JP2022151802A - 抗t細胞ナノボディ及びその核酸コード配列、並びにその使用 - Google Patents
抗t細胞ナノボディ及びその核酸コード配列、並びにその使用 Download PDFInfo
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Abstract
Description
ウエスタンブロッティング(Western blotting)により、抗CD3εナノボディがヒトT細胞の細胞溶解物中のCD3εタンパク質を特定できることを証明し、
免疫組織化学的染色(immunohistochemistry staining、IHC staining)及びフローサイトメトリー(flow cytometric analysis)により、抗CD3εナノボディがフローサイトメトリーによって細胞サンプルにおけるCD3εの発現を検出できることを証明し、
表面プラズモン共鳴結合分析(surface plasmon resonance binding assay、SPR binding assay)により、抗CD3εナノボディが0.5056nM以内のKD値でCD3ε/CD3δヘテロ二量体に有効に結合することを証明し、及び、
免疫細胞化学の分析により、抗CD3εナノボディが細胞サンプルにおけるCD3εの発現を検出するために用いられることを証明する。
本明細書に記載の数値は、概算値である。全ての実験データは、その数値の±20%、好ましいは±10%、より好ましくは±5%を示す。
本実施例において、抗CD3ε(CD3 epsilon)ナノボディ(nanobody、NB)、及び重鎖可変ドメイン(heavy chain variable domain、VHH)の製造プロセスは、以下の通りである。
本実施例において、抗CD3εナノボディのT細胞(即ち、末梢血単核細胞)の増殖及び活性化の分析(T cell(i.e. peripheral blood mononuclear cell、PBMC) proliferation and activation assay)の操作手順は、以下の通りである。1×106のPBMC細胞を12ウェルプレートに接種する。抗CD3εナノボディ(1μg/ml)又は臨床用CD3εモノクローナル抗体OKT3(10mg/ml、Invitrogen、Cat:MA1-10175)は、存在しても存在しなくてもよい。
本実施例において、抗CD3εナノボディによるPBMCにおけるCD3陽性T細胞の増殖の向上効果の評価の操作手順は、以下の通りである。1×106のPBMC細胞を12ウェルプレートに接種する。抗CD3εナノボディ(10、100、1000、5000ng/ml)は、存在しても存在しなくてもよい。
フローサイトメトリーによって分析する。写真は、40倍の顕微鏡で撮影した画像である。CD3陽性細胞の数は、CD3細胞パーセンテージ(%)×総細胞数である。
本実施例において、抗CD3εナノボディによるγδT(GDT)細胞におけるCD3陽性T細胞の増殖の向上効果の評価の操作手順は、以下の通りである。1×106の一次γδT(GDT)細胞を12ウェルプレートに接種する。CD3εナノボディ(10、100、1000、5000ng/ml)は、存在しても存在しなくてもよい。
本実施例において、抗CD3εナノボディのウエスタンブロッティング(Western blotting)の操作手順は、以下の通りである。プロテアーゼ阻害剤カクテルを含有するPRO-PREPタンパク質抽出溶液(iNtRON、台北、台湾)から細胞を取り、4℃で15分間激しく振とうした後、遠心分離をする。上清を回収し、Bio-Rad BCA試薬(Bio-Rad Hercules、CA、USA)を使用してタンパク質の濃度を測定する。
本実施例において、抗CD3εナノボディの免疫組織化学的染色(immunohistochemistry staining、IHC staining)の操作手順は、以下の通りである。
本実施例において、抗CD3εナノボディのフローサイトメトリー(flow cytometric analysis)の操作手順は、以下の通りである。ヒトPBMCを氷上でFITC共役CD3εナノボディ(1μg/ml)及びOKT3抗体(抗CD3モノクローナル抗体)(10μg/ml)によって45分間染色する。洗浄した後、FL1チャネルを使用してフローサイトメトリーによって細胞を分析する。
本実施例において、CD3ε/CD3δヘテロ二量体に対する抗CD3εナノボディ表面プラズモン共鳴結合分析(surface plasmon resonance binding assay、SPRbinding assay)の操作手順は、以下の通りである。
本実施例において、抗CD3εナノボディの免疫細胞化学(immunocytochemistry)の分析の操作手順は、以下の通りである。
ウエスタンブロッティング(Western blotting)により、抗CD3εナノボディがヒトT細胞の細胞溶解物中のCD3εタンパク質を特定できることを証明し、
免疫組織化学的染色(immunohistochemistry staining、IHC staining)及びフローサイトメトリー(flow cytometric analysis)により、抗CD3εナノボディがフローサイトメトリーによって細胞サンプルにおけるCD3εの発現を検出できることを証明し、
表面プラズモン共鳴結合分析(surface plasmon resonance binding assay、SPR binding assay)により、抗CD3εナノボディが0.5056nM以内のKD値でCD3ε/CD3δヘテロ二量体に有効に結合することを証明し、及び、
免疫細胞化学の分析により、抗CD3εナノボディが細胞サンプルにおけるCD3εの発現を検出するために用いられることを証明する。
Claims (10)
- CD3ε(CD3 epsilon)に特異的に結合する抗T細胞ナノボディであって、
配列番号1、配列番号2、配列番号3、及びそれらの任意の結合からなる群から選ばれるアミノ酸配列を含むことを特徴とする、
抗T細胞ナノボディ。 - 前記アミノ酸配列は、前記抗T細胞ナノボディの重鎖可変ドメイン(heavy chain variable domain、VHH)のアミノ酸配列であることを特徴とする、
請求項1に記載の抗T細胞ナノボディ。 - フラグメント結晶化可能領域(fragment crystallizable region、Fc region)をさらに含むことを特徴とする、
請求項2に記載の抗T細胞ナノボディ。 - 第2の抗体に共役されることにより、二重特異性T細胞エンゲージャー(bispecific T-cell engager、BiTE)、三重特異性T細胞エンゲージャー(triple specific T-cell engager、TriTE)、二重特異性キラー細胞エンゲージャー(bispecific killer cell engager、BiKE)、三重特異性キラー細胞エンゲージャー(triple specific killer cell engager、TriKE)、又は任意の二重特異性抗体を形成することを特徴とする、
請求項3に記載の抗T細胞ナノボディ。 - CD3ε陽性細胞を活性化及び/又は凝集させることを特徴とする、
請求項4に記載の抗T細胞ナノボディ。 - 請求項1~5のいずれか1項に記載の抗T細胞ナノボディのアミノ酸配列をコードする単離された核酸であって、
配列番号4、配列番号5、配列番号6、及びそれらの任意の結合からなる群から選ばれるヌクレオチド配列を含むことを特徴とする、
単離された核酸。 - 請求項1~5のいずれか1項に記載の抗T細胞ナノボディ、及び医薬上許容可能な担体を含むことを特徴とする、
医薬組成物。 - がんの治療、免疫調節及び免疫細胞の活性化のための医薬品を製造するための請求項1~5のいずれか1項に記載の抗T細胞ナノボディの使用。
- サンプルに請求項1~5のいずれか1項に記載の抗T細胞ナノボディを投与することを含むことを特徴とする、
CD3εの発現レベルを検出するための方法。 - 前記サンプルは、血液、尿液、痰液、唾液、体液、腫瘍、臓器、組織又は細胞であることを特徴とする、
請求項9に記載の方法。
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