JP6419693B2 - 腫瘍抗原ペプチド - Google Patents
腫瘍抗原ペプチド Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Description
いずれにせよin silicoによるエピトープの解析は今まで報告されているデータに基づき行われるため、HLAクラスIにおいては、12mer以上のペプチドについては十分なデータベースがなく、in silicoでの解析をすることができない。12mer以上のペプチドはその配列中に含まれる8〜11merのエピトープ候補を予測解析することは可能であるが、HLAクラスIに結合する際のプロセシングにより期待されるエピトープが結合できるようなプロセシングを受けるかどうかの予測は困難であり、未だ有用なペプチドを見いだすための実用に耐えるものではない。
Lck33 KGRLLIRNGSEVRDP(配列番号1)
Lck374 DTLSCKIADFGLARL(配列番号2)
Lck487 FDYLRSVLEDFFTAT(配列番号3)
本発明者らはHLA−A2結合性ならびにHLA−DR結合性を指標にin silicoスクリーニングを行い、94種類のペプチドを特定し、これらのペプチドをペプチド純度70%以上となるよう作製した。
実施例1でがん患者において抗ペプチド抗体を産生しやすいことが分かった17ペプチドを、純度95%以上で合成した。ペプチドはDMSO溶液として培地へ添加した。各ペプチドのCD8陽性CFN−γ陽性T細胞の誘導能をIntracellular cytokine stainingにより検討した。まず、凍結保存していたHLA−A2陽性のがん患者由来のPBMCを解凍し、10μg/mLのペプチドを含む培養液にて5日間培養した(Day0−5)。次いでDay5、8、11および14に10μg/mLのペプチドを含む培養液で培地交換を行い、細胞をペプチドで刺激した。
細胞傷害活性を検討するにあたり、PBMCからのペプチド反応性CTLの誘導は既報の方法に一部変更を加えて検出した(Hida N, Maeda Y, Katagiri K, Takasu H, Harada M, Itoh K., Cancer Immunol Immunother 2002;51:219-28.)。具体的にはU底96ウェルマイクロカルチャープレート(Nunc, Roskilde, Denmark)において各ペプチド(10μg/mL)を含む培養液200μLを用いて、がん患者由来のPBMC(1 x 105 細胞/ウェル)を2ウェル一組にて培養した。患者はいずれもHLA−A2陽性の前立腺がん患者である。
配列番号2または3のペプチドにより誘導したCTLの、その部分ペプチドを提示したT2細胞に対する細胞傷害活性を調べた。
ペプチドによるCTL誘導ならびに細胞傷害活性の測定は実施例3に示す方法に従って行った。
PBMCとしては患者番号757および728の患者(いずれもHLA−A2、A24陽性の大腸がん患者)から得たPBMCを用いた。実施例3と同じスケジュールで配列番号2または3のペプチドで刺激して細胞傷害性T細胞を誘導した。
BALB/cマウスにLckを発現する腫瘍細胞株Colon26を移植した。コントロール群としては腫瘍移植を行わないマウスを用いた。
ペプチドを10−20mg/mlとなるよう調製したプレートへ添加した。Mock群には何も添加しなかった。プレートを37℃、5%CO2の条件下で16時間以上培養した。
癌患者由来PBMCを各ペプチドで刺激した場合のCTL誘導能を、各細胞からのIFN−γの放出をELISPOT法にて測定することにより確認した。
ELISPOT法はIFN−γELISPOTキット(mAb-1-D1K、Cat#3420-3-1000, MABTECH)を用いて行った。
各患者の末梢血リンパ球(PBMC)を10%AB型ヒト血清(Gemini Biosciences)を添加したIMDM(Iscove's Modified Dulbecco's Medium, Gibco)で調整し、5×105/wellとなるよう96穴丸底プレートへ添加した。各ウエルへLck374−388(配列番号2)またはLck487−501(配列番号3)ペプチドを10−20μg/mlとなるよう添加し、37℃、5%CO2の条件下で4日間培養した。Mock群には何も添加せずに同様に培養した。
一方、ELISPOT用ニトロセルロースメンブレンプレート(Multi Screen HTS Filter Plate Ca#MSHAS4510, Millipore)に抗ヒトIFN−γモノクローナル抗体を固相化し、37℃、1時間ブロッキングした。
培養後のPBMCをカウントし、新たな10% AB型ヒト血清/IMDM中に懸濁し、5×105/wellとなるよう抗ヒトIFN−γ抗体を固相化したニトロセルロースメンブレンプレートへ添加した。ここへペプチドを10−20μg/ml添加し、16時間以上、37℃、5%の条件下で培養した。
培養終了後、細胞を洗浄した。洗浄後、キット製造元の説明にしたがい、ビオチン結合抗ヒトIFN−γmAb、ストレプトアビジン−ALP、およびBCIP/NBT基質を用いてIFN−γが結合したスポットを可視化した。各フィルターの画像を撮影し、スポット数を計測した。スポット数は、ELISPOTリーダー(CTL-ImmunoSpot, Cellular Technology, Ltd.)を用いて計測した。
がん患者のHLAならびに罹患しているがん種を下記表5に示す:
Claims (11)
- 配列番号1〜3いずれかに記載のペプチド。
- 配列番号1〜3いずれかに記載のペプチドをコードする核酸分子。
- 請求項2記載の核酸分子を含むベクター。
- 請求項1に記載のペプチドまたは請求項3に記載のベクターの少なくとも1種を含む、細胞傷害性T細胞を誘導するための薬剤。
- 請求項4に記載の細胞傷害性T細胞がHLA−A2拘束性T細胞である、薬剤。
- がん患者より採取された末梢血単核細胞を請求項1記載のペプチドまたは請求項3記載のベクターの少なくとも1種と接触させることを含む、がん反応性細胞傷害性T細胞を誘導する方法。
- がん患者より採取された末梢血単核細胞を請求項1記載のペプチドまたは請求項3記載のベクターの少なくとも1種と接触させることを含む、抗原提示細胞を調製する方法。
- がん患者がHLA−A2陽性がん患者である、請求項6または7記載の方法。
- 請求項1に記載のペプチドまたは請求項3に記載のベクターの少なくとも1種を含む、がんを治療または予防するための医薬組成物。
- がんワクチンである、請求項9記載の医薬組成物。
- HLA−A2陽性がん患者の治療またはHLA−A2陽性対象におけるがんの予防のためのものである、請求項9または10に記載の医薬組成物。
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