JP4925033B2 - Stress-induced anti-apoptotic molecule (IEX-1) derived peptide - Google Patents
Stress-induced anti-apoptotic molecule (IEX-1) derived peptide Download PDFInfo
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- JP4925033B2 JP4925033B2 JP2005517705A JP2005517705A JP4925033B2 JP 4925033 B2 JP4925033 B2 JP 4925033B2 JP 2005517705 A JP2005517705 A JP 2005517705A JP 2005517705 A JP2005517705 A JP 2005517705A JP 4925033 B2 JP4925033 B2 JP 4925033B2
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Description
本発明は、癌患者に対する免疫療法に利用可能なストレス誘導抗アポトーシス分子（ＩＥＸ−１）由来ペプチドに関する。詳細には、細胞傷害性Ｔ細胞（ＣＴＬ）により認識され、特異的なＣＴＬを誘導する、ＩＥＸ−１由来ペプチド、該ペプチドを含むポリペプチドおよびこれらペプチドを含む癌ワクチン等に関する。 The present invention relates to a stress-induced anti-apoptotic molecule (IEX-1) -derived peptide that can be used in immunotherapy for cancer patients. Specifically, the present invention relates to an IEX-1-derived peptide that is recognized by cytotoxic T cells (CTL) and induces a specific CTL, a polypeptide containing the peptide, a cancer vaccine containing the peptide, and the like.
胃癌は、世界で最も普遍的に発生する悪性腫瘍の一つである（文献１−６）。本疾患は、早期では予後は一般的に良好であるが、進行癌においては、外科的切除、化学療法、および放射線療法などの既存の治療法が最近顕著に進歩しているにも係らず、予後が非常に悪い。それゆえ、進行段階の胃癌患者を処置するため、新しい特異的免疫療法のような新規な治療法の開発が必要とされている。 Gastric cancer is one of the most universally occurring malignant tumors in the world (References 1-6). The disease has a generally good prognosis at an early stage, but in advanced cancer, despite recent advances in existing therapies such as surgical resection, chemotherapy, and radiation therapy, Prognosis is very bad. Therefore, there is a need to develop new therapies such as new specific immunotherapy to treat patients with advanced stage gastric cancer.
近年、ヒト腫瘍がＣＴＬに認識される抗原性ペプチドを発現していることが明らかになってきており、このようなペプチドはＨＬＡ−Ａ２またはＡ２４アレルを有する癌患者に対するペプチドワクチンとして使用されている（文献１−７）。ＨＬＡ−Ａ２またはＡ２４拘束性ＣＴＬにより認識されるエピトープペプチドについては多くの報告がなされており（文献８−１１）、本発明者らも、最近、ペプチドワクチン処置がＨＬＡ−Ａ２またはＡ２４重症胃癌患者の全体的生存率を延長することを報告した（文献４）。
一方、ＨＬＡ−Ａ３３アレルは世界中の様々な人種において比較的広く発現しているにも拘わらず（文献１４、１５）、ＨＬＡ−Ａ３３拘束性ＣＴＬにより認識される抗原およびペプチドについての報告は非常に限られている（文献１２、１３）。このことは、ＨＬＡ−Ａ３３＋癌患者に対するペプチド基盤特異的免疫療法の開発を妨げている。In recent years, it has become clear that human tumors express antigenic peptides recognized by CTLs, and such peptides are used as peptide vaccines for cancer patients with HLA-A2 or A24 alleles. (References 1-7). Many reports have been made on epitope peptides recognized by HLA-A2 or A24-restricted CTL (References 8-11), and the present inventors have recently also been treated with peptide vaccine treatment for patients with severe gastric cancer of HLA-A2 or A24. Reported an increase in the overall survival rate (Reference 4).
On the other hand, despite the relatively wide expression of HLA-A33 alleles in various races around the world (References 14 and 15), there are reports on antigens and peptides recognized by HLA-A33-restricted CTLs. Very limited (12, 13). This has hindered the development of peptide-based specific immunotherapy for HLA-A33 + cancer patients.
ＩＥＸ−１（immediate early response gene X-1)(ｐ２２／ＰＲＧ１（文献２０）、Ｄｉｆ−２（文献２１）、またはマウスホモログｇｙｌ９６（文献２２）としてもまた知られる）は、ストレス誘導遺伝子であり、細胞周期進行およびアポトーシスの調節に関与する。ＩＥＸ−１は、ＴＮＦおよびＦａｓのような様々なアポトーシス誘発因子により誘導されるアポトーシスに対する細胞抵抗性に重要な役割を果たすこと（文献２３）、およびいくつかの細胞株において細胞周期進行を加速すること（文献２４−２６）が報告されている。また、in vivoにおけるＴリンパ球の活性化誘導細胞死に対するＩＥＸ−１の抗アポトーシス効果も報告されている（文献２７）。
ＩＥＸ−１の発現は、放射線照射、成長因子、ウィルス感染、ＴＮＦ−αおよびＩＬ−１βのような炎症性サイトカイン、リポポリサッカライド、およびステロイドホルモンを含むいくつかの細胞ストレスによって急速に活性化され得る（文献２７）。ＩＥＸ−１はもともとＮＦ−κＢ／ｒeｌ標的遺伝子として同定されたが（文献２３）、ＩＥＸ−１プロモーターには例えばｐ５３、ＳＰ−１およびｃ−Ｍｙｃのような他の転写因子のためのコンセンサス配列がいくつか含まれる（文献７）。例えば、腫瘍細胞に共通する癌抑制遺伝子ｐ５３の変異がＩＥＸ−１発現を上昇させることが示されている（文献２９）。
以上のように、ＩＥＸ−１は腫瘍細胞の悪性形質転換に関与することが示唆されているが、腫瘍細胞において抗原性ペプチドとして機能するＩＥＸ−１エピトープペプチドは知られていなかった。IEX-1 (immediate early response gene X-1) (also known as p22 / PRG1 (Reference 20), Dif-2 (Reference 21), or mouse homolog gyl96 (Reference 22)) is a stress-inducing gene. Involved in the regulation of cell cycle progression and apoptosis. IEX-1 plays an important role in cell resistance to apoptosis induced by various pro-apoptotic factors such as TNF and Fas (Ref. 23) and accelerates cell cycle progression in several cell lines (References 24-26) have been reported. In addition, the anti-apoptotic effect of IEX-1 on T-lymphocyte activation-induced cell death in vivo has been reported (Reference 27).
IEX-1 expression is rapidly activated by several cellular stresses including irradiation, growth factors, viral infections, inflammatory cytokines such as TNF-α and IL-1β, lipopolysaccharides, and steroid hormones. Obtain (Reference 27). Although IEX-1 was originally identified as an NF-κB / rel target gene (Reference 23), the IEX-1 promoter contains consensus sequences for other transcription factors such as p53, SP-1 and c-Myc. Are included (Reference 7). For example, it has been shown that a mutation in the tumor suppressor gene p53 common to tumor cells increases IEX-1 expression (Reference 29).
As described above, it has been suggested that IEX-1 is involved in malignant transformation of tumor cells, but no IEX-1 epitope peptide that functions as an antigenic peptide in tumor cells has been known.
本発明は、ＨＬＡ−Ａ３３＋癌患者に対する癌ワクチンの開発に有用な抗原ペプチドを提供することを目的とする。An object of the present invention is to provide an antigenic peptide useful for developing a cancer vaccine for HLA-A33 + cancer patients.
本発明者らは、重症胃癌患者の腫瘍組織浸潤リンパ球（ＴＩＬ）からＨＬＡ−Ａ３３拘束性ＣＴＬ株を樹立し、ＩＥＸ−１由来の三つのＨＬＡ−Ａ３３結合エピトープを、本細胞株により認識される腫瘍抗原として同定した。さらに、これらの抗原性エピトープが悪性腫瘍疾患患者のＰＢＭＣ（末梢血単核細胞）においてペプチド特異的ＣＴＬを誘導することを確認したことにより、本発明を完成した。 The present inventors established an HLA-A33-restricted CTL line from tumor tissue-infiltrating lymphocytes (TIL) of patients with severe gastric cancer, and three HLA-A33 binding epitopes derived from IEX-1 were recognized by this cell line. Identified as a tumor antigen. Furthermore, the present invention was completed by confirming that these antigenic epitopes induce peptide-specific CTLs in PBMC (peripheral blood mononuclear cells) of patients with malignant tumor diseases.
即ち本発明は、以下を包含する。
（１）細胞傷害性Ｔ細胞（ＣＴＬ）によって認識され、特異的なＣＴＬを誘導する、ストレス誘導抗アポトーシス分子（ＩＥＸ−１）由来のペプチドまたはその変異ペプチド。
（２）ＣＴＬがＨＬＡ−Ａ３３拘束性に認識する、（１）記載のペプチド。
（３）連続する８〜１１個のアミノ酸残基からなる、（１）または（２）に記載のペプチド。
（４）配列番号１〜３のいずれかに示されるアミノ酸配列からなるペプチド、または配列番号１〜３のいずれかに示されるアミノ酸配列において１若しくは複数のアミノ酸が欠失、置換および/または付加されたアミノ酸配列からなり、かつＨＬＡ−Ａ３３分子と結合して特異的なＣＴＬを誘導するペプチドである、（１）〜（３）のいずれかに記載のペプチド。
（５）上記（１）〜（４）のいずれかに記載のペプチドを含むポリペプチド。
（６）単離された抗原提示能を有する細胞の表面に、ＨＬＡ−Ａ３３分子と（１）〜（４）のいずれかに記載のペプチドとの複合体を提示させてなる、抗原提示細胞。
（７）上記（１）〜（４）のいずれかに記載のペプチド、または（５）記載のポリペプチドをコードする核酸分子。
（８）上記（７）記載の核酸分子を含有するベクター。
（９）上記（１）〜（４）のいずれかに記載のペプチド、（５）記載のポリペプチド、（６）記載の抗原提示細胞、（７）記載の核酸分子、または（８）記載のベクターを含む、特異的なＣＴＬを誘導するための医薬組成物。
（１０）癌ワクチンである、（９）記載の医薬組成物。
（１１）上記（１）〜（４）のいずれかに記載のペプチドとＨＬＡとの複合体、または（６）記載の抗原提示細胞に提示された複合体を認識する、ＩＥＸ−１反応性ＣＴＬ。
（１２）上記（１）〜（４）のいずれかに記載のペプチド、（５）記載のポリペプチド、または（６）記載の抗原提示細胞を用いてＩＥＸ−１反応性ＣＴＬを誘導する方法。
（１３）上記（１）〜（４）のいずれかに記載のペプチドまたは（５）記載のポリペプチドを特異的に認識する抗体。
（１４）次の１）または２）に記載のポリペプチドを含む、特異的なＣＴＬを誘導するための医薬組成物：
１）配列番号４に示されるアミノ酸配列からなるポリペプチド、または
２）配列番号４に示されるアミノ酸配列において１若しくは複数のアミノ酸が欠失、置換および/または付加されたアミノ酸配列からなり、かつＨＬＡ−Ａ３３分子と結合して特異的なＣＴＬを誘導するペプチドを与えるポリペプチド。
（１５）癌ワクチンである、（１４）記載の医薬組成物。That is, the present invention includes the following.
(1) A peptide derived from a stress-induced anti-apoptotic molecule (IEX-1) or a mutant peptide thereof that is recognized by cytotoxic T cells (CTL) and induces a specific CTL.
(2) The peptide according to (1), wherein CTL recognizes HLA-A33 restricted.
(3) The peptide according to (1) or (2), comprising 8 to 11 consecutive amino acid residues.
(4) A peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3, or one or more amino acids in the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 are deleted, substituted and / or added The peptide according to any one of (1) to (3), which is a peptide that has a specific amino acid sequence and induces a specific CTL by binding to an HLA-A33 molecule.
(5) A polypeptide comprising the peptide according to any one of (1) to (4) above.
(6) An antigen-presenting cell obtained by presenting a complex of the HLA-A33 molecule and the peptide according to any one of (1) to (4) on the surface of an isolated cell having an antigen-presenting ability.
(7) A nucleic acid molecule encoding the peptide according to any one of (1) to (4) above or the polypeptide according to (5).
(8) A vector containing the nucleic acid molecule according to (7) above.
(9) The peptide according to any one of (1) to (4), the polypeptide according to (5), the antigen-presenting cell according to (6), the nucleic acid molecule according to (7), or the description according to (8) A pharmaceutical composition for inducing specific CTL, comprising a vector.
(10) The pharmaceutical composition according to (9), which is a cancer vaccine.
(11) An IEX-1 reactive CTL that recognizes a complex of the peptide according to any one of (1) to (4) above and HLA, or a complex presented on an antigen-presenting cell according to (6) .
(12) A method for inducing IEX-1-reactive CTL using the peptide according to any one of (1) to (4), the polypeptide according to (5), or the antigen-presenting cell according to (6).
(13) An antibody that specifically recognizes the peptide according to any one of (1) to (4) or the polypeptide according to (5).
(14) A pharmaceutical composition for inducing specific CTL, comprising the polypeptide according to the following 1) or 2):
1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4, or 2) an amino acid sequence comprising one or more amino acids deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4, and HLA A polypeptide that gives a peptide that binds to the A33 molecule and induces a specific CTL.
(15) The pharmaceutical composition according to (14), which is a cancer vaccine.
本発明は、悪性腫瘍、特にＨＬＡ−Ａ３３陽性の癌患者の治療に適したＩＥＸ−１基盤免疫療法を可能にする。 The present invention enables IEX-1-based immunotherapy suitable for the treatment of malignant tumors, particularly HLA-A33 positive cancer patients.
本発明についてさらに詳細に説明する。
ＨＬＡ拘束性ＣＴＬ株
ＨＬＡ拘束性ＣＴＬ株は、目的とするＨＬＡアレルを有する癌患者の腫瘍組織に浸潤しているリンパ球（ＴＩＬ）を採取し、Ｔ細胞の増殖を促すサイトカインであるＩＬ−２と共に培養して増殖させることにより得られる。本方法は当業界において周知である。
遺伝子発現クローニング法
ＨＬＡ拘束性ＣＴＬ株によって認識される腫瘍抗原は、遺伝子発現クローニング法により同定する。本方法は、ｃＤＮＡライブラリーにコードされるタンパク質を哺乳類細胞で一過性に発現させ、樹立したＣＴＬ株が特異的に認識する目的のタンパク質をコードするｃＤＮＡをスクリーニングし、当該タンパク質をコードする遺伝子を単離するものである。本発明では、樹立したＣＴＬ株がタンパク質発現細胞を認識した際に産生するＩＦＮ−γを指標としてスクリーニングを行う。The present invention will be described in further detail.
HLA-restricted CTL line HLA-restricted CTL line is IL-2 which is a cytokine that collects lymphocytes (TIL) infiltrating tumor tissues of cancer patients having the target HLA allele and promotes T cell proliferation. It is obtained by culturing and growing together. This method is well known in the art.
Gene Expression Cloning Method Tumor antigens recognized by HLA restricted CTL lines are identified by gene expression cloning methods. In this method, the protein encoded by the cDNA library is transiently expressed in mammalian cells, the cDNA encoding the target protein specifically recognized by the established CTL line is screened, and the gene encoding the protein Is to be isolated. In the present invention, screening is performed using IFN-γ produced when an established CTL line recognizes a protein-expressing cell as an index.
ペプチドおよびポリペプチド
本発明のＩＥＸ−１由来ペプチドは、ＨＬＡと結合して特異的ＣＴＬを誘導しうるペプチドである。本発明ペプチドはＩＥＸ−１の連続する８〜１１個のアミノ酸残基からなることが好ましい。このようなＩＥＸ−１の部分ペプチドの具体例として、ＩＥＸ４７−５６、（配列番号：１）、ＩＥＸ６１−６９（配列番号：２）、またはＩＥＸ６５−７３（配列番号：３）が挙げられる。ＩＥＸ−１遺伝子の全ヌクレオチド配列および推定のアミノ酸配列は、受入番号ＮＭ＿００３８９７としてGeneBankに登録されている（配列番号：４）。特異的なＣＴＬを誘導する他のＩＥＸ−１由来ペプチドは、本明細書の実施例に準じた方法により容易に決定および選択できる。 Peptides and polypeptides The IEX-1-derived peptides of the present invention are peptides that can bind to HLA and induce specific CTLs. The peptide of the present invention preferably comprises 8 to 11 consecutive amino acid residues of IEX-1. Specific examples of such a partial peptide of IEX-1 include IEX 47-56, (SEQ ID NO: 1), IEX 61-69 (SEQ ID NO: 2), or IEX 65-73 (SEQ ID NO: 3). The entire nucleotide sequence and deduced amino acid sequence of the IEX-1 gene are registered in GeneBank as accession number NM_003897 (SEQ ID NO: 4). Other IEX-1-derived peptides that induce specific CTLs can be readily determined and selected by methods according to the examples herein.
「ＨＬＡと結合して特異的なＣＴＬを誘導しうる」とは、本発明のペプチドがＨＬＡと結合して複合体を形成し、かかる複合体をＣＴＬが認識できることをいう。換言すれば、本発明のペプチドが、ＨＬＡとの結合活性を有し、かつ、ＨＬＡとの複合体の形で、ペプチド特異的なＣＴＬを誘導する活性を有することを意味する。本発明において好ましいＨＬＡは、ＨＬＡ−Ａ３３である。 The phrase “can bind to HLA to induce specific CTL” means that the peptide of the present invention binds to HLA to form a complex, and CTL can recognize the complex. In other words, it means that the peptide of the present invention has an activity of inducing peptide-specific CTL in the form of a complex with HLA, as well as binding activity with HLA. A preferred HLA in the present invention is HLA-A33.
また、本発明は、ＨＬＡと結合して特異的なＣＴＬを誘導しうるＩＥＸ−１由来ペプチドの変異ペプチドであって、同等のＣＴＬ誘導能を有する変異ペプチドも包含する。変異は、本発明のＩＥＸ−１由来ペプチドに対して一個または数個のアミノ酸の欠失、置換、付加、挿入などを行うことにより導入することができ、その手段は当業界にて周知である。変異が他のアミノ酸による置換または付加を含む場合、他のアミノ酸は、天然のアミノ酸またはアミノ酸アナログであってよく、アミノ酸アナログとしては、種々のアミノ酸のＮ−アシル化物、Ｏ−アシル化物、エステル化物、酸アミド化物、アルキル化物等が挙げられる。本発明のペプチドの変異体を得るには、例えば、配列番号１〜３に記載のアミノ酸配列の１〜数個、好ましくは１〜４個、より好ましくは１〜３個、さらに好ましくは１個または２個のアミノ酸残基が欠失しているか、他のアミノ酸残基またはアミノ酸アナログで置換された、またはそれが付加された候補ペプチドを合成し、該候補ペプチドとＨＬＡ−Ａ３３分子との複合体がＣＴＬにより認識されるか否かをアッセイすることにより、同定することができる。 The present invention also includes a mutant peptide of an IEX-1-derived peptide capable of inducing specific CTL by binding to HLA, and having a similar CTL inducing ability. Mutations can be introduced into the IEX-1-derived peptide of the present invention by deletion, substitution, addition, insertion, or the like of one or several amino acids, and the means are well known in the art. . When the mutation includes substitution or addition with other amino acids, the other amino acids may be natural amino acids or amino acid analogs, and examples of amino acid analogs include N-acylated products, O-acylated products, and esterified products of various amino acids. , Acid amidated products, alkylated products and the like. In order to obtain a variant of the peptide of the present invention, for example, 1 to several amino acid sequences represented by SEQ ID NOs: 1 to 3, preferably 1 to 4, more preferably 1 to 3, more preferably 1 Alternatively, a candidate peptide in which two amino acid residues are deleted, substituted with another amino acid residue or amino acid analog, or added thereto is synthesized, and the candidate peptide is combined with the HLA-A33 molecule. It can be identified by assaying whether the body is recognized by CTL.
アッセイは例えば、後述するＣＴＬ誘導法に準じて行うことができる。即ち、in vitroで候補ペプチドを添加して刺激した場合に、該候補ペプチドをパルスしたＨＬＡ−Ａ３３陽性細胞を特異的に認識するＣＴＬが誘導されるか否かを調べる。ここで、ＣＴＬ誘導の有無は、例えば、抗原ペプチド提示細胞に反応してＣＴＬが産生する種々のサイトカイン（IFN-γ等）の量を酵素免疫測定法（ELISA）等により測定することによって調べることができる。または、51Crで標識した抗原ペプチド提示細胞に対するＣＴＬの傷害性を測定する方法（51Cr リリースアッセイ、Int．J．Cancer，58:p317,1994）によっても調べることができる。前記アッセイで用いるＨＬＡ−Ａ３３陽性細胞としては、実施例に記載のＨＬＡ−Ａ３３陽性細胞が挙げられる。
変異ペプチドのアミノ酸残基数は、抗原提示細胞表面上に提示され、かつＣＴＬ認識エピトープとしての性質を有する数であればよく、通常少なくとも約８個以上であり、好ましくは約９個以上であって、１２個以下、好ましくは１１以下、さらに好ましくは１０個以下である。特に好ましアミノ酸残基数は９個ないし１０個である。The assay can be performed, for example, according to the CTL induction method described below. That is, when a candidate peptide is added and stimulated in vitro, it is examined whether CTL that specifically recognizes HLA-A33 positive cells pulsed with the candidate peptide is induced. Here, the presence or absence of CTL induction is examined, for example, by measuring the amount of various cytokines (IFN-γ, etc.) produced by CTL in response to antigen peptide-presenting cells by enzyme immunoassay (ELISA) or the like. Can do. Or a method of measuring the cytotoxicity of CTL against labeled antigen peptide presenting cells in 51 Cr (51 Cr release assay, Int.J.Cancer, 58: p317,1994) can be examined by. Examples of HLA-A33 positive cells used in the assay include HLA-A33 positive cells described in Examples.
The number of amino acid residues of the mutant peptide may be any number as long as it is displayed on the antigen-presenting cell surface and has a property as a CTL recognition epitope, and is usually at least about 8 and preferably about 9 or more. 12 or less, preferably 11 or less, and more preferably 10 or less. Particularly preferred is 9 to 10 amino acid residues.
本発明はさらに、特異的なＣＴＬの誘導活性を有する、本発明のＩＥＸ−１由来ペプチドまたはその変異ペプチドを含有するポリペプチドを包含する。ポリペプチドを構成するアミノ酸数は、特に限定されず、本発明が属する分野での技術常識に従う。通常、アミノ酸残基数約１００個以下の長さであり、好ましくは約５０個以下、より好ましくは約３０個以下程度である。
本発明のポリペプチドはＨＬＡ−Ａ３３陽性細胞内で断片化されて特異的ＣＴＬ誘導活性を有するペプチド断片を与えることができるものである。そのようなポリペプチドは、配列番号：１、配列番号：２、または配列番号：３で示される本発明のペプチドに相当する部分配列、または本発明ペプチドの変異体に相当する配列を含有することが特に好ましい。The present invention further includes a polypeptide containing the IEX-1-derived peptide of the present invention or a mutant peptide thereof having specific CTL inducing activity. The number of amino acids constituting the polypeptide is not particularly limited, and follows the common general technical knowledge in the field to which the present invention belongs. Usually, the length is about 100 or less amino acid residues, preferably about 50 or less, more preferably about 30 or less.
The polypeptide of the present invention can be fragmented in HLA-A33 positive cells to give a peptide fragment having specific CTL inducing activity. Such a polypeptide contains a partial sequence corresponding to the peptide of the present invention represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a sequence corresponding to a variant of the peptide of the present invention. Is particularly preferred.
また、機能を著しく障害しない程度に構成アミノ酸またはカルボキシル基などを修飾して、本発明のペプチドおよびポリペプチドを改変することもできる。例えば、Ｎ末端や遊離のアミノ基には、ホルミル基、アセチル基、ｔ−ブトキシカルボニル（ｔ−Ｂｏｃ）基等が結合していてもよく、抗原ペプチドのＣ末端や遊離のカルボキシル基には、メチル基、エチル基、ｔ−ブチル基、ベンジル基等が結合していてもよい。さらに、本発明のペプチドは、生体内への導入を容易にするように、修飾されていてもよい。
一般に、ＨＬＡ分子と結合する腫瘍抗原 ペプチドのアミノ酸配列には、ＨＬＡの型により異なるモチーフ（規則的配列）が存在することが知られており、変異または改変は、そのモチーフ上、許容されるものであることが好ましい。変異の導入において、ペプチドまたはポリペプチドの基本的な性質（物性、機能、生理活性または免疫学的活性等）を変化させないという観点からは、例えば、同族アミノ酸（極性アミノ酸、非極性アミノ酸、疎水性アミノ酸、親水性アミノ酸、陽性荷電アミノ酸、陰性荷電アミノ酸および芳香族アミノ酸等）の間での相互置換は容易に想定される。
本発明のペプチドおよびポリペプチドは、ペプチド化学における一般的な公知方法により製造できる。Furthermore, the peptides and polypeptides of the present invention can be altered by modifying the constituent amino acids or carboxyl groups to such an extent that the function is not significantly impaired. For example, a formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group or the like may be bonded to the N-terminus or a free amino group, and the C-terminus or free carboxyl group of an antigen peptide may be A methyl group, an ethyl group, a t-butyl group, a benzyl group or the like may be bonded. Furthermore, the peptide of the present invention may be modified so as to facilitate introduction into the living body.
In general, it is known that there are different motifs (regular sequences) depending on the type of HLA in the amino acid sequences of tumor antigen peptides that bind to HLA molecules, and mutations or modifications are permitted on the motifs. It is preferable that From the viewpoint of not changing the basic properties (physical properties, functions, physiological activities or immunological activities, etc.) of peptides or polypeptides in the introduction of mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobicity) Mutual substitution between amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned.
The peptides and polypeptides of the present invention can be produced by general known methods in peptide chemistry.
「抗原提示細胞（ＡＰＣ）」とは、ＨＬＡと抗原ペプチドとの複合体を細胞表面に提示する細胞を意味する。従って、抗原提示細胞は、本発明の腫瘍抗原ペプチドとＨＬＡ−Ａ３３分子との複合体を細胞表面に提示することにより、ＨＬＡ−Ａ３３拘束性ＣＴＬの活性化をもたらす機能を有する。そのような細胞には、ＣＴＬ細胞障害作用の標的である腫瘍細胞も含まれる。
本発明の抗原提示細胞は、単離された抗原提示能を有する細胞の表面に、ＨＬＡ−Ａ３３分子と本発明の腫瘍抗原ペプチドとの複合体を提示している。そのような細胞は、in vitroで誘導することもでき、具体的には、ＨＬＡ−Ａ３３陽性の抗原提示能を有する細胞に本発明の腫瘍抗原ペプチドをパルスしてＨＬＡ−Ａ３３と該ペプチドとの複合体をその細胞表面に提示させることにより得られる。「抗原提示能を有する細胞」とは、本発明の腫瘍抗原ペプチドを提示可能なＨＬＡ-Ａ２４抗原を細胞表面に発現している細胞であれば特に限定されないが、特に抗原提示能が高いとされる樹状細胞が好ましい。特に、ＨＬＡ−Ａ３３陽性腫瘍患者由来の単離された抗原提示能を有する細胞の表面に、ＨＬＡ−Ａ３３分子と本発明の腫瘍抗原ペプチドとの複合体を提示させた抗原提示細胞が好ましい。“Antigen-presenting cell (APC)” means a cell that presents a complex of HLA and an antigen peptide on the cell surface. Therefore, the antigen-presenting cell has a function of bringing about activation of HLA-A33-restricted CTL by presenting the complex of the tumor antigen peptide of the present invention and the HLA-A33 molecule on the cell surface. Such cells also include tumor cells that are targets for CTL cytotoxic effects.
The antigen-presenting cell of the present invention presents a complex of the HLA-A33 molecule and the tumor antigen peptide of the present invention on the surface of an isolated cell having the ability to present antigen. Such cells can also be induced in vitro. Specifically, cells having the antigen-presenting ability of HLA-A33 positive are pulsed with the tumor antigen peptide of the present invention, and HLA-A33 and the peptide are used together. It is obtained by presenting the complex on the cell surface. The “cell having antigen-presenting ability” is not particularly limited as long as it is a cell that expresses the HLA-A24 antigen capable of presenting the tumor antigen peptide of the present invention on the cell surface. Dendritic cells are preferred. In particular, antigen-presenting cells in which a complex of an HLA-A33 molecule and the tumor antigen peptide of the present invention is presented on the surface of an isolated cell having an antigen-presenting ability derived from an HLA-A33-positive tumor patient is preferable.
核酸分子
本発明の核酸分子は、本発明のＩＥＸ−１由来ペプチドまたはその変異ペプチドおよび該ぺプチドを含むポリペプチドの、アミノ酸配列をコードする一本鎖（相補鎖を含む）および二本鎖ポリヌクレオチドを含む。本発明の核酸分子はＤＮＡであってもＲＮＡであってもよい。これら核酸分子がコードするアミノ酸配列を有するペプチドは、それ自体がＣＴＬにより認識され、該ＣＴＬを活性化するか、そのような活性を有するペプチド断片を与えることができ、腫瘍抗原として機能し得る。
また、本発明の核酸分子は、本発明のペプチドをコードする領域に対応する少なくとも２４個以上の塩基からなるポリヌクレオチドおよびその相補鎖であってよい。このようなポリヌクレオチドは、例えば公知のタンパク質発現系を利用して発現ペプチドを確認することにより選択できる。 Nucleic Acid Molecules The nucleic acid molecules of the present invention comprise single-stranded (including complementary strands) and double-stranded polypec- tides encoding amino acid sequences of the IEX-1-derived peptide of the present invention or a mutant peptide thereof and a polypeptide comprising the peptide. Contains nucleotides. The nucleic acid molecule of the present invention may be DNA or RNA. Peptides having the amino acid sequence encoded by these nucleic acid molecules are recognized by CTLs themselves and can activate the CTLs or give peptide fragments having such activities, and can function as tumor antigens.
Further, the nucleic acid molecule of the present invention may be a polynucleotide comprising at least 24 bases corresponding to the region encoding the peptide of the present invention and its complementary strand. Such a polynucleotide can be selected, for example, by confirming the expressed peptide using a known protein expression system.
抗体
本発明の抗体は、モノクローナル抗体またはポリクローナル抗体のいずれであってもよい。本発明のＩＥＸ−１由来ペプチドまたはポリペプチドの中から選ばれる１つのペプチドまたはポリペプチドのアミノ酸配列中、連続する少なくとも５個のアミノ酸残基からなるエピトープペプチドまたはポリペプチドを特異的に認識するものである。
抗体はそれらのエピトープペプチドを使用して作製でき、そのエピトープペプチドは少なくとも５個、好ましくは少なくとも８〜１０個のアミノ酸で構成される。本発明は、この少なくとも５個のアミノ酸残基からなるペプチドおよびそれをコードする核酸分子も包含する。
本発明の抗体は、エピトープペプチドを単独で、または担体と結合した形で、アジュバントの存在または非存在下に例えばマウス、ラット、ウサギ、ヤギ等に免疫し産生を誘導することができる。得られたポリクローナル抗体は、公知の方法により血清から回収することができる。
一方、モノクローナル抗体は、上記のように免疫応答を誘導した動物から回収した抗体産生細胞を、永久増殖性細胞と融合することで生産できる。本方法は当業界において周知である。
これらのポリクローナル抗体およびモノクローナル抗体は、精製用抗体、試薬、標識マーカー等として利用することができる。また、ヒト型化して治療用に供する場合もあり得る。 Antibody The antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody. A peptide or polypeptide that specifically recognizes an epitope peptide or polypeptide comprising at least 5 consecutive amino acid residues in the amino acid sequence of one peptide or polypeptide selected from the IEX-1-derived peptides or polypeptides of the present invention It is.
Antibodies can be generated using these epitope peptides, which epitope peptides are composed of at least 5, preferably at least 8-10 amino acids. The present invention also includes a peptide consisting of at least 5 amino acid residues and a nucleic acid molecule encoding the peptide.
The antibody of the present invention can induce production by immunizing, for example, mice, rats, rabbits, goats and the like, in the presence or absence of an adjuvant, with the epitope peptide alone or in a form bound to a carrier. The obtained polyclonal antibody can be recovered from the serum by a known method.
On the other hand, monoclonal antibodies can be produced by fusing antibody-producing cells collected from animals that have induced immune responses as described above with permanently proliferating cells. This method is well known in the art.
These polyclonal and monoclonal antibodies can be used as purifying antibodies, reagents, labeling markers and the like. Moreover, it may be humanized and used for treatment.
医薬組成物
本発明の医薬組成物は、本発明のＩＥＸ−１由来ペプチド、変異体ペプチド、ポリペプチド、ＨＬＡ分子とペプチドとの複合体を細胞表面に提示している抗原提示細胞、ペプチドまたはポリペプチドをコードする核酸分子、該核酸分子の塩基配列情報に基づき作製した組換えベクター、または本発明の抗体を、単独または複数組み合わせて利用することにより調製できる。
具体的には、本発明のＩＥＸ−１由来ペプチドまたはその変異体、該ペプチドを含むポリペプチド、抗原提示細胞は癌ワクチンとして使用することができる。一種類のペプチドでも癌ワクチンとして有効であるが、複数種類のペプチドを組み合わせて使用するのが好ましい。これは、癌患者のＣＴＬが複数の異なる種類の腫瘍抗原を認識する細胞の集団であることから、複数種類の腫瘍抗原を組み合わせて癌ワクチンとして使用する方がより効果的であると期待されるからである。本発明に係るペプチド等を他のペプチドと共に複数種類組み合わせて使用してもよい。 Pharmaceutical Composition The pharmaceutical composition of the present invention comprises an IEX-1-derived peptide, mutant peptide, polypeptide, antigen-presenting cell, peptide or polyporoid presenting a complex of an HLA molecule and a peptide on the cell surface. It can be prepared by using a nucleic acid molecule encoding a peptide, a recombinant vector prepared based on the nucleotide sequence information of the nucleic acid molecule, or an antibody of the present invention alone or in combination.
Specifically, the IEX-1-derived peptide of the present invention or a variant thereof, a polypeptide containing the peptide, and antigen-presenting cells can be used as a cancer vaccine. Although one type of peptide is effective as a cancer vaccine, it is preferable to use a combination of multiple types of peptides. This is because the CTL of a cancer patient is a population of cells that recognize a plurality of different types of tumor antigens, and it is expected that it is more effective to use a combination of a plurality of types of tumor antigens as a cancer vaccine. Because. A plurality of types of peptides according to the present invention may be used in combination with other peptides.
本発明の癌ワクチンは、適当なアジュバントの存在または非存在下で、単独で、または製薬的に許容される担体と結合して使用することができる。担体は、人体に有害な作用を起こさない限り限定されるものではなく、例えば、セルロース、重合アミノ酸、アルブミン等が使用できる。剤形は、ペプチド製剤について周知の剤形が選択可能である。投与量は、ＣＴＬによる認識性、治療すべき疾患、患者の年齢、体重等により変化するが、ペプチドの場合、活性本体として、通常、0.0001mg〜1000mg、好ましくは0.001mg〜1000mg、より好ましくは0.1mg〜100mg、さらに好ましくは0.1〜10ｍｇ／日／成人ヒトである。これを数日ないし数周あるいは数ヶ月に一回投与する。 The cancer vaccine of the present invention can be used alone or in combination with a pharmaceutically acceptable carrier in the presence or absence of a suitable adjuvant. The carrier is not limited as long as it does not adversely affect the human body. For example, cellulose, polymerized amino acid, albumin and the like can be used. As the dosage form, a known dosage form for the peptide preparation can be selected. The dose varies depending on the recognizability by CTL, the disease to be treated, the age of the patient, the body weight, etc. In the case of peptides, the active substance is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 100 mg, more preferably 0.1 to 10 mg / day / adult human. This is administered once every few days or several weeks or months.
本発明の医薬組成物はまた、本発明に係るペプチドをコードする核酸配列を適当なベクターに組み込み、in vivoまたはex vivoで導入するのに利用することができる。ベクターとしては、例えばレトロウィルス、アデノウィルス、ワクシニアウィルス等が挙げられるが、レトロウィルス系が好ましい。投与量は、ＣＴＬによる認識性により変化するが、ＤＮＡ含量として０．１μg-１００ｍｇ／日／成人ヒト、好ましくは１μg−５０ｍｇ／日／成人ヒトである。これを数日ないし数ヶ月に一回投与する。 The pharmaceutical composition of the present invention can also be used to incorporate a nucleic acid sequence encoding the peptide of the present invention into an appropriate vector and introduce it in vivo or ex vivo. Examples of the vector include retrovirus, adenovirus, vaccinia virus and the like, but a retrovirus system is preferable. Although the dose varies depending on the recognition by CTL, the DNA content is 0.1 μg-100 mg / day / adult human, preferably 1 μg-50 mg / day / adult human. This is administered once every few days or months.
ＩＥＸ−１反応性ＣＴＬおよびその誘導方法
「ＩＥＸ−１反応性ＣＴＬ」とは、本発明のＩＥＸ−１由来ペプチドまたはその変異体とＨＬＡとの複合体を認識し、誘導されるＣＴＬを意味する。該ＣＴＬはＨＬＡ−Ａ３３拘束性ＣＴＬである。
そのようなＣＴＬは、例えばＨＬＡ−Ａ３３＋胃癌患者の末梢血単核球（ＰＢＭＣ）から本発明に係るペプチドを用いて誘導することができる。
つまり、本発明のペプチドでパルスした抗原提示細胞（ＡＰＣ）とともにＨＬＡ−Ａ３３＋胃癌患者のＰＢＭＣをインキュベートしてＣＴＬを誘導し、ＩＦＮ-γ産生を指標として評価する。さらに、誘導されたＣＴＬの活性は、51Ｃｒ放出試験等により腫瘍細胞傷害性を指標として確認できる。 The IEX-1-reactive CTL and the induction method “IEX-1-reactive CTL” mean a CTL that recognizes and induces a complex of the IEX-1-derived peptide of the present invention or a variant thereof and HLA. . The CTL is HLA-A33 restricted CTL.
Such CTLs can be derived, for example, from peripheral blood mononuclear cells (PBMC) of HLA-A33 + gastric cancer patients using the peptides according to the present invention.
That is, HLA-A33 + gastric cancer patient's PBMC is incubated with antigen-presenting cells (APC) pulsed with the peptide of the present invention to induce CTL, and IFN-γ production is evaluated as an index. Furthermore, the induced CTL activity can be confirmed by a 51 Cr release test or the like using tumor cytotoxicity as an index.
上記の方法は、in vitroで誘導した抗原特異的ＣＴＬを患者体内に戻し腫瘍細胞を傷害する、養子免疫療法に利用できる。すなわち、メラノーマにおいては、患者本人の腫瘍内浸潤Ｔ細胞を体外で大量に培養して、これを患者に戻す養子免疫療法に治療効果が認められている（J. Natl.Cancer.Inst.,86:1159、1994）。またマウスのメラノーマにおいては、脾細胞をin vitroで腫瘍抗原ペプチドTRP-2で刺激し、腫瘍抗原ペプチドに特異的なＣＴＬを増殖させ、該ＣＴＬをメラノーマ移植マウスに投与することにより、転移抑制が認められている（J. Exp.Med.，185:453, 1997）。これは、抗原提示細胞のＨＬＡ抗原と腫瘍抗原ペプチドとの複合体を特異的に認識するＣＴＬをin vitroで増殖させた結果に基づくものである。本発明の腫瘍抗原ペプチドを用いて、in vitroで患者末梢血リンパ球を刺激して腫瘍特異的ＣＴＬを増やした後、このＣＴＬを患者に戻すことにより腫瘍を治療することが可能である。 The above method can be used for adoptive immunotherapy in which antigen-specific CTLs induced in vitro are returned to the body of a patient to injure tumor cells. That is, in melanoma, a therapeutic effect has been observed in adoptive immunotherapy in which a patient's own tumor infiltrating T cells are cultured in large amounts outside the body and returned to the patient (J. Natl. Cancer. Inst., 86 : 1159, 1994). In mouse melanoma, spleen cells are stimulated with tumor antigen peptide TRP-2 in vitro, CTL specific for the tumor antigen peptide is proliferated, and the CTL is administered to melanoma transplanted mice to suppress metastasis. (J. Exp. Med., 185: 453, 1997). This is based on the result of in vitro proliferation of CTL that specifically recognizes a complex of an antigen-presenting cell HLA antigen and a tumor antigen peptide. The tumor antigen peptide of the present invention is used to stimulate a patient's peripheral blood lymphocytes in vitro to increase tumor-specific CTL, and then return the CTL to the patient to treat the tumor.
以下、本発明を実施例によってより詳細に説明するが、本発明は下記実施例によっていかなる意味においても制限されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited in any meaning by the following Example.
本発明には、以下の癌細胞株を使用した。
胃腺癌 ＭＫＮ−２８、ＭＫＮ−４５、ＳＳＴＷ−９、ＫＡＴＯ−ＩＩＩ、ＫＷＳ、およびＨＧＣ−２７；肺腺癌 ＬＣ−１；肺偏平上皮癌 ＱＧ−５６；頭頸部癌 ＫＵＭＡ−１；大腸腺癌 ＳＷ６２０およびＣＯＬＯ２０１；膵臓腺癌 Ｐａｎｃ−１；ヒト慢性骨髄性白血病 Ｋ５６２。これらの腫瘍細胞のＨＬＡクラスＩ遺伝子型は以前に示されている（文献１０、１１）。
これらの細胞上のＨＬＡクラスＩまたはＨＬＡ−Ａ３３抗原の発現は、抗ＨＬＡクラスＩ（Ｗ６／３２）モノクローナル抗体（ｍＡｂ）（ＨＬＡクラスＩ分子の単一形領域を認識）、または抗ＨＬＡ−Ａ３３ｍＡｂ（ＨＬＡ−Ａ３３分子の多形領域を認識）（ＩｇＭ、One Lamda, Canoga Park, CA）を用いて、ＦＡＣＳｃａｎ（Becton Dickinson, San Jose, CA）でのフローサイトメトリーによって測定した。
統計学的解析には、本発明すべてにおいて両側 Student ｔ検定を用いた。The following cancer cell lines were used in the present invention.
Gastric adenocarcinoma MKN-28, MKN-45, SSTW-9, KATO-III, KWS, and HGC-27; lung adenocarcinoma LC-1; lung squamous cell carcinoma QG-56; head and neck cancer KUMA-1; SW620 and COLO201; pancreatic adenocarcinoma Panc-1; human chronic myeloid leukemia K562. The HLA class I genotype of these tumor cells has been shown previously (10, 11).
The expression of HLA class I or HLA-A33 antigen on these cells is anti-HLA class I (W6 / 32) monoclonal antibody (mAb) (recognizing a single region of the HLA class I molecule), or anti-HLA-A33 mAb. (Recognizing the polymorphic region of the HLA-A33 molecule) (IgM, One Lamda, Canoga Park, Calif.) And measured by flow cytometry on a FACScan (Becton Dickinson, San Jose, Calif.).
For statistical analysis, a two-sided Student t test was used in all of the present invention.
実施例１
（８５０Ｂ−ＣＴＬ株の樹立）
ＨＬＡ−Ａ３３拘束性および腫瘍特異的ＣＬＴ株（８５０Ｂ−ＣＴＬ）は、重症胃腺癌患者（ＨＬＡ−Ａ＊２４０２／Ａ＊３３０３、Ｂ７／Ｂ４４、Ｃｗ７／Ｃｗ１４）のＴＩＬを１０％ＦＣＳ（Equitech Bio, Ingram, TX）、１００Ｕ／ｍｌＩＬ−２（Shionogi Pharmaceutical, Osaka, Japan）、および１０μｇ／ｍｌＰＨＡ（Difco, Detroit, MI）含有培養培地（４５％ＲＰＩＭ１６４０培地、４５％ＡＩＭ−Ｖ培地；Life Technologies, Walkersville, MA）で14日間インキュベートし、続いて支持細胞としての放射線照射（３０Ｇｙ）アロジェニック末梢血単核細胞（ＰＢＭＣ）の存在下でさらに３０日より長く培養することにより樹立した。本ＣＴＬ株の表現型をＦＩＴＣ結合抗ＣＤ３、ＣＤ４、またはＣＤ８モノクローナル抗体（ｍＡｂ）を用いて免疫学的蛍光試験により検討したところ、ＣＤ３＋ＣＤ４−ＣＤ８＋（＞９５％）であった（データ非提示）。 Example 1
(Establishment of 850B-CTL strain)
HLA-A33-restricted and tumor-specific CLT strain (850B-CTL) has a TIL of 10% FCS (Equitech Bio) for patients with severe gastric adenocarcinoma (HLA-A * 2402 / A * 3303, B7 / B44, Cw7 / Cw14). , Ingram, TX), 100 U / ml IL-2 (Shionogi Pharmaceutical, Osaka, Japan), and 10 μg / ml PHA (Difco, Detroit, MI) -containing culture medium (45% RPIM1640 medium, 45% AIM-V medium; Life Technologies, It was established by incubation for 14 days in Walkersville, MA, followed by further culturing for more than 30 days in the presence of irradiated (30 Gy) allogeneic peripheral blood mononuclear cells (PBMC) as support cells. The phenotype of this CTL line was examined by immunological fluorescence test using FITC-conjugated anti-CD3, CD4, or CD8 monoclonal antibody (mAb) and found to be CD3 + CD4 − CD8 + (> 95%) (data) Not shown).
（８５０Ｂ−ＣＴＬ株の特性）
文献記載の方法に従い、８５０Ｂ−ＣＴＬ株を特性化した（文献９）。
標的細胞を認識することによりＩＦＮ−γを産生する能力について、様々なＥ：Ｔ比（エフェクター細胞：標的細胞）において８５０Ｂ−ＣＴＬ細胞株を試験した。値はＥＬＩＳＡ（検出限界１０ｐｇ／ｍｌ）によるトリプリケート測定の平均を表す。図１Ａに示すように、このＣＴＬ株はＨＬＡ−Ａ３３＋上皮癌細胞、ＬＣ−１およびＫＵＭＡ−１を認識することにより有意なレベルのＩＦＮ−γを産生したが、ＨＬＡ−Ａ３３−標的細胞に対しては反応しなかった。(Characteristics of 850B-CTL strain)
The 850B-CTL strain was characterized according to literature methods (Reference 9).
The 850B-CTL cell line was tested at various E: T ratios (effector cells: target cells) for the ability to produce IFN-γ by recognizing target cells. The value represents the average of triplicate measurements by ELISA (detection limit 10 pg / ml). As shown in FIG. 1A, this CTL line produced significant levels of IFN-γ by recognizing HLA-A33 + epithelial cancer cells, LC-1 and KUMA-1, but it was found in HLA-A33 - target cells. There was no reaction.
次に、様々な標的細胞に対する８５０Ｂ−ＣＴＬの細胞傷害活性を、相異なるＥ：Ｔ比において６時間51Ｃｒ放出試験により試験した。本方法は既知である（文献９）。値はトリプリケート測定の平均を表す。８５０Ｂ−ＣＴＬは、ＨＬＡ−Ａ３３＋ＬＣ−１およびＫＵＭＡ−１細胞に対してより強い細胞傷害性を示したが、ＨＬＡ−Ａ３３−標的細胞、ＣＯＳ７細胞、ＮＫ標的細胞株、Ｋ５６２、または健常提供者のＰＢＭＣから得たＨＬＡ−Ａ３３＋ＰＨＡ活性化正常Ｔ細胞（ＰＨＡ幼若化細胞）のいずれに対しても示さなかった。Next, the cytotoxic activity of 850B-CTL against various target cells was tested by a 6 hr 51 Cr release test at different E: T ratios. This method is known (Reference 9). The value represents the average of triplicate measurements. 850B-CTL showed stronger cytotoxicity against HLA-A33 + LC-1 and KUMA-1 cells, but HLA-A33 - target cells, COS7 cells, NK target cell lines, K562, or healthy donors None of the HLA-A33 + PHA-activated normal T cells (PHA immature cells) obtained from human PBMCs.
さらにｍＡｂを用いた阻害実験によって、８５０Ｂ−ＣＴＬの反応性を検討した。抗ＨＬＡクラスＩ（Ｗ６／３２、ＩｇＧ２ａ）、抗ＣＤ８（Ｎｕ−Ｔｓ／ｃ、ＩｇＧ２ａ）、抗ＨＬＡ−Ａ２４（００４１ＨＡ、ＩｇＧ２ａ）、抗ＣＤ４（Ｎｕ−Ｔｈ／ｉ、ＩｇＧ１）、抗ＨＬＡクラスＩＢ,Ｃ（Ｂ１−２３、ＩｇＧ２ａ）、および抗ＨＬＡクラスＩＩ（Ｈ−ＤＲ、ＩｇＧ２ａ）ｍＡｂ（２０μｇ／ｍｌ）を文献記載の方法と同様に使用した（文献１０、１１）。アイソタイプ適合対照ｍＡｂとして、抗ＣＤ１４（ＪＭＬ−Ｈ１４、ＩｇＧ１）または抗ＣＤ１３（ＭＣＳ2、ＩｇＧ２ａ）を準備した。
ＨＬＡ−Ａ３３＋ＬＣ−１細胞の認識による８５０Ｂ−ＣＴＬからのＩＦＮ−γ産生は、抗ＨＬＡクラスＩおよび抗ＣＤ８ｍＡｂ（２０μｇ／ｍｌ）によって阻害されたが、抗ＨＬＡ−Ｂ, Ｃ、抗ＨＬＡクラスＩＩ、抗ＨＬＡ−Ａ２４、抗ＣＤ４、または無関係なアイソタイプ適合抗ＣＤ１３または抗ＣＤ１４ｍＡｂによっては阻害されなかった（図１Ｃ）。
これらの結果は、８５０Ｂ−ＣＴＬ株が腫瘍細胞に対してはＨＬＡ−Ａ３３拘束性細胞傷害性を示すが、正常細胞に対しては示さないことを意味している。Furthermore, the reactivity of 850B-CTL was examined by an inhibition experiment using mAb. Anti-HLA class I (W6 / 32, IgG2a), anti-CD8 (Nu-Ts / c, IgG2a), anti-HLA-A24 (0041HA, IgG2a), anti-CD4 (Nu-Th / i, IgG1), anti-HLA class IB , C (B1-23, IgG2a), and anti-HLA class II (H-DR, IgG2a) mAb (20 μg / ml) were used in the same manner as described in the literature (10, 11). Anti-CD14 (JML-H14, IgG1) or anti-CD13 (MCS2, IgG2a) was prepared as an isotype matched control mAb.
IFN-γ production from 850B-CTL by recognition of HLA-A33 + LC-1 cells was inhibited by anti-HLA class I and anti-CD8 mAb (20 μg / ml), but anti-HLA-B, C, anti-HLA class It was not inhibited by II, anti-HLA-A24, anti-CD4, or an irrelevant isotype-matched anti-CD13 or anti-CD14 mAb (FIG. 1C).
These results indicate that the 850B-CTL line shows HLA-A33 restricted cytotoxicity against tumor cells but not normal cells.
実施例２
（ＩＥＸ−１遺伝子の同定）
遺伝子発現クローニング法（文献９）により、８５０Ｂ−ＣＴＬ株によって認識される腫瘍抗原をコードする遺伝子を同定した。
ＬＣ−１肺腺癌細胞のポリ（Ａ）＋ＲＮＡをｃＤＮＡに変換し、ＳａｌＩアダプターをライゲートし、そして発現ベクターｐＳＶ−ＳＰＯＲＴ−６（Invitrogen, San Diego, CA）に挿入した。ＨＬＡ−Ａ＊３３０３またはＨＬＡ−Ａ＊２６０１のｃＤＮＡをそれぞれＫＵＭＡ−１またはＫＥ４細胞より回収したＲＮＡからＲＴ−ＰＣＲによって作製し、真核細胞発現ベクターｐＣＲ３（Invitrogen）へ挿入してクローニングした。ＬＣ−１ｃＤＮＡライブラリーのプラスミドＤＮＡプールまたはクローン（２００ｎｇ）、およびＨＬＡ−Ａ＊３３０３またはＨＬＡ−Ａ＊２６０１（陰性対照）ｃＤＮＡ（２００ｎｇ）、の両方を、１μｌのリポフェクタミン（Invitrogen）と１２０μｌのＯｐｔｉ−ＭＥＭ（Invitrogen）中で４０分間混合した。ＣＯＳ７細胞（５ｘ１０３）をこの混合物５０μｌと６時間インキュベートし、続いて１０％ＦＣＳ含有ＲＰＭＩ１６４０培地１５０μｌを添加した。２日間培養した後８５０Ｂ−ＣＴＬ（２ｘ１０５細胞／ウェル）を添加し、その後１８時間インキュベートし、上清１００μｌを回収してＥＬＩＳＡによりデュプリケート試験でＩＦＮ−γを測定した（文献９）。 Example 2
(Identification of IEX-1 gene)
A gene encoding a tumor antigen recognized by the 850B-CTL strain was identified by a gene expression cloning method (Reference 9).
LC-1 lung adenocarcinoma cell poly (A) + RNA was converted to cDNA, SalI adapters were ligated and inserted into the expression vector pSV-SPORT-6 (Invitrogen, San Diego, Calif.). HLA-A * 3303 or HLA-A * 2601 cDNA was prepared by RT-PCR from RNA recovered from KUMA-1 or KE4 cells, respectively, and inserted into a eukaryotic cell expression vector pCR3 (Invitrogen) for cloning. Both the plasmid DNA pool or clone (200 ng) of the LC-1 cDNA library, and HLA-A * 3303 or HLA-A * 2601 (negative control) cDNA (200 ng), 1 μl Lipofectamine (Invitrogen) and 120 μl Opti Mix for 40 minutes in MEM (Invitrogen). COS7 cells (5 × 10 3 ) were incubated with 50 μl of this mixture for 6 hours, followed by addition of 150 μl of RPMI 1640 medium containing 10% FCS. After culturing for 2 days, 850B-CTL (2 × 10 5 cells / well) was added, followed by incubation for 18 hours, and 100 μl of the supernatant was recovered, and IFN-γ was measured by a duplicate test by ELISA (Reference 9).
一次スクリーニングにおいて、ＬＣ−１ｃＤＮＡライブラリーから得た１ｘ１０５クローンすべてを、ＨＬＡ−Ａ＊３３０３ｃＤＮＡとともにＣＯＳ７細胞にトランスフェクションした後に、８５０Ｂ−ＣＴＬによるＩＦＮ−γ産生を刺激する能力について試験した。すなわち、ｃＤＮＡプール（１ｘ１０５クローン）を９６平底プレート中の約２０００の相違するウェルにデュプリケートで分割した（各ウェルの期待されるクローン数：１００クローン／ウェル）。一次スクリーニングでは、１０の異なるウェルで有意なレベルのＩＦＮ−γ産生が得られた。二次スクリーニングとして、陽性ウェルからクローン化して得た各ｃＤＮＡプールを９６平底プレートの約２００の異なるウェルにデュプリケートで分割し、ＩＦＮ−γ産生刺激活性について試験した。
二次スクリーニングの後、更なるアッセイのための二つの陽性クローンを同定した。ＤＮＡシークエンスキットおよびABI PRISM 377 DNA シークエンサー（Perkin-Elmer, Foster, CA）を使用したジデオキシヌクレオチドシークエンス法によりＤＮＡシークエンスを行った。単離された遺伝子の一つについて以下の検討を行った。In the primary screen, all 1 × 10 5 clones from the LC-1 cDNA library were tested for their ability to stimulate IFN-γ production by 850B-CTL after transfection into COS7 cells with HLA-A * 3303 cDNA. That is, the cDNA pool (1 × 10 5 clones) was divided in duplicate into about 2000 different wells in 96 flat bottom plates (expected number of clones in each well: 100 clones / well). The primary screen resulted in significant levels of IFN-γ production in 10 different wells. As a secondary screen, each cDNA pool obtained by cloning from positive wells was divided in duplicate into about 200 different wells of 96 flat bottom plates and tested for IFN-γ production stimulating activity.
After secondary screening, two positive clones for further assay were identified. DNA sequencing was performed by the dideoxynucleotide sequencing method using a DNA sequencing kit and an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Foster, CA). The following examination was performed on one of the isolated genes.
図２に示されるように、クローン１およびＨＬＡ−Ａ＊３３０３をトランスフェクトしたＣＯＳ７細胞は、用量依存的に８５０Ｂ−ＣＴＬにおけるＩＦＮ−γ産生を誘導したが、陰性対照としてクローン１とＨＬＡ−Ａ＊２６０１をトランスフェクトした細胞は誘導しなかった。それに対し、クローン１またはＨＬＡ−Ａ＊３３０３のいずれかを単独でトランスフェクトしたＣＯＳ７細胞は８５０Ｂ−ＣＴＬに認識されなかった（データ非提示）。さらに、ＬＣ−１ｃＤＮＡライブラリーから得られた陰性対照として使用した他のクローンは、ＨＬＡ−Ａ＊３３０３とともにＣＯＳ７細胞にトランスフェクトした場合に８５０Ｂ−ＣＴＬにおけるＩＦＮ−γ産生を誘導できなかった（データ非提示）。このことはクローン１が８５０Ｂ−ＣＴＬによって特異的に認識される腫瘍抗原をコードすることを示唆している。
GeneBankの検索によって、クローン１のヌクレオチド配列は、ストレス誘導抗アポトーシス遺伝子として報告されているＩＥＸ−１（文献１７）の配列と同一であることがわかった。As shown in FIG. 2, COS7 cells transfected with
A search of GeneBank revealed that the nucleotide sequence of
実施例３
（正常および癌組織におけるＩＥＸ−１ｍＲＮＡおよびタンパク質の発現）
様々な腫瘍または正常組織（Multiple Tissue Northern Blots, Clontech, Tokyo, Japan）におけるＩＥＸ−１ｍＲＮＡの発現を、以前に記載した方法に従い３２Ｐ標識ＩＥＸ−１プローブを用いてノーザンブロット解析によって調べた（文献９）（図３Ａ、レーン１：脳、レーン２：心臓、レーン３：骨格筋、レーン４：大腸、レーン５：胸腺、レーン６：脾臓、レーン７：腎臓、レーン８：肝臓、レーン９：小腸、レーン１０：胎盤、レーン１１：肺、レーン１２：ＰＢＬ）。β-アクチンプローブを対照として使用した。図３Ａに示すように、脳（レーン１）を除き試験したすべての正常組織において約〜１．３ｋｂのバンドがはっきりと検出され、心臓（レーン２）、腎臓（レーン７）、肺（レーン１１）、または末梢血リンパ球（ＰＢＬ）（レーン１２）において特に発現が高く、胸腺（レーン５）、脾臓（レーン６）、肝臓（レーン８）、または小腸（レーン９）においては発現が低かった。 Example 3
(Expression of IEX-1 mRNA and protein in normal and cancerous tissues)
Expression of IEX-1 mRNA in various tumors or normal tissues (Multiple Tissue Northern Blots, Clontech, Tokyo, Japan) was examined by Northern blot analysis using a 32 P-labeled IEX-1 probe according to previously described methods (references) 9) (FIG. 3A, lane 1: brain, lane 2: heart, lane 3: skeletal muscle, lane 4: colon, lane 5: thymus, lane 6: spleen, lane 7: kidney, lane 8: liver, lane 9: Small intestine, lane 10: placenta, lane 11: lung, lane 12: PBL). A β-actin probe was used as a control. As shown in FIG. 3A, a band of approximately ˜1.3 kb was clearly detected in all normal tissues except the brain (lane 1), and the heart (lane 2), kidney (lane 7), lung (lane 11). ), Or peripheral blood lymphocytes (PBL) (lane 12), and particularly low expression in the thymus (lane 5), spleen (lane 6), liver (lane 8), or small intestine (lane 9) .
次に、正常および癌細胞におけるＩＥＸ−１遺伝子のｍＲＮＡ発現をノーザンブロット解析により検討した（図３Ｂ、レーン1：ＰＢＬ、レーン２：ＭＫＮ４５、レーン３：ＭＫＮ２８、レーン４：ＳＳＴＷ、レーン５：ＨＧＣ２７、レーン６：ＬＣ−１、レーン７：ＱＧ５６、レーン８：ＫＵＭＡ−１、レーン９：Ｐａｎｃ−１、レーン１０：ＳＷ６２０、レーン１１：ＣＯＬＯ２０１、レーン１２：ＫＡＴＯ−ＩＩＩ）。ＩＥＸ−１は、ＨＣＧ２７胃癌細胞株（レーン５）を除き、胃（レーン２−４、１２）、肺（レーン６、７）、頭頸部（レーン８）、膵臓（レーン９）、および大腸（レーン１０、１１）を含む様々な臓器に由来する、被験腺癌およびＳＣＣ細胞株のほとんどにおいて高発現していた。
これらの結果は、この遺伝子があらゆる癌および正常組織において発現していることを意味する。Next, mRNA expression of IEX-1 gene in normal and cancer cells was examined by Northern blot analysis (FIG. 3B, lane 1: PBL, lane 2: MKN45, lane 3: MKN28, lane 4: SSTW, lane 5: HGC27. Lane 6: LC-1, Lane 7: QG56, Lane 8: KUMA-1, Lane 9: Panc-1, Lane 10: SW620, Lane 11: COLO201, Lane 12: KATO-III). IEX-1 except for the HCG27 gastric cancer cell line (lane 5), stomach (lanes 2-4, 12), lungs (
These results imply that this gene is expressed in all cancers and normal tissues.
さらに、様々な腫瘍組織におけるこの遺伝子の発現をタンパク質レベルで検討した。ＩＥＸ−１タンパク質の発現は、抗ＩＥＸ−１抗体（Santa-Cruz biotechnology, Santa-Cruz, CA）とともにVentana Medical Systems 自動化装置（Tucson, AZ）を使用して、ホルマリン固定パラフィン包埋組織切片上で免疫組織化学によって評価した。
胃癌組織における代表的染色を示す（図３Ｃ）。胃癌においてＩＥＸ−１タンパク質の発現は癌細胞で選択的に増強されていたが、周囲の正常上皮または結合組織においては増強されていなかった。ＩＥＸ−１タンパク質は、乳癌（図３Ｄ）、肺癌（図３Ｅ）、および大腸癌（データ非提示）を含む様々な型の癌組織においてもまた、高くかつ選択的に発現していた。なお、図３Ｃ〜Ｅにおいて、ＩＥＸ−１タンパク質は茶色に染まっており、正常細胞および結合組織は茶色に染まっていない。これら、茶色に染まった数箇所を、便宜上、矢印で示した。
以上の結果より、ＩＥＸ−１は癌の治療において理想的な標的分子の一つであるといえる。Furthermore, the expression of this gene in various tumor tissues was examined at the protein level. Expression of IEX-1 protein was performed on formalin-fixed paraffin-embedded tissue sections using a Ventana Medical Systems automation device (Tucson, AZ) with anti-IEX-1 antibody (Santa-Cruz biotechnology, Santa-Cruz, CA). Assessed by immunohistochemistry.
Representative staining in gastric cancer tissue is shown (FIG. 3C). In gastric cancer, IEX-1 protein expression was selectively enhanced in cancer cells but not in surrounding normal epithelium or connective tissue. IEX-1 protein was also highly and selectively expressed in various types of cancer tissues including breast cancer (FIG. 3D), lung cancer (FIG. 3E), and colon cancer (data not shown). 3C to 3E, IEX-1 protein is stained brown, and normal cells and connective tissue are not stained brown. For convenience, some of these spots stained brown are indicated by arrows.
From the above results, it can be said that IEX-1 is one of the ideal target molecules in the treatment of cancer.
実施例４
（８５０−Ｂ ＣＴＬによって認識されるＩＥＸ−１由来抗原性ペプチドの同定）
ＩＥＸ−１の抗原性エピトープとしてＣＴＬに認識され得るペプチドを同定するため以下の実験を行った。
ＩＥＸ−１の推定アミノ酸配列においてＨＬＡ−Ａ３３分子に結合するためのモチーフ（文献１８、１９）を有する可能性のあるペプチド配列の中で、コンピューター解析（Bioinformatics and Molecular Analysis Section (BIMAS), NIH, Bethesda, MD）ではＨＬＡ−Ａ３３に対してより強い結合活性を有する８の相異なるペプチドを使用した。BioSynthesis, Lewisville, TXより純度＞９５％のペプチドを得た。ペプチド結合アッセイには、ＲＭＡ−Ｓ−Ａ３３細胞（ＨＬＡ−Ａ＊３３０３ｃＤＮＡを安定的にトランスフェクトしたＲＭＡ−Ｓタップ（ペプチドプロセシングに関与するトランスポーター）欠損マウスリンパ腫細胞（Hiroko Takedatsu, et al., Identification of Peptide Vaccine Candidates Sharing Among HLA-A3, -A11, -A31, and -A33 Cancer Patients., Clin Can Res, 2004, in press）（１ｘ１０４細胞／ウェル）を使用した。簡単に言うと、細胞を２６℃で１８時間インキュベートした。ＰＢＳで洗浄した後、細胞（１ｘ１０６細胞）をOpti-MEM（ヒトβ２-ミクログロブリン３μｇ／ｍｌおよびペプチド１０μｇ／ｍｌ含有）に懸濁し、続いて２６℃で３時間そして３７℃で３時間インキュベートした。ＰＢＳで洗浄した後、細胞を抗ＨＬＡ−Ａ３３ｍＡｂと４℃で３０分間インキュベートし、続いてＦＩＴＣ結合ウサギ抗マウスＩｇＭ抗体（Cappel, Aurora, OH）と４℃で３０分間インキュベートした。細胞をＦＡＣＳｃａｎで解析し、平均蛍光強度（ＭＦＩ）により結合活性を評価した。２６℃で、ＴＲＰ２−１９７ペプチド（参考ペプチド）でパルスした細胞、およびパルスしていない細胞も使用した。表１に示すように、若干親和性は異なるが、８ペプチド全てがＲＭＡ−Ｓ−Ａ３３細胞に結合できた。
(Identification of IEX-1-derived antigenic peptide recognized by 850-B CTL)
The following experiment was conducted to identify a peptide that can be recognized by CTL as an antigenic epitope of IEX-1.
Among peptide sequences that may have a motif (References 18 and 19) for binding to the HLA-A33 molecule in the deduced amino acid sequence of IEX-1, computer analysis (Bioinformatics and Molecular Analysis Section (BIMAS), NIH, Bethesda, MD) used 8 different peptides with stronger binding activity to HLA-A33. Peptides with> 95% purity were obtained from BioSynthesis, Lewisville, TX. For peptide binding assays, RMA-S-A33 cells (RMA-S tap (transporter involved in peptide processing) stably transfected with HLA-A * 3303 cDNA) mouse lymphoma cells (Hiroko Takedatsu, et al., Identification of Peptide Vaccine Candidates Sharing Among HLA-A3, -A11, -A31, and -A33 Cancer Patients., Clin Can Res, 2004, in press) (1 × 10 4 cells / well). Was incubated for 18 hours at 26 ° C. After washing with PBS, cells (1 × 10 6 cells) were suspended in Opti-MEM (containing 3 μg / ml human β2-microglobulin and 10 μg / ml peptide) followed by 26 ° C. Incubated for 3 hours and 3 hours at 37 ° C. After washing with PBS, cells were incubated with anti-HLA-A33 mAb for 30 minutes at 4 ° C. Followed by incubation with FITC-conjugated rabbit anti-mouse IgM antibody (Cappel, Aurora, OH) for 30 minutes at 4 ° C. Cells were analyzed by FACScan and binding activity was assessed by mean fluorescence intensity (MFI) .26 Cells pulsed with TRP2-197 peptide (reference peptide) and non-pulsed cells were also used at 0 ° C. As shown in Table 1, all of the 8 peptides were RMA-S-A33 cells with slightly different affinity. Could be combined.
８５０Ｂ-ＣＴＬ株によって認識される抗原性ペプチドの検出のため、Ｃ１Ｒ−Ａ３３細胞（ＨＬＡ−Ａ＊３３０３ｃＤＮＡをトランスフェクトして安定的に発現させたＣ１Ｒヒト多発性骨髄腫細胞（Hiroko Takedatsu, et al., Clin Can Res, 2004, in press）を指示濃度のペプチドとともに培養した。二時間後、８５０Ｂ−ＣＴＬ（２ｘ１０５細胞/ウェル）を添加し、さらに１８時間インキュベートした。培養上清中のＩＦＮ−γの産生はＥＬＩＳＡにより測定した。
ペプチド非ロードＣ１Ｒ−Ａ３３細胞に応答した８５０Ｂ−ＣＴＬによるＩＦＮ−γ産生をバックグラウンドとして、その値から差し引いた。値はトリプリケート試験の平均を示す。For detection of antigenic peptides recognized by the 850B-CTL line, C1R-A33 cells (C1R human multiple myeloma cells stably transfected with HLA-A * 3303 cDNA transfected (Hiroko Takedatsu, et al , Clin Can Res, 2004, in press) with the indicated concentration of peptide, 2 hours later, 850B-CTL (2 × 10 5 cells / well) was added and incubated for another 18 hours. -Γ production was measured by ELISA.
IFN-γ production by 850B-CTL in response to peptide non-loaded C1R-A33 cells was subtracted from that value as background. Values represent the average of triplicate tests.
これらペプチドのうち３つ、ＩＥＸ４７−５６、ＩＥＸ６１−６９、およびＩＥＸ６５−７３が、有意なレベルのＩＦＮ−γ産生を用量依存的に誘導した（図４Ａ、４Ｂ）。ＨＬＡ−Ａ３３トランスフェクトＣ１Ｒ細胞上にロードするのに最適な三つのペプチドの濃度は、各ペプチドにおいて０．１−１μＭの範囲にわたり様々であったが（図４Ｂ）、ＲＭＡ−Ｓ−Ａ３３細胞により決定されたＨＬＡ−Ａ３３分子に対するそれらの結合親和性（表１）には依存していなかった。
以上の結果に基づき、ＩＥＸ４７−５６、ＩＥＸ６１−６９、およびＩＥＸ６５−７３を、８５０Ｂ−ＣＴＬ株によって認識されるＩＥＸ−１由来抗原性ペプチドとして同定した。Three of these peptides, IEX 47-56, IEX 61-69, and IEX 65-73, induced significant levels of IFN-γ production in a dose-dependent manner (FIGS. 4A, 4B). The optimal concentration of the three peptides to load on HLA-A33 transfected C1R cells varied over the range of 0.1-1 μM for each peptide (FIG. 4B), but by RMA-S-A33 cells It was not dependent on their determined binding affinity for HLA-A33 molecules (Table 1).
Based on the above results, IEX47-56, IEX61-69, and IEX65-73 were identified as IEX-1-derived antigenic peptides recognized by the 850B-CTL strain.
実施例５
（ＩＥＸ−１由来ペプチドによるＣＴＬの誘導）
ＩＥＸ４７−５６、ＩＥＸ６１−６９、およびＩＥＸ６５−７３ペプチドのＨＬＡ−Ａ３３拘束性および腫瘍特異的ＣＴＬ誘導能について、ＨＬＡ−Ａ３３＋上皮癌患者（ｎ＝４、胃癌患者（２）、肺癌患者（１）、前立腺癌（１））およびＨＬＡ−Ａ３３＋健常人（ＨＤ）のＰＢＭＣにおいて試験した。
ＨＬＡ−Ａ３３＋癌患者およびＨＬＡ−Ａ３３＋健常人ＰＢＭＣ（１ｘ１０５／ウェル）を、９６穴マイクロカルチャープレート(Nunc, Roskiide, Denmark)においてＩＬ−２含有培養液２００μｌ中で各ペプチド（１０μＭ）とインキュベートした（文献１３）。１４日目に各ウェルから別個にペプチド刺激ＰＢＭＣ（８０−１２０ｘ１０４／ウェル）を回収し、洗浄し、４等分した。２つは対応するペプチドをロードしたＣ１Ｒ−Ａ３３細胞で、残りの二つは陰性対照のＨＩＶペプチドをロードしたＣ１Ｒ−Ａ３３細胞で刺激した。１８時間後、上清を回収しそれらのＩＦＮ−γ産生活性について試験した。
４人の患者の代表例を図５に示す。ＨＩＶペプチドに対するＩＦＮ−γ産生（＜５０ｐｇ／ｍｌ）をバックグラウンドとして差し引いた。これら３つのぺプチドで刺激した癌患者由来ＰＢＭＣは、ほとんどの場合において対応するペプチドをロードしたＨＬＡ−Ａ３３トランスフェクトＣ１Ｒ細胞を認識して有意な量のＩＦＮ−γを産生した（図５）。それに対して、５人のＨＤから得たＰＢＭＣはそれらに対して有意な量のＩＦＮ−γを産生しなかった（データ非提示）。Example 5
(Induction of CTL by IEX-1-derived peptide)
Regarding the HLA-A33-restricted and tumor-specific CTL inducing ability of IEX47-56, IEX61-69, and IEX65-73 peptides, HLA-A33 + epithelial cancer patients (n = 4, gastric cancer patients (2), lung cancer patients (1 ), Prostate cancer (1)) and HLA-A33 + healthy individuals (HD) PBMC.
HLA-A33 + cancer patients and HLA-A33 + healthy human PBMC (1 × 10 5 / well) were combined with each peptide (10 μM) in 200 μl of IL-2 containing culture medium in a 96-well microculture plate (Nunc, Roskiide, Denmark). Incubated (Reference 13). On day 14, peptide-stimulated PBMC (80-120 × 10 4 / well) were collected separately from each well, washed and divided into 4 equal parts. Two were stimulated with C1R-A33 cells loaded with the corresponding peptide and the other two were C1R-A33 cells loaded with the negative control HIV peptide. After 18 hours, supernatants were collected and tested for their IFN-γ producing activity.
A representative example of four patients is shown in FIG. IFN-γ production against HIV peptides (<50 pg / ml) was subtracted as background. Cancer patient-derived PBMC stimulated with these three peptides recognized HLA-A33 transfected C1R cells loaded with the corresponding peptide in most cases and produced significant amounts of IFN-γ (FIG. 5). In contrast, PBMCs obtained from 5 HD did not produce significant amounts of IFN-γ against them (data not shown).
次に、ペプチド誘導ＣＴＬの腫瘍細胞傷害活性を検討した。
有意な量のＩＦＮ−γを産生できた細胞を回収し、ＩＬ−２単独でさらに１０−１４日間培養し、相異なるＥ：Ｔ比で６時間５１Ｃｒ放出試験を行った（文献１３）。ＬＣ−１（ＨＬＡ−Ａ３３＋ＩＥＸ−１＋）、ＱＧ５６（ＨＬＡ−Ａ３３−ＩＥＸ−１＋）、およびＨＧＣ２７（ＨＬＡ−Ａ３３−ＩＥＸ−１−）に対する細胞傷害活性を計測した。対照として、非抗原性ＩＥＸ−１由来ペプチド、ＩＥＸ４３−５１によって刺激した癌患者のＰＢＭＣを使用した。値はトリプリケート測定の平均を示す。
図６に示すように、ＩＥＸ−１由来ペプチドで刺激されたＰＢＭＣは、ＨＬＡ−Ａ３３＋ＩＥＸ−１＋ＬＣ−１腫瘍細胞に対して有意なレベルの細胞傷害性を示したが、ＨＬＡ−Ａ３３−ＨＧＣ２７またはＱＧ５６細胞に対しては示さなかった。また、ＩＥＸ４３−５１（陰性対照ペプチド）は特異的ＣＴＬ活性を示さなかった。この結果は、ＩＥＸ４７−５６、ＩＥＸ６１−６９、およびＩＥＸ６５−７３が、上皮癌患者のＰＢＭＣにおいてＨＬＡ−Ａ３３拘束性に特異的ＣＴＬを誘導できる抗原性エピトープペプチドであることを示唆している。Next, the tumor cytotoxic activity of peptide-derived CTLs was examined.
Cells capable of producing a significant amount of IFN-γ were collected, cultured for additional 10-14 days with IL-2 alone, and subjected to a 51 Cr release test at different E: T ratios for 6 hours (Reference 13). LC-1 (HLA-A33 + IEX-1 +), QG56 (HLA-A33 - IEX-1 +), and HGC27 (HLA-A33 - IEX- 1 -) for the measurement of the cytotoxic activity. As a control, PBMCs from cancer patients stimulated with a non-antigenic IEX-1-derived peptide, IEX 43-51, were used. Values indicate the average of triplicate measurements.
As shown in FIG. 6, PBMC stimulated with IEX-1 derived peptides showed a significant level of cytotoxicity against HLA-A33 + IEX-1 + LC-1 tumor cells, but HLA-A33. - it did not show against HGC27 or QG56 cells. IEX43-51 (negative control peptide) did not show specific CTL activity. This result suggests that IEX47-56, IEX61-69, and IEX65-73 are antigenic epitope peptides capable of inducing specific CTL in an HLA-A33-restricted manner in PBMC of epithelial cancer patients.
さらに、細胞傷害性の拘束性およびペプチド特異性を阻害試験および競合試験により確認した。
阻害試験には、抗ＨＬＡクラスＩ（Ｗ６／３２、ＩｇＧ２ａ）、抗ＨＬＡクラスＩＩ（Ｈ−ＤＲ、ＩｇＧ２ａ）、抗ＣＤ８（Ｎｕ−Ｔｓ／ｃ、ＩｇＧ２ａ）、抗ＣＤ４（Ｎｕ−Ｔｈ／ｉ、ＩｇＧ１）（２０μｇ／ｍｌ）を使用した。抗ＣＤ１４（ＪＭＬ−Ｈ１４、ＩｇＧ２ａ）ｍＡｂを対照として使用した。
これらペプチド刺激ＰＢＭＣの細胞傷害性は、試験したすべてのケースで抗ＨＬＡクラスＩまたは抗ＣＤ８抗体によって有意に阻害されたが、他のｍＡｂよっては阻害されなかった（図７）。
競合試験では、対応ペプチドまたはＨＩＶペプチド（陰性対照）でパルスした非標識Ｃ１Ｒ細胞を５１Ｃｒ放出試験に非標識細胞対標識細胞の比率を１０対１で添加した。１０：１のＥ／Ｔ比で５１Ｃｒ放出試験を行った。値は特異的傷害活性（％）の平均±ＳＤを示す。
対応ペプチドパルスＣ１Ｒ−Ａ３３細胞を添加することにより細胞傷害性は阻害されたが、ＨＩＶペプチドパルス細胞では阻害されなかった（図８）。
以上の結果は、ペプチド特異的ＣＴＬ活性が、主としてＨＬＡ−ＡクラスＩ拘束性にＣＤ８＋Ｔ細胞によって発揮されることを示唆している。Furthermore, the cytotoxic restraint and peptide specificity were confirmed by inhibition tests and competition tests.
Inhibition tests include anti-HLA class I (W6 / 32, IgG2a), anti-HLA class II (H-DR, IgG2a), anti-CD8 (Nu-Ts / c, IgG2a), anti-CD4 (Nu-Th / i, IgG1) (20 μg / ml) was used. Anti-CD14 (JML-H14, IgG2a) mAb was used as a control.
The cytotoxicity of these peptide-stimulated PBMCs was significantly inhibited by anti-HLA class I or anti-CD8 antibodies in all cases tested, but not by other mAbs (FIG. 7).
In competition experiments, and the ratio of non-labeled C1R cells pulsed with the corresponding peptide or HIV peptide (negative control) to a 51 Cr release test unlabeled cells versus labeled cells were added at 10: 1. A 51 Cr release test was conducted at an E / T ratio of 10: 1. Values represent the mean ± SD of specific injury activity (%).
Cytotoxicity was inhibited by adding corresponding peptide pulsed C1R-A33 cells, but not in HIV peptide pulsed cells (FIG. 8).
These results suggest that peptide-specific CTL activity is exerted by CD8 + T cells mainly in HLA-A class I restriction.
本発明は、ＩＥＸ−１が、胃腺癌に浸潤しているＴ細胞より樹立されたＨＬＡ拘束性および腫瘍特異的ＣＴＬにより認識される腫瘍抗原性エピトープをコードしていることを開示するものである。また、本発明はＩＥＸ−１由来抗原性ペプチドが癌患者のＰＢＭＣ培養においてＨＬＡ拘束性に腫瘍特異的ＣＴＬを誘導できることをも開示するものである。
ＩＥＸ−１は、正常組織、特に、心臓、腎臓、肺およびＰＢＬにおいても発現しているので、これらの臓器はＩＥＸ−１由来抗原性エピトープによる特異的免疫療法の有害事象となる恐れがある。しかしながら、本発明は８５０−ＢＣＴＬ株およびＩＥＸ−１由来ペプチドにより誘導されたＣＴＬのいずれもＨＬＡ−Ａ３３＋腫瘍細胞を溶解する一方、過剰量の対応ペプチドが培養中に存在するにも拘わらずＰＨＡ活性化正常ＨＬＡ−Ａ３３＋Ｔ細胞は傷害しないことを明らかにした。また、発明者らが行っている腫瘍抗原由来ペプチドワクチンを用いる臨床試験では、腫瘍抗原のいくつかは正常組織または臓器に広く発現しているにも拘わらず、深刻な有害事象は観察されていない（文献４−７）。
従って、以上の結果は、本発明のＩＥＸ−１由来ペプチドが癌治療に適したペプチドワクチンとして使用可能であることを示唆している。The present invention discloses that IEX-1 encodes a tumor antigenic epitope recognized by HLA-restricted and tumor-specific CTL established from T cells infiltrating gastric adenocarcinoma. . The present invention also discloses that the IEX-1-derived antigenic peptide can induce tumor-specific CTL in an HLA-restricted manner in PBMC cultures of cancer patients.
Since IEX-1 is also expressed in normal tissues, particularly heart, kidney, lung and PBL, these organs may be adverse events of specific immunotherapy with IEX-1-derived antigenic epitopes. However, the present invention shows that both the 850-BCTL strain and the CTL induced by the IEX-1-derived peptide lyse HLA-A33 + tumor cells, while PHA is present in culture despite the presence of an excess of the corresponding peptide. It was revealed that activated normal HLA-A33 + T cells were not damaged. In clinical trials using peptide vaccines derived from tumor antigens conducted by the inventors, no serious adverse events have been observed even though some tumor antigens are widely expressed in normal tissues or organs. (References 4-7).
Therefore, the above results suggest that the IEX-1-derived peptide of the present invention can be used as a peptide vaccine suitable for cancer treatment.
また、放射線照射およびいくつかの化学療法薬が、比較的高いレベルでＩＥＸ−１発現を誘導することが報告されているので（文献２６、３０）、本発明のＩＥＸ−１分子を標的とした特異的免疫療法は、特に化学療法または放射線療法抵抗性癌を患う患者の処置にとって新規で魅力的な方法となり得る。
ＨＬＡ−Ａ３３は、アジア人および黒人において最も一般的なＨＬＡ−Ａアレルの一つであり、日本人の１３％、韓国人の１４％、白人の１４％、および黒人の１６％に見られる（文献１４、１５）。またＩＥＸ−１は癌組織に高発現している。従って、本発明の抗原ペプチドは、ＨＬＡ−Ａ３３＋癌患者に対する特異的免疫療法に広く利用可能であろう。In addition, radiation and some chemotherapeutic drugs have been reported to induce IEX-1 expression at relatively high levels (Refs. 26, 30), thus targeting the IEX-1 molecule of the present invention. Specific immunotherapy can be a novel and attractive method especially for the treatment of patients suffering from chemotherapy or radiation resistant cancer.
HLA-A33 is one of the most common HLA-A alleles in Asians and blacks and is found in 13% of Japanese, 14% of Koreans, 14% of whites, and 16% of blacks ( References 14, 15). IEX-1 is highly expressed in cancer tissues. Therefore, the antigenic peptides of the present invention will be widely available for specific immunotherapy against HLA-A33 + cancer patients.
文献リスト
文献１：Rosenberg, S. A. A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity, 10: 281-287, 1999.
文献２：Rosenberg, S. A. Progress in human tumour immunology and immunotherapy. Nature, 411: 380-384, 2001.
文献３：Ribas, A., Butterfield, L. H., Glaspy, J. A., and Economou, J. S. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol, 21: 2415-2432, 2003.
文献４：Sato, Y., Shomura, H., Maeda, Y., Mine, T., Une, Y., Akasaka, Y., Kondo, M., Takahashi, S., Shinohara, T., Katagiri, K., Sato, M., Okada, S., Matsui, K., Yamada, A., Yamana, H., Itoh, K., and Todo, S. Immunological evaluation of peptide vaccination for patients with gastric cancer based on pre-existing cellular response to peptide. Cancer Sci, 94: 802-808, 2003.
文献５：Mine, T., Gouhara, R., Hida, N., Imai, N., Azuma, K., Rikimaru, T., Katagiri, K., Nishikori, M., Sukehiro, A., Nakagawa, M., Yamada, A., Aizawa, H., Shirouzu, K., Itoh, K., and Yamana, H. Immunological evaluation of ＣＴＬ precursor-oriented vaccines for advanced lung cancer patients. Cancer Sci, 94: 548-556, 2003.
文献６：Tsuda, N., Mochizuki, K., Harada, M., Sukehiro, A., Kawano, K., Yamada, A., Ushijima, K., Sugiyama, T., Nishida, T., Yamana, H., Itoh, K., and Kamura, T. Vaccination with pre-designated or evidence-based peptides for patients with recurrent gynecologic cancers. J Immunother, in press.
文献７：Noguchi, M., Kobayashi, K., Suetsugu, N., Tomiyasu, K., Suekane, S., Yamada, A., Itoh, K., and Noda, S. Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination. Prostate, 57: 80-92, 2003.
文献８：Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med, 183: 527-534, 1996.
文献９：Shichijo, S., Nakao, M., Imai, Y., Takasu, H., Kawamoto, M., Niiya, F., Yang, D., Toh, Y., Yamana, H., and Itoh, K. A gene encoding antigenic peptides of human squamous cell carcinoma recognized by cytotoxic T lymphocytes. J Exp Med, 187: 277-288, 1998.
文献１０：Gomi, S., Nakao, M., Niiya, F., Imamura, Y., Kawano, K., Nishizaka, S., Hayashi, A., Sobao, Y., Oizumi, K., and Itoh, K. A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific ＣＴＬs. J Immunol, 163: 4994-5004, 1999.
文献１１：Ito, M., Shichijo, S., Tsuda, N., Ochi, M., Harashima, N., Saito, N., and Itoh, K. Molecular basis of T cell-mediated recognition of pancreatic cancer cells. Cancer Res, 61: 2038-2046, 2001.
文献１２：Wang, R. F., Johnston, S. L., Southwood, S., Sette, A., and Rosenberg, S. A. Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by ＣＴＬ in the context of HLA-A31 and -A33. J Immunol, 160: 890-897, 1998.
文献１３：Takedatsu, H., Shichijo, S., Katagiri, K., Sawamizu, H., Sata, M., and Itoh, K. Identification of peptide vaccine candidates sharing among HLA-A3+, -A11+, -A31+, -A33+ cancer patients. Clin Cancer Res, in press.
文献１４：Imanishi I, A. T., Kimura A, Tokunaga K, Gojobori T. Allele and haprotype frequencies for HLA and complement loci in various ethnic groups. In, pp. 1065-1220: Oxford University Press, 1992.
文献１５：Aizawa, M. Allele and haplotype frequencies for HLA and ccomplement loci in various ethnic groups. Oxford: Oxford University Press, 1992.
文献１６：Macdonald, J. S. Advances in the therapy of gastric cancer. Gastric Cancer, 5 Suppl 1: 35-40, 2002.
文献１７：Wu, M. X. Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis. Apoptosis, 8: 11-18, 2003.
文献１８：Takiguchi, M., Matsuda, T., Tomiyama, H., and Miwa, K. Analysis of three HLA-A*3303 binding peptide anchors using an HLA-A*3303 stabilization assay. Tissue Antigens, 55: 296-302, 2000.
文献１９：Takiguchi, M., Matsuda, T., and Tomiyama, H. Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303. Tissue Antigens, 56: 501-506, 2000.
文献２０：Schafer, H., Trauzold, A., Siegel, E. G., Folsch, U. R., and Schmidt, W. E. PRG1: a novel early-response gene transcriptionally induced by pituitary adenylate cyclase activating polypeptide in a pancreatic carcinoma cell line. Cancer Res, 56: 2641-2648, 1996.
文献２１：Pietzsch, A., Buchler, C., Aslanidis, C., and Schmitz, G. Identification and characterization of a novel monocyte/macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide, and lysophosphatidylcholine. Biochem Biophys Res Commun, 235: 4-9, 1997.
文献２２：Charles, C. H., Yoon, J. K., Simske, J. S., and Lau, L. F. Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene, 8: 797-801, 1993.
文献２３：Wu, M. X., Ao, Z., Prasad, K. V., Wu, R., and Schlossman, S. F. IEX-1L, an apoptosis inhibitor involved in NF-kappaB-mediated cell survival. Science, 281: 998-1001, 1998.
文献２４：Kumar, R., Kobayashi, T., Warner, G. M., Wu, Y., Salisbury, J. L., Lingle, W., and Pittelkow, M. R. A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes. Biochem Biophys Res Commun, 253: 336-341, 1998.
文献２５：Schafer, H., Lettau, P., Trauzold, A., Banasch, M., and Schmidt, W. E. Human PACAP response gene 1 (p22/PRG1): proliferation-associated expression in pancreatic carcinoma cells. Pancreas, 18: 378-384, 1999.
文献２６：Grobe, O., Arlt, A., Ungefroren, H., Krupp, G., Folsch, U. R., Schmidt, W. E., and Schafer, H. Functional disruption of IEX-1 expression by concatemeric hammerhead ribozymes alters growth properties of 293 cells. FEBS Lett, 494: 196-200, 2001.
文献２７：Zhang, Y., Schlossman, S. F., Edwards, R. A., Ou, C. N., Gu, J., and Wu, M. X. Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice. Proc Natl Acad Sci U S A, 99: 878-883, 2002.
文献２８：Garcia, J., Ye, Y., Arranz, V., Letourneux, C., Pezeron, G., and Porteu, F. IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation. Embo J, 21: 5151-5163, 2002.
文献２９：Huang, Y. H., Wu, J. Y., Zhang, Y., and Wu, M. X. Synergistic and opposing regulation of the stress-responsive gene IEX-1 by p53, c-Myc, and multiple NF-kappaB/rel complexes. Oncogene, 21: 6819-6828, 2002.
文献３０：Kondratyev, A. D., Chung, K. N., and Jung, M. O. Identification and characterization of a radiation-inducible glycosylated human early-response gene. Cancer Res, 56: 1498-1502, 1996.
文献３１：Kittlesen, D. J., Thompson, L. W., Gulden, P. H., Skipper, J. C., Colella, T. A., Shabanowitz, J., Hunt, D. F., Engelhard, V. H., Slingluff, C. L., Jr., and Shabanowitz, J. A. Human melanoma patients recognize an HLA-A1-restricted ＣＴＬ epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development. J Immunol, 160: 2099-2106, 1998.
Literature list Literature 1: Rosenberg, SA A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity, 10: 281-287, 1999.
Reference 2: Rosenberg, SA Progress in human tumour immunology and immunotherapy. Nature, 411: 380-384, 2001.
Reference 3: Ribas, A., Butterfield, LH, Glaspy, JA, and Economou, JS Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol, 21: 2415-2432, 2003.
Reference 4: Sato, Y., Shomura, H., Maeda, Y., Mine, T., Une, Y., Akasaka, Y., Kondo, M., Takahashi, S., Shinohara, T., Katagiri, K., Sato, M., Okada, S., Matsui, K., Yamada, A., Yamana, H., Itoh, K., and Todo, S. Immunological evaluation of peptide vaccination for patients with gastric cancer based on pre-existing cellular response to peptide. Cancer Sci, 94: 802-808, 2003.
Reference 5: Mine, T., Gouhara, R., Hida, N., Imai, N., Azuma, K., Rikimaru, T., Katagiri, K., Nishikori, M., Sukehiro, A., Nakagawa, M., Yamada, A., Aizawa, H., Shirouzu, K., Itoh, K., and Yamana, H. Immunological evaluation of CTL precursor-oriented vaccines for advanced lung cancer patients. Cancer Sci, 94: 548-556 , 2003.
Reference 6: Tsuda, N., Mochizuki, K., Harada, M., Sukehiro, A., Kawano, K., Yamada, A., Ushijima, K., Sugiyama, T., Nishida, T., Yamana, H., Itoh, K., and Kamura, T. Vaccination with pre-designated or evidence-based peptides for patients with recurrent gynecologic cancers.J Immunother, in press.
Reference 7: Noguchi, M., Kobayashi, K., Suetsugu, N., Tomiyasu, K., Suekane, S., Yamada, A., Itoh, K., and Noda, S. Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination. Prostate, 57: 80-92, 2003.
Reference 8: Skipper, JC, Hendrickson, RC, Gulden, PH, Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, CL, Jr., Boon, T., Hunt, DF, and Engelhard, VH An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.J Exp Med, 183: 527-534, 1996.
Reference 9: Shiichijo, S., Nakao, M., Imai, Y., Takasu, H., Kawamoto, M., Niiya, F., Yang, D., Toh, Y., Yamana, H., and Itoh , K. A gene encoding antigenic peptides of human squamous cell carcinoma recognized by cytotoxic T lymphocytes.J Exp Med, 187: 277-288, 1998.
Reference 10: Gomi, S., Nakao, M., Niiya, F., Imamura, Y., Kawano, K., Nishizaka, S., Hayashi, A., Sobao, Y., Oizumi, K., and Itoh , K. A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs. J Immunol, 163: 4994-5004, 1999.
Reference 11: Ito, M., Shichijo, S., Tsuda, N., Ochi, M., Harashima, N., Saito, N., and Itoh, K. Molecular basis of T cell-mediated recognition of pancreatic cancer cells Cancer Res, 61: 2038-2046, 2001.
Reference 12: Wang, RF, Johnston, SL, Southwood, S., Sette, A., and Rosenberg, SA Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33 J Immunol, 160: 890-897, 1998.
Reference 13: Takedatsu, H., Shichijo, S., Katagiri, K., Sawamizu, H., Sata, M., and Itoh, K. Identification of peptide vaccine candidates sharing among HLA-A3 +, -A11 +, -A31 +, -A33 + cancer patients. Clin Cancer Res, in press.
Reference 14: Imanishi I, AT, Kimura A, Tokunaga K, Gojobori T. Allele and haprotype frequencies for HLA and complement loci in various ethnic groups. In, pp. 1065-1220: Oxford University Press, 1992.
Reference 15: Aizawa, M. Allele and haplotype frequencies for HLA and ccomplement loci in various ethnic groups. Oxford: Oxford University Press, 1992.
Reference 16: Macdonald, JS Advances in the therapy of gastric cancer. Gastric Cancer, 5 Suppl 1: 35-40, 2002.
Reference 17: Wu, MX Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis. Apoptosis, 8: 11-18, 2003.
Reference 18: Takiguchi, M., Matsuda, T., Tomiyama, H., and Miwa, K. Analysis of three HLA-A * 3303 binding peptide anchors using an HLA-A * 3303 stabilization assay. Tissue Antigens, 55: 296 -302, 2000.
Reference 19: Takiguchi, M., Matsuda, T., and Tomiyama, H. Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A * 3101 and HLA-A * 3303. Tissue Antigens, 56: 501-506, 2000.
Reference 20: Schafer, H., Trauzold, A., Siegel, EG, Folsch, UR, and Schmidt, WE PRG1: a novel early-response gene transcriptionally induced by pituitary adenylate cyclase activating polypeptide in a pancreatic carcinoma cell line. , 56: 2641-2648, 1996.
Reference 21: Pietzsch, A., Buchler, C., Aslanidis, C., and Schmitz, G. Identification and characterization of a novel monocyte / macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide, and lysophosphatidylcholine. Biochem Biophys Res Commun, 235: 4-9, 1997.
Reference 22: Charles, CH, Yoon, JK, Simske, JS, and Lau, LF Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene, 8 : 797-801, 1993.
Reference 23: Wu, MX, Ao, Z., Prasad, KV, Wu, R., and Schlossman, SF IEX-1L, an apoptosis inhibitor involved in NF-kappaB-mediated cell survival. Science, 281: 998-1001, 1998.
Reference 24: Kumar, R., Kobayashi, T., Warner, GM, Wu, Y., Salisbury, JL, Lingle, W., and Pittelkow, MR A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes. Biochem Biophys Res Commun, 253: 336-341, 1998.
Reference 25: Schafer, H., Lettau, P., Trauzold, A., Banasch, M., and Schmidt, WE Human PACAP response gene 1 (p22 / PRG1): proliferation-associated expression in pancreatic carcinoma cells. Pancreas, 18 : 378-384, 1999.
Reference 26: Grobe, O., Arlt, A., Ungefroren, H., Krupp, G., Folsch, UR, Schmidt, WE, and Schafer, H. Functional disruption of IEX-1 expression by concatemeric hammerhead ribozymes alters growth properties of 293 cells. FEBS Lett, 494: 196-200, 2001.
Reference 27: Zhang, Y., Schlossman, SF, Edwards, RA, Ou, CN, Gu, J., and Wu, MX Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1- transgenic mice.Proc Natl Acad Sci USA, 99: 878-883, 2002.
Reference 28: Garcia, J., Ye, Y., Arranz, V., Letourneux, C., Pezeron, G., and Porteu, F. IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation Embo J, 21: 5151-5163, 2002.
Reference 29: Huang, YH, Wu, JY, Zhang, Y., and Wu, MX Synergistic and opposing regulation of the stress-responsive gene IEX-1 by p53, c-Myc, and multiple NF-kappaB / rel complexes. Oncogene , 21: 6819-6828, 2002.
Reference 30: Kondratyev, AD, Chung, KN, and Jung, MO Identification and characterization of a radiation-inducible glycosylated human early-response gene. Cancer Res, 56: 1498-1502, 1996.
Reference 31: Kittlesen, DJ, Thompson, LW, Gulden, PH, Skipper, JC, Colella, TA, Shabanowitz, J., Hunt, DF, Engelhard, VH, Slingluff, CL, Jr., and Shabanowitz, JA Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development.J Immunol, 160: 2099-2106, 1998.
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