WO2021060343A1 - T-cell receptor expressed by human t-cells that recognize peptide derived from urlc10 or peptide derived from depdc1 - Google Patents

T-cell receptor expressed by human t-cells that recognize peptide derived from urlc10 or peptide derived from depdc1 Download PDF

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WO2021060343A1
WO2021060343A1 PCT/JP2020/035947 JP2020035947W WO2021060343A1 WO 2021060343 A1 WO2021060343 A1 WO 2021060343A1 JP 2020035947 W JP2020035947 W JP 2020035947W WO 2021060343 A1 WO2021060343 A1 WO 2021060343A1
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seq
tcr
cells
urlc10
cdr3
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PCT/JP2020/035947
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French (fr)
Japanese (ja)
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在賢 朴
祥子 山下
哲郎 引地
陳之 井手
盛男 柳樂
啓司 土肥
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オンコセラピー・サイエンス株式会社
塩野義製薬株式会社
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Priority claimed from JP2019172706A external-priority patent/JP2022191532A/en
Priority claimed from JP2019172705A external-priority patent/JP2022191531A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the field of biological science, more specifically to the field of cancer treatment.
  • the present invention utilizes the amino acid sequence of a T cell receptor (TCR) that recognizes a cancer antigen-derived peptide presented with a major histocompatibility complex (MHC) class I molecule on the surface of cancer cells and the TCR.
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • immune checkpoint inhibitors anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA-4 antibody
  • Typical examples of immune checkpoint inhibitors are new for various cancers such as malignant melanoma, non-small cell lung cancer and Hodgkin lymphoma. Approved as a therapeutic drug.
  • the response rate is not high at about 20 to 30%. Therefore, it is desired to develop further immunotherapy for many patients who do not have the therapeutic effect of immune checkpoint inhibitors.
  • Non-Patent Document 1 TCR gene transfer T cell therapy. So far, the therapeutic effect of TCR gene transfer T cell therapy targeting the cancer antigen NY-ESO-1 on multiple myeloma has been reported (Non-Patent Document 1). However, as a result of TCR gene-introduced T cell administration, there have been reports of serious adverse events that may have been caused by T cells recognizing peptides presented on MHC class I molecules of normal cells through TCR. (Non-Patent Document 2; Non-Patent Document 3).
  • URLC10 Up-regulated in lung cancer 10, alias: lymphocyte antigen 6 complex, locus K (LY6K), GenBank accession number: NM_017527, or accession number: BC117142
  • LY6K lymphocyte antigen 6 complex
  • GenBank accession number: NM_017527 accession number: BC117142
  • URLC10 is genome-wide by cDNA microarray for 27,648 genes. From the results of gene expression profiles, it has been reported as a gene whose expression is upregulated in non-small cell lung cancer and esophageal squamous cell carcinoma (Non-Patent Document 4). Expression of URLC10 was observed in cancer tissues in 88.2% of patients with non-small cell lung cancer (358 of 406 cases) and 95.1% of patients with esophageal cancer (252 of 265 cases). On the other hand, the only normal organ in which URLC10 expression was observed was the testis.
  • DEPDC1 (DEP domain containing 1, GenBank accession number: NM_001114120, or accession number: BM683578) has been reported as a gene whose expression is upregulated in bladder cancer (Non-Patent Document 5). Expression of DEPDC1 was observed in cancer cells in 81% of bladder cancer patients (17 of 21 cases). On the other hand, the testis was the only normal tissue in which DEPDC1 expression was observed. That is, cancer cell-specific molecules such as URLC10 and DEPDC1 are suitable therapeutic targets. against this background, a peptide that induces URLC10-specific cytotoxic T cells (CTL) has been identified (Patent Document 1). The identified URLC10 peptide is useful for cancer vaccine therapy targeting URLC10.
  • CTL cytotoxic T cells
  • a DEPDC1 peptide that enables cancer vaccine therapy has been identified (Patent Document 2).
  • the URLC10 peptide and DEPDC1 peptide have antitumor effects by inducing cytotoxic T cells (CTL).
  • CTL cytotoxic T cells
  • An object of the present invention is the identification of a TCR capable of imparting a cytotoxic effect specific to URLC10 or DEPDC1.
  • URLC10 and DEPDC1 Due to its cancer-specific high expression, URLC10 and DEPDC1 have excellent properties as target antigens for cancer immunotherapy.
  • the URLC10 peptide, or DEPDC1 peptide, identified as CTL-inducible peptides from these molecules has been shown to enable cancer immunotherapy via CTL induction.
  • the present inventors considered that if the TCR possessed by CTLs induced by these peptides could be isolated, TCR gene transfer T cell therapy could be realized instead of the induction of CTLs by peptides. Then, in fact, they succeeded in identifying a TCR having a cytotoxic effect specific to URLC10 and DEPDC1, and came to the present invention. That is, the present invention relates to each of the following aspects.
  • T cell receptor ⁇ chain containing CDR3 identified by any of the selected amino acid sequences, or a CDR3 functionally equivalent thereto.
  • a T cell receptor comprising a combination of any T cell receptor ⁇ chain according to [1] and any T cell receptor ⁇ chain according to [2] or [3].
  • T cell receptor according to [4], wherein the amino acid sequence of CDR3 of the T cell receptor ⁇ chain and the T cell receptor ⁇ chain is any combination of the following: CDR3 of T cell receptor ⁇ chain CDR3 of T cell receptor ⁇ chain SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 2 SEQ ID NO: 3, SEQ ID NO: 14 SEQ ID NO: 15, SEQ ID NO: 16 SEQ ID NO: 10, SEQ ID NO: 17 SEQ ID NO: 26, SEQ ID NO: 60 SEQ ID NO: 63, SEQ ID NO: 69 SEQ ID NO: 71, SEQ ID NO: 77 SEQ ID NO: 80, and SEQ ID NO: 84 SEQ ID NO: 86.
  • [6] A polynucleotide encoding any T cell receptor ⁇ chain according to [1] and any T cell receptor ⁇ chain according to [2] or [3].
  • [7] An expression vector containing the polynucleotide according to [6].
  • [8] The expression vector according to [7], which contains both a polynucleotide encoding a T cell receptor ⁇ chain and a polynucleotide encoding a T cell receptor ⁇ chain.
  • An expression vector comprising the expression vector according to [8] or [9] for inducing cytotoxic T cells having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells.
  • a composition for inducing cytotoxic T cells which comprises the expression vector according to any one of [8] to [10].
  • the expression vector described in any of [8] to [10] is introduced into CD8 + (CD8 positive) T cells collected from a subject requiring treatment for cancer, and HLA for URLC10 or DEPDC1-expressing cells is introduced.
  • -A24 A method for producing cytotoxic T cells, which comprises a step of recovering cytotoxic T cells having a binding cytotoxic effect.
  • the present invention is a cell having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells of a polynucleotide encoding either the T cell receptor ⁇ chain or the T cell receptor ⁇ chain of the present invention.
  • the present invention has an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells of the polynucleotide encoding either the T cell receptor ⁇ chain or the T cell receptor ⁇ chain of the present invention.
  • compositions for inducing cytotoxic T cells With respect to use in the manufacture of compositions for inducing cytotoxic T cells.
  • the TCR of the present invention was obtained from T cells considered to have a cytotoxic effect specific to URLC10 or DEPDC1. Therefore, by introducing the TCR of the present invention into T cells, it is possible to impart a cytotoxic effect specific to URLC10 or DEPDC1. Since URLC10 or DEPDC1 is a tumor antigen that is specifically overexpressed in cancer, the T cells into which the TCR of the present invention has been introduced can be expected to have a cancer-specific cytotoxic effect.
  • the TCR of the present invention is an index of T cells having a cytotoxic effect specific to URLC10 or DEPDC1. That is, it is possible to analyze the frequency of TCR of the present invention in a subject to which the URLC10 or DEPDC1 vaccine has been administered, and to know that effective T cells are induced by vaccination when the ratio is increased. ..
  • FIG. 1 consists of the results (a) and (b) of the tetramer assay performed on PBMCs after culture (in vitro stimulation).
  • HLA-A * 24: 02 A URLC10 tetramer-positive CD8-positive T cell population recognizing the URLC10-derived peptide presented above was detected.
  • the URLC10 tetramer-positive CD8-positive T cell population was collected using the sorting function of the flow cytometer and then used as a sample for TCR repertoire analysis.
  • FIG. 2 consists of pie charts (a) and (b) showing the results of TCR repertoire analysis of the URLC10 tetramer-positive CD8-positive T cell population. The pie chart shows the frequency of detection of TCR chronotypes detected by TCR repertoire analysis.
  • FIG. 3 is composed of line graphs (a)-(c) showing IFN- ⁇ production of CTL clones established by the limiting dilution method from PBMC after culture (in vitro stimulation). While IFN- ⁇ production of CTL clones was observed in target cells pulsed with URLC10-derived peptide, no significant IFN- ⁇ production of CTL clones was observed in target cells not pulsed with peptide.
  • FIG. 4A consists of the results (a)-(f) of the URLC10 tetramer assay performed on PBMCs after culture (in vitro stimulation).
  • HLA-A * 24: 02 A URLC10 tetramer-positive CD8-positive T cell population recognizing the URLC10-derived peptide presented above was detected. See the description in Figure 4A-1. See the description in Figure 4A-1.
  • FIG. 4A
  • HLA-A * 24 02
  • a DEPDC1 tetramer-positive CD8-positive T cell population recognizing the DEPDC1-derived peptide presented above was detected.
  • the tetramer-positive CD8-positive T cell population was collected using the sorting function of the flow cytometer and then used as a sample for TCR repertoire analysis.
  • FIG. 5A is composed of pie charts (a)-(g) showing the results of TCR repertoire analysis of the URLC10 tetramer-positive CD8-positive T cell population. See the description in Figure 5A-1. See the description in Figure 5A-1. See the description in Figure 5A-1. See the description in Figure 5A-1.
  • FIG. 5B consists of a pie chart showing the results of TCR repertoire analysis of the DEPDC1 tetramer-positive CD8-positive T cell population. The pie chart shows the frequency of detection of TCR chronotypes detected by TCR repertoire analysis. If the occupied area in the pie chart is large, it means that it was detected frequently. It was identified as a TCR that recognizes TCR chronotype peptides with a detection frequency of 1% or more. TCR chronotypes with a detection frequency of less than 1% are collectively shown in the same pattern (* in the figure).
  • FIG. 6A consists of line graphs (a)-(d) showing IFN- ⁇ production of URLC10-derived peptide-specific CTL clones established by limiting dilution from PBMC after culture (in vitro stimulation). While IFN- ⁇ production of CTL clones was observed in target cells pulsed with URLC10-derived peptide, no significant IFN- ⁇ production of CTL clones was observed in target cells not pulsed with peptide. From this, it was confirmed that the CTL clone recognized the URLC10-derived peptide presented on HLA-A * 24: 02.
  • FIG. 6A consists of line graphs (a)-(d) showing IFN- ⁇ production of URLC10-derived peptide-specific CTL clones established by limiting dilution from PBMC after culture (in vitro stimulation). While IFN- ⁇ production of CTL clones was observed in target cells pulsed with URLC10-derived peptide, no significant IFN- ⁇ production of CTL clones was observed
  • 6B consists of a line graph showing IFN- ⁇ production of DEPDC1-derived peptide-specific CTL clones established by limiting dilution from PBMCs after culture (in vitro stimulation). While IFN- ⁇ production of CTL clones was observed in target cells pulsed with DEPDC1-derived peptide, no significant IFN- ⁇ production of CTL clones was observed in target cells not pulsed with peptide. From this, it was confirmed that the CTL clone recognized the DEPDC1-derived peptide presented on HLA-A * 24: 02.
  • the R / S ratio represents the ratio of the number of CTL clones, which are responder cells, to the number of target cells (Stimulator cells) that stimulate them.
  • FIG. 7 consists of the results (a) and (b) of the URLC10 tetramer assay performed on PBMCs.
  • No URLC10 tetramer-positive T cell population was detected in untreated PBMCs (Fig. 7a).
  • a URLC10 tetramer-positive T cell population was detected in PBMCs into which a URLC10-derived peptide-reactive TCR gene was introduced by a retroviral vector (Fig. 7b).
  • TCR gene transfer produced T cells that recognize the URLC10-derived peptides presented on HLA-A * 24: 02.
  • FIG. 8 is composed of line graphs (a) and (b) showing peptide-specific IFN- ⁇ production and cytotoxic granule (Granzyme B) production of TCR transgenic T cells.
  • IFN- ⁇ production Fig. 8a
  • Granzyme B production Fig. 8b
  • IFN- ⁇ production Fig. 8a
  • Fig. 8b Granzyme B production of TCR gene-introduced T cells against HLA-A * 24: 02 expression target cells pulsed with URLC10-derived peptide were observed, while the peptide was pulsed. No significant IFN- ⁇ production or Granzyme B production was observed for non-target cells. From this, it was confirmed that the TCR transgenic T cells have the function of recognizing the URLC10-derived peptide presented on HLA-A * 24: 02 and further producing IFN- ⁇ and Granzyme B.
  • the R / S ratio represents the ratio of the number of TCR gene-introduced T cells, which are responding cells (Responder cells), to the number of target cells (Stimulator cells) that stimulate them.
  • FIG. 9 consists of a line graph showing the response of TCR transgenic T cells to the esophageal cancer cell line. IFN- ⁇ production by TCR-transfected T cells was observed when co-cultured with TE-1 (URLC10 positive, HLA-A * 24: 02 positive), while TE-9 (URLC10 positive, HLA-A *). No significant IFN- ⁇ production was observed when co-cultured with (24:02 negative).
  • TCR transgenic T cells can recognize cancer cells via the URLC10-derived peptide presented on HLA-A * 24: 02.
  • the E / T ratio represents the ratio of the number of TCR gene-introduced T cells (Effector cells) to the number of target cells (Target cells).
  • an isolated or purified peptide refers to a peptide that is substantially free of other cellular materials from the cell or tissue source from which the peptide is derived, such as sugars, lipids, and other contaminating proteins.
  • an isolated or purified peptide refers to a peptide that is substantially free of precursors or other chemicals.
  • substantially free of cellular material includes a preparation of a peptide in which the peptide has been isolated from the cellular components of the cell in which it was isolated or recombinantly produced.
  • peptides that are substantially free of cellular material contain less than about 30%, 20%, 10%, or 5%, 3%, 2% or 1% (dry weight basis) of other cellular material. Includes peptide preparations.
  • the isolated or purified peptide is substantially free of culture medium, and the peptide which is substantially free of culture medium is such that the culture medium is approximately 20% of the volume of the peptide preparation. Includes peptide preparations containing less than 10%, or 5%, 3%, 2% or 1% (dry weight basis).
  • the isolated or purified peptide is substantially free of precursors and other chemicals, and the peptide is substantially free of precursors and other chemicals. Preparations of peptides containing precursors and other chemicals in less than about 30%, 20%, 10%, 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide preparation. Including.
  • a particular peptide preparation is an isolated or purified peptide is due, for example, by the appearance of a single band after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the gel. Can be confirmed.
  • SDS sodium dodecyl sulfate
  • the peptides and polynucleotides of the invention have been isolated or purified.
  • polypeptide polypeptide
  • peptide protein
  • protein polymers of amino acid residues.
  • the term applies to natural amino acid polymers as well as non-natural amino acid polymers containing one or more unnatural amino acid residues.
  • Non-natural amino acids include amino acid analogs and amino acid mimetics.
  • amino acid refers to natural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids.
  • Natural amino acids are amino acids encoded by the genetic code and amino acids that have been intracellularly modified after translation (eg, hydroxyproline, ⁇ -carboxyglutamic acid, and O-phosphoserine).
  • amino acid analog has the same basic chemical structure as a natural amino acid (a hydrogen, a carboxy group, an amino group, and an alpha carbon attached to an R group), but with a modified R group or a modified skeleton. Refers to compounds such as homoserine, norleucine, methionine sulfoxide and methionine methyl sulfonium.
  • amino acid mimetic refers to a compound that has a structure different from that of common amino acids but has the same function as an amino acid.
  • the amino acid may be either L-amino acid or D-amino acid, but the peptide of the present invention is preferably a polymer of L-amino acid.
  • polynucleotide oligonucleotide
  • nucleic acid a polymer of nucleotides
  • composition is intended to include products that contain a particular amount of a particular ingredient, and any product that results directly or indirectly from a combination of a particular amount of a particular ingredient.
  • composition refers to a product containing an active ingredient and an Inactive ingredient, as well as a combination of any two or more ingredients, complex formation, or aggregation. From, it is intended to include any product that results directly or indirectly from the dissociation of one or more components, or from other types of reactions or interactions of one or more components.
  • the pharmaceutical compositions of the invention include any composition made by mixing a compound or cell of the invention with a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein include liquid or solid bulking agents, diluents, excipients, solvents and encapsulating materials. Means a pharmaceutically or physiologically acceptable material, composition, substance, or vehicle without limitation.
  • cancer refers to cancer that overexpresses the URLC10 gene or the DEPDC1 gene.
  • examples of cancers that overexpress the URLC10 gene include bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, and soft tissue. Includes, but is not limited to, tumors and head and neck malignancies (HNMT).
  • HNMT head and neck malignancies
  • cancers that overexpress the DEPDC1 gene include, for example, bladder cancer, breast cancer, cervical cancer, bile duct cell cancer, chronic myelogenous leukemia (CML), NSCLC, lymphoma, osteosarcoma, prostate cancer, and small cell carcinoma. Includes cell lung cancer (SCLC), and soft tissue tumors.
  • the "cancer” is a cancer that expresses HLA-A24 with either or both of URLC10 and DEPDC1.
  • cytotoxic T lymphocytes cytotoxic T cells
  • CTL non-autologous cells
  • HLA-A24 refers to the HLA-A24 type, including the sub-type, such as 01, HLA-A * 24:02.
  • the phrase "the subject's (or patient's) HLA antigen is HLA-A24" as used herein means that the subject or patient is an MHC (major histocompatibility complex) class I. It refers to carrying the HLA-A24 antigen gene as a molecule in a homozygous or heterozygous manner, and expressing the HLA-A24 antigen as an HLA antigen in the cells of a subject or a patient.
  • treatment has clinical benefits such as reduction of cancer size, spread, or metastatic potential in a subject, cancer progression.
  • Treatment is considered “effective” if it results in delay, alleviation of clinical symptoms of cancer, prolongation of survival, suppression of postoperative recurrence, and the like.
  • “effective” means that the treatment delays or prevents the formation of cancer or prevents or alleviates the clinical manifestations of the cancer. Efficacy is determined in connection with any known method for diagnosing or treating a particular tumor type.
  • prevention has any function herein to reduce the burden of disease mortality or morbidity.
  • Prevention can be done at "primary, secondary, and tertiary levels of prevention".
  • Primary prophylaxis avoids the development of the disease, whereas secondary and tertiary levels of prophylaxis prevent the progression of the disease and the appearance of symptoms, as well as restore function and are disease-related. Includes work aimed at reducing the adverse effects of existing diseases by reducing the complications of.
  • prophylaxis may include a wide range of prophylactic treatments aimed at alleviating the severity of a particular disorder, eg, reducing tumor growth and metastasis.
  • treatment of cancer, prevention of cancer, or prevention of recurrence of cancer after surgery may include inhibition of cancer cell growth, tumor regression or regression, induction of remission, and so on. Includes one or more of the events selected from suppression of cancer development, tumor regression, and reduction or inhibition of metastasis, suppression of postoperative recurrence of cancer, and prolongation of survival.
  • Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces the level of tumor markers in the blood, and is associated with cancer.
  • Relieve detectable symptoms For example, symptomatic relief or amelioration constitutes either or both of effective treatment and prevention, including 10%, 20%, 30%, or more alleviation or symptom-stable conditions.
  • antibody refers to an immunoglobulin and a fragment thereof that specifically react with a designated protein or peptide thereof.
  • Antibodies can include human antibodies, primated antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments.
  • antibody is used in a broad sense, specifically, a multispecific antibody formed from an intact monoclonal antibody, a polyclonal antibody, and two or more intact antibodies (for example, a bispecific antibody). And include antibody fragments as long as they exhibit the desired biological activity.
  • the “antibody” may be an antibody of any class (eg, IgA, IgD, IgE, IgG, and IgM).
  • T Cell Receptors The present invention also comprises compositions comprising one or more polynucleotides encoding one or more polypeptides capable of forming subunits of the T cell receptor (TCR), and the like. Provides a way to use.
  • TCR subunits have the ability to form TCRs that confer specificity on CD8 + T cells for the URLC10 peptide, or URLC10 or DEPDC1 for APCs that present the DEPDC1 peptide.
  • ⁇ - and ⁇ -chain nucleic acids can be identified as TCR subunits of T cells induced by the URLC10 peptide or DEPDC1 peptide (International Publication No.
  • the derivative TCR can bind to APCs presenting the URLC10 peptide or DEPDC1 peptide with high binding force and, in some cases, effectively mediate cytokines.
  • One or more polynucleotides encoding TCR subunits in a suitable vector, eg, retro. It can be incorporated into a viral vector. These vectors are well known in the art. Polynucleotides or vectors containing them can be usefully transferred to CD8 + T cells, such as patient-derived CD8 + T cells. Conveniently, the present invention allows rapid modification of a patient's own T cells (or T cells of another subject) to quickly and easily produce modified T cells with excellent cancer cell killing properties. To provide a readily available composition.
  • T cells stimulated by the URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) in an HLA-A24 restraint manner SEQ ID NOs: 1-2, 5-8, 14, 16, 17-25, 33-40, respectively. , 49-54, 60-62, 65-70, 77-79, TCR- ⁇ subunits, each having a CDR3 consisting of the amino acid sequences, were detected.
  • TCR- ⁇ subunit each of which has a CDR3 consisting of amino acid sequences, was detected (Fig. 2, Fig. 5A).
  • T cells stimulated by the DEPDC1 peptide EYYELFVNI / SEQ ID NO: 178 in the HLA-A24 restraint mode, for example, TCR- ⁇ subs each having a CDR3 consisting of the amino acid sequence of SEQ ID NO: 84-85.
  • a TCR- ⁇ subunit having a unit and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 86 was detected (Fig. 5B).
  • T cells expressing TCR formed between the ⁇ and ⁇ subunits, each of which has a CDR3 consisting of the amino acid sequences of these SEQ ID NOs are also preferred embodiments of the present invention.
  • the antigen specificity of TCRs depends primarily on their CDR3s. Therefore, it is also possible to reconstruct the URLC10 or DEPDC1-specific TCR from the known TCR by replacing the CDR3 of the ⁇ subunit and the ⁇ subunit with the above SEQ ID NOs, respectively.
  • Such a TCR with a CDR3 ported from another subunit can be referred to as a chimeric TCR.
  • the present invention further provides cytotoxic T cells prepared by transfection with a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits, wherein the TCR subunits It can bind to URLC10 peptide and DEPDC1 peptide in the HLA-A24 restraint manner.
  • Transduced T cells can be homing to cancer cells in vivo and can be grown in vitro by well-known culture methods (eg Kawakami et al., J Immunol 1989, 142: 3452-61). T cells prepared as described above can be used to form immunogenic compositions useful in treating or preventing cancer in patients in need of treatment or prevention.
  • modifications of one, two, or more amino acids in a polypeptide do not affect the function of the polypeptide, and in some cases even enhance the desired function of the original polypeptide. is there.
  • the modified polypeptide ie, from the group consisting of one, two, or several amino acid residues modified (ie, substitutions, deletions, insertions, and additions compared to the original reference sequence).
  • the CDR3 of the present invention comprises a group consisting of one, two, or several amino acids substituted, deleted, inserted, and added to the amino acid sequence selected from the above SEQ ID NOs.
  • the binding specificity of HLA-A24 binding to the URLC10 peptide or DEPDC1 peptide on the TCR is similar to that of CDR3, which is derived by modifying the amino acid sequence specified by the above-mentioned SEQ ID NO: and consists of the original amino acid sequence.
  • the CDR3 that grants is called the functionally equivalent CDR3.
  • N, C, E, Q, G, H, K, S, T as well as side chains that share the following functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur amino acid-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W).
  • each of the following eight groups contains amino acids recognized in the art as mutually conservative substitutions: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine (C), methionine (M) (see, for example, Creighton, Proteins 1984).
  • CDR3 including such a conservative modification is also included in CDR3 of the present invention.
  • the peptides of the invention are not limited to these and may include non-conservative modifications as long as the modified CDR3 retains the functionality of the original CDR3.
  • the original function of CDR3 was retained in the modified CDR3 when the same antigen recognition specificity as the TCR from which the original CDR3 was derived was imparted to the transplanted TCR.
  • Such specific recognition can be confirmed by any known method, and preferred methods include, for example, a tetramer assay using a peptide administered to a subject who has obtained an HLA molecule and TCR (eg, Altman et al. Science. 1996, 274, 94-6; McMichael et al. J Exp Med. 1998, 187, 1367-71), as well as the ELISPOT assay.
  • T cells expressing TCR on the cell surface recognize the cells by TCR, and signals are transmitted intracellularly, and then cytokines such as IFN- ⁇ are released from the T cells. It can be confirmed that it will be done.
  • the cytotoxic activity of T cells against target cells can be investigated using methods well known in the art. Preferred methods include, for example, a chromium release assay using HLA-positive cells expressing URLC10 or DEPDC1 as target cells.
  • URLC10 GenBank accession number NM_017527 or accession number: BC117142
  • DEPDC1 GenBank accession number: NM_001114120, or BM683578
  • the URLC10 gene is also known as the LY6K gene.
  • the LY6K gene is a bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, soft tissue tumor, and head and neck malignancies (HNMT).
  • NSCLC non-small cell lung cancer
  • HNMT head and neck malignancies
  • the present invention relates to the lymphocyte antigen 6 complex, the gene product of the K gene locus (lymphocyte antigen 6 complex) (LY6K), and more specifically to GenBank.
  • the polypeptide encoded by the gene of accession number NM__017527 is targeted for further analysis.
  • DEPDC1 protein (DEP domain containing 1; GenBank accession number NM_001114120, BM683578) is still unclear.
  • the DEP region contained in this protein is found in Dishevelled, Egl-10 and Pleckstrin.
  • the Drosophila dishevelled DEP domain plays an essential role in the relief of planar polarity defects and the induction of JNK signaling; nevertheless, its function in humans has not yet been elucidated.
  • DEPDC1 In House No .: B5860N
  • B5860N is a two different transcriptome consisting of 12 and 11 exons corresponding to DEPDC1 V1 and V2, respectively.
  • the V2 variant does not have the exon 8 of V1, but produces the same stop codon within the last exon.
  • the full-length cDNA sequence of the B5860NV1 and B5860NV2 variants consists of 5318 and 4466 nucleotides, respectively.
  • the ORFs of these variants begin within each exon 1.
  • the transcripts of V1 and V2 encode 811 and 527 amino acids, respectively.
  • siRNA suppressed the growth of cancer cells.
  • the amino acid sequence of SEQ ID NO: 178 which is a recognition epitope of DEPDC1-specific TCR provided by the present invention, is contained in both variants of V1 and V2. Therefore, the TCR of the present invention provides a CTL that can be expected to have a damaging effect on cells expressing either V1 or V2 variant of DEPDC1.
  • the present invention relates to a TCR expressed by a T cell that recognizes a URLC10-derived peptide or a DEPDC1-derived peptide.
  • TCR is a protein molecule consisting of ⁇ -chain and ⁇ -chain dimers.
  • Human T cells recognize peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules through TCR.
  • MHC class I also known as HLA, Human Leukocyte Antigen
  • the TCR- ⁇ gene includes the V ⁇ gene, the J ⁇ gene and the C ⁇ gene.
  • the TCR- ⁇ gene includes a V ⁇ gene, a D ⁇ gene, a J ⁇ gene and a C ⁇ gene.
  • CDRs Complementarity Determining Regions
  • CDR1 CDR2, and CDR3 are present.
  • CDR3 comes into direct contact with peptides, so its amino acid sequence is very important for determining the antigen recognition specificity of TCR.
  • VJs and in the TCR- ⁇ chain, VDs and DJs correspond to CDR3, and diversity occurs due to the insertion or deletion of bases.
  • the URLC10 or URLC10 shown in the present specification is used.
  • the amino acid sequence of TCR (particularly the amino acid sequence in CDR3) identified from T cells that recognize peptides derived from DEPDC1 is extremely valuable.
  • An immunogenic epitope peptide derived from a tumor antigen induces a tumor antigen-specific CTL and produces a therapeutic effect in cancer patients.
  • it often takes a long time (several months) to induce a therapeutically effective amount of CTL in vivo by inoculating a peptide.
  • vaccine-based cancer treatment may limit the applicable patients.
  • TCR gene transfer T cell therapy can be realized.
  • In vitro TCR-recombined T cells can be easily grown in the amount required for treatment by culturing. Therefore, compared to the peptide vaccine, a faster and sufficient therapeutic effect can be expected.
  • the present invention enables personalized cancer immunotherapy.
  • Tumor antigens that are overexpressed in a wide range of cancers are useful targets for TCR transgenic T cell therapy. Identification of a TCR that targets a suitable tumor antigen will enable personalized cancer immunotherapy in other patients with the same type of HLA.
  • URLC10 is highly expressed in many non-small cell lung cancers and esophageal cancers.
  • overexpression of DEPDC1 in bladder cancer is also known. Therefore, URLC10 and DEPDC1-specific TCR transgenic T cells are useful for adoptive immunotherapy.
  • chimeric antigen receptor (CAR) T cell therapy and TCR gene transfer T cell therapy are limited to some hematological cancers.
  • the TCR of the present invention enables adoption immunotherapy in solid tumors.
  • the invention provides an immunologically active TCR, or engineered T lymphocyte, which presents a portion thereof.
  • T lymphocytes are, for example, CD8 + cytotoxic T cells.
  • the recombinant T cells of the present invention recognize antigen-presenting cells that have presented an epitope peptide bound to MHC.
  • the recombinant T lymphocytes can be CD8 + T lymphocytes, CD4 + helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells and the like.
  • a nucleic acid or vector encoding the amino acid sequence of the TCR can be designed.
  • recombinant T lymphocytes that present a target antigen-specific TCR or part thereof can be obtained by the following steps; (i) The nucleic acid sequence encoding the target antigen-specific TCR was cloned into a vector. (ii) The vector was introduced into host T lymphocytes (eg CD8 + cytotoxic T cells) and (iii) The TCR is cultured under conditions that can be expressed and presented on recombinant T lymphocytes.
  • a part of the TCR can be, for example, one or both of the ⁇ chain and the ⁇ chain of the TCR.
  • some of the TCRs are one, two, or three complementarity determining regiions (CDRs) of either or both of the ⁇ and ⁇ chains of the TCR.
  • CDRs complementarity determining regiions
  • the portion of the TCR comprises CDR3 of either or both of the ⁇ and ⁇ chains of the TCR.
  • the preferred amino acid sequence of CDR3 identified in the present invention is: SEQ ID NO: Any selected from the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70, 77-79 and 84-85.
  • CDR3 of the human T cell receptor ⁇ chain identified by the amino acid sequence and SEQ ID NO: 3-4, 9-13, 15, 26-32, 41-48, 55-59, 63-64, 71- CDR3 of the human T cell receptor ⁇ chain identified by any amino acid sequence selected from the group consisting of 76, 80-83 and 86.
  • the amino acid sequences of the T cell receptor ⁇ chain and the T cell receptor ⁇ chain CDR3 can be combined, for example, as follows; CDR3 of T cell receptor ⁇ chain CDR3 of T cell receptor ⁇ chain Sequence number: 1 Sequence number: 3 (URLC10), SEQ ID NO: 2 SEQ ID NO: 3 (URLC10), SEQ ID NO: 14 SEQ ID NO: 15 (URLC10), SEQ ID NO: 16 SEQ ID NO: 10 (URLC10), SEQ ID NO: 17 SEQ ID NO: 26 (URLC10), SEQ ID NO: 60 SEQ ID NO: 63 (URLC10), SEQ ID NO: 69 SEQ ID NO: 71 (URLC10), SEQ ID NO: 77 SEQ ID NO: 80 (URLC10), and SEQ ID NO: 84 SEQ ID NO: 86 (DEPDC1).
  • URLC10 Sequence number: 3
  • SEQ ID NO: 2 SEQ ID NO: 3 (URLC10)
  • SEQ ID NO: 14 SEQ ID NO: 15
  • TRBV9 SEQ ID NO: 4, 9, 12-13, 15, 41, 42, 44-48, 55-59, 63, 71, 73-76, 80-82 was identified by 11-13 amino acid sequences.
  • the sequence of CASS- [X] nF (X is an arbitrary amino acid and n is 6-8 / SEQ ID NO: 179) is common.
  • the present invention provides, in some embodiments, a method for constructing recombinant T lymphocytes (eg, CD8 + T cells) containing TCRs that recognize antigen-presenting cells presented with an epitope peptide-bound MHC.
  • the antigen-binding region of the TCR can contain, for example, the above CDR3 in the ⁇ and ⁇ chains.
  • the TCR can also additionally include a transmembrane domain or a signaling domain.
  • the transmembrane domain can, in some embodiments, be the transmembrane domain of a T cell. Examples of the transmembrane domain of T cells include the transmembrane domain of CD28.
  • the signaling domains constituting the TCR can contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • Immunoreceptor-activated tyrosine motifs can include T cell receptor signaling domains such as the CD3 ⁇ chain.
  • the TCR can also include one or more stimulation domains.
  • the stimulation domain provides, for example, a secondary signal that stimulates T cells.
  • recombinant T lymphocytes can be CD8 + T cells, CD4 + helper T cells, NK cells, NKT cells, B cells, or dendritic cells.
  • host T lymphocytes for introducing a nucleic acid encoding TCR or a vector containing the same can be collected from a healthy donor or a subject in need of cancer treatment.
  • TCR of the present invention By expressing the TCR of the present invention in T lymphocytes collected from a subject in need of cancer treatment, it is possible to artificially impart a cytotoxic effect specific to URLC10 or DEPDC1 to the T cells of the subject. ..
  • the invention provides recombinant T cells that express active TCR.
  • Methods for obtaining recombinant cells are known.
  • recombinant T cells can be obtained by nucleic acids encoding active TCR.
  • a nucleic acid encoding an active TCR, or nucleic acid sequence is provided.
  • the nucleic acid encoding the TCR is usually a synthesized nucleic acid.
  • Nucleic acids can also include DNA, RNA, PNA (peptidenucleic acid), or hybrid molecules thereof.
  • the recombinant T cells of the present invention expressing active TCR can be evaluated for properties such as cytotoxic activity and proliferative activity, if necessary.
  • properties such as cytotoxic activity and proliferative activity, if necessary.
  • Techniques for evaluating these properties are known. Examples of methods for evaluating cytotoxic effects include tetramer assays using peptides administered to subjects who have acquired HLA molecules and TCRs (eg, Altman et al. Science. 1996, 274, 94-6; McMichael et al. JExpMed.1998,187,1367-71), ELISPOT assay, or chromium release assay using HLA-positive cells expressing URLC10 or DEPDC1 as target cells.
  • cell proliferation can be evaluated by applying a method such as MTT assay. As a result of the evaluation, it is possible to select cells having more practically desirable properties, such as cells having a large cytotoxic effect or cells having further excellent proliferative properties.
  • the nucleic acid encoding an immunologically active TCR can be determined, for example, by the following method.
  • the nucleotide sequence information of the nucleic acids encoding them has already been determined and can be used as a database. By assembling them, the positions of the three CDRs of the antigen binding site can be clarified. Of the CDRs contained in the antigen-binding site of TCR, CDR1 and CDR2 are said to recognize HLA. Therefore, in the case of the present invention, a nucleic acid encoding HLA-A24-binding TCR may be used. .. Then, by substituting the remaining CDR3 with the base sequence information encoding the above amino acid sequence information identified in the present invention, a nucleic acid encoding TCR required for induction of recombinant T lymphocytes can be obtained.
  • TCR repertoire analysis based on the entire nucleotide sequence information of the antigen-binding site of TCR including CDR1 and CDR2 as well as CDR3 has already been performed. From the sequence information of the antigen-binding site accumulated through such analysis, one that matches the sequence information of the target CDR3 can be selected, and the sequence information of CDR1 and CDR2 can be obtained. By transplanting the sequence information of CDR1, CDR2, and CDR3 thus selected to the antigen-binding site of another TCR, the antigen specificity of interest can also be transplanted.
  • a nucleic acid encoding an immunologically active TCR may be obtained from human lymphocytes that were actually vaccinated with either or both of the URLC10 peptide and the DEPDC1 peptide. it can.
  • the CDR3 of the TCR provided by the present invention will occupy a predominant population in subjects inoculated with these peptides with the response of the CTL of interest. .. Therefore, it is easy to obtain a nucleic acid encoding a TCR containing CDR3 of the TCR provided by the present invention from a CTL recovered from such a subject.
  • a cDNA library of nucleic acids encoding TCR can be constructed from mRNA. By cloning from the cDNA library the amino acid sequence corresponding to CDR3 found in the present invention as the amino acid sequence of CDR3, the nucleic acid encoding the target TCR can be isolated.
  • a nucleic acid encoding the TCR of interest can be isolated after recovering a CTL having a cytotoxic effect specific to the URLC10 peptide or DEPDC1 peptide from the subject and cloning it.
  • the nucleic acid encoding the TCR usually contains a secretory signal.
  • the secretory signal can be originally contained in the TCR mRNA or can be replaced by another secretory signal.
  • the nucleic acid encoding the TCR in the present invention can comprise one or more control sequences. Specific examples thereof include promoters, transcriptional elements, and sequences capable of inducing expression.
  • the nucleic acid can be introduced into the cell with a suitable vector that allows expression in the cell.
  • a recombinant vector comprising an immunologically active TCR of the invention, or a nucleic acid encoding a portion thereof, is provided.
  • Many vectors are known to those skilled in the art, and the appropriate one can be selected according to the required function.
  • vectors such as plasmids, cosmids, viruses, bacteriophages, etc. are known and are widely used for gene recombination.
  • the construction of vectors is well known to those skilled in the art (Sambrock and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989)).
  • a polynucleotide or vector is encapsulated in a liposome to deliver the target cell.
  • the DNA of interest can also be isolated using a cloning vector.
  • the desired nucleic acid sequence can be expressed by incorporating the nucleic acid sequence of interest into an expression vector and introducing it into cells.
  • Known typical cloning vectors include, for example, pBluescriptSK, pGEM, pUC9, pBR322, and pGBT9.
  • the expression vector includes pTRE, pCAL-n-EK, pESP-I, pOPI3CAT and the like.
  • the vector can comprise a regulatory sequence operably linked to a nucleic acid sequence encoding TCR (or a portion thereof). Regulatory sequences (regulatory units) are known to those of skill in the art and may include, for example, promoters, splice cassettes, transcription initiation codons, insertion sites for insertion into vectors, and the like.
  • the nucleic acid is operably linked to an expression regulatory sequence to allow expression in eukaryotic or prokaryotic cells. Nucleic acids encoding the ⁇ and ⁇ chains of the TCR can be loaded in combination in one vector. Alternatively, they can be integrated into separate vectors for co-transformation.
  • a vector in which a nucleic acid encoding an ⁇ chain and a ⁇ chain is mounted in a tandem can correctly reproduce the combination of both, so that higher introduction efficiency can be expected.
  • the base sequence of the nucleic acid encoding the ⁇ -chain and ⁇ -chain of the TCR can be expected to improve the expression efficiency by optimizing the codons.
  • the structures of the ⁇ and ⁇ chains of the TCR other than CDR3 can be replaced with structures of different origins as long as their cytotoxic activity is maintained.
  • the C region which does not affect the antigen specificity, may be replaced with one obtained from any source.
  • CDR1 and CDR2 are supposed to mainly recognize HLA molecules, they can be replaced with the HLA-restricted (that is, HLA-A24) TCR structure common to the TCR of the present invention.
  • HLA-A24 HLA-restricted TCR structure common to the TCR of the present invention.
  • Its origin is not limited to humans, and sources can be sought from other species.
  • mouse HLA has a high degree of commonality with humans, it can be replaced with mouse D region or J region. That is, the present invention provides a chimeric TCR molecule in which the CDR3 identified by the present invention is transplanted into the ⁇ chain or ⁇ chain of TCRs of different origins.
  • a viral vector is used as the vector
  • a lentiviral vector or an adenovirus vector can be used.
  • nucleic acids and vectors are utilized to express immunologically active TCR in cells.
  • the nucleic acid or vector contains a DNA sequence encoding an immunologically active TCR and is introduced into a cell to express the immunologically active TCR.
  • the present invention relates to a method for obtaining a vector usually used in the field of genetic engineering such as plasmid, cosmid, bacteriophage, virus, etc., which contains a nucleic acid sequence encoding an immunologically active TCR.
  • the vector can be an expression vector, a gene transfer vector, or a gene targeting vector.
  • Viral vectors derived from retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, bovine papillomaviruses, etc. are useful for introducing expressed polynucleotides and vectors into cell populations. Many viral vectors integrate their genetic information into the host cell's genome after introduction into the cell. Therefore, the target genetic information is stably maintained in the cell population as the cells proliferate. Methods of constructing recombinant vectors are well known to those of skill in the art.
  • the vector can be gene-introduced by a well-known method depending on the type of host cell.
  • the present invention provides a host cell transformed or transgenic with an immunologically active TCR-encoding nucleic acid.
  • the host cell can be obtained by introducing at least one of the above-mentioned vectors and nucleic acid molecules, and in some cases, a plurality of them into the host cell.
  • the host cell can express immunologically active TCR or a part thereof by the introduced nucleic acid molecule or vector. Nucleic acid molecules and vectors introduced into host cells can be integrated into the genome or maintained extranuclearly.
  • the present invention provides a method of culturing the above host cells into which a nucleic acid molecule or vector has been introduced.
  • the present invention comprises culturing a host cell under conditions capable of expressing the introduced construct (including immunologically active TCR and nucleic acids encoding a portion thereof).
  • the cultured cells are provided to a subject from which the host cells have been obtained or a second subject separate from the subject from which the host cells are derived. Methods for culturing cells carrying the construct for expression are well known.
  • lymphocytes collected from a patient to be treated or a healthy donor can be used as a host cell.
  • Lymphocytes collected from multiple different sources can also be mixed into host cells.
  • Specific examples of the source for collecting lymphocytes include peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, ascites, pleural effusion, and spleen tissue.
  • lymphocytes can be recovered from infected lesions or tumor tissue. Techniques for selectively recovering the required type of lymphocytes (eg, cytotoxic T cells or T cells without antigen specificity) are known. Specifically, a method of recovering a target cell by cell sorting using a specific cell marker such as CD8 or CD4 as an index is common. The recovered lymphocytes can be subsequently cultured.
  • the recombinant lymphocytes obtained by the present invention can be administered by a general delivery system.
  • recombinant lymphocytes can be administered as a composition dispersed in a pharmaceutically acceptable carrier.
  • the genetic information encoding an immunologically active TCR can be integrated into the genome of interest.
  • the methods or compositions of the present invention can be used to prevent, treat, or ameliorate symptoms of cancer.
  • a method for preventing, treating, or ameliorating symptoms of cancer according to the present invention comprises the step of administering an effective amount of the composition to a subject in need thereof.
  • the composition used in the method of the present invention contains, for example, a nucleic acid encoding a recombinant lymphocyte immunologically active TCR, a vector containing the nucleic acid, and the like as an active ingredient.
  • cells such as recombinant lymphocytes are administered, they can also be administered locally to the treatment site.
  • composition or method for treating or preventing cancer of the present invention can be used alone or in combination with other compositions or methods for treating or preventing cancer.
  • the composition or method of the present invention can be utilized in combination with the composition or method for chemotherapy or immunological treatment methods.
  • physicochemical treatment methods such as radiation therapy, proton beam therapy, and hyperthermia therapy can be used in combination.
  • the TCR provided by the present invention specifically recognizes cells expressing either or both of URLC10 and DEPDC1. Therefore, any cancer expressing URLC10 or DEPDC1 can be treated or prevented.
  • URLC10 includes, for example, bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, soft tissue tumor, and It is known to be highly expressed in head and neck malignancies (HNMT). Therefore, the T cells expressing the TCR of the present invention are useful for the treatment or prevention of these cancers.
  • DEPDC1 is highly expressed in bladder cancer, breast cancer, cervical cancer, bile duct cell cancer, CML, NSCLC, lymphoma, osteosarcoma, prostate cancer, small cell lung cancer (SCLC), and soft tissue tumor.
  • TCR provided in the present invention has an HLA-A24 restrictive effect. Therefore, the T cells expressing the TCR of the present invention can be effectively used for HLA-A24 positive subjects.
  • the expression of URLC10 or DEPDC1 in the target cancer can be confirmed in advance. That is, the present invention provides methods for the treatment and prevention of cancer, or both, including the following steps: (1) The process of collecting cancer tissue from the target of treatment or prevention; (2) Step of detecting the expression of URLC10 or DEPDC1 in the collected cancer tissue; and (3) A step of selecting a subject having cancer having a high expression level of either or both of URLC10 and DEPDC1 as compared with normal tissue and administering the composition of the present invention.
  • the invention detects the expression of URLC10 or DEPDC1 in cancer tissue taken from a subject to be treated or prevented, and the expression level of either or both of URLC10 and DEPDC1 is compared to normal tissue. It relates to a composition for selectively administering a subject having high cancer.
  • the present invention detects the expression of URLC10 or DEPDC1 in cancer tissues collected from a subject to be treated or prevented, and can detect cancers in which the expression level of either or both of URLC10 and DEPDC1 is higher than that of normal tissues.
  • TCR is the use of TCR in the manufacture of compositions for selective administration of subjects having.
  • the present invention detects the expression of URLC10 or DEPDC1 in cancer tissues collected from a subject to be treated or prevented, and can detect cancers in which the expression level of either or both of URLC10 and DEPDC1 is higher than that of normal tissues.
  • TCR in the treatment and prevention of cancer, including the step of selecting and administering a subject to have.
  • the treatment or prevention of the present invention is usually effective in subjects who have the HLA-A24 allele homo or heterozygous.
  • the therapeutic effect of cancer can be expected by introducing the TCR of the present invention into T cells collected from a subject and then administering the TCR to the subject. That is, the present invention (1) Subjects who have detected the expression of URLC10 or DEPDC1 in cancer tissues collected from the subject of treatment or prevention and have cancers in which the expression level of either URLC10 and / or DEPDC1 is higher than that of normal tissues.
  • the process of selecting (2) The process of collecting T cells from the selected subject, (3) A step of introducing a nucleic acid encoding the TCR of the present invention into the collected T cells, and (4)
  • the present invention relates to a method for treating cancer, which comprises a step of administering a T cell having a nucleic acid encoding the TCR of the present invention into a subject.
  • T cells into which a nucleic acid encoding TCR has been introduced can be administered to a subject after being proliferated as needed. Therefore, the amount of T cells recovered from peripheral blood does not necessarily have to be large.
  • the amino acid sequences of the T cell receptor ⁇ chain and the T cell receptor ⁇ chain CDR3 can be combined, for example, as follows; CDR3 of T cell receptor ⁇ chain CDR3 of T cell receptor ⁇ chain Sequence number: 1 Sequence number: 3 (URLC10), SEQ ID NO: 2 SEQ ID NO: 3 (URLC10), SEQ ID NO: 14 SEQ ID NO: 15 (URLC10), SEQ ID NO: 16 SEQ ID NO: 10 (URLC10), SEQ ID NO: 17 SEQ ID NO: 26 (URLC10), SEQ ID NO: 60 SEQ ID NO: 63 (URLC10), SEQ ID NO: 69 SEQ ID NO: 71 (URLC10), SEQ ID NO: 77 SEQ ID NO: 80 (URLC10), and SEQ ID NO: 84 SEQ ID NO: 86 (DEPDC1).
  • URLC10 Sequence number: 3
  • SEQ ID NO: 2 SEQ ID NO: 3 (URLC10)
  • SEQ ID NO: 14 SEQ ID NO: 15
  • the invention in another aspect, relates to the use of the nucleic acid encoding the TCR of the invention in the treatment of cancer, including the steps described above.
  • the present invention also relates to the use of the nucleic acid encoding the TCR of the present invention in the production of a composition for the treatment of cancer, which comprises the above steps.
  • the present invention relates to a composition for treating cancer, which comprises the above steps, comprising a nucleic acid encoding the TCR of the present invention.
  • the invention introduces an expression vector containing a nucleic acid encoding the TCR of the invention into CD8 + (CD8 positive) T cells taken from a subject in need of cancer treatment and URLC10 or DEPDC1.
  • a method for producing a cytotoxic T cell which comprises a step of recovering a cytotoxic T cell having an HLA-A24-binding cytotoxic effect on an expressing cell. From T cells into which an expression vector containing a nucleic acid encoding the TCR of the present invention has been introduced, the HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells can be evaluated, and active cells can be isolated. it can. Any target cell is used to evaluate the cytotoxic effect.
  • HLA-A24-positive cells expressing URLC10 or DEPDC1 include HLA-A24-positive cells expressing URLC10 or DEPDC1, and HLA-A24-positive cells transformed with an expression vector of URLC10 or DEPDC1.
  • T cells that recognize the HLA-peptide complex can be identified in advance by tetramer assay.
  • the method for producing cytotoxic T cells additionally isolates HLA-A24-binding cytotoxic effects on URLC10 or DEPDC1-expressing cells, and expands the isolated T cells.
  • a method for expanding and culturing T cells is also known. For example, maintaining T cells in the coexistence of feeder cells can support their proliferation.
  • Non-proliferative lymphocyte cells and the like can be used as the feeder cells. It is common to preliminarily irradiate lymphocyte cells with gamma rays to control mitosis. After the proliferated T cells are collected, washed if necessary, they can be redispersed in an appropriate medium such as physiological saline and used for administration to a subject.
  • the number of recombinant T cells to be administered to a subject, the schedule of administration, and the administration method are appropriately determined by those skilled in the art depending on the condition of the subject and the type and condition of the cancer to be treated. You can choose.
  • the dose and schedule can be adjusted by observing the clinical symptoms of the administration subject.
  • administration schedules for recombinant T cells One intravenous dose of 2 x 10 8 cells, 1 x 10 9 cells or 5 x 10 9 cells (Clin Cancer Res. 2015, 21 (10): 2268-77); 5.3 x 10 9 cells in 2 divided doses or 2.4 x 10 9 cells in 1 dose (Blood.
  • Non-Patent Document 1 Median 5.5 x 10 10 cells (range 0.9-13 x 10 10 cells) given once (Clin Cancer Res. 2015, 21 (5): 1019-27.); Or mean 2.4 x 10 9 cells given once (Non-Patent Document 1)
  • a "peptide-specific CTL response" is a complex in which the TCR formed between a pair of ⁇ and ⁇ subunits is formed between the peptide of the present invention and an HLA molecule. It is understood to mean the specific recognition of the body. As discussed above, the CTL cell inducibility of peptides defined by the specific sequences of the invention can be maintained even after amino acid modification. Therefore, in addition to stimulation with a specific peptide, even when T cells are derived from a mutant peptide, as long as the TCR specifically recognizes such a complex formed by the original peptide. Its antigen specificity is considered "peptide specific”.
  • TCR- ⁇ and TCR- ⁇ subunits with CDR3 consisting of the following amino acid sequences were increased in the HLA-A24 constrained manner in a T cell population stimulated by a particular URLC10 or DEPDC1 peptide ( Figure). 2, Fig. 5A, Fig. 5B).
  • URLC10 peptide SEQ ID NO:: Any amino acid sequence selected from the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70 and 77-79.
  • CDR3 of the T cell receptor ⁇ chain identified by any amino acid sequence selected from the group consisting of. DEPDC1 peptide: CDR3 of the T cell receptor ⁇ chain identified by the amino acid sequence of SEQ ID NO: 84 or 85, and CDR3 of the T cell receptor ⁇ chain identified by SEQ ID NO: 86. Therefore, their accumulation in the T cell population of the subject means that the T cell response mediated by the URLC10 peptide or DEPDC1 peptide was successfully induced in the peptide-inoculated subject.
  • the presence of CDR3 consisting of the above amino acid sequence can be detected by antibody-based analysis.
  • specific pairs of TCR- ⁇ and TCR- ⁇ subunits can also be evaluated by detecting the polynucleotides encoding them.
  • the polynucleotides encoding each of the CDR3s identified in this application (SEQ ID NOs: 1-86 in Tables 1, 3A and 3B) are represented by the nucleotide sequences of SEQ ID NOs: 87-173, respectively.
  • each of the cDNAs synthesized from the ⁇ and ⁇ subunits of the URLC10 or DEPDC1-specific TCR may each contain the nucleotide sequence of the above SEQ ID NO:.
  • Such polynucleotides can be detected by PCR-based analysis.
  • the invention is a method for monitoring, evaluating, or assessing a peptide-specific T cell response in a peptide-immunized subject.
  • a method comprising a step in which the induction of a T cell response specific to the peptide is exhibited when detected in.
  • T cells specific for URLC10 or DEPDC1 in the HLA-A24 restraint mode can be detected.
  • any biological sample obtained from the subject can be used to monitor the CTL response as long as the sample contains T cells.
  • blood or blood-derived samples can be used as biological samples.
  • the blood-derived sample comprises a cell population containing T cells. Methods for obtaining cell populations containing T cells are well known to those of skill in the art.
  • a tissue or lymph node infiltrated with T cells is also useful as a biological sample.
  • Cytotoxic T cells can also be proliferated prior to analysis.
  • Peripheral blood mononuclear cells (PBMCs) collected from the subject also contain several different cell types. In order to analyze cytotoxic T cells with immunologically active TCR, it is advantageous to increase the number of target cells in advance. Post-proliferation cytotoxic T cells can also be collected and used as samples for TCR repertoire analysis.
  • a cell population containing T cells can be immobilized for antibody-based analysis in the evaluation of TCR subunits.
  • the antibody recognizes the CDR3 of the ⁇ and ⁇ subunits of the fixed cell, and if the antibody binds to both subunits of TCR on a single cell, a particular sub.
  • the TCR-antibody complex formed with the subunit pair can be detected.
  • the accumulation of amplicon synthesized from polynucleotides encoding TCR subunits in a single cell means that a particular pair of subunits is present.
  • a specific pair of polynucleotides encoding the TCR subunit for each of the peptides to be vaccinated can be obtained by deep cDNA sequencing as shown in Fang H, et al., Oncoimmunology 2015, 3: e968467. Can be monitored.
  • the TCR- ⁇ subunit and / or both of the TCR- ⁇ subunits, or both at least once or multiple times after vaccination, to monitor or assess the CTL response. Can be detected. If the pair increases in a time-dependent manner through monitoring, it means that the peptide-mediated CTL response in the subject is well induced. Alternatively, if a particular pair is detected at least once, it indicates that a CTL response has occurred in the subject. Alternatively, it is possible to obtain a repertoire of TCRs in the subject before vaccination and compare it with the repertoire after vaccination to confirm the increase in TCRs of the present invention. If the TCR of the invention is not detected in the pre-vaccination repertoire, a response to the vaccine in the subject can be detected if its appearance or increase is seen after vaccination. The TCR of the subject after vaccination can be analyzed at any time after vaccination.
  • the monitoring method of the present invention can include pre-administering either or both of the URLC10 peptide and the DEPDC1 peptide to the subject. That is, the present invention (1) Step of administering URLC10 peptide and / or DEPDC1 peptide to a subject (2) A step of collecting T cells from the subject at least once after administration of the peptide, and (3) A step of determining the frequency of the TCR of the present invention in the TCR of T cells. Then, it can be seen that the peptide-mediated CTL response in the subject is sufficiently induced when the frequency of the TCR of the present invention is increased. By determining the frequency multiple times and following the changes over time, it is possible to know the increase in the frequency of TCR.
  • the frequency occupies a certain frequency after determining the frequency at least once, it can be known that the immune response to the pre-inoculated peptide is induced.
  • the constant frequency can be exemplified by, for example, the ratio of the analyzed TCR to the entire repertoire is usually 1%, or 2% or 3% or more.
  • the subject of analysis is preferably T cells specific for the administered peptide. Peptide-specific T cells can be identified, for example, by tetramer assay.
  • the URLC10 peptide and the DEPDC1 peptide can be administered alone or in combination with other peptides effective as a cancer vaccine.
  • a so-called cocktail vaccine containing the DEPDC1 peptide (EYYELFVNI / SEQ ID NO: 178) in addition to the URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) can also be administered.
  • a TCR repertoire analysis is useful for assessing the frequency of TCRs of the present invention.
  • the amino acid sequences constituting the CDR3 of the TCR in a certain cell population are usually comprehensively analyzed. It has already been mentioned that CDR3 is primarily the region that determines the antigen recognition specificity of the TCR. Therefore, by analyzing its structure, it is possible to evaluate the proportion of TCR that constitutes the cell population.
  • TCR repertoire analysis is performed by comprehensively analyzing the CDR3s of a large number of TCRs and comparing the detection frequencies of each CDR3.
  • TCR The T cell receptor
  • VDJ VDJ genes
  • CDR3 VDJ genes
  • TCR repertoire analysis When performing TCR repertoire analysis, in order to amplify various TCR genes evenly by the PCR method, cDNA with an adapter added to the 5'end is synthesized from RNA derived from the T cell population.
  • a next-generation sequencer is used to sequence a large amount of DNA fragments (sequence library) obtained by using an adapter-specific forward primer and a TCR- ⁇ or TCR- ⁇ -specific reverse primer.
  • Next-generation sequencers are devices that have the ability to sequence millions of DNA fragments in parallel. In TCR repertoire analysis, it is necessary to determine a long base sequence across the V gene, D gene, J gene and C gene that compose TCR. Therefore, among next-generation sequencers, MiSeq (Illumina), which is good at long read analysis (determination of a base sequence of about 300 bp), is often used.
  • Forward primer (adapter sequence common to TCR- ⁇ and TCR- ⁇ , SEQ ID NO: 174): 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3' Reverse primer (for TCR- ⁇ , SEQ ID NO: 175): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3' Reverse primer (for TCR- ⁇ , SEQ ID NO: 176): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3'
  • HLA-A * 24 02-positive esophageal cancer patients who received a total of five or more doses of peptide cocktail vaccine containing URLC10-derived peptide once a week to patients with cellular esophageal cancer (case 1001, case 1001)
  • PBMC Peripheral blood mononuclear cells
  • TISI cells a human B lymphoblastoid cell line, were purchased from the International Histocompatibility Working Group.
  • Peptide HLA-A * 24: 02 A restrictive URLC10-derived peptide (RYCNLEGPPI: SEQ ID NO: 177: WO2006 / 090810) was used. The lyophilized peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg / ml) and stored at -80 ° C until use.
  • PBMCs were stimulated with peptides and cytokines (IL-2) for the purpose of proliferating peptide-specific cytotoxic T cells (CTLs) in PBMCs.
  • CTLs cytotoxic T cells
  • PBMCs collected from esophageal cancer patients (HLA-A * 24: 02 positive) who received a peptide cocktail vaccine containing a URLC10-derived peptide were seeded on a 48-well plate (Corning), and a URLC10-derived peptide was added (final peptide). Concentration: 10 ⁇ g / ml). The next day, IL-2 (Novartis) was added (IL-2 final concentration: 120 IU / ml). Half of the medium (supernatant) was removed every 2 to 3 days, the same amount of medium containing IL-2 was added, and the medium was replaced (IL-2 final concentration: 120 IU / ml).
  • PBMCs were used in the tetramer assay and limiting dilution method.
  • Tetramer assay A tetramer assay was performed to detect T cells that recognize the HLA-peptide complex from PBMCs after culture (in vitro stimulation). PE-labeled URLC10 tetramer and PE-labeled HIV tetramer (negative control) synthesized by Medical Biology Laboratory Co., Ltd. were used. PBMC was stained with tetramer and then further stained with FITC-labeled anti-CD8 antibody, APC-labeled anti-CD3 antibody and PE-Cy7-labeled anti-CD4 antibody (all BD Biosciences). Finally, the cells were stained with 0.1 mg / ml DAPI (BD Biosciences) solution and analyzed with a flow cytometer (SH800 cell sorter, Sony). Tetramer-positive CD8-positive T cells in the DAPI-negative CD3-positive and CD4-negative cell population were collected and used as samples for TCR repertoire analysis.
  • CTLs were cultured in 5% ABS / AIM-V medium with -2 (final concentration: 144 IU / ml) (culture solution volume 25 ml / flask). Every 2-3 days, media exchange was performed with 5% ABS / AIM-V medium containing 72 IU / ml IL-2 (IL-2 final concentration: 36 IU / ml) (Uchida N et al., Clin Cancer).
  • IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed to confirm peptide-specific IFN- ⁇ production of CTL clones.
  • TISI cells pulsed with the peptide were prepared as target cells.
  • the IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed according to the procedures recommended by the assay kit manufacturer.
  • TCR analysis RNA was extracted from tetramer-positive CD8-positive T cells using the R Easy micro kit (QIAGEN). SMARTScribe Reverse Transcriptase (Clontech) synthesized cDNA with an adapter added to the 5'end. Sequence live of TCR- ⁇ chain and TCR- ⁇ chain using a method similar to the method reported by Choudhury et al. (Choudhury NJ et al., Eur Urol Focus 2016, 2 (4): 445-52). A rally was prepared. A 300 bp paired end sequence was performed with MiSeq (Illumina).
  • the obtained sequence reads were used for the TCR- ⁇ gene and TCR- registered in the TCR database IMGT / GENE-DB (Giudicelli V et al., Nucleic Acids Res 2005, 33 (Database issue): D256-61). Mapped to the base sequence of the ⁇ gene.
  • RNA was extracted from peptide-specific CTL clones using the R Easy micro kit, and then cDNA was synthesized. The nucleotide sequences of the TCR- ⁇ chain and the TCR- ⁇ chain were decoded by Sanger sequence analysis.
  • TCR repertoire analysis of URLC10 tetramer-positive CD8-positive T cells Comprehensive TCR analysis (TCR repertoire analysis) of URLC10 tetramer-positive CD8-positive T cells was performed.
  • the TCR chronotype detection frequency is represented by a pie chart (Fig. 2). TCR chronotypes with a detection frequency of less than 1% are shown together in the same pattern (* in the figure).
  • TCR- ⁇ of case 1002 Fig. 2-a
  • the TCR chronotypes SEQ ID NO: 1 and SEQ ID NO: 2 whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides.
  • TCR- ⁇ the TCR chronotypes (SEQ ID NO: 3 and SEQ ID NO: 4) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides.
  • TCR- ⁇ of case 1003 the TCR chronotype (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) whose detection frequency was 1% or more was derived from URLC10. It was identified as a TCR that recognizes peptides.
  • TCR- ⁇ URLC10-derived peptides were recognized for TCR chronotypes (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13) whose detection frequency was 1% or more. Identified as TCR.
  • Table 1 shows the CDR3 amino acid sequence of the TCR that recognizes the URLC10-derived peptide detected at a frequency of 1% or more in the URLC10 tetramer-positive CD8-positive T cells.
  • HLA-A * 24 02-positive esophageal cancer patients who received a total of five or more doses of peptide cocktail vaccine containing URLC10 and DEPDC1-derived peptides once a week to patients with cellular esophageal cancer (cases)
  • Peripheral blood mononuclear cells (PBMC) were collected from the blood of 1004, 1005, 1006, 1007, 1008 and 1009).
  • TISI cells a human B lymphoblastoid cell line, were purchased from the International Histocompatibility Working Group.
  • the lyophilized peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg / ml) and stored at -80 ° C until use.
  • PBMCs were stimulated with peptides and cytokines (IL-2) for the purpose of proliferating peptide-specific cytotoxic T cells (CTLs) in PBMCs.
  • CTLs cytotoxic T cells
  • PBMCs collected from esophageal cancer patients who received a peptide cocktail vaccine containing a URLC10-derived peptide and a DEPDC1-derived peptide were seeded on a 48-well plate (Corning), and the URLC10-derived peptide or DEPDC1 was seeded.
  • the derived peptide was added (final peptide concentration: 10 ⁇ g / ml).
  • IL-2 Novartis
  • IL-2 final concentration: 120 IU / ml was added.
  • Tetramer assay A tetramer assay was performed to detect T cells that recognize the HLA-peptide complex from PBMCs after culture (in vitro stimulation). PE-labeled URLC10 tetramer, PE-labeled DEPDC1 tetramer and PE-labeled HIV tetramer (negative control) synthesized by Medical Biology Laboratory Co., Ltd. were used. PBMC was stained with tetramer and then further stained with FITC-labeled anti-CD8 antibody, APC-labeled anti-CD3 antibody and PE-Cy7-labeled anti-CD4 antibody (all BD Biosciences).
  • CTLs were cultured in 5% ABS / AIM-V medium with -2 (final concentration: 144 IU / ml) (culture solution volume 25 ml / flask). Every 2-3 days, media exchange was performed with 5% ABS / AIM-V medium containing 72 IU / ml IL-2 (IL-2 final concentration: 36 IU / ml) (Uchida N et al., Clin Cancer).
  • IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed to confirm peptide-specific IFN- ⁇ production of CTL clones.
  • TISI cells pulsed with the peptide were prepared as target cells.
  • the IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed according to the procedures recommended by the assay kit manufacturer.
  • TCR analysis RNA was extracted from tetramer-positive CD8-positive T cells using the R Easy micro kit (QIAGEN). SMARTScribe Reverse Transcriptase (Clontech) synthesized cDNA with an adapter added to the 5'end. Sequence live of TCR- ⁇ chain and TCR- ⁇ chain using a method similar to the method reported by Choudhury et al. (Choudhury NJ et al., Eur Urol Focus 2016, 2 (4): 445-52). A rally was prepared. A 300 bp paired end sequence was performed with MiSeq (Illumina).
  • the obtained sequence reads were used for the TCR- ⁇ gene and TCR- registered in the TCR database IMGT / GENE-DB (Giudicelli V et al., Nucleic Acids Res 2005, 33 (Database issue): D256-61). Mapped to the base sequence of the ⁇ gene.
  • RNA was extracted from peptide-specific CTL clones using the R Easy micro kit, and then cDNA was synthesized. The nucleotide sequences of the TCR- ⁇ chain and the TCR- ⁇ chain were decoded by Sanger sequence analysis.
  • TCR repertoire analysis of tetramer-positive CD8-positive T cells
  • the frequency of detection of TCR chronotypes is represented by a pie chart (Fig. 5A and Fig. 5B). TCR chronotypes with a detection frequency of less than 1% are shown together in the same pattern (* in the figure). In TCR- ⁇ of case 1004 (Fig. 5A-a), the detection frequency was 1% or more.
  • TCR chronotype SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25
  • TCR- ⁇ the detection frequency was 1% or more for the TCR chronotypes (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and sequence. Number: 32) was identified as a TCR that recognizes URLC10-derived peptides.
  • TCR- ⁇ of case 1005 Fig. 5A-b
  • the detection frequency was 1% or more.
  • TCR chronotype SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40
  • the detection frequency was 1% or more for the TCR chronotype (SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: Number: 47 and SEQ ID NO: 48) were identified as TCRs that recognize URLC10-derived peptides.
  • TCR- ⁇ of case 1006 Fig.
  • TCR chronotype SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:: 53 and SEQ ID NO: 54
  • TCR- ⁇ URLC10-derived peptides were recognized for TCR chronotypes (SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) whose detection frequency was 1% or more.
  • TCR chronotypes SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59
  • the TCR chronotype (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) whose detection frequency was 1% or more was derived from URLC10. It was identified as a TCR that recognizes peptides.
  • TCR- ⁇ the TCR chronotypes (SEQ ID NO: 63 and SEQ ID NO: 64) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides.
  • TCR chronotypes (SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68) whose detection frequency was 1% or more in TCR- ⁇ of case 1007 (Fig. 5A-e, High population).
  • TCR- ⁇ the TCR chronotype (SEQ ID NO: 64) whose detection frequency was 1% or more was identified as a TCR that recognizes URLC10-derived peptides.
  • TCR chronotypes SEQ ID NO: 69 and SEQ ID NO: 70 whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides.
  • TCR- ⁇ the detection frequency was 1% or more for the TCR chronotypes (SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 41, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and sequence. Number: 76) was identified as a TCR that recognizes URLC10-derived peptides.
  • TCR chronotypes SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79
  • TCR- ⁇ the TCR chronotypes (SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides. ..
  • TCR- ⁇ of case 1007 the TCR chronotypes (SEQ ID NO: 84 and SEQ ID NO: 85) whose detection frequency was 1% or more were identified as TCRs that recognize DEPDC1-derived peptides.
  • TCR chronotype SEQ ID NO: 86
  • TCR chronotype whose detection frequency was 1% or more was identified as a TCR that recognizes DEPDC1-derived peptides.
  • Table 3A shows the CDR3 amino acid sequence of the TCR that recognizes the URLC10-derived peptide detected at a frequency of 1% or more in the URLC10 tetramer-positive CD8-positive T cells.
  • Table 3B shows the CDR3 amino acid sequence of TCR that recognizes DEPDC1-derived peptides detected at a frequency of 1% or more in DEPDC1 tetramer-positive CD8-positive T cells.
  • CTL clones that recognize URLC10-derived peptides were established by the limiting dilution method.
  • CTL clones # 1, # 2, # 3 and # 4 showed peptide-specific IFN- ⁇ production (Fig. 6A, ad). From this, it was confirmed that CTL clones # 1, # 2, # 3 and # 4 recognized the URLC10-derived peptide presented on HLA-A * 24: 02.
  • CTL clone # 5 showed peptide-specific IFN- ⁇ production (Fig. 6B). From this, it was confirmed that CTL clone # 5 recognized the DEPDC1-derived peptide presented on HLA-A * 24: 02.
  • TCR gene-introduced T cells Nucleotide sequence of T cell receptor ⁇ chain (Amino acid sequence of CDR3: SEQ ID NO: 17) and T cell receptor ⁇ chain (amino acid sequence of CDR3: SEQ ID NO: 26) that recognize URLC10-derived peptides After performing codon optimization, they were incorporated into the pMP71-PRE retroviral vector (Leisegang M et al., Clin Cancer Res 2010, 16 (8): 2333-43).
  • the V region (including CDR1 and CDR2) and J region of the ⁇ chain and ⁇ chain are as follows.
  • V region J region ⁇ chain TRAV26-1 -CDR3-TRAJ23 (SEQ ID NO: 180) ⁇ -chain TRBV10-2-CDR3-TRBJ1-5 (SEQ ID NO: 181)
  • TRAC and TRBC1 the nucleotide sequences of the C gene regions (TRAC and TRBC1) of the ⁇ chain and ⁇ chain were modified to the nucleotide sequences derived from mice, respectively (Cohen CJ et al., Cancer). Res 2006, 66 (17): 8878-86).
  • a transient recombinant retroviral solution was used to introduce the T cell receptor (TCR) gene into PBMCs collected from HLA-A24 positive humans.
  • TCR transgenic T cells were evaluated by tetramer assay.
  • CD8-positive T cells detected by PE-labeled URLC10 tetramer (Institute of Medical Biology Co., Ltd.) and Alexa Fluor 700-labeled anti-mouse TCR ⁇ antibody (eBioscience) were designated as TCR-transfected T cells.
  • URLC10-derived peptide-specific IFN- ⁇ production and Granzyme B production by TCR-transfected T cells were confirmed by ELISA.
  • TISI cells pulsed with peptides HLA-A * 24: 02 positive
  • the response of TCR transgenic T cells to tumor cell lines was verified by ELISA.
  • Esophageal cancer cell lines TE-1 (URLC10 positive, HLA-A * 24: 02 positive) and TE-9 (URLC10 positive, HLA-A * 24: 02 negative) were targeted cells.
  • the esophageal cancer cell line was purchased from the Medical Cell Resources Center of the Institute of Aging Medicine, Tohoku University.
  • TCR-transfected T cells IFN- ⁇ ELISA and Granzyme B ELISA were performed to analyze the function of TCR-transfected T cells.
  • TISI cells pulsed with the peptide were prepared as target cells.
  • ELISA was performed according to the procedure recommended by the assay kit manufacturer.
  • TCR-introduced T cells showed URLC10-derived peptide-specific IFN- ⁇ production (Fig. 8a) and cytotoxic granule (Granzyme B) production (Fig. 8a). Figure 8b). From this, it was confirmed that the TCR transgenic T cells have a function of recognizing the URLC10-derived peptide presented on HLA-A * 24: 02 and further producing IFN- ⁇ and Granzyme B.
  • TCR-transfected T cells showed IFN- ⁇ production when co-cultured with TE-1 (URLC10 positive, HLA-A * 24: 02 positive) ( Figure 9). No significant IFN- ⁇ production was observed when co-cultured with TE-9 (URLC10 positive, HLA-A * 24: 02 negative). Therefore, it was shown that TCR transgenic T cells can recognize cancer cells that present URLC10-derived peptides on HLA-A * 24: 02.
  • a TCR that imparts HLA-A24-restricting cytotoxic effects specific to URLC10 or DEPDC1 to T cells has been found.
  • a CTL specific for URLC10 or DEPDC1 can be obtained, enabling TCR gene transfer T cell therapy.
  • the TCR of the present invention is also useful as a monitoring index of therapeutic effect in a subject to which a CTL-induced peptide has been administered.
  • the CTL equipped with the TCR of the present invention is actually a cell having cytotoxic activity against a target cell. Therefore, if the proportion of CTLs equipped with the TCR of the present invention increases through TCR repertoire analysis, it means that the desired CTL response is induced in the subject, and it is possible that the therapeutic effect of the peptide can be achieved. Can be known.

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Abstract

The present invention provides a T-cell receptor (TCR) that has URLC10- or DEPDC1-specific HLA-A24-restricted cytotoxic activity. Introducing this TCR into T cells makes it possible to produce cytotoxic T cells (CTL) that can be used to treat cancer. This TCR can also be used to monitor immune response in subjects that have been inoculated with a URLC10 or DEPDC1 peptide vaccine.

Description

ＵＲＬＣ１０由来ペプチドまたはＤＥＰＤＣ１由来ペプチドを認識するヒトＴ細胞が発現するＴ細胞受容体T cell receptor expressed by human T cells that recognize URLC10-derived peptides or DEPDC1-derived peptides
　本発明は、生物科学の分野、より具体的にはがん治療の分野に関する。特に本発明は、がん細胞表面の主要組織適合遺伝子複合体（MHC）クラスI分子とともに提示されるがん抗原由来ペプチドを認識するT細胞受容体（TCR）のアミノ酸配列および該TCRを利用したTCR遺伝子導入T細胞療法に関する。 The present invention relates to the field of biological science, more specifically to the field of cancer treatment. In particular, the present invention utilizes the amino acid sequence of a T cell receptor (TCR) that recognizes a cancer antigen-derived peptide presented with a major histocompatibility complex (MHC) class I molecule on the surface of cancer cells and the TCR. TCR gene transfer T cell therapy.
　がんに対する三大療法とは手術療法、放射線療法および化学療法であるが、近年、第四の治療法として免疫療法が注目されている。その代表例である免疫チェックポイント阻害剤（抗PD-1抗体、抗PD-L1抗体あるいは抗CTLA-4抗体）は、悪性黒色腫をはじめ非小細胞肺がんやホジキンリンパ腫等様々ながんに対する新しい治療薬として承認されている。しかしながら、その奏効率は20～30%程度と決して高くはない。そこで、免疫チェックポイント阻害剤による治療効果が認められない多くの患者に対するさらなる免疫療法の開発が望まれる。 The three major therapies for cancer are surgery, radiation therapy and chemotherapy, but in recent years, immunotherapy has been attracting attention as the fourth treatment method. Typical examples of immune checkpoint inhibitors (anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA-4 antibody) are new for various cancers such as malignant melanoma, non-small cell lung cancer and Hodgkin lymphoma. Approved as a therapeutic drug. However, the response rate is not high at about 20 to 30%. Therefore, it is desired to develop further immunotherapy for many patients who do not have the therapeutic effect of immune checkpoint inhibitors.
　がんに対する新たな免疫療法の一つとして、TCR遺伝子導入T細胞療法が挙げられる。これまでに、がん抗原NY-ESO-1を標的としたTCR遺伝子導入T細胞療法の多発性骨髄腫に対する治療効果が報告されている（非特許文献1）。しかし、TCR遺伝子導入T細胞投与の結果、T細胞がTCRを通じて正常細胞のMHCクラスI分子上に提示されたペプチドを認識したことが原因と考えられる重篤な有害事象が発生した例も報告されている（非特許文献2；非特許文献3）。がんにおいて発現の亢進が認められる一方で正常組織ではほとんど発現が認められない抗原を標的とし、その抗原に由来するペプチドを認識するTCRを同定すれば、重篤な有害事象を引き起こさないTCR遺伝子導入T細胞療法を開発することが可能になると期待される。 One of the new immunotherapies for cancer is TCR gene transfer T cell therapy. So far, the therapeutic effect of TCR gene transfer T cell therapy targeting the cancer antigen NY-ESO-1 on multiple myeloma has been reported (Non-Patent Document 1). However, as a result of TCR gene-introduced T cell administration, there have been reports of serious adverse events that may have been caused by T cells recognizing peptides presented on MHC class I molecules of normal cells through TCR. (Non-Patent Document 2; Non-Patent Document 3). If you target an antigen that is upregulated in cancer but rarely expressed in normal tissues and identify a TCR that recognizes peptides derived from that antigen, a TCR gene that does not cause serious adverse events It is expected that it will be possible to develop transduced T cell therapy.
　URLC10（Up-regulated in lung cancer 10, 別名：lymphocyte antigen 6 complex, locus K (LY6K), GenBankアクセッション番号：NM_017527、またはアクセッション番号：BC117142）は、27,648遺伝子を対象としたcDNAマイクロアレイによるゲノムワイドな遺伝子発現プロファイルの結果から、非小細胞肺がんや食道扁平上皮がんにおいて発現が亢進している遺伝子として報告されている（非特許文献4）。非小細胞肺がん患者の88.2%（406症例中358症例）および食道がん患者の95.1%（265症例中252症例）において、がん組織でのURLC10の発現が認められた。一方、URLC10の発現が認められた正常臓器は精巣のみであった。 URLC10 (Up-regulated in lung cancer 10, alias: lymphocyte antigen 6 complex, locus K (LY6K), GenBank accession number: NM_017527, or accession number: BC117142) is genome-wide by cDNA microarray for 27,648 genes. From the results of gene expression profiles, it has been reported as a gene whose expression is upregulated in non-small cell lung cancer and esophageal squamous cell carcinoma (Non-Patent Document 4). Expression of URLC10 was observed in cancer tissues in 88.2% of patients with non-small cell lung cancer (358 of 406 cases) and 95.1% of patients with esophageal cancer (252 of 265 cases). On the other hand, the only normal organ in which URLC10 expression was observed was the testis.
　DEPDC1 (DEP domain containing 1, GenBankアクセッション番号：NM_001114120、またはアクセッション番号：BM683578)は膀胱がんにおいて発現の亢進が認められる遺伝子として報告されている(非特許文献5)。膀胱がん患者の81%（21症例中17症例）において、がん細胞でのDEPDC1の発現が認められた。一方、DEPDC1の発現が認められた正常組織は精巣のみであった。
　つまりURLC10やDEPDC1のような、がん細胞に特異的な分子は、治療標的に好適である。このような背景の下、URLC10特異的な細胞傷害性T細胞（CTL）を誘導するペプチドが同定された（特許文献１）。同定されたURLC10ペプチドは、URLC10を標的とするがんワクチン療法に有用である。そして、DEPDC1についても同様に、がんワクチン療法を可能とするDEPDC1ペプチドが同定されている（特許文献２）。URLC10ペプチドやDEPDC1ペプチドは、細胞傷害性T細胞（CTL）の誘導によって抗腫瘍効果をもたらす。 DEPDC1 (DEP domain containing 1, GenBank accession number: NM_001114120, or accession number: BM683578) has been reported as a gene whose expression is upregulated in bladder cancer (Non-Patent Document 5). Expression of DEPDC1 was observed in cancer cells in 81% of bladder cancer patients (17 of 21 cases). On the other hand, the testis was the only normal tissue in which DEPDC1 expression was observed.
That is, cancer cell-specific molecules such as URLC10 and DEPDC1 are suitable therapeutic targets. Against this background, a peptide that induces URLC10-specific cytotoxic T cells (CTL) has been identified (Patent Document 1). The identified URLC10 peptide is useful for cancer vaccine therapy targeting URLC10. Similarly, for DEPDC1, a DEPDC1 peptide that enables cancer vaccine therapy has been identified (Patent Document 2). The URLC10 peptide and DEPDC1 peptide have antitumor effects by inducing cytotoxic T cells (CTL).
WO2006/090810WO2006 / 090810 WO2008/047473WO2008 / 074773
　本発明は、URLC10、あるいはDEPDC1特異的な細胞傷害作用を付与することができる、TCRの同定を課題とする。 An object of the present invention is the identification of a TCR capable of imparting a cytotoxic effect specific to URLC10 or DEPDC1.
　URLC10やDEPDC1は、そのがん特異的な高発現のために、がんの免疫療法のための標的抗原として優れた特性を備える。実際、これらの分子からCTL誘導性ペプチドとして同定されたURLC10ペプチド、あるいはDEPDC1ペプチドは、CTLの誘導を介するがん免疫療法を可能とすることが明らかにされている。本発明者らは、これらペプチドによって誘導されたCTLが有するTCRを単離することができれば、ペプチドによるCTLの誘導に代わる、TCR遺伝子導入T細胞療法を実現することができると考えた。そして、実際に、URLC10やDEPDC1特異的な細胞傷害作用を有するTCRの同定に成功し本発明に至った。すなわち本発明は、以下の各態様に関する。
〔１〕配列番号：1-2、5-8、14、16、17-25、33-40、49-54、60-62、65-70、77-79、および84-85からなる群から選択されるいずれかのアミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含むT細胞受容体α鎖。
〔２〕CASS-[X]n-F（Xは任意のアミノ酸でnが6-8／配列番号：179）を含む11-13アミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含むT細胞受容体β鎖。
〔３〕配列番号：3-4、9-13、15、26-32、41-48、55-59、63-64、71-76、80-83、および86からなる群から選択されるいずれかのアミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含む〔２〕に記載のT細胞受容体β鎖。
〔４〕〔１〕に記載のいずれかのT細胞受容体α鎖と、〔２〕または〔３〕に記載のいずれかのT細胞受容体β鎖の組み合わせからなるT細胞受容体。
〔５〕T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列が、以下のいずれかの組み合わせである〔４〕に記載のT細胞受容体：
T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
配列番号：1　　　　　　　　　　　　　　配列番号：3、
配列番号：2　　　　　　　　　　　　　　配列番号：3、
配列番号：14　　　　　　　　　　　　　 配列番号：15、
配列番号：16　　　　　　　　　　　　　 配列番号：10、
配列番号：17　　　　　　　　　　　　　 配列番号：26、
配列番号：60　　　　　　　　　　　　　 配列番号：63、
配列番号：69　　　　　　　　　　　　　 配列番号：71、
配列番号：77　　　　　　　　　　　　　 配列番号：80、および
配列番号：84　　　　　　　　　　　　　 配列番号：86。
〔６〕〔１〕に記載のいずれかのT細胞受容体α鎖および、〔２〕または〔３〕に記載のいずれかのT細胞受容体β鎖のいずれかをコードするポリヌクレオチド。
〔７〕〔６〕に記載のポリヌクレオチドを含む発現ベクター。
〔８〕T細胞受容体α鎖をコードするポリヌクレオチドと、T細胞受容体β鎖をコードするポリヌクレオチドの両方を含む〔７〕に記載の発現ベクター。
〔９〕T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列が、以下のいずれかの組み合わせである〔８〕に記載の発現ベクター：
T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
配列番号：1　　　　　　　　　　　　　　配列番号：3、
配列番号：2　　　　　　　　　　　　　　配列番号：3、
配列番号：14　　　　　　　　　　　　　 配列番号：15、
配列番号：16　　　　　　　　　　　　　 配列番号：10、
配列番号：17　　　　　　　　　　　　　 配列番号：26、
配列番号：60　　　　　　　　　　　　　 配列番号：63、
配列番号：69　　　　　　　　　　　　　 配列番号：71、
配列番号：77　　　　　　　　　　　　　 配列番号：80、および
配列番号：84　　　　　　　　　　　　　 配列番号：86。
〔１０〕〔８〕または〔９〕に記載の発現ベクターからなる、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞を誘導するための発現ベクター。
〔１１〕〔８〕～〔１０〕いずれかに記載の発現ベクターを含む、細胞傷害性T細胞の誘導用組成物。
〔１２〕がんの治療を必要とする対象から採取されたCD8+（CD8陽性）T細胞に〔８〕～〔１０〕いずれかに記載の発現ベクターを導入し、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞を回収する工程を含む、細胞傷害性T細胞の製造方法。
〔１３〕〔８〕～〔１０〕いずれかに記載の発現ベクターで形質転換されたURLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞。
〔１４〕URLC10およびDEPDC1のいずれか、または両方の細胞傷害性T細胞の誘導処置を受けた対象から採取された末梢血リンパ球のT細胞受容体のCDR3のアミノ酸配列を決定する工程を含む、URLC10またはDEPDC1に対するHLA-A24拘束性の細胞傷害性T細胞の誘導をモニタリングする方法であって、CDR3のアミノ酸配列が配列番号：1-86からなる群から選択される少なくとも一つのアミノ酸配列である方法。
〔１５〕CDR3のアミノ酸配列のレパトア解析によって、CDR3のアミノ酸配列の検出頻度を決定する工程を含む〔１４〕に記載の方法。 Due to its cancer-specific high expression, URLC10 and DEPDC1 have excellent properties as target antigens for cancer immunotherapy. In fact, the URLC10 peptide, or DEPDC1 peptide, identified as CTL-inducible peptides from these molecules has been shown to enable cancer immunotherapy via CTL induction. The present inventors considered that if the TCR possessed by CTLs induced by these peptides could be isolated, TCR gene transfer T cell therapy could be realized instead of the induction of CTLs by peptides. Then, in fact, they succeeded in identifying a TCR having a cytotoxic effect specific to URLC10 and DEPDC1, and came to the present invention. That is, the present invention relates to each of the following aspects.
[1] SEQ ID NO: From the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70, 77-79, and 84-85. A T cell receptor α chain containing CDR3 identified by any of the selected amino acid sequences, or a CDR3 functionally equivalent thereto.
[2] Contains CDR3 specified by the 11-13 amino acid sequence including CASS- [X] nF (X is an arbitrary amino acid and n is 6-8 / SEQ ID NO: 179), or CDR3 functionally equivalent thereto. T cell receptor β chain.
[3] SEQ ID NO: Any selected from the group consisting of 3-4, 9-13, 15, 26-32, 41-48, 55-59, 63-64, 71-76, 80-83, and 86. The T cell receptor β chain according to [2], which comprises CDR3 specified by the amino acid sequence or CDR3 functionally equivalent thereto.
[4] A T cell receptor comprising a combination of any T cell receptor α chain according to [1] and any T cell receptor β chain according to [2] or [3].
[5] The T cell receptor according to [4], wherein the amino acid sequence of CDR3 of the T cell receptor α chain and the T cell receptor β chain is any combination of the following:
CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
SEQ ID NO: 1 SEQ ID NO: 3,
SEQ ID NO: 2 SEQ ID NO: 3,
SEQ ID NO: 14 SEQ ID NO: 15,
SEQ ID NO: 16 SEQ ID NO: 10,
SEQ ID NO: 17 SEQ ID NO: 26,
SEQ ID NO: 60 SEQ ID NO: 63,
SEQ ID NO: 69 SEQ ID NO: 71,
SEQ ID NO: 77 SEQ ID NO: 80, and SEQ ID NO: 84 SEQ ID NO: 86.
[6] A polynucleotide encoding any T cell receptor α chain according to [1] and any T cell receptor β chain according to [2] or [3].
[7] An expression vector containing the polynucleotide according to [6].
[8] The expression vector according to [7], which contains both a polynucleotide encoding a T cell receptor α chain and a polynucleotide encoding a T cell receptor β chain.
[9] The expression vector according to [8], wherein the amino acid sequence of CDR3 of the T cell receptor α chain and the T cell receptor β chain is any combination of the following:
CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
SEQ ID NO: 1 SEQ ID NO: 3,
SEQ ID NO: 2 SEQ ID NO: 3,
SEQ ID NO: 14 SEQ ID NO: 15,
SEQ ID NO: 16 SEQ ID NO: 10,
SEQ ID NO: 17 SEQ ID NO: 26,
SEQ ID NO: 60 SEQ ID NO: 63,
SEQ ID NO: 69 SEQ ID NO: 71,
SEQ ID NO: 77 SEQ ID NO: 80, and SEQ ID NO: 84 SEQ ID NO: 86.
[10] An expression vector comprising the expression vector according to [8] or [9] for inducing cytotoxic T cells having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells.
[11] A composition for inducing cytotoxic T cells, which comprises the expression vector according to any one of [8] to [10].
[12] The expression vector described in any of [8] to [10] is introduced into CD8 + (CD8 positive) T cells collected from a subject requiring treatment for cancer, and HLA for URLC10 or DEPDC1-expressing cells is introduced. -A24 A method for producing cytotoxic T cells, which comprises a step of recovering cytotoxic T cells having a binding cytotoxic effect.
[13] Cytotoxic T cells having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells transformed with the expression vector according to any one of [8] to [10].
[14] The step of determining the amino acid sequence of CDR3 of the T cell receptor of peripheral blood lymphocytes collected from a subject who has undergone induction treatment of either or both of URLC10 and DEPDC1. A method of monitoring the induction of HLA-A24-restrained cytotoxic T cells against URLC10 or DEPDC1 in which the amino acid sequence of CDR3 is at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-86. Method.
[15] The method according to [14], which comprises a step of determining the detection frequency of the amino acid sequence of CDR3 by repertoire analysis of the amino acid sequence of CDR3.
　あるいは本発明は、本発明のT細胞受容体α鎖および、T細胞受容体β鎖のいずれかをコードするポリヌクレオチドの、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞の製造における使用に関する。加えて本発明は、本発明のT細胞受容体α鎖および、T細胞受容体β鎖のいずれかをコードするポリヌクレオチドの、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞誘導用組成物の製造における使用に関する。 Alternatively, the present invention is a cell having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells of a polynucleotide encoding either the T cell receptor α chain or the T cell receptor β chain of the present invention. For use in the production of cytotoxic T cells. In addition, the present invention has an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells of the polynucleotide encoding either the T cell receptor α chain or the T cell receptor β chain of the present invention. With respect to use in the manufacture of compositions for inducing cytotoxic T cells.
　本発明のTCRは、URLC10あるいはDEPDC1に特異的な細胞傷害作用を持つと考えられるT細胞から取得された。したがって、本発明のTCRをT細胞に導入することによって、URLC10あるいはDEPDC1に特異的な細胞傷害作用を付与することができる。URLC10あるいはDEPDC1は、がんに特異的に過剰発現している腫瘍抗原であることから、本発明のTCRを導入したT細胞には、がん特異的な細胞傷害作用を期待できる。 The TCR of the present invention was obtained from T cells considered to have a cytotoxic effect specific to URLC10 or DEPDC1. Therefore, by introducing the TCR of the present invention into T cells, it is possible to impart a cytotoxic effect specific to URLC10 or DEPDC1. Since URLC10 or DEPDC1 is a tumor antigen that is specifically overexpressed in cancer, the T cells into which the TCR of the present invention has been introduced can be expected to have a cancer-specific cytotoxic effect.
　あるいは、本発明のTCRは、URLC10あるいはDEPDC1に特異的な細胞傷害作用を持つT細胞の指標となる。すなわち、URLC10あるいはDEPDC1のワクチンを投与された対象において、本発明のTCRの頻度を解析し、その割合が高まる場合に、有効なT細胞がワクチンの接種によって誘導されていることを知ることができる。 Alternatively, the TCR of the present invention is an index of T cells having a cytotoxic effect specific to URLC10 or DEPDC1. That is, it is possible to analyze the frequency of TCR of the present invention in a subject to which the URLC10 or DEPDC1 vaccine has been administered, and to know that effective T cells are induced by vaccination when the ratio is increased. ..
図1は、培養（インビトロ刺激）後のPBMCについて実施したテトラマーアッセイの結果（a）および（b）で構成される。HLA-A*24:02上に提示されたURLC10由来ペプチドを認識するURLC10テトラマー陽性CD8陽性T細胞集団が検出された。URLC10テトラマー陽性CD8陽性T細胞集団は、フローサイトメーターのソーティング機能を利用して回収されたのち、TCRレパトア解析用検体として用いられた。FIG. 1 consists of the results (a) and (b) of the tetramer assay performed on PBMCs after culture (in vitro stimulation). HLA-A * 24: 02 A URLC10 tetramer-positive CD8-positive T cell population recognizing the URLC10-derived peptide presented above was detected. The URLC10 tetramer-positive CD8-positive T cell population was collected using the sorting function of the flow cytometer and then used as a sample for TCR repertoire analysis. 図2は、URLC10テトラマー陽性CD8陽性T細胞集団のTCRレパトア解析の結果を示すパイチャート（a）および（b）で構成される。パイチャートは、TCRレパトア解析によって検出されたTCRクロノタイプの検出頻度を表している。パイチャートにおける専有面積が広ければ、高頻度で検出されたことを意味する。検出頻度が1%以上のTCRクロノタイプをURLC10由来ペプチドを認識するTCRとして同定した。検出頻度が1%未満のTCRクロノタイプはまとめて同じパターンで示した（図中*）。FIG. 2 consists of pie charts (a) and (b) showing the results of TCR repertoire analysis of the URLC10 tetramer-positive CD8-positive T cell population. The pie chart shows the frequency of detection of TCR chronotypes detected by TCR repertoire analysis. If the occupied area in the pie chart is large, it means that it was detected frequently. TCR chronotypes with a detection frequency of 1% or more were identified as TCRs that recognize URLC10-derived peptides. TCR chronotypes with a detection frequency of less than 1% are collectively shown in the same pattern (* in the figure). 図3は、培養（インビトロ刺激）後のPBMCから限界希釈法によって樹立されたCTLクローンのIFN-γ産生を示す折れ線グラフ（a）-（c）で構成される。URLC10由来ペプチドをパルスした標的細胞に対するCTLクローンのIFN-γ産生が認められた一方で、ペプチドをパルスしていない標的細胞に対するCTLクローンの有意なIFN-γ産生は認められなかった。このことから、CTLクローンがHLA-A*24:02上に提示されたURLC10由来ペプチドを認識したことが確認された。R/S比は、応答細胞（Responder cells）であるCTLクローンの細胞数とそれを刺激する標的細胞（Stimulator cells）の細胞数の比を表す。FIG. 3 is composed of line graphs (a)-(c) showing IFN-γ production of CTL clones established by the limiting dilution method from PBMC after culture (in vitro stimulation). While IFN-γ production of CTL clones was observed in target cells pulsed with URLC10-derived peptide, no significant IFN-γ production of CTL clones was observed in target cells not pulsed with peptide. From this, it was confirmed that the CTL clone recognized the URLC10-derived peptide presented on HLA-A * 24: 02. The R / S ratio represents the ratio of the number of CTL clones, which are responder cells, to the number of target cells (Stimulator cells) that stimulate them. 図4Aは、培養（インビトロ刺激）後のPBMCについて実施したURLC10テトラマーアッセイの結果（a）-（f）で構成される。HLA-A*24:02上に提示されたURLC10由来ペプチドを認識するURLC10テトラマー陽性CD8陽性T細胞集団が検出された。FIG. 4A consists of the results (a)-(f) of the URLC10 tetramer assay performed on PBMCs after culture (in vitro stimulation). HLA-A * 24: 02 A URLC10 tetramer-positive CD8-positive T cell population recognizing the URLC10-derived peptide presented above was detected. 図4A-1の説明を参照。See the description in Figure 4A-1. 図4A-1の説明を参照。See the description in Figure 4A-1. 図4Bは、培養（インビトロ刺激）後のPBMCについて実施したDEPDC1テトラマーアッセイの結果で構成される。HLA-A*24:02上に提示されたDEPDC1由来ペプチドを認識するDEPDC1テトラマー陽性CD8陽性T細胞集団が検出された。テトラマー陽性CD8陽性T細胞集団は、フローサイトメーターのソーティング機能を利用して回収されたのち、TCRレパトア解析用検体として用いられた。FIG. 4B consists of the results of the DEPDC1 tetramer assay performed on PBMCs after culture (in vitro stimulation). HLA-A * 24: 02 A DEPDC1 tetramer-positive CD8-positive T cell population recognizing the DEPDC1-derived peptide presented above was detected. The tetramer-positive CD8-positive T cell population was collected using the sorting function of the flow cytometer and then used as a sample for TCR repertoire analysis.
図5Aは、URLC10テトラマー陽性CD8陽性T細胞集団のTCRレパトア解析の結果を示すパイチャート（a）-（g）で構成される。FIG. 5A is composed of pie charts (a)-(g) showing the results of TCR repertoire analysis of the URLC10 tetramer-positive CD8-positive T cell population. 図5A-1の説明を参照。See the description in Figure 5A-1. 図5A-1の説明を参照。See the description in Figure 5A-1. 図5A-1の説明を参照。See the description in Figure 5A-1. 図5Bは、DEPDC1テトラマー陽性CD8陽性T細胞集団のTCRレパトア解析の結果を示すパイチャートで構成される。パイチャートは、TCRレパトア解析によって検出されたTCRクロノタイプの検出頻度を表している。パイチャートにおける専有面積が広ければ、高頻度で検出されたことを意味する。検出頻度が1%以上のTCRクロノタイプのペプチドを認識するTCRとして同定した。検出頻度が1%未満のTCRクロノタイプはまとめて同じパターンで示した（図中*）。FIG. 5B consists of a pie chart showing the results of TCR repertoire analysis of the DEPDC1 tetramer-positive CD8-positive T cell population. The pie chart shows the frequency of detection of TCR chronotypes detected by TCR repertoire analysis. If the occupied area in the pie chart is large, it means that it was detected frequently. It was identified as a TCR that recognizes TCR chronotype peptides with a detection frequency of 1% or more. TCR chronotypes with a detection frequency of less than 1% are collectively shown in the same pattern (* in the figure).
図6Aは、培養（インビトロ刺激）後のPBMCから限界希釈法によって樹立されたURLC10由来ペプチド特異的CTLクローンのIFN-γ産生を示す折れ線グラフ（a）-（d）で構成される。URLC10由来ペプチドをパルスした標的細胞に対するCTLクローンのIFN-γ産生が認められた一方で、ペプチドをパルスしていない標的細胞に対するCTLクローンの有意なIFN-γ産生は認められなかった。このことから、CTLクローンがHLA-A*24:02上に提示されたURLC10由来ペプチドを認識したことが確認された。FIG. 6A consists of line graphs (a)-(d) showing IFN-γ production of URLC10-derived peptide-specific CTL clones established by limiting dilution from PBMC after culture (in vitro stimulation). While IFN-γ production of CTL clones was observed in target cells pulsed with URLC10-derived peptide, no significant IFN-γ production of CTL clones was observed in target cells not pulsed with peptide. From this, it was confirmed that the CTL clone recognized the URLC10-derived peptide presented on HLA-A * 24: 02. 図6Bは、培養（インビトロ刺激）後のPBMCから限界希釈法によって樹立されたDEPDC1由来ペプチド特異的CTLクローンのIFN-γ産生を示す折れ線グラフで構成される。DEPDC1由来ペプチドをパルスした標的細胞に対するCTLクローンのIFN-γ産生が認められた一方で、ペプチドをパルスしていない標的細胞に対するCTLクローンの有意なIFN-γ産生は認められなかった。このことから、CTLクローンがHLA-A*24:02上に提示されたDEPDC1由来ペプチドを認識したことが確認された。R/S比は、応答細胞（Responder cells）であるCTLクローンの細胞数とそれを刺激する標的細胞（Stimulator cells）の細胞数の比を表す。FIG. 6B consists of a line graph showing IFN-γ production of DEPDC1-derived peptide-specific CTL clones established by limiting dilution from PBMCs after culture (in vitro stimulation). While IFN-γ production of CTL clones was observed in target cells pulsed with DEPDC1-derived peptide, no significant IFN-γ production of CTL clones was observed in target cells not pulsed with peptide. From this, it was confirmed that the CTL clone recognized the DEPDC1-derived peptide presented on HLA-A * 24: 02. The R / S ratio represents the ratio of the number of CTL clones, which are responder cells, to the number of target cells (Stimulator cells) that stimulate them. 図7は、PBMCについて実施したURLC10テトラマーアッセイの結果（a）および（b）で構成される。無処置のPBMCからURLC10テトラマー陽性T細胞集団は検出されなかった（図7a）。一方、レトロウイルスベクターによってURLC10由来ペプチド反応性TCR遺伝子を導入したPBMCからはURLC10テトラマー陽性T細胞集団が検出された（図7b）。TCR遺伝子導入により、HLA-A*24:02上に提示されたURLC10由来ペプチドを認識するT細胞が作製された。FIG. 7 consists of the results (a) and (b) of the URLC10 tetramer assay performed on PBMCs. No URLC10 tetramer-positive T cell population was detected in untreated PBMCs (Fig. 7a). On the other hand, a URLC10 tetramer-positive T cell population was detected in PBMCs into which a URLC10-derived peptide-reactive TCR gene was introduced by a retroviral vector (Fig. 7b). TCR gene transfer produced T cells that recognize the URLC10-derived peptides presented on HLA-A * 24: 02. 図8は、TCR遺伝子導入T細胞のペプチド特異的なIFN-γ産生および細胞傷害顆粒（Granzyme B）産生を示す折れ線グラフ（a）および（b）で構成される。URLC10由来ペプチドをパルスしたHLA-A*24:02発現標的細胞に対するTCR遺伝子導入T細胞のIFN-γ産生（図8a）およびGranzyme B産生（図8b）が認められた一方で、ペプチドをパルスしていない標的細胞に対する有意なIFN-γ産生およびGranzyme B産生は認められなかった。このことから、TCR遺伝子導入T細胞がHLA-A*24:02上に提示されたURLC10由来ペプチドを認識し、さらにはIFN-γおよびGranzyme Bを産生する機能を持つことが確認された。R/S比は、応答細胞（Responder cells）であるTCR遺伝子導入T細胞の細胞数とそれを刺激する標的細胞（Stimulator cells）の細胞数の比を表す。FIG. 8 is composed of line graphs (a) and (b) showing peptide-specific IFN-γ production and cytotoxic granule (Granzyme B) production of TCR transgenic T cells. IFN-γ production (Fig. 8a) and Granzyme B production (Fig. 8b) of TCR gene-introduced T cells against HLA-A * 24: 02 expression target cells pulsed with URLC10-derived peptide were observed, while the peptide was pulsed. No significant IFN-γ production or Granzyme B production was observed for non-target cells. From this, it was confirmed that the TCR transgenic T cells have the function of recognizing the URLC10-derived peptide presented on HLA-A * 24: 02 and further producing IFN-γ and Granzyme B. The R / S ratio represents the ratio of the number of TCR gene-introduced T cells, which are responding cells (Responder cells), to the number of target cells (Stimulator cells) that stimulate them. 図9は、TCR遺伝子導入T細胞の食道がん細胞株に対する反応を示す折れ線グラフで構成される。TE-1（URLC10陽性, HLA-A*24:02陽性）と共培養した場合にTCR遺伝子導入T細胞によるIFN-γ産生が認められた一方で、TE-9（URLC10陽性, HLA-A*24:02陰性）と共培養した場合には有意なIFN-γの産生は認められなかった。このことから、TCR遺伝子導入T細胞 はHLA-A*24:02上に提示されたURLC10由来ペプチドを介してがん細胞を認識しうることが確認された。E/T比は、TCR遺伝子導入T細胞（Effector cells）の細胞数とその標的細胞（Target cells）の細胞数の比を表す。FIG. 9 consists of a line graph showing the response of TCR transgenic T cells to the esophageal cancer cell line. IFN-γ production by TCR-transfected T cells was observed when co-cultured with TE-1 (URLC10 positive, HLA-A * 24: 02 positive), while TE-9 (URLC10 positive, HLA-A *). No significant IFN-γ production was observed when co-cultured with (24:02 negative). From this, it was confirmed that TCR transgenic T cells can recognize cancer cells via the URLC10-derived peptide presented on HLA-A * 24: 02. The E / T ratio represents the ratio of the number of TCR gene-introduced T cells (Effector cells) to the number of target cells (Target cells).
Ｉ．定義
　本明細書で用いる「1つの（a）」、「1つの（an）」および「その（the）」という単語は、他に特記されない限り「少なくとも１つの」を意味する。
　物質（例えば、ペプチド、抗体、ポリヌクレオチド等）に関して用いる「単離された」および「精製された」という用語は、該物質がそうでなければ天然源中に含まれ得る少なくとも1種の物質を実質的に含まないことを示す。したがって、単離または精製されたペプチドは、そのペプチドが由来する細胞もしくは組織源からの他の細胞材料、例えば糖質、脂質、および他の混入タンパク質を実質的に含まないペプチドを指す。またはペプチドが化学合成される場合には、単離または精製されたペプチドは前駆体物質もしくは他の化学物質を実質的に含まないペプチドを指す。「細胞材料を実質的に含まない」という用語は、それが単離された細胞または組換え産生された細胞の細胞成分から、ペプチドが分離されたペプチドの調整物を含む。したがって、細胞材料を実質的に含まないペプチドは、約30%、20%、10%、または5%、3%、2%または1%（乾燥重量ベース）未満の他の細胞材料を含有する、ペプチドの調製物を包含する。ペプチドを組換え産生する場合、単離または精製されたペプチドは、培養培地も実質的に含まず、培養培地を実質的に含まないペプチドは、培養培地をペプチド調製物の容量の約20%、10%、または5%、3%、2%または1%（乾燥重量ベース）未満で含有する、ペプチドの調製物を包含する。ペプチドを化学合成によって生成する場合、単離または精製されたペプチドは、前駆体物質および他の化学物質を実質的に含まず、前駆体物質および他の化学物質を実質的に含まないペプチドは、前駆体物質および他の化学物質をペプチド調製物の容量の約30%、20%、10%、5%、3%、2%または1%（乾燥重量ベース）未満で含有する、ペプチドの調製物を包含する。特定のペプチド調製物が単離または精製されたペプチドであることは、例えば、ドデシル硫酸ナトリウム（SDS）－ポリアクリルアミドゲル電気泳動およびゲルのクーマシーブリリアントブルー染色等の後の単一バンドの出現によって確認することができる。好ましい態様では、本発明のペプチドおよびポリヌクレオチドは単離または精製されている。 I. Definitions The words "one (a)", "one (an)" and "the" as used herein mean "at least one" unless otherwise specified.
The terms "isolated" and "purified" used with respect to a substance (eg, peptides, antibodies, polynucleotides, etc.) refer to at least one substance that could otherwise be contained in a natural source. Indicates that it is practically not included. Thus, an isolated or purified peptide refers to a peptide that is substantially free of other cellular materials from the cell or tissue source from which the peptide is derived, such as sugars, lipids, and other contaminating proteins. Alternatively, when a peptide is chemically synthesized, an isolated or purified peptide refers to a peptide that is substantially free of precursors or other chemicals. The term "substantially free of cellular material" includes a preparation of a peptide in which the peptide has been isolated from the cellular components of the cell in which it was isolated or recombinantly produced. Thus, peptides that are substantially free of cellular material contain less than about 30%, 20%, 10%, or 5%, 3%, 2% or 1% (dry weight basis) of other cellular material. Includes peptide preparations. When the peptide is recombinantly produced, the isolated or purified peptide is substantially free of culture medium, and the peptide which is substantially free of culture medium is such that the culture medium is approximately 20% of the volume of the peptide preparation. Includes peptide preparations containing less than 10%, or 5%, 3%, 2% or 1% (dry weight basis). When the peptide is produced by chemical synthesis, the isolated or purified peptide is substantially free of precursors and other chemicals, and the peptide is substantially free of precursors and other chemicals. Preparations of peptides containing precursors and other chemicals in less than about 30%, 20%, 10%, 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide preparation. Including. The fact that a particular peptide preparation is an isolated or purified peptide is due, for example, by the appearance of a single band after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the gel. Can be confirmed. In a preferred embodiment, the peptides and polynucleotides of the invention have been isolated or purified.
　「ポリペプチド」、「ペプチド」、および「タンパク質」という用語は、本明細書で互換的に用いられ、アミノ酸残基のポリマーを指す。本用語は、天然型アミノ酸ポリマーのほか、1個もしくは複数個の非天然型アミノ酸残基を含む非天然型アミノ酸ポリマーにも適用される。非天然型アミノ酸には、アミノ酸類似体およびアミノ酸模倣体などが含まれる。 The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to natural amino acid polymers as well as non-natural amino acid polymers containing one or more unnatural amino acid residues. Non-natural amino acids include amino acid analogs and amino acid mimetics.
　本明細書で用いる「アミノ酸」という用語は、天然アミノ酸、ならびに天然アミノ酸と同様に機能するアミノ酸類似体およびアミノ酸模倣体を指す。天然アミノ酸とは、遺伝暗号によってコードされるアミノ酸、および細胞内で翻訳後に修飾されたアミノ酸（例えば、ヒドロキシプロリン、γ-カルボキシグルタミン酸、およびO-ホスホセリンなど）である。「アミノ酸類似体」という語句は、天然アミノ酸と同じ基本化学構造（水素、カルボキシ基、アミノ基、およびR基に結合したα炭素）を有するが、修飾されたR基または修飾された骨格を有する化合物（例えば、ホモセリン、ノルロイシン、メチオニンスルホキシドおよびメチオニンメチルスルホニウムなど）を指す。「アミノ酸模倣体」という語句は、一般的なアミノ酸とは異なる構造を有するが、アミノ酸と同様の機能を有する化合物を指す。アミノ酸はL-アミノ酸またはD-アミノ酸のいずれであってもよいが、本発明のペプチドは、L-アミノ酸のポリマーであることが好ましい。 The term "amino acid" as used herein refers to natural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are amino acids encoded by the genetic code and amino acids that have been intracellularly modified after translation (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase "amino acid analog" has the same basic chemical structure as a natural amino acid (a hydrogen, a carboxy group, an amino group, and an alpha carbon attached to an R group), but with a modified R group or a modified skeleton. Refers to compounds such as homoserine, norleucine, methionine sulfoxide and methionine methyl sulfonium. The phrase "amino acid mimetic" refers to a compound that has a structure different from that of common amino acids but has the same function as an amino acid. The amino acid may be either L-amino acid or D-amino acid, but the peptide of the present invention is preferably a polymer of L-amino acid.
　「ポリヌクレオチド」、「オリゴヌクレオチド」、および「核酸」という用語は、本明細書において互換的に用いられ、ヌクレオチドのポリマーを指す。 The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides.
　本明細書で使用する「組成物」という用語は、特定量の特定成分を含む生成物、および特定量の特定成分の組み合わせから直接または間接的に生じる任意の生成物を包含することが意図される。組成物が薬学的組成物である場合には、組成物という用語は、有効成分および不活性成分とを含む生成物、ならびに任意の2つもしくはそれ以上の成分の組み合わせ、複合体形成、もしくは凝集から、1つもしくは複数の成分の解離から、または1つもしくは複数の成分の他の種類の反応もしくは相互作用から直接または間接的に生じる任意の生成物を包含することが意図される。したがって、本発明の薬学的組成物は、本発明の化合物または細胞と薬学的または生理学的に許容される担体とを混合することにより作製される任意の組成物を包含する。本明細書で使用する「薬学的に許容される担体」または「生理学的に許容される担体」という語句は、液体もしくは固体の増量剤、希釈剤、賦形剤、溶媒および封入材料を含むがこれらに限定されない、薬学的または生理学的に許容される材料、組成物、物質、または媒体を意味する。 As used herein, the term "composition" is intended to include products that contain a particular amount of a particular ingredient, and any product that results directly or indirectly from a combination of a particular amount of a particular ingredient. To. When the composition is a pharmaceutical composition, the term composition refers to a product containing an active ingredient and an Inactive ingredient, as well as a combination of any two or more ingredients, complex formation, or aggregation. From, it is intended to include any product that results directly or indirectly from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Accordingly, the pharmaceutical compositions of the invention include any composition made by mixing a compound or cell of the invention with a pharmaceutically or physiologically acceptable carrier. Although the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein include liquid or solid bulking agents, diluents, excipients, solvents and encapsulating materials. Means a pharmaceutically or physiologically acceptable material, composition, substance, or vehicle without limitation.
　特記しない限り、「がん」という用語は、URLC10遺伝子やDEPDC1遺伝子を過剰発現するがんを指す。URLC10遺伝子を過剰発現するがんの例としては、膀胱がん、子宮頸がん、胆管細胞がん、食道がん、胃がん、非小細胞肺がん（NSCLC）、骨肉腫、膵がん、軟部組織腫瘍、および頭頸部悪性腫瘍（HNMT）などが含まれるが、これらに限定されない。一方、DEPDC1遺伝子を過剰発現するがんには、たとえば膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病（CML）、NSCLC、リンパ腫、骨肉腫、前立腺がん、小細胞肺がん（SCLC）、および軟部組織腫瘍などが含まれる。また、例示的な態様において、「がん」は、URLC10およびDEPDC1のいずれか、または両方とHLA-A24を発現するがんである。 Unless otherwise specified, the term "cancer" refers to cancer that overexpresses the URLC10 gene or the DEPDC1 gene. Examples of cancers that overexpress the URLC10 gene include bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, and soft tissue. Includes, but is not limited to, tumors and head and neck malignancies (HNMT). On the other hand, cancers that overexpress the DEPDC1 gene include, for example, bladder cancer, breast cancer, cervical cancer, bile duct cell cancer, chronic myelogenous leukemia (CML), NSCLC, lymphoma, osteosarcoma, prostate cancer, and small cell carcinoma. Includes cell lung cancer (SCLC), and soft tissue tumors. Also, in an exemplary embodiment, the "cancer" is a cancer that expresses HLA-A24 with either or both of URLC10 and DEPDC1.
　特記しない限り、「細胞傷害性Tリンパ球」、「細胞傷害性T細胞」、および「CTL」という用語は本明細書において互換的に用いられ、特に別段の定めのない限り、非自己細胞（例えば、腫瘍／がん細胞、ウイルス感染細胞）を認識し、そのような細胞の死滅を誘導することができるTリンパ球の亜群を指す。 Unless otherwise stated, the terms "cytotoxic T lymphocytes," "cytotoxic T cells," and "CTL" are used interchangeably herein, and unless otherwise specified, non-autologous cells (unless otherwise specified). For example, it refers to a subgroup of T lymphocytes that can recognize tumor / cancer cells, virus-infected cells) and induce the death of such cells.
　特記しない限り、「HLA-A24」という用語は、HLA-A*24:01、HLA-A*24:02などのサブタイプを含むHLA-A24型を指す。 Unless otherwise indicated, the term "HLA-A24" is, HLA-A * 24: refers to the HLA-A24 type, including the sub-type, such as 01, HLA-A * 24:02.
　対象または患者との関連において、本明細書で使用される「対象の（または患者の）HLA抗原はHLA-A24である」という表現は、対象または患者がMHC（主要組織適合複合体）クラスＩ分子としてのHLA-A24抗原遺伝子をホモ接合的またはヘテロ接合的に保有し、かつHLA-A24抗原が対象または患者の細胞においてHLA抗原として発現されることを指す。 In the context of a subject or patient, the phrase "the subject's (or patient's) HLA antigen is HLA-A24" as used herein means that the subject or patient is an MHC (major histocompatibility complex) class I. It refers to carrying the HLA-A24 antigen gene as a molecule in a homozygous or heterozygous manner, and expressing the HLA-A24 antigen as an HLA antigen in the cells of a subject or a patient.
　本発明の方法および組成物ががんの「治療」との関連において有用である限り、治療が臨床的利点、例えば対象におけるがんの大きさ、広がり、もしくは転移能の減少、がんの進行遅延、がんの臨床症状の緩和、生存期間の延長、術後再発の抑制等をもたらす場合に、治療は「有効である」とみなされる。治療を予防的に適用する場合、「有効な」とは、治療によって、がんの形成が遅延されるもしくは妨げられるか、またはがんの臨床症状が妨げられるもしくは緩和されることを意味する。有効性は、特定の腫瘍の種類を診断または治療するための任意の公知の方法と関連して決定される。 As long as the methods and compositions of the invention are useful in the context of "treatment" of cancer, treatment has clinical benefits such as reduction of cancer size, spread, or metastatic potential in a subject, cancer progression. Treatment is considered "effective" if it results in delay, alleviation of clinical symptoms of cancer, prolongation of survival, suppression of postoperative recurrence, and the like. When the treatment is applied prophylactically, "effective" means that the treatment delays or prevents the formation of cancer or prevents or alleviates the clinical manifestations of the cancer. Efficacy is determined in connection with any known method for diagnosing or treating a particular tumor type.
　本発明の方法および組成物ががんの「予防」との関連において有用である限り、「予防」という用語は本明細書において、疾患による死亡率または罹患率の負荷を軽減させる任意の働きを含む。予防は、「第一次、第二次、および第三次の予防レベル」で行われ得る。第一次の予防は疾患の発生を回避するのに対し、第二次および第三次レベルの予防は、疾患の進行および症状の出現を予防することに加え、機能を回復させ、かつ疾患関連の合併症を減少させることによって、既存の疾患の悪影響を低下させることを目的とした働きを包含する。あるいは、予防は、特定の障害の重症度を緩和すること、例えば腫瘍の増殖および転移を減少させることを目的とした広範囲の予防的治療を含み得る。 As long as the methods and compositions of the invention are useful in the context of "prevention" of cancer, the term "prevention" has any function herein to reduce the burden of disease mortality or morbidity. Including. Prevention can be done at "primary, secondary, and tertiary levels of prevention". Primary prophylaxis avoids the development of the disease, whereas secondary and tertiary levels of prophylaxis prevent the progression of the disease and the appearance of symptoms, as well as restore function and are disease-related. Includes work aimed at reducing the adverse effects of existing diseases by reducing the complications of. Alternatively, prophylaxis may include a wide range of prophylactic treatments aimed at alleviating the severity of a particular disorder, eg, reducing tumor growth and metastasis.
　本発明との関連において、がんの治療、がんの予防、または術後のがんの再発の予防は、それぞれが、がん細胞の増殖阻害、腫瘍の退行または退縮、寛解の誘導およびがんの発生の抑制、腫瘍退縮、ならびに転移の低減または阻害、がんの術後の再発抑制、および生存期間の延長などから選択される事象のいずれか、または複数を含む。がんの効果的な治療および予防のいずれか、または両方は、死亡率を減少させ、がんを有する個体の予後を改善し、血中の腫瘍マーカーのレベルを低下させ、かつがんに伴う検出可能な症状を緩和する。例えば、症状の軽減または改善は効果的な治療および予防のいずれかまたは両方を構成し、10%、20%、30%、もしくはそれ以上の軽減もしくは症状が安定した状態を含む。 In the context of the present invention, treatment of cancer, prevention of cancer, or prevention of recurrence of cancer after surgery, respectively, may include inhibition of cancer cell growth, tumor regression or regression, induction of remission, and so on. Includes one or more of the events selected from suppression of cancer development, tumor regression, and reduction or inhibition of metastasis, suppression of postoperative recurrence of cancer, and prolongation of survival. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces the level of tumor markers in the blood, and is associated with cancer. Relieve detectable symptoms. For example, symptomatic relief or amelioration constitutes either or both of effective treatment and prevention, including 10%, 20%, 30%, or more alleviation or symptom-stable conditions.
　本発明との関連において、「抗体」という用語は、指定のタンパク質またはそのペプチドと特異的に反応する免疫グロブリンおよびその断片を指す。抗体には、ヒト抗体、霊長類化抗体、キメラ抗体、二重特異性抗体、ヒト化抗体、他のタンパク質または放射標識と融合させた抗体、および抗体断片が含まれ得る。さらに、本明細書において「抗体」は広義で使用され、具体的にはインタクトなモノクローナル抗体、ポリクローナル抗体、2以上のインタクトな抗体から形成される多重特異性抗体（例えば、二重特異性抗体）を包含し、また所望の生物活性を示す限り、抗体断片を包含する。「抗体」は、いずれのクラス（例えば、IgA、IgD、IgE、IgG、およびIgM）の抗体であってもよい。 In the context of the present invention, the term "antibody" refers to an immunoglobulin and a fragment thereof that specifically react with a designated protein or peptide thereof. Antibodies can include human antibodies, primated antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Further, in the present specification, "antibody" is used in a broad sense, specifically, a multispecific antibody formed from an intact monoclonal antibody, a polyclonal antibody, and two or more intact antibodies (for example, a bispecific antibody). And include antibody fragments as long as they exhibit the desired biological activity. The "antibody" may be an antibody of any class (eg, IgA, IgD, IgE, IgG, and IgM).
　特記しない限り、本明細書で使用する技術用語および科学用語はすべて、本発明が属する技術分野の当業者によって共通して理解されている用語と同じ意味を有する。 Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs.
ＩＩ．T細胞受容体
　本発明はまた、T細胞受容体（TCR）のサブユニットを形成することができる１つまたは複数のポリペプチドをコードする１つまたは複数のポリヌクレオチドを含有する組成物、およびこれを使用する方法を提供する。そのようなTCRサブユニットは、URLC10ペプチド、あるいはDEPDC1ペプチドを提示するAPCに対するURLC10やDEPDC1に対する特異性をCD8+T細胞に付与するTCRを形成する能力を有する。当技術分野において公知の方法を用いることにより、URLC10ペプチドあるいはDEPDC1ペプチドによって誘導されるT細胞のTCRサブユニットとしてα鎖およびβ鎖の核酸を同定することができる（国際公開第2007/032255号およびMorgan et al., J Immunol 2003, 171: 3288）。誘導体TCRは、URLC10ペプチドあるいはDEPDC1ペプチドを提示するAPCに高い結合力で結合することができ、場合により効果的にサイトカインを媒介することができる。 II. T Cell Receptors The present invention also comprises compositions comprising one or more polynucleotides encoding one or more polypeptides capable of forming subunits of the T cell receptor (TCR), and the like. Provides a way to use. Such TCR subunits have the ability to form TCRs that confer specificity on CD8 + T cells for the URLC10 peptide, or URLC10 or DEPDC1 for APCs that present the DEPDC1 peptide. By using methods known in the art, α- and β-chain nucleic acids can be identified as TCR subunits of T cells induced by the URLC10 peptide or DEPDC1 peptide (International Publication No. 2007/032255 and Morgan et al., J Immunol 2003, 171: 3288). The derivative TCR can bind to APCs presenting the URLC10 peptide or DEPDC1 peptide with high binding force and, in some cases, effectively mediate cytokines.
　TCRサブユニットをコードする１つまたは複数のポリヌクレオチド（すなわち両方のTCRサブユニットをコードする単一ポリヌクレオチドまたは各々が別々のTCRサブユニットをコードする複数のポリヌクレオチド）を適切なベクター、例えばレトロウイルスベクターに組み込むことができる。これらのベクターは当技術分野において周知である。ポリヌクレオチドまたはそれらを含むベクターは、CD8+T細胞、例えば患者由来のCD8+T細胞に有用に移入することができる。好都合には、本発明は、患者自身のT細胞（または別の対象のT細胞）の速やかな改変を可能にし、優れたがん細胞殺傷特性を有する改変されたT細胞を迅速かつ容易に生産する、すぐに入手可能な組成物を提供する。 One or more polynucleotides encoding TCR subunits (ie, a single polynucleotide encoding both TCR subunits or multiple polynucleotides each encoding a separate TCR subunit) in a suitable vector, eg, retro. It can be incorporated into a viral vector. These vectors are well known in the art. Polynucleotides or vectors containing them can be usefully transferred to CD8 + T cells, such as patient-derived CD8 + T cells. Conveniently, the present invention allows rapid modification of a patient's own T cells (or T cells of another subject) to quickly and easily produce modified T cells with excellent cancer cell killing properties. To provide a readily available composition.
　例えば、HLA-A24拘束様式でURLC10ペプチド(RYCNLEGPPI／配列番号：177)によって刺激されたT細胞においては、それぞれ配列番号：1-2、5-8、14、16、17-25、33-40、49-54、60-62、65-70、77-79のアミノ酸配列からなるCDR3を各々が有するTCR-αサブユニットが検出された。同様にHLA-A24拘束様式でURLC10ペプチド(RYCNLEGPPI／配列番号：177)によって刺激されたT細胞においては、例えば、それぞれ配列番号：3-4、9-13、15、26-32、41-48、55-59、63-64、71-76、80-83のアミノ酸配列からなるCDR3を各々が有するTCR-βサブユニットが検出された（図2、図5A）。
　一方、HLA-A24拘束様式でDEPDC1ペプチド(EYYELFVNI／配列番号：178)によって刺激されたT細胞においては、例えば、それぞれ配列番号：84－85のアミノ酸配列からなるCDR3をそれぞれが有するTCR-αサブユニットと、配列番号：86のアミノ酸配列からなるCDR3を有するTCR-βサブユニットが検出された（図5B）。
　それゆえ、それらをコードするポリヌクレオチドは、本発明の細胞傷害性T細胞誘導に好ましい可能性がある。同様に、それぞれこれら配列番号のアミノ酸配列からなるCDR3を各々が有するαサブユニットとβサブユニットとの間で形成されるTCRを発現するT細胞もまた、本発明の好ましい態様である。一般に、TCRの抗原特異性は、主にそれらのCDR3に依存している。したがって、αサブユニットおよびβサブユニットのCDR3をそれぞれ、上記の配列番号で置き換えることによって、公知のTCRからURLC10あるいはDEPDC1特異的TCRを再構築することもできる。別のサブユニットから移植されたCDR3を有するそのようなTCRは、キメラTCRと称され得る。特異的なCDRを移植するための方法は、当技術分野において周知である。 For example, in T cells stimulated by the URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) in an HLA-A24 restraint manner, SEQ ID NOs: 1-2, 5-8, 14, 16, 17-25, 33-40, respectively. , 49-54, 60-62, 65-70, 77-79, TCR-α subunits, each having a CDR3 consisting of the amino acid sequences, were detected. Similarly, in T cells stimulated by the URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) in an HLA-A24 restraint manner, for example, SEQ ID NOs: 3-4, 9-13, 15, 26-32, 41-48, respectively. , 55-59, 63-64, 71-76, 80-83 TCR-β subunit, each of which has a CDR3 consisting of amino acid sequences, was detected (Fig. 2, Fig. 5A).
On the other hand, in T cells stimulated by the DEPDC1 peptide (EYYELFVNI / SEQ ID NO: 178) in the HLA-A24 restraint mode, for example, TCR-α subs each having a CDR3 consisting of the amino acid sequence of SEQ ID NO: 84-85. A TCR-β subunit having a unit and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 86 was detected (Fig. 5B).
Therefore, the polynucleotides encoding them may be preferred for the induction of cytotoxic T cells of the present invention. Similarly, T cells expressing TCR formed between the α and β subunits, each of which has a CDR3 consisting of the amino acid sequences of these SEQ ID NOs, are also preferred embodiments of the present invention. In general, the antigen specificity of TCRs depends primarily on their CDR3s. Therefore, it is also possible to reconstruct the URLC10 or DEPDC1-specific TCR from the known TCR by replacing the CDR3 of the α subunit and the β subunit with the above SEQ ID NOs, respectively. Such a TCR with a CDR3 ported from another subunit can be referred to as a chimeric TCR. Methods for transplanting specific CDRs are well known in the art.
　本発明はさらに、両方のTCRサブユニットをコードするポリヌクレオチドまたはTCRサブユニットの各々をコードするポリヌクレオチドでの形質導入によって調製される細胞傷害性T細胞を提供し、ここで、TCRサブユニットはURLC10ペプチドやDEPDC1ペプチドにHLA-A24拘束様式で結合することができる。形質導入されたT細胞は、インビボでがん細胞にホーミングすることができ、インビトロで周知の培養方法によって増殖させることができる（例えばKawakami et al., J Immunol 1989, 142: 3452-61）。上述したように調製したT細胞は、治療または予防を必要とする患者においてがんを治療するまたは予防する上で有用な免疫原性組成物を形成するために使用できる。 The present invention further provides cytotoxic T cells prepared by transfection with a polynucleotide encoding both TCR subunits or a polynucleotide encoding each of the TCR subunits, wherein the TCR subunits It can bind to URLC10 peptide and DEPDC1 peptide in the HLA-A24 restraint manner. Transduced T cells can be homing to cancer cells in vivo and can be grown in vitro by well-known culture methods (eg Kawakami et al., J Immunol 1989, 142: 3452-61). T cells prepared as described above can be used to form immunogenic compositions useful in treating or preventing cancer in patients in need of treatment or prevention.
　一般的に、あるポリペプチド中の1個、2個、またはそれ以上のアミノ酸の改変は該ポリペプチドの機能に影響を及ぼさず、場合によっては元のポリペプチドの所望の機能を増強することさえある。実際に、改変ポリペプチド（すなわち、元の参照配列と比較して、1個、2個、または数個のアミノ酸残基が改変された（すなわち、置換、欠失、挿入および付加からなる群から選択される少なくとも一つの改変を含む）アミノ酸配列から構成されるポリペプチド）は、元のポリペプチドの生物活性を保持することが知られている（Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6；Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500；Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13）。したがって、一態様において、本発明のCDR3は、前記配列番号の中より選択されるアミノ酸配列に対して1個、2個、または数個のアミノ酸が置換、欠失、挿入および付加からなる群から選択される改変を含むアミノ酸配列を有するCDR3であって、かつTCRにURLC10ペプチドあるいはDEPDC1ペプチドに対するHLA-A24拘束性の結合特異性を付与するCDR3であり得る。本発明において、前記配列番号によって特定されるアミノ酸配列を改変することによって導かれ、かつ元のアミノ酸配列からなるCDR3と同様に、TCRにURLC10ペプチドあるいはDEPDC1ペプチドに対するHLA-A24拘束性の結合特異性を付与するCDR3を、機能的に等価なCDR3という。 In general, modifications of one, two, or more amino acids in a polypeptide do not affect the function of the polypeptide, and in some cases even enhance the desired function of the original polypeptide. is there. In fact, the modified polypeptide (ie, from the group consisting of one, two, or several amino acid residues modified (ie, substitutions, deletions, insertions, and additions compared to the original reference sequence). A polypeptide composed of an amino acid sequence (including at least one modification selected) is known to retain the biological activity of the original polypeptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Therefore, in one embodiment, the CDR3 of the present invention comprises a group consisting of one, two, or several amino acids substituted, deleted, inserted, and added to the amino acid sequence selected from the above SEQ ID NOs. It can be a CDR3 having an amino acid sequence containing the modification of choice and that confer an HLA-A24 binding specificity on the TCR to the URLC10 peptide or DEPDC1 peptide. In the present invention, the binding specificity of HLA-A24 binding to the URLC10 peptide or DEPDC1 peptide on the TCR is similar to that of CDR3, which is derived by modifying the amino acid sequence specified by the above-mentioned SEQ ID NO: and consists of the original amino acid sequence. The CDR3 that grants is called the functionally equivalent CDR3.
　当業者は、元のアミノ酸側鎖の特性の保存をもたらす傾向がある、単一のアミノ酸またはわずかな割合のアミノ酸を変更する、アミノ酸配列に対する個々の置換を認識することができる。したがって、それらはしばしば「保存的置換」または「保存的改変」と称され、「保存的置換」または「保存的改変」によるタンパク質の改変は、元のタンパク質と類似の機能を有する改変タンパク質を生じ得る。機能的に類似しているアミノ酸を提示する保存的置換の表は、当技術分野において周知である。機能的に類似しているアミノ酸側鎖の特性の例には、例えば、疎水性アミノ酸（A、I、L、M、F、P、W、Y、V）、親水性アミノ酸（R、D、N、C、E、Q、G、H、K、S、T）、ならびに以下の官能基または特徴を共通して有する側鎖が含まれる：脂肪族側鎖（G、A、V、L、I、P）；ヒドロキシル基含有側鎖（S、T、Y）；硫黄原子含有側鎖（C、M）；カルボン酸およびアミド含有側鎖（D、N、E、Q）；塩基含有側鎖（R、K、H）；および芳香族含有側鎖（H、F、Y、W）。加えて、以下の8群はそれぞれ、相互に保存的置換であるとして当技術分野で認められているアミノ酸を含む：
1）アラニン（A）、グリシン（G）；
2）アスパラギン酸（D）、グルタミン酸（E）；
3）アスパラギン（N）、グルタミン（Q）；
4）アルギニン（R）、リジン（K）；
5）イソロイシン（I）、ロイシン（L）、メチオニン（M）、バリン（V）；
6）フェニルアラニン（F）、チロシン（Y）、トリプトファン（W）；
7）セリン（S）、スレオニン（T）；および
8）システイン（C）、メチオニン（M）（例えば、Creighton，Proteins 1984を参照されたい）。 One of ordinary skill in the art can recognize individual substitutions on an amino acid sequence that alter a single amino acid or a small proportion of amino acids that tend to result in preservation of the properties of the original amino acid side chain. Therefore, they are often referred to as "conservative substitutions" or "conservative modifications", and modification of a protein by "conservative substitution" or "conservative modification" results in a modified protein having a function similar to that of the original protein. obtain. A table of conservative substitutions that present functionally similar amino acids is well known in the art. Examples of functionally similar amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D,). N, C, E, Q, G, H, K, S, T), as well as side chains that share the following functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur amino acid-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W). In addition, each of the following eight groups contains amino acids recognized in the art as mutually conservative substitutions:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) Phenylalanine (F), tyrosine (Y), tryptophan (W);
7) Serine (S), threonine (T); and
8) Cysteine (C), methionine (M) (see, for example, Creighton, Proteins 1984).
　このような保存的改変を含むCDR3もまた、本発明のCDR3に包含される。しかしながら、本発明のペプチドはこれらに限定されず、改変CDR3が元のCDR3の機能を保持する限り、非保存的な改変を含み得る。 CDR3 including such a conservative modification is also included in CDR3 of the present invention. However, the peptides of the invention are not limited to these and may include non-conservative modifications as long as the modified CDR3 retains the functionality of the original CDR3.
　本発明において、元のCDR3が由来するTCRと同様の抗原認識特異性が、それを移植したTCRに付与されたとき、改変CDR3においてCDR3の元の機能が保持されたことを意味する。そのような特異的認識は任意の公知の方法によって確認することができ、好ましい方法には、例えば、HLA分子とTCRを取得した対象に投与されたペプチドを用いるテトラマーアッセイ（例えば、Altman et al. Science. 1996, 274, 94-6 ;McMichael et al. J Exp Med.1998, 187, 1367-71）、ならびにELISPOTアッセイが含まれる。ELISPOTアッセイを行うことにより、細胞表面上にTCRを発現しているT細胞がTCRによって細胞を認識すること、およびシグナルが細胞内で伝達され、次にIFN-γなどのサイトカインがT細胞から放出されることを確認することができる。当技術分野で周知の方法を用いて、標的細胞に対するT細胞の細胞傷害活性を調べることができる。好ましい方法には、例えば、URLC10やDEPDC1を発現するHLA陽性細胞を標的細胞として用いるクロム放出アッセイが含まれる。 In the present invention, it means that the original function of CDR3 was retained in the modified CDR3 when the same antigen recognition specificity as the TCR from which the original CDR3 was derived was imparted to the transplanted TCR. Such specific recognition can be confirmed by any known method, and preferred methods include, for example, a tetramer assay using a peptide administered to a subject who has obtained an HLA molecule and TCR (eg, Altman et al. Science. 1996, 274, 94-6; McMichael et al. J Exp Med. 1998, 187, 1367-71), as well as the ELISPOT assay. By performing the ELISPOT assay, T cells expressing TCR on the cell surface recognize the cells by TCR, and signals are transmitted intracellularly, and then cytokines such as IFN-γ are released from the T cells. It can be confirmed that it will be done. The cytotoxic activity of T cells against target cells can be investigated using methods well known in the art. Preferred methods include, for example, a chromium release assay using HLA-positive cells expressing URLC10 or DEPDC1 as target cells.
　cDNA マイクロアレイ技術の最近の発展により、正常細胞と比べた、がん細胞における遺伝子発現の包括的プロファイルの作成が可能になった（Okabe, H. et al., Cancer Res.2001, 61, 2129-37；Lin YM. et al.,Oncogene 2002, 21；4120-8；Hasegawa S. et al.,Cancer Res 2002, 62：7012-7）。このアプローチにより、がん細胞の複雑な性質および発癌のメカニズムをより完全に理解することが可能になり、かつ、腫瘍において発現が亢進している遺伝子の同定が容易になる（Bienz M. et al., Cell 2000, 103, 311-20）。がんにおいて発現の亢進が認められた転写物のうちでは、URLC10（GenBankアクセッション番号NM_017527またはアクセッション番号：BC117142）、DEPDC1（GenBankアクセッション番号：NM_001114120、またはBM683578）が近年発見された。参考文献の内容全体は、参照により本明細書に組み入れられる。 Recent developments in cDNA microarray technology have made it possible to create a comprehensive profile of gene expression in cancer cells compared to normal cells (Okabe, H. et al., Cancer Res. 2001, 61, 2129- 37; Lin YM. Et al., Oncogene 2002, 21; 4120-8; Hasegawa S. et al., Cancer Res 2002, 62: 7012-7). This approach allows a more complete understanding of the complex nature of cancer cells and the mechanism of carcinogenesis, and facilitates the identification of genes that are upregulated in tumors (Bienz M. et al). ., Cell 2000, 103, 311-20). Among the transcripts that were found to be upregulated in cancer, URLC10 (GenBank accession number NM_017527 or accession number: BC117142) and DEPDC1 (GenBank accession number: NM_001114120, or BM683578) were recently discovered. The entire content of the reference is incorporated herein by reference.
　URLC10遺伝子は、別名LY6K遺伝子とも呼ばれている。LY6K遺伝子は、膀胱がん、子宮頸がん、胆管細胞がん、食道がん、胃がん、非小細胞肺がん（NSCLC）、骨肉腫、膵がん、軟部組織腫瘍、および頭頸部悪性腫瘍（HNMT）を含む多くの癌腫において発現の亢進が認められているので、本発明は、リンパ球抗原6複合体、K遺伝子座（lymphocyte antigen 6 complex）（LY6K）遺伝子の遺伝子産物、より詳細にはGenBankアクセッション番号NM__017527の遺伝子によってコードされるポリペプチドをさらなる分析のための標的とする。 The URLC10 gene is also known as the LY6K gene. The LY6K gene is a bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, soft tissue tumor, and head and neck malignancies (HNMT). ) Has been observed to be upregulated in many cancers, the present invention relates to the lymphocyte antigen 6 complex, the gene product of the K gene locus (lymphocyte antigen 6 complex) (LY6K), and more specifically to GenBank. The polypeptide encoded by the gene of accession number NM__017527 is targeted for further analysis.
　DEPDC1タンパク質（DEP領域含有1（DEP domain containing 1）；GenBank　アクセッション番号NM_001114120、BM683578）の機能は、いまだ明らかでない。このタンパク質に含まれるDEP領域はDishevelled、Egl-10およびPleckstrinにおいて見られる。特に、ショウジョウバエのdishevelledのDEPドメインは、平面極性欠損の救済およびJNKシグナリングの誘導において欠くことのできない役割を果たしている；にもかかわらず、ヒトにおけるその機能は、まだ明らかにされていない。しかしながら、WO2006/085684（PCT/JP2006/302684）に開示されているように、DEPDC1（In House No.：B5860N）は、DEPDC1V1およびV2に対応するそれぞれ12と11のエキソンからなる2つの異なるトランスクリプショナルバリアントを有する。V1のエキソン8における変異が認められており、そして、他の残りのエキソンは両方のバリアントに共通であることが見いだされた。V2異型は、V1のエキソン8を有しないが、最後のエキソン内で同じ終止コドンを生成する。B5860NV1およびB5860NV2バリアントの全長cDNAシーケンスは、それぞれ5318および4466のヌクレオチドから成る。これらのバリアントのORFはそれぞれのエキソン１内で始まる。最終的に、V1およびV2の転写産物は、それぞれ811および527のアミノ酸をコードする。siRNAはがん細胞の増殖を抑制した。これらの結果はDEPDC1が殆どのがん細胞の増殖において重要な役割を果たしていることを示す。なお本発明によって提供されるDEPDC1特異的なＴＣＲの認識エピトープである配列番号：178のアミノ酸配列は、V1とV2のいずれのバリアントにも含まれている。したがって、本発明のＴＣＲは、DEPDC1のV1とV2のいずれのバリアントを発現する細胞に対しても傷害作用が期待できるＣＴＬを与える。 The function of the DEPDC1 protein (DEP domain containing 1; GenBank accession number NM_001114120, BM683578) is still unclear. The DEP region contained in this protein is found in Dishevelled, Egl-10 and Pleckstrin. In particular, the Drosophila dishevelled DEP domain plays an essential role in the relief of planar polarity defects and the induction of JNK signaling; nevertheless, its function in humans has not yet been elucidated. However, as disclosed in WO2006 / 085684 (PCT / JP2006 / 302684), DEPDC1 (In House No .: B5860N) is a two different transcriptome consisting of 12 and 11 exons corresponding to DEPDC1 V1 and V2, respectively. Has a tional variant. Mutations in exon 8 of V1 were found, and the other remaining exons were found to be common to both variants. The V2 variant does not have the exon 8 of V1, but produces the same stop codon within the last exon. The full-length cDNA sequence of the B5860NV1 and B5860NV2 variants consists of 5318 and 4466 nucleotides, respectively. The ORFs of these variants begin within each exon 1. Finally, the transcripts of V1 and V2 encode 811 and 527 amino acids, respectively. siRNA suppressed the growth of cancer cells. These results indicate that DEPDC1 plays an important role in the growth of most cancer cells. The amino acid sequence of SEQ ID NO: 178, which is a recognition epitope of DEPDC1-specific TCR provided by the present invention, is contained in both variants of V1 and V2. Therefore, the TCR of the present invention provides a CTL that can be expected to have a damaging effect on cells expressing either V1 or V2 variant of DEPDC1.
　本発明は、URLC10由来ペプチドあるいはDEPDC1由来ペプチドを認識するT細胞が発現するTCRに関する。TCRはα鎖およびβ鎖の二量体からなるタンパク分子である。ヒトT細胞はTCRを通じてMHCクラスI（別名：HLA, Human Leukocyte Antigen）分子上に提示されたペプチドを認識する。その結果、T細胞の増殖、分化、サイトカインの産生あるいは細胞傷害性物質（パーフォリンやグランザイム）の分泌等が誘導される。
　TCR-α遺伝子とはVα遺伝子、Jα遺伝子およびCα遺伝子を含む。TCR-β遺伝子とはVβ遺伝子、Dβ遺伝子、Jβ遺伝子およびCβ遺伝子を含む。TCRのペプチドに対する特異性を決定する領域は相補性決定領域（Complementarity Determining Region: CDR）と呼ばれており、CDR1、CDR2およびCDR3が存在する。なかでもCDR3はペプチドと直接接触することから、そのアミノ酸配列はTCRの抗原認識特異性決定の上で非常に重要である。TCR-α鎖においてはV-J間、TCR-β鎖においてはV-DおよびD-J間がCDR3にあたり、塩基の挿入や欠失により多様性が生じる。
　T細胞療法を目的として、がん細胞のHLA分子上に提示されたペプチドを認識し、細胞傷害性物質を分泌するようなT細胞を遺伝子改変によって調製する場合、本明細書に示されるURLC10またはDEPDC1に由来するペプチドを認識するT細胞から同定されたTCRのアミノ酸配列（特にCDR3におけるアミノ酸配列）は、非常に利用価値が高い。 The present invention relates to a TCR expressed by a T cell that recognizes a URLC10-derived peptide or a DEPDC1-derived peptide. TCR is a protein molecule consisting of α-chain and β-chain dimers. Human T cells recognize peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules through TCR. As a result, T cell proliferation, differentiation, cytokine production or secretion of cytotoxic substances (perforin and granzyme) are induced.
The TCR-α gene includes the Vα gene, the Jα gene and the Cα gene. The TCR-β gene includes a Vβ gene, a Dβ gene, a Jβ gene and a Cβ gene. The regions that determine the specificity of TCRs for peptides are called Complementarity Determining Regions (CDRs), and CDR1, CDR2, and CDR3 are present. Among them, CDR3 comes into direct contact with peptides, so its amino acid sequence is very important for determining the antigen recognition specificity of TCR. In the TCR-α chain, VJs and in the TCR-β chain, VDs and DJs correspond to CDR3, and diversity occurs due to the insertion or deletion of bases.
For the purpose of T cell therapy, when a T cell that recognizes a peptide presented on the HLA molecule of a cancer cell and secretes a cytotoxic substance is prepared by genetic modification, the URLC10 or URLC10 shown in the present specification is used. The amino acid sequence of TCR (particularly the amino acid sequence in CDR3) identified from T cells that recognize peptides derived from DEPDC1 is extremely valuable.
　腫瘍抗原に由来する免疫原性を有するエピトープペプチドは腫瘍抗原に特異的なCTLを誘導し、がん患者において治療効果を生む。しかしペプチドの接種によって生体中で治療に有効な量のCTLを誘導するには、しばしば長い時間（数カ月）を要する。そのため、ワクチンによるがん治療は、適用可能な患者が制限される場合があった。腫瘍抗原特異的なTCRが同定できれば、TCR遺伝子導入T細胞療法を実現することができる。生体外でTCRを組み換えたT細胞は、治療に必要な量を培養によって容易に増殖させることができる。そのため、ペプチドワクチンに比べると、より早く、十分な治療効果を期待できる。また、ペプチドワクチンによって誘導されるCTLの数を予測することは通常は難しいので、期待される治療効果を予測することを困難にしている。しかしTCR遺伝子導入T細胞療法においては、治療効果を持つCTLの数が明らかなので、その治療効果も予測が容易である。 An immunogenic epitope peptide derived from a tumor antigen induces a tumor antigen-specific CTL and produces a therapeutic effect in cancer patients. However, it often takes a long time (several months) to induce a therapeutically effective amount of CTL in vivo by inoculating a peptide. As a result, vaccine-based cancer treatment may limit the applicable patients. If a tumor antigen-specific TCR can be identified, TCR gene transfer T cell therapy can be realized. In vitro TCR-recombined T cells can be easily grown in the amount required for treatment by culturing. Therefore, compared to the peptide vaccine, a faster and sufficient therapeutic effect can be expected. Also, predicting the number of CTLs induced by a peptide vaccine is usually difficult, making it difficult to predict the expected therapeutic effect. However, in TCR gene transfer T cell therapy, since the number of CTLs having a therapeutic effect is clear, the therapeutic effect is also easy to predict.
　本発明によって、個別化がん免疫療法(personalized cancer immunotherapy)が可能となる。幅広いがんで過剰発現している腫瘍抗原は、TCR遺伝子導入T細胞療法の標的として有用である。好適な腫瘍抗原を標的とするTCRが同定できれば、同じタイプのHLAを持つ他の患者において、個別化がん免疫療法を可能とする。たとえばURLC10は非小細胞肺がんや食道がんの多くで高発現している。あるいはDEPDC1の膀胱がんにおける過剰発現も公知である。したがって、URLC10やDEPDC1特異的なTCR遺伝子導入T細胞は、養子免疫療法に有用である。現在のところ、キメラ抗原受容体(chimeric antigen receptor ；CAR)T細胞療法や、TCR遺伝子導入T細胞療法は、一部の血液がんへの適用に限られている。しかし本発明のTCRにより、固形癌における養子免疫療法を可能とできる。 The present invention enables personalized cancer immunotherapy. Tumor antigens that are overexpressed in a wide range of cancers are useful targets for TCR transgenic T cell therapy. Identification of a TCR that targets a suitable tumor antigen will enable personalized cancer immunotherapy in other patients with the same type of HLA. For example, URLC10 is highly expressed in many non-small cell lung cancers and esophageal cancers. Alternatively, overexpression of DEPDC1 in bladder cancer is also known. Therefore, URLC10 and DEPDC1-specific TCR transgenic T cells are useful for adoptive immunotherapy. At present, chimeric antigen receptor (CAR) T cell therapy and TCR gene transfer T cell therapy are limited to some hematological cancers. However, the TCR of the present invention enables adoption immunotherapy in solid tumors.
　ある態様において、本発明は、免疫学的に活性なTCR、またはその一部を提示した組み換えTリンパ球 (engineered T lymphocyte)を提供する。Tリンパ球は、たとえばCD8+細胞傷害性T細胞である。本発明の組み換えT細胞は、MHCと結合したエピトープペプチドを提示した抗原提示細胞を認識する。あるいは組み換えTリンパ球は、CD8+Tリンパ球、CD4+ヘルパーリンパ球、NK細胞、NKT細胞、B細胞、樹状細胞等であることができる。ある態様において、TCRのアミノ酸配列に基づいて、それをコードする核酸やベクターをデザインすることができる。たとえば標的抗原特異的なTCRあるいはその一部を提示した組み換えTリンパ球は、次の工程によって得ることができる；
(i) 標的抗原特異的なTCRをコードする核酸配列をベクターにクローニングし、
(ii) 当該ベクターを宿主Tリンパ球（たとえばCD8+細胞傷害性T細胞）に導入し、および
(iii) 当該TCRを、組み換えTリンパ球上に発現、および提示可能な条件下で培養する。本発明において、TCRの一部は、例えばTCRのα鎖、およびβ鎖のいずれか、あるいは両方であることができる。あるいは本発明のある態様においては、TCRの一部は、TCRのα鎖、およびβ鎖のいずれか、または両方の、1つ、2つ、あるいは3つの相補性決定領域(complementarity determining regiions; CDRs)を含むこともできる。好ましい態様においては、TCRの一部とは、TCRのα鎖、およびβ鎖の、いずれかまたは両方のCDR3を含む。本発明において同定された、好ましいCDR3のアミノ酸配列は、次のとおりである；
配列番号：1-2、5-8、14、16、17-25、33-40、49-54、60-62、65-70、77-79および84-85からなる群から選択されるいずれかのアミノ酸配列で特定されるヒトT細胞受容体α鎖のCDR3、および
配列番号：3-4、9-13、15、26-32、41-48、55-59、63-64、71-76、80-83および86からなる群から選択されるいずれかのアミノ酸配列で特定されるヒトT細胞受容体β鎖のCDR3。
　本発明の、ある態様において、T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列は、たとえば以下のように組み合わせることができる；
T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
配列番号：1　　　　　　　　　　　　　　配列番号：3（URLC10）、
配列番号：2　　　　　　　　　　　　　　配列番号：3（URLC10）、
配列番号：14　　　　　　　　　　　　　 配列番号：15（URLC10）、
配列番号：16　　　　　　　　　　　　　 配列番号：10（URLC10）、
配列番号：17　　　　　　　　　　　　　 配列番号：26（URLC10）、
配列番号：60　　　　　　　　　　　　　 配列番号：63（URLC10）、
配列番号：69　　　　　　　　　　　　　 配列番号：71（URLC10）、
配列番号：77　　　　　　　　　　　　　 配列番号：80（URLC10）、および
配列番号：84　　　　　　　　　　　　　 配列番号：86（DEPDC1）。 In some embodiments, the invention provides an immunologically active TCR, or engineered T lymphocyte, which presents a portion thereof. T lymphocytes are, for example, CD8 + cytotoxic T cells. The recombinant T cells of the present invention recognize antigen-presenting cells that have presented an epitope peptide bound to MHC. Alternatively, the recombinant T lymphocytes can be CD8 + T lymphocytes, CD4 + helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells and the like. In some embodiments, a nucleic acid or vector encoding the amino acid sequence of the TCR can be designed. For example, recombinant T lymphocytes that present a target antigen-specific TCR or part thereof can be obtained by the following steps;
(i) The nucleic acid sequence encoding the target antigen-specific TCR was cloned into a vector.
(ii) The vector was introduced into host T lymphocytes (eg CD8 + cytotoxic T cells) and
(iii) The TCR is cultured under conditions that can be expressed and presented on recombinant T lymphocytes. In the present invention, a part of the TCR can be, for example, one or both of the α chain and the β chain of the TCR. Alternatively, in some aspects of the invention, some of the TCRs are one, two, or three complementarity determining regiions (CDRs) of either or both of the α and β chains of the TCR. ) Can also be included. In a preferred embodiment, the portion of the TCR comprises CDR3 of either or both of the α and β chains of the TCR. The preferred amino acid sequence of CDR3 identified in the present invention is:
SEQ ID NO: Any selected from the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70, 77-79 and 84-85. CDR3 of the human T cell receptor α chain identified by the amino acid sequence, and SEQ ID NO: 3-4, 9-13, 15, 26-32, 41-48, 55-59, 63-64, 71- CDR3 of the human T cell receptor β chain identified by any amino acid sequence selected from the group consisting of 76, 80-83 and 86.
In some embodiments of the invention, the amino acid sequences of the T cell receptor α chain and the T cell receptor β chain CDR3 can be combined, for example, as follows;
CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
Sequence number: 1 Sequence number: 3 (URLC10),
SEQ ID NO: 2 SEQ ID NO: 3 (URLC10),
SEQ ID NO: 14 SEQ ID NO: 15 (URLC10),
SEQ ID NO: 16 SEQ ID NO: 10 (URLC10),
SEQ ID NO: 17 SEQ ID NO: 26 (URLC10),
SEQ ID NO: 60 SEQ ID NO: 63 (URLC10),
SEQ ID NO: 69 SEQ ID NO: 71 (URLC10),
SEQ ID NO: 77 SEQ ID NO: 80 (URLC10), and SEQ ID NO: 84 SEQ ID NO: 86 (DEPDC1).
　URLC10ペプチドで誘導されたT細胞受容体β鎖のうち、V領域のTRBV9では、共通した配列が多く同定された。TRBV9（配列番号：4、9、12－13、15、41、42、44－48、55－59、63、71、73－76、80－82）はアミノ酸配列11-13個で特定され、CASS-[X]n-F（Xは任意のアミノ酸でnが6-8／配列番号：179）という配列が共通している。 Among the T cell receptor β chains induced by the URLC10 peptide, many common sequences were identified in TRBV9 in the V region. TRBV9 (SEQ ID NO: 4, 9, 12-13, 15, 41, 42, 44-48, 55-59, 63, 71, 73-76, 80-82) was identified by 11-13 amino acid sequences. The sequence of CASS- [X] nF (X is an arbitrary amino acid and n is 6-8 / SEQ ID NO: 179) is common.
　本発明は、ある態様において、エピトープペプチドを結合したMHCが提示された抗原提示細胞を認識するTCRを含む組み換えTリンパ球（たとえばCD8+T細胞）の構築方法を提供する。TCRの抗原結合領域は、たとえば上記のCDR3をα鎖とβ鎖に含むことができる。TCRには、さらに付加的に、膜貫通ドメイン(transmembrane domain)やシグナリングドメインを含むこともできる。膜貫通ドメインは、ある態様において、T細胞の膜透過ドメインであることができる。T細胞の膜透過ドメインとしては、たとえばCD28の膜貫通ドメインを挙げることができる。一方、TCRを構成するシグナリングドメインには、1または複数の、免疫受容活性化チロシンモチーフ(immunoreceptor tyrosine-based activation motif; ITAMs)を含むことができる。免疫受容活性化チロシンモチーフとしては、CD3 ζ鎖(zeta chain)のようなT細胞受容体シグナリングドメインを挙げることができる。更にTCRは、一つまたは複数の刺激ドメインを含むこともできる。刺激ドメインは、たとえば、T細胞を刺激する二次的なシグナルを与える。本発明において、組み換えTリンパ球は、CD8+T細胞、CD4+ヘルパーT細胞、NK細胞、NKT細胞、B細胞、あるいは樹状細胞であることができる。
　一方、TCRをコードする核酸あるいは、それを含むベクターを導入するための宿主Tリンパ球は、健常者ドナーや、がんの治療を必要とする対象から採取することができる。がんの治療を必要とする対象から採取したTリンパ球に本発明のTCRを発現させることにより、対象のT細胞にURLC10あるいはDEPDC1に特異的な細胞傷害作用を人為的に付与することができる。 The present invention provides, in some embodiments, a method for constructing recombinant T lymphocytes (eg, CD8 + T cells) containing TCRs that recognize antigen-presenting cells presented with an epitope peptide-bound MHC. The antigen-binding region of the TCR can contain, for example, the above CDR3 in the α and β chains. The TCR can also additionally include a transmembrane domain or a signaling domain. The transmembrane domain can, in some embodiments, be the transmembrane domain of a T cell. Examples of the transmembrane domain of T cells include the transmembrane domain of CD28. On the other hand, the signaling domains constituting the TCR can contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Immunoreceptor-activated tyrosine motifs can include T cell receptor signaling domains such as the CD3 ζ chain. In addition, the TCR can also include one or more stimulation domains. The stimulation domain provides, for example, a secondary signal that stimulates T cells. In the present invention, recombinant T lymphocytes can be CD8 + T cells, CD4 + helper T cells, NK cells, NKT cells, B cells, or dendritic cells.
On the other hand, host T lymphocytes for introducing a nucleic acid encoding TCR or a vector containing the same can be collected from a healthy donor or a subject in need of cancer treatment. By expressing the TCR of the present invention in T lymphocytes collected from a subject in need of cancer treatment, it is possible to artificially impart a cytotoxic effect specific to URLC10 or DEPDC1 to the T cells of the subject. ..
　ある態様において、本発明は、活性なTCRを発現する組み換えT細胞を提供する。組み換え細胞を得る方法は公知である。たとえば、組み換えT細胞は、活性なTCRをコードする核酸によって得ることができる。ある態様において、活性なTCRをコードする核酸、あるいは核酸配列が提供される。TCRをコードする核酸は、通常は合成された核酸である。また核酸は、DNA、RNA，PNA（peptide nucleic acid）、あるいはそれらのハイブリッド分子を含むことができる。 In some embodiments, the invention provides recombinant T cells that express active TCR. Methods for obtaining recombinant cells are known. For example, recombinant T cells can be obtained by nucleic acids encoding active TCR. In some embodiments, a nucleic acid encoding an active TCR, or nucleic acid sequence, is provided. The nucleic acid encoding the TCR is usually a synthesized nucleic acid. Nucleic acids can also include DNA, RNA, PNA (peptidenucleic acid), or hybrid molecules thereof.
　ある態様において、活性なTCRを発現する本発明の組み換えT細胞は、必要に応じてその細胞傷害活性や増殖活性などの性状を評価することができる。これらの性状を評価する手法は公知である。細胞傷害作用を評価する手法には、例えば、HLA分子とTCRを取得した対象に投与されたペプチドを用いるテトラマーアッセイ（例えば、Altman et al. Science.1996, 274, 94-6 ;McMichael et al. J Exp Med.1998, 187, 1367-71）、ELISPOTアッセイあるいは、URLC10またはDEPDC1を発現するHLA陽性細胞を標的細胞として用いるクロム放出アッセイ等が含まれる。一方、細胞の増殖については、MTTアッセイなどの手法を応用して評価することができる。評価の結果、細胞傷害作用の大きな細胞、あるいはさらに増殖性に優れる細胞などの、より実用上望ましい性状を備えた細胞を選択することができる。 In some embodiments, the recombinant T cells of the present invention expressing active TCR can be evaluated for properties such as cytotoxic activity and proliferative activity, if necessary. Techniques for evaluating these properties are known. Examples of methods for evaluating cytotoxic effects include tetramer assays using peptides administered to subjects who have acquired HLA molecules and TCRs (eg, Altman et al. Science. 1996, 274, 94-6; McMichael et al. JExpMed.1998,187,1367-71), ELISPOT assay, or chromium release assay using HLA-positive cells expressing URLC10 or DEPDC1 as target cells. On the other hand, cell proliferation can be evaluated by applying a method such as MTT assay. As a result of the evaluation, it is possible to select cells having more practically desirable properties, such as cells having a large cytotoxic effect or cells having further excellent proliferative properties.
　本発明のある態様において、免疫学的に活性なTCRをコードする核酸は、たとえば次のような手法で決定することができる。たとえば、多くのTCRについて、それらをコードする核酸の塩基配列情報が既に決定され、データベースとして利用できるようになっている。それらをアセンブルすることによって、抗原結合部位の3つのCDRの位置を明らかにすることができる。TCRの抗原結合部位に含まれるCDRのうち、CDR1とCDR2は、HLAを認識しているとされているので、本発明の場合、HLA-A24拘束性のTCRをコードする核酸を利用すればよい。そして、残るCDR3を本発明において同定された上記アミノ酸配列情報をコードする塩基配列情報に置換することによって、組み換えＴリンパ球の誘導に必要なTCRをコードする核酸とすることができる。 In one aspect of the present invention, the nucleic acid encoding an immunologically active TCR can be determined, for example, by the following method. For example, for many TCRs, the nucleotide sequence information of the nucleic acids encoding them has already been determined and can be used as a database. By assembling them, the positions of the three CDRs of the antigen binding site can be clarified. Of the CDRs contained in the antigen-binding site of TCR, CDR1 and CDR2 are said to recognize HLA. Therefore, in the case of the present invention, a nucleic acid encoding HLA-A24-binding TCR may be used. .. Then, by substituting the remaining CDR3 with the base sequence information encoding the above amino acid sequence information identified in the present invention, a nucleic acid encoding TCR required for induction of recombinant T lymphocytes can be obtained.
　あるいは、CDR3のみならずCDR1とCDR2を含むTCRの抗原結合部位の全塩基配列情報に基づくTCRのレパトア解析も既に行われている。このような解析を通じて蓄積される抗原結合部位の配列情報の中から、目的とするCDR3の配列情報に一致するものを選択し、そのCDR1とCDR2の配列情報を得ることもできる。こうして選択されたCDR1、CDR2、およびCDR3の配列情報を、他のTCRの抗原結合部位に移植することによって、目的とする抗原特異性を移植することもできる。 Alternatively, TCR repertoire analysis based on the entire nucleotide sequence information of the antigen-binding site of TCR including CDR1 and CDR2 as well as CDR3 has already been performed. From the sequence information of the antigen-binding site accumulated through such analysis, one that matches the sequence information of the target CDR3 can be selected, and the sequence information of CDR1 and CDR2 can be obtained. By transplanting the sequence information of CDR1, CDR2, and CDR3 thus selected to the antigen-binding site of another TCR, the antigen specificity of interest can also be transplanted.
　あるいは、本発明の別の態様において、免疫学的に活性なTCRをコードする核酸を、実際にURLC10ペプチドおよびDEPDC1ペプチドのいずれか、または両方をワクチンとして接種されたヒトのリンパ球から得ることもできる。本発明において明らかにされたように、本発明によって提供されるTCRのCDR3は、これらのペプチドを接種された対象において、目的とするCTLの応答に伴って、優勢なポピュレーションを占めるようになる。したがって、このような対象から回収されるCTLから、本発明によって提供されるTCRのCDR3を含むTCRをコードする核酸を得ることは容易である。具体的には、ワクチンを接種された対象のCTL応答を確認し、URLC10ペプチドおよびDEPDC1ペプチド特異的でHLA-A24拘束性の作用を有するCTLが確認された対象からリンパ球を回収すれば、そのmRNAからTCRをコードする核酸のcDNAライブラリーを構築することができる。cDNAライブラリーからCDR3のアミノ酸配列として本発明において見出されたCDR3に相当するアミノ酸配列をコードしているものをクローニングすれば、目的とするTCRをコードする核酸を単離することができる。あるいは、対象からURLC10ペプチドまたはDEPDC1ペプチド特異的な細胞傷害作用を有するCTLを回収し、クローン化した後に、目的とするTCRをコードする核酸を単離することもできる。TCRをコードする核酸は、通常、分泌シグナルを含む。分泌シグナルは、もともとTCRのmRNAに含まれたものであることもできるし、それを他の分泌シグナルに置き換えることもできる。 Alternatively, in another aspect of the invention, a nucleic acid encoding an immunologically active TCR may be obtained from human lymphocytes that were actually vaccinated with either or both of the URLC10 peptide and the DEPDC1 peptide. it can. As demonstrated in the present invention, the CDR3 of the TCR provided by the present invention will occupy a predominant population in subjects inoculated with these peptides with the response of the CTL of interest. .. Therefore, it is easy to obtain a nucleic acid encoding a TCR containing CDR3 of the TCR provided by the present invention from a CTL recovered from such a subject. Specifically, if the CTL response of the vaccinated subject is confirmed and the lymphocytes are collected from the subject in which the URLC10 peptide- and DEPDC1 peptide-specific and HLA-A24-binding CTL is confirmed, the lymphocytes are collected. A cDNA library of nucleic acids encoding TCR can be constructed from mRNA. By cloning from the cDNA library the amino acid sequence corresponding to CDR3 found in the present invention as the amino acid sequence of CDR3, the nucleic acid encoding the target TCR can be isolated. Alternatively, a nucleic acid encoding the TCR of interest can be isolated after recovering a CTL having a cytotoxic effect specific to the URLC10 peptide or DEPDC1 peptide from the subject and cloning it. The nucleic acid encoding the TCR usually contains a secretory signal. The secretory signal can be originally contained in the TCR mRNA or can be replaced by another secretory signal.
　ある態様において、本発明におけるTCRをコードする核酸は、一つあるいは複数の制御配列を含むことができる。具体的には、プロモーターや転写要素(transcriptional element)、あるいは発現誘導を可能とする配列などを挙げることができる。核酸は、細胞における発現を可能とする適当なベクターで細胞に導入することができる。 In some embodiments, the nucleic acid encoding the TCR in the present invention can comprise one or more control sequences. Specific examples thereof include promoters, transcriptional elements, and sequences capable of inducing expression. The nucleic acid can be introduced into the cell with a suitable vector that allows expression in the cell.
　ある態様において、本発明の免疫学的に活性なTCR、あるいはその一部をコードする核酸を含む組み換えベクターが提供される。当業者には多くのベクターが知られていて、必要とされる機能に応じて適切なものを選択することができる。たとえば、プラスミド、コスミド、ウイルス、バクテリオファージ、等のベクターが知られており、遺伝子組み換えのために広く利用されている。ベクターの構築は、当業者には周知である（Sambrock and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989)）。あるいはポリヌクレオチドやベクターをリポソームに封入して目的の細胞の送達することも知られている。クローニングベクターを利用して目的のDNAを単離することもできる。目的の核酸配列は発現ベクターに組み込んで、細胞に導入することにより、所望のポリペプチドを発現させることができる。代表的なクローニングベクターには、たとえば、pBluescript SK、pGEM、pUC9、pBR322、あるいはpGBT9などが知られている。一方、発現ベクターには、pTRE、pCAL-n-EK、pESP-I、あるいはpOPI3CATなどが含まれる。 In some embodiments, a recombinant vector comprising an immunologically active TCR of the invention, or a nucleic acid encoding a portion thereof, is provided. Many vectors are known to those skilled in the art, and the appropriate one can be selected according to the required function. For example, vectors such as plasmids, cosmids, viruses, bacteriophages, etc. are known and are widely used for gene recombination. The construction of vectors is well known to those skilled in the art (Sambrock and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989)). Alternatively, it is also known that a polynucleotide or vector is encapsulated in a liposome to deliver the target cell. The DNA of interest can also be isolated using a cloning vector. The desired nucleic acid sequence can be expressed by incorporating the nucleic acid sequence of interest into an expression vector and introducing it into cells. Known typical cloning vectors include, for example, pBluescriptSK, pGEM, pUC9, pBR322, and pGBT9. On the other hand, the expression vector includes pTRE, pCAL-n-EK, pESP-I, pOPI3CAT and the like.
　ある態様において、ベクターは、TCR（あるいはその一部）をコードする核酸配列と作動可能に連結された調節配列を含むことができる。調節配列（調節単位）は当業者には公知で、たとえば、プロモーター、スプライスカセット、転写開始コドン、ベクターへの挿入用の挿入サイトなどを挙げることができる。ある態様において、真核細胞や原核細胞での発現を可能とするように、核酸が発現調節配列と作動可能に連結される。ＴＣＲのα鎖とβ鎖をコードする核酸は、１つのベクターに組み合わせて搭載することができる。あるいは別々のベクターに組み込んで、共形質転換することもできる。α鎖とβ鎖をコードする核酸をタンデムに搭載するベクターは、両者の組み合わせを正しく再現することができるので、より高い導入効率が期待できる。
　ＴＣＲのα鎖とβ鎖をコードする核酸の塩基配列は、コドンを最適化することにより発現効率の向上を期待することができる。更に、ＴＣＲのα鎖とβ鎖のＣＤＲ３以外の構造は、その細胞傷害活性が維持される限り、由来の異なる構造に置き換えることもできる。具体的には、抗原特異性に影響を与えないＣ領域は、任意のソースから得たもので置き換えられていてもよい。またＣＤＲ１とＣＤＲ２は、主にＨＬＡ分子を認識するとされているため、本発明のＴＣＲと共通のＨＬＡ拘束性（すなわちＨＬＡ－Ａ２４）のＴＣＲの構造と置き換えることもできる。その由来は、ヒトに限らず、他の種にソースを求めることができる。たとえば、マウスのＨＬＡはヒトとの共通性が高いので、マウスのＤ領域やＪ領域に置き換えることもできる。すなわち本発明は、本発明によって同定されたＣＤＲ３を、由来の異なるＴＣＲのα鎖、あるいはβ鎖に移植したキメラＴＣＲ分子を提供する。
　ベクターとしてウイルスベクターを用いる場合には、レンチウイルスベクターやアデノウイルスベクターを用いることができる。
　本発明において、核酸やベクターは、免疫学的に活性なTCRを細胞に発現させるために利用される。この場合、核酸やベクターは免疫学的に活性なTCRをコードするDNA配列を含み、細胞に導入することによって免疫学的に活性なTCRを発現する。 In some embodiments, the vector can comprise a regulatory sequence operably linked to a nucleic acid sequence encoding TCR (or a portion thereof). Regulatory sequences (regulatory units) are known to those of skill in the art and may include, for example, promoters, splice cassettes, transcription initiation codons, insertion sites for insertion into vectors, and the like. In some embodiments, the nucleic acid is operably linked to an expression regulatory sequence to allow expression in eukaryotic or prokaryotic cells. Nucleic acids encoding the α and β chains of the TCR can be loaded in combination in one vector. Alternatively, they can be integrated into separate vectors for co-transformation. A vector in which a nucleic acid encoding an α chain and a β chain is mounted in a tandem can correctly reproduce the combination of both, so that higher introduction efficiency can be expected.
The base sequence of the nucleic acid encoding the α-chain and β-chain of the TCR can be expected to improve the expression efficiency by optimizing the codons. Furthermore, the structures of the α and β chains of the TCR other than CDR3 can be replaced with structures of different origins as long as their cytotoxic activity is maintained. Specifically, the C region, which does not affect the antigen specificity, may be replaced with one obtained from any source. Further, since CDR1 and CDR2 are supposed to mainly recognize HLA molecules, they can be replaced with the HLA-restricted (that is, HLA-A24) TCR structure common to the TCR of the present invention. Its origin is not limited to humans, and sources can be sought from other species. For example, since mouse HLA has a high degree of commonality with humans, it can be replaced with mouse D region or J region. That is, the present invention provides a chimeric TCR molecule in which the CDR3 identified by the present invention is transplanted into the α chain or β chain of TCRs of different origins.
When a viral vector is used as the vector, a lentiviral vector or an adenovirus vector can be used.
In the present invention, nucleic acids and vectors are utilized to express immunologically active TCR in cells. In this case, the nucleic acid or vector contains a DNA sequence encoding an immunologically active TCR and is introduced into a cell to express the immunologically active TCR.
　本発明は、免疫学的に活性なTCRをコードする核酸配列を含む、プラスミド、コスミド、バクテリオファージ、ウイルスなどの通常遺伝子工学分野で用いられるベクターを得る方法に関する。 The present invention relates to a method for obtaining a vector usually used in the field of genetic engineering such as plasmid, cosmid, bacteriophage, virus, etc., which contains a nucleic acid sequence encoding an immunologically active TCR.
　ある態様において、ベクターは、発現ベクターや遺伝子導入用ベクター、あるいは遺伝子標的化ベクターであることができる。レトロウイルス、ワクシニアウイルス、アデノ随伴ウイルス、ヘルペスウイルス、あるいは牛パピローマウイルスなどに由来するウイルス性のベクターは、発現ポリヌクレオチドやベクターを細胞集団への導入に有用である。ウイルスベクターの多くは、細胞への導入後、その遺伝情報を宿主細胞のゲノムに組み込む。そのため、細胞の増殖に伴って目的の遺伝情報が安定して細胞集団中に維持される。組み換えベクターの構築方法は当業者によく知られている。ベクターは、宿主細胞の種類に応じて周知の方法で遺伝子導入することができる。 In some embodiments, the vector can be an expression vector, a gene transfer vector, or a gene targeting vector. Viral vectors derived from retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, bovine papillomaviruses, etc. are useful for introducing expressed polynucleotides and vectors into cell populations. Many viral vectors integrate their genetic information into the host cell's genome after introduction into the cell. Therefore, the target genetic information is stably maintained in the cell population as the cells proliferate. Methods of constructing recombinant vectors are well known to those of skill in the art. The vector can be gene-introduced by a well-known method depending on the type of host cell.
　ある態様において、本発明によって、免疫学的に活性なTCRをコードする核酸によって、形質転換された、あるいは遺伝子導入した宿主細胞が提供される。宿主細胞は、上記のようなベクターや核酸分子の少なくとも一つ、場合によっては複数を宿主細胞に導入することによって得ることができる。宿主細胞は、導入された核酸分子やベクターによって、免疫学的に活性なTCRやその一部を発現することができる。宿主細胞に導入された核酸分子やベクターは、ゲノムに組み込まれて、あるいは核外において維持することができる。 In some embodiments, the present invention provides a host cell transformed or transgenic with an immunologically active TCR-encoding nucleic acid. The host cell can be obtained by introducing at least one of the above-mentioned vectors and nucleic acid molecules, and in some cases, a plurality of them into the host cell. The host cell can express immunologically active TCR or a part thereof by the introduced nucleic acid molecule or vector. Nucleic acid molecules and vectors introduced into host cells can be integrated into the genome or maintained extranuclearly.
　ある態様において、本発明は、核酸分子やベクターを導入した上記の宿主細胞を培養する方法を提供する。たとえば、本発明は、導入されたコンストラクト（免疫学的に活性なTCRやその一部をコードする核酸を含む）を発現可能な条件下で宿主細胞を培養する工程を含む。特定の態様において、培養された細胞は、宿主細胞を得た対象や、あるいは宿主細胞が由来する対象とは別の第2の対象に提供される。発現用のコンストラクトを保持した細胞を培養する方法は周知である。 In some embodiments, the present invention provides a method of culturing the above host cells into which a nucleic acid molecule or vector has been introduced. For example, the present invention comprises culturing a host cell under conditions capable of expressing the introduced construct (including immunologically active TCR and nucleic acids encoding a portion thereof). In a particular embodiment, the cultured cells are provided to a subject from which the host cells have been obtained or a second subject separate from the subject from which the host cells are derived. Methods for culturing cells carrying the construct for expression are well known.
　本発明において、組み換え用のリンパ球の由来は任意である。たとえば、治療の対象となる患者や、健常なドナーから回収したリンパ球を宿主細胞として利用することができる。複数の異なるソースから回収したリンパ球を混合して宿主細胞とすることもできる。リンパ球を回収するためのソースとしては、具体的には、末梢血の単核球細胞、骨髄、リンパ節組織、臍帯血、胸腺組織、腹水、胸水、脾臓組織などを示すことができる。あるいは、感染病巣や腫瘍組織からリンパ球を回収することもできる。必要な種類のリンパ球（たとえば細胞傷害性T細胞、あるいは抗原特異性を持たないＴ細胞）を選択的に回収する手法は公知である。具体的には、ＣＤ８やＣＤ４等の特定の細胞マーカーを指標として、目的の細胞をセルソーティングによって回収する方法が一般的である。回収されたリンパ球は、続いて培養することができる。 In the present invention, the origin of lymphocytes for recombination is arbitrary. For example, lymphocytes collected from a patient to be treated or a healthy donor can be used as a host cell. Lymphocytes collected from multiple different sources can also be mixed into host cells. Specific examples of the source for collecting lymphocytes include peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, ascites, pleural effusion, and spleen tissue. Alternatively, lymphocytes can be recovered from infected lesions or tumor tissue. Techniques for selectively recovering the required type of lymphocytes (eg, cytotoxic T cells or T cells without antigen specificity) are known. Specifically, a method of recovering a target cell by cell sorting using a specific cell marker such as CD8 or CD4 as an index is common. The recovered lymphocytes can be subsequently cultured.
　本発明によって得られる組み換えリンパ球は、一般的な送達システムにより投与することができる。一般的には、薬学的に許容される担体に分散された組成物として組み換えリンパ球を投与することができる。あるいは核酸分子やベクターを投与する場合には、免疫学的に活性なTCRをコードする遺伝情報を対象のゲノムに組み込むこともできる。 The recombinant lymphocytes obtained by the present invention can be administered by a general delivery system. In general, recombinant lymphocytes can be administered as a composition dispersed in a pharmaceutically acceptable carrier. Alternatively, when a nucleic acid molecule or vector is administered, the genetic information encoding an immunologically active TCR can be integrated into the genome of interest.
　ある態様において、本発明の方法、あるいは組成物は、がんの予防、治療、あるいは症状の改善に利用することができる。たとえば、本発明によるがんの予防、治療、あるいは症状の改善のための方法は、それを必要とする対象に有効量の組成物を投与する工程を含む。ここで、本発明の方法に用いられる組成物は、たとえば、組み換えリンパ球免疫学的に活性なTCRをコードする核酸、それを含むベクターなどを有効成分として含む。組み換えリンパ球等の細胞を投与する場合、治療部位に局所的に投与することもできる。 In some embodiments, the methods or compositions of the present invention can be used to prevent, treat, or ameliorate symptoms of cancer. For example, a method for preventing, treating, or ameliorating symptoms of cancer according to the present invention comprises the step of administering an effective amount of the composition to a subject in need thereof. Here, the composition used in the method of the present invention contains, for example, a nucleic acid encoding a recombinant lymphocyte immunologically active TCR, a vector containing the nucleic acid, and the like as an active ingredient. When cells such as recombinant lymphocytes are administered, they can also be administered locally to the treatment site.
　本発明のがんの治療あるいは予防のための組成物、あるいは方法は、単独で、あるいは他のがんの治療あるいは予防のための治療や予防のための組成物あるいは方法と組み合わせることができる。具体的には、化学療法や免疫学的治療方法のための組成物あるいは方法と合わせて、本発明の組成物あるいは方法を利用することができる。あるいは、放射線療法、陽子線治療、温熱治療などの理化学的治療方法を併用することもできる。 The composition or method for treating or preventing cancer of the present invention can be used alone or in combination with other compositions or methods for treating or preventing cancer. Specifically, the composition or method of the present invention can be utilized in combination with the composition or method for chemotherapy or immunological treatment methods. Alternatively, physicochemical treatment methods such as radiation therapy, proton beam therapy, and hyperthermia therapy can be used in combination.
　本発明によって提供されるTCRは、URLC10およびDEPDC1のいずれか、または両方を発現する細胞を特異的に認識する。したがって、URLC10あるいはDEPDC1を発現している任意のがんを、治療あるいは予防の対象とすることができる。具体的には、URLC10は、たとえば、膀胱がん、子宮頸がん、胆管細胞がん、食道がん、胃がん、非小細胞肺がん（NSCLC）、骨肉腫、膵がん、軟部組織腫瘍、および頭頸部悪性腫瘍（HNMT）などにおいて高発現していることが知られている。したがって、本発明のTCRを発現するT細胞は、これらのがんの治療または予防に有用である。一方DEPDC1は、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、CML、NSCLC、リンパ腫、骨肉腫、前立腺がん、小細胞肺がん（SCLC）、および軟部組織腫瘍において高発現していることが報告されている。したがって、本発明のTCRを発現するT細胞によって、これらのがんの治療あるいは予防が期待できる。本発明において提供されるＴＣＲは、ＨＬＡ－Ａ２４拘束性の作用を有している。したがって、本発明のTCRを発現するT細胞は、ＨＬＡ-Ａ２４陽性の対象に対して有効に利用することができる。 The TCR provided by the present invention specifically recognizes cells expressing either or both of URLC10 and DEPDC1. Therefore, any cancer expressing URLC10 or DEPDC1 can be treated or prevented. Specifically, URLC10 includes, for example, bladder cancer, cervical cancer, bile duct cell cancer, esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), osteosarcoma, pancreatic cancer, soft tissue tumor, and It is known to be highly expressed in head and neck malignancies (HNMT). Therefore, the T cells expressing the TCR of the present invention are useful for the treatment or prevention of these cancers. On the other hand, DEPDC1 is highly expressed in bladder cancer, breast cancer, cervical cancer, bile duct cell cancer, CML, NSCLC, lymphoma, osteosarcoma, prostate cancer, small cell lung cancer (SCLC), and soft tissue tumor. Has been reported. Therefore, the T cells expressing the TCR of the present invention can be expected to treat or prevent these cancers. The TCR provided in the present invention has an HLA-A24 restrictive effect. Therefore, the T cells expressing the TCR of the present invention can be effectively used for HLA-A24 positive subjects.
　本発明のTCRによるがんの治療や予防に先立って、予め対象とするがんにおけるURLC10あるいはDEPDC1の発現を確認することができる。すなわち本発明は、以下の工程を含む、がんの治療および予防の、いずれかまたは両方のための方法を提供する：
(1) 治療あるいは予防の対象からがん組織を採取する工程；
(2) 採取されたがん組織におけるURLC10あるいはDEPDC1の発現を検出する工程；および
(3) 正常組織と比較してURLC10およびDEPDC1のいずれか、または両方の発現レベルが高いがんを有する対象を選択して本発明の組成物を投与する工程。 Prior to the treatment or prevention of cancer by the TCR of the present invention, the expression of URLC10 or DEPDC1 in the target cancer can be confirmed in advance. That is, the present invention provides methods for the treatment and prevention of cancer, or both, including the following steps:
(1) The process of collecting cancer tissue from the target of treatment or prevention;
(2) Step of detecting the expression of URLC10 or DEPDC1 in the collected cancer tissue; and
(3) A step of selecting a subject having cancer having a high expression level of either or both of URLC10 and DEPDC1 as compared with normal tissue and administering the composition of the present invention.
　別の態様において、本発明は、治療あるいは予防の対象から採取されたがん組織におけるURLC10あるいはDEPDC1の発現を検出し、正常組織と比較してURLC10およびDEPDC1のいずれか、または両方の発現レベルが高いがんを有する対象を選択して投与するための、組成物に関する。あるいは本発明は、治療あるいは予防の対象から採取されたがん組織におけるURLC10あるいはDEPDC1の発現を検出し、正常組織と比較してURLC10およびDEPDC1のいずれか、または両方の発現レベルが高いがんを有する対象を選択して投与するための組成物の製造におけるTCRの使用を提供する。更に本発明は、治療あるいは予防の対象から採取されたがん組織におけるURLC10あるいはDEPDC1の発現を検出し、正常組織と比較してURLC10およびDEPDC1のいずれか、または両方の発現レベルが高いがんを有する対象を選択して投与する工程を含む、がんの治療および予防の、いずれかまたは両方におけるTCRの使用を提供する。本発明の治療あるいは予防は、通常、ＨＬＡ－Ａ２４アレルをホモあるいはヘテロに有している対象において有効である。 In another embodiment, the invention detects the expression of URLC10 or DEPDC1 in cancer tissue taken from a subject to be treated or prevented, and the expression level of either or both of URLC10 and DEPDC1 is compared to normal tissue. It relates to a composition for selectively administering a subject having high cancer. Alternatively, the present invention detects the expression of URLC10 or DEPDC1 in cancer tissues collected from a subject to be treated or prevented, and can detect cancers in which the expression level of either or both of URLC10 and DEPDC1 is higher than that of normal tissues. Provided is the use of TCR in the manufacture of compositions for selective administration of subjects having. Furthermore, the present invention detects the expression of URLC10 or DEPDC1 in cancer tissues collected from a subject to be treated or prevented, and can detect cancers in which the expression level of either or both of URLC10 and DEPDC1 is higher than that of normal tissues. Provided is the use of TCR in the treatment and prevention of cancer, including the step of selecting and administering a subject to have. The treatment or prevention of the present invention is usually effective in subjects who have the HLA-A24 allele homo or heterozygous.
　あるいは、対象から回収したT細胞に本発明のTCRを導入後、それを対象に投与することでがんの治療効果を期待することもできる。すなわち本発明は、
(1) 治療あるいは予防の対象から採取されたがん組織におけるURLC10あるいはDEPDC1の発現を検出し、正常組織と比較してURLC10およびDEPDC1のいずれか、または両方の発現レベルが高いがんを有する対象を選択する工程、
(2) 選択された対象からT細胞を回収する工程、
(3) 回収したT細胞に本発明のTCRをコードする核酸を導入する工程、および
(4) 本発明のTCRをコードする核酸を導入したT細胞を対象に投与する工程、を含むがんの治療方法に関する。
　本発明において、末梢血から回収するＴ細胞は、抗原特異性を有していない（未刺激）ものであることが望ましい。なお、TCRをコードする核酸を導入したT細胞は、必要に応じて増殖させたのちに対象に投与することができる。したがって、末梢血から回収されるＴ細胞は、必ずしも大量である必要はない。
　本発明の、ある態様において、T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列は、たとえば以下のように組み合わせることができる；
T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
配列番号：1　　　　　　　　　　　　　　配列番号：3（URLC10）、
配列番号：2　　　　　　　　　　　　　　配列番号：3（URLC10）、
配列番号：14　　　　　　　　　　　　　 配列番号：15（URLC10）、
配列番号：16　　　　　　　　　　　　　 配列番号：10（URLC10）、
配列番号：17　　　　　　　　　　　　　 配列番号：26（URLC10）、
配列番号：60　　　　　　　　　　　　　 配列番号：63（URLC10）、
配列番号：69　　　　　　　　　　　　　 配列番号：71（URLC10）、
配列番号：77　　　　　　　　　　　　　 配列番号：80（URLC10）、および
配列番号：84　　　　　　　　　　　　　 配列番号：86（DEPDC1）。 Alternatively, the therapeutic effect of cancer can be expected by introducing the TCR of the present invention into T cells collected from a subject and then administering the TCR to the subject. That is, the present invention
(1) Subjects who have detected the expression of URLC10 or DEPDC1 in cancer tissues collected from the subject of treatment or prevention and have cancers in which the expression level of either URLC10 and / or DEPDC1 is higher than that of normal tissues. The process of selecting,
(2) The process of collecting T cells from the selected subject,
(3) A step of introducing a nucleic acid encoding the TCR of the present invention into the collected T cells, and
(4) The present invention relates to a method for treating cancer, which comprises a step of administering a T cell having a nucleic acid encoding the TCR of the present invention into a subject.
In the present invention, it is desirable that the T cells recovered from peripheral blood do not have antigen specificity (unstimulated). In addition, T cells into which a nucleic acid encoding TCR has been introduced can be administered to a subject after being proliferated as needed. Therefore, the amount of T cells recovered from peripheral blood does not necessarily have to be large.
In some embodiments of the invention, the amino acid sequences of the T cell receptor α chain and the T cell receptor β chain CDR3 can be combined, for example, as follows;
CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
Sequence number: 1 Sequence number: 3 (URLC10),
SEQ ID NO: 2 SEQ ID NO: 3 (URLC10),
SEQ ID NO: 14 SEQ ID NO: 15 (URLC10),
SEQ ID NO: 16 SEQ ID NO: 10 (URLC10),
SEQ ID NO: 17 SEQ ID NO: 26 (URLC10),
SEQ ID NO: 60 SEQ ID NO: 63 (URLC10),
SEQ ID NO: 69 SEQ ID NO: 71 (URLC10),
SEQ ID NO: 77 SEQ ID NO: 80 (URLC10), and SEQ ID NO: 84 SEQ ID NO: 86 (DEPDC1).
　別の態様において、本発明は、本発明のTCRをコードする核酸の、上記工程を含むがんの治療における使用に関する。また本発明は、本発明のTCRをコードする核酸の、上記工程を含むがんの治療のための組成物の製造における使用に関する。あるいは本発明は、本発明のTCRをコードする核酸を含む、上記工程を含むがんの治療のための組成物に関する。 In another aspect, the invention relates to the use of the nucleic acid encoding the TCR of the invention in the treatment of cancer, including the steps described above. The present invention also relates to the use of the nucleic acid encoding the TCR of the present invention in the production of a composition for the treatment of cancer, which comprises the above steps. Alternatively, the present invention relates to a composition for treating cancer, which comprises the above steps, comprising a nucleic acid encoding the TCR of the present invention.
　別の態様において、本発明は、がんの治療を必要とする対象から採取されたCD8+（CD8陽性）T細胞に本発明のTCRをコードする核酸を含む発現ベクターを導入し、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞を回収する工程を含む、細胞傷害性T細胞の製造方法を提供する。
　本発明のTCRをコードする核酸を含む発現ベクターを導入したT細胞からは、そのURLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を評価し、活性を有する細胞を単離することができる。細胞傷害作用の評価には、任意の標的細胞が用いられる。具体的には、URLC10またはDEPDC1を発現するHLA-A24陽性細胞や、URLC10またはDEPDC1の発現ベクターで形質転換したHLA-A24陽性細胞などを挙げることができる。細胞傷害作用の評価に先立って、予めテトラマーアッセイによってHLA-ペプチド複合体を認識するT細胞を識別することもできる。
　本発明のある態様において、細胞傷害性T細胞の製造方法は、さらに付加的に、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を単離し、そして単離したT細胞を拡大培養によって増殖させる工程を含むことができる。T細胞を拡大培養する方法も公知である。たとえば、フィーダー細胞の共存化でT細胞を維持することによって、その増殖を支持することができる。フィーダー細胞には、非増殖性のリンパ球細胞などを利用することができる。リンパ球細胞を予めガンマ線照射して***性を制御するのが一般的である。増殖後のT細胞を回収し、必要に応じて洗浄したのち、生理食塩水などの適当な媒体に再分散して対象への投与に供することができる。 In another embodiment, the invention introduces an expression vector containing a nucleic acid encoding the TCR of the invention into CD8 + (CD8 positive) T cells taken from a subject in need of cancer treatment and URLC10 or DEPDC1. Provided is a method for producing a cytotoxic T cell, which comprises a step of recovering a cytotoxic T cell having an HLA-A24-binding cytotoxic effect on an expressing cell.
From T cells into which an expression vector containing a nucleic acid encoding the TCR of the present invention has been introduced, the HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells can be evaluated, and active cells can be isolated. it can. Any target cell is used to evaluate the cytotoxic effect. Specific examples thereof include HLA-A24-positive cells expressing URLC10 or DEPDC1, and HLA-A24-positive cells transformed with an expression vector of URLC10 or DEPDC1. Prior to the evaluation of cytotoxic effects, T cells that recognize the HLA-peptide complex can be identified in advance by tetramer assay.
In certain aspects of the invention, the method for producing cytotoxic T cells additionally isolates HLA-A24-binding cytotoxic effects on URLC10 or DEPDC1-expressing cells, and expands the isolated T cells. Can include the step of growing by. A method for expanding and culturing T cells is also known. For example, maintaining T cells in the coexistence of feeder cells can support their proliferation. Non-proliferative lymphocyte cells and the like can be used as the feeder cells. It is common to preliminarily irradiate lymphocyte cells with gamma rays to control mitosis. After the proliferated T cells are collected, washed if necessary, they can be redispersed in an appropriate medium such as physiological saline and used for administration to a subject.
　本発明の、ある態様において、対象に投与される組み換えT細胞の数、投与のスケジュール、投与方法は、対象の状態や、治療を目的とするがんの種類や状態に応じて当業者は適宜選択することができる。あるいは、投与対象の臨床症状を観察して、投与量やスケジュールを調節することもできる。たとえば、組み換えT細胞の投与スケジュールとして次のような例が知られている：
2 x 108 cells, 1 x 109 cellsまたは5 x 109 cellsの静脈内投与1回（Clin Cancer Res. 2015, 21(10):2268-77）；
5.3 x 109 cellsを2回に分けて投与または2.4 x 109 cellsを1回投与（Blood. 2013, 122:863-71）；
中央値5.5 x 1010 cells（範囲0.9-13 x 1010 cells）を1回投与（Clin Cancer Res. 2015, 21(5):1019-27.）；あるいは
平均2.4 x 109 cellsを1回投与（非特許文献1） In one embodiment of the present invention, the number of recombinant T cells to be administered to a subject, the schedule of administration, and the administration method are appropriately determined by those skilled in the art depending on the condition of the subject and the type and condition of the cancer to be treated. You can choose. Alternatively, the dose and schedule can be adjusted by observing the clinical symptoms of the administration subject. For example, the following are known examples of administration schedules for recombinant T cells:
One intravenous dose of 2 x 10 8 cells, 1 x 10 9 cells or 5 x 10 9 cells (Clin Cancer Res. 2015, 21 (10): 2268-77);
5.3 x 10 9 cells in 2 divided doses or 2.4 x 10 9 cells in 1 dose (Blood. 2013, 122: 863-71);
Median 5.5 x 10 10 cells (range 0.9-13 x 10 10 cells) given once (Clin Cancer Res. 2015, 21 (5): 1019-27.); Or mean 2.4 x 10 9 cells given once (Non-Patent Document 1)
ＩＩＩ．ペプチドによって媒介されるCTL応答のモニタリング
　TCR-α遺伝子およびTCR-β遺伝子のディープcDNA配列解析を通して、当該遺伝子の特定のペアの頻度が、特定のURLC10ペプチド（RYCNLEGPPI ／配列番号：177）あるいはDEPDC1ペプチド（EYYELFVNI／配列番号：178）によって刺激されたT細胞において増加したことが明らかになった。それゆえ、そのような遺伝子のペアがURLC10あるいはDEPDC1ペプチド接種後に対象において検出される場合、それは、対象におけるペプチド特異的CTL応答が誘導されたことを意味する。したがって、URLC10あるいはDEPDC1ペプチドによって刺激されたT細胞集団における遺伝子の特定のペアの増加は、刺激後の対象におけるCTL応答をモニタリングまたは検出するための代用マーカーとして有用であり得る。本発明に関連して、「ペプチド特異的CTL応答」とは、αサブユニットおよびβサブユニットのペアの間に形成されるTCRが、本発明のペプチドとHLA分子との間に形成される複合体を特異的に認識することを意味するように理解される。上記で論じたように、本発明の特異的配列で定義されるペプチドのCTL細胞誘導能は、アミノ酸改変後でさえも維持され得る。それゆえ、特異的ペプチドによる刺激に加えて、T細胞が変異ペプチドから誘導される場合でさえも、そのTCRがもとのペプチドによって形成されるそのような複合体を特異的に認識する限り、その抗原特異性は「ペプチド特異的」とみなされる。 III. Monitoring Peptide-mediated CTL Response Through deep cDNA sequence analysis of the TCR-α and TCR-β genes, the frequency of a particular pair of genes is determined by a particular URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) or DEPDC1 peptide. It was revealed that it was increased in T cells stimulated by (EYYELFVNI / SEQ ID NO: 178). Therefore, if such a pair of genes is detected in a subject after inoculation with URLC10 or DEPDC1 peptide, it means that a peptide-specific CTL response in the subject has been induced. Therefore, an increase in a particular pair of genes in a T cell population stimulated by the URLC10 or DEPDC1 peptide may be useful as a substitute marker for monitoring or detecting the CTL response in the stimulated subject. In the context of the present invention, a "peptide-specific CTL response" is a complex in which the TCR formed between a pair of α and β subunits is formed between the peptide of the present invention and an HLA molecule. It is understood to mean the specific recognition of the body. As discussed above, the CTL cell inducibility of peptides defined by the specific sequences of the invention can be maintained even after amino acid modification. Therefore, in addition to stimulation with a specific peptide, even when T cells are derived from a mutant peptide, as long as the TCR specifically recognizes such a complex formed by the original peptide. Its antigen specificity is considered "peptide specific".
　例えば、以下のアミノ酸配列からなるCDR3を有するTCR-αサブユニットおよびTCR-βサブユニットが、HLA-A24拘束様式で、特定のURLC10ペプチドあるいはDEPDC1ペプチドによって刺激されたT細胞集団において増加した（図2、図5A、図5B）。
URLC10ペプチド：
配列番号：1-2、5-8、14、16、17-25、33-40、49-54、60-62、65-70および77-79からなる群から選択されるいずれかのアミノ酸配列で特定されるT細胞受容体α鎖のCDR3、および
配列番号：3-4、9-13、15、26-32、41-48、55-59、63-64、71-76および80-83
からなる群から選択されるいずれかのアミノ酸配列で特定されるT細胞受容体β鎖のCDR3。
DEPDC1ペプチド：
配列番号：84または85のアミノ酸配列で特定されるT細胞受容体α鎖のCDR3、および配列番号：86で特定されるT細胞受容体β鎖のCDR3。
　それゆえ、対象のT細胞集団におけるそれらの蓄積は、URLC10ペプチドあるいはDEPDC1ペプチドによって媒介されるT細胞応答が、ペプチドを接種した対象において成功裡に誘導されたことを意味する。上記アミノ酸配列からなるCDR3の存在は、抗体に基づく解析によって検出することができる。本出願の代替的な態様において、TCR-αサブユニットおよびTCR-βサブユニットの特定のペアはまた、それらをコードするポリヌクレオチドを検出することによっても評価することができる。例えば、本出願において同定されたCDR3の各々（表１、表３Ａおよび３Ｂの配列番号：1-86）をコードするポリヌクレオチドは、それぞれ配列番号：87－173のヌクレオチド配列によって代表される。
　したがって、URLC10あるいはDEPDC1特異的TCRのαサブユニットおよびβサブユニットをコードするmRNAから合成されるcDNAの各々は、それぞれ上記配列番号のヌクレオチド配列を含み得る。そのようなポリヌクレオチドは、PCRに基づく解析によって検出することができる。 For example, TCR-α and TCR-β subunits with CDR3 consisting of the following amino acid sequences were increased in the HLA-A24 constrained manner in a T cell population stimulated by a particular URLC10 or DEPDC1 peptide (Figure). 2, Fig. 5A, Fig. 5B).
URLC10 peptide:
SEQ ID NO:: Any amino acid sequence selected from the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70 and 77-79. CDR3 of the T cell receptor α chain identified in, and SEQ ID NOs: 3-4, 9-13, 15, 26-32, 41-48, 55-59, 63-64, 71-76 and 80-83.
CDR3 of the T cell receptor β chain identified by any amino acid sequence selected from the group consisting of.
DEPDC1 peptide:
CDR3 of the T cell receptor α chain identified by the amino acid sequence of SEQ ID NO: 84 or 85, and CDR3 of the T cell receptor β chain identified by SEQ ID NO: 86.
Therefore, their accumulation in the T cell population of the subject means that the T cell response mediated by the URLC10 peptide or DEPDC1 peptide was successfully induced in the peptide-inoculated subject. The presence of CDR3 consisting of the above amino acid sequence can be detected by antibody-based analysis. In an alternative aspect of the application, specific pairs of TCR-α and TCR-β subunits can also be evaluated by detecting the polynucleotides encoding them. For example, the polynucleotides encoding each of the CDR3s identified in this application (SEQ ID NOs: 1-86 in Tables 1, 3A and 3B) are represented by the nucleotide sequences of SEQ ID NOs: 87-173, respectively.
Thus, each of the cDNAs synthesized from the α and β subunits of the URLC10 or DEPDC1-specific TCR may each contain the nucleotide sequence of the above SEQ ID NO:. Such polynucleotides can be detected by PCR-based analysis.
　好ましい態様において、本発明は、ペプチドで免疫された対象におけるペプチド特異的T細胞応答をモニタリングする、評価する、または評定するための方法であって、
　（ａ）該ペプチドを投与された対象から得られた試料を提供する段階であって、該試料がT細胞を含む、段階；
　（ｂ）該試料における、MHCクラスＩ分子と該ペプチドまたはその断片との複合体に結合するTCRを発現するT細胞の存在を検出する段階；および
　（ｃ）該T細胞の存在が（ｂ）において検出される場合に、該ペプチドに特異的なT細胞応答の誘導が示される段階
を含む方法を提供する。例えば、上記配列番号のアミノ酸配列からなるCDR3を含むαサブユニットおよびβサブユニットの、いずれか、または両方を検出することによって、HLA-A24拘束様式でのURLC10あるいはDEPDC1に特異的なT細胞の誘導を検出することができる。 In a preferred embodiment, the invention is a method for monitoring, evaluating, or assessing a peptide-specific T cell response in a peptide-immunized subject.
(A) A step of providing a sample obtained from a subject to which the peptide has been administered, wherein the sample contains T cells;
(B) The step of detecting the presence of TCR expressing TCR in the sample that binds to the complex of MHC class I molecule and the peptide or fragment thereof; and (c) the presence of the T cell is (b). Provided is a method comprising a step in which the induction of a T cell response specific to the peptide is exhibited when detected in. For example, by detecting one or both of the α and β subunits containing CDR3 consisting of the amino acid sequence of the above SEQ ID NO:, T cells specific for URLC10 or DEPDC1 in the HLA-A24 restraint mode. Induction can be detected.
　本発明において、対象から得られる任意の生物学的試料を、その試料にT細胞が含まれる限り、CTL応答をモニタリングするために使用することができる。例えば、本発明のために、血液または血液由来試料を、生物学的試料として使用することができる。本発明において、血液由来試料は、T細胞を含む細胞集団を含む。T細胞を含む細胞集団を得るための方法は、当業者に周知である。あるいは、いくつかの態様において、T細胞が浸潤した組織またはリンパ節もまた、生物学的試料として有用である。解析に先立ち、細胞傷害性T細胞をあらかじめ増殖させることもできる。また、対象から採取された末梢血単核球（PBMC）は、いくつかの異なった細胞種を含む。免疫学的に活性なTCRを備えた細胞傷害性T細胞を解析するには、予め目的の細胞を増やしておくのが有利である。増殖後の細胞傷害性T細胞を集めて、TCRのレパトア解析の試料とすることもできる。 In the present invention, any biological sample obtained from the subject can be used to monitor the CTL response as long as the sample contains T cells. For example, for the present invention, blood or blood-derived samples can be used as biological samples. In the present invention, the blood-derived sample comprises a cell population containing T cells. Methods for obtaining cell populations containing T cells are well known to those of skill in the art. Alternatively, in some embodiments, a tissue or lymph node infiltrated with T cells is also useful as a biological sample. Cytotoxic T cells can also be proliferated prior to analysis. Peripheral blood mononuclear cells (PBMCs) collected from the subject also contain several different cell types. In order to analyze cytotoxic T cells with immunologically active TCR, it is advantageous to increase the number of target cells in advance. Post-proliferation cytotoxic T cells can also be collected and used as samples for TCR repertoire analysis.
　本発明のいくつかの態様において、TCRサブユニットの評定における抗体に基づく解析のために、T細胞を含む細胞集団を固定することができる。抗体は、固定された細胞と接触した後、固定された細胞のαサブユニットおよびβサブユニットのCDR3を認識し、抗体が単一細胞上のTCRの両サブユニットに結合する場合、特定のサブユニットペアと共に形成されるTCR-抗体複合体を検出することができる。あるいは、ヌクレオチド配列に基づく解析において、単一細胞におけるTCRサブユニットをコードするポリヌクレオチドから合成されたアンプリコンの蓄積は、サブユニットの特定のペアが存在していることを意味する。あるいは、ワクチン接種されるペプチドの各々についてのTCRサブユニットをコードするポリヌクレオチドの特定のペアを、Fang H, et al., Oncoimmunology 2015, 3: e968467に示されているようなディープｃDNA配列解析によってモニタリングすることができる。 In some embodiments of the invention, a cell population containing T cells can be immobilized for antibody-based analysis in the evaluation of TCR subunits. After contacting the fixed cell, the antibody recognizes the CDR3 of the α and β subunits of the fixed cell, and if the antibody binds to both subunits of TCR on a single cell, a particular sub. The TCR-antibody complex formed with the subunit pair can be detected. Alternatively, in nucleotide sequence-based analysis, the accumulation of amplicon synthesized from polynucleotides encoding TCR subunits in a single cell means that a particular pair of subunits is present. Alternatively, a specific pair of polynucleotides encoding the TCR subunit for each of the peptides to be vaccinated can be obtained by deep cDNA sequencing as shown in Fang H, et al., Oncoimmunology 2015, 3: e968467. Can be monitored.
　本局面において、T細胞上のTCRの構造多様性は、主にそれらのCDR3に依存していることが周知である。その結果、それによる抗原特異性もまた、CDR3に依存している。それゆえ、単一細胞上のαサブユニットおよびβサブユニットの特定のペアの存在を、CDR3の検出を通してモニタリングすることができる。 In this aspect, it is well known that the structural diversity of TCRs on T cells depends mainly on their CDR3. As a result, the resulting antigen specificity is also CDR3 dependent. Therefore, the presence of specific pairs of α and β subunits on a single cell can be monitored through the detection of CDR3.
　本発明のいくつかの態様において、CTL応答をモニタリングするかまたは評定するために、TCR-αサブユニットおよびTCR-βサブユニットのいずれか、または両方を、ワクチン接種後に少なくとも１回または複数回、検出することができる。ペアがモニタリングを通して時間依存的に増加する場合、それは、対象におけるペプチド媒介性CTL応答が十分に誘導されていることを意味する。あるいは、特定のペアが少なくとも１回検出される場合、それは、対象におけるCTL応答が生じたことを示す。
　あるいは、ワクチン接種前の対象におけるTCRのレパートリーを取得し、接種後のレパートリーと比較して、本発明のTCRの増加を確認することもできる。接種前のレパートリーに本発明のTCRが検出されない場合には、ワクチン接種後にその出現や増加が見られた場合に、対象におけるワクチンへの応答を検出することができる。ワクチン接種後の対象のTCRは、接種後の、任意のタイミングで解析することができる。 In some embodiments of the invention, the TCR-α subunit and / or both of the TCR-α subunits, or both, at least once or multiple times after vaccination, to monitor or assess the CTL response. Can be detected. If the pair increases in a time-dependent manner through monitoring, it means that the peptide-mediated CTL response in the subject is well induced. Alternatively, if a particular pair is detected at least once, it indicates that a CTL response has occurred in the subject.
Alternatively, it is possible to obtain a repertoire of TCRs in the subject before vaccination and compare it with the repertoire after vaccination to confirm the increase in TCRs of the present invention. If the TCR of the invention is not detected in the pre-vaccination repertoire, a response to the vaccine in the subject can be detected if its appearance or increase is seen after vaccination. The TCR of the subject after vaccination can be analyzed at any time after vaccination.
　ある態様において、本発明のモニタリング方法は、予めURLC10ペプチドおよびDEPDC1ペプチドのいずれか、または両方を対象に投与する工程を含むことができる。すなわち本発明は、
(1) URLC10ペプチドおよびDEPDC1ペプチドのいずれか、または両方を対象に投与する工程
(2) ペプチド投与後の対象から少なくとも一度T細胞を回収する工程、および
(3) T細胞のTCRに占める本発明のTCRの頻度を決定する工程。
　そして、本発明のTCRの頻度が高まった場合に、対象におけるペプチド媒介性CTL応答が十分に誘導されていることを知ることができる。頻度を複数回決定して、経時的な変化を追うことにより、TCRの頻度の高まりを知ることができる。あるいは、少なくとも1度の頻度の決定後、一定の頻度を占めることがわかれば、予め接種したペプチドに対する免疫応答が誘導されたことを知ることができる。一定の頻度とは、たとえば解析したTCRの全レパートリーに占める割合が通常1％、あるいは2％もしくは3％以上を例示することができる。解析の対象は、投与されたペプチドに特異的なＴ細胞であることが望ましい。ペプチド特異的なＴ細胞は、たとえばテトラマーアッセイで同定することができる。 In some embodiments, the monitoring method of the present invention can include pre-administering either or both of the URLC10 peptide and the DEPDC1 peptide to the subject. That is, the present invention
(1) Step of administering URLC10 peptide and / or DEPDC1 peptide to a subject
(2) A step of collecting T cells from the subject at least once after administration of the peptide, and
(3) A step of determining the frequency of the TCR of the present invention in the TCR of T cells.
Then, it can be seen that the peptide-mediated CTL response in the subject is sufficiently induced when the frequency of the TCR of the present invention is increased. By determining the frequency multiple times and following the changes over time, it is possible to know the increase in the frequency of TCR. Alternatively, if it is found that the frequency occupies a certain frequency after determining the frequency at least once, it can be known that the immune response to the pre-inoculated peptide is induced. The constant frequency can be exemplified by, for example, the ratio of the analyzed TCR to the entire repertoire is usually 1%, or 2% or 3% or more. The subject of analysis is preferably T cells specific for the administered peptide. Peptide-specific T cells can be identified, for example, by tetramer assay.
　本発明において、URLC10ペプチドおよびDEPDC1ペプチドは、単独で投与することもできるし、がんワクチンとして有効な他のペプチドと組み合わせて投与することもできる。たとえばURLC10ペプチド(RYCNLEGPPI／配列番号：177)に加え、DEPDC1ペプチド(EYYELFVNI／配列番号：178)を含む、いわゆるカクテルワクチンを投与することもできる。 In the present invention, the URLC10 peptide and the DEPDC1 peptide can be administered alone or in combination with other peptides effective as a cancer vaccine. For example, a so-called cocktail vaccine containing the DEPDC1 peptide (EYYELFVNI / SEQ ID NO: 178) in addition to the URLC10 peptide (RYCNLEGPPI / SEQ ID NO: 177) can also be administered.
　ある態様において、本発明のTCRの頻度の評価のために、TCRのレパトア解析が有用である。レパトア解析においては、通常、ある細胞集団におけるTCRのCDR3を構成するアミノ酸配列を網羅的に解析する。CDR3は、主としてTCRの抗原認識特異性を決定する領域であることはすでに述べた。したがってその構造を解析することにより、細胞集団を構成するTCRが、どのような割合で構成されているのかを評価することができる。多数のTCRのCDR3を網羅的に解析して、各CDR3の検出頻度を比較することで、TCRのレパトア解析が実施される。 In some embodiments, a TCR repertoire analysis is useful for assessing the frequency of TCRs of the present invention. In the repertoire analysis, the amino acid sequences constituting the CDR3 of the TCR in a certain cell population are usually comprehensively analyzed. It has already been mentioned that CDR3 is primarily the region that determines the antigen recognition specificity of the TCR. Therefore, by analyzing its structure, it is possible to evaluate the proportion of TCR that constitutes the cell population. TCR repertoire analysis is performed by comprehensively analyzing the CDR3s of a large number of TCRs and comparing the detection frequencies of each CDR3.
　T細胞受容体（TCR）はV遺伝子、D遺伝子、J遺伝子およびC遺伝子からなる。
V遺伝子やJ遺伝子の再構成さらにはV-D-J遺伝子間（CDR3）にランダムに発生する塩基の挿入や欠失により、TCRには10の18乗にもおよぶ多様性が生じると考えられている。したがってヒト体内には様々なTCRを発現するT細胞が存在する。
　あるT細胞集団におけるTCRの多様性（どのようなTCRがどれくらいの頻度で検出されるか）を調べることをTCRレパトア解析と呼ぶ。
　TCRレパトア解析を行う場合、様々なTCR遺伝子をPCR法によって偏りなく増幅するために、T細胞集団に由来するRNAから5’末端にアダプターが付加されたcDNAを合成する。アダプター特異的なフォワードプライマーとTCR-αもしくはTCR-β特異的なリバースプライマーを用いて得られた大量のDNAフラグメント（シーケンスライブラリー）の塩基配列決定には次世代シーケンサーが利用される。
　次世代シーケンサーは数百万ものDNAフラグメントの塩基配列を並列的に決定する能力を備える機器である。TCRレパトア解析では、TCRを構成するV遺伝子、D遺伝子、J遺伝子およびC遺伝子にまたがって長い塩基配列を決定する必要がある。そこで、次世代シーケンサーの中でもロングリード解析（およそ300bpの塩基配列の決定）に長けているMiSeq（Illumina）が用いられることが多い。
　試料中に含まれるTCRをコードするmRNAの塩基配列を網羅的に解析し、試料を構成しているT細胞集団に占める各CDR3の検出頻度を容易に知ることができる。CDR3の塩基配列を決定するために有用なプライマーの塩基配列の例を以下に示す。
フォワードプライマー（TCR-α, TCR-β共通のアダプター配列、配列番号：174）：
5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3'
リバースプライマー（TCR-α用、配列番号：175）：
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3'
リバースプライマー（TCR-β用、配列番号：176）：
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3' The T cell receptor (TCR) consists of the V, D, J and C genes.
It is thought that the rearrangement of the V and J genes, as well as the insertion and deletion of randomly occurring bases between the VDJ genes (CDR3), cause as much diversity as 10 to the 18th power in the TCR. Therefore, there are T cells that express various TCRs in the human body.
Examining the diversity of TCRs in a T cell population (what TCRs are detected and how often) is called TCR repertoire analysis.
When performing TCR repertoire analysis, in order to amplify various TCR genes evenly by the PCR method, cDNA with an adapter added to the 5'end is synthesized from RNA derived from the T cell population. A next-generation sequencer is used to sequence a large amount of DNA fragments (sequence library) obtained by using an adapter-specific forward primer and a TCR-α or TCR-β-specific reverse primer.
Next-generation sequencers are devices that have the ability to sequence millions of DNA fragments in parallel. In TCR repertoire analysis, it is necessary to determine a long base sequence across the V gene, D gene, J gene and C gene that compose TCR. Therefore, among next-generation sequencers, MiSeq (Illumina), which is good at long read analysis (determination of a base sequence of about 300 bp), is often used.
By comprehensively analyzing the nucleotide sequence of the TCR-encoding mRNA contained in the sample, it is possible to easily know the detection frequency of each CDR3 in the T cell population constituting the sample. An example of the base sequence of the primer useful for determining the base sequence of CDR3 is shown below.
Forward primer (adapter sequence common to TCR-α and TCR-β, SEQ ID NO: 174):
5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3'
Reverse primer (for TCR-α, SEQ ID NO: 175):
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3'
Reverse primer (for TCR-β, SEQ ID NO: 176):
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3'
材料および方法
細胞
　食道がん患者に対し、週1回のURLC10由来ペプチドを含むペプチドカクテルワクチン投与を合計5回以上行い、投与を受けたHLA-A*24:02陽性食道がん患者3名（症例1001、1002および1003）の血液から、末梢血単核球（PBMC）を採取した。
　ヒトBリンパ芽球様細胞株であるTISI細胞は、International Histocompatibility Working Groupから購入した。 Materials and methods
Three HLA-A * 24: 02-positive esophageal cancer patients who received a total of five or more doses of peptide cocktail vaccine containing URLC10-derived peptide once a week to patients with cellular esophageal cancer (case 1001, case 1001) Peripheral blood mononuclear cells (PBMC) were collected from the blood of 1002 and 1003).
TISI cells, a human B lymphoblastoid cell line, were purchased from the International Histocompatibility Working Group.
ペプチド
　HLA-A*24:02拘束性URLC10由来ペプチド（RYCNLEGPPI：配列番号：177：WO2006/090810）を使用した。凍結乾燥ペプチドをジメチルスルホキシドで溶解し（最終濃度：20mg/ml）、使用するまで-80℃で保存した。 Peptide HLA-A * 24: 02 A restrictive URLC10-derived peptide (RYCNLEGPPI: SEQ ID NO: 177: WO2006 / 090810) was used. The lyophilized peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg / ml) and stored at -80 ° C until use.
細胞培養（インビトロ刺激）
　PBMC中のペプチド特異的細胞傷害性T細胞（CTL）を増殖させる目的で、PBMCをペプチドおよびサイトカイン（IL-2）で刺激した。AIM-V培地（Invitrogen）およびRPMI1640培地（Invitrogen）を等量混合後、調製量に対して10%の非働化済みウシ胎児血清（Invitrogen）および1%のMEM non-essential amino acids（Invitrogen） を添加し、培地として使用した。URLC10由来ペプチドを含むペプチドカクテルワクチンの投与を受けた食道がん患者（HLA-A*24:02陽性）から採取したPBMCを48ウェルプレート（Corning）へ播き、URLC10由来ペプチドを添加した（ペプチド最終濃度：10μg/ml）。翌日、IL-2（Novartis）を添加した（IL-2最終濃度：120IU/ml）。2～3日おきに培地の半量（上清）を取り除き、IL-2を含む同量の培地を添加し、培地交換を行った（IL-2最終濃度：120IU/ml）。培養開始から7日後に、培地の半量（上清）を除去したのち、ペプチドを含む同量の培地を添加した（ペプチド最終濃度：10μg/ml）。14日目以降、PBMCをテトラマーアッセイおよび限界希釈法に用いた。 Cell culture (in vitro stimulation)
PBMCs were stimulated with peptides and cytokines (IL-2) for the purpose of proliferating peptide-specific cytotoxic T cells (CTLs) in PBMCs. After mixing equal amounts of AIM-V medium (Invitrogen) and RPMI1640 medium (Invitrogen), add 10% deactivated fetal bovine serum (Invitrogen) and 1% MEM non-essential amino acids (Invitrogen) to the prepared amount. It was added and used as a medium. PBMCs collected from esophageal cancer patients (HLA-A * 24: 02 positive) who received a peptide cocktail vaccine containing a URLC10-derived peptide were seeded on a 48-well plate (Corning), and a URLC10-derived peptide was added (final peptide). Concentration: 10 μg / ml). The next day, IL-2 (Novartis) was added (IL-2 final concentration: 120 IU / ml). Half of the medium (supernatant) was removed every 2 to 3 days, the same amount of medium containing IL-2 was added, and the medium was replaced (IL-2 final concentration: 120 IU / ml). Seven days after the start of culturing, half of the medium (supernatant) was removed, and then the same amount of medium containing the peptide was added (final peptide concentration: 10 μg / ml). From day 14 onwards, PBMCs were used in the tetramer assay and limiting dilution method.
テトラマーアッセイ
　培養（インビトロ刺激）後のPBMCからHLA-ペプチド複合体を認識するT細胞を検出するために、テトラマーアッセイを実施した。株式会社医学生物学研究所で合成されたPE標識URLC10テトラマーおよびPE標識HIVテトラマー（陰性コントロール）を使用した。PBMCをテトラマーで染色したのち、さらにFITC標識抗CD8抗体、APC標識抗CD3抗体およびPE-Cy7標識抗CD4抗体（すべてBD Biosciences）で染色した。最後に0.1mg/mlのDAPI（BD Biosciences）溶液で染色し、フローサイトメーター（SH800 cell sorter, Sony）による解析を実施した。DAPI陰性CD3陽性CD4陰性細胞集団におけるテトラマー陽性CD8陽性T細胞を回収し、TCRレパトア解析の検体とした。 Tetramer assay A tetramer assay was performed to detect T cells that recognize the HLA-peptide complex from PBMCs after culture (in vitro stimulation). PE-labeled URLC10 tetramer and PE-labeled HIV tetramer (negative control) synthesized by Medical Biology Laboratory Co., Ltd. were used. PBMC was stained with tetramer and then further stained with FITC-labeled anti-CD8 antibody, APC-labeled anti-CD3 antibody and PE-Cy7-labeled anti-CD4 antibody (all BD Biosciences). Finally, the cells were stained with 0.1 mg / ml DAPI (BD Biosciences) solution and analyzed with a flow cytometer (SH800 cell sorter, Sony). Tetramer-positive CD8-positive T cells in the DAPI-negative CD3-positive and CD4-negative cell population were collected and used as samples for TCR repertoire analysis.
CTLクローンの樹立（限界希釈法）
　96穴丸底マイクロプレート（Corning）において、1個/ウェルとなるように培養（インビトロ刺激）後の細胞を播種した。マイトマイシンCで処理した2種類のヒトBリンパ芽球様細胞株（各1 x 104個）、抗CD3抗体（最終濃度：30ng/ml）およびIL-2（最終濃度：150IU/ml）とともに、細胞を培養した（培養液量150μl/ウェル）。培地には非働化済みAB型血清（MP Biomedicals）を含むAIM-V培地を用いた（5%ABS/AIM-V培地）。10日後、600IU/mｌのIL-2を含む5%ABS/AIM-V培地50μlを該培養物に添加した（Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86；Suda T et al., Cancer Sci 2006, 97(5):411-9；Watanabe T et al., Cancer Sci 2005, 96(8):498-506）。14日目以降、ELISPOTアッセイにおいてURLC10由来ペプチド特異的なIFN-γ産生を示したCTLを後述の方法を利用して増殖させた。 Establishment of CTL clone (limit dilution method)
In a 96-well round-bottomed microplate (Corning), cells after culturing (in vitro stimulation) were seeded so as to be 1 cell / well. With two human B lymphoblastoid cell lines treated with mitomycin C (1 x 10 4 each), anti-CD3 antibody (final concentration: 30 ng / ml) and IL-2 (final concentration: 150 IU / ml) The cells were cultured (culture solution volume 150 μl / well). AIM-V medium containing deactivated AB type serum (MP Biomedicals) was used as the medium (5% ABS / AIM-V medium). Ten days later, 50 μl of 5% ABS / AIM-V medium containing 600 IU / ml IL-2 was added to the culture (Uchida N et al., Clin Cancer Res 2004, 10 (24): 8577-86; Suda. T et al., Cancer Sci 2006, 97 (5): 411-9; Watanabe T et al., Cancer Sci 2005, 96 (8): 498-506). From day 14 onwards, CTLs showing URLC10-derived peptide-specific IFN-γ production in the ELISPOT assay were grown using the method described below.
CTL増殖手順
　Riddellら（Walter EA et al., N Engl J Med 1995, 333(16): 1038-44；Riddell SR et al., Nat Med 1996, 2(2):216-23）によって報告されている方法と類似の方法を利用して、CTLを増殖させた。組織培養用フラスコ（Falcon）において、マイトマイシンCで処理した2種類のヒトBリンパ芽球様細胞株（各5 x 106個）、抗CD3抗体（BD biosciences, 最終濃度：40ng/ml）およびIL-2（最終濃度：144IU/ml）とともにCTLを5%ABS/AIM-V培地中で培養した（培養液量25ml/フラスコ）。2～3日おきに、72IU/mlの IL-2を含む5%ABS/AIM-V培地による培地交換を行った(IL-2最終濃度：36IU/ml)（Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86；Suda T et al., Cancer Sci 2006, 97(5):411-9；Watanabe T et al., Cancer Sci 2005, 96(8):498-506；Tanaka H et al., Br J Cancer 2001, 84(1):94-9；Umano Y et al., Br J Cancer 2001, 84(8):1052-7）。 CTL Proliferation Procedure Reported by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996, 2 (2): 216-23) CTLs were grown using a method similar to that used. Two human B lymphoblastoid cell lines (5 x 10 6 each) treated with mitomycin C, anti-CD3 antibody (BD biosciences, final concentration: 40 ng / ml) and IL in a tissue culture flask (Falcon). CTLs were cultured in 5% ABS / AIM-V medium with -2 (final concentration: 144 IU / ml) (culture solution volume 25 ml / flask). Every 2-3 days, media exchange was performed with 5% ABS / AIM-V medium containing 72 IU / ml IL-2 (IL-2 final concentration: 36 IU / ml) (Uchida N et al., Clin Cancer). Res 2004, 10 (24): 8577-86; Suda T et al., Cancer Sci 2006, 97 (5): 411-9; Watanabe T et al., Cancer Sci 2005, 96 (8): 498-506; Tanaka H et al., Br J Cancer 2001, 84 (1): 94-9; Umano Y et al., Br J Cancer 2001, 84 (8): 1052-7).
IFN-γ産生の確認
　CTLクローンのペプチド特異的IFN-γ産生を確認するために、IFN-γ ELISPOTアッセイおよびIFN-γ ELISAを実施した。ペプチドをパルスしたTISI細胞を標的細胞として調製した。IFN-γ ELISPOTアッセイおよびIFN-γ ELISAは、アッセイキット製造業者の推奨する手順に従って実施した。 Confirmation of IFN-γ production IFN-γ ELISPOT assay and IFN-γ ELISA were performed to confirm peptide-specific IFN-γ production of CTL clones. TISI cells pulsed with the peptide were prepared as target cells. The IFN-γ ELISPOT assay and IFN-γ ELISA were performed according to the procedures recommended by the assay kit manufacturer.
TCR解析
　RNeasy micro kit（QIAGEN）を用いて、テトラマー陽性CD8陽性T細胞からRNAを抽出した。SMARTScribe Reverse Transcriptase (Clontech)によって5’末端にアダプターが付加されたcDNAを合成した。Choudhury ら（Choudhury NJ et al., Eur Urol Focus 2016, 2(4):445-52）によって報告されている方法と類似の方法を利用して、TCR-α鎖およびTCR-β鎖のシーケンスライブラリーを調製した。MiSeq (Illumina)で 300bp ペアエンドシーケンスを実施した。得られたシーケンスリードを、TCRのデータベースであるIMGT/GENE-DB（Giudicelli V et al., Nucleic Acids Res 2005, 33(Database issue):D256-61）に登録されたTCR-α遺伝子およびTCR-β遺伝子の塩基配列へマップした。
　また、ペプチド特異的CTLクローンからRNeasy micro kitを用いてRNAを抽出後、cDNAを合成した。サンガーシーケンス解析によりTCR-α鎖およびTCR-β鎖の塩基配列を解読した。 TCR analysis RNA was extracted from tetramer-positive CD8-positive T cells using the R Easy micro kit (QIAGEN). SMARTScribe Reverse Transcriptase (Clontech) synthesized cDNA with an adapter added to the 5'end. Sequence live of TCR-α chain and TCR-β chain using a method similar to the method reported by Choudhury et al. (Choudhury NJ et al., Eur Urol Focus 2016, 2 (4): 445-52). A rally was prepared. A 300 bp paired end sequence was performed with MiSeq (Illumina). The obtained sequence reads were used for the TCR-α gene and TCR- registered in the TCR database IMGT / GENE-DB (Giudicelli V et al., Nucleic Acids Res 2005, 33 (Database issue): D256-61). Mapped to the base sequence of the β gene.
In addition, RNA was extracted from peptide-specific CTL clones using the R Easy micro kit, and then cDNA was synthesized. The nucleotide sequences of the TCR-α chain and the TCR-β chain were decoded by Sanger sequence analysis.
結果
URLC10テトラマー陽性CD8陽性T細胞の検出と回収
　「材料および方法」に記載したテトラマーアッセイのプロトコールに従って、培養（インビトロ刺激）後のPBMCからURLC10テトラマー陽性CD8陽性T細胞を検出し、回収した（図1）。 result
Detection and Recovery of URLC10 Tetramer-Positive CD8-Positive T Cells URLC10 Tetramer-Positive CD8-Positive T Cells were detected and recovered from PBMCs after culture (in vitro stimulation) according to the Tetramer Assay Protocol described in "Materials and Methods" (Fig. 1). ).
URLC10テトラマー陽性CD8陽性T細胞のTCRレパトア解析
　URLC10テトラマー陽性CD8陽性T細胞の網羅的なTCR解析（TCRレパトア解析）を実施した。TCRクロノタイプの検出頻度をパイチャートで表した（図2）。検出頻度が1%未満のTCRクロノタイプをまとめて同じパターンで示した（図中*）。
　症例1002（図2-a）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：1および配列番号：2）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：3および配列番号：4）をURLC10由来ペプチドを認識するTCRとして同定した。症例1003（図2-b）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：5、配列番号：6、配列番号：7および配列番号：8）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：9、配列番号：10、配列番号：11、配列番号：12および配列番号：13）をURLC10由来ペプチドを認識するTCRとして同定した。 TCR repertoire analysis of URLC10 tetramer-positive CD8-positive T cells Comprehensive TCR analysis (TCR repertoire analysis) of URLC10 tetramer-positive CD8-positive T cells was performed. The TCR chronotype detection frequency is represented by a pie chart (Fig. 2). TCR chronotypes with a detection frequency of less than 1% are shown together in the same pattern (* in the figure).
In TCR-α of case 1002 (Fig. 2-a), the TCR chronotypes (SEQ ID NO: 1 and SEQ ID NO: 2) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides. In TCR-β, the TCR chronotypes (SEQ ID NO: 3 and SEQ ID NO: 4) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides. In TCR-α of case 1003 (Fig. 2-b), the TCR chronotype (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) whose detection frequency was 1% or more was derived from URLC10. It was identified as a TCR that recognizes peptides. In TCR-β, URLC10-derived peptides were recognized for TCR chronotypes (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13) whose detection frequency was 1% or more. Identified as TCR.
URLC10テトラマー陽性CD8陽性T細胞から検出されたTCR（検出頻度1%以上）
　URLC10テトラマー陽性CD8陽性T細胞から1%以上の頻度で検出されたURLC10由来ペプチドを認識するTCRのCDR3アミノ酸配列を表1に示す。 TCR detected in URLC10 tetramer-positive CD8-positive T cells (detection frequency 1% or more)
Table 1 shows the CDR3 amino acid sequence of the TCR that recognizes the URLC10-derived peptide detected at a frequency of 1% or more in the URLC10 tetramer-positive CD8-positive T cells.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
URLC10由来ペプチド特異的CTLクローンの樹立
　限界希釈法によって、URLC10由来ペプチドを認識するCTLクローンを樹立した。ELISAによるIFN-γ測定の結果、CTLクローン#1、#2および#3はペプチド特異的なIFN-γ産生を示した（図3）。このことから、CTLクローン#1、#2および#3がHLA-A*24:02上に提示されたURLC10由来ペプチドを認識したことが確認された。 Establishment of URLC10-derived peptide-specific CTL clones CTL clones that recognize URLC10-derived peptides were established by the limiting dilution method. As a result of IFN-γ measurement by ELISA, CTL clones # 1, # 2 and # 3 showed peptide-specific IFN-γ production (Fig. 3). From this, it was confirmed that CTL clones # 1, # 2 and # 3 recognized the URLC10-derived peptide presented on HLA-A * 24: 02.
URLC10由来ペプチド特異的CTLクローンが発現するTCRの同定
　サンガーシーケンス解析によって、URLC10由来ペプチド特異的CTLクローンが発現するTCRのCDR3アミノ酸配列を同定した。その結果を表2に示す。 Identification of TCRs Expressed by URLC10-Derived Peptide-Specific CTL Clone By Sanger sequence analysis, the CDR3 amino acid sequence of TCR expressed by URLC10-derived peptide-specific CTL clones was identified. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
材料および方法
細胞
　食道がん患者に対し、週1回のURLC10およびDEPDC1由来ペプチドを含むペプチドカクテルワクチン投与を合計5回以上行い、投与を受けたHLA-A*24:02陽性食道がん患者6名（症例1004、1005、1006、1007、1008および1009）の血液から、末梢血単核球（PBMC）を採取した。
　ヒトBリンパ芽球様細胞株であるTISI細胞は、International Histocompatibility Working Groupから購入した。 Materials and methods
Six HLA-A * 24: 02-positive esophageal cancer patients who received a total of five or more doses of peptide cocktail vaccine containing URLC10 and DEPDC1-derived peptides once a week to patients with cellular esophageal cancer (cases) Peripheral blood mononuclear cells (PBMC) were collected from the blood of 1004, 1005, 1006, 1007, 1008 and 1009).
TISI cells, a human B lymphoblastoid cell line, were purchased from the International Histocompatibility Working Group.
ペプチド
　HLA-A*24:02拘束性URLC10由来ペプチド（RYCNLEGPPI／配列番号：177：WO2006/090810）およびHLA-A*24:02拘束性DEPDC1由来ペプチド（EYYELFVNI／配列番号：178：WO2008/047473）を使用した。凍結乾燥ペプチドをジメチルスルホキシドで溶解し（最終濃度：20mg/ml）、使用するまで-80℃で保存した。 Peptide HLA-A * 24: 02 Restrictive URLC10-derived peptide (RYCNLEGPPI / SEQ ID NO: 177: WO2006 / 090810) and HLA-A * 24:02 Restrictive DEPDC1-derived peptide (EYYELFVNI / SEQ ID NO: 178: WO2008 / 047473) It was used. The lyophilized peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg / ml) and stored at -80 ° C until use.
細胞培養（インビトロ刺激）
　PBMC中のペプチド特異的細胞傷害性T細胞（CTL）を増殖させる目的で、PBMCをペプチドおよびサイトカイン（IL-2）で刺激した。AIM-V培地（Invitrogen）およびRPMI1640培地（Invitrogen）を等量混合後、調製量に対して10%の非働化済みウシ胎児血清（Invitrogen）および1%のMEM non-essential amino acids（Invitrogen） を添加し、培地として使用した。URLC10由来ペプチドおよびDEPDC1由来ペプチドを含むペプチドカクテルワクチンの投与を受けた食道がん患者（HLA-A*24:02陽性）から採取したPBMCを48ウェルプレート（Corning）へ播き、URLC10由来ペプチドまたはDEPDC1由来ペプチドを添加した（ペプチド最終濃度：10μg/ml）。翌日、IL-2（Novartis）を添加した（IL-2最終濃度：120IU/ml）。2～3日おきに培地の半量（上清）を取り除き、IL-2を含む同量の培地を添加し、培地交換を行った（IL-2最終濃度：120IU/ml）。培養開始から7日後に、培地の半量（上清）を除去したのち、ペプチドを含む同量の培地を添加した（ペプチド最終濃度：10μg/ml）。14日目以降、PBMCをテトラマーアッセイおよび限界希釈法に用いた。 Cell culture (in vitro stimulation)
PBMCs were stimulated with peptides and cytokines (IL-2) for the purpose of proliferating peptide-specific cytotoxic T cells (CTLs) in PBMCs. After mixing equal amounts of AIM-V medium (Invitrogen) and RPMI1640 medium (Invitrogen), add 10% deactivated fetal bovine serum (Invitrogen) and 1% MEM non-essential amino acids (Invitrogen) to the prepared amount. It was added and used as a medium. PBMCs collected from esophageal cancer patients (HLA-A * 24: 02 positive) who received a peptide cocktail vaccine containing a URLC10-derived peptide and a DEPDC1-derived peptide were seeded on a 48-well plate (Corning), and the URLC10-derived peptide or DEPDC1 was seeded. The derived peptide was added (final peptide concentration: 10 μg / ml). The next day, IL-2 (Novartis) was added (IL-2 final concentration: 120 IU / ml). Half of the medium (supernatant) was removed every 2 to 3 days, the same amount of medium containing IL-2 was added, and the medium was replaced (IL-2 final concentration: 120 IU / ml). Seven days after the start of culturing, half of the medium (supernatant) was removed, and then the same amount of medium containing the peptide was added (final peptide concentration: 10 μg / ml). From day 14 onwards, PBMCs were used in the tetramer assay and limiting dilution method.
テトラマーアッセイ
　培養（インビトロ刺激）後のPBMCからHLA-ペプチド複合体を認識するT細胞を検出するために、テトラマーアッセイを実施した。株式会社医学生物学研究所で合成されたPE標識URLC10テトラマー、PE標識DEPDC1テトラマーおよびPE標識HIVテトラマー（陰性コントロール）を使用した。PBMCをテトラマーで染色したのち、さらにFITC標識抗CD8抗体、APC標識抗CD3抗体およびPE-Cy7標識抗CD4抗体（すべてBD Biosciences）で染色した。最後に0.1mg/mlのDAPI（BD Biosciences）溶液で染色し、フローサイトメーター（SH800 cell sorter, Sony）による解析を実施した。DAPI陰性CD3陽性CD4陰性細胞集団におけるテトラマー陽性CD8陽性T細胞を回収し、TCRレパトア解析の検体とした。 Tetramer assay A tetramer assay was performed to detect T cells that recognize the HLA-peptide complex from PBMCs after culture (in vitro stimulation). PE-labeled URLC10 tetramer, PE-labeled DEPDC1 tetramer and PE-labeled HIV tetramer (negative control) synthesized by Medical Biology Laboratory Co., Ltd. were used. PBMC was stained with tetramer and then further stained with FITC-labeled anti-CD8 antibody, APC-labeled anti-CD3 antibody and PE-Cy7-labeled anti-CD4 antibody (all BD Biosciences). Finally, the cells were stained with 0.1 mg / ml DAPI (BD Biosciences) solution and analyzed with a flow cytometer (SH800 cell sorter, Sony). Tetramer-positive CD8-positive T cells in the DAPI-negative CD3-positive and CD4-negative cell population were collected and used as samples for TCR repertoire analysis.
CTLクローンの樹立（限界希釈法）
　96穴丸底マイクロプレート（Corning）において、1個/ウェルとなるように培養（インビトロ刺激）後の細胞を播種した。マイトマイシンCで処理した2種類のヒトBリンパ芽球様細胞株（各1 x 104個）、抗CD3抗体（最終濃度：30ng/ml）およびIL-2（最終濃度：150IU/ml）とともに、細胞を培養した（培養液量150μl/ウェル）。培地には非働化済みAB型血清（MP Biomedicals）を含むAIM-V培地を用いた（5%ABS/AIM-V培地）。10日後、600IU/mｌのIL-2を含む5%ABS/AIM-V培地50μlを該培養物に添加した（Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86；Suda T et al., Cancer Sci 2006, 97(5):411-9；Watanabe T et al., Cancer Sci 2005, 96(8):498-506）。14日目以降、ELISPOTアッセイにおいてペプチド特異的なIFN-γ産生を示したCTLを後述の方法を利用して増殖させた。 Establishment of CTL clone (limit dilution method)
In a 96-well round-bottomed microplate (Corning), cells after culturing (in vitro stimulation) were seeded so as to be 1 cell / well. With two human B lymphoblastoid cell lines treated with mitomycin C (1 x 10 4 each), anti-CD3 antibody (final concentration: 30 ng / ml) and IL-2 (final concentration: 150 IU / ml) The cells were cultured (culture solution volume 150 μl / well). AIM-V medium containing deactivated AB type serum (MP Biomedicals) was used as the medium (5% ABS / AIM-V medium). Ten days later, 50 μl of 5% ABS / AIM-V medium containing 600 IU / ml IL-2 was added to the culture (Uchida N et al., Clin Cancer Res 2004, 10 (24): 8577-86; Suda. T et al., Cancer Sci 2006, 97 (5): 411-9; Watanabe T et al., Cancer Sci 2005, 96 (8): 498-506). From day 14 onwards, CTLs showing peptide-specific IFN-γ production in the ELISPOT assay were grown using the method described below.
CTL増殖手順
　Riddellら（Walter EA et al., N Engl J Med 1995, 333(16): 1038-44；Riddell SR et al., Nat Med 1996, 2(2):216-23）によって報告されている方法と類似の方法を利用して、CTLを増殖させた。組織培養用フラスコ（Falcon）において、マイトマイシンCで処理した2種類のヒトBリンパ芽球様細胞株（各5 x 106個）、抗CD3抗体（BD biosciences, 最終濃度：40ng/ml）およびIL-2（最終濃度：144IU/ml）とともにCTLを5%ABS/AIM-V培地中で培養した（培養液量25ml/フラスコ）。2～3日おきに、72IU/mlの IL-2を含む5%ABS/AIM-V培地による培地交換を行った(IL-2最終濃度：36IU/ml)（Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86；Suda T et al., Cancer Sci 2006, 97(5):411-9；Watanabe T et al., Cancer Sci 2005, 96(8):498-506；Tanaka H et al., Br J Cancer 2001, 84(1):94-9；Umano Y et al., Br J Cancer 2001, 84(8):1052-7）。 CTL Proliferation Procedure Reported by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996, 2 (2): 216-23) CTLs were grown using a method similar to that used. Two human B lymphoblastoid cell lines (5 x 10 6 each) treated with mitomycin C, anti-CD3 antibody (BD biosciences, final concentration: 40 ng / ml) and IL in a tissue culture flask (Falcon). CTLs were cultured in 5% ABS / AIM-V medium with -2 (final concentration: 144 IU / ml) (culture solution volume 25 ml / flask). Every 2-3 days, media exchange was performed with 5% ABS / AIM-V medium containing 72 IU / ml IL-2 (IL-2 final concentration: 36 IU / ml) (Uchida N et al., Clin Cancer). Res 2004, 10 (24): 8577-86; Suda T et al., Cancer Sci 2006, 97 (5): 411-9; Watanabe T et al., Cancer Sci 2005, 96 (8): 498-506; Tanaka H et al., Br J Cancer 2001, 84 (1): 94-9; Umano Y et al., Br J Cancer 2001, 84 (8): 1052-7).
IFN-γ産生の確認
　CTLクローンのペプチド特異的IFN-γ産生を確認するために、IFN-γ ELISPOTアッセイおよびIFN-γ ELISAを実施した。ペプチドをパルスしたTISI細胞を標的細胞として調製した。IFN-γ ELISPOTアッセイおよびIFN-γ ELISAは、アッセイキット製造業者の推奨する手順に従って実施した。 Confirmation of IFN-γ production IFN-γ ELISPOT assay and IFN-γ ELISA were performed to confirm peptide-specific IFN-γ production of CTL clones. TISI cells pulsed with the peptide were prepared as target cells. The IFN-γ ELISPOT assay and IFN-γ ELISA were performed according to the procedures recommended by the assay kit manufacturer.
TCR解析
　RNeasy micro kit（QIAGEN）を用いて、テトラマー陽性CD8陽性T細胞からRNAを抽出した。SMARTScribe Reverse Transcriptase (Clontech)によって5’末端にアダプターが付加されたcDNAを合成した。Choudhury ら（Choudhury NJ et al., Eur Urol Focus 2016, 2(4):445-52）によって報告されている方法と類似の方法を利用して、TCR-α鎖およびTCR-β鎖のシーケンスライブラリーを調製した。MiSeq (Illumina)で 300bp ペアエンドシーケンスを実施した。得られたシーケンスリードを、TCRのデータベースであるIMGT/GENE-DB（Giudicelli V et al., Nucleic Acids Res 2005, 33(Database issue):D256-61）に登録されたTCR-α遺伝子およびTCR-β遺伝子の塩基配列へマップした。
　また、ペプチド特異的CTLクローンからRNeasy micro kitを用いてRNAを抽出後、cDNAを合成した。サンガーシーケンス解析によりTCR-α鎖およびTCR-β鎖の塩基配列を解読した。 TCR analysis RNA was extracted from tetramer-positive CD8-positive T cells using the R Easy micro kit (QIAGEN). SMARTScribe Reverse Transcriptase (Clontech) synthesized cDNA with an adapter added to the 5'end. Sequence live of TCR-α chain and TCR-β chain using a method similar to the method reported by Choudhury et al. (Choudhury NJ et al., Eur Urol Focus 2016, 2 (4): 445-52). A rally was prepared. A 300 bp paired end sequence was performed with MiSeq (Illumina). The obtained sequence reads were used for the TCR-α gene and TCR- registered in the TCR database IMGT / GENE-DB (Giudicelli V et al., Nucleic Acids Res 2005, 33 (Database issue): D256-61). Mapped to the base sequence of the β gene.
In addition, RNA was extracted from peptide-specific CTL clones using the R Easy micro kit, and then cDNA was synthesized. The nucleotide sequences of the TCR-α chain and the TCR-β chain were decoded by Sanger sequence analysis.
結果
テトラマー陽性CD8陽性T細胞の検出と回収
　「材料および方法」に記載したテトラマーアッセイのプロトコールに従って、培養（インビトロ刺激）後のPBMCからURLC10テトラマー陽性CD8陽性T細胞を検出し、回収した（図4A, a-f）。また、DEPDC1テトラマー陽性CD8陽性T細胞を検出し、回収した（図4B）。 result
Detection and Recovery of Tetramer-Positive CD8-Positive T Cells URLC10 Tetramer-Positive CD8-Positive T Cells were detected and recovered from PBMCs after culture (in vitro stimulation) according to the Tetramer Assay Protocol described in “Materials and Methods” (Fig. 4A, FIG. af). In addition, DEPDC1 tetramer-positive CD8-positive T cells were detected and recovered (Fig. 4B).
テトラマー陽性CD8陽性T細胞のTCRレパトア解析
　テトラマー陽性CD8陽性T細胞の網羅的なTCR解析（TCRレパトア解析）を実施した。TCRクロノタイプの検出頻度をパイチャートで表した（図5Aおよび図5B）。検出頻度が1%未満のTCRクロノタイプをまとめて同じパターンで示した（図中*）。
　症例1004（図5A-a）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：17、配列番号：18、配列番号：19、配列番号：20、配列番号：21、配列番号：22、配列番号：23、配列番号：24および配列番号：25）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：26、配列番号：27、配列番号：28、配列番号：29、配列番号：30、配列番号：31および配列番号：32）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1005（図5A-b）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：33、配列番号：34、配列番号：35、配列番号：36、配列番号：37、配列番号：38、配列番号：39および配列番号：40）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：41、配列番号：42、配列番号：43、配列番号：44、配列番号：45、配列番号：46、配列番号：47および配列番号：48）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1006（図5A-c）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：49、配列番号：50、配列番号：51、配列番号：52、配列番号：53および配列番号：54）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：55、配列番号：56、配列番号：57、配列番号：58および配列番号：59）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1007（図5A-d, High and Low population）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：60、配列番号：61および配列番号：62）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：63および配列番号：64）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1007（図5A-e, High population）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：65、配列番号：66、配列番号：67および配列番号：68）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：64）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1008（図5A-f）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：69および配列番号：70）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：71、配列番号：72、配列番号：41、配列番号：73、配列番号：74、配列番号：75および配列番号：76）をURLC10由来ペプチドを認識するTCRとして同定した。
　症例1009（図5A-g）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：77、配列番号：78および配列番号：79）をURLC10由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：80、配列番号：81、配列番号：82および配列番号：83）をURLC10由来ペプチドを認識するTCRとして同定した。
　また、症例1007（図5B）のTCR-αにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：84および配列番号：85）をDEPDC1由来ペプチドを認識するTCRとして同定した。またTCR-βにおいて、検出頻度が1%以上であったTCRクロノタイプ（配列番号：86）をDEPDC1由来ペプチドを認識するTCRとして同定した。 TCR repertoire analysis of tetramer-positive CD8-positive T cells Comprehensive TCR analysis (TCR repertoire analysis) of tetramer-positive CD8-positive T cells was performed. The frequency of detection of TCR chronotypes is represented by a pie chart (Fig. 5A and Fig. 5B). TCR chronotypes with a detection frequency of less than 1% are shown together in the same pattern (* in the figure).
In TCR-α of case 1004 (Fig. 5A-a), the detection frequency was 1% or more. TCR chronotype (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25) were identified as TCRs that recognize URLC10-derived peptides. In TCR-β, the detection frequency was 1% or more for the TCR chronotypes (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and sequence. Number: 32) was identified as a TCR that recognizes URLC10-derived peptides.
In TCR-α of case 1005 (Fig. 5A-b), the detection frequency was 1% or more. TCR chronotype (SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40) were identified as TCRs that recognize URLC10-derived peptides. In TCR-β, the detection frequency was 1% or more for the TCR chronotype (SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: Number: 47 and SEQ ID NO: 48) were identified as TCRs that recognize URLC10-derived peptides.
In TCR-α of case 1006 (Fig. 5A-c), the detection frequency was 1% or more. TCR chronotype (SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:: 53 and SEQ ID NO: 54) were identified as TCRs that recognize URLC10-derived peptides. In TCR-β, URLC10-derived peptides were recognized for TCR chronotypes (SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59) whose detection frequency was 1% or more. Identified as TCR.
In TCR-α of case 1007 (Fig. 5A-d, High and Low population), the TCR chronotype (SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62) whose detection frequency was 1% or more was derived from URLC10. It was identified as a TCR that recognizes peptides. In TCR-β, the TCR chronotypes (SEQ ID NO: 63 and SEQ ID NO: 64) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides.
TCR chronotypes (SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68) whose detection frequency was 1% or more in TCR-α of case 1007 (Fig. 5A-e, High population). Was identified as a TCR that recognizes URLC10-derived peptides. In TCR-β, the TCR chronotype (SEQ ID NO: 64) whose detection frequency was 1% or more was identified as a TCR that recognizes URLC10-derived peptides.
In TCR-α of case 1008 (Fig. 5A-f), TCR chronotypes (SEQ ID NO: 69 and SEQ ID NO: 70) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides. In TCR-β, the detection frequency was 1% or more for the TCR chronotypes (SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 41, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and sequence. Number: 76) was identified as a TCR that recognizes URLC10-derived peptides.
In TCR-α of case 1009 (Fig. 5A-g), TCR chronotypes (SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79) whose detection frequency was 1% or more are recognized as URLC10-derived peptides. Identified as. In TCR-β, the TCR chronotypes (SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83) whose detection frequency was 1% or more were identified as TCRs that recognize URLC10-derived peptides. ..
In TCR-α of case 1007 (Fig. 5B), the TCR chronotypes (SEQ ID NO: 84 and SEQ ID NO: 85) whose detection frequency was 1% or more were identified as TCRs that recognize DEPDC1-derived peptides. In TCR-β, the TCR chronotype (SEQ ID NO: 86) whose detection frequency was 1% or more was identified as a TCR that recognizes DEPDC1-derived peptides.
テトラマー陽性CD8陽性T細胞から検出されたTCR（検出頻度1%以上）
　URLC10テトラマー陽性CD8陽性T細胞から1%以上の頻度で検出されたURLC10由来ペプチドを認識するTCRのCDR3アミノ酸配列を表3Aに示す。
　また、DEPDC1テトラマー陽性CD8陽性T細胞から1%以上の頻度で検出されたDEPDC1由来ペプチドを認識するTCRのCDR3アミノ酸配列を表3Bに示す。 TCR detected in tetramer-positive CD8-positive T cells (detection frequency 1% or more)
Table 3A shows the CDR3 amino acid sequence of the TCR that recognizes the URLC10-derived peptide detected at a frequency of 1% or more in the URLC10 tetramer-positive CD8-positive T cells.
Table 3B shows the CDR3 amino acid sequence of TCR that recognizes DEPDC1-derived peptides detected at a frequency of 1% or more in DEPDC1 tetramer-positive CD8-positive T cells.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000005
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
ペプチド特異的CTLクローンの樹立
　限界希釈法によって、URLC10由来ペプチドを認識するCTLクローンを樹立した。ELISAによるIFN-γ測定の結果、CTLクローン#1、#2、#3および#4はペプチド特異的なIFN-γ産生を示した（図6A, a-d）。このことから、CTLクローン#1、#2、#3および#4がHLA-A*24:02上に提示されたURLC10由来ペプチドを認識したことが確認された。
　また、DEPDC1由来ペプチドを認識するCTLクローンを樹立した。ELISAによるIFN-γ測定の結果、CTLクローン#5はペプチド特異的なIFN-γ産生を示した（図6B）。このことから、CTLクローン#5がHLA-A*24:02上に提示されたDEPDC1由来ペプチドを認識したことが確認された。 Establishment of peptide-specific CTL clones CTL clones that recognize URLC10-derived peptides were established by the limiting dilution method. As a result of IFN-γ measurement by ELISA, CTL clones # 1, # 2, # 3 and # 4 showed peptide-specific IFN-γ production (Fig. 6A, ad). From this, it was confirmed that CTL clones # 1, # 2, # 3 and # 4 recognized the URLC10-derived peptide presented on HLA-A * 24: 02.
We also established a CTL clone that recognizes DEPDC1-derived peptides. As a result of IFN-γ measurement by ELISA, CTL clone # 5 showed peptide-specific IFN-γ production (Fig. 6B). From this, it was confirmed that CTL clone # 5 recognized the DEPDC1-derived peptide presented on HLA-A * 24: 02.
ペプチド特異的CTLクローンが発現するTCRの同定
　サンガーシーケンス解析によって、URLC10由来ペプチド特異的CTLクローンが発現するTCRのCDR3アミノ酸配列を同定した（表4A）。
　また、DEPDC1由来ペプチド特異的CTLクローンが発現するTCRのCDR3アミノ酸配列を同定した（表4B）。 Identification of TCRs Expressed by Peptide-Specific CTL Clone Sanger sequence analysis identified the CDR3 amino acid sequence of TCRs expressed by URLC10-derived peptide-specific CTL clones (Table 4A).
We also identified the CDR3 amino acid sequence of the TCR expressed by the DEPDC1-derived peptide-specific CTL clone (Table 4B).
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
材料および方法
TCR遺伝子導入T細胞の作製
　URLC10由来ペプチドを認識するT細胞受容体α鎖（ＣＤＲ３のアミノ酸配列：配列番号１７）とT細胞受容体β鎖（ＣＤＲ３のアミノ酸配列：配列番号２６）の塩基配列についてコドン最適化を行った上、それらをpMP71-PREレトロウイルスベクターに組み込んだ（Leisegang M et al., Clin Cancer Res 2010, 16(8):2333-43）。α鎖とβ鎖のＶ領域（ＣＤＲ１とＣＤＲ２を含む）とＪ領域はそれぞれ次のとおりである。
　　　　　　Ｖ領域　　　　　　　　　　　　Ｊ領域
　α鎖　ＴＲＡＶ２６－１　－ＣＤＲ３－　ＴＲＡＪ２３（配列番号180）
　β鎖　ＴＲＢＶ１０－２　－ＣＤＲ３－　ＴＲＢＪ１－５（配列番号181）
　T細胞受容体の発現量を最大化する目的で、α鎖とβ鎖のC遺伝子の領域（TRACおよびTRBC1）の塩基配列をそれぞれマウス由来の塩基配列に改変した（Cohen CJ et al., Cancer Res 2006, 66(17):8878-86）。一過性の組み換えレトロウイルス液を用いて、ＨＬＡ－Ａ２４陽性のヒトから採取されたPBMCへT細胞受容体（TCR）遺伝子を導入した。
　TCR遺伝子導入T細胞をテトラマーアッセイによって評価した。PE標識URLC10テトラマー（株式会社医学生物学研究所）およびAlexa Fluor 700標識抗マウスTCRβ抗体（eBioscience）によって検出されたCD8陽性T細胞をTCR遺伝子導入T細胞とした。
　TCR遺伝子導入T細胞によるURLC10由来ペプチド特異的なIFN-γ産生およびGranzyme B産生を ELISAで確認した。ペプチドをパルスしたTISI細胞（HLA-A*24:02陽性）を標的細胞として調製した。
　さらにTCR遺伝子導入T細胞の腫瘍細胞株に対する反応をELISAで検証した。食道がん細胞株TE-1（URLC10陽性, HLA-A*24:02陽性）およびTE-9（URLC10陽性, HLA-A*24:02陰性）を標的細胞とした。食道がん細胞株は東北大学加齢医学研究所附属医用細胞資源センターより購入した。 Materials and methods
Preparation of TCR gene-introduced T cells Nucleotide sequence of T cell receptor α chain (Amino acid sequence of CDR3: SEQ ID NO: 17) and T cell receptor β chain (amino acid sequence of CDR3: SEQ ID NO: 26) that recognize URLC10-derived peptides After performing codon optimization, they were incorporated into the pMP71-PRE retroviral vector (Leisegang M et al., Clin Cancer Res 2010, 16 (8): 2333-43). The V region (including CDR1 and CDR2) and J region of the α chain and β chain are as follows.
V region J region α chain TRAV26-1 -CDR3-TRAJ23 (SEQ ID NO: 180)
β-chain TRBV10-2-CDR3-TRBJ1-5 (SEQ ID NO: 181)
In order to maximize the expression level of the T cell receptor, the nucleotide sequences of the C gene regions (TRAC and TRBC1) of the α chain and β chain were modified to the nucleotide sequences derived from mice, respectively (Cohen CJ et al., Cancer). Res 2006, 66 (17): 8878-86). A transient recombinant retroviral solution was used to introduce the T cell receptor (TCR) gene into PBMCs collected from HLA-A24 positive humans.
TCR transgenic T cells were evaluated by tetramer assay. CD8-positive T cells detected by PE-labeled URLC10 tetramer (Institute of Medical Biology Co., Ltd.) and Alexa Fluor 700-labeled anti-mouse TCRβ antibody (eBioscience) were designated as TCR-transfected T cells.
URLC10-derived peptide-specific IFN-γ production and Granzyme B production by TCR-transfected T cells were confirmed by ELISA. TISI cells pulsed with peptides (HLA-A * 24: 02 positive) were prepared as target cells.
Furthermore, the response of TCR transgenic T cells to tumor cell lines was verified by ELISA. Esophageal cancer cell lines TE-1 (URLC10 positive, HLA-A * 24: 02 positive) and TE-9 (URLC10 positive, HLA-A * 24: 02 negative) were targeted cells. The esophageal cancer cell line was purchased from the Medical Cell Resources Center of the Institute of Aging Medicine, Tohoku University.
TCR遺伝子導入T細胞の機能解析
　TCR遺伝子導入T細胞の機能を解析するために、IFN-γ ELISAおよびGranzyme B ELISAを実施した。ペプチドをパルスしたTISI細胞を標的細胞として調製した。ELISAは、アッセイキット製造業者の推奨する手順に従って実施した。 Functional analysis of TCR-transfected T cells IFN-γ ELISA and Granzyme B ELISA were performed to analyze the function of TCR-transfected T cells. TISI cells pulsed with the peptide were prepared as target cells. ELISA was performed according to the procedure recommended by the assay kit manufacturer.
結果
TCR遺伝子導入T細胞の検出
　レトロウイルスベクターによってURLC10由来ペプチド反応性TCR遺伝子を導入した結果、PBMCからURLC10テトラマー陽性T細胞集団が検出された（図7b）。 result
Detection of TCR-introduced T cells As a result of introducing a URLC10-derived peptide-reactive TCR gene by a retroviral vector, a URLC10 tetramer-positive T cell population was detected in PBMC (Fig. 7b).
TCR遺伝子導入T細胞の抗原特異性および機能の確認
　ELISAの結果、TCR遺伝子導入T細胞はURLC10由来ペプチド特異的なIFN-γ産生（図8a）および細胞傷害顆粒（Granzyme B）産生を示した（図8b）。このことから、TCR遺伝子導入T細胞がHLA-A*24:02上に提示されたURLC10由来ペプチドを認識し、さらにはIFN-γおよびGranzyme Bを産生する機能を有することが確認された。 Confirmation of antigen specificity and function of TCR-introduced T cells As a result of ELISA, TCR-introduced T cells showed URLC10-derived peptide-specific IFN-γ production (Fig. 8a) and cytotoxic granule (Granzyme B) production (Fig. 8a). Figure 8b). From this, it was confirmed that the TCR transgenic T cells have a function of recognizing the URLC10-derived peptide presented on HLA-A * 24: 02 and further producing IFN-γ and Granzyme B.
TCR遺伝子導入T細胞の食道がん細胞株に対する反応
　TCR遺伝子導入T細胞はTE-1（URLC10陽性, HLA-A*24:02陽性）と共培養した場合にIFN-γの産生を示した（図9）。TE-9（URLC10陽性, HLA-A*24:02陰性）と共培養した場合に有意なIFN-γの産生は認められなかった。したがってTCR遺伝子導入T細胞がHLA-A*24:02上にURLC10由来ペプチドを提示するがん細胞を認識しうることが示された。 Response of TCR-transfected T cells to esophageal cancer cell lines TCR-transfected T cells showed IFN-γ production when co-cultured with TE-1 (URLC10 positive, HLA-A * 24: 02 positive) ( Figure 9). No significant IFN-γ production was observed when co-cultured with TE-9 (URLC10 positive, HLA-A * 24: 02 negative). Therefore, it was shown that TCR transgenic T cells can recognize cancer cells that present URLC10-derived peptides on HLA-A * 24: 02.
　本発明によって、T細胞にURLC10あるいはDEPDC1に特異的なＨＬＡ－Ａ２４拘束性の細胞傷害作用を付与するTCRが見出された。本発明のTCRによって、URLC10あるいはDEPDC1に特異的なCTLを得ることができ、TCR遺伝子導入T細胞療法を可能とする。
　あるいは、本発明のTCRは、CTL誘導性のペプチドを投与した対象における治療効果のモニタリング指標としても有用である。本発明のTCRを備えたCTLは、実際に標的細胞に対する細胞傷害活性を備えた細胞である。したがって、TCRレパトア解析などを通じて、本発明のTCRを備えたCTLの割合が高まれば、対象において目的とするCTL応答が誘導されていることを意味し、ペプチドによる治療効果を達成できている可能性を知ることができる。 INDUSTRIAL APPLICABILITY According to the present invention, a TCR that imparts HLA-A24-restricting cytotoxic effects specific to URLC10 or DEPDC1 to T cells has been found. With the TCR of the present invention, a CTL specific for URLC10 or DEPDC1 can be obtained, enabling TCR gene transfer T cell therapy.
Alternatively, the TCR of the present invention is also useful as a monitoring index of therapeutic effect in a subject to which a CTL-induced peptide has been administered. The CTL equipped with the TCR of the present invention is actually a cell having cytotoxic activity against a target cell. Therefore, if the proportion of CTLs equipped with the TCR of the present invention increases through TCR repertoire analysis, it means that the desired CTL response is induced in the subject, and it is possible that the therapeutic effect of the peptide can be achieved. Can be known.

Claims (15)

  1. 　配列番号：1-2、5-8、14、16、17-25、33-40、49-54、60-62、65-70、77-79および84-85からなる群から選択されるいずれかのアミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含むT細胞受容体α鎖。 SEQ ID NO: Any selected from the group consisting of 1-2, 5-8, 14, 16, 17-25, 33-40, 49-54, 60-62, 65-70, 77-79 and 84-85. A T cell receptor α chain containing CDR3 identified by the amino acid sequence, or CDR3 functionally equivalent thereto.
  2. 　CASS-[X]n-F（Xは任意のアミノ酸でnが6-8／配列番号：179）を含む11-13アミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含むT細胞受容体β鎖。 T cell receptor containing CDR3 identified by the 11-13 amino acid sequence containing CASS- [X] nF (X is an arbitrary amino acid with n 6-8 / SEQ ID NO: 179), or a CDR3 functionally equivalent thereto Body β chain.
  3. 　配列番号：3-4、9-13、15、26-32、41-48、55-59、63-64、71-76、80-83および86からなる群から選択されるいずれかのアミノ酸配列で特定されるCDR3、またはそれと機能的に等価なCDR3を含むT細胞受容体β鎖。 SEQ ID NO:: Any amino acid sequence selected from the group consisting of 3-4, 9-13, 15, 26-32, 41-48, 55-59, 63-64, 71-76, 80-83 and 86. A T cell receptor β chain containing CDR3 identified in, or a CDR3 functionally equivalent thereto.
  4. 　請求項1に記載のいずれかのT細胞受容体α鎖と、請求項2または3に記載のいずれかのT細胞受容体β鎖の組み合わせからなるT細胞受容体。 A T cell receptor comprising a combination of any T cell receptor α chain according to claim 1 and any T cell receptor β chain according to claim 2 or 3.
  5. 　T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列が、以下のいずれかの組み合わせである請求項4に記載のT細胞受容体：
    T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
    配列番号：1　　　　　　　　　　　　　　配列番号：3、
    配列番号：2　　　　　　　　　　　　　　配列番号：3、
    配列番号：14　　　　　　　　　　　　　 配列番号：15、
    配列番号：16　　　　　　　　　　　　　 配列番号：10、
    配列番号：17　　　　　　　　　　　　　 配列番号：26、
    配列番号：60　　　　　　　　　　　　　 配列番号：63、
    配列番号：69　　　　　　　　　　　　　 配列番号：71、
    配列番号：77　　　　　　　　　　　　　 配列番号：80、および
    配列番号：84　　　　　　　　　　　　　 配列番号：86。     The T cell receptor according to claim 4, wherein the amino acid sequence of CDR3 of the T cell receptor α chain and the T cell receptor β chain is any combination of the following:
    CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
    SEQ ID NO: 1 SEQ ID NO: 3,
    SEQ ID NO: 2 SEQ ID NO: 3,
    SEQ ID NO: 14 SEQ ID NO: 15,
    SEQ ID NO: 16 SEQ ID NO: 10,
    SEQ ID NO: 17 SEQ ID NO: 26,
    SEQ ID NO: 60 SEQ ID NO: 63,
    SEQ ID NO: 69 SEQ ID NO: 71,
    SEQ ID NO: 77 SEQ ID NO: 80, and SEQ ID NO: 84 SEQ ID NO: 86.
  6. 　請求項1に記載のいずれかのT細胞受容体α鎖および、請求項3に記載のいずれかのT細胞受容体β鎖のいずれかをコードするポリヌクレオチド。 A polynucleotide encoding any of the T cell receptor α chain according to claim 1 and any T cell receptor β chain according to claim 3.
  7. 　請求項6に記載のポリヌクレオチドを含む発現ベクター。 An expression vector containing the polynucleotide according to claim 6.
  8. 　T細胞受容体α鎖をコードするポリヌクレオチドと、T細胞受容体β鎖をコードするポリヌクレオチドの両方を含む請求項7に記載の発現ベクター。 The expression vector according to claim 7, which contains both a polynucleotide encoding a T cell receptor α chain and a polynucleotide encoding a T cell receptor β chain.
  9. 　T細胞受容体α鎖とT細胞受容体β鎖のCDR3のアミノ酸配列が、以下のいずれかの組み合わせである請求項8に記載の発現ベクター：
    T細胞受容体α鎖のCDR3　　　　　　　　 T細胞受容体β鎖のCDR3
    配列番号：1　　　　　　　　　　　　　　配列番号：3、
    配列番号：2　　　　　　　　　　　　　　配列番号：3、
    配列番号：14　　　　　　　　　　　　　 配列番号：15、
    配列番号：16　　　　　　　　　　　　　 配列番号：10、
    配列番号：17　　　　　　　　　　　　　 配列番号：26、
    配列番号：60　　　　　　　　　　　　　 配列番号：63、
    配列番号：69　　　　　　　　　　　　　 配列番号：71、
    配列番号：77　　　　　　　　　　　　　 配列番号：80、および
    配列番号：84　　　　　　　　　　　　　 配列番号：86。     The expression vector according to claim 8, wherein the amino acid sequences of CDR3 of the T cell receptor α chain and the T cell receptor β chain are any combination of the following:
    CDR3 of T cell receptor α chain CDR3 of T cell receptor β chain
    SEQ ID NO: 1 SEQ ID NO: 3,
    SEQ ID NO: 2 SEQ ID NO: 3,
    SEQ ID NO: 14 SEQ ID NO: 15,
    SEQ ID NO: 16 SEQ ID NO: 10,
    SEQ ID NO: 17 SEQ ID NO: 26,
    SEQ ID NO: 60 SEQ ID NO: 63,
    SEQ ID NO: 69 SEQ ID NO: 71,
    SEQ ID NO: 77 SEQ ID NO: 80, and SEQ ID NO: 84 SEQ ID NO: 86.
  10. 　請求項8または9に記載の発現ベクターからなる、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞を誘導するための発現ベクター。 An expression vector for inducing cytotoxic T cells having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells, which comprises the expression vector according to claim 8 or 9.
  11. 　請求項8～10いずれかに記載の発現ベクターを含む、細胞傷害性T細胞の誘導用組成物。 A composition for inducing cytotoxic T cells, which comprises the expression vector according to any one of claims 8 to 10.
  12. 　がんの治療を必要とする対象から採取されたCD8+T細胞に請求項8～10いずれかに記載の発現ベクターを導入し、URLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞を回収する工程を含む、細胞傷害性T細胞の製造方法。 The expression vector according to any one of claims 8 to 10 is introduced into CD8 + T cells collected from a subject requiring treatment for cancer, and an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells is exhibited. A method for producing a cytotoxic T cell, which comprises a step of recovering the cytotoxic T cell having the cell.
  13. 　請求項8～10いずれかに記載の発現ベクターで形質転換されたURLC10またはDEPDC1発現細胞に対するHLA-A24拘束性の細胞傷害作用を有する細胞傷害性T細胞。 Cytotoxic T cells having an HLA-A24-binding cytotoxic effect on URLC10 or DEPDC1-expressing cells transformed with the expression vector according to any one of claims 8 to 10.
  14. 　URLC10およびDEPDC1のいずれか、または両方の細胞傷害性T細胞の誘導処置を受けた対象から採取された末梢血リンパ球のT細胞受容体のCDR3のアミノ酸配列を決定する工程を含む、URLC10またはDEPDC1に対するHLA-A24拘束性の細胞傷害性T細胞の誘導をモニタリングする方法であって、CDR3のアミノ酸配列が配列番号：1-86からなる群から選択される少なくとも一つのアミノ酸配列である方法。 URLC10 or DEPDC1 comprises the step of determining the amino acid sequence of CDR3 of the T cell receptor of peripheral blood lymphocytes taken from a subject who has undergone induction treatment of either or both of URLC10 and DEPDC1. A method of monitoring the induction of HLA-A24-restricted cytotoxic T cells against, wherein the amino acid sequence of CDR3 is at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-86.
  15. 　CDR3のアミノ酸配列のレパトア解析によって、CDR3のアミノ酸配列の検出頻度を決定する工程を含む請求項14に記載の方法。 The method according to claim 14, which comprises a step of determining the detection frequency of the amino acid sequence of CDR3 by repertoire analysis of the amino acid sequence of CDR3.
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