Receptor gene for peptide cancer antigen-specific T cell

#The present invention relates to a polynucleotide contained in the gene for a cancer antigen-specific T-cell receptor, and the peptide encoded by the polynucleotide, and to cancer tests, e.g., diagnosis, prognosis, or treatment monitoring using the same, cancer therapy, and the like.

#Cancer treatments include extirpation of cancer by surgical operation, radiotherapy, treatment with anti-cancer medication, and immunotherapy. Among these treatments, immunotherapy in particular has drawn attention in recent years because it is a treatment more selective and specific against cancer with least side effects. Inter alia, attempts have been extensively made to treat cancer and leukemia by targeting WT1 protein, which is abundantly present in many types of cancer cells and leukemia cells. In order to study the mechanism of the WT1-targeted anti-cancer therapy and to further increase the effect of the therapy, it is necessary to identify the nucleotide sequences of the T-cell receptor (TCR) genes of WT1-specific cytotoxic T-cells (CTL) and the amino acid sequences of the receptor peptides encoded by the nucleotide sequences. However, up to the present date, there is little information about the sequences of those receptor genes and the receptor peptides, and uses thereof although a number of researches have been conducted (see, e.g., patent document 1 and non-patent documents 1-5).

#Patent Document 1: WO 2008/108257 A1

#FIG. 1-1 shows the nucleotide and amino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs), the number of clone (clonality) thereof and the like of WT1-specific cytotoxic T-cells (CTLs) from healthy individuals and HLA-A*0201-positive solid cancer patients, which sequences have been identified in the present invention (DNA disclosed as SEQ ID NOS: 1-68 and peptides disclosed as SEQ ID NOS: 757-824, respectively, in order of appearance). In the figure, “clone #” indicates the clone numbers, the numbers to the left of the dot indicate the family numbers of Vβ chains, and the numbers to the right of the dot indicate the reference numbers. The column “name” indicates the subjects from which the clone derived, HD1, HD2, HD3, HD4, and HD5 indicate healthy individuals, respectively, and PT1, PT2, PT3, PT4, PT5 and PT6 indicate solid cancer patients and indicate the patients with glioblastoma, primitive neuroectodermal tumor, glioblastoma, ovarian cancer, cecal cancer, and glioblastoma, respectively. V name, J name, and D name indicate the details of the V region, J region, and D region, respectively. The nucleotide sequences are indicated by portion which derives from V region, N1 region, D region, (P)N2 region, or J region. The column to the right of the nucleotide sequence indicates what kind of region the sequence which constitutes the nucleotide sequence was derived from. The amino acid sequence encoded by each nucleotide sequence is indicated by 1 letter code known to those skilled in the art. Two columns to the right of the amino acid sequence indicate clonality in each healthy individual and clonality in each cancer patient. The rightmost column indicates the total of clonality of each clone.

#FIG. 1-2 shows the nucleotide and amino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs), the number of clone (clonality) thereof and the like of WT1-specific cytotoxic T-cells (CTLs) from healthy individuals and HLA-A*0201-positive solid cancer patients, which sequences have been identified in the present invention (DNA disclosed as SEQ ID NOS: 69-141 and peptides disclosed as SEQ ID NOS: 825-897, respectively, in order of appearance). In the figure, “clone #” indicates the clone numbers, the numbers to the left of the dot indicate the family numbers of Vβ chains, and the numbers to the right of the dot indicate the reference numbers. The column “name” indicates the subjects from which the clone derived, HD1, HD2, HD3, HD4, and HD5 indicate healthy individuals, respectively, and PT1, PT2, PT3, PT4, PT5 and PT6 indicate solid cancer patients and indicate the patients with glioblastoma, primitive neuroectodermal tumor, glioblastoma, ovarian cancer, cecal cancer, and glioblastoma, respectively. V name, J name, and D name indicate the details of the V region, J region, and D region, respectively. The nucleotide sequences are indicated by portion which derives from V region, N1 region, D region, (P)N2 region, or J region. The column to the right of the nucleotide sequence indicates what kind of region the sequence which constitutes the nucleotide sequence was derived from. The amino acid sequence encoded by each nucleotide sequence is indicated by 1 letter code known to those skilled in the art. Two columns to the right of the amino acid sequence indicate clonality in each healthy individual and clonality in each cancer patient. The rightmost column indicates the total of clonality of each clone.

#FIG. 1-3 shows the nucleotide and amino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs), the number of clone (clonality) thereof and the like of WT1-specific cytotoxic T-cells (CTLs) from healthy individuals and HLA-A*0201-positive solid cancer patients, which sequences have been identified in the present invention (DNA disclosed as SEQ ID NOS: 142-214 and peptides disclosed as SEQ ID NOS: 898-970, respectively, in order of appearance). In the figure, “clone #” indicates the clone numbers, the numbers to the left of the dot indicate the family numbers of Vβ chains, and the numbers to the right of the dot indicate the reference numbers. The column “name” indicates the subjects from which the clone derived, HD1, HD2, HD3, HD4, and HD5 indicate healthy individuals, respectively, and PT1, PT2, PT3, PT4, PT5 and PT6 indicate solid cancer patients and indicate the patients with glioblastoma, primitive neuroectodermal tumor, glioblastoma, ovarian cancer, cecal cancer, and glioblastoma, respectively. V name, J name, and D name indicate the details of the V region, J region, and D region, respectively. The nucleotide sequences are indicated by portion which derives from V region, N1 region, D region, (P)N2 region, or J region. The column to the right of the nucleotide sequence indicates what kind of region the sequence which constitutes the nucleotide sequence was derived from. The amino acid sequence encoded by each nucleotide sequence is indicated by 1 letter code known to those skilled in the art. Two columns to the right of the amino acid sequence indicate clonality in each healthy individual and clonality in each cancer patient. The rightmost column indicates the total of clonality of each clone.

#The inventors have stained the peripheral lymphocytes from cancer patients with WT1 tetramer that consists of WT1 peptide/HLA-A*0201 complexes, and has separated WT1 tetramer-positive cells one by one using a FACS; cDNAs have been generated from each separated cell, and the nucleotide sequences encoding CDR3 contained in the Vβ chain of T-cell receptors (hereinafter may be referred to as “TCR”) of WT1-specific cytotoxic T-cells, hereinafter may be referred to as “WT1-specific CTL”, have been determined by applying PCR method (FIGS. 1-1 to 1-9 and FIGS. 2-1 to 2-13; SEQ ID Nos.: 1-756). From these results, the amino acid sequences of the CDR3 have been also determined (FIGS. 1-1 to 1-9 and FIGS. 2-1 to 2-13; SEQ ID Nos.: 757-1512). These sequences have been determined for the first time in the present invention.

#Thus, in one aspect, the present invention provides a polynucleotide having the nucleotide sequence encoding CDR3 of the Vβ chain of T-cell receptors (TCRs) of WT1-specific cytotoxic T-cells (CTLs) obtained from healthy individuals and HLA-A*0201-positive patients, wherein the polynucleotide has DNA having any of the CDR3 nucleotide sequences shown in SEQ ID Nos.: 1-756, RNA complementary to the DNA, or a complementary sequence thereof. As used herein, these DNA and RNA molecules and the complementary polynucleotides thereof are collectively referred to as “CDR3 polynucleotides.” For example, the CDR3 polynucleotides include, in addition to the DNAs comprising the nucleotide sequences shown in SEQ ID Nos.: 1-756, DNAs comprising these sequences. Also, for example, the CDR3 polynucleotides include, in addition to the RNAs complementary to the DNAs comprising the nucleotide sequences shown in SEQ ID Nos.: 1-756, RNAs comprising these sequences. Further, for example, the CDR3 polynucleotides include polynucleotides having the sequences of the DNAs or RNAs, and polynucleotides complementary to the RNAs. The “CDR polynucleotides” include those having degenerate sequences encoding “CDR3 peptides.”

#In another aspect, the present invention provides peptides having the amino acid sequences of CDR3 of the Vβ chain of T-cell receptors (TCRs) of WT1-specific cytotoxic T-cells (CTLs), which CDR3 amino acid sequences being shown in any of SEQ ID Nos.: 757-1512. As used herein, these peptides are collectively referred to as “CDR3 peptides.” For example, the CDR3 peptides include, in addition to a peptide comprising any of the CDR3 amino acid sequences shown in SEQ ID Nos.: 757-1512, peptides comprising these CDR3 amino acid sequences (such as, for example, Vβ chain peptides or a portion thereof). In addition, a peptide consisting or comprising the amino acid sequence shown in SEQ ID Nos.: 757-1512 in which one or a few, preferably one to three, one or two, or one amino acid is substituted, added, or deleted is included in the “CDR3 peptides.” However, these peptides are required to have equivalent functions to the original peptide. These CDR3 peptides are encoded by the above-mentioned CDR3 polynucleotides.