Scientists at the University of Southampton have used Diamond’s long-wavelength beamline for protein crystallography (I23) to gain unprecedented new insight into the key properties of an antibody, needed to fight off cancer. The interdisciplinary study, published in Science Immunology, revealed how an antibody can be engineered to change its flexibility, which could stimulate a stronger immune response. The findings have enabled the Southampton team to design antibodies to activate important receptors on immune cells to “fire them up” and deliver more powerful anti-cancer effects. The researchers believe their findings could pave the way to improve antibody drugs that target cancer as well as other autoimmune diseases.
In the study, the team investigated antibody drugs targeting the receptor CD40 for cancer treatment. Clinical development has been hampered by a lack of understanding of how to stimulate the receptors to the right level. The problem being that if antibodies are too active they can become toxic.
Previous Southampton research has shown that a specific type of antibody called IgG2 is uniquely suited as a template for pharmaceutical intervention, since it is more active than other antibody types. However, the reason why it is more active had not been determined. What was known, however, is that the structure between the antibody arms, the so called hinges, is important. This latest research harnesses hinge properties and explains how it works: the researchers call this process “disulfide-switching”.
In their study, the Southampton team analysed the effect of modifying the hinge and used a combination of biological activity assays, structural biology – using Diamond MX beamlines I03 and I23 – and computational chemistry to study how disulfide switching alters antibody structure and activity.
Our approach was to analyse the structure of the antibody in atomic detail, using the method of X-ray crystallography. Diamond’s I23 beamline was particularly important to determine the structure of the hinge region, required for activity. We also needed an image of the antibody in solution, for which we used an X-ray scattering approach called SAXS and a chemical-computing approach to analyse the data, using the Southampton High Performance Computing cluster IRIDIS.
Dr Ivo Tews, Associate Professor in Structural Biology at the University of Southampton
Through this detailed study of the hinge the team revealed that more compact, rigid antibodies are more active than their flexible counterparts. Using beamline I23, the team were able to see why this is the case.
We knew that the hinges incorporate disulfide bonds. The long-wavelength X-rays on I23 enable us to study light elements such as sulfur, so we used it to pinpoint the disulfide bonds. We were able to look at the bonds in incredible detail and discovered that they form a specific cross-over, rather than simply joining the antibody’s arm as previously thought. This makes the structure more rigid and apparently promotes cell signalling that conveys the anticancer response.
Dr Christian Orr, first author of the publication and now Beamline Scientist on I23
This study has given us new information about how to engineer antibodies to deliver a better immune response. We propose that more rigid antibodies enable the receptors to be bound closer together on the cell surface, promoting receptor clustering and stronger signalling for activity. This means by modifying the hinge we can now generate more or less active antibodies in a more predictable way. Excitingly, our findings could have wider implications as it may provide a highly controlled and tractable means of developing antibodies for clinical use in future immunostimulatory antibody drugs.
Professor Mark Cragg, of the Centre for Cancer Immunology at the University of Southampton
The study was funded by Cancer Research UK and brought together structural biologists, immunologists, chemists and computer experts from across the University of Southampton. Collaboration with Diamond Light Source in Oxfordshire and the University of Hamburg, that Southampton is partnered with, were instrumental to these studies.
This is the first time such a cross-link has been revealed. Beamline I23 is a one-of-a-kind instrument which has been specifically designed to access a unique wavelength range to perform these delicate experiments - the video below shows how I23 captures atomic detail. In this case, the sulfur atoms in the cross-link could be easily identified with the data collected on I23. These results will help in the design of future antibody models which can pave the way to tailored treatments by inducing the right immune response.
Orr C. et al. Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility. Science Immunology (Jul 2022). DOI: 10.1126/sciimmunol.abm3723
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