利用非天然氨基酸构建位点特异性IL-2/Sorafenib偶联物及其抗肿瘤作用和机制研究

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) has limited its use in tumor immunotherapy. Sorafenib, a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases, inhibitis tumor cell proliferation and angiogenesis. Here we demonstrate the use of genetically encoded unnatural amino acids (UAA) with orthogonal chemical reactivity to synthesize homogeneous IL-2/Sorafenib conjugates with precise control of conjugation site. p-Acetylphenylalanine was site-specifically incorporated into IL-2. The mutant protein was selectively and efficiently conjugated to Sorafenib through a stable oxime linkage. The conjugates allow selective targeting of IL-2 to tumor microenvironment by Sorafenib, thereby minimizing systemic toxicity, and the direct antitumor activity of sorafenib may combine in an additive manner with the immunomodulatory effects of IL-2. The IL-2/Sorafenib conjugates with optimized conjugation site and linker structure are obtained by in vitro and in vivo activity against cancer cells such as RCC, HCC and melanoma. The tumor targeting activity and inhibition of tumor angiogenesis are studied, and immunocyte subsets, cytokines and signaling pathway such as RAF/MEK/ERK are assayed to discover the cooperative effects of IL-2/Sorafenib conjugates in action mechanism. The site-specific conjugations of IL-2 and Sorafenib break through the bottleneck of random modification, and obtain homogeneous conjugates with optimized pharmacological properties, pharmacokinetics and toxicity.

IL-2是肿瘤免疫治疗最成功的细胞因子之一，但其副作用“毛细血管渗漏综合症”制约其应用。Sorafenib能靶向抑制多种肿瘤细胞激酶，抑制肿瘤增殖及血管形成。本项目利用扩展密码子和生物正交反应将IL-2特定位点突变为非天然氨基酸(UAA)对乙酰苯丙氨酸（pAcF），通过肟键将Sorafenib与pAcF上酮基偶联，构建位点特异性IL-2/Sorafenib偶联物，利用Sorafenib的靶向作用将IL-2递送至肿瘤微环境降低系统毒性，并将直接抗肿瘤活性与免疫调节作用结合。通过体内外抗肿瘤活性筛选获得偶联位点和linker优化的IL-2/Sorafenib偶联物；研究肿瘤组织靶向性及抗肿瘤血管生成作用，检测免疫效应细胞、细胞因子和相关信号通路，揭示偶联物在作用机制上的协同效应。基于UAA定位的IL-2/Sorafenib位点特异性偶联突破随机修饰的瓶颈，获得在疗效、药代等方面优化的单一偶联物

IL-2是肿瘤免疫治疗最成功的细胞因子之一，但其副作用“毛细血管渗漏综合症”制约其应用。Sorafenib能靶向抑制多种肿瘤细胞激酶，抑制肿瘤增殖及血管形成。本项目利用扩展密码子和生物正交反应将IL-2特定位点突变为非天然氨基酸获得IL-2突变体；通过分子模拟将Sorafenib的非药效基团吡啶-2-酰胺改造为巯基衍生物，Autodock Vina 1.1.2分子对接表明该衍生物能够与靶分子很好的结合；进一步采用MAL-PEG-NHS为linker分子，合成IL-2/Sorafenib偶联物，利用Sorafenib的靶向作用将IL-2递送至肿瘤微环境降低系统毒性，并将直接抗肿瘤活性与免疫调节作用结合。移植瘤小鼠模型实验表明，获得的IL-2/Sorafenib偶联物在小鼠肿瘤组织显示上调的IL-2浸润和CD8+T细胞募集，以及下调的CD31表达，发挥更有效的抗肿瘤血管生成和抗肿瘤免疫效应，提示偶联物在作用机制上的具有协同效应。