A Practical Approach to Tumor-Specific Aptamers for B-Cell Hematologic Malignancies

个性化医疗将极大地提高癌症护理的有效性；然而，开发可实际实现的针对患者的具体策略仍然具有挑战性。一种独特的机会存在于B细胞来源的恶性肿瘤中，这种肿瘤通常在其B细胞受体(BCR)内表达具有可变区域(独特型，ID)的表面免疫球蛋白(SIGs)。由于恶性细胞来自单个克隆，因此这种Sig-ID分子是肿瘤特有的，对每个患者都是独特的。因此，以SIG-ID为目标可以实现个性化的疾病识别和治疗策略。使用患者特异性抗SIG-ID抗体的早期研究取得了有希望的结果，但被认为是不可持续的。在基于SIG-ID的诊断和治疗中，以节省时间和成本效益的方式生成个性化配体的技术仍然是一个未得到满足的需求。 适配子，即与生物靶标特异结合的单链寡核苷酸，为这一未得到满足的需求提供了一个有吸引力的解决方案。通过一种称为SELEX的体外过程从随机寡核苷酸文库中分离适体，这一过程传统上是劳动密集型、耗时(长达一个月)的，并且对于个性化的适体生成是不现实的。相反，我们开发了一个称为MicroSELEX(μSELEX)的微流体平台，它已被用于分离蛋白质生物标记物的适体，包括来自多发性骨髓瘤和新冠肺炎患者的单抗的ID区。给出患者样本中的单克隆蛋白，该平台能够在约10小时内快速分离个性化的抗ID适配子。 我们建议探索针对肿瘤B细胞SIG-ID的针对B细胞恶性血液病患者特异性μ适体的时间效率和成本效益DNASELEX分离。我们将首先利用B细胞来源的细胞系建立最佳的μSELEX方案，然后从B细胞淋巴瘤患者的外周血样本中分离出针对肿瘤B细胞的抗SIG适配子，最后通过使用该适配子检测循环中的肿瘤B细胞来展示基于外周血的微小残留疾病的无创监测。 除了能够及时识别最小残留疾病，以便更准确地做出临床决策外，抗Sig-ID适配子还可以用作治疗配体，以实现个性化和精确的靶向治疗，从而更有效地治疗疾病。因此，这种个性化的适配子可能会导致B细胞恶性血液病患者的护理发生变革性的变化。

Personalized medicine will greatly improve the effectiveness of cancer care; however, the development of practically attainable patient-specific strategies has remained challenging. One unique opportunity exists with B cell-derived malignancies, which often express surface immunoglobulins (sIgs) with variable regions (idiotypes, Ids) within their B-cell receptors (BCRs). As malignant cells originate from a single clone, such sIg-Id molecules are specific to the tumor and unique to each patient. Targeting sIg-Ids can hence enable personalized disease identification and treatment strategies. Early studies using patient-specific anti-sIg-Id antibodies yielded promising results but were deemed unsustainable. A technology to generate personalized ligands in a time-efficient and cost-effective manner remains an unmet need in sIg-Id-based diagnostics and therapeutics. Aptamers, i.e., single-stranded oligonucleotides that specifically bind to biological targets, offer an attractive solution to this unmet need. Aptamers are isolated from a randomized oligonucleotide library via an in vitro process known as SELEX, which is traditionally labor-intensive, time-consuming (up to a month), and impractical for personalized aptamer generation. In contrast, we have developed a microfluidic platform, called microSELEX (μSELEX), which has been used to isolate aptamers for protein biomarkers, including Id regions of monoclonal antibodies from patients with multiple myeloma and COVID-19. Given a monoclonal protein from a patient sample, the platform is capable of rapidly isolating personalized anti-Id aptamers within ~10 hours. We propose to explore time-efficient and cost-effective μSELEX isolation of patient-specific DNA aptamers targeting sIg-Ids of tumor B cells for B-cell hematologic malignancies. We will first establish an optimal μSELEX protocol using B cell-derived cell lines, then isolate anti-sIg aptamers against tumor B cells obtained from peripheral blood samples of B cell lymphoma patients, and finally demonstrate noninvasive peripheral blood-based monitoring of minimal residual disease by using the aptamers to detect circulating tumor B cells. In addition to enabling timely identification of minimal residual disease for more precise clinical decision making, anti-sIg-Id aptamers can also be used as therapeutic ligands to enable personalized and precisely targeted therapy for more effective disease treatment. Such personalized aptamers can hence potentially lead to transformative changes in the care of patients with B-cell hematologic malignancies.