Validating Rapid Microfluidic Isolation of Personalized Aptamers for Monitoring Minimal Residual Disease in Multiple Myeloma

 描述（由申请人提供）：多发性骨髓瘤（MM）是一种浆细胞来源的血液癌症，可产生单克隆免疫球蛋白（M-Ig）。微小残留病（MRD）的检测对于MM护理非常重要。用于MRD检测的所有方法均基于对从骨髓（BM）活检获得的浆细胞的评价。这些方法容易失败，并且由于其侵入性而不允许频繁监测。检测外周血中的MRD是最理想的，但仍然是一个未满足的医学挑战。循环M-Ig蛋白是MM的标志，通过目前可用的基于血清的方法（包括血清蛋白电泳（SPEP）和免疫固定（IFE））检测M-Ig蛋白用于诊断和随访，但由于灵敏度不足，未用于MRD监测。这个问题可以通过利用M-Ig蛋白的可变区（称为“独特型”）来解决，M-Ig蛋白是肿瘤特异性的，对患者来说是独特的。监测治疗后血液中相同独特型的存在和再现的能力将导致灵敏的MRD检测。这可以通过独特型靶向适体作为个性化试剂来实现。 适体是特异性结合靶标的单链寡核苷酸，并且已被证明允许对包括免疫球蛋白在内的分析物进行灵敏的基于血清的检测。通过称为指数富集配体系统进化（SELEX）的体外过程，从寡核苷酸的随机文库产生适体。MRD检测需要生成靶向患者独特独特型的适体。这是不切实际的传统的SELEX方法，是劳动密集型和耗时的，但可以通过我们的创新微流体技术，允许快速生成适体。我们的初步微流体装置已经证明了在约10小时内完成SELEX过程以成功分离免疫球蛋白靶向适体的能力。 我们的长期目标是实现一个范例，敏感检测MRD在外周血中使用个性化的适体靶向个别MM患者的M-Ig蛋白的患者和肿瘤特异性独特型。本项目的目标是验证我们的微流控技术快速生成个体患者独特型靶向适体的能力，用于外周血中敏感的MRD检测。我们将首先使用容易获得的IgG 1/κ单克隆抗体优化SELEX方案，然后使用该方案来验证靶向患者M-Ig蛋白的适体的微流体分离，用于基于血清的M-Ig检测。 在三年资助期结束时，我们将准备启动一项临床研究，包括纵向、实时监测具有完整免疫球蛋白多发性骨髓瘤（IIMM）且IgG 1/kappa水平增加的实际患者。单克隆抗体，同时我们将扩展和推广我们的程序到其他类别/亚类，使个性化试剂能够显著改善MRD检测，为几乎所有MM患者提供最佳护理。

 DESCRIPTION (provided by applicant): Multiple myeloma (MM) is a plasma cell derived blood cancer that produces monoclonal immunoglobulins (M-Ig). Detection of minimal residual disease (MRD) is of great importance for MM care. All methods used for MRD detection are based on evaluation of plasma cells obtained from bone marrow (BM) biopsies. These methods are susceptible to failure and do not allow frequent monitoring because of their invasiveness. Detection of MRD in peripheral blood is most ideal but remains an unmet medical challenge. Circulating M-Ig proteins are the hallmark of MM, and their detection by currently available serum-based methods, including serum protein electrophoresis (SPEP) and immunofixation (IFE), is used in diagnosis and follow-up, but not MRD monitoring due to insufficient sensitivity. This problem can be addressed by exploiting variable regions (called "idiotypes") of M-Ig proteins, which are tumor-specific and unique to the patient. The ability to monitor the presence and reappearance of the same idiotype in blood after therapy would lead to sensitive MRD detection. This can be enabled by idiotype-targeting aptamers as personalized reagents. Aptamers are single-stranded oligonucleotides that specifically bind to targets, and have been demonstrated to allow sensitive serum-based detection of analytes including immunoglobulins. Through an in vitro process termed systematic evolution of ligands by exponential enrichment (SELEX), aptamers are generated from randomized libraries of oligonucleotides. MRD detection requires generation of aptamers targeting the unique idiotype of the patient. This is impractical with conventional SELEX methods that are labor-intensive and time-consuming, but can be enabled by our innovative microfluidic technology that allows rapid generation of aptamers. Our preliminary microfluidic devices have demonstrated the capability of completing the SELEX process within ~10 hours for successful isolation of immunoglobulin-targeting aptamers. Our long-term objective is to realize a paradigm for sensitive detection MRD in peripheral blood using personalized aptamers targeting patient- and tumor-specific idiotypes of M-Ig proteins of individual MM patients. The goal of this project is to validate the ability of our microfluidic technology to rapidly generate idiotype- targeting aptamers for individual patients for sensitive MRD detection in peripheral blood. We will first optimize a SELEX protocol using readily available IgG1/kappa monoclonal antibodies, and then use this protocol to validate microfluidic isolation of aptamers targeting patient M-Ig proteins for serum-based M-Ig detection. At the end of the three-year grant period, we would be ready to initiate a clinical study, including longitudinal, real-time monitoring of actual patients with intact immunoglobulin multiple myeloma (IIMM) with increased levels of the IgG1/kappa monoclonal antibody, while in parallel we will expand and generalize our procedure to other classes/subclasses, enabling personalized reagents capable of dramatically improving MRD detection for optimal care for nearly all MM patients.