JP4645312B2 - Nk細胞活性化方法、これを用いたnk細胞増殖方法及び細胞製造方法並びにnk細胞を含む単核球 - Google Patents
Nk細胞活性化方法、これを用いたnk細胞増殖方法及び細胞製造方法並びにnk細胞を含む単核球 Download PDFInfo
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Description
1) 末梢血リンパ球または磁気ビーズで単離したNK細胞を、IL−2あるいはIL−15によって刺激し、増殖させる。斯かる増殖は、多くの報告によれば10倍前後であるため、末梢血単核球(以下、PBMCと称する)を大量に用意する必要がある(Dunne J, et al., J. Immunol.2001, 167:3129, Klingemann HG, et al., Cytotherapy 2004, 6:15)。
2) 末梢血リンパ球またはNK細胞とBリンパ腫細胞株であるK562またはダウジ(Daudi)との混合培養をIL−2存在下で行うことで、IL−2単独の場合よりも多く増殖し、数10倍に増えることが報告されている。
3) プラスチックプレートに接着したLymphokine−activated killer(LAK)細胞(実際はNK細胞)に、IL−2とフィーダ細胞(feeder細胞)としてのCon−A刺激末梢血リンパ球またはEBウイルス感染リンパ芽球細胞株とを加え、増殖させる(Rabinowich H, et al., Cell Immunol. 1991, 135:454)。しかし、プラスチックプレートに接着するLAK細胞が少ないため、増殖させても最初に採取したNK細胞の36倍にとどまる。
4) PBMC48時間刺激培養上清
PBMC、EBウイルス感染リンパ芽球細胞株、PMA及びPHAとイオノマイシン、IL−2を加えることで、IL−2単独刺激よりもNK細胞を9倍に増やす。しかし手技が煩雑なわりに大量に得ることは難しい(Robertson MJ, et al., J Immunol 1993, 150:1705)。
5) PBMCと接着性腫瘍細胞株HFWTをIL−2存在下で培養すると、NK細胞が10日間から21日間で58倍から401倍に増殖する(Harada, et al., Jpn J Cancer Res 2002, 93:313)。
6) 4−1BB遺伝子及びIL−15遺伝子を組み込んだK562とPBMCとを3週間混合培養する場合には、NK細胞が平均1000倍以上に増殖する(Imai C, et al., Blood 2005)。
Claims (7)
- 末梢血単核球を、ヒトCD52抗体(Alemtuzumab)の存在下において培養してCD16+NK細胞を増殖させるNK細胞増殖方法。
- 末梢血単核球を、ヒトCD52抗体(Alemtuzumab)及びヒトCD3抗体(ORTHO CLONE OKT3)の存在下において培養してCD16+NK細胞を増殖させるNK細胞増殖方法。
- 末梢血単核球を、ヒトCD52抗体(Alemtuzumab)及びIL−2の存在下において培養してCD16+NK細胞を増殖させるNK細胞増殖方法。
- 末梢血単核球を、ヒトCD52抗体(Alemtuzumab)、ヒトCD3抗体(ORTHO CLONE OKT3)及びIL−2の存在下において培養してCD16+NK細胞を増殖させるNK細胞増殖方法。
- ヒトCD3抗体(ORTHO CLONE OKT3)は、0.001μg/ml以上の濃度である請求項2又は4に記載のNK細胞増殖方法。
- ヒトCD52抗体(Alemtuzumab)は、2μg/ml以上の濃度である請求項1から5のいずれか一項に記載のNK細胞増殖方法。
- 請求項1から6のいずれか一項に記載のNK細胞増殖方法を用いて増殖されたCD16+NK細胞を含む末梢血単核球。
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