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.
免疫治疗领域的最新进展揭示了CTLA-4与PD-1/PD-L1通路在现代肿瘤学中的关键作用，其在治疗反应率和不良反应方面既展现出前景也面临挑战。本研究采用计算生物学工具（计算机模拟方法）设计能够同时结合抑制性受体CTLA4与NKG2A的适配体，从而解除T细胞和NK细胞的抑制状态，增强CD8+T细胞与NK细胞的肿瘤细胞杀伤功能。计算分析显示AYA22T-R2-13的HADDOCK结合评分分别为：与CTLA4结合−78.2 ± 10.2，与NKG2A结合−60.0 ± 4.2，与CD94/NKG2A复合物结合−77.5 ± 5.6。通过ELISA和流式细胞术验证了适配体与靶蛋白的特异性结合。采用乳酸脱氢酶（LDH）细胞毒性实验评估体外生物学功能。ELISA与流式细胞术的直接结合及竞争实验表明，AYA22T-R2-13能选择性结合CTLA4和NKG2A蛋白，以及IL-2刺激的T细胞和NK细胞表面受体，该结合作用在CTLA4或NKG2A竞争蛋白存在时受到抑制。值得注意的是，AYA22T-R2-13对CTLA4或NKG2A的阻断作用在体外显著增强了人CD8+T细胞和NK细胞介导的肿瘤细胞裂解。我们的研究结果凸显了AYA22T-R2-13对CTLA4-B7-1/B7-2（CD80/CD86）或CD94/NKG2A-HLA-E相互作用具有精确的结合特异性，使其成为小鼠肿瘤模型中免疫检查点阻断适配体研究的重要工具。这些体外研究为进一步提升结合能力、验证动物模型中的疗效与安全性奠定了良好基础。因此，本研究结果揭示了AYA22T-R2-13在癌症免疫治疗中的应用潜力，其具有高特异性、低毒性及成本效益优势。
肿瘤(癌症)患者之家