JP2001522603A - Tumor-specific antigens, methods for their production and their use for immunization and diagnosis - Google Patents

Abstract

  (57) [Summary] In the tumor-specific peptide antigen, at least a part thereof is encoded by an intron of a tumor antigen encoded by an exon, and (a) in reverse transcriptase PCR from mRNA isolated from a soluble cytoplasmic fraction of a tumor cell, By reverse transcriptase PCR using as primers a nucleic acid fragment that hybridizes under stringent conditions with the intron sequence of the tumor antigen encoded by the exon, and (b) isolating the PCR product, A tumor-specific peptide antigen obtained by expressing a fragment in a host cell and isolating the tumor-specific antigen encoded by the PCR product or a fragment thereof that also hybridizes to the exon sequence of the tumor-specific antigen Is useful for the diagnosis and treatment of tumor-related diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to novel tumor-specific antigens, methods for their production, and to activate immunity, in particular cytotoxic, tumor-specific T lymphocytes, and to the tumors of MHC class I complexes. It relates to the use of them to specifically diagnose tumor cells presenting specific antigens.

The immune system plays an important role in immunological surveillance for cancer and in tumor regression. Anti-tumor immune responses may be generated by B and T cells that recognize tumor antigens expressed on tumor cells. Production of cytotoxic T lymphocytes (CTLs) from either peripheral blood lymphocytes or tumor-infiltrating T lymphocytes (TILs) taken from patients with cancer in the past several years during tumor regression The role of T cells can be assessed.

In humans, adoptive transfer of tumor infiltrating T lymphocytes with interleukin 2 (IL-2) to patients with autologous cancer can cause tumor regression (Rosenbe
rg et al., New Engl. J. Med. 319 (1988) 1676-1680; Rosenberg et al., J. Natl. Canc.
er Inst. 86 (1994) 1159-1166). This suggests that T cells play an important role in tumor regression in vivo. The ability of TIL to cause tumor regression in vivo was associated with the ability to cause specific cell lysis and cytokine release when co-cultured with syngeneic tumor cells in vitro (Barth et al., J. Am. Exp. Med. 173 (1991) 647-658).

To understand the molecular basis of T cell-mediated antitumor responses, various genes recognized by T cells and 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; Par
doll, Nature 369 (1994) 357-358; Rosenberg, Cancer J. Sci. Am. 1 (1995)
90-100). These antigens can be divided into several classes based on their expression patterns.

[0005] The first class of tumor antigens is antigens present on melanomas and tumors of various other tissue types, but not on normal cells other than testis and placenta (eg, MAGE
, BAGE, GAGE) (Van der Bruggen et al., Science 254 (1991) 1643-164).
7; Boel et al., Immunity 2 (1995) 167-175; Van den Eynde et al., J. Exp. Med. 182
(1995) 689-698). Clinical trials using these antigens, which are recognized by CTL controlled by various HLA class I alleles, in patients affected by melanoma and other oncogenic diseases are ongoing (Marchand et al., Int. J
Cancer). Considering the frequency of expression of each of these antigens and the frequency of HLA class I alleles, more than 60% of Caucasian melanoma patients and 40% of head and neck cancer patients
These, and more than 28% of bladder cancer patients, may be suitable for immunization with at least one antigen of this group. No side effects have been detected in patients receiving this treatment. In fact, the MAGE, BAGE, and GAGE genes are normally expressed in testis cells, such as spermatogonia and spermatocytes (II), but these cells use the classic MHC required for antigen presentation. No expression of class I molecules (Haas et al., Am. J.
Repord. Immunol. Microbiol. 18 (1988) 47-57) and therefore would not be a target for T cells.

[0006] The second class of tumor antigens includes tissue-specific antigens expressed on normal and tumorigenic cells of the melanocyte lineage. Tyrosinase (Brichard et al., J. Exp.
Med. 178 (1993) 489-49513), MelanA ^Mart1 (Coulie et al., J. Exp. Med. 180 (
1994) 35-42; Castelli et al., J. Exp.Med. 181 (1995) 363-368; Kawakami et al., Pr.
oc. Natl. Acad. Sci. USA 91 (1991) 3515-3519), gp100 ^Pme17 (Bakker et al., J. ^Biol .
Exp. Med. 179 (1994) 1005-1009; Kawakami et al., Proc. Natl. Acad. Sci. USA
91 (1994) 6458-6462), gp75 ^TRP1 (Wang et al., J. Exp. Med. 181 (1995) 799-80).
4), and C from TRP-2 (Wang et al., J. Exp. Med. 184 (1996) 2207-2216).
TL recognition epitopes can develop in vitro on cells from many melanoma patients. Thus, the majority of melanoma patients may benefit from immunotherapy trials aimed at eliciting a T cell response to these antigens. In fact, differentiated antigens are expressed on almost all melanomas,
Most of them are presented to immune effectors by HLA-A2 alleles, which occur frequently in various ethnic groups. However, normal tissue (
That is, it is necessary to carefully consider the possibility that the side effects of these treatments may be caused by the occurrence of cross-reactions against skin melanoma cells and retinal pigment cells.

[0007] The third class of tumor antigens includes antigens that are expressed only by the tumor cells from which they were isolated. Such antigens are not expressed in other normal tissues or in tumorigenic tissues of different origin, and their antigenic epitopes are usually point mutations in proteins that are universally expressed without the mutation. Caused by For tumor antigens belonging to this group, mouse strains (Boon et al., Annu.
Rev. Immunol. 12 (1994) 337-365), and some human tumors (Wolfel et al., S
ciul 269 (1995) 1281-1284; Coulie et al., Proc. Natl. Acad. Sci. USA 92 (19
95) 7976-7980; Robbins et al., J. Exp. Med. 183 (1996) 1185-1192). Lack of natural resistance to these antigens avoids potential autoimmune responses, but provokes strong immune responses. However, until an extensive hotspot panel of mutations was discovered, the clinical application of such antigens would have resulted in the individual therapy of patients, or at least the very fact that the tumor had the mutation. Limited to treatment of a small number of patients (Wolfel et al., Science 269).
(1995) 1281-1284).

[0008] A fourth tumor antigen class arises from transcripts that have been selectively processed. Robbins et al., In J. Immunol. 159 (1997) 303-308, corresponded to a portion of the fourth intron of gp100, which was not found in the gp100 glycoprotein of normal patients, with an additional 35 Transcripts encoding amino acids are described. However, this epitope is only expressed at low levels in melanoma. Robbins et al. In J. Immunol. 154 (1995) 5944-5950 describe the cloning of a gene encoding an antigen recognized by melanoma-specific HLA-A24 that restricts tumor infiltrating lymphocytes. . This antigen is a fragment of a full-length clone that is not encoded by an intron, or part thereof.

Describe in J. Immunol. 153 (1994) 5516-5524 a soluble form of the non-tumorigenic HLA-G antigen encoded by mRNA containing the fourth intron. . However, at this time, the specific function of the soluble HLA-G protein is unknown.

[0010] The presence of a fifth class of tumor antigens is a panel in CTL clones that can recognize self and HLA-matched allogeneic melanomas, but not melanocytes or targets of different histological origin. (Anichini et al., J. Immunol. 156 (1996) 208-217).

[0011] It is an object of the present invention to provide a novel tumor-specific antigen that is not expressed on normal cells and thus can specifically distinguish normal cells from tumor cells.

SUMMARY OF THE INVENTION The present invention is directed to the soluble cytoplasm of tumor cells, partially encoded by the intron sequence of the polypeptide gene presented by the MHC class I complex (tumor antigen encoded by exons) on tumor cells Reverse transcriptase PC from mRNA isolated from fraction
R is a tumor-specific polypeptide having an antigenic effect, characterized by being obtained by R. A nucleic acid fragment that hybridizes under stringent conditions to an intron sequence of a tumor antigen encoded by an exon is used as a primer. If the PCR product is obtained, the PCR product is isolated, expressed in host cells, and
And polypeptides obtained by isolating the tumor-specific antigen encoded by the R product.

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

[0014] Another object of the present invention is a method for identifying a tumor-specific polypeptide having an antigenic effect, the method comprising: -Reverse transcriptase PCR in tumor cells, using as primers a nucleic acid fragment that hybridizes under stringent conditions with the intron sequence of the exon-encoded tumor antigen;-If a PCR product is obtained, the PCR product Isolation, expression in host cells, and isolation of a tumor-specific antigen encoded by the PCR product that also hybridizes to the exon sequence of the antigen.

[0015] After isolation, the hybridized product, or a fragment thereof, is inserted into an expression vector, and the vector is introduced into a suitable host cell and expressed in the host cell. The resulting recombinant polypeptide is isolated.

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

Thus, a subject of the present invention is a tumor-specific polypeptide antigen which is partially encoded by introns of a tumor antigen which is encoded by an exon, obtained by the following (a) and (b) Antigens: a) Reverse transcription of mRNA isolated from the soluble cytoplasmic fraction of tumor cells using as primers a nucleic acid fragment that hybridizes under stringent conditions to the intron sequence of the tumor antigen encoded by the exon. Enzymatic PCR, and b) isolation of the PCR product, expression of the PCR product or a fragment thereof in a host cell, and the tumor encoded by the PCR product or a fragment thereof and also hybridizing to the exon sequence of the antigen Isolation of specific antigen.

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

Yet another subject of the invention is that the exon-encoded tumor antigen is MAGE
, BAGE, and CTL recognition antigens such as GAGE, and tyrosinase, MelanA ^Mart1 ,
The tumor-specific antigen according to the present invention, which is a CTL recognition epitope derived from gp100 ^Pme17 , gp75 ^TRP1 or TRP-2.

[0020] The present invention further relates to a tumor-specific polypeptide antigen encoded by SEQ ID NO: 1.

A further subject of the invention is a method for isolating the mRNA of a tumor-specific antigen encoded by an intron of a tumor antigen encoded by an exon,
The method is carried out according to the following (a) and (b): a) The use of a primer for a nucleic acid fragment that hybridizes under stringent conditions to an intron sequence of a tumor antigen encoded by an exon as a primer. Reverse transcriptase PCR of mRNA isolated from the soluble cytoplasmic fraction, and b) isolation of the PCR product that also hybridizes to the exon sequence of the antigen.

Furthermore, the present invention relates to a method for measuring the proliferation of tumor-specific cytotoxic T cells, which comprises using the tumor-specific antigen according to the present invention to separate antigen-presenting cells and cytotoxic T cells. In addition to a patient body fluid sample containing, preferably, cytokine release (TNF, IFNγ,
Measurement of cytokines such as GM-CSF) to measure the proliferation of cytotoxic T cells.

Another subject of the present invention comprises the step of using a nucleic acid encoding a tumor-specific antigen according to the present invention for the manufacture of a therapeutic for treating a tumor disease.

Furthermore, the invention is the use of a tumor-specific antigen according to the invention for activating cytotoxic T cells from T cell precursors in vivo or in vitro.

Furthermore, a subject of the present invention is a method for producing a tumor-specific polypeptide antigen, wherein said tumor-specific antigen is obtained according to the following (a) and (b): a) reverse transcriptase PCR of mRNA isolated from the soluble cytoplasmic fraction of tumor cells, using as primers a nucleic acid fragment that hybridizes under stringent conditions to the intron sequence of the tumor antigen encoded by the exon, and b) isolation of the PCR product, expression of the PCR product or a fragment thereof in a host cell, and expression of the tumor-specific antigen encoded by the PCR product or a fragment thereof and also hybridizing to the exon sequence of the antigen. Isolation.

Further, another subject of the present invention is a nucleic acid which hybridizes under stringent conditions with the intron sequence of the tumor antigen encoded by the exon, a primer for reverse transcriptase PCR from mRNA. The method includes a step of combining two types of nucleic acids that can be used as a pair.

The subject of the present invention also includes the nucleic acids encoded by SEQ ID NO: 3 to SEQ ID NO: 9.

The tumor antigen according to the present invention is not found on the surface of normal cells such as melanocytes. However, this antigen is specific for tumor cells, for example, because it is found in over 80% of melanoma cells.

The peptide is therefore particularly suitable for immunizing patients with tumor cells and for diagnosing melanoma cells from normal melanocytes.

[0030] Cytolytic T lymphocyte clones (CTL128) derived from peripheral blood lymphocytes of metastatic melanoma patients are responsible for autologous tumors and allogeneic melanomas controlled by HLA A ^* 6801. Some could be dissolved. A cDNA library constructed from autologous melanoma mRNA was transfected into Cos-7 cells expressing the HLA A ^* 6801 allele to identify the gene encoding the antigen recognized by CTL128. In contrast, it has surprisingly been found that splicing errors in melanocytes occur during mRNA maturation. This results in partial splicing of the melanocyte differentiation antigen (TRP) -2, including exons 1 through 4 that carry the second intron and part of the fourth intron (TRP-2-INT2). Translation of the peptide constituted by the morphology is performed. Using the same reading frame as TRP-2, TRP-2-INT2 is a terminator site (at position 113) in the second intron, just 18 nucleotides downstream of the peptide coding region from the 400th start codon. Encoding a protein consisting of a putative 238 amino acids, up to and including nucleotides).

[0031] The differentiation antigen TRP-2 is obtained from RF Wang, J. Experimental Medicine 184 (199).
6) It is described in 2207-2216. TRP-2 is one of the most strongly expressed in human pigmented melanocytes and melanomas (Wang et al., J. Exp. Med. 184 (
1996) 2207-2216). It is present on human chromosome 13 and is known to be a member of the tyrosinase-related gene family.
gp75 / TRP-1 has 40-45% amino acid homology (Yokohama et al., Biochim. Bio
phys. Acta. 1217 (1994), 317-321; Robbins et al., J. Immunol. 154 (1995), 594.
4-5950). TRP-2 encodes a protein consisting of 519 amino acids and has been demonstrated to have dopa (DOPA) chromotautomerase activity involved in melanin synthesis (Bouchard et al., Eur. J. Biochem. 219 (1994). ), 127-134).

In contrast to the fully spliced TRP-2 mRNA described by Wang et al. As found in melanoma, normal skin melanocytes and retinal cells, TRP-2-INT2 mRNA Was detected only in melanoma. These results indicate that melanoma antigens recognized by CTL may be regulated by related proteins in known cell lines due to splicing differences that occur in tumors but not in normal cells. This mechanism gives rise to a new group of antigens that are not present in normal tissues in the same cell line, are truly tumor-specific and are considered to be common only to tumors of the same tissue type. Therefore, the novel antigen group is characterized by being controlled by known cell line-related proteins by tumor-specific alternative splicing that is not present in normal cells. Normal melanocytes,
Skin samples, and retinal cells, proved to be negative in reverse transcriptase (RT) PCR analysis. These characteristics are the characteristics of antigens that enable the development of safer and more effective immunotherapy tests.

Thus, the present invention relates to a tumor-specific antigen, partially encoded by introns, obtained by reverse transcriptase PCR of mRNA isolated from the soluble cytoplasmic fraction of tumor cells. These antigens are recognized in the MHC class I complex by specific T lymphocytes that lyse tumor cells presenting the antigen according to the invention. Surprisingly, the mRNA encoding the antigen of the invention, which is predominantly present in the cytoplasm of such tumor cells, is particularly tumor-specific.

The “tumor-specific antigen encoded by the intron” includes not only exon sequences but also a part (preferably about 30% or more, more preferably about 50% or more, and most preferably about 80% Above) means a tumor antigen that is also encoded by an intron sequence and specifically recognizes a tumor antigen presented by MHC class I. Intron expression has been implicated in the mechanism of alternative splicing of cell line-related proteins. "Part" preferably means that 30-50%, or 30-80%, is encoded by the intron. The sequence of the antigen according to the invention, encoded by exons and encoded by introns, is directly linked in the relevant genomic sequence, since the antigen was generated by alternative splicing.

“Tumor antigens encoded by exons” include, for example, MAGE, BAGE, and GAGE (Van der Bruggen et al., Science 254 (1991) 1643-1647; Boel et al., Imm
unity 2 (1995) 167-175; Van den Eynde et al., J. Exp.Med. 182 (1995) 689-698.
Or tyrosinase (Brichard et al., J. Exp. Med. 178 (1993) 48
9-49513), MelanA ^Mart1 (Coulie et al., J. Exp. Med. 180 (1994) 35-42; Castell
i et al., J. Exp.Med. 181 (1995) 363-368; Kawakami et al., Proc. Natl. Acad. Sci.
USA 91 (1991) 3515-3519), gp100 ^Pme17 (Bakker et al., J. Exp. Med. 179 (1994)
1005-1009; Kawakami et al., Proc. Natl. Acad. Sci. USA 91 (1994) 6458-6462.
), Gp75 ^TRP1 (Wang et al., J. Exp. Med. 181 (1995) 799-804), and TRP-2 (Wa
ng et al., J. Exp. Med. 184 (1996) 2207-2216) means the antigen described in the introduction such as a CTL recognition epitope.

“Peptide or polypeptide according to the invention” preferably refers to a polypeptide of 8 to 12 amino acids, most preferably of at least 10 amino acids, but also of protein size. May be included. The peptide may be part of a protein, such as a fusion protein.

“Polypeptide with an antigenic effect” means a polypeptide that induces a specific immune response in vivo and in vitro.

The term “hybridizes under stringent conditions” is described in Sambrook et al., Molecular Cloning: Experimental Manual.
A Laboratory Manual) (1989), Cold Spring Harbor Press (Cold Sp
ring Harbor Press), New York, USA, 9.47-9
.62 and 11.45-11.61, "Expression of Cloned Genes in E. coli (Expre
ssion of cloned genes in E. coli) and two nucleic acid fragments that can hybridize to each other under standard hybridization conditions.

More specifically, “stringent conditions” as used herein refers to 6.
Hybridization in which hybridization is performed at about 45 ° C. in 0 × SSC, and then washed at 50 ° C. with 2.0 × SSC. In order to select stringency, the salt concentration in the washing step can be selected, for example, from low stringency conditions of about 2.0 × SSC at 50 ° C. to high stringency conditions of about 0.2 × SSC at 50 ° C. . Further, the temperature in the cleaning process is
From low stringency conditions at room temperature about 22 ° C, it can be increased to high stringency conditions at about 65 ° C.

[0040] For expression in a prokaryote or eukaryote, such as a prokaryote or eukaryote host cell, the nucleic acid sequence is incorporated into a suitable expression vector by methods known to those of skill in the art. Such expression vectors preferably include a regulatable / inducible promoter. In order to express such a recombinant vector, for example, Escherichia coli is used as a prokaryotic host cell, or Saccharomyces cerevisiae, CHO cell or COS cell is used as a eukaryotic host cell. The transformed or transduced host cells are cultured under conditions that allow expression of the heterologous gene. The peptide can be isolated from the host cell or a culture supernatant of the host cell by a known method. Such methods are described, for example, in Ausubel I.
, The latest protocol in molecular biology by Frederick M. (Curr
ent Protocols in Mol. Biol. (1992), Jon Wiley & Sons, New York.

“Host cell” refers to a prokaryotic or eukaryotic cell, preferably a CHO cell or COS
Means cell. Production in prokaryotes is preferred when glycosylation is less important with respect to the action of the protein.

By transfecting a cDNA library constructed from autologous tumor mRNA into eukaryotic cells, preferably into eukaryotic cells expressing the appropriate HLA allele of the patient, specific cells Genes encoding tumor-specific antigens recognized by intoxicating T lymphocytes (CTLs) can be identified.

To determine the HLA antigen, total RNA was isolated from the patient's tumor cells or tumor tissue and cD by reverse transcriptase PCR using primers specifically encoding the HLA antigen.
Obtain the NA and clone it into a eukaryotic expression vector such as, for example, pcDNA3 (Invitrogen Corporation, Oxon, UK). To determine a tumor-specific antigen encoded by an intron according to the present invention, poly A ⁺ RNA is isolated from mRNA isolated from the soluble cytoplasmic fraction of tumor cells, e.g., MAGE, BAGE,
And GAGE, and reverse transcriptase PCR using primers that specifically encode tumor antigens encoded by exons such as CTL recognition epitopes derived from tyrosinase, MelanA ^Mart1 , gp100 ^Pme17 , gp75 ^TRP1 , or TRP-2 To construct a cDNA library. A cDNA fragment from a cDNA library is used, for example, pcDNA3.1
(Invitrogen Corporation, Oxon, UK) and cloned into a eukaryotic expression vector. For example, an expression vector in eukaryotic cells such as Cos-7 cells (expressing an MHC-related gene capable of displaying a peptide fragment of an antigen capable of activating a specific tumor antigen T cell). After co-transfection, the clones that give rise to tumor-specific cytotoxic T lymphocyte stimulation can be determined. The stimulatory effect on tumor-specific cytotoxic T lymphocytes was
rsari) et al., Immunogenetics 35 (1992), 145-152 (measurement of TNF-α, IFNγ, GM-CSF).

In this way, a cell clone that specifically activates cytotoxic T cells that cause lysis of each tumor cell can be obtained.

This cell clone can be directly used for immunization / vaccination, either as a full-length polypeptide or in the form of shortened peptide fragments, since the antigenic polypeptide according to the invention is processed in vivo. To express a polypeptide that can be administered to a patient in an isolated and purified form.

To this end, according to established methods (Sambrook et al., Molecular Cloning (1989), Cold Spring Harbor Laboratory Press) according to the present invention. The nucleic acid encoding the repeptide is isolated from this cell clone and shortened by restriction enzyme digestion. These truncated fragments can be cloned into eukaryotic expression vectors and then transfected into eukaryotic cells such as, for example, Cos-7 cells (supra), and used by Traversari et al. Immunogenetics 35 (1992),
Cytotoxic T cell stimulation is measured by the CTL stimulation assay as described in 145-152. In this way, the coding sequence of the nucleic acid can be limited to peptide epitopes essential for stimulating cytotoxic T cells.

In a preferred embodiment, the nucleic acid sequence encoding the protein is associated with a nucleic acid sequence that can facilitate processing of the protein in a host cell.

By the way, many sequences, such as ubiquitin, are known to promote protein transport, protein degradation, and introduction into MHC class I complexes (Bachmair et al., Science 234 (1989)). 179-186; Gonda et al., J. Biol. Chem. 264 (1
989) 16700-16712; Bachmair et al., Cell 56 (1989) 1019-1032. By binding a protein containing the antigen polypeptide according to the present invention to ubiquitin, more rapid proteolysis and more efficient introduction into an MHC class I complex can be performed. As a result, peptide presentation on the tumor cell surface is particularly efficient. In this regard, the amino acids between the protein containing the antigenic peptide and ubiquitin are particularly important for binding the protein containing the antigenic peptide to ubiquitin. By selecting appropriate amino acids, recognition of melanoma cells by specific cytotoxic T cells can be significantly improved.

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

In another preferred embodiment, the polypeptide EVISCKLIK, which is an amino acid sequence encoded by the DNA sequence shown in SEQ ID NO: 2 in an MHC class I complex
A tumor-specific antigen, TRP-2-INT2 (hereinafter TR), partially encoded by introns, recognized by specific T lymphocytes lysing melanoma cells presenting R
P-2-INT2 fragment or TRP-2-INT2 peptide) is used.

To obtain the coding sequence for the tumor-specific antigen TRP-2-INT2 peptide, metastatic metastases taken from surgical specimens of patients undergoing surgery at the National Cancer Institute (Milan, Italy) From the cytoplasm of the melanoma cell line established from the lesion, HLA-
The cDNA encoding the A ^* 6801 allele and the TRP-2-INT2 antigen was isolated.

After transfecting the two cDNAs into COS-7 cells, a clone capable of stimulating the cytotoxic effect of cytotoxic T lymphocyte (CTL128) was isolated (TR
P-2-INT2, HLA-A ^* 6801). By DNA sequencing analysis, the second intron, and
Exon-encoded cDNA fragments of the TRP-2 antigen, including exon 1 to exon 4, including part of the four introns, were revealed. Molecular cloning: A Laboratory Manual (1989), Cold Spring Harbor Press, New York, USA, by methods well known to those skilled in the art or in Sambrook et al. York, USA), 5.3-5.10 "Expression of cloned genes in E. coli (E
xpression of cloned genes in E. coli), after digestion of TRT-2-INT-2 DNA with a restriction enzyme, or a fragment-specific primer (SEQ ID NO: 6). , 7, 8), a partial fragment of the TRP-2-INT2 fragment was obtained. After transfection into Cos-7 cells, Traversari et al.
The CTL stimulation effect is measured by a CTL stimulation assay as described in mmunogenetics 35 (1992), 145-152. The nucleic acid coding sequence was determined by DNA sequence analysis.

Another subject of the invention comprises the use of an antigen according to the invention on tumor cells presenting by MHC class I.

Another subject of the present invention comprises a method for detecting the expression of an antigen according to the present invention from a patient's body fluid, preferably blood or tissue specimens. For this purpose, a tumor-specific cDNA encoded by an intron, encoding the antigen according to the invention, is used before, during and after treatment to diagnose tumor cells expressing the antigen according to the invention.

Since treatment can be performed only when the tumor actually expresses the antigen according to the present invention, whether the tumor antigen according to the present invention is expressed in the tumor cells of the patient before the treatment. Check whether or not. In the course of treatment, it can be observed whether the expression of the antigen according to the present invention, which is a marker for the presence of tumor cells, could be reduced, and after treatment, the expression of the antigen according to the present invention was measured. This makes it possible to control whether the tumor cells have been removed as far as possible.

In a preferred embodiment, the nucleic acid encoding the tumor-specific TRP-2-INT2 peptide is
Used for diagnosis of melanoma cells displaying the TRP-2-INT2 peptide on the surface.

The TRP-2-INT2 peptide sequence is specifically expressed in melanoma. Therefore, the TRP-2-INT2 sequence can be used as a specimen for identifying tumor cells. The identification is performed by a PCR method, a method using a labeled hybridization sample, or a method using any of various nucleic acid probes based on assays known in the art.

Measurement of TRP-2-INT2 expression was determined by reverse transcription PCR (RT-PCR), and specific labeled TRP-2-I
Performed at the mRNA level by hybridization with the NT2 probe.

[0059] TRP-2-INT2 mRNA can be specifically determined to distinguish it from TRP-2 mRNA by the following methods: 1) cDNA transcription; 2) use of intron-specific primers; 3) Comparison of amplified cDNA fragment lengths (by determining the length of exon-intron-exon fragment of amplified cDNA along with exon cDNA)
4) hybridization with intron-specific probes.

Another subject of the present invention is a method for measuring the amplification of tumor-specific cytotoxic T lymphocytes. This method is used to detect cytotoxic T cells that can be activated by the antigen according to the invention before or during the treatment. Prior to treatment, patients can be selected that already have cytotoxic T lymphocytes that can be activated by the antigens of the invention.

To check for the presence of T cells that can be activated in a tumor-specific manner in a patient, T cells and antigen presenting cells are isolated from the blood of the patient and the antigen of the present invention Contact ex vivo with antigen presenting cells ("pulse"). When T cells that can be activated by the antigen according to the present invention are present in the blood of a patient, it is likely that specific cytotoxic T lymphocytes that can be detected, for example, by a CTL stimulation assay will proliferate. . Such activatable T cells can be used for therapy after being stimulated by the antigen according to the present invention. This diagnostic method must be performed before treatment.

Another diagnostic method performed during treatment helps control the formation of cytotoxic T lymphocytes by allowing tumor-specific T cell activation to be determined quantitatively.

[0063] Proliferation of cytotoxic T lymphocytes can be measured, for example, by a CTL stimulation assay. For stimulator cell lines, see Traversari et al., Immuno
As described in genetics 35 (1992), 145-152, it is examined whether TNF production can be induced by CTL. WEHI-164.3 cells (Espevic and Niessen-Meye
r, J. Immunol. Methods 95 (1986) 99-105)
The TNF content is measured by a T colorimetric assay (CTL stimulation assay).

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

Another subject of the present invention involves the use of a nucleic acid encoding an intron-specific antigen according to the present invention for the manufacture of a therapeutic agent for treating a tumor disease.

In this context, the nucleic acids according to the invention can be used for gene therapy. When this nucleic acid is introduced into a patient with the help of a viral or non-viral vector, the coding sequence is specifically expressed and the peptide according to the invention binds to the antigen-presenting cell, thereby Induces a potent cytotoxic T cell response. Then, the induced immune response will be elicited against all tumor cells that express the peptide of the present invention on the cell surface. Vector coded
The DNA sequence can be applied subcutaneously, intramuscularly, or intratumorally, in the form of bare DNA, with liposomes, or with a suitable adjuvant (known in the art).

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 immunizing and / or vaccinating tumor patients. Immunization is based on the activation of specific cytotoxic T cells by presenting the antigen according to the invention by antigen presenting cells. Immunization can be performed ex vivo or in vivo.

At this time, antigen presenting cells (macrophages, dendritic cells, or B cells) and T lymphocytes are collected from the patient's blood and contacted ex vivo with the peptide of the invention (“pulsing”). ). These antigen presenting cells carry the peptide according to the present invention, thereby causing specific cytotoxic T cell activation and then returning to the patient's blood.

Immunization is performed by subcutaneously administering the polypeptide according to the invention to the patient in vivo, whereby the activation of specific cytotoxic T cells can take place directly in the patient. Binding of a peptide according to the invention to the corresponding HLA molecule on the surface of an antigen presenting cell causes the proliferation of specific cytotoxic T lymphocytes.

The immunogenic effect of the peptides according to the invention during application can be enhanced by the following methods: 1) binding to bacterial toxins (superantigens); 2) administration with Freund's adjuvant. 3) mixing the peptide according to the invention with liposomes.

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

Another subject of the present invention comprises primers for detecting the expression of a tumor-specific antigen according to the present invention by RT-PCR. In this regard, primers can be selected by the following method. 1) A sense primer and an antisense primer that hybridize to two different exon sequences of a tumor antigen. 2) The sense primer hybridizes with the exon sequence, and the antisense primer (oligo dT primer) hybridizes with the polyA tail of the mRNA sequence of the tumor antigen.

In a preferred embodiment, SEQ ID NO: 2 (PRIT-3) and SEQ ID NO: 4 (INT2-126
Specific primers are used to detect the expression of a TRP-2-INT fragment having the amino acid encoded by the DNA sequence shown in 0).

The following examples, references, sequence listings, and figures are intended to more specifically illustrate various aspects and embodiments of the present invention, and are intended to limit the scope of the invention. is not.

Description of the sequence: SEQ ID NO: 1: coding of an antigenic peptide recognized by CTL 128 (nucleic acid)
Array. SEQ ID NO: 2: coding (amino acid) sequence of the antigenic peptide recognized by CTL 128. SEQ ID NO: 3: (PRIT-1) sense primer used to identify unspliced intron TRP-2-INT; located at 5′UTR of TRP-2 gene. SEQ ID NO: 4: (INT2-1260) Antisense primer used for confirming unspliced intron TRP-2-INT; located in 5′UTR of TRP-2 gene and second intron. SEQ ID NO: 5: A partial fragment of (KS-INT2) cDNA 131 was prepared, and TRP-2-I was prepared from genomic DNA.
The sense primer used to clone NT2; located in the second exon of the TRP-2 gene. SEQ ID NO: 6: (INT2-as1) antisense primer used for preparing partial fragment INT2-107 of cDNA 131; located in second exon of TRP-2 gene SEQ ID NO: 7: (INT2-as2) antisense primer used for preparing partial fragment INT2-166 of cDNA 131; located in second exon of TRP-2 gene. SEQ ID NO: 8: (Sp6) Antisense primer used to prepare partial fragment INT2-434 of cDNA 131; located in pCDNA1 plasmid. SEQ ID NO: 9: (PR2) antisense primer used for cloning TRP-2-INT2 from genomic DNA; located in exon 3 SEQ ID NO: 10: (PR3) sense primer used for amplifying TRP-2 DNA;
Located in 2 exons. SEQ ID NO: 11: (TRP-2L) Antisense primer used for amplifying TRP-2 DNA; located in exon 8 SEQ ID NO: 12: nucleic acid sequence of a 5'-terminal 1500-base fragment including the coding region of an antigenic peptide The 45 bases upstream from the start position of cDNA 131 are excluded because they are sequences of the pcDNA3.1 vector.

Example 1 Identification of HLA-A ^* 6801 as a Factor Restricting CTL 128 The melanoma cell line Me18732 was established from metastatic lesions in patients and was serologically typed into HLA-A2 and HLA-A28 And then HLA-A ^* 0201 and HLA-A ^* 68011 (and even HLA-A ^* 6801) by the sequence-specific oligonucleotide probe (SSOP) subtyping method (Oh et al., Genomics 29 (1995) 24-34). Was typed). Anti-tumor CTL clones are available from Anichini et al., J. Immu.
nol. 156 (1996) 208-217.

The CTL clone 128 had its cytolytic activity reduced by anti-HLA class I mAB W6 / 32, but not by anti-HLA-B, -C mAb 4E (FIG. 1). Self-related melanoma was recognized in relation to the locus allele. Anti-HLA-A2,
Inhibition of cell lysis was observed only with -A28 mAb CR1 1.351, anti-HLA-A2, -A69
HLA-A ^* 0201 could be excluded from the antigen-presenting molecules recognized by CTL 128, since they were not observed when mAb BB7.2 was used (FIG. 1). Therefore, the inhibitory activity of CR11.351 indicated that A28 (A ^* 6801) was an HLA presenting molecule for CTL128.

Culture of Cell Lines The melanoma cell line Me18732 was established from metastatic lesions taken from surgical specimens of patients admitted to the National Cancer Institute (Milan, Italy) for surgery. Was. The PBL of this patient was serologically typed as follows: HLA-A2, -A28, -B44, -B51, -C2, -C5. Human metastatic potential (Me17697, Me2559 / 1, M
e12657, Me17088 / 1, Me4023) and primary (Me20842) melanoma cell lines have been established and cultured in 10% FCS / RPMI 1640. Melanoma cell lines LB-33, LB-4
The 0 and Cos-7 (ATCC: CRL 1651) cell lines were maintained in 10% FCS / DMEM. American Type Culture Collection (Rockville, MD) ATCC,
Rockville, MD) purchased tumor cell lines CALU3, SKBR3, and SKOV3 with 10% FC
Culture was continued in S / RPMI-1640. CRA ^* 03301 transformant, homozygous EBV transformant L
CL, cell line SCHU, is characterized as HLA ^* 0301, B ^* 0702, -C7, AMA-1 is characterized as HLA ^* 6802, B ^* 5301, -C4, and WT-100-bis is , HLA-A11, -B35,
Characterized as -C4. EBV-LCL JHAF (HLA-A ^* 31011, -B51, -C8) and LB
(HLA-A ^* 68011, B ^* 40011, -C2, -C3) were obtained from ATCC. EBV-LCL is 10% FCS
/ RPMI-1640.

Evaluation of CTL 128 Antigen Specificity To assess the frequency of expression of the antigen recognized by CTL 128 in another tumor, a panel of HLA-A ^* 6801 melanoma cell lines was evaluated by CTL stimulation assay. Examined. Five of the eight melanoma cell lines induced TNF release by CTL 128 (FIG. 2). On the other hand, no reactivity was seen in the three HLA-A ^* 6801 tumor cell lines from different tissues. HLA-A ^* 6801-negative melanoma, melanocytes, and another histological tumor cell line failed to stimulate cytokine release by CTL128. The response pattern exhibited by CTL 128 on the melanoma cell lines examined did not correlate with the expression of the aforementioned melanoma antigens in these cell lines as measured by RT-PCR. This indicates that the plasmid pcDNA3 / A ^* 6801 containing the HLA-A ^* 6801 gene of the patient No. 18732, and a gene known to encode a melanoma antigen in both cases: Melan-A / MART-1, Tyrosigenase, gp 1
00, TRP-1, 2, MAGE-1, 2, -3, -4, -12, BAGE-1, -2, GAGE-1, -2, -3, -4, -5
-6, confirmed by the absence of TNF release by CTL 128 in the presence of Cos-7 cells co-transfected with each. These results are consistent with the hypothesis that CTL 128, regulated by HLA-A ^* 6801, recognizes a novel antigen common to many melanomas.

A lymphocyte-tumor cell culture solution (MLTC) mixed and cultured for 4 weeks was subjected to limiting dilution and proliferated under the same conditions as those described above (Anichini et al., J. Immunol. 156 (1996) 208-217). Thus, CTL clone 128 was obtained. CTL 128 showed a CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , TCR- ⁺ phenotype as determined by flow cytometry with specific mAbs.

Measurement of cytolytic activity: Chromium release assay described above (Anichini et al., J. Immunol. 156 (1996) 208-2.
17) tested the cytolytic activity of CTL128. The result was shown by the following equation: Here, the spontaneous release was measured by incubating the target cells in the absence of the effector, and the maximum release was measured in the presence of 1% NP-40 surfactant (BDH Biochemicals, Poole, UK). The following mAbs reported in (Anichini et al., J. Immunol. 156 (1996) 208-217) inhibited lysis against autologous melanoma: anti-HLA-A, -B, -C W6 / 32 ( Parham et al., J. Immunol. 123 (1979) 342-349), anti-HLA
-A2, -A69, BB7.2 (Parham and Brodsky, Hum. Immunol. 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 HLA-A ^* 6801 Allele Total RNA was prepared from Me18732 cells by the guanidine-isothiocyanate method using RNAzolB (Cinna / Biotex, South Loop East, TX). Using an oligo-dT primer and a reverse transcriptase derived from Moloney murine leukemia virus without RNase-H activity (MMLV-RT RNase-H Superscript; GIBCO BRL, Gaithersburg, MD) according to the manufacturer's disclosure, Single-stranded cDNA was synthesized for 2 μg of total RNA.

1 U of DynaZyme® (Finnzymes OY, Espoo, Finland) and a primer pair suitable for specifically amplifying the entire coding region of the HLA-A allele and directional cloning PCR corresponding to 300 ng of total RNA
DNA was amplified. After digestion with BamHI and HindIII, the 1.1 kb PCR product was subcloned into the eukaryotic expression vector pcDNA3 (Invitrogen Corporation, Oxon, UK) at the BamHI / EcoRV site.

Using diagnostic restriction enzymes, HLA-A ^* 68011 or A ^* 0201 (18732 patient HLA-A2
Plasmid clones encoding the 8 and -A2 alleles) were identified. And
The sequence of the HLA-A ^* 68011 gene was determined to confirm its association with the published DNA sequence. This plasmid was named pcDNA3 / HLA-A ^* 6801.

[0086] Poly isolated from the cloning Me18732 cells of cDNA encoding melanoma antigen recognized by Example 2 CTL 128 (A) ^- the RNA, an appropriate expression vector (pcDNA3
A cDNA library was constructed in .1). Fast Track
Poly (A) ⁺ RNA was isolated from Me18732 cells using an mRNA extraction kit (Invitrogen). Superscript Choice System Kit using oligo dT primers with a NotI site at the 5 'end (GIBCO BRL, Gai
Thersburg, MD) converted a library by converting 5 μg of poly (A) ⁺ RNA to cDNA. The cDNA was ligated to a BstXI adapter (Invitrogen) and digested with NotI by restriction enzymes. After size fractionation, the mammalian expression vector pcDNA3.1 (Invi
cDNA was unidirectionally cloned into the BstXI / NotI site of C. trogen). After electroporation of the recombinant plasmid into E. coli DH5, ampicillin (100 mg
/ ml).

The library was divided into 1,300 pools of about 100 cDNA clones. Each pool of bacteria is grown to saturation, plasmid DNA is extracted, pcDNA
3 / HLA-A ^* 6801 (100 ng) together with 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) into 1.2 × 10 ⁴ Cos-7 cells. In another experiment, Cos-7 cells were co-transfected with 100 ng of pcDNA3 / A ^* 6801 vector and 100 ng of pcDNAI or pcDSR plasmid containing one cDNA from the following melanoma antigens using the same technique. Performed: Melan-A / MART-1, tyrosigenase, gp100, MAGE-1, 2, -3, -4, -12, BA
GE-1, -2, GAGE-1, -2, -3, -4, -5, -6, TRP-1. 5 ′ untranslated region (UTR) and specificity located at the end of exon 8 Full-length TRP by RT-PCR using specific primers
-2 cDNA was amplified, cloned into pcDNA3 and sequenced to confirm its relevance to published DNA sequences (Yokohama et al., Bioch. Bioph. Acta 1271 (1994
) 317-321). Transfected Cos-7 cells were examined 48 hours later by CTL stimulation assay.

CTL Stimulation Assay The ability to induce TNF production by CTL 128 has been described previously (Traversari et al., Immunogenetics 35 (1992), 145-152) to transform cells, or The stimulator cell line was tested. Briefly, 10% pooled human serum (PHS) and 25 U / ml r-hu-IL2 (EuroCetus, Amsterdam, The
1,500 CTLs were added to microwells containing target cells in 100 l IMDM (BioWhittaker, Walkersville, MD) supplemented with Netherlands). Twenty-four hours later, the supernatant was collected, and WEHI-164.13 (Espevic and Niessen-Meyer, J. Mol.
The TNF content was determined by examining the cytotoxic effect on WEHI cells such as Immunol. Methods 95 (1986) 99-105). WEHI-164.13 cells are 10% FCS / RP
Maintained in MI 1640 culture.

Two days after transfection of replicated microcultures, CTL 128
We screened whether TNF release could be stimulated. DNA from one of the 1,300 pools (pool A255) induced high levels of TNF production (FIG. 3), a finding that was confirmed by a second transfection experiment. The positive pool of bacteria was cloned and the plasmid DNA was co-transfected with the HLA-A ^* 6801 construct as described above. 25 out of 159 clones stimulated TNF release by CTL128. The result obtained from one of these, namely cDNA131, is shown in FIG.

Transfection of melanoma cell line pcDNA3.1 (neomycin resistance gene)
The plasmid melanoma cell line LB-33 was transformed with the cDNA 131 cloned into Invitrogen). Cloned subcell lines were isolated from the G418 resistant transformed population. Using a similar method, melanoma cell lines LB-40, SK23-MEL, and MZ2-MEL, maintained in 10% FCS / DMEM, were transfected with HLA-A ^* 6801 cDNA into G418. Was selected. The expression of the transformed HLA-A ^* 6801 allele in stable transformants was confirmed by flow cytometry with specific mAbs.

HLA-A ^* 6801 ⁺ melanoma cell line LB-33 not recognized by CTL 128 (see FIG. 2)
Acquired the property of inducing TNF release when transformed with cDNA 131 (FIG. 4A) and became susceptible to cell lysis by CTL 128 (FIG. 4B), indicating that tumor cells This indicates that antigen recognition occurs and is distinct from the artificially high expression levels that occur in Cos-7 cells.

The sequence of cDNA 131 proved to be 3548 bp in length. Genbank (G
enBank), the nucleotides 1-994 and 3081-3347 were recently identified as melanoma antigens in the melanocyte lineage, TRP-2 (Wang et al., J. Exp. Med. 184 (1996) 2207-2216) was found to correspond to two non-contiguous regions of the cDNA (Yokohama et al., Bioch. Bioph. Acta.
1217 (1994), 317-321). The first region is the 5'-UTR of the TRP-2 gene, the first exon,
And the second region contained exons 3 and 4, respectively. The nucleotide sequence between nucleotides 995-3080 and the sequence downstream of nucleotide 3347 did not show significant homology to any of the sequences recorded in the databank. Present in cDNA 131 but TRP-2 cDNA
These two regions, which are not present in, carry intron sequences. The sequence of the 10 amino acids adjacent to the exon site was completely identical to the known sequence of the exon-intron binding site of the TRP-2 gene (Sturm et al., Genomics 29 (1995) 24-34). Furthermore, as estimated from a published genome map (Sturm et al., Genomics 29 (1995) 24-34), the sequence at positions 995-3080 (2986 nucleotides) was found to be similar to the sequence of the second intron of TRP-2. It was similar. Therefore, the nucleotide sequence at positions 995 to 3080 matched the second intron of TRP-2. The sequence downstream from nucleotide 3347 of cDNA 131 indicated that the 5 'splice donor sequence was consistent with the fourth intron sequence of TRP-2 (Sturm et al., Genomics 29 (1995) 24-34). ). This sequence lacks the 3 'terminal splicing acceptor site sequence and is considerably shorter than the length estimated from the published genomic map, so that the sequence of the fourth intron of TRP-2 is at nucleotide 200. It has been deleted. Thus, the cDNA 131 is composed of a spliced form of the melanocyte differentiation antigen TRP-2 containing the first to fourth exons while retaining the first part of the second intron and the first part of the fourth intron. (Figure 5).

DNA sequencing and homology search DNA sequencing analysis was performed using the synthetic oligonucleotides as specific primers. The sequencing reaction was performed by dye-labeled dideoxynucleotides and ABI's PRISM® Dye Terminator Cycle Sequencing Ready Reaction Kit (Dye Terminator Cycle Sequencing Ready Reaction Kit).
(Perkin Elmer, Foster City CA). The DNA sequence of plasmid clone cDNA 131 was determined by an automatic DNA sequencer (ABI Prism 377 DNA sequencer: Perkin Elmer). Sequence homology was searched by computer using the EMBL FASTA program in Heidelberg.

Identification of the Sequence Encoding the Antigen Peptide Recognized by CTL 128 To determine the location of the sequence encoding the antigenic peptide recognized by CTL 128, cDNA 131 was cut with HindIII and cut to 1500 and 200, respectively. , And 2000 bp (5′-end-1500, 200 bp, and 3′-end in FIG. 5).

Preparation of partial fragment of cDNA 131 The plasmid was digested with HindIII and BstUI to obtain partial fragments of cDNA 131 (5′-end-1500, 5′-end-800, 200 bp, and 3′-end). After purification on an agarose gel, the fragment was cloned into pcDNAI. From the 5 'end-1500, small fragments INT-2-434, INT-2-166 and INT-2-107 were generated by PCR amplification. Increase all fragments
For breadth, the sense primer KS-INT2 (SEQ ID NO: 5) was used. This ply
Ma also put Kozak's consensus sequence in the same reading frame as TRP-2.
One ATG initiation codon (underlined) is produced. Amplifies INT-2-434, -166, and -107
The antisense primers used for
SP6 (SEQ ID NO: 8), located in the second intron of TRP-2
They are INT2-as1 (SEQ ID NO: 6) and INT2-as2 (SEQ ID NO: 7). Antisense
Primers INT2-as1 and -as2 are used for directional cloning.
It had an XhoI restriction site. Dynazyme (registered trademark) for facilitating ligation and for the terminal transferase activity of DNA polymerase.
The 5 'end of the fragment amplified by (marker) has one A overhang toward the 3' end.
used. The pcDNAI plasmid was digested with EcoRV and then digested with 2 mM dTTP as described by Marchuck et al. in Nucl. Acids Res. 19 (1991) 1154.
By incubating with Dynazyme® in the presence,
One thymidine was added to the 3 'end of each fragment. Cut T-vector and PCR product with XhoI
, Purified on agarose gel and ligated. After ligation,
The plasmid was electroporated into E. coli DH-5 and ampicillin (50 mg /
ml). Isolate clones, extract plasmid DNA, and use HLA-A ^* It was transfected into Cos-7 cells together with the 6801 gene.

In this step, the presence of an initiation codon that regulates translation was not detected in the 5 ′ terminal fragment. In the presence of Cos-7 cells transfected with the 5′-end-1500 fragment (SEQ ID NO: 12), the levels of TNF released by CTL 128 were comparable when stimulated by cDNA 131 (FIG. 5). This indicates that the antigenic peptide is encoded in this region. In the nucleotide sequence of this partial fragment,
SEQ ID NO: 12 contains the first 410 exons of exon 2, exon 2, and intron 2. CTL 128 contains fully spliced TRP-2 cDNA and HLA
It was not stimulated by Cos-7 cells co-transfected with both -A ^* 6801 (FIG. 5). This indicates that all or part of the sequence encoding the antigen recognized by CTL 128 may be located in the intron of the TRP-2 gene present in cDNA 131. The idea is to recognize Cos-7 cells transfected with the 5'-end -800 cDNA fragment containing the first exon and half of the second exon, obtained by cutting the 5'-end-1500 fragment at the BstUI site. This is also supported by the fact that it does not (Figure 5). The presence of the intron in the sequence encoding the antigenic peptide was confirmed by the fact that a PCR-amplified fragment (INT-2-343) containing the first 434 bp of the second intron could result in antigen expression. (Figure 5). In the same reading frame as the exon preceding this region, the HLA-A ^* 6801 peptide binding motif (
An anchor residue (2) corresponding to Rammensee et al., Immunogenetics 41 (1995) 178-228).
The sequence encoding 10 peptides (EVISCKLIKR) (SEQ ID NO: 2) having the 9th, 9th and 10th positions was found.

To further define the region containing the epitope, the sense primer KS-INT2 (SEQ ID NO: 5), which produces ATG with the appropriate Kozak consensus sequence, and the reverse primer INT2-as1 (SEQ ID NO: 5) : 6) and INT2-as2 (SEQ ID NO: 7) to amplify a PCR fragment. The first fragment (INT-2-166) contains the full-length putative peptide sequence, partially deleted in the second fragment (INT-2-107). Only the INT-2-166 fragment, which contains the complete deduced peptide sequence,
NF release could be stimulated (FIG. 5). 10-mer and 9-mer peptides,
EVISCKLIKR (SEQ ID NO: 2) and EVISCKLIK (9-mer peptides have the difference that the last peptide (R) of the 10-mer peptide (SEQ ID NO: 2) is deleted) are synthesized by HLA Incubated with -A ^* 6801 homozygous LCL cell line LB. The 10 peptide EVISCKLIKR was able to sensitize LB cells to be lysed by CTL 128, resulting in a half-maximal cell solubility at a concentration of about 100 pM, but with 9 peptides the efficiency was very low. The control peptide was negative (Fig. 6).

In order to exclude the possibility that the epitope recognized by CTL 128 may be caused by a mutation made in the tumor, the genomic DNA of CTL 128 and another melanoma, MZ2-mel, span the entire second intron (cDNA 131 Nucleotides 995-3080)
The 52 bp fragment was amplified by PCR.

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). The second intron of the TRP-2 gene is designated as KS-INT2 (SEQ ID NO: 5) located in the second intron as a sense primer, and PR2 (SEQ ID NO: 9) located in the third intron. Was used as an antisense primer, and PCR was amplified from 100 ng of genomic DNA. According to the above,
The TRP-2-INT2 fragment was cloned into the pcDNAI vector, sequenced, and HLA-
It was transfected into Cos-7 cells together with the A ^* 6801 gene.

The PCR product was cloned, sequenced and transfected into Cos-7 cells. All clones had the expected sequence and were able to express the antigen.

The sense primer PRIT-I located in the first exon and the second intron (SEQ ID NO:
RT-PCR analysis is performed using 3) and the antisense primer INT2-1260 (SEQ ID NO: 4) to amplify a 977 bp fragment only from the TRP-2 transcript carrying the second intron.

Example 4 PCR Analysis for Expression of TRP-2-INT2 and TRP2 Total RNA from cultured cell lines, freshly harvested skin, retina, and tumor samples using RNAzolB by guanidine-isothiocyanate method Was prepared. CDNA corresponding to 300 ng of total RNA was combined with 1 U of DynaZyme® (Finnzymes)
Amplification was performed by PCR using mM primers in a final volume of 50 ml. The reaction mixture was subjected to 30 cycles of amplification. To amplify the TRP-2 cDNA, PR3 (SEQ ID NO: 10) located in the second exon and TRP-2L (SEQ ID NO:
11) was used as a sense primer and an antisense primer, respectively.
PCR was performed for 30 cycles (94 ° C for 1 minute, 58 ° C for 1 minute, and 72 ° C for 1 minute). To confirm the expression of the second intron without splicing, 5'U
The sense primer PRIT-1 (SEQ ID NO: 3) and the antisense primer INT2-1260 (SEQ ID NO: 4) located in TR and the second intron were used. PCR was performed for 30 cycles (94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute). To detect TRP-2, the primer should be located in a remote exon,
In order to prevent amplification from contaminated genomic DNA and amplify TRP-2-INT2, a 10 kb long first intron exists between the first and second exons (Sturm et al., Ge
nomics 29 (1995) 24-34), amplification from contaminated genomic DNA was avoided. Actin cDNA was amplified by PCR using specific primers, and the quality of the RNA preparation was checked.

[0103] Fully spliced TRP-2 by sense primer PR3 (SEQ ID NO: 10) and antisense primer TRP-2L (SEQ ID NO: 11) located in exons 2 and 8, respectively. Messenger expression was detected.

Among the tumors examined, only melanoma proved to be positive for the TRP-2 and TRP-2-INT2 antigens (Table 1). When TRP-2 is not expressed, TRP-
No expression of 2-INT2 was seen. This result indicates that TRP-2 is a melanocyte differentiation antigen (Wang et al., J. Exp. Med. 184 (1996) 2207-2216) and that the same promoter synthesizes a common messenger that produces both antigens. Is consistent with the concept of working. When analyzing the melanoma sample immediately after collection, 69% expressed TRP-2,
78% of TRP-2 ⁺ melanomas also expressed TRP-2-INT2 (Table 1). This result was also observed in melanoma cell lines, with normal TRP-2 and those retaining the second intron expressed in 84% and 68% of the analyzed cell lines, respectively. Normal tissues known to have TRP-2 were analyzed for TRP-2-INT2 expression. Three melanocyte cell lines, four skin samples, and one retinal sample were negative in the RT-PCR assay (Table 1). When four skin samples were compared with a biopsy of a primary lesion taken from the same patient, TRP-2 was detected in all samples, but TRP-2-I
NT2 was exclusively present in tumor specimens only.

[0105]

[Table 1] Expression of TRP-2-INT2 and TRP2 in tumor tissue and normal tissue * TRP-2-INT2 expression was detected only in TRP-2 positive samples.

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

Presentation of antigenic peptides by HLA-A3-like supertype alleles HLA-A ^* 6801, together with A3, A11: A31, and A ^* 3301, are HLA-A alleles with similar peptide binding properties Belongs to the HLA-A3-like super type (Sidney
JM et al., Immunol. 154 (1995) 247-259). Peptide EVISCKLIKR (SEQ ID NO: 2)
EBV LCs expressing such alleles were tested to determine if they could be represented in CTL 128 by the HLA-A ^* 6802 allele of the A3-like supertype.
L was pulsed with this peptide and used as a target for CTL 128 (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, A ^* 3301
Only was able to present the TRP-2-INT _222-223 peptide with the same efficiency as A ^* 6801. When the peptide was presented by A ^* 6802, the level of recognition was observed to decrease with increasing concentration.

Example 5 Antigen Peptide and CTL Assay Peptides were synthesized on a solid phase using Fmoc to temporarily protect the NH ₂ terminus and characterized by mass spectrometry (Primm, Milan) , Italy). All peptides were greater than 90% pure, as indicated by analytical HPLC. The freeze-dried peptide was dissolved at 10 mM concentration to contain 10% DMSO and stored at -80 ° C. ^{The 51} Cr-labeled target cells were incubated at room temperature in a 96-well microplate with various concentrations of peptide before adding CTL 128 at an effector / target ratio of 20: 1.
The peptides were tested by an 1 hour incubation assay. After 4 hours, cell lysis was measured. Antigen peptide presentation was also tested in a TNF release assay. Briefly, stimulator cells are incubated with a fixed concentration of peptide for 1 hour at room temperature, followed by extensive washing, CTL 128, and WEHI
TNF release was measured on 164.13 cells after 18-20 hours.

[Sequence list]

[Brief description of the drawings]

FIG. 1. CTL 128 is a form of autologous melanoma (M) restricted to HLA-A ^* 6801.
e18732). The target cells were incubated with the following anti-HLA monoclonal antibody (mAb) for 1 hour at room temperature, and then the effector was added to a constant E: T ratio (A: Me18732 only; B: Me18732 + HLA class I; C ： Me18732 + HLA-A2, -A
69; D: ME18732 + HLA-A2, -A28; E: Me18732 + HLA-B, -C).

FIG. 2. Recognition of autologous melanoma Me18732 and HLA-A ^* 6801 + melanoma cell lines by CTL 128. 1,500 CTLs were added to 25,000 stimulatory cells, and the TNF content in the supernatant was examined on WEHI-164.13 cells after 24 hours (melanoma: A: Me18732)
B: Me20842; C: Me17697; D: Me2559 / 1; E: Me12657; F: Me17088; G: Me4023
H: LB-33; I: Lung cancer Calu 3; K: Breast cancer SKBR3; L: Ovarian cancer SKOV3).

FIG. 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 A255 pools or with cDNA131. The A255 pool is a group of 100 cDNA clones from the Me18732 cDNA library, which were amplified to saturation and plasmid DNA was extracted. cDNA 131 is a single clone subcloned from the A255 pool. Twenty hours after co-culture with the transfected cells, TNF production by CTL 128 was measured using the TNF-sensitive cell line WEHI-
Measured using 164.13. As a control, Cos-7 cells were treated with cDNA 131 or HLA-A ^* 680.
Transfection with only 1 (A: Me18732; B: COS; C: COS + A ^* 6801; D: COS + CDNA131; E: COS + A ^* 6801 + cDNA131).

FIG. 4. cDNA 131 encodes an antigen recognized by CTL 128. (A) Recognition by the geneticin-resistant population CTL128 of LB33 in the HLA-A ^* 6801 melanoma cell line by CTL128 after transfection with the pcDNA3.1 / cDNA131 construct, and (B) cell lysis. TNF secretion by CTL 128 was measured 24 hours after co-culturing 1,500 responder cells with 20,000 stimulator cells. The lytic activity of CTL 128 was measured on ⁵¹ Cr-labeled target cells after 4 hours of co-incubation with CTL with different effector to target (E / T) ratios.

FIG. 5: Identification of the sequence encoding the antigenic peptide recognized by CTL128. The exon / intron structure of cDNA 131 is shown at the top of panel (A). Exons and introns are indicated by black and white squares, respectively. The horizontal lines at both ends indicate the pcDNA3.1 vector, and the numbers in the sequence are those counted from the 5 'end of cDNA 131. cDNA 1
31 and partial fragments from the PCR product are shown as white rectangles below cDNA 131, which were cloned into the expression vector and transferred to Cos-7 cells for HLA-A ^* 680.
Transfected with 1 cDNA. Screening of the 18732 cDNA library using the 8th exon-specific oligonucleotide probe,
A spliced full-length TRP-2 cDNA was obtained. TNF release by CTL 128 was assessed on WEHI-164.13 cells (B). The sequence coding for the peptide present in the PCR fragments INT-2-I66 and INT-2-I07 is described.

FIG. 6. CTL of HLA-A ^* 6801 cells pulsed with synthetic antigenic peptide
Lysis by 128. 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 synthetic peptides at the indicated concentrations on the left. After 4 hours, ⁵¹ Cr release was measured. As a negative control, a peptide (M3A1) derived from MAGE-3 that can bind to HLA-A1 was used.

FIG. 7. T when presented by the A3-like supertype HLA allele.
RP-2-INT2_221-231Recognition of peptides by CTL 128.⁵¹Cr-labeled EBV-LCL is TRP-2-INT2 _221-231 The peptides were incubated with CTL 128 at an E / T ratio of 20: 1 in various concentrations of the peptide. Chromium release was measured after 4 hours. c: peptide-free shade
Sex control.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/705 C12Q 1/68 A 4H045 C12Q 1/68 C12N 15/00 ZNAA (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, (KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU , CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Sensi Maria Luisa Milano Italy Piazza Donegani 6 (72) Inventor Traversari Katia Sesto S. Italy. Giovanni Ferris Cavarotti 229 F-term (reference) 4B024 AA01 AA12 BA36 CA04 DA02 EA04 GA11 GA18 HA01 HA12 HA17 4B063 QA01 QA19 QQ02 QQ08 QQ13 QQ53 QR32 QR40 QR48 QR62 QR69 QR72 QR77 QR80 QS12 QS25 4C08A A012A 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 ZB02 ZB26 4H045 AA10 AA30 BA10 CA41 DA86 EA28 EA51 FA74

Claims (11)

[Claims] 1. A tumor-specific polypeptide antigen which is partially encoded by an intron of an exon-encoded tumor antigen, comprising the following (a) and (
Antigens obtained by b): a) Nucleic acids that hybridize under stringent conditions to the intron sequences of tumor antigens encoded by exons in reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of tumor cells Reverse transcriptase using fragments as primers
PCR, and b) isolating the PCR product, expressing the PCR product or a fragment thereof in a host cell, and encoding the PCR product or a fragment thereof that also hybridizes to an exon sequence of the tumor-specific antigen. Isolation of tumor-specific antigen. 2. The tumor-specific polypeptide antigen according to claim 1, wherein a fragment of 8 to 12 codons of the PCR product is used for expression. 3. The exon-encoded tumor antigen is MAGE, BAGE, and GAGE, and a CTL recognition epitope derived from tyrosinase, MelanA ^Mart1 , gp100 ^Pme17 , gp75 ^TRP1 , and TRP-2. 3. The tumor-specific antigen according to 1 or 2. 4. A tumor-specific polypeptide antigen encoded by SEQ ID NO: 1. 5. A method for isolating mRNA of a tumor-specific antigen, a part of which is encoded by an intron of a tumor antigen encoded by an exon, comprising the following steps (a) and (b): Methods performed: a) In reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of tumor cells, a primer is used to prime a nucleic acid fragment that hybridizes under stringent conditions to the intron sequence of the tumor antigen encoded by exons. Transcriptase used as
PCR, and b) isolation of the PCR product that also hybridizes to the exon sequence of the antigen. 6. A method for measuring the proliferation of a tumor-specific cytotoxic T cell, which comprises using the tumor-specific antigen according to claim 1 and an antigen-presenting cell and a cytotoxic T cell. A method for measuring the proliferation of cytotoxic T cells by adding to a patient body fluid sample containing the same. 7. Use of a nucleic acid encoding a tumor-specific antigen according to claim 1 for the manufacture of a therapeutic agent for treating a tumor disease. 8. Use of a tumor-specific antigen according to claims 1 to 4 for activating cytotoxic T cells from T cell precursors in vivo or in vitro. 9. A method for producing a tumor-specific polypeptide antigen, wherein said tumor-specific antigen is obtained by the following (a) and (b): a) from a soluble cytoplasmic fraction of tumor cells In reverse transcriptase PCR from isolated mRNA, reverse transcriptase PC using as a primer a nucleic acid fragment that hybridizes under stringent conditions to an intron sequence of a tumor antigen encoded by an exon
R, and b) isolating the PCR product, expressing the PCR product or a fragment thereof in a host cell, and encoding the PCR product or a fragment thereof that hybridizes to an exon sequence of the tumor-specific antigen. Isolation of said tumor-specific antigen. 10. A combination of two nucleic acids that hybridizes under stringent conditions to an intron sequence of a tumor antigen encoded by an exon and can be used as a primer pair for reverse transcriptase PCR from mRNA. 11. The nucleic acid encoded by SEQ ID NO: 3 to SEQ ID NO: 9.