Tumor associated peptide and uses thereof

Abstract

The invention relates to a peptide with the sequence of RLLEFYLAM, methods for the use of the peptide, and antisera and monoclonal antibodies against the peptide. The peptide is derived from the NY-ESO-1 molecule, which form complexes with HLA molecules, leading to lysis of cells presenting these complexes, by cytolytic T lymphocytes.

Description

FIELD OF THE INVENTION

[0002] This invention relates to the isolation and cloning of genes which are members of the “SSX” family, which is discussed herein, and the uses thereof, including determination of cancer. Also a part of the invention are peptides derived from these SSX genes, as well as from the NY-ESO-1 gene. These peptides stimulate proliferation of cytolytic T cells, and thus are useful as markers for presence of disorders such as cancer, for HLA-A2 cells, and as therapeutic agents for treating cancer.

BACKGROUND AND PRIOR ART

[0003] It is fairly well established that many pathological conditions, such as infections, cancer, autoimmune disorders, etc., are characterized by the inappropriate expression of certain molecules. These molecules thus serve as “markers” for a particular pathological or abnormal condition. Apart from their use as diagnostic “targets,” i.e., materials to be identified to diagnose these abnormal conditions, the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents. A by no means limiting example of this is the use of cancer markers to produce antibodies specific to a particular marker. Yet another non-limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytolytic T cells against abnormal cells.

[0004] Preparation of such materials, of course, presupposes a source of the reagents used to generate these. Purification from cells is one laborious, far from sure method of doing so. Another preferred method is the isolation of nucleic acid molecules which encode a particular marker, followed by the use of the isolated encoding molecule to express the desired molecule.

[0005] To date, two strategies have been employed for the detection of such antigens, in e.g., human tumors. These will be referred to as the genetic approach and the biochemical approach. The genetic approach is exemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275 (1988), incorporated by reference. In this approach, several hundred pools of plasmids of a cDNA library obtained from a tumor are transfected into recipient cells, such as COS cells, or into antigen-negative variants of tumor cell lines. Transfectants are screened for the expression of tumor antigens via their ability to provoke reactions by anti-tumor cytolytic T cell clones. The biochemical approach, exemplified by, e.g., Mandelboim, et al., Nature 369: 69 (1994) incorporated by reference, is based on acidic elution of peptides which have bound to MHC-class I molecules of tumor cells, followed by reversed-phase high performance liquid chromography (HPLC). Antigenic peptides are identified after they bind to empty MHC-class I molecules of mutant cell lines, defective in antigen processing, and induce specific reactions with cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a 51Cr release assay.

[0006] These two approaches to the molecular definition of antigens have the following disadvantages: first, they are enormously cumbersome, time-consuming and expensive; second, they depend on the establishment of cytotoxic T cell lines (CTLs) with predefined specificity; and third, their relevance in vivo for the course of the pathology of disease in question has not been proven, as the respective CTLs can be obtained not only from patients with the respective disease, but also from healthy individuals, depending on their T cell repertoire.

[0007] The problems inherent to the two known approaches for the identification and molecular definition of antigens are best demonstrated by the fact that both methods have, so far, succeeded in defining only very few new antigens in human tumors. See, e.g., van der Bruggen et al., Science 254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

[0008] Further, the methodologies described rely on the availability of established, permanent cell lines of the cancer type under consideration. It is very difficult to establish cell lines from certain cancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg. Clin. North. Am. 10: 607-637 (1990). It is also known that some epithelial cell type cancers are poorly susceptible to CTLs in vitro, precluding routine analysis. These problems have stimulated the art to develop additional methodologies for identifying cancer associated antigens.

[0009] One key methodology is described by Sahin, et al., Proc. Natl. Acad. Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, see U.S. patent application Ser. No. 08/580,980, and filed on Jan. 3, 1996, and U.S. Pat. No. 5,698,396. All three of these references are incorporated by reference. To summarize, the method involves the expression of cDNA libraries in a prokaryotic host. (The libraries are secured from a tumor sample). The expressed libraries are then immunoscreened with absorbed and diluted sera, in order to detect those antigens which elicit high titer humoral responses. This methodology is known as the SEREX method (“Serological identification of antigens by Recombinant Expression Cloning”). The methodology has been employed to confirm expression of previously identified tumor associated antigens, as well as to detect new ones. See the above referenced patent applications and Sahin, et al., supra as well as Crew, et al., EMBO J 144: 2333-2340 (1995).

[0010] The SEREX methodology has been applied to esophageal cancer samples, and an esophageal cancer associated antigen has now been identified, and its encoding nucleic acid molecule isolated and cloned, as per U.S. patent application Ser. No. 08/725,182, filed Oct. 3, 1996, incorporated by reference herein.

[0011] The relationship between some of the tumor associated genes and a triad of genes, known as the SSX genes, is under investigation. See Sahin, et al., supra; Tureci, et al., Cancer Res 56:4766-4772 (1996). One of these SSX genes, referred to as SSX2, was identified, at first, as one of two genes involved in a chromosomal translocation event (t(X; 18)(p11.2; q 11.2)), which is present in 70% of synovial sarcomas. See Clark, et al., Nature Genetics 7:502-508 (1994); Crew et al., EMBO J 14:2333-2340 (1995). It was later found to be expressed in a number of tumor cells, and is now considered to be a tumor associated antigen referred to as HOM-MEL-40 by Tureci, et al, supra. Its expression to date has been observed in cancer cells, and normal testes only. Thus parallels other members of the “CT” family of tumor antigens, since they are expressed only in cancer and testis cells. Crew et al. also isolated and cloned the SSX1 gene, which has 89% nucleotide sequence homology with SSX2. Sequence information for SSX1 and SSX2 is presented as SEQ ID NOS: 1 and 2 respectively. See Crew et al., supra. Additional work directed to the identification of SSX genes has resulted in the identification of SSX3, as is described by DeLeeuw, et al., Cytogenet. Genet 73:179-183 (1996). The fact that SSX presentation parallels other, CT antigens suggested to the inventors that other SSX genes might be isolated. The parent application, supra discloses this work, as does Gure, et al. Int. J. Cancer 72:965-971 (1997), incorporated by reference.

[0012] With respect to additional literature on the SSX family, most of it relates to SSX1. See PCT Application W/96 02641A2 to Cooper, et al, detailing work on the determination of synovial sarcoma via determination of SSX1 or SSX2. Also note DeLeeuw, et al. Hum. Mol. Genet 4(6):1097-1099 (1995). also describing synovial sarcoma and SYT-SSX1 or SSX2 translocation. Also see Kawai, et al, N. Engl. J. Med 338(3):153-160 (1998); Noguchi, et al. Int. J. Cancer 72(6):995-1002 (1997), Hibshoosh, et al., Semin. Oncol 24(5):515-525 (1997), Shipley, et al., Am. J. Pathol. 148(2):559-567 (1996); Fligman, et al. Am. J. Pathol. 147(6); 1592-1599 (1995). Also see Chand, et al., Genomics 30(3):545-552 (1995), Brett, et al., Hum. Mol Genet 6(9): 1559-1564 (1997), deBruyn, et al, Oncogene (1313):643-648. The SSX3 gene is described by deLeeuw, et al, Cytogenet Cell Genet 73(3):179-1983 (1966).

[0013] Application of a modification of the SEREX technology described supra has been used, together with other techniques, to clone two, additional SSX genes, referred to as SSX4 and SSX5 hereafter as well as an alternate splice variant of the SSX4 gene. Specifically, while the SEREX methodology utilizes autologous serum, the methods set forth infra use allogenic serum.

[0014] Motif analysis is a tool which permits one to ascertain what regions of a longer protein may in fact be of particular interest as binders of MHC or HLA molecules. Essentially, one works with an amino acid motif, which generally includes at least two, and sometimes more, defined amino acids in a sequence of 8-12 amino acids. This motif is then used to screen a longer sequence to determine which sequences within the longer sequence constitute peptides which would bind to an HLA or MHC molecule, and possibly stimulate proliferation of cytolytic T lymphocytes with specificity to complexes of the peptide and MHC/HLA molecule. Motifs differ for different MHC/HLA molecules. Much work has been done in this area, but it is ongoing. As will be seen in the disclosure which follows, the inventors have used motif analysis to identify peptides which bind to HLA molecules, HLA-A2 molecules in particular.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 depicts a peptide titration experiment showing recognition by the CTL of COS-A201 cells pulsed with increasing amounts of the SEQ ID NO: 19. The four lines represent separate assays using 4 different CTL lines derived from a stimulation culture.

[0016] FIG. 2 depicts the results of cytotoxicity assays to determine HLA specificity of SEQ ID NO: 19.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] One embodiment of the invention is a novel immunogenic peptide, SEQ ID NO: 19 (Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met), that is capable of stimulating proliferation of cytolytic T cells and inducing a cellular immune response against cells with SEQ ID NO: 19 complex on the cell surface.

[0018] SEQ ID NO: 19 may be used as a vaccine either therapeutically or prophylactically. It may be administered, for example, to a patient suffering from a disorder to stimulate one or more components of the patient's immune system, such as cytotoxic T lymphocytes, to mount a cellular immune response against the cells presenting the complex. The disorder may be a neoplasia, such as a melanoma. SEQ ID NO: 19 may be administered directly. Preferably, SEQ ID NO: 19 is administered as a complex with an HLA molecule or an HLA molecule fragment to stimulate HLA specific cytotoxic lymphocyte reaction.

[0019] In prophylactic usage, SEQ ID NO: 19 or SEQ ID NO: 19 conjugated to a HLA molecule is provided to a patient who is in a high risk group for developing a disorder such as a neoplastic disorder. High risk groups may include patients with a family history of neoplastic disorders, patients with a genetic predisposition to certain neoplastic disorders, and patients with habits and lifestyles which predispose them to a high risk of such disorders. Examples of high risk groups include, for example, Xeroderma Pigmentosum patients who have an increased risk for melanoma, cigarette smokers who have an increased risk for small cell lung carcinoma, and Beckwith-Wiedemann syndrome patients who have an increased risk for hepatoblastoma, and adrenal carcinoma. In prophylactic usage, SEQ ID NO: 19 by itself or complexed with an HLA molecule is administered in advance of any indications of neoplastic disorder to stimulate the patient's immune system to prevent neoplastic disorder or to attenuate any undiscovered neoplastic disorder. Prophylactic usage also includes the administration of SEQ ID NO: 19 to a patient recovering from a neoplastic disorder treatment such as a bone marrow transplant or tumor excision to prevent re-emergence of the neoplasia.

[0020] In a preferred embodiment, mammals, including humans, who are at high risk for a neoplastic disorder are treated with vaccines comprising SEQ ID NO: 19 or SEQ ID NO: 19 complexed with HLA. The vaccine may be in the form of a virus which can infect a cell and induce presentation of SEQ ID NO: 19 on the cell surface. Alternatively, the vaccine may be an attenuated bacterium, which expresses SEQ ID NO: 19 and a HLA molecule on its cell surface. In one embodiment, the vaccine may be a mammalian cell transfected by a nucleic acid molecule that encodes SEQ ID NO: 19.

[0021] Another embodiment of the invention is directed to the use of SEQ ID NO: 19 to generate antisera for prophylactic or treatment purposes. Antisera may be produced using methods known in the art. For example, SEQ ID NO: 19 by itself or in complex with HLA molecules may be injected into animals. Antibody titer to SEQ ID NO: 19 may be monitored by withdrawing blood from the animals at regular intervals and analyzing serum titer. Booster shots of additional SEQ ID NO: 19 or SEQ ID NO: 19/HLA complexes to stimulate antibody production may be administered if needed. Alternatively, a blood sample from a patient treated with SEQ ID NO: 19 either therapeutically or prophylactically may be used to prepare antisera.

[0022] In addition, monoclonal antibodies to SEQ ID NO: 19 may be made using conventional monoclonal antibody techniques. Further, to improve the efficacy of the monoclonal antibody, it may be humanized. In an alternative embodiment, antibodies or antisera may be made in a nonhuman mammal and used. It is understood that all antibodies and antisera may be purified by antigen immunoaffinity column. In antigen immunoaffinity purification, the antigen which is immobilized on the solid phase may be SEQ ID NO: 19 alone or SEQ ID NO: 19 complexed with a HLA molecule or HLA molecule fragment. Preferred HLA molecules include HLA-A2 molecules such as HLA-A*0201.

[0023] The antibodies and antisera produced may be conjugated to toxic molecules or a detectable label and used to target neoplastic cells for destruction or detection. A toxin may include, for example, ricin. Detectable labels may include, for example, radioisotopes 32P, 125I, and 111In. It is understood that some molecules, such as 131I, can be both a detectable label and a toxin.

[0024] Another embodiment of the invention is directed to a method of diagnosing a neoplastic disorder using an antibody that specifically binds SEQ ID NO: 19. For example, a SEQ ID NO: 19 specific antibody may be coupled to a detectable label and used in immunocytochemistry to determine the presence of neoplastic disorder in a biopsy, in an in-situ hybridization, and in fluorescent activator sorting.

[0025] In another embodiment of the invention, the antibody to SEQ ID NO: 19 may be used to purify SEQ ID NO: 19 by immunoaffinity chromatography. Protocols for antibody purification and antibody affinity purification are commonly known. Chromatography media for antibody purification and affinity purification are available commercially (e.g., Pharmacia Biotech, Uppsala, Sweden) and detailed protocols for performing these techniques are supplied with the media.

[0026] In another embodiment of the invention, the antibody to SEQ ID NO: 19 may be used as a fluorescent activated cell sorter marker during bone marrow sorting. For example, in an autologous bone marrow transplant as a treatment for cancer, a patient's extracted bone marrow cells are sorted to separate the neoplastic cells from normal cells. The neoplastic cells are discarded while the normal cells are reintroduced into a patient after the patient has undergone cancer treatment. The sorting (or multiple sortings) is performed with a fluorescent activated cell sorter after the cells are contacted with fluorescent labeled neoplastic cell specific antibody. The antibodies and antisera of the invention, specific for SEQ ID NO: 19, may be fluorescent labeled and used as the fluorescent antibody for cell sorting.

[0027] Another embodiment of the invention is directed to a method of enhancing stem cell transplantation using SEQ ID NO: 19. Stem cell transplantation is used in the treatment of neoplastic disorder where the treatment (i.e., chemotherapy) destroys the stem cells of a patient. Without a stem cell transplant, the patient may die. In a stem cell transplantation, stem cells (marrow cells) are isolated from a histocompatible donor and injected into a host. The transplanted stem cells, obtained from the marrow, generally contain a fair portion of T lymphocytes. The T lymphocytes do not affect the stem cell transplant and are not removed prior to transplantation. Thus, T lymphocytes are a part of most stem cell transplants. If the T lymphocytes are properly stimulated, they may initiate an immune response against any possible residual neoplastic cells in the host. In the method, the donor stem cell population comprising T lymphocytes is treated with SEQ ID NO: 19 or SEQ ID NO: 19 complexed to an HLA peptide before it is deposited into the patient. The treatment may stimulate the immune response of the T lymphocyte in the donor stem cell.

[0028] Another embodiment of the invention is a novel, substantially purified and isolated nucleic acid molecule encoding a peptide with an amino acid sequence of SEQ ID NO: 19 (Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met). The nucleic acid that encodes SEQ ID NO: 19 may be deduced from the amino acid sequence of SEQ ID NO: 19. It is known that the nucleic acid code is degenerate. In this case, for example, Arginine is encoded by the codons, CGT, CGC, CGA, CGG, AGA and AGG. Leucine is encoded by the codons TTA, TTG, CTT, CTC, CTA and CTG. Glutamic Acid is encoded by the codons GAA or GAG. Phe is encoded by the codons TTT and TTC. Tyr is encoded by the codons TAT and TAC. Alanine is encoded by the codons GCT, GCC, GCA, and GCG. Met is encoded by the codon ATG. Thus, the nucleic acid molecule may have any sequence which encodes a peptide of SEQ ID NO: 19. Preferably, the nucleic acid is in operable linkage with a promoter which can express SEQ ID NO: 19 constitutively or upon induction in a eukaryotic or prokaryotic host. More preferably, the plasmid may have a wide host range allowing replication multiple hosts such as yeast and bacteria.

[0029] Another embodiment of the invention is directed to novel pharmaceutical compositions useful for treating a neoplastic disorder. The pharmaceutical composition contains between 0.001% to 100% by weight of SEQ ID NO: 19 in a pharmaceutically acceptable carrier and/or diluent. Suitable carriers may be bovine serum albumin and suitable diluents may be phosphate buffered saline or distilled water. In a preferred embodiment, SEQ ID NO: 19 is bound to an HLA molecule for stimulation of the recipient's immune response. Preferred HLA molecules include HLA-A2 molecules such as HLA-A*0201.

[0030] Another embodiment of the invention is a method for ameliorating the symptoms of a neoplastic disorder by administering a pharmaceutical composition comprising SEQ ID NO: 19 to a patient. Administration may be performed by means known in the art including topical administration, injection, intravenous drip, implantation of a slow release device, topical administration and aerosol administration. The therapeutic effects of SEQ ID NO: 19 may be enhanced by the addition of an adjuvant, a T cell booster, or a cytokine. Adjuvants and cytokines that can enhance T lymphocyte response to antigens are known and include, for example, microorganisms such as BCG, T cell boosters such as lentinan, and cytokines such as IL-1, IL-2, IL-4, IL-6, IL-12 and TNF.

[0031] Another embodiment of the invention is directed to a host cell or virus containing a DNA molecule which encodes the peptide of the invention. The host cell or virus may be used in a method to produce the peptide. Further, the host cell or virus may be used as a live, or attenuated vaccine to vaccinate against a neoplastic disorder.

[0032] Another embodiment of the invention is directed to a method to provide a vaccine for preventing a neoplastic disorder. The vaccine may be a virus or bacterium containing a DNA molecule that encodes a peptide of the invention.

[0033] Another embodiment of the invention is directed to a method of making a peptide of the invention by culturing a virus or bacteria containing a DNA molecule that encodes a peptide of the invention. Methods for peptide expression are known. For example, the peptide may be produced by transfecting an expression vector containing a nucleic acid sequence that encodes SEQ ID NO: 19 into a host cell and inducing expression. The host cell may be a prokaryotic (bacterium), yeast, insect, or mammalian cell. Various methods of host cell expression are well known. Reagents, vectors, cell lines and detailed expression protocols are commercially available (e.g., Invitrogen (Carlsbad, Calif.), Stratagene (La Jolla, Calif.)). For example, SEQ ID NO: 19 may be expressed as a thioredoxin-SEQ ID NO: 19 fusion protein under the ThioFusion™ Expression system of Invitrogen. Following expression, the fusion peptide may be purified by a metal binding resin with specific affinity for thioredoxin part of the fusion protein. The fusion protein, still bound to the metal binding resin, may be cleaved by enterokinase to specifically release intact SEQ ID NO: 19. Alternatively, SEQ ID NO: 19 may be synthesized and purified by commercially available peptide synthesis machines (e.g., PE Corporation (Norwalk, Conn.); Advanced ChemTech (Louisville, Ky.)).

[0034] Another embodiment of the invention is directed to a polytope molecule comprising a plurality of sequences corresponding to amino acid sequences which bind to MHC molecules, at least one of which is the amino acid sequence of SEQ ID NO: 19. A polytope is a polypeptide comprising two or more MHC binding sequence. A nucleic acid molecule encoding a polytope may be made by ligating together oligonucleotides encoding different peptides. The polytope encoding nucleic acid molecule may be placed in an expression vector in functional linkage with a promoter for expressing a recombinant polytope in a host cell. The recombinant polytope polypeptide of the invention may comprise from 2 to 1000, preferably 4 to 200 and most preferably between 4 and 20 peptides; wherein at least one of them is SEQ ID NO: 19. In addition, the peptides may be sandwiched between recognition sites for a sequence specific cleavage enzyme. Sequence specific cleavage enzymes may be, for example, enterokinase or thrombin which recognizes and cleaves at known sequences. Thus, cleavage of the polytope will result in multiple peptides which can bind to a MHC molecule.

[0035] Another embodiment of the invention is directed to a method of treating a patient with neoplastic disorder with SEQ ID NO: 19. In the method, T lymphocytes isolated from a patient are treated with SEQ ID NO: 19 to sensitize the T cells to SEQ ID NO: 19. The treated T lymphocytes are reintroduced into the patient to stimulate the patient's response to neoplastic cells. Optionally, the treated T lymphocytes may be cultured and amplified before reintroduction to enhance the effects of SEQ ID NO: 19.

[0036] Another embodiment of the invention is directed to a composition of matter comprising a peptide of SEQ ID NO: 19. The composition of matter may include, in addition to SEQ ID NO: 19, one or more disorder associated antigens, HLA molecules or HLA molecule fragments. The compositions may be tailored to elicit a CTL reaction against specific cells or cell populations when the composition is administered to a patient. For example, the composition may comprise multiple cell surface markers expressed by a targeted cell population. The cell surface markers may include, for example, HLA antigens and disorder associated antigens.

EXAMPLE 1

[0037] A human testicular cDNA expression library was obtained, and screened, with serum from a melanoma patient identified as MZ2. See e.g., parent application U.S. patent application Ser. No. 08,479,328 incorporated by reference; also see U.S. patent application Ser. No. 08/725,182 also incorporated by reference; Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995). This serum had been treated using the methodology described in these references. Briefly, serum was diluted 1:10, and then preabsorbed with transfected E. coli lysate. Following this preabsorption step, the absorbed serum was diluted 11:10,

[0038] for a final dilution of 1:100. Following the final dilution the samples were incubated overnight at room temperature, with nitrocellulose membranes containing phage plaques prepared using the methodology referred to supra. The nitrocellulose membranes were washed, incubated with alkaline phosphatase conjugated goat anti-human Fcγsecondary antibodies, and the reaction was observed with the substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. In a secondary screen, any phagemids which encoded human immunoglobulin were eliminated.

[0039] A total of 3.6×105 pfus were screened, resulting in eight positive clones. Standard sequencing reactions were carried out, and the sequences were compared to sequence banks of known sequences.

[0040] Of the eight clones, two were found to code for known autoimmune disease associated molecules, i.e., Golgin-95 (Fritzler, et al., J. Exp. Med.178:49-62 (1993)), and human upstream binding factor (Chan, et al., J. Exp. Med. 174:1239-1244 (1991)). Three other clones were found to encode for proteins which are widely expressed in human tissue, i.e., ribosomal receptor, collagen type VI globular domain, and rapamycin binding protein. Of the remaining three sequences, one was found to be non-homologous to any known sequence, but was expressed ubiquitously in human tissues (this was found via RT-PCR analysis, but details are not provided herein). The remaining two were found to be identical to full length HOM-MEL-40, described in Ser. No. 08/479,328, while the eighth clone was found to be almost identical to “SSX3,” as described by DeLeeuw, et al., Cytogenet. Cell Genet 73:179-183 (1996), differing therefrom in only two base pair differences in the coding region. These differences are probably artifactual in nature; however, the clone also included a 43 base pair 3′-untranslated region.

EXAMPLE 2

[0041] In order to carry out Southern blotting experiments, described infra, the SSX genes were amplified, using RT-PCR.

[0042] To do this, two primers were prepared using the published SSX2 sequence i.e., MEL-40A:

5′-CACACAGGAT CCATGAACGG AGA, (SEQ ID NO: 3)

[0043] and

[0044] MEL-40B:

(SEQ. ID NO: 4)
5′-CACACAAAGC TTTGAGGGGA GTTACTCGTC ATC

[0045] See Crew, et al., EMBO J 14:2333-2340 (1995). Amplification was then carried out using 0.25 U Taq polymerase in a 25 μl reaction volume, using an annealing temperature of 60° C. A total of 35 cycles were carried out.

EXAMPLE 3

[0046] The RT-PCR methodology described supra was carried out on testicular total RNA, and the amplification product was used in southern blotting experiments.

[0047] Genomic DNA was extracted from non-neoplastic tissue samples, and then subjected to restriction enzyme digestion, using BamHI, Eco RI, or HindIII in separate experiments and then separated on a 0.7% agarose gel, followed by blotting onto nitrocellulose filters. The amplification products described supra were labeled with 32P, using well-known methods, and the labeled materials were then used as probes under high stringency conditions (65° C., aqueous buffer), followed by high stringency washes, ending with a final wash at 0.2×SSC, 0.2% SDS, 65° C.

[0048] The Southern blotting revealed more than 10 bands, in each case (i.e., each of the BamHI, EcoRI, and HindIII digests), strongly suggesting that there is a family of SSX genes which contained more than the three identified previously. In view of this observation, an approach was designed which combined both PCR cloning, and restriction map analysis, to identify other SSX genes.

EXAMPLE 4

[0049] When the sequences of SSX1, 2 and 3 were compared, it was found that they shared highly conserved 5′ and 3′ regions, which explained why the olignucleotides of SEQ ID NOS: 3 and 4 were capable of amplifying all three sequences under the recited conditions, and suggested that this homology was shared by the family of SSX genes, whatever its size. Hence, the oligonucleotides of SEQ ID NOS: 3 and 4 would be sufficient to amplify the other members of the SSX gene family.

[0050] An analysis of the sequences of SSX1, 2 and 3 revealed that SSX1 and 2 contained a BglII site which was not shared by SSX3. Similarly, SSX3 contained an EcoRV site not shared by the other genes.

[0051] In view of this information, testicular cDNA was amplified, using SEQ ID NOS: 3 and 4, as described supra, and was then subjected to BglII digestion. Any BglII resistant sequences were then cloned, sequenced, and compared with the known sequences.

[0052] This resulted in the identification of two previously unidentified sequences, referred to hereafter as SSX4 and SSX5, presented as SEQ ID NOS: 5 and 6 herein. A search of the GenBank database found two clones, identified by Accession Number N24445 and WO0507, both of which consisted of a sequence—tag—derived cDNA segment. The clone identified by N24445 contained the 3′-untranslated region of SSX4, and part of its coding sequence, while the one identified as WO0507 contained a shorter fragment of the 3′-untranslated region of SSX4, and a longer part of the coding sequence. Specifically, N24445 consists of base 344 of SSX4 (SEQ ID NO:5), through the 3-end, plus 319 bases 3′ of the stop codon. The W00507 sequence consists of a 99 base pair sequence, showing no homology to SSX genes followed by a region identical to nucleotides 280 through the end of SEQ ID NO:5, through 67 bases 3′ of the stop codon of SEQ ID NO: 1.

[0053] Two forms of SSX4 (SEQ ID NO: 5) were identified. One of these lacked nucleotides 331 to 466 but was otherwise identical to SSX4 as presented in SEQ ID NO: 5. As is described infra, the shorter form is an alternatively spliced variant.

[0054] In Table 1, which follows, the nucleotide and amino acid sequences of the 5 known members of the SSX family are compared. One reads the table horizontally for nucleotide homology, and vertically for amino acid homology.

TABLE 1
Nucleotide and amino acid homology among SSX family members
	Nucleotide Sequence Homology (%)
		SSX1	SSX2	SSX3	SSX4	SSX5
	SSX1		89.1	89.6	89.4	88.7
	SSX2	78.2		95.1	91.5	92.9
	SSX3	77.7	91.0		91.1	92.7
	SSX4	79.3	79.8	80.9		89.8
	SSX5	76.6	83.5	84.0	77.7
	Amino Acid Sequence Homology (%)

[0055] Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide level, and 79.3% homology on the amino acid level.

[0056] When the truncated form of SSX4 is analyzed, it has an amino acid sequence completely different from others, due to alternate splicing and shifting of a downstream open reading frame. The putative protein is 153 amino acids long, and the 42 carboxy terminal amino acids show no homology to the other SSX proteins.

EXAMPLE 5

[0057] The genomic organization of the SSX2 genes was then studied. To do this, a genomic human placental library (in lambda phage) was screened, using the same protocol and probes described supra in the discussion of the southern blotting work. Any positive primary clones were purified, via two additional rounds of cloning.

[0058] Multiple positive clones were isolated, one of which was partially sequenced, and identified as the genomic clone of SSX2. A series of experiments carrying out standard subcloning and sequencing work followed, so as to define the exon-intron boundaries.

[0059] The analysis revealed that the SSX2, gene contains six exons, and spans at least 8 kilobases. All defined boundaries were found to observe the consensus sequence of exon/intron junctions, i.e. GT/AG.

[0060] The alternate splice variant of SSX4, discussed supra, was found to lack the fifth exon in the coding region. This was ascertained by comparing it to the SSX2 genomic clone, and drawing correlations therefrom.

EXAMPLE 6

[0061] The expression of individual SSX genes in normal and tumor tissues was then examined. This required the construction of specific primers, based upon the known sequences, and these follow, as SEQ ID NOS: 7-16:

TABLE 2
Gene-specific PCR primer sequences for individual
SSX genes
SSX 1A (5′):
5′-CTAAAGCATCAGAGAAGAGAAGC    [nt.44-66]
SSX 1B (3′):
5′-AGATCTCTTATTAATCTTCTCAGAAA [nt.440-65]
SSX 2A (5′):
5′-GTGCTCAAATACCAGAGAAGATC    [nt.41-63]
SSX 2B (3′):
5′-TTTTGGGTCCAGATCTCTCGTG     [nt.102-25]
SSX 3A (5′):
5′-GGAAGAGTGGGAAAAGATGAAAGT   [nt.454-75]
SSX 3B (3′):
5′-CCCCTTTTGGGTCCAGATATCA     [nt.458-79]
SSX 4A (5′):
5′-AAATCGTCTATGTGTATATGAAGCT  [nt.133-58]
SSX 4B (3′):
5′-GGGTCGCTGATCTCTTCATAAAC    [nt.526-48]
SSX 5A (5′):
5′-GTTCTCAAATACCACAGAAGATG    [nt.39-63]
SSX 5B (3′):
5′-CTCTGCTGGCTTCTCGGGCCG      [nt.335-54]

[0062] The specificity of the clones was confirmed by amplifying the previously identified cDNA for SSX1 through SSX5. Taq polymerase was used, at 60° C. for SSX1 and 4, and 65° C. for SSX2, 3 and 5. Each set of primer pairs was found to be specific, except that the SSX2 primers were found to amplify minute (less than {fraction (1/20)} of SSX2) amounts of SSX3 plasmid DNA.

[0063] Once the specificity was confirmed, the primers were used to analyze testicular mRNA, using the RT-PCR protocols se t forth supra.

[0064] The expected PCR products were found in all 5 cases, and amplification with the SSX4 pair did result in two amplification products, which is consistent with alternative splice variants.

[0065] The expression of SSX genes in cultured melanocytes was then studied. RT-PCR was carried out, using the protocols set forth supra. No PCR product was found. Reamplification resulted in a small amount of SSX4 product, including both alternate forms, indicating that SSX4 expression in cultured melanocytes is inconsistent and is at very low levels when it occurs.

[0066] This analysis was then extended to a panel of twelve melanoma cell lines. These results are set forth in the following table.

TABLE 3
SSX expression in melanoma cell lines detected by RT-PCR*
		SSX1	SSX2	SSX3	SSX4	SSX5
	MZ2-Mel 2.2	+	+	−	−	−
	MZ2-Mel 3.1	+	+	−	−	−
	SK-MEL-13	−	−	−	−	−
	SK-MEL-19	−	−	−	−	−
	SK-MEL-23	−	−	−	−	−
	SK-MEL-29	−	−	−	−	−
	SK-MEL-30	−*	−*	−	−*	−
	SK-MEL-31	−	−	−	−	−
	SK-MEL-33	−	−	−	−	−
	SK-MEL-37	+	+	−	+	+
	SK-MEL-179	−	−	−	−	−
	M24-MET	−	−	−	−	−
	*Positive (+) denotes strong expression. Weak positivity was observed inconsistently in SK-MEL-30 for SSX 1, 2, and 4, likely representing low level expression.

EXAMPLE 7

[0067] Additional experiments were carried out to analyze expression of the members of the SSX family in various tumors. To do this, total cellular RNA was extracted from frozen tissue specimens using guanidium isothiocyanate for denaturation followed by acidic phenol extraction and isopropanol precipitation, as described by Chomczynski, et al, Ann. Biochem 162: 156-159 (1987), incorporated by reference. Samples of total RNA (4 ug) were primed with oligoDT(18) primers, and reverse transcribed, following standard methodologies. The integrity of the cDNA thus obtained was tested via amplifying B-acin transcripts in a 25 cycle, standard PCR, as described by Tureci, et al, Canc. Res. 56: 4766-4772 (1996).

[0068] In order to carry out PCR analyses, the primers listed as SEQ ID NOS: 5-14, supra were used, as well as SEQ ID NOS: 17 and 18, i.e.:

ACAGCATTAC CAAGGACAGC AGCCACC
GCCAACAGCA AGATOCATAC CAGGGAC

[0069] These two sequences were each used with both SEQ ID NOS: 6 and 8 in order to detect the SYT/SSX fusion transcript reported for synovial sarcoma by Clark et al, supra, and Crew, et al, supra. The amplification was carried out by amplifying 1 μl of first strand cDNA with 10 pMol of each dNTP, and 1.67 mN MgCl2 in a 30 μl reaction. Following 12 minutes at 94° C. to activate the enzyme, 35 cycles of PCR were performed. Each cycle consisted of 1 minute for annealing (56° C. for SEQ ID NOS: 7 & 8; 67° C. for SEQ ID NOS: 9 & 10; 65° C. for SEQ ID NOS: 11& 12; 60° C. for SEQ ID NOS: 13 & 14; 66° C. for SEQ ID NOS: 15 & 16; 60° C. for SEQ ID NOS: 17 & 8 and 18 & 10), followed by 2 minutes at 72° C., 1 minute at 94° C., and a final elongation step at 72° C. for 8 minutes. A 15 μl aliquot of each reaction was size fractionated on a 2% agarose gel, visualized with ethidium bromide staining, and assessed for expected size. The expected sizes were 421 base pairs for SEQ ID NOS: 7 & 8; 435 base pairs for SEQ ID NOS: 9 & 10; 381 base pairs for SEQ ID NOS 11 & 12; 413 base pairs for SEQ ID NOS: 13 & 14, and 324 base pairs for SEQ ID NOS: 15 & 16. The conditions chosen were stringent, so as to prevent cross anneling of primers to other members of the SSX family. Additional steps were also taken to ensure that the RT-PCR products were derived from cDNA, and not contaminating DNA. Each experiment was done in triplicate. A total of 325 tumor specimens were analyzed. The results are presented in Tables 4 & 5 which follow.

[0070] It is to be noted that while most of the SSX positive tumors expressed only one member of the SSX family, several tumor types showed coexpression of two or more genes.

[0071] Expression of SSX genes in synovial sarcoma was analyzed, because the literature reports that all synovial sarcoma cases analyzed have been shown to carry either the SYT/SSX1 or SYT/SSX2 translocation, at breakpoints flanked by the primer sets discussed herein, i.e., SEQ ID NO: 17/SEQ ID NO: 8; SEQ ID NO: 17/SEQ ID NO: 10; SEQ ID NO. 17/SEQ ID NO: 8; SEQ ID NO: 18/SEQ ID NO: 10. The PCR work described supra showed that SYT/SSX1 translocations were found in three of the synovial sarcoma samples tested, while SYT/SSX2 was found in one. The one in which it was found was also one in which SYT/SSX1 was found. Expression of SSX appeared to be independent of translocation.

TABLE 4
Expression of SSX genes by human neoplasms
	Tissues						at lease one
Tumor entity	tested	SSX1	SSX2	SSX3	SSX4	SSX5	positive	%
Lymphoma	11	—	4	—	—	—	4	36
Breast cancer	67	5	5	—	10	—	16	23
Endometrial cancer	8	1	2	—	1	1	1	13
Colorectal cancer	58	3	7	—	9	1	16	27
Ovarian cancer	12	—	—	—	6	—	6	50
Renal cell cancer	22	—	1	—	—	—	1	4
Malignant melanoma	37	10	13	—	10	2	16	43
Glioma	31	—	2	—	3	—	5	16
Lung cancer	24	1	4	—	1	1	5	21
Stomach cancer	3	—	—	—	1	—	1	33
Prostatic cancer	5	—	2	—	—	—	2	40
Bladder cancer	9	2	4	—	2	—	5	55
Head-Neck cancer	14	3	5	—	4	1	8	57
Synovial sarcoma	4	—	2	—	1	1	3	75
Leukemia	23	—	—	—	—	—	0	0
Leiomyosarcoma	6	—	—	—	—	—	0	0
Thyroid cancer	4	—	—	—	—	—	0	0
Seminoma	2	—	—	—	—	—	0	0
Total	325	25	50	0	48	7	89

[0072]

TABLE 5
Expression pattern of individual SSX genes
in SSX-positive tumor samples.J
		SSX1	SSX2	SSX4	SSX5
	Breast Cancer
	(67 specimens)
	51 specimens	−	−	−	−
	7 specimens	−	−	+	−
	4 specimens	−	+	−	−
	2 specimens	+	−	−	−
	2 specimens	+	−	+	−
	1 specimen	+	+	+	−
	Melanoma
	(37 specimens)
	21 specimens	−	−	−	−
	5 specimens	+	+	+	−
	4 specimens	−	+	−	−
	2 specimens	−	+	+	−
	1 specimen	+	−	−	−
	1 specimen	+	+	−	−
	1 specimen	+	−	+	−
	1 specimen	+	−	+	+
	1 specimen	+	+	+	+
	Endomet. Cancer
	(8 specimens)
	7 specimens	−	−	−	−
	1 specimen	+	+	+	+
	Glioma
	(31 specimens)
	25 specimens	−	−	−	−
	3 specimens	−	+	−	−
	2 specimens	−	−	+	−
	Lung Cancer
	(24 specimens)
	19 specimens	−	−	−	−
	3 specimens	−	+	−	−
	1 specimen	−	−	−	+
	1 specimen	+	+	+	−
	Colorectal
	Cancer
	(58 specimens)
	42 specimens	−	−	−	−
	7 specimens	−	+	−	−
	5 specimens	−	−	+	−
	3 specimens	+	−	+	−
	1 specimen	−	−	+	+
	Bladder Cancer
	(9 specimens)
	4 specimens	−	−	−	−
	2 specimens	−	+	−	−
	1 specimen	−	−	+	−
	1 specimen	+	+	−	−
	1 specimen	+	+	+	−
	Head-Neck
	Cancer
	(14 specimens)
	6 specimens	−	−	−	−
	2 specimens	+	−	−	−
	2 specimens	−	+	+	−
	1 specimen	−	+	−	−
	1 specimen	−	−	+	−
	1 specimen	+	+	−	−
	1 specimen	−	+	+	+
	SSX1	SSX2	SSX4	SSX5	SYT/SSX1	SYT/SSX5
Synovial
Sarcoma
(4 specimens)
Sy1	−	−	+	−	+	−
Sy2	−	+	−	+	+	−
Sy3	−	−	−	−	−	+
Sy4	−	+	−	−	+	−

EXAMPLE 8

[0073] This example details further experiments designed to identify additional peptides which bind to HLA-A2 molecules, and which stimulate CTL proliferation.

[0074] First, peripheral blood mononuclear cells (“PBMCs” hereafter) were isolated from the blood of healthy HLA-A*0201+ donors, using standard Ficoll-Hypaque methods. These PBMCs were then treated to separate adherent monocytes from non-adherent peripheral blood lymphocytes (“PBLs”), by incubating the cells for 1-2 hours, at 37° C., on plastic surfaces. Any non-adherent PBLs were cryopreserved until needed in further experiments. The adherent cells were stimulated to differentiate into dendritic cells by incubating them in AIMV medium supplemented with 1000 U/ml of IL-4, and 1000 U/ml of GM-CSF. The cells were incubated for 5 days.

[0075] Seven days after incubation began, samples of the dendritic cells (8×105) were loaded with 50 μg/ml of exogenously added peptide. (Details of the peptides are provided infra). Loading continued for 2 hours, at 37° C., in a medium which contained 1000 U/ml of TNF-α, and 10,000 U/ml IL-1β. The peptide pulsed dendritic cells were then washed, twice, in excess, peptide free medium. Autologous PBLs, obtained as described, supra, were thawed, and 4×107 PBLs were then combined with 8×105 peptide leaded dendritic cells, (ratio: 50:1), in a medium which contained 5 ng/ml of IL-7 and 20 U/ml of IL-2. The cultures were then incubated at 37° C.

[0076] Lymphocyte cultures were restimulated at 14, 21, and 28 days, in the same manner as the experiment carried out after 7 days. Cytotoxicity assays were carried out, at 14, 21, and 28 days, using a europium release assay, as described by Blomberg, et al., J. Immunol. Meth. 114: 191-195 (1988), incorporated by reference, or the commercially available ELISPOT assay, which measures IFN-γ release.

[0077] The peptides which were tested were all derived from the amino acid sequence of NY-ESO-1 as is described in U.S. Pat. No. 5,804,381, to Chen, et al., incorporated by reference, or the amino acid sequences of SSX-4. The peptides tested were:

[0078] RLLEFYLAM (SEQ ID NO: 19) and

[0079] SLAQDAPPL (SEQ ID NO: 20)

[0080] both of which are derived from NY-ESO-1, and

[0081] STLEKINKT (SEQ ID NO: 21)

[0082] derived from SSX-4. The two NY-ESO-1 derived peptides were tested in ELISPOT assays. The results follow. In summary, three experiments were carried out. The results are presented in terms of the number of spots (positives) secured when the HLA-A2 positive cells were pulsed with the peptide minus the number of spots obtained using non-pulsed cells. As indicated, measurements were taken at 14, 21 and 28 days.

[0083] The following results are for peptide RLLEFYLAM (SEQ ID NO. 19).

Day Measured
(Pulsed Cells - Unpulsed Cells)
	14	21	28
	Expt 1	30	8	*
	Expt 2	22	*	12
	Expt 3	6	*	12
	* not determined

EXAMPLE 9

[0084] In follow up experiments, the T cell cultures described supra were tested on both COS cells which had been transfected with HLA-A*0201 encoding cDNA and were pulsed with endogenous peptide, as described supra, or COS cells which had been transfected with both HLA-A*0201 and NY-ESO-1 encoding sequences. Again, the ELISPOT assay was used, for both types of COS transfectants. Six different cultures of T cells were tested, in two experiments per culture.

	Pulsed with	Endogenous
	Peptide	NY-ESO-1 Production
	Culture 1	Expt 1	64	44
		Expt 2	44	52
	Culture 2	Expt 1	48	45
		Expt 2	100	64
	Culture 3	Expt 1	20	37
		Expt 2	16	16
	Culture 4	Expt 1	17	40
		Expt 2	28	34
	Culture 5	Expt 1	36	26
		Expt 2	4	36
	Culture 6	Expt 1	12	62
		Expt 2	44	96

[0085] The fact that the endogenous NY-ESO-1 led to lysis suggests that NY-ESO-1 is processed to this peptide via HLA-A2 positive cells.

[0086] Similar experiments were carried out with the second NY-ESO-1 derived peptide, i.e., SLAQDAPPL (SEQ ID NO. 20). These results follow:

	Pulsed with	Endogenous
	Peptide	NY-ESO-1 Production
	Culture 1	Expt 1	28	16
		Expt 2	30	14
	Culture 2	Expt 1	31	75
		Expt 2	30	70
	Culture 3	Expt 1	32	44

EXAMPLE 10

[0087] In further experiments, the specificity of the CTLs generated in the prior experiment was tested by combining these CTLs with COS cells, transfected with HLA-A*0201 encoding sequences, which were then pulsed with peptide. First, the peptide RLLEFYLAM (SEQ ID NO. 19) was tested, in three experiments, and then SLAQDAPPL (SEQ ID NO. 20) was tested, in six experiments. Europium release was measured, as described supra, and the percent of target cells lysed was determined. The results follow:

	% LYSIS
	Peptide Added	No Peptide
	PEPTIDE RLLEFYLAM
	(SEQ ID NO. 19)
	Expt 1	43	0
	Expt 2	8	0
	Expt 3	9	0
	PEPTIDE SLAQDAPPL
	(SEQ ID NO. 20)
	Expt 1	11	0
	Expt 2	13	0
	Expt 3	13	0
	Expt 4	21	0
	Expt 5	12	0
	Expt 6	42	0

[0088] In additional experiments, the CTLs specific to RLLEFYLAM (SEQ ID NO. 19)/HLA-A2 complexes also recognized and lysed melanoma cell line SK-Mel-37 which is known to express both HLA-A2 and NY-ESO-1. This recognition was inhibited via preincubating the target cells with an HLA-A2 binding monoclonal antibody, BB7.2. This confirmed that the CTLs were HLA-A2 specific for the complexes of the peptide and HLA-A2.

[0089] In a second set of experiments, a peptide titration experiment was performed to further determine the ability of SEQ ID NO: 19 to induce a CTL response. Experiments were conducted substantially in accordance with the protocol of Example 8. Samples of the cells (8×105) were loaded with 1 μM, 5 μM, 10 μM and 50 μM of SEQ ID NO: 19 for 2 hours at 37° C. in a medium containing 1000 U/ml of TNF-α. Cytotoxicity assays were performed using a europium release assay as described supra. In four separate sets of experiments, increasing concentrations of SEQ ID NO: 19 cause an increase in lysis. The data is plotted in FIG. 1. The results indicate that cells pulsed with SEQ ID NO: 19 can elicit a CTL mediated lysis.

[0090] To determine if the lysis is HLA specific, the CTL assays were performed using COS cells transfected with a cDNA encoding HLA-A*0201 as described supra and pulsed with SEQ ID NO: 19. As shown in FIG. 2, transfected COS cells that were not pulsed with peptides had a lysis rate of 5%. HLA-A*0201 transfected COS cells pulsed with SEQ ID NO: 19, showed 55% lysis. This is consistent with the hypothesis that SEQ ID NO: 19, when complexed with an HLA molecule, is responsible for increased lysis. To determine if the effect was HLA restricted, HLA-A*0201 transfected COS cells pulsed with SEQ ID NO: 19 were assayed in the presence of anti-HLA-A2 antibodies. The lysis decreased to about 10%. This indicated that the cytotoxicity is HLA-A2 restricted because the anti-HLA-A2 antibody bound to the HLA-A2 molecule, interfering with the recognition of the HLA-A2/SEQ ID NO: 19 complex by the TCR (T cell receptor) of the specific T cell, thus inhibiting lysis. Similar experiments using the SK-MEL-37 cell line, which is known to express both NY-ESO-1 and HLA-A2, as the target demonstrated that the peptide was endogenously processed and presented in tumor cells. SK-MEL-37 cells showed about 23% lysis whereas lysis was reduced to 6% in the presence of anti-HLA-A2, which is consistent with the findings using COS-A201 cells, described supra.

[0091] To determine the cellular location of NY-ESO-1, several cell lines known to express NY-ESO-1 were stained by immunofluorescence with a NY-ESO-1 specific monoclonal antibody (Stockert et al., J. Exp Med 187 1349-54, 1998; incorporated herein by reference. Also see U.S. patent application Ser. No. 09/062,422 filed Apr. 17, 1998, incorporated herein by reference). Upon analysis of the stained cell samples, it was found that NY-ESO-1 was localized to the endoplasmic reticulum. This is consistent with the immunogenicity of NY-ESO-1.

EXAMPLE 11

[0092] An additional peptide derived from SSX-4, i.e., STLEKINKT (SEQ ID NO: 21) was also tested, in the same way the NY-ESO-1 derived peptides were tested. First, ELISPOT assays were carried out, using COS cells which expressed HLA-A*0201, and which either expressed full length SSX-4, due to transfection with cDNA encoding the protein, or which were pulsed with the peptide. Three cultures were tested, in two experiments. The results follow:

	Pulsed with	Endogenous
	Peptide	NY-ESO-1 Production
	Culture 1	Expt 1	50	100
		Expt 2	20	138
	Culture 2	Expt 1	8	12
		Expt 2	6	14
	Culture 3	Expt 1	15	47
		Expt 2	14	54

[0093] Further, as with the NY-ESO-1 peptides, specificity of the CTLs was confirmed, using the same assay as described supra, i.e., combining the CTLs generated against the complexes with COS cells, transfected with HLA-A*0201, and pulsed with peptide. The europium release assay described supra was used. The results follow:

	% LYSIS
	Peptide Added	No Peptide
	Expt 1	22	0
	Expt 2	14	0
	Expt 3	46	0
	Expt 4	16	0

[0094] As with the NY-ESO-1 derived peptides, CTL recognition was inhibited via preincubation with the monoclonal antibody BB7.2, confirming specificity of the CTL for complexes HLA-A2 and peptides.

EXAMPLE 12

[0095] Additional experiments were carried out on peptides derived from SSX-2 i.e, KASEKIFYV, and peptides derived from NY-ESO-1, i.e., SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. In each case, the same type of assays as were carried out in examples 8-11 were carried out. The results were comparable, in that for each peptide, CTL were generated which were specific for the respective peptide/HLA-A2 complex.

EXAMPLE 13

[0096] The amino acid sequence of the proteins encoded by the SSX genes were analyzed for peptide sequences which correspond to HLA binding motifs. This was done using the algorithm taught by Parker et al., J. Immunol. 142: 163 (1994), incorporated by reference, augmented by using, as an additional motif, nonamers where position 2 is Thr or Ala, and position 9 is Thr or Ala. In the information which follows, the amino acid sequence, the HLA molecule to which it presumably binds, and the positions in the relevant SSX molecule are given. The resulting complexes should provoke a cytolytic T cell response. This could be determined by one skilled in the art following methods taught by, e.g., van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994), incorporated by reference, as well as the protocols set forth in Examples 8-11, supra.

	SSX-5
	A2	KASEKIIYV	41-49	(SEQ ID NO: 22)
		DAFVRRPRV	5-13	(SEQ ID NO: 23)
		QIPQKMQKA	16-24	(SEQ ID NO: 24)
		MTKLGFKAT	58-66	(SEQ ID NO: 25)
		MTFGRLQGI	99-107	(SEQ ID NO: 26)
		NTSEKVNKT	146-154	(SEQ ID NO: 27)
		YVYMKRKYEA	48-57	(SEQ ID NO: 28)
		YMKRKYEAMT	50-59	(SEQ ID NO: 29)
		EAMTKLGFKA	56-65	(SEQ ID NO: 30)
		MTKLGFKATL	58-67	(SEQ ID NO: 31)
		RLQGIGPKIT	103-112	(SEQ ID NO: 32)
		QLRPSGKLNT	138-147	(SEQ ID NO: 33)
	A3	GIFPKITPEK	106-115	(SEQ ID NO: 34)
		KLNTSEKVNK	144-153	(SEQ ID NO: 35)
	A24	KYEAMTKLGF	54-63	(SEQ ID NO:36)
	B7	HPQMTFGRL	96-104	(SEQ ID NO: 37)
		GPQNNGKQL	131-139	(SEQ ID NO: 38)
	B8	RVRERKQL	167-174	(SEQ ID NO: 39)
	B44	YEAMTKLGF	55-63	(SEQ ID NO: 40)
		RERKQLVIY	169-177	(SEQ ID NO: 41)
	B52	KQLVIYEEI	172-180	(SEQ ID NO: 42)
		MTFGRLQGIF	99-108	(SEQ ID NO: 43)
	SSX-4
	A2	KSSEKIVYV	41-49	(SEQ ID NO: 44)
		VMTKLGFKV	57-65	(SEQ ID NO: 45)
		YVYMKLNYEV	48-57	(SEQ ID NO: 46)
		KLNYEVMTKL	52-61	(SEQ ID NO: 47)
		FARRPRDDA	7-13	(SEQ ID NO: 48)
		QISEKLRKA	16-24	(SEQ ID NO: 49)
		MTFGSLQRI	99-107	(SEQ ID NO: 50)
		SLQRIFPKI	103-111	(SEQ ID NO: 51)
		KJVYVYMKL	45-53	(SEQ ID NO: 52)
		KLRKAFDDI	20-28	(SEQ ID NO: 53)
		KLRKAFDDIA	20-29	(SEQ ID NO: 54)
		YMKLNYEVMT	50-59	(SEQ ID NO: 55)
		MTKLGFKVTL	58-67	(SEQ ID NO: 56)
		QLCPPGNPST	138-147	(SEQ ID NO: 57)
	A3	KLNYEVMTK	52-60	(SEQ ID NO: 58)
	A24	NYEVMTKLGF	54-63	(SEQ ID NO:59)
	B7	RPQMTFGSL	96-104	(SEQ ID NO: 60)
		KPAEEENGL	115-123	(SEQ ID NO: 61)
		GPQNDGKQL	131-139	(SEQ ID NO: 62)
		CPPGNPSTL	140-148	(SEQ ID NO: 63)
	B8	RLRERKQL	167-174	(SEQ ID NO: 64)
	B35	RPRDDAQI	10-17	(SEQ ID NO: 65)
		KPAEEENGL	115-123	(SEQ ID NO: 66)
	B44	YEVMTKLGF	55-63	(SEQ ID NO: 67)
		RERKQLVVY	169-177	(SEQ ID NO: 68)
	B52	KQLVVYEEI	172-180	(SEQ ID NO:69)
		MTFGSLQRIF	99-108	(SEQ ID NO: 70)
	SSX-2
	A2	KIQKAFDDI	20-28	(SEQ ID NO: 71)
		KASEKIFYV	41-49	(SEQ ID NO: 72)
		AMTKLGFKA	57-65	(SEQ ID NO: 73)
		RLQGISPKI	103-111	(SEQ ID NO: 74)
		RLRERKQLV	167-175	(SEQ ID NO: 75)
		DAFARRPTV	5-13	(SEQ ID NO: 76)
		FARRPTVGA	7-15	(SEQ ID NO: 77)
		QIPEKIQKA	16-24	(SEQ ID NO: 78)
		MTFGRLQGI	99-107	(SEQ ID NO: 79)
		ELCPPGKPT	138-146	(SEQ ID NO: 80)
		YVYMKRKYEA	48-57	(SEQ ID NO: 81)
		EAMTKLGFKA	56-65	(SEQ ID NO: 82)
		MTKLGFKATL	58-67	(SEQ ID NO: 83)
		RAEDFQGNDL	75-84	(SEQ ID NO: 84)
		ELCPPGKPTT	138-147	(SEQ ID NO: 85)
	A3	TLPPFMCNK	66-74	(SEQ ID NO:86)
		KIFYVYMKRK	45-54	(SEQ ID NO: 87)
	A24	KYEAMTKLGF	54-63	(SEQ ID NO:88)
	B7	RPQMTFGRL	96-104	(SEQ ID NO: 89)
		GPQNDGKEL	131-139	(SEQ ID NO: 90)
	B8	RLRERKQL	167-174	(SEQ ID NO: 91)
	B35	FSKEEWEKM	32-40	(SEQ ID NO: 92)
	B44	YEAMTKLGF	55-63	(SEQ ID NO: 93)
		RERKQLVIY	169-177	(SEQ ID NO: 94)
	B52	LQGISPKIM	104-112	(SEQ ID NO: 95)
		KQLVIYEEI	172-180	(SEQ ID NO: 96)
	SSX-1
	A2	AMTKLGEKV	57-65	(SEQ ID NO: 97)
		AMTKLGFKV	56-65	(SEQ ID NO: 98)
		FAKRPRDDA	7-15	(SEQ ID NO: 99)
		KASEKRSKA	16-24	(SEQ ID NO: 100)
		YVYMKRNYKA	48-57	(SEQ ID NO: 101)
		KAMTKLGFKV	56-65	(SEQ ID NO: 102)
		MTKLGFKVT	58-66	(SEQ ID NO: 103)
		MTKLGFKVTL	58-67	(SEQ ID NO: 104)
		RIQVEHPQMT	91-100	(SEQ ID NO: 105)
		MTFGRLHRI	99-107	(SEQ ID NO: 106)
	A3	TLPPFMCNK	66-74	(SEQ ID NO: 107)
	A24	NYKAMTKLGF	54-63	(SEQ ID NO: 108)
	B7	HPQMTFGRL	96-104	(SEQ ID NO: 109)
		GPQNDGKOL	131-139	(SEQ ID NO: 110)
	B8	RLRERKQL	167-174	(SEQ ID NO: 111)
	B44	RERKQLVIY	169-177	(SEQ ID NO: 112)
	B52	KQLVIYEEI	172-180	(SEQ ID NO: 113)
		MTFGRLHRII	99-108	(SEQ ID NO: 114)
	NY-
	A2	SISSCLQQL	148-156	(SEQ ID NO: 115)
		GTGGSTGDA	7-15	(SEQ ID NO: 116)
		RASGPGGGA	52-60	(SEQ ID NO: 117)
		GARGPESRL	79-87	(SEQ ID NO: 118)
		ATPMEAELA	97-105	(SEQ ID NO: 119)
		FTVSGNILT	126-134	(SEQ ID NO: 120)
		LTAADHRQL	137-145	(SEQ ID NO: 121)
		QLSLLMWJT	155-163	(SEQ ID NO: 122)
		LMWJTQCFL	159-167	(SEQ ID NO: 123)
		FATPMEAEL	96-104	(SEQ ID NO: 124)
		TVSGNTLTI	127-135	(SEQ ID NO: 125)
		ATGGRGPRGA	39-48	(SEQ ID NO: 126)
		GAPRGPHGGA	59-68	(SEQ ID NO: 127)
		LARRSLAQDA	104-113	(SEQ ID NO: 128)
		ITQCFLPVFL	162-171	(SEQ ID NO: 129)

[0097] The foregoing examples describe the isolation and cloning of nucleic acid molecules for the SSX4, splice variant of SSX4, and SSX5 genes as well as methods for determining expression of the various SSX genes as a possible indication of cancer. As was indicated, supra, these genes are expressed in tumor cells, thereby enabling the skilled artisan to utilize these for, e.g., assaying for cancer. The determination of expression can be carried out via, e.g., determination of transcripts of an SSX gene or genes, via nucleic acid hybridization, such as via polymerase chain reaction. In a preferred embodiment, one determines presence of a transcript of an SSX gene by contacting a sample with a nucleic acid molecule which specifically hybridizes to the transcript.

[0098] The hybridization of the nucleic acid molecule to a target is indicative of expression of an SSX gene, and of the possibility of cancer. Preferably, this is done with two primer molecules, as in a polymerase chain reaction. Determination of expression of more than one SSX gene in the context by these assays also a part of the invention. For the convenience of the artisan, the nucleotide sequences of SSX 1 and SSX2, which are known, are presented herein as SEQ ID NOS: 1 & 2.

[0099] Alternate assays are also a part of the invention. Members of the CT family are known to provoke antibodies in the individual who expresses a CT family member. Hence, one can carry out the assays described herein via, e.g., determining antibodies in a sample taken from a subject in question. Most preferably, the sample being analyzed is serum. Such assays can be carried out in any of the standard ways one determines antibodies, such as by contacting the sample with an amount of protein or proteins, and any additional reagents necessary to determine whether or not the antibody binds. One approach involves the use of immobilized protein, where the protein is immobilized in any of the standard ways known to the art, followed by contact with the sample and then, e.g., anti-IgG, anti-Fc antibodies, and so forth. Conversely, presence of an SSX protein can also be determined, using antibodies in the place of the proteins of the above described assays.

[0100] The correlation of SSX expression with cancer also suggests various therapeutic methods and compositions useful in treating conditions associated with abnormal SSX expression. “Abnormal SSX expression” in this context may mean expression per se, or levels which differ from those in a normal individual, i.e., they may be lower or higher.

[0101] The invention envisions therapeutic approaches such as the use of antisense molecules to inhibit or block expression. This antisense molecules are oligonucleotides which hybridize to the nucleic acid molecules and inhibit their expression. Preferably these are 17-50 nucleotides in length. These antisense oligonucleotides are preferably administered in combination with a suitable carrier, such as a cationic liposome.

[0102] Other therapeutic approaches include the administration of SSX proteins per se, one or more antigenic peptides derived therefrom, as well as so-called polytopic vaccines. These include a plurality of antigenic peptides, untied together, preferably by linker sequences. The resulting peptides may bind to either MHC-Class I or Class II molecules. These proteins, peptides, or polytopic vaccines may be administered in combination with an appropriate adjuvant. They may also be administered in the form of genetic constructs which are designed to permit expression of the protein, the peptide, the polytopic structures, etc. Peptides and polytopic structures can be expressed by so-called “minigenes” i.e., DNA molecules designed to express portions of the entire SSX molecule, or the various portions of the molecules, linked together as described supra. One can formulate the therapeutic compositions and approaches described herein such that one, or more than one SSX protein, is used as the source of the compositions. In other words, if a whole protein approach is used, one SSX molecule may be used, or two or more may be combined in one formulation. For peptides, these can all be taken from one SSX molecule, or be combinations of peptides taken from more than one. The polytopic structures described herein can also be made up of components of one, or more than one, SSX molecule.

[0103] The amount of agent administered and the manner in which it is administered will, vary, based on the condition being treated and the individual. Standard forms of administration, such as intravenous, intradermal, subcutaneous, oral, rectal and transdermal administration can be used. With respect to formulations, the proteins and or peptides may be combined with adjuvant and/or carriers such as a saponin, GM-CSF, one or more interleukin, an emulsifying oil such as vitamin E, one or more heat shock protein, etc.

[0104] When the nucleic acid approach is utilized, various vectors, such as Vaccinia or adenovirus based vectors can be used. Any vector useful in eukaryotic transfection, such as in transfection of human cells, can be used. These vectors can be used to produce, e.g., cells such as dendritic cells which present relevant peptide/MHC complexes on their surface. The cells can then be rendered non-proliferative prior to their administration, using standard methodologies.

[0105] Also a part of the invention are peptides which consist of amino acid sequences corresponding to portions of SSX molecules, or the NY-ESO-1 molecule, such as those peptide sequences described supra. As has been shown, such peptides bind to MHC molecules, such as HLA-A2 molecules, and provoke proliferation of cytolytic T cells against the formed complexes. As it has been shown that cells which express the full length molecules (NY-ESO-1, or SSX molecules) are in fact recognized by CTLs which were generated following pulsing of cells with relevant peptides. This result indicates that both the peptides and CTLs should be useful therapeutic agents. Hence, an additional aspect of the invention is the administration of one or more peptides, derived from NY-ESO-1 or an SSX molecule as described, alone or in combination, such as in antigen “cocktails.” Such cocktails can include a mixture of peptides, which have been formulated following typing of a particular patient's HLA type. Similarly, CTLs, developed in vitro, can be administered to the patient, in view of the recognition that the peptides are presented following endogenous expression of the full length molecule.

[0106] It is to be pointed out that when an MHC molecule is mentioned, such as HLA-A2, this is meant to include all allelic forms of that molecule. There are various types of HLA-A2 molecules which are known, and while these differ in a few amino acids, the degree of disparity is generally less than 10 amino acids over the full length of the molecule, and the differences are not expected to impact the ability of the form of the molecule to bind to peptides. Hence, a peptide which binds to an HLA-A*0201 molecule may by presumed to also bind to HLA-A*0202, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0209, and so forth.

[0107] Other aspects of the invention will be clear to the skilled artisan and need not be reiterated herein.

[0108] 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.

Claims

1. An isolated peptide consisting of 9 to 20 amino acids, comprising the sequence of ArgLeuLeuGluPheTyrLeuAlaMet (SEQ ID NO: 19) wherein said isolated peptide binds to an HLA molecule to form a complex.

2. The isolated peptide of claim 1, wherein said complex stimulates proliferation of cytolytic T lymphocytes.

3. The isolated peptide of claim 1, wherein said HLA molecule is an HLA-A2 molecule.

4. The isolated peptide of claim 3, wherein said HLA molecule is HLA-A*0201.

5. A method for stimulating proliferation of cytolytic T lymphocytes comprising combining a cell which presents an MHC or HLA molecule on its surface with the isolated peptide of claim 1, and a T lymphocyte containing sample, under conditions favoring formation of a complex between said HLA molecule and said peptide, and proliferation of cytolytic T lymphocytes specific for said complex.

6. The method of claim 5, wherein said HLA molecule is HLA-A2.

7. The method of claim 5, comprising administering said peptide to a subject in need of stimulation of cytolytic T lymphocytes.

8. The method of claim 7, wherein said peptide is administered with an adjuvant.

9. The method of claim 7, wherein said peptide is administered with an interleukin.

10. The method of claim 7, wherein said subject has a neoplastic disorder.

11. The method of claim 10, wherein said neoplastic disorder is melanoma.

12. The method of claim 5, comprising combining said cell, said peptide, and said T lymphocyte containing sample in vitro.

13. Isolated cytolytic T lymphocyte which specifically recognizes a complex of an MHC or HLA molecule and the isolated peptide of claim 1.

14. The isolated cytolytic T lymphocyte of claim 13, wherein said HLA molecule is HLA-A2.

15. A monoclonal antibody that specifically bind the peptide of claim 1 in complex with a carrier protein.

16. The monoclonal antibody of claim 15 wherein said antibody is humanized.

17. The monoclonal antibody of claim 15 wherein said carrier protein is an HLA molecule.

18. A purified antisera that specifically bind the peptide of claim 1 in complex with an HLA molecule.

19. The antisera of claim 16 which is purified by antigen affinity column.

20. Composition of matter comprising at least one peptide, the amino acid sequence of which is set forth at SEQ ID NO: 1, and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid molecule with a sequence which encodes a peptide of claim 1.

22. Expression vector comprising the isolated nucleic acid molecule of claim 21, operably linked to a promoter.

23. The expression vector of claim 22 wherein said promoter is an inducible promoter.

24. The expression vector of claim 22 wherein said promoter is an eukaryotic constitutive promoter.

25. Recombinant cell comprising the isolated nucleic acid molecule of claim 21.

26. The recombinant cell of claim 25, further comprising a recombinant nucleic acid molecule which encodes an HLA molecule.

27. A polytope comprising a plurality of amino acid sequences which correspond to peptides which bind to MHC molecules, wherein at least one of said amino acid sequence is the amino acid sequence of SEQ ID NO: 19.

28. An isolated nucleic acid molecule with a sequence which encodes a polytope of claim 27.

29. Expression vector comprising the isolated nucleic acid molecule of claim 28, operably linked to a promoter.

30. The expression vector of claim 29 wherein said promoter is an inducible promoter.

31. The expression vector of claim 29 wherein said promoter is an eukaryotic constitutive promoter.

32. Recombinant cell comprising the isolated nucleic acid molecule of claim 28.

33. The recombinant cell of claim 32, further comprising a recombinant nucleic acid molecule which encodes an HLA molecule.