTTCGTGAAT ACTGGCTCAT AGGATTAGTA GGGTATTAAG CCTCTAAGAT GAGAGTGGTC AACTGCATTT AGGGCCAGAT TCTGAGCAGT GGG TAGTTAACGT TTTATTCAGA ACAGCAGTGG AGATAAATTA TAAAATTTAA GTAAGAGAAA TTCTTCTCTA GTGGAGATAC AGTCTAGGAG GTAGGGGAAA GGGTGTAAAT TCTTCTGAGG TCTCAGAGGG TCCTTTGTGA ATATTGCTGT TTGGGAAATC AGTAAGTATT AAAGATACTT AAATATATAT TTAAATCTGA TGCACTGAGC TAGGGTAAGC CGCGGTCATA AGTAACGAGT TCCCTGTGTG GATCTGATTC AGAGGGAAAA CAATGGATGA TAATATAGTT CGTTCTATTT TAGAAGTGTG AATTCCCGCC GCATACCTGG ATAAATAATT ATCTGCTCTG CAGACACACA TAATTAAGGT GTGGGTATGG" GGGCCTTTTG TAATGAAGCT GCCCAGATTG ACAGAGAGGA TAGGAGTAAG TGTGAATTTG AATTCACCGT TTATGCCTCA ATTTCCTTGG CTTTCTGTTA TGGAAGGCCC CCTACCGATA GACAAGATGT GGCTCCAGGT GGCTTTGGGA TGGTGGGTCC GAAAAGTTGC GCCTCTACCT 1978 2028 2078 2128 2178 2228 2278 2328 2378 2428 2478 2528 2531 INFORMATION FOR SEQUENCE ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear MAGE-41 (xi) SEQUENCE DESCRIPTION: GGATCCAGGC GGGATCATCC TCTTGATGGC GGCCCATGGA TGGTCTGAGA GCCAGCAGTG CAAGACACAT CCTGCAGAAT CTTCCTCCTT TGGAGGCCAC TAGAGCCTCT TCTCCGTAGG CCTGCTGCCC CCTGCCTGGA ACTCCATGAG ACTGAGGGAC TTCCTCTCCT CAGTGTCCTC AATGTTTGCC AGGACTCCAA CGACCTCTGC CAGGTTCTGA AGAGGAGCAC AAGATTTGGT CCTGTGGGTC TGAGCAGAGT GAAATGTGAG AGTGGGGACC CGGGGCTGTG AGGAGCTCCA AGGTTACAGA CTGAATGCAC AGAGTCTGGC TGGCCGGCTA GCAGACAGGC CAAGGAGAAG TCTCAGCTGA CCCATTGCCC CATC genomic DNA gene SEQ ID NO: GGCCCTGAGT TCACAGAGTC CTTACAGTCT GGAACAAGGC GCAGAGGATG ACCAAGGGCC CTCACCTCCC TACCCTGAGG CAACCGGAGA ATCTGTAAGT GGTCTCTCAC AGCTTTTGCC GAACACAGTG CAGCCTACCC GCACCCTAAG AGTGAGGCCT CACAGGCTGT CCACCTGCCA TACCATCAAT TGCTCTCTCA CAGGATTCCC AAGCCTTTGT ATGCTCCCTC TGCACTCTTG ATG GGC CAG TCC GCT GCC AAT TCG AAG GCC ATC GCC GAA CTG TTT GCA GAG GTC CAG CCT GAA AAT GCT TCT GTT GCT TCT GAG TTA GAG CCT GTG AAG AAA TCC GTG GGC CCC ATG CTG TAT GAA GCG ACC GCA TTG TCT GAG CCT CCT TCA CCC GGT GAC GAT GAG AAT GAG GAC CTT AAG GAG GGT GGG AAC CGC AGC AGA TTA GAG GCC ACT CTG GCA ACT TCC GCA GAG CTG TAC TCC CCC TCC ACA GGC GTG GAG TAC TAT TAT GTT GAG CAG CAA ACT GTC GGT ACC AGC GAG TTG GTC AAG CTG ACC TAT GGC GAC ATG CCC CTG GAG GTG CGC GAG AAG GAA GAG CCT CCT ATC AGC TCC GCT ACA CGC AAG AGC GAT CTT AGC GGG AGG GAG TTC AAA ATT GAA AGT GAG GAG GGC CCC AGC CAA TTG CAT AAG TGC ATG AAC GGC CTG GCC GTG AAA TAC CTG GTC GCC GAG CAG GCC CAG ACC CAG TTC GAA TTC TTT GCA TTT ATC ACC CTG ATA TCT TAT CTG CGG TGG CTG TAC GGA CAC CTG GAG CTG AGT ACT GAG CGA CTG GAA CCT TTT TAC CTG ATC GAG GAT CTC CAG GGT GAG CCA GTC TGC GGC GCT GAG CCT TGC GAG GAA CTC ATG GTG GGC ACC GGT GTC GAG GGG ACC GTA CCA CAT TCC TGA AAG CTG GCT GAA CAG TGG GGG GCA CGC CTG ATC ATT CTT AAT CTG GAA AGG CAA CCC AGG GTG CTG CCT GTG GTC GTG GGA AGG CCA CTC AAG GAG TTC GAC GTC AAT GGC ATC GAG GAT GGC GCT GTC CGT GAG GGT TCC CCT GCC CAA AGC AGT TAT AGA GGC GTG ACC CAG ACA TGG CAC TGG AGT CTG AGG GAA GAA GCG TCC GCT TCT CCC ACC AAC CGA GTC AAA AAG TGC ATC ATT GAG ACT GTG AAT GCT GTC GCA GCATGAGTTG ATCTAACAGC CATTCTTCAC TCTATTTTGT GTTGAAATGT TTATGAATCG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA TCTGTTTGAA GAAAATAGTC AGTGTTCTTA TGGATGACTT GGAGATTTAT CTCTGTTTCC TCCTTTTAAT GGATGGTTGA ATTAACTTCA TAGTTAACGT ATATTGCTGT TAATATAGTT GGGCCAGTGC ACATGAGGCC GTAGTGGGTT TTTTACAATT GCATCCAAGT TAGGAGTAAG 150 200 250 300 350 400 450 500 550 600 624 666 708 750 792 834 876 918 960 1086 1128 1170 1212 1254 1296 1338 1380 1422 1464 1506 1548 1578 1628 1678 1728 1778 1828 1878 AGTCTTGTTT GGACATAATA GAAATAGGTG GTCTATTCTG CTTCGTGAAT ACTGGCTCAT AGGATTAGTA GGGTATTAAG CCTCTAAGAT GAGAGTGGTC AACTCCATTT AGGGCCAGAT TCTGAGCGGT GGG TTTATTCAGA ACAGCAGTGG AGATAAATTA TAAAATTTAA GTAAGAGAAA TTCTTCTCTA GTGGAGATAC AGTCTAGGAG GTAGGGGAAA GGGTGTAAAT TCTTCTGAGG TCTCAGAGGG TCCTTTGTGA TTGGGAAATC AGTAAGTATT AAAGATACTT AAATATATAT TTAAATCTGA TGCACTGAGC TAGGGTAAGC CGCGGTCATA AGTAACGAGT TCCCTGTGTG GATCTGATTC AGAGGGAAAA CAATGGATGA CGTTCTATTT TAGAAGTGTG AATTCCCGCC GCATACCTGG ATAAATAATT ATCTGCTCTG CAGACACACA TAATTAAGGT GTGGGTATGG GGGCCTTTTG TAATGAAGCT GCCCAGATTG ACAGAGAGGA TGTGAATTTG AATTCACCGT TTATGCCTCA ATTTCCTTGG CTTTCTGTTA TGGAAGGCCC CCTACCGATA GACAAGATGT GGCTCCAGGT GGCTTTGGGA TGGTGGGTCC GAAAAGTTGC GCCTCTACCT 2028 2078 2128 2178 2228 2278 2328 2378 2428 2478 2528 2531 G GAA CTC ATG GTG GGC ACC GGT GTC GAG GGG ACC GTA CCA CAT TCC INFORMATION FOR SEQUENCE ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) (ix) (xi) GGG GCA CGC CTG ATC ATT CTT AAT CTG GAA AGG CAA CCC AGG GTG CTG CCA CTC AAG GAG TTC GAC GTC AAT GGC ATC GAG GAT GGC GCT GTC CGT AGC AGT TAT AGA GGC GTG ACC CAG ACA TGG CAC TGG AGT CTG AGG GAA ACC AAC CGA GTC AAA AAG TGC ATC ATT GAG ACT GTG AAT GCT GTC GCA nucleic acid linear CDNA to mRNA MOLECULE TYPE: FEATURE: (A) NAME/KEY: SEQUENCE DESCRIPTION: TCG AAG GCC ATC GCC GAA CTG TTT GCA GAG GTC CAG CCT GAA AAT GCT base pairs CDNA MAGE-4 SEQ ID CCT GTG AAG AAA TCC GTG GGC CCC ATG CTG TAT GAA GCG ACC GCA TTG GAC GAT GAG AAT GAG GAC CTT AAG GAG GGT GGG AAC CGC AGC AGA TTA GCA GAG CTG TAC TCC CCC TCC ACA GGC GTG GAG TAC TAT TAT GTT GAG GAG TTG GTC AAG CTG GCC TAT GGC GAC ATG CCC CTG GAG GTG CGC GAG TCC GCT ACA CGC AAG AGC GAT CTT AGC GGG AGG GAG TTC AAA ATT GAA TTG CAT AAG TGC ATG AAC GGC CTG GCC GTG AAA TAC CTG GTC GCC GAG TTC TTT GCA TTT ATC ACC CTG ATA TCT TAT CTG CGG TGG CTG TAC GGA CGA CTG GAA CCT TTT TAC CTG ATC GAG GAT CTC CAG GGT GAG CCA GTC TGAGCATGAG TGCATCTAAC GCCCATTCTT GTTTCTATTT ATTGTTGAAA AGTTTATGAA AAGAGTCTTG TTGGGACATA TTGCAGCCAG AGCCCTGTGC CACTCTGTTT TGTTGGATGA TGTTCCTTTT TCGTAGTTAA TTTTTTATTC ATAACAGCAG GGCTGTGGGG AGCAGCTTCC GAAGAAAATA CTTGGAGATT AATGGATGGT CGTATATTGC AGATTGGGAA TGGAGTAAGT AAGGGGCAGG CTTGCCTCGT GTCAGTGTTC TATCTCTGTT TGAATTAACT TGTTAATATA ATCCGTTCTA ATTTAGAAGT GCTGGGCCAG GTAACATGAG TTAGTAGTGG TCCTTTTACA TCAGCATCCA GTTTAGGAGT TTTTGTGAAT GTGAATTC 208 250 292 334 376 418 460 502 544 586 628 670 720 770 820 870 920 970 1020 1068 (2) INFORMATION FOR s (i) SEQUENCE c (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: EQUENCE ID base N0: 16: HARACTERISTICS: pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: GGATCCAGGC GGGACCATTC TCCTGTTAGC GGCCCATGCA TGGTCTGAGG TAGTGCCAGC GCCCCAGAAC TCAGTCCTGC CTCACTTTTT GAAGCTCCAG ATCTGTAAGT GGCTTCTCAC AGCTCCTGCC ATG TCT CTT GAG CAG AA CTC CTC TGG TCC CAG GC GGT CAC CAG GTC CTC TC TCC CCA CTG CCA TCG AT AGG GCT CCA GCA ACC AA CTG ACC CAG AGT CTG TG CTTGCCAGGA ACCCCAAGAG ACTGGGGGCC TTCCTCTTCC CCGTGCCCTC AGTGAACGTT ATATGGGACT AGAATCAGCC CCTTCAGGTT AGGATCCCCA AAGCCTTTGT ATGCTCCCTC CACACTCCTG TGG CTG ACT TGA TTCATTTTCT GAAATGCTGG CTTCGGCAAA AGGAAGCGGA CTCCTATGAT GCCTCCTGAT CCTGAGGAGA GAGGGAGCAC TGGTGCAGGA ATATGCTATG CTGGAGCACG CCTGCGTGAA CTGCAGCCAG CTCCGTCCAG TCTCTTTGAA TGGATGACTT TTCTTTTAAT CAGTAGTCAC TTTTTATTCA TACAGCAGTG TGATGACATA GCTCCTCAAG AGAGCGTCAT GCCTCCGAGT CCCCACCAGC GGCCTGCTGG AATCGTCTTG AAATCTGGGA AGTGTCTGTG AAACTACCTG AGTTACTGTG TGGTCAGGGT GCAGCTTTGA GGCCACTGCG TAGTTTCCCC GAGAGCAGTC TGAGATTTGT GGGTGGTTGA ACATAGTGCT GATTGGGAAA GAATAAGTAT AAGAAATTAA GAAAGGTGAG GGTGGAGACC TGAGGCTGTG AGGAGCTCCA AGGTCACAGA TGCCTTGAAT CCAGAGCACC TCTGCTTGCT CTCAGGGGAC GGAGGCCCTA TAGAGCCTCC TCTCTCCAGG CCTGTTGCGG genomic DNA MAGE-5 gene SEQ ID NO: GGCCCTGTGT TCACAGATTC CTTGCAGTCT GGAAACAGAC GCAGAGGAGA GCACACTAAT TGGCCTCACC TGTGTACCCT AGGCTGACCA GAGGAGCACC AAGGTTCAGT CCAGTGGGTC TGACCAGAGT G AGT CAG CAC TGC AAG A CCC TGG GGG AGG TGC A AGA GTC CTC AGG GAG T TCA CTC TAT GGA GGC G AAG AGG AGG GGC CAA T TCC GAG CAG CAC TCA TATTAAGTCA CAAAAATTAC CCTTGCAGCT AACACCTACA TTGATAATAA GGCATGATTG GGAGCTGAGT GGGAGCCCAG GAGTACCGGC GGGTCCAAGG CAATGCAAGA GAGAGGAGGA AGGGGGGCTG TGCCTTAATG AACATTCTTA CTTTGTTTCC ATGAACTTCA GTTTATATAG TCCATTCCAT TCATTTAGAA AAGATATTTA AGGAGCTGGT AAGCGCTGCT GGTCTTTGGC CCCTTGTCAC TCAGATCATG CAATGGAGGG GTGATGAAGG GAAGCTGCTC AGGTGCCCAG GCACTCGCTG GTTCTCATTT AGAGGGAGTC GGCCAGTGCA TGACATGAGG GTAGTGGGTT TTTTGGAATT GCATTCAAAT TTTAGGAGTA TTTGTGAATT ATGTGAATGA ATTCTTGCTT GAGCACAGAG CAGCCTACCC GCACCCTGAG ACTGAGGCCT TGCAGACGTC GGCCCCCATC CTCTCTACTG GAGGTGCCCT GGATCACCAG AAAGGAGAAG TTTTAGCTGA TCCATTGCCC CGTC CCT GAG GAA CTG CTG CTG CCT CCG CCA AAT CCA TTA GCA CCT CCC GTA AGA AGG CACAAAGGCA TTCCTGAGAT ATTGACGTGA CTGCCTGGGA CCCAAGACGG CAAATGCGTC TGTATGTTGG ACCCAAGATT CAGTGATCCC CTTGAAAGTA CCTACCCATC TGAGCATGAG CCTTCCAGGG CCCATTCTTC TCTGTTCTAT GTTCAAATGT TTATGAATGA AGAGTCTTGT GGGACATAGT GCAGTAAAAC ATACTCAGTC 150 200 250 300 350 400 450 500 550 600 644 684 728 770 812 854 896 908 958 1108 1158 1208 1258 1308 1358 1408 1458 1508 1558 1608 1658 1708 1758 1808 1858 1908 19 TATTCGGTAA AATTTTTTTT AAAAAATGTG CATACCTGGA TTTCCTTGGC TTCTTTGAGA ATGTAAGACA AATTAAATCT GAATAAATCA TTCTCCCTGT TCACTGGCTC ATTTATTCTC TATGCACTGA GCATTTGCTC TGTGGAAGGC CCTGGGTTAA TAGTGGAGAT GCTAAGGTAA GCCAGACTCA CCCCTACCCA CAGGGTAGTA AAGTCTAGGA GCAGCAGTCA TATAATTAAG GTGGAGAGAT GCCCTCTAAG ATGTAGAG 2108 2158 2208 22 (2) INFORMATION FOR SEQUENCE ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear MAGE-51 (xi) SEQUENCE DESCRIPTION: GGATCCAGGC GGGACCATTC TCCTGTTAGC GGCCCATGCA TGGTCTGAGG TAGTGCCAGC GCCCCAGAAC TCAGTCCTGC CTCACTTTTT GAAGCTCCAG ATCTGTAAGT GGCTTCTCAC AGCTCCTGCC CTTGCCAGGA ACCCCAAGAG ACTGGGGGCC TTCCTCTTCC CCGTGCCCTC AGTGAACGTT ATATGGGACT AGAATCAGCC CCTTCAGGTT AGGATCCCCA AAGCCTTTGT ATGCTCCCTC CACACTCCTG GAAAGGTGAG GGTGGAGACC TGAGGCTGTG AGGAGCTCCA AGGTCACAGA TGCCTTGAAT CCAGAGCACC TCTGCTTGCT CTCAGGGGAC GGAGGCCCTA TAGAGCCTCC TCTCTCCAGG CCTGTTGCGG genomic DNA gene SEQ ID NO:~ GGCCCTGTGT TCACAGATTC CTTGCAGTCT GGAAACAGAC GCAGAGGAGA GCACACTAAT TGGCCTCACC TGTGTACCCT AGGCTGACCA GAGGAGCACC AAGGTTCAGT CCAGTGGGTC TGACCAGAGT GAGCACAGAG CAGCCTACCC GCACCCTGAG ACTGAGGCCT TGCAGACGTC GGCCCCCATC CTCTCTACTG GAGGTGCCCT GGATCACCAG AAAGGAGAAG TTTTAGCTGA TCCATTGCCC CGTC ATG GGC AGG CTC GGT TCC AGG CTG TCT CTT CTG CTC CAC CCA GCT ACC CTT GAC CCA TGG CAG CTG CCA CAG GAG CAG ACC CAA CTA CTG TCC CAG GTC CTC CCA TCG GCA ACC AGT CTG AAG GAA AGG GCA TCA ATT AAG TGT AGT GAG AGC CCC AGA TCA AAG TCC CAG CCC AGG TGG GTC CTC AGG GAG CAC TGG AGG GGG CTC TAT AGG CAG TGC GCC CTG AGG AGG GGA GGC CAC AAG TGG TGT TGC GAG GGC CAA TCA CCT TGG CCT CTG CCT AAT GCA GTA GAG GTG CCT CTG CCG CCA CCT AGA GAA TGC CCT CTG CCA TTA CCC AGG TGG CTG ACT TGA TTCATTTTCT GAAATGCTGG CTTCGGCAAA AGGAAGCGGA CTCCTATGAT CTGATAATCG GGAGAAAATC AGCACAGTGT CAGGAAAACT TATGAGTTAC CACGTGGTCA TGAAGCAGCT CCAGGGCCAC CCAGTAGTTT TGAAGAGAGC ACTTTGAGAT TAATGGGTGG TCACACATAG TTCAGATTGG GCTCCTCAAG AGAGCGTCAT GCCTCCGAGT CCCCACCAGC GGCCTGGTGG TCTTGGGCAT TGGGAGGAGC CTGTGGGGAG ACCTGGAGTA TGTGGGGTCC GGGTCAATGC TTGAGAGAGG TGCGAGGGGG CCCCTGCCTT AGTCAACATT TTGTCTTTGT TTGAATGAAC TGCTGTTTAT GAAATCCATT TATTAAGTCA CAAAAATTAC CCTTGCAGCT AACACCTACA TTTAATCAGA GATTGCAATG TGGGTGTGAT CCCAGGAAGC CCGCAGGTGC AAGGGCACTC AAGAGTTCTC AGGAAGAGGG GCTGGGCCAG AATGTGACAT CTTAGTAGTG TTCCTTTTGG TTCAGCATTC ATAGTTTAGG CCATTTTGTG AGGAGCCGGT AAGCGCTGCT GGTCTTTGGC CCCTTGTCAC TCATGCCCAA GAGGGCAAAT GAAGGTGTAT TGCTCACCCA CCAGCAGTGA GCTGCTTGAA ATTTCCTACC AGTCTGAGCA TGCACCTTCC GAGGCCCATT GGTTTCTGTT AATTGTTCAA AAATTTATGA AGTAAGAGTC AATTGGGACA CACAAAGGCA TTCCTGAGAT ATTGACGTGA CTGCCTGGGA GACGGGCCTC GCGTCCCTGA GTTGGGAGGG AGATTTGGTG TCCCATATGC AGTACTGGAG CATCCCTGCA TGAGCTGCAG AGGGCTCCGT CTTCTCTCTT CTATTGGATG ATGTTCCTTT ATGACAGTAG TTGTTTTTTA TAGTTACAGC 150 200 250 300 350 400 450 500 550 600 644 686 728 770 812 854 896 938 980 992 1142 1192 1242 1292 1342 1392 1442 1492 1542 1592 1642 1692 1742 1792 1842 1892 1942 AGTGGAATAA GATAAAGAAA GGTAAAATTT TGAGAATGTA GGCTCATTTA GTTAATAGTG TAGTAAAGTC CTAAGATGTA GTATTCATTT AGAAATGTGA TTAAAAGATA TTTAATTCTT TTTTTTAAAA ATGTGCATAC AGACAAATTA AATCTGAATA TTCTCTATGC ACTGAGCATT GAGATGCTAA GGTAAGCCAG TAGGAGCAGC AGTCATATAA GAG ATGAGCAGTA GCCTTATACT CTGGATTTCC AATCATTCTC TGCTCTGTGG ACTCACCCCT TTAAGGTGGA AAACTGATGA CAGTCTATTC TTGGCTTCTT CCTGTTCACT AAGGCCCTGG ACCCACAGGG GAGATGCCCT . 2142 2192 2242 2292 23 (2) INFORMATION FOR SEQUENCE ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 225 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (ix) FEATURE: (A) NAME/KEY: MAGE-6 gene (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: TAT TTC TTT CCT GTG ATC TTC AGC AAA GCT TCC GAT TCC TTG CAG CTG GTC TTT GGC ATC GAG CTG ATG GAA GTG GGC CAC GTG TAC ATC TTT GCC ACC TGC CTG GGC GAT GGC CTG CTG GGT GAC AAT CAG ATC ATG CCC TTC CTG ATA ATC ATC CTG GCC ATA ATC GCA AGA TGT GCC CCT GAG GAG GAC CTC AGG GAG CCC ATC TCC TAC ACA GGC GGC GAC 126 168 210 2 (2) INFORMATION FOR SEQUENCE ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: TGAATGGACA GAGCCCAGCC TGGCCGGCTG GCGGACAGGC ACCGAAGGAG GGTTCACAAA GTTCCTCCCC CCAGAGTCAT GATGCCTTGA GCTCCCGCCA AGGCACCCTG GTCTCAGGGT GCCAATCCAG TAGACACACC ACAAGGGCCC TCACCTTCCC TACCCTGAGG CGGCCAGGAG AAGATCTGTA TGAGGCCCCT ATCGCCCAGC CATGTCTTCT GGCCCAAGGA CCGAGGAGCA GAGGAGGTGC TCCTCCTTTT TGAGGGCACC CCGCTCACCT CACACTCCCC TACTGTCAGT TGCCCTCTCA GTCAGAAGCC AGTAGGCCTT CACAAGCTCC TGCTGCCCGC GAGCAGAGGA CAGGAGGCTC CGAGGCTGCC CTGCTGCTGG CCCTGACCAT AGCAGCCGGG GGCGTCCTTG ATG GGA AGG TGG CTG AGT TGG TTC ATC GAG TCA AGG AGC TGG GTG TCA TCA AAA ATT ACA AGC ACT GGC AAA GCC TCA GAG TGC ATG CAG ATG AAG GAA GTG GAC CCC GCG GCC ACT CCT ACG CCT GCT TGG GCC TCT CCT ACA ATG AGA GCA TGC CCG AGA CCG GCC TTC TTATGGTCTT GCAATCTGGG TCTTTGGGCA TACCTGCAAT CCTGTGGGGT AGTATGCAGC CATGAAGAGG AGCCAGGGCC CACACATCCA CTGTGTTTGA GTGTGAGGGA GATTTGGAGG TTAATGGATG GCAGACTTAC TTATGTAAGA CAGAGGATTA GTGAGATAAA CCTGTAATCC GGAGATCGAG AATACAAAAC GACCATGATC AAGCGTTGAG GCTGAGGAAG ACCGCCAGGT CCAAGGGCCC CAGGGTCAGT CTTTGGGAGA AGTGGGGCAG CCACCTTCCC AGAGAGCAGT ATACAAGGTG TTTATCTTTG GTGTAATGAA TGTTTTTTAT AAATCTATGT AGTACCTTTT GAAATAAAGA CAGCACTTTA ACCATTCTGG TTAGCCGGGC TTAATGGAGG TGTAATGGTG CTGCTCACCC GCCCAGCAGT TCATTGAAAC ACTAAAGAGA GGAGGAAGAG ATTGGGGGAG TGTCCTGTTA CAATGTTCTC GACCATCTCT TTTCCTTTTG CTTCAACATT ATAGTTAAAA TATTTCTTGA ATAATGTGAA AATTAAATTG GGAGGCAGAG CTAACACAGT GTGGTGGCGG genomic DNA MAGE-7 gene SEQ ID NO: AGAACACAAG CCTGCAGCCT CTTCCTCCTT CCAGGAGGCC TGTTAGGGCC TTCTCTCCCC ACTCCAGCCT GTCAGCACTG TGGGCCTGGT TCCTCCTTCA GTCCCCCAGT CAGCAACAAC AAGAGGAGGG TTCCA GCT TCC TGC TCA CAA AGG CAG AAA AGT TTC CTT GTG ATG TTT GCC TGC TGG TGA GCCACTGTGC TATGATGGGA AAGATTGGGT GATCCCCCGT CAGCTATGTG GCATTTCCTA GGAGTCTGAG GGCCTGGGCA CATGAGGCCC AGTAGCGGGG CAGTTCCTGT CAGTCGTTCA CATTTCATGT GTAAGTGCAT ATTGGGACAA AGAACAAAGC GCTGGGCACG GCACGGGGAT GAAACACCAT GTG GGACTCCAGA CAGCCTCTGC CAGGTTCTCA CCAGAGGAGC TCCAGGGCGT AGATCTGTGG GCTGCCCTGA CAAGCCTGAG GGGTGCGCAG CTCTGATTGA CCTCCCCTGA ACTCTATGGA GCCAACCACC TGC ACA AGT TGC TGG ACA GTG ATC TAT GGC ATT GAC TCC TTG TCA GTG ATG ATC CCCTGAGGAG TGGAGCAGTT GCAGGAAAAC GCTACCAGTT AAAGTCCTGG CCCATCCCTG CAGAAGTTGC GTGCACGTTC ATTCTTCACT AGTGTGTTGG TCTCTTGGGC AATGTTCCTT ATGACAGTAG TGTTTTTTAT CATAACATAG GGTAAAATGG GTGGCTCACG CACGAGGTCA CTCTATTAAA 150 200 250 300 350 400 450 500 550 600 650 685 727 769 811 853 895 937 964 1114 1164 1214 1264 1314 1364 1414 1464 1514 1564 1614 1664 1714 1764 1814 1864 1914 19 (2) INFORMATION FOR SEQUENCE ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (Xi) SEQUENCE DESCRIPTION: GAGCTCCAGG TCACAGAGCA GTTTCCCCTG ACCCCACAGT GCCTTTGCCA GGTTCGCAGA CTGAAGAAGA CCCAGCTGAG CAATTGCCCA C AACCAGGCTG TAAGAGGCCC TATGTATACC TCCTGGCCCT GGAGGCTGCA GAACAGGCCA CCTGTAAGTA GCCTCTCACA GCTCCGGCCC TGAGGTCTTG AGGCAGTAGT AGAGGCCCCT ACCAGCCCTT CCCTGAGATG GCCAGGAGGT GACCTTTGTT CGCTTCCTCT ACACTCTCCT genomic DNA MAGE-8 gene SEQ ID NO: GTCTGAGGCA AGCAGTCAAG CTGGCATCAG TTGTCAGTCC CCCTCTCAAT CAGGAGGCCC AGGGCATCCA CTCCCCAGGC GCTGCCCTGA GTATCTTCAA CTGAGGTGGT AACAGCAGGA TGGAGCCTTG TTCTCCTTCA CAGAGAAGCA GGGTGTAGTA CTGTGGGTCT CCTGAGTCAT ATG GGC CAG TCT GGG TCC GAG CCG GAT TAT AGT AGC GTG ACC CAG ATG TGG CTT CTT ATT ACT TCA CTG GGT GAC GAG CAA GTC AAA AAG TGC AGT ATC GAA CTT CAG CCC CTG CCA ACT TCC CCA AAA ATT ATC GCC GAA CTG ACG TTA GCA GGG GCC ACA ATC AGT GTC AGC GCT GTG AAG AAA TCT GTG GGC CCC ATG TTG CAG CAA GCT ATG CCT ACC AGC CAC GCT GAG AAT GAG GAC CTC AAG GAG AGT AAG GGA GAG GGA CCC GAC AAT CTG GAG CCG TAC TGC CCT TCC ACC GGC GTG AGT GAG GAG ACC CAG AGC GAA GAG TTA GTC AAG ATG GCC TAT GGC AGC ATG CAG GCA CAG CTT AGT ACT GAG TCC GTT ACA AAC CAG GGC GAT CTC CGC GGG CGC CCA AAG GAG CCT CTG GAG CTG CGT AAG CAC GTG CAC GGC CTG GCC GCT TAC GGG GCT GAG GAG TGG GGG TTC TTC GCA TTT ATC TCC CTG ATA CCG GTA AAG CTT GCA GTG GGT AGC CCA CGG CTG GAA CCT TTT TAC CTG ATC GAG TGA GCT ATG TCC ACT GCC CAA AGC GAA CTC ATG GAT GGC ATC GGT GTC GAG GAG GAT TCC GAT TCC TCC ACC GCA CGC CTT ATC ATT CTT GAT CTG GCA GAA GTG TCC TCT TCT GAT TCC CTT AAA GAG TTC GAT GTC GAT GGC ATC TGGGAGGGAG AGTGGGTGCA CCTGTGCGCT CTATGTGAAA TTTCCTACCC TGAGCAGGAG GGCCAGTGCA ATGAGGCCCA GTAGTGGGGA AGTTCCTGTT AATTGTTCCA ATTTTATGTA GTAAGAGTCT ATTC CACAGTGTCT GGAGAACTAC ACGAGTTCCT GTCCTGGAGC ATCCCTGCAT TTGCAGCTAG CGTTCCAGGG TTCTTCACTC GCATGTTGGG CTATTGGGCG ATGTTCCTTC TGACAGTAGA TGCTTTTCAT ATTGGAAGCT CTGGAGTACC GTGGGGTCCA ATGTGGTCAG GAAGAGGCTT GGCCAGTGGG CCACATCCAC TGTGTTTGAA TGTGAGGGAA ATTTGGAGGT TAATGGATGG CAGACTTACT TTATACTGGG CAGGAAGCTG GCCAGGCGCC AGGGCCCTTG GGTCAATGCA TGGGAGAGGA GCAGGTTGTG CACTTTCCCT GAGAGCAGTC CACAGTGTGG TTATCTTTGT TGTAATGAAC GCTTTTTATA AAACCCATGT CTCACCCAAG CGGCAGTGAT CTGAAACCAG AGAGTTCGCA GAAAGGAGTT GGAGGGCCTG GCTCTGTTAC ACAGTTCTCA ACCATCTCTC TTCCTTTTGG TTCAACATTC TAGTTTAGGA TATTTCTTGA 150 200 250 300 350 400 450 451 493 535 577 619 661 703 745 787 829 871 913 955 997 1123 1156 1206 1256 1306 1356 1406 1456 1506 1556 1606 1656 1706 1756 1806 1810 INFORMATION FOR SEQUENCE ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: TCTGAGACAG AGCAGTGAAG GCCCCAGCAC TCACTCATAG TCTCACTTCC GCCCCAAGAG TGTTAGAACC CTCTCTCCCC CTGACTGCTG TGTCCTCAGG GTGAAGTGTT ACATGGGACC AGCCTTGATC TCCCTCAGGT GCCCCAGAGC TCCAAGGTTC AGGCCTGTGG CCCTGACCAG TCGCAGAGCA CACCCTGAAT CCATAGCACC TCTGCAGGCT TCTCGGGACA AGCACTGACG GGTTCTCAGC GTCTCCATCG AGTCATC genomic DNA MAGE-9 gene SEQ ID NO: GAGGAGACCC GTGCACCAAG TGGCCCCATT AGCTGCACGC GGCTAACCAG AAGACCTGTA TGAAGTCTCT CCCAGCTCCT AGGCAGTGTC GGCCCCACCT CCCCCTACTG TGAGTAGCCC GAGGACAGGA AGTCAGCCTT CACACACTCC GCCCACGCTC ATG GAC CAG GAC CCT GTC AGT CAG GCT GAG AAT GAG GAC CTC AAG AAA AGT GGG AAC CAC AGC AGA GGA TCT CTT GAA AGC CCC TAC CAA CTG GAG CCG TAC TTC CCC TCG GCC GAC GTG GAG TAC TAC TAT GAG GAG CTC GAA CCC AAG CAG TAC GAA GAG TTG GTC AAG ATG GCC TGC GCC AAC ATG CCC CTG GAG GAG CCC GAG GAG GCC ACA GAG AGT ACT GAG TTC GTT ACA CGC CAG GGC GAT CTC TGC GGG AGG GAG TTC AAG ATC CAA CAG CAA GGC GAG CCT TTA GAA ATG CAT AAG TAC GTG CAC AGC CTG GCC GTG AAG TAC CTG GTC TGC GAG AGG GGA GAG GAG CAG TGG GAG TTC TTC GCA TTT ATC TCC ATG ATC CCT TAT CTG CGG TGG ATA TAC GGA AGT GAG GAG GTG GGA AGC CCA CAA CTG GAA CCT TTT TAC CTG ATT GAA GTT CTC CAG GGT AAT CCA GTC CCG GAC GAG TCT GGC CAA AGC GAA CTC ATG GTG GGC ATC GGT GTC GAG GGG ACC GTG TCC TAT TCC TGA CAC TTG GAG GCT GCT TTC TCC GCA CAC CTG ATC ACT CTT GAT CTG GTT AAG CAA CCC AAG TTG CTT TGC GGC ACT GCT TCC GAT TCG CTG AAA GAG TTC GAT GTC GGT GGT ATC GAG GAT GGC GCC GTC TAT AAG CTG ACC GGG TCC GAG GTC AAA TAT AGC GGC GTG ACT CAT GTG TGG CAC TGG AGT CAC ATG GAA GCACCAGCCG CAGCCGGGGC CAAAGTTTGT GGGGTCA CCT ATG TCC TCA TCC GGC GAC TTG CGA GTC AAA AAG GCT AGC ATC GAA ATG GTG GAT GCT CTC GAG GAT GGT TCC TCA ATT TCC CCA AAG GTC ATC GCC GAG CTT ATG CTA GCG TTC CAG CCT GAA AAT GTT GAA GCA TCT AGT TCC AGC GCT GTG AAG AAA TCC GTG GGC CCC ACC TTG TAC GAA GCG ACC GCA TTG 150 200 250 300 350 400 427 469 511 553 595 637 679 721 763 805 847 889 931 973 1099 1141 1183 1225 1267 1309 1351 1375 1412 INFORMATION FOR SEQUENCE ID NO: 22: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: (Xi) SEQUENCE DESCRIPTION: ACCTGCTCCA CTGTCAGTCC CTCTCACTTC AGAGCTGTGG GGCCTTTGTT CCCTCCCTCT ACACTCCCAC ATG GAT CAG TCC TCT CCA CAG TCC AAG GAG TTG ATC GAA ATG CCT CTT GCT ACC TCC GAG AGT CTT GAG TCT GTG ACA GAC CTG CGA CAA CCC AGC TCC GAG GCT CCA GAG TTA CAG AAG CAC CTG GCT TCC CTG TCC TCC GTT CAG TTA AGT CCC TTT GCA TTC GTC GGACAAAGTG TGGAGCCTTG CTTCTTCAGG GACACCACAG AGAACCTCCA CTCCCCAGGC CTGCTACCCT CCA CAA GCT TCT TCC TCT ATA GAT CCA AGA CTG GAA CCT TTT nucleic acid linear genomic DNA AAG AGT GTG TTT TCC GCT GCC CAA AGC AGT CTC ATA TTG GGC base pairs MAGE—1O gene CGT GAG GAG CCA TGC GAT TGC TCT ACC GAG TTC CTG TTG ATT GACCCCACTG GCCTCTGCCG TTCTCAGGGG AGCAGCACTG GGGTGTGGTT CTGTGGGTCC GATCAGAGTC CAG ACA GAG TCC TAT GAT TCC GAT CTA ATA AAG GAG TTT GAT SEQ ID NO:" CATCAGCTCC GCTGCATCCT ACAGGGAGAG CAAGAGGTCA AAGGAGAAGA CCTGTAAGTT CTCAGCTGTG CCATCGCCCA AGTCCTGCCC ATC CGC CAG GAT TCT CCT GAG TCC GAG CAG GAT TAT AGT AGT GTA TGC GGC GCT TTT CTA ACA CCC GGC GTC GAA CAA GTC GAA AAG ATG CTC TCA CCC ATA CCA TCG TCC CTG AAG ATG ATA GCC GAA ACCTACCCTA GAGGAGCCAT GCCACTTACA CCT GAG TCA TCC CCA AAT GTC AGC CCA GTG AAG AAA TCC GTG GAA GGT TCC TCC AGC CCT GTT AGC GAC ACT GAG AAT GAG GAT GAA GCA ACT TCC ACC CCC GCT CAA AGT GAT CCG TAT TGC CC 150 200 250 300 333 375 417 459 501 543 585 627 669 711 753 795 837 879 920 (B) TYPE: (D) TOPOLOGY: (ii) MOLECULE TYPE: (ix) FEATURE: (A) NAME/KEY: base INFORMATION FOR SEQUENCE ID No: 23: (i) SEQUENCE CHARA (A) LENGTH: CTERISTICS: pairs nucleic acid linear MAGE-11 (Xi) SEQUENCE DESCRIPTION: AGAGAACAGG CACTGGAGGA CATATCTCAT GGCCCCATCA AGTCATCATG CCTTCAGGCC AAGCTGAGGA ACTCTAGAGG TCAGGAAGAG TATCTGATGA CCTGACCTGA GATAATTGAT GATCACAAAG CCAACCTGGA GAACAAGTGT CTGAGTCTGT CCCAGATATT CCTCTTGAGC CAAGAAGAAG GCAGGAGGCT AGTTGCCTGC TCCTTCTCTC GGGCTCTGGC TAGACCCTGA TTGGTTCATT GCAGAA GGACAGGAGT AAGTAGGCCT TCTCACGCTC TCCCACAGTT AAAGAAGTCA ACCTGGGCCT GCCTTCTTCT TGCTGAGTCA CCACTGCCAT AGCCAAGAAA GTCCTTTTCC TATTCTCCGC genomic DNA gene SEQ ID NO: CCCAGGAGAA' TTGTTAGATT CCTCTCTCCC CGGCCTGCTG GCACTGCAAG GGTGGGTGCA CCTCTACTCT CCAAGTCCTC GGATGCCATC AGGAGGGGCC CAAGATATAC AAGTATCGAG CCCAGAGGAT CTCCATGGTT CAGGCTGTGG ACCTAACCAG CCTGAGGAAG CAGGCTCTCC GAATGTGGGC CCCAGAGTCC TTTGGGAGCC AAGTACCTCG TACATGACAA TCAAGGGGCT ATG GAG GGC GTC TGT GTC GAG GGA ACC GTG CCA CTG ATA ATT CTT AAT CTG GTT AGG CAA CCC AGG GGG TTT GAT GTC GAG GGT ATG GAG AAT GGC GCC TAC ATA GCC AGT AGG GTG ACC CAG GTA TGG CAC TGG ACT CAC AAT GTC GAA AAG TCC AGC ATC GAA TTC GTG GAT GCT GCC ATC GCC GAA CTC ATG TTC GTC CTC CAG CCT GAG AAT AAA TCT GTG AAC CCC ATG CTG TTT GAA GCA ACC GGG AAT GTA GAC CTC AAG GAG AGC GGG AAG TGC AGC AGG TAT TGC CCC TCT TCT GGG ATT GAG TAC TAT GAG ATG ACT TAT GGC AAC ATG CCC CTG GAG GAC CAA AGC GAT CTC TGC GGG AAG GTG TTC TAC CTG CAC GGC CTG ATC GTG AGG TAC CTG AAG ATG AAA GTT TTT CTC TCC ATA ATA CCT TAT CTC CGG TGG CTT CCT TTT TAT CAG ATA GAA GCT CTT CAG GGT GAG 150 200 250 300 350 400 450 500 550 600 616 658 700 742 784 826 868 910 952 994 11 (2) INFORMATION FOR SEQUENCE ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2150 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: _ (A) NAME/KEY: smage-I (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TCTGTCTGCA TATGCCTCCA CTTGTGTGTA GCAGTCTCAA ATGGATCTCT 50 CTCTACAGAC CTCTGTCTGT GTCTGGCACC CTAAGTGGCT TTGCATGGGC 100 ACAGGTTTCT GCCCCTGCAT GGAGCTTAAA TAGATCTTTC TCCACAGGCC 150 TATACCCCTG CATTGTAAGT TTAAGTGGCT TTATGTGGAT ACAGGTCTCT 200 GCCCTTGTAT GCAGGCCTAA GTTTTTCTGT CTGCTTAACC CCTCCAAGTG 250 AAGCTAGTGA AAGATCTAAC CCACTTTTGG AAGTCTGAAA CTAGACTTTT 300 ATGCAGTGGC CTAACAAGTT TTAATTTCTT CCACAGGGTT TGCAGAAAAG 350 AGCTTGATCC ACGAGTTCAG AAGTCCTGGT ATGTTCCTAG AAAG 394 ATG TTC TCC TGG AAA GCT TCA AAA GCC AGG TCT CCA TTA AGT 436 CCA AGG TAT TCT CTA CCT GGT AGT ACA GAG GTA CTT ACA GGT 478 TGT CAT TCT TAT CCT TCC AGA TTC CTG TCT GCC AGC TCT TTT 520 ACT TCA GCC CTG AGC ACA GTC AAC ATG CCT AGG GGT CAA AAG 565 AGT AAG ACC CGC TCC CGT GCA AAA CGA CAG CAG TCA CGC AGG 604 GAG GTT CCA GTA GTT CAG CCC ACT GCA GAG GAA GCA GGG TCT 646 TCT CCT GTT GAC CAG AGT GCT GGG TCC AGC TTC CCT GGT GGT 688 TCT GCT CCT CAG GGT GTG AAA ACC CCT GGA TCT TTT GGT GCA 730 GGT GTA TCC TGC ACA GGC TCT GGT ATA GGT GGT AGA AAT GCT 772 GCT GTC CTG CCT GAT ACA AAA AGT TCA GAT GGC ACC CAG GCA 814 GGG ACT TCC ATT CAG CAC ACA CTG AAA GAT CCT ATC ATG AGG 856 AAG GCT AGT GTG CTG ATA GAA TTC CTG CTA GAT AAA TTT AAG 898 ATG AAA GAA GCA GTT ACA AGG AGT GAA ATG CTG GCA GTA GTT 940 AAC AAG AAG TAT AAG GAG CAA TTC CCT GAG ATC CTC AGG AGA 982 ACT TCT GCA CGC CTA GAA TTA GTC TTT GGT CTT GAG TTG AAG 1024 GAA ATT GAT CCC AGC ACT CAT TCC TAT TTG CTG GTA GGC AAA 1066 CTG GGT CTT TCC ACT GAG GGA AGT TTG AGT AGT AAC TGG GGG 1108 TTG CCT AGG ACA GGT CTC CTA ATG TCT GTC CTA GGT GTG ATC 1150 TTC ATG AAG GGT AAC CGT GCC ACT GAG CAA GAG GTC TGG CAA 1192 TTT CTG CAT GGA GTG GGG GTA TAT GCT GGG AAG AAG CAC TTG 1234 ATC TTT GGC GAG CCT GAG GAG TTT ATA AGA GAT GTA GTG CGG 1276 GAA AAT TAC CTG GAG TAC CGC CAG GTA CCT GGC AGT GAT CCC 1314 CCA AGC TAT GAG TTC CTG TGG GGA CCC AGA GCC CAT GCT GAA 1360 ACA ACC AAG ATG AAA GTC CTG GAA GTT TTA GCT AAA GTC AAT 1402 GGC ACA GTC CCT AGT GCC TTC CCT AAT CTC TAC CAG TTG GCT 1444 CTT AGA GAT CAG GCA GGA GGG GTG CCA AGA AGG AGA GTT CAA 1486 GGC AAG GGT GTT CAT TCC AAG GCC CCA TCC CAA AAG TCC TCT 1528 AAC ATG TAG 1537 TTGAGTCTGT TCTGTTGTGT TTGAAAAACA GTCAGGCTCC TAATCAGTAG 1587 AGAGTTCATA GCCTACCAGA ACCAACATGC ATCCATTCTT GGCCTGTTAT 1637 ACATTAGTAG AATGGAGGCT ATTTTTGTTA CTTTTCAAAT GTTTGTTTAA 1687 CTAAACAGTG CTTTTTGCCA TGCTTCTTGT TAACTGCATA AAGAGGTAAC 1737 TGTCACTTGT ACATTATTTT GATTGTCATG GGAAAGTTTA TACTTTTTTC GACTTTACTC TTATTTTCTT GTAGCACAGG GTTATCAGAG CAGATTAGGA CTTGTTTTGT GTTTTTACTA AAACATTGTG GCAATGTGAT ATCATACAGT TATTGTTAAT TTTGAAAATT TTTTTTGTAT AATGCTAAGT AAATTCATTA GAAAGTAAAT CAATTATGAA TTAAGCATTG ATCTAGTATG AAATGTATCT TCT TATTTGCAAC AAACTGGAAA TAACATTGCA TTGGAGAAGG GGTGAAACAA CAGTGAAGTG TTATGAGTGT GATTGCTGTA GAAATAAAGT TGGATTTGAT CGTAAAACTC TATTACTTTA GTTATCTGGA AGTTTCTCCA AGTATAGGCA CTGACAGTGA 1887 1937 1987 2037 2087 2137 21 (2) INFORMATION FOR SEQUENCE ID NO: 25: (i) SEQUENCE CHARA (A) LENGTH: (B) TYPE: TOPOLOGY: (ii) MOLECULE TYPE: (13) (ix) FEATURE: (A) NAME/KEY: base CTERISTICS: pairs nucleic acid linear (xi) SEQUENCE DESCRIPTION: ACCTTATTGG AATGGATCTC TTTGCATGGG CTCCACAGGC TACAGGTCTC CCCTCCAAGT ACTAGACTTT TTGCAGAAAA GAAAGATGTT AGGTATTCTC TCTTTCCAGA TCAACATGCC CAGCAGTCAC AGGGTCTTCT CTGCTCCTCA TGCACAGGCT AAAAAGTTCA AAGATCCTAT AAGTTTAAGA TAACAAGAAG CACGCCTAGA ACTCATTCCT TTTGAGTAGT TAGGTGTGAT CAATTTCTGC TGGCGAGCCT AGTACCGCCA GGACCCAGAG AGCTAAAGTC TGGCTCTTAG AAGGGTGTTC GAGTCTGTTC AGTTCATAGC ATTAGTAGAA AAACAGTGCT TCACTTGTCA ATTATTTTGT TTGTCATGGC AAAGTTTATA CTTTTTTCTT GTCTGTCTGC TCTCTACAGA CACAGGTTTC CTATACCCCT TGCCCTTGTA GAAGCTAGTG TATGCAGTGG GAGCTTGATC CTCCTGGAAA TACCTGGTAG TTCCTGTCTG TAGGGGTCAA GCAGGGAGGT CCTGTTGACC GGGTGTGAAA CTGGTATAGG GATGGCACCC CATGAGGAAG TGAAAGAAGC TATAAGGAGC ATTAGTCTTT ATTTGCTGGT AACTGGGGGT CTTCATGAAG ATGGAGTGGG GAGGAGTTTA GGTACCTGGC CCCATGCTGA AATGGCACAG AGATCAGGCA ATTCCAAGGC TGTTGTGTTT CTACCAGAAC TGGAGGCTAT TTTTGCCATG GATTAGGACT TTTTACTAAA AATGTGATAT TTGTTAGTTT TTTTGTATAA ATATGCCTCC CCTCTGTCTG TGCCCCTGCA GCATTGTAAG TGCAGGCCTA AAAGATCTAA CCTAACAAGT CACGAGTTCG GCTTCAAAAG TACAGAGGTA CCAGCTCTTT AAGAGTAAGA TCCAGTAGTT AGAGTGCTGG ACCCCTGGAT TGGTAGAAAT AGGCAGGGAC GCTAGTGTGC AGTTACAAGG AATTCCCTGA GGTCTTGAGT AGGCAAACTG TGCCTAGGAC GGTAACCGTG GGTATATGCT TAAGAGATGT AGTGATCCCC AACAACCAAG TCCCTAGTGC GGAGGGGTGC CCCATCCCAA GAAAAACAGT CAACATGCAT TTTTGTTACT CTTCTTGTTA TGTTTTGTTA ACATTGTGTA CATACAGTGG TGAAAATTTT TGCTAAGTGA smage-II genomic DNA SEQ ID NO: ACTTGTGTGT TGTCTGGCAC TGGAGCTTAA TTTAAGTGGC AGTTTTTCTG CCCACTTTTG TTTAATTTCT GAAGTCCTGG CCAGGTCTCC CTTACAGGTT TACTTCAGCC CCCGCTCCCG CAGCCCACTG GTCCAGCTTC CTTTTGGTGC GCTGCTGTCC TTCCATTCAG TGATAGAATT AGTGAAATGC GATCCTCAGG TGAAGGAAAT GGTCTTTCCA AGGTCTCCTA CCACTGAGCA GGGAAGAAGC AGTGCGGGAA CAAGCTATGA ATGAAAGTCC CTTCCCTAAT CAAGAAGGAG AAGTCCTCTA CAGGCTCCTA CCATTCTTGG TTTCAAATGT ACTGCATAAA TTTGCAACAA ACATTGCATT TGAAACAACA ATGAGTGTGA AATAAAGTTG AGCAGTCTCA CCTAAGTGGC ATAGATCTTT TTTATGTGGA TCTGCTTAGC GAAGTCTGAA TCCACAGGGT TATGTTCCTA ATTAAGTCCA GTCATTCTTA CTGAGCACAG TGCAAAACGA CAGAGGAAGC CCTGGTGGTT AGGTGTATCC TGCCTGATAC CACACACTGA CCTGCTAGAT TGGCAGTAGT AGAACTTCTG TGATCCCAGC CTGAGGGAAG ATGTCTGTCC AGAGGTCTGG ACTTGATCTT AATTACCTGG GTTCCTGTGG TGGAAGTTTT CTCTACCAGT AGTTCAAGGC ACATGTAGTT ATCAGTAGAG CCTGTTATAC TTGTTTAACT GAGGTAACTG ACTGGAAAAC GGAGAAGGGA GTGAAGTGGG TTGCTGTATA GATTTGATGA 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 CTTTACTCAA ATTCATTAGA AAGTAAATCA TAAAACTCTA TTACTTTATT 2050 G TTTCTCCAG 2099 ATTTTCTTCA ATTATTAATT AAGCATTGGT TATCTGGAA (2) INFORMATION FOR SEQUENCE ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Glu Ala Asp Pro Thr Gly His Ser Tyr

Claims (28)

1. An isolated nucleic molecule, wherein said molecule is:- (a) a DNA molecule comprising a nucleotide sequence selected from SEQ.ID.NOS. 7, 8, 9, ll, 12, 13, 15, 16, 18 and 19, equal length complementary sequences and portions thereof; or, (b) hybridizable under stringent conditions to a DNA molecule having a nucleotide sequence selected from SEQ.ID.NOS. 7, 8, 9, 11, 12, 13, 15, 16, 18 and 19 and equal length complementary sequences; and codes for, or is an equal length complement of a nucleic acid molecule which codes for a polypeptide or protein capable, when presented on a cell surface, of eliciting a cytolytic response from human T-lymphocytes, or a precursor for such a polypeptide or protein.

2. An isolated nucleic acid molecule as claimed in claim 1, wherein said molecule is a CDNA, genomic DNA, or an RNA molecule.

3. An isolated nucleic acid molecule comprising an RNA transcript of a DNA molecule as claimed in claim 2.

4. An isolated nucleic acid molecule which comprises a nucleic acid sequence coding for a polypeptide or protein capable, when presented on a cell surface, of eliciting a cytolytic response from human T-lymphocytes, or a precursor for such a polypeptide or protein, that is coded for by a nucleic acid molecule as claimed in any one of claims 1-3, or an equal length complementary nucleic acid sequence.

5. An isolated nucleic acid molecule as claimed in any one of claims 1 - 4, wherein said polypeptide or protein comprises the amino acid sequence set out in SBQ.ID.NO. 26.

6. An isolated nucleic acid molecule as claimed in any of claims 1-4 which is hybridizable under stringent conditions to a nucleic acid molecule having a nucleotide sequence as set out in any one of SEQ.lD.NOs. 7, 8, 9 and 11, or to an equal length complementary nucleotide sequence.

7. An isolated nucleic acid molecule as claimed in claim 6 and having a nucleotide sequence as set out in figure 9, or in any one of SEQ.lD.NOs. 7, 8, 9 and 11, or an equal length complementary nucleotide sequence.

8. An expression vector comprising a nucleic acid molecule as claimed in any of claims 1-7.

9. An expression vector as claimed in claim 8, wherein the nucleic acid molecule is operably linked to a promoter.

10. An expression vector as claimed in claim 8 or claim 9, further comprising a nucleic acid sequence coding for a major histocompatability antigen (MHC) or a human leukocyte antigen (I-ILA), a cytokine, or a bacterial or viral genome or a portion thereof.

11. An expression vector as claimed in claim 10, wherein the cytokine is an interleukin, preferably IL-2 or IL-4.

12. A cell transfected with a nucleic acid molecule as claimed in any of claims 1-7, or an expression vector as claimed in any of claims 8-11.

13. A cell as claimed in claim 12, transfected with a nucleic acid molecule coding for an MHC or HLA, or a cytoltine.

14. A cell as claimed in claim 12 and capable of expressing an Ml-lC or HLA, or a cytoltine.

15. A cell as claimed in claim 13 or claim 14, wherein the cytoltine is an interleukin, preferably IL-2 or IL-4.

16. A cell as claimed in any of claims 12-15 and being non-proliferative.

17. A polypeptide or protein capable of eliciting a cytolytic response from human lymphocytes, or a precursor polypeptide or protein, coded for by a nucleic acid molecule as claimed in any of claims 1-7.

18. A polypeptide or protein, as claimed in claim 17, having an amino acid sequence as set out in SEQ.ID.NO. 26.

19. A virus containing a nucleic acid molecule as claimed in any of claims 1-7.

20. A virus as claimed in claim 19, wherein said virus is mutated or etiolated.

21. An antibody which specifically binds a polypeptide or protein as claimed in claim 17 or claim 18, or a complex of a polypeptide or protein as claimed in claim 17 or claim 18 and an MHC or HLA, but which does not bind to the MHC or HLA alone.

22. An antibody as claimed in claim 21, wherein said antibody is a monoclonal antibody.

23. An isolated nucleic acid molecule as claimed in any of claims 1-7, an expression vector as claimed in any of claims 8-11, a cell as‘ claimed in any of claims 12-16, a polypeptide or protein as claimed in either of claims 17 and 1-8, a virus as claimed in either of claims 19 and 211, or an antibody as claimed in either of claims 21 and 22, for use in the therapy, prophylaxis or diagnosis of tumors.

24. A pharmaceutical composition for the prophylaxis, therapy or diagnosis of tumors comprising a nucleic acid molecule as claimed in any of claims 1-7, an expression vector as claimed in any -of claims 8-11, a cell as claimed in any of claims 12-16, a polypeptide or protein as claimed in either of claims 17 and 18, a virus as claimed in either of claims 19 and 20, or an antibody as claimed in either of claims 21 and 22, optionally in admixture with a pharmaceutically acceptable carrier and optionally further comprising an MHC or HLA.

25. A method of producing a cytolytic T-cell culture reactive against autologous tumor cells of an individual, comprising the step of culturing a sample of lymphocytes from the individual with cells as claimed in any of claims 12-16.

26. Use of an isolated nucleic acid molecule as claimed in any of claims 1-7, an expression vector as claimed in any of claims 8-11, a cell as claimed in any of claims 12-16, a polypeptide or protein as claimed in either of claims 17 and 18, a virus as claimed in either ofclaims 19 and 20, an antibody as claimed in either of claims 21 and 22, or a pharmaceutical composition as claimed in claim 24, for the preparation of a medicament for the prophylaxis, therapy or diagnosis of tumors.

27. A use as claimed in claim 26, wherein the tumor is a melanoma, a sarcoma, or a carcinoma such as a small cell lung, non-small cell lung, squamous cell, thyroid, colon, pancreatic, prostate, breast or larynx carcinoma.

28. A method for determining the regression, progress or onset of a tumor in an individual comprising the steps of:- (a) determining the amount of polypeptide or protein as claimed in claim 17 or 18 present in a sample of body fluid, tissue, or tumor from said individual, optionally by employing an immunoassay involving an antibody as claimed in either of claims 21 and 22, (b) determining the number of cytolytic T-cells responsive to a cell as claimed in any of claims 12-16, or a polypeptide or protein as claimed in either of claims 17 and 18, present in a sample of body fluid, tissue, or tumor from said individual, or (c) determining the expression of a polypeptide as claimed in either of claims 17 and 18, opuonally by nucleic acid hybridization employing a nucleic acid molecule as claimed in any one of claims 1-7 as a probe, present in a sample of body fluid, tissue, or tumor from said individual.