Ex Vivo Culture And Characterization Of Dendritic Cells For Clinical Immunotherap

该项目的目标是开发和评估用于临床免疫治疗试验的树突状细胞(DC)的制造方法。1999财年，我们建立并优化了一种完整的GMP培养方法，用于在RPMI、自体血浆或同种异体血清、IL4和GMCSF中培养5天的自体DC，首先通过分离收集外周血单核细胞，然后通过淋洗纯化。然后，在临床给药之前，生成的未成熟DC可用于进一步的操作(例如，肽脉冲)。这种制造方法被结合到几项临床试验中，并于2001年初发表了一篇描述这种方法的手稿。在2000财年，由于我们对开发封闭式系统和消除难以标准化的试剂的兴趣，我们在一种蛋白质定义的无血清培养液(XVIVO15)中评估了一个7天的培养系统，从从洗脱的单核细胞开始，到使用Isolex 300i的阴性免疫磁性选择，在袋子和烧瓶中。我们证明了两种不同的单核细胞分离方法产生了相当于未成熟的DC群体，而袋子相当于培养瓶。此外，历史比较表明，对于未成熟DC的生成，无血清培养液与无血清培养液相当，甚至可能优于有血清培养液。描述这项工作的手稿于2002年1月出版。在2001财年，我们重点评估了在IL4和GMCSF中培养后使用CD40配体生成成熟DC的培养条件。2001年初，成功地开发了一个过程，并将其纳入几项癌症免疫治疗试验。在2002财年，我们完成了原材料(单个核细胞浓缩物)在4℃下过夜后产生未成熟和成熟DC的能力的稳定性研究。我们还完成了成熟多肽冲击的DC的稳定性测试，并建立了2小时的室温保存期，以适应运输或产品给药延迟。此外，我们建立了一个程序来记录用于培养成熟DC的CD40配体试剂的批次到批次的效力。正在进行的研究集中在通过流式细胞仪表型来表征DC。2003财年，完成了一项评估DC冷冻保存可行性的初步研究。用上述方法获得的成熟DC在5%DMSO和6%五倍体、5%人血清白蛋白的PlatmaLyte A中成功地冷冻保存，较高的蛋白质浓度并不能显著提高冻融过程后DC的回收率。还计划进行更多的冷冻保存研究。在2004财年和2005财年，由于缺乏CD40Ligand，对不同的DC成熟剂进行了评估。内毒素和干扰素联合应用是可行的。通过CCMD可获得脂多糖，从而可以计划与NCI进行三项DC临床试验。进行了三种相似的DC程序的开发。在2006财年，一项涉及制造自体肿瘤裂解物脉冲DC的临床试验启动。

The goal of this project is to develop and evaluate methods for manufacturing dendritic cells (DCs) for clinical immunotherapy trials. In FY 1999, we developed and optimized a full-scale GMP method for 5-day flask culture of autologous DCs in RPMI, autologous plasma or allogeneic serum, IL4 and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The immature DCs generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. This manufacturing method was incorporated into several clinical trials, and a manuscript describing this method was published in early 2001. In FY2000, because of our interest in developing closed systems and eliminating reagents that are difficult to standardize, we evaluated a 7-day culture system in a protein-defined, serum-free medium (XVIVO15) starting with monocytes from elutriation vs. negative immunomagnetic selection using the Isolex 300I, in bags vs. flasks. We demonstrated that the 2 different isolation methods for monocytes product equivalent immature DC populations, and that bags were equivalent to flasks. Furthermore, historical comparison showed that serum-free medium was equivalent and perhaps even superior to serum-containing medium for generation of immature DCs. A manuscript describing this work was published in January 2002. In FY2001, we focused on evaluating culture conditions for generating mature DCs using CD40 ligand after culture in IL4 and GMCSF. A process was successfully developed and incorporated into several cancer immunotherapy trials in early 2001. During FY2002, we completed studies demostrating stability after overnight hold at 4C of the raw material (mononuclear cell concentrates) in terms of ability to generate immature and mature DCs. We also completed stability testing of mature peptide-pulsed DCs and established a 2 hour room temperature hold period to accomodate transport or delays in administration of the product. In addition, we established a program to document lot-to-lot potency of the CD40ligand reagent used to mature DCs in culture. Ongoing studies are focused on characterizing DCs by flow cytometric phenotyping. In FY2003, a pilot study evaluating the feasibility of DC cryopreservation was completed. Mature DCs generated by the method previously described were successfully cryopreserved in 5% DMSO and 6%Pentastarch, with 5% human serum albumin in Plasmalyte A. Higher protein concentrations did not significantly improve DC recovery after the freeze/thaw process. Additional cryopreservation studies are planned. During FY04 and FY05, evaluation of different maturing agents for DC was conducted due to lack of availability of CD40Ligand. The combination of LPS and IFN was found to be feasible. Availability of LPS thru the CCMD allowed for planning of three DC clinical trials with NCI. Development of three similar but DC procedures were performed. During FY06, one clinical trial involving manufacturing autologous tumor lysate-pulsed DC was initiated.