JPWO2012141201A1 - Peptides for cancer immunotherapy and use thereof - Google Patents
Peptides for cancer immunotherapy and use thereof Download PDFInfo
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- JPWO2012141201A1 JPWO2012141201A1 JP2013509939A JP2013509939A JPWO2012141201A1 JP WO2012141201 A1 JPWO2012141201 A1 JP WO2012141201A1 JP 2013509939 A JP2013509939 A JP 2013509939A JP 2013509939 A JP2013509939 A JP 2013509939A JP WO2012141201 A1 JPWO2012141201 A1 JP WO2012141201A1
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Abstract
【課題】腎臓癌の免疫療法に有用なペプチドを見出し、有効な治療法や早期診断方法を提供すること。【解決手段】下記(1)〜(6)のいずれかのアミノ酸配列から成り、免疫誘導活性を有することを特徴とするペプチド。AYPMPFITTI ‥‥‥(1)AYCETHYNQL ‥‥‥(2)FLVQSSDFKV ‥‥‥(3)ILFVQYFHRV ‥‥‥(4)KLTLKNKFV ‥‥‥(5)ELYHEQCFV ‥‥‥(6)【選択図】図1An object of the present invention is to find a peptide useful for immunotherapy of kidney cancer and to provide an effective treatment method and early diagnosis method. [MEANS FOR SOLVING PROBLEMS] A peptide comprising the amino acid sequence of any one of (1) to (6) below and having immunity-inducing activity. AYPMPFITTI (1) AYCETHYNQL (2) FLVQSSDFKV (3) ILFVQYFHRV (4) KLTLKNKFV (5) ELYHEQCFV (6) [Selection] Figure 1
Description
本発明は、腎臓癌の免疫療法に有用な腎臓癌特異的細胞傷害性リンパ球を誘導しうるペプチドと、そのペプチドの利用に関する。詳細には、腎臓癌に特異的な抗原蛋白質、その部分ペプチド、それらをコードするDNA、抗原蛋白質をエピトープとする抗体、誘導活性された免疫細胞、腎臓癌ワクチン、腎臓癌診断薬、腎臓癌発症素因評価方法、更には、腎臓癌等の免疫療法に有用な標的遺伝子の探索方法に関する。 The present invention relates to a peptide capable of inducing kidney cancer-specific cytotoxic lymphocytes useful for immunotherapy of kidney cancer, and use of the peptide. In detail, antigen protein specific to kidney cancer, partial peptide thereof, DNA encoding them, antibody having antigen protein as epitope, inducible immune cell, kidney cancer vaccine, kidney cancer diagnostic agent, kidney cancer onset The present invention also relates to a predisposition evaluation method and a method for searching for a target gene useful for immunotherapy such as kidney cancer.
癌は、発生機序の解明や、診断法、治療法が進歩しつつあるが、多くの進行癌を治療できないのが現状である。これを改善するために、有効な早期診断法と治療法の開発が求められている。
腎臓癌には、特異的な腫瘍マーカーがないのが現状であるので、早期診断が困難である。Elucidation of the mechanism of cancer, diagnostic methods, and therapeutic methods are progressing, but at present, many advanced cancers cannot be treated. In order to improve this, development of an effective early diagnosis method and treatment method is demanded.
Since renal cancer currently has no specific tumor marker, early diagnosis is difficult.
癌の治療法のひとつに免疫療法がある。従来の免疫療法は非特異的免疫療法を中心として行われてきたが、近年、T細胞が生体内での腫瘍拒絶に重要な役割を果たすことが明らかになり、細胞傷害性T細胞(CTL:Cytotoxic T Lymphocyte)を誘導しうるT細胞認識腫瘍抗原の単離とMHCクラスI拘束性エピトープの特定が求められている。しかし、多くの腫瘍抗原の単離として細胞傷害性T細胞を用いたcDNA発現クローニング法が行われてきたが、腫瘍の細胞株化と細胞傷害性T細胞の樹立が必要であることから、メラノーマ以外の癌腫からの腫瘍抗原の単離は困難であった。 One therapy for cancer is immunotherapy. Conventional immunotherapy has been performed centering on non-specific immunotherapy, but in recent years it has become clear that T cells play an important role in tumor rejection in vivo, and cytotoxic T cells (CTL: There is a need for isolation of T cell-recognized tumor antigens that can induce Cytotoxic T Lymphocyte) and identification of MHC class I-restricted epitopes. However, although a cDNA expression cloning method using cytotoxic T cells has been performed as an isolation of many tumor antigens, it is necessary to establish a tumor cell line and establish cytotoxic T cells. Isolation of tumor antigens from other carcinomas was difficult.
ペプチドワクチンを利用した免疫療法は、肺癌に対するSART、MAGEペプチドワクチン療法や、脾臓癌に対するHLA−A24SARTペプチドワクチン療法や、血液悪性腫瘍・消化器・胸部・泌尿器・生殖器等の殆どの固形癌に対するWT1ペプチドワクチン療法など、多数の報告があり、腎臓癌に関連するものもある(非特許文献1〜6)。
非特許文献1〜3は、CA9ペプチドワクチン療法を開示している。しかし、ワクチン接種により特異的細胞傷害性T細胞は誘導できるものの、極めて限られた臨床効果であり、23例の患者のうち癌の部分的縮小を2名で認めたのみである。CA9は腎臓癌(淡明細胞癌)でほぼ100%の発現であるが、ワクチンとしての有用性は現時点では高くない。
非特許文献4は、変異VHLペプチドワクチン療法、非特許文献5は、WT1ペプチドワクチン療法を開示している。しかし、小規模な臨床研究があるのみで、極めて限られた臨床効果である。
非特許文献6は、HIFPH3ペプチドによる細胞傷害性T細胞の誘導を開示している。しかし、特異的細胞傷害性T細胞誘導が証明されているものの、臨床での検討はこれからである。Immunotherapy using peptide vaccines includes SART and MAGE peptide vaccine therapy for lung cancer, HLA-A24SART peptide vaccine therapy for spleen cancer, and WT1 for most solid cancers such as hematological malignancies, digestive organs, chest, urinary organs, and genitals There are many reports such as peptide vaccine therapy, and some are related to kidney cancer (
Non-patent
Non-Patent
Non-Patent Document 6 discloses the induction of cytotoxic T cells by HIFPH3 peptide. However, although specific cytotoxic T cell induction has been demonstrated, clinical studies are now on.
このように臨床効果が限られている理由は、癌抗原に対する免疫が、実際には生体でうまく機能していないからであると考えられる。癌組織での発現量の多さでスクリーニングされた抗原は、実際には免疫療法の良いターゲットであるとは限らない。 The reason why the clinical effect is limited in this way is considered that immunity against cancer antigens does not actually function well in the living body. Antigens screened for high expression levels in cancer tissues are not always good targets for immunotherapy.
関連する従来技術には、特許文献1〜6もある。
特許文献1は、主に食道癌に関するものであるが、cDNAマイクロアレイを用いて食道癌の抗原を発見し、その抗原アミノ酸配列由来のペプチドを用いたワクチン療法を開示している。特許文献2〜6も、腎臓癌またはワクチンに関連するものである。しかしながら、従来技術では、腎臓癌の免疫療法に有用なペプチドは発見されず、ワクチンや早期診断方法も得られていない。Related arts also include Patent Documents 1-6.
そこで、本発明は、腎臓癌の免疫療法に有用なペプチドを見出し、有効な治療法や早期診断方法を提供すること、更には、腎臓癌等の免疫療法に有用な標的遺伝子の探索方法を提供することを課題とする。 Therefore, the present invention finds a peptide useful for immunotherapy of kidney cancer, provides an effective treatment method and early diagnosis method, and further provides a method for searching a target gene useful for immunotherapy of kidney cancer and the like. The task is to do.
本発明のペプチドは、下記(1)〜(6)のいずれかのアミノ酸配列から成り、免疫誘導活性を有することを特徴とする。
AYPMPFITTI ‥‥‥(1)
AYCETHYNQL ‥‥‥(2)
FLVQSSDFKV ‥‥‥(3)
ILFVQYFHRV ‥‥‥(4)
KLTLKNKFV ‥‥‥(5)
ELYHEQCFV ‥‥‥(6)The peptide of the present invention comprises the amino acid sequence of any one of the following (1) to (6), and has immunity-inducing activity.
AYPMPFITTI (1)
AYCETHYNQL (2)
FLVQSSDFKV (3)
ILFVQYFHRV (4)
KLTLKNKFV (5)
ELYHEQCFV (6)
上記(1)〜(6)のいずれかのアミノ酸配列において、1または数個のアミノ酸の置換、欠失、挿入、付加を含むアミノ酸配列から成り、免疫誘導活性を有するペプチドでもよい。 The amino acid sequence of any one of the above (1) to (6) may be a peptide having an immunity-inducing activity, comprising an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids.
本発明の蛋白質は、上記のいずれかに記載のペプチドを含み、腎臓癌抗原蛋白質を認識する細胞傷害性T細胞を活性化しうることを特徴とする。 The protein of the present invention includes any of the peptides described above, and is characterized in that it can activate cytotoxic T cells that recognize the renal cancer antigen protein.
本発明の細胞は、上記のいずれかに記載のペプチドまたはそれらの混合物を用いてインビトロ刺激により誘導されたことを特徴とするヘルパーT細胞、細胞傷害性T細胞、または、インビトロでパルスされたことを特徴とする抗原提示細胞、樹状細胞、或いは、これらを含む免疫細胞集団である。 The cell of the present invention is a helper T cell, cytotoxic T cell, or pulsed in vitro characterized by being induced by in vitro stimulation using any of the peptides described above or a mixture thereof An antigen-presenting cell, a dendritic cell, or an immune cell population containing these.
本発明の腎臓癌ワクチンは、上記のいずれかに記載のペプチドを含むことを特徴とする。 The kidney cancer vaccine of the present invention is characterized by including any of the peptides described above.
本発明の抗体は、上記のいずれかに記載のペプチドに対して抗原抗体反応を生じ得ることを特徴とする。 The antibody of the present invention is characterized by being capable of causing an antigen-antibody reaction against any of the above-described peptides.
本発明の腎臓癌診断薬は、上記の抗体を含むことを特徴とする。 The diagnostic agent for renal cancer of the present invention is characterized by containing the above-mentioned antibody.
本発明の腎臓癌発症素因評価方法は、腎臓癌を発症する素因を有するか否かを評価する方法であって、被験者由来の生体試料において請求項1に記載のペプチド関連遺伝子の発現レベルを求めるステップと、その発現レベルを正常対照レベルと比較し所定の閾値より高ければ、腎臓癌を発症する素因が高いと評価するステップとを有することを特徴とする。
The method for evaluating a predisposition to developing kidney cancer according to the present invention is a method for evaluating whether or not a predisposition to develop kidney cancer is present, and the expression level of the peptide-related gene according to
本発明の標的遺伝子の探索方法は、免疫療法に有用な標的遺伝子の探索方法であって、 サイトカイン療法が有効であった癌患者から血清を採取するステップと、その血清をプローブとして用いたSEREX法によりcDNA発現ライブラリーをスクリーニングするステップとを有することを特徴とする。 The method for searching for a target gene of the present invention is a method for searching for a target gene useful for immunotherapy, comprising the steps of collecting serum from a cancer patient for which cytokine therapy was effective, and a SEREX method using the serum as a probe Screening the cDNA expression library.
本発明によると、腎臓癌に有効な免疫療法と腎臓癌の発症素因評価方法が得られるので、腎臓癌の治療や予防に寄与する。 According to the present invention, an immunotherapy effective for kidney cancer and a method for evaluating the predisposition to developing kidney cancer can be obtained, which contributes to the treatment and prevention of kidney cancer.
以下に、本発明の実施形態を説明する。実施形態は、前記特許文献など従来公知の技術を援用して適宜設計変更可能であり、また、薬剤等の製造方法及び装置についても従来公知の技術を適宜適用可能である。 Hereinafter, embodiments of the present invention will be described. The embodiment can be appropriately changed in design by using a conventionally known technique such as the above-mentioned patent document, and a conventionally known technique can be appropriately applied to a method and an apparatus for manufacturing a drug or the like.
進行性腎臓癌では、少数ながらサイトカイン療法が著効する患者がいる。このような患者の血清には、癌転移巣の縮小や消失など関わる抗体が産生され、免疫が獲得されたと考えられる。実際に今まで報告されたペプチドワクチン療法奏功例では、IgGレベルが高いという報告が多い。 In advanced kidney cancer, there are a small number of patients who can benefit from cytokine therapy. It is considered that immunity was acquired by producing antibodies related to the reduction or disappearance of cancer metastasis in the serum of such patients. In fact, there have been many reports that IgG levels are high in the cases of successful peptide vaccine therapy reported so far.
本発明者は、腎臓癌の中のどの蛋白質が抗原となり免疫に関わる抗体を産生するに至ったかを解明するために、サイトカイン療法が奏功した患者の血清と反応する抗原蛋白質を探索した。そして、2種類の抗原遺伝子(36-6-1, 113-3-1)を同定し、これら抗原より高い活性を有する特異的細胞傷害性T細胞を誘導する6種類のペプチドを見出して、本発明を完成するに至った。
なお、サイトカイン療法が有効であった進行性腎臓癌患者血清を用いた解析は、本発明が初めてである。In order to elucidate which protein in kidney cancer became an antigen and produced an antibody related to immunity, the present inventor searched for an antigen protein that reacts with the serum of a patient who succeeded in cytokine therapy. Then, two types of antigen genes (36-6-1, 113-3-1) were identified, and six types of peptides that induce specific cytotoxic T cells having higher activity than these antigens were found. The invention has been completed.
The present invention is the first analysis using serum from patients with advanced renal cancer who have been effective for cytokine therapy.
本発明によるペプチド及び蛋白質は、以下の通りである。
本発明のペプチドは、下記(1)〜(6)のいずれかのアミノ酸配列から成り、免疫誘導活性を有する。
AYPMPFITTI ‥‥‥(1)
AYCETHYNQL ‥‥‥(2)
FLVQSSDFKV ‥‥‥(3)
ILFVQYFHRV ‥‥‥(4)
KLTLKNKFV ‥‥‥(5)
ELYHEQCFV ‥‥‥(6)The peptides and proteins according to the present invention are as follows.
The peptide of the present invention comprises any one of the following amino acid sequences (1) to (6) and has immunity-inducing activity.
AYPMPFITTI (1)
AYCETHYNQL (2)
FLVQSSDFKV (3)
ILFVQYFHRV (4)
KLTLKNKFV (5)
ELYHEQCFV (6)
本発明の蛋白質は、上記(1)〜(6)のいずれかのアミノ酸配列を含み、免疫誘導活性を有する。 The protein of the present invention comprises any one of the amino acid sequences (1) to (6) above and has immunity-inducing activity.
ここで、上記(1)〜(6)のいずれかのアミノ酸配列において、1または数個のアミノ酸の置換、欠失、挿入、付加を含んでもよい。 Here, the amino acid sequence of any one of the above (1) to (6) may include substitution, deletion, insertion, or addition of one or several amino acids.
特に、腎臓癌抗原蛋白質を認識する細胞傷害性T細胞を活性化しうるものが好ましい。 In particular, those capable of activating cytotoxic T cells that recognize kidney cancer antigen protein are preferred.
上記(1)は、細胞性免疫にかかわる糖鎖結合蛋白質ガレクチン9(36-6-1)(非特許文献7) のアミノ酸配列由来のペプチドA24/10mer190である。ガレクチン9は腎臓癌の大多数を占める淡明細胞癌において、正常組織に比して著しく高発現している。遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も公知でない。 The above (1) is peptide A24 / 10mer190 derived from the amino acid sequence of sugar chain binding protein galectin 9 (36-6-1) (Non-patent Document 7) involved in cellular immunity. Galectin 9 is remarkably expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues. Although the gene sequence is known, this peptide is not known and its use is not known.
上記(2)は、細胞接着のシグナリングに関するアダプター蛋白PINCH(113-3-1)(非特許文献8)のアミノ酸配列由来のペプチドA24/10mer238である。PINCHも、腎臓癌の大多数を占める淡明細胞癌において、正常組織に比して著しく高発現し、遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も公知でない。 The above (2) is peptide A24 / 10mer238 derived from the amino acid sequence of adapter protein PINCH (113-3-1) (Non-patent Document 8) relating to cell adhesion signaling. PINCH is also highly expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues, and the gene sequence is known, but this peptide is not known and its use is not known.
同様に、上記(3)及び(4)は、細胞性免疫にかかわる糖鎖結合蛋白質ガレクチン9(36-6-1)(非特許文献7) のアミノ酸配列由来のペプチド10m-103及び10m-117である。ガレクチン9は腎臓癌の大多数を占める淡明細胞癌において、正常組織に比して著しく高発現している。遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も公知でない。 Similarly, (3) and (4) above are peptides 10m-103 and 10m-117 derived from the amino acid sequence of sugar chain binding protein galectin 9 (36-6-1) (Non-patent Document 7) involved in cellular immunity. It is. Galectin 9 is remarkably expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues. Although the gene sequence is known, this peptide is not known and its use is not known.
同様に、上記(5)及び(6)は、細胞接着のシグナリングに関するアダプター蛋白PINCH(113-3-1)(非特許文献8)のアミノ酸配列由来のペプチド9m-284及び9m-29である。PINCHも、腎臓癌の大多数を占める淡明細胞癌において、正常組織に比して著しく高発現し、遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も公知でない。 Similarly, (5) and (6) above are peptides 9m-284 and 9m-29 derived from the amino acid sequence of adapter protein PINCH (113-3-1) (Non-patent Document 8) relating to signaling of cell adhesion. PINCH is also highly expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues, and the gene sequence is known, but this peptide is not known and its use is not known.
これらの腎臓癌抗原蛋白質は、例えば、腎臓癌患者から採取した癌細胞からcDNAマイクロアレイ解析法などにより検出することができる。なお、cDNAマイクロアレイ解析法とは、被験者から摘出された組織を癌部と非癌部とに分けてmRNAを調製し、それから蛍光標識したcDNAを作成し、そのcDNAを全遺伝子を所定割合スポットしてあるスライドガラスに載せ、ハイブリダイズ後、スキャナーでシグナルを取込み、遺伝子発現を解析する方法である。 These kidney cancer antigen proteins can be detected by, for example, cDNA microarray analysis from cancer cells collected from kidney cancer patients. In the cDNA microarray analysis method, mRNA is prepared by dividing a tissue extracted from a subject into a cancerous part and a non-cancerous part, and then fluorescence-labeled cDNA is prepared. This is a method of analyzing the gene expression by placing the signal on a glass slide and hybridizing, and then capturing the signal with a scanner.
腎臓癌抗原蛋白質の製造方法は特に限定されず、天然由来の蛋白質でも、化学合成した蛋白質でも、遺伝子組み換え技術により作成した組み換え蛋白質でもよい。比較的容易な操作で大量に製造できるという点では、組み換え蛋白質が好ましい。
天然由来の蛋白質を入手する場合には、その蛋白質を発現している細胞または組織から蛋白質の単離精製方法を適宜組み合わせて単離することができる。化学合成の蛋白質を入手する場合には、Fmoc法やtBoc法などの化学合成法に従って合成することができる。また、各種の市販のペプチド合成機を利用して合成することもできる。
組み替え蛋白質として産生するには、その蛋白質をコードする塩基配列を有するDNAまたはその変異体または相同体を入手し、好適な発現系に導入することにより製造することができる。The method for producing the renal cancer antigen protein is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein prepared by a gene recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities by a relatively easy operation.
When a naturally-derived protein is obtained, it can be isolated from cells or tissues expressing the protein by appropriately combining methods for protein isolation and purification. When a chemically synthesized protein is obtained, it can be synthesized according to a chemical synthesis method such as the Fmoc method or the tBoc method. It can also be synthesized using various commercially available peptide synthesizers.
To produce it as a recombinant protein, it can be produced by obtaining a DNA having a base sequence encoding the protein or a mutant or homologue thereof and introducing it into a suitable expression system.
発現ベクターとしては、好ましくは宿主細胞において自立複製可能であるか、宿主細胞の染色体中へ組込み可能であるものであればよく、遺伝子を発現できる位置にプロモーターを含有しているものが使用される。また、蛋白質をコードする遺伝子を有する形質転換体は、発現ベクターを宿主に導入することにより作製することができる。宿主は、細菌、酵母、動物細胞、昆虫細胞のいずれでもよく、また宿主への発現ベクターの導入は、各宿主に応じた従来公知の定法により行えばよい。遺伝子を有する形質転換体を培養し、培養物中に所望の蛋白質を生成蓄積させ、培養物より蛋白質を採取することにより組み換え蛋白質を単離することができる。
形質転換体が大腸菌等の原核生物、酵母菌等の真核生物である場合、これら微生物を培養する培地は、微生物が資化しうる炭素原、窒素源、無機塩類等を含有し、形質転換体の培養を効率的に行える培地であれば、天然培地でも合成培地でもよい。また、培養条件も微生物を培養するのに通常用いられる条件で行なえばよい。培養後、所望の蛋白質を単離精製するには、従来公知の蛋白質の単離精製法を用いればよい。The expression vector is preferably any vector that can replicate autonomously in the host cell or can be integrated into the chromosome of the host cell, and a vector containing a promoter at a position where the gene can be expressed is used. . Moreover, the transformant which has the gene which codes a protein can be produced by introduce | transducing an expression vector into a host. The host may be any of bacteria, yeast, animal cells, and insect cells, and the introduction of the expression vector into the host may be performed by a conventionally known standard method according to each host. A recombinant protein can be isolated by culturing a transformant having a gene, producing and accumulating a desired protein in the culture, and collecting the protein from the culture.
When the transformant is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism. A natural medium or a synthetic medium may be used as long as the medium can efficiently be cultured. Further, the culture conditions may be the conditions usually used for culturing microorganisms. In order to isolate and purify a desired protein after culturing, a conventionally known protein isolation and purification method may be used.
なお、上記(1)〜(6)に記載のアミノ酸配列において、1または数個のアミノ酸の置換、欠損、挿入及び/又は付加を含むアミノ酸配列を有する蛋白質は、そのアミノ酸配列をコードするDNA配列の情報に基づいて従来公知の定法により製造することができる。すなわち、所望のアミノ酸配列を有する蛋白質をコードする塩基配列を有する変異遺伝子は、化学合成、遺伝子工学的手法、突然変異誘発などで作製することもできる。例えば、変位前のDNAに対し、変異原となる薬剤と接触作用させる方法、紫外線を照射する方法、遺伝子工学的手法を用いて行なうことができる。 In addition, in the amino acid sequence described in the above (1) to (6), a protein having an amino acid sequence including substitution, deletion, insertion and / or addition of one or several amino acids is a DNA sequence encoding the amino acid sequence Based on this information, it can be produced by a conventionally known method. That is, a mutant gene having a base sequence encoding a protein having a desired amino acid sequence can also be prepared by chemical synthesis, genetic engineering techniques, mutagenesis, and the like. For example, it can be carried out by using a method in which the DNA before displacement is brought into contact with a mutagen agent, a method of irradiating with ultraviolet rays, or a genetic engineering technique.
ペプチドの合成は、通常のペプチド化学において用いられる方法に準じて行うことができる。例えば、Fmoc法やtBoc法等の化学合成法に従って合成することができる。また、各種の市販ペプチド合成機を利用して本発明のペプチドを合成することもできる。本発明によるペプチドは、HLA−A*2402またはHLA−A*0201に対する結合モチーフを有する。このようなHLA−A*2402またはHLA−A*0201に対する結合モチーフを有するペプチドの選択は、種々のペプチドとHLA抗原との結合親和性の算出を含め、従来公知の定法に基づいて行うことができる。 Peptide synthesis can be performed according to methods used in normal peptide chemistry. For example, it can be synthesized according to a chemical synthesis method such as the Fmoc method or the tBoc method. Moreover, the peptide of this invention can also be synthesize | combined using various commercially available peptide synthesizers. The peptides according to the invention have a binding motif for HLA-A * 2402 or HLA-A * 0201. Selection of peptides having binding motifs for HLA-A * 2402 or HLA-A * 0201 can be performed based on conventionally known methods including calculation of binding affinity between various peptides and HLA antigens. it can.
本発明によるDNAは、以下の通りである。
本発明のDNAは、上記に記載した蛋白質またはペプチドをコードするDNAであり、好ましくは、下記(a)、(b)、(c)のいずれかに記載のDNAである。
(a)上記(1)〜(6)のいずれかに記載の塩基配列を有するDNA。
(b)前記(a)のDNAとストリンジェントな条件下でハイブリダイズし、免疫誘導活性を有する蛋白質またはペプチドをコードするDNA。
(c)前記(a)または(b)のDNAの部分配列を有し、免疫誘導活性を有する蛋白質またはペプチドをコードするDNA。The DNA according to the present invention is as follows.
The DNA of the present invention is a DNA encoding the protein or peptide described above, and is preferably the DNA described in any one of (a), (b) and (c) below.
(A) DNA having the base sequence according to any one of (1) to (6) above.
(B) DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein or peptide having immunity-inducing activity.
(C) DNA encoding a protein or peptide having a partial sequence of the DNA of (a) or (b) and having immunity-inducing activity.
なお、ストリンジェントな条件下でハイブリダイズするとは、DNAをプローブとして使用し、コロニー・ハイブリダイゼーション法、プラークハイブリダイゼーション法、サザンブロットハイブリダイゼーション法などを用いることにより得られるDNAの塩基配列を意味し、従来公知の定法によって同定でき、プローブとして使用するDNAの塩基配列と一定以上の相同性を有するDNAが挙げられる。 Hybridization under stringent conditions means a DNA base sequence obtained by using DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Examples thereof include DNA that can be identified by a conventionally known method and has a certain degree of homology with the base sequence of DNA used as a probe.
DNAの取得方法は特に限定されず、上記(1)〜(6)のいずれかに記載の配列情報に基づいて適当なプローブやプライマーを調製し、それらを用いてヒトなどのcDNAライブラリーをスクリーニングすることにより単離することができる。また、PCR法により取得することもでき、ヒト染色体DNA又はcDNAライブラリーを鋳型として使用し、所望の塩基配列を増幅できるように設計した1対のプライマーを用いてPCRを行い、次いで、増幅されたDNA断片を、大腸菌等の宿主で増幅可能な適切なベクター中にクローニングすることができる。 The DNA acquisition method is not particularly limited, and appropriate probes and primers are prepared based on the sequence information described in any of (1) to (6) above, and cDNA libraries such as humans are screened using them. Can be isolated. It can also be obtained by the PCR method. Using a human chromosomal DNA or cDNA library as a template, PCR is performed using a pair of primers designed to amplify a desired base sequence, and then amplified. DNA fragments can be cloned into an appropriate vector that can be amplified in a host such as E. coli.
本発明による抗体及び細胞傷害性細胞は、以下の通りである。
本発明は、本発明の蛋白質またはペプチドの一部もしくは全部をエピトープとして認識する抗体、並びに、本発明の蛋白質またはペプチドを用いてインビトロ刺激により誘導された細胞傷害性T細胞にも関する。一般的には、細胞傷害性T細胞の方が抗体よりも強い抗腫瘍活性を示す。The antibodies and cytotoxic cells according to the present invention are as follows.
The present invention also relates to an antibody that recognizes part or all of the protein or peptide of the present invention as an epitope, and cytotoxic T cells induced by in vitro stimulation using the protein or peptide of the present invention. In general, cytotoxic T cells show stronger antitumor activity than antibodies.
本発明の抗体は、ポリクローナル抗体でもモノクローナル抗体でもよく、また、その断片、更に、抗体の標識抗体でもよい。その作製は従来公知の定法により行うことができる。
ポリクローナル抗体は、本発明の蛋白質を抗原として哺乳動物を免疫感作して血液を採取し、採取した血液から抗体を分離精製することにより得られる。
哺乳動物としては、マウス、ハムスター、モルモット、ニワトリ、ラット、ウサギ、イヌ、ヤギ、ヒツジ、ウシなどが挙げられ、例えば、抗原約0.05〜2mg程度を7〜30日間隔で2〜3回投与する。抗原は完全フロインドアジュバントや水酸化アルミニウム等のアジュパントを含有する適当な緩衝液に溶解して用い、投与経路としては、皮下投与、皮内投与、腹膜腔内投与、静脈内投与、筋肉内投与などが挙げられる。免疫感作した哺乳動物から血液を採取して、遠心分離、硫酸アンモニウムやポリエチレングリコールを用いた沈殿、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー等のクロマトグラフィーなどによって分離精製することによりポリクローナル抗血清として、本発明の蛋白質を認識するポリクローナル抗体を得ることができる。The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, a fragment thereof, or a labeled antibody of the antibody. The production can be performed by a conventionally known method.
A polyclonal antibody is obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood, and separating and purifying the antibody from the collected blood.
Examples of mammals include mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, cows, and the like. For example, about 0.05 to 2 mg of antigen is administered 2 to 3 times at intervals of 7 to 30 days. Administer. Antigen is dissolved in an appropriate buffer containing adjuvant such as complete Freund's adjuvant or aluminum hydroxide, and the administration route includes subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. Is mentioned. By collecting blood from the immunized mammal and separating and purifying it by centrifugation, precipitation with ammonium sulfate or polyethylene glycol, chromatography such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. As an antiserum, a polyclonal antibody that recognizes the protein of the present invention can be obtained.
モノクローナル抗体は、ハイブリドーマを調製して得ることができる。
例えば、抗体産生細胞とミエローマ細胞株との細胞融合によりハイブリドーマが得られる。抗体産生細胞としては、免疫された動物からの脾細胞、リンパ節細胞、Bリンパ球等を使用する。抗原としては、本発明の蛋白質またはペプチドを使用する。免疫動物としては、マウス、ラット等を用い、アジュバントと抗原の蛋白質またはペプチドとの懸濁液を動物の静脈、皮下、皮内、腹腔内に数回投与することによって動物を免疫する。免疫動物から抗体産生細胞として例えば脾細胞を取得し、これとミエローマ細胞とを従来公知の定法により融合してハイブリドーマを作製する。細胞融合に使用するミエローマ細胞株としては、例えばマウスではP3X63Ag8、P3U1株、Sp2/0株などが挙げられる。細胞融合に際しては、ポリエチレングリコール、センダイウイルスなどの融合促進剤を用い、細胞融合後のハイブリドーマの選択にはヒポキサンチン・アミノプテリン・チミジン培地などを使用する。細胞融合により得られるハイブリドーマは限界希釈法等によりクローニングする。酵素免疫測定法でスクリーニングを行うことにより、所望の蛋白質を特異的に認識するモノクローナル抗体を産生する細胞株を得ることができる。
ハイブリドーマから目的とするモノクローナル抗体を製造するには、細胞培養法や腹水形成法によりハイブリドーマを培養し、培養上清あるいは腹水からモノクローナル抗体を精製する。精製には、硫安分画、ゲルろ過、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーなどを適宜組み合わせて使用する。Monoclonal antibodies can be obtained by preparing hybridomas.
For example, a hybridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line. As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the protein or peptide of the present invention is used. As the immunized animal, mice, rats and the like are used, and the animal is immunized by administering a suspension of an adjuvant and an antigen protein or peptide several times into the vein, subcutaneous, intradermal or intraperitoneal cavity of the animal. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and this and myeloma cells are fused by a conventionally known method to prepare a hybridoma. Examples of myeloma cell strains used for cell fusion include P3X63Ag8, P3U1 strain, Sp2 / 0 strain and the like in mice. For cell fusion, fusion promoters such as polyethylene glycol and Sendai virus are used, and hypoxanthine / aminopterin / thymidine medium or the like is used for selection of hybridomas after cell fusion. Hybridomas obtained by cell fusion are cloned by limiting dilution or the like. By screening by enzyme immunoassay, a cell line producing a monoclonal antibody that specifically recognizes a desired protein can be obtained.
In order to produce a target monoclonal antibody from a hybridoma, the hybridoma is cultured by a cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. For purification, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, etc. are used in appropriate combination.
本発明の抗体は標識して使用することもできる。例えば、酵素標識、蛍光標識、呈色物質による標識、アフィニティ標識、同位体標識などを挙げることができる。本発明の標識抗体を用いた蛋白質またはペプチドの分析には、酵素抗体法、免疫組織染色法、免疫ブロット法、直接蛍光抗体法、間接蛍光抗体法などが挙げられる。 The antibody of the present invention can also be used after labeling. For example, an enzyme label, a fluorescent label, a label with a coloring substance, an affinity label, an isotope label and the like can be mentioned. Examples of protein or peptide analysis using the labeled antibody of the present invention include enzyme antibody method, immunohistochemical staining method, immunoblot method, direct fluorescent antibody method, indirect fluorescent antibody method and the like.
本発明はまた、本発明の蛋白質またはペプシドを用いたインビトロ刺激により誘導された活性化T細胞に関する。例えば、末梢血リンパ球や腫瘍浸潤リンパ球を、本発明の蛋白質またはペプチドでインビトロ刺激すると、腫瘍反応性活性化T細胞が誘導され、この活性化されたT細胞は免疫療法に有効に用いることができる。また本発明の蛋白質またはペプチドを、強力な抗原提示細胞である樹状細胞にインビボまたはインビトロで発現させて、その抗原発現樹状細胞の投与により免疫誘導を行うこともできる。 The present invention also relates to activated T cells induced by in vitro stimulation using the protein or pepsid of the present invention. For example, when peripheral blood lymphocytes or tumor-infiltrating lymphocytes are stimulated in vitro with the protein or peptide of the present invention, tumor-reactive activated T cells are induced, and these activated T cells are effectively used for immunotherapy. Can do. In addition, the protein or peptide of the present invention can be expressed in vivo or in vitro in dendritic cells which are strong antigen-presenting cells, and immunity induction can be performed by administration of the antigen-expressing dendritic cells.
本発明によるワクチンは、以下の通りである。
本発明の蛋白質、ペプチド、DNAは、抗原特異的に腎臓癌細胞株を傷害することのできるT細胞を誘導することができるので、腎臓癌の治療薬、予防薬として使用可能である。例えば、本発明のDNAを適当なベクターに組み込み、組換えDNAで形質転換されたBCG菌の細菌、または本発明のDNAをゲノムに組込まれたワクシニアウイルス等のウイルスをワクチンとする。アジュバントとしては、フロイントの不完全アジュバント、BCG、トレハロースダイマイコレート、リポ多糖、ミョウバンアジュバント、シリカアジュバント等が挙げられる。The vaccine according to the present invention is as follows.
Since the protein, peptide, and DNA of the present invention can induce T cells that can specifically damage kidney cancer cell lines, they can be used as therapeutic or preventive agents for kidney cancer. For example, BCG bacteria transformed with recombinant DNA by incorporating the DNA of the present invention into an appropriate vector, or viruses such as vaccinia virus integrated with the DNA of the present invention in the genome are used as vaccines. Examples of adjuvants include Freund's incomplete adjuvant, BCG, trehalose dimycolate, lipopolysaccharide, alum adjuvant, silica adjuvant and the like.
本発明による腎臓癌診断用プローブ、診断薬は、以下の通りである。
本発明のDNAは、腎臓癌のDNAを取り出してその相同性を調べることで診断用プローブとして使用することができ、また、このプローブや上記抗体を使用し、腎臓癌診断薬として使用することもできる。
診断用プローブとしては、本発明の蛋白質をコードするDNAまたはRNAのアンチセンス鎖の全部または一部であり、プローブとして成立する程度の長さを有するものが好ましい。例えば、アンチセンス鎖を用いて検体から得られた腎臓癌抗原のmRNAを検出することにより診断が可能となる。検出に用いられる検体としては、被験者の腎臓等の細胞や、血液、尿、唾液、組織等の生検から得ることができるゲノムDNAや、RNAなどを挙げられる。The probe and diagnostic agent for diagnosing kidney cancer according to the present invention are as follows.
The DNA of the present invention can be used as a diagnostic probe by taking out the DNA of kidney cancer and examining its homology, and can also be used as a diagnostic agent for kidney cancer using this probe or the above-mentioned antibody. it can.
As the diagnostic probe, a probe which is the whole or a part of the antisense strand of DNA or RNA encoding the protein of the present invention and has a length sufficient to be established as a probe is preferable. For example, diagnosis can be performed by detecting mRNA of kidney cancer antigen obtained from a specimen using an antisense strand. Examples of the sample used for detection include cells such as kidneys of a subject, genomic DNA that can be obtained from a biopsy of blood, urine, saliva, tissue, and RNA.
本発明の蛋白質やペプチドに特異的に結合するモノクローナル抗体、ポリクローナル抗体、キメラ抗体、一本鎖抗体、ヒト化抗体等の免疫特異的な抗体は、腎臓癌の診断に利用することができる。
本発明の蛋白質、ペプチド、または抗体をそのまま、或いは医薬的に許容される担体や希釈剤や補助剤と共に、注射や経皮吸収などで投与可能である。Immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies that specifically bind to the proteins and peptides of the present invention can be used for the diagnosis of kidney cancer.
The protein, peptide, or antibody of the present invention can be administered as it is, or together with a pharmaceutically acceptable carrier, diluent or adjuvant, by injection or percutaneous absorption.
本発明による腎臓癌予防薬、治療薬は、以下の通りである。
本発明の蛋白質またはペプチドは、T細胞エピトープとして腎臓癌細胞特異的細胞傷害性T細胞を誘導できるので、腎臓癌の予防薬、治療薬として有用である。細胞傷害性T細胞の誘導活性を高めるための補助剤としては、サポニン系のQS−21、リポソーム、水酸化アルミニウムなどが挙げられる。また、レンチナン、シゾフィラン、ピシバーニールなどの免疫賦活剤を補助剤として用いることもできる。また、IL−2、IL−4、IL−12、IL−1、IL−6、TNF、IFNなどのT細胞の増殖、分化を増強するサイトカイン等も補助剤として用いることができる。The renal cancer preventive and therapeutic agents according to the present invention are as follows.
Since the protein or peptide of the present invention can induce a renal cancer cell-specific cytotoxic T cell as a T cell epitope, it is useful as a preventive or therapeutic agent for kidney cancer. Examples of the adjuvant for enhancing the inducing activity of cytotoxic T cells include saponin-based QS-21, liposome, aluminum hydroxide and the like. In addition, immunostimulants such as lentinan, schizophyllan, and picibanil can be used as adjuvants. In addition, cytokines that enhance proliferation and differentiation of T cells such as IL-2, IL-4, IL-12, IL-1, IL-6, TNF, and IFN can also be used as adjuvants.
また、腎臓癌患者から採取した細胞、または、同一のHLAパプロタイプをもつ細胞に試験管内で抗原ペプチドを加え、抗原提示させた後、患者血管内に投与し、患者体内で効果的に細胞障害性T細胞を誘導することもできる。また、患者末梢血リンパ球に抗原ペプチドを加えて試験管内で培養することにより試験管内で細胞傷害性T細胞を誘導した後に患者血管内に戻すこともできる。 In addition, antigen peptides are added to cells collected from patients with kidney cancer or cells having the same HLA paprotype in vitro and presented to the antigen, then administered into the patient's blood vessels and effectively cytotoxic in the patient's body. T cells can also be induced. In addition, cytotoxic T cells can be induced in a test tube by adding an antigen peptide to the patient's peripheral blood lymphocytes and cultured in a test tube, and then returned to the patient's blood vessel.
特異的抗腫瘍免疫療法の標的抗原となるためには、その抗原が細胞傷害性T細胞の認識抗原であることが必要である。本発明の抗原は日本人に多いHLA−A*2402において、また欧米人で多いHLA−A*0201において、インビトロにおけるキラーT細胞誘導活性を増大させるので、本発明の抗原を体内に注入することにより、細胞傷害性T細胞を誘導活性化し抗腫瘍に用いられる。また、本発明の抗原で刺激するとインビトロにおいて活性化T細胞が誘導されるので、活性化されたT細胞を体内に注入することによる免疫療法にも有効に用いることができる。 In order to be a target antigen for specific anti-tumor immunotherapy, it is necessary that the antigen is a recognition antigen for cytotoxic T cells. The antigen of the present invention increases in vitro killer T cell-inducing activity in HLA-A * 2402, which is common in Japanese, and in HLA-A * 0201, which is common in Westerners. Thus, cytotoxic T cells are induced and activated and used for antitumor. Moreover, since activated T cells are induced in vitro when stimulated with the antigen of the present invention, they can be effectively used for immunotherapy by injecting activated T cells into the body.
本発明によるRNAiは、以下の通りである。
本発明の蛋白質の発現をRNAi現象により抑制できる核酸としては、siRNA、shRNA、またはそれらの発現ベクターなどが挙げられる。RNAi according to the present invention is as follows.
Examples of the nucleic acid capable of suppressing the expression of the protein of the present invention by the RNAi phenomenon include siRNA, shRNA, or expression vectors thereof.
本発明の抗腫瘍剤は、必要に応じて薬学的に許容可能な添加剤を配合することができる。薬学的に許容可能な添加剤としては、抗酸化剤、保存剤、着色料、風味料、希釈剤、乳化剤、懸濁化剤、溶媒、フィラー、増量剤、緩衝剤、送達ビヒクル、希釈剤、キャリア、賦形剤、薬学的アジュバントなどが挙げられる。 The antitumor agent of this invention can mix | blend a pharmaceutically acceptable additive as needed. Pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, Examples include carriers, excipients, and pharmaceutical adjuvants.
また、本発明のペプチドは、ペプチド誘導体として提供してもよい。
この誘導体には、合成や精製を促進するための修飾、物理、化学的安定化を促進するための修飾、生体内の代謝に対する安定性と不安定性、条件付けの等の活性化修飾などを含む。
ペプチド誘導体におけるその他の修飾には、アセチル化、アシル化、ADP−リボシル化、アミド化、フラビンの共有結合、ヘム部分の共有結合、ヌクレオチドまたはヌクレオチド誘導体の共有結合、脂質または脂質誘導体の共有結合、ホスファチジルイノシトールの共有結合、交差架橋、環化、ジスルフィド結合、脱メチル化、交差架橋共有結合形成、シスチン形成、ピログルタメート形成、ホルミル化、ガンマーカルボキシル化、グリコシル化、GPIアンカー形成、水酸化、ヨウ素化、メチル化、ミリストイル化、酸化、タンパク質加水分解プロセッシング、リン酸化、プレニル化、ラセミ化、脂質結合、硫酸化、セレノイル化などが含まれる。
具体的には、ペプチド誘導体は、本発明のペプチドの活性を破壊せず、またこれを含有する組成物に毒性を与えない範囲において、残基の側鎖またはN末端基またはC末端基として生じる機能性基として調製することができる。例えば、体液中でペプチドの残存を延長するポリエチレングリコール側鎖を含む誘導体、カルボキシル基の脂肪族エステル、アンモニアまたはアミンと反応することによるカルボキシル基のアミド、アシル部分と形成されるアミノ酸残基の遊離アミノ基のN−アシル誘導体またはアシル部分と形成される遊離の水酸基のO−アシル誘導体などが挙げられる。Moreover, you may provide the peptide of this invention as a peptide derivative.
Such derivatives include modifications to promote synthesis and purification, modifications to promote physical and chemical stabilization, stability and instability to metabolism in vivo, and activation modifications such as conditioning.
Other modifications in peptide derivatives include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, Phosphatidylinositol covalent bond, cross-linking, cyclization, disulfide bond, demethylation, cross-linking covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodine , Methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid binding, sulfation, selenoylation and the like.
Specifically, the peptide derivative is generated as a side chain of a residue or an N-terminal group or a C-terminal group as long as the activity of the peptide of the present invention is not destroyed and the composition containing the peptide is not toxic It can be prepared as a functional group. For example, derivatives containing polyethylene glycol side chains that extend the residue of peptides in body fluids, aliphatic esters of carboxyl groups, amides of carboxyl groups by reacting with ammonia or amines, liberation of amino acid residues formed with acyl moieties Examples include N-acyl derivatives of amino groups or O-acyl derivatives of free hydroxyl groups formed with acyl moieties.
また、薬理学的に許容し得る塩としてペプチドを提供してもよい。
この塩には、ポリペプチドのカルボキシル基の塩とアミノ基の酸付加塩の双方を含む。
カルボキシル基の塩は、例えば、ナトリウム、カルシウム、アンモニウム、鉄、亜鉛などの無機塩や、トリエタノールアミン、アルギニン、リジン、ピペリジン、プロカインなどのアミンを用いて形成された有機塩基との塩が挙げられる。酸付加塩としては、例えば塩酸や硫酸などの鉱酸との塩、酢酸やシュウ酸などの有機酸との塩が挙げられる。Alternatively, the peptide may be provided as a pharmacologically acceptable salt.
This salt includes both a carboxyl group salt and an amino acid addition salt of a polypeptide.
Examples of the salt of the carboxyl group include inorganic salts such as sodium, calcium, ammonium, iron, and zinc, and salts with organic bases formed using amines such as triethanolamine, arginine, lysine, piperidine, and procaine. It is done. Examples of acid addition salts include salts with mineral acids such as hydrochloric acid and sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid.
本発明による腎臓癌発症素因評価方法は、以下の通りである。
被験者由来の生体試料において、本発明のペプチド関連遺伝子の発現レベルを求めるステップと、その発現レベルを正常対照レベルと比較し所定の閾値より高ければ、腎臓癌を発症する素因が高いと評価するステップとを有する。生体試料としては、腎臓細胞や血液などが挙げられ、その採取調製方法やペプチド関連遺伝子の発現レベルの測定方法や評価方法は、従来公知の定法が利用できる。The method for evaluating the predisposition to developing kidney cancer according to the present invention is as follows.
A step of obtaining the expression level of the peptide-related gene of the present invention in a biological sample derived from a subject, and a step of evaluating that the predisposition to develop kidney cancer is high if the expression level is higher than a predetermined threshold by comparing with the normal control level. And have. Examples of the biological sample include kidney cells and blood. Conventionally known conventional methods can be used as a method for collecting and preparing the same and a method for measuring and evaluating the expression level of peptide-related genes.
サイトカイン療法が有効であった進行性腎臓癌患者から採取した血清に、治療奏効に関わる抗体を想定し、SEREX法を用いて淡明細胞癌の発現ライブラリーをスクリーニングすることによって、免疫療法に有用な標的遺伝子を探索した。
なお、SEREX(serological analysis of cancer antigens by
recombinant cDNA expression cloning)法とは、癌患者より摘出した癌組織から直接mRNAを抽出し、作製したcDNA発現ライブラリーから癌患者の血清を用いて癌抗原を検索する方法である。マイクロアレイ等とは異なった網羅的解析法であり、データベースにSEREX抗原として多くの遺伝子が登録されているが殆どは未評価で、腎臓癌での報告は極めて少ない。Useful for immunotherapy by screening an expression library of clear cell carcinoma using the SEREX method assuming antibodies related to therapeutic response in serum collected from patients with advanced renal cancer who were effective with cytokine therapy We searched for a target gene.
SEREX (serological analysis of cancer antigens by
The recombinant cDNA expression cloning method is a method in which mRNA is directly extracted from a cancer tissue extracted from a cancer patient, and a cancer antigen is searched from the prepared cDNA expression library using the serum of the cancer patient. It is a comprehensive analysis method different from microarrays, and many genes are registered as SEREX antigens in the database, but most are not yet evaluated, and there are very few reports on kidney cancer.
具体的には、次のようにSEREX法を活用して免疫療法に有用な標的遺伝子を探索した。
E coli phage lysateにニトロセルロース(NC)膜を浸し、E coliやファージで発現している蛋白質をNC膜に吸着させた。そのNC膜を、TBSTで5倍に希釈した腎臓癌患者血清に浸して取り出し、血清中から大腸菌やファージと交差反応する抗体を除去して、SEREX法によるスクリーニングに使用した。Specifically, a target gene useful for immunotherapy was searched using the SEREX method as follows.
Nitrocellulose (NC) membrane was immersed in E. coli phage lysate, and proteins expressed in E. coli and phage were adsorbed on the NC membrane. The NC membrane was soaked in kidney cancer patient serum diluted 5-fold with TBST, and antibodies cross-reacting with E. coli and phage were removed from the serum and used for screening by the SEREX method.
ライブラリーは、患者の腎癌細胞(淡明細胞癌)を細胞培養株にして樹立したもの(OCUU1)よりRNAを抽出し、mRNAを分離した後、λZAPにクローニングされた形のcDNAライブラリーを作製した(Stratagene社製、ZAP-cDNA synthesis kit、Gigapack III gold cloning kit)。 The library was extracted from RNA (OCUU1) established from a patient's kidney cancer cells (clear cell carcinoma) as a cell culture strain, and after mRNA was isolated, a cDNA library was cloned into λZAP. (ZAP-cDNA synthesis kit, Gigapack III gold cloning kit, manufactured by Stratagene).
大腸菌細胞XL1-Blue MRF’をホストセルとして、10×14cmのNZYプレート1枚当たり19000プラークとなるようにcDNAライブラリーを播いた。42℃で4.5時間培養後、IPTGを含ませたNC膜をプレートに乗せ更に37℃で3.5時間培養し、ファージのプラークで蛋白質発現をさせた。4℃で一晩冷やし、NC膜をプレートより剥がして、プラークリフトを取った。
TBSTで洗浄の後1時間ブロッキングを行い、1次抗体として100倍希釈の上記処理済みの患者血清と室温で2時間反応させた。TBSTで洗浄のあと、2次抗体としてgoat anti-human IgG(ALP-conjugated)と室温で1時間反応させた。TBST及びTBSで洗った後、BCIP及びNBTを用いて発色させた。プラークリフトを取った元のファージのプレートより、NC膜で陽性のシグナルが出ている場所と一致する場所のプラークを含む培地を打ち抜いて、SMバッファーに浮かべ、2次スクリーニングに供した。Using the E. coli cell XL1-Blue MRF ′ as a host cell, a cDNA library was seeded so that there would be 19000 plaques per 10 × 14 cm NZY plate. After culturing at 42 ° C. for 4.5 hours, an NC membrane containing IPTG was placed on the plate and further cultured at 37 ° C. for 3.5 hours, and protein expression was carried out by phage plaques. The plate was cooled overnight at 4 ° C., the NC membrane was peeled off from the plate, and a plaque lift was taken.
After washing with TBST, blocking was performed for 1 hour, and reaction was performed at room temperature for 2 hours with the above-treated patient serum diluted 100 times as a primary antibody. After washing with TBST, it was reacted with goat anti-human IgG (ALP-conjugated) as a secondary antibody at room temperature for 1 hour. After washing with TBST and TBS, color was developed using BCIP and NBT. From the original phage plate from which the plaque lift was taken, the medium containing the plaque at a location that coincided with the location where a positive signal appeared on the NC membrane was punched out, floated in SM buffer, and subjected to secondary screening.
2次スクリーニングとして、直径10cmのNZYプレートに1プレート当たり500個になるようにファージを播き、同様の操作でスクリーニングを行った。
3次スクリーニングで単一のクローンを得て、ファージよりin-vivo excisionを行い、プラスミド(pBluescript)に遺伝子がクローニングされている形にして、シーケンスを行った。As secondary screening, phages were seeded on an NZY plate having a diameter of 10 cm so that the number of phages per plate was 500, and screening was performed in the same manner.
A single clone was obtained by the third screening, and in-vivo excision was performed from the phage, and the gene was cloned into a plasmid (pBluescript) and sequenced.
その結果、230万個のプラークをスクリーニングし(1次スクリーニングでは121枚のプレート)、重複も含めて43個の陽性クローンを得た。それらはchromatin modulator、ubiquitin関連、癌抑制遺伝子関連、mitosis関連、接着関連蛋白など15の遺伝子をコードしていた。その中にIFN治療有効例の血清とのみ反応する遺伝子蛋白が1つ、腎癌組織において正常腎組織とくらべ著しく発現の亢進している遺伝子が2種類得られた。 As a result, 2.3 million plaques were screened (121 plates in the primary screening), and 43 positive clones including duplicates were obtained. They encoded 15 genes including chromatin modulator, ubiquitin-related, tumor suppressor gene-related, mitosis-related and adhesion-related proteins. Among them, one gene protein that reacts only with the serum of IFN treatment effective cases was obtained, and two genes whose expression was remarkably increased in renal cancer tissues compared with normal kidney tissues were obtained.
得られた遺伝子について、腎臓癌組織と正常腎臓組織との発現の比較、また、各組織における発現をRT−PCR法により調べた。
その結果、全ての臨床検体の腎臓癌組織において、正常腎臓組織と比べて著しく発現の亢進している遺伝子が2種類得られた。About the obtained gene, the expression of a kidney cancer tissue and a normal kidney tissue was compared, and the expression in each tissue was examined by RT-PCR method.
As a result, two types of genes whose expression was remarkably increased in kidney cancer tissues of all clinical specimens as compared with normal kidney tissues were obtained.
図1は、その遺伝子のRT−PCRを示す。遺伝子36-6-1は、細胞性免疫にかかわる糖鎖結合蛋白質ガレクチン9(非特許文献7) の遺伝子と同一であり、もう一方の遺伝子113-3-1は、細胞接着のシグナリングに関するアダプター蛋白PINCH(非特許文献8)の遺伝子と同一であることが分かった。いずれも、遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も未知のものであった。 FIG. 1 shows RT-PCR of the gene. Gene 36-6-1 is the same as the gene of sugar chain binding protein galectin 9 (Non-patent Document 7) involved in cellular immunity, and the other gene 113-3-1 is an adapter protein related to cell adhesion signaling. It was found to be identical to the PINCH gene (Non-Patent Document 8). In any case, the sequence of the gene is known, but this peptide is not known, and the use is unknown.
PCRのプライマーには、36-6-1のFW及びRVについて、それぞれ下記(3)及び(4)の配列を用い、変性94℃、30秒、アニーリング65℃、30秒、ポリメラゼーション72℃、 3分、30サイクル行った(TaKaRa社製、RNA PCR kit)。
ATGGCCTTCAGCGGTTCCCAG ‥‥‥(7)
CTATGTCTGCACATGGGTCAG ‥‥‥(8)For PCR primers, the following sequences (3) and (4) were used for 36-6-1 FW and RV, respectively, denaturation 94 ° C., 30 seconds, annealing 65 ° C., 30 seconds, polymerization 72 ° C. , 3 minutes, 30 cycles (TaKaRa, RNA PCR kit).
ATGGCCTTCAGCGGTTCCCAG (7)
CTATGTCTGCACATGGGTCAG (8)
113-3-1のFW及びRVについては、それぞれ下記(5)及び(6)の配列を用い同様に、変性94℃、30秒、アニーリング55℃、30秒、ポリメラゼーション72℃、 3分、33サイクル行った。
AAGATAATTCGCAGTGATGTGAA ‥‥‥(9)
GTAGATCAAGACAAGTAATGTTG ‥‥‥(10)For FW and RV of 113-3-1, using the sequences (5) and (6) below, respectively, denaturation 94 ° C, 30 seconds, annealing 55 ° C, 30 seconds, polymerization 72 ° C, 3 minutes 33 cycles were performed.
AAGATAATTCGCAGTGATGTGAA (9)
GTAGATCAAGACAAGTAATGTTG (10)
図1では、腎臓癌の特定の培養細胞株のデーターを参考に示しているが、培養細胞株では、ある一定の条件で培養され続けることにより、遺伝子発現に選択がかかり、発現がなくなってしまう蛋白質もありうる。36-6-1は、そのような蛋白質、遺伝子であり、113-3-1は、培養の継続にも関わらず比較的いつまでも残る蛋白質、遺伝子と考えられる。
なお、図1では、正常部分と腎臓癌部分の遺伝子の発現を比較した5例のみの結果を示したが、症例を815例に増やして検討しても、36-6-1及び113-3-1も同様の結果であった。In FIG. 1, data of a specific cultured cell line of kidney cancer is shown as a reference. In a cultured cell line, the gene expression is selected and the expression is lost by continuing to culture under certain conditions. There can also be protein. 36-6-1 is such a protein or gene, and 113-3-1 is considered to be a protein or gene that remains relatively indefinitely despite continuation of culture.
FIG. 1 shows the results of only 5 cases in which the expression of genes in the normal part and the kidney cancer part are compared. However, even if the number of cases is increased to 815, 36-6-1 and 113-3 -1 gave similar results.
なお、サイトカイン療法が有効であったことに関しては、36-6-1は、腎臓癌の多発性骨転移があり、インターフェロンαで完全に骨転移が消失した稀な症例の血清を用いてスクリーニングして得られたものである。113-3-1は、クローニングするのに用いた血清が、腎臓癌の肺転移がインターフェロンαで明らかに縮小した患者由来のものである。 Regarding the effectiveness of cytokine therapy, 36-6-1 was screened using the serum of a rare case of multiple bone metastases of kidney cancer that completely disappeared with interferon α. It was obtained. 113-3-1 is derived from a patient whose sera used for cloning had lung cancer metastasis clearly reduced by interferon α.
この2種の遺伝子36-6-1(ガレクチン9)及び113-3-1(PINCH)に基づいて、それぞれペプチドを合成した。36-6-1由来のペプチドA24/10mer190(配列(1))、113-3-1由来のペプチドA24/10mer238(配列(2))を用いて正常人末梢血単核球(PBMC)より誘導した細胞傷害性T細胞について、HLA−A*2402拘束性と抗原特異的細胞傷害活性を調べた。なお、HLA−A*2402は、日本人で最も多いタイプのHLAである。
その結果、図2(a)及び(b)に示すように、各抗原を発現している腎臓癌細胞株TUHR−10TKBのみ、誘導された細胞傷害性T細胞により、HLA−A*2402拘束性に障害されることが明らかになった。
なお、細胞傷害性T細胞の誘導に用いた細胞は、後述のようにHLA−A*2402/A*0201のヘテロの細胞であるので、これらペプチドのHLA−A*0201に対する結合が安定な条件下で、このタイプのHLAも同様に細胞性免疫を誘導可能である。
従来の細胞障害性T細胞を誘導するペプチドは、本発明のペプチドとは異なった方法でスクリーニングされた遺伝子由来であり、腎臓癌患者血液のリンパ球を刺激して細胞障害性T細胞の誘導を示していたが、本発明のペプチドでは、正常人のリンパ球から、高い活性の細胞障害性T細胞を誘導できた。Peptides were synthesized based on these two genes 36-6-1 (galectin 9) and 113-3-1 (PINCH), respectively. Induced from normal human peripheral blood mononuclear cells (PBMC) using peptide A24 / 10mer190 (sequence (1)) derived from 36-6-1 and peptide A24 / 10mer238 (sequence (2)) derived from 113-3-1 The cytotoxic T cells thus obtained were examined for HLA-A * 2402 restriction and antigen-specific cytotoxic activity. HLA-A * 2402 is the most common type of HLA among Japanese.
As a result, as shown in FIGS. 2 (a) and (b), only the renal cancer cell line TUHR-10TKB expressing each antigen was restricted by HLA-A * 2402 due to the induced cytotoxic T cells. It became clear that it was disturbed.
Since the cells used for the induction of cytotoxic T cells are heterogeneous cells of HLA-A * 2402 / A * 0201, as described later, the conditions under which the binding of these peptides to HLA-A * 0201 is stable Below, this type of HLA can induce cellular immunity as well.
The conventional peptide that induces cytotoxic T cells is derived from a gene screened by a method different from the peptide of the present invention, and induces cytotoxic T cells by stimulating lymphocytes in kidney cancer patient blood. As shown, the peptide of the present invention was able to induce highly active cytotoxic T cells from normal human lymphocytes.
図1で触れたのと同様に、図3からも、36-6-1は、ある一定の条件で培養され続けることにより、遺伝子発現に選択がかかり、発現がなくなってしまう蛋白質、遺伝子であり、113-3-1は、培養の継続にも関わらず比較的いつまでも残る蛋白質、遺伝子と考えられる。図3で示されるように、36-6-1は腎臓癌特異的であるが、113-3-1は他の癌細胞でも発現している(PC3, DU145, LNCaPは前立腺癌、T24は膀胱癌、NEC8は精巣腫瘍、HeLaは子宮頸癌、OCUB1及びMCF7は乳癌の培養細胞株)。 Similar to that mentioned in FIG. 1, from FIG. 3, 36-6-1 is a protein or gene that loses its expression when it is selected for gene expression by continuing to be cultured under certain conditions. , 113-3-1 is considered to be a relatively long-lasting protein and gene despite continued culture. As shown in FIG. 3, 36-6-1 is specific to kidney cancer, but 113-3-1 is also expressed in other cancer cells (PC3, DU145, LNCaP is prostate cancer, T24 is bladder Cancer, NEC8 is testicular tumor, HeLa is cervical cancer, OCUB1 and MCF7 are breast cancer cell lines).
具体的には、次のように細胞傷害性T細胞を誘導した。
HLA−A*2402/A*0201の健常人の血液より、Ficoll-Paque PLUS(GE Healthcare Bio-Science AB)を使用してPBMCを分離した(responder細胞)。
抗原提示細胞は、B−LCL(EB virusにより形質変換させ不死化したB-cell)のうち、JTK−LCL細胞(RCB1873)(HLA−A*2402/A*0206)を用いた。
HLA−A*2402ドナーよりPBMCを分離し、24wellプレートに3 x 106/ml/well個ずつ分注し、ペプチドを5μM濃度で添加し、5日間培養した。Specifically, cytotoxic T cells were induced as follows.
PBMCs were isolated from the blood of healthy individuals with HLA-A * 2402 / A * 0201 using Ficoll-Paque PLUS (GE Healthcare Bio-Science AB) (responder cells).
Among antigen-presenting cells, JTK-LCL cells (RCB1873) (HLA-A * 2402 / A * 0206) among B-LCLs (B-cells transformed and immortalized by EB virus) were used.
PBMCs were separated from the HLA-A * 2402 donor, dispensed at 3 × 10 6 / ml / well in 24-well plates, added with a peptide at a concentration of 5 μM, and cultured for 5 days.
JTK−LCL細胞を用いて刺激を入れた。すなわちJTK−LCL細胞を回収し、セルをカウントし0.8 x 106 /mlの密度に調製した後、ペプチドを3μM濃度で添加した(ペプチドパルス)。増殖しないように放射線照射したJTK−LCL細胞を500μLずつ、上述のresponder細胞に重層し共培養した。2日後より、2日毎にIL2を最終濃度20IU/mLになるように調製した。
その後、7〜14日後に、同様の刺激を4回(ここからの刺激より各wellのrespondor細胞を2x106/mL/well個に調製する)入れ、次いで、responder細胞中のCD8+T細胞を90%程度まで濃縮し、JTK−LCL細胞を用いて同様に刺激した。最終刺激の5〜6日後に、51Cr releasing assayにてCD8+T細胞のターゲット細胞に対する細胞傷害活性を測定した。Stimulation was applied using JTK-LCL cells. That is, JTK-LCL cells were collected, the cells were counted and adjusted to a density of 0.8 × 10 6 / ml, and then the peptide was added at a concentration of 3 μM (peptide pulse). 500 μL of JTK-LCL cells irradiated so as not to proliferate were layered on the above-mentioned responder cells and co-cultured. From 2 days later, IL2 was prepared to a final concentration of 20 IU / mL every 2 days.
Thereafter, after 7 to 14 days, the same stimulation was applied 4 times (preparing 2 × 10 6 / mL / well of responder cells in each well based on the stimulation from here), and then 90% of CD8 + T cells in the responder cells were added. It concentrated to the extent and stimulated similarly using JTK-LCL cell. Five to six days after the final stimulation, the cytotoxic activity of CD8 + T cells against target cells was measured by 51Cr releasing assay.
細胞傷害試験には、腫瘍細胞(5種類)をプレートより回収し、各々5×106 cells/mlとして51Crを加え、インキュベーター内で培養し腫瘍細胞に51Crを取り込ませ、試料を調製した。
誘導したCTLであるエフェクター細胞を回収し、2×106cells/mlの細胞濃度の液を用いて96 wellのマイクロプレートに希釈系列を作った(2×106cells/ml、その3/10及び1/10)。51Crでラベルした標的細胞100μlをエフェクター細胞に加え、4時間共培養した後、CTLの攻撃を受けて傷害された標的細胞から遊離される放射活性を求めた。In the cytotoxicity test, tumor cells (5 types) were collected from the plate, 51Cr was added as 5 × 10 6 cells / ml each, cultured in an incubator, 51Cr was taken up into the tumor cells, and a sample was prepared.
Effector cells are induced with CTL recovered, 2 × 10 6 cells / ml using the cell concentration in the liquid was made a dilution series microplate 96 well (2 × 10 6 cells / ml, the 3/10 And 1/10). After adding 100 μl of 51Cr-labeled target cells to effector cells and co-culturing for 4 hours, the radioactivity released from the target cells damaged by CTL attack was determined.
また、図2(a)及び(b)に示すように、各細胞障害性T細胞によるTUHR−10TKBに対する細胞傷害活性は、低いE:T比でも高い値であった。本発明の2つのペプチドによる正常人末梢血リンパ球より誘導した細胞障害性T細胞はいずれも、低いE:T比(10:1)で60%を超える程度の癌細胞の傷害活性が認められ、従来技術(特許文献1)の報告などに比べても、著しく有効性の高いペプチドである。
このことは、これら抗原が、腎臓癌抗原として高い抗原性を有し、有用な免疫療法の標的であることを示すものである。なお、本発明の2つのペプチドは、併用も可能である。Further, as shown in FIGS. 2 (a) and (b), the cytotoxic activity against TUHR-10TKB by each cytotoxic T cell was high even at a low E: T ratio. Both cytotoxic T cells derived from normal human peripheral blood lymphocytes by the two peptides of the present invention have cancer cell cytotoxicity of over 60% at a low E: T ratio (10: 1). Compared with the report of the prior art (Patent Document 1) and the like, the peptide is remarkably highly effective.
This indicates that these antigens have high antigenicity as kidney cancer antigens and are useful immunotherapy targets. The two peptides of the present invention can be used in combination.
また、本発明において同定された2種類の遺伝子は、図1にも示したように、腎臓癌細胞において高発現しているため、RT−PCR法を用いた循環血中の腎臓癌細胞の検出など、診断にも適用可能である。 Further, since the two types of genes identified in the present invention are highly expressed in kidney cancer cells as shown in FIG. 1, detection of kidney cancer cells in circulating blood using the RT-PCR method. It is also applicable to diagnosis.
上記実施例と同様に、HLA−A*0201に対するペプチドを、SYFPEITHIエピトープ予測プログラム及びBIMASデータベースを用いて設計した。
その結果、細胞性免疫にかかわる糖鎖結合蛋白質ガレクチン9(遺伝子36-6-1)(非特許文献7) のアミノ酸配列からは、下記(3)及び(4)の配列が、細胞接着のシグナリングに関するアダプター蛋白PINCH(遺伝子113-3-1)(非特許文献8)のアミノ酸配列からは、下記(5)及び(6)の配列が得られた。いずれも、遺伝子の配列は公知であるが、このペプチドは公知でなく、また用途も未知のものであった。
FLVQSSDFKV ‥‥‥(3)
ILFVQYFHRV ‥‥‥(4)
KLTLKNKFV ‥‥‥(5)
ELYHEQCFV ‥‥‥(6)Similar to the above examples, peptides for HLA-A * 0201 were designed using the SYFPEITHI epitope prediction program and the BIMAS database.
As a result, from the amino acid sequence of sugar chain binding protein galectin 9 (gene 36-6-1) (non-patent document 7) involved in cell-mediated immunity, the following sequences (3) and (4) indicate that cell adhesion signaling From the amino acid sequence of adapter protein PINCH (gene 113-3-1) (Non-patent Document 8), the following sequences (5) and (6) were obtained. In any case, the sequence of the gene is known, but this peptide is not known, and the use is unknown.
FLVQSSDFKV (3)
ILFVQYFHRV (4)
KLTLKNKFV (5)
ELYHEQCFV (6)
この4種のペプチドをそれぞれ合成し、それらを用いて正常人末梢血単核球(PBMC)より誘導した細胞傷害性T細胞について、HLA−A*0201拘束性と抗原特異的細胞傷害活性を調べた。
図4(a)及び(b)は、ガレクチン9(36-6-1)に基づくペプチド(3)及びペプチド(4)を用いて誘導した細胞傷害性T細胞におけるHLA−A*0201拘束性と抗原特異的細胞傷害活性を示し、図5(a)及び(b)は、PINCH(113-3-1)に基づくペプチド(5)及びペプチド(6)を用いて誘導した細胞傷害性T細胞におけるHLA−A*0201拘束性と抗原特異的細胞傷害活性を示す。
図4(a)及び(b)、図5(a)及び(b)に示すように、各抗原を発現している腎臓癌細胞株A498が、誘導された細胞傷害性T細胞により、HLA−A*0201拘束性に障害されることが明らかになり、また、図4(b)及び5(b)に示すように、上記(4)及び(6)の配列を有する抗原を発現している腎臓癌細胞株A498及びTUHR−10TKBが、誘導された細胞傷害性T細胞により、HLA−A*0201拘束性に障害されることが明らかになった。Each of these four peptides was synthesized, and using them, cytotoxic T cells derived from normal human peripheral blood mononuclear cells (PBMC) were examined for HLA-A * 0201 restriction and antigen-specific cytotoxic activity. It was.
FIGS. 4 (a) and (b) show HLA-A * 0201 restriction in cytotoxic T cells induced by using peptide (3) based on galectin 9 (36-6-1) and peptide (4). 5 shows antigen-specific cytotoxic activity, and FIGS. 5 (a) and (b) show cytotoxicity T cells induced using peptide (5) and peptide (6) based on PINCH (113-3-1). It shows HLA-A * 0201 restriction and antigen-specific cytotoxic activity.
As shown in FIGS. 4 (a) and 4 (b) and FIGS. 5 (a) and 5 (b), the renal cancer cell line A498 expressing each antigen was transformed into HLA- by the induced cytotoxic T cells. It becomes clear that A * 0201 restriction is impaired, and as shown in FIGS. 4 (b) and 5 (b), the antigens having the sequences (4) and (6) are expressed. Kidney cancer cell lines A498 and TUHR-10TKB were found to be impaired by HLA-A * 0201 restriction by induced cytotoxic T cells.
本発明の抗原蛋白、ペプチド、或いはそれらをコードするDNAは自己傷害性等の副作用が少なく優れた腎臓癌ワクチンとして使用することができる。また、抗体は診断薬として使用することができ、抗原により刺激、活性化された細胞障害性T細胞は抗癌剤として使用でき、本発明の抗原の発現を抑制するRNAiは、本発明の抗原を高発現している腎臓癌の治療に使用できる。また、本発明の探索方法は、癌免疫療法に有用な標的遺伝子の探索に有用である。
そのため、腎臓等の癌の治療や早期発見に寄与し、産業上有用である。The antigenic protein, peptide of the present invention, or DNA encoding them can be used as an excellent kidney cancer vaccine with few side effects such as self-injury. In addition, antibodies can be used as diagnostic agents, cytotoxic T cells stimulated and activated by antigens can be used as anticancer agents, and RNAi that suppresses the expression of the antigen of the present invention can enhance the antigen of the present invention. It can be used to treat developing kidney cancer. Moreover, the search method of the present invention is useful for searching for target genes useful for cancer immunotherapy.
Therefore, it contributes to the treatment and early detection of cancer such as kidney and is industrially useful.
Claims (9)
ことを特徴とするペプチド。
AYPMPFITTI ‥‥‥(1)
AYCETHYNQL ‥‥‥(2)
FLVQSSDFKV ‥‥‥(3)
ILFVQYFHRV ‥‥‥(4)
KLTLKNKFV ‥‥‥(5)
ELYHEQCFV ‥‥‥(6)A peptide comprising the amino acid sequence of any one of (1) to (6) below and having immunity-inducing activity.
AYPMPFITTI (1)
AYCETHYNQL (2)
FLVQSSDFKV (3)
ILFVQYFHRV (4)
KLTLKNKFV (5)
ELYHEQCFV (6)
ことを特徴とするペプチド。
AYPMPFITTI ‥‥‥(1)
AYCETHYNQL ‥‥‥(2)
FLVQSSDFKV ‥‥‥(3)
ILFVQYFHRV ‥‥‥(4)
KLTLKNKFV ‥‥‥(5)
ELYHEQCFV ‥‥‥(6)A peptide comprising an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids in the amino acid sequence of any one of the following (1) to (6), and having immunity-inducing activity.
AYPMPFITTI (1)
AYCETHYNQL (2)
FLVQSSDFKV (3)
ILFVQYFHRV (4)
KLTLKNKFV (5)
ELYHEQCFV (6)
ことを特徴とする蛋白質。A protein comprising the peptide according to claim 1 or 2 and capable of activating cytotoxic T cells recognizing a renal cancer antigen protein.
ことを特徴とするヘルパーT細胞、細胞傷害性T細胞、
または、インビトロでパルスされたことを特徴とする
抗原提示細胞、樹状細胞、或いは、これらを含む免疫細胞集団。Helper T cells, cytotoxic T cells, which are induced by in vitro stimulation using the peptide according to any one of claims 1 to 3 or a mixture thereof,
Alternatively, an antigen-presenting cell, a dendritic cell, or an immune cell population containing these, characterized by being pulsed in vitro.
ことを特徴とする腎臓癌ワクチン。A kidney cancer vaccine comprising the peptide according to any one of claims 1 to 3.
ことを特徴とする抗体。An antibody capable of producing an antigen-antibody reaction against the peptide according to any one of claims 1 to 3.
ことを特徴とする腎臓癌診断薬。A diagnostic agent for renal cancer comprising the antibody according to claim 6.
被験者由来の生体試料において請求項1に記載のペプチド関連遺伝子の発現レベルを求めるステップと、
その発現レベルを正常対照レベルと比較し所定の閾値より高ければ、腎臓癌を発症する素因が高いと評価するステップとを有する
ことを特徴とする腎臓癌発症素因評価方法。A method for assessing whether or not to have a predisposition to develop kidney cancer,
Obtaining an expression level of the peptide-related gene according to claim 1 in a biological sample derived from a subject;
A method for evaluating the predisposition to developing kidney cancer, comprising: comparing the expression level with a normal control level and evaluating that the predisposition for developing kidney cancer is high if the expression level is higher than a predetermined threshold value.
サイトカイン療法が有効であった癌患者から血清を採取するステップと、
その血清をプローブとして用いたSEREX法によりcDNA発現ライブラリーをスクリーニングするステップとを有する
ことを特徴とする標的遺伝子の探索方法。A method for searching for a target gene useful for immunotherapy,
Collecting serum from cancer patients for whom cytokine therapy was effective;
Screening a cDNA expression library by a SEREX method using the serum as a probe.
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PCT/JP2012/059878 WO2012141201A1 (en) | 2011-04-11 | 2012-04-11 | Peptide for cancer immunotherapy and method using same |
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WO2002037114A1 (en) * | 2000-11-01 | 2002-05-10 | Galpharma Co., Ltd. | Agent for detecting cancer's ability to metastasize |
JP2010151822A (en) * | 2010-01-04 | 2010-07-08 | Agensys Inc | Nucleic acid and correspondence protein called 24p4c12 useful in treating and detecting cancer |
JP2010215639A (en) * | 2001-09-06 | 2010-09-30 | Agensys Inc | Nucleic acid and corresponding protein entitled steap-1 useful in treatment and detection of cancer |
JP2010220621A (en) * | 2002-09-06 | 2010-10-07 | Agensys Inc | Nucleic acid entitled as 98p4b6 and corresponding protein useful in treatment and detection of cancer |
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EP0996857B1 (en) * | 1997-07-17 | 2009-09-09 | Ludwig Institute For Cancer Research | Cancer associated nucleic acids and polypeptides |
WO2001089204A1 (en) * | 2000-04-21 | 2001-11-22 | Lockheed Martin Corporation | Wide-field extended-depth doubly telecentric catadioptric optical system for digital imaging |
JP2009502112A (en) * | 2005-07-28 | 2009-01-29 | オンコセラピー・サイエンス株式会社 | Methods for diagnosing and treating renal cell carcinoma |
JP5267969B2 (en) * | 2007-12-04 | 2013-08-21 | 学校法人慶應義塾 | Cancer vaccine |
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WO2002037114A1 (en) * | 2000-11-01 | 2002-05-10 | Galpharma Co., Ltd. | Agent for detecting cancer's ability to metastasize |
JP2010215639A (en) * | 2001-09-06 | 2010-09-30 | Agensys Inc | Nucleic acid and corresponding protein entitled steap-1 useful in treatment and detection of cancer |
JP2010220621A (en) * | 2002-09-06 | 2010-10-07 | Agensys Inc | Nucleic acid entitled as 98p4b6 and corresponding protein useful in treatment and detection of cancer |
JP2010151822A (en) * | 2010-01-04 | 2010-07-08 | Agensys Inc | Nucleic acid and correspondence protein called 24p4c12 useful in treating and detecting cancer |
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