Lamparski Henry G, Metha-Damani Anita, Yao Jenq-Yuan, Patel Sanjay, Hsu Di-Hwei, Ruegg Curtis, Le Pecq Jean-Bernard
Anosys Inc., 1014 Hamilton Court, Menlo Park, CA 94025, USA.
J Immunol Methods. 2002 Dec 15;270(2):211-26. doi: 10.1016/s0022-1759(02)00330-7.
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 I, MHC class II, 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 2-3 l 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 1.210 g/cm3) 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 II 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 II) 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) 11a].
我们描述了从抗原呈递细胞（APC）中生产、纯化和鉴定临床级（cGMP）外泌体的方法。外泌体已被证明通过呈递生物学相关抗原而具有免疫治疗特性[《自然医学》4（1998年）594]，并且正在被开发作为细胞疗法的替代方案。外泌体是直径为50 - 90纳米的囊泡，由存在于多种造血细胞和肿瘤细胞中的多泡体（MVB）分泌。这些颗粒包含抗原呈递分子（MHC I类、MHC II类和CD1）、四跨膜分子（CD9、CD63、CD81）、黏附分子（CD11b和CD54）以及共刺激分子（CD86）；因此，为它们提供了产生有效免疫反应所需的必要机制[《生物化学杂志》273（1998年）20121；《细胞科学杂志》113（2000年）3365；《免疫方法杂志》247（2001年）163；《免疫学杂志》166（2001年）7309]。基于其独特的大小和密度，来自单核细胞衍生树突状细胞（MDDC）的外泌体能够快速纯化（例如，2 - 3升培养物4 - 6小时）。通过500 kDa的膜对澄清的上清液进行超滤，并超速离心至30%蔗糖/氧化氘（D2O）（98%）垫层（密度1.210 g/cm³），分别使体积和蛋白质浓度降低约200倍和1000倍。基于起始澄清上清液中外泌体MHC II类浓度，外泌体的回收率在40%至50%之间。该方法扩展到小规模过程时也得到了类似的结果。相反，传统的差速离心技术是一个更长且变化较大的过程，导致外泌体被培养基蛋白污染，并且仅含有起始外泌体MHC II类浓度的5 - 25%；因此，使其难以用于临床开发。最后，我们开发了以下质量控制检测方法来规范外泌体疫苗：数量（MHC II类浓度）和蛋白质鉴定（流式细胞术）。快速且可重复的外泌体纯化方法与质量控制检测方法相结合，使得其能够作为癌症疫苗在临床试验中进行评估[《美国肿瘤学会会议论文集》21（2002年）11a]。
