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

 描述(申请人提供)：多发性骨髓瘤(MM)是一种浆细胞来源的血癌，可产生单抗免疫球蛋白(M-Ig)。微小残留病(MRD)的检测对MM的治疗具有重要意义。所有用于检测MRD的方法都是基于对从骨髓活检获得的浆细胞的评估。这些方法很容易失败，而且由于其侵入性，不允许频繁监测。检测外周血中的MRD是最理想的，但仍然是一个尚未满足的医学挑战。循环M-Ig蛋白是多发性骨髓瘤的标志，目前已有的血清M-Ig蛋白检测方法，包括血清蛋白电泳法(SPEP)和免疫固定法(IFE)，用于诊断和随访，但由于敏感性不足，不能用于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，这种可变区是肿瘤特有的，对患者来说是独一无二的。监测治疗后血液中同一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。适配子是一种单链寡核苷酸，能与靶标特异结合，已被证明可以对包括免疫球蛋白在内的分析物进行敏感的基于血清的检测。通过一种称为指数富集型配体系统进化(SELEX)的体外过程，从随机的寡核苷酸文库中产生适配子。检测MRD需要产生针对患者独特独特型的适体。这对于传统的SELEX方法来说是不切实际的，因为SELEX方法是劳动密集型和耗时的，但我们的创新微流控技术可以快速生成适体。我们的初步微流控设备已经证明，能够在10小时内完成SELEX过程，成功分离免疫球蛋白靶向适配子。我们的长期目标是实现一种灵敏的检测外周血中MRD的范例，使用针对患者和个体MM患者的肿瘤特异性M-Ig蛋白的个性化适配子。该项目的目标是验证我们的微流控技术为个别患者快速产生独特型靶向适体的能力，以检测外周血中灵敏的MRD。我们将首先使用现成的IgG1/kappa单抗优化SELEX方案，然后使用该方案验证微流控分离针对患者M-Ig蛋白的适配子，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，包括对免疫球蛋白多发性骨髓瘤(IIMM)的实际患者进行纵向、实时监测，这些患者的IgG1/kappa单抗水平增加，同时我们将把我们的程序扩展和推广到其他类别/子类别，使能够显著改进MRD检测的个性化试剂能够为几乎所有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.