JP5267969B2 - Cancer vaccine - Google Patents
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- JP5267969B2 JP5267969B2 JP2007313856A JP2007313856A JP5267969B2 JP 5267969 B2 JP5267969 B2 JP 5267969B2 JP 2007313856 A JP2007313856 A JP 2007313856A JP 2007313856 A JP2007313856 A JP 2007313856A JP 5267969 B2 JP5267969 B2 JP 5267969B2
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Abstract
Description
本発明は、癌ワクチンに関する。 The present invention relates to a cancer vaccine.
近年、腫瘍免疫学において、免疫細胞による腫瘍抗原認識機構がかなり解明されてきた。それによると、まず、抗原提示細胞である樹状細胞（dendritic cells または ＤＣ）は、細胞内で、腫瘍が発現するタンパク質を分解する際に生じた８〜１０個のアミノ酸からなる抗原ペプチドを、主要組織適合性抗原複合体（major histocompatibility complex または ＭＨＣ；ヒトでは、human leukocyte antigen または ＨＬＡ）と共に細胞表面に提示する。細胞障害性Ｔ細胞（cytotoxic T lymphocyte または ＣＴＬ）は、樹状細胞表面のＨＬＡクラスＩに結合した抗原ペプチドを認識し、活性化・増殖し、腫瘍内に侵入し、抗原ペプチドが由来するタンパク質を有する腫瘍細胞に対し細胞障害を生じる（例えば、非特許文献１参照）。 In recent years, the mechanism of tumor antigen recognition by immune cells has been elucidated considerably in tumor immunology. According to this, first, dendritic cells (DCs), which are antigen-presenting cells, express an antigenic peptide consisting of 8 to 10 amino acids generated when degrading a protein expressed by a tumor in the cell, Presented on the cell surface together with a major histocompatibility complex or MHC (human leukocyte antigen or HLA in humans). Cytotoxic T cells (cytotoxic T lymphocytes or CTLs) recognize antigen peptides bound to HLA class I on the surface of dendritic cells, activate and proliferate, invade tumors, and produce proteins derived from antigen peptides Cytotoxicity is caused to tumor cells (see, for example, Non-Patent Document 1).
この機構を利用して、腫瘍の治療方法として癌ワクチンが開発されてきた。例えば、腫瘍特異的タンパク質由来の抗原ペプチドを細胞表面に提示する樹状細胞をin vitroで作製し、増殖させ、腫瘍患者に投与したり、その樹状細胞によって教育された細胞障害性Ｔ細胞を投与したりすることにより、腫瘍患者の体内で腫瘍免疫を誘導させる。あるいは、腫瘍特異的タンパク質を腫瘍患者に投与し、患者の体内で、腫瘍免疫機構の全過程を誘導させるのである（例えば、非特許文献２〜７参照）。 By utilizing this mechanism, cancer vaccines have been developed as a method for treating tumors. For example, dendritic cells that present tumor-specific protein-derived antigenic peptides on the cell surface are generated in vitro, proliferated, administered to tumor patients, or cytotoxic T cells educated by the dendritic cells. In other words, tumor immunity is induced in the body of a tumor patient. Alternatively, a tumor-specific protein is administered to a tumor patient, and the whole process of the tumor immune mechanism is induced in the patient's body (see, for example, Non-Patent Documents 2 to 7).
メラノーマ特異的腫瘍抗原については臨床試験における成果が報告されている。例えば、メラノーマ抗原ｇｐ１００ペプチドをメラノーマ患者に皮下投与し、インターロイキン−２を静脈内投与することにより、４２％の患者で腫瘍の縮小が認められている（例えば、非特許文献８参照）。
しかし、腫瘍免疫を効率的に誘導することのできる腫瘍特異的タンパク質は、一部の腫瘍において、ほんの少数の例が知られているだけである。
そこで、本発明は、腫瘍免疫を効率的に誘導できるペプチド、そのペプチドを含有する組成物、そのペプチドを提示した抗原提示細胞、この抗原提示細胞によって刺激されたＴ細胞、およびこれらのペプチドや細胞を利用した癌ワクチン、及びそれらを用いた腫瘍患者の治療方法を提供することを目的としてなされた。
However, only a few examples of tumor-specific proteins that can efficiently induce tumor immunity are known in some tumors.
Therefore, the present invention provides a peptide capable of efficiently inducing tumor immunity, a composition containing the peptide, an antigen-presenting cell presenting the peptide, a T cell stimulated by the antigen-presenting cell, and these peptides and cells The purpose of the present invention was to provide a cancer vaccine utilizing the above and a method for treating tumor patients using the same.
本発明者らは、転写因子であるＳＯＸ６が、様々な腫瘍で高度に発現していることを既に見いだしている（Ueda et al., Oncogene 23, 1420-1427, 2004；国際公開番号WO2006/077941明細書）。この知見に基づき、ＳＯＸ６の様々な部分ペプチドに対し、ＨＬＡクラスＩ拘束性に細胞障害性Ｔ細胞に認識されるかどうか調べたところ、樹状細胞に添加したとき、効率よく細胞障害性Ｔ細胞によって認識されるいくつかの部分ペプチドを同定することができ、本発明の完成に至った。 The present inventors have already found that the transcription factor SOX6 is highly expressed in various tumors (Ueda et al., Oncogene 23, 1420-1427, 2004; International Publication No. WO2006 / 077941). Specification). Based on this finding, it was investigated whether various partial peptides of SOX6 were recognized by cytotoxic T cells in an HLA class I-restricted manner. When they were added to dendritic cells, they were efficiently cytotoxic T cells. Several partial peptides recognized by can be identified, and the present invention has been completed.
本発明にかかるペプチドは、配列番号１（ＡＬＦＧＤＱＤＴＶ）または配列番号２（ＫＬＧＰＧＶＩＤＬ）の配列を有する。これらのペプチドを提示した抗原提示細胞、及びこの抗原提示細胞によって誘導され、ＳＯＸ６を発現する細胞を認識するＴ細胞も、本発明の技術的範囲に属する。このＴ細胞は細胞障害性Ｔ細胞であることが好ましく、ＳＯＸ６を発現する細胞は腫瘍細胞であることが好ましく、例えば、グリオーマ細胞、ニューロブラストーマ細胞、膵臓癌細胞、肝癌細胞、肺癌細胞、食道癌細胞、メラノーマ細胞、前立腺癌細胞、乳癌細胞、腎癌細胞、胃癌細胞、大腸癌細胞、または白血病細胞が例示される。 The peptide according to the present invention has the sequence of SEQ ID NO: 1 (ALGFGDQDTV) or SEQ ID NO: 2 (KLGPGVIDL). Antigen presenting cells that present these peptides and T cells that are induced by these antigen presenting cells and recognize cells that express SOX6 also belong to the technical scope of the present invention. The T cell is preferably a cytotoxic T cell, and the cell expressing SOX6 is preferably a tumor cell. For example, a glioma cell, a neuroblastoma cell, a pancreatic cancer cell, a liver cancer cell, a lung cancer cell, an esophagus Examples are cancer cells, melanoma cells, prostate cancer cells, breast cancer cells, kidney cancer cells, gastric cancer cells, colon cancer cells, or leukemia cells.
さらに、本発明にかかる癌ワクチンは、配列番号１（ＡＬＦＧＤＱＤＴＶ）を有するペプチドまたは配列番号２（ＫＬＧＰＧＶＩＤＬ）を有するペプチドのいずれか又は両方のペプチド、上記抗原提示細胞、上記Ｔ細胞のうち、少なくとも一つを含有する。この癌ワクチンは、神経膠腫（グリオーマ）、神経芽腫（ニューロブラストーマ）、膵臓癌、肝癌、肺癌、膵癌、食道癌、黒色腫（メラノーマ）、前立腺癌、乳癌、腎癌、胃癌、大腸癌、または白血病に対する癌ワクチンであることが好ましい。 Furthermore, the cancer vaccine according to the present invention is at least one of the peptide having SEQ ID NO: 1 (ALFGGDQDTV) or the peptide having SEQ ID NO: 2 (KLGPGVIDL), the antigen-presenting cell, and the T cell. Contains one. This cancer vaccine is glioma, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, stomach cancer, colon A cancer vaccine against cancer or leukemia is preferred.
本発明にかかる腫瘍治療方法は、ヒト及びヒト以外の脊椎動物に対しこの癌ワクチンを用いることを特徴とする。 The tumor treatment method according to the present invention is characterized by using this cancer vaccine against humans and non-human vertebrates.
本発明によって、腫瘍免疫を効率的に誘導できるペプチド、そのペプチドを含有する組成物、そのペプチドを提示した抗原提示細胞、この抗原提示細胞によって刺激されたＴ細胞、およびこれらのペプチドや細胞を利用した癌ワクチン、及びそれらを用いた腫瘍患者の治療方法を提供することが可能になった。 According to the present invention, a peptide capable of efficiently inducing tumor immunity, a composition containing the peptide, an antigen-presenting cell presenting the peptide, a T cell stimulated by the antigen-presenting cell, and these peptides and cells are used. It has become possible to provide a cancer vaccine and a method for treating a tumor patient using the same.
実施の形態及び実施例に特に説明がない場合には、J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.などの標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いる場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。
なお、本発明の目的、特徴、利点、及びそのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば、容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的に実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をそれらに限定するものではない。本明細書で開示されている本発明の意図並びに範囲内で、本明細書の記載に基づき、様々な改変並びに修飾ができることは、当業者にとって明らかである。
Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.
＝＝ＨＬＡクラスＩと結合性が高いペプチドのスクリーニング＝＝
ウエブサイト上のデータベースであるBioInformatics & Molecular Analysis Section (BIMAS) のHLA Peptide Binding predictions Program を用い、ＳＯＸ６遺伝子がコードするアミノ酸配列から、Ａ０２０１タイプのＨＬＡクラスＩと結合性が高い、９アミノ酸残基のペプチド配列を特定した。表１に、このスクリーニングによって得られたスコアの高いペプチド配列の例（ＳＯＸ６−２５２、ＳＯＸ６−５２１、ＳＯＸ６−４４７）を示す。
== Screening for peptides with high binding to HLA class I ==
Using the HLA Peptide Binding predictions Program of BioInformatics & Molecular Analysis Section (BIMAS), which is a database on the website, 9 amino acid residues that have high binding properties to A0201 type HLA class I from the amino acid sequence encoded by the SOX6 gene The peptide sequence was identified. Table 1 shows examples of high-scoring peptide sequences (SOX6-252, SOX6-521, SOX6-447) obtained by this screening.
これらのペプチドのうち、実際に合成し、抗原として抗原提示細胞である樹状細胞に投与したとき、ＨＬＡクラスＩ分子に結合することにより細胞表面に提示され、細胞障害性Ｔ細胞に認識されることで特異的な細胞障害性Ｔ細胞を誘導できるペプチドを同定するため、樹状細胞と共培養された細胞障害性Ｔ細胞のヒト・グリオーマ細胞または膵臓癌細胞に対する反応性を調べた。その結果、ＳＯＸ６−４４７（配列番号１）及びＳＯＸ６−５２１（配列番号２）のペプチドを細胞表面に提示する樹状細胞が効率よく細胞障害性Ｔ細胞を刺激し、また各ペプチドの刺激により樹立された細胞障害性Ｔ細胞がＳＯＸ６を発現するグリオーマ細胞または膵臓癌細胞を効率よく認識することが明らかになった。 Among these peptides, when they are actually synthesized and administered as antigens to dendritic cells that are antigen-presenting cells, they are presented on the cell surface by binding to HLA class I molecules and recognized by cytotoxic T cells. In order to identify peptides capable of inducing specific cytotoxic T cells, the reactivity of cytotoxic T cells co-cultured with dendritic cells to human glioma cells or pancreatic cancer cells was examined. As a result, dendritic cells presenting SOX6-447 (SEQ ID NO: 1) and SOX6-521 (SEQ ID NO: 2) peptides on the cell surface efficiently stimulate cytotoxic T cells, and are established by stimulation of each peptide. It was revealed that the cytotoxic T cells thus recognized efficiently recognize glioma cells or pancreatic cancer cells that express SOX6.
以下の実施例には、これらの実験結果を示すことにより、配列番号１または配列番号２を有するペプチドを抗原提示細胞に添加すると、それらのペプチドがＨＬＡ上に提示され、共培養したＴ細胞を刺激することにより抗原特異的なＴ細胞を誘導すること、そして、この抗原特異的なＴ細胞はＳＯＸ６を発現する細胞と反応し、その細胞に対し細胞障害性を有することを示す。 In the following examples, by showing the results of these experiments, when peptides having SEQ ID NO: 1 or SEQ ID NO: 2 were added to antigen-presenting cells, the peptides were presented on HLA and co-cultured T cells were Stimulation induces antigen-specific T cells and indicates that the antigen-specific T cells react with cells expressing SOX6 and are cytotoxic to the cells.
ここで、Ｔ細胞による障害の対象とする細胞は、ＳＯＸ６を発現していれば特に限定されない。例えば、ヒト正常組織においては胎児脳または成人精巣しかＳＯＸ６の発現が検出されないものの、腫瘍細胞においては、様々な腫瘍や腫瘍細胞株で、その発現が検出されている（国際公開番号WO2006/077941明細書）ので、腫瘍細胞、例えば、グリオーマ細胞、ニューロブラストーマ細胞、膵臓癌細胞、肝癌細胞、肺癌細胞、膵癌細胞、食道癌細胞、メラノーマ細胞、前立腺癌細胞、乳癌細胞、腎癌細胞、胃癌細胞、大腸癌細胞、または白血病細胞をＴ細胞による障害の対象とすることができる。従って、配列番号１または配列番号２を有するペプチド、そのペプチドを細胞表面に提示した抗原提示細胞、及びその抗原提示細胞の刺激によって樹立された細胞障害性Ｔ細胞は、例えば、神経膠腫（グリオーマ）、神経芽腫（ニューロブラストーマ）、膵臓癌、肝癌、肺癌、膵癌、食道癌、黒色腫（メラノーマ）、前立腺癌、乳癌、腎癌、胃癌、大腸癌、または白血病に対する癌ワクチンとして有用である。 Here, the cells to be damaged by T cells are not particularly limited as long as they express SOX6. For example, although expression of SOX6 is detected only in fetal brain or adult testis in normal human tissues, its expression is detected in various tumors and tumor cell lines (International Publication No. WO2006 / 077941). Tumor cells such as glioma cells, neuroblastoma cells, pancreatic cancer cells, liver cancer cells, lung cancer cells, pancreatic cancer cells, esophageal cancer cells, melanoma cells, prostate cancer cells, breast cancer cells, kidney cancer cells, gastric cancer cells Colorectal cancer cells or leukemia cells can be targeted for damage by T cells. Therefore, a peptide having SEQ ID NO: 1 or SEQ ID NO: 2, an antigen-presenting cell presenting the peptide on the cell surface, and a cytotoxic T cell established by stimulation of the antigen-presenting cell are, for example, glioma ), Neuroblastoma (neuroblastoma), pancreatic cancer, liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, stomach cancer, colon cancer, or cancer vaccine against leukemia is there.
＝＝癌ワクチンの投与方法＝＝
現在、癌ワクチンとして、腫瘍特異的癌抗原、癌抗原提示抗原提示細胞、または癌抗原反応性細胞障害性Ｔ細胞を腫瘍患者に投与する方法が開発されている。本発明においては、ＳＯＸ６の部分ペプチドを用いているので、治療（予防も含まれる）対象となる腫瘍は、ＳＯＸ６が発現している腫瘍であれば何でもよく、例えば、神経膠腫（グリオーマ）、神経芽腫（ニューロブラストーマ）、膵臓癌、肝癌、肺癌、膵癌、食道癌、黒色腫（メラノーマ）、前立腺癌、乳癌、腎癌、胃癌、大腸癌、または白血病も治療対象の一つとなる。主な治療対象は、こうした腫瘍を有するヒト患者であるが、腫瘍を有するヒト以外の脊椎動物でもかまわない。
== Method of administration of cancer vaccine ==
Currently, methods for administering tumor-specific cancer antigens, cancer antigen-presenting antigen-presenting cells, or cancer antigen-reactive cytotoxic T cells to tumor patients are being developed as cancer vaccines. In the present invention, since a partial peptide of SOX6 is used, the tumor to be treated (including prevention) may be any tumor that expresses SOX6. For example, glioma (glioma), Neuroblastoma (neuroblastoma), pancreatic cancer, liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, gastric cancer, colon cancer, or leukemia are also treated. The main treatment target is a human patient having such a tumor, but it may be a vertebrate other than a human having a tumor.
治療対象となる腫瘍を有する患者に対し投与する癌ワクチンは、腫瘍特異的癌抗原となりうる配列番号１及び配列番号２を有するペプチドを含有してもよい。この場合、あらかじめ患者のＨＬＡクラスＩのタイプを調べ、Ａ０２０１である場合に、この癌ワクチンを投与する。投与するペプチドは、一種類であっても複数種類であってもよい。投与部位に関しては、皮内投与、皮下投与、静脈内投与、腹腔内投与などが考えられ、特に限定されることはない。また、投与する際には、免疫誘導能を高めるアジュバントなどとともにペプチドを投与してもよい。また、投与されるペプチドは、生体内で分解されにくくするような修飾が施されていてもよい。 A cancer vaccine to be administered to a patient having a tumor to be treated may contain a peptide having SEQ ID NO: 1 and SEQ ID NO: 2, which can be a tumor-specific cancer antigen. In this case, the patient's HLA class I type is examined in advance, and if it is A0201, this cancer vaccine is administered. The peptide to be administered may be one type or a plurality of types. Regarding the administration site, intradermal administration, subcutaneous administration, intravenous administration, intraperitoneal administration, and the like can be considered, and there is no particular limitation. Moreover, when administering, you may administer a peptide with the adjuvant etc. which improve immunity induction ability. Moreover, the peptide to be administered may be modified so as not to be degraded in vivo.
また、上記癌ワクチンは配列番号１または配列番号２を有するペプチドを提示した抗原提示細胞を含有してもよい。ここで、細胞表面に提示されているペプチドは、配列番号１または配列番号２を有するペプチドそのものでもよく、糖やリン酸などで修飾されてもよい。抗原提示細胞としては、樹状細胞の他にマクロファージ、Ｂ細胞等が考えられるが、抗原提示能の高さなどから、樹状細胞が好ましい。以下、樹状細胞の単離方法の例を記述する。 Further, the cancer vaccine may contain antigen-presenting cells presenting a peptide having SEQ ID NO: 1 or SEQ ID NO: 2. Here, the peptide displayed on the cell surface may be the peptide itself having SEQ ID NO: 1 or SEQ ID NO: 2, or may be modified with sugar, phosphate, or the like. In addition to dendritic cells, macrophages, B cells, and the like can be considered as antigen-presenting cells. Dendritic cells are preferred because of their high antigen-presenting ability. Hereinafter, an example of a method for isolating dendritic cells will be described.
まず、脊椎動物個体の末梢血から単核球を単離する。この単核球は、治療対象となる個体自身から分離することが好ましいが、他の個体から単離してもよい。また、この単核球はＣＤ１４陽性であることが好ましい。単離した単核球を、ＧＭ−ＣＳＦとＩＬ−４で7 日前後培養すると、未熟な樹状細胞に分化誘導することができる。このようにして分化誘導された樹状細胞は、抗原提示分子であるＭＨＣ分子を高発現している。この未熟な樹状細胞に、配列番号１または配列番号２を有するペプチドを添加し結合させる。樹状細胞のＨＬＡクラスＩタイプがＡ０２０１である場合に、配列番号１または配列番号２を有するペプチドを添加する。添加するのは、人工合成ペプチドに限らず、ペプチドを発現させた細胞の抽出物（extractやlysate）や精製物などでもよい。こうして得られた抗原提示樹状細胞を、腫瘍を有する個体に投与する。投与部位に関しては、皮内投与、皮下投与、静脈内投与、リンパ節内投与などが考えられ、特に限定されることはないが、生理的な樹状細胞の抗原提示が、樹状細胞投与部位の所属リンパ節にて行なわれることを考えると、リンパ節内への直接投与が好ましい。 First, mononuclear cells are isolated from the peripheral blood of vertebrate individuals. The mononuclear cells are preferably separated from the individual to be treated, but may be isolated from other individuals. The mononuclear cells are preferably CD14 positive. When the isolated mononuclear cells are cultured with GM-CSF and IL-4 for 7 days later, differentiation can be induced into immature dendritic cells. The dendritic cells thus induced to differentiate highly express MHC molecules that are antigen-presenting molecules. A peptide having SEQ ID NO: 1 or SEQ ID NO: 2 is added to and bound to the immature dendritic cells. When the HLA class I type of dendritic cells is A0201, a peptide having SEQ ID NO: 1 or SEQ ID NO: 2 is added. What is added is not limited to an artificially synthesized peptide, but may be an extract (extract or lysate) of a cell in which the peptide is expressed or a purified product. The antigen-presenting dendritic cells thus obtained are administered to an individual having a tumor. With regard to the administration site, intradermal administration, subcutaneous administration, intravenous administration, intralymphatic administration, etc. are conceivable, and although there is no particular limitation, physiological dendritic cell antigen presentation is In view of the fact that it is performed in the regional lymph nodes, direct administration into the lymph nodes is preferable.
また、上記癌ワクチンは、配列番号１または配列番号２を有するペプチドに由来するペプチドを提示した抗原提示細胞の刺激によって樹立されたＴ細胞を含有してもよい。配列番号１または配列番号２を有するペプチドに由来するペプチドを提示した抗原提示細胞に対し、Ｔ細胞を共培養し、抗原提示細胞で刺激する。このようにして樹立されたＴ細胞を腫瘍を有する個体に投与してもよい。ここでのＴ細胞は、細胞障害性Ｔ細胞が好ましいが、ヘルパーＴ細胞等でもよい。投与部位に関しては、皮内投与、皮下投与、静脈内投与、腫瘍内投与などが考えられ、特に限定されることはないが、細胞障害性Ｔ細胞の場合、抗原を発現する細胞を直接攻撃できるため、腫瘍内投与が好ましい。 Further, the cancer vaccine may contain T cells established by stimulation of antigen-presenting cells presenting peptides derived from the peptide having SEQ ID NO: 1 or SEQ ID NO: 2. T cells are co-cultured with antigen-presenting cells presenting a peptide derived from the peptide having SEQ ID NO: 1 or SEQ ID NO: 2, and stimulated with the antigen-presenting cells. The T cells thus established may be administered to an individual having a tumor. The T cell here is preferably a cytotoxic T cell, but may be a helper T cell or the like. Regarding the administration site, intradermal administration, subcutaneous administration, intravenous administration, intratumoral administration, and the like are conceivable. Although there is no particular limitation, in the case of cytotoxic T cells, cells expressing the antigen can be directly attacked. Therefore, intratumoral administration is preferred.
〈実施例１〉抗原刺激によって樹立された細胞障害性Ｔ細胞の細胞傷害活性の評価
＝＝実験初日＝＝
ＨＬＡクラス１のタイプがＡ０２０１の健常人末梢血から、以下のように単核球を分離した。まず、ヘパリン５ｍｌで洗浄したシリンジで、末梢血を５０ｍｌ採血した。等量のLymphoprep (Fresenius kabi Norge AS, Axis-Shield PoC AS, Oslo, Norway) を転倒混和し、遠心（２０℃、２０００ｒｐｍ、３５分間 、ブレーキなし）し、中間層を吸引して採取した。これにＰＢＳを加えて再混濁し、遠心（20℃、2000 rpm、10分間）する操作を３回繰り返して、得られた単核球を洗浄した。
この単核球を初代細胞培養用培養皿(FALCON MULTIWELL PRIMARIA 24 well)に５ｘ１０６個/ウエルの密度で播種し、３７℃、５％ＣＯ２存在下で４時間培養した。培養液はAIM-V(GIBCO)とRPMI-1640を1:1に混合したもの（基本培養液）を用いた。培養皿の底面に接着した細胞を回収し、４ｘ１０５ 個/ウエルの密度で新しい２４ウエルの培養皿に播種し、GM-CSF(10ng/ml), IL-4(1ng/ml)添加した基本培養液を用いて、３７℃、５％ＣＯ２存在下で７日間培養し、樹状細胞を分化させた。
<Example 1> Evaluation of cytotoxic activity of cytotoxic T cells established by antigen stimulation == First day of experiment ==
Mononuclear cells were isolated from the peripheral blood of healthy individuals with
The mononuclear cells were seeded in a culture dish for primary cell culture (FALCON MULTIWELL PRIMARIA 24 well) at a density of 5 × 10 6 cells / well and cultured at 37 ° C. in the presence of 5% CO 2 for 4 hours. The culture solution used was a mixture of AIM-V (GIBCO) and RPMI-1640 in 1: 1 (basic culture solution). Cells adhering to the bottom of the culture dish were collected, seeded in a new 24-well culture dish at a density of 4 × 10 5 cells / well, and GM-CSF (10 ng / ml) and IL-4 (1 ng / ml) were added. Using the culture solution, the cells were cultured for 7 days at 37 ° C. in the presence of 5% CO 2 to differentiate the dendritic cells.
＝＝実験７日目＝＝
培養７日目に分化した樹状細胞を抗原提示細胞（antigen presenting cells;ＡＰＣｓ）として用いた。得られた樹状細胞をirradiate (60Gy)したのち、合成・精製したペプチド（それぞれ配列番号１〜３を有するＳＯＸ６由来のペプチドであるＳＯＸ６-２５１、ＳＯＸ６-４４７、及びＳＯＸ６-５２１）を１０μＭ添加し、３７℃、５％ＣＯ２存在下で２時間培養して細胞に結合させ、ＰＢＳで２回洗浄し、刺激細胞（stimulator cells）とした。
一方、この日（培養７日目当日）、実験１５日目に樹状細胞を得るために、初日と同じ操作で単核球を培養した。
その過程で、単核球を４時間培養後、培養皿に接着せず、培養上清に浮遊している細胞を回収し、１ｘ１０７ 個の細胞に対しIMag anti-human CD8 particules −DM (BD Biosciences社)１００μlを、４℃、３０分間反応させた。磁石を用いてＣＤ８陽性細胞を吸着・回収し、応答細胞（responder cells）とした。
このようにして得られた応答細胞２ｘ１０５個を、上記刺激細胞のウエルに添加し、共培養した。なお、培養液として、IL-1α(10 unit/ml), IL-2(20 unit/ml), IL-4(1 ng/ml), IL-6(125 unit/ml), IL-12(1 ng/ml)を添加した基本培養液を用いた。
== Experiment day 7 ==
Dendritic cells differentiated on the seventh day of culture were used as antigen presenting cells (APCs). After the obtained dendritic cells are irradiated (60 Gy), 10 μM of synthesized and purified peptides (SOX6-251, SOX6-447, and SOX6-521, which are peptides derived from SOX6 having SEQ ID NOS: 1 to 3, respectively) are added. The cells were cultured for 2 hours at 37 ° C. in the presence of 5% CO 2 to bind to the cells, washed twice with PBS, and used as stimulator cells.
On the other hand, in order to obtain dendritic cells on this day (the 7th day of culture) and the 15th day of experiment, mononuclear cells were cultured by the same operation as the first day.
In the process, after 4 hours of culture Mononuclear cells, does not adhere to the culture dish, the cells floating in the culture supernatant was collected, 1x10 7 cells to IMag anti-human CD8 particules -DM ( BD Biosciences) 100 μl was reacted at 4 ° C. for 30 minutes. CD8 positive cells were adsorbed and collected using a magnet, and used as responder cells.
2 × 10 5 responding cells thus obtained were added to the wells of the stimulating cells and co-cultured. In addition, IL-1α (10 unit / ml), IL-2 (20 unit / ml), IL-4 (1 ng / ml), IL-6 (125 unit / ml), IL-12 ( A basic culture solution supplemented with 1 ng / ml) was used.
＝＝実験１５日目＝＝
培養７日目から培養し、分化させた樹状細胞を用いて、上記と同様の操作で刺激細胞を調整した。
また、培養７日目より刺激細胞と共培養した応答細胞を回収し、新たに調整した刺激細胞と、同様に共培養した。ただし、培養液は培養７日目に用いた上記培養液においてＩＬ−１２を含まない培養液を用いた。
なお、実験２２日目に樹状細胞を得るための単核球の培養を、同様にして、新たに始めておいた。
==
Stimulatory cells were prepared in the same manner as described above using dendritic cells cultured and differentiated from day 7 of culture.
Moreover, the response cells co-cultured with the stimulator cells were collected from the 7th day of culture, and co-cultured with the newly prepared stimulator cells in the same manner. However, the culture solution used was a culture solution not containing IL-12 in the culture solution used on the seventh day of culture.
In addition, the culture of mononuclear cells to obtain dendritic cells on the 22nd day of the experiment was newly started in the same manner.
＝＝実験２２日目＝＝
実験１５日目と同様に、刺激細胞と共培養を続けている応答細胞を回収し、新たに調整した刺激細胞と共培養した。
なお、樹状細胞を得るための単核球の培養を始めたが、９６ウエルの培養皿を用い、４ｘ１０４個／ウエルに調整して播種し、培養した。
== Experiment 22 day ==
As in the 15th day of the experiment, the responder cells that had been co-cultured with the stimulator cells were collected and co-cultured with the newly prepared stimulator cells.
In addition, although the culture | cultivation of the mononuclear cell for obtaining a dendritic cell was started, it seed | inoculated and culture | cultivated by adjusting to 4x10 < 4 > piece / well using a 96 well culture dish.
＝＝実験２８日目＝＝
この日まで、培養７日目より刺激細胞と共培養した応答細胞を回収し、エフェクター細胞として、ＳＯＸ６由来ペプチドＳＯＸ６−２５１の刺激により樹立したＣＴＬ（ＣＴＬ−ＳＯＸ６−２５１）、ＳＯＸ６由来ペプチドＳＯＸ６−４４７の刺激により樹立したＣＴＬ（ＣＴＬ−ＳＯＸ６−４４７）、ＳＯＸ６由来ペプチドＳＯＸ６−５２１の刺激により樹立したＣＴＬ（ＣＴＬ−ＳＯＸ６−５２１）を準備した。
また、ターゲット細胞として、ＨＬＡ−Ａ，Ｂ欠損ヒトＢ細胞株ＣＩＲにＨＬＡ−Ａ０２０１のｃＤＮＡを導入したＣＩＲ−Ａ０２０１、ＣＩＲ−Ａ０２０１株にＳＯＸ６遺伝子を導入したＣＩＲ−Ａ０２０１（ＳＯＸ６）、ＨＬＡ−Ａ０２０１とＳＯＸ６が共に発現しておりＨＬＡ−Ａ２４は発現していないグリオーマ細胞Ｕ８７、ＨＬＡ−Ａ２４０２とＳＯＸ６が共に発現しておりＨＬＡ−Ａ０２０１は発現していないＭａｒｃｕｓ、ＨＬＡ−Ａ０２０１とＳＯＸ６が共に発現しておりＨＬＡ−Ａ２４は発現していない他の癌細胞（膵臓癌由来Ｐａｎｃ−１）を準備した。これらの細胞５Ｘ１０６個に対して５００μｌのFetal Bovine Serum（ＦＢＳ）、５１Ｃｒ（１．８５ＭＢｑ／５０μｌ）を５０μｌ添加し、３７℃、５％ＣＯ２下で６０分振蕩培養した。その後洗浄を３回行い、５ｘ１０３／１００μｌに調整して９６穴プレート（COSTER 3595-96 well）の各ウエルに１００μｌずつ添加した。
E/T ratio (エフェクター細胞／ターゲット細胞の比)は、60:1(エフェクター細胞は3x105/ウエル) 、30:1(エフェクター細胞は1.5x105/ウエル) 、15:1 (エフェクター細胞は7.5X104/ウエル)の３群を設けて、それぞれの数のエフェクター細胞を51Crでラベルした９６穴プレート上のターゲット細胞に添加した。これらを３７℃、５％ＣＯ２下で４時間培養した。
各ウエルのcpm(cpm experimental release)を計測し、特異的溶解率（Percentage of specific lysis)を以下の式から算出した。
特異的溶解率= (cpm−csr)/(cmr−csr) x100
（なお、csr(cpm spontaneous release)はエフェクター細胞を含まない培地を添加したウエルのcpmであり、cmr(cpm maximal release)はエフェクター細胞を含まない０．１％ Triton Xを添加したウエルのcpmである。）
== Experiment 28th day ==
Until this day, the responder cells co-cultured with the stimulating cells from day 7 were collected, and as effector cells, CTL (CTL-SOX6-251) and SOX6-derived peptide SOX6- established by stimulation with SOX6-derived peptide SOX6-251. CTL established by stimulation of 447 (CTL-SOX6-447) and CTL established by stimulation of SOX6-derived peptide SOX6-521 (CTL-SOX6-521) were prepared.
Further, as target cells, CIR-A0201 in which HLA-A0201 cDNA was introduced into HLA-A, B-deficient human B cell line CIR, CIR-A0201 (SOX6) in which SOX6 gene was introduced into CIR-A0201, and HLA-A0201 And SOX6 are expressed together, but HLA-A24 is not expressed glioma cell U87, HLA-A2402 and SOX6 are both expressed, and HLA-A0201 is not expressed Marcus, HLA-A0201 and SOX6 are both expressed And other cancer cells not expressing HLA-A24 (pancreatic cancer-derived Panc-1) were prepared. 50 μl of 500 μl of Fetal Bovine Serum (FBS) and 51 Cr (1.85 MBq / 50 μl) was added to 6 of 5 × 10 6 cells, and the mixture was shaken and cultured at 37 ° C. under 5% CO 2 for 60 minutes. Performed subsequently washed 3 times, was added in 100 [mu] l to each well of a 96-well plate was adjusted to 5x10 3 / 100μl (COSTER 3595-96 well ).
E / T ratio (effector cell / target cell ratio) is 60: 1 (effector cell is 3 × 10 5 / well), 30: 1 (effector cell is 1.5 × 10 5 / well), 15: 1 (effector cell is 7.5 × 10) 3 groups of X10 4 / well) and each number of effector cells was added to target cells on a 96-well plate labeled with 51 Cr. These were cultured at 37 ° C. under 5% CO 2 for 4 hours.
The cpm (cpm experimental release) of each well was measured, and the specific lysis rate (Percentage of specific lysis) was calculated from the following equation.
Specific dissolution rate = (cpm-csr) / (cmr-csr) x100
(Note that csr (cpm spontaneous release) is the cpm of the well added with the medium not containing the effector cell, and cmr (cpm maximal release) is the cpm of the well added with 0.1% Triton X not containing the effector cell. is there.)
＝＝結果＝＝
健常人（ＨＬＡクラスＩ：Ａ０２０１／Ａ２４０２）の末梢血リンパ球から樹立したＣＴＬ-ＳＯＸ６-４４７とＣＴＬ-ＳＯＸ６-５２１は、ＨＬＡ-Ａ０２０１とＳＯＸ６を共に発現しているＣＩＲ-Ａ０２０１-ＳＯＸ６（図１Ａ）及びグリオーマ細胞由来のＵ８７（図１Ｂ）をE/T ratio依存性に傷害した。一方、ＣＴＬ-ＳＯＸ６-２５２はそれらを傷害しなかった（図１Ａ、Ｂ）。このＣＴＬ-ＳＯＸ６-４４７とＣＴＬ-ＳＯＸ６-５２１はＨＬＡ-Ａ０２０１を発現しているがＳＯＸ６を発現していないＣＩＲ-Ａ０２０１を傷害しなかった（図１Ｃ）。
次に別のタイプのＨＬＡクラスＩを有する健常人（ＨＬＡクラスＩ：Ａ０２０１／ - ）の末梢血リンパ球をＳＯＸ６-４４７によって刺激してＣＴＬ-ＳＯＸ６-４４７を樹立し、それぞれの細胞傷害活性を検証した。ＣＴＬ-ＳＯＸ６-４４７は、ＨＬＡ-Ａ０２０１とＳＯＸ６を共に発現している細胞であるＣＩＲ-Ａ０２０１-ＳＯＸ６、グリオーマ細胞由来のＵ８７、膵臓癌細胞由来のＰａｎｃ-１をE/T ratio依存性に傷害した（図２）。一方、ＨＬＡ-Ａ０２０１を発現しているがＳＯＸ６を発現していない細胞ＣＩＲ-Ａ０２０１を傷害しなかった（図２）。
このように、ＳＯＸ６に由来するペプチドＳＯＸ６-４４７とＳＯＸ６-５２１を用いて、それらのペプチドを提示した抗原提示細胞を作製することができ、さらに、その抗原提示細胞によって刺激することにより、各ペプチドのみならずＳＯＸ６を発現した細胞を認識する特異的Ｔ細胞が誘導される。
== Result ==
CTL-SOX6-447 and CTL-SOX6-521 established from peripheral blood lymphocytes of healthy individuals (HLA class I: A0201 / A2402) are CIR-A0201-SOX6 expressing both HLA-A0201 and SOX6 (Fig. 1A) and U87 derived from glioma cells (FIG. 1B) were damaged in an E / T ratio-dependent manner. On the other hand, CTL-SOX6-252 did not damage them (FIGS. 1A, B). CTL-SOX6-447 and CTL-SOX6-521 expressed HLA-A0201, but did not damage CIR-A0201 not expressing SOX6 (FIG. 1C).
Next, peripheral blood lymphocytes of healthy individuals having another type of HLA class I (HLA class I: A0201 //) were stimulated with SOX6-447 to establish CTL-SOX6-447, and each cytotoxic activity was determined. Verified. CTL-SOX6-447 damages CIR-A0201-SOX6, a cell that expresses both HLA-A0201 and SOX6, U87 derived from glioma cells, and Panc-1 derived from pancreatic cancer cells in an E / T ratio-dependent manner (FIG. 2). On the other hand, cells CIR-A0201 expressing HLA-A0201 but not SOX6 were not damaged (FIG. 2).
Thus, using the peptides SOX6-447 and SOX6-521 derived from SOX6, antigen-presenting cells presenting those peptides can be prepared, and further, each peptide can be stimulated by the antigen-presenting cells, Not only specific T cells that recognize cells expressing SOX6 are induced.
〈実施例２〉ペプチド刺激によって樹立された細胞障害性Ｔ細胞（エフェクター細胞）の、刺激ペプチドに対する反応特異性の評価
本実施例では、ＳＯＸ６−４４７の刺激により樹立されたＨＬＡ拘束性ＳＯＸ６特異的細胞傷害活性を有するＣＴＬは、ＳＯＸ６−４４７を提示した抗原提示細胞をペプチド特異的に傷害するかどうかを検証した。
Example 2 Evaluation of Response Specificity of Cytotoxic T Cells (Effector Cells) Established by Peptide Stimulation to Stimulating Peptides In this example, HLA-restricted SOX6-specific established by stimulation with SOX6-447 It was verified whether CTL having cytotoxic activity was able to specifically damage antigen-presenting cells presenting SOX6-447.
＝＝実験方法＝＝
まず、ターゲット細胞として、２種類の細胞を準備した。まず、ＨＬＡ−Ａ０２０１とＳＯＸ６を発現しているグリオーマ細胞ＳＦ−１２６を実施例１と同様の手順にて５１Ｃｒでラベルした（Hot target cell）。また、コンペティターのターゲット細胞として、ＨＬＡ−Ａ０２０１を発現しているがＳＯＸ６を発現していないＣＩＲ−Ａ０２０１に対し、ＳＯＸ６由来ペプチド（ＳＯＸ６−４４７）をパルスしたＣＩＲ−Ａ０２０１（ＳＯＸ６−４４７）、ＳＯＸ６と無関係な配列を有するインフルエンザウィルス（Ｆｌｕ）由来ペプチドＦｌｕ−Ｍ１５８−６６（ＧＩＬＧＦＶＦＴＬ：配列番号４）をパルスしたＣＩＲ−Ａ０２０１（Ｆｌｕ−Ｍ１）、ペプチドをパルスしないＣＩＲ−Ａ０２０１（ｎｏ ｐｅｐｔｉｄｅ）を準備し、５１Ｃｒでラベルしないで、９６穴プレートに各ウエル１ｘ１０３／５０μｌずつ播種した（Cold target cell）。
一方、実施例１と同様に、エフェクター細胞として、ＳＯＸ６由来ペプチドＳＯＸ６−４４７の刺激により樹立したＣＴＬ−ＳＯＸ６−４４７を準備した。
E/T ratio は80:1（エフェクター細胞は8×104/100μl/ウエル）、40:1（エフェクター細胞は4×104/100μl/ウエル）、20:1（エフェクター細胞は2×104/100μl/ウエル）の３群を設けた。それぞれの数のエフェクター細胞をＣＩＲ−Ａ０２０１ (Cold target cell) に添加し、３７℃、５％ＣＯ２下で１時間培養したのち、１ｘ１０３個のＳＦ−１２６ (Hot target cell)を添加し、３７℃、５％ＣＯ２下でさらに４時間培養した。
培養後、各ウエルのcpmを計測し、実施例１と同様に特異的溶解率（Percentage of specific lysis)を算出した。
== Experimental method ==
First, two types of cells were prepared as target cells. First, glioma cells SF-126 expressing HLA-A0201 and SOX6 were labeled with 51 Cr in the same procedure as in Example 1 (Hot target cell). Further, CIR-A0201 (SOX6-447), SOX6 pulsed with SOX6-derived peptide (SOX6-447) against CIR-A0201 expressing HLA-A0201 but not expressing SOX6 as target cells of the competitor CIR-A0201 (Flu-M1) pulsed with an influenza virus (Flu) -derived peptide Flu-M1 58-66 (GILGFVFTL: SEQ ID NO: 4) having a sequence unrelated to CIR-A0201 (no peptide) prepared, not labeled with 51 Cr, were seeded by each well 1x10 3/50 [mu] l in 96 well plates (Cold target cell).
On the other hand, as in Example 1, CTL-SOX6-447 established by stimulation with SOX6-derived peptide SOX6-447 was prepared as an effector cell.
E / T ratio is 80: 1 (effector cells 8 × 10 4 / 100μl / well), 40: 1 (effector cells 4 × 10 4 / 100μl / well), 20: 1 (effector cells 2 × 10 4 / 100 μl / well) were provided. After adding each number of effector cells to CIR-A0201 (Cold target cell) and culturing at 37 ° C. under 5% CO 2 for 1 hour, 1 × 10 3 SF-126 (Hot target cell) were added, The cells were further cultured at 37 ° C. and 5% CO 2 for 4 hours.
After culturing, the cpm of each well was measured, and the specific lysis rate (Percentage of specific lysis) was calculated in the same manner as in Example 1.
＝＝結果＝＝
以上のようにして、各種ペプチドをパルスしたコンペティターＣＩＲ−Ａ０２０１(Cold target cell)の存在下で、グリオーマ細胞ＳＦ−１２６(Hot target cell)に対するＣＴＬ−ＳＯＸ６−４４７の細胞傷害活性を調べたところ、ＣＴＬ−ＳＯＸ６−４４７は、E/T ratio依存性に５１ＣｒでラベルしたＳＦ−１２６に対する細胞傷害活性を有し、かつその細胞傷害活性は、ネガティブコントロールであるＣＩＲ−Ａ０２０１（ｎｏ ｐｅｐｔｉｄｅ）の存在下の場合と比べ、ＣＩＲ−Ａ０２０１（Ｆｌｕ−Ｍ１）の存在下では抑制されなかったが、ＣＩＲ−Ａ０２０１（ＳＯＸ６−４４７）の存在下で顕著に抑制された（図３）。
このように、ＣＴＬ−ＳＯＸ６−４４７は、提示されたペプチド特異的にＳＯＸ６−４４７をパルスしたＣＩＲ−Ａ０２０１を認識し、傷害し得た。
== Result ==
As described above, the cytotoxic activity of CTL-SOX6-447 against glioma cell SF-126 (Hot target cell) was examined in the presence of a competitor CIR-A0201 (Cold target cell) pulsed with various peptides. CTL-SOX6-447 has cytotoxic activity against SF-126 labeled with 51 Cr in an E / T ratio dependency, and the cytotoxic activity is the presence of CIR-A0201 (no peptide) which is a negative control Compared with the lower case, it was not suppressed in the presence of CIR-A0201 (Flu-M1), but was significantly suppressed in the presence of CIR-A0201 (SOX6-447) (FIG. 3).
Thus, CTL-SOX6-447 was able to recognize and damage CIR-A0201 pulsed with SOX6-447 specifically for the presented peptide.
〈実施例３〉ペプチド刺激によって樹立された細胞障害性Ｔ細胞（エフェクター細胞）のターゲット細胞に対する障害活性の、ターゲット細胞作製時にパルスするペプチド濃度に対する濃度依存性
本実施例では、ＣＴＬ−ＳＯＸ６−４４７は、どの程度の刺激ペプチド濃度でパルスするとそのペプチドを提示した抗原提示細胞を認識して傷害するのかを検証した。
<Example 3> Dependence of cytotoxicity of cytotoxic T cells (effector cells) established by peptide stimulation on target cells on concentration of peptide pulsed at the time of preparation of target cells In this example, CTL-SOX6-447 Examined the concentration of stimulating peptide at which the antigen-presenting cells presenting the peptide were recognized and damaged when pulsed.
＝＝実験方法＝＝
ターゲット細胞として、ＳＯＸ６−４４７ペプチドを様々な濃度（０ｎＭ、０．１ｎＭ、１ｎＭ、１０ｎＭ、１００ｎＭ）でパルスしたＣＩＲ−Ａ０２０１（ＳＯＸ６−４４７）を準備した。これらの細胞５ｘ１０６個に対して５００μｌのＦＢＳ、５１Ｃｒ（１．８５ＭＢｑ／５０μｌ）を５０μｌ添加し、３７℃、５％ＣＯ２下で６０分振蕩培養後、洗浄を３回行い、５ｘ１０３／１００μｌに調整して９６穴プレート（COSTER 3595-96 well）の各ウエルに１００μｌずつ添加した。
エフェクター細胞として、実施例１と同様に、ＳＯＸ６由来ペプチドＳＯＸ６−４４７の刺激により樹立したＣＴＬ−ＳＯＸ６−４４７を準備した。
E/T ratio は30:1（エフェクター細胞は1.5x105/100μl）及び15:1（エフェクター細胞は7.5x104/100μl）とし、それぞれの数のエフェクター細胞を、９６穴プレート上の５１ＣｒでラベルしたＣＩＲ−Ａ０２０１（ＳＯＸ６−４４７）に添加し、３７℃、５％ＣＯ２下で４時間培養した。
培養後、各ウエルのcpmを計測し、実施例と同様に特異的溶解率（Percentage of specific lysis)を算出した。
== Experimental method ==
As target cells, CIR-A0201 (SOX6-447) pulsed with SOX6-447 peptide at various concentrations (0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM) was prepared. 50 μl of 500 μl of FBS, 51 Cr (1.85 MBq / 50 μl) was added to 5 × 10 6 of these cells, and after shaking culture at 37 ° C. under 5% CO 2 for 60 minutes, washing was performed 3 times, and 5 × 10 3 / 100 μl, and 100 μl was added to each well of a 96-well plate (COSTER 3595-96 well).
As effector cells, as in Example 1, CTL-SOX6-447 established by stimulation with SOX6-derived peptide SOX6-447 was prepared.
The E / T ratio 30: 1 (effector cells 1.5 × 10 5/100 [mu] l) and 15: 1 (effector cells 7.5 × 10 4/100 [mu] l) and, respectively, of the number of effector cells with 51 Cr on 96-well plates was added to CIR-A0201 were labeled (SOX6-447), 37 ℃, and incubated for 4 hours under 5% CO 2.
After culturing, the cpm of each well was measured, and the specific lysis rate (Percentage of specific lysis) was calculated in the same manner as in the Examples.
＝＝結果＝＝
図４に示すように、ＣＴＬ−ＳＯＸ６−４４７は、ＳＯＸ６−４４７ペプチド濃度０．１ｎＭ以上でパルスした場合に、ペプチド濃度依存性にＣＩＲ−Ａ０２０１を傷害した。この結果はE/T ratio=15とE/T ratio=30において同様であった。このように、ペプチド濃度０．１ｎＭ以上の場合、ＣＴＬ−ＳＯＸ６−４４７はＳＯＸ６−４４７／ＨＬＡ−Ａ０２０１複合体を認識して標的細胞を傷害し得た。
== Result ==
As shown in FIG. 4, CTL-SOX6-447 damaged CIR-A0201 in a peptide concentration-dependent manner when pulsed at a SOX6-447 peptide concentration of 0.1 nM or higher. This result was the same when E / T ratio = 15 and E / T ratio = 30. Thus, when the peptide concentration was 0.1 nM or higher, CTL-SOX6-447 was able to recognize the SOX6-447 / HLA-A0201 complex and damage target cells.
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| WO2003018629A1 (en) * | 2001-08-23 | 2003-03-06 | In2Gen Co., Ltd. | Cage antigen |
| WO2006077941A1 (en) * | 2005-01-19 | 2006-07-27 | Keio University | Cancer vaccine |
| JP4840858B2 (en) * | 2006-03-16 | 2011-12-21 | 学校法人慶應義塾 | Cancer vaccine |
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| WO2009072555A1 (en) | 2009-06-11 |
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