CN1278865A - Tumor-specific antigens, methods for their production and their use for immunization and diagnosis - Google Patents

Abstract

A tumor-specific polypeptidic antigen, which is coded at least 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 antigens 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, is useful for diagnosis and therapeutic use in connection with tumor diseases.

Description

Tumor-specific antigens, methods for their preparation and their use in immunization and diagnosis

The present invention relates to novel tumor-specific antigens, methods for their preparation, and their role in immunization, especially in the activation of cytotoxic, tumor-specific T-lymphocytes, and to the presentation of said tumor-specific antigens in MHC class I complexes Antigen-specific diagnostic applications of tumor cells.

The immune system plays an important role in immune surveillance against cancer and tumor regression. Antitumor immune responses can be mediated by B and T cells, which recognize tumor antigens expressed on tumor cells. Generation of cytotoxic T lymphocytes (CTLs) from peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TILs) from the last years of cancer patients allowed the assessment of the role of T cells in tumor regression in humans.

Adoptive transfer of tumor-infiltrating lymphocytes and interleukin 2 (IL-2) into cancer patients themselves can mediate regression of human tumors (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 the process of tumor rejection in vivo. The ability to mediate tumor regression in vivo correlates with the ability of TILs to mediate specific lysis and cytokine release when co-cultured with syngeneic tumors in vitro (Barth et al., J. Exp. Med. 173 (1991) 647-658) .

In an effort to understand the molecular basis of T cell-mediated antitumor responses, a variety of genes encoding tumor antigens recognized by T cells have been identified (Boon et al., Annual Review of Immunology 12 (1994) 337-365; Houghton, J. Experimental Med. 180 (1994) 1-4; Tsomides and Eisen, Proceedings of the American Academy of Sciences 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 categories:

Class I tumor antigens include those common to melanoma and other tumors of various histotypes, but absent from normal tissues other than testis and placenta, (e.g., MAGE, BAGE, and GAGE) (Vander Bruggen et al., Science 254 (1991) 1643-1647; Boel et al., Immunity 2 (1995) 167-175; Van der Bruggen et al., J. Experimental Med. 182 (1995) 689-698). Clinical trials based on the use of antigens recognized by CTLs defined by different HLA class I alleles are ongoing (Marchand et al., Int. J. Cancer 63 (1995) 883-885; Rosenberg, Immunology Today (1997) 175-182) , using patients with melanoma and other neoplastic diseases. Taking into account the frequency of expression of these antigens and HLA class I alleles, more than 60% of Caucasian melanoma patients, 40% of head and neck, and 28% of bladder cancer patients are candidates for at least one of these classes. immunization against antigens. No side effects were detected in the tested patients. Indeed, MAGE, BAGE and GAGE gene expression normally occurs in testes like spermatogonia and spermatocytes (II), which do not express the classical MHC class I molecules required for antigen presentation (Haas et al., Am. J. Reprod. Immunol. Microbio. 18 (1988) 47-57), 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 melanocytic lineage. Tyrosinase from melanoma and normal cultured melanocytes (Brichard et al., J. Experimental Med. 178 (1993) 489-49513), MelanA ^Martl (Coulie et al., J. Experimental Med. 180 (1994) 35-42; Castelli et al. , J. Experimental Med. 181 (1995) 363-368; Kawakami et al., PNAS 91 (1991) 3515-3519), gp100P ^mel7 (Bakker et al., J. Experimental Med. 179 (1994) 1005-1009; Kawakami et al., NAS Proc. 91 (1994) 6458-6462), gp75 ^TRP1 (Wang et al., J. Experimental Med. 181 (1995) 799-804) and TRP-2 (Wang et al., J. Experimental Med. 184 (1996) 2207-2216 ) CTLs recognize antigens The determinant can be amplified in vitro from many melanoma patients. Thus, a significant proportion of melanoma patients may benefit from immunotherapy that induces T cell responses against these antigens. In fact, antigens of differentiation are expressed in almost all melanomas, and most of them are presented to immune effectors through the HLA-A2 allele, which is present at extremely high frequency in all ethnic groups middle. However, the side effects of cross-reactivity against normal tissues (ie, skin melanocytes and pigmented retinal cells) in these treatments must be carefully considered.

A third class of tumor antigens includes antigens expressed only by tumor cells that have been isolated. These antigens are not expressed in otherwise normal or tumor tissues of different origin, and the epitopes are usually generated by point mutations in ubiquitously expressed proteins. Tumor antigens belonging to this group have been identified in murine systems (Boon et al., Annual Review of Immunoblood 12 (1994) 337-365) and in some human tumors (Wolfel et al., Science 269 (1995) 1281-1284; Coulie et al., American Academy of Sciences Described in Proc. 92 (1995) 7976-7980; Robbins et al., J. Experimental Med. 183 (1996) 1185-1192). The lack of natural tolerance to these antigens allows a strong immune response to be induced while avoiding strong autoimmune responses. However, until a series of widespread hotspot mutations are discovered, the clinical utility of these antigens will be limited to a few or a few patients whose tumors carry a given mutation (Wolfel et al., Science 269 (1995) 1281-1284) .

A fourth class of tumor antigens arises from differential processing of transcripts. Robbin et al., J. Immunoblood 159 (1997) 303-308 describe a gp100 transcript that corresponds to part of the fourth intron and encodes an additional 35 amino acids not present in the normal gp100 glycoprotein. However, this epitope is only expressed at low levels in melanoma. Robbins et al. in Immunoblood 154 (1995) 5944-5950 describe the cloning of a gene encoding an antigen recognized by lymphocytes, tumor-infiltrating lymphocytes defined by melanoma-specific HLA-A24. The antigen is a fragment of a full-length clone, not encoded by an intron or part thereof.

Fujii et al., J. Immunol. 153 (1994) 5516-5524 describe a soluble form of the non-tumoricidal HLA-G antigen encoded by intron 4-containing mRNA. However, the specific function of this soluble HLA-G protein is not yet known.

The existence of a fifth class of tumor antigens has recently been proposed based on the reactive profile of a series of CTL clones that recognize autologous and HLA-matched allogeneic melanoma, but not melanocytes or other targets of different tissue origin ( Anichini et al., J. Immunol. 156 (1996) 208-217).

An object of the present invention is to provide novel tumor-specific antigens which are not expressed in normal cells and which can specifically distinguish tumor cells from normal cells.

Summary of the invention

The present invention comprises a tumor-specific polypeptide having an antigenic effect, characterized in that it is partly encoded by an intron sequence of a gene derived from a polypeptide presented on tumor cells by an MHC class I complex ( an exon-encoded tumor antigen) and can be obtained by reverse-transcriptase PCR from an mRNA isolated from the soluble cytoplasm of tumor cells in which an exon-encoded tumor antigen is associated under stringent conditions Nucleic acid fragments to which the intronic sequences of the antigen hybridize are used as primers; and, if the PCR product is obtained by isolation of the PCR product, expression in a host cell, and isolation of the tumor-specific antigen encoded by the PCR product.

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

Another object of the present invention is a method for identifying such tumor-specific polypeptides with antigenic effects, comprising the following steps:

- performing reverse transcriptase PCR from mRNA of a tumor cell, wherein a nucleic acid fragment hybridizing under stringent conditions to an intronic sequence of an exon-encoded tumor antigen is used as a primer;

- If a PCR product is obtained: Isolate the PCR product, express it in a host cell, and isolate the tumor-specific antigen encoded by the PCR product, which also hybridizes to the exon sequence of said antigen.

After isolation, the hybridization products or fragments thereof are inserted into expression vectors, which are transferred into appropriate host cells and expressed in said host cells. Subsequently, the resulting recombinant polypeptide is isolated.

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

Thus, the subject of the present invention is a tumor-specific polypeptide antigen partially encoded by the introns of an exon-coded tumor antigen and obtained by:

a) mRNA isolated from the soluble cytoplasmic fraction of a tumor cell was subjected to reverse transcriptase PCR in which nucleic acid fragments hybridizing under stringent conditions to the intronic sequence of an exon-encoded tumor antigen were used as primers ;

b) isolating said PCR product, expressing said PCR product or a fragment thereof in a host cell, and isolating a tumor-specific antigen encoded by said PCR product or a fragment thereof, which is also associated with an exon of said antigen sequence hybridization.

Another subject of the invention is the tumor-specific polypeptide antigen of the invention, wherein a fragment of said PCR product of 8 to 12 codons is used for expression.

Another subject of the present invention is the tumor-specific antigen of the present invention, wherein said exon-coded tumor antigen is a CTL recognition antigen class of MAGE, BAGE and GAGE, derived from tyrosinase, MelanA ^Martl , CTLs of gp100 ^Pmel7 , gp75 ^TRP1 or TRP-2 recognize epitopes.

The present invention also relates to a tumor-specific polypeptide antigen encoded by SEQ ID NO:1.

Yet another subject of the invention is a method for isolating the mRNA of a tumor-specific antigen encoded by an intron of an exon-encoded tumor antigen, comprising the following steps:

a) reverse transcriptase PCR of mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, in which nucleic acid fragments that hybridize under stringent conditions to an intronic sequence of an exon-encoded tumor antigen are used as primers ;and

b) isolating said PCR product which also hybridizes to the exon sequence of said antigen.

The present invention also relates to a method for determining the proliferation of tumor-specific cytotoxic T-cells, wherein a tumor-specific antigen of the present invention is added to a sample of a patient's body fluid, which contains antigen-presenting cells and cytotoxic T-cells, Proliferation of cytotoxic T cells is measured, preferably by release of cytokines (measurement of cytokines such as TNF, IFNy, GM-CSF).

Another subject of the invention is the use of nucleic acids encoding tumor-specific antigens of the invention for the preparation of medicaments for the treatment of neoplastic diseases.

The invention also relates to the use of the tumor-specific antigens of the invention in vivo or in vivo to activate cytotoxic T cells from T precursor cells.

Another subject of the present invention is a method for preparing tumor-specific polypeptide antigens, wherein said tumor-specific antigens are obtained by the following steps:

a) mRNA isolated from the soluble cytoplasmic fraction of a tumor cell was subjected to reverse transcriptase PCR in which nucleic acid fragments hybridizing under stringent conditions to the intronic sequence of an exon-encoded tumor antigen were used as primers ;

b) isolating said PCR product, expressing said PCR product or a fragment thereof in a host cell, and isolating a tumor-specific antigen encoded by said PCR product or a fragment thereof, which is also associated with an exon of said antigen sequence hybridization.

Yet another subject of the invention is the combination of two nucleic acids which hybridize under stringent conditions to the intronic sequence of an exon-coded tumor antigen and which can be used as primers for reverse transcriptase PCR from mRNA right.

Yet another subject of the invention is the nucleic acid encoded by SEQ ID NO: 3 to SEQ ID NO: 9.

The tumor antigens of the present invention are not found on the surface of normal cells such as melanocytes. However, it is present on more than 80% of eg melanoma cells and is thus specific for tumor cells.

Thus, the peptide is particularly suitable for tumor cell-specific immunity in patients and can also be used for the diagnosis of differentiation of melanoma cells and normal melanocytes.

A cytolytic T lymphocyte clone (CTL 128) derived from peripheral blood lymphocytes of patients with metastatic melanoma was able to lyse autologous tumors and several allogeneic melanomas in an HLA A*6801-defined manner. Genes encoding antigens recognized by CTL 128 were identified by transfection of a cDNA library constructed from autologous melanoma mRNA into Cos-7 cells expressing the HLA-A*6801 allele. It was surprisingly found that, in contrast to melanocytes, splicing errors occur in mRNA maturation. This produces in translation a peptide consisting of a partially spliced form of melanocyte differentiation antigen (TRP)-2, which contains exons 1-4 and retains intron 2 and part of intron 4 (TRP-2- INT2). TRP-2-INT2 encodes a putative 238 amino acid protein using the same reading frame as TRP-2, which extends from the start codon at position 400 to the stop site in intron 2 (nucleoside acid 1113), at 18 nucleotides downstream of the peptide coding region.

The differentiation antigen TRP-2 is described in R. F． Wang, Journal of Experimental Medicine 184 (1996) 2207-2216. TRP-2 is one of the most highly expressed glycoproteins in human pigmented melanocytes and melanomas (Wang et al., J. Experimental Med. 184 (1996), 2207-2216). It is located on human chromosome 13 and has been shown to be a member of the tyrosinase-related gene family, sharing 40-45% amino acid 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 of 519 amino acids and has been shown to have DOPA chromium tautomerase activity, which is involved in the synthesis of melanin (Bouchard et al., European Journal of Biochemistry 219 (1994), 127-134).

In contrast to fully spliced TRP-2 mRNA found in melanoma, normal skin melanocytes, and retina as described by Wang et al., TRP-2-INT2 mRNA was only detected in melanoma. These results suggest that melanoma antigens recognized by CTLs may be derived from known lineage-associated proteins through differential splicing that occurs in tumors but not in normal cells. This mechanism generates a new set of antigens that can be considered to be truly tumor-specific and present only in tumors of the same tissue type, but not in normal tissues of the same kind. This novel group of antigens is thus characterized by its derivation from known lineage-associated proteins by tumor-specific and alternative splicing that is not present in normal cells. Normal melanocytes, skin samples and retinas were confirmed negative in reverse transcriptase (RT) PCR analysis. These features define an antigen that can be used to develop safer and more effective immunotherapeutic regimens.

Thus, the present invention relates to partially intron-encoded tumor-specific antigens obtained by reverse transcriptase PCR of mRNA isolated from the soluble cytoplasmic fraction of tumor cells. These antigens are recognized by specific T lymphocytes which then lyse tumor cells presenting the antigens of the invention in MHC class I complexes. Surprisingly, it was found that the mRNAs encoding the antigens of the invention enriched in the cytoplasm of such tumor cells are tumor-specific.

"Intron-encoded tumor-specific antigen" refers to a tumor antigen that consists not only of an exon sequence but also of a portion (preferably about or greater than 30%, more preferably about or greater than 50%, most preferably about or greater than 80%) intronic sequences, and it specifically recognizes tumor antigens presented by MHC class I. Expression of introns is associated with altered splicing mechanisms in strain-associated proteins. "Partially" means preferably 30-50% or 30-80% intronic coding. The exon-encoded and intron-encoded sequences of the antigens of the invention are directly linked in the relevant genomic sequence, since said antigens result from different splicing.

"Exon-encoded antigen" refers to the antigens described in the introduction, eg, MAGE, BAGE and GAGE (Van der Bruggen et al., Science 254 (1991) 1643-1647; Boel et al., Immunity 2 (1995) 167-175 ; Van der Bmggen et al., Journal of Experimental Medicine 182 (1995) 689-698) or, for example, CTL recognition epitopes from tyrosinase (Brichard et al., Journal of Experimental Medicine 178 (1993) 489-49513), MelanA ^Martl ( Coulie et al., J. Experimental Med. 180 (1994) 35-42; Castelli et al., J. Experimental Med. 181 (1995) 363-368; Kawakami et al., PNAS 91 (1991) 3515-3519), gp100 ^Pmel7 (Bakker et al. Journal of Experimental Medicine 179 (1994) 1005-1009; Kawakami et al., Proceedings of the American Academy of Sciences 91 (1994) 6458-6462), gp75 ^TRP1 (Wang et al., Journal of Experimental Medicine 181 (1995) 799-804) and TRP-2 (Wang et al., Journal of Experimental Medicine 184 (1996) 2207-2216).

"Peptide or polypeptide of the invention" refers to a polypeptide which preferably consists of 8 to 12 amino acids, most preferably at least 10 amino acids, but may contain the entirety of a protein. The peptide can also be part of a protein, such as a fusion protein.

"Polypeptide having an antigenic effect" refers to a polypeptide that elicits a specific immune response in vivo and in vitro.

The term "hybridizes under stringent conditions" means that two nucleic acid fragments can be expressed in the same manner as 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 under standard hybridization conditions.

More specifically, "stringent conditions" as used herein refers to hybridization in 6.0xSSC at about 45°C, followed by washing in 2.0xSSC at 50°C. To select for stringency, the salt concentration in the wash step can be selected, for example, from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature during the washing steps can range from low stringency conditions at room temperature, about 22°C, to high stringency conditions, about 65°C.

For expression in prokaryotic or eukaryotic organisms, eg prokaryotic host cells or eukaryotic host cells, the nucleic acid sequence is incorporated into an appropriate expression vector using methods familiar to those of ordinary skill in the art. Such expression vectors preferably contain a regulatable/inducible promoter. These recombinant vectors are then introduced into appropriate host cells for expression, such as Escherichia coli as a prokaryotic host cell, or Saccharomyces cerevisiae, CHO or COS cells as a eukaryotic host cell, and the transformed or transduced host cells are cultured in a permissible Conditions under which heterologous genes can be expressed. Isolation of the peptide can be carried out from the host cell or from the culture supernatant of the host cell according to a known method. These methods are described, for example, in Ausubel I, Frederick M, Current Protocols in Mol Biol, (1992) John Wiley and Sons, New York.

"Host cell" refers to prokaryotic and eukaryotic cells, preferably COS or CHO cells. Production in prokaryotic cells is preferred, as demonstrated by glycation to be less critical for protein activity.

Genes encoding tumor-specific antigens recognized by specific cytotoxic T lymphocytes (CTLs) can be identified by transfection of a cDNA library constructed from autologous tumor mRNA into eukaryotic cells, preferably expressing the patient's Appropriate HLA alleles.

In order to determine the HLA antigens, the total RNA is isolated from the patient's tumor cells or tumor tissues, and cDNA is obtained by reverse transcriptase PCR, using primers that specifically encode HLA antigens, and cloned into eukaryotic cell expression vectors, such as pcDNA3 (Invitrogen Corporation, Oxon, UK). In order to determine the tumor-specific antigen encoded by the intron of the present invention, the mRNA is isolated from the soluble cytoplasmic fraction of the tumor cells to isolate the poly A+ RNA, and the cDNA library is established by reverse transcriptase PCR, and the primers used are specific coding exome Sub-encoded tumor antigens, eg, MAGE, BAGE and GAGE, epitopes from CTL recognition of tyrosinase, MelanA ^Martl , gp100, gp75 ^TRP1 and TRP-2. The cDNA fragments from the cDNA library are cloned into a eukaryotic expression vector, eg, pcDNA3.1 (Invitrogen Corporation, Oxon, UK). After the expression vector is co-transfected into eukaryotic cells, such as Coa-7 cells (expressing the relevant MHC gene, which can present a peptide fragment of the antigen that can activate specific tumor antigen T cells), one can then measure the effect on tumor Those clones that are stimulated by specific cytotoxic T lymphocytes. The stimulating effect on tumor-specific cytotoxic T lymphocytes can be determined by CTL stimulation assay (measurement of TNF-α, IFN-γ, GM-CSF), such as Traversari et al., Immunogenetics 35 (1992), 145-152 stated.

In this way it is possible to obtain cell clones which specifically activate those cytotoxic T cells which cause lysis of the corresponding tumor cells.

This cell clone expresses a polypeptide which can be administered to a patient in an isolated and purified form for immunization/vaccination, or as a full-length polypeptide, since the antigenic polypeptide of the present invention is processed in vivo, Alternatively, it may be administered in the form of a truncated polypeptide fragment.

To this end, nucleic acids encoding polypeptides of the invention are isolated from such cell clones according to established methods (Sambrook et al., Molecular Cloning (1989), Cold Spring Harbor Laboratory Press) and carried out by restriction digestion. truncate. These truncated fragments are then transfected into eukaryotic cells, e.g., COS-7 cells (supra), after cloning into eukaryotic expression vectors, and the stimulation of cytotoxic T cells is then measured in a CTL stimulation assay, such as Traversari et al., Immunogenetics 35 (1992) 145-152. Nucleic acids encoding in this manner can be restricted to peptide epitopes necessary for the stimulation of cytotoxic T cells.

In a preferred embodiment, the nucleic acid sequence encoding the protein is coupled to a nucleic acid sequence which enhances the processing of the protein in the host cell.

At the same time, some sequences, such as ubiquitin, are known to improve protein transport, protein degradation and import into MHC class I complex (Bachmair et al., Science 234 (1986) 179-186; Gonda et al., Biochem. J. 264 (1989 ) 16700-16712; Bachmair et al., Cell 56 (1989) 1019-1032). By coupling the protein containing the antigenic polypeptide of the present invention to ubiquitin, the degradation of the protein can be accelerated and the introduction into the MHC class I complex can be more efficient. As a result, the presentation of this peptide on the surface of tumor cells was particularly effective. In this regard, the amino acids between the containing antigenic peptide and ubiquitin are particularly important in the conjugation of the protein containing the antigenic peptide to ubiquitin. By selecting appropriate amino acids, one can additionally obtain a considerable improvement in the recognition of melanoma cells by specific cytotoxic T cells.

In a preferred embodiment a fragment of 8 to 12 codons of a PCR product is used.

In another preferred embodiment, a partially intron-encoded tumor-specific antigen TRP-2-INT2 (hereinafter referred to as TRP-2-INT2 fragment or TRP-2-INT2 peptide), which consists of Recognized by specific T lymphocytes, they lyse those melanoma cells presenting the polypeptide EVISCKLIKR in the MHC class I complex and whose amino acid sequence is encoded by the DNA sequence shown in SEQ ID NO:2.

To obtain the coding sequence for the tumor-specific antigen of the TRP-2-INT2 peptide, cDNA encoding the HLA-A*6801 allele and the TRP-2-INT2 antigen was isolated from the cytoplasm of a melanoma cell line that was Established from metastatic lesions obtained from a surgical sample of a patient who underwent surgery at the Istituto Nazionale Tumori (Milan, Italy). After the two cDNAs were transfected into COS-7 cells, one clone (TRP-2-INT2, HLA-A*6801) was isolated, which could stimulate the cytotoxic effect of cytotoxic T lymphocytes (CTL128). DNA sequence analysis revealed an exon-encoded cDNA fragment of the TRP-2 antigen, which contained exons 1-4 and retained intron 2 and part of intron 4 (TRP-2-INT2). Subfragments of the TRP-2-INT2 fragment were obtained by digesting the TRP-2-INT2 DNA with restriction enzymes according to methods known to those of ordinary skill, Sambrook et al., "Expression of Cloned Genes in E. coli" Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York, USA, 5.3-5.10, or by PCR amplification with fragment-specific primers (SEQ ID NO: 6, 7, 8). After transfection into COS-7 cells, the CTL-stimulatory effect was determined by the CTL stimulation assay as described by Traversari et al., Immunogenetics 35 (1992) 145-152. The sequence of the encoding nucleic acid is determined by DNA sequence analysis.

Another subject of the invention is the use of tumor cells presenting the antigens of the invention via MHC class I.

Another subject of the invention is a method for detecting the expression of an antigen of the invention from a body fluid of a patient, preferably a blood or tissue sample. For this purpose, tumor cells expressing an antigen of the invention are diagnosed before, during, and after treatment using a tumor-specific cDNA encoded by an intron encoding the antigen of the invention.

Before treatment, it is determined whether the tumor antigen of the present invention is expressed in the patient's tumor cells, because the treatment can only be carried out if the tumor actually expresses the antigen of the present invention. During the course of treatment, one can monitor whether the expression of the antigen of the invention as a marker of the presence of tumor cells is reduced, and after treatment, the determination of the expression of the antigen of the invention allows one to monitor whether the tumor cells have maximally decreased.

In a preferred embodiment, a nucleic acid encoding a tumor-specific TRP-2-INT2 peptide is used to diagnose melanoma cells presenting the TRP-2-INT2 peptide on their surface.

Sequence-specific expression of the TRP-2-INT2 peptide in melanoma. Thus, the TRP-2-INT2 sequence can be used as a sample to identify tumor cells. Identification can be by PCR or labeled hybridized samples or by various nucleic acid probe-based tests known in the art.

The expression of TRP-2-INT2 was determined at the mRNA level by reverse transcription PCR (RT-PCR) and by hybridization with a specifically labeled TRP-2-INT2 probe.

In order to differentiate from TRP-2 mRNA, the specificity of TRP-2-INT2 mRNA is determined by:

1) transcribed into cDNA;

2) using intron-specific primers;

3) Compare the lengths of the amplified cDNA fragments (use cDNA exon to exon to determine the length of the amplified

Increased cDNA exon-intron-exon fragment length);

4) Hybridization with intron-specific probes.

Another subject of the invention is a method for determining the proliferation of tumor-specific cytotoxic T lymphocytes. This method is used to detect cytotoxic T cells that can be activated by the antigens of the invention before or during treatment. Patients who already have cytotoxic T lymphocytes that can be activated by the antigens of the invention can be selected prior to treatment.

To test whether a patient has tumor-specifically activatable T cells, T cells and antigen-presenting cells are isolated from the patient's blood, and the antigens of the invention are contacted ("pulsed") with the antigen-presenting cells in vitro. If T cells that can be activated by the antigens of the invention are present in the patient's blood, specific cytotoxic L lymphocytes proliferate, which can be detected, for example, by a CTL stimulation test. Such activatable T cells can be used therapeutically after stimulation with the antigens of the invention. This diagnostic process must be performed prior to treatment.

During treatment, another diagnostic procedure is used as a control for the formation of cytotoxic L lymphocytes, making it possible to quantify the induction of tumor-specific T cell activation.

Proliferation of cytotoxic T lymphocytes can be determined, for example, by a CTL stimulation assay. Stimulator cell lines were assayed for their ability to induce TNF production by CTL as described by Traversari et al., Immunogenetics 35 (1992) 145-152. TNF content was determined by detecting its cytotoxic effect on WEHI-164.13 cells in an MTT chromogenic assay (CTL stimulation test) (Espevic and Nissen-Meyer, Journal of Immunological Methods 95 (1986) 99- 105).

In a preferred embodiment, TRP-2-INT2 peptides are used to measure the proliferation of TRP-2-INT2 specific cytotoxic T lymphocytes.

Another subject of the invention is the use of nucleic acids encoding the intron-specific antigens of the invention for the preparation of medicaments for the treatment of neoplastic diseases.

In this regard, the nucleic acids of the invention may be used in gene therapy. This nucleic acid is introduced into the patient by means of a viral or non-viral vector, wherein the coding sequence should be specifically expressed and binding of the peptides of the invention by means of antigen-presenting cells should generate a specific cytotoxic T cell response. The resulting immune response should be directed against all tumor cells expressing the peptides of the invention on their cell surface. The DNA sequence encoded on the vector can be administered subcutaneously, intramuscularly, or intratumorally in the form of naked DNA, in combination with liposomes, or with appropriate adjuvants (known in the art).

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

In another embodiment, the peptides of the invention can be used for immunization and/or vaccination of tumor patients. This immunization is based on the activation of specific cytotoxic T cells by presentation of antigens of the invention by antigen presenting cells. Immunization can be performed in vitro or in vivo.

In this procedure, antigen-presenting cells (giant cells, dendritic cells, or B cells) and T lymphocytes are isolated from the patient's blood and contacted ("pulsed") with the antigen of the invention ex vivo. These antigen-presenting cells equipped with the peptides of the invention are brought about in this way to activate specific cytotoxic T cells and then infused back into the patient's blood.

The immunization process can be carried out in vivo by subcutaneously administering the polypeptides of the invention to the patient, wherein the activation of specific cytotoxic T cells is achieved directly in the patient. Binding of the peptides of the invention to the corresponding HLA molecules on the surface of antigen-presenting cells results in the proliferation of specific cytotoxic T lymphocytes.

The immunogenic effect of the peptides of the present invention during use can be enhanced by:

1) Conjugated with bacterial toxin (superantigen);

2) Used in combination with Freund's adjuvant;

3) The peptide of the present invention is mixed with liposomes.

In a preferred embodiment, the TRP-2-INT2 peptide is used to immunize and vaccinate melanoma patients.

Another subject of the invention are primers for detecting the expression of specific tumor antigens by RT-PCR. In this regard, the primers can be selected by the following measures:

1) The sense and antisense primers hybridize to two different exon sequences of the tumor antigen.

2) The sense primer hybridizes to the exon sequence, and the antisense primer (oligo dT primer) hybridizes to the

The poly-A of the mRNA sequence of the tumor antigen is hybridized.

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

The following examples, references, sequence listing and figures are provided to further illustrate various aspects of embodiments of the invention without limiting the scope thereof.

Brief description of the figures: Figure 1: CTL 128 recognizes autologous melanoma (Me18732) in an HLA-A*6801-defined manner. Target cells were treated at a fixed E:T ratio (A.: Me 18732+none; B: Me 18732+HLA class I; C: Me18732+HLA-A2, -A69; D: ME 18732+HLA-A2, - A28; Me 18732+HLA-B, -C) Incubated with the indicated anti-HLA monoclonal antibodies at room temperature prior to addition of effectors. Figure 2: CTL 128 recognizes autologous melanoma Me18732 and HLA-A*6801+ melanoma cell lines. Add 1500 CTL to 25000 stimulated cells, and detect TNF content in the supernatant of WEHI-164.13 cells after 24 hours (melanoma: A: Me 18732 + none; B: Me 20842; C: Me 17697 D : Me 2559/1; E: ME 12657; F: Me 17088; G: Me 4023; H: LB-33; I: lung cancer Calu 3; K: breast cancer SKBR3; L: ovarian cancer SKOV3). Figure 3: Stimulation of CTL clone 128 by Cos-7 cells transfected with cDNA131 and HLA-A*6801 cDNA. Cos-7 cells were transfected with HLA-A*6801 and A255 or cDNA131 pools. Pool A255 is a set of 100 cDNA clones from the Me 18732 cDNA library from which plasmid DNA was extracted. cDNA131 is a single clone subcloned from the A255 pool. TNF production by CTL 128 was measured 20 hours after co-culture with transfected cells using the TNF sensitive cell line WEHI-164.13. As a control, Cos-7 cells were transfected with cDNA 131 or HLA-A*6801 alone (A: Me18732; B: COS; C: COS+A*6801; D: COS+eDNA 131; E: COS+A* 6801+cDNA 131). Figure 4: cDNA 131 encodes an antigen recognized by CTL 128. (A) Recognition and (B) cleavage of CTL 128 by a Geneticin-resistant population of the HLA-A*6801 melanoma cell line LB33, followed by transfection with the pcDNA3.1/cDNA131 construct. TNF secretion by CTL 128 was determined after 24 hours of co-culture of 1500 responder cells with 20000 stimulator cells. The lytic activity of CTL 128 was determined on ⁵¹ Cr-labeled target cells after co-culture with CTL for 4 hours at different effector:target (E/T) ratios. Figure 5: Identification of sequences encoding antigenic peptides recognized by CTL128. The exon/intron organization of cDNA131 is shown in the upper part of (A). Exons and introns are represented as solid and open boxes, respectively, the horizontal line at the end indicates the pcDNA3.1 vector, and the sequence numbering is relative to the 5' end of cDNA131. Subfragments from cDNA 131, shown as open boxes below cDNA 131, were cloned into expression vectors and transfected into Cos-7 cells together with HLA-A*6801 cDNA. Exon 8-specific oligonucleotide probes were used to screen the 18732 cDNA library to obtain the cleaved full-length form of TRP-2 cDNA. TNF release by CTL128 was assessed on WEHI164.12 (B). Peptide coding sequences present in PCR fragments INT-2-166 and INT-2-107 are indicated. Figure 6: HLA-A*6801 cells pulsed with synthetic antigenic peptides by CTL 128 lysis. ⁵¹ Cr-labeled HLA-A*6801 EBV-LCL (LB-EBV) was incubated with CTL 128 at an E/T ratio of 20:1 in the presence of the indicated concentrations of synthetic peptides shown on the left. ⁵¹ Cr-release was measured after 4 hours. As a negative control, MAGE-3 derived from an HLA-A1-binding peptide (M3A1) was used. Figure 7: Response of CTL 128 to TRP-2-INT _221- Identification of _231. ⁵¹ Cr-labeled EBV-LCL was incubated with CTL 128 at an E/T ratio of 20:1 in the presence of different concentrations of the peptide TRP-2-INT _221-231 . Chromium release was measured after 4 hours. C: negative control without peptide. Sequence description: 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 for confirming non-spliced intron TRP2-INT; located in 5'UTR of TRP2-gene.SEQ ID NO:4: for confirming (INT2-1260) antisense primer for the spliced intron TRP2-INT; located in the 5'UTR of the TRP2-gene and intron 2. SEQ ID NO: 5: Subfragment used to prepare cDNA 131 and use (KS-INT2) sense primer for cloning TRP-2-INT2 from genomic DNA; located in exon 2 of the TRP2-gene. SEQ ID NO: 6: ( INT2-as1) antisense primer; located in intron 2 of the TRP2-gene. SEQ ID NO: 7: (INT2-as2) antisense primer for the preparation of subfragment INT2-166 of cDNA 131; located in the TRP2-gene in intron 2 of . SEQ ID NO: 8: (Sp6) antisense primer for preparation of subfragment INT2-434 of cDNA 131; located in pCDNA1-plasmid. SEQ ID NO: 9: for cloning from genomic DNA (PR2) antisense primer for TRP-2-INT2; located in exon 3. SEQ ID NO: 10: (PR3) sense primer for amplifying TRP-2 DNA; located in exon 2. SEQ ID NO: 10: (PR3) sense primer for amplifying TRP-2 DNA; located in exon 2. SEQ ID NO: 10: ID NO: 11: (TPR-2L) antisense primer for amplifying TRP-2 DNA; located in exon 8. SEQ ID NO: 12: 5'-1500 fragment including the coding region of the antigenic peptide Nucleic acid sequence. The first 45 bases preceding the start of cDNA 131 and belonging to pcDNA3.1 were omitted.

Embodiment 1CTL 128 is to the recognition of HLA-A*6801 as restriction element

The melanoma cell line Me18732, established from a patient's metastatic lesion, was serologically typed to LA-A2 and HLA-A28, and then HLA-A by a sequence-specific oligonucleotide probe (SSOP) subtyping method *0201 and HLA*68011 (also known as HLA-A*6801) (Oh et al., Genome 29 (1995) 24-34). Antitumor CTL clones were obtained as described by Anichini et al., J. Immunol. 156 (1996) 208-217.

CTL clone 128 recognized autologous melanoma in the context of the HLA-A locus, as its cytolytic activity was reduced by the anti-HLA class I mAb W6/23 but not by the anti-HLA-B,-C mAb (Fig. 1). HLA-A*0201 can be excluded from the presentation molecules for antigens recognized by CTL 128, since inhibition of cleavage was only observed with anti-HLA-A2,-A28 mAb CR1 1.351, whereas with anti- HLA-A2, -A69 mAb BB7.2 was not observed (Figure 1). Thus, the inhibitory activity of CR1 1.351 revealed that A28(A*6801) is the HLA presenting molecule of CTL128. cell line culture

The melanoma cell line Me18732 was established from metastatic lesions from surgical samples of patients from the Istituto Nazionale Tumori (Milan, Italy). The patient's PBL was serotyped as; HLA-A2, -A28, -B44, -B51, -C2, -C5. Human metastatic (Me17697, Me2559/1, Me17088/1, Me4023) and primary (Me20842) melanoma cell lines were established and cultured in 10% FCS/PRMI 1640. Melanoma cell lines LB-33, LV-40 and Cos-7 (ATCC: CRL 1651 ) cell lines were maintained in 10% FCS/DMEM. Cancer cell lines CALU3, SDBR3 and SKOV3 purchased from the American Type Culture Collection (ATCC, Rockville, MD) were maintained in 10% FCS/RPMI-1640. C1RA*03301 transfectants, homozygous EBV-transformed LCL, cell line SCHU typed for HLA-A*0301, B*0702, -C7, AMA-1 typed for HLA-A*6802, B*5301, -C4, and WT-100-bis were typed as HLA-A11, -B35, -C4. EBV-LCL JHAF (HLA-A*31011, -B51, -C8) and LB (HLA-A*68011, B*40011, -C2, -C3) were obtained from ATCC. EBV-LCL was maintained in 10% FCS/RPMI-1640. Antigen specificity assessment for CTL 128

To assess the frequency of expression of antigens recognized by CTL 128 on other tumor cells, a panel of HLA-A*6801 melanocyte cell lines was tested in a CTL stimulation assay. TNF release was induced by CTL 128 in 5 of 8 melanoma cell lines (Fig. 2). Reactivity was observed in HLA-A*6801 cancer cell lines from three different tissue origins (Figure 2). HLA-A*6801-negative melanomas of other tissue types, melanocytes and tumor cell lines were not stimulated by CTL128 to release cytokines. The type of response shown by CTL 128 to the tested melanoma cell lines did not correlate with the expression of already described melanoma antigens in these cell types, as assessed by RT-PCR. This was confirmed by the absence of TNF release by CTL 128 in Cos-7 cells co-transfected with the plasmid pcDNA3/A*6801, which contains HLA-A*6801 from patient 18732, as well as the known coding common Every gene of melanoma antigen: Melan-A/MART-1, tyrosinase, gp100, TRP-1, -2, MAGE-1, 2, -3, -4, -12, BAGE-1, - 2, GAGE-1, -2, -3, -4, -5, -6. These results are consistent with the hypothesis that HLA-A*6801-defined CTL 128 recognizes a neoantigen shared by multiple tumors.

CTL clone 128 was obtained by limiting dilution of 4-week-old mixed lymphocytic tumor culture (MLTC) and cultured under conditions similar to those described previously (Anichini et al., J. Immunol. 156 (1996) 208-217) . CTL 128 expressed CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , TCR- ⁺ phenotype, which was assessed by flow cytometry with specific monoclonal antibodies. Tests 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). The result is represented as follows: Where spontaneous release is assessed by incubating target cells in the presence of effectors, maximal release is determined in the presence of 1% NP-40 detergent (BDH Biochemicals, Poole, UK). Inhibition of lysis against autologous melanoma was performed using the following monoclonal antibody that has been reported (Anichini et al., J. Immunol. 156 (1996) 208-217): Anti-HLA-A, -B, -C W6/32( Parhan et al., Journal of Immunology 123 (1979) 342-349), anti-HLA-A2, -A69 BB7.2 (Parham and Brodsky, Human Immunology 3 (1981) 277-299), anti-HLA-A2, -A28 CR11 .351 (RUsso et al., Immunogenetics 18 (1983) 23-35), and anti-HLA-B,-C4E (Yang et al., Immunogenetics 19 (1984) 217-231). Subcloning of the HLA-A*6801 allele

Using RNAzolz by the guanidine-isothiocyanate method. B (Cinna/Biotecx, SouthLoop East, TX) Total RNA was prepared from Me18732 cells. Single-stranded cDNA synthesis was performed on 2 micrograms of total RNA using oligo-dT primers and a Moloney murine leukemia virus-derived reverse transcriptase (MMLV-RT RNase-H-Superscript; GIBCO BRL, Gaithersburg, MD) without RNase-H activity Follow 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 coding region of the full-length HLA-A allele . After BamHI and HindIII digestion, the 1.1 Kb PCR product was subcloned into the BamHI/EcorRV site of the eukaryotic expression vector pcDNA3 (Invitrogen Corporation, Oxon, UK).

Plasmid clones encoding HLA-A*68011 or A*0201 (HLA-A28 and -A2 alleles of patient 18732) were identified using diagnostic restriction enzymes. The HLA-A*68011 gene was then sequenced to confirm correspondence to published DNA sequences. This plasmid was named pcDNA3/HLA-A*6801. Example 2 cDNA clones encoding melanoma antigens recognized by CTL 128

A cDNA library was constructed in an appropriate expression vector using poly(A) ⁺ RNA extracted from Me18732 cells. Poly(A) ⁺ RNA was isolated from Me18732 cells using the Fast Track mRNA extraction kit (Invitrogen). The library was constructed by converting 5 micrograms of poly(A) ⁺ RNA to cDNA using the SuperScript ChoiceSystem kit (GIBCO BRL, Gaithersburg, MD) using an oligo-dT primer containing a NotI site at the 5'-end. The cDNA was then ligated with BstXI adapter (Invitrogen) and digested with NotI. After size fractionation, the cDNA was cloned in a single orientation into the BstXI/NotI site of the mammalian expression vector pcDNA3.1 (Invitrogen). The recombinant plasmid was electroporated into DH5-E. coli and selected with ampicillin (100 mg/ml).

The library was divided into 1300 pools of approximately 100 cDNA clones. Each bacterial pool was amplified to saturation, plasmid DNA was extracted and transfected (100ng) together with pcDNA3/A*6801 (100ng) into 1.2×10 ⁴ Cos-7 cells using DEAE-dextran-chloroquine Methods (Coulie et al., J. Experimental Med. 180 (1994) 35-42; Seed and Aruffe, PNAS 84 (1987) 3365-3369). Using the same technique, in other experiments Cos-7 cells were co-transfected 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 melanin antigens: Melan A/MART-I, tyrosinase, gp100, MAGE-1, -2, -3, -4, -12, BAGE-1, -2, GAGE-1, -2, -3, -4, - 5, -6, TRP-1. The full-length TRP-2 cDNA was amplified by RT PCR, and the primers used were specific primers located in the 5'-untranslated region (UTR) and the end of exon 8. It was cloned into pcDNA3 and sequenced, confirming that it was consistent with the Identity of published cDNA sequences (Yokohama et al., Bioch. Bioph. Acta 1271 (1994) 317-321). Transfected Cos-7 cells were detected by CTL stimulation assay after 48 hours. CTL stimulation test

Transfectants or stimulated cell lines were tested for their ability to induce TNF production by CTL 128 as previously described (Traversari et al., Immunogenetics 35 (1992) 145-152). 1500 CTL were added to microwells containing target cells containing 100 l of IMDM (BioWhittaker, Walkersville, MD) and 10% pooled human serum (PHS) and 25 U/ml r-hu-IL2 (EuroCetus, Amsterdam, The Netherlands ). After 24 hours, the supernatant was collected and detected by a chromogenic assay on WEHI cells, such as WEHI-164.13 (Espevic and Nissen-Meyer, J Immunol Methods 95 (1986) 99-105). Toxic effect to determine its TNF content. WEHI-164.13 cells were maintained in 10% FCS/RPMI-1640.

Duplicate microcultures were transfected and screened two days later for their ability to stimulate TNF release by CTL128. DNA from one of the 1300 sets (set A255) induced high levels of TNF production (Figure 3), a finding that was confirmed in a second transfection experiment. Bacteria from the positive pool were cloned and their plasmids co-transfected with the HLA-A*6801 construct as previously described. 25 of 159 clones stimulated TNF release by CTL 128. The results obtained with one of them, cDNA 131, are shown in Fig. 3 . Transfection of melanoma cell lines

The melanoma cell line LB-33, which contains the neomycin resistance gene, was transfected by the calcium phosphate precipitation technique with cDNA 131 cloned in plasmid pcDNA3.1 (Invitrogen). A clonal subline was isolated from the G418-resistant transfected population. Using the same method, melanoma cell lines LB-40, SK23-MEL and MZ2-MEL (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 confirmed by flow cytometry with specific monoclonal antibodies.

The HLA-A*6801+ melanoma cell line LB-33 is not recognized by CTL 128 (Fig. 2), and when transfected with cDNA 131 then obtains the ability to induce TNF release (Fig. 4A), and is sensitive to the lysis of CTL 128 (Fig. 4B), showing that antigen recognition can occur in tumor cells and is not dependent on the high artificial expression levels achieved in Cos-7 cells.

The sequence of cDNA 131 was confirmed to be 3548 base pairs long. By searching Genbank, it was found that nucleotides (nt) 1-994 and nt 3081-3347 are identical to two discontinuous regions of TRP-2 encoded by cDNA (Yokohama et al., Bioch. Bioph. Acta 1217 (1994) 317-321 ), which was recently identified as a melanoma antigen of a melanocyte line (Wang et al., J. Experimental Med. 184 (1996) 2207-2216). The first region contains the 5'-UTR of the TRP-2 gene, exon 1 and exon 2, while the second region contains exon 3 and exon 4. Sequences between nt995-3080 and downstream of nt3347 showed no significant homology to any of the sequences recorded in the database. These two regions present in cDNA 131 but not in TRP-2 cDNA are preserved intronic sequences. A 10-amino acid stretch flanking the exon portion completely matched the sequence described at the exon-intron junction of the TRP-2 gene (Sturm et al., Genome 29 (1995) 24-34). Furthermore, the length of sequence 995-3080 (2086 nucleotides) is compatible with the length of intron 2 of TRP-2, as deduced from published genome maps (Sturm et al., Genomics 29 (1995) 24-34). Thus, the identity of nucleotides 995-3080 is consistent with intron 2 of TRP-2. The sequence downstream of nucleotide 3347 of cDNA 131 provides a 5' donor splice site sequence identical to intron 4 of TRP-2 (Sturm et al., Genomics 29 (1995) 24-34). Since it lacks the 3' acceptor splice site sequence and is significantly shorter in length than estimated from published genome maps, this is the sequence of intron 4 of TRP-2, truncated at nucleotide 200. Thus, cDNA 131 is composed of a partially spliced form of the melanocyte differentiation antigen TRP-2, which contains exons 1-4 and retains the beginning of intron 2 and intron 4 (Fig. 5). DNA Sequencing and Homology Search

DNA sequence analysis is performed by specific priming using synthetic oligonucleotides. Sequencing reactions were performed by the dideoxy chain termination method using dye-labeled dideoxynucleotides and the ABIPRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA). The DNA sequence of plasmid clone cDNA 131 was determined using an automated DNA sequencer (ABI Prism 377 DNA sequencer; perkin Elmer). Computer searches for sequence homology were performed using the program FASTA EMBL-Heidelberg. Identification of sequences encoding antigenic peptides recognized by CTL 128

In order to locate the sequence encoding the antigenic peptide recognized by CTL 128, cDNA131 was digested with HindII to obtain subfragments of 1500, 200 and 2000 base pairs respectively (5'-ends in Figure 5-1500, 200 base pairs and 3 '-end). Preparation of subfragments of cDNA 131

Subfragments of cDNA 131 (5'-end-1500, 5'-end-800, 200 bp and 3'-end) were obtained by digesting the plasmid with HindII and BstUI. After purification on agarose gel, these fragments were cloned into the pcDNAI plasmid. From the 5'-end-1500, smaller fragments INT-2-434, INT-2-166 and INT-2-107 were generated by PCR amplification. Sense primer KS-INT2 (SEQ ID NO: 5) was used for amplification of all fragments. This primer generates an ATG initiation codon (underlined) with an appropriate Kozak consensus sequence in the same reading frame as that of TRP-2. The antisense primers that are used to amplify INT-2-434 respectively,-166 and-107 fragments are SP6 (SEQ ID NO:8) that is positioned at pcDNAI plasmid, INT2-as1 ( SEQ ID NO: 6) and INT2-as2 (SEQ ID NO: 7). The antisense primers INT2-asl and -as2 contain an XhoI restriction site for directional cloning. To facilitate ligation, we take advantage of a single 3'A-overhang present at the 5' end of Dynazyme™ amplified fragments, which is generated by the terminal transferase activity of DNA polymerase. The pcDNAI plasmid was digested with EcoRV, and then a single thymine was added at the 3' end of each fragment by incubating with DynazymeTM in the presence of 2 mM dTTP, as described by Marchuk et al., Nucleic Acids Res. 19 (1991) 1154. The T-vector and PCR product were digested with XhoI, purified on agarose gel and ligated. After ligation, the plasmid was electroporated into DH-5 E. coli and selected with ampicillin (50 mg/ml). Clones were isolated, plasmid DNA was extracted and transfected with HLA-A*6801 gene into Cos-7 cells.

In this step, the presence of the 5'-terminal fragments of the two initiation codons regulating their translation was not investigated. The level of TNF release 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 is encoded in this region. The nucleotide sequence of this subfragment, including the first 410 base pairs of exon 1, exon 2 and intron 2, is shown in SEQ ID NO:12. CTL 128 was not stimulated by Cos-7 cells co-transfected with fully sheared TRP-2 cDNA and HLA-A*6801 (Fig. 5). This suggests that the sequence encoding the antigen recognized by CTL 128 may be located in whole or in part in the intronic portion of the TRP-2 gene present in cDNA 131. This conclusion is supported by the lack of recognition in Cos-7 cells transfected with the 5'-end-800 cDNA fragment truncated at the BstUI site from the 5'-end-1500 fragment (Figure 5). and contains half of exon 1 and exon 2. The localization of the intron of the sequence encoding the antigenic peptide was confirmed by the ability of a PCR-amplified fragment including the first 434 base pairs of intron 2 (INT-2-434), which is capable of conveying the antigen expression (Figure 5). In the same reading frame as the preceding exons of this region, the presence of a sequence encoding a decapeptide (EVISCKLIKR) (SEQ ID NO: 2) with anchor residues (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 epitope-containing region, the sense primer KS-INT2 (SEQ ID NO: 5), which generates an ATG with the appropriate Kozak consensus sequence, and the antisense primer INT2-asl (SEQ ID NO: 6) were used Or INT2-as2 (SEQ ID NO: 7) amplifies the PCR fragment. The first fragment (INT-2-166) included the entire putative peptide coding sequence, which was partially deleted in the second fragment (INT-2-107). Only fragment INT-2-166 containing the complete putative peptide sequence was able to stimulate TNF release by CTL 128 (Figure 5). Then synthesize 10-mer and 9-mer peptides, the difference between EVISCKLIKR (SEQ ID NO: 2) and EVISCKLIK (9-mer peptide and 10-mer state (SEQ ID NO: 2) is the last amino acid (R) deletion) and incubated with HLA-A*6801 homozygous LB. The decapeptide EVISCKLIKR sensitizes LB cells for lysis by CTL 128, giving a half-maximal lysis at a concentration of about 100 pM, whereas the nonapeptide had very low potency, and the control peptide was negative (Figure 6).

To rule out that the epitope recognized by CTL 128 could arise through mutations occurring in the tumor, a 2152 bp fragment was amplified by PCR from genomic DNA of CTL 128 and from a different melanoma, MZ2-mel , which spans the complete intron 2 (nucleotides 995-3080 in cDNA 131). Example 3 Cloning of TRP-2-INT2 from Genomic DNA

Genomic DNA was purified from MZ2 melanoma cells and CTL 128 using the QLAamp blood kit (QIAGEN, Hilden, Germany). From 100 ng of genomic DNA by PCR using KS-INT2 (SEQ ID NO: 5) located in exon 2 as a sense primer and PR2 (SEQ ID NO: 9) located in exon 3 as an antisense primer Intron 2 of TRP-2 was amplified. The TRP-2-INT2 fragment was 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 possessed the desired sequence and conveyed expression of the antigen.

RT-PCR using sense primer PRIT-I (SEQ ID NO: 3) and antisense primer INT2-1260 (SEQ ID NO: 4) located in exon 1 and intron 2 only from the retained intron A fragment of 977 base pairs was amplified in the TRP2 transcript of 2. The PCR analysis of the expression of embodiment 4TRP-2-INT2 and TRP2

Total RNA was prepared from cultured cell lines, fresh skin, retina, and tumor samples by the guanidine-isothiocyanate method using RNAzol B. cDNA corresponding to 300 ng of total RNA was amplified in a final volume of 50 ml by PCR using 1 U of Dynazyme™ (Finnzymes) and 1 mM of each primer. The reaction mixture was subjected to 30 cycles of amplification. 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 antisense primers, respectively. PCR was performed for 30 cycles (94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute). In order to confirm the expression of non-spliced intron 2, the sense primer PRIT-1 (SEQ ID NO: 13) and antisense primer INT2-1260 (SEQ ID NO: 4), which are located in intron 2 and 5'-UTR. PCR was performed for 30 cycles (94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute). Detection of TRP-2 by positioning primers in distal exons and amplification of TRP-2-INT2 by the presence of a 10 kb long intron 1 between exon 1 and exon 2 (Sturm et al., Genome 29 (1995) 24-34) avoids amplification of contaminating genomic DNA. The quality of the RNA preparation was checked by PCR amplification using specific primer pair-actin cDNA.

Expression of fully cleaved TRP-2 message was detected using sense primer PR3 (SEQ ID NO: 10) and antisense primer TRP-2L (SEQ ID NO: 11), which are located in exons 2 and 8, respectively.

Of the tumors examined, only melanomas proved positive for TRP-2 and TRP-2-INT2 antigens (Table 1). Expression of TRP-2-INT2 was never observed in the absence of TRP-2. This result is consistent with the conclusion that TRP-2 is a melanocyte differentiation antigen (Wang et al., Journal of Experimental Medicine 184 (1996) 2207-2216), and that the same promoter drives the synthesis of a common messenger, Two antigens are thus produced. 69% of fresh melanoma samples analyzed expressed TRP-2, and 78% of TRP-2 ⁺ melanomas also expressed TRP-2-INT2 (Table 1). This was also observed in melanoma cell lines, where the normal form of TRP-2 and the one retaining intron 2 were expressed in 84% and 68% of the samples analyzed, respectively. Analysis of TRP-2-INT2 expression was performed on normal tissues in which TRP2 is known to be present. Three melanocyte lines, four skin samples and one retina were negative in RT-PCR assays (Table 1). Four skin samples were analyzed and compared to biopsied primary lesions from the same patients, and while TRP-2 was detected in all samples, TRP-2-INT2 was only present in tumor samples. Table 1 Expression of TRP-2-INT2 in tumor and normal tissues Number of samples with antigen expression/number of samples tested TRP-2-INT2 TRP-2 melanoma cell line 13/19 (68%)* 16/19 (84%) Melanocytes 0/3 3/3 Fresh Melanoma Samples 7/13 (54%)* 9/13 (69%) skin 0/4 4/4 retina 0/1 1/1 Tumor (non-melanoma) 0/3 0/3 *TRP-2-INT2 expression was only detected in TRP-2 positive samples

A strong correlation was found between the expression of TRP-2-INT2 mRNA in melanoma cell lines and their ability to stimulate TNF release through CTL128. Presentation of antigenic peptides by HLA-A3-class supertype alleles

HLA-A*6801, as well as A3, A11: A31 and A*3301 belong to the A3-class supertype of HLA-A alleles, which have similar peptide binding properties (Sidney JM et al., Immunology 154 (1995) 247- 259). To investigate whether the peptide EVISCKLIR (SEQ ID NO: 2) can be presented by the A3-class supertype of the HLA-A86802 allele phase CTL 128, EBV expressing this allele will be expressed after pulsing with this peptide LCL was used as a target for CTL 128 (Figure 7).

This analysis also included the HLA-A*6802 allele, a subtype of lHLA-A28 belonging to the A2-class supertype. Among the A3-like supertype alleles, only A*3301 was able to present the TRP-2-INT _222-223 peptide with the same potency as A*6801. When this peptide was presented by A*6802, a low level of recognition was observed at higher concentrations. Example 5 Antigen Peptide and CTL Test

Peptides were synthesized on solid phase using Fmoc for transient NH2-terminal protection and identified by mass spectrometry (Primm, Milan, Italy). All peptides were greater than 90% pure by HPLC analysis. Freeze-dried peptides were dissolved in 10% DMSO at a concentration of 10 mM and stored at -80°C. These peptides were tested in an assay in which ^51Cr -labeled target cells were incubated in 96-well microplates with different concentrations for 1 hour at room temperature before adding CTL 128 at an effector/target ratio of 20:1 . Lysis was measured after 4 hours. The presentation of antigenic peptides was also detected in a TNF release assay. Briefly, stimulator cells were incubated with a fixed concentration of peptide for 1 hour at room temperature; then washed extensively, CTL 128 added and TNF release assessed on WEHI-164.13 cells after 18-20 hours.

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Sequence listing (1) total information:

(i) Applicant:

(A) Name: BOEHRINGER MANNHEIM GMBH

(B) Street: Sandhofer str. 116

(C) City: Mannheim

(E) Country: Germany

(F) Zip code: D-68305

(G) Tel: 08856／60-3446

(H) Fax: 08856／60-3451

(ii) Title of Invention: Tumor-specific antigens, methods for their preparation, and their use in immunization and diagnosis

(ⅲ) Number of sequences: 12

(iv) Computer-readable form

(A) Media type: floppy disk

(B) Computer IBM PC compatible

(C) Operating system PC-DOS/MS-DOS

(D) Software: PatentIn Release #1.0, Version #1.30B (EPO) (2) Information on SEQ ID NO: 1:

(i) Sequence features

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: cDNA

(ⅸ) Features:

(A) Name/keyword: CBS

(B) Site: 1. ． 30

(ⅹⅰ) Sequence Description: SEQ ID NO: 1: GAA GTA ATT TCA TGC AAG TTA NO ATT AAG AGA 30Glu Val Ile Ser Cys Lys Leu Ile Lys Arg1 Information of SE ID: 2 Q 10 (

(i) Sequence features

(A) Length: 10 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecule type: protein

(ⅹⅰ) Sequence description: SEQ ID NO: 2: Glu Val Ile Ser Cys Lys Leu Ile Lys Arg1 5 10 (2) Information of SEQ ID NO: 3:

(i) Sequence features

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 3: GGAGA AAAGT ACGACAG (2) Information of SEQ ID NO: 4:

(i) Sequence features

(A) Length: 20 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 4: ACCTCACCAA CTCACATCTT (2) Information of SEQ ID NO: 5:

(i) Sequence features

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 5: GCCGCCATGT ATTCTGTTAG AGATACA (2) Information of SEQ ID NO: 6:

(i) Sequence features

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 6: ATCCGCTCGA GCATGAAATT ACTTCCC (2) Information of SEQ ID NO: 7:

(i) Sequence features

(A) Length: 25 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 7: ATCCGCTCGA GGATAATTCT ACGAC (2) Information of SEQ ID NO: 8:

(i) Sequence features

(A) Length: 18 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 8: ATTTAGGTGA CACTATAG (2) Information on SEQ ID NO: 9:

(i) Sequence features

(A) Length: 20 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 9: GAGAAATCTA TGGCCCTGTA (2) Information of SEQ ID NO: 10:

(i) Sequence features

(A) Length: 20 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 10: TTCGGCAGAA CATCCATTCC (2) Information of SEQ ID NO: 11:

(i) Sequence features

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="primer"

(ⅹⅰ) Sequence description: SEQ ID NO: 11: ACCCTAGGCT TCTTCTGTGT ATCTCTT (2) Information of SEQ ID NO: 12:

(i) Sequence features

(A) Length: 1409 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: DNA (genome)

(ⅸ) Features:

(A) Name/Keyword: Exon

(B) Site: 1. ． 994

(ⅸ) Features:

(A) Name/Keyword: Intron

(B) site: 995. ． 1409

(ⅸ) Features:

(A) Name/keyword: misc signal

(B) Site: 399. ． 402

(C) Other information: /function="start codon"

(ⅸ) Features:

(A) Name/keyword: misc signal

(B) Site: 1111. ． 1113

(C) Other information: /function="the first stop codon"

(ⅸ) Features:

(A) Name/keyword: mat peptide

(B) Site: 1063. ． 1093

(ⅹⅰ)序列描述：SEQ ID NO：12：AGCTAAGGAG GGAGGGAGAG GGTTTAGAAA TACCAGCATA ATAAGTAGTA TGACTGGGTG     60CTCTGTAAAT TAACTCAATT AGACAAAGCC TGACTTAACG GGGGAAGATG GTGAGAAGCG    120CTACCCTCAT TAAATTTGGT TGTTAGAGGC GCTTCTAAGG AAATTAAGTC TGTTAGTTGT    180TTGAATCACA TAAAATTGTG TGTGCACGTT CATGTACACA TGTGCACACA TGTAACCTCT    240GTGATTCTTG TGGGTATTTT TTTAAGAAGA AAGGAATAGA AAGCAAAGAA AAATAAAAAA    300TACTGAAAAG AAAAGACTGA AAGAGTAGAA GATAAGGAGA AAAGTACGAC AGAGACAAGG    360AAAGTAAGAG AGAGAGAGAG CTCTCCCAAT TATAAAGCCA TGAGCCCCCT TTGGTGGGGG    420TTTCTGCTCA GTTGCTTGGG CTGCAAAATC CTGCCAGGAG CCCAGGGTCA GTTCCCCCGA    480GTCTGCATGA CGGTGGACAG CCTAGTGAAC AAGGAGTGCT GCCCACGCCT GGGTGCAGAG    540TCGGCCAATG TCTGTGGCTC TCAGCAAGGC CGGGGGCAGT GCACAGAGGT GCGAGCCGAC    600ACAAGGCCCT GGAGTGGTCC CTACATCCTA CGAAACCAGG ATGACCGTGA GCTGTGGCCA    660AGAAAATTCT TCCACCGGAC CTGCAAGTGC ACAGGAAACT TTGCCGGCTA TAATTGTGGA   720GACTGCAAGT TTGGCTGGAC CGGTCCCAAC TGCGAGCGGA AGAAACCACC AGTGATTCGG   780CAGAACATCC ATTCCTTGAG TCCTCAGGAA AGAGAGCAGT TCTTGGGCGC CTTAGATCTC   840GCGAAGAAGA GAGTACACCC CGACTACGTG ATCACCACAC AACACTGGCT GGGCCTGCTT   900GGGCCCAATG GAACCCAGCC GCAGTTTGCC AACTGCAGTG TTTATGATTT TTTTGTGTGG   960CTCCATTATT ATTCTGTTAG AGATACATTA TTAGGTGGGT TTTTTCCTTG GCTGAAGGTA  1020TATTATTACA GGTTTGTGAT TGGGTTGAGG GTATGGCAGT GGGAAGTAAT TTCATGCAAG  1080TTAATTAAGA GAGCAACCAC AAGGCAGCCT TAGGTTTATG AAAGTCGTAG AATTATCAAA  1140TACCGCCTGG AGTTAGAAGG AAGCAGTTTC TTCCTGTGCA TTGGATGCAG ACACTTTAAA  1200TGTTCTCTCC TCTACCGTAT GTTCTTGGTT CAAAGTGTAA ACTTTTCTCT GTGAAGCTGT  1260TAATCATCAA AGATGTGAGT TGGTGAGGTG GAGGCGAATT CCTTTTGATT TCAGAAGAAA  1320ATATTTGCGA ATCTGGCCAT GGAAGCCCTC TCTGACCTTT TCTCCAAATT AGAGGAATTA 1380ACTGAACATG TGCTAAGGCA CATGAAGCT 1409

Claims (11)

1. A tumor-specific polypeptide antigen partially encoded by an intron of an exon-encoded tumor antigen and obtained by: a) Reverse transcriptase PCR of mRNA isolated from a soluble cytoplasmic fraction of a tumor cell, wherein a nucleic acid fragment that hybridizes under stringent conditions to an intronic sequence of an exon-encoded tumor antigen is used as Primer; b) isolating said PCR product, expressing said PCR product or a fragment thereof in a host cell, and isolating a tumor-specific antigen encoded by said PCR product or a fragment thereof, which is also associated with an exon of said antigen sequence hybridization. 2. The tumor-specific polypeptide antigen according to claim 1, wherein a fragment of 8 to 12 codons of said PCR product is used for expression. 3. According to claim 1 or 2 described tumor-specific polypeptide antigens, wherein, the tumor antigen encoded by said exon is MAGE, BAGE and GAGE, from tyrosinase, MelanA ^Martl , gp100 ^Pmel7 , gp75 ^TRP1 or TRP -2 CTLs recognize epitopes. 4. A tumor-specific polypeptide antigen encoded by SEQ ID NO:1. 5. A method of isolating mRNA of a tumor-specific antigen encoded by an intronic portion of an exon-encoded tumor antigen comprising the steps of: a) Reverse transcriptase PCR of mRNA isolated from a soluble cytoplasmic fraction of a tumor cell, wherein a nucleic acid fragment that hybridizes under stringent conditions to an intronic sequence of an exon-encoded tumor antigen is used as primers; and b) isolating said PCR product which also hybridizes to the exon sequence of said antigen. 6. A method for determining the proliferation of tumor-specific cytotoxic T-cells, wherein the tumor-specific antigen according to any one of claims 1 to 4 is added to a sample of a patient's body fluid, which contains antigen-presenting cells and cytotoxic T cells, measuring the proliferation of cytotoxic T cells. 7. The nucleic acid encoding the tumor-specific antigen according to any one of claims 1 to 4 is used in the preparation of medicaments for treating tumor diseases. 8. Use of a tumor-specific antigen according to any one of claims 1 to 4 for in vivo or in vivo activation of cytotoxic T cells from T precursor cells. 9. A method for preparing a tumor-specific polypeptide antigen, wherein said tumor-specific antigen is obtained through the following steps: a) Reverse transcriptase PCR of mRNA isolated from a soluble cytoplasmic fraction of a tumor cell, wherein a nucleic acid fragment that hybridizes under stringent conditions to an intronic sequence of an exon-encoded tumor antigen is used as Primer; b) isolating said PCR product, expressing said PCR product or a fragment thereof in a host cell, and isolating a tumor-specific antigen encoded by said PCR product or a fragment thereof, and combining said antigen under stringent conditions Exon sequence hybridization. 10. A combination of two nucleic acids that hybridize under stringent conditions to an intronic sequence of an exon-encoded tumor antigen and that can be used as a primer pair for reverse transcriptase PCR from mRNA. 11. Nucleic acids encoded by SEQ ID NO: 3 to SEQ ID NO: 9.

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