AU664560B2 - Tumor rejection antigen precursors, tumor rejection antigens and uses thereof - Google Patents

Description

L- r OI'I DATE 30/12/92 APPLN. ID 21583/92 AOJP DATE 11/02/93 PCT NUMBER PCT/US92/04354 I l lli ll lll llAU9221 583i AU9221583 wc LI3 4s 4-1 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/20356 A61K 35/14, 39/00, 37/22 C07K 3/00, 13/00, 15/00 Al C07K 17/00, C12Q 1/68, 1/00 (43) International Publication Date: 26 November 1992 (26.11.92) C12 el4Q 15/00, C12N 1/20, 1/00 (21) International Application Number: PCT/US92/04354 (74) Agent: HANSON, Norman, Felfe Lynch, 805 Third Avenue, New York, NY 10022 (US).

(22) International Filing Date: 22 May 1992 (22.05.92) (81) Designated States: AT (European patent), AU, BB, BE (Eu- (30) Priority data: ropean patent), BF (OAPI patent), BG, BJ (OAPI pa- 705,702 23 May 1991 (23.05.91) US tent), BR, CA, CF (OAPI patent), CG (OAPI patent), 728,838 9 July 1991 (09.07.91) US CH (European patent), CI (OAPI patent), CM (OAPI 761f4Sl 23 September 1991 (23.09.91) US patent), CS, DE (European patent), DK (European pa- 807,043 12 December 1991 (12.12.91) US tent), ES (European patent), FI, FR (European patent), GA (OAPI patent), GB (European patent), GN (OAPI patent), GR (European patent), HU, IT (European pa- (71) Applicant (for all designated States except US): LUDWIG IN- tent), JP, KP, KR, LK, LU (European patent), MC (Eu- STITUTE FOR CANCER RESEARCH [US/US]; 1345 ropean patent), MG, ML (OAPI patent), MR (OAPI pa- Avenue of the Americas, New York, NY 10105 tent), MW, NL (European patent), NO, PL, RO, RU, SD, SE (European patent), SN (OAPI patent), TD (OA- (72) Inventors; and PI patent), TG (OAPI patent), US.

Inventors/Applicants (for US only) BOON, Thierry [BE/BE]; VAN DER BRUGGEN, Pierre [BE/BE]; VAN DEN EYNDE, Benoit [BE/BE]; VAN PEL, Aline [BE/BE]; Published DE PLAEN, Etienne [BE/BE]; LURQUIN, Christophe With international search report.

[BE/BE]; CHOMEZ, Patrick [BE/BE]; Avenue Hippoc- Before the expiration of the time limit for amending the claims rate 74, UCL 7459, B-1200 Brussels TRAVERSA- and to be republished in the event of the receipt of amendments.

RI, Catia [IT/IT]; Sesto S. Giovanni, 1-20099 Milano 664 (54) Title: TUMOR REJECTION ANTIGEN PRECURSORS, TUMOR REJECTION ANTIGENS AND USES THEREOF (57) 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.

p WO 92/20356 PCT/US92/04354 1 TUMOR REJECTION ANTIGEN PRECURSORS, TUMOR REJECTION ANTIGENS AND USES THEREOF Number 7,043, filed December 12, 1991, which is a continuation-in- t of Serial Number 764,364, filed September 23, 1991, whicf a continuation-in-part of Serial Number 728,838, filed July 1991, which is a continuation-in-part of Serial Number 705,702, iled May 10 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- Scalled tumor rejection antigens, and the expression of what will be referred to herein as "tumor rejection antigen precursors".

SBACKGROUND AND PRIOR ART S 20 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.

WO 92/20356 PCT/US92/04354 2 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-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced in vitro by chemical carcinogens, such as methylcholanthrene. The antigens j expressed by the tumors and which elicited the T-cell i 10 response were found to be different for each tumor. See Prehn, et al., 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. 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as "tumor specific transplantation antigens" or "TSTAs".

Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst.

53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types of tumor described 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).

WO 92/20356 PCT/US92/04354 3 The family of tum- antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp.

Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum- antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors "tum+" cells). When these tum cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus 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, Frost et al., Cancer Res. 43: 125 (1983).

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

The evidence in favor of this hypothesis includes the ability of "tum-" variants of tumors, those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum- cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory WO 92/20356 PCT/US92/04354 4 which permits them to resist subsequent challenge to the same tum" variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., 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.

i A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized i 20 by cytotoxic T cells, leading to lysis. This class of SIantigens will be referred to as "tumor rejection antigens" or "TRAs" hereafter. TRAs may or may not elicit antibody Sresponses. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro 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 I WO 92/20356 PCT/US92/04354 the cells presenting the antigen are lysed. Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysis is required for other types of antigens recognized by CTLs, including minor histocompatibility antigens, the male specific H-Y antigens, and a class of antigens, referred to as "tum-" antigens, and discussed herein.

A tumor exemplary of the subject matter described 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 methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum" variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast to tumor rejection antigens and this is a key distinction the tum- antigens are -r;ii ?ii-iii.- i WO 92/20356 PCT/US92/04354 6 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 and can be provoked to produce tumvariants. Since the tum- phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum- cell lines as compared to their tum+ parental lines, I and this difference can be exploited to locate the gene of interest in tum- cells. As a result, it was found that genes of tum" variants such as P91A, 35B and P198 differ I 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 tumantigen are presented by the Ld molecule for recognition i 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.

WO 92/20356 PCT/US92/04354 7 The gene is useful as a source for the isolated and purified tumor rejection antigen precursor and the TRA themselves, either of which can be used as an agent for treating the cancer for which the antigen is a "marker", as well as in various diagnostic and surveillance approaches to oncology, discussed infra. It is known, for example, that tum cells can be used to generate CTLs which lyse cells presenting different tum- antigens as well as tum cells. See, 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.

WO 92/20356 PCT/US92/04354 8 158: 240 (1983); H6rin et all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol 6: 839-853 (1988).

Stable cytotoxic T cell clones ("CTLs" hereafter) have been derived from MLTC responder cells, and these clones are specific for the tumor cells. See Mukherji et al., supra, 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 murine tumor rejection antigen precursors, have led to the isolation of nucleic acid sequences coding for tumor rejection antigen l precursors of TRAs presented on human tumors. It is now possible to isolate the nucleic acid sequences which code Sfor 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 I 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.

WO 92/20356 PCT/US92/04354 9 BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts detection of transfectants expressing antigen P815A.

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

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

Figure 4 shows Northern Blot analysis of expression of gene P1A.

Figure 5 sets forth the structure of gene P1A with its restriction sites.

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

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

1 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.

WO 92/20356 PCT/US92/04354 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 P1A.

I SEQ ID NO: 3 shows non coding DNA for P1A cDNA which is 3' to the coding region of SEQ ID NO: 2.

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

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

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

i 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.

I ~4 WO 92/20356 PCr/US92/04354

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SE Q

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

SEQ

ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO' cDNA for MAGE-3.

the gene for MAGE-31.

the gene for MAGE-4.

the gene for MAGE-41.

cDNA for MAGE-4.

cDNA for genomic DNA for MAGE-51.

cDNA for MAGE-6.

genomic DNA for MAGE-7.

genomic DNA for MAGE-8.

genomic DNA for MAGE-9.

genomic DNA for MAGE-lO.

genomic DNA for MAGE-li.

genomic DNA for smage-I.

genomic DNA for smage-II.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Many different "RAGE" genes have been identified, as will be seen from the sequences which follow the application. The protocols described in the following .~lrr i ~*Ra~ia -12examples were used to isolate these genes and cDNA sequences.

"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".

The acronym "smage" is used to described 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.

|I

Example 1 In order to identify and isolate the gene coding for antigen P815A, gene transfection was used. This approach requires both a source of the gene, and a recipient cell S. 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 recognised P815 tumor antigens. See Van Pel et ,a al., Molecular Genetics 11: 467-475 (1985). Thus, screening experiments were carried out to isolate cell lines which did not express the antigen and which nonetheless possessed P1.HTR's desirable qualities.

staffHahleoekeep/21583.92.vs 13.4 c 12a To do this, P1.HTR was screened with CTLs which were specific for each tumor antigens A, B, C and D. Such CTLs are described by Uyttenhove et al., J. Exp. Med. 157: 1040-1052 (1983).

4 4 .4

S

4

I'

.4, a, staff/ahleer4keepJ2l 583.92.vs 13.4 WO 92/20356 PCT/US92/04354 13 To carry out the selection, 10 cells of P1.HTR were mixed with 2-4xi0 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 0 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.

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

Additional selections with CTLs specific for antigen C then Iyielded 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 Snumber 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, antigens A, B, C and D, where the variants rn WO 92/20356 PCT/US92/04354 14 present none of antigens A, B and C. P1.HTR is a mastocytoma cell line, so it will be seen that the protocol enables the isolation of biologically pure mastocytoma cell lines which express none of P815 antigens A, B and C, but which are highly transfectable. Other tumor types may also be screened in this fashion to secure desired, biologically pure cell lines. The resulting cell lines should be at least as transfectable with foreign DNA as is P1.HTR, and should be selected so as to 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 i is incorporated by reference herein had shown the efficacy of using cosmid library transfection to recover genes coding for tum- antigens.

Selective plasmid and genomic DNA of P1.HTR were i prepared, following Wl1fel et al., Immunogenetics 26: 178- !i 187 (1987). The transfection procedure followed Corsaro et al., Somatic Cell Molec. Genet 7: 603-616 (1981), with some i 20 modification. Briefly, 60 Mg of cellular DNA and 3 ig 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 0.1 mM EDTA; and 310 ul 1M CaC1 2 WO 92/20356 PCT/US92/04354 The solution was added slowly, and under constant agitation to 1.25 ml of 50 mM Hepes, 280 mM NaCl, 1.5 mM Na 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 thisy fifteen groups of PO.HTR cells (5x10 6 per group were centrifuged for 10 minutes at 400 g. Supe:natants were removed, and pellets were resuspended directly into the medium containing the DNA precipitates. This mixture was incubated for 20 minutes at 37 0 C, after which it was added to an 80 cm 2 tissue culture flask containing 22.5 ml DMEM, supplemented with 10% fetal S calf serum. After 24 hours, medium was replaced. Forty- I eight hours after transfection, cells were collected and counted. Transfected cells were selected in mass culture i using culture medium supplemented with hygromycin B (350 Sug/ml). This treatment selected cells for hygromycin resistance.

IFor each group, two flasks were prepared, each I containing 8x10 6 cells in 40 ml of medium. In order to I 20 estimate the number of transfectants, 1x16 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 L WO 92/20356 PCT/US92/04354 16 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 4 cells and replicate plates were prepared by transferring 1/10 of the wells to microplates which were then incubated at 300C.

One day later, master plates were centrifuged, medium l removed, and 750 CTLs against P815 antigen A I were added to each well together with 106 irradiated syngeneic feeder spleen cells in CTL culture medium containing 40 U/ml recombinant human IL-2, and HAT medium to kill stimulator cells. Six days later, plates were 1 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 P1.HTR target cells (2x10 3 4x10 3 per well), and chromium release r^ WO 92/20356 PCT/US92/04354 17 was measured after 4 hours. Replicate microcultures corresponding to those showing high CTL activity were expanded and cloned by limited dilution in DMEM with FCS. Five days later, about 200 clones were collected and screened with the CTL.P1:5 cell line, described supra, in a visual lysis assay. See figure 1A for these results.

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

This activity was well above background, as shown in figure lB. This figure summarizes data wherein two groups of cells (groups and 400 and 300 microwells were j seeded with 30 hygromycin resistant transfected cells.

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

Duplicate microcultures corresponding to several Spositive 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.

i WO 92/20356 PCT/US92/04354 18 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 P1.HTR. To that end, this cell line was used as a source for the gene for the antigen precursor in the following experiments.

Prior work had shown that genes coding for tumantigens could be recovered directly from transfectants i 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 kb DNA fragments, following Grosveld et al., Gene 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 I reference. These cosmid arms had been obtained by cleavage with SmaI and treatment with calf intestinal phosphatase, followed by digestion with BamHI. Ligated DNA was packaged into lambda phage components, and titrated on E. coli ED 8767, following Grosveld et al., supra. Approximately 9x10 i i I *urr"R SWO 92/20356 PCT/US92/04354 19 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 0 C, diluted with 20 ml of Luria Bertani 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 2x10 8 cells/ml (OD 6 0 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 Using the twenty-one groups of cosmids alluded to supra, (60 ug) and 4 ug of pHMR272, described supra, groups of 5x106 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 I I -i th 1v WO 92/20356 PCT/US92/04354 11 transfectants. The transfectants, as with P1A.T, also showed expression of both antigen A and B. The pattern of expression of transfectant PlA.TC3.1 is shown in figure 2.

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

High frequencies of -transfection iwere observed, as described in Table 1, which follows: Table LTransfer of the expression of antigen PSISA by cos-nids ob-.2ined by direct packaging Transfeciant No. of cosrnids obtiainre' No. of transfectanis obiaintd w*%ith b)y direct packaging of expressing PE15A no.

the cosmid library) 0.5 pig of D)NA of HmBT trznsferiants 7C31] 32 E7/192 TC3.2 32000 49/364 TC3.3 .44 25/72

L

P

I 4 1 WO 92/20356 PCr/US92/04354 The cosmids were analyzed with restriction enzymes and it was found that directly packaged transfectant P1A.TC3.1 contained 32 cosmids, 7 of which were different. Each of these 7 cosmids was transfected into PO.HTR, in the manner described supra, and again, following the protocols described above, transfectants were studied for presentation of P815A. Four of the cosmid transfectants showed P815A presentation and, as with all experiments described herein, P815B was co-expressed.

Of the four cosmids showing presentation of the two antigens, cosmid C1A.3.1 was only 16,7 kilobases long, and was selected for further analysis as described infra.

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

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

This fragment the 4.1 kb PstI fragment), was digested with SmaI, 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, the transfected host cell presented both antigen A and antigen B.

i; WO 92/20356 PCT/US92/04354 22 Example 7 The 900 base fragment described above was used as a probe to detect the expression of the P815A gene in parent cell line P1.HTR. To accomplish this, total cellular RNA was first isolated using the guanidine-isothiocyanate procedure of Davis et al., Basic Methods In Molecular Biology (Elseview Science Publishing Co, New York) (1986).

The same reference was the source of the method used to isolate and purify polyA mRNA using oligodT cell-lose 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 (SSC: 0.15 M NaCI; 0.015 M sodium citrate, pH 7.0) for 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 0 C in a solution containing 10% dextran sulfate, 1% SDS and 1M NaCl. Hybridization was then carried out using denatured probe (the 900 base fragment), together with 100 ug/ml salmon sperm DNA.

When this protocol was carried out using P1.HTR poly 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 1. k-_ WO 92/20356 PCT/US92/04354 23 a clone with a 1kb 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 0 C. The blots were washed twice at room temperature with 2xSSC and twice at 60 0 C with 2xSSC supplemented with 1% SDS.

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

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

a gZxample 8 The Northern analysis described supra suggested that SI the 5' end of the cDNA was missing. To obtain this sequence, cDNA was prepared from P1.HTR RNA using a primer corresponding to positions 320-303. The sequence was then i 20 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 8998-9002 (1988). A band of the expected size (270 bases) was found, which hybridized to the 900 bp SmaI/Xbal fragment described supra on a Southern blot. Following cloning into ml3tg 130 X tg 131, the small, 270 bp fragment was sequenced. The sequence is shown in sequence id no: 1.

)~aPI~rC

I

WO 92/20356 PCT/US92/04354 24 Example 9 Following the procurement of the sequences described in Examples 7 and 8 and depicted in seq id no: 4, a 5.7 kb Sregion of cosmid C1A.3.1 was sequenced. This fragment was fknown to contain the 900 base fragment which expressed P815A in transfectants. This experiment permitted delineation of introns and exons, since the cosmid is genomic in origin.

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

A computer homology search was carried out, using program FASTA with K-triple parameters of 3 and 6, as suggested by Lipman et al., Science 227: 1435-1441 (1985), and using Genbank database release 65 (October 1990). No homology was found except for a stretch of 95 bases corresponding to part of an acid region coded by exon 1 (positions 524-618), which is similar to sequences coding r c i r WO 92/20356 PCT/US92/04354 for acidic regions in mouse nucleolar protein N038/B23, as described by Bourbo.i 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 j nucleolin, while the 1.5 kb cDNA corresponded to mouse Snucleolar protein N038/B23.

Analysis of the nucleotide sequence of the gene, referred to as "P1A" hereafter, suggests that its coded product has a molecular mass of 25 kd. Analysis of the sequence id no: 4 shows a potential nuclear targeting signal at residues 5-9 (Dingwall et al., Ann. Rev. Cell SBiol. 2: 367-390 (1986)), as well as a large acidic domain at positions 83-118. As indicated supra, this contains the Sregion of homology between P1A and the two nucleolar proteins. A putative phosphorylation site can be found at position 125 (serine). Also, a second acidic domain is found close to the C-terminus as an uninterrupted stretch of 14 glutamate residues. A similar C-terminal structure has been found by Kessel et al. Proc. Natl. Acad. Sci. USA 84: 5306-5310 (1987), in a murine homeodomain protein having nuclear localization.

w WO 92/20356 PCT/US92/04354 26 In studies comparing the sequence of gene PlA to the sequences for P91A, 35B and P198, no similarities were found, showing that P1A is indicative of a different class of genes and antigens.

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

Hybridization conditions were as described supra, and a hybridizing clone was found. The clone contained exons one and two of the P1A gene, and corresponded to positions 0.7 to 3.8 of figure 5. Following localization of this sequence, PCR amplification was carried out to obtain the sequence corresponding to 3.8 to 4.5 of figure Sequence analysis was carried out, and no differences were found between the gene from normal kidneys and the P1A 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 PlA gene isolated from normal kidney cells.

IsP WO 92/20356 PCT/US92/04354 27 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 rather than the normal 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.

i Example 11 IAdditional experiments were carried out with other i 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 P1A gene could be found. The Northern blot data are presented I in figure 4 and, as can be seen, there is no evidence of expression.

The murine P815 cell line from which P1A was isolated is a mastocytoma. Therefore, mast cell lines were studied to determine if they expressed the gene. Mast cell line MC/9, described by Nabel et al., Cell 23: 19-28 (1981), and short term cultures of bone marrow derived mast cells were tested in the manner described supra (Northern blotting), but no transcript was found. In contrast when a Balb/C derived IL-3 dependent cell line L138.8A (Hltner et al., WO 92/20356 PCT/US92/04354 28 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, teratocarcinoma cell line PCC4 (Boon et al., Proc. Natl. Acad. Sci. USA 74: 272-275 (1977), and leukemias LEC and WEH1-3B. Expression could not be detected in any of these samples.

Example 12 The actual presentation of the P1A antigen by MHC molecules was of interest. To test this, cosmid C1A.3.1 was transfected into fibroblast cell line DAP, which shows phenotype H-2 The cell lines were transfected with genes expr ssing one of the Kd, Dd, and Ld 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 Ld is required for presentation of the P1A antigens A and B.

^r WO 92/20356 2 PCT'/US92/04354 T2,2 H-2-yest-i-ticr, or an-,igms PEISA z-.d PE)SB i e. I No of clones ysed b th~e CTL/ no. of )J:B clcesl C-7L vdtiA CiT La1i.B DAP (H-2k) 0/208 0/1 9A DA p 4 Xd 0/165 0/162 Dd 0/157 0/229 D)A? d 25/33 15/20 OCosnid CIA.3.1 containing ite entire PIA gene -Is trznsfecied in DAP cells previou.sly transfecied with R[ 2 d class I genes as indicated.

ORIndependent d.ugresisi'nt colonies w'ere lested for Jy-sis by IniA or a-zsi.B CTL i: rsa a SS sy.

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

EVxam~le 13 Using the sequence of the PlA gene as well as the amino acid sequence derivable therefrom, antigenic peptides which were A+ B+ 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 This peptide when administered to samples of PO.HTR cells WO 92/20356 PCT/US92/04354 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 E, F, and In addition, two other antigens and are expressed by some sublines of the tumor.

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

Among the recognized subclones of MZ2-MEL are MEL.43, 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: 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, it must be a "good" recipient.

WO 92/20356 PCT/US92/04354 31 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 This subclone is also HPRT, 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 The genomic DNA of MEL3.0 was prepared following W8lfel et al., Immunogenetics 26: 178-187 (1987), the disclosure of which is incorporated by reference. The plasmid pSVtkneoB, as described by Nicolas et al., Cold Spring Harb., Conf. Cell Prolif. 10: 469-485 (1983) confers geneticin resistance, so it can be used as a marker for cotransfection, as it was in this experiment.

Following a procedure similar but not identical to that of Corsao et al., Somatic Cell Molec. Genet 7: 603- 616 (1981), total genomic DNA and the plasmid were cotransfected. The genomic DNA (60 Mg) and plasmid DNA (6 h g) were mixed in 940 Ml of 1 mM Tris-HCl (pH 0.1 mM EDTA, after which 310 Ml of 1M CaC1 2 was added. This solution was slowly added, under constant agitation, to 1.25 ml of 2xHBS (50 mM HEPES, 280 mM NaC1 1.5 mM Na 2

HPO

4 adjusted to pH 7.1 with NaOH). The calcium phosphate DNA precipitates were allowed to form for 30-45 minutes at room WO 92/20356 PCT/US92/04354 32 temperature, after which they were applied to 80 cm 2 tissue culture flasks which had been seeded 24 hours previously with 3x10 6 MEL2.2 cells, in 22.5 ml of melanoma culture medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum. After 24 hours, the medium was replaced. Forty eight hours after transfection, the cells were harvested and seeded at 4x10 cells per 80 cm flask in melanoma culture medium supplemented with 2 mg/ml of I geneticin. The geneticin serves as a selection marker.

1 10 Example 16

I

1 -i Thirteen days after transfection, geneticin-resistant colonies were counted, harvested, and cultured in Snonselective 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 j redundancy, such that every independent transfectant Sshould be represented at least four times.

I 20 After 10 days, wells contained approximately 6x10 4 b cells. These cells were detached, and 1/3 of each microculture was transferred to a duplicate plate. After 6 hours, after readherence, medium was removed and 1500 anti-E CTL (CTL 82/30), were added to each well in 100 pl of CTL culture medium with 35 U/ml of IL-2. One day later, the supernatant (50 pl) was harvested and examined t WO 92/20356 PCT/US92/04354 33 for TNF concentration, for reasons set forth in the following example.

Example 17 The size of the mammalian-genome is 6x10 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 Sreduce the number of assays by a factor of 30. While an anti-E CTL assay, as described supra, in mixed 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 j necrosis factor 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 (4x10 4 had readhered, the CTLs and IL-2 were added thereto. The 50 pl of supernatant was removed 24 hours later and transferred to a microplate containing 3x10 4 W13 (WEHI-164 clone 13; WO 92/20356 PCT/US92/04354 34 Espevik et al., J. Immunol. Meth. 95: 99-105 (1986)) cells in 50 1l of W13 culture medium (RPMI-1640, supplemented with L-arginine (116 mg/1), L-asparagine (36 mg/l), Lglutamine (216 mg/1), and 10% FCS supplemented with 2 pg of actinomycin D at 37% in an 8% CO 2 at- sphere. The cell line W13 is a mouse fibrosarcoma cell line sensitive to TNF.

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

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

Immunol. Meth. 119: 203-210 (1989). This involved adding ml of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide at 2.5 mg/ml in PBS, followed by two j hours of incubation at 37 0 C. Dark blue formazan crystals were dissolved by adding 100 Al of lysis solution (1 volume N,N dimethyl formamide mixed at 37 0 C with two volumes of water containing 30% sodium dodecyl sulphate, at pH 4.7 from 1.6% acetic acid and 2.5% 1N HC1). Plates were incubated at 37 0 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) 100 x 1

OD

5 70 well medium __1

I

WO 92/20356 PCT/US92/04354 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 Example 18 Cells were tested for TNF production as discussed in Example 17, supra. A total of 100 groups of E- cells (4x10 6 cells/group) were tested following transfection, and 7x10 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.T1, discussed herein, had the same lysis pattern as did MEL2.2 for CTLs i 20 against antigens B,C,D and F.

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

I

i; i f WO 92/20356 PC'/US92/04354 36 Example 19 Once transfectant E.T1 was found, analysis had to address several questions including whether an E contaminant of the cell population was the cause. The analysis of antigen presentation, described supra, shows that E.T1 is B- and just like the recipient cell MEL2.2.

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

i 10 It was also possible that an E revertant of MEL2.2 was I the source for E.T1. To test this, the observation by I Perucho et al., Cell 22: 309-317 (1980), that cotransfected sequences usually integrate together at a single location I of recipient genome was employed. If antigen E in a Stransfectant results from cotransfec-tion with pSVtkneoB, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoB sequences.

W8lfel et al., supra, has shown this to be true. If a normally E cell is transfected with pSVtkneoB, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoB sequences. If a normally E cell transfected with pSVtkneoB is E.T1, however, "co-deletion" should not take place. To test this, the transfectant E.T1 was subjected to immunoselection with 82/30, as described supra. Two antigen loss variants were obtained, which resisted lysis by this CTL. Neither of these had lost geneticin b WO 92/20356 PCT/US92/04354 37 resistance; however, Southern blot analysis showed loss of several neor sequences in the variants, showing close i linkage between the E gene and neor qene in E.T1, leading to the conclusion that E.Ti was a transfectant.

V Example 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 Si 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 j 20 restriction endonuclease digestion via XmaI, or by BamHI it digestion of a large, 12 kb XmaI transfected fragment. The fragments were cloned into vector pTZ 18R, and then transfected into MEL2.2. Again, TNF production was the measure by which successful transfection was determined.

The experiments led to the determination of a gene sequence capable of transfecting antigen E on the 12 kb XmaI WO 92/20356 PCT/US92/04354 38 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.

iI S1 p.r

F;

WO 92/20356 PCI'/US92/04354 39 The sequence f or the E antigen precursor gene has been determined, and is presented herein: 1 10 301 CA ~CA 12: CGACCC7G 421 VGSAZXV.G 41 GACG~X 302 121 GT1CACZ 1142 AC7C=-.

62 0=?0.CC 121 r?:jkkCc 2441 AACG 1502 Cc.=XCJ 21 1?V-kOT 2.321 GCACA .2651 GCCK 1501 TCTTC.0CAC 1621 X7CATT 2162 &%M7A: 2042 GT~~ 2101 CcG 2261 C=VCTTA =22 AUMOCAV 2211 AA"="W.

2341 CGGIZ4 2401 ATWCT=?GO I %o 1 20 CT G GAT &C7ACCT

AXGGCCGC

C? C? CCCCAG ?G7GA.?CT

GC%GCT

~.CACJ.C

gA=:ZCT

?CAAGGC?.

CACGCAC

GCALWGG

I 20AAA

AC

A.ATG

!ACC

M CA

CT

CA?

1 30-, 1 4 C so COMAG.A LCACCC T A=-.AAo 7- ACU?'C :CWNC AC ah CO TCTC7 )LCN.?C CL TCCA Mx 3 M07: G=oCTTKGTD CXTCCG, UCCC CT?"AI A~bh.N.N0C "3v c=V4 wJA~ TC-LLA?0 CACT CC COCATCU AGL? OCAMGG~ 70CGTSUTG ak3CT GAzx"C3aG CGA-V.CP)= TOAGIOC CATCTTaCA CJ-*=A 'SAxvGG 2GCGIU.? OLCCA.-=C% &CT?2a CT3C:'QA hT:C?).AT tG.k??GG-A ~C OT'JLW7 AGM~hG A

GCTC:?

TCTGO.AJXA

CA=.=cC:? c-.TTCW???: ACCAAOs??

AV--.

2 4 3C: 420 4E: 540 11.0 540 I CC 20200 100 114: 226:

IS::

:120 2610 :14 0 28^.0 2660 1520 1950o

LCTCA

1 30

CCU??C?;

CATV-k"CTG

?CCCGAO

WwWThOhrC 1 40 ZLLMT=?! AATA2?A? A.LLA?&C ?GATLU.ZA AACTO=CGC ??CTGGAAOG iAT)6CC.LCO ALTXa.GGCT TOCCCM 36ATA= &CTOGUG MC?"?CC ??CGGMCMA WC~TTC?.A I so I s0 204D 2100 2260 2220 2250 2340 2400 2411 ii'" i I. IX~ WO 92/20356 PCF/US92/04354 Example 21 After the 2.4 kb genomic segment had been identified, studies were carried out to determine if an "E subline expressed any homologous DNA. Cell line MZ2-MEL 3.0 was used as a source, and a cDNA library was prepared from its mRNA, using art known techniques. The 2.4 kb segment was used as a probe, and mRNA of about 1.8 kb was identified as homologous, using Northern blot analysis. When cDNA was screened, clones were obtained showing almost complete identity to parts of the 2.4 kb fragment. Two exons were thus identified. An additional exon was located upstream of these, via sequencing segments of cosmid B3 located in front of the 2.4 kb BamHI fragment. The gene extends over about 4.5 kb, as shown in Figure 8. The starting point of the transcribed region was confirmed using PCR for the 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.

WO 92/20356 PCT/US92/04354 41 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.

Example 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 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 S' other than the first (18.1 and 18.9% difference compared to the first; 12% with each other). Out of 9 cDNA clones i obtained, three of each type were obtained, suggesting S equal expression.

re 9 EEli 1 Arj WO 92/20356 PCT/US92/04354 42 r, AwCE ref crc to a family 99 tumor rcjcotin. antigen precursors and eic acid sequences coding therefore.

The antigens resulting therefrom ar red to herein as "MAGE TRAY' sr melanoma antigen tumor rjootion ant&g 'I 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 I 10 from the activation of genes that are not expressed in most I 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.

WO 92/20356 PCT/US92/04354 43 Example 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 phytohemagglutininactivated 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 WO 92/20356 PCT/US92/04354 44 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 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 oligonucleotides. 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.

WO 92/20356 111~ PCT'/US92/04354 Exammple 26 The availability of a sequence that transfects at high efficiency and efficiently expresses a TRAP made it possible to search for the associated major histocompatibility complex (MHC) class I molecule. The class I specificities of patient MZ2 are HLA-A1, A29, B37, B44 and C6. Four other melanomas of patients that had Al in common with MZ2 were cotransfected with the 2.4 kb fragment and pSVtkneoB. Three of them yielded neor transfectants that stimulated TNF release by anti-E CTL clone 82/30, which is CD8+ (Figure 10). No E- transfectant was obtained with four other melanomas, some of which shared A29, B44 or C6 with MZ2. This suggests that the presenting molecule for antigen MZ2-E is HLA-A1. In confirmation, it was found that, out of 6 melanoma cell lines derived from tumors of HLA-A1 patients, two stimulated TNF release by anti-E CTL clone 82/30 of patient MZ2. One of these tumor cell lines, MI13443-MEL also showed high sensitivity to lysis by these anti-E CTL. These two melanomas were those that expressed mage-1 gene (Figure 13). Eight melanomas of patients with HLA haplotypes that did not include 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 WO 92/20356 PCT/US92/04354 46 mage 2 and 3 can also be presented to autologous CTL by HLA-A1 or other class I molecules, especially in view of the similar results found with murine tumors, as elaborated upon supra.

Example 27 As indicated supra, melanoma MZ2 expressed antigens F, D and in addition to antigen E. Following the isolation of the nucleic acid sequence coding for antigen I E, similar experiments were carried out to isolate the t 10 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 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 antiil WO 92/20356 PC/US92/04354 47 F CTLs. Lysis assays, also following protocols described supra, confirmed 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 Sthis, 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 Al/cm 2 of 3.5xSSC, IxDenhardt's solution; 25 mM sodium phosphate buffer (pH 0.5% SDS, 2mM EDTA, where the 2.4 kb probes had been labelled with [a32pJdCTP (2-3000 i ni j111 .i -rn11,11 L-f- r-fl rr lr WO 92/20356 PCT/US92/04354 48 Ci/mole), at 3x10 6 cpm/ml. Hybridization was carried out for 18 hours at 65 0 C. After this, the membranes were washed at 65 0 C four times for one hour each in 2xSSC, 0.1% SDS, and finally for 30 minutes in 0.1xSSC, 0.1% SDS. To identify hybridization, membranes were autoradiographed using Kodak X-AR film and Kodak X-Omatic fine 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 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 m WO 92/20356 PCT/US92/04354 49 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 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 -I amplified with oligos CH09: (ACTCAGCTCCTCCCAGATTT), and (GAAGAGGAGGGGCCAAG). These oligos correspond to regions of exon 3 that are common to previously described mage 1, 2 and 3.

To do this, 1 Ag of RNA was diluted to a total volume of 20 Ml, using 2 p4l t lox PCR buffer, 2 Al of each of mM dNTP, 1.2 p1 of 25 mM MgCl 2 1 p1 of an 80 mM solution of CHO9, described supra, 20 units of RNAsin, and 200 units of M-MLV reverse transcriptase. This was followed by incubation for 40 minutes at 42 0 C. PCR amplification Sfollowed, using 8 tl of 10x PCR buffer, 4.8 4l of 25 mM MgC12, 1 4l of CHO10, 2.5 units of Thermus acquaticus polymerase, and water to a total volume of 100 l1.

Amplification was then carried out for 30 cycles (1 minute 94 0 C; 2 minutes at 52°C, 3 minutes at 72 0 Ten 41 of each reaction were then size fractionated on agarose gel, r WO 92/20356 PCT/US92/04354 followed by nitrocellulose blotting. The product was found to hybridize with oligonucleotide probe CHO18 (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 oligonucleotides 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 ;age 1 was shown to transfer expression of antigen E. As a result, it was decided to test whether WO 92/20356 PCT/US92/04354 51 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 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 0 C to identify the smage material.

SExample 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 WO 92/20356 PCT/US92/04354 52 pancreatic tumor (MAGE non-small cell lung (MAGE 1, 2, 3 and prostate (MACE sarcomas (MAGE 1, 2, 3 and 4), breast (MAGE 1, 2 and 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 iLethodology 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 Spoint where, given a strand of DNA, the skilled artisan can I isolate its complement, or synthesize it.

"Nucleic acid molecule" as used herein refers to all it species of DNA and RNA which possess the properties 1 discussed supra. Genomic and complementary DNA, or "cDNA" Sboth 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, in r WO 92/20356 PCT/US92/04354 53 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, 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 IL-2 or IL-4), or major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules. Cytoy ne 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 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, i WO 92/20356 PCT/US92/04354 54 additional transfection may not be necessary although further transformation could be used to cause overexpression 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 H 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, 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

L_

WO 92/20356 PCT/US92/04354 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 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 WO 92/20356 PCT/US92/04354 56 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, 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 Bcell response, this involves, inter alia, the generation of antibodies to the TRA, 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, a Balb/C mouse or in a test tube, using, EBV transformants. In addition, antiserum may be isolated from a subject afflicted with a cancerous condition where certain cells present a TRA. Such WO 92/20356 PCT/US92/04354 57 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 a subject, binding of antibodies to TRAs, or the activity of anti-TRA CTLs in connection with subject samples.

i Similarly, the expression of nucleic acid molecules for I TRAPs can be monitored via amplification "polymerase j 3 chain reaction"), anti-sense hybridization, probe technologies, and so forth. Various subject samples, including body fluids (blood, serum, and other exudates, 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 WO 92/20356 PCT/US92/04354 58 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.

SWhere "subject" is used, the term embraces any species iwhich can be afflicted with a cancerous condition. This i 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 WO 92/20356 PCT/US92/04354 59 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.

jis WO 92/20356 PCY/US92I04354 GENERAL INFORMATION: 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: ADDRESSEE: Felfe Lynch STREET: 805 Third Avenue CITY: New York City STATE: New York ZIP: 10022 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage COMPUTER: IBM OPERATING SYSTEM: PC-DOS SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 07/807,043 FILING DATE: 12-DECEMBER-1991 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 07/764,364 FILING DATE: 23-SEPTEMBER-1991 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 07/728,838 FILING DATE: 9-JULY-1991 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 07/705,702 FILING DATE: 23-May-1991 (viii) ATTORNEY/AGENT INFORMATION: NAME: Hanson, Norman D.

REGISTRATION NUMBER: 30,946 REFERENCE/DOCKET NUMBER: LUD 253.4 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 688-9200 TELEFAX: (212) 838-3884 SUBSTITUTE SHEET WO 92/20356 INFORMATION FOR SEQUENCE ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 462 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE% genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: PCr/US92/04354

ACCACAGGAG

GAAGATCCTG

CAGCCAATGA

AAGTTTTGCA

CACGTA.AAAA

CCTCGTGCTG

AGAACTCTTC

CATGCATTGT

CTAGCTTGCG

ACCCTTTGTG

AATGAAAAGA

ATCACTCATT

GCTTACTGTT

AGTTCCGCCT

AGTAGTCCAG

TGCTGAGTTT

CGGAGGAAGG

GTCAACGCCA

ACTCTACTCT

cc

ACCCGGGACT

GGGTGTCTGA

CTCGTGGGGG

ACAGCTCTAG

AGTTTACTAC

AGAAGTCTTC

AGGGAGGACC

TTGCACTGAG

TATCTTAACT

CCCAAAGACG

GTTCTGCGAT

GTTTGTGAGC

CTTGTGAATT

ACCCTCCCTC

CTTATAGAAG

CCCCCCCTTT

CTGGTCGAAG

TAGCTCGGCT

CTAGATGTGT

ATTCATCCCT

CTTGGGTAGG

TGTACCCTTT

CCCCCTCCCA

TCTTCCGTAT

GCTCTCCCAG

AAGTAAGCCG

TCCTGCTGGT

so 100 150 200 250 300 350 400 450 462 t i 4 11 SUBSTITUTE SHEET p WO 92/20356 PCr/US92/04354 INFORMATION FOR SEQUENCE ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 675 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC AGT GOC TCA Met Ser Asp Asn Lys Lys Pro Asp Lys Ala His Ser Gly Ser GGT GGT Gly Gly GAC GGT GAT GGG AAT AGG Asp Gly Asp Gly Asn Arg TGC AAT TTA Cys Asn Leu 25 TTG CAC COG TAC Leu His Arg Tyr TCC CTG GAA Ser Leu Olu GAA ATT CTG Glu Ile Leu CCT TAT CTA 000 Pro Tyr Leu Gly CTG GTC TTC OCT GTT GTC ACA ACA Leu Val Phe Al.a Val Val Thr Thr AOT TTT Ser Phe so CTG OCO CTC CAG ATO TTC ATA GAC GCC Leu Ala Leu Oln Met Phe Ile Asp Ala TAT GAG GAG CAG Tyr Glu Olu Gln

TAT

Tyr GAA AGG GAT GTO Glu Arg Asp Val TGG ATA 0CC AGO Trp Ile Ala Arg CAA AGC AAG CGC Gln Ser Lys Arg GAT GAG GAT GAC Asp 0Th Asp Asp

ATG

Met TCT GTC GAT GAG Ser Val Asp Glu GAA GAC OAT GAG Olu Asp Asp Glu TAC TAC Tyr Tyr GAC GAC GAG Asp Asp Glu GAG GAA GAA Glu Glu Glu 115 GAC GAC GAC OAT Asp Asp Asp Asp TTC TAT GAT OAT Phe Tyr Asp Asp GAG OAT OAT Olu Asp Asp 110 OAA OAT GAO Glu Asp Olu GAA TTO GAG AAC Glu Leu Olu Asn ATO OAT OAT OAA Met Asp Asp Olu 0CC GAA Ala Glu 130 GAA GAO ATO AOC Olu Olu Met Ser OAA ATG GOT GCC Glu Met Gly Ala

GT

Gly 145 OCT GOC OCT AAC Ala Oly Ala Asn 0CC TOT OTT CCT Ala Cys Val Pro GGA OCT GAG GAA ATG oly Ala Glu Olu Met 140 CAT CAT TTA AGO AAG, His His Leu Arg Lys 160 CAC GAC CCT AAT TTC His Asp Pro Asn Phe 175 AAT OAA OTO AAG Asn Olu Val Lys TOT AG4 ATO ATT Cys Arg Met Ile 165 TAT TTC TTC Tyr Phe Phe 170 SUBSTITUTE SHEET WO 92/20356 WO 9220356PC1'/US92/04354 CTG GTG TCT ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT Leu Val Ser Ile Pro Val Asn Pro Lys Glu Gin Met Glu Cys Arg Cys 180 183 190 GAA AAT GCT GAT GAA Glu Asri Ala Asp Glu 195 GAG GTT GCA ATG GAA Glu Val Ala Met Glu 200 GAG GAA GAA GAA GAA GAG Glu Glu Glu Glu Glu Glu 210 GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCA CCT Glu Glu Giu Glu Glu Glu Glu Met Gly Asn Pro Asp Gly Phe Ser Pro 220 225 230 235

TAG

SUBSTITUTE SHEET WO 92/20356 WO 9220356PCV'US92/04354 INFORMATION F *OR SEQUENCE ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 228 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCA'TGCAGTT GCAAAGCCCA GAAGAAAGAA ATG47ACAGCG GAAGAAGTGG TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTT'm C T i-ATC.CAGAAA ATTTGATTTT GTTCTAAAGT TCATTATGCA AAGATGTCAC CAACAGACTT CTL~ACTGCAT GGTGAACTTT CATATGATAC ATAGGATTAC ACTTGTACCT GTTAAAAATA AAAGTTTGAC TTGCATAC SUBSTITUTE

SHE~ET

I

WO 92/20356 PCT'/US92/04354 INFORMATION FOR SEQUENCE ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 1365 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACCACAGGAG AATGAAhAGA GAAGATCCTG ATCACTCATT CAGCCAATGA OCTTACTGTT AAGTTTTGCA AGTTCCGCCT CACGTAAAAA AGTACTCCAG CCTCGTGCTG TGCTGAGTTT AGAACTCTTC CGGAGGAAG CATGCATTOT OTCAACGOCA CTAGCTTGCG ACTCTACTCT ACCCTTTGTG CC

ACCCGGGACT

GGGTGTCTGA

CTCGTGGGGG

ACAGCTCTAG

AGTTTACTAC

AGAAGTCTTC

AGGGAGGACC

TTGCACTGAG

TATCTTAACT

CCCAAAGACG

GTTCTGCGAT

GTTTGTGAGC

CTTGTGAATT

ACCCTCCCTC

CTTATAGAAG

CCCCCCCTTT

CTGGTCGAAG

TAGCTCGGCT

CTAGATGTGT

ATTCATCCCT

CTTGGGTAGG

TGTACCCTTT

CCCCCTCCCA

TCTTCCCTAT

CCTCTCCCAG

AAGTAAGCCG

TCCTGCTGGT

1., ATG TCT GGT COT TAC TCC TTC GCT ATA GAC TOG ATA GAT GAA GAG GAC GAG CAA CAT GAG OCT GAG GGC CAT TAT TTC AAC CCT GAA GAG GAG GAG

GAT

GAC

CTG

OTT

CC

GCC

GAC

GAC

GAA

CC

GAA

CAT

TTC

AAG

GT'I

CAA

AAC

GGT

OAA

OTC

CTT

AG

GAT

GAC

GAA

AAA

G

ATT

ACA

GAG

AGC

CAT

CAT

GAG

CAC AAA AGO TGC CCT TAT TTT CTO CAC TAT CGC ATG GAG CAT TTC TAT CTO ATO ACC OTO OCT AAC CAA OTO TTC CTG TGT AGO GAA GAA

CAC

TTA

G

CTC

AGO

TCT

TAC

CAT

CAT

ATG

CC

TOT

TCT

OAA

GAA

ACT

TTC

TOO

CAG

CAT

GTC

TAC

GAG

OAA

OCT

TO-T

AGO

ATA

AAT

GAG

TCA

CGC

GTC

TTC

CC

GAG

GAC

CAT

GAA

GGA

CCT

ATT

OTO

CAT

GAG

CAA CAA GAO ATG GOT OCT TTA AGO, AAO CAC GAC CCT OAA CAA ATO OCA ATO OAA GAO CAA ATO GCAAAGCCCA G TTCCCCTTCA T GTTCTAA.AGT Ti GGTGAACTTT C.

AAAOTTTOAC T 504 546 588 630 672 714 756 798 840 882 924 966 1008 1050 1092 1134 1137 1187 1237 1287 1337 1365 AAC CCC CAT CCC TTC TCA CCT

TAG

CCATGCACTT

TTGTTTTTTT

ATTTCATTTT

CTOACTCCAT

GTTAAAAATA

PAOGAAAGAA ATOGACAC TAATTTTCT AGTTTTTAOT CATTATOCA AAOATGTCAC PTATCATAC ATAGGATTAC

TGCATAC

CAACAACTCO

AATCCAOAAA

CAACAGACTT

ACTTGTACCT

SUBSTITUTE SHEET WO 92/20356 INFORMATION FOR SEQUENCE ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 4698 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: PCT/US92/04354 ACCACAGGAG AATGAAAAGA ACCCGGGACT

GAAGATCCTG

CAGCCAATGA

AAGTTTTGCA

CACGTAAAAA

CCTCGTGCTG

AGAACTCTTC

CATGCATTGT

CTAGCTTGCG

ATCACTCATT

GCTTACTGTT

AGTTCCGCCT

AGTAGTCCAG

TGCTGAGTTT

CGGAGGAAGG

GTCAACGCCA

ACTCTACTCT

GGGTGTCTGA

CTCGTGGGGG

ACAGCTCTAG

AGTTTACTAC

AGAAGTCTTC

AGGGAGGACC

TTGCACTGAG

TATCTTAACT

CCCAAAGACG

GTTCTGCGAT

GTTTGTGAGC

CTTGTGAATT

ACCCTCCCTC

CTTATAGAAG

CCCCCCCTTT

CTGGTCGAAG

TAOCTCGGCT

CTAGATGTGT

ATTCATCCCT

CTTGGGTAGG

TGTACCCTTT

CCCCCTCCCA

TCTTCCGTAT

GCTCTCCCAG

AAGTAAGCCG

TCCTGCTGGT

ACCCTTTGTG CC ATG TCT GAT OGT GGT GAC TAC TCC CTG TTC GCT GTT ATA GAC GCC TGG ATA GCC GAT GAA GAC GAG GAC GAC GAG GAA GAP GAT GAG GCC GCT GAG GAP~

GTGAGTAACC

CTCTTGCCCA

TGGAGCCATT

CCCCACTCCT

TTCAGTCCAT

TCCCCCTCGO

TTCAGGCTTC

CCTTTTCGCG

TCACCAGCTT

TCCTGCTCCC

CTACCTGCTT

TGCTCCTCCC

CCTCCCTCCC

TTGGTTTTTC

TCACTCTGTA

CCTCCCAAAT

GCCTTTCTTT

AACTCCCCTT

TTCCCTTCCG

CCTCCCCCTC

GCCCCGTTCC

AGCTCACCTT

TTTTTTTTTT

CCTCTGTGTG,

AAC

GGT

GAA

OTC

CTT

AGO

OAT

GAC

OAA

GAA

ATG

AAG

OAT

GAA

ACA

TAT

CAA

GAG

GAC

TTG

GAA

GGT

AAA

GG

ATT

ACA

GAG

AOC

GAT

OAT

GAG

GAG

GCT

GAC

AGO

CCT

TTT

CAG

CGC

GAG

TTC

CTG

AGC

OCT

AAA

TOC

TAT

CTG

TAT

ATG

GAT

TAT

ATG

OTO

AAC

AGT GGC TTG CAC TGG CTO CAG ATO OAT GTG GTC GAT TAC GAC GAG GAT OAA TCA OGT GCC

T

CGTOGTCTTT

CATCTGTAGT

CCTGOCTCTC

TGCTCCGCTC

CCTGCTCTGC

CTCAACTTTT

CCCATTTGCT

CCTTTTCTTT

TOCTCTCCCT

CTCCCCCTCC

CCCTCCCCCT

TCCCCCTCCC

CCTCCCCAG

GAGACAGGGT

GACCAGGCTG

GCTGGGATTA

TTTCTCCTCT

TTGGCACCTT

GCACCCTTCC

TTTC" ."CGAC

CCTTTTTTGT

TTTGTTTGTT

GCACCTTGTT

CCTTTCCTGT

ACTCTAGATT

AAAGACCACA

CTOTCCACGC

TCTTTCCTTT

TCCCTTTCCC

CGTGCCTTCT

CCTCTCCCOA

CCTGCTCCCC

GCTCCCCTCC

CCTCCCTOTT

TGCTGCTCCC

CCTCCCTCCC

CCTTTTTTTT

TTCTCTTTGT

GCCTCAAACT

AAGGCTTGCA

CTGGTCTCCC

TCCTTTACAG

TAGCCCTGCT

.TTTTAGCAGC

GCCTTTCCTC

TGGTTGTTTG,

TTCCAAGATC

TCCCTCCCCC

CAGGTGGOGT

TTTTGGTTOG

CTATCCCCGC

TCCCACCTTG

CTTTOCTCTC

OCTCTCTGAT

AACCCTCCCC

TCCCCCTCCC

CCCTTTTOCA

TACCCTTCAC

TCCCTATTTG

TATTTGCATT

TTTTTTTTTT

ATCCCTGGCT

CAGAAATCTG

CCAGGACTGC

TAATCCCTTT

GACCCCCTCC

CTGTTCCCTC

CTTACCTCTC

CTGGCTCCCC

GTTGTTTGGT

CCCCTCCCCC

TCGCTGGCTC

GCATTCTTTA

GTCATTGC

TCCTCCCATC

CCTCTGGAGC

CTTGCTCCCC

CCC CACCCTC

TTCCTOTTCC

TATTTACCTT

CCTTTTCTTT

CGCTTTTCCT

CATTTTCGGG

TTCGGGTGCT

TTTTTTTTTT

GTCCTGGCAC

CCTGCCTCTG

CCCAGTGCAG

TCTGCATGTT

CCCTCCCTGT

TCCCTGCTCC

CCTGCTTTCT

TCCACCTTCC

TTGCTTTTTT

TCCGGCTTCC

CCCCTCCCTT

504 546 588 630 672 714 756 798 840 882 916 966 1016 1066 1116 1166 1216 1266 1316 1366 1416 1466 1516 1566 1616 1666 1716 1766 1816 1866 1916 1966 2016 2066 2116 SUBSTITUTE SHEET 4 WO 92/20356 PCr/US92/04354

TCTGCCTTTC

CTTTTCTAGA.

CCTGACCCTG

CCTTTCTCCA

TCCTGCTTCC

GACTTCCTCT

CTCTCTGTCC

ATGTGTCTCT

CCATCACCTC

CCTGCTTCTT

TCCATGTCCC

ATTTCCCTCT

TTCCCTTTGC

TACTTGATCT

CTTTGTCCCC

ATCAACAACA

AAGGCTGGAT

AAGTGGCTCC

CTTGATCCTT

CAGGCCATGC

GAATCTGAAA

TAGTGATATT

TCCTTCTACA

GGCTAAAGAT

TTGCTAAAAT

GT GTT CC'I AGG ATG ATI ATA CCA GTC AAT GCT GAI GAG GAG GAG.

TTC TCA CCI

GCATGCAGGT

GCTAAGAGCA

TCTTTTTACA

CCCTAAGTTA

GTAGTGAGAC

GACCAGTAAA

TTCTTATAGT

TTCAAGAAAG

TTCTGATTTT

CTTAAAATTT

TAGAATTCAA

AATGTTTTTT

GTAACTGGOG

GTTCTGGTCT

CAGTAGGTTA

ATAAATACTC

ATTTTAGTTT

AGTCAGGAGT

AGTTGCAAAG

TTTTTTCCCC

TTTTGTTCTA

GCATGGTGAA

AATAAAAGTT

CTGTCCCTGC

CTCCCCCCTC

CTCCCCTTCC

GCCTGTCACC

TTTACCCCTT

CCAGCCGCCC

ATCACTTCCC

CTTCCTATCT

TCTCCTCCCT

TACCCTGCCT

CTCTCAATTC

TTCTCCCTTA

TTCTCCCTCC

TCTCTCCTCT

AGACCCTACA

AGGAGGCAAG

GAAAATAAGG

TATAACCCTA

GCTGCTTCTT

TCCATGCTTG

ACTAGGGGCC

TCCCCCTAAA

GGTGAGAAGT

ACTTGGAACC

ATTCTTTCTC

GGC CAT Cl TAT TTC TJ AAC CCT Al GAA GAG GIJ GAG GAG GI

TAG

ACTGGCTTCA

TCTTTTTAAA

TTAATAAGTA

AACAGAAGTC

TACTTACTAC

AGATCATGCA

ACCTTTGAGA

ATCACACGCC

TTTCATTTCT

CCTTCATCTT

TTCAAATTCT

AAAAAAAATG

GGCTTAGGGA

CTGAGAAGCA

GTGAGGTTGA

TAACAGCTAA

CTCCTTGAGA

GTATTCTAAT

CCCAGAAGAA

TTCATTAATT

AAGTTCATTA

CTTTCATATG

TGACTTGCAT

TCCCTTCTCT

CAGGCTTGCT

CCTCCCAGCT

CCTCCTTCTC

CCCTCTCCCT

AGTTCCCTGC

CCTAGTTTCA

ATCCCTTCCT

TCCCTTTCCT

CTCCCATTGC,

CCTGTCCCAT

GCCTCTTCTT

TCCTTTCCCC

CCACATACCC

GTATCCTGTG

AAACAGAGCA

CCAGGTTCTG

AGTACCAAGG

TTACATATGT

GCGCTTGCTC

AGTGGTTTGT

AATTATAACA

GGAAAAATTG

ATAGAAGCGT

ACATATTCAT

~T TTA AGG ~C CAC GAC ~G GAA C.AA ~T GCA ATG A GAG GAA

CTAACCAACC

AAATATTATT

TTAAATTAAT

AATGATGTCT

AGATGAGAAG

GTGAAATGTG

CAGCTGATAA

ATGGTTCACA

AGACCTGTGG

TAATTTTCCT

TAATTCAATC

CAAATCTCAT

ATCTGTAGGG

GTCAGAGAGA

TATGATCAGA

GGATCTCTGA

AACAATGACA

AAGTGTTGCT

AGAAATGGAC

TTCTAGTTTT

TGCAAAGATG

ATACATAGGA

AC

GCTAACCTTT

GTTTGCTTCT

CCCCCCTCTT

TCCTCTCTGT

ACTCTCCTCC

AGTCCTGGAG

CTTCCCTTTC

TTCTGTCCCC

CTCTCTTCCA

CCTCTTACCT

TGTGCTCCCT

CCTCTTCTCT

TTCCCCTATG

TTTTTCCTTT

CACAGGAAGT

AAATCCCAAA

AGGACAGCTG

GAGAAAGTGA

TGGCACATCT

AGCGTGGTTA

TTTGGGGACA

AACAGATTCA

TCACTATGAA

TGTTAAAATA

ATTCTCCAG

AG AAT GAA ,CT AAT TTC MT GAG TGT ;AA GAG GAA k.TG GGA AAC

TAATGCCTTT

GTGCACTTTT

TTCCCACCTC

TTCTCCCACT

CTGCCTGCTG

TCTTTCCTGC

ACTCTCCCCT

TCTCCTCTGT

TTTTCTTCCA

TTATGCCCAT

CACATCTTCC

TGTATCTCCC

CCCTCTACTC

CCACCCTGCC

GGGAGGTGCC

ATCAGCAGGA

GAATCTAGCC

TGGTGAAGTT

TTCTCAAATG

AGTAATGGGA

AATTAGCACG

TGATTTGAGA

GTTCTTTTTA

CTGCTTTCTT

AAG TGT GTG TCT TGT GAA GAA GAA GAT GGC 2166 2216 2266 2316 2366 2416 2466 2516 2566 2616 2666 2716 2766 2816 2866 2916 2966 3016 3066 3116 3166 3216 3266 3316 3355 3396 3438 3480 3522 3564 3576 3626 3676 3726 3776 3826 3876 3926 3976 4026 4076 4126 4176 4226 4276 4326 4376 4426 4476 4526 4576 4626 4676 4698

ATTCCTAACA

GGTAAACTAA

CCAGTATACA

AGATGCCTGT

TTGTTAGACT

GCCATGGAAA

CAGCTGACAA

TGCAAATTAT

TTTTAAAGAG

TAACTTTAGT

TTAATTTTTA

TTTTAAGAGA

TTGCGGTATA

ATGGAAAACC

TTATGGACAC

GGGAAACACA

AGACATAAAA

TATCTCTTAT

AGCGGAAGAA

TAGTAATCCA

TCACCAACAG

TTACACTTGT

TATGCCTGTA

ACAATTGTTA

GTTTTAAGAA.

TCTTTAGATT

CGGGAGTAGA

TCGCATATTG

AAATAAGTGT

TATTTTGTCG

ATGAAAATCT

TTTTTTCACT

GATTTCTTAA

TGAAAGCAGA

GCAATAGGGA

AGGCCCTTGC

TCTCCAAATC

ACAGGGAAAT

TTGGCAAGAA

TTTCTTCTAC

GTGGTTGTTT

GAAAATTTGA

ACTTCTGACT

ACCTGTTAAA

SUBSTITUTE SHEET

A

r PCT/US92/04354 WO 92/20356 INFORMATION FOR SEQUENCE ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Leu Pro Tyr Leu Gly Trp Leu Val Phe SUBSTITUTE SHEET i- WO 92/20356 PCr/US92/04354 INFORMATION FOR SEQUENCE ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 2418 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGATCCAGGC CCTGCCAGGA GGGGTCATCC ACTGCATGAG GGCCCGTGGA TTCCTCTTCC TGGTCTGAGA CAGTATCCTC GCCAGCAGTG AATGTTTGCC CAGGACACAT AGGACTCCAC CCTGTAGAAT CGACCTCTGC VTTCCTCCTTC AGGTTTTCAG CTGGAGGCCA CAGAGGAGCA TTAGAGTCTC CAAGGTTCAG CTCTCCCCAG GCCTGTGGGT GCCTGCTGCC CTGACGAGAG ACTGCAAGCC TGAGGAAGCC GTGTGTGTGC AGGCTGCCAC CCTGGAGGAG GTGCCCACTG AGGGAGCCTC CGCCTTTCCC CCCAGTGAGG GTTCCAGCAG TATCCTGGAG TCCTTGTTCC TGGTTGGTTT TCTGCTCCTC GCAGAAATGC TGGAGAGTGT GATCTTCGGC AAAGCCTCTG TGAAGGAAGC AGACCCCACC GGTCTCTCCT ATGATGGCCT AGGCTTCCTG ATAATTGTCC CTCCTGAGGA GGAAATCTGG GGGAGGGAGC ACAGTGCCTA TTTGGTGCAG GAAAAGTACC CGCACGCTAT GAGTTCCTGT ATGTGAAAGT CCTTGAGTAT TTCTTCCCAT CCCTGCGTGA CTGAGCATGA GTTGCAGCCA ACCTTCCAGG GCCGCGTCCA SCCCATTCTTC ACTCTGAAGA TGTTCTATTG GGTGACTTGG TCAAATGTTT TTTTTTAAGG, TATGAATGAC AGCAGTCACA GTCTTGTGTT TTATTCAGAT ATAATAACAG CAGTGGAATA AAATAGATGA GATAAAGAAC CTTATACCTC AGTCTATTCT GATTTCCTTG, GCTTCTTTGA AATTCTTCCT GTTCACTGGC TTTTTGGAAG GCCCTGGGTT

AAAATATAAG

AGTGGGGATG

CAGGGCTGTG

TGGAGCTCCA

AGGTCACAGA

CTGAATGCAC

AGAGTCTGGC

TGGCCGGCTG

GGGACAGGCC

CCAAGGAGAA

TTCTCAGCTG

CTTCATTGCC

TCATCATGTC

CTTGAGGCCC

CTCCTCCTCC

CTGGGTCAAC

ACTACCATCA

CCGTGAAGAG

GAGCAGTAAT

AAATATCGAG

CATCAAAAAT

AGTCCTTdCA

GGCCACTCCT

GCTGGGTGAT

TGGTCATGAT

GAGGAGCTGA

TGGGGAGCCC

TGGAGTACGG

GGGGTCCAAG

GTGATCAAGG

AGCAGCTTTG

AGGCCAGTGG

GCAGCTTCCC

GAGCGGTCAG

AGATTTATCT

GATGGTTGAA

CAGTTCTGTG

TGGGAAATCC

AGTACTTAGA

TAAAGAAATT

GTAAAATTTT

GAATGTAAGA

TCTTTTCTTC

AGTAGTGGAG

GGCCCTGCGT

TCACAGAGTC

CTTGCGGTCT

GGAACCAGGC

GCAGAGGATG

ACCAAGGGCC

CTCACCTCCC

TACCCTGAGT

AACCCAGAGG

GATCTGTAAG

AGGCCTCTCA

CAGCTCCTGC

TCTTGAGCAG

AACAAGAGGC

TCTCCTCTGG

AGATCCTCCC

ACTTCACTCG

GAGGGGCCAA

CACTAAGAAG

CCAGGGAGCC

TACAAGCACT

GCTGGTCTTT

ATGTCCTTGT

AATCAGATCA

TGCAATGGAG

GTGTGATGGA.

AGGAAGCTGC

CAGGTGCCGG

GGCCCTCGCT

TCAGTGCAAG

AGAGAGGAGG

GAGGGGGACT

CTGCCTCGTG

TGTTCTCAGT

TTGTTCTCTT

TGAACTTCAG

TATATAGTTT

ATTCTATTTT

AATGTGAAAA

AAGAGATAGT

TAAAGATATA

GAAATTAAAT

TCCATG;CACT

ATGCTAAGGT

GAGAACAGAG

CAGCCCACCC

GCACCCTGAG

AGTGAGGCCT

CACAGGGTGT

CCACCTGCCA

TACTGTCAGT

ACCCTCTCAC

ACAGGATTCC

TAGGCCTTTG

CACACTCCCT

CCACACTCCT

AGGAGTCTGC

CCTGGGCCTG

TCCTGGGCAC

CAGAGTCCTC

ACAGAGGCAA

GCACCTCTTG

GTGGCTGATT

AGTCACAAAG

GTTTTCCTGA

GGCATTGACG

CACCTGCCTA

TGCCCAAGAC

GGCGGCCATG

GGTGTATGAT

TCACCCAAGA

ACAGTGATCC

GAAACCAGCT

AGTTCGCTTT

AAGAGGGAGT

GGGCCAGTGC

TGACATGAGG

AGTAGGTTTC

TTGGAATTGT

CATCCAAGTT

AAGGGTAAGA

GTGAATTGGG

ATGAGCAGTA

CAATTCTTGC

TGCATACCTG

CTGAATAAAG

GAGCATCTGC

AAGCCAGACT

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 SUBSTITUTE SHEET -4

I-

WO 92/20356 WO 920356Pr/US92/04354

CATACCCACC

AGGTGGCAAG

GGGTGTGGGG

GGCATTTTGG

AATGATCTTG

CATAGGGTCG TAGAGTCTAG GAGCTGCAGT CACGTAATCG ATGTCCTCTA AAGATGTAGG GAAAAGTGAG AGAGGGGTGA CTCCGGGTGA GAGTGGTGGA GTGTCAATGC CCTGAGCTGG GCTTTGGGAA ACTGCAGTTC CTTCTGGGGG AGCTGATTGT

GGTGGATCC

2250 2300 2350 2400 2418 SUBSTITUTE SHEET WO 92/20356 PCr/US92/04354 INFORMATION FOR SEQUENCE ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 5724 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-1 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CCCGGGGCAC

TACGCCACCC

AGAATCCGGT

ATGTGACGCC

CGGTCTGAGG

TAAGGAGGCA

AGATAGAGGA

GGTGGACTTC

CTGGGGACTC

AGAGGGCAGC

AGGGCTGAGG

ATGCTCACTC

CCCCACATCC

ATTCCACCCT

CAGGCAGGAT

GCCACTGACT

AGGGACGGCG

AGGCAAGGTG

GAGAGCCCCA

CGCGGGAAGA

CCTTGAGAGA

TGTGACCAGG

GGCATCAAGA

ACTCCAATCC

CCCATCTCC7

CCTGACCACC

TCACCCTCAC

CCCATCGCC7

CCAGGGAAGC

AGATCTGAGP

ATCCACTGAC

CTGAGGGAGC

ATCCAGTACC

GTCTCAGCTC

GGCAATCTGJ

GGCAGGGCC(

ACCCTGGGA(

CACCGCCAC(

TCTTGTCAG]

CAGGCACTC(

GCTTGAACA(

GGGAGGCCT1

ACTGAGGCTI

CACTGGCATC

ATCCAAACAT

TCCACCCCTG

ACTGACTTGA

GGCGGCTTGA

AGGTGACATG

CCCCAAATAA

TCAGGCTGGG

GAAGTCAGAG

GTCCAGGCTC

GTCCCTAAGA

CCGTGACCCA

CCCACCCCAT

CACCCCCACC

CCGGTTCCCG

TGCGCATTGT

TAGAGTTCGG

AGAGGCTGAG

AATATTCCAG

CGTCTCAGCC

CACCAGGTTC

GCAGGACTGG

TCAGCACCCA

CCACTCCCAC

CAGCTACACC

ACCCTCCAGC

TGCCCCCAAC

CCCCCATTCI

CCTGGTAGGC

GAAGCCAGG

GGGAGTGGTJ

ACTGAGGAGC

ACCCCTGCT(

GACCACCCC(

AGTCATAGC'.

AGO CATCAA(

GGAACTGAGI

CCACTCACA'

k ATCCCTGCT1

GATCTTGAC,

SGGCCTCAGG,

AGAGGACCC

7CCACTTCTG

CCTCCCCCTA

CTTCACGCTC

CTCTCAACCC

GCATTAGTGG

GATCGGTGGA

CTGAGGGAGG

TCCCTTCATG

CCACCCCCAG

CTCCGTGTGA

TGCCAGACAT

CCCCACTCCC

ACCCCCTCTT

CCCTCAACCC

CCCACCCCCA

CCAGGAAACA

GGGGCAGAGA

CCGAAGGAAC

GGAGGACTGA

CCCCGCCCTT

TGGGCTGCCC

TTCTCCCCAA

TTAGGAGAGG

AGAGGGAGGG

*CCCATTCGCA

TCCACCCCCA

-CCCAGCACCA

CCCACCCTCA

7GGCAGAATCC

:CCGATGTGAP.

TCATTTAATG.

r' TTAGGCTCTC

;CACACACCCC

;CCAGCCCTG(

'CGTCCCGTCC

r' TATGTGACCC

GTCCAGCAT(

GTTCCCCACC

r TCCCATACC! aTCAACCCACC G TCCCCATCCJ G GAGCAGAGG4 A GCACCCTAG4 G CCTCAAGAA'

CCACCCCCAA

ACCCCCAGCC

AGGGAAGCCC

TTAGAGAGAA

GGGAAGCGGG

ACTGAGGACC

CCAGTCCTGG

CCCCCTTGCT

TCAGGGAAGG

CATGCTCAGG

GTGACCCAAC

CATTGTCATT

TGATGCCCAT

CGCCCACTCC

TCCGGGTGCC

GAAGCGAGGT

CTGACCCAGG

GGACCCCGCC

GCTGCCAGCC

CCAGACCCCT

GCTCTGGAAT

GCAGGGCACA

CTGTGGGCCC

TTCCCATTCC

*TCCCTACTCC

GCCCCAACCC

,TCTCTCTCA1

GGTTTGCCCC

ACCACTGACI

GTTCTGAGGC

TGAGGAGGCI

AGGTAGATGC

ACCACCCGG(

CACTGCCAC'

GG4GCAGGGT'

CGCCCGGCA

CACACCTGT(

V ACCCCCTACI GAAGCCACG4 k GGGTCTGAT1

;AGGGCCCTA,

;ACACCGCAC,

r CAGAACGAT,

TCCCTCCCTT

CAAGCCAGGC

AGGTGCCCAG

GCGAGGTTTT

CCCAGCTCTG

CACTTACCCC

ACCATCTGGT

GCTTAAACCA

GCTGCTTAGG,

ATTCTCAAGG

CCCCACTCCA

CCAACCCCCA

CCGCCCAGCC

CACCCCCACC

CGGATGTGAC

TTCCATTCTG

CTCTGTGAGG

ACTCCAAATA

CTGGCCCACC

GCTCCAAAAG

CAGAGGTTGC

GGCTCTGCCA

CCAAGACTGC

CCACCCAACC

TACTCCGTCA

TTCTGCCACC

GTGCCCCACT

TGCTCTCAAC

TGAACCTCAC

GCGGCTTGAG

k AGGTGAGATG

;CCCCAAAATG

'CAGGACAGAT

C TAACCCACAG r GGTCAGGAGA r TAGGGTCAGG

TCCTCATCTC

CCCAACCTCA

3 GAATGGCGGC a GAGGGAAGGG C TGCGAGATGA C CCTGTCTGAG G GGGACTCAGA 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 SUBSTITUTE SHEET pp. WO 92/20356 PC1'/US92/04354 77

TTGCATGGGG

GGGAGGACTC

GAGAGGTCCA

TGAGGTGACA

AGAGGGAGGA

AGGACTGGGG

CTGTCCCCTT

TTCCATTCTC

ATGGGGTCTT

GGTTGAGGAA

AGGCTATTGG

TCACCCAGGA

GTGAGGACCT

TCTGGTCTAA

GAACATGAGG

CTGCACAGAA

GGGCCGTCTG

ACGGGGAGGC

GTCCCAGGCC

CAGGACACAT

AAGGACCTAG

TCCTTATCAT

GGGCAGGATC

CAGAGGGGGT

CACCCTCCTG

CTGAGGGCCC

GGCCTTGGTC

GGTGTGCCAG

TGCCACAGGA

TCAGTCCTGT

CTCACTTCCT

ATTCCCTGGA

CTTTGTTAGA

TCCCTCTCTC

CTCCTGCCTG

GTGGGACCCA

AGGGGACCTT

GGGCACGGTG

GGACAGAGCT

GTTCCAGGAT

ATATCCCCGG

TTAGTAGCTC

ACTTGTACCA

GGGGTAAAGG

GCACAGGCGC

AATCCACACC

TGTGGCTTCT

CATTCTCAGA

AGACAGAGCG

GAGGACTGAG

ATCAGCCCTG

CCGAGGTCCT

CTTGGTCTGA

CTGCCAGGAG

TAATTCCAAT

GCACGTGTGG

GGATGTGAAC

CAGGCCCTGC

CATCCACTGC

GTAGCACTGA

GTGGATTCCT

TGAGACAGTA

CAGTGAATGT

CACATAGGAC

AGAATCGACC

CCTTCAGGTT

GGCCACAGAG

*GTCTCCAAGG

CCCAGGCCTG

*CTGCCCTGAC

GGCCTGCAAG

GGAATCCAGA

GCCACATATG

GTGGTCTGAG

CCATATGGCC

CTCAGAAAGA

TAGGGGGACC

CAGGCAGGAA

GGGGATGTCT

TGGCAGGAAT

CCAGAACCAA

TTTTCACTCC

GGGTGACTCA

GTCCCAGGAT

GGTACCCCAG

CCCCTGCTGT

TCCGTTATCC

GAAGGCTGCG

TCAAGGTGAG

GAATTTTGAT

CCAGATGTTT

TCTTGATTTG

CAGGAAAAAT

ATGAGAGTGG

GAAGCCAGGG

CTTCCTGGAG

TCCTCAGGTC

TTGCCCTGAA

TCCACAGAGT

TCTGCTGGCC

TTCAGGGGAC

GAGCACCAAG

TTCAGTT CT C

TGGGTCTTCA

GAGAGTCATC

GCTTACGCGG

TCAGTGTGGA

GCCCATATTT

AAGTGGGGCC

CAAGATGTGC

AGGGACTCCA

AGATCAGGGA

GTTGGGGGGC

ACTCATGTCA

!4AAGATGAGT

AGGGGTCAGC

TGTTTCCAGA

GGTCAACGTA

CTGCCATGCG,

GACCAGAACA

CACCCCAGAG

TGGGATCATT

CTCAGGTCAG

GACCAAGCGG

ATCTCTTGCT

GTCCCCTCCT

GATTTCTCAG

ATAAGGGCCC

GGATGTCACA

CTGTGCTTGC

CTCCAGGAAC

ACAGAGCAGA

TGCACACCAA

CTGGCCTCAC

GGCTGTACCC

AGGCCAACCC

GAGAAGATC7

AGCTGAGGCC

TTGCCCAGC7

AGGAAGAGGA

CCTCGGCCCT

CCTGCATCTT

TCAGGTCAAC

CCCCTTCATG

CACAGTCTGG

TGGCGGTATG

CCTCAGGGAG

GGGAATTGGG

GAGACAGACA

CCTGGACACC

TCTGGGGCAG

GGGACCCCCA

TTCGGGTGAG,

CTGAGGGAGA

AGCATGGGCT

GATGTCAGGG

TAGAGGGAGC

GCACCTCACC

GCCCTTCCCC

GTCCTTCCAT

ACCAGCAAAA

TGCGTGAGAA

GAGTCCAGCC

GGTCTGCACC

CAGGCAGTGA

GGATGCACAG

GGGCCCCACC

CTCCCTACTG

TGAGTACCCT

AGAGGACAGG

GTAAGTAGGC

TCTCACACAC

CCTGCCCACA

2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3930 3972 4014 4056 4098 4140 4182 4224 4266 4308 4350 4392 4434 4476 4518 4560 4602 4644 4686 4728 4761

ATG

GCC

CAG

CTG

AGT

ACT

GAG

CGA

CTG

GAA

CCT

TTT

TAT

CTG

ATT

GAG

GAT

CTC

AGG

GTC

TCT

CTT

OCT

GAG

CCT

CGA

GAG

GCA

CTC

ATG

GAG

GGC

GTC

GOT

GTC

GAG

000

ACC

TGC

CAA

CTT

GAG

GCC

GAG

CAG

CAG

000

GTA

CTC

CTG

ATC

ATT

CTT

GAT

CTG

GAA

AGO

CAA

CGG

GG

GAG

GCC

ACC

GTG

GGA

AGG

CCA

ATC

AAA

GAG

TTC

GAC

GTC

AAT

GTC

ATC

GAG

GAT

ACA

CCC

CAG

CAA

TCC

CCC

GCC

CAA

AGC

ACT

TAT

AGT

GGC

GTG

ACC

CAG

ATG

TG

CAC

TTG

GTG

TCG

AGG

CAA

TCC

ACT

TCC

CCC

ACC

AAG

CGA

GTC

AAA

AAG

TGC

ATC

ATT

GAG

AGT

GTG

ATC

CTG

AGT

GAG

TCC

GCT

GCC

AGT

TCT

AAG

GCC

ATC

GCC

GAA

CTA

ATG

OCA

GAG

GCC

CAG

CCG

AAA

CTG

GCC

TCT

GGG

TTT

GAG

TGT

GTG

AGG

AAA

TCT

OCA

GOT

CCC

ATG

CTG

TAT

GAA

CAC

CCA

CAC

CTG

CCT

TCA

CCC

GGT

ATC

GCT

GAG

AAT

GAG

GAC

CTC

AAG

GAG

AGT

GG

AAG

OCT

GCT

TGC

GOC

CTG

ACA

ACT

TCC

CTG

GAT

CCA

TAC

TCC

CCC

TCC

ACA

GGC

OTG

GAG

TAC

ATG,

ATG

AAG

CTG

OTC

OAT

ACC

AGC

GAG

TTG

GTC

AAG

TTG

ACC

TAT

GGC

GOC

ATG

CCC

CTG,

AGT

TGA

CCT

GTG

CTG

CCT

ATC

AGC

TCC

OTT

ACA

CAC

CAG

GGC

OAT

TTC

CAT

GAG

AGG

GAG

TCC

GAG

TGT

GOC

CCC

AAC

COT

TTG

GT

AAO,

TGT

CTG

CAC

GGC

CTO

OCT

GTG

AAG

TAC

TGT

GAA

GTG

ACC

CAG

TTC

GAA

TTC

TTT

GCA

TTT

GTC

TCC

CTG

ATA

CCT

TAT

CTG

GOC

GG

SUBSTITUTE SHEET -7 WO 92/20356 PCr/US92/04354 73

AAGTCCTTGA

GCTTTTTCTT

GGAGTCTGAG

AGTGCACCTT

TGAGGCCCAT

GTTTCTGTTC

ATTGTTCAAA

AAGTTTATGA

TAAGAGTCTT

TTGGGATAAT

CAGTAAAATA

CTTGCCTTAT

ACCTGGATTT

TAAAGAATTC

TCTGCTTTTT

AGACTCATAC

AATCGAGGTG

GGTGAGGGTG

GCTGGGGCAT

ATTGTAATGA

GTATGTGATC

CCCATCCCTG

CATGAGTTGC

CCAGGGCCGC

TCTTCACTCT

TATTGGGTGA

TGTTTTTTTT

ATGACAGCAG

GTGTTTTATT

AACAGCAGTG

GATGAGATAA

ACCTCAGTCT

CCTTGGCTTC

TTCCTGTTCA

GGAAGGCCCT

CCACCCATAG

GCAAGATGTC

TGGGGCTCCG

TTTGGGCTTT

TCTTGGGTGG

AAGGTCAGTG

CGTGAAGCAG

AGCCAAGGCC

GTCCAGCAGC

GAAGAGAGCG

CTTGGAGATT

TAAGGGATGG

TCACACAGTT

CAGATTGGGA

GAATAAGTAC

AGAACTAAAG

ATTCTGTAAA

TTTGAGAATG

CTGGCTCTTT

GGGTTAGTAG

GGTCGTAGAG

CTCTAAAGAT

GGTGAGAGTG

GGGAAACTGC

ATCC

CAAGAGTTC

CTTTGAGAGA

AGTGGGAGG4G

TTCCCCTGCC

GTCAGTGTTC

TATCTTTGTT

TTGAATGAAC

CTGTGTATAT

AATCCATTCT

TTAGAAATGT

AAATTAAGAG

ATTTTTAAAG

TAAGAGAAAT

TCTTCTCCAT

TGGAGATGCT

TCTAGGAGCT

GTAGGGAAAA

GTGGAGTGTC

AGTTCCTTCT

GGAGGAAGAG

GGACTGGGCC

TCGTGTGACA

TCAGTAGTAG

CTCTTTTGGA

TTCAGCATCC

AGTTTAAGGG

ATTTTGTGAA

GAAAAATGAG

ATAGTCAATT

ATATATGCAT

TAAATCTGAA

GCACTGAGCA

AAGGTAAGCC

GCAGTCACGT

GTGAGAGAGG

AATGCCCTGA

GGGGG.AGCTG

4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450 5500 5550 5600 5650 5700 5724 SUBSTITUTE SHEET L- L_

I-

pr PCr/US92/04354 WO 92/20356 794 INFORMATION FOR SEQUENCE ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 4157 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-2 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCCATCCAGA

CCCAGGGAAG

GGTCAGAGGA

GAGGGAAGCA

AGGACTGAGG

GCTGCCTCTG

GCTCAGTCGC

GGAACTCTGG

TGCTCAGGGC

GACTGAGGGC

ACCAACCCCA

TCCCATCTCC

CAATCAACCC

ACGTTCACAT

GGCCTTTGGG

TCCTTAGGGG

TCAAACTGAG

CCCACTTCAG

AAGAGGGAGG

GCTGGGGGAT

CTTCAGGGTG

GGTCAGCAGA

CTTCATGAGG

AGTCTGGAAG

CCCTAAGTGA

CAGGGAGATA

GGTTCCCCCT

CCACAGGAGG

GGACAACGCA

CAGATCTCAG

ACAGGGGCCC

ATCCAGGTGG

GCAGCAAGGG

AGACCCTGGG

TGATGTCAGC

GTAGAGGGAG

GACTCGTCAC

GTCCTTCGCC

TCTCCTTCTC

CAAGCCAGC;

CCTGAGTGAC

CGGAGTCTG(

GCAGTCTGC)

TCCCCATCCG

TCACGGGCCC

CAGCGAGATT

GGCGCAGGCT

CGGGCCTCAC

CTGCCGGGCC

CACCACCTCA

CGTAAGAGCT

CCAGACTCAG

AACCCACCCC

CCCCCATCCC

TCCCCCACCA

ACGGAAGCTC

GTACGGCTAA

ATGCAGAGGA

ACCCAGCATG

CCACCTTTTC

GGGGTTGGGG

ACTGAGGGGA

CCTGGGCACA

ACAGAGAGTT

GGGAGGAATC

ACTCCCCATA

TAAATTGTTC

CAATCTCATT

AGGTGTTGG7

TGAGAAAGGG

CCATCATAAC

CGTGGGGTAP,

GGAGTTGATG

CTCTGGTCG;

AGAGCCTGAG.

GGCCCCATAC

CAGGGCTGTC

SGAAGGGGAG(

SGGTCTCAGGC

CCAGGACACC

SGAGGACCTGC

STACCATATC)

i AAAGGGTGGC

SCACAGAGGG(

SCCAACCCTG(

k CACTGAAGG(

GGCAGAATCC

GGATGTGACG

CTCGCCCTGA

CCGTGAGGAG

CCCAGACAGA

TGGACCACCC

CCCCGCCACC

TTGTGTGACC

CCAGGAATCA

CTACCCTCAC

TCAAACACCA

CCATCCTGGC

CGGGAATGGC

GGGAGGGAAG

AGGGCCCAGG

CCAGGACAGG

ATTCAGCCGA

CCCAGCCTGC

CCTTGGAGTC

GTGGCCGAAT

GAGGGCTGTG

CCAGGATCTG

CCCCCGGCCC

TTAGCTCTGG

TGTACCACAG

GTAAAGAGGA

CAGTCCCTGG

GTTCACCCTA

CAGGATGTGG

*ACCTTGTTTT

CAGATGCAGT

*GTAGGATTGA

AAATCTGCCC

*AGCTGAAGTC

CCTTGGTCTG

CCTGCCAGGA

TGGACTCCAA

STCACGTATGG

k GGGATGTGAG ;ATTAGGCCC72

ACCCTCCACC

TGAGACTTC1

SCCGTGCATTC

GGTTCCACCC

CCACTGACTT

GCAACGGCCT(

GCAAGGTAAG

GGGCCCCCAA

TGCAGGGGAA

CCCCGCCGCT

AGGGCAGGGC

AGGTCAGGAC

TACCAATCCC

ACCCCACCCC

AGAATCCGGC

GGCCAAGCAC

GGGTTGGGTC

CCTCCTGGAA

GGGCCCACTG

GGGAATCCTA

GAGGAGTCAA

CAGATCAGTG

GTGCCCCGTG

GTCTGAGGGC

CCGGACCCAA

AGAAAGAAGG

GGGAACCTGA

GCAGGAGGTT

GCTGTCTGCT

CAGGAGTAAA

GAACCAAAGG

CCCCTCCTCA

CAGAAGGTGA

GGTTCTAGGA

GGGTACCCCT

TGCCCCTGCG,

CCTCCATTAT

AAGGGGCTGG

GTGGACGTGA

TGAATTTGAC

CCAGATGTGG

TTCTTGACAT

ACAAGGAGAA

SCAAGTAGAGT

'GGGAATCCGT

CTCTCCCAGG

rTGCCGTGAA

;CACATTGGA

;ACGTCGGCG

DiCGCCGAGGG

TTAATCCAGC

GACTTCTCAG

TTAACCGCAG

TGGTTAGAAG

CCCAAGAGGG

A~TCCCCCAAC

CAAACCCCAT

TTTGCCCCTG

GCGGATCCTG

TCGTGAGTAT

GACAGTGGAG

TACCCCTGTC

GGGATGCAGA

GGGGAGGAAG

GCAACCTTGG

CTCATTGCAC

TGGGACTTCA

GGTGTGCCCC

GATOCCACAG

TCAGGGATGG

GGGGAACCCT

CATTTCAGGG

GATGAGTAAC

GGTCAGCCCT

CTTGTCTTTC

CTCAGTCAAC

TCTGCCAAGC

GGGCCAGAAT

GTTACTTCAG

CTGGGATCTT

AGTCAGGTCA

GGACCAAGCG

ATCTCTCGTT

GTCCCCTCTA

GAGAGATTCT

AGGTGAGGGC

GGGGACCTCA

GGCTGTGCTT

AATCAGGAGC

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 SUBSTITUTE SHEET WO 92/20356 PGI'/US92/04354

TCCAGGAACC

AGAGCAGAGG

CACACCAAGG

GCCTCACCCT

CTGTACCCTG

AGGCTGACAA

CTGTAAGTAA

TAAGGCCTCA

CCCAGCTCCT

AGGCAGTGAO

GGACGCAGAC

GCCCCACCCG

CCCTATTCTC

AGGTGCCCTC

GTAGGACCCG

GCCTTTGTCA

CACACGCTCC

GCCCGCACTC

GCCTTGGTCT

AGTGCCAACA

CCCAGAACAA

AGTCCTGCAG

CCACTTCCTC

AGGCACTOGA

GAGCCTCCAA

TTCTCTCCCC

CTGCCTGCTG

GAGTCAGTGC

CTGAAGGTTT

ATGGGACTCC

CCTGAGCATG

CTTCAGGTTC

OGAGCATTGA

GGTTCAGTTC

AGGCCTGTOG

CCCTGACCAG

CTCAGGTCAC

GCCTGGAATG

AGAGGGCCTG

TGCTGGCCOG

TGAGGGGGAC

AGGAGAAGAT

AGTTCTCACC

GTCTTCATTG

AGTCATC

CCT GAA GAK GTG GOT GO TCT TCC TC' CCT OCT GO

ATG

GGC

CAG

TCT

OAC

TTC

,GAG

ATG

AGG

AGA

TCC

GTG

GGC

CCC

ATA

CTG

TTC

GAA

GCA

ACC

OGA

TTG

CCT

CTT

GCT

ACT

TCA

TCG,

GGC

GAC

OTT

GAG

AAT

GAG

OTC

CTC

AAO

GAG

ACT

OCA

AAC

TOC

AOC

OAA

AGA

CTT

GAG

CCT

CTA

CCO

ACT

TCC

CTO

GAO

CCG

TOC

TAC

CCC

TCC

ACA

GOC

ATO

CAT

TAC

TAC

TAT

CCT

GAG

GAG

0CC

OCT

GTG

AOT

ACC

AOC

GAG

TTG

GTC

CAO

TTO

ATC

TAC

OOC

GAC

TTO

CCC

CTO

GAG

OTO

CAC

GGA

CAO

COA

ACT

OAA

CCT

ATC

AAC

TCC

OTT

ACA

OAC

CAO

AOC

OAT

CTC

TOT

GAG

AG

GAG

TTC

AAA

ATT

GAA

AG

GGA

GAG

OTT

CCC

AAC

CAA

GAG

CAT

AAO

TTC

CTG

CAC

OOC

CTG

0CC

GTO

AAG

TAC

CTG

OTC

TCC

GAG

AOT

GAG

GAG

ACC

CAC

TAC

OAA

TTC

TTT

OCA

TTT

OTC

TTO

CTG

ATA

CCT

TTT

CTG

COO

TOO

CTO

TAC

TGA

CAO

0CC

CAO

CTO

AOT

ACT

GAG

CAA

CTG

OAA

CCC

TTT

TAC

CTO

ATC

GAG

GAG

CTC

CAG

GGT

CAC

CCA

CAC

CTO

CAO

000

CCT

CTT

GAG

OCA

CTC

ATO

OTO

OGC

ATC

OOC

OTC

GAO

GG

ATO

OTO

CCA

CAT

CCC

TOC

OGC

ACC

GAO

CAO

TOO

000

OCA

CTC

CTG

ATC

ATC

CTT

OAC

CTO

AAA

AG

CAA

CCC

AG

ACA

CTG

AAG

CTO

OCT

OTO

GGA

AGA

CCA

ATC

AAG

GAG

TTC

GAG

OTC

AAT

0CC

ATC

GAG

OAT

OOC

0CC

CTA

CAT

0CC

CAA

AGA

AOT

TAT

AOT

AOC

OTO

ACC

CAO

ATA

TOO

GAC

CTG

AOT

CTC

AAG

GAA

TCC AOC TCC OAT ATO TTT AOG AAO COA 0CC OTC CTC AAA 0CC GTO OAA TOC CTO OTC ATG ATC OCA GAO GAG AOT OTC OTO CAO OAT CCT ATT GAA ATC OOT COO OCT 2200 2250 2300 2350 2400 2450 2500 2550 2597 2639 2681 2723 2765 2807 2849 2891 2933 2975 3017 3059 3101 3143 3185 3227 3269 3311 3353 3395 3437 3479 3521 3542 3592 3642 3692 3742 3792 3842 3892 3942 3992 4042 4092 4142 4157

OTCTCAOCAC

OCACCTTCCA

OGCCCATTCC

TTTCTGTTCT

TTOTTCAAAT

OTTTATOAAT

TAAGAOTCCT

TTOTCACATA

AATTAOCAOT

TGCCTTATAC

TOCTTCTTTO

TCACTOGCTC

CCTGGTAGTA

ATGTTGCNGC

GGOCCCCATC

TOCCTCTTTG

OTTOGATOAC

GTTCCTTTTA

GACAGTAGTC

OTTTTTTATT

ATAACAGCAO

AAAATACATO

CTCAGTCTAT

AGAATOCAAA.

ATTTCTTTAC

GTOG

CAGGGCCAGT

CATTAOCTTC

AAOAOAOCAO

TTTOAOATTT

ACAAATGOTT

ACACATAOTG

CAGATTGGGA

TGOAATATOT

ATACAAOOAA

TATOTAAAAT

AOAAATTAAA

CATTCACTCA

GGGAGGGGGT

CACTOCCTCO

TCAGCATTCT

ATCTTTCTTT

G0ATGAACTT

CTOTTTATAT

AATCCATTCC

ATTTOCCTAT

CTCAAAAGAT

TAAAAATATG

TCTOAATAAA

GCATCTGCTC

CTGOOCCAGT

TGTOATATOA

TAOCAOTGAG

CCTOTTGGAA

CAGCATCCAA

AOTTTAGGGG

ATTTTOTOAO

ATTGTOAACO

AOTTAATTCT

TGTATOTTTT

TTCTTCCTOT

TGTGGAAGGC

SUBSTITUTE SHEET I WO 92/20356 PCT/US92/04354 76 INFORMATION FOR SEQUENCE ID NO: SEQUENCE CHAP.ACTERISTIC6: LENGTH: 662 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-21 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

I>

GGATCCCCAT

AGGGAhGTCA

CAGAGAACAG

GGAAGCAGGC

ACTGAGGCGG

CCTCTGCTGC

AGTCGCCACC

CTCTGGTGTA

CAGGGCCCAG

GAGGGTAACC

CCCCACCCCC

ACGGCACCCC

GAGCTTTGCC

GCACGCGGAT

GGATCCAGGA

CGGGGCCGGA

CGAGATTCTC

GCAGGCTCCG

GCCTCACCCC

CAGGCCTGGA

ACCTCACCCC

AGAGCTTTGT

ACTCAGCCAG

CCCCCGCACC

A5'CCCCCAAC

CAAACCCCGA

CCTGCAATCA

cc

AGAATCCAGT

TGTGACGCCA

GCCCTGAGCA

TGAGGAGGCA

AGACAGAGGG

CCACCCTGCA

GCCACCCCCC

GTGACCAGGG

GAATCAAGGT

CCCACCACCA

ACCAAACCCA

TTCCCATCCC

ACCCACGGAA

TCCACCCCTG

CTGACTTGCG

ACGGCCTGAC

AGGTAAGATG

CCCCCAATAA

GGGGAAGACT

GCCGCTTTAA

CAGGGCTGGT

CAGGACCCCA

TTCCCATCCC

CCACCATCGC

CACCCATCCT

GCTCCGGGAA

CTGTGAACCC

CGTTGGAGGT

GTCGGCGGAG

CCGAGGGAGG

TCCAGCGCTG

TCTCAGGCTC

CCGCAGGGAA

TAGAAGTGCT

AGAGGGGACT

CCAACACCAA

TCAAACATCA

GGCAGAATCG

TGGCGGCCAA

1 1

I

SUBSTITUTE SHEET WO 92/20356 77 INFORMATION FOR SEQUENCE ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 1640 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: cDNA MAGE-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NOi 11: PCT/US92/04354

GCCGCGAGGG

GACAGGCTGA

AGATCTGCCA

AAGCCGGCCC AGGCTCGGTG CCTGGAGGAC CAGAGGCCCC GTGGGTCTCC ATTGCCCAGC

AGGAGGCAAG

CGGAGGAGCA

TCCTGCCCAC

GTTCTGAGGG

CTGAAGGAGA

ACTCCCGCCT

GTTGCCCTGA CCAGAGTCAT C

ATG

GC

CAG

TCT

GAG

CTC

GAG

CCT

GTG

AGG

GGA

TCC

GTG

GGC

CCC

AGA

CTG

TTG

GAA

GCA

ACC

GGA

TTG

CCT CTT GAG CTT GAG GCC GCT CCT GCT ACT CTA GTT TCA CCA GAT CCC ACT ACC GAC TCC AGC GAC CTG GAG GCC GAG TTG GAG CCG GTC AAT TGG CAG AGT TCC TTG GAC CCC ATC CTC TCC TAC AAG GCA GOC

AGG

GGA

GAG

GTC

CCC

AAC

CAA

GAG

CAT

AAG

TTC

CTG

CAC

GGC

CTG

GCC

GTG

AAG

TAC

CTG

GTC

TCC

GAG

AGT

GAG

GAG

ACC

CAG

TAC

GAA

TTC

TTT

GCA

TTT

GTC

TTG

CTG

ATA

CCT

TTT

CTG

CGG

TGG

CTG

TAC

TGA

CAC

CTG

GAG

GGG

CCT

CTC

GAG

GCA

CTC

ATG

GTG

GGC

ATC

GGT

GTC

GAG

GGG

ACC

GTC

CCA

CAT

CCC

TGC

GGC

GCT

GAG

CAG

TGG

GGG

GCA

CTC

CTG

ATC

ATC

TTT

GAC

CTG

AAA

AGG

CAA

CCC

AGG

ATG

CTG

AAG CCT GAA CTG GTG GGT GCC TCC TCC GTG CCT GCT GGA GCC TCC AGC CAA TCC CCA AGC ACC CTC AGT AGG AAG, TAT CGA GGG AGT GTC TTC AGC AAA GAG CTO ATG GCC ACC TGC AAT CAG ATC GCC ATA ATC ATC TGG GAG GAA GAC AGT CAT TTC GTG GGC AGT GAT 0CC CTC GTT GTA AAG ATC CAT GAG TOG 100 150 171 213 255 297 339 381 423 465 507 549 591 633 675 717 759 801 843 885 927 969 1011 1053 1095 1116 1166 1216 1266 1316 1366 1416 1466 1516 1566 1616 1640

GTCTGAGCAC

GCACCTTCCG

GGCCCAXTCT

TTTCTGTTCT

TTGTTCA.AAT

GTTTATGAAT

TAAGAGTCTT

TTGTGACATA

GAATTAGCAA

ATTCTTGCCT

ACCAGGATTT

GAGTTGCAGC

GGGCCGCATC

TCACTCTTTG

GTTGGATGAC

GTTCCTTTTA

GACAGTAGTC

GttTTTTACT

ATAATAGCAG

TAACATACAT

TGTACCTCAA

CCTTGACTTC

CAGGGCCAGT

CCTTAGTTTC

AAGCGAGCAG

TTTGAGATTA

ACGGATGGTT

ACACATAGTG

CAAATTgGGA

TGGTAAAAGT

GAGATAACTC

TCTATTCTGT

TTTG

GGGAGGGGGT

CACTGCCTCC

TCAGCATTCT

TTCTTTGTTT

GAATGAGCGT

CTGTTTATAT

AATCCATTCC

ATTTGCTTAA

AAGAAATCAA

AAAATTAAAC

CTGGGCCAGT

TGTGACGTGA

TAGTAGTGGG

CCTGTTGGAG

CAGCATCCAG

AGTTTAGGAG

ATTTTGTGAA.

AATTGTGAGC

AAGATAGTTG

AAATATGCAA

SUBSTITUTE SHEET mb- F- -7 WO 92/20356 ~78 INFORMATION FOR SEQUENCE ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 943 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-31 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: PCT/US92/04354

GGATCCTCCA

CCTGACAGTT

GCCCGTGGAT

AGGACTTGGT

GATAGTGCCA

CTGCCCCAGA

TTCAGTCCTG

CTCTCACTTC

AGAGGCCCCC

TTAGAGCCTC

TCCCTCTCTC

CTCCCGCCTG

CCCCAGTAGA

CTGGGAATCC

TCCTCTCCCA

OTGAGGCAGT

ACGGTGAAGG

ACACATGGAC

CAGCCTCAGC

CTCCTTCAGG

GGAGGAGCAC

CAAGGTTCCA

CCCAGGCCAG

TTGCCCTGAC

GAG CAG A( GCC CGA G( GCT ACT G1 k GTT GAA G.

k GAT CCT C( r ACC ATG Al AGC AAC C; ;GAG TCT Gi STTG OTT Ci

GTGGGGACCT

GTGGCTGCGT

GGAATCAGGA

GTCCTCAGGT

TTTGCCTTGG,

TCCAGAGCGC

ATGCGCTGGC

TTCTGAGGGG

TGAAGGAGAA

TTCAGTACTC

TGGGTCTCCA

CAGAGTCATC

CACAGAGTCT

TTGCTGTCTG

GCTCCAGGAA

CACAGAGTAG

ATTCAAACCA

CTGGCCTCAC

CGGATGTACC

ACAGGCTGAC

GATCTGTAAG

AGCTGAGGTC

TTGCCCAGCT

GGCCAACCCT

CACATTGGGG

CAAGGCAGTG

AGGGGgCTCA

AGGGCCCCAC

CCTCAATACT

CTGAGGTGCC

CTGGAGGACC

TAAGCCTTTG

TCTCACATGC

CCTGCCCACA

CCT GAA GAA GTG GOT GCG SUBSTITUTE SHEET

BW

)92/20356 PCU/US92/04354 -7-9 INFORMATION FOR SEQUENCE ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 2531 base pairs TYPE: nucleic acid TOPOLOGY: linear (1i) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-4 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GGATCCAGGC

GGGATCATCC

TCTTGATGGC

GGCCCATGGA

TGGTCTGAGA

GCCAGCAGTG

CAAGACACAT

CCTGCAGAAT

CTTCCTCCTT

TGGAGGCCAC

TAGAGCCTCT

CCTGCCTGGA

ACTCCATGAG

ACTGAGGGAC

TTCCTCTCCT

CAGTGTCCTC

AATGTTTGCC

AGGACTCCAA

CGACCTCTGC

CAGGTTCTGA

AGAGGAGCAC

AAGATTTGGT

GAAATGTGAG

AGTGGGGACC

CGGGGCTGTG

AGGAGCTCCA

AGGTTACAGA

CTGAATGCAC

AGAGTCTGGC

TGGCCGGCTA

GCAGACAGGC

CAAGGAGAAG

TCTCAGCTGA

CCCATTGCCC

GGCCCTGAGT

TCACAGAGTC

CTTACAGTCT

GGAACAAGGC

GCAGAGGATG

ACCAAGGGCC

CTCACCTCCC

TACCCTGAGG

CAACCGGAGA

ATCTGTAAGT

GGTCTCTCAC

AGCTTTTGCC

GAACACAGTG

CAGCCTACCC

GCACCCTAAG

AGTGAGGCCT

CACAGGCTGT

CCACCTGCCA

TACCATCAAT

TGCTCTCTCA

CAGGATTCCC

AAGCCTTTGT

ATGCTCCCTC

TGCACTCTTG

TCTCCGTAGG CCTGTGGGTC CCTGCTGCCC TGACCAGAGT CATC

ATG

GGC

CAG

TCC

GCT

GCC

AAT

TCG

A.AG

GCC

ATC

GCC

GAA

CTG

TTT

GCA

GAG

GTC

CAG

CCT

GAA

AAT

GCT

TCT

GTT

GCT

TCT

GAG

TTA

GAG

CCT

GTG

AAG

AAA

TCC

GTG

GGC

CCC

ATG

CTG

TAT

GAIN

GCG

ACC

GCA

TTG

TCT

GAG

CCT

CCT

TCA

CCC

GGT

GAC

GAT

GAG

AAT

GAG

GAC

CTT

AAG

GAG

GGT

GGG

AAC

coc

AGC

AGA

TTA

GAG

0CC

ACT

CTG

GCA

ACT

TCC

OCA

GAG

CTG

TAC

TCC

CCC

TCC

ACA

GGC

GTG

GAG

TAC

TAT

TAT

GTT

GAG

CAG

CAA

ACT

GTC

GGT

ACC

AGC

GAG

TTG

GTC

AAG

CTG

GCC

TAT

GC

GAC

ATG

CCC

CTG

GAG

GTC

CGC

GAG

AAG

GAA

GAG

CCT

CCT

ATC

AGC

TCC

GCT

ACA

CGC

AAG

AGC

GAT

CTT

AGC

GGG

AGG

GAG

TTC

ATT

GAA

PAGT

GAG

GAG

GGC

CCC

AGC

CAA

TTG

CAT

AAG

TGC

ATG

AAC

GGC

CTG

GCC

GTG

AAA

TAC

CTG

GTC

0CC

GAG

CAG

GCC

CAG

ACC

CAG

TTC

GAA

TTC

TTT

GCA

TTT

ATC

ACC

CTG

ATA

TCT

TAT

CTG

CGG

TG

CTG

TAC

GGA

CAC

CTG

GAG

CTG

AGT

ACT

GAG

CGA

CTG

GAA

CCT

TTT

TAC

CTG

ATC

GAG

GAT

CTC

CAG

GGT

GAG

CCA

GTC

TOC

GC

GCT

GAG

CCT

TGC

GAG

GAA

CTC

ATG

GTG

GC

ACC

GGT

GTC

GAG

GG

ACC

GTA

CCA

CAT

TCC

TGA

P.AG

CTG

GCT

GAA

CAG

TGG

GGG

GCA

CGC

CTG

ATC

ATT

CTT

AAT

CTG

GAA

AGG

CAA

CCC

AGG

GTG

CTG

CCT

GTG

GTC

GTG

GGA

AGG

CCA

CTC

AAG

GAG

TTC

GAC

GTC

AAT

GGC

ATC

GAG

GAT

GGC

GCT

GTC

CGT

GAG

GT

TCC

CCT

GCC

CAA

AGC

AOT

TAT

AGA

GGC

GTG

ACC

CAG

ACA

TG

CAC

TOG

AGT

CTG

AGG

GAA

GAA

GCA

TCC

GCT

TCT

CCC

ACC

AAC

CGA

GTC

AAA

AAG

TGC

ATC

ATT

GAG

ACT

GTG

AAT

OCT

GTC

GCA

100 150 200 250 300 350 400 450 500 550 600 624 666 708 750 792 834 876 918 960 1002 1044 1086 1128 1170 1212 1254 1296 1338 1380 1422 1464 1506 1548 1578 1628 1678 1728 1778 1828 1878 1928

OCATGAGTTG

ATCTAACAGC

CATTCTTCAC

TCTATTTTGT

GTTGAAATGT

TTATGAATCG

AGTCTTGTTT

CAGCCAGGGC

CCTGTGCAGC

TCTGTTTGAA

TGGATGACTT

TCCTTTTAAT

TAGTTAACGT

TTTATTCAGA

TGTGGGGAAG,

AGCTTCCCTT

GAAAATAGTC

GGAGATTTAT

GGATGGTTGA

ATATTGCTGT

TTGGGAAATC

GGGCAGGGCT

GCCTCGTGTA

AGTGTTCTTA

CTCTGTTTCC

ATTAACTTCA

TAATATAGTT

CGTTCTATTT

GGGCCAGTGC

ACATGAGGCC

GTAGTOGGTT

TTTTACAATT

GCATCCAAGT

TAGGAGTAAG

TGTGAATTTG

SUBSTITUTE SHEET r WO 92/20356 PGI'/US92/04354

GGACATAATA

GAAATAGGTG

GTCTATTCTG

CTTCGTGAAT

ACTGGCTCAT

AGGATTAGTA

GGGTATTAAG

CCTCTAAGAT

GAGAGTGGTC

AACTGCATTT

AGGGCCAGAT

TCTGAGCAGT

GGG

ACAGCAGTGG

AGATAAATTA

TAAAATTTAA

GTAAGAGAAA

TTCTTCTCTA

GTGGAGATAC

AGTCTAGGAG

GTAGGGGAALA

GGGTGTAAAT

TCTTCTGAGG

TCTCAGAGGG

TCCTTTGTGA

AGTAAGTATT

AAAGATACTT

AAATATATAT

TTAAATCTGA

TGCACTGAGC

TAGG4GTAAGC

CGCGGTCATA

AGTAACGAGT

TCCCTGTGTG

GATCTGATTC

AGAGGGAAAA

CAATGGATGA

TAGAAGTGTG

AATTCCCGCC

GCATACCTGG

ATAAATAATT

ATCTGCTCTG

CAGACACACA

TAATTAAG4GT

GTGGGTATGG

GGGCCTTTTG

TAATGAAGCT

GCCCAGATTG

ACAGAGAGGA

AATTCACCGT

TTATGCCTCA

ATTTCCTTGG

CTTTCTGTTA

TGGAAGGCCC

CCTACCGATA

GACAAGATGT

GGCTCCAGGT

GGCTTTGGGA

TGGTGGGTCC

GAAAAGTTGC

GCCTCTACCT

1978 2028 2078 2128 2178 2228 2278 2328 2378 2428 2478 2528 2531 SUBSTITUTE SHEET p WO 92/20356 PCT/US92/04354 INFORMATION FOR SEQUENCE ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 2531 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-41 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GGATCCAGGC

GGGATCATCC

TCTTGATGGC

GGCCCATGGA

TGGTCTGAGA

GCCAGCAGTG

CAAGACACAT

CCTGCAGAAT

CTTCCTCCTT

TGGAGGCCAC

TAGAGCCTCT

TCTCCGTAGG

CCTGCTGCCC

CCTGCCTGGA

ACTCCATGAG

ACTGAGGGAC

TTCCTCTCCT

CAGTGTCCTC

AATGTTTGCC

AGGACTCCAA

CGACCTCTGC

CAGGTTCTGA

AGAGGAGCAC

AAGATTTGGT

CCTGTGGGTC

TGAGCAGAGT

GAAATGTGAG

AGTGGGGACC

CGGGGCTGTG

AGGAGCTCCA

AGGTTACAGA

CTGAATGCAC

AGAGTCTGGC

TGGCCGGCTA

GCAGACAGGC

CAAGGAGAAG

TCTCAGCTGA

CCCATTGCCC

CATC

GGCCCTGAGT

TCACAGAGTC

CTTACAGTCT

GGAACAAGGC

GCAGAGGATG

ACCAAGGGCC

CTCACCTCCC

TACCCTGAGG

CAACCGGAGA

ATCTGTAAGT

GGTCTCTCAC

AGCTTTTGCC

GAACACAGTG

CAGCCTACCC

GCACCCTAAG

AGTGAGGCCT

CACAGGCTGT

CCACCTGCCA

TACCATCAAT

TGCTCTCTCA

CAGGATTCCC

AAGCCTTTGT

ATGCTCCCTC

TGCACTCTTG

I 4

ATG

GGC

CAG

TCC

GCT

GCC

AAT

TCG

AAG

GCC

ATC

GCC

GAA

CTG

TTT

GCA

GAG

GTC

CAG

CCT

GAA

AAT

GCT

TCT

OTT

OCT

TCT

GAG

TTA

GAG

CCT

GTG

AAG

AAA

TCC

GTG

GC

CCC

ATG

CTG

TAT

GAA

GCG

ACC

GCA

TTG

TCT

GAG

CCT

CCT

TCA

CCC

GOT

GAC

GAT

GAG

AAT

GAG

GAC

CTT

AAG

GAG

GOT

GGG

AAC

CGC

AGC

AGA

TTA

AGT

GAG

GAG

GGC

CCC

AGC

CAA

TTG

CAT

AAG

TGC

ATG

AAC

GC

CTG

GCC

GTG

AAA

TAC

CTG

GTC

GCC

GAG

CAC

CTG

GAG

CTG

AGT

ACT

GAG

CGA

CTG

OAA

CCT

TTT

TAC

CTG

ATC

GAG

GAT

CTC

CAG

GGT

GAG

CCA

GTC

100 150 200 250 300 350 400 450 500 550 600 624 666 708 750 792 834 876 918 960 1002 1044 1086 1128 1170 1212 1254 1296 1338 1380 1422 1464 1506 1548 1578 1628 1678 1728 1778 1828 1878 1928 1978

GCATGAGTTO

ATCTAACAGC

CATTCTTCAC

TCTATTTTGT

GTTGAA.ATGT

TTATGAATCG

AGTCTTGTTT

GGACATAATA

CAGCCAGGGC

CCTGTGCAGC

TCTGTTTGAA

TGGATGACTT

TCCTTTTAAT

TAGTTAACGT

TTTATTCAGA

ACAGCAGTGG

TGTGGGGAAG,

AGCTTCCCTT

GAAAATAGTC

GGAGATTTAT

GGATGGTTGA

ATATTGCTGT

TTGGGAAATC

AGTAAGTATT

GGGCAGGGCT

GCCTCGTGTA

AGTGTTCTTA

CTCTGTTTCC

ATTAACTTCA

TAATATAGTT

COTTCTATTT

TAGAAGTGTG

GGGCCAGTGC

ACATGAGGCC

GTAGTGGGTT

TTTTACAATT

GCATCCAAGT

TAGGAGTAAG

TGTGAATTTG

AATTCACCGT

SUBSTITUTE SHEET h

I

WO 92/20356 PCT/US92/04354

GAAATAGGTG

GTCTATTCTG

CTTCGTGAAT

ACTGGCTCAT

AGGATTAGTA

GGGTATTAAG

CCTCTAAGAT

GAGAGTGGTC

AACTCCATTT

AGG4GCCAGAT

TCTGAGCGGT

GGG

AGATAAATTA

TAAAATTTAA

GTAAGAGAAA

TTCTTCTCTA

GTGGAGATAC

AGTCTAGGAG

GTAGGGGAAA

GGGTGTAAAT

TCTTCTGAGG

TCTCAGAGGG

TCCTTTGTGA

AAAGATACTT

AAATATATAT

TTAAATCTGA

TGCACTGAGC

TAGGGTAAGC

CGCGGTCATA

AGTAACGAGT

TCCCTGTGTG

GATCTGATTC

AGAGGGAAAA

CAATGGATGA

AATTCCCGCC

GCATACCTGG

ATAAATAATT

ATCTGCTCTG

CAGACACACA

TAATTAAGGT

GTGGGTATGG

GGGCCTTTTG

TAATGAAGCT

GCCCAGATTG

ACAGAGAGGA

TTATGCCTCA

ATTTCCTTGG

CTTTCTGTTA

TGGAAGGCCC

CCTACCGATA

GACAAGATGT

GGCTCCAGGT

GGCTTTGGGA

TGGTGGGTCC

GAAAAGTTGC

GCCTCTACCT

2028 2078 2128 2178 2228 2278 2328 2378 2428 2478 2528 2531 SUBSTITUTE SHEET WO 92/20356 PC1/US92/04354 INFORMATION FOR SEQUENCE ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1068 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: cDNA MAGE-4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

G

GAA

CTC

ATG

GTG

GGC

ACC

GGT

GTC

GAG

GGG

ACC

GTA

CCA

CAT

TCC

GGG

GCA

CGC

CTG

ATC

ATT

CTT

AAT

CTG

GAA

AGG

CAA

CCC

AGG

GTG

CTG

CCA AGC CTC AGT AAG TAT GAG AGA TTC GOC GAC GTG GTC ACC AAT CAG GGC ACA ATC TGG GAG CAC GAT TGG GGC AGT OCT CTG GTC AGG COT GAA

ACC

AAC

CGA

GTC

AAA

AAG

TGC

ATC

ATT

GAG

ACT

GTG

AAT

GCT

GTC

GCA

TCG

AAG

GCC

ATC

0CC

GAA

CTG

TTT

OCA

GAG

GTC

CAG

CCT

GAA

AAT

GCT

CCT

GTG

AAG

AAA

TCC

GTG

GGC

CCC

ATG

CTG

TAT

GAA

GCG

ACC

GCA

TTG

GCA GAG GAG TTG CTG GTC TAC AAO TCC CTG CCC 0CC TCC TAT ACA GOC GOC GAC GTG ATG GAG CCC TAC CTG TAT GAG TAT GTG GTT COC GAG GAG TTG TTC CGA CAT TTT CTG AAG GCA GAA TOC TTT CCT ATO ATC TTT AAC ACC TAC GGC CTG CTO CTG ATA ATC GCC TCT GAG GTG TAT GAT AAA CTG CTC TAC CGG CAG CTO TOG GOT GTC CTG GAG GCC TAC CCA GAG GGA GTC

GCTGGGCCAG

GTAACATGAG

TTAGTAGTGG

TCCTTTTACA

TCAGCATCCA

GTTTAGGAGT

TTTTGTGAAT

GTGAATTC

82 124 166 208 250 292 334 376 418 460 502 544 586 628 670 720 770 820 870 920 970 1020 1068

*TGAGCATGAO

TGCATCTAAC

OCCCATTCTT

OTTTCTATTT

ATTGTTGAAA

AGTTTATGAA

AAGAGTCTTG

TTGGGACATA

TTGCAGCCAG

AGCCCTGTOC

CACTCTGTTT

TGTTGGATOA

TOTTCCTTTT

TCGTAGTTAA

TTTTTTATTC

ATAACAGCAG

GGCTGTGGGG

AGCAGCTTCC

GAAGAAAATA

CTTGGAGATT

AATGGATGGT

COTATATTGC

AGATTGGGAA

TGGAGTAAGT

AAGGGGCAGG

CTTGCCTCOT

GTCAGTGTTC

TATCTCTGTT

TGAATTAACT

TGTTAATATA

ATCCGTTCTA

ATTTAGAAGT

SUBSTITUTE SHEET WO 92/20356 PC1/US92/04354 INFORMATION FOR SEQUENCE ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 2226 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-5 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GGATCCAGGC

GGGACCATTC

TCCTGTTAGC

GGCCCATGCA

TGGTCTGAGG,

TAGTGCCAGC

GCCCCAGAAC

TCAGTCCTGC

CTCACTTTTT

GAAGCTCCAG

ATCTGTAAGT

GGCTTCTCAC

AGCTCCTGCC

CTTGCCAGGA

ACCCCAAGAG

ACTGGGGGCC

TTCCTCTTCC

CCGTGCCCTC

AGTGAACGTT

ATATGGGACT

AGAATCAGCC

CCTTCAGGTT

AGGATCCCCA

AAGCCTTTGT

ATGCTCCCTC

CACACTCCTG

GAAAGGTGAG

GGTGGAGACC

TGAGGCTGTG

AGGAGCTCCA

AGGTCACAGA

TGCCTTGAAT

CCAGAGCACC

TCTGCTTGCT

CTCAGGGGAC

GGAGGCCCTA

TAGAGCCTCC

TCTCTCCAGG

CCTGTTGCGG,

kG AGT CAG 'A CCC TGG 'A AGA GTCI C~T TCA CTC kG AAG AGG ;T TCC GAG

GGCCCTGTGT

TCACAGATTC

CTTGCAGTCT

GGAAACAGAC

GCAGAGGAGA

GCACACTAAT

TGGCCTCACC

TGTGTACCCT

AGGCTGACCA

GAGGAGCACC

AAGGTTCAGT

CCAGTGGGTC

TGACCAGAGT

CTT GAG TGG TCC CAG GTC CTG CCA CCA GCA CAG AGT ACT TGA

TTCATTTTCT

GAAATGCTGG

CTTCGGCAAA

AGGAAGCGGA

CTCCTATGAT

GCCTCCTGAT

CCTGAGGAGA

GAGGGAGCAC

TGGTGCAGGA

ATATGCTATG,

CTGGAGCACG

CCTGCGTGAA

CTGCAGCCAG

CTCCGTCCAG

TCTCTTTGAA

TGGATGACTT

TTCTTTTAAT

CAGTAGTCAC

TTTTTATTCA

TACAGCAGTG

TGATGACATA

TATTCGGTAA

TTCTTTGAGA

GCTCCTCAAG

AGAGCGTCAT

GCCTCCGAGT

CCCCACCAGC

GGCCTGCTGG

AATCGTCTTG

AAATCTGGGA

AGTGTCTGTG

AAACTACCTG

AGTTACTGTG

TGGTCAGGGT

GCAGCTTTGA

GGCCACTGCG

TAGTTTCCCC

GAGAGCAGTC

TGAGATTTGT

GGGTGGTTGA

ACATAGTGCT

GATTGGGAAA

GAATAAGTAT

AAGAAATTAA

AATTTTTTTT

ATGTAAGACA

TATTAAGTCA

CAAAAATTAC

CCTTGCAGCT

AACACCTACA

TTGATAATAA

GGCATGATTG

GGAGCTGAGT

GGGAGCCCAG

GAGTACCGGC

GGGTCCAAGG

CAATGCAAGA

GAGAGGAGGA

AGGGGGGCTG,

TGCCTTAATG

AACATTCTTA

CTTTGTTTCC

ATGAACTTCA

GTTTATATAG

TCCATTCCAT

TCATTTAGAA

AAGATATTTA

AAAAAATGTG

AATTAAATCT

AGGAGCTGGT

AAGCGCTGCT

GGTCTTTGGC

CCCTTGTCAC

TCAGATCATG

CAATGGAGGG

GTGATGAAGG

GAAGCTGCTC

AGGTGCCCAG

GCACTCGCTG

GTTCTCATTT

AGAGGGAGTC

GGCCAGTGCA

TGACATGAGG

GTAGTGGGTT

TTTTGGAATT

GCATTCAAAT

TTTAGGAGTA

TTTGTGAATT

ATGTGAATGA

ATTCTTGCTT

CATACCTGGA

GAATAAATCA

GAGCACAGAG

CAGCCTACCC

GCACCCTGAG

ACTGAGGCCT

TGCAGACGTC

GGCCCCCATC

CTCTCTACTG

GAGGTGCCCT

GGATCACCAG

AAAGGAGAAG,

TTTTAGCTGA

TCCATTGCCC

CGTC

CCT GAG GAA CTG CTG CTG CCT CCG CCA AAT CCA TTA GCA CCT CCC OTA AGA AGG

CACAAAGGCA

TTCCTGAGAT

ATTGACGTGA

CTGCCTGGGA

CCCAAGACGG

CAAATGCGTC

TGTATGTTGG

ACCCAAGATT

CAGTGATCCC

CTTGAAAGTA

CCTACCCATC

TGAGCATGAG

CCTTCCAGGG

CCCATTCTTC

TCTGTTCTAhT

GTTCAAATGT

TTATGAATGA

AGAGTCTTGT

GGGACATAGT

GCAGTAAAAC

ATACTCAGTC

TTTCCTTGGC

TTCTCCCTGT

100 150 200 250 300 350 400 450 500 550 600 644 684 728 770 812 854 896 908 958 1008 1058 1108 1158 1208 1258 1308 1358 1408 1458 1508 1558 1608 1658 1708 1758 1808 1858 1908 1958 2008 2058 SUBSTITUTE SHEET WO 92/20356 PCr/US92/04354 TCACTGGCTC ATTTATTCTC TATGCACTGA GCATTTGCTC TGTGGAAGGC 2108 CCTGGGTTAA TAGTGGAGAT GCTAAGGTAA GCCAGACTCA CCCCTACCCA 2158 CAGGGTAGTA AAGTCTAGGA GCAGCAGTCA TATAATTAAG GTG(GAGAGAT 2208 GCCCTCTAAG ATGTAGAG 2226 SUBSTITUTE SHEET WO 92/20356 PCr/US92/04354 INFORMATION FOR SEQUENCE ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 2305 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-51. gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GGATCCAGGC

GGGACCATTC

TCCTGTTAGC

GGCCCATGCA

TGGTCTGAGG

TAGTGCCAGC

GCCCCAGAAC

TCAGTCCTGC

CTCACTTTTT

GAAGCTCCAG

ATCTGTAAGT

GGCTTCTCAC

AGCTCCTGCC

CTTGCCAGGA

ACCCCAAGAG

ACTGGGGGCC

TTCCTCTTCC

CCGTGCCCTC

AGTGAACGTT

ATATGGGACT

AGAATCAGCC

CCTTCAGGTT

AGGATCCCCA

AAGCCTTTGT

ATGCTCCCTC

CACACTCCTG

GAAAGGTGAG

GGTGGAGACC

TGAGGCTGTG

AGGAGCTCCA

AGGTCACAGA

TGCCTTGAAT

CCAGAGCACC

TCTGCTTGCT

CTCAGGGGAC

GGAGGCCCTA

TAGAGCCTCC

TCTCTCCAGG

CCTGTTGCGG

GGCCCTGTGT

TCACAGATTC

CTTGCAGTCT

GGAAACAGAC

GCAGAGGAGA

GCACACTAAT

TGGCCTCACC

TGTGTACCCT

AGGCTGACCA

GAGGAGCACC

AAGGTTCAGT

CCAGTGGGTC

TGACCAGAGT

GAGCACAGAG

CAGCCTACCC

GCACCCTGAG

ACTGAGGCCT

TGCAGACGTC

GGCCCCCATC

CTCTCTACTG

GAGGTGCCCT

GGATCACCAG

AAAGGAGAAG

TTTTAGCTGA

TCCATTGCCC

CGTC

CCT GAG GAA TGG GTG TGC CCT CCT CCT CTG CTG CTG CCT CCG CCA AAT CCA TTA GCA CCT CCC GTA AGA AGG

ATG

GGC

AGG

CTC

GGT

TCC

AGG

CTG

TCT CTT GAG CAG AAG AGT CTT GAC ACC CAA GAA GAG CTG CCA CTA CTG AGG AGC CTC TGG TCC CAG GCA CCC CAC CAG GTC CTC TCA AGA CCA CTG CCA TCG ATT TCA GCT CCA GCA ACC AAG AAG, ACC CAG AGT CTG TOT TCC 100 150 200 250 300 350 450 500 550 600 644 686 728 770 812 854 896 938 980 992 1042 1092 1142 1192 1242 1292 1342 1392 1442 1492 1542 TGG CTG ACT T TTCATTTTCT GC GAAATGCTGG AG CTTCGGCAAA GC AGGAAGCGGA CC CTCCTATGAT GG CTGATAATCG TC GGAGAAAATC TG AGCACAGTGT CT CAGGAAAACT AC TATGAGTTAC TG CACGTGGTCA GC TGAAGCAGCT T1 CCAGGGCCAC TC.

CCAGTAGTTT CC TGAAGAGAGC AC ACTTTGAGAT T1 TAATGGGTGG T1 TCACACATAG TC TTCAGATTGG G AGTGGAATAA G~ GATAAAGAAA T1

GA

TCCTCAAG

AGCGTCAT

~CTCCGAGT

~CCACCAGC

ICCTGGTGG

~TTGGGCAT

rGGAGGAGC

~GTGGGGAG

CTGGAGTA

TGGGGTCC

GTCAATGC

TATTAAGTCA

CAAAAATTAC

CCTTGCAGCT

AACACCTACA

TTTAATCAGA

GATTGCAATG

TGGGTGTGAT

CCCAGGAAGC

CCGCAGGTGC

AAGGGCACTC

AAGAGTTCTC

AGGAGCCGGT

AAGCGCTGCT

GGTCTTTGGC

CCCTTGTCAC

TCATGCCCAA

GAGGGCAAAT

GAAGGTGTAT

TGCTCACCCA

CCAGCAGTGA

GCTGCTTGAA

ATTTCCTACC

CACAAAGGCA

TTCCTGAGAT

ATTGACGTGA

CTGCCTGGGA

GACGGGCCTC

GCGTCCCTGA

GTTGGGAGGG

AGATTTGGTG

TCCCATATGC

AGTACTGGAG

CATCCCTGCA

GAGiAGA AGAGGG AGTCTG~AGCA TGAGCTGiCAG 19 ;CGAGGGGG GCTGGGCCAG TGCACCTTCC AGGGCTCCGT 1642 CCTGCCTT AATGTGACAT GAGGCCCATT CTTCTCTCTT 1692 ;TCAACATT CTTAGTAGTG GGTTTCTGTT CTATTGGATG 1742 'GTCTTTGT TTCCTTTTGG AATTGTTCAA ATGTTCCTTT 1792 ~GAATGAAC TTCAGCATTC AAATTTATGA ATGACAGTAG 1842 CTGTTTAT ATAGTTTAGG AGTAPLGAGTC TTGTTTTTTA 1892 LAATCCATT CCATTTTGTG AATTGGGACA TAGTTACAGC 1942 LATTCATTT AGAAATGTGA ATGAGCAGTA AAACTGATGA 1992 ~AAAAGATA TTTAATTCTT GCCTTATACT CAGTCTATTC 2042 SUBSTITUTE SHEET r WO 92/20356 PCr/US92/04354

GGTAAAATTT

TGAGAATGTA

GGCTCATTTA

GTTAATAGTG

TAGTAAAGTC

CTAAGATGTA

TTTTTTAAAA

AGACAAATTA

TTCTCTATGC

GAGATGCTAA

TAGGAGCAGC

GAG

ATGTGCATAC

AATCTGAATA

ACTGAGCATT

GGTAAGCCAG

AGTCATATAA

CTGGATTTCC

AATCATTCTC

TGCTCTGTGG

3RCTCACCCCT

TTAAGGTGGA

TTGGCTTCTT

CCTGTTCACT

AAGGCCCTGG,

ACCCACAGGG

GAGATGCCCT

2092 2142 2192 2242 2292 2305 SUBSTITUTE SHEET I-s~ WO 92/20356 PCT/US92/04354 INFORMATION FOR SEQUENCE ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 225 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: MAGE-6 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

TAT

CAG

GGC

GAT

TTC

TGT

TTT

GTC

GTG

CTG

ATA

CCT

CCT GTG TTT GGC TAC ATC CTG GGT ATC ATC GAG GAG SUBSTITUTE SHEET WO 92/20356 PCT/US92/04354 INFORMATION FOR SEQUENCE ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 1947 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-7 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

TGAATGGACA

GAGCCCAGCC

TGGCCGGCTG

GCGGACAGGC

ACCGAAGGAG

GGTTCACAAA

GTTCCTCCCC

CCAGAGTCAT

GATGCCTTGA

GCTCCCGCCA

AGGCACCCTG

GTCTCAGGGT

GCCAATCCAG

TAGACACACC

ACAAGGGCCC

TCACCTTCCC

TACCCTGAGG

CGGCCAGGAG

AAGATCTGTA

TGAGGCCCCT

ATCGCCCAGC

CATGTCTTCT

GGCCCAAGGA

CCGAGGAGCA

GAGGAGGTGC

TCCTCCTTTT

TGAGGGCACC

CCGCTCACCT

CACACTCCCC

TACTGTCAGT

TGCCCTCTCA

GTCAGAAGCC

AGTAGGCCTT

CACAAGCTCC

TGCTGCCCGC

GAGCAGAGGA

CAGGAGGCTC

CGAGGCTGCC

CTGCTGCTGG,

CCCTGACCAT

AGCAGCCGOG

GGCGTCCTTG

AGAACACAAG

CCTGCAGCCT

CTTCCTCCTT

CCAGGAGGCC

TGTTAGGGCC

TTCTCTCCCC

ACTCCAGCCT

GTCAGCACTG

TGGGCCTGGT

TCCTCCTTCA

GTCCCCCAGT

CAGCAACAAC

AAGAGGAGGG

TTCCA

GGACTCCAGA

CAGCCTCTGC

CAGGTTCTCA

CCAGAGGAGC

TCCAGGGCGT

AGATCTGTGG

GCTGCCCTGA

CA.AGCCTGAG

GGGTGCGCAG

CTCTGATTGA

CCTCCCCTGA

ACTCTATGGA

GCCAACCACC

ATG

ATC

GTG

GGC

ATG

CCT

AGA

GGA

GAG

TCA

AAA

AAG

GCT

GCA

AGG

TCA

TCA

GCC

GAA

TG

TGC

TGG

AGG

AAA

TCA

GTG

GCC

CCG

TTATGGTCTT

GCAATCTGGG

TCTTTGGGCA

TACCTGCAAT

CCTGTGGGGT

AGTATGCAGC

CATGAAGAGG,

AGCCAGGGCC

CACACATCCA

CTGTGTTTGA

GTGTGAGGGA

GATTTGGAGG

TTAATGGATG

GCAGACTTAC

TTATGTAAGA

CAGAGGATTA.

GTGAGATAAA

CCTGTAATCC

GGAGATCGAG

AATACAAAAC

GACCATGAI

AAGCGTTG;

GCTGAGGAI

ACCGCCAGC

CCAAGGGC(

CAGGGTCAC

CTTTGGGAC

AGTGGGGC~

CCACCTTC(

AGAGAGCA(

ATACAAGG~

TTTATCTT

GTGTAATG

TGTTTTTT

AAATCTAT4

AGTACCTT'

GAAATAAAI

CAGCACTT

ACCATTCT'

TTAGCCGG

AGT TGG TTC OCT TOG TCA CAA AGG ACA AGC ACT AGT TGC ATG CAG GTG CCC GCG GCC ACT CCT ACA ATG 0CC CCG GCC TTC TGA 'C TTAATGGAGG 0CC ~G TGTAATGGTG TAI ~G CTGCTCACCC AAC T GCCCAGCAGT GAI C TCATTGAAAC CAC T ACTAAAGAGA GCI ;A GGAGGAAGAG GGJ kG ATTGGGGGAG GG( C TGTCCTGTTA CA.

GT CAATGTTCTC AG' TG GACCATCTCT CA( TG TTTCCTTTTG, CA AiA CTTCAACATT CA ALT ATAGTTAAAA GT GT TATTTCTTGA AT TT ATAATGTGAA AG~ ~A AATTAAATTG GC TA GGAGGCAGAG GC, GG CTAACACAGT GA ~C GTGGTGGCGG GT TCC TGC TGC ACA CAG AAA TGC TG TTC CTT GTG ATC ATO TTT GGC ATT CCT ACO TCC TTG TGC TGG GTG ATG

ACTGTGC

~GATGGGA

ATTGGGT

6CCCCCGT

CTATGTG

kTTTCCTA kGTCTGAG

.CTGGGCA

LGAGGCCC

rAGCGGGG

~TTCCTGT

3TCGTTCA

~TTCATGT

PiAGTGCAT

TGGGACAA

PiACAAAGC

TGGGCACG

~CGGGGAT

NACACCAT

G

CCCTGAGGAG

TGGAGCAGTT

GCAGGAAAAC

GCTACCAGTT

AAAGTCCTGG

CCCATCCCTG

CAGAAGTTGC

GTGCACGTTC

ATTCTTCACT

AGTGTGTTGG

TCTCTTGGGC

AATGTTCCTT

ATGACAGTAG

TGTTTTTTAT

CATAACATAG

GGTAAAATGG

GTGGCTCACG

CACGAGGTCA

CTCTATTAAA

964 1014 1064 1114 1164 1214 1264 1314 1364 1414 1464 1514 1564 1614 1664 1714 1764 1814 1864 1914 1947 SUBSTITUTE SHEET

I

I

WO 92/20356 PCT/US92/04354 INFORMATION FOR SEQUENCE ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1810 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-8 gene (xi) S8QUENCE DESCRIPTION: SEQ ID NO:

GAGCTCCAGG

TCACAGAGCA

GTTTCCCCTG

ACCCCACAGT

GCCTTTGCCA

GGTTCGCAGA

CTGAAGAAGA

CCCAGCTGAG

CAATTGCCCA

AACCAGGCTG

TAAGAGGCCC

TATGTATACC

TCCTGGCCCT

GGAGGCTGCA

GAACAGGCCA

CCTGTAAGTA

GCCTCTCACA

TGAGGTCTTG

AGGCAGTAGT

AGAGGCCCCT

ACCAGCCCTT

CCCTGAGATG

GCCAGGAGGT

GACCTTTGTT

CGCTTCCTCT

GTCTGAGGCA

AGCAGTCAAG

CTGGCATCAG

TTGTCAGTCC

CCCTCTCAAT

CAGGAGGCCC

AGGGCATCCA

CTCCCCAGGC

GTATCTTCAA

CTGAGGTGGT

AACAGCAGGA

TGGAGCCTTG

TTCTCCTTCA

CAGAGAAGCA

GGGTGTAGTA

CTGTGGGTCT

CCTGAGTCAT GCTCCGGCCC ACACTCTCCT GCTGCCCTGA

ATG

GGC

CAG

TCT

GGG

TCC

GAG

CCG

GAT

TAT

AGT

AGC

GTG

ACC

CAG

ATG

TG

CTT

CAG

CCC

CTG

CCA

ACT

TCC

CCA

AAA

ATT

ATC

GCC

GAA

CTG

ACG

TTA

GCA

GGG

GCC

ACA

ATC

AGT

GTC

AGC

GCT

GTG

AAG

AAA

TCT

GTG

GGC

CCC

ATG

TTG

AAG

GGA

GAG

GGA

CCC

GAC

AAT

CTG

GAG

CCG,

TAC

TGC

CCT

TCC

ACC

GGC

GTG

AGT

GAr(j

GAG

ACC

CAG

AGC

GAA

GAG

TTA

GTC

AAG

ATG

GCC

TAT

GGC

AGC

ATG

CGC

CCA

AAG

GAG

CCT

CTG

GAG

CTG

CGT

AAG

CAC

GTG

CAC

GGC

CTG

GCC

GCT

TAC

GGG

GCT

GAG

GAG

TOG

GGG

TTC

TTC

GCA

TTT

ATC

TCC

CTG

ATA

CCG

GTA

AAG GCT CTT ATG GCA TCC GTG ACT GGT GCC AGC CAA CCA AGC CGG GAA CTG CTC GAA ATG CCT GAT TTT GGC TAC ATC CTG GOT ATC GTC GAG GAG

TGA

GAG

GAT

TCC

GAT

TCC

TCC

ACC

GCA

CGC

CTT

ATC

ATT

CTT

GAT

CTG

GCA

GAA

GTG

TCC

TCT

TCT

OAT

TCC

CTT

AAA

GAG

TTC

GAT

GTC

GAT

GGC

ATC

493 535 577 619 661 703 745 787 829 871 913 955 997 1039 1081 1123 1156 1206 1256 1306 1356 1406 1456 1506 1556 1606 1656 1706 1756 1806 1810 TGGGAGC GAG

AGTGGGTGCA

CCTGTGCGCT

CTATGTGAAA

TTTCCTACCC

TGAGCAGGAG

GGCCAGTGCA

ATGAGGCCCA

GTAGTGGGGA

AGTTCCTGTT

AATTGTTCCA

ATTTTATGTA

GTAAGAGTCT

ATTC

CACAGTGTCT

GGAGAACTAC

ACGAGTTCCT

GTCCTGGAGC

ATCCCTGCAT

TTGCAGCTAG

CGTTCCAGGG

TTCTTCACTC

GCATGTTGGG

CTATTGGGCG

ATGTTCCTTC

TGACAGTAGA

TGCTTTTCAT

ATTGGAAGCT

CTGGAGTACC

GTGGGGTCCA

ATGTGGTCAG

GAAGAGGCTT

GGCCAGTGGG,

CCACATCCAC

TGTGTTTGAA

TGTGAGGGAA

ATTTGGAGGT

TAATGGATG

CAGACTTACT

TTATACTGGG

CAGGAAGCTG

GCCAGGCGCC

AGGGCCCTTG

GGTCAATGCA

TGGGAGAGGA

GCAGGTTGTG

CACTTTCCCT

GAGAGCAGTC

CACAGTGTGG

TTATCTTTGT

TGTAATGAAC

GCTTTTTATA

AAACCCATGT

CTCACCCAAG

CGGCAGTGAT

CTGAAACCAG

AGAGTTCGCA

GAAAGGAGTT

GGAGGGCCTG

GCTCTGTTAC

ACAGTTCTCA

ACCATCTCTC

TTCCTTTTGG,

TTCAACATTC

TAGTTTAGGA

TATTTCTTGA

SUBSTITUTE SHEET pp.~ WO 92/20356 PC7/US92/04354 INFORMATION FOR SEQUENCE ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 1412 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-9 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

TCTGAGACAG

AGCAGTGAAG

GCCCCAGCAC

TCACTCATAG

TCTCACTTCC

GCCCCAAGAG

TGTTAGAACC

CTCTCTCCCC

TGTCCTCAGG

GTGAAGTGTT

ACATGGGACC

AGCCTTGATC

TCCCTCAGGT

GCCCCAGAGC

TCCAAGGTTC

AGGCCTGTGG,

TCGCAGAGCA

CACCCTGAAT

CCATAGCACC

TCTGCAGGCT

TCTCGGGACA

AGCACTGACG

GGTTCTCAGC

GTCTCCATCG

GAGGAGACCC

GTGCACCAAG

TGGCCCCATT

AGCTGCACGC

GGCTAACCAG

AAGACCTGTA

TGAAGTCTCT

CCCAGCTCCT

AGGCAGTGTC

GGCCrCACCT

CCCCCTACTG

TGAGTAGCCC

GAGGACAGGA

AGTCAGCCTT

CACACACTCC

GCCCACGCTC

CTGACTGCTG CCCTGACCAG AGTCATC ATG TCT GAC CTT CAG GAA GAC AGC CCT CCC GTC TAC AGT CAA CAG CTG GCT GAG GAG CCG AAT TAC GAG TTC GAC CCC CTC TCG AAG, GCC AAA GAC AGT GTG GGG GAG AAC TAC CAC TAC AGC TAT AGA GAG GGA GAG

CTC

GAA

CCC

AAG

CAG

TAC

GAA

GAG

TTG

GTC

AAG

ATO

GCC

TGC

GCC

AAC

ATG

CCC

CTG

GAG

GAG

CCC

GAG

GAG

GCC

ACA

GAG

AGT

ACT

GAG

TTC

GTT

ACA

CGC

CAG

GGC

GAT

CTC

TGC

GG

AGG

GAG

TTC

AAG

ATC

CAA

CAG

CAA

GGC

GAG

CCT

TTA

GAA

ATG

CAT

AAG

TAC

GTG

CAC

AGC

CTG

GCC

GTG

AAG

TAC

CTG

GTC

TGC

GAG

AGO,

GGA

GAG

GAG

CAG

TGG

GAG

TTC

TTC

GCA

TTT

ATC

TCC

ATG

ATC

CCT

TAT

CTG

CG

TGG

ATA

TAC

GGA

AGT

GAG

GAG

GTG

OGA

AGC

CCA

CAA

CTG

GAA

CCT

TTT

TAC

CTG

ATT

GAA

OTT

CTC

CAG

GT

AAT

CCA

GTC

CCG CAC GAC TTG GAG GAG TCT GCT GGC GCT CAA TTC AOC TCC GAA GCA CTC CAC ATO CTG OTG ATC GGC ACT ATC CTT GGT GAT GTC CTG GAG GTT GGG AAG ACC CAA GTG CCC TCC AAG TAT TTG TCC CTT

TGA

AAG

CTG

ACC

GGG

TCC

GAG

GTC

AAA

TAT

AGC

GGC

GTG

ACT

CAT

GTG

TGG

CAC

TGG

AGT

CAC

ATO

GAA

CCT

ATG

TCC

TCA

TCC

GC

GAC

TG

CGA

GTC

AAA

AAG

GCT

AGC

ATC

GAA

ATG

GTG

GAT

GCT

CTC

GAG

GAT

GGT

TCC

TCA

ATT

TCC

CCA

AAG

GTC

ATC

GCC

GAG

CTT

ATG

CTA

GCG

TTC

CAG

CCT

GAA

AAT

GTT

GAA

GCA

TCT

AGT

TCC

AGC

GCT

GTG

AAG

AAA

TCC

GTG

GGC

CCC

ACC

TTG

TAC

GAA

GCG

ACC

GCA

TTG

100 150 200 250 300 350 400 427 469 511 553 595 637 679 721 763 805 847 889 931 973 1015 1057 1099 1141 1183 1225 1267 1309 1351 1375 1412 GCACCAGCCO CAGCCGGGGC CAAAGTTTGT GGGGTCA SUBSTITUTE SHEET Ii :ew.

~1a.l Wj

A

r WO 92/20356 PCT/US92/04354 INFORMATION FOR SEQUENCE ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 920 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-10 gene (xi) SEQUENCE DEf:CRIPTION: SEQ ID NO: 22:

ACCTGCTCCA

CTGTCAGTCC

CTCTCACTTC

AGAGCTGTGG

GGCCTTTGTT

CCCTCCCTCT

ACACTCCCAC

GGACAAAGTG

TGGAGCCTTG

CTTCTTCAGG

GACACCACAG

AGAACCTCCA

CTCCCCAGGC

CTGCTACCCT

GACCCCACTG

GCCTCTGCCG

TTCTCAGGGG

AGCAGCACTG

GGGTGTGGTT

CTGTGGGTCC

GATCAGAGTC

CATCAGCTCC

GCTGCATCCT

ACAGGGAGAG

AAGGAGAAGA

CTCAGCTGTG

CCATCGCCCA

ATC

ACCTACCCTA

GAGGAGCCAT

CAAGAGGTCA

CCTGTAAGTT

GCCACTTACA

AGTCCTGCCC

ATG CCT GAT CTT CAG GCT TCC ACC TCT TCC CCA GAG CAG AGT TCC CTT AAG GAG GAG TCT TTG GTG ATC ACA GAA GAC ATG CTG CGA GCT CAA TCC CCC CTG AGC TCC TCC TCC GAG GTT GCT CAG CCA TTA GAG AGT TTA CCC CAG TTT AAG GCA CAC TTC CTG GTC

CCA

CAA

OCT

TCT

TCC

TCT

ATA

GAT

CCA

AGA

CTG

GAA

CCT

TTT

AAG COT AGT GAG GTG GAG TTT CCA TCC TGC GCT GAT GCC TOC CAA TCT AGC ACC AGT GAG CTC TTC ATA CTG TTG TTG GGC ATT

CAG

ACA

GAG

TCC

TAT

GAT

TCC

GAT

CTA

ATA

AAG

GAG

TTT

GAT

COC

CAG

GAT

TCT

CCT

GAG

TCC

GAG

CAG

GAT

TAT

AGT

AGT

GTA

ATG

CTC

TCA

CCC

ATA

CCA

TCG

TCC

CTG

AAG

ATG

ATA

(ICC

G AA

CCT

GAG

TCA

TCC

CCA

AAT

GTC

AGC

CCA

GTG

AAG

AAA

TCC

GTG

s0 100 150 200 250 300 333 375 417 459 501 543 585 627 669 711 753 795 837 879 920 SUBSTITUTE SHEET p WO 92/20356 PCI'/US92/04354 9~3 INFORMATION FOR SEQUENCE ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 1107 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: MAGE-1l gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

AGAGAACAGG

CACTGGAGGA

CATATCTCAT

GGCCCCATCA

AGTCATCATG

CCTTCAGGCC

AAGCTGAGGA

ACTCTAGAGG

TCAGGAAGAG

TATCTGATGA

CCTGACCTGA

GATAATTGAT

CCAACCTGGA

GAACAAGTGT

CTGAGTCTGT

CCCAGATATT

CCTCTTGAGC

CAAGAAGAAG

GCAGGAGGCT

AGTTGCCTGC

TCCTTCTCTC

GGGCTCTGGC

TAGACCCTGA

TTGGTTCATT

GGACAGGAGT

AAGTAGGCCT

TCTCACGCTC

TCCCACAGTT

AAAGAAGTCA

ACCTGGGCCT

GCCTTCTTCT

TGCTGAGTCA

CCACTGCCAT

AGCCAAGAAA

GTCCTTTTCC

TATTCTCCGC

CCCAGGAGAA

TTGTTAGATT

CCTCTCTCCC

CGGCCTGCTG

GCACTGCAAG

GGTGGGTGCA

CCTCTACTCT

CCAAGTCCTC

GGATGCCATC

AGGAGGGGCC

CAAGATATAC

AAGTATCGAG

CCCAGAGGAT

CTCCATGGTT

CAGGCTGTGG

ACCTAACCAG

CCTGAGGAAG

CAGGCTCTC

GAATGTGCLGC

CCCAGAGTCC

TTTGGGAGCC

AAGTACCTCG

TACATGACAA

TCAAGGGGCT

GATCACAAAG, GCAGAA 100 150 200 250 300 350 400 450 500 550 600 616 658 700 742 784 826 868 910 952 994 1036 1078 1107

ATG

GAG

GGC

GTC

TGT

GTC

GAG

GGA

ACC

GTG

CCA

TAC

AGT

AGG

GTG

ACC

CAG

GTA

TGG

CAC

TGG

ACT

CAC

AAT

GTC

GAA

AAG

TCC

AGC

ATC

GAA

TTC

GTG

GAT

GCT

GCC

AAA

TCT

GTG

AAC

CCC

ATG

CTG

TTT

GAA

GCA

ACC

GG

AAT

GTA

GAC

CTC

AAG

GAG

AGC

GGG

AAG

TOC

AGC

AGG

TAT

TGC

CCC

TCT

TCT

GGG

ATT

GAG

TAC

TAT

AAG

GAT

GAG

ATG

ACT

TAT

GGC

AAC

ATG

CCC

CTG

GAG

ATG

cc

TTT

CTC

TCC

ATA

ATA

CCT

TAT

CTC

CG

TG

CTT

SUBSTITUTE SHEET im-.E~,

S

WO 92/20356 PCr/US92/04354 9 L INFORMATION FOR SEQUENCE ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 2150 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: smage-I (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

TCTGTCTGCA

CTCTACAGAC

ACAGGTTTCT

TATACCCCTG

GCCCTTGTAT

AAGCTAGTGA

ATGCAGTGGC

AGCTTGATCC

TATGCCTCCA

CTCTGTCTGT

GCCCCTGCAT

CATTGTAAGT

GCAGGCCTAA

AAGATCTAAC

CTAACAAGTT

ACGAGTTCAG

CTTGTGTGTA

GTCTGGCACC

GGAGCTTAAA

TTAAGTGGCT

GTTTTTCTGT

CCACTTTTGG

TTAATTTCTT

AAGTCCTGGT

GCAGTCTCAA

CTAAGTGGCT

TAGATCTTTC

TTATGTGGAT

CTGCTTAACC

AAGTCTGAAA

CCACAGGGTT

ATGTTCCTAG

ATG

CCA

TGT

ACT

AGT

GAG

TCT

TCT

GGT

GCT

GG

AAG

ATG

AAC

ACT

GAA

CTG

TTG

TTC

TTT

ATC

GAA

CCA

ACA

GGC

CTT

GC

AAC

TTC

AGG

CAT

TCA

AAG

GTT

CCT

GCT

GTA

GTC

ACT

GCT

AAA

AAG

TCT

ATT

GGT

CCT

ATG

CTG

TTT

AAT

AGC

ACC

ACA

AGA

AAG

ATO

TCC

TAT

TCT

GCC

ACC

CCA

GTT

CCT

TCC

CTG

TCC

AGT

GAA

AAG

GCA

GAT

CTT

AGO

AAG

CAT

GGC

TAC

TAT

AAG

GTC

GAT

GGT

TAG

AAA

CTA

CCT

AGC

TCC

GTT

CAG

GOT

ACA

GAT

GAG

CTG

GTT

AAG

CTA

AGC

ACT

GGT

AAC

GTG

CCT

GAG

TTC

AAA

AGT

GCA

CAT

GCT

CCT

TCC

ACA

CGT

CAG

AGT

GTG

GGC

ACA

CAC

ATA

ACA

GAG

GAA

ACT

GAG

CTC

CGT

GGG

GAG

TAC

CTG

GTC

GCC

GGA

TCC

TCA AAA GGT AGT AGA TTC GTC AAC GCA AAA CCC ACT OCT GGG AAA ACC TCT GGT AAA AGT ACA CTG GAA TTC AGG AGT CAA TTC TTA GTC CAT TCC GGA AGT CTA ATG GCC ACT GTA TAT GAG TTT CGC CAG TGG GGA CTG GAA TTC CCT GGG GTG AAG GCC

GCC

ACA

CTG

ATG

CGA

GCA

TCC

CCT

ATA

TCA

AAA

CTG

GAA

CCT

TTT

TAT

TTG

TCT

GAG

GCT

ATA

GTA

CCC

GTT

AAT

CCA

CCA

TCT

GTA

GCC

AGG,

CAG

GAA

TTC

TCT

GT

GGC

CCT

GAT

CTG

ATC

CTT

CTG

AGT

CTA

GAG

AAG

GAT

GGC

GCC

GCT

TAC

AGG

CAA

ATGGATCTCT

TTGCATGGGC

TCCACAGGCC

ACAGGTCTCT

CCTCCAAGTG

CTAGACTTTT

TGCAGAAAAG

AAAG

CCA TTA AG7 CTT ACA GG'I AGC TCT TT7 GGT CAA AAG TCA CGC AGG GCA GGG TC7 CCT GGT GGI TTT GGT GCP AGA AAT GC7 ACC CAG GCP ATC ATG AGG AAA TTT AAG GCA GTA GT7 CTC AGG, AGP GAG TTG AAC GTA GGC AAP AAC TOG GGG GGT GTG ATC GTC TGG CAP AAG CAC TTC GTA GTG CGC.

AGT GAT CCC CAT GCT GAP AAA GTC AA1I CAG TTG GC1 AGA OTT CAP AAG TCC TC1

TAATCAGTAG

GGCCTGTTAT

GTTTGTTTAA

AAGAGGTAAC

AAACTGGAAA

394 436 478 520 565 604 646 688 730 772 814 856 898 940 982 1024 1066 1108 1150 1192 1234 1276 1314 1360 1402 1444 1486 1528 1537 1587 1637 1687 1737 1787

TTGAGTCTGT

AGAGTTCATA

ACATTAGTAG

CTAAACAGTG

TGTCACTTGT

TCTGTTOTOT

GCCTACCAGA

AATGGAGGCT

CTTTTTGCCA

CAGATTAGGA

TTGAAAAACA

ACCAACATGC

ATTTTTGTTA

TGCTTCTTGT

CTTGTTTTOT

GTCAGGCTCC

ATCCATTCTT

CTTTTCAAAT

TAACTGCATA

TATTTGCAAC

SUBSTITUTE SHEET WO 92/20356 PCI'/US92/04354

ACATTATTTT

GATTGTCATG

GGAAAGTTTA

TACTTTTTTC

GACTTTACTC

TTATTTTCTT

GTAGCACAGG

GTTATCAGAG

GTTTTTACTA

GCAATGTGAT

TATTGTTAAT

TTTTTTGTAT

AAATTCATTA

CAATTATGAA

ATCTAGTATG

TCT

AAACATTGTG

ATCATACAGT

TTTGAAAATT

AATGCTAAGT

GAAAGTAAAT

TTAAGCATTG

AAATGTATCT

TAACATTGCA

GGTGAAACAA

TTATGAGTGT

GAAATAAAGT

CGTAAAACTC

GTTATCTGGA

AGTATAGGCA

TTGGAGAAGG

CAGTGAAGTG

GATTGCTGTA

TGGATTTGAT

TATTACTTTA

AGTTTCTCCA

CTGACAGTGA

1837 1887 1937 1987 2037 2087 2137 2150 SUBSTITUTE SHEET tm.u,~ PMTUS92/04354 WO 92/20356 96 INFORMATION FOR SEQUENCE ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 2099 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA (ix) FEATURE: NAME/KEY: Bmage-II (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

ACCTTATTGG

AATGGATCTC

TTTGCATGGG

CTCCACAGGC

TACAGGTCTC

CCCTCCAAGT

ACTAGACTTT

TTGCAGAAAA

GAAAGATGTT

AGGTATTCTC

TCTTTCCAGA

TCAACATGCC

CAGCAGTCAC

AGGGTCTTCT

CTGCTCCTCA

TGCACAGGCT

AAAAAGTTCA

AAGATCCTAT

AAGTTTAAGA

TAACAAGAAG

CACCCCTAGA

ACTCATTCCT

TTTGAGTAGT

TAGGTGTGAT

CAATTTCTGC

TGGCGAGCCT

AGTACCGCCA

GGACCCAGAG

AGCTAAAGTC

TGGCTCTTAG

AAGGGTGTTC

GAGTCTGTTC

AGTTCATAGC

ATTAGTAGAA

AAACAGTGCT

TCACTTGTCA

ATTATTTTGT

TTGTCATGGC

AAAGTTTATA

CTTTTTTCTT

CTTTACTCAA

ATTTTCTTCA

GTCTGTCTGC

TCTCTACAGA

CACAGGTTTC

CTATACCCCT

TGCCCTTGTA

GAAGCTAGTG

TATGCAGTGG

GAGCTTGATC

CTCCTGGAAA.

TACCTGGTAG

TTCCTGTCTG

TAGGGGTCAA

GCAGGGAGGT

CCTGTTGACC

GGGTGTGAAA

CTGGTATAGG

GATGGCACCC

CATGAGGAAG

TGAAAGAAGC

TATAAGGAGC

ATTAGTCTTT

ATTTGCTGGT

AACTGGGGGT

CTTCATGAAG,

ATGGAGTGGG

GAGGAGTTTA

GGTACCTGGC

CCCATGCTGA

AATGGCACAG

AGATCAGGCA

ATTCCAAGGC

TGTTGTGTTT

CTACCAGAAC

TGGAGGCTAT

TTTTGCCATG,

GATTAGGACT

TTTTACTAAA

AATGTGATAT

TTGTTAGTTT

TTTTGTATAA

ATTCATTAGA

ATTATTAATT

ATATGCCTCC

CCTCTGTCTG

TGCCCCTGCA

GCATTGTAAG

TGCAGGCCTA

AAAGATCTAA

CCTAACAAGT

CACGAGTTCG

GCTTCAAAAG

TACAGAGGTA

CCAGCTCTTT

AAGAGTAAGA

TCCAGTAGTT

AGAGTGCTGG

ACCCCTGGAT

TGGTAGAAAT

AGGCAGGGAC

GCTAGTGTGC

AGTTACAAGG

AATTCCCTGA

GGTCTTGAGT

AGGCAAACTG

TGCCTAGGAC

GGTAACCGTG

GGTATATGCT

TAAGAGATGT

AGTGATCCCC

AACAACCAAG

TCCCTAGTGC

GGAGGGGTGC

CCCATCCCAA

GAAAAACAGT

CAACATGCAT

TTTTGTTACT

CTTCTTGTTA

TGTTTTGTTA

ACATTGTGTA

CATACAGTGG

TGAAAATTTT

TGCTAAGTGA

AAGTAAATCA

AAGCATTGGT

ACTTGTGTGT

TGTCTGGCAC

TGGAGCTTAA

TTTAAGTGGC

AGTTTTTCTG

CCCACTTTTG

TTTAATTTCT

GAAGTCCTGG

CCAGGTCTCC

CTTACAGGTT

TACTTCAGCC

CCCGCTCCCG

CAGCCCACTG

GTCCAGCTTC

CTTTTGGTGC

GCTGCTGTCC

TTCCATTCAG

TGATAGAATT

AGTGAAATGC

GATCCTCAGG

TGAAGGAAAT

GGTCTTTCCA

AGGTCTCCTA

CCACTGAGCA

GGGAAGAAGC

AGTGCGGGAA

CAAGCTATGA

ATGAAAGTCC

CTTCCCTAAT

CAAGAAGGAG

AAGTCCTCTA

CAGGCTCCTA

CCATTCTTGG

TTTCAAATGT

ACTGCATAAA

TTTGCAACAA

ACATTGCATT

TGAAACAACA.

ATGAGTGTGA

AATAAAGTTG

TAAAACTCTA

TATCTGGAAG

AGCAGTCTCA

CCTAAGTGGC

ATAGATCTTT

TTTATGTGGA

TCTGCTTAGC

GAAGTCTGAA.

TCCACAGGGT

TATGTTCCTA

ATTAAGTCCA

GTCATTCTTA

CTGAGCACAG

TGCAAAACGA

CAGAGGAAGC

CCTGGTGGTT

AGGTGTATCC

TGCCTGATAC

CACACACTGA

CCTGCTAGAT

TGGCAGTAGT

AGAACTTCTG

TGATCCCAGC

CTGAGGGAAG

ATGTCTGTCC

AGAGGTCTGG

ACTTGATCTT

AATTACCTGG

GTTCCTGTGG

TGGAAGTTTT

CTCTACCAGT

AGTTCAAGGC

ACATGTAGTT

ATCAGTAGAG

CCTGTTATAC

TTGTTTAACT

GAGGTAACTG

ACTGGAAAAC

GGAGAAGGGA

GTGAAGTGGG

TTGCTGTATA

GATTTGATGA

TTACTTTATT

TTTCTCCAG

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2099 SUBSTITUTE SHEET

-L

WO 92/20356 57 INFORMATION FOR SEQUENCE ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acids TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Glu Ala Asp Pro Thr Gly His Ser Tyr PCr/US92/04354 SUBSTITUTE SHEET

Claims (28)

1. Isolated nucleic acid molecule which codes for a tumor rejection antigen precursor, or (ii) is complementary to a nucleic acid molecule which codes for a tumor rejection antigen precursor, wherein said tumor rejection antigen precursor hybridizes to the nucleotide sequence set forth in SEQ ID NO: 8, under stringent conditions.

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 i, 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. 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.

8. The isolated nucleic acid molecule of claim 5, wherein said DNA is genomic DNA. :B n i' 1 r 1 staffaWahleenlkeep/21583.92.vs 13.4 r-98a

9. The isolated nucleic acid molecule of claim 5, wherein said DNA is cDNA. staff/ahIeer/keept21 5B3.92.vs 13.4 (r Scl ri/r Iv JUN 1995 PCT/jS 92/04354 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 an isolated nucleic acid molecule which codes for a tumor rejection antigen precursor, under stringent conditions.

12. The isolated nucleic acid molecule of claim 1, wherein IN#an 4urvno- refu, rbn said molecule codes for a 'MAG antigen precursor or is uman f4rvo ry'enoCb' complementary to a molecule which codes for aVWAGN antigen precursor.

13. The isolated nucleic acid molecule of claim 12, humwi -tumor miec-hbe wherein said +4-A 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 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. The isolated nucleic acid molecule of wherein said molecule is complementary to which codes for a MAGE antigen precursor.

16. The isolated nucleic acid molecule of wherein said molecule is DNA.

17. The isolated nucleic acid molecule of wherein said molecule is RNA. claim 12, a molecule claim 12, claim 12,

18. The isolated nucleic acid molecule wherein said molecule is a gene. of claim 12, SUBSTITUTE SHEET IPEA/US L p r p. ib i' fcu fui/-u i JUN 1995 PCT /JS 92/043 54 /OO

19. The isolated nucleic acid molecule of claim 16, wherein said DNA is genomic DNA. 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 wherein said molecule hybridizes to a molecule which codes for a MAGE antigen precursor under stringent Sconditions. i 24. The isolated nucleic acid molecule of claim 1, wherein Ssaid molecule codes for a tumor rejection antigen precursor for mastocytoma. The isolated nucleic acid molecule of claim 1, wherein said molecule codes for tumor rejection antigen precursor P1A. -II

26. The isolated nucleic acid molecule of claim 1, having Sthe nucleotide sequence of figure

27. Biologically pure culture of a cell line transfected with the nucleic acid molecule of claim 2.

28. Biologically pure culture of a cell line transfected with the nucleic acid molecule of claim 12.

29. Biologically pure culture of a cell line transfected with the nucleic acid molecule of claim 22. SUBSTITUTE SHEET IPEA/US i p..7 11 16 Rec'd PCT/PIU I I n 1993 PCT, US 92/04354 101 Biologically pure culture of a cell line of claim 27, selected from the group consisting of P1A.T2 and P1A.TC3.1.

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 rejection antigen precursor.

34. Biologically pure culture of a cell line of claim 33, wherein said human tumor rejection antigen precursor is found in melanoma cells. SUBSTITUTE SHEET IENUS i 16 R 06C'd FCJ/FITO I1 JUN~ Jtq

9204-354 lo?-. Biologically pure cell line of claim wherein said tumor rejection antigen precursor is mage-l and said isolated nucleic acid molecule consists of nucleotide sequence: 12 121 301 3C: 421 4E2 1721 1911 301 U21 2M21 12241 1561 3741 21 191 2042 2101 22 242 I it CAC2.Z-.CA &CC7GC!15CC CA7:==.AA CT0ZCAX: A7CAC-%.TT M41= 1; 1 20 1 30- tMC"CArou AMUIC tGC=.0CT WACCCGAre =Mk=ACACAC. C1OMM=?? UvT?ATC AXOA070.? Ch~i A3CA CTATUOUT AT=CTGXa" 70. 6 G~CA *=CACG4 xakC?~ma A "TC CGACCO GCATW-U.? CC~~NOCA oIW4CTOC4UA 1:c 20 30o 1 40 CV=O.*= CA 1 .CA CC TWCA ACC? I.OCCTO C .11A VA h X, CI60AZc= I s0 CA LCA"; 17CC? CCT= C=O.&CJGC &GC"DOC? to: cG-= CA C CT ?6C TGCC?7T? ?CCOAOCJ.C CDAOTOCCA? SCCCT=C-TG aT=cCT"! DJJ.CJJ.ATTA ATAO.37TC=2? to :2D 260 24C ICC 36: 420 54 61.0 1720 1710 840 9.60 2060 1.140 1320 2360 1620 210 2140 28.^0 35"0 1320 11910 2040 2.100 2160 2220 2250 2340 2400 2411 I 40 1 s0 1 SUBSTITUTE SHEET IPEA/US i m f C w w i PCT'US 92/04354 /03+' .36. The biologically pure.,culture of claim 27, wherein said cell line isVtransfected by a nucleic acid molecule coding for a cytokine. 37. The biologically pure culture of claim 36, wherein said cell line is further transfected by a nucleic acid molecule 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. 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 molecule 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 molecule transfected into said cell line. f 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. Transfected bacteria containing the nucleic acid molecule of claim 2. SUBSTITUTE SHEET y I PEA/US W W M M I U W M i L's^' I i CT/uS 92/04354 /0f .46. Mutated virus containing the nucleic acid molecule 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 molecule coding for a tumor rejection antigen which is not a tum- antigen, operably linked to a promoter. 49. Expression vector of claim 47, wherein sa:d is a strong promoter. Expression vector of claim 47, wherein said is a differential promoter. 51. Expression vector useful in transfecting comprising the isolated nucleic acid molecule 7 operably linked to a promoter. 52. Expression vector useful in transfecting comprising the isolated nucleic acid molecule 13 operably linked to a promoter. 53. Expression vector useful in transfecting comprising the isolated nucleic acid molecule 14 operably linked to a promoter. 54. Expression vector useful in transfecting comprising the isolated nucleic acid molecule 18 operably linked to a promoter. Expression vector useful in transfecting comprising the isolated nucleic acid molecule 22 operably linked to a promoter. SUBSTITUTE SHEET IPEA/US promoter promoter a cell of claim a cell of claim a cell of claim a cell of claim cell claim 105 56. The expression vector of claim 47, further comprising a nucleic acid molecule which codes for an MHC or HLA. S57. 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. 59. The expression vector of claim 58, wherein said interleukin is IL-2. 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 is BCG DNA. I 63. Expression system useful in transfecting a cell, compiising a first vector containing a nucleic acid molecule which codes for the tumor rejection antigen precursor of claim 1, and (ii) a second vector selected from the group consisting of 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 a vector containing a nucleic acid molecule which codes for an interleukin. 64. Isolated tumor rejection antigen precursor for a non- 6 tum- antigen encoded by the isolated nucleic acid molecule of claims 1 to 3, 12 to 14 or 22 to 26. 11 j 3 xreso yse sfu ntanfcig el comiiin (i irtvcorcnaiiganuli i 106 The isolated tumor rejection antigen precursor of claim 64 which is a human tumor rejection antigen precursor. 66. Isolated tumor rejection antigen precursor of claim 65, wherein said precursor is mage-l. 67. Isolated tumor rejection antigen precursor of claim 65, wherein said precursor is a precursor for antigen F. 68. Isolated tumor by the nucleic 69. Isolated tumor by the nucleic Isolated tumor by the nucleic 71. Isolated tumor by the nucleic 72. Isolated tumor antigen and is rejection antigen precursor acid molecule of claim 2. rejection antigen precursor acid molecule of claim 12. rejection antigen precursor acid molecule of claim 13. rejection antigen precursor acid molecule of claim 22. coded for coded for coded for coded for rejection antigen which is not a tum' derived from the isolated tumor 0 0 rejection antigen precursor of any of claims 64 to 71. 73. The isolated tumor rejection antigen of claim 72 which is a human tumor rejection antigen. 74. Isolated tumor rejection antigen of claim 72 having the amino acid sequence of SEQ ID NO: 4. Isolated tumor rejection antigen of claim 72, wherein said tumor rejection antigen is antigen E. 76. Isolated tumor rejection antigen of claim 72, wherein said tumor rejection antigen is antigen F. PCT'US 92/04354 /07 77. Vaccine useful in treating a subject afflicted with a cancerous condition .comprising a tumor rejection accwrd,,n, lb aaiin roq antigen precursorVfor n tu. antigon 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 -fr accordl/ -fo cdarmbq Va nn tum- antigonr., 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 tumor rejection antigen precursor is a human tumor rejection antigen precursor. Vaccine of claim 77 wherein said tumor rejection antigen precursor is mage-l. 81. Vaccine of claim 79, wherein said tumor rejection antigen precursor is antigen F precursor. 82. Vaccine useful in treating a patient with a cancer comprising the 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. A- SUBSTITUTE SHEET SIPEA/US b PCT. US 92/04354 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 *pacteric comprising the transfected agoteNeid 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 t pharmacologically acceptable adjuvant. 89. Vaccine useful in treating a subject afflicted with a cancerous condition comprising a complex of a tumor accord",g ?b c/az?, 64L 4 r <,lSd rejection antigenVwhiel- is not a tum- 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. r 93. Composition of matter useful in treating a cancerous condition comprising a non-proliferative cell line SSUBSTITUTE SHEET IPEA/JS I havil anti a tu and V:T'US 9 2/04354 /'09 ng expressed on its surface a tumor rejection accciq kC/oa;,n 6 gen precursotVfor a non tum antigen specific for mor characteristic of said cancerous condition, a pharmaceutically acceptable carrier. 94. Composition of matter of claim 93, wherein said cell line is a human cell line. 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 acczrdi' Vfo ccuwn 7 2 antigen which is not a tum- antigen specific for a tumor characteristic of said cancerous condition, and a pharmaceutically 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 pharmaceutically acceptable carrier is a liposome. 99. Composition of matter useful in treating a cancerous accordMiAIe -t condition, comprising a tumor rejection antigenV car' 7a, Vwnhch is not a tum- antigen or a tumor rejection accotd/h dr/1n antigen precursorVfor a non tum antigen, (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 accord/'v -focdav 69 antigen precursorv or a non tum antigen. SUBSTITUTE SHEET IPEANUS LD ne fo, tr/ r i u Jun i7>j PCTiJS 92/04354 //0 102. 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 I monoclonal antibody. S107. Antibody of claim 101, wherein said tumor rejection jlman, -funx rqcfhcbo ant'Ce' antigen precursor is aV+SME 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 1I II. 110. Antibody of claim 109, wherein said antibody is a monoclonal antibody. 111. Antibody which specifically binds to a tumor rejection accorc(d- -flvclat, 72 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. SSUBSTITUTE SHEET IPE AUS lb ecd't PCi/PiO 1 I Jui 19; PC/US 92/0435 1/1 114. 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. l 118. Antibody of claim 117, wherein said antibody is a ii monoclonal antibody. 119. Antibody which specifically binds to a complex of (i) Saccotdt' A cain 72 tumor rejection antigenVwhch is not a tum- antigen and (ii) HLA molecule, but does not bind to or (ii) i alone. 120. The antibody of claim 119, wherein said antibody is a monoclonal antibody. i 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 precursorVTor a non tum- antigen 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 which is not a tum- antigen derived from said tumor rejection antigen precursor, said lysis being indicative of said cancerous condition. SUBSTITUTE SHEET IPE A US LIroi 6 w4 Wi d 4 SCT'US 92,04354 //12 122. Method of claim 121, wherein said tumor rejection human -unfo/- re'echlon cuvbae&, piVcuso# antigen precursor is aVMAGE antigon. 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 aco irP -lq o alda,'n t tumor rejection antigen precursorVfor a non tum- antigen, (ii) tumor rejection antigen and (iii) cytolytic T cells specific for a tumor rejection j accorcdtqfo daim 12 antigen which is not a tum- antigen associated with said car ous condition, wherein amount of said parameter is indicative of progression or regression or onset of said cancerous condition. I 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 jy 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. SUBSTITUTE SHEET SIPEANU PCT/S 92/04354 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. 133. Method for diagnosing a cancerous condition comprising assaying a sample taken from a subject for a cytolytic aor~di c(lar' 72, T cell specific for a tumor rejection antigenVwhich is not a tum- 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: removing a lymphocyte containing sample from i. 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 p accor n c/m'nn t j 1 precursorVfor a non tum- antigen expressed by cancer cells associated with said conditions, under conditions favoring production of cytotoxic T cells I against a tumor rejection antigen derived from said tumor rejection antigen precursor, and (iii) introducing said cytotoxic T cells to said r subject in an amount sufficient to lyse said cells. 135. Method for treating a subject afflicted with a cancerous condition, comprising: e er- -c identifying a MAGE geneVexpressed by cancer cells associated with said condition; (ii) identifying an HLA molecule which presents a portion of an expression product of said MAGE gene; I' SUBSTITUTE SHEET IPEA/US £C/Uo 92/04354 (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; 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: (s herebe rededed) identifying a MAGE gene expressed by said tumor; (ii) transfecting a host cel1 having the same HLA type as said patient with said MAGE gene; (iii) culturing said transfected cells to express said MAGE gene, and; S(iv) introducing an amount of said cells to said SUBSTITUTE SHEET JPEAUS p m PCT/U 92/04'354 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 a nucleic acid sequence which codes for a tumor rejection antigen a C C rK~l g 9a/. n CO precursorVfor a non tum- antigen and (ii) a nucleic acid sequence which codes for an MHC or HLA molecule which presents a tumor rejection antigen which is not a tum 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: transfecting a host cell with a nucleic acid molecule which codes for or expresses a tumor rejection antigen precursorVfor a non tum antigen; SUBSTITUTE SHEET IIPEAJUS L .JUa1 PCT'US 92/04354 /16 (ii) transfecting said host cell with a nucleic acid molecule which codes for an HLA molecule which presents a tumor rejection antigen which -s not a tum- 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-A1. 150. Method of claim 148, wherein said interleukin is IL- 2. SUBSTITUTE SHEET IPEA/US PC T .IUS 92/04354 /17 151. 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 accordi;/ fDo cla/m 72- surface a tumor rejection antigenVwhich is not a tum- Santigen characteristic of cancerous cells in an amount i 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 accordt~i 7 dcl 72 tumor rejection antigenVwhich is not a tum- antigen expressed on a cancer cell associated with said condition, said antibody being coupled to an Santicancer 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 jaccvrdiha97 v dla/'n 64' tumor rejection antigen precurscrV for a non tum antigen expressed by a cancer cell associated with said condition, said antibody being coupled to an SUBSTITUTE SHEET S IPEAUS PCT'US 92/04354 i anticancer agent, in an amount sufficient to treat said cancerous condition. 155. Method fay 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. 4 157. 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 Sin a subject comprising administering an amount of 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. SUBSTITUTE SHEET IPEA/US LY hrl. V I id i iv1 1" -CT'us 92/04354 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. 151. 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 i 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. SUBSTITUTE SHEET IPE4US i4 P92/04-354 165. Method for treating a subject afflicted with a cancerous condition, comprising: identifying cells from said subject which accgtcOni' Yo cJcvi 6 express a tumor rejection antigen precursorVfor a non tum- antigen and present a tumor rejection antigen which is not a tumr 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. 1 167. Method for identifying a cytotoxic T cell useful in treating a subject afflicted with a cancerous condition, comprising: accordA-clg o dclio 12- S(i) identifying a tumor rejection antigenVwhich is not a tunm antigen presented by cells associated j with said cancerous condition derived from a tumor rejection antigen precursor for a non tum antigen expressed by said cells, prior to introducing them to -said subject; SUBSTITUTE SHEET IPEA/US PCT.US 92/04354 (21 (ii) contacting a cell presenting said antigen to a cytotoxic T cell, and; (iii) measuring a parameter selected from the group consisting of 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: assaying a sample from a subject to determine level of a parameter selected from the group accordt' 9 f Claor 72 consisting of tumor rejection antigenlwhich is not a tum- 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; assaying level of the parameter selected in at a second period of time and comparing it to the level determined in 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 SUBSTITUTE SHEET V IPEANUS 1' y nch U rt/ i iv PVIL i,. 9C/ZS 92/04354 /22. of a tumor rejection antigen precursorVfor a non tum- antigen 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 which is not a tum- antigen sufficient to generate a delayed type response in a subject. 'A 'V SUBSTITUTE SHEET !PEA/US