The invention relates to new tumor-specific antigens, methods for their production, and their use for immunization, in particular, for the activation of cytotoxic, tumor-specific T- lymphocytes, and for specific diagnosis of tumor cells presenting said tumor-specific antigen in MHC class I-complex.

The immune system plays an important role in immunosurveillance against cancer and in tumor regression. The anti-tumor immune responses can be mediated through B and T cells which recognize tumor antigens expressed on tumor cells. The generation of cytotoxic T lymphocytes (CTLs) from either peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TLs) derived from patients with cancer in the last few years has allowed one to evaluate the role of T cells in the process of tumor regression in humans.

The adoptive transfer of tumor-infiltrating lymphocytes along with interleukin 2 (ici.-2) into autologous patients with cancer can mediate the regression of tumor in humans (Rosenberg et al., New Engl. J. Med. 319 (1988) 1676-1680; Rosenberg et al., J. Natl. Cancer Inst. 86 (1994) 1159-1166), suggesting that T cells play an important role in tumor rejection in vivo. The ability to mediate tumor regression in vivo was associated with the ability of TELs to mediate specific lysis and cytokine release when co-cultivated with syngenic tumor cells in vitro (Barth et al., J. Exp. Med. 173 (1991) 647-658).

To understand the molecular basis of T cell-mediated anti-tumor responses, a variety of genes recognized by T cells encoding tumor antigens have been identified (Boon et al., Annu. Rev. Immunol. 12 (1994) 337-365; Houghton, J. Exp. Med. 180 (1994) 1-4; Tsomides and Eisen, Proc. Natl. Acad Sci. USA 91 (1994) 3487-3489; Pardoll, Nature 369 (1994) 357-358; Rosenberg, Cancer J. Sci. Am. 1 (1995) 90-100). Based on their expression pattern these antigens can be divided into several classes: The first class of tumor antigens includes antigens (e. g. MAGE, BAGE and GAGE) that are shared between melanomas and other tumors of various histological types, but not by normal tissues other than testis and placenta (Van der Bruggen et al., Science 254 (1991) 1643-1647; Boel et al., Immunity 2 (1995) 167-175; Van den Eynde et al., J. Exp. Med 182 (1995) 689-698). Clinical trials based on the use of these antigens recognized by CTL- restricted by different HLA-class I alleles are in progress (Marchand et al., Int. J. Cancer

63 (1995) 883-885; Rosenberg, Immunology Today (1997) 175-182) in patients affected by melanoma and other neoplastic diseases. Taking into account the frequency of expression of each of these antigens and that of the HLA class I alleles, more than 60% of Caucasian melanoma patients, 40% of the head and neck, and 28% of bladder cancer patients could be eligible for immunization with at least one antigen of this group. No side effects have been detected in the treated patients. Indeed, MAGE, BAGE and GAGE gene expression normally occurs in cells of testis like spermatogonia and spermatocytes (II), which do not express the classical MHC class I molecules (Haas et al., Am. J. Reprod. Immunol.

Microbiol. 18 (1988) 47-57) needed for antigen presentation and thus will not be targeted by T cells.

The second class of tumor antigens contains tissue-specific antigens expressed in normal and neoplastic cells of the melanocyte lineage. CTL recognizing epitopes from tyrosinase (Brichard et al., J. Exp. Med. 178 (1993) 489-49513), MelanAMa"1 (Coulie et al., J. Exp.

Med. 180 (1994) 35-42; Castelli et al., J. Exp. Med 181 (1995) 363-368; Kawakami et al., Proc. Natl. Acad. Sci. USA 91 (1991) 3515-3519), gplOO' (Bakker et al., J. Exp. Med.

179 (1994) 1005-1009; Kawakami et al., Proc. Natl. Acad Sci. USA 91 (1994) 6458-6462), <BR> <BR> gp75 p75 (Wang et al., J. Exp. Med. 181 (1995) 799-804) and TRP-2 (Wang et al., J. Exp.

Med. 184 (1996) 2207-2216) on melanoma and normal cultured melanocytes can be expanded in vitro from many melanoma patients. Therefore, a large fraction of melanoma patients could potentially benefit of immunotherapy trials aimed at inducing a T cell response against these antigens. Indeed, differentiation antigens are expressed in almost all melanomas and the majority of them are presented to the immune effectors by the HLA-A2 allele that has a high frequency in various ethnic groups. However, the potential side effects of these treatments due to the development of cross-reacting responses against normal tissues (i. e., skin melanocytes and pigmente retinal cells) must be carefully considered.

The third class of tumor antigens includes antigens expressed only by the tumor cells from which they have been isolated. Such antigens are not expressed in other normal or neoplastic tissues of different origin and the antigenic epitope is usually generated by a point-mutation occurring in an otherwise ubiquitously expressed protein. Tumor antigens belonging to this group have been described in the murine system (Boon et al., Annu. Rev.

Immunol. 12 (1994) 337-365) and in some human tumors (Wolfel et al., Science 269 (1995) 1281-1284; Coulie et al., Proc. Natl. Acad Sci. USA 92 (1995) 7976-7980; Robbins et al., J. Exp. Med. 183 (1996) 1185-1192). The lack of natural tolerance against these antigens may allow the induction of a strong immune response, while avoiding the development of potential auto-immune reactions. However, until a panel of broad hot-spot

mutations will be discovered, the clinical application of such antigens should be limited to the treatment of individual patients or at least to very few individuals whose tumor carries the given mutation (Wolfel et al., Science 269 (1995) 1281-1284).

A fourth class of tumor antigens results from alternatively processed transcripts. Robbins et al. describe in J. Immunol. 159 (1997) 303-308 a gplOO transcript corresponding to a part of the fourth intron and coding for additional 35 amino acids not found in the normal gplOO glycoprotein. However, this epitope is expressed only in low levels in melanomas.

Robbins et al., in J. Immunol. 154 (1995) 5944-5950, describe the cloning of gene encoding an antigen recognized by melanoma-specific HLA-A24 restricted tumor- infiltrating lymphocytes. This antigen is a fragment of a full-length clone not encoded by an intron or a part thereof.

Fujii et al., in J. Immunol. 153 (1994) 5516-5524, describe a soluble form of the non- tumoricidal HLA-G antigen, which is coded by mRNA containing intron 4. However, no specific function for the soluble HLA-G protein is known at this time.

The existence of a fifth set of tumor antigens has been recently suggested based on the pattern of reactivity of a panel of CTL clones able to recognize the autologous and HLA- matched allogeneic melanomas, but not melanocytes or other targets of different histological origin (Anichini et al., J. Immunol. 156 (1996) 208-217).

It is an object of the invention to provide new tumor-specific antigens which are not expressed in normal cells and are capable of specifically distinguishing tumor cells from normal cells.

Summary of the invention The invention comprises a tumor-specific polypeptide having an antigenic effect, characterized in that it is partly coded by intron sequences from the gene of a polypeptide which is presented by the MHC class I complex on tumor cells (an exon-coded tumor antigen) and is obtainable by reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigen are used as a primer; and, if a PCR product is obtained, by isolation of the PCR product, expression in a host cell, and isolation of the tumor-specific antigen coded by the PCR product.

Said antigen can be presented as a fragment by an antigen-presenting cell (APC) in order to induce specific CTL response.

Another object of the invention is a method for the identification of such a tumor-specific polypeptide having an antigenic effect, whereby the following is carried out: -reverse transcriptase PCR from mRNA of a tumor cell, whereby nucleic acid fragments which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigen are used as a primer; -if a PCR product is obtained: isolation of the PCR product, expression in a host cell, and isolation of the tumor-specific antigen coded by the PCR product which hybridizes also with exon sequences of said antigen.

After isolation, the hybridization product, or a fragment thereof, is inserted into an expression vector, the vector is transferred into an appropriate host cell and expressed in said host cell. Subsequently, the resultant recombinant polypeptide is isolated.

In a preferred embodiment of the invention, a fragment of 8 to 12 codons of the hybridization product is used for the expression of the antigen.

The subject-matter of the invention therefore is a tumor-specific polypeptidic antigen which is coded partially by an intron of an exon-coded tumor antigen and which is obtained by a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigen are used as a primer, b) isolation of the product of said PCR, expression of said PCR product or of a fragment thereof in a host cell, and isolation of said tumor-specific antigen which is coded by said PCR product or a fragment thereof and which hybridizes also with exon sequences of said antigen.

Another subject-matter of the invention is a tumor-specific polypeptidic antigen according <BR> <BR> <BR> to the invention, wherein a fragment of 8 to 12 codons of said PCR product is used for the expression.

Yet another subject-matter of the invention is a tumor-specific antigen according to the invention, wherein said exon-coded tumor antigen is a CTL recognizing antigen like MAGE, BAGE and GAGE, CTL recognizing epitopes from tyrosinase, MelanAMart', gplOO gp75 orTRP-2.

The present invention further relates to a tumor-specific polypeptidic antigen which is coded by SEQ ID NO: 1.

A further subject-matter of the invention is a method for the isolation of mRNA of a tumor- specific antigen coded by an intron of an exon-coded tumor antigen, whereby there is carried out a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigens are used as a primer, and b) the product of said PCR is isolated which hybridizes also with exon sequences of said antigen.

The invention further relates to a method for measurement of proliferation of tumor- specific cytotoxic T-cells, wherein a tumor-specific antigen according to the invention is added to a sample of a body fluid of a patient, which contains antigen-presenting cells and cytotoxic T cells, and the proliferation of the cytotoxic T cells is measured, preferably via cytokine release (measurement of cytokines like TNF, IFNy, GM-CSF).

Another subject-matter of the present invention is the use of a nucleic acid coding for a tumor-specific antigen according to the invention for the manufacture of a therapeutic agent for the treatment of a tumor disease.

The invention in addition relates to the use of a tumor-specific antigen according to the invention for the activation of cytotoxic T cells from T precursor cells in vivo or in vitro.

A further subject-matter of the invention is a method for the production of a tumor-specific polypeptidic antigen, wherein said tumor-specific antigen is obtained by a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments which hybridize under stringent

conditions with intron sequences of an exon-coded tumor antigen are used as a primer, b) isolation of the product of said PCR, expression of said PCR product or of a fragment thereof in a host cell, and isolation of said tumor-specific antigen which is coded by said PCR product or a fragment thereof which hybridizes also with exon sequences of said antigen.

Yet another subject-matter of the invention is a combination of two nucleic acids which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigen and which can be used as a primer pair for reverse transcriptase PCR from mRNA.

Nucleic acids which are coded by SEQ ID NO: 3 to SEQ ID NO: 9 are a further subject- matter of this invention.

The tumor antigen according to the invention is not found on the surface of normal cells such as melanocytes. However, it is found on more than 80% of, e. g., melanoma cells and hence is specific for tumor cells.

Thus, this peptide is particularly suitable for the tumor cell-specific immunization of patients, and also for the diagnostic differentiation of melanoma cells and normal melanocytes.

A cytolytic T lymphocyte clone (CTL 128), derived from peripheral blood lymphocytes of a patient with metastatic melanoma, was able to lyse the autologous tumor and several allogenic melanomas in an HLA A*6801 restricted fashion. The gene coding for the antigen recognized by CTL 128 was identified by transfection of a cDNA library, constructed from autologous melanoma mRNA, into Cos-7 cells expressing the HLA-A*6801 allele. It has surprisingly been found that in contrast to melanocytes splicing- errors occur in mRNA maturation. This results in the translation of a peptide which was composed by a partially spliced form of the melanocyte differentiation antigen (TRP)-2 containing exon 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). TRP-2- INT2 codes for a putative protein of 238 amino acids which runs, using the same reading frame of TRP-2, from the start codon in position 400 to the terminator site (nt 1113) located in intron 2, just 18 nt downstream the peptide coding region.

The differentiation antigen TRP-2 is described by R. F. Wang, J. Experimental Medicine 184 (1996) 2207-2216. TRP-2 is one of the most highly expressed glycoproteins in human pigmente melanocytic cells and melanoma (Wang et al., J. Exp. Med. 184 (1996), 2207-

2216). It is located on the human chromosome 13 and has been shown to be a member of the tyrosinase-related gene family and shares a 40-45% amino acid sequence identity with tyrosinase and gp75/TRP-1 (Yokoyama et al., Biochim. Biophys. Acta. 1217 (1994), 317- 321; Robbins et al., J. Immunol. 154 (1995), 5944-5950). TRP-2 encodes a protein with 519 amino acids and has been demonstrated to have DOPAchrome tautomerase activity which is involved in melanin synthesis (Bouchard et al., Eur. J. Biochem. 219 (1994), 127- 134).

In contrast to the fully spliced TRP-2 mRNA which is described by Wang et al. to be found in melanomas, normal skin melanocytes and in retina, the TRP-2-INT2 mRNA was detected exclusively in melanomas. These results indicate that melanoma antigens recognized by CTL may derive from known lineage-related proteins by differences in splicing occurring in tumor but not in normal cells. This mechanism results in a new group of antigens that can be considered as true tumor specific and shared only by tumors of the same histotype while absent in normal tissues of the same lineage. This new group of antigens is therefore characterized by being derived from known lineage-related proteins by a tumor-specific and alternative splicing which does not occur in normal cells. Normal melanocytes, skin samples and retina proved negative in a reverse transcriptase (RT) PCR analysis. These features define an antigen that may allow the development of safer and more efficacious immunotherapy trials.

The present invention therefore relates to partially intron-coded tumor-specific antigens that are obtained by reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell. These antigens are recognized by specific T lymphocytes which then lyse the tumor cells presenting, in the MHC class I complex, the antigen according to the invention. It was surprisingly found that the mRNA coding for the antigens of the invention enriched in the cytoplasm of such tumor cells is particularly tumor-specific.

By"intron-coded tumor-specific antigen"is meant a tumor antigen which is coded not only by an exon sequence but partly (preferably about or more than 30%, more preferably about or more than 50%, most preferably about or more than 80%), by an intron sequence and which specifically recognizes tumor antigens that are presented via MHC class I. The expression of the intron is related to mechanism (s) of altered splicing in lineage related proteins.,. Partly" means preferably 30 to 50% or 30 to 80% intron coded. The exon coded and intron coded sequences of the antigens according to the invention are directly linked in the related genomic sequence because said antigens are caused by alternative splicing.

By"exon-coded tumor antigens"are meant the antigens described in the introductory part, such as, e. g., MAGE, BAGE and GAGE (Van der Bruggen et al., Science 254 (1991) 1643-1647; Boel et al., Immunity 2 (1995) 167-175; Van den Eynde et al., J. Exp. Med 182 (1995) 689-698) or such as e. g. CTL recognizing epitopes from tyrosinase (Brichard et al., J. Exp. Med. 178 (1993) 489-49513), MelanA'' (Couhe et al., J. Exp. Med. 180 (1994) 35-42; Castelli et al., J. Exp. Med 181 (1995) 363-368; Kawakami et al., Proc. Natl. Acad.

Sci. USA 91 (1991) 3515-3519), gplOO (Bakker et al., J. Exp. Med. 179 (1994) 1005- 1009; Kawakami et al., Proc. Natl. Acad Sci. USA 91 (1994) 6458-6462), gp7STRP (Wang et al., J. Exp. Med. 181 (1995) 799-804) and TRP-2 (Wang et al., J. Exp. Med. 184 (1996) 2207-2216).

By"peptide or polypeptide according to the invention"is meant a polypeptide which preferably consists of 8 to 12 amino acids, and most preferably at least 10 amino acids, but may also comprise the size of a protein. The peptide may be also a part of a protein, such as a fusion protein.

By"polypeptide having an antigenic effect"is meant a polypeptide that elicits, in vivo and in vitro, a specific immune response.

The term"hybridize under stringent conditions"means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al.,"Expression of cloned genes in E. coli" in Molecular cloning: A laboratory manual (1989), Cold Spring Harbor Press, New York, USA, 9.47-9.62 and 11.45-11.61.

More specifically,"stringent conditions"as used herein refer to hybridization in 6.0 x SSC at about 45°C, followed by a wash of 2.0 x SSC at 50°C. For selection of the stringency the salt concentration in the wash step can be selected, for example, from about 2.0 x SSC at 50°C, for low stringency, to about 0.2 x SSC at 50°C, for high stringency. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperatures, about 22°C, to high stringency conditions at about 65°C.

For expression in a prokaryotic or eukaryotic organism, such as prokaryotic host cells or eukaryotic host cells, the nucleic acid sequence is integrated into suitable expression vectors, according to methods familiar to a person skilled in the art. Such an expression vector preferably contains a regulatable/inducible promoter. These recombinant vectors are then introduced for the expression into suitable host cells, such as, e. g., E. coli as a prokaryotic host cell, or Saccharomyces cerevisiae. CHO or COS cells as eukaryotic host

cells, and the transformed or transduced host cells are cultured under conditions which allow the expression of a heterologous gene. The isolation of the peptide can be carried out according to known methods from the host cell or from the culture supernatant of the host cell. Such methods are described, for example, by Ausubel I., Frederick M., Current Protocols in Mol. Biol. (1992) John Wiley and Sons, New York.

By"host cells"are meant prokaryotic and eukaryotic cells, preferably COS or CHO cells.

Production in prokaryotic cells will be preferred if glycosylation proves to be of minor importance with respect to the action of the protein.

The gene coding for the tumor-specific antigen recognized by a specific cytotoxic T lymphocyte (CTL) can be identified by transfection of a cDNA library, constructed from autologous tumor mRNA, into eukaryotic cells, preferably expressing a suitable HLA allele of the patient.

In order to determine the HLA antigen the entire RNA is isolated from the patient's tumor cells or tumor tissue, and the cDNA is obtained by reverse transcriptase PCR with primers that specifically code for the HLA antigens, and is cloned in eukaryotic expression vectors, such as, e. g., pcDNA3 (Invitrogen Corporation, Oxon, U. K.). In order to determine the intron-coded tumor-specific antigen according to the invention the poly A+ RNA is isolated from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, and a cDNA library is established by reverse transcriptase PCR with primers that specifically code for an exon-coded tumor antigen, such as, e. g., MAGE, BAGE and GAGE, CTL <BR> <BR> recognizing epitopes from tyrosinase, MelanA "'gplOO gp75 and TRP-2. The cDNA fragments from the cDNA library are cloned into eukaryotic expression vectors, such as, e. g., pcDNA3.1 (Invitrogen Corporation, Oxon, U. K.). After co-transfection of the expression vectors into eukaryotic cells, such as, e. g., Cos-7 cells (expressing relevant MHC genes which are able to present the peptide fragment of the antigen which is capable of activating specific tumor antigen T cells), one may then determine those clones that cause stimulation of the tumor-specific cytotoxic T lymphocytes. The stimulatory effect upon the tumor-specific cytotoxic T lymphocytes can be determined by a CTL stimulation assay (determination of TNF-a, IFN-y, GM-CSF) as described by Traversari et al., Immunogenetics 35 (1992), 145-152.

In this manner it is possible to obtain a cell clone which specifically activates those cytotoxic T cells that cause lysis of the respective tumor cells.

This cell clone expresses a polypeptide which, in isolated and purified form, may be administered to the patient for immunization/vaccination, either directly as a full length polypeptide since the antigenic polypeptide according to the invention is processed in vivo, or in the form of shortened polypeptide fragments.

To this end, the nucleic acid coding for the polypeptide according to the invention is isolated from this cell clone according to established methods (Sambrook et al., Molecular cloning (1989), Cold Spring Harbor Laboratory Press) and is shortened by restriction digestion. These shortened fragments, after cloning into eukaryotic expression vectors, are then transfected into eukaryotic cells, such as, e. g., COS-7 cells (supra), and the stimulation of the cytotoxic T cells is determined in a CTL stimulation assay as described by Traversari et al. in Immunogenetics 35 (1992) 145-152. In this manner the coding nucleic acid sequence can be restricted to the peptide epitope essential for the stimulation of the cytotoxic T cells.

In a preferred embodiment, the nucleic acid sequence coding for the protein is coupled with a nucleic acid sequence capable of enhancing the processing of the protein in the host cell.

Meanwhile a number of sequences, such as, e. g., ubiquitin, are known which enhance the transport of the protein, the degradation of the protein and the introduction into the MHC class I complex (Bachmair et al., Science 234 (1986) 179-186; Gonda et al., J. Biol. Chem.

264 (1989) 16700-16712; Bachmair et al., Cell 56 (1989) 1019-1032). A faster degradation of the protein and a more effective introduction into the MHC class I complex can be achieved by coupling a protein containing the antigenic polypeptide according to the invention with ubiquitin. As a result of this, the presentation of the peptide on the cell surface of the tumor cells is particularly efficient. In this connection, the amino acid between the protein containing the antigenic peptide and ubiquitin is of special importance regarding the coupling of a protein containing the antigenic peptide with ubiquitin. By selecting the appropriate amino acid one can additionally achieve a considerable improvement in the recognition of the melanoma cell by the specific cytotoxic T cell.

As a preferred embodiment a fragment of 8 to 12 codons of the PCR products is used.

In another preferred embodiment there is used a partly intron-coded tumor-specific antigen TRP-2-INT2 (hereafter referred to as TRP-2-INT2 fragment or TRP-2-INT2 peptide) which is recognized by specific T lymphocytes which then lyse those melanoma cells that present in the MHC class I complex the polypeptide EVISCKLIKR and whose amino acid sequence is coded by the DNA sequence shown in SEQ ID NO: 2.

To obtain the coding sequences of the tumor-specific antigen of the TRP-2-INT2-peptide the cDNA coding for the HLA-A*6801 allele and the TRP-2-INT2 antigen were isolated from the cytoplasm of a melanoma cell line which was established from a metastatic lesion obtained from a surgical specimen of a patient, who was admitted to surgery to the Istituto Nazionale Tumori (Milan, Italy). After transfection of both cDNAs in COS-7 cells a clone was isolated (TRP-2-INT2, HLA-A*6801), which was able to stimulate the cytotoxic effect of a cytolytic T lymphocyte (CTL128). The DNA sequencing analysis presented a cDNA fragment of the exon-coded TRP-2 antigen which contains exon 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). Subfragments of the TRP-2-INT2 fragment were obtained by digestion of the TRP-2-INT2 DNA with restriction enzymes according to methods familiar to a person skilled in the art or according to methods which are described by Sambrook et al.,"Expression of cloned genes in E-coli"in Molecular cloning: A laboratory manual (1989), Cold Spring Harbor Press, New York, USA, or by PCR amplification with fragment-specific primers (SEQ ID NOS: 6,7,8). After transfection into COS-7 cells the CTL-stimulating effect is determined by a CTL Stimulation Assay as described in Traversari et al., Immunogenetics 35 (1992) 145-152.

The coding nucleic acid sequence was determined by DNA sequence analysis.

Another subject-matter of the invention is the use of tumor cells presenting the antigen according to the invention via MHC class I.

Another subject-matter of the invention is a method for the detection of the expression of the antigen according to the invention from the patient's body fluid, preferably a blood or tissue specimen. To this end, an intron-coded tumor-specific cDNA coding for the antigen according to the invention is used prior to, during, and after therapy to diagnose tumor cells which express the antigen according to the invention.

Prior to therapy, it is examined whether the tumor antigen according to the invention is expressed in the patient's tumor cells, because only if the tumor does indeed express the antigen according to the invention can the therapy be carried out. During the course of therapy, one can monitor whether the expression of the antigen according to the invention, being a marker for tumor cells present, can be reduced, and after therapy, measurement of the expression of the antigen according to the invention allows controlling of whether the tumor cells have been eliminated to the fullest possible extent.

In a preferred embodiment, a nucleic acid coding for the tumor-specific TRP-2-INT2 peptide is used for the diagnosis of melanoma cells presenting on their surface the TRP-2- INT2 peptide.

The sequence for the TRP-2-INT2 peptide is specifically expressed in melanomas. Hence, TRP-2-INT2 sequences may be used as specimens for identifying tumor cells.

Identification is by means of PCR or labelled hybridization samples or by any of the various nucleic acid probe based assays known in the art.

The determination of the TRP-2-INT2 expression is carried out at the mRNA level by reverse transcription PCR (RT-PCR) and by hybridization with specific labelled TRP-2-INT2 probes.

TRP-2-INT2 mRNA can be specifically determined, for distinction against TRP-2 mRNA, by 1) transcription to cDNA; 2) the use of intron-specific primers; 3) comparing the length of the amplified cDNA fragment (determining the length of the amplified cDNA exon-intron-exon fragment with cDNA exon to exon); 4) hybridizing with intron-specific probes.

Another subject-matter of the invention is a method for the determination of proliferation of tumor-specific cytotoxic T lymphocytes. This method is used for the detection of cytotoxic T cells which can be activated by the antigen according to the invention, prior or during the course of therapy. Prior to therapy, it is possible to select patients who already have cytotoxic T lymphocytes that can be activated by the antigen according to the invention.

To check whether there are tumor-specific activatable T cells in the patient, T cells and antigen-presenting cells are isolated from the patient's blood, and the antigen according to the invention is brought into contact ("pulsed"), ex vivo, with the antigen-presenting cells.

If T cells capable of being activated by the antigen according to the invention are present in the patient's blood, there will be a proliferation of the specific cytotoxic L lymphocytes which can be detected then by a CTL stimulation assay, for instance. Such activatable T cells, after being stimulated with the antigen according to the invention, can be used for therapy then. This diagnostic procedure must be carried out prior to therapy.

Another diagnostic procedure, during therapy, serves as a control of the cytotoxic L lymphocytes formed, making it possible to quantitatively determine the induction of tumor- specific T cell activation.

The proliferation of the cytotoxic T lymphocytes can be determined by a CTL stimulation assay, for instance. The stimulating cell lines are tested for their ability to induce the production of TNF by the CTLs as described in Traversari et al., Immunogenetics 35 (1992) 145-152. The TNF content is determined by testing its cytotoxic effect on WEHI- 164.13 cells (Espevic and Nissen-Meyer, J. Immunol. Methods 95 (1986) 99-105) in an MTT colorimetric assay (CTL Stimulation Assay).

In a preferred embodiment, the TRP-2-INT2 peptide is used for the determination of proliferation of TRP-2-INT2-specific cytotoxic T lymphocytes.

Another subject-matter of the invention is the use of a nucleic acid, which codes for the intron-specific antigen according to the invention, for the manufacture of a therapeutic agent for the treatment of tumor diseases.

In this connection, nucleic acids according to the invention can be used for gene therapy.

The nucleic acid is introduced into the patient's body with the help of viral or non-viral vectors, whereby the coding sequence should be specifically expressed and the peptide according to the invention should, by virtue of the binding to antigen-presenting cells, elicit a specific cytotoxic T cell response. The immune response elicited should then be directed against all tumor cells that express on their cell surface the peptide according to the invention. The DNA sequences coded on vectors can be applied in the form of nude DNA, in combination with liposomes, or together with a suitable adjuvant (as well-known in the art), either subcutaneously, intramuscularly, or intratumorally.

In a preferred embodiment, the TRP-2-INT2 peptide is used for gene therapy.

In another embodiment, the antigen according to the invention can be used for the immunization and/or vaccination of tumor patients. The immunization is based on the activation of specific cytotoxic T cells by presenting the antigen according to the invention via antigen-presenting cells. Immunization can be carried out both ex vivo and in vivo.

In doing so, antigen-presenting cells (macrophages, dendritic cells, or B cells) and T lymphocytes are taken from the patient's blood and brought into contact ("pulsed), ex vivo, with the peptide according to the invention. These antigen-presenting cells equipped with the peptide according to the invention, which in this manner cause an activation of specific cytotoxic T cells, are subsequently returned to the patient's blood.

Immunization can be carried out in vivo by subcutaneously administering to the patient the polypeptide according to the invention, whereby the activation of specific cytotoxic T cells directly in the patient is achieved. The binding of the peptide according to the invention to the corresponding HLA molecule on the surface of antigen-presenting cells leads to proliferation of specific cytotoxic T lymphocytes.

The immunogenic effect of the peptide according to the invention during application can be enhanced by the following measures: 1) coupling with bacterial toxins (superantigens); 2) administration in combination with Freud's adjuvant; 3) mixing the peptide according to the invention with liposomes.

In a preferred embodiment, the TRP-2-INT2 peptide is used for the immunization and vaccination of melanoma patients.

Another subject-matter of the invention is a primer for the detection of the expression of the specific tumor antigen according to the invention by RT-PCR. In this connection, the primer can be selected by the following measures: 1) The sense and anti-sense primer hybridizes with two difference exon sequences of the tumor antigen.

2) The sense primer hybridizes with an exon sequence and the anti-sense primer (oligo- dT primer) hybridizes with the poly-A tail of the mRNA sequence of the tumor antigen.

In a preferred embodiment, a specific primer is used for the detection of the expression of the TRP-2-INT fragment whose amino acid sequence is coded by the DNA sequence shown in SEQ ID NO: 2 (PRIT-3) and SEQ ID NO: 4 (INT2-1260).

The following examples, references, sequence listing and the drawings are provided for further illustrating various aspects and embodiments of the present invention and are in no way intended to be limiting in scope.

Description of the drainas: Figure 1: CTL 128 recognized the autologous melanoma (Me 18732) in an HLA- A*6801 restricted fashion. Target cells were incubated with the

indicated anti-HLA mAb for lh at room temperature before addition of effectors at fixed E: T ratio (A.: Me 18732 + none; B: Mel8732 + HLA Class 1; C: Mel8732 + HLA-A2,-A69; D: ME18732 + HLA- A2,-A28; Mel8732 + HLA-B,-C).

Figure 2: Recognition by CTL 128 of autologous melanoma Mel8732 and HLA-A*6801+ melanoma cell lines. 1,500 CTL were added to 25,000 stimulator cells and the TNF content of the supernatant was tested 24 h later on WEHI-164.13 cells (Melanomas: A: Mel8732; B: Me 20842; C: Mel7697; D: Me 2559/1; E: Mel2657; F: Mel7088; G: Me 4023; H: LB-33; I: Lung carcinoma Calu 3; K: Breast carcinoma SKBR3; L: Ovarian carcinoma SKOV3).

Figure 3: Stimulation of CTL clone 128 by Cos-7 cells transfected with cDNA 131 and HLA-A*6801 cDNA. Cos-7 cells were transfected with HLA- A*6801 and with pool A255 or cDNA 131. Pool A255 is a (group of 100 cDNA clones of the Mel8732 cDNA library which was amplified to saturation and from which plasmid DNA was extracted. cDNA 131 was a single clone subcloned from pool A255. The production of TNF by CTL 128 was measured after 20 h of co-culture with the transfected cells, using the TNF sensitive cell line WEHI-164.13. As control Cos- 7 cells were transfected with cDNA 131 or HLA-A*6801 alone (A: Mel8732; B: COS; C: COS + A*6801; D: COS + cDNA 131; E: COS + A*6801 + cDNA 131).

Figure 4: cDNA 131 codes for the antigen recognized by CTL 128. (A) Recognition and (B) lysis by CTL 128 of a geneticin resistant population of the HLA-A*6801 melanoma cell line LB33, following transfection with the pcDNA3.1/cDNA 131 construct. TNF secretion by CTL 128 was measured after 24 h of co-culture of 1,500 responder cells with 20,000 stimulating cells. Lytic activity of CTL 128 was measured on''cor labeled target cells after 4 h of co-incubation with the CTL at different effector-to-target (E/T) ratios.

Figure 5: Identification of the sequence coding for the antigenic peptide recognized by CTL 128. Exon/intron organization of cDNA 131 is shown in the upper part of the panel (A). Exon and introns are indicated as solid and open boxes respectively, the horizontal line at

the extremities represents pcDNA3.1 vector, while the numbering of the sequence is relative to the 5'end of cDNA 131. Subfragments derived from cDNA 131 and PCR products, shown below cDNA 131 as open boxes, were cloned in expression vectors and transfected into Cos-7 cells with HLA-A*6801 cDNA. Spliced full-length form of TRP-2 cDNA was obtained by screening the 18732 cDNA library with an exon 8 specific oligonucleotide probe. TNF release by CTL 128 was evaluated on WEHI164.13 cells (B). The peptide encoding sequence present in the PCR fragments INT-2-I66 and INT-2-107 are pointed out.

Figure 6: Lysis by CTL 128 of HLA-A*6801 cells pulsed with the synthetic antigenic peptide. 5'Cr-labeled HLA-A*6801 EBV-LCL (LB-EBV) were incubated with CTL 128 at an E/T ratio of 20: 1, in the presence of the synthetic peptides shown on the left, at the concentration indicated. 5lCr-release was measured after 4 h. As a negative control, a MAGE-3 derived peptide (M3A1) able to bind HLA-A1 was used.

Figure 7: Recognition by CTL 128 of TRP-2-INT2-) 21-231 peptide when presented by HLA alleles of the A3-like supertype. 5'Cr-labeled EBV- LCLs were incubated with CTL 128 at an E/T ratio of 20: 1, in the presence of peptide TRP-2-INT2221-23) different concentrations.

Chromium release was measured after 4 h. c: negative control without peptide.

Description of the sequences: SEQ ID NO: 1: coding sequence (nucleic acid) of the antigenic peptide recognized by CTL 128.

SEQ ID NO: 2: coding sequence (amino acid) of the antigenic peptide recognized by CTL 128.

SEQ ID NO: 3: (PRIT-1) sense primer used for the verification of unspliced intron TRP2-INT; located in the 5'UTR of the TRP2-gene.

SEQ ID NO: 4: (INT2-1260) anti-sense primer used for the verification of unspliced intron TRP2-INT; located in the 5'UTR and intron 2 of the TRP2- gene.

SEQ ID NO: 5: (KS-INT2) sense primer used for the production of the subfragments of cDNA 131 and for cloning of TRP-2-INT2 from genomic DNA; located in exon 2 of the TRP2-gene.

SEQ ID NO: 6: (INT2-asl) anti-sense primer used for the production of subfragment INT2-107 of cDNA 131; located in intron 2 of the TRP2-gene.

SEQ ID NO: 7: (INT2-as2) anti-sense primer used for the production of subfragment INT2-166 of cDNA 131; located in intron 2 of the TRP2-gene.

SEQ ID NO: 8: (Sp6) anti-sense primer used for the production of subfragment INT2-434 of cDNA 131; located in the pCDNAI-plasmid.

SEQ ID NO: 9: (PR2) anti-sense primer used for cloning of TRP-2-INT2 from genomic DNA; located in exon 3.

SEQ ID NO: 10: (PR3) sense primer used for amplification of TRP-2 DNA; located in exon 2.

SEQ ID NO: 11: (TRP-2L) anti-sense primer used for amplification of TRP-2 DNA; located in exon 8.

SEQ ID NO: 12: Nucleic acid sequence of the 5'end-1500 fragment including the coding region for the antigenic peptide. The first 45 bp before the start of cDNA 131 and belonging to pcDNA3.1 vector are omitted.

Example 1 Identification of HLA-A*6801 as a restriction element for CTL 128 Melanoma cell line Me 18732 was established from a metastatic lesion of a patient, typed as LA-A2 and HLA-A28 by serological methods and then as HLA-A*0201 and HLA *68011 (further referred to as HLA-A*6801) by sequence-specific oligonucleotide probe (SSOP)

subtyping (Oh et al., Genomics 29 (1995) 24-34). Anti-tumor CTL clones were obtained as described by Anichini et al., J. Immunol. 156 (1996) 208-217.

CTL clone 128 recognized the autologous melanoma in the context of an allele of the HLA-A locus, since its cytolytic activity was reduced by the anti-HLA Class I mAB W6/32, but not by the anti-HLA-B,-C mAb 4E (Fig. 1). HLA-A*0201 could be excluded as presenting molecule for the antigen recognized by CTL 128 since inhibition of lysis was observed only with the anti-HLA-A2,-A28 mAb CR1 1.351, but not with the anti-HLA-A2,-A69 mAb BB7.2 (Fig. 1). The inhibitory activity of CR1 1.351, therefore, indicated that A28 (A*6801) was the HLA presenting molecule for the CTL 128.

Cultivation of cell lines The melanoma cell line Mel8732 was established from a metastatic lesion obtained from a surgical specimen of a patient who was admitted for surgery to the Istituto Nazionale Tumori (Milan, Italy). PBLs of this patient were serologically typed as: HLA-A2,-A28,- B44,-B51,-C2,-C5. Human metastatic (Mel7697, Me2559/1, Mel2657, Mel7088/1, Me4023) and primary (Me20842) melanoma cell lines were established and cultured in 10% FCS/RPMI 1640. The melanoma line LB-33, LB-40 and the Cos-7 (ATCC: CRL 1651) cell line were maintained in 10% FCS/DMEM. The carcinoma lines CALU3, SKBR3 and SKOV3, purchased from the American Type Culture Collection (ATCC, Rockville, MD), were kept in culture in 10% FCS/RPMI-1640. C1RA*03301 transfectant, the homozygous EBV-transformed LCL, the cell lines SCHU is characterized as HLA- A*0301, B*0702,-C7, AMA-1 is characterized as HLA-A*6802, B*5301,-C4, and WT- 100-bis is characterized as HLA-A11,-B35,-C4. The EBV-LCL JHAF (HLA-A*31011,- B51,-C8) and LB (HLA-A*68011, B*40011,-C2,-C3) were obtained from ATCC. EBV- LCL were maintained in 10% FCS/RPMI-1640.

Evaluation of the antigenic specificity of CTL 128 To evaluate the frequency of expression of the antigen recognized by CTL 128 on other tumors, a panel of HLA-A*6801 melanoma lines was tested in a CTL stimulation assay.

Five out of eight melanoma cell lines induced TNF release by CTL 128 (Fig. 2). No reactivity was instead observed with three HLA-A*6801 carcinoma lines of different histological origin (Fig. 2). HLA-A*6801 negative melanomas, melanocytes and tumor lines of other histological type failed to stimulate cytokine release by CTL 128. The pattern of reactivity displayed by CTL 128 towards the melanoma cell lines tested did not correlate with expression of already described melanoma antigens in these cell lines, as assessed by

RT-PCR. This was confirmed by lack of TNF release by CTL 128 in the presence of Cos-7 cells cotransfected with plasmid pcDNA3/A*6801, containing the HLA-A*6801 gene of patient 18732, together with each of the genes known to encode shared melanoma antigens: Melan-A/MART-1, tyrosinase, gp 100, TRP-1,-2, MAGE-1,2,-3,-4,-12, BAGE-l,-2, GAGE-1,-2,-3,-4,-5,-6. These results were consistent with the hypothesis that HLA- A*6801 restricted CTL 128 recognized a new antigen shared by a number of melanomas.

The CTL clone 128 was derived by limiting dilution of 4 week-old mixed lymphocyte- tumor cultures (MLTC) and grown in conditions similar to those previously described (Anichini et al., J. Immunol. 156 (1996) 208-217). CTL 128 expressed a CD3+, CD4-, CD8+, TCR-+ phenotype, as assessed by flow cytometry with specific mAbs.

Assay for cytolytic activity: The lytic activity of CTL 128 was tested in a chromium release assay as previously described (Anichini et al., J. Immunol. 156 (1996) 208-217). Results were expressed as: (experimental release-spontaneous release) % lysis (maximum release-spontaneous release) where spontaneous release was assessed by incubating target cells in the absence of effectors, and the maximum release was determined in the presence of 1 % NP-40 detergent (BDH Biochemicals, Poole, U. K.). Inhibition of lysis against the autologous melanoma was performed with the following mAbs as reported (Anichini et al., J.

Immunol. 156 (1996) 208-217): the anti-HLA-A,-B,-C W6/32 (Parham et al., J.

Immunol. 123 (1979) 342-349), the anti-HLA-A2,-A69 BB7.2 (Parham and Brodsky, Hum. Immunol. 3 (1981) 277-299), the anti-HLA-A2,-A28 CR11.351 (Russo et al., Immunogenetics 18 (1983) 23-35), and the anti-HLA-B,-C 4E (Yang et al., Immunogenetics 19 (1984) 217-231).

Subcloning of the HLA-A*6801 allele Total RNA was prepared from Mel8732 cells by the guanidine-isothiocyanate method using RNAzol. B (Cinna/Biotecx, South Loop East, TX). Single-stranded cDNA synthesis was carried out on 2 jjg of total RNA using oligo-dT primer and Moloney murine leukemia virus-derived reverse transcriptase without RNase-H activity (MMLV-RT RNase-H- Superscript; GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions.

cDNA corresponding to 300 ng of total RNA was amplified by PCR using 1 U of DynaZymeTM (Finnzymes OY, Espoo, Finland) and a primer pair suitable for specific amplification and directional cloning of the full length coding region of HLA-A alleles.

Following BamHI and HindE digestion, the 1.1 Kb PCR-product was subcloned into the BamHI/EcoRV site of the eukaryotic expression vector pcDNA3 (Invitrogen Corporation, Oxon, UK).

Plasmid clones encoding the HLA-A*68011 or the A*0201 (the HLA-A28 and-A2 alleles of patient 18732) were identified using diagnostic restriction enzymes. The HLA-A*68011 gene was then sequenced to verify the correspondence to the published DNA sequence.

This plasmid was called pcDNA3/HLA-A*6801.

Example 2 Cloning of a cDNA encoding the melanoma antigen recognized by CTL 128 A cDNA library was constructed in a suitable expression vector (pcDNA3.1) with poly (A) + RNA extracted from Me 18732 cells. Poly (A) + RNA was isolated from Me 18732 cells using the Fast Track mRNA extraction kit (Invitrogen). The library was constructed converting 5 ut ouf poly (A) + RNA to cDNA with the SuperScript Choice System kit (GIBCO BRL, Gaithersburg, MD) using an oligo-dT primer containing a NotI site at its 5'- end. cDNAs were then ligated to BstXI adapters (Invitrogen) and digested with NotI. After size fractionation, cDNAs were unidirectionally cloned into the BstXI/NotI site of the mammalian expression vector pcDNA3.1 (Invitrogen). Recombinant plasmids were electroporated into DH5-Escherichia coli and selected with ampicillin (100 mg/ml).

The library was divided into 1,300 pools of about 100 cDNA clones. Each pool of bacteria was amplified to saturation, plasmid DNA was extracted and transfected (100 ng) together with pcDNA3/A*6801 (100 ng) into 1.2x104 Cos-7 cells by the DEAE-dextran- chloroquine method (Coulie et al., J. Exp. Med. 180 (1994) 35-42; Seed and Aruffo, Proc.

Natl. Acad. Sci. USA 84 (1987) 3365-3369). Using the same technique, in other experiments Cos-7 cells were cotransfected with 100 ng of pcDNA3/A*6801 vector and 100 ng of pcDNAI or pcD SR plasmids containing the cDNA of one of the following melanoma antigens: Melan A/MART-I, tyrosinase, gpl00, MAGE-l,-2,-3,-4,-12, BAGE-1,-2, GAGE-1,-2,-3,-4,-5,-6, TRP-I. Full length TRP-2 cDNA was amplified by RT PCR using specific primers located in the 5'-untranslated region (UTR) and at the end of exon 8, cloned into pcDNA3 and sequenced to verify the correspondence to the

published cDNA sequence (Yokohama et al., Bioch. Bioph. Acta 1271 (1994) 317-321).

Transfected Cos-7 cells were tested in a CTL stimulation assay after 48 h.

CTL stimulation assay Transfectants or stimulating cell lines were tested for their ability to induce the production of TNF by CTL 128 as previously described (Traversari et al., Immunogenetics 35 (1992) 145-152). Briefly, 1,500 CTL were added to microwells containing target cells, in 100 1 of IMDM (BioWhittaker, Walkersville, MD) with 10% pooled human serum (PHS) and 25 U/ml r-hu-IL2 (EuroCetus, Amsterdam, The Netherlands). After 24 h, the supernatant was collected and its TNF content was determined by testing its cytotoxic effect on WEHI cells, such as WEHI-164.13 (Espevic and Nissen-Meyer, J. Immunol. Methods 95 (1986) 99-105) in an MTT colorimetric assay. The WEH1-164.13 cells were kept in culture in 10% FCS/RPMI-1640.

Duplicate microcultures were transfected and screened two days later for their ability to stimulate TNF release by CTL 128. The DNA of one of the 1,300 pools (pool A255) induced the production of a high level of TNF (Fig. 3), a finding confirmed in a second transfection experiment. Bacteria of the positive pool were cloned and their plasmid DNA was cotransfected with the HLA-A*6801 construct as before. 25 out of 159 clones stimulated TNF release by CTL 128. The results obtained with one of these, namely cDNA 131, are shown in Fig. 3.

Transfection of melanoma cell lines The melanoma cell line LB-33 was transfected by the calcium phosphate precipitation technique with cDNA 131 cloned in plasmid pcDNA3.1 (Invitrogen), which contains the neomycin resistance gene. A clonal subline was isolated from a G418-resistant transfected population. Using the same method, the melanoma cell lines LB-40, SK23-MEL and MZ2- MEL and maintained in 10% FCS/DMEM) were transfected with HLA-A*6801 cDNA and selected in G418. Expression of the transfected HLA A*6801 allele in stable transfectants was verified by flow cytometry with specific mAbs.

The HLA-A*6801+ melanoma cell line LB-33, which was not recognized by CTL 128 (see Fig. 2), when transfected with cDNA 131 acquired the property capable of inducing TNF release (Fig. 4A) and became sensitive to lysis by CTL 128 (Fig : 4B), indicating that recognition of the antigen may occur in a tumor cell and was independent of the high, artificial expression level achieved in Cos-7 cells.

The sequence of the cDNA 131 proved to be 3548 bp long. By searching Genbank, it was found that nucleotides (nt) 1-994 and nt 3081-3347 were identical to two non-contiguous regions of the cDNA coding TRP-2 (Yokohama et al., Bioch. Bioph. Acta 1217 (1994) 317-321), which has been recently identified as a melanoma antigen of the melanocyte lineage (Wang et al., J. Exp. Med. 184 (1996) 2207-2216). The first region contained the 5'-UTR, exon 1 and exon 2, whereas the second region exon 3 and exon 4 of the TRP-2 gene, respectively. The sequence between nt 995-3080 and that downstream nt 3347 showed no significant homology with any sequence recorded in databanks. The two regions which were present in cDNA 131 but absent in the TRP-2 cDNA were retained intron sequences. A stretch of ten amino acids flanking the exonic portions perfectly matched the described sequences at exon-intron junctions of the TRP-2 gene (Sturm et al., Genomics 29 (1995) 24-34). Moreover, the length of sequence 995-3080 (2086 nt) was compatible with that of the intron 2 of TRP-2, as deduced from the published genomic map (Sturm et al., Genomics 29 (1995) 24-34). The identity of nt 995-3080 was therefore consistent with that of the intron 2 of TRP-2. The sequence downstream nt 3,347 of cDNA 131 presented a 5' donor splice site sequence identical to that of the intron 4 of TRP-2 (Sturm et al., Genomics 29 (1995) 24-34). Since it lacks the 3'acceptor splice site sequence and its length is considerably shorter than that estimated from the published genomic map, this is the sequence of intron 4 of TRP-2, truncated at nt 200. Thus, cDNA 131 is composed of a partially spliced form of the melanocyte differentiation antigen TRP-2 containing exon 1-4 with retention of intron 2 and of the initial portion of intron 4 (Fig. 5).

DNA sequencing and homology search DNA sequencing analysis was performed by specific priming with synthetic oligonucleotides. The sequencing reactions were performed by the dideoxy-chain termination method using dye-labeled dideoxynucleotides and the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Foster City, CA). DNA sequence of the plasmid clone cDNA 131 was determined with an automated DNA sequencer (ABI Prism 377 DNA Sequencer; Perkin Elmer). The computer search for the sequence homology was done with the program FASTA EMBL-Heidelberg.

Identification of the sequence encoding the antigenic peptide recognized by CTL 128 To localize the sequence coding for the antigenic peptide recognized by CTL 128, cDNA 131 was digested with Hindis and three subfragments of 1500,200 and 2000 bp, respectively (5'-end-1500,200 bp and 3-end in Fig. 5) were obtained.

Production of subfragments of cDNA 131 Subfragments of cDNA 131 (5'-end-1500,5'-end-800,200 bp and 3'-end) were obtained by digestion of the plasmid with HindE and BstUI. After purification on agarose gel, the fragments were cloned into the pcDNAI plasmid. From the 5'-end-1500, the smaller fragments INT-2-434, INT-2-166 and INT-2-107 were generated by PCR amplification.

The sense primer KS-INT2 (SEQ ID NO: 5) was used for amplification of all the fragments.

This primer generates an ATG start codon (underlined), with an appropriated Kozak consensus sequence, in the same frame as TRP-2. The anti-sense primer used for amplification of the INT-2-434,-166 and-107 fragments respectively were SP6 (SEQ ID NO: 8) located in the pcDNAI plasmid, INT2-asl (SEQ ID NO: 6) and INT2-as2 (SEQ ID NO: 7), located in intron 2 of TRP-2. The anti-sense primers INT2-asl and-as2 contained a XhoI restriction site for directional cloning. To facilitate ligations, we took avantage of the presence of a single 3'A-overhang, due to the terminal transferase activity of the DNA polymerase, at the 5'-end of the DynazymeTM-amplified fragments. The pcDNAI plasmid was digested with EcoRV and a single thymidine was then added at the 3'end of each fragment by incubation with DynazymeTM in the presence of 2 mM dTTP, as described by Marchuk et al., Nucl. Acids Res. 19 (1991) 1154. The T-vector as well as the PCR products were digested with XhoI, purified on agarose gel and ligated. After ligation the plasmids were electroporated into DH-5 E. coli and selected with ampicillin (50 mg/mI). Clones were isolated, plasmid DNA was extracted and transfected into Cos-7 cells along with the HLA-A*6801 gene.

At this step the presence in the two 5'-end fragments of start codons regulating their translation was not investigated. The level of TNF released by CTL 128 in the presence of Cos-7 cells transfected with the 5'-end-1500 fragment (SEQ ID NO: 12) was comparable to that stimulated by cDNA 131 (Fig. 5), indicating that the antigenic peptide was encoded within this region. The nucleotide sequence of this subfragment, which encompassed exon 1, exon 2 and the first 410 bp of intron 2, is shown in SEQ ID NO: 12. CTL 128 was not stimulated by Cos-7 cells cotransfected with both the fully spliced TRP-2 cDNA and HLA- A*6801 (Fig. 5). This shows that the sequence coding for the antigen recognized by CTL 128 could be entirely or partially located in the intronic portion of the TRP-2 gene present in cDNA 131. This notion received support by lack of recognition of Cos-7 cells transfected with the 5'-end-800 cDNA fragment (Fig. 5), derived by truncation of the 5'- end-1500 fragment at a BstUI site and comprising exon 1 and half of exon 2. The intronic localization of the sequence encoding the antigenic peptide was confirmed by the ability of a PCR amplified fragment, encompassing the first 434 bp of intron 2 (INT-2-434), to convey the expression of the antigen (Fig. 5). In the same reading frame of the previous

exons of this region, there was observed the presence of a sequence that coded for a decapeptide (EVISCKLIKR) (SEQ ID NO: 2) which possessed anchor residues (position 2, 9 and 10) corresponding to the HLA-A*6801 peptide binding motif (Rammensee et al., Immunogenetics 41 (1995) 178-228).

To further define the region containing the epitope, PCR fragments were amplified using the sense primer KS-INT2 (SEQ ID NO: 5), which generates an ATG that has the appropriate Kozak consensus sequence and the reverse primers INT2-asl (SEQ ID NO: 6) or INT2-as2 (SEQ ID NO: 7). The first fragment (INT-2-166) includes the entire putative peptide encoding sequence that is partially deleted in the second one (INT-2-107). Only fragment INT-2-166, containing the intact putative peptide sequence was able to stimulate TNF release by CTL 128 (Fig. 5). Both 10-mer and 9-mer peptides, EVISCKLIKR (SEQ Nô: 2) and EVISCKLIK (the 9-mer peptide differs from the 10-mer peptide (SEQ ID NO: 2) by the deletion of the last peptide (R)), were then synthesized and incubated with the HLA-A*6801 homozygous LCL line LB. Decapeptide EVISCKLIKR was able to sensitize LB cells to lysis by CTL 128, with half-maximum lysis obtained at a concentration of about 100 pM, whereas the nonapeptide had a very low efficiency and a control peptide was negative (Fig. 6).

To exclude that the epitope recognized by CTL 128 may be generated by a mutation occurred in the tumor, a 2152 bp fragment, spanning the entire intron 2 (nt 995-3080 in cDNA 131), was amplified by PCR from genomic DNA of CTL 128 and from a different melanoma, MZ2-mel.

Example 3 Cloning of TRP-2-INT2 from genomic DNA Genomic DNA was purified from MZ2 melanoma cells and CTL 128, by use of the QIAamp blood kit (QIAGEN, Hilden, Germany). Intron 2 of TRP-2 gene was amplified by PCR from 100 ng of genomic DNA with KS-INT2 (SEQ ID NO: 5), located in exon 2 as <BR> <BR> <BR> sense primer and PR2 (SEQ ID NO: 9), located in exon 3. as anti-sense primer. TRP-2- INT2 fragments were cloned into the pcDNAI vector as described above, sequenced and transfected into Cos-7 cells together with the HLA-A*6801 gene.

The PCR products were cloned, sequenced and transfected into Cos-7 cells. All clones had the expected sequences and were able to transfer the expression of the antigen.

An RT-PCR analysis, performed with the sense primer PRIT-1 (SEQ ID NO: 3) and the anti-sense primer INT2-1260 (SEQ ID NO: 4), located in exon 1 and intron 2, amplifies a 977 bp fragment only from TRP 2 transcripts that retain intron 2.

Example 4 PCR analysis for the expression of TRP-2-INT2 and TRP2 Total RNA was prepared by the guanidine-isothiocyanate method, using RNAzoI B, from cultured cell lines, fresh skin, retina and tumor samples. cDNA corresponding to 300 ng of total RNA was amplified by PCR using 1 U of DynazymeTM (Finnzymes) and 1 mM of each primer, in a final volume of 50 ml. Reaction mixtures were subjected to 30 amplification cycles. For amplification of TRP-2 cDNA, PR3 (SEQ ID NO: 10) located in exon 2 and TRP-2L (SEQ ID NO: 11), located in exon 8, were used as sense and anti-sense primers respectively. PCR were performed for 30 cycles (1 min at 94°C, 1 min at 58°C and 1 min at 72°C). To verify the expression of the unspliced intron 2, there were used the sense primer PRIT-1 (SEQ ID NO: 3) and the anti-sense primer INT2-1260 (SEQ ID NO: 4), located in the 5'-UTR and in intron 2, respectively. PCR were performed for 30 cycles (1 min at 94°C, 1 min at 55°C and 1 min at 72°C). Amplification from contaminating genomic DNA was avoided by localization of the primers in distant exons for detection of TRP-2 and by the presence of a 10 kb long intron 1 (Sturm et al., Genomics 29 (1995) 24-34) between exon 1 and exon 2, for the amplification of TRP-2- INT2. The quality of RNA preparations was checked by PCR amplification of-actin cDNA with specific primers.

Expression of the completely spliced TRP-2 messenger was detected with the sense primer PR3 (SEQ ID NO: 10), and the anti-sense primer TRP-2L (SEQ ID NO: 11), located in exon 2 and 8, respectively.

Among the tumors tested only melanomas proved positive for TRP-2 and TRP-2-INT2 antigens (Table 1). Expression of TRP-2-INT2 in the absence of TRP-2 was never observed. This result is in agreement with the notion that TRP-2 is a melanocytic differentiation antigen (Wang et al., J. Exp. Med. 184 (1996) 2207-2216) and that the same promoter drives the synthesis of a common messenger from which both the antigens arise.

69% of fresh melanoma samples analyzed expressed TRP-2 with 78% of the TRP-2+ melanomas also expressing TRP-2-1NT2 (Table 1). This was also observed in melanoma cell lines, where the normal form of TRP-2 and the one retaining intron 2 are expressed in 84% and in 68% of the analyzed samples, respectively. Normal tissues in which TRP 2 is

known to be present were analyzed for TRP-2-INT2 expression. Three melanocytes cell lines, four skin samples and one retina were negative in the RT-PCR assay (Table 1). The four skin samples were analyzed in comparison with bioptic primary lesions derived from the same patient and whereas TRP-2 was detected in all samples, TRP-2-INT2 was exclusively present in tumor samples.

Table 1 Expression of TRP-2-INT2 and TRP-2 in tumors and normal tissues Number of samples with antigen expression/ number of samples tested TRP-2-INT2 TRP-2 Melanoma cell lines 13/19 (68%) * 16/19 (84%) Melanocyte cell lines 0/3 3/3 Fresh melanoma samples 7/13 (54%) * 9/13 (69%) Skins 0/4 4/4 Retina 0/1 1/1 Tumors (non melanomas) 0/3 0/3 *TRP-2-INT2 expression was detected only in samples positive for TRP-2.

A strong correlation was found between expression of TRP-2-INT2 mRNA in melanoma lines and their ability to stimulate TNF release by CTL 128 (Fig. 7).

Presentation of the antigenic peptide by alleles of the HLA-A3-like Supertype HLA-A*6801, along with A3, All; A31 and A*3301, belongs to an A3-like supertype of HLA-A alleles with similar peptide-binding characteristics (Sidney J. M. et al., Immunol.

154 (1995) 247-259). To investigate whether the peptide EVISCKLIR (seq ID NO: 2) can be presented to CTL 128 by HLA-A*6802 alleles of the A3-like supertype, EBV LCL expressing such alleles were used as targets for CTL128 after pulsing with the peptide (Fig.

7).

The analysis also included the HLA-A*6802 allele, a subtype of HLA-A28 belonging to the A2-like supertype. Among the A3-like supertype alleles only A*3301 was able to present the TRP-2-INT222-223 peptide with the same efficiency as A*6801. A low level of recognition, at the higher concentration, was observed when the peptide was presented by A*6802.

Example 5 Antigenic peptide and CTL assay Peptides were synthesized on solid phase using Fmoc for transient NH2-terminal protection and characterized by mass spectrometry (Primm, Milan, Italy). All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were dissolved at 10 mM concentration in 10% DMSO and stored at-80°C. Peptides were tested in an assay, where 5iCr-labeled target cells were incubated for 1 h at room temperature in 96-wells microplates with various concentration of the peptide before addition of CTL 128 at an effector/target ratio of 20: 1. Lysis was measured 4 h later. Presentation of the antigenic peptides was also tested in a TNF-release assay. Briefly, stimulator cells were incubated for 1 h at room temperature with a fixed concentration of peptides; following extensive washing, CTL 128 was added and the TNF release was evaluated 18-20 h later on WEHI- 164.13 cells.

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