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

摘要/摘要 树突状细胞(DC)治疗代表了一种新的、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 30多种基于DC的疫苗正在为其他癌症的临床开发管道中。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增长 从2010年的2000万美元增加到2018年的20亿美元。这突显了开发临床级别技术的必要性 采集DC前体细胞，生成DC，用于肿瘤疫苗接种。通常，DC与 单核细胞，用不同的方法从外周血单核细胞中分离出来，包括 塑料黏附、逆流淋洗和免疫磁性细胞选择或耗尽。其中 技术，CD14+单核细胞的免疫磁选是最常用的技术 诊所。虽然与另一种技术相比，这种技术产生了相对纯净的单核细胞群 技术上，单核细胞的回收率/得率较低。此外，使用抗体来纯化单核细胞使其 对于大规模生产单核细胞来说，成本过高。此外，上述所有技术都是 无法从全血中分离单核细胞，在分离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，有一种未得到满足的需求需要发展 一种简单、快速、有效的方法，能够分离出高度纯净和静止的种群 直接从全血中提取单核细胞。亲和能源公司已经鉴定出一组能特异性结合的多肽 单核细胞，但不与其他血细胞结合。当与磁珠结合时，这些多肽可以 直接从全血中分离单核细胞，回收率和纯度高，不需要任何额外的 处理步骤。在第一阶段结束时，我们将建立一种快速有效的方法来 从全血中纯化单核细胞，并证明浓缩的单核细胞能够 分化为DC。在第二阶段，我们将优化多肽结合珠的规模化生产， 建立货架期和储存条件，并证明我们的技术纯化单核细胞的可行性 在更大的范围内。

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.