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。在1999财政年度，我们开发和优化了一种全规模GMP方法，用于在RPMI、自体血浆或同种异体血清、IL 4和GMCSF中培养自体DC 5天，从通过单采术收集的外周血单核细胞开始，并通过淘洗纯化。然后产生的未成熟DC可用于进一步操作（例如，肽脉冲）。这种制造方法被纳入了几项临床试验，并于2001年初发表了描述这种方法的手稿。在2000财年，由于我们对开发封闭系统和消除难以标准化的试剂感兴趣，我们评价了在蛋白质确定的无血清培养基（XVIVO 15）中的7天培养系统，从使用Isolex 300 I进行淘洗的单核细胞开始，在袋中与烧瓶中进行阴性免疫磁性选择。我们证明了用于单核细胞的2种不同分离方法产生等同的未成熟DC群体，并且袋等同于烧瓶。此外，历史比较显示，无血清培养基与含血清培养基在产生未成熟DC方面相当，甚至可能上级。2002年1月发表了一份介绍这项工作的手稿。在2001财政年度，我们重点评估了在IL 4和GMCSF中培养后使用CD 40配体产生成熟DC的培养条件。2001年初，成功开发了一种方法，并将其纳入了几项癌症免疫治疗试验。在2002财年期间，我们完成了证明原材料（单核细胞浓缩物）在4 ℃下放置过夜后在生成未成熟和成熟DC的能力方面稳定性的研究。我们还完成了成熟肽脉冲DC的稳定性测试，并建立了2小时室温保持期，以适应产品的运输或给药延迟。此外，我们建立了一个程序来记录培养中用于成熟DC的CD 40配体试剂的批间效价。正在进行的研究集中于通过流式细胞术表型来表征DC。 在2003财政年度，完成了一项评估DC冷冻保存可行性的试点研究。 将通过前述方法产生的成熟DC成功地冷冻保存在含5% DMSO和6% Pentastarch以及5%人血清白蛋白的Plasmalyte A中。 较高的蛋白质浓度并没有显着提高冷冻/解冻过程后的DC回收率。 计划进行额外的冷冻保存研究。在2004和2005财年期间，由于缺乏CD 40配体，对不同的DC成熟剂进行了评价。LPS与IFN联合应用是可行的。通过CCMD获得LPS允许计划三项NCI DC临床试验。进行了三种类似但DC程序的开发。在2006财年，启动了一项涉及制造自体肿瘤裂解物脉冲DC的临床试验。

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.