CA2201327A1

CA2201327A1 - Compositions containing tumor rejection antigen precursors or tumor rejection antigens, and an adjuvant and/or growth factor

Info

Publication number: CA2201327A1
Application number: CA002201327A
Authority: CA
Inventors: Pierre Van Der Bruggen; Thierry Boon-Falleur; Benoit Van Den Eynde
Original assignee: Individual
Current assignee: Ludwig Institute for Cancer Research Ltd
Priority date: 1994-09-30
Filing date: 1995-09-27
Publication date: 1996-04-11
Also published as: WO1996010413A1; ZA958229B; JPH10511639A; AU690371B2; AU3886495A; EP0782453A1; CN1159759A

Abstract

Compositions with improved immunogenic effect are disclosed. The compositions contain one or more peptides which, when complexed with a cell surface molecule, such as an MHC, HLA or B cell receptor, provoke an immune response. The compositions contain adjuvants such as saponins, which potentiate the immune response. Especially preferred are compositions which stimulate cytolytic T cell responses, such as peptides which satisfy criteria for MHC binding, such as peptides derived from tumor rejection antigen precursors, including MAGE, BAGE, and GAGE derived peptides.

Description

2~0 ~3~7~

3 PCT~S95/12463 COMPOSITIONS CONTAINING TUMOR REJECTION ANTIGEN
PREC~R.s OR TUMOR REJECTION ANTIGENS, AND AN ADJUVANT AND/OR GROWTH FACTOR
RELATED APPLICATIONS
This application is a continuation in part of all of the following applications, each of which discuss the provocation of cytolytic T cells in the presence of complexing tumor rejection antigens and MHC/HLA molecules: PCT application PCT/US92/04354 (designating the United States); Serial No.
938,334, filed August 31, 1992; Serial No. 008,446, filed January 22, 1993; Serial No. 54,714, filed April 28, 1993;
Serial No. 203,054, filed February 28, 1994; Serial No.
233,305, filed April 26, 1994; Serial No. 195,186, filed February 14, 1994; Serial No. 196,630, filed February 15, 1994; Serial No. 32,978, filed March 18, 1993; Serial Nos.
217,186; 217,187; and 217,188, all of which were filed on March 24, 1994; Serial No. 190,411, filed on April 1, 1994 and Serial No. 253,503, filed June 3, 1994.

FIELD OF THE lNv~.l-lON
This invention relates to compositions useful in the generation of an immune response against the classes of molecules referred to as tumor rejection antigen precursors ("TRAPs") and tumor rejection antigens ("TRAs"). The immune response includes, inter alia, the production of antibodies against the TRAPs and TRAs, as well as T cells specific for complexes of TRA and major histocompatibility molecules ("MHCs"). Such T cells and antibodies may be generated, e.g., in a mouse, rat, rabbit, sheep, goat or other non-human animal, and then used in diagnostic methods to identify tumor presence. The compositions may also be used, therapeutically, via administration to a subject afflicted with a cancerous condition or one where cell transformation has taken place, such as melanoma or dysplastic nevi, to provoke an SUBSTITUTE SHEET (RULE 26) WO96/10413 - 2 ~ PCT~S95/12463 immune response against tumors, cancer cells, and transformed cells.

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

SUBSTITUTE SHEET (RULE 26) WO96/10413 PCT~S95112463 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum~ antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., "tum+" cells).
When these tum+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "tum~"). See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).
It appears that 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, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad.
Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum~ cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tum~ variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977), Van Pel et al., 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 SUBSTITUTE SHEET (RULE 26) W096tlO413 2 2 ~ PCT~S95/12463 spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.
A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytotoxic T cells, leading to lysis. This class of antigens will be referred to as "tumor rejection antigens" or "TRAs"
hereafter. TRAs may or may not elicit antibody responses.
The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cell ("CTL" hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed.
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 major 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 P9lA
(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 only present after SUBSTITUTE SHEET (RULE 26) .... ..

~ ~ 2 0 ~ 3 ~
WO96/10413 PCT~S95/12463 the tumor cells are mutagenized. Tumor re~ection 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 "Pl", and can be provoked to produce tum~variants. Since the tum~ phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum~ cell lines as compared to their tum+ parental lines, and this difference can be exploited to locate the gene of interest in tum~ cells. As a result, it was found that genes of tum~ variants such as P9lA, 35B and Pl98 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989). This has proved not to be the case with the TRAs of this invention. These papers also demonstrated that peptides derived from the tum~ antigen are presented by the Ld molecule for recognition by CTLs. P9lA is presented by Ld, P35 by Dd and Pl98 by Kd.
Prior patent applications PCT/US92/04354, U.S. Serial No.
807,043; 764,364; 728,838 and 705,702, all of which are incorporated by reference and U.S. Patent No. 5,342,774, describe inventions involving, inter alia, genes and other nucleic acid molecules which code for various TRAPs, which are in turn processed to tumor rejection antigen, or "TRAs". SEQ
ID NOS: 1-26 which are a part of the subject application present sequences of genes coding for various TRAPs, and the TRA referred to hereafter as MZ2E, which is derived from MAGE-l TRAP (SEQ ID NO: 26).
The genes are useful as a source for the isolated and purified tumor rejection antigen precursor and the TRA
themselves, either of which can be used as an agent for treating the cancer for which the antigen is a "marker", as well as in various diagnostic and surveillance approaches to oncology, discussed infra. It is known, for example, that tum~ cells can be used to generate CTLs which lyse cells presenting different tum~ antigens as well as tum+ cells. See, e.g., Maryanski et al., Eur. J. Immunol 12: 401 (1982); and Van den Eynde et al., Modern Trends in Leukemia IX (June SUBSTITUTE SHEET (RULE 26) WO96110413 ~ 2 ~= PCT~S95/12463 1990), the disclosures of which are incorporated by reference.
The tumor rejection antigen precursor may be expressed in cells transfected by the gene, and then used to generate an immune response against a tumor of interest.
In the parallel case of human neoplasms, it has been observed that autologous mixed lymphocyte-tumor cell cultures ("MLTC" hereafter) frequently generate responder lymphocytes which lyse autologous tumor cells and do not lyse natural killer targets, autologous EBV-transformed B cells, or autologous fibroblasts (see Anichini et al., Immunol. Today 8:
385-389 (1987)). This response has been particularly well studied for melanomas, and MLTC have been carried out either with peripheral blood cells or with tumor infiltrating lymphocytes. Examples of the literature in this area including Knuth et al., Proc. Natl. Acad. Sci. USA 86: 2804-2802 (1984); Mukherji et al., J. Exp. Med. 158: 240 (1983);
Hérin et all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol 6: 839-853 (1988). Stable cytolytic T cell clones have been derived from MLTC responder cells, and these clones are specific for the tumor cells. See Mukherji et al., supra, Hérin 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 tumor cells in vivo. 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 precursors of TRAs presented on human tumors. It is now possible to isolate the nucleic acid sequences which code for tumor rejection antigen precursors, including, but not being limited to those most characteristic of a particular tumor, with ramifications that are described infra.
Additional work has focused upon the presentation of TRAs by the class of molecules known as major histocompatibility complexes, or "MHCs". Human forms of these molecules are SUBSTITUTE SHEET (RULE 26) ~ r 2 ~ ~ ~ 3 2 ;r8 WO96/10413 PCT~S95112463 "human leukocyte antigens" or "HLAs". This work has resulted in several unexpected discoveries regarding the field.
Specifically in U.S. patent application Serial Number 938,334, now U.S. Patent No. the disclosure of which is - incorporated by reference, nonapeptides are taught which are presented by the HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect particular peptides to bind one HLA molecule. These peptides, presented herein as SEQ ID NOS: 27-34 are also presented in Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). This is important, because different individuals possess different HLA
phenotypes. As a result, while identification of particular peptides or of particular motifs, and the peptides which are members thereof, as being partners for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype.
There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA
phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
In U.S. Patent Application Serial Number 008,446, filed January 22, 1993 and incorporated by reference, the fact that the MAGE-l expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-Cw* 1601 molecules. The disclosure shows that a given TRAP can yield a plurality of TRAs.
In U.S. Patent Application Serial Number 994,928, filed December 22, 1992, and incorporated by reference herein, tyrosinase is described as a tumor rejection antigen precursor. This is a well known molecule as per Kwon, U.S.
Patent No. 4,898,814. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules. The peptide presented thereby is described in U.S. Application Serial No. 54,714, filed April 28, 1993. SEQ ID NO: 35 sets SUBSTITUTE SH EET (RULE 26) WO96/10413 r 2 ~ 7 PCT~S95/12463 forth this peptide. Additional tyrosinase derived peptides presented by HLA molecules are set forth in Serial Nos.
203,054, and 233,305 filed February 28, 1994 and April 26, 1994 and incorporated by reference (SEQ ID NOS: 36-41).
Other peptides which are TRAs are described in additional patent applications. U.S. Patent Application Serial No.
195,186, filed February 14, 1994, and incorporated by reference herein, sets forth three peptides (SEQ ID NOS: 42-44 herein), which are derived from MAGE-1 and which complex with HLA-Cw~ 1601. Serial No. 196,630, filed February 15, 1994, discloses an unrelated tumor rejection antigen precursor, the so-called "BAGE" gene, and peptides derived therefrom, which are processed and then presented by HLA-Cw* 1601. These are set forth as SEQ ID NOS: 45-48, and this application is incorporated by reference. SEQ ID NO: 48 is the tumor re~ection antigen. Additional coding sequences for a tumor rejection antigen precursor are set forth in Serial No.
32,978, filed March 18, 1993 and incorporated by reference.
These are included herein as SEQ ID NOS: 49 and 50. A more extended sequence for this gene is set forth in Serial No.
272,351, filed July 8, 1994 incorporated by reference, and is SEQ ID NO: 51. In Serial No. 96,039, filed July 22, 1993, the sequence of tumor rejection antigen precursor GAGE is set forth. See SEQ ID NO: 52 for this information.
A series of peptides which provoke lysis by cytolytic T
cells when presented by MHC molecules are set forth in Serial No. 217,186, Serial No. 08/217,188, and Serial No. 217,187, all filed on March 24, 1994, and all of which are incorporated by reference herein. The first of these applications discloses MAGE-3 derived peptides presented by HLA-A2. Five peptides are of interest. These are repeated here as SEQ ID
NOS: 53-57. The second application presents 11 sequences derived from MAGE-2, believed to complex with HLA-A2.1 molecules (SEQ ID NOS: 58-68). The last of these applications discloses two additional peptides (SEQ ID NOS: 69 and 70) derived from MAGE-3 which complex to HLA-A2. Serial No.
190,411, filed April 1, 1994 and incorporated by reference, SUBSTITUTE SHEET (RULE 26) ~ WO96/10413 ~ 3 2 7 PCT~S95/12463 sets forth three peptides (SEQ ID NO: 71-73), derived from MAGE-l, which are immunogenic in that they provoke production of antibodies in a host animal to which they have been administered. Seial No. 253,503, filed June 3, 1994 and incorporated by reference, teaches a further tumor rejection antigen precursor gene (SEQ ID N0:74), and a peptide, derived therefrom (SEQ ID NO:75), whch is presented by HLA-B44 molecules. Further in the application of Coulie, Ikeda and Boon-Falleur, filed concurrently, a sequence coding for a tumor rejection antigen precursor known as DAGE (SEQ ID NO:76) is set forth. DAGE is found almost universally on tumor cells, and only on testis cells with respect to normal cell expression. This makes it especially useful for cancer diagnosis and in the applications disclosed herein. The above listing should not be presumed to be exhaustive of the TRAP
and TRA literature, but is presented to show its diversity and the fact that these materials not only provoke T cell proliferation, but also stimulate production of antibodies.
It is well known that antibody producing cells can be used as a source to produce hybridomas, which in turn produce monoclonal antibodies. Thus, when the term "antibodies" is used herein, it encompasses both polyclonal and monoclonal antibodies.
The parent applications to the present case, including Serial No. 142,368 and Serial No. 190,411, both discuss the usefulness of combining TRAPs or TRAs with various materials as adjuvants, to produce vaccines, immunogenic compositions, etc. Adjuvants, broadly defined, are substances which promote immune responses. Frequently, the adjuvant of choice if Freund's complete adjuvant, or killed B. pertussis organisms, used in combination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (Second edition, 1986), at pages 61-63, which are incorporated by reference herein.
Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Such molecules, according SUBSTITUTE SH EET (RULE 26) 2 2 ~ ~ 3 ~ 7 W096/10413 PCT~S95112463 to Goding, generally have molecular weights below about 1000.
Among the carriers suggested by Goding, at page 283, are keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, and fowl immunoglobulin.
What is problematic about such carriers, however, is that frequently they are also immunogenic themselves. Thus, the immune response may be a general one, with part, most, or all of it being directed against the carrier molecule rather than the immunogen itself.
Exemplary of developments in the art as they relate to adjuvants is U.S. Patent No. 5,057,540 to Kensil, et al, the disclosure of which is incorporated by reference herein.
Kensil et al disclose the preparation of various saponin extracts, which are useful as adjuvants in immunogenic compositions. As natural products, the extracts are not completely defined. Kensil, et al do provide a complete and enabling disclosure for how various extracts, including QA-7, QA-19, and QA-21 (also referred to as QS-21) are prepared.
Experiments are set forth in which bovine serum albumin ("BSA") was combined with various extracts (examples 8 and 9), and where feline leukemia virus recombinant glycoprotein "gp70R~ was tested, following absorption to aluminum hydroxide (alum). The two immunogens tested, however, are expected to be immunogenic in their own right (gp70R~ has a molecular weight of 70 kd, and serum albumin has about the same molecular weight). No experiments were carried out at all on molecules which should, per se, be considered to be poorly or even non-immunogenic, and thus would be expected to require the use of alum absorption or the use of haptenic carriers for provocation of a response.
In PCT Application W09219758, which corresponds to defensive publication 7697275, which is incorporated by reference herein, an adjuvant referred to as "MTP-MF59" is disclosed. This adjuvant is used in connection with a Plasmodium falciparum protein, "Pfs-25-B". This combination is described as a transmission blocking vaccine. The P.
falciparum protein is itself large enough to be immunogenic.

SUBSTITUTE SHEET (RULE 26) .

~ WO96/10413 2 ~ ~ ~ 3 ~ 7 PCT~S95/12463 Thus, none of the art shows that the improved adjuvants can be used in combination with presumptively non-immunogenic proteins and peptides to yield immunologically effective compositions. This is especially true for TRAP and TRA
molecules, as outlined supra.
It has now been found, surprisingly, that compositions comprising tumor rejection antigen precursors or tumor rejection antigens can be made which, when administered to a subject animal, provoke an immunogenic response. In especially preferred embodiments the immunogenic portion of the composition consists of TRAP or, more preferably TRA
molecules, of one or more types, and an adjuvant. Especially preferred are compositions where the adjuvant is QS21, as is disclosed in the Kensil, et al patent, incorporated by reference supra.
The immunogens of this invention consist of TRAPs or TRAs, meaning that they do not include haptens, carriers, precipitated alum, or any of the materials normally associated with materials which are or are expected to be poorly immunogenic. In especially preferred embodiments, the compositions consist essentially of the immunogen and the adjuvant.
The invention is described in greater detail in the disclosure which follows.
D~TATT.F~n DESCRIPTION OF PREFERRED EMBOD
Example The following example demonstrates the use of peptide MZ2E (SEQ ID NO: 26) in connection with adjuvant QS-21. It is to be understood, however, that one may routinely substitute proteins or peptides (the first component of the composition) and adjuvants (the second component of the composition). The unexpected effect of the combination of MZ2E and QS21 should occur in the alternate combinations, i.e., when other peptides are used.
Subjects tested are patients with stage IV melanoma or high risk stage III malignant melanoma. Stage IV patients have a median survival time of one year following diagnosis, SUBSTITUTE SH EET (RULE 26) WO96/10413 2 ~ O ~ 3 2 7 PCT~S95/12463 ~

and only a 15~ chance of long-term survival (Balch, et al, Cutaneous Melanoma, J.b. Lippincott, Philadelphia, 1992). The standard therapy for these patients includes treatment with decarbazine or drug combinations with decarbazine; however, response rates only range from 8-25%, and there is no evidence of the treatment extending survival. Balch et al, supra.
Patients with high risk Stage III melanoma (pT4 thick truncal primary tumors or extremity melanomas, with five or more positive regional lymph nodes) have a median survival of 1-2 years following onset, and a 19% chance of long term survival.
Balch, et al, supra.
Twelve patients are used in the study, all of whom have either Stage IV or high risk Stage III malignant melanoma in accordance with the criteria provided supra, confirmed by review of histology.
The patients fulfil the following additional criteria:
(i) complete recovery from surgery;
(ii) no chemotherapy or immunotherapy, within the four weeks preceding immunization;
(iii) expected survival of at least three months;
(iv) Karnofsky Performance Status of 60 or more;
(v) Laboratory results as follows:
granulocytes 2 2,500/min3 lymphocytes 2 700/min3 platelets 2 lO0,000/min3 serum creatinine ~ 2.0 mg/lO0 ml serum bilirubin < 2.0 mg/lO0 ml (vi) for MZ2E immunization, patients are positive for HLA-Al;
(vii) for MZ2E immunization, the patients' tumors express MAGE-l;
(viii) they be over l9 years old and be capable of giving informed consent in writing.

Any potential subjects who satisfy any of the following criteria are excluded:
(i) clinically significant heart disease;

SUBSTITUTE SH EET (RULE 26) WO96/10413 ~ ~ a ~ 3 ~ 7= PCT~S~5/12463 (ii) other serious illness, such as active infection requiring antibiotics, or bleeding disorders;

(iii) treatment with antihistamines, N-SAIDS, or corti-costeroids;
(iv) immunodeficiency, splenectomy, or radiation therapy to the spleen;
(v) pregnancy or lactation;
(vi) women of childbearing age who do not use effective methods of contraception.
All subjects are treated as outpatients. They are immunized, subcutaneously, with MZ2E (30 ug or 300 ug) and QS21 (lO0 ug), in 0.3 ml phosphate buffered saline, pH 7.4. Six patients receive 30 ug of peptide, and six receive 300 ug. The first injection is into the deltoid region of the anterior aspect of the thigh, and the site of injection is changed with follow up injections. No injections are given into limbs where draining lymph nodes have been surgically removed or irradiated.
Injections are given at day l, and then at days 8, 15, 22, and 57. Patients are monitored over a course of 12 weeks, unless intervention is required (as determined by the investigator). Any patients who show stabilization or tumor response remain in the study until disease progression is evidenced. Patients may also be removed from the study or receive different doses of the peptide, if toxicity reactions are observed.
The patients show response as follows. In a complete response, all signs, symptoms, biochemical and imaging evidence of tumor disappear for a period of at least 30 days.
In a partial response, there is a decrease in size of all measurable tumors of at least 50% of the sum of products of the greatest and perpendicular diameters for at least 30 days, without the appearance of new lesions or progression of any new lesions. In a minor response, there is a decrease in size of all measurable tumors of at least 25~ of the sum of products of greatest and perpendicular diameters, for at least 30 days, without appearance of new lesions or progression o~

SUBSTITUTE SH EET (RULE 26) WO96/10413 PCT~S95/12463 any lesion. In stable disease, there is a change of less than 25% of the sum of products of greatest and perpendicular diameters, for at least 30 days, without the appearance of new lesions or progression of lesions. In progression of the disease, there is appearance of new tumors, or an increase in the size of measurable tumors of at least 50% of the sum of the product of greatest and perpendicular diameter.
The foregoing example demonstrates a composition comprising an amount of a tumor rejection antigen, i.e., MZ2E, and an adjuvant, i.e., QS21, and the use of the composition in the n vivo treatment of cancer (i.e., melanoma). The tumor rejection antigen is used in an amount sufficient to provoke an immune response against tumor cells which present it on their surface.
The compositions of the invention comprise any tumor rejection antigen precursor ("TRAP") or tumor rejection antigen ("TRA"), in combination with a pharmaceutically acceptable adjuvant. Preferred embodiments of the invention utilize the TRAPs and TRAs discussed supra and set forth in SEQ ID N0: 65, as well as the adjuvants described in the Background section.
As will be seen from the foregoing discussion, an important aspect of the invention is stimulation of proliferation of T cells. This can be an initial stimulation or an augmentation of a prior stimulation. In particular, it is desirable to stimulate cytolytic T cells which present peptides, such as the TRAs described herein, on their surfaces. The cytolytic T cells recognize these complexes of MHC and peptide, bind thereto via their receptor, and proliferate. They also lyse the recognized cells. This response can be used not only in vivo, but in vitro, as it is well established that cytolytic T cells specific for particular complexes of MHC and peptide are present in the blood of subjects who have experienced cell transformation.
By c~tacting a blood sample of individuals in vitro with the peptide of interest and cells which present the MHC molecule of interest, any cytolytic T cells in the blood sample will SUBSTITUTE SH EET (RULE 26) WO96/10413 ~ ~ ~ 0 ~ 3 ~ 7- PCT~S95/12463 expand, i.e., proliferate. This proliferation an be measured via any of the well known assays designed therefor.
~ Especially preferred are the radioactive chromium (5lCr) release assay, and the measurement of release of tumor necrosis factor (TNF).
lOThe compositions are also useful as stimulators of B cell proliferation, or antibody production. Again, it is well known that B cells produce antibodies, and the size of their targets are well within the sizes of the tumor rejection antigens, and certainly the tumor rejection antigen precursors. As with T cells, the stimulation may be "ab initio", or an augmentation of a prior response, in vitro or in vivo.
The amount of TRAP or TRA used will vary, depending upon the purpose of the immunization and the subject to which it is administered. For example, in the case of generating murine antibodies which can then be used, e.g., to diagnose for the presence of cancer cells presenting a TRA, the amount of protein or peptide may be less than that used in a course of in vivo therapy, such as that described in the example, supra.
In general, a preferred dose can range from about l ug to about 750 ug of protein or peptide per dose. In a preferred embodiment, the range is from about lO ug to about 500 ug.
Most preferably, anywhere from about 30 ug to about 300 ug per dose may be used. Of course, in the context of the therapeutic aspect of the invention, the investigator will modify the dose, as a six month old infant will require dosing different from a full grown man, e.g. The mode of administration may vary, with preferred forms being oral, subcutaneous, intramuscular, intravenous and intraperitoneal adminstration.
The choice of TRAP or TRA protein or peptide in the composition will depend upon parameters determinable by the artisan. It is art recognized, for example, that different TRAs are presented by the various MHC molecules. As such, if a subject is typed, using well known t~chn;ques, as presenting HLA-AZ molecules on the surface of tumor cells, one will use SUBSTITUTE SHEET (RULE 26) WO96/10413 2 2 0 ~ 3 ~ ~ PCT~S95/12463 a TRA presented by HLA-A2 molecules rather than one presented by, e.g., HLA-Cw~ 1601. Similarly, using techniques such as polymerase chain reaction ("PCR"), lysis studies, and other assay methodologies which are well known in the art, one can determine which tumor rejection antigen precursor gene or genes are being expressed by a subject patient. This will lead to the decision as to what protein or peptide to use.
Again, by way of example, if a subject's tumor cells are expressing MAGE-3 but not MAGE-1, the peptide used in immunization should be derived from MAGE-3, and not MAGE-1.
While the molecules discussed herein are referred to as "tumor" rejection antigens and "tumor" rejection antigen precursors, it is intended that their use, in a therapeutic and also a diagnostic context, extends beyond cancer per se.
The art is familiar with pathological conditions, such as displastic nevis, which are not cancer per se, but where the cells of the afflicted individuals are in fact characterized by transformation. Any and all such conditions are within the intended ambit of the invention.
Other aspects of the invention will be clear to the skilled artisan and need not be reiterated here.
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 po~sible within the scope of the invention.

SUBSTITUTE SHEET (RULE 26) ~ WO 96/10413 2 ~ O ~ ~ ~ 7 PCTIUS95/12463 (1) GENERAL INFORMATION:

(i) APPLICANTS: Boon-Falleur, Thierry; van den Eynde, Benôit;
van der Bruggen, Pierre (ii) TITLE OF INVENTION: COMPOSITIONSCONTAININGTUMORREJECTION
ANTIGEN PRECURSORS OR TUMOR REJECTION ANTIGENS, AND AN
ADJUVANT AND/OR GROWTH FACTOR
(iii) NUMBER OF SEQUENCES: 76 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10022 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.Z5 inch, 360 kb storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/315,961 (B) FILING DATE: 30-SEPTEMBER-1994 (C) CLASSIFICATION: 435 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US92/04354 (B) FILING DATE: 22-MAY-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 07/938,334 (B) FILING DATE: 31-AUGUST-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/008,446 (B) FILING DATE: 22-JANUARY-1993 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/054,714 (B) FILING DATE: 28-APRIL-1993 SUBSTITUTE SHEET (RULE 26) 2 ~ 7 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/203,054 (B) FILING DATE: 28-FEBRUARY-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/233,305 (B) FILING DATE: 26-APRIL-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/195,186 (B) FILING DATE: 14-FEBRUARY-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/196,630 (B) FILING DATE: 15-FEBRUARY-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/032,978 (B) FILING DATE: 18-MARCH-1993 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/217,186 (B) FILING DATE: 24-MARCH-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/217,187 (B) FILING DATE: 24-MARCH-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/217,188 (B) FILING DATE: 24-MARCH-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/190,411 (B) FILING DATE: l-APRIL-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/253,503 (B) FILING DATE: 3-JUNE-1994 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCKET NUMBER: LUD 5370 (ix) TELECOMMUNICATION INFORMATION:
~o (A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 SUBSTITUTE SHEET (RULE 26) 3 ~ 7 (2) INFORMATION FOR SEQUENCE ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 462 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQUENCE ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Asp Asn Lys Lys Pro Asp Lys Ala His Ser Gly Ser Gly Gly Asp Gly Asp Gly Asn Arg Cys Asn Leu Leu His Arg Tyr Ser Leu Glu Glu Ile Leu Pro Tyr Leu Gly Trp Leu Val Phe Ala Val Val Thr Thr SUBSTITUTE SH EET (RULE 26) WO 96/10413 i ~ 3 ~ 7 PCTtUS95tl2463 Ser Phe Leu Ala Leu Gln Met Phe I le Asp Ala Leu Tyr Glu Glu Gln Tyr Glu Arg Asp Val Ala Trp Ile Ala Arg Gln Ser Lys Arg Met Ser Ser Val Asp Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Tyr Tyr Asp Asp Glu Asp Asp Asp Asp Asp Ala Phe Tyr Asp Asp Glu Asp Asp Glu Glu Glu Glu Leu Glu Asn Leu Met Asp Asp Glu Ser Glu Asp Glu Ala Glu Glu Glu Met Ser Val Glu Met Gly Ala Gly Ala Glu Glu Met Gly Ala Gly Ala Asn Cys Ala Cys Val Pro Gly His His Leu Arg Lys Asn Glu Val Lys Cys Arg Met Ile Tyr Phe Phe His Asp Pro Asn Phe 3 o CTG GTG TCT ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT 576 Leu Val Ser Ile Pro Val Asn Pro Lys Glu Gln Met Glu Cys Arg Cys Glu Asn Ala Asp Glu Glu Val Ala Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Met Gly Asn Pro Asp Gly Phe Ser Pro 4 o TAG 675 SUBSTITUTE SHEET (RULE 26) ~ 22~32~

(2) INFORMATION FOR SEQUENCE ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 base pairs (B) TYPE: nucleic acid lo (C) STRANDEDNESS: single (D ) TOPOLOGY: 1 i near ( i i ) MOLECULE TYPE: genomic DNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQUENCE ID NO: 4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1365 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngl e ( D ) TOPOLOGY: 1 i near ( i i ) MOLECULE TYPE: genomic DNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

SUBSTITUTE SH EET (RULE 26) 2 2 ~ ~ 3 ~ 7 (2) INFORMATION FOR SEQUENCE ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4698 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

SUBSTITUTE SH EET (RULE 26) -r 2 2 ~ ~ 3 ~ 7 wo 96/10413 PCT/USg5/12463 CCTCCCTCCC CCTCCCCAGG CC I I I I I I I I l l l l l l l l l l l l l l l l l l l l 1566 SUBSTITUTE SHEET (RULE 26) wo 96/10413 ~ ~ O ~ 3 2 7 PCT/US95/12463 ~1 l l l l l l l l l l GCACCTTGTT TTCCAAGATC CCCCTCCCCC TCCGGCTTCC 2066 4 o GCATGCAGGT ACTGGCTTCA CTAACCAACC ATTCCTAACA TATGCCTGTA 3626 SUBSTITUTE SHEET (RULE 26) .

1~ WO 96/10413 ~ 3 ~ PCT/US95/12463 lo TTCAAGAAAG ATCACACGCC ATGGTTCACA TGCAAATTAT TATTTTGTCG 3976 (2) INFORMATION FOR SEQUENCE ID NO: 6:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid ( C ) STRANDEDNESS: s i ngle (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Leu Pro Tyr Leu Gly Trp Leu Val Phe SU BSTITU TE SH EET (R U LE 26) wo 96/10413 ~ ~ 2 ~ ~ 3 ~ 7 PcT/usss (2) INFORMATION FOR SEQUENCE ID NO: 7:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2419 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle (D) TOPOLOGY: 1 inear ( i i ) MOLECULE TYPE: genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

2 o GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 3 o CCTGGAGGAG GTGCCCACTG CTGGGTCAAC AGATCCTCCC CAGAGTCCTC 800 GATCTTCGGC MAGCCTCTG AGTCCTTGCA GCTGGTCTTT GGCATTGACG l lOO

S U BSTIT U TE S H EET (R U LE 26) ~ WO 96/10413 ~ 3 2 7 PCT/US95/12463 lo ACCTTCCAGG GCCGCGTCCA GCAGCTTCCC CTGCCTCGTG TGACATGAGG 1650 2 o AATTCTTCCT GTTCACTGGC TCTTTTCTTC TCCATGCACT GAGCATCTGC 2150 (2) INFORMATION FOR SEQUENCE ID NO: 8:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5674 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle 3 5 ( D ) TOPOLOGY: 1 i near ( i i ) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: MAGE- 1 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

SUBSTITUTE SHEET (RULE 26) wo 96/10413 ~ ~ O ~ 3 2 7 PCT/US95/12463 SUBSTITUTE SHEET (RULE 26) WO 96110413 r ~! 2 t~ ~ 3 ~ 7 PCTIUS95/12463 SUBSTITUTE SHEET (RULE 26) .

wo 96/10413 ~ ~ 0 '~ 3 ~ ~ PCT/US95/12463 3 o GCTTTTTCTT CCCATCCCTG CGTGMGCAG CTTTGAGAGA GGAGGAAGAG 4800 SUBSmUTE S~tEET (RULE 26) WO 96/10413 ~ 3 2 ~ PCT/US95/12463 lo GCTGGGGCAT TTTGGGCTTT GGGMACTGC AGTTCCTTCT GGGGGAGCTG 5650 (2) INFORMATION FOR SEQUENCE ID NO: 9:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4157 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: MAGE-2 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCC/\CCCMG TCACGGGCCC GGATGTGACG CCACTGACTT GCACATTGGA 100 3 o AGGACTGAGG CGGGCCTCAC CCCAGACAGA GGGCCCCCM TTAATCCAGC 250 SVBSTITUTE SHEET (RULE 26) wo 96/10413 2 2 ~ ~ 3 2 ~ 3 2 PCT/US95/12463 3 o CGGAGTCTGG CCMCCCTGC TGAGACTTCT GGGMTCCGT GGCTGTGCTT2100 CTGTACCCTG AGGTGCCCTC CCACTTCCTC CTTCAGGTTC TGACCCCCI\C 2400 SUBSTITUTE SHEE~ (RULE 26) ~ 2 ~ 11 32 7 3 o TTTCTGTTCT GTTGGATGAC TTTGAGATTT ATCTTTCTTT CCTGTTGGAA 3742 ;uBS, ,TV, E ~iEt I ~RULE 26) WO 96/10413 2 2 ~ 2 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 10:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 662 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear ( i i ) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-21 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQUENCE ID NO: 11:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1640 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA
(ix) FEATURE:
(A) NAME/KEY: cDNA MAGE-3 SUBS I ITUTE S~tEEl (RULE 2~i) ? ~0 ~ 32 7 W 096/10413 PCTrUS95/l2463 =
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

TMGAGTCTT GttTTTTACT CMMTTgGGA AATCCATTCC ATTTTGTGAA1466 SUBSTITUTE SHEE~ (RULE 26~

WO96/10413 7 2 G ~ 3 ~ 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 12:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 943 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l i near ( i i ) MOLECULE TYPE: genomi c DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-31 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

AGGACTTGGT CTGAGGCAGT GTCCTCAGGT CACAGAGTAG AGGGGgCTCA 200 3 o CTCTCACTTC CTCCTTCAGG TTCTGAGGGG ACAGGCTGAC CTGGAGGACC 400 AGAGGCCCCC GGAGGAGCAC TGAACCAC/\A GATCTGTMG TMGCCTTTG 450 GGC CTT GAG GCC CGA GGA GAg GCC CTG GGC CTG GTG GGT GCG 664 SUBSTITUTE SHE~ (RULE 2~) Wo 96/10413 2 2 ~ ~ ~ 2 ~. PcTlus9sll2463 2) INFORMATION FOR SEQUENCE ID NO: 13:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2531 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng 1 e (D) TOPOLOGY: 1 inear ( i i ) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-4 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

3 o CTTCCTCCTT CAGGTTCTGA GCAGACAGGC CMCCGGAGA CAGGATTCCC 450 SUBSmUTE SHEET (RULE 26) WO 96/10413 ~ 2 ~ ~ 3 2 7 PCT/US95/12463 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAf~AGTTGC 2478 SUBSTITUTE St{EET (RULE 26) wo 96/10413 ' ~ 3 2 7 PcTIusssll2463 (2) INFORMATION FOR SEQUENCE ID NO: 14:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2531 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle ( D ) TOPOLOGY: l i near (ii) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: MAGE-41 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID N0: 14:

3 o TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC TGCACTCTTG 600 SUBSTITUTE SHE~ tRULE 26) wo 96/10413 ~ ~ O ~ 3 2 7 PCT/USg5/12463 ~

SUBS 111 UTE SHEET (RULE 26) WO 96/10413 ~ 3 2 7 PCTIUS95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 15:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1068 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng l e (D) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA to mRNA
( ix) FEATURE:
(A) NAME/KEY: cDNA MAGE-4 (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

2 o ATG CTG GAG AGA GTC ATC MM MT TAC AAG CGC TGC TTT CCT 166 3 o CCA AGG GCT CTG GCT GAA ACC AGC TAT GTG AM GTC CTG GAG 586 SUBSTtTUTE SHEET (RULE 26) =

2 2 ~ ~1 3 ~ 7 wo 96/10413 PCT/US95112463 (2) INFORMATION FOR SEQUENCE ID NO: 16:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2226 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle lo (D) TOPOLOGY: 1 inear ( i i ) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-5 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GCCTCCTGAT AATCGTCTTG GGCAT~ATTG CMTGGAGGG CAMTGCGTC 1208 SUBSTlTUTE SHEET tRULE 26) 3 ~ ~

TATTCGGT M M llllllll AAAAAATGTG CATACCTGGA TTTCCTTGGC 2008 (2) INFORMATION FOR SEQUENCE ID N0: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2305 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
(ix) FEATURE:
(A) NAME/KEY: MAGE-51 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

SUBSTITUTE SHE~ tRULE 26) wo 96/10413 ~ 2 7 PCTIUS95/12463 3 o CTCCTATGAT GGCCTGGTGG TTTMTCAGA TCATGCCCM GACGGGCCTC 1242 SUBSTlTUTE SHEET (RULE 26) WO96/1041~ 3 2 7 PCI'IUS95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 18:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: MAGE-6 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR SEQUENCE ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:

SUBSTITUTE SHEET (RULE 26) WO96110413 2 2 ~ ~ 3 2 7 PCrlUSg5/12463 ~

(A) LENGTH: 1947 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
(ix) FEATURE:
(A) NAME/KEY: MAGE-7 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GCCAATCCAG TGAGGGCACC AGCAGCCGGG AAC/\CCACCC GCCAACCACC 650 3 o ATC GAG TCA AGG AGC TGG TCA CAA AGG CAG MA TGC TGG ACA 769 SUBSTlTUTE SHEET (RULE 26) WO 96/10413 ~ 2 7 PCT/US95/12463 CTGTGTTTGA AC/\CACCAGT CAATGTTCTC AGTAGCGGGG AGTGTGTTGG1464 1 o GATTTGGAGG TTTATCTTTG TTTCCTTTTG CAGTCGTTCA AATGTTCCTT1564 GCAGACTTAC TG l l l l l l AT ATAGTTAAAA GTAAGTGCAT TGTTTTTTAT 1664 (2) INFORMATION FOR SEQUENCE ID NO: 20:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1810 base pairs (B) TYPE: nucleic acid 2 5 ( C ) STRANDEDNESS: s i ngle ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: MAGE-8 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GGTTCGCAGA GMCAGGCCA GCCAGGAGGT CACC/\CCCCC CACACMCCA 300 SUBSTITUTE SHEET (RULE 26) wo 96/10413 ~ ~ ~ 3 ~ 7 PcTlus9sll2463 (2) INFORMATION FOR SEQUENCE ID NO: 21:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1412 base pairs (B) TYPE: nucleic acid SUBSTITUTE SHEET (RULE 2~) 2 ~ ~ ~ 3 2 7 ~ Wo 96/104I3 PCT/US95/12463 ( C ) STRANDEDNESS: s i ng 1 e ( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: MAGE-9 gene (xi ) SEQUENCE DESCRIPTION: SEQ ID N0: 21:

MM GAC MC TGC GCC ccr GM GAG GTT ATC TGG GAA GCG TTG1099 4 o CAC TAC GAG TTC CTG TGG GGT TCC AAG GCC CAC GCT GAA ACC 1267 SUBSTITUTE SH~ET ~RULE 26) wo 96/10413 1 2 2 ~ ~ 3 2 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID N0: 22:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 920 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle 1 5 ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-10 gene (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 22:

SUBSTI~VTE SHE~ (RULE 26) ~I WO96110413 2 ~ ~ ~1 3 ~ 7 Pcr/uss5ll2463 (2) INFORMATION FOR SEQUENCE ID NO: 23:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1107 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng 1 e ( D ) TOPOLOGY: 1 i near ( i i ) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: MAGE-ll gene (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

3 o TATCTGATGA GGGCTCTGGC AGCCMGMM AGGAGGGGCC MGTACCTCG500 GATMTTGAT TTGGTTCATT TATTCTCCGC MGTATCGAG TCMGGGGCT6û0 SUBSTITUTE SHEE~ (RULE 26) WO 96/10413 2 2 û ~ 3 2 7 PCT/US95/12463 ~

(2) INFORMATION FOR SEQUENCE ID NO: 24:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2150 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: 1 i near ( i i ) MOLECULE TYPE: genomic DNA
( ix) FEATURE:
(A) NAME/KEY: smage-I
(Xi ) SEQUENCE DESCRIPTION: SEQ ID N0: 24:

3 o AGCTTGATCC ACGAGTTCAG AAGTCCTGGT ATGTTCCTAG MMG 394 SUBSTITUTE S}~EEl' (RULE 26) WO96/10413 ~ 2 ~ O ~ 3 2 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 25:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2099 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng l e SUBS 1 1 1 UTE SHEE~ (RULE 2~i) , . .

WO96/10413 2 ~ O ~ 3 ~ 7 PCT/US95/12463 ~?.

( D ) TOPOLOGY: 1 i near (ii) MOLECULE TYPE: genomic DNA
( i x ) FEATURE:
(A) NAME/KEY: smage-II
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

2 o GAAAGATGTT CTCCTGGMA GCTTCMMG CCAGGTCTCC ATTAAGTCCA 450 SUBSTITUTE SHEET (RULE 26) ~ W 096/10413 2 2 0 ~ 3 ~ 7 PCT~US95/12463 lo TCACTTGTCA GATTAGGACT TGTTTTGTTA TTTGCMCAA ACTCCAAMC 1800 (2) INFORMATION FOR SEQUENCE ID NO: 26:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 ami no acids (B) TYPE: amino acids (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Glu Ala Asp Pro Thr Gly His Ser Tyr (2) INFORMATION FOR SEQUENCE ID NO: 27:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids ( D ) TOPOLOGY: l i near ( i i ) MOLECULE TYPE: prote i n (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

4 o G l u Va l Va l Pro I l e Ser H i s Leu Tyr SUBSTITUTE SHEET (RULE 26) WO 96110413 2 ~ ~ ~ 3 ~ 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ~8:

Glu Val Val Arg Ile Gly His Leu Tyr (2) INFORMATION FOR SEQUENCE ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Glu Val Asp Pro Ile Gly His Leu Tyr (2) INFORMATION FOR SEQUENCE ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Gly Val Asp Pro Ala Ser Asn Thr Tyr SUBSTITUTE SHEET (RULE 26) WO 96/10413 ~ 3 2 ~ PCTIUS95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino ac;ds (B) TYPE: amino acids (D) TOPOLOGY: linear o (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Glu Val Asp Pro Thr Ser Asn Thr Tyr (2) INFORMATION FOR SEQUENCE ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Glu Ala Asp Pro Thr Ser Asn Thr Tyr (2) INFORMATION FOR SEQUENCE ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

Glu Ala Asp Pro Thr Ser Asn Thr Tyr SU85TITUTE S}tE~T tRULE 26) (2) INFORMATION FOR SEQUENCE ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Glu Val Asp Pro Ile Gly His Val Tyr (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: single (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Tyr Met Asn Gly Thr Met Ser Gln Val 2s 5 (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

Met Leu Leu Ala Val Leu Tyr Cys Leu Leu SVBSTmJTE SHEE~ (RULE 26) WO 96/10413 2 2 ~ ~ 3 2 ~ PCT/US95/12463 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: l;near (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Met Leu Leu Ala Val Leu Tyr Cys Leu (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Leu Leu Ala Val Leu Tyr Cys Leu Leu (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala SUBSTITUTE SHEET (RULE 26) WO 96/10413 2 2 0 ~ 3 2 7 PCT/US95/12463 (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Ser Glu Ile Trp Arg Asp Ile Asp Phe Ala (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Ser Glu Ile Trp Arg Asp Ile Asp Phe (2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr Gln Asp Leu SUBSTITUTE SHEET tRULE 26) (Z) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

Ser Ala Tyr Gly Glu Pro Arg Lys Leu (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1032 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

SUBSTITUTE SltEET (RULE 26) WO 96/10413 ~ 2 0 ~ 3 2 7 PCT/US95/12463 (2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln Ala Arg Leu Met Lys Glu (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln SUBST~TUTE SHEET (RULE 26) W 096/10413 ~ ~ 2 0 ~ 3 2 ~= PCTAUS95112463 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid - (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Ala Ala Arg Ala Val Phe Leu Ala Leu (2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Val Leu Leu leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val SUBSTITUTE 5HEET tRULE 26) 2 2 0 ~ 3 2 ~

Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro (2) INFORMATION FOR SEQ ID N0: 50:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 676 base pairs (B) TYPE: nucleic acid 2 5 ( C ) STRANDEDNESS: s i ng l e (D) TOPOLOGY: l inear (xi ) SEQUENCE DESCRIPTION: SEQ ID N0: 50:

SUBSTITVTE SHEET (RULE 26~

(2) INFORMATION FOR SEQ ID NO: 51:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13585 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: doub l e ( D ) TOPOLOGY: l i near ( ix) FEATURE:
(D) OTHER INFORMATION: at positions 9421-9456, the "Ns"
refer to an unsequenced portion of from 4.7 to 5.3 kilobases (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

3 o TGAGTTTGAC TTTCATTTCT TTTTTACMC TGGGMGGGC MMTTACACA TMMTGTCC 660 AGTAAGAGAG CAC/\MAATA CACAATMTA AGGMMGGAG CTTCCATTAT TGGTTTTTAA 840 TGAGCGTACT TGMTTACGG CCACTGCaGT TTATGGATAT TTTTTGTTGT TCATTTGTAT 900 SUBSTlTUTE StlEET ~RULE 26) WO 96/10413 ~ 2 0 ~ 3 2 ~ PCTrUS95/12463 GTGGGAMGG GACATACTCT GTACGTGCTG GGTGGCAGGG GCACCCC/\CC CCCCACCCTC 2520 AGCMGATGT AMGCCCMC MMGGCACTG ACCAACACTT C/\CCI\AC/\CA GCATTTCCTC 2700 ATCCTCTCAG MMTGTCCT GACAGCMM ACATACTCTT TCACCC/\GGG GAGCCCATTG 2820 SUBSTITUTE SH EET (RULE 26) ~ ~ ~ 0 ~ ~ ~ 7 TCAGGCCTCT GAGGTCAGCC AGTGCTCTGC TCCCC/\CCCC CATAATGMG CTGGCTCTGA 3840 SUBSmVTE SHEET (RULE 26) WO96/10413 2 2 0 1 3 2 ~ PCT/US95/12463 GAAGGCATCA CATGGGCMG GGAGTGAGAG AGCAAGAGGG AGCTGAACTC Al l l l l l l l l 7260 SUE~STITUTE SHEET ~RULE 26) w 096/10413 ~ ~ 3 ~ PCT~US95/12463 Tr~ J~ J~J~J~J~:~J~J~J ~J~J~J~J~ J~J~J~J~:~J~ J~JGAGT GCAGTGGTAT GATCTGGGCT 9480CACTGCAAGC TCTGCCTCCT GGGTTCATGC CATTCTCCTG ACTCAGCCTC CCAAGTAGCT 9540 SUBSTtTUTE SHEET (RULE 26) CCATGGTCAT TCATATCGGC ACAG M TGAA ACCTCTTT M M TATTTTGC AG~ IIC 10560 SUBSTITUTE SltEET tRULE 26) ~ 3 ~ 7 lo AGTGCCACTG CACTCCAGCC TGGGAGACAA GAGCMAACT CCATCTCAM AATAAATMA 12600 (2) INFORMATION FOR SEQUENCE ID NO: 52:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 648 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng 1 e ( D ) TOPOLOGY: 1 i near (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

4 o MCCAGCAAC TCAACGTCAG GATCCTGCAG CTGCTCAGGA CCCACACCAT CACCCACCAT 240 SUBSTITUTE SHEET (RULE 26) WO 96/10413 2 2 0 ~ ~ 2 7 PCT/US95/12463 lo TTAAAAGAAG ACATGCTGAA ATGTTGCAGG CTGCTCCTAT GTTGG M AAT TCTTCATTGA 600 (2) INFORMATION FOR SEQUENCE ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

Ala Leu Ser Arg Lys Val Ala Glu Leu (2) INFORMATION FOR SEQUENCE ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

Phe Leu Trp Gly Pro Arg Ala Leu Val (2) INFORMATION FOR SEQUENCE ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid SUBSTITUTE SHEET (RUI E ~6) WO 96/10413 F~ 3 ~ 7 PCT/US95/12463 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Thr Leu Val Glu Val Thr Leu Gly Glu Val (2) INFORMATION FOR SEQUENCE ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino ac;d residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Ala Leu Ser Arg Lys Val Ala Glu Leu Val (2) INFORMATION FOR SEQUENCE ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57 Ala Leu Val Glu Thr Ser Tyr Yal Lys Val (2) INFORMATION FOR SEQUENCE ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acid residues SUBSTITUTE SHEET (RULE 26) F 2 2 0 ~ 3 ~ 7 ~
WO 96/10413 PCTtUS95/12463 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

Ser Thr Leu Val Glu Val Thr Leu Gly Glu Val (2) INFORMATION FOR SEQUENCE ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

Leu Val Glu Val Thr Leu Gly Glu Val (2) INFORMATION FOR SEQUENCE ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Lys Met Val Glu Leu Val His Phe Leu (2) INFORMATION FOR SEQUENCE ID NO: 61:

SUBSTlTUTE SHEET (RULE 26) ~ WO 96/10413 ~ 3 2 7~ PCT/US95/12463 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein lo (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Val Ile Phe Ser Lys Ala Ser Glu Tyr Leu (2) INFORMATION FOR SEQUENCE ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

Tyr Leu Gln Leu Val Phe Gly Ile Glu Val (2) INFORMATION FOR SEQUENCE ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

Gln Leu Val Phe Gly Ile Glu Val Val (2) INFORMATION FOR SEQUENCE ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:

5U BS m UTE 5~ EET (R U LE 26) ~ 2 0 1 3 2 ~

(A) LENGTH: 11 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

Gln Leu Val Phe Gly Ile Glu Val Val Glu Val (2) INFORMATION FOR SEQUENCE ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

Ile Ile Val Leu Ala Ile Ile Ala Ile (2) INFORMATION FOR SEQUENCE ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

Lys Ile Trp Glu Glu Leu Ser Met Leu Glu Val SUBST~TUTE SHEET tRULE 26) r ~ 7 (2) INFORMATION FOR SEQUENCE ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
< (A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

Ala Leu Ile Glu Thr Ser Tyr Val Lys Val (2) INFORMATION FOR SEQUENCE ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

Leu Ile Glu Thr Ser Tyr Val Lys Val Leu (2) INFORMATION FOR SEQUENCE ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (D) TOPOLOGY linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: M3-195.203 OR SEQ ID NO: 69:

Ile Met Pro Lys Ala Gly Leu Leu Ile SUBSTilTUTE SH E~ (RULE 26) WO96/10413 ~ 2 9 1 3 ~ 7 PCT/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids lo `(D) TOPOLOGY linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: M3-220.228 OR SEQ ID NO: 70:

Lys Ile Trp Glu Glu Leu Ser Val Leu (2) INFORMATION FOR SEQUENCE ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser Ser (2) INFORMATION FOR SEQUENCE ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp SUBSTITUTE SHEET (RULE 26) ~ WO 96/10413 ~ 7 PCI/US95/12463 (2) INFORMATION FOR SEQUENCE ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 am;no acid res;dues (B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:

Asp Val Lys Glu Ala Asp Pro Thr Gly His Ser Tyr (2) INFORMATION FOR SEQ ID NO: 74 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1896 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (x;) SEQUENCE DESCRIPTION: SEQ ID NO: 74 5U B5lllU TE S~ EET (R U LE 26) ~ 2 ~ O t 3 ~ 7 ATAGGGATAT TTCCIIllll 1Illllllll TTTTGACTGT TTCTTAATCA GTGCCATGCC 1140 (2) INFORMATION FOR SEQUENCE ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:

Glu Glu Lys Leu Ile Val Val Leu Phe (2) INFORMATION FOR SEQUENCE ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1554 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE St~EET tRULE 26) 0 ~ ~ ~ 7~

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE:
(A) NAME/KEY: DAGE
r (D) OTHER INFORMATION Xaa is Arg when V is C or A or Gly when Y is G
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

Xaa Leu Arg Pro Arg Asn Pro Ser Val Gly Gly Pro Glu Ala Ser Leu Ser Arg Phe Lys Met Glu Arg Arg Arg Leu Arg Gly Ser Ile Gln Ser Arg Tyr Ile Ser Met Ser Val Trp Thr Ser Pro Arg Arg Leu Val Glu Leu Ala Gly Gln Ser Leu Leu Lys Asp Glu Ala Leu Ala Ile Ala Ala Leu Glu Leu Leu Pro Arg Glu Leu Phe Pro Pro Leu Phe Met Ala Ala Phe Asp Gly Arg His Ser Gln Thr Leu Lys Ala Met Val Gln Ala Trp Pro Phe Thr Cys Leu Pro Leu Gly Val Leu Met Lys Gly Gln His Leu 100 105 llo SUBSl 11 UTE SHEET (RULE 26) WO 96/10413 2 2 ~ ~ 3 ~ 7 PCT/US95/12463 ~

His Leu Glu Thr Phe Lys Ala Val Leu Asp Gly Leu Asp Val Leu Leu Ala Gln Glu Val Arg Pro Arg Arg Trp Lys Leu Gln Val Leu Asp Leu Arg Lys Asn Ser His Gln Asp Phe Trp Thr Val Trp Ser Gly Asn Arg Ala Ser Leu Tyr Ser Phe Pro Glu Pro Glu Ala Ala Gln Pro Met Thr Lys Lys Arg Lys Val Asp Gly Leu Ser Thr Glu Ala Glu Gln Pro Phe Ile Pro Val Glu Val Leu Val Asp Leu Phe Leu Lys Glu Gly Ala Cys Asp Glu Leu Phe Ser Tyr Leu I le Glu Arg Val Lys Arg Lys Lys Asn Val Leu Arg Leu Cys Cys Lys Lys Leu Lys Ile Phe Ala Met Pro Met G l n Asp I l e Lys Met I l e Leu Lys Met Va l G l n Leu Asp Ser I l e G l u SUBSmUTE S}}EET (RULE 26) 1~ WO 96/10413 ~ 3 2 ~= PCT/US95112463 Asp Leu Glu Val Thr Cys Thr Trp Lys Leu Pro Thr Leu Ala Lys Phe Ser Pro Tyr Leu Gly Gln Met Ile Asn Leu Arg Arg Leu Leu Leu Ser His Ile His Ala Ser Ser Tyr Ile Ser Pro Glu Lys Glu Glu Ser Ile Ser Pro Ser Ser Pro Leu Ser Ser Ser Val Cys Ser Ala Cys Arg Leu Ser Met Trp Thr Leu Tyr Phe Ser Leu Glu Ala Ala Trp Thr Gln Leu Leu Arg His Val Met Asn Phe Leu Glu Thr Leu Ser Ile Thr Asn Cys Arg Leu Ser Glu Gly Asp Val Met His Leu Ser Gln Ser Pro Ser Val Ser Gln Leu Ser Val Leu Ser Leu Ser Gly Val Met Leu Thr Asp Val Ser Pro Glu Pro Leu Gln Ala Leu Leu Glu Arg Ala Ser Ala Thr Leu Gln Asp Leu Val Phe Asp Glu Cys Gly Ile Thr Asp Asp Gln Leu Leu SUBST~TUTE S~tEET (RULE 26) WO 96/10413 r 2 ~ O ~ 3 ~ 7 PCTrUS95/12463 ~

Ala Leu Leu Pro Ser Leu Ser His Cys Ser Gln Leu Thr Thr Leu Ser Phe Tyr Gly Asn Ser Ile Ser Ile Ser Ala Leu Gln Ser Leu Leu Gln His Leu Ile Gly Leu Ser Asn Leu Thr His Val Leu Tyr Pro Val Pro Leu Glu Ser Tyr Glu Asp Ile His Gly Thr Leu His Leu Glu Arg Leu Ala Tyr Leu His Ala Arg Leu Arg Glu Leu Leu Cys Glu Leu Gly Arg Pro Ser Met Val Trp Leu Ser Ala Thr Pro Val Leu Thr Val Gly Thr Glu Pro Ser Met Thr Arg SUB5TITUTE SHEET (RULE 26

Claims

1. Composition of matter comprising:
(i) a tumor rejection antigen precursor or a tumor rejection antigen, and at least one of:
(ii) a pharmaceutically acceptable adjuvant, and (iii) at least one T cell growth factor or B cell growth factor.

2. The composition of matter of claim 1, wherein said tumor rejection antigen precursor is a MAGE protein.

3. The composition of matter of claim 1, wherein said tumor rejection antigen precursor is a BAGE protein.

4. The composition of matter of claim 1, wherein said tumor rejection antigen precursor is a GAGE protein.

5. The composition of claim 1, wherein said tumor rejection antigen is derived from a MAGE protein.

6. The composition of matter of claim 5, wherein said MAGE protein is MAGE-1, MAGE-2, or MAGE-3.

7. The composition of matter of claim 6, wherein said tumor rejection antigen consists of one of SEQ ID
NO: 1 through SEQ ID NO: 5.

8. The composition of matter of claim 1, wherein said pharmaceutically acceptable adjuvant is a substantially pure saponin derived from Ouillaja saponaria.

9. The composition of matter of claim 8, wherein said substantially pure saponin is selected from the group consisting of QA-7, QA-21, QA-17, and QA-18.

10. The composition of matter of claim 1, wherein said pharmaceutically acceptable adjuvant is MTP-MF59.

11. Method for stimulating an immune response in a subject comprising administering the composition of matter of claim 1 to said subject in an amount sufficient to provoke an immune response to said tumor rejection antigen precursor or tumor rejection antigen.

12. The method of claim 11, wherein said immune response comprises proliferation of T cells specific for complexes of said tumor rejection antigen and a major histocompatibility complex molecule to which said tumor rejection antigen binds.

13. The method of claim 11, wherein said T cells are cytolytic T cells.

14. The method of claim 11, wherein said immune response comprises production of antibodies against said tumor rejection antigen precursor or tumor rejection antigen.

15. The composition of claim 1, in intravenous form.

16. The composition of claim 1, in the form of a liposome.