利用低pH耐受型抗体构建高效抗体-毒素偶联物的研究

Liver cancer is one of the most lethal malignancies in China. There is an urgent need to develop effective strategy for liver cancer therapy. GPC3 is specifically overexpressed in hepatocellular carcinoma (HCC) and internalizes efficiently, which makes it a potential target for immunotherapy, especially for internalized drug such as antibody-drug conjugate and antibody-toxin conjugate. However, the internalized drug, especially those protein-based antibody conjugate, normally goes degradation under the low-pH condition in endosome and lysosome, which significantly reduces its anti-tumor activity. Therefore, improving the low-pH stability of these protein-based antibody conjugates is the major issue to solve this problem. In the current study, we are planning to target GPC3, screening specific antibody with stable function under low-pH, and constructing antibody-toxin conjugate to improve its protein stability and anti-tumor activity in vivo. Our findings will reveal the feasibility of low pH-resistant antibody for construction of antibody-conjugates with high stability, and provide an alternative strategy and drug candidate for liver cancer therapy. Moreover, the observations in the current study would also be helpful for designing and improving other internalized drugs.

肝癌是我国高发的恶性肿瘤之一，缺乏有效的治疗手段。GPC3是肝细胞癌特异性的细胞膜表面分子标志物，能够快速内吞，是抗体-药物和抗体-毒素偶联物等内吞类药物的理想靶标。然而，蛋白类内吞药物如抗体-毒素偶联物进入细胞后往往被转运至内体和溶酶体较为酸性的环境中，导致蛋白质构象被破坏，发生大量降解，极大地降低了其抗肿瘤效果。因此，寻找合适的手段增强该类药物在酸性环境下的蛋白质稳定性是解决上述问题的关键。本项研究选取GPC3作为肝癌治疗的靶标，拟通过筛选在酸性环境下功能稳定的抗体来构建抗体-毒素偶联物，降低其在酸性环境下的降解，借以提高抗体-毒素偶联物在肿瘤细胞内的稳定性，促进其对肿瘤细胞的杀伤效率。研究结果将明确低pH耐受型抗体在构建高稳定性抗体-毒素偶联物上的可行性，为肝癌的治疗提供新策略和可能的候选药物；同时也为其他蛋白类内吞药物的设计和优化提供参考，具有重要的认识意义和转化应用价值。

抗体-毒素偶联物是一种高效的肿瘤杀伤分子，可通过与肿瘤表面抗原结合后内化到肿瘤细胞内，在溶酶体中降解释放游离菌素，发挥细胞毒杀伤功能。大部分内化的抗体-毒素偶联物在溶酶体中降解，只有约5%的游离毒素逃逸到胞质中发挥杀伤作用。 因此，如何提高细胞内抗体-毒素偶联物的利用率是提升其抗肿瘤活性的关键问题。已有研究针对偶联物的毒素片段进行了优化，但是抗体片段对偶联物活性的影响尚缺乏研究。.本项目以肝癌特异性细胞膜表面分子GPC3为靶点，通过噬菌体展示技术在不同pH条件下进行抗体筛选，成功获得了低pH耐受型抗体42A1和低pH敏感型抗体32A9。我们分析了两种抗体的亲和力、特异性和抗原表位等特征，并构建了低pH耐受型抗体-毒素偶联物42A1-PE24以及低pH敏感型抗体-毒素偶联物32A9-PE24。42A1-PE24能够拮抗溶酶体水解，具有更佳的蛋白质稳定性；32A9-PE24对溶酶体降解敏感，拥有更高效的游离菌素释放能力。体外细胞毒实验及裸鼠皮下移植瘤实验证明低pH敏感型抗体-毒素偶联物32A9-PE24具有更佳的抗肿瘤活性。我们的研究证明了抗体的pH响应性对抗体-毒素偶联物肿瘤杀伤功能的调控作用，发现了相较于提升偶联物的蛋白质稳定性，促进偶联物在溶酶体中快速降解，加速游离菌素释放能够更直接的引发高效的细胞杀伤活性，对设计高效的抗体-毒素偶联物具有重要指导意义。同时，我们的研究结果也为设计其他溶酶体降解依赖的抗体偶联药物提供了新的实验证据和理论依据。