PDF(Pubmed)
Abstract：
Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of -78.2 ± 10.2 (with CTLA4), -60.0 ± 4.2 (with NKG2A), and -77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.
摘要：
免疫疗法的最新进展揭示了CTLA-4和PD-1/PD-L1通路在当代肿瘤学中的关键作用。在回应率和不利影响方面提出承诺和挑战。本研究采用计算生物学工具（计算机模拟方法）来制作能够与双受体结合的适体，即,抑制性CTLA4和NKG2A，从而释放T和NK细胞并增强CD8+T和NK细胞用于肿瘤细胞裂解的功能。计算分析突出显示AYA22T-R2-13，HADDOCK评分为-78.2±10.2（CTLA4），-60.0±4.2(含NKG2A),和-77.5±5.6（CD94/NKG2A）。通过ELISA和流式细胞术方法获得适体与靶向蛋白结合的确认。使用乳酸脱氢酶(LDH)细胞毒性测定评估体外生物学功能。使用ELISA和流式细胞术的直接和竞争性测定法证明AYA22T-R2-13与CTLA4和NKG2A蛋白的选择性结合，以及IL-2刺激的T细胞和NK细胞的细胞表面受体。在存在来自CTLA4或NKG2A蛋白的竞争的情况下,这种结合被抑制。值得注意的是,AYA22T-R2-13阻断CTLA4或NKG2A可在体外增强人CD8T细胞和NK细胞介导的肿瘤细胞裂解。我们的发现突出了AYA22T-R2-13对CTLA4-B7-1/B7-2（CD80/CD86）或CD94/NKG2A-HLA-E相互作用的精确结合特异性，将其定位为小鼠肿瘤模型中免疫检查点阻断适体研究的有价值的工具。这些体外研究为进一步增强结合能力以及在动物模型中建立功效和安全性奠定了有希望的基础。因此,我们的结果强调了AYA22T-R2-13在癌症免疫治疗中的潜力，提供高特异性，低毒性,以及具有成本效益的生产潜力。
