基于内源白蛋白搭乘式“大分子前药”肿瘤靶向输送策略改善蛋白毒素的类药性质

A crucial issue for biologics research is how to improve the drug-like properties of active macromolecular candidates, in term of the biopharmaceutics profiles. The significant progress on molecular biology, structure biology, and genetic engineering has provided many new technologies and methods for the research of protein drug delivery. Rational design of protein structure for improved in vivo delivery has become a cutting-edge area in pharmaceutical research. Herein proposed is a conceptual work of molecular structure pharmaceutics of proteins. Trichosanthin, a ribosome-inactivated protein, will be investigated as protein drug candidate. Rational design of "structure-delivery" based on the drug delivery principles will be studied in order to improve the drugability-associated PK, PD, and immunology profiles. A macromolecular prodrug strategy based on albumin-hitchhiking delivery and intein-mediated cleavage will be developed. Some specific functional domains (i.e., albumin-binding domain and intein domain) will be fused to trichosanthin by a recombinant method. The trichosanthin fusion protein will hitchhike onto serum albumin in systemic circulation via the interaction of its fused albumin-binding domain. The binding with albumin and its shielding effect on trichosanthin will render poor accessibility to the active domains of trichosanthin, thus yielding a "prodrug-like" feature. Importantly, albumin can function as targeting motif which binds with the tumor-associated overexpression of albumin receptors such as SPARC, gp60, gp30, and gp18, thus achieving albumin-mediated tumor targeting trichosanthin delivery. Once entering the tumor cells, the highly reducing environment will trigger the intein-mediated cleavage and the consequent release of free trichosanthin for carrying out its antitumor activity. In summary, we aim to overcome the poor drug-like properties of ribosome-inactivated proteins (e.g., trichosanthin) via a research strategy of molecular structure pharmaceutics of proteins, and explore the therapeutic application of the drug delivery system on drug-resistant glioma.

生物药研究的关键问题之一是提高候选活性​大分子的类药性（尤其是生物药剂学特性）。分子生物学、结构生物学、基因工程的飞速发展为蛋白递药研究带来了新技术与方法。对蛋白结构进行合理设计及生物改造正成为药剂学的重要方向。我们提出了蛋白药物的分子结构药剂学研究的新概念，以蛋白毒素天花粉蛋白为研究对象，基于药物输送原则进行蛋白的“结构-输送”理性设计，来改善与类药性密切相关的药动学、药效学、免疫学性质。基于内源白蛋白搭乘式、内含肽介导自剪切的大分子前药输送策略，通过重组技术在天花粉蛋白上引入特定功能域，令其搭乘于血浆白蛋白。白蛋白的结合及掩蔽作用使该给药系统处于灭活“前药型”；同时白蛋白介导肿瘤靶向输送，进入肿瘤细胞后，响应高还原性环境，融合蛋白的内含肽域发生剪切，释放出游离天花粉蛋白。本项目通过分子结构药剂学研究新思路，来改善以天花粉蛋白为例的核糖体失活蛋白的类药性质及其治疗耐​药脑胶质瘤的作用。

本项目已顺利完成所提出的目标，发展了一种基于生物工程方法来制备的靶向蛋白给药系统。相较于化学合成方法，生物工程法更加高效、简便和可控。采用基因工程技术进行蛋白质的功能构建并基于药物输送 （Drug Delivery）原则进行合理设计，针对蛋白药物的三个主要类药性质进行改造或优化。以天花粉蛋白作为研究对象，提出了基于内源白蛋白“搭乘式”的大分子前药靶向输送策略以改善蛋白毒素的类药性。通过基因重组技术在天花粉蛋白结构上引入三段功能域，分别为白蛋白结合域ABD(albumin binding domain), 肿瘤酶Legumain相应的底物肽(PTN)和细胞穿膜肽（TAT），通过ABD与白蛋白的结合搭载天花粉蛋白进入肿瘤组织。由于白蛋白在血浆丰度高且在体内半衰期较长，能有效减缓天花粉蛋白的体内清除。并且肿瘤新生血管内皮细胞表面及肿瘤细胞表面，具有高表达的白蛋白结合蛋白(Albumin-binding proteins)，例如 SPARC 和 gp60 等。白蛋白与其结合后，可通过受体介导内吞入胞，从而实现内源白蛋白“搭乘式”大分子前药靶向输送作用。肿瘤酶底物肽的引入有助于天花粉蛋白进入肿瘤部位后，通过酶响应机制释药，增强天花粉蛋白肿瘤细胞杀伤能力。.本项目的科学意义及创新性：.1. 成功构建了内源性白蛋白“搭乘式”输送的大分子前药靶向系统，并进行了较为系统的生物学评价，显示了所构建的系统具有良好的体内外抗肿瘤活性和安全性。.2. 基于白蛋白结合域（ABD）的内源白蛋白“搭乘”的策略，避免了传统蛋白化学修饰的复杂步骤，通过生物工程技术制备，具有较好的工艺转化价值。.3. 揭示了由于白蛋白分子与天花粉蛋白-ABD 结合，会形成纳米尺寸复合物，有利药物肿瘤被动靶向。该方法为改善核糖体失活蛋白的类药性及治疗提供一个有用的解决方案。