Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-12-07
2004-11-09
Helms, Larry R. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S327000, C530S328000
Reexamination Certificate
active
06815531
ABSTRACT:
TECHNICAL FIELD
The present invention relates to medicines for activating antitumor immunity and for treating autoimmune diseases as well as to diagnosis of tumors or autoimmune diseases. In particular, the present invention relates to novel tumor antigen proteins, novel genes therefor, novel tumor antigen peptides, and the like.
PRIOR ART
It is known that the immune system, particularly T cells, plays an important role in vivo in tumor rejection. Indeed, infiltration of lymphocytes having cytotoxic effects on tumor cells has been observed in human tumor foci (
Arch. Surg.,
126:200-205, 1990), and cytotoxic T lymphocytes (CTLs) recognizing autologous tumor cells have been isolated from melanomas without great difficulties (e.g.,
Immunol. Today,
8:385, 1987;
J. Immunol.,
138:989, 1987; and
Int. J. Cancer,
52:52-59, 1992). In addition, the results of clinical treatment of melanomas by T cell introduction also suggest the importance of T cells in tumor rejection (
J. Natl. Cancer. Inst.,
86:1159, 1994).
Although it has long been unknown about target molecules for CTLs attacking autologous tumor cells, the recent advance in immunology and molecular biology has gradually begun elucidating such target molecules. Specifically, it has been found that using T cell receptors (TCRs), CTL recognizes a complex consisting of tumor antigen peptide and major histocompatibility complex (MHC) class I antigen, and thereby attacks autologous tumor cells.
Tumor antigen peptides are generated from tumor antigen proteins. Thus, the proteins are intracellularly synthesized and then degraded in cytoplasm into the peptides by proteasome. On the other hand, MHC class I antigens formed at endoplasmic reticulum bind to the above tumor antigen peptides, and are transported via cis Golgi to trans Golgi, i.e., the mature side, and expressed on the cell surface (
Rinsho-Menneki,
27(9):1034-1042, 1995).
As such a tumor antigen protein, T. Boon et al. identified a protein named MAGE from human melanoma cells for the first time in 1991 (
Science,
254:1643-1647, 1991), and thereafter several additional tumor antigen proteins have been identified from melanoma cells.
As described in the review by T. Boon et al. (
J. Exp. Med.,
183, 725-729, 1996), tumor antigen proteins hitherto identified can be divided into the following four categories.
Tumor antigen proteins belonging to the first category are those proteins which are expressed only in testis among normal tissues, while they are expressed in melanoma, head and neck cancer, non-small cell lung cancer, bladder cancer and others, among tumor tissues. Among tumor antigen proteins in this category are the above-described MAGE and analogous proteins constituting a family of more than 12 members (
J. Exp. Med.,
178:489-495, 1993), as well as BAGE (
Immunity,
2:167-175, 1995) and GAGE (
J. Exp. Med.,
182:689-698, 1995), all of which have been identified from melanoma cells.
Although some of such tumor antigen proteins in this category are highly expressed in melanoma, their expression is observed in only 10 to 30% of patients having a particular tumor other than melanoma, and therefore, they can not be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the second category are those proteins which are expressed only in melanocytes and retina among normal tissues, while the expression is observed only in melanomas among tumor tissues. Since these tissue-specific proteins are highly expressed in melanomas, they function as tumor antigen proteins specific for melanomas. Among tumor antigen proteins in this category are tyrosinase (
J. Exp. Med.,
178:489-495, 1993), MART-1 (
Proc. Natl. Acad. Sci. USA,
91:3515, 1994), gp100 (
J. Exp. Med.,
179:1005-1009, 1994), and gp75 (
J. Exp. Med.,
181:799-804, 1995), genes for which have all been cloned from melanoma cells. Additionally and separately identified Melan-A (
J. Exp. Med.,
180:35, 1994) has proved to be the same molecule as MART-1.
However, the tumor antigen proteins in this category can not be used widely for treatments or diagnoses of various tumors, since they are not expressed in tumors other than melanoma.
Tumor antigen proteins belonging to the third category are those proteins which yield, through tumor-specific mutations, tumor antigen peptides newly recognized by CTL. Among tumor antigen proteins in this category are mutated CDK4 (
Science,
269:1281-1284, 1995), &bgr;-catenin (
J. Exp. Med.,
183:1185-1192, 1996), and MUM-1 (
Proc. Natl. Acad. Sci. USA,
92:7976-7980, 1995). In CDK4 and &bgr;-catenin, a single amino acid mutation increases the binding affinity of the peptides to MHC class I antigen, and allows them to be recognized by T cells. In MUM-1, its intron normally untranslated is translated due to mutation, and the peptide thus generated is recognized by T cells. However, since such mutations occur at low frequency, they can not be applied widely to treatments or diagnoses of various tumors.
As a tumor antigen protein belonging to the fourth category, P15 has been identified from melanoma cells, which is a protein widely expressed in normal tissues and which is also recognized by CTL (
J. Immunol.
154:5944-5955, 1995).
Tumor antigen proteins or peptides hitherto known have been identified as follows.
In such identification, a set of tumor cells and CTLs attacking the tumor cells (usually established from lymphocytes of the same patient from whom the tumor cells are obtained) are firstly provided. Then, the cells from this set are used to directly identify tumor antigen peptides, or used to determine the gene encoding the tumor antigen protein from which corresponding tumor antigen peptides are identified.
Specifically, in the case where tumor antigen peptides are directly identified, tumor antigen peptides bound to MHC class I antigens in tumor cells are extracted under acidic conditions, and separated into various peptides using high-performance liquid chromatography. Cells expressing MHC class I antigen, but not expressing tumor antigen protein (for example, B cells from the same patient), are then pulsed with such various peptides, and examined for their reactivity with CTL to identify tumor antigen peptides. Then, the sequences of the peptides thus identified are further determined by, for example, mass spectrometry. In this way, tumor antigen peptides derived from Pmel 17 which is the same molecule as gp100 have been identified from melanoma cells (
Science,
264:716-719, 1994).
In order to firstly determine the gene encoding tumor antigen protein and then to identify therefrom corresponding tumor antigen peptides, the gene encoding tumor antigen protein may be cloned using molecular biological techniques. cDNAs are prepared from tumor cells, and cotransfected with MHC class I antigen gene into cells not expressing tumor antigen proteins (for example, COS cells), in order to express them transiently. The products thus expressed are then repeatedly screened on the basis of their reactivity with CTL, until the gene encoding tumor antigen protein may finally be isolated. In this way, the genes for the above-mentioned MAGE, tyrosinase, MART-1, gp100, and gp75 have been cloned.
In order to deduce and identify the presented tumor antigen peptides actually bound to MHC class I antigens on the basis of the information about such tumor antigen gene, the methods as described below are used. Firstly, fragments of the gene encoding tumor antigen protein, having various sizes, are prepared using, for example, PCR, exonucleases, or restriction enzymes, and cotransfected with MHC class I antigen gene into cells not expressing tumor antigen proteins (e.g., COS cells), in order to express them transiently. The region(s) which include tumor antigen peptides are then identified on the basis of their reactivity with CTL. Subsequently, peptides are synthesized. Cells expressing MHC class I antigen but not expressing tumor antigen proteins are then pulsed with the synthesized peptides, and examined for their reactions with CTL to identify the tumor antig
Imai Yasuhisa
Itoh Kyogo
Shichijo Shigeki
Birch & Stewart Kolasch & Birch, LLP
Helms Larry R.
Itoh Kyogo
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