基于非天然氨基酸的定点原位生长技术研究及其在长效干扰素中的应用

Therapeutic proteins and peptides have been widely used in clinical practice, but they have conspicuous limitations such as rapid clearance from the blood circulation and high immunogenicity. Covalent conjugation of hydrophilic polymer to a protein is often used as a means to address the above shortcomings. The representative techniques include PEGylation and site-specific in situ growth (SIG). However, since attachment of PEG to a protein involves the reaction between two large macromolecules, PEGylation has drawbacks such as low yield, various positional isomers and decreased bioactivity. The first generation of SIG for in situ synthesis of site-specific protein-polymer conjugates currently has some limitations that it is not favorable for further application. For example, the methodology of site-specific protein modification is complicated and poor in universality; the reaction site is restricted. In this study, we propose to develop the second generation of site-specific in situ growth via combination of SIG and unnatural amino acid (UAA) technique, ultimately realize the randomly desired and site-specific in situ protein modification. We call this methodology UAA-SIG. Interferon (IFN) is chosen as the model protein and in our previous work, IFN was successfully modified with “chemical hander”-UAA- at desired sites and the biological activities were measured. In the future work, we will screen long-acting IFN with excellent characteristics through systematic synthesis of site-specific IFN-polymer conjugates at desired modification sites and investigation of the influence rule on biological properties of IFN, providing theoretical basis for the application of UAA-SIG technology in medicine.

目前蛋白药物已被广泛地应用于临床，但仍然存在半衰期短、免疫原性强等共性问题。将蛋白药物与水溶性高分子偶联，是解决上述问题的有效途径之一。代表性技术包括聚乙二醇（PEG）化和定点原位生长技术（SIG）。但蛋白的PEG化存在修饰效率低、修饰位点不均一等问题；而现有SIG技术由于修饰位点局限、反应条件复杂等，亦不利于进一步的生产应用。通过将非天然氨基酸（UAA）介导的可任意位点修饰的技术优势与SIG技术相结合，本项目拟以干扰素为模型，建立基于UAA的第二代SIG技术（UAA-SIG），从而实现蛋白质表面定点且任意位点的原位生长修饰。前期的工作中，本项目已经实现了干扰素任意位点含有修饰“把手”UAA的插入及活性评价。在进一步工作中，我们将系统性地探索基于UAA-SIG技术的干扰素可修饰位点及其性质影响规律，从而筛选并获得性质最优的高分子原位生长的长效干扰素产品，为该技术在医药中的应用提供理论依据。

目前蛋白药物已被广泛地应用于临床，但仍然存在半衰期短、稳定性差、免疫原性强等共性问题。将蛋白药物与水溶性高分子偶联，或者与长循环蛋白融合，是解决上述问题的常用途径。代表性技术包括已商业化的如聚乙二醇（PEG）化、白蛋白（HSA）融合技术等，以及本人开发的定点原位生长（SIP）、ELP融合技术等。然而，以上策略均需要引入外源大分子，不可避免产生免疫原性、降低生物活性，以及其他潜在的毒性。本项目以绿色荧光蛋白（GFP）和干扰素（IFN）为模型蛋白，开发一系列蛋白融合串联技术，包括自融合串联技术（SEC）和基于穿膜肽和环化的融合串联技术，在不引入外源大分子的情况下，实现蛋白在体内的长时间滞留，且显著提高蛋白的生物活性。本项目以GFP为模型蛋白，成功完成了以上技术开发。进一步，本项目验证了SEC在IFN修饰中的可行性，并系统性地探究串联数量对蛋白药学性质的影响规律，从而筛选并获得性质最优的长效IFN产品，为以上技术在医药中的应用提供理论依据。此外，本项目参与开发基于非天然氨基酸的PEG化技术应用于白介素-2的修饰改良，实现更安全有效的自身免疫病治疗效果。