Peptide-based tool for the rapid isolation of quiescent monocytes from peripheral blood

总结/摘要 树突状细胞（DC）疗法代表了一种新的和有前途的免疫治疗方法， 晚期癌症，DC疫苗已被批准用于晚期前列腺癌等 其他癌症的临床开发管道中有30多种基于DC的疫苗。尤其是美国市场 癌症疫苗（包括基于DC的疫苗）将以78.1%的复合年增长率大幅扩张， 从2010年的2000万美元到2018年的20亿美元。这突出表明需要开发临床级技术， 收集DC前体细胞并产生用于癌症疫苗接种的DC。典型地，DC分化自 单核细胞，其使用不同的方法从外周血单核细胞分离，包括 塑性粘附、逆流淘析和免疫磁性选择或细胞耗竭。这些 技术，CD 14+单核细胞的免疫磁性选择是免疫学中最常用的技术。 诊所虽然与其他方法相比，该技术产生相对纯的单核细胞群， 然而，在这些技术中，单核细胞的回收率/产率低。此外，使用抗体纯化单核细胞使其 这对于大规模生产单核细胞是成本过高的。此外，所有上述技术都是 不能从全血中分离单核细胞，并且在分离单核细胞之前需要密度梯度步骤， 单核细胞，从而增加了成本、时间和所涉及的技术技能。因此，有一个未得到满足的需要， 一种简单、快速和有效的方法，能够分离高纯度和静止的群体， 单核细胞直接来自全血。Affinergy已经鉴定了一个肽家族， 单核细胞，而不与其他血细胞结合。当与磁珠缀合时，这些肽可以 直接从全血中分离单核细胞，回收率和纯度高，无需任何额外的 加工步骤。在第一阶段结束时，我们将建立一种快速有效的方法， 从全血中纯化单核细胞，并证明富集的单核细胞能够 分化为DC。在第二阶段，我们将优化肽缀合珠的大规模生产， 建立有效期和储存条件，并证明我们的技术纯化单核细胞的可行性 in a larger更大scale规模.

SUMMARY/ABSTRACT Dendritic cell (DC) therapy represents a new and promising immunotherapeutic approach for treatment of advanced stage cancers, with DC vaccines already approved for use in advanced prostate cancer, and more than 30 DC-based vaccines in the clinical development pipeline for other cancers. In particular, the US market for cancer vaccines (including DC-based vaccines) is set to expand tremendously at a CAGR of 78.1% from $20 million in 2010 to $2 billion by 2018. This highlights the need to develop clinical grade technologies for collecting DC precursor cells and generating DCs for cancer vaccination. Typically, DCs are differentiated from monocytes, which are isolated from peripheral blood mononuclear cells using different methods including plastic adherence, counter-flow elutriation, and immunomagnetic selection or depletion of cells. Of these techniques, immunomagnetic selection of CD14+ monocytes is the most commonly employed technique in the clinic. While this technique results in a relatively pure population of monocytes as compared to the other techniques, the recovery/yield of monocytes is low. Also, the use of antibodies to purify monocytes makes it cost-prohibitive for large-scale production of monocytes. In addition, all the above mentioned techniques are unable to isolate monocytes from whole blood, and require a density-gradient step prior to isolation of monocytes, thus increasing costs, time and technical skill involved. Thus, there is an unmet need to develop a simple, rapid and efficient method capable of isolating a highly pure and quiescent population of monocytes directly from whole blood. Affinergy has identified a family of peptides that bind specifically to monocytes, while not binding to the other blood cells. When conjugated to magnetic beads, these peptides can directly isolate monocytes from whole blood, and result in high recovery and purity, without any additional processing steps. At the conclusion of Phase I, we will have established a rapid and efficient method for purifying monocytes from whole blood and demonstrated that the enriched monocytes are capable of differentiating into DCs. In Phase II, we will optimize large-scale production of peptide-conjugated beads, establish shelf-life and storage conditions and demonstrate the feasibility of our technique to purify monocytes at a larger scale.