Production and characterization of clinical grade exosomes derived from dendritic cells
Production and characterization of clinical grade exosomes derived from dendritic cells
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DOI：
10.1016/s0022-1759(02)00330-7
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发表时间：
2002-12-15
影响因子：
2.2
通讯作者：
Le Pecq, JB
中科院分区：
文献类型：
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作者：
Lamparski, HG;Metha-Damani, A;Le Pecq, JB
我们描述了由抗原提呈细胞(APC)衍生的临床级(CGMP)外切体的生产、纯化和特性的方法。外切体已被证明具有免疫治疗特性，通过它们呈递与生物相关的抗原[NAT。地中海医院。4(1998)594]，并正在开发作为细胞疗法的替代方案。Exosome是一种直径为50-90 nm的囊泡，由多种造血细胞和肿瘤细胞中的多泡小体分泌而成。这些颗粒含有抗原提呈分子(MHC 1类、MHC 11类和CD1类)、四环素分子(CD9、CD63、CD81)、黏附分子(CD11b和CD54)和共刺激分子(CD86)；因此，为它们提供了产生强大免疫反应所需的必要机制[J.Biol]。化学。273(1998年)20121；J.SCI。113(2000)3365；J.免疫。方法247(2001)163；J.免疫。166(2001)7309]。基于其独特的大小和密度，单核细胞来源的树突状细胞(MDDC)的外切体被快速纯化(例如，2-31培养4-6h)。澄清的上清液通过500 kDa的膜超滤和超速离心入30%的蔗糖/氧化氢(D2O)(98%)垫(密度为1.210g/cm(3))中，体积和蛋白质浓度分别减少约200倍和1000倍。根据起始澄清上清液中外切体MHC类H的浓度，外切体的回收率在40%~50%之间。这一方法被扩展到一个小规模的过程，得到了类似的结果。相反，经典的差速离心法是一个更漫长和多变的过程，导致外切体被介质蛋白污染，并且只含有起始外切体MHC II类浓度的5-25%，从而使其难以用于临床开发。最后，我们建立了以下质量控制方法来标准化外切体疫苗：数量(MHC 11类的浓度)和蛋白质特性(FACS)。一种快速和可重复性的纯化方法与外切体的质量控制分析相结合，使其能够在临床试验中作为癌症疫苗进行评估。上午好。SoC。奥科尔。21(2002)11 a]。(C)2002 Elsevier Science B.V.保留所有权利。
We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50-90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class 1, MHC class 11, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. Biol. Chem. 273 (1998) 20121; J. Cell. Sci. 113 (2000) 3365; J. Immunol. Methods 247 (2001) 163; J. Immunol. 166 (2001) 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4-6 h of a 2-31 culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/deuterium oxide (D2O) (98%) cushion (density 1.210g/cm(3)) reduced the volume and protein concentration approximately 200- and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class H concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5-25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class 11) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11 a]. (C) 2002 Elsevier Science B.V. All rights reserved.