Design and generation of new absorption-enabling peptides for oral delivery of biologics

该项目与BBSRC的战略研究重点3“生物科学促进健康”(生物技术促进健康挑战)保持一致，因为它旨在发展基础生物科学，为有利于维护和促进健康的生物制剂的制定和验证奠定基础。在申请者先前工作的基础上，受益于协同性、多学科和Proxima的技术，该项目将开发用于口服生物制剂的新一代材料。生物制剂改变了疗法(2014年最畅销的8种药物中有7种)。他们的给药仅限于注射，因为肠道吸收可以忽略不计。这些药物不太被患者接受(痛苦/潜在的副作用)，而且制造和管理成本很高。有几个障碍限制了生物制品的全身吸收，但肠道上皮细胞是最难克服的，也是最具挑战性的。目前改善口服生物制品吸收的方法通常使用非选择性地干扰和增加肠道通透性的“吸收促进剂”，但安全问题(例如。许多表面活性剂)阻碍了临床翻译。因此，对安全有效的口服生物制剂技术的需求尚未得到满足。安全有效口服生物制剂的关键是在不破坏这一生理上重要的屏障的情况下，有选择地通过肠道粘膜走私药物的材料。它们可以结合到药物或药物输送系统(例如纳米颗粒)上。该项目旨在开发新的跨细胞激活材料，在不破坏上皮细胞的情况下促进生物制剂的口服吸收。这些都是以多肽为基础的，设计灵感来自于一种天然的大分子配体，通过其受体，即免疫球蛋白G和新生儿Fc受体(FcRN)，通过细胞转运很容易穿过肠道粘膜。跨细胞激活化合物的制备将由受体和配体结合的表位Ig G和FcRn决定。Vllasaliu以前证明，FcRN通过细胞穿梭运送Ig G Fc包裹的纳米颗粒。Proxima的发现平台将用于制备跨细胞激活的多肽结构。在Mozaic中，原型多肽是由表面带有氨基酸(AA)组合的胶束纳米颗粒创建的。构建筛选文库，经细胞培养检测，筛选出最有效的多肽合成组合。氨基酸的选择是由免疫球蛋白受体结合域中的序列决定的。同时，Almanac(来自蛋白质-蛋白质相互作用结构分析数据的算法)将被用于根据受体结合结构域序列推断配体结合肽的结构。用两种互补的方法开发FcRN结合配体可以减少失败的风险。项目阶段Yr 1：项目相关技术的学生培训和研究Ig G-FcRN序列Yr 1和2：用合适的荧光基团制定Mozaic筛选文库和肽以进行定量。纯化(高效液相色谱)和MSYr3&4表征：单独测试化合物和连接到模型蛋白(白蛋白)的细胞通过Caco-2单分子层的摄取和吸收(肠道模型)。竞争分析测试FcRN受体介导的效应。通过抑制特定途径的siRNA和共聚焦显微镜确定摄取机制(共定位研究)YR4：测试大多数成功化合物在体外大鼠肠道中的吸收

The project aligns with BBSRC's strategic research priority 3, 'Bioscience for Health' (Biotechnology for Health challenge) as it aims to develop basic bioscience underpinning the formulation and validation of biologics that benefit the maintenance and promotion of health. Building on applicants' prior work and benefiting from synergy, multidisciplinarity and Proxima's technologies, the project will develop a new generation of materials for oral delivery of biologics.Biologics have transformed therapeutics (7 out of 8 top selling drugs in 2014). Their administration is limited to injection due to negligible intestinal absorption. These are less accepted by patients (painful/potential side effects), and expensive to manufacture and administer. Several barriers limit systemic absorption of biologics, but the intestinal epithelium is the most formidable and challenging to overcome. Current approaches to improve oral biologics absorption typically employ 'absorption enhancers' that non-selectively disrupt and increase intestinal permeability, but safety concerns (eg. many surfactants) have hindered clinical translation. Therefore, there is an unmet need in safe and effective technologies for oral delivery of biologics.Key to safe and effective oral delivery of biologics are materials that 'smuggle' drugs selectively across the intestinal mucosa without disrupting this physiologically important barrier. These can be conjugated to the drug or drug delivery systems (e.g. nanoparticle). This project aims to develop novel transcytosis-enabling materials that promote oral absorption of biologics without disrupting the epithelium. These are peptide-based, designed taking inspiration from a natural macromolecular ligand that readily crosses the intestinal mucosa by transcytosis via its receptor, namely IgG and neonatal Fc receptor (FcRn). Preparation of transcytosis-enabling compounds will be informed by the receptor and ligand binding epitopes of IgG and FcRn.Vllasaliu previously demonstrated that FcRn shuttles IgG Fc-coated nanoparticles transepithelially. Proxima's discovery platforms will be used to prepare transcytosis-enabling peptide constructs. In Mozaic, prototype peptides are created from micellar nanoparticles bearing combinations of amino acids (AA) on surface. A screening library is constructed and after cell culture testing, most effective combination selected for peptide synthesis. AA choice is informed by sequences in IgG receptor-binding domains. In parallel, Almanac (algorithms derived from data of structural analyses of protein-protein interactions) will be used to infer the structure of ligand-binding peptides based on receptor binding domain sequence. Developing FcRn-binding ligands using two complementary approaches reduces the risk of failure.Project stagesYr 1: Student training in project relevant techniques and study of IgG-FcRn sequences Yr 1&2: Formulation of Mozaic screening libraries and peptides with a suitable fluorophore for quantitation. Purification (HPLC) and characterisation by MSYr 3&4: Testing compounds alone and conjugated to a model protein (albumin) for cell uptake and absorption across Caco-2 monolayers (intestinal model). Competition assays testing an FcRn receptor-mediated effect. Uptake mechanisms determined by siRNA inhibition of specific pathways and confocal microscopy (co-localisation studies)Yr 4: Testing most successful compounds' absorption ex vivo in excised rat intestine