TUMOR REJECTION ANTIGEN PRECURSORS, TUMOR REJECTION ANTIGENS AND USES THEREOF

Abstract: The invention relates to an isolated DNA sequence which codes for an antigen expressed by tumor cells which is recognized by cytotoxic T cells, leading to lysis of the tumor which expresses it. Also described are cells transfected by the DNA sequence, and various therapeutic and diagnostic uses arising out of the properties of the DNA and the antigen for which it codes.

Description: TUMOR REJECTION ANTIGEN PRECURSORS, TUMOR REJECTION ANTIGENS AND USES THEREOF This application is a continuation-in-part of Serial Number 807,043, filed December 12, 1991, which is a continuation-in-part of Serial Number 764,364, filed September 23, 1991, which is a continuation-in-part of Serial Number 728,838, filed July 9, 1991, which is a continuation-in-part of Serial Number 705,702, filed May 23, 1991, and now abandoned. FIELD OF THE INVENTION 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". BACKGROUND AND PRIOR ART 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 when transplanted into syngeneic animals. These molecules are "recognized" by T-cellε 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. r J. Natl. Cane. 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. Cane. Inst. 53: 333-1336 (1974). While T-cell mediated immune responses were observed for the types of tumor described supra. 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 turn " antigen presenting cell lines are immunogenic variants obtained by utagenesis 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, turn " 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 "turn " "). 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 turn " variants fail to form progressive tumors because they elicit an immune rejection process. The evidence in favor of this hypothesis includes the ability of "turn "11 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 turn " 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 turn" 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., supra: 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.. Cane. 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 "turn-" antigens, and discussed herein. A tumor exemplary of the subject matter described supra 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 turn " variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra) . In contrast to tumor rejection antigens - and this is a key distinction - the turn " 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 "PI", and can be provoked to produce turn " variants. Since the turn " phenotype differs from that of the parent cell line, one expects a difference in the DNA of turn " cell lines as compared to their turn* parental lines, and this difference can be exploited to locate the gene of interest in turn " cells. As a result, it was found that genes of turn " 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, supra. and Lurquin et al. , Cell 58: 293-303 (1989). This has proved not to be the case with the TRAs of this invention. These papers also demonstrated that peptides derived from the turn " antigen are presented by the L molecule for recognition by CTLs. P91A is presented by L d , P35 by D d and P198 by K d . 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 turn " cells can be used to generate CTLs which lyse cells presenting different turn" antigens as well as turn "1" 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. 158: 240 (1983); Herin et all. Int. J. Cane. 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. Herin et all, supra. 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. Topalian et al. , supra; Degiovanni et al. , Eur. J. Immunol. 20: 1865-1868 (1990). These observations, coupled with the techniques used herein to isolate the genes for specific urine 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. These and various 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 Pl.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 CIA.3.1. Figure 4 shows Northern Blot analysis of expression of gene PIA. Figure 5 sets forth the structure of gene PIA with its restriction sites. Figure 6 shows the results obtained when cells were transfected with the gene from PIA, 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 SEQUENCES 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 PIA. SEQ ID NO: 5 is the genomic DNA sequence for PIA. SEQ ID NO: 6 shows the amino acid sequence for the antigenic peptides for PIA 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 E. SEQ ID NO: 8 is a nucleic acid sequence coding for MAGE- 1. 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 MAGE-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 l 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 Pl.HTR's desirable qualities. To do this, Pl.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: 1040-1052 (1983). To carry out the selection, 10 cells of Pl.HTR were mixed with 2-4xl0 6 cells of the CTL clone in a round bottom tube in 2 ml of medium, and centrifuged for three minutes at 150xg. After four hours at 37°C, the cells were washed and resuspended in 10 ml 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 C 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 1-1117. 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. Pl.HTR is a mastocyto a 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 Pl.HTR, and should be selected so as to not 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 turn- antigens. Selective plasmid and genomic DNA of Pl.HTR were prepared, following Wolfel et al. , Immunogenetics .26.: 178- 187 (1987) . The transfection procedure followed Corsaro et al.. Somatic Cell Molec. Genet 7: 603-616 (1981), with some modification. Briefly, 60 μg of cellular DNA and 3 μg 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 1 mM Tris-HCl (pH 7.5) , 0.1 mM EDTA; and 310 ul IM CaCl 2 . The solution was added slowly, and under constant agitation to 1.25 ml of 50 mM Hepes, 280 mM NaCl, 1.5 mM Na 2 HP0 4 , 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 PO.HTR cells (5xl0 6 ) 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 37°C, after which it was added to an 80 cm 2 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 8xl0 6 cells in 40 ml of medium. In order to estimate the number of transfectants, lxlO 6 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, supra, 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 6x10* 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 10 6 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 51 Cr labeled Pl.HTR target cells (2xl0 3 - 4xl0 3 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.PI:5 cell line, described supra. in a visual lysis assay. See figure IA 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 Pl.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 IB. 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 PI:5. Six days later, lytic activity against Pl.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 supra. Example 4 The CTL assays carried out for P1A.T2 demonstrated that it presented antigen A ("P815A") , and therefore had received the gene from Pl.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 turn " 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 10: 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 Smal 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 8767, following Grosveld et al., supra. Approximately 9xl0 5 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 MgCl 2 , 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 2xl0 8 cells/ml (OD 600 =0.8), a 10 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 21 groups of approximately 30,000 cosmids. Example 5 Using the twenty-one groups of cosmids alluded to supra. (60 ug) and 4 ug of pHMR272, described supra. groups of 5xl0 6 PO.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 P1A.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, supr . 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 0 (19 8 8) . The resulting product was titrated on E. coli ED 8 7 6 7 with ampicillin selection, as in Example 5. Similarly, amplification of the cosmids and transfection followed Example 5, again using PO.HTR. High frequencies of transfection were observed, as described in Table l, which follows: Table 1. Transfer of the expression of antigen PE15A by cosmids obtained by direct r p>_aι cc kk aag r ii nng £ Transfectant No. of cosmids obtained No. of ιr2nsfecιaπιs cbi≥ined with by direct packaging of expressing PS15A / no the cosmid library 0.5 μg of DNA of Hr_B r tr2nsfeci 2nιs TCS.l 32 E7/1 2 C3_. 32000 < /3S4 TC3.3 <4 25/72 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 CIA.3.1 was only 16.7 kilobases long, and was selected for further analysis as described infra. The cosmid CIA.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 Pstl 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 Pstl 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 Smal/Xbal, 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 Pl.HTR. To accomplish this, total cellular RNA was first isolated using the guanidine-isothiocyanate procedure of Davis et al. , Basic Methods In Molecular Bioloσy (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 lOxSSC (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 IM 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 Pl.HTR poly A + 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 β 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. Exam le 8 The Northern analysis described supra suggested that the 5' end of the cDNA was missing. To obtain this sequence, cDNA was prepared from Pl.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 S al/Xbal fragment described supra on a Southern blot. Following cloning into ml3tg 130 λ tg 131, the small, 270 bp fragment was sequenced. The sequence is shown in sequence id no: 1. y_r_mple 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 CIA.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 "PIA" 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); Ki ura 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 "PIA" 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 supra. this contains the region of homology between PIA 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 84: 5306-5310 (1987), in a urine homeodomain protein having nuclear localization. In studies comparing the sequence of gene PIA to the sequences for P91A, 35B and P198, no similarities were found, showing that PIA is indicative of a different class of genes and antigens. Example 10 With the PIA 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 zapll 10 and genomic DNA of DBA2 murine kidney cells. PIA was used as a probe. Hybridization conditions were as described supra. and a hybridizing clone was found. The clone contained exons one and two of the PIA 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 supra. 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 PIA gene isolated from normal kidney 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 mutation; 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 "PIA". 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 supra. RNA of normal liver and spleen cells was tested to determine if a transcript of the PIA 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 PIA 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 supra (Northern blotting) , but no transcript was found. In contrast when a Balb/C derived IL-3 dependent cell line L138.8A (Hϋltner et al.. J. Immunol. 142: 3440-3446 (1989)) was tested, a strong signal was found. The mast cell work is shown in figure 4. It is known that both BALB/C and DBA/2 mice share H- 2 d haplotype, and thus it was possible to test sensitivity to lysis using the CTLs described supra. Figure 8 shows these results, which essentially prove that anti-A and anti-B CTLs lysed the cells strongly, whereas anti-C and anti-D lines did not. 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 PIA antigen by MHC molecules was of interest. To test this, cosmid CIA.3.1 was transfected into fibroblast cell line DAP, which shows phenotype H-2 k . The cell lines were transfected with genes expressing one of the K d , D d , and L d antigen. Following transfection with both the cosmid and the MHC gene, lysis with CTLs was studied, again as described supra. These studies, summarized in Table 2, show that L d is required for presentation of the PIA antigens A and B. Tzb!c 2. H-2-ressricticn of antigens PE 15A and PE15B Recipient cell ' No of clones lysed b> the CTL/ no. of H.τ.B r de es' DAP (K-2 k ) DA? + K d D. _P -t D d DAP L d •Cosmid CIA.3.1 containing the entire PIA gene was tra_sfecιεd in DAP cells previously transfected with H-2 d class I genes as indic-ied. •Independent drug-resistant colonies were tested for lysis by ar.ti-A or asti-B CTL ic a 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 PIA 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 PO.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. Cane. 44: 634-640 (1989). Among the recognized subclones of MZ2-MEL are MEL.43, MEL3.0 and MEL3.1. (Van den Eynde et al., supra) . Cell line MEL3.1 expresses antigen E, as determined by CTL studies as described for P815 variants, supra■ 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 antigen precursor, the techniques developed supra- 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, supra. 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 "4 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 Wδlfel et al., Immunogenetics 26: 178-187 (1987), the disclosure of which is incorporated by reference. The plasmid pSVtkneoβ, 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 μg) and plasmid DNA (6 μg) were mixed in 940 μl of 1 mM TrisΗCl (pH 7.5), 0.1 mM EDTA, after which 310 μl of IM CaCl 2 was added. This solution was slowly added, under constant agitation, to 1.25 ml Of 2XHBS (50 mM HEPES, 280 mM NaCl 1.5 mM Na 2 HP0 4 , 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 3xl0 6 MEL2.2 cells, in 22.5 ml of melanoma culture medium (Dulbecco'ε 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 4xl0 6 cells per 80 cm 2 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, harvested, and cultured in 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 6xl0 4 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 μl of CTL culture medium with 35 U/ml of IL-2. One day later, the supernatant (50 μl) was harvested and examined for TNF concentration, for reasons set forth in the following example. Example 17 The size of the mammalian-genome is 6xl0 6 kb. As the average amount of DNA integrated in each drug-resistant transfectant was expected to be about 200 kb, a minimum of 30,000 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 supra. 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 15, 1500 CTL 82/30 cells had been added per well of transfectants. These CTLs were collected 6 days after stimulation. As indicated supra. after 1/3 of the cells in each well had been removed and the remaining 2/3 (4xl0 4 ) had readhered, the CTLs and IL-2 were added thereto. The 50 μl 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 μl of W13 culture medium (RPMI-1640, supplemented with L-arginine (116 mg/1) , L-asparagine (36 mg/1) , L- glutamine (216 mg/1) , and 10% FCS supplemented with 2 μg of actinomycin D at 37% in an 8% C0 2 atmosphere. The cell line W13 is a mouse fibrosarcoma cell line sensitive to TNF. Dilutions of recombinant TNF-β 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-dimethylthiazol-2-yl)-2,5-diphenyl 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 μl 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% IN HCl). Plates were incubated at 37°C overnight, and ODs were taken at 570 nm using 650 nm as control. Dead cell percentage was determined via the formula: 100- (OD 570 sample well) OD 570 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 E" cells (4xl0 6 cells/group) were tested following transfection, and 7xl0 4 independent geneticin resistant 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 51 Cr release assay, and were found to be lysed as efficiently as the original E + cell line. The transfectant E.Tl, 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 5 times less DNA than Pl.HTR. Example 19 Once transfectant E.Tl 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 supra, shows that E.Tl 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.Tl. 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 pSVtkneoβ, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoβ sequences. Wolfel et al., supra. has shown this to be true. If a normally E~ cell is transfected with pSVtkneoβ, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoβ sequences. If a normally E + cell transfected with pSVtkneoβ is E.Tl, however, "co-deletion" should not take place. To test this, the transfectant E.Tl 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 r sequences in the variants, showing close linkage between the E gene and neo r gene in E.Tl, leading to the conclusion that E.Tl 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 protocols described supra. 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 Xmal 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/Smal digested DNA) . The band is absent from E " antigen loss variants of MZ2- MEL, as seen in Figure 12. The sequence for the E antigen precursor gene has been determined , and is presented herein : I 10 I 20 ! 90 I 4 C I S 3 GOλTCCΛM- CCTG CλSOA AΛΛA7Λ7JU.G CGcεcr cσr GASAACAGA Cl ACrGCλTCλδ AG7G£&Sλ?G TCACASACTC CλGCCCλCCC TCCTG-Tλ& 121 CAOG-J TGΓG TTO OGTCT OCACCCTC_.G 65CCCG7G3_ TTCC7C77C iti GSAACCAGGC ΛCTOλsoccT T&srcr&λ&λ CA57A7CC7C AGCTCλlλ. 241 CλCλS5_TG7 SC_A&:λ57G Aλ767T7GCC C7Gλλ7GCΛC ACCλλS&SC 3 CA_-__ . _Λ__ .. AWXCTCCAC ASAS?C7KC C7CACC7CCC _ΛC7S7CλG7 3.1 CGλCCTCTG TGGCCG6C7G _ACeC7GA57 ACCCT TCΛC T7CC7CC77C 421 GSSλSAGSCC JUCCCλSλSG ACAG0A77 C C7tt_ .ft3CO CΛSλSG SCλ El &λ7C7G7λλG Tλ&00C7??G TIASAStCTC CλλGv_ CAG T7C7CAGC7C 541 CλCλcrccc? C. CTCCCCXG 6CC G7GW? • C77CAT7GCC CA5C7CC7ftC €01 GCC7G 7&CC CTGλCSλSλG TCA.CλTG- . TC77&λG2λG AG£λG7C7GC « . : T<_ΛS_λASCC C?70λGGCCC AACλλOλGS CC7GSGC7&3 _G7GTG7GCλ "521 TCCTCCTCCT C7CC7C7GG7 CC7GβSCλCC dSλs_λGG TGcccxrrac "7*1 GλϊCCT CCC At_AG7CC7CA <»_A5CC7CC CCC7?7CCCA CTACCATCAλ • 41 CASλGG AAC CCAG70A535 T7_XΛG _&C CGT_AA__._0 AGO 3CCAAG »01 A?CCTGSXG? CCTTCTTCCG &cλsrλλTC C7AΛSU& TK. _j.rr: sε C7K7CC7CA ΛλtλrCGλGC GλGΦSASCCλ G70.CΛΛ 3G CAGA-A7&C7 2021 λTCAAλλATT ACλλG2AC7G T777CCTGAG A7CT7 55CA AλKX7CT_A i&t: C7&S;C??ΓG GCA_?<_A2G GAAG-:_-.GC_ SλCCCCACCG GCCΛC7CC7A 2141 ACCTSCCΓAG DTCTCTCCΓA TC_».TGGC ?G CT&G_7(_A7A AT__V_λ7CA7 1201 β_errec- β TAATTGTCCΪ G57Cλ Gλ7 ccAλ?Gθλ_ e cwcc rac 2261 C_>..:π&35 AGOXGCTOAC- TCTGATGGλG 67G7Λ7&7.7G OSAPSSASCA 2321 GGGSAGCCCλ CSAAGC7GC7 C-CCCAΛGA7 T7GG75CA55 AλλλG7ACC7 2381 A&GTGCCWSλ _AG.SA.7CCC GCλC&C7λ?G AG77CC7G75 &&37CCAΛ&G 2441 AλλCGA&CTλ TG7GAλAG7C CT70λG2__7G TOλ7CAXGG7 CAST&CAAGA 2S01 TCT7CCCATC CC7GCG7GAA GCλ&C_ _*7GA SλSλSGλSOλ AGASSGΛGTC IS 61 TTGCAGCCλλ C&CC_S7GdS A9∞S0λT7G GGCCA97&C1 CCT7CCAM. 2 £21 AGCTTCCCC ΪG CTCO. CT GACATGλ∞C CCAT7CT7CA CTCrOλλύAG CB1 677C7CASTΛ C7AOG777C7 CT7C_λT7GG OTC LCTTSSλ _y__ _ _Α7CT7 1 * ,41 *aθλλ?7C~7 Cλλλ7G7r77 TJ777AJUWG A7G_TT_AA7 SλλCTTCΛGC 2101 ATGAΛ70λ£λ CCXST ACAC AST7C7G7G7 ATλ7λσ777λ A&GCTA>(__G 21 Cl Sλ7T__ .SλT7 OrøUA7CCλ T_Cϊλr_T7G 96U.77GSSA XAΛ7ΛΛCA&C 2J21 07λC?7λGAλ A7G7__UAλλ TCΛG_AG7AA AA7AGλ7_ΛG A7AΛAOΛΛC7 2l . _ ASAffA7AG7C AATTCTTGCC T7A7J C7CA β 7CSA7TC7G ΣAAλλTTT?? 2041 eCA7ACC7GG A777CCT7«G CT7C7_ , 7GAG AλrSTλλ&λG AAA7-AAATC 2101 AΪ7CT7CCTG TTCλCTβGCT CTTΓTCTTCT CCATGCλCTG A5 ATCTSC7 21 Cl CCC7GSG7TA CTXGTOOA-A *ΘCΪλAGGTλ AOCCAG TC ATλCCCλCCC 2221 At__37C__.&5 ___ C70CλG7C ACS7λA7COλ «αTG OJ _λ 9G7CC7C7AA 2211 AλλλffTGΑS.. CA∞∞70λG 05TGTSSSSC »CC&5-T-_λG AGTtXTT&SλG 2341 CTfiλGC7GSG CCATTTTftSC CtTTOSOi Λ CTSC_ST7CC TTCTββSSOλ 2401 ATttWCTTβG C700X7CC I 10 I 20 I 20 I 40 I so ! €6 Example 21 After the 2.4 b genomic segment had been identified, studies were carried out to determine if an H 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. Transfer of antigen expression in this manner indicates that the gene codes for the antigen precursor, rather than coding for a protein which activates the antigen. yam le 23 The probing of cDNA described supra revealed, surprisingly, two different but closely related cDNAs. These cDNAs, when tested, did not transfer expression of antigen E, but they do show substantial homology to the first cDNA segment. The three segments, appear to indicate a newly recognized family of genes, referred to as "MAGE" for "melanoma antigen". In Figure 9, "mage -1" directs expression of the antigen from MZ2 cells. Portions of the third exon of each gene are presented in Figure 9. The second and third sequences are more closely related to each other than the first (18.1 and 18.9% difference compared to the first; 12% with each other) . Out of 9 cDNA clones obtained, three of each type were obtained, suggesting equal expression. "MAGE" as used hereafter refers to a family of molecules, and the nucleic acids coding for them. These nucleic acids share a certain degree of homology and are expressed in tumor cells including several types of human tumor cells as well as in human tumors. The family is referred to as "MAGE" because the first members were identified in human melanoma cells. As the experiments which follow indicate, however, the members of the MAGE family are not at all restricted to melanoma tumors; 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 MZ2 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 oligo- 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 MZ2- 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 1/300 th that of the MZ2 melanoma cell line (Figure 11) . 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. T__aττvmp_ fi g fi 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-Al, A29, B37, B44 and C6. Four other melanomas of patients that had Al in common with MZ2 were cotransfected with the 2.4 kb fragment and pSVtkneoβ. Three of them yielded neo r 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-Al. In confirmation, it was found that, out of 6 melanoma cell lines derived from tumors of HLA-Al 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 Al 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-Al or other class I molecules, especially in view of the similar results found with murine tumors, as elaborated upon supra. Example 27 As indicated supra. 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-MEL2.2, an E " cell line described supra. 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, "MZ2- 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 supra. 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 supra. The library was divided into 14 groups of about 50,000 cosmids, and DNA from each group was transfected into MZ2-MEL2.2.5. 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 μl/cm 2 of 3.5xSSC, lxDenhardt's solution; 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, 2mM EDTA, where the 2.4 kb probes had been labelled with [α 32 p]dCTP (2-3000 Ci/mole) , at 3xl0 6 cpm/ml. Hybridization was carried out for 18 hours at 65°C. After this, the membranes were washed at 65°C four times for one hour each in 2xSSC, 0.1% SDS, and finally for 30 minutes in O.lxSSC, 0.1% SDS. To identify hybridization, membranes were autoradiographed using Kodak X-AR film and Kodak X-Omatic ine intensifying screens. In the following examples, whenever "hybridization" is referred to, the stringency conditions used were similar to those described supra. "Stringent conditions" as used 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 LB-33-MEL was tested. Total mRNA from the cell line was used to prepare cDNA, which was then amplified with oligos CH09: (ACTCAGCTCCTCCCAGATTT) , and CHO10: (GAAGAGGAGGGGCCAAG) . These oligos correspond to regions of exon 3 that are common to previously described mage l, 2 and 3. To do this, 1 μg of RNA was diluted to a total volume of 20 μl, using 2 μl of lOx PCR buffer, 2 μl of each of 10 mM dNTP, 1.2 μl of 25 mM MgCl 2 , 1 μl of an 80 mM solution of CH09, described supra. 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 μl of lOx PCR buffer, 4.8 μl of 25 mM MgCl 2 , 1 μl of CH010, 2.5 units of Thermus acguaticus ("Taq") polymerase, and water to a total volume of 100 μl. Amplification was then carried out for 30 cycles (1 minute 94°C; 2 minutes at 52°C, 3 minutes at 72°C) . Ten μl 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 MZ2, 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 synthetic peptides derived from this exon 3 could be used to confer sensitivity to anti-E CTL. To do this, and using standard protocols, cells normally insensitive to anti-E/CTLs were incubated with the synthetic peptides derived from Exon 3.1. Using the CTL lytic assays described supra on P815A, and a peptide concentration of 3 mM, the peptide Glu-Ala-Asp-Pro-Thr- Gly-His-Ser-Tyr was shown to be best. The assay showed lysis of 30%, indicating conferring of sensitivity to the anti-E CTL. Example 35 Nucleic acid sequences referred to as "smage" were isolated from murine cells. Using the protocols described supra. a cosmid library was prepared from the DNA of normal DBA/2 kidney cells, using cosmid vector C2RB. As a probe, the 2.4 kb BamHI fragment of MAGE-1 was used. The DNA was blotted to nylon filters, and these were washed in 2xSSC at 65°C to identify the smage material. Example 36 Further tissue samples were tested for the presence of MAGE genes, using the protocols discussed supra. Some of these results follow. There was no expression of the MAGE genes in brain or kidney tumor tissue. Colon tumor tissue showed expression of MAGE 1, 2, 3 and 4, although not all tumors tested showed expression of all MAGE 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) . 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 supr . 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 in vivo. 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 MHC/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 supra 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 linked 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 isolated cells which present the TRA molecule on their surface, as well as TRAP fragments, mutated viruses, especially etiolated forms, and transfected bacteria. "Fragments" as used herein refers to peptides which are smaller than the TRA, but which possess the properties required of a vaccine, as discussed supra. Another vaccine comprises or consists of complexes of TRA and HLA molecule. Vaccines of the type described herein may be used preventively, i.e., via administration to a subject in an amount sufficient to prevent onset of a cancerous condition. 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 alia, 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 include all events involved in 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. There are therapeutic aspects of this invention as well. The efficacy of administration of effective amounts of TRAPs and TRAs as vaccines has already been discussed supra. Similarly, one may develop the specific CTLs .in 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. (1) GENERAL INFORMATION: (i) APPLICANTS: Boon, Thierry, Van den Eynde, Benoit (ii) TITLE OF INVENTION: Isolated And Purified DNA Sequence Coding Antigen Expressed By Tumor Cells And Recognized By Cytotoxic T Cells, And Uses Thereof (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Felfe _ Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (F) ZIP: 10022 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMPUTER: IBM (C) OPERATING SYSTEM: PC-DOS (D) SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/807,043 (B) FILING DATE: 12-DECEMBER-1991 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/764,364 (B) FILING DATE: 23-SEPTEMBER-1991 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/728,838 (b) FILING DATE: 9- ULY-1991 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/705,702 (B) FILING DATE: 23-May-1991 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Hanson, Norman D. (B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCKET NUMBER: LUD 253.4 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 (2) INFORMATION FOR SEQUENCE ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT 50 GAAGATCCTG ATCACTCATT GGGTGTCTGA GTTCTGCGAT ATTCATCCCT 100 CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGC CTTGGGTAGG 150 AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT TGTACCCTTT 200 CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCCCCTCCCA 250 CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG 350 CATGCATTGT GTCAACGCCA TTGCACTGAG CTGGTCGAAG AAGTAAGCCG 400 CTAGCTTGCG ACTCTACTCT TATCTTAACT TAGCTCGGCT TCCTGCTGGT 450 ACCCTTTGTG CC 462 SUBSTITUTE SHEET b2 (2) INFORMATION FOR SEQUENCE ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 675 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC AGT GGC TCA GGT GGT 48 Met Ser Asp Asn Lys Lys Pro Asp Lys Ala His Ser Gly Ser Gly Gly 5 10 15 GAC GGT GAT GGG AAT AGG TGC AAT TTA TTG CAC CGG TAC TCC CTG GAA 96 Asp Gly Asp Gly Asn Arg Cys Asn Leu Leu His Arg Tyr Ser Leu Glu 20 25 30 GAA ATT CTG CCT TAT CTA GGG TGG CTG GTC TTC GCT GTT GTC ACA ACA 144 Glu lie Leu Pro Tyr Leu Gly Trp Leu Val Phe Ala Val Val Thr Thr 35 40 45 AGT TTT CTG GCG CTC CAG ATG TTC ATA GAC GCC CTT TAT GAG GAG CAG 192 Ser Phe Leu Ala Leu Gin Met Phe lie Asp Ala Leu Tyr Glu Glu Gin 50 55 60 TAT GAA AGG GAT GTG GCC TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC 240 Tyr Glu Arg Asp Val Ala Trp lie Ala Arg Gin Ser Lys Arg Met Ser 65 70 75 80 TCT GTC GAT GAG GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC TAC 288 Ser Val Asp Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Tyr Tyr 85 90 95 GAC GAC GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAT GAT 336 Asp Asp Glu Asp Asp Asp Asp Asp Ala Phe Tyr Asp Asp Glu Asp Asp 100 105 110 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCA GAA GAT GAG 384 Glu Glu Glu Glu Leu Glu Asn Leu Met Asp Asp Glu Ser Glu Asp Glu 115 120 125 GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA GCT GAG GAA ATG 432 Ala Glu Glu Glu Met Ser Val Glu Met Gly Ala Gly Ala Glu Glu Met 130 135 140 GGT GCT GGC GCT AAC TGT GCC TGT GTT CCT GGC CAT CAT TTA AGG AAG 480 Gly Ala Gly Ala Asn Cys Ala Cys Val Pro Gly His His Leu Arg Lys 145 150 155 160 AAT GAA GTG AAG TGT AGG ATG ATT TAT TTC TTC CAC GAC CCT AAT TTC 528 Asn Glu Val Lys Cys Arg Met lie Tyr Phe Phe His Asp Pro Asn Phe 165 170 175 CTG GTG TCT ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT 576 Leu Val Ser lie Pro Val Asn Pro Lys Glu Gin Met Glu Cys Arg Cys 180 185 190 GAA AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA GAG 624 Glu Asn Ala Asp Glu Glu Val Ala Met Glu Glu Glu Glu Glu Glu Glu 195 200 210 GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCA CCT 672 Glu Glu Glu Glu Glu Glu Glu Met Gly Asn Pro Asp Gly Phe Ser Pro 220 225 230 235 TAG 675 SUBSTITUTE SHEET (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 60 TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA ATTTGATTTT GTTCTAAAGT 120 TCATTATGCA AAGATGTCAC CAACAGACTT CTGACTGCAT GGTGAACTTT CATATGATAC 180 ATAGGATTAC ACTTGTACCT GTTAAAAATA AAAGTTTGAC TTGCATAC 228 h5 (2) INFORMATION FOR SEQUENCE ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1365 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG GAAGATCCTG ATCACTCATT GGGTGTCTGA GTTCTGCGAT CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGC AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT CATGCATTGT GTCAACGCCA TTGCACTGAG CTGGTCGAAG CTAGCTTGCG ACTCTACTCT TATCTTAACT TAGCTCGGCT ACCCTTTGTG CC ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC GGT GGT GAC GGT GAT GGG AAT AGG TGC AAT TTA TAC TCC CTG GAA GAA ATT CTG CCT TAT CTA GGG TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC ATA GAC GCC CTT TAT GAG GAG CAG TAT GAA AGG TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC TCT GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GCT GAG GAA ATG GGT GCT GGC GCT AAC TGT GCC GGC CAT CAT TTA AGG AAG AAT GAA GTG AAG TGT TAT TTC TTC CAC GAC CCT AAT TTC CTG GTG TCT AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT GAA GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TAG GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTTTTTAGT ATTTGATTTT GTTCTAAAGT TCATTATGCA AAGATGTCAC CTGACTGCAT GGTGAACTTT CATATGATAC ATAGGATTAC GTTAAAAATA AAAGTTTGAC TTGCATAC SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4698 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG GAAGATCCTG ATCACTCATT GGGTGTCTGA GTTCTGCGAT CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGC AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT CATGCATTGT GTCAACGCCA TTGCACTGAG CTGGTCGAAG CTAGCTTGCG ACTCTACTCT TATCTTAACT TAGCTCGGCT ACCCTTTGTG CC ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC GGT GGT GAC GGT GAT GGG AAT AGG TGC AAT TTA TAC TCC CTG GAA GAA ATT CTG CCT TAT CTA GGG TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC ATA GAC GCC CTT TAT GAG GAG CAG TAT GAA AGG TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC TCT GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GCT GAG GAA ATG GGT GCT GGC GCT AAC TGT GCC GTGAGTAACC CGTGGTCTTT ACTCTAGATT CAGGTGGGGT CTCTTGCCCA CATCTGTAGT AAAGACCACA TTTTGGTTGG TGGAGCCATT CCTGGCTCTC CTGTCCACGC CTATCCCCGC CCCCACTCCT TGCTCCGCTC TCTTTCCTTT TCCCACCTTG TTCAGTCCAT CCTGCTCTGC TCCCTTTCCC CTTTGCTCTC TCCCCCTCGG CTCAACTTTT CGTGCCTTCT GCTCTCTGAT TTCAGGCTTC CCCATTTGCT CCTCTCCCGA AACCCTCCCC CCTTTTCGCG CCTTTTCTTT CCTGCTCCCC TCCCCCTCCC TCACCAGCTT TGCTCTCCCT GCTCCCCTCC CCCTTTTGCA TCCTGCTCCC CTCCCCCTCC CCTCCCTGTT TACCCTTCAC CTACCTGCTT CCCTCCCCCT TGCTGCTCCC TCCCTATTTG TGCTCCTCCC TCCCCCTCCC CCTCCCTCCC TATTTGCATT CCTCCCTCCC CCTCCCCAGG CCTTTTTTTT TITTTTTTTT TTGGTTTTTC GAGACAGGGT TTCTCTTTGT ATCCCTGGCT TCACTCTGTA GACCAGGCTG GCCTCAAACT CAGAAATCTG CCTCCCAAAT GCTGGGATTA AAGGCTTGCA CCAGGACTGC GCCTTTCTTT TTTCTCCTCT CTGGTCTCCC TAATCCCTTT AACTCCCCTT TTGGCACCTT TCCTTTACAG GACCCCCTCC TTCCCTTCCG GCACCCTTCC TAGCCCTGCT CTGTTCCCTC CCTCCCCCTC TTTGCTCGAC TTTTAGCAGC CTTACCTCTC GCCCCGTTCC CCTTTTTTGT GCCTTTCCTC CTGGCTCCCC AGCTCACCTT TTTGTTTGTT TGGTTGTTTG GTTGTTTGGT TTTTTTTTTT GCACCTTGTT TTCCAAGATC CCCCTCCCCC CCTCTGTGTG CCTTTCCTGT TCCCTCCCCC TCGCTGGCTC TCTGCCTTTC CTGTCCCTGC TCCCTTCTCT GCTAACCTTT CTTTTCTAGA CTCCCCCCTC CAGGCTTGCT GTTTGCTTCT CCTGACCCTG CTCCCCTTCC CCTCCCAGCT CCCCCCTCTT CCTTTCTCCA GCCTGTCACC CCTCCTTCTC TCCTCTCTGT TCCTGCTTCC TTTACCCCTT CCCTCTCCCT ACTCTCCTCC GACTTCCTCT CCAGCCGCCC AGTTCCCTGC AGTCCTGGAG CTCTCTGTCC ATCACTTCCC CCTAGTTTCA CTTCCCTTTC ATGTGTCTCT CTTCCTATCT ATCCCTTCCT TTCTGTCCCC CCATCACCTC TCTCCTCCCT TCCCTTTCCT CTCTCTTCCA CCTGCTTCTT TACCCTGCCT CTCCCATTGC CCTCTTACCT TCCATGTCCC CTCTCAATTC CCTGTCCCAT TGTGCTCCCT ATTTCCCTCT TTCTCCCTTA GCCTCTTCTT CCTCTTCTCT TTCCCTTTGC TTCTCCCTCC TCCTTTCCCC TTCCCCTATG TACTTGATCT TCTCTCCTCT CCACATACCC TTTTTCCTTT CTTTGTCCCC AGACCCTACA GTATCCTGTG CACAGGAAGT ATCAACAACA AGGAGGCAAG AAACAGAGCA AAATCCCAAA AAGGCTGGAT GAAAATAAGG CCAGGTTCTG AGGACAGCTG AAGTGGCTCC TATAACCCTA AGTACCAAGG GAGAAAGTGA CTTGATCCTT GCTGCTTCTT TTACATATGT TGGCACATCT CAGGCCATGC TCCATGCTTG GCGCTTGCTC AGCGTGGTTA GAATCTGAAA ACTAGGGGCC AGTGGTTTGT TTTGGGGACA TAGTGATATT TCCCCCTAAA AATTATAACA AACAGATTCA TCCTTCTACA GGTGAGAAGT GGAAAAATTG TCACTATGAA GGCTAAAGAT ACTTGGAACC ATAGAAGCGT TGTTAAAATA TTGCTAAAAT ATTCTTTCTC ACATATTCAT ATTCTCCAG 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 TTC TCA CCT TAG GCATGCAGGT ACTGGCTTCA CTAACCAACC ATTCCTAACA GCTAAGAGCA TCTTTTTAAA AAATATTATT GGTAAACTAA TCTTTTTACA TTAATAAGTA TTAAATTAAT CCAGTATACA CCCTAAGTTA AACAGAAGTC AATGATGTCT AGATGCCTGT GTAGTGAGAC TACTTACTAC AGATGAGAAG TTGTTAGACT GACCAGTAAA AGATCATGCA GTGAAATGTG GCCATGGAAA TTCTTATAGT ACCTTTGAGA CAGCTGATAA CAGCTGACAA TTCAAGAAAG ATCACACGCC ATGGTTCACA TGCAAATTAT TTCTGATTTT TTTCATTTCT AGACCTGTGG TTTTAAAGAG CTTAAAATTT CCTTCATCTT TAATTTTCCT TAACTTTAGT TAGAATTCAA TTCAAATTCT TAATTCAATC TTAATTTTTA AATGTTTTTT AAAAAAAATG CAAATCTCAT TTTTAAGAGA GTAACTGGGG GGCTTAGGGA ATCTGTAGGG TTGCGGTATA GTTCTGGTCT CTGAGAAGCA GTCAGAGAGA ATGGAAAACC CAGTAGGTTA GTGAGGTTGA TATGATCAGA TTATGGACAC ATAAATACTC TAACAGCTAA GGATCTCTGA GGGAAACACA ATTTTAGTTT CTCCTTGAGA AACAATGACA AGACATAAAA AGTCAGGAGT GTATTCTAAT AAGTGTTGCT TATCTCTTAT AGTTGCAAAG CCCAGAAGAA AGAAATGGAC AGCGGAAGAA TTTTTTCCCC TTCATTAATT TTCTAGTTTT TAGTAATCCA TTTTGTTCTA AAGTTCATTA TGCAAAGATG TCACCAACAG GCATGGTGAA CTTTCATATG ATACATAGGA TTACACTTGT AATAAAAGTT TGACTTGCAT AC SUB TITUTESHEET (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: Leu Pro Tyr Leu Gly Trp Leu Val Phe 5 - 0 (2) INFORMATION FOR SEQUENCE ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2418 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: SUB TIT E ΎO CATACCCACC CATAGGGTCG TAGAGTCTAG GAGCTGCAGT CACGTAATCG 2250 AGGTGGCAAG ATGTCCTCTA AAGATGTAGG GAAAAGTGAG AGAGGGGTGA 2300 GGGTGTGGGG CTCCGGGTGA GAGTGGTGGA GTGTCAATGC CCTGAGCTGG 2350 GGCATTTTGG GCTTTGGGAA ACTGCAGTTC CTTCTGGGGG AGCTGATTGT 2400 AATGATCTTG GGTGGATCC 2418 (2) INFORMATION FOR SEQUENCE ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5724 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-1 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: SUBSTITUTE SHEET Z TTGCATGGGG GTGGGACCCA GGCCTGCAAG GCTTACGCG GGGAGGACTC AGGGGACCTT GGAATCCAGA TCAGTGTGG GAGAGGTCCA GGGCACGGTG GCCACATATG GCCCATATTT TGAGGTGACA GGACAGAGCT GTGGTCTGAG AAGTGGGGCC AGAGGGAGGA GTTCCAGGAT CCATATGGCC CAAGATGTGC AGGACTGGGG ATATCCCCGG CTCAGAAAGA AGGGACTCC CTGTCCCCTT TTAGTAGCTC TAGGGGGACC AGATCAGGGA TTCCATTCTC ACTTGTACCA CAGGCAGGAA GTTGGGGGGC ATGGGGTCTT GGGGTAAAGG GGGGATGTCT ACTCATGTCA GGTTGAGGAA GCACAGGCGC TGGCAGGAAT AAAGATGAGT AGGCTATTGG AATCCACACC CCAGAACCAA AGGGGTCAGC TCACCCAGGA TGTGGCTTCT TTTTCACTCC TGTTTCCAGA GTGAGGACCT CATTCTCAGA GGGTGACTCA GGTCAACGTA TCTGGTCTAA AGACAGAGCG GTCCCAGGAT CTGCCATGCG GAACATGAGG GAGGACTGAG GGTACCCCAG GACCAGAACA CTGCACAGAA ATCAGCCCTG CCCCTGCTGT CACCCCAGAG GGGCCGTCTG CCGAGGTCCT TCCGTTATCC TGGGATCATT ACGGGGAGGC CTTGGTCTGA GAAGGCTGCG CTCAGGTCAG GTCCCAGGCC CTGCCAGGAG TCAAGGTGAG GACCAAGCGG CAGGACACAT TAATTCCAAT GAATTTTGAT ATCTCTTGCT AAGGACCTAG GCACGTGTGG CCAGATGTTT GTCCCCTCCT TCCTTATCAT GGATGTGAAC TCTTGATTTG GATTTCTCAG GGGCAGGATC CAGGCCCTGC CAGGAAAAAT ATAAGGGCCC CAGAGGGGGT CATCCACTGC ATGAGAGTGG GGATGTCACA CACCCTCCTG GTAGCACTGA GAAGCCAGGG CTGTGCTTGC CTGAGGGCCC GTGGATTCCT CTTCCTGGAG CTCCAGGAAC GGCCTTGGTC TGAGACAGTA TCCTCAGGTC ACAGAGCAGA GGTGTGCCAG CAGTGAATGT TTGCCCTGAA TGCACACCAA TGCCACAGGA CACATAGGAC TCCACAGAGT CTGGCCTCAC TCAGTCCTGT AGAATCGACC TCTGCTGGCC GGCTGTACCC CTCACTTCCT CCTTCAGGTT TTCAGGGGAC AGGCCAACCC ATTCCCTGGA GGCCACAGAG GAGCACCAAG GAGAAGATCT CTTTGTTAGA GTCTCCAAGG TTCAGTTCTC AGCTGAGGCC TCCCTCTCTC CCCAGGCCTG TGGGTCTTCA TTGCCCAGCT CTCCTGCCTG CTGCCCTGAC GAGAGTCATC ATG TCT CTT GAG CAG AGG AGT CTG CAC TGC AAG GCC CTT GAG GCC CAA CAA GAG GCC CTG GGC CTG CAG GCT GCC ACC TCC TCC TCC TCT CCT CTG GTC CTG GAG GAG GTG CCC ACT GCT GGG TCA ACA GAT AGT CCT CAG GGA GCC TCC GCC TTT CCC ACT ACC ACT CGA CAG AGG CAA CCC AGT GAG GGT TCC AGC GAG GAG GGG CCA AGC ACC TCT TGT ATC CTG GAG CGA GCA GTA ATC ACT AAG AAG GTG GCT GAT TTG CTG CTC CTC AAA TAT CGA GCC AGG GAG CCA GTC GAA ATG CTG GAG AGT GTC ATC AAA AAT TAC AAG CCT GAG ATC TTC GGC AAA GCC TCT GAG TCC TTG TTT GGC ATT GAC GTG AAG GAA GCA GAC CCC ACC TAT GTC CTT GTC ACC TGC CTA GGT CTC TCC TAT CTG GGT GAT AAT CAG ATC ATG CCC AAG ACA GGC ATT GTC CTG GTC ATG ATT GCA ATG GAG GGC GGC GAG GAG GAA ATC TGG GAG GAG CTG AGT GTG ATG GAT GGG AGG GAG CAC AGT GCC TAT GGG GAG CCC CTC ACC CAA GAT TTG GTG CAG GAA AAG TAC CTG AGG TGC CGG ACA GTG ATC CCG CAC GCT ATG AGT GTC CAA GGG CCC TCG CTG AAA CCA GCT ATG TGA S B E Ύ (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: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-2 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCCAGGAACC AGGCAGTGAG GCCTTGGTCT GAGTCAGTGC AGAGCAGAGG GGACGCAGAC AGTGCCAACA CTGAAGGTTT CACACCAAGG GCCCCACCCG CCCAGAACAA ATGGGACTCC GCCTCACCCT CCCTATTCTC AGTCCTGCAG CCTGAGCATG CTGTACCCTG AGGTGCCCTC CCACTTCCTC CTTCAGGTTC AGGCTGACAA GTAGGACCCG AGGCACTGGA GGAGCATTGA CTGTAAGTAA GCCTTTGTCA GAGCCTCCAA GGTTCAGTTC TAAGGCCTCA CACACGCTCC TTCTCTCCCC AGGCCTGTGG CCCAGCTCCT GCCCGCACTC CTGCCTGCTG CCCTGACCAG ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG CAG GCT CCT GCT ACT GAG GAG CAG CAG ACC GCT TCT ACT CTA GTG GAA GTT ACC CTG GGG GAG GTG GAC TCA CCG AGT CCT CCC CAC AGT CCT CAG GGA TTC TCG ACT ACC ATC AAC TAC ACT CTT TGG AGA GAG GGC TCC AGC AAC CAA GAA GAG GAG GGG CCA CCC GAC CTG GAG TCC GAG TTC CAA GCA GCA ATC ATG GTT GAG TTG GTT CAT TTT CTG CTC CTC AAG AGG GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGA AAT TGC CAG GAC TTC TTT CCC GTG ATC TTC TCC GAG TAC TTG CAG CTG GTC TTT GGC ATC GAG GTG GTC CCC ATC AGC CAC TTG TAC ATC CTT GTC GGC CTC TCC TAC GAT GGC CTG CTG GGC GAC AAT CCC AAG ACA GGC CTC CTG ATA ATC GTC CTG GCC ATA GAG GGC GAC TGT GCC CCT GAG GAG AAA ATC CTG AGT ATG TTG GAG GTG TTT GAG GGG AGG GAG TTC GCA CAT CCC AGG AAG CTG CTC ATG CAA GAT GAA AAC TAC CTG GAG TAC CGG CAG GTG CCC GGC GCA TGC TAC GAG TTC CTG TGG GGT CCA AGG GCC ACC AGC TAT GTG AAA GTC CTG CAC CAT ACA CTA GGA GAA CCT CAC ATT TCC TAC CCA CCC CTG CAT TTG AGA GAG GGA GAA GAG TGA GTCTCAGCAC ATGTTGCAGC CAGGGCCAGT GGGAGGGGGT GCACCTTCCA GGGCCCCATC CATTAGCTTC CACTGCCTCG GGCCCATTCC TGCCTCTTTG AAGAGAGCAG TCAGCATTCT TTTCTGTTCT GTTGGATGAC TTTGAGATTT ATCTTTCTTT TTGTTCAAAT GTTCCTTTTA ACAAATGGTT GGATGAACTT GTTTATGAAT GACAGTAGTC ACACATAGTG CTGTTTATAT TAAGAGTCCT GTTTTTTATT CAGATTGGGA AATCCATTCC TTGTCACATA ATAACAGCAG TGGAATATGT ATTTGCCTAT AATTAGCAGT AAAATACATG ATACAAGGAA CTCAAAAGAT TGCCTTATAC CTCAGTCTAT TATGTAAAAT TAAAAATATG TGCTTCTTTG AGAATGCAAA AGAAATTAAA TCTGAATAAA TCACTGGCTC ATTTCTTTAC CATTCACTCA GCATCTGCTC CCTGGTAGTA GTGGG SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 662 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-21 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGATCCCCAT GGATCCAGGA AGAATCCAGT TCCACCCCTG CTGTGAACCC 50 AGGGAAGTCA CGGGGCCGGA TGTGACGCCA CTGACTTGCG CGTTGGAGGT 100 CAGAGAACAG CGAGATTCTC GCCCTGAGCA ACGGCCTGAC GTCGGCGGAG 150 GGAAGCAGGC GCAGGCTCCG TGAGGAGGCA AGGTAAGATG CCGAGGGAGG 200 ACTGAGGCGG GCCTCACCCC AGACAGAGGG CCCCCAATAA TCCAGCGCTG 250 CCTCTGCTGC CAGGCCTGGA CCACCCTGCA GGGGAAGACT TCTCAGGCTC 300 AGTCGCCACC ACCTCACCCC GCCACCCCCC GCCGCTTTAA CCGCAGGGAA 350 CTCTGGTGTA AGAGCTTTGT GTGACCAGGG CAGGGCTGGT TAGAAGTGCT 400 CAGGGCCCAG ACTCAGCCAG GAATCAAGGT CAGGACCCCA AGAGGGGACT 450 GAGGGTAACC CCCCCGCACC CCCACCACCA TTCCCATCCC CCAACACCAA 500 CCCCACCCCC ATCCCCCAAC ACCAAACCCA CCACCATCGC TCAAACATCA 550 ACGGCACCCC CAAACCCCGA TTCCCATCCC CACCCATCCT GGCAGAATCG 600 GAGCTTTGCC CCTGCAATCA ACCCACGGAA GCTCCGGGAA TGGCGGCCAA 650 GCACGCGGAT CC 662 (2) INFORMATION FOR SEQUENCE ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1640 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: cDNA MAGE-3 ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCCGCGAGGG AAGCCGGCCC AGGCTCGGTG AGGAGGCAAG GACAGGCTGA CCTGGAGGAC CAGAGGCCCC CGGAGGAGCA AGATCTGCCA GTGGGTCTCC ATTGCCCAGC TCCTGCCCAC GTTGCCCTGA CCAGAGTCAT C ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCT ACT CTA GTT GAA GTC ACC CTG GGG GAG GTG GAG TCA CCA GAT CCT CCC CAG AGT CCT CAG GGA CTC CCC ACT ACC ATG AAC TAC CCT CTC TGG AGC GAG GAC TCC AGC AAC CAA GAA GAG GAG GGG CCA CCT GAC CTG GAG TCC GAG TTC CAA GCA GCA CTC GTG GCC GAG TTG GTT CAT TTT CTG CTC CTC AAG AGG GAG CCG GTC ACA AAG GCA GAA ATG CTG GGG GGA AAT TGG CAG TAT TTC TTT CCT GTG ATC TTC TCC AGT TCC TTG CAG CTG GTC TTT GGC ATC GAG GTG GAC CCC ATC GGC CAC TTG TAC ATC TTT GCC GGC CTC TCC TAC GAT GGC CTG CTG GGT GAC AAT CCC AAG GCA GGC CTC CTG ATA ATC GTC CTG GCC AGA GAG GGC GAC TGT GCC CCT GAG GAG AAA ATC CTG AGT GTG TTA GAG GTG TTT GAG GGG AGG GAA TTG GGG GAT CCC AAG AAG CTG CTC ACC CAA CAT GAA AAC TAC CTG GAG TAC CGG CAG GTC CCC GGC GCA TGT TAT GAA TTC CTG TGG GGT CCA AGG GCC ACC AGC TAT GTG AAA GTC CTG CAC CAT ATG GTA GGA GGA CCT CAC ATT TCC TAC CCA CCC CTG CAT TTG AGA GAG GGG GAA GAG TGA GTCTGAGCAC GAGTTGCAGC CAGGGCCAGT GGGAGGGGGT GCACCTTCCG GGGCCGCATC CCTTAGTTTC CACTGCCTCC GGCCCATTCT TCACTCTTTG AAGCGAGCAG TCAGCATTCT TTTCTGTTCT GTTGGATGAC TTTGAGATTA TTCTTTGTTT TTGTTCAAAT GTTCCTTTTA ACGGATGGTT GAATGAGCGT GTTTATGAAT GACAGTAGTC ACACATAGTG CTGTTTATAT TAAGAGTCTT GttTTTTACT CAAATTgGGA AATCCATTCC TTGTGACATA ATAATAGCAG TGGTAAAAGT ATTTGCTTAA GAATTAGCAA TAACATACAT GAGATAACTC AAGAAATCAA ATTCTTGCCT TGTACCTCAA TCTATTCTGT AAAATTAAAC ACCAGGATTT CCTTGACTTC TTTG SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 943 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-31 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGATCCTCCA CCCCAGTAGA GTGGGGACCT CACAGAGTCT GGCCAACCCT 50 CCTGACAGTT CTGGGAATCC GTGGCTGCGT TTGCTGTCTG CACATTGGGG 100 GCCCGTGGAT TCCTCTCCCA GGAATCAGGA GCTCCAGGAA CAAGGCAGTG 150 AGGACTTGGT CTGAGGCAGT GTCCTCAGGT CACAGAGTAG AGGGGgCTCA 200 GATAGTGCCA ACGGTGAAGG TTTGCCTTGG ATTCAAACCA AGGGCCCCAC 250 CTGCCCCAGA ACACATGGAC TCCAGAGCGC CTGGCCTCAC CCTCAATACT 300 TTCAGTCCTG CAGCCTCAGC ATGCGCTGGC CGGATGTACC CTGAGGTGCC 350 CTCTCACTTC CTCCTTCAGG TTCTGAGGGG ACAGGCTGAC CTGGAGGACC 400 AGAGGCCCCC GGAGGAGCAC TGAAGGAGAA GATCTGTAAG TAAGCCTTTG 450 TTAGAGCCTC CAAGGTTCCA TTCAGTACTC AGCTGAGGTC TCTCACATGC 500 TCCCTCTCTC CCCAGGCCAG TGGGTCTCCA TTGCCCAGCT CCTGCCCACA 550 CTCCCGCCTG TTGCCCTGAC CAGAGTCATC 580 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG CCT GAA GAA 622 GGC CTT GAG GCC CGA GGA GAg GCC CTG GGC CTG GTG GGT GCG 664 CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCC TCC TCT 706 TCT AGT GTA GTT GAA GTC ACC CTG GGG GAG GTG CCT GCT GCC 748 GAG TCA CCA GAT CCT CCC CAG AGT CCT CAG GGA GCC TCC AGC 790 CTC CCC ACT ACC ATG AAC TAC CCT CTC TGG AGC CAA TCC TAT 832 GAG GAC TCC AGC AAC CAA GAA GAG GAG GGG CCA AGC ACC TTC 874 CCT GAC CTG GAG TCT GAG TTC CAA GCA GCA CTC AGT AGG AAG 916 GTG GCC AAG TTG GTT CAT TTT CTG CTC 943 ~ T3 (2) INFORMATION FOR SEQUENCE ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2531 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATUR : (A) NAME/KEY: MAGE-4 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13! GGATCCAGGC CCTGCCTGGA GAAATGTGAG GGCCCTGAGT GGGATCATCC ACTCCATGAG AGTGGGGACC TCACAGAGTC TCTTGATGGC ACTGAGGGAC CGGGGCTGTG CTTACAGTCT GGCCCATGGA TTCCTCTCCT AGGAGCTCCA GGAACAAGGC TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATG GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC CCTGCAGAAT CGACCTCTGC TGGCCGGCTA TACCCTGAGG CTTCCTCCTT CAGGTTCTGA GCAGACAGGC CAACCGGAGA TGGAGGCCAC AGAGGAGCAC CAAGGAGAAG ATCTGTAAGT TAGAGCCTCT AAGATTTGGT TCTCAGCTGA GGTCTCTCAC TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC CCTGCTGCCC TGACCAGAGT CATC ATG TCT TCT GAG CAG AAG AGT CAG CAC TGC AAG GGC GTT GAG GCC CAA GAA GAG GCC CTG GGC CTG CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GCT GAG TCA GCA GGT CCT CCC CAG AGT CCT CAG GCC TTA CCC ACT ACC ATC AGC TTC ACT TGC TGG AAT GAG GGT TCC AGC AGC CAA GAA GAG GAG GGG TCG CCT GAC GCA GAG TCC TTG TTC CGA GAA GCA AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG ATC AAA AAT TAC AAG CGC TGC TTT CCT GTG ATC GCC TCC GAG TCC CTG AAG ATG ATC TTT GGC ATT GAA GTG GAC CCC GCC AGC AAC ACC TAC ACC CTT CTG GGC CTT TCC TAT GAT GGC CTG CTG GGT AAT TTT CCC AAG ACA GGC CTT CTG ATA ATC GTC CTG GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA GAG CTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GTC TAT GGG GAG CCC AGG AAA CTG CTC ACC CAA CAG GAA AAC TAC CTG GAG TAC CGG CAG GTA CCC CCT GCG CGC TAT GAG TTC CTG TGG GGT CCA AGG GAA ACC AGC TAT GTG AAA GTC CTG GAG CAT GTG AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA GCATGAGTTG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT ATCTAACAGC CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTA TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT AGTCTTGTTT TTTATTCAGA TTGGGAAATC CGTTCTATTT SUBSTITUTE SHEET GGACATAATA ACAGCAGTGG AGTAAGTATT TAGAAGTGTG AATTCACCGT 1978 GAAATAGGTG AGATAAATTA AAAGATACTT AATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATAT GCATACCTGG ATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATT CTTTCTGTTA 2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC 2178 AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228 GGGTATTAAG AGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGAT GTAGGGGAAA AGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAAT TCCCTGTGTG GGGCCTTTTG GGCTTTGGGA 2378 AACTGCATTT TCTTCTGAGG GATCTGATTC TAATGAAGCT TGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAAAGTTGC 2478 TCTGAGCAGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT 2528 GGG 2531 (2) INFORMATION FOR SEQUENCE ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2531 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-41 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGATCCAGGC CCTGCCTGGA GAAATGTGAG GGCCCTGAGT GGGATCATCC ACTCCATGAG AGTGGGGACC TCACAGAGTC TCTTGATGGC ACTGAGGGAC CGGGGCTGTG CTTACAGTCT GGCCCATGGA TTCCTCTCCT AGGAGCTCCA GGAACAAGGC TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATG GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC CCTGCAGAAT CGACCTCTGC TGGCCGGCTA TACCCTGAGG CTTCCTCCTT CAGGTTCTGA GCAGACAGGC CAACCGGAGA TGGAGGCCAC AGAGGAGCAC CAAGGAGAAG ATCTGTAAGT TAGAGCCTCT AAGATTTGGT TCTCAGCTGA GGTCTCTCAC TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC CCTGCTGCCC TGAGCAGAGT CATC ATG TCT TCT GAG CAG AAG AGT CAG CAC TGC AAG GGC GTT GAG GCC CAA GAA GAG GCC CTG GGC CTG CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GCT GAG TCA GCA GGT CCT CCC CAG AGT CCT CAG GCC TTA CCC ACT ACC ATC AGC TTC ACT TGC TGG AAT GAG GGT TCC AGC AGC CAA GAA GAG GAG GGG TCG CCT GAC GCA GAG TCC TTG TTC CGA GAA GCA AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG ATC AAA AAT TAC AAG CGC TGC TTT CCT GTG ATC GCC TCC GAG TCC CTG AAG ATG ATC TTT GGC ATT GAA GTG GAC CCC ACC AGC AAC ACC TAC ACC CTT CTG GGC CTT TCC TAT GAT GGC CTG CTG GGT AAT TTT CCC AAG ACA GGC CTT CTG ATA ATC GTC CTG GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA GAG CTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GTC TAT GGG GAG CCC AGG AAA CTG CTC ACC CAA CAG GAA AAC TAC CTG GAG TAC CGG CAG GTA CCC CCT GCG CGC TAT GAG TTC CTG TGG GGT CCA AGG GAA ACC AGC TAT GTG AAA GTC CTG GAG CAT GTG AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA GCATGAGTTG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT ATCTAACAGC CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTA TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT AGTCTTGTTT TTTATTCAGA TTGGGAAATC CGTTCTATTT GGACATAATA ACAGCAGTGG AGTAAGTATT TAGAAGTGTG SUBSTITUTE SHEET 8_ GAAATAGGTG AGATAAATTA AAAGATACTT AATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATAT GCATACCTGG ATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATT CTTTCTGTTA 2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC 2178 AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228 GGGTATTAAG AGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGAT GTAGGGGAAA AGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAAT TCCCTGTGTG GGGCCTTTTG GGCTTTGGGA 2378 AACTCCATTT TCTTCTGAGG GATCTGATTC TAATGAAGCT TGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAAAGTTGC 2478 TCTGAGCGGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT 2528 GGG 2531 , (2) INFORMATION FOR SEQUENCE ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1068 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: cDNA MAGE-4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: G GGG CCA AGC ACC TCG CCT GAC GCA GAG TCC TTG TTC CGA 40 GAA GCA CTC AGT AAC AAG GTG GAT GAG TTG GCT CAT TTT CTG 82 CTC CGC AAG TAT CGA GCC AAG GAG CTG GTC ACA AAG GCA GAA 124 ATG CTG GAG AGA GTC ATC AAA AAT TAC AAG CGC TGC TTT CCT 166 GTG ATC TTC GGC AAA GCC TCC GAG TCC CTG AAG ATG ATC TTT 208 GGC ATT GAC GTG AAG GAA GTG GAC CCC GCC AGC AAC ACC TAC 250 ACC CTT GTC ACC TGC CTG GGC CTT TCC TAT GAT GGC CTG CTG 292 GGT AAT AAT CAG ATC TTT CCC AAG ACA GGC CTT CTG ATA ATC 334 GTC CTG GGC ACA ATT GCA ATG GAG GGC GAC AGC GCC TCT GAG 376 GAG GAA ATC TGG GAG GAG CTG GGT GTG ATG GGG GTG TAT GAT 418 GGG AGG GAG CAC ACT GTC TAT GGG GAG CCC AGG AAA CTG CTC 460 ACC CAA GAT TGG GTG CAG GAA AAC TAC CTG GAG TAC CGG CAG 502 GTA CCC GGC AGT AAT CCT GCG CGC TAT GAG TTC CTG TGG GGT 544 CCA AGG GCT CTG GCT GAA ACC AGC TAT GTG AAA GTC CTG GAG 586 CAT GTG GTC AGG GTC AAT GCA AGA GTT CGC ATT GCC TAC CCA 628 TCC CTG CGT GAA GCA GCT TTG TTA GAG GAG GAA GAG GGA GTC 670 TGAGCATGAG TTGCAGCCAG GGCTGTGGGG AAGGGGCAGG GCTGGGCCAG 720 TGCATCTAAC AGCCCTGTGC AGCAGCTTCC CTTGCCTCGT GTAACATGAG 770 GCCCATTCTT CACTCTGTTT GAAGAAAATA GTCAGTGTTC TTAGTAGTGG 820 GTTTCTATTT TGTTGGATGA CTTGGAGATT TATCTCTGTT TCCTTTTACA 870 ATTGTTGAAA TGTTCCTTTT AATGGATGGT TGAATTAACT TCAGCATCCA 920 AGTTTATGAA TCGTAGTTAA CGTATATTGC TGTTAATATA GTTTAGGAGT 970 AAGAGTCTTG TTTTTTATTC AGATTGGGAA ATCCGTTCTA TTTTGTGAAT 1020 TTGGGACATA ATAACAGCAG TGGAGTAAGT ATTTAGAAGT GTGAATTC 1068 SUBSTITUTE SHEE V ! (2) INFORMATION FOR SEQUENCE ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2226 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-5 gene ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGT GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC TCCTGTTAGC ACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GGCCCATGCA TTCCTCTTCC AGGAGCTCCA GGAAACAGAC TGGTCTGAGG CCGTGCCCTC AGGTCACAGA GCAGAGGAGA TAGTGCCAGC AGTGAACGTT TGCCTTGAAT GCACACTAAT GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACC TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT CTCACT_ΓTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GAAGCTCCAG AGGATCCCCA GGAGGCCCTA GAGGAGCACC ATCTGTAAGT AAGCCTTTGT TAGAGCCTCC AAGGTTCAGT GGCTTCTCAC ATGCTCCCTC TCTCTCCAGG CCAGTGGGTC AGCTCCTGCC CACACTCCTG CCTGTTGCGG TGACCAGAGT ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG CTC CTC TGG TCC CAG GCA CCC TGG GGG AGG TGC GGT CAC CAG GTC CTC TCA AGA GTC CTC AGG GAG TCC CCA CTG CCA TCG ATT TCA CTC TAT GGA GGC AGG GCT CCA GCA ACC AAG AAG AGG AGG GGC CAA CTG ACC CAG AGT CTG TGT TCC GAG CAG CAC TCA TGG CTG ACT TGA TTCATTTTCT GCTCCTCAAG TATTAAGTCA AGGAGCTGGT GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCT CTTCGGCAAA GCCTCCGAGT CCTTGCAGCT GGTCTTTGGC AGGAAGCGGA CCCCACCAGC AACACCTACA CCCTTGTCAC CTCCTATGAT GGCCTGCTGG TTGATAATAA TCAGATCATG GCCTCCTGAT AATCGTCTTG GGCATGATTG CAATGGAGGG CCTGAGGAGA AAATCTGGGA GGAGCTGAGT GTGATGAAGG GAGGGAGCAC AGTGTCTGTG GGGAGCCCAG GAAGCTGCTC TGGTGCAGGA AAACTACCTG GAGTACCGGC AGGTGCCCAG ATATGCTATG AGTTACTGTG GGGTCCAAGG GCACTCGCTG CTGGAGCACG TGGTCAGGGT CAATGCAAGA GTTCTCATTT CCTGCGTGAA GCAGCTTTGA GAGAGGAGGA AGAGGGAGTC CTGCAGCCAG GGCCACTGCG AGGGGGGCTG GGCCAGTGCA CTCCGTCCAG TAGTTTCCCC TGCCTTAATG TGACATGAGG TCTCTTTGAA GAGAGCAGTC AACATTCTTA GTAGTGGGTT TGGATGACTT TGAGATTTGT CTTTGTTTCC TTTTGGAATT TTCTTTTAAT GGGTGGTTGA ATGAACTTCA GCATTCAAAT CAGTAGTCAC ACATAGTGCT GTTTATATAG TTTAGGAGTA TTTTTATTCA GATTGGGAAA TCCATTCCAT TTTGTGAATT TACAGCAGTG GAATAAGTAT TCATTTAGAA ATGTGAATGA TGATGACATA AAGAAATTAA AAGATATTTA ATTCTTGCTT TATTCGGTAA AA__TTTTTT AAAAAATGTG CATACCTGGA TTCTTTGAGA ATGTAAGACA AATTAAATCT GAATAAATCA β TCACTGGCTC ATTTATTCTC TATGCACTGA GCATTTGCTC TGTGGAAGGC 2108 CCTGGGTTAA TAGTGGAGAT GCTAAGGTAA GCCAGACTCA CCCCTACCCA 2158 CAGGGTAGTA AAGTCTAGGA GCAGCAGTCA TATAATTAAG GTGGAGAGAT 2208 GCCCTCTAAG ATGTAGAG 2226 S B v b (2) INFORMATION FOR SEQUENCE ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2305 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-51 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGT GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC TCCTGTTAGC ACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GGCCCATGCA TTCCTCTTCC AGGAGCTCCA GGAAACAGAC TGGTCTGAGG CCGTGCCCTC AGGTCACAGA GCAGAGGAGA TAGTGCCAGC AGTGAACGTT TGCCTTGAAT GCACACTAAT GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACC TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT CTCACTTTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GAAGCTCCAG AGGATCCCCA GGAGGCCCTA GAGGAGCACC ATCTGTAAGT AAGCCTTTGT TAGAGCCTCC AAGGTTCAGT GGCTTCTCAC ATGCTCCCTC TCTCTCCAGG CCAGTGGGTC AGCTCCTGCC CACACTCCTG CCTGTTGCGG TGACCAGAGT ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG GGC CTT GAC ACC CAA GAA GAG CCC TGG GCC TGG AGG CTG CCA CTA CTG AGG AGC AGG AGG CTG TGT CTC CTC TGG TCC CAG GCA CCC TGG GGG AGG TGC GGT CAC CAG GTC CTC TCA AGA GTC CTC AGG GAG TCC CCA CTG CCA TCG ATT TCA CTC TAT GGA GGC AGG GCT CCA GCA ACC AAG AAG AGG AGG GGC CAA CTG ACC CAG AGT CTG TGT TCC GAG CAG CAC TCA TGG CTG ACT TGA TTCATTTTCT GCTCCTCAAG TATTAAGTCA AGGAGCCGGT GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCT CTTCGGCAAA GCCTCCGAGT CCTTGCAGCT GGTCTTTGGC AGGAAGCGGA CCCCACCAGC AACACCTACA CCCTTGTCAC CTCCTATGAT GGCCTGGTGG TTTAATCAGA TCATGCCCAA CTGATAATCG TCTTGGGCAT GATTGCAATG GAGGGCAAAT GGAGAAAATC TGGGAGGAGC TGGGTGTGAT GAAGGTGTAT AGCACAGTGT CTGTGGGGAG CCCAGGAAGC TGCTCACCCA CAGGAAAACT ACCTGGAGTA CCGCAGGTGC CCAGCAGTGA TATGAGTTAC TGTGGGGTCC AAGGGCACTC GCTGCTTGAA CACGTGGTCA GGGTCAATGC AAGAGTTCTC ATTTCCTACC TGAAGCAGCT TTGAGAGAGG AGGAAGAGGG AGTCTGAGCA CCAGGGCCAC TGCGAGGGGG GCTGGGCCAG TGCACCTTCC CCAGTAGTTT CCCCTGCCTT AATGTGACAT GAGGCCCATT TGAAGAGAGC AGTCAACATT CTTAGTAGTG GGTTTCTGTT ACTTTGAGAT TTGTCTTTGT TTCCTTTTGG AATTGTTCAA TAATGGGTGG TTGAATGAAC TTCAGCATTC AAATTTATGA TCACACATAG TGCTGTTTAT ATAGTTTAGG AGTAAGAGTC TTCAGATTGG GAAATCCATT CCATTTTGTG AATTGGGACA AGTGGAATAA GTATTCATTT AGAAATGTGA ATGAGCAGTA GATAAAGAAA TTAAAAGATA TTTAATTCTT GCCTTATACT GGTAAAATTT TTTTTTAAAA ATGTGCATAC CTGGATTTCC TTGGCTTCTT 2092 TGAGAATGTA AGACAAATTA AATCTGAATA AATCATTCTC CCTGTTCACT 2142 GGCTCATTTA TTCTCTATGC ACTGAGCATT TGCTCTGTGG AAGGCCCTGG 2192 GTTAATAGTG GAGATGCTAA GGTAAGCCAG ACTCACCCCT ACCCACAGGG 2242 TAGTAAAGTC TAGGAGCAGC AGTCATATAA TTAAGGTGGA GAGATGCCCT 2292 CTAAGATGTA GAG 2305 S B UTE SHE 86 (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: 18: TAT TTC TTT CCT GTG ATC TTC AGC AAA GCT TCC GAT TCC TTG 42 CAG CTG GTC TTT GGC ATC GAG CTG ATG GAA GTG GAC CCC ATC 84 GGC CAC GTG TAC ATC TTT GCC ACC TGC CTG GGC CTC TCC TAC 126 GAT GGC CTG CTG GGT GAC AAT CAG ATC ATG CCC AGG ACA GGC 168 TTC CTG ATA ATC ATC CTG GCC ATA ATC GCA AGA GAG GGC GAC 210 TGT GCC CCT GAG GAG 225 (2) INFORMATION FOR SEQUENCE ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1947 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-7 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TGAATGGACA ACAAGGGCCC CACACTCCCC AGAACACAAG GAGCCCAGCC TCACCTTCCC TACTGTCAGT CCTGCAGCCT TGGCCGGCTG TACCCTGAGG TGCCCTCTCA CTTCCTCCTT GCGGACAGGC CGGCCAGGAG GTCAGAAGCC CCAGGAGGCC ACCGAAGGAG AAGATCTGTA AGTAGGCCTT TGTTAGGGCC GGTTCACAAA TGAGGCCCCT CACAAGCTCC TTCTCTCCCC GTTCCTCCCC ATCGCCCAGC TGCTGCCCGC ACTCCAGCCT CCAGAGTCAT CATGTCTTCT GAGCAGAGGA GTCAGCACTG GATGCCTTGA GGCCCAAGGA CAGGAGGCTC TGGGCCTGGT GCTCCCGCCA CCGAGGAGCA CGAGGCTGCC TCCTCCTTCA AGGCACCCTG GAGGAGGTGC CTGCTGCTGG GTCCCCCAGT GTCTCAGGGT TCCTCCTTTT CCCTGACCAT CAGCAACAAC GCCAATCCAG TGAGGGCACC AGCAGCCGGG AAGAGGAGGG TAGACACACC CCGCTCACCT GGCGTCCTTG TTCCA ATG GGA AGG TGG CTG AGT TGG TTC GCT TCC TGC ATC GAG TCA AGG AGC TGG TCA CAA AGG CAG AAA GTG TCA TCA AAA ATT ACA AGC ACT AGT TTC CTT GGC AAA GCC TCA GAG TGC ATG CAG GTG ATG TTT ATG AAG GAA GTG GAC CCC GCG GCC ACT CCT ACG CCT GCT TGG GCC TCT CCT ACA ATG GCC TGC TGG AGA GCA TGC CCG AGA CCG GCC TTC TGA TTATGGTCTT GACCATGATC TTAATGGAGG GCCACTGTGC GCAATCTGGG AAGCGTTGAG TGTAATGGTG TATGATGGGA TCTTTGGGCA GCTGAGGAAG CTGCTCACCC AAGATTGGGT TACCTGCAAT ACCGCCAGGT GCCCAGCAGT GATCCCCCGT CCTGTGGGGT CCAAGGGCCC TCATTGAAAC CAGCTATGTG AGTATGCAGC CAGGGTCAGT ACTAAAGAGA GCATTTCCTA CATGAAGAGG CTTTGGGAGA GGAGGAAGAG GGAGTCTGAG AGCCAGGGCC AGTGGGGCAG ATTGGGGGAG GGCCTGGGCA CACACATCCA CCACCTTCCC TGTCCTGTTA CATGAGGCCC CTGTGTTTGA AGAGAGCAGT CAATGTTCTC AGTAGCGGGG GTGTGAGGGA ATACAAGGTG GACCATCTCT CAGTTCCTGT GATTTGGAGG TTTATCTTTG TTTCCTTTTG CAGTCGTTCA TTAATGGATG GTGTAATGAA CTTCAACATT CATTTCATGT GCAGACTTAC TGTTTTTTAT ATAGTTAAAA GTAAGTGCAT TTATGTAAGA AAATCTATGT TATTTCTTGA ATTGGGACAA CAGAGGATTA AGTACCTTTT ATAATGTGAA AGAACAAAGC GTGAGATAAA GAAATAAAGA AATTAAATTG GCTGGGCACG CCTGTAATCC CAGCACTTTA GGAGGCAGAG GCACGGGGAT GGAGATCGAG ACCATTCTGG CTAACACAGT GAAACACCAT AATACAAAAC TTAGCCGGGC GTGGTGGCGG GTG SUBSTITUTE SHEET (2) INFORMATION FOR SEQUENCE ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1810 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-8 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GAGCTCCAGG AACCAGGCTG TGAGGTCTTG GTCTGAGGCA TCACAGAGCA TAAGAGGCCC AGGCAGTAGT AGCAGTCAAG GTTTCCCCTG TATGTATACC AGAGGCCCCT CTGGCATCAG ACCCCACAGT TCCTGGCCCT ACCAGCCCTT TTGTCAGTCC GCCTTTGCCA GGAGGCTGCA CCCTGAGATG CCCTCTCAAT GGTTCGCAGA GAACAGGCCA GCCAGGAGGT CAGGAGGCCC CTGAAGAAGA CCTGTAAGTA GACCTTTGTT AGGGCATCCA CCCAGCTGAG GCCTCTCACA CGCTTCCTCT CTCCCCAGGC CAATTGCCCA GCTCCGGCCC ACACTCTCCT GCTGCCCTGA C ATG CTT CTT GGG CAG AAG AGT CAG CGC TAC AAG GGC CTT CAG GCC CAA GGA GAG GCA CCA GGG CTT CAG ATT CCC ACA GCT GAG GAG CAG AAG GCT GCA TCT ACT CTG ATC ATG GGA ACC CTT GAG GAG GTG GGG TCA CCA AGT CCT CCC CAG AGT CCT GAG GGT TCC CTG ACT GTC ACC GAC AGC ACT CTG TGG AGC GAG GGT TCC AGC AGC AAT GAA GAG GAG GGG CCA CCG GAC CCA GCT CAC CTG GAG TCC CTG TTC CGG GAT GAG AAA GTG GCT GAG TTA GTT CGT TTC CTG TAT CAA ATT AAG GAG CCG GTC ACA AAG GCA GAA AGT GTC ATC AAA AAT TAC AAG AAC CAC TTT CCT AGC AAA GCC TCT GAG TGC ATG CAG GTG ATC TTT GTG AAG GAA GTG GAC CCT GCC GGC CAC TCC TAC ACC TGC CTG GGC CTC TCC TAT GAT GGC CTG CTG CAG AGT ACG CCC AAG ACC GGC CTC CTG ATA ATC ATG ATC TTA ATG GAG GGC AGC CGC GCC CCG GAG TGG GAA GCA TTG AGT GTG ATG GGG GCT GTA TGA TGGGAGGGAG CACAGTGTCT ATTGGAAGCT CAGGAAGCTG AGTGGGTGCA GGAGAACTAC CTGGAGTACC GCCAGGCGCC CCTGTGCGCT ACGAGTTCCT GTGGGGTCCA AGGGCCCTTG CTATGTGAAA GTCCTGGAGC ATGTGGTCAG GGTCAATGCA TTTCCTACCC ATCCCTGCAT GAAGAGGCTT TGGGAGAGGA TGAGCAGGAG TTGCAGCTAG GGCCAGTGGG GCAGGTTGTG GGCCAGTGCA CGTTCCAGGG CCACATCCAC CACTTTCCCT ATGAGGCCCA TTCTTCACTC TGTGTTTGAA GAGAGCAGTC GTAGTGGGGA GCATGTTGGG TGTGAGGGAA CACAGTGTGG AGTTCCTGTT CTATTGGGCG ATTTGGAGGT TTATCTTTGT AATTGTTCCA ATGTTCCTTC TAATGGATGG TGTAATGAAC ATTTTATGTA TGACAGTAGA CAGACTTACT GCTTTTTATA GTAAGAGTCT TGCTTTTCAT TTATACTGGG AAACCCATGT ATTC 9 (2) INFORMATION FOR SEQUENCE ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1412 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-9 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TCTGAGACAG TGTCCTCAGG TCGCAGAGCA GAGGAGACCC AGCAGTGAAG GTGAAGTGTT CACCCTGAAT GTGCACCAAG GCCCCAGCAC ACATGGGACC CCATAGCACC TGGCCCCATT TCACTCATAG AGCCTTGATC TCTGCAGGCT AGCTGCACGC TCTCACTTCC TCCCTCAGGT TCTCGGGACA GGCTAACCAG GCCCCAAGAG GCCCCAGAGC AGCACTGACG AAGACCTGTA TGTTAGAACC TCCAAGGTTC GGTTCTCAGC TGAAGTCTCT CTCTCTCCCC AGGCCTGTGG GTCTCCATCG CCCAGCTCCT CTGACTGCTG CCCTGACCAG AGTCATC ATG TCT CTC GAG CAG AGG AGT CCG CAC TGC AAG GAC CTT GAA GCC CAA GGA GAG GAC TTG GGC CTG CAG GAA CCC ACA GGC GAG GAG GAG GAG ACT ACC GAC AGC AAG GAG GAG GAG GTG TCT GCT GCT GGG CCT CCC CAG AGT CCT CAG GGA GGC GCT TCC TCC GTC TAC TAC ACT TTA TGG AGC CAA TTC GAT GAG AGT CAA GAA GAG GAA GAG CCA AGC TCC TCG GTC CAG CTG GAG TTC ATG TTC CAA GAA GCA CTG AAA GCT GAG TTG GTT CAT TTC CTG CTC CAC AAA TAT GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGC AAT TAC AAG CGC TAC TTT CCT GTG ATC TTC GGC GAG TTC ATG CAG GTG ATC TTT GGC ACT GAT GTG GAC CCC GCC GGC CAC TCC TAC ATC CTT GTC ACT CTC TCG TGC GAT AGC ATG CTG GGT GAT GGT CAT AAG GCC GCC CTC CTG ATC ATT GTC CTG GGT GTG AAA GAC AAC TGC GCC CCT GAA GAG GTT ATC TGG AGT GTG ATG GGG GTG TAT GTT GGG AAG GAG CAC GGG GAG CCC AGG AAG CTG CTC ACC CAA GAT TGG AAC TAC CTG GAG TAC CGG CAG GTG CCC GGC AGT CAC TAC GAG TTC CTG TGG GGT TCC AAG GCC CAC AGC TAT GAG AAG GTC ATA AAT TAT TTG GTC ATG AGA GAG CCC ATC TGC TAC CCA TCC CTT TAT GAA GGA GAG GAG CAA GAG GGA GTC TGA GCACCAGCCG CAGCCGGGGC CAAAGTTTGT GGGGTCA SUB TIT TE HE (2) INFORMATION FOR SEQUENCE ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 920 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-10 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ACCTGCTCCA GGACAAAGTG GACCCCACTG CATCAGCTCC ACCTACCCTA 50 CTGTCAGTCC TGGAGCCTTG GCCTCTGCCG GCTGCATCCT GAGGAGCCAT 100 CTCTCACTTC CTTCTTCAGG TTCTCAGGGG ACAGGGAGAG CAAGAGGTCA 150 AGAGCTGTGG GACACCACAG AGCAGCACTG AAGGAGAAGA CCTGTAAGTT 200 GGCCTTTGTT AGAACCTCCA GGGTGTGGTT CTCAGCTGTG GCCACTTACA 250 CCCTCCCTCT CTCCCCAGGC CTGTGGGTCC CCATCGCCCA AGTCCTGCCC 300 ACACTCCCAC CTGCTACCCT GATCAGAGTC ATC 333 ATG CCT CGA GCT CCA AAG CGT CAG CGC TGC ATG CCT GAA GAA 375 GAT CTT CAA TCC CAA AGT GAG ACA CAG GGC CTC GAG GGT GCA 417 CAG GCT CCC CTG GCT GTG GAG GAG GAT GCT TCA TCA TCC ACT 459 TCC ACC AGC TCC TCT TTT CCA TCC TCT TTT CCC TCC TCC TCC 501 TCT TCC TCC TCC TCC TCC TGC TAT CCT CTA ATA CCA AGC ACC 543 CCA GAG GAG GTT TCT GCT GAT GAT GAG ACA CCA AAT CCT CCC 585 CAG AGT GCT CAG ATA GCC TGC TCC TCC CCC TCG GTC GTT GCT 627 TCC CTT CCA TTA GAT CAA TCT GAT GAG GGC TCC AGC AGC CAA 669 AAG GAG GAG AGT CCA AGC ACC CTA CAG GTC CTG CCA GAC AGT 711 GAG TCT TTA CCC AGA AGT GAG ATA GAT GAA AAG GTG ACT GAT 753 TTG GTG CAG TTT CTG CTC TTC AAG TAT CAA ATG AAG GAG CCG 795 ATC ACA AAG GCA GAA ATA CTG GAG AGT GTC ATA AAA AAT TAT 837 GAA GAC CAC TTC CCT TTG TTG TTT AGT GAA GCC TCC GAG TGC 879 ATG CTG CTG GTC TTT GGC ATT GAT GTA AAG GAA GTG GAT CC 920 (2) INFORMATION FOR SEQUENCE ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1107 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: MAGE-11 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AGAGAACAGG CCAACCTGGA GGACAGGAGT CCCAGGAGAA CACTGGAGGA GAACAAGTGT AAGTAGGCCT TTGTTAGATT CATATCTCAT CTGAGTCTGT TCTCACGCTC CCTCTCTCCC GGCCCCATCA CCCAGATATT TCCCACAGTT CGGCCTGCTG AGTCATCATG CCTCTTGAGC AAAGAAGTCA GCACTGCAAG CCTTCAGGCC CAAGAAGAAG ACCTGGGCCT GGTGGGTGCA AAGCTGAGGA GCAGGAGGCT GCCTTCTTCT CCTCTACTCT ACTCTAGAGG AGTTGCCTGC TGCTGAGTCA CCAAGTCCTC TCAGGAAGAG TCCTTCTCTC CCACTGCCAT GGATGCCATC TATCTGATGA GGGCTCTGGC AGCCAAGAAA AGGAGGGGCC CCTGACCTGA TAGACCCTGA GTCCTTTTCC CAAGATATAC GATAATTGAT TTGGTTCATT TATTCTCCGC AAGTATCGAG GATCACAAAG GCAGAA ATG CTG GGG AGT GTC ATC AAA AAT TAT GAG GAC GAG ATA TTT AGG GAA GCC TCT GTA TGC ATG CAA GGC ATT GAT GTG AAG GAA GTG GAC CCC ACT AGC GTC CTT GTC ACC TCC CTC AAC CTC TCT TAT GAT TGT AAT GAG CAG AGC ATG CCC AAG TCT GGC CTC GTC CTG GGT GTA ATC TTC ATG GAG GGG AAC TGC GAG GTT ATG TGG GAA GTC CTG AGC ATT ATG GGG GGA AGG GAG CAC TTC CTC TTT GGG GAG CCC AAG ACC CAA AAT TGG GTG CAG GAA AAG TAC CTG GTG GTG CCC GGC ACT GAT CCT GCA TGC TAT GAG TTC CCA AGG GCC CAC GCT GAG ACC AGC AAG ATG AAA TAC ATA GCC AAT GCC AAT GGG AGG GAT CC SUBSTITUTE SHEET 9 . (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 CTCTACAGAC CTCTGTCTGT GTCTGGCACC CTAAGTGGCT ACAGGTTTCT GCCCCTGCAT GGAGCTTAAA TAGATCTTTC TATACCCCTG CATTGTAAGT TTAAGTGGCT TTATGTGGAT GCCCTTGTAT GCAGGCCTAA GTTTTTCTGT CTGCTTAACC AAGCTAGTGA AAGATCTAAC CCACTTTTGG AAGTCTGAAA ATGCAGTGGC CTAACAAGTT TTAATTTCTT CCACAGGGTT AGCTTGATCC ACGAGTTCAG AAGTCCTGGT ATGTTCCTAG ATG TTC TCC TGG AAA GCT TCA AAA GCC AGG TCT CCA AGG TAT TCT CTA CCT GGT AGT ACA GAG GTA TGT CAT TCT TAT CCT TCC AGA TTC CTG TCT GCC ACT TCA GCC CTG AGC ACA GTC AAC ATG CCT AGG AGT AAG ACC CGC TCC CGT GCA AAA CGA CAG CAG GAG GTT CCA GTA GTT CAG CCC ACT GCA GAG GAA TCT CCT GTT GAC CAG AGT GCT GGG TCC AGC TTC TCT GCT CCT CAG GGT GTG AAA ACC CCT GGA TCT GGT GTA TCC TGC ACA GGC TCT GGT ATA GGT GGT GCT GTC CTG CCT GAT ACA AAA AGT TCA GAT GGC GGG ACT TCC ATT CAG CAC ACA CTG AAA GAT CCT AAG GCT AGT GTG CTG ATA GAA TTC CTG CTA GAT ATG AAA GAA GCA GTT ACA AGG AGT GAA ATG CTG AAC AAG AAG TAT AAG GAG CAA TTC CCT GAG ATC ACT TCT GCA CGC CTA GAA TTA GTC TTT GGT CTT GAA ATT GAT CCC AGC ACT CAT TCC TAT TTG CTG CTG GGT CTT TCC ACT GAG GGA AGT TTG AGT AGT TTG CCT AGG ACA GGT CTC CTA ATG TCT GTC CTA TTC ATG AAG GGT AAC CGT GCC ACT GAG CAA GAG TTT CTG CAT GGA GTG GGG GTA TAT GCT GGG AAG ATC TTT GGC GAG CCT GAG GAG TTT ATA AGA GAT GAA AAT TAC CTG GAG TAC CGC CAG GTA CCT GGC CCA AGC TAT GAG TTC CTG TGG GGA CCC AGA GCC ACA ACC AAG ATG AAA GTC CTG GAA GTT TTA GCT GGC ACA GTC CCT AGT GCC TTC CCT AAT CTC TAC CTT AGA GAT CAG GCA GGA GGG GTG CCA AGA AGG GGC AAG GGT GTT CAT TCC AAG GCC CCA TCC CAA AAC ATG TAG TTGAGTCTGT TCTGTTGTGT TTGAAAAACA GTCAGGCTCC AGAGTTCATA GCCTACCAGA ACCAACATGC ATCCATTCTT ACATTAGTAG AATGGAGGCT ATTTTTGTTA CTTTTCAAAT CTAAACAGTG CTTTTTGCCA TGCTTCTTGT TAACTGCATA TGTCACTTGT CAGATTAGGA CTTGTTTTGT TATTTGCAAC ACATTATTTT GTTTTTACTA AAACATTGTG TAACATTGCA TTGGAGAAGG 1837 GATTGTCATG GCAATGTGAT ATCATACAGT GGTGAAACAA CAGTGAAGTG 1887 GGAAAGTTTA TATTGTTAAT TTTGAAAATT TTATGAGTGT GATTGCTGTA 1937 TACTTTTTTC TTTTTTGTAT AATGCTAAGT GAAATAAAGT TGGATTTGAT 1987 GACTTTACTC AAATTCATTA GAAAGTAAAT CGTAAAACTC TATTACTTTA 2037 TTATTTTCTT CAATTATGAA TTAAGCATTG GTTATCTGGA AGTTTCTCCA 2087 GTAGCACAGG ATCTAGTATG AAATGTATCT AGTATAGGCA CTGACAGTGA 2137 GTTATCAGAG TCT 2150 SUBSTITUTE SHEET g (2) INFORMATION FOR SEQUENCE ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2099 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: (A) NAME/KEY: smage-II (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 37 (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 5 SUB TIT TE SHEET . _ Claims ; 1. Isolated nucleic acid molecule which codes for a tumor rejection antigen precursor or is complementary to a nucleic acid molecule which codes for a tumor rejection antigen precursor. 2. The isolated nucleic acid molecule of claim 1, wherein said molecule codes for a tumor rejection antigen precursor. 3. Isolated nucleic acid molecule of claim 1, wherein said molecule codes for a human tumor rejection antigen precursor. 4. The isolated nucleic acid molecule of claim 1, wherein said molecule is complementary to a nucleic acid molecule which codes for tumor rejection antigen precursor. 5. The isolated nucleic acid molecule of claim 1, wherein said molecule is DNA. 6. The isolated nucleic acid molecule of claim 1, wherein said molecule is RNA. 7. The isolated nucleic acid molecule of claim 1, wherein said molecule is a gene.

Claims: 8. The isolated nucleic acid molecule of claim 5, wherein said DNA is genomic DNA. 9. The isolated nucleic acid molecule of claim 5, wherein said DNA is cDNA. 10. The isolated nucleic acid molecule of claim 6, wherein said RNA is mRNA. 11. The isolated nucleic acid molecule of claim 4, wherein said molecule hybridizes to isolated nucleic acid which codes for tumor rejection antigen precursor under stringent conditions. 12. The isolated nucleic acid molecule of claim 1, wherein said molecule codes for a MAGE antigen precursor or is complementary to a molecule which codes for a MAGE antigen precursor. 13. The isolated nucleic acid molecule of claim 12, wherein said MAGE antigen precursor is selected from the group consisting of mage 1, mage 2, mage 3, mage 4, mage 5, mage 6, mage 7, mage 8, mage 9, mage 10, mage 11, smage I and smage II. 14. The isolated nucleic acid molecule of claim 12, wherein said molecule codes for a MAGE antigen precursor. tec 15. The isolated nucleic acid molecule of claim 12, wherein said molecule is complementary to a molecule which codes for a MAGE antigen precursor. 16. The isolated nucleic acid molecule of claim 12, wherein said molecule is DNA. 17. The isolated nucleic acid molecule of claim 12, wherein said molecule is RNA. 18. The isolated nucleic acid molecule of claim 12, wherein said molecule is a gene. 19. The isolated nucleic acid molecule of claim 16, wherein said DNA is genomic DNA. 20. The isolated nucleic acid molecule of claim 16, wherein said DNA is cDNA. 21. The isolated nucleic acid molecule of claim 17, wherein said RNA is mRNA. 22. The isolated nucleic acid molecule of claim 12, comprising a nucleotide sequence set forth in figure 9. 23. The isolated nucleic acid molecule of claim 15, wherein said molecule hybridizes to a molecule which codes for a MAGE antigen precursor under stringent conditions. 24. Isolated nucleic acid molecule of claim 1, coding for a tumor rejection antigen precursor for mastocytoma. 25. Isolated nucleic acid molecule of claim 1, coding for tumor rejection antigen precursor PIA. 26. Isolated nucleic acid molecule of claim __. , having the nucleotide sequence of figure 5. 27. Biologically pure culture of a cell line transfected with the nucleic acid sequence of claim 2. 28. Biologically pure culture of a cell line transfected with the nucleic acid sequence of claim 12. 29. Biologically pure culture of a cell line transfected with the nucleic acid sequence of claim 22. 30. Biologically pure culture of a cell line of claim 27, selected from the group consisting of P1A.T2 and P1A.TC3.1. l t 31. Biologically pure culture of a highly transfectable cell line derived from a parent cell line which expresses at least one P815 tumor antigen, wherein said highly transfectable cell line does not express any of P815 tumor antigens A, B and C. 32. Biologically pure cell line of claim 31, comprising cell line PO.HTR. 33. Biologically pure culture of a cell line of claim 27, wherein said tumor rejection antigen precursor is a human tumor antigen precursor. 34. Biologically pure culture of a cell line of claim 33, wherein said human tumor antigen precursor is found in melanoma cells. /o3 35. Biologically pure cell line of claim 34, said tumor rejection antigen precursor is mage-1 and said isolated DNA has nucleic acid sequence: 1 10 I 20 I 30 I 40 I 50 ' I CO 2 GSλTCCXSaC CCTGCGASSΛ _A -λ7AiAΛG ' C6CCC7& G7 GλSλλCAGλG GG3G7CA7CC £C f_ ACrff- TOλS AG7<__OSλ7G TCACACAC7C C-GCCCACCC TCC73.TAGC AC.Oλi__._._ 120 221 CA&GOC7G7G CTrftCCWTCT ©CACCC7GAG 4WCCCG7SOλ T7CC7C77CC T&OA&C7CCA leo Itl CSAACCAOG {.?αλ.s5ccτ rosrersksx CA37A7CC-C λGGTCASλSλ GCA_λ&_λ7G 240 241 CλCλG557ST GCCλS^iSTfi AλTCTTT&CC CTGλλTGCAC ACCϋ.»_:C CCACC7GCO. 300 301 CA&GA __.__.. AG5AC7CCAC A_λ.TCTS3C CTCACC7CCC ΪAC7G7CAGT CC7GTASAAT 3ε: 362 CGACC7C7GC G CCaGCTG IACCC7_AG7 ACCC7 7CAC T7CCTCC77C A&GR777CA5 420 421 (KSACATCCC -U-Cca.t_.es ACAGGATTCC CTGS G&CC* CA&AGOASCA CCAASOXG^A 4 O 4«ι GλTC Ciλλδ ixascctrTG TTASASTCTC CXAG ΓTCAG TΓCTCA&CXG AS5CC7C7CA 540 541 CACAC CCC? CTC7CCCCΛG ©CCTGTGWT • CT70.TT6CC CAI_ 7CC7GC CCλCACTCC7 £00 €01 ocw&cr&cc cτ&λCG_.Sλδ _CA.-CATC C _ T?__.___Λ_ A5&AG7C7SC ACTSCAAGCC no «£_ ΪGASSλλ&CC CrTβAGGCCC λλCAΛOλ&G C 7GSG TOS _G-C7G7ύr_Λ ©GC7GCCASC 720 -21 TCC7CCTCC7 CTCCTCTGG? CC7___CACC CTdGASOAGG T0CCCAC7GC T∞G7Cλλ_λ 7.0 til GλϊCCTCCCC ASλSTCCTCΛ OOOλGCCTCC CCCTT7CCCA CIACCA7CAA C77CAC7C0A S40 • 41 CASλ∞CAλC CCAC70AS5G T7CCAOCA(S CG7GλλC_.∞ Λ&∞GCGAΛG CACC7CT7G7 SCO jci __?ccτffθλe? ccTTCTxccδ AGCASΓAATC __C7ΛA5λλG T&σ raArr? GGT7M TΓ $£1 CTG 7CC7CA J__7A7C_ASC CA∞OASCCA G7CACAAΛSG Cλ(_»Aλ7(3 T &_AGAGTG-C 202 2021 λTCλλλλλTT λ_ΛλS=λ?7G T777CCTGAG ATCTTCGGCA XA5CC7 T(__ 07CC77GCAG 106 lot: CTGCTCTΓΓG GO.TTG_CGT &U. SXAG=A GλCCCCACCG GCCAC7CC7A 7G7CCTTS7C 114 1343 ACrrCCCtAG G7CTCΪCC7A TOA7GSCC7G CTGG 7GA7A ATCAS__7CA7 OCCCAAG Λ 120 1201 c&c?τceraλ T_ATTGTCCT GG CA?GATT GCAΛ79SA55 GCGGCC-7SC TCCTOAOOAG 22 C 12(1 Cλλλ7CT&SS λ&SλθCTOλδ TGTC_λTGSλS C7C7λ7Gλ7G OSA&GGXGCA CA07OCC7A7132 2321 ©GGGAG CCΛ COAAG TOTT CACCCAASλ? Ϊ7GG7GCA&G ΛλλΛG?ACC7 CGΛG7ACG& 23E 23 β AGGTGCCWA CAG7SA7CCC GCACGCTATG AG77CC7G7G e&S7CCAΛ9G OCCC7CG TG 144 2441 JAACCAG IA 7870AA_-G?C CT70AG7A7G T0λ7CAλ&G7 CAS SGAλOλ C77CQC7777 250 2S03 TCT7CCCA7C CCTGCGTGAA GCAGCTTCGλ Cλ5ΛS_ΛS_. JLSλSSSAG7C TOAGCATGAG 15 C 15.1 TTOCλSCCλλ G&C-A-7&-G AΦO∞OλCTG GGCCAOTGCA CC77CCA65G CC&CS7CC1G 1C2 1423 CAGC7TCCCC TOCC7C07G7 GλCA7GAGGC CCAT7CT CA TCTOAAGAG λtS ∞7__K57 €B 1«1 GTTCTCASTA CTA&β?7-C7 CT7C7A7 GG OTOAc rβαλ CAT tATcrr τc__'C C--' 17 2741 *fi__U.«STT CAAA7G7777 *7777AλS* ».TGGTTGA_.Γ CAACT7CAG AT CAλOTTΪ It C 2ID1 __7CAA7G___λ GCAGTCACAC AS77C7G7GT __Tλ7λGT77A AG GTAAOλG TC77C7CT_ I .O ll€l ΪATTCACAT7 OQgXλXTCC* TT__AT_T7G Oλ TTGSαλ ZAATAACAGC AGT&3Aλ7Aλ 3520 3.521 07AertλSXA XTGTOAλλλ-. TGΛ&-XG7AA JLλ7AB_7fiA6 _L__ A&λAC7 AAASλλλTTA 1$£0 2. «: λ___ 7A_.rc AA _C:I\_ C TTATACCTCA CTCIλTTCTG tAAAArT777 KλλSλTΛ.?Λ? 2040 2043 ' eCATACCTdS λ777CCTTO0 CrTCT7T__AG jαrsuu-SAG a__.τ_λλλ?c τfiλAtϋϋ__λ 2100 2102 JLTTCTΪCCT. _TCACT©SC_ CTTTΪCTTCT CCATSCλCTβ J__ λTCTG 7 T77TGOAA__- 22 €0 22(1 CC TGGSTΣλ CTAGTβSλOλ føCΣλλβGTλ J.9C£AOACTC AZλCCCACCC ___ASG57CG72220 2221 ASλff77λGG AG TGASTC J__S7λλ7CSλ βGTG&CUGΛ TGTCCTC7A-. ASATGTλSGG 22 tO 2213 JUλλffTfiASA CA&GGβϊβλβ OGTSTGϋSG TCCe&TTS G AOTOarSGJlG TCTCλλTGCC 2340 2)42 CTOAOCtβOG CCλTTTTGdG CTTrSSSAAA CTGCAOTtCC *-CTGG∞αλ O T&A77ff7λ 2400 2401 ATβATCTTβG 0-Oβλ?CC 2411 I 10 I 20 1 20 I 40 1 50 I «e 36. The biologically pure culture of claim 27, wherein said cell line is transfected by a nucleic acid sequence coding for a cytokine. 37. The biologically pure culture of claim 36, wherein said cell line is further transfected by a nucleic acid sequence coding for an HLA molecule. 38. The biologically pure culture of claim 36, wherein said cytokine is an interleukin. 39. The biologically pure culture of claim 38, wherein said interleukin is IL-2. 40. The biologically pure culture of claim 38, wherein said interleukin is IL-4. 41. The biologically pure culture of claim 27, wherein said cell line is transfected by a nucleic acid sequence which codes for an MHC molecule or an HLA molecule. 42. The biologically pure culture of claim 27, wherein said cell line expresses an MHC or HLA molecule which presents a tumor rejection antigen derived from a tumor rejection antigen precursor (TRAP) , wherein said TRAP is coded for by a nucleic acid sequence transfected into said cell line. 43. The biologically pure culture of claim 27, wherein said culture is non-proliferative. 44. The biologically pure culture of claim 27, wherein said cell line is a fibroblast cell line. 45. Transfected bacteria containing the nucleic acid sequence of claim 2. 46. Mutated virus containing the nucleic acid sequence of claim 2. 47. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 2 operably linked to a promoter. 48. Expression vector useful in transfecting a cell comprising a nucleic acid sequence coding for a tumor rejection antigen operably linked to a promoter. 49. Expression vector of claim 47, wherein said promoter is a strong promoter. 50. Expression vector of claim 47, wherein said promoter is a differential promoter. fo6 51. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 7 operably linked to a promoter. 52. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 13 operably linked to a promoter. 53. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 14 operably linked to a promoter. 54. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 18 operably linked to a promoter. 55. Expression vector useful in transfecting a cell comprising the isolated nucleic acid molecule of claim 22 operably linked to a promoter. 56. The expression vector of claim 47, further comprising a nucleic acid molecule which codes for an MHC or HLA. 57. The expression vector of claim 47, further comprising a nucleic acid molecule which codes for a cytokine. 58. The expression vector of claim 57, wherein said cytokine is an interleukin. fe 59. The expression vector of claim 58, wherein said interleukin is IL-2. 60. The expression vector of claim 58, wherein said interleukin is IL-4. 61. The expression vector of claim 47, further comprising a bacterial or viral genome or portion thereof. 62. The expression vector of claim 61, wherein said viral genome vaccinia virus DNA and said bacterial genome or portion thereof in BCG DNA. 63. Expression system useful in transfecting a cell, comprising (i) a first vector containing a nucleic acid molecule which codes for a tumor rejection antigen precursor, and (ii) a second vector selected from the group consisting of (a) a vector containing a nucleic acid molecule which codes for an MHC or HLA molecule which presents a tumor rejection antigen derived from said tumor rejection antigen precursor, and (b) a vector containing a nucleic acid sequence which codes for an interleukin. 64. Isolated tumor rejection antigen precursor. 65. Isolated human tumor rejection antigen precursor. 66. Isolated tumor rejection antigen precursor of claim 65, wherein said precursor is mage-1. 67. Isolated tumor rejection antigen precursor of claim 65, wherein said precursor is a precursor for antigen F. 68. Isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 2. 69. Isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 12. 70. Isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 13. 71. Isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 22. 72. Isolated tumor rejection antigen. 73. Isolated human tumor rejection antigen. 74. Isolated tumor rejection antigen of claim 72 having amino acid sequence of SEQ ID NO: 4. 75. Isolated tumor rejection antigen of claim 72, wherein said tumor rejection antigen is antigen E. lo . 76. Isolated tumor rejection antigen of claim 72, wherein said tumor rejection antigen is antigen F. 77. Vaccine useful in treating a subject afflicted with a cancerous condition comprising a tumor rejection antigen precursor which provokes an immune response when administered to a subject. 78. Vaccine useful in treating a subject afflicted with a cancerous condition comprising a peptide fragment derived from a tumor rejection antigen precursor, wherein said fragment is larger than the tumor rejection antigen derived from said tumor rejection antigen precursor and smaller than said tumor rejection antigen precursor and which provokes an immune response when administered to a subject. 79. Vaccine of claim 77, wherein said TRAP is a human TRAP. 80. Vaccine of claim 77 wherein said precursor is mage- 1. 81. Vaccine of claim 79, wherein said precursor is antigen F precursor. MO 82. Vaccine useful in treating a patient with a cancer comprising a tumor rejection antigen of claim 72 which provokes an immune response when administered to a subject. 83. Vaccine of claim 82, wherein said tumor rejection antigen has amino acid sequence of SEQ ID NO: 4. 84. The vaccine of claim 81, wherein said tumor rejection antigen is antigen E. 85. The vaccine of claim 81, wherein said tumor rejection antigen is antigen F. 86. The vaccine of claim 77, wherein said tumor rejection antigen precursor is the expression product of an expression vector containing a viral genome or portion thereof. 87. Vaccine useful in treating a patient with a cancer comprising the transfected bacterial of claim 45 and a pharmaceutically acceptable adjuvant. 88. Vaccine useful in treating a cancerous condition comprising the mutated virus of claim 46, and a pharmacologically acceptable adjuvant. 89. Vaccine useful in treating a subject afflicted with a cancerous condition comprising a complex of a tumor rejection antigen and an HLA molecule. 90. Isolated peptide useful in treating a subject afflicted with a cancerous condition, said peptide having the amino acid of SEQ ID NO: 26. 91. Vaccine useful in treating a subject afflicted with a cancerous condition comprising the isolated cell line of claim 27 and a pharmacologically acceptable adjuvant. 92. Vaccine useful in treating a subject afflicted with a cancerous condition comprising the isolated cell line of claim 37 and a pharmacologically acceptable adjuvant. 93. Composition of matter useful in treating a cancerous condition comprising a non proliferative cell line having expressed on its surface a tumor rejection antigen precursor specific for a tumor characteristic of said cancerous condition, and a pharmaceutically acceptable carrier. 94. Composition of matter of claim 93, wherein said cell line is a human cell line. 0356 ,, χ C 9 95. Composition of matter of claim 93, wherein said pharmaceutically acceptable carrier is a liposome. 96. Composition of matter useful in treating a cancerous condition comprising a non proliferative cell line having expressed on its surface a tumor rejection antigen specific for a tumor characteristic of said cancerous condition, and a pharma- ceutically acceptable carrier. 97. Composition of matter of claim 96, wherein said cell line is a human cell line. 98. Composition of matter of claim 96, wherein said pharma ceutically acceptable carrier is a liposome. 99. Composition of matter useful in treating a cancerous condition, comprising (i) a tumor rejection antigen or tumor rejection antigen precursor, (ii) an MHC or HLA molecule, and (iii) a pharmaceutically acceptable carrier. 100. Composition of matter of claim 99, wherein said pharmaceutically acceptable carrier is a liposome. 101. Antibody which specifically binds to a tumor rejection antigen precursor. M_ 102. Antibody of claim 101, wherein said antibody is a monoclonal antibody. 103. Antibody of claim 101, wherein said tumor rejection antigen precursor is mage-1. 104. Antibody of claim 103, wherein said antibody is a monoclonal antibody. 105. Antibody of claim 101, wherein said tumor rejection antigen precursor is antigen F precursor. 106. Antibody of claim 105, wherein said antibody is a monoclonal antibody. 107. Antibody of claim 101, wherein said tumor rejection antigen precursor is a MAGE precursor. 108. Antibody of claim 107, wherein said antibody is a monoclonal antibody. 109. Antibody of claim 107, wherein said MAGE precursor is mage 1, mage 2, mage 3, mage 4, mage 5, mage 6, mage 7, mage 8, mage 9, mage 10, mage 11, smage I and smage II. 110. Antibody of claim 109, wherein said antibody is a monoclonal antibody. 11 111. Antibody which specifically binds to a tumor rejection antigen. 112. Antibody of claim 111, wherein said antibody is a monoclonal antibody. 113. Antibody of claim 111, wherein said tumor rejection antigen is that set forth in SEQ ID NO: 4. 114. Antibody of claim 113, wherein said antibody is a monoclonal antibody. 115. Antibody of claim 111, wherein said tumor rejection antigen is antigen E. 116. Antibody of claim 115, wherein said antibody is a monoclonal antibody. 117. Antibody of claim 111, wherein said tumor rejection antigen is antigen F. 118. Antibody of claim 117, wherein said antibody is a monoclonal antibody. 119. Antibody which specifically binds to a complex of (i) tumor rejection antigen and (ii) HLA molecule, but does not bind to (i) or (ii) alone. , 120. The antibody of claim 119, wherein said antibody is a monoclonal antibody. 121. Method for diagnosing a cancerous condition in a subject, comprising contacting a lymphocyte containing sample of said subject to a cell line transfected with a DNA sequence coding for a tumor rejection antigen precursor expressed by cells associated with said cancerous condition, and determining lysis of said transfected cell line by a cytotoxic T cell line specific for a tumor rejection antigen derived from said tumor rejection antigen precursor, said lysis being indicative of said cancerous condition. 122. Method of claim 121, wherein said tumor rejection antigen precursor is a MAGE antigen. 123. Method for determining regression, progression or onset of a cancerous condition comprising monitoring a sample from a patient with said cancerous condition for a parameter selected from the group consisting of (i) tumor rejection antigen precursor, (ii) tumor rejection antigen and (iii) cytolytic T cells specific for a tumor rejection antigen associated with said cancerous condition, wherein amount of said parameter is indicative of progression or regression or onset of said cancerous condition. 124. Method of claim 123, wherein said sample is a body fluid. 125. Method of claim 123, wherein said sample is a tissue. 126. Method of claim 123, comprising contacting said sample with an antibody which specifically binds with said tumor rejection antigen or tumor rejection antigen precursor. 127. Method of claim 126, wherein said antibody is labelled with a radioactive label or an enzyme. 128. Method of claim 126, wherein said antibody is a monoclonal antibody. 129. Method of claim 123, comprising amplifying RNA which codes for said tumor rejection antigen precursor. 130. Method of claim 129, wherein said amplifying comprises carrying out polymerase chain reaction. 131. Method of claim 123, comprising contacting said sample with a nucleic acid molecule which specifically hybridizes to a nucleic acid molecule which codes for or expresses said tumor rejection antigen precursor. 132. Method of claim 123, comprising assaying said sample for shed tumor rejection antigen. .. . . ' 1 T 133. Method for diagnosing a cancerous condition comprising assaying a sample taken from a subject for a cytolytic T cell specific for a tumor rejection antigen, presence of said cytolytic T cell being indicative of said cancerous condition. 134. Method for treating a subject afflicted with a cancerous condition, comprising: (i) removing a lymphocyte containing sample from said subject, (ii) contacting the lymphocyte containing sample to a cell line transfected with a gene coding for and expressing a gene for a tumor rejection antigen precursor expressed by cancer cells associated with said conditions, under conditions favoring production of cytotoxic T cells against a tumor rejection antigen derived from said tumor rejection antigen precursor, and (iii) introducing said cytotoxic T cells to said subject in an amount sufficient to lyse said cells. 135. Method for treating a subject afflicted with a cancerous condition, comprising: (i) identifying a MAGE gene expressed by cancer cells associated with said condition; (ii) identifying an HLA molecule which presents a portion of an expression product of said MAGE gene; (iii) transfecting a host cell having the same HLA molecule as identified in (ii) with said MAGE gene; (iv) culturing said transfected cells to express said MAGE-gene, and; (v) introducing an amount of said cells to said subject sufficient to provoke an immune response against said tumor. 136. Method of claim 135, wherein said immune response comprises a B-cell response. 137. Method of claim 135, wherein said immune response is a T-cell response. 138. Method of claim 136, wherein said B cell response comprises production of antibodies specific to said tumor rejection antigen or tumor rejection antigen precursor. 139. Method of claim 137, wherein said T-cell response comprises generation of cytolytic T-cells specific for cells presenting said tumor rejection antigen. 140. Method of claim 139, further comprising treating said cells to render them non-proliferative. ( 141. Method for treating a subject with a cancerous condition, comprising: (i) identifying a MAGE gene expressed by said tumor; (ii) transfecting a host cell having the same HLA type as said patient with said MAGE gene; (iii) culturing said transfected cells to express said MAGE gene, and; (iv) introducing an amount of said cells to said subject sufficient to provoke an immune response against said tumor. 142. Method of claim 141, further comprising treating said cells to render them non proliferative. 143. Method for treating a subject with a cancerous condition, comprising administering to said subject an amount of a cell transfected with (i) a nucleic acid sequence which codes for a tumor rejection antigen precursor (TRAP) and (ii) a nucleic acid sequence which codes for an MHC or HLA molecule which presents a tumor rejection antigen derived from said TRAP, wherein said tumor rejection antigen is presented by cells associated with said cancerous condition, sufficient to alleviate said cancerous condition. 144. Method of claim 143, further comprising treating said cell to render it non-proliferative. 145. Method for preparing a biological material useful in treating a subject afflicted with a cancerous condition, comprising: (i) transfecting a host cell with a nucleic acid molecule which codes for or expresses a tumor rejection antigen precursor; (ii) transfecting said host cell with a nucleic acid molecule which codes for an HLA molecule which presents a tumor rejection antigen derived from said tumor rejection antigen precursor on a cell surface, and; (iii) treating said host cells under conditions favoring expression of said nucleic acid molecules, and presentation of said tumor rejection antigen by said human leukocyte antigen. 146. Method of claim 145, further comprising treating said host cells to render them non proliferative following presentation of said tumor rejection antigen. 147. Method of claim 146, further comprising transfecting said host cell with a nucleic acid molecule which codes for or expresses a cytokine. 148. Method of claim 146, wherein said cytokine is an interleukin. 149. Method of claim 146, wherein said human leukocyte antigen is HLA-Al. 150. Method of claim 148, wherein said interleukin is IL- 2. 151. Method of claim 146, wherein said interleukin is IL- 4. 152. Method for treating a subject afflicted with a cancerous condition comprising administering to said subject an amount of a reagent consisting essentially of non-proliferative cell having expressed on its surface a tumor rejection antigen characteristic of cancerous cells in an amount sufficient to elicit an immune response thereto. 153. Method for treating a subject afflicted with a cancerous condition comprising administering to said subject an antibody which specifically binds to a tumor rejection antigen expressed on a cancer cell associated with said condition, said antibody being coupled to an anticancer agent, in an amount sufficient to treat said cancerous condition. 154. Method for treating a subject afflicted with a cancerous condition comprising administering to said subject an antibody which specifically binds to a 111 tumor rejection antigen precursor expressed by a cancer cell associated with said condition, said antibody being coupled to an anticancer agent, in an amount sufficient to treat said cancerous condition. 155. Method for treating a subject afflicted with a cancerous condition comprising administering to said subject a biological sample prepared in accordance with claim 142 in an amount sufficient to alleviate said cancerous condition. 156. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 77 in an amount sufficient to prevent onset of said cancerous condition in said subject. 157. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 78 in an amount sufficient to prevent onset of said cancerous condition in said subject. 158. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 82 in an amount sufficient to prevent onset of said cancerous condition in said subject. 159. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 86 in an amount sufficient to prevent onset of said cancerous condition in said subject. 160. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 87 in an amount sufficient to prevent onset of said cancerous condition in said subject. 161. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 88 in an amount sufficient to prevent onset of said cancerous condition in said subject. 162. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 89 in an amount sufficient to prevent onset of said cancerous condition in said subject. 163. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 89 in an amount sufficient to prevent onset of said cancerous condition in said subject. 164. Method for preventing onset of a cancerous condition in a subject comprising administering an amount of the vaccine of claim 90 in an amount sufficient to prevent onset of said cancerous condition in said subject. 165. Method for treating a subject afflicted with a cancerous condition, comprising: (i) identifying cells from said subject which express a tumor rejection antigen precursor and present a tumor rejection antigen derived from said precursor on their surface; (ii) isolating a sample of said cells; (iii) cultivating said cell, and; (iv) introducing said cells to said subject in an amount sufficient to provoke an immune response against said cells. 166. Method of claim 165, further comprising rendering said cells non proliferative, prior to introducing them to said subject. 167. Method for identifying a cytotoxic T cell useful in treating a subject afflicted with a cancerous condition, comprising: (i) identifying a tumor rejection antigen presented by cells associated with said cancerous condition derived from a tumor rejection antigen precursor expressed by said cells, prior to introducing them to said subject; (ii) contacting a cell presenting said antigen to a cytotoxic T cell, and; (iii) measuring a parameter selected from the group consisting of (i) proliferation of said cytotoxic T cell and (ii) release of a cytotoxic T cell produced factor, wherein increase in said parameter is indicative of said cancerous condition. 168. Method of claim 167, wherein said factor is tumor necrosis factor. 169. Method for following progress of a therapeutic regime designed to alleviate a cancerous condition, comprising: (a) assaying a sample from a subject to determine level of a parameter selected from the group consisting of (i) tumor rejection antigen, (ii) a cytolytic T cell specific for cells presenting said tumor rejection antigen, and (iii) an antibody which specifically binds to said tumor rejection antigen at a first time period; (b) assaying level of the parameter selected in (a) at a second period of time and comparing it to the level determined in (a) as a determination of effect of said therapeutic regime. 170. Method for diagnosing a cancerous condition comprising assaying a sample taken from a subject for expression of a TRAP molecule, and comparing levels of expression to a normal level, wherein variance there between is indicative of a cancerous condition. 171. Method of claim 164, comprising measuring expression via polymerase chain reaction. 172. Method of claim 123, comprising intradermally administering an amount of a tumor rejection antigen sufficient to generate a delayed type response in a subject.

Inventors: BOON THIERRY, CHOMEZ PATRICK, DE PLAEN ETIENNE, LURQUIN CHRISTOPHE, TRAVERSARI CATIA, VAN DEN EYNDE BENOIT, VAN DER BRUGGEN PIERRE, VAN PEL ALINE, BOON, THIERRY, CHOMEZ, PATRICK, DE PLAEN, ETIENNE, LURQUIN, CHRISTOPHE, TRAVERSARI, CATIA, VAN DEN EYNDE, BENOIT, VAN DER BRUGGEN, PIERRE, VAN PEL, ALINE
Applicants: LUDWIG INST CANCER RES, LUDWIG INSTITUTE FOR CANCER RESEARCH
Application year: 1992
Main classification: A61K35
IPC codes: A61K31, A61K35, A61K38, A61K39, A61K48, A61P35, C07K7, C07K14, C07K16, C12N1, C12N5, C12N7, C12N15, C12P21, C12Q1, C12R1, G01N33