Their results give unprecedented insight into the mechanics of a trending topic in immunology.
Since their development in the 1970's, monoclonal antibodies (mAbs) have assumed their role as a vital player in the field of bio-therapeutics. Over the past decade, they have become the dominant recombinant therapeutic proteins used in the clinic, applicable in the treatment of cancer and autoimmune diseases such as Crohn's and Rheumatoid arthritis.
An antibody is a protein that binds to an antigen – a cell surface protein that elicits an immune response. They are secreted by B cells during the adaptive immune response to infection. Composed of two heavy – and two light – polypeptide chains, these proteins fold up to form a Y-shaped molecule, held together by intermolecular disulphide bonds. Variable domains at the tip of each arm of the Fab (fragment antigen-binding) allows antibodies to bind to antigens expressed on the surface of pathogens, such as bacteria and viruses. Binding induces a plethora of responses including neutralisation of bacterial toxins and activation of the classical arm of the complement system. Whilst your body produces lots of antibodies from various B cells during an immune response, each with alternative Fab sequences, mAbs are a bit different in that they are identical in sequence, hence 'clonal' and produced by genetic engineering and culture of mammalian cells in industry. A mAb can therefore be designed to bind to a specific therapeutic target in order to treat certain cancers, autoimmune, infectious or cardiovascular diseases.
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Left: Depicting the specificity of binding of mAbs to antigens on target cells.Right: A diagram illustrating the shape of an antibody with the variable (Fab) and constant (Fc) regions highlighted.
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This collaboration studied a mAb produced by MedImmune, based on the immunoglobulin-1 (IgG1) antibody subclass, which they called 'COE-3'. The research aimed to understand what happens when a mAb sticks to a glass surface. This is important to the industry because liquid formulations of mAbs are often filled into glass vials or syringes for shipping to the pharmacy. A better understanding of the sticking (or adsorption) process may help the industry in its formulation design. While there are several analytical methods available to study the adsorption of a protein to a surface, each lacks the resolution and information that can be obtained by neutron reflectivity (NR.) For this reason, ISIS was chosen as a key collaborator for the research.
Using the SURF reflectometer at ISIS, a team led by Dr Fang Pan from the University of Manchester studied the adsorption of monoclonal antibody COE-3 at the hydrophilic silicon oxide (Si02)/water interface, aptly chosen as SiO2 surface is a good model for glass. By investigating the adsorption of mAbs at the solid/water interfaces, we can gain a greater understanding of the impact that sequence modifications may have.
Their results, published in the journal 'Applied Materials and Interfaces' successfully report experimental and molecular modelling data together in one study. NR consistently determined an adsorbed monolayer thickness of 50-52 Å, which corresponds to the short axial lengths of the Fab and Fc (fragment crystallisation). This implies minimal structural perturbation. The data acquired using SURF could then be compared to simulations based on Derjaguin-Landau-Verwey-Overbeek (DVLO) theory, which explains the stability of a particle in solution.
Neutron reflectometry was the technique of choice in this experiment as it is a powerful tool that can reveal the thickness and composition of multiple adsorbed protein layers. The instrument in question, SURF, is a high flux, high resolution reflectometer which allows scientists to gather structural information on adsorbed protein layers in timely, reproducible, precise experiments. It is a world-leading instrument for liquid interface research, with the added benefit that it can offer horizontal sample geometry to users.
Combined with spectroscopy ellipsometry data, neutron reflectivity has allowed Pan et al to piece together a mechanistic interpretation of the adsorption process. By collaborating with scientists from the Formulation Sciences department of MedImmune – the global biologics research and development arm of AstraZeneca – the team was able to exchange ideas and make use of cutting-edge facilities. Partnerships like this one therefore demonstrate the value of academic-industrial relations within the scientific community.
Further information
For further information about the research, please contact Professor Jian Ren Lu (j.lu@manchester.ac.uk).
The research was supported by MedImmune Ltd whilst neutron beam times were awarded to undertake the work at ISIS, under the support of STFC. The full publication is available to view in Applied Materials and Interfaces.
Learn more on the SURF instrument.
