Structural changes of interfacially adsorbed antibody molecules

在过去的几十年里，癌症的治疗经历了三个革命性的阶段：化疗，针对突变基因的生物标记物，以及生物标记物靶向和免疫过程调节的联合治疗。这些治疗的每一个阶段都得益于我们对癌症行为的理解的进步，特别是在分子和细胞水平上。我们对癌细胞高生长率的早期认识导致了化学/药物疗法和放射治疗的发展，以杀灭癌细胞。然而，在临床部署之前，这种治疗方法必须经过严格的临床试验，并满足监管要求，例如FDA的批准。这一过程加上进一步的改进以完善治疗可能需要十多年的时间。目前，化疗仍然是癌症患者的主流治疗方法，但化疗的毒性仍然是影响患者生存率的主要限制因素。在过去的5-8年里，已经实现了调节免疫过程的能力，基于这一新思路的一些开创性治疗最近取得了临床成功。这一前景推动了一项重大的全球努力，以抗体技术为基础开发新的治疗方法，不仅涵盖肿瘤学，还涵盖其他主要疾病，包括心血管、呼吸系统、自身免疫和传染病。用于癌症或其他疾病治疗的抗体必须经过设计、制造、分离、纯化，并最终制成可供临床使用的医疗产品。一种流行的给药方法是静脉注射。这一选择要求抗体药物在瓶子(玻璃或塑料)或准备注射的注射器套装中以稳定的蛋白质溶液的形式配制，保质期在1-2年之间。由于这些生物工程抗体必须具备两种或两种以上的生物功能，它们的氨基酸序列(即所谓的初级序列)必须改变。因此，我们不知道它们的折叠结构域有多稳定，以及修饰结构域的不稳定性会对整个抗体的稳定性产生多大影响。由于表面同时存在极性和非极性氨基酸，所有蛋白质都是两亲性的。这种两亲性驱动蛋白质自发地在不同的界面上吸附和解吸。在这些界面过程中，蛋白质与底物表面和自身相互作用，根据底物表面的性质和吸附后它们之间的接近程度，球状结构可能发生变形，甚至局部展开，导致疏水斑块暴露，可能导致聚集和沉淀，损害此类抗体药物的生物活性。新的生物工程抗体往往是不稳定的，吸附会加速它们的不稳定。使用一系列在Fab和Fc区域具有良好控制序列修改的生物工程抗体，这个链接项目建立了一个新的合作团队，由MedImmune、曼彻斯特大学和帝国理工学院组成，目的是通过结合中子反射实验和分子动力学模拟来发展新的理解。我们将研究Fab和Fc结构域中某些控制良好的序列修改如何影响其吸附的球状结构，以及这些结构域中的不稳定性如何影响整个mAb的结构。另一项工作将是研究具有代表性的衬底表面如何影响结构变形和展开。这些研究将导致新的结果，这将对生物制药行业和学术研究具有重要价值。该项目的成功实施将导致新的一级结构-稳定性关系，这将有助于MedImmune和其他蛋白质药物开发商在他们的生物疗法中提高他们的抗体稳定性。其结果最终将惠及广大公众。

Cancer treatments have undergone three revolutionary stages over the past few decades: chemotherapies, biomarkers targeted at mutated genes, and combined treatments of biomarker targeting and immune process mediation. Each stage of these treatments was facilitated by advances in our understanding of the behaviour of cancers, especially at the molecular and cell levels. Our early understanding of the high growth rates of cancerous cells led to the development of chemical/drug therapies together with radiation treatments to kill cancerous cells. However, such medical treatments must undergo rigorous clinical trials and meet regulatory requirements, e.g., FDA approval, before clinical deployment. This process plus further improvement to perfect the treatments can take more than a decade. Currently, chemotherapy is still the mainstream cancer treatment for patients, but their toxicity remains a major limiting factor to survival rates. Over the past 5-8 years, the ability to moderate immune processes has been realised, and a number of pioneering treatments based on this new line of thinking have very recently achieved clinical success. This prospect has driven a major global effort to develop new treatments based on antibody technologies, covering not only oncology but also other major diseases including cardiovascular, respiratory, autoimmunity and infectious diseases. Antibodies used for cancer or other disease treatments must be designed, manufactured, separated, purified and eventually formulated into medical products ready for clinical use. A popular means of administration is to apply via intravenous injection. This option requires the antibody drugs to be formulated as a stable protein solution in a bottle (glass or plastic) or a ready-to-inject syringe set, with a shelf-life between 1-2 years. Because these bioengineered antibodies have to be equipped with two or more biological functions, their amino acid sequences (so called primary sequences) must be altered. As a result, we do not know how stable their folded domains are and how instability from the modified domains will affect the stability of the whole antibody.All proteins are amphiphilic due to the presence of both polar and apolar amino acids on their surfaces. This amphiphilic character drives proteins to adsorb and desorb at different interfaces spontaneously. During these interfacial processes, proteins interact with the substrate surface and with themselves, and depending on the nature of the substrate surface and the close proximity between them once adsorbed, deformation of the globular structures and even local unfolding can occur, causing exposure of hydrophobic patches that may induce aggregation and precipitation, compromising the bioactivity of such antibody drugs. Newly bioengineered antibodies are often unstable, and adsorption can accelerate their instability.Using a series of bioengineered antibodies with well-controlled sequence modifications in Fab and Fc domains, this LINK project forges a new collaborative team involving MedImmune, Manchester University and Imperial College London, with the aim to develop new understanding by combining neutron reflection experiments with molecular dynamics simulation. We will examine how certain well-controlled sequence modifications in Fab and Fc domains affect their adsorbed globular structures and how instability in these domains affects the structure of the whole mAb. Another line of work will be to examine how representative substrate surfaces affect structural deformation and unfolding. These studies will lead to new results that will be of great value to the biopharmaceutical industry and to academic research. The successful delivery of this project will lead to new primary structure-stability relationships that will assist MedImmune and other protein drug developers to improve their antibody stability in their biotherapeutics. The outcome will ultimately benefit the general public.