基于S-层蛋白表面展示技术研究多靶位纳米抗体增强肿瘤靶向的作用机制

Tumor development depends on a series of pathways and key proteins such as ubiquitin ligase Trim39, programmed cell death protein PD-L1, and vascular endothelial growth factor receptors VEGFR-1/2. However, current single-target drugs are frequently frustrated by tumor resistance generated by target mutations and other alternative pathways. It has been reported that dual-targeted antibody drugs can enhance inhibition of the tumor bypass mechanism. Based on Bacillus S-layer protein surface display technology, we have developed Trim39 antibody heavy chain variable region VH as an intrabody to block the target intracellular Trim39 protein in tumor cells, and on this base, the current project will fuse or couple the single chain antibodies to multi-targeted nanobodies with specificity for several key tumor specific proteins, using genetic engineering (such as the S-layer protein surface display) and material chemical methods, along with optimization, and then, drug delivery systems targeting multiple sites will be constructed. Through study of the affinity of the single chain antibodies or multi-targeted nano-antibodies (nanobodies) to the antigens and related conformation informatino, and through study of tumor suppression with multi-targeted drug systems, the mechanism of enhancing tumor target efficacy using the multi-targeted nanobodies will be proven, and a scientific method and a theoretical basis will be provided for optimization of engineered multi-target antibodies and study of their target mechanisms.

肿瘤发展有赖于泛素连接酶Trim39、细胞程序性死亡蛋白PD-L1、血管内皮生长因子受体VEGFR-1/2等一系列通路和关键蛋白。然而，目前的单靶位药物往往面临靶标突变等旁路途径产生的肿瘤耐药性问题。最近研究表明双靶位抗体药物可加强抑制肿瘤旁路机制。基于芽胞杆菌S-层蛋白表面展示，我们已开展了Trim39抗体重链可变区VH封闭肿瘤细胞内Trim39靶蛋白的研究，以此为基础，本项目将通过S-层蛋白表面展示等基因工程和材料化学方法将肿瘤关键蛋白的单链抗体融合或嵌合构建为多靶位纳米抗体，同时对其优化，进而制备多靶位药物传递体系。通过单链抗体、多靶位纳米抗体对抗原的亲和作用及其构象研究、多靶位药物体系靶向肿瘤治疗研究，探明多靶位纳米抗体增强肿瘤靶向机理，为工程抗体多靶向优化策略及靶向机制研究提供科学方法和理论依据。

癌症，即恶性肿瘤，因为其细胞的易变性，例如通过旁路途径突变或下调靶标分子的表达量，而常常使单靶点药物失去靶标。因此亟待发展双靶或是多靶向的药物体系。通常选择肿瘤特异性高表达的蛋白作为靶标分子，而采用能特异性识别并结合靶标蛋白的分子作为靶向基团。传统抑制剂虽能结合靶标，但特异性普遍不够。DNA适体特异性虽好，但是对高通量的筛选过程要求较高。迄今为止，抗体是与蛋白有着最强的特异性识别与结合能力的生物大分子，且相对易于获得。但传统的抗体存在抗原性，其Fc区段能引发免疫反应，此外，天然抗体或多克隆抗体体积较大，运动性及可调性较弱。本研究针对抗体的上述问题，研发了去免疫原性的系列单链抗体，包括针对重要肿瘤相关蛋白TIM39、VEGFR-2、PD-L1的特异性单链抗体，并且以VEGFR-2和PD-L1双靶点为试验，发展了双靶向的单链纳米抗体。基于抗VEGFR-2-抗PD-L1双靶单链抗体基因，实现了该双靶抗体在肿瘤细胞内表达，极大地提高了抑制肿瘤迁移和抗肿瘤的能力。实验过程中，研究了肿瘤耐药性相关的PI3K 110α/β、CDK6靶点与CDK6-PI3K调节通路，有望在此基础上获得更精确的抗耐药癌细胞的双/多向纳米抗体，为解决当前癌症容易耐药、复发、药物治疗副作用大等难题提供新的解决途径，包括选取可用的靶点、采用安全的抗体治疗方案，以及提高肿瘤靶向的精确性和效率，并为治疗-检测一体的技术提供准确捕获-杀死肿瘤细胞的优良嵌合抗体。