Structural changes of interfacially adsorbed antibody molecules

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.

Natural proteins such as antibodies (mAbs) have vastly different sequences, sizes, shapes and globular stability. It is difficult to establish common features linking their sequences to domain stability and that of the whole mAb. Thus, current knowledge of protein adsorption and desorption is very limited at predicting how bioengineered mAbs behave.

MedImmune has designed and bioengineered a group of mAbs from the native human IgG1, with careful sequence modifications in Fab, Fc, other receptor binding domains and connection domains. Because of their well-controlled sequence changes and stability design, these mAbs are not only important for biotherapeutic development but also crucial to the development of new scientific understanding.

Specifically, we will have two lines of work tasks to pursue. Firstly, we will examine how sequence modifications affect the adsorbed structures of Fab and Fc with regard to the extent of deformation of globular domains and the impact on the whole mAb. Secondly, we will examine how the different nature of substrate surfaces affects adsorption and deformation, again focusing on the extent of structural deformation and possible unfolding. It is especially useful to explore how the extent of deformation and unfolding which is likely to be affected by sequence modification, solution conditions and adsorption time, is affected by the nature of the different substrates.

An important technical advance that will emerge from this project is the development of the capability of molecular dynamics (MD) simulation to analyse neutron reflectivity data, providing structural conformations of adsorbed mAbs with unprecedented resolution. Concomitantly, MD will also be able to develop its own predictive power in modelling mAb adsorption and desorption. The combined use of the two techniques will lead to development of a software package with the capability to predict adsorption and desorption of mAbs and other proteins.

Societal impact and responsibility: In spite of the huge advances, chemotherapy combined with radiotherapy currently remains the mainstream cancer treatment for patients. Unfortunately, toxicity and other related side-effects constrain survival rates. However, new treatments combining biomarker-based gene therapies and immune therapies are rapidly increasing, with these new drugs dominating the market sales. These new drugs are clearly performing better in avoiding the drawbacks from chemotherapy, but are still far from perfect, and further research is needed to overcome these technical hurdles. With MedImmune's support, we will develop online materials to highlight the pros and cons of current cancer treatments, seeking public support towards developing new and better treatments through collaborative research. Cancer prevention and early diagnosis are better options than treatment. As the research project develops, we will write popular cancer educational articles targeting the general public, explaining how to prevent developing cancer by changing lifestyles, using existing literature on prostate cancer as an example. With our expertise in reliable data treatment, we will be able to critically review the relevant publications on this topic, presenting a balanced view of the statistical significance (or otherwise) of the literature.

Cancer screening - Prevention by lifestyle changes is clearly the best option, but screening is preferable to treatment. There are several UK websites devoted to prostate cancer, and there is now a national awareness of the need for men below 55 years old to undertake prostate screening. Cancers usually have approximately 20-year incubation periods, and early screening could save lives and resources for the NHS. We will look into the difficulties in implementing early screening programmes within the NHS, and explore possible alternatives by reviewing practices used in the USA, again aiming at the general public in these articles.

International development - Cancer rates are strongly correlated with life expectancy and wealth: highly developed countries such as Denmark, France and Italy have 40% more cancer cases per head of population than poor countries. In addition, the top five cancers in rich nations are in the order of breast, lung, prostate, colon, and stomach, whilst in poor countries these are in the different order of lung, stomach, breast, liver and colon. Apart from environmental factors, aspects such as smoking, alcohol, food, exercise, exposure to radiation, chronic infection, immune compromise, etc have been identified as causal factors in cancer. We will introduce examples of classical research publications in this area to the general public, e.g., the effect of the smoking ban in the USA on the significant reduction of lung cancer incidence.

Business development and wealth creation - MedImmune is one of the major antibody biotherapeutic companies in the world. As the key joint partner in this project they will be the first to consider the implications of the new results on their formulation and product development. The new knowledge will help support redesign and reformulation of existing products, or new product development. UoM and IC will strengthen their links with MedImmune and could develop new collaborations. Any new IP in surface modifications (UoM) and software capability (IC) will be protected by their respective university IP bodies.

Skills training opportunities - The two RAs will receive interdisciplinary training between physics and biology. They will also receive training from the collaborating industrial partner, and have opportunities to organise and participate in workshops. They will also disseminate their results to research students and academics in other fields, through joint project seminars, group seminars, department/school open days and visits to local schools.