The best fit IgG4 model generated by neutron analysis; the Fab regions of the antibody are shown in blue and the Fc region is shown in red. This Image was originally published in the Journal of Biological Chemistry. Lucy E. Rayner, Gar Kay Hui, Jayesh Gor, Richard K. Heenan, Paul A. Dalby and Stephen J. Perkins. The Fab conformations in the solution structure of human IgG4 restricts access to its Fc region: implications for functional activity. Journal of Biological Chemistry 2014; Vol:pp-pp. Copyright the American Society for Biochemistry and Molecular Biology.
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In recent years a swarm of new antibody-based drugs have hit the market, offering treatment for cancer, autoimmunity, and infectious diseases. As these magic bullets gain momentum, many scientists are acquiring a new enthusiasm in their pursuit to discover the “next generation” of therapeutic antibodies.
The human IgG4 antibody is just one of these promising biopharmaceutical candidates. It’s an antibody with personality - unique in its structure, function and activity. And because of these characteristics, it’s already making its break, in drugs to treat conditions such as leukaemia and multiple sclerosis. However, structural studies of this antibody are few and far between. And so, in order to get a deeper understanding of this antibody and clarify its function and therapeutic application, scientists from University College London (UCL) and ISIS have been using neutron scattering to reveal the solution structure of IgG4.
Antibodies and antigens are like two pieces of a puzzle; they have a specific structure which allows them to recognise each other and fit together. They are ‘Y’ shaped proteins and each arm is known as a Fab (fragment, antigen-binding region). Antibodies trigger the immune response in our bodies when they bind to foreign objects using these Fab arms. The base of the ‘Y’ however, is known as the Fc region, and this is specific to the kind of immune response triggered. For example, the Fc region binds to proteins from a part of the immune response called complement; this leads to the destruction of the antigen by the immune system. The antibody’s ability to recognise a specific antigen is essential in biopharmaceuticals. For example, they may bind to certain molecules in the body and block them from binding to their normal targets and therefore stop a perhaps unwanted response.
Monoclonal antibodies (mAbs) comprise one-third of drugs in the pharmaceutical industry, however their administration to patients can carry the risk of immune reactions. For this reason, antibodies such as IgG4, which do not activate the immune system, are ideal for use in some therapeutics as they are likely to have less side effects.
In a recent paper published in the Journal of Biological Chemistry, UCL scientists have shown the structural reasons for this inactivity. They used SANS2d instrument at ISIS for neutron scattering experiments as well as X-ray scattering at ESRF to reveal that the conformations of the Fab arms of the IgG4 antibody in solution restrict access to the Fc region, and therefore block some proteins of the immune response system from binding to IgG4.
Prof Steve Perkins at UCL explains, “One of the human antibodies, IgG4 does not interact with the human immune system. From the industrial point of view this is an advantage because the pharmaceutical industry is looking for antibodies without side effects so the fact that this antibody does not trigger an immune response gives it great potential.”
The IgG4 mAb is currently used in the drug Natalizumab to treat multiple sclerosis (MS) and reduces the activity of the disease and the risk of disability progression in patients with relapsing MS. It is directed against a surface molecule found on white blood cells. It blocks the interaction between another antigen and adhesion proteins on endothelial cells to control the white blood cell adhesion attachment and migration across the blood brain barrier. By stopping the white blood cells from crossing the blood brain barrier, natalizumab reduces the inflammatory response and subsequent myelin loss in the brain and spinal cord of patients.
Importantly in this treatment IgG4 does not activate complement and does not illicit an immune inflammatory reaction. This is significant. If the antibody did activate complement it would counteract the anti-inflammatory effect of the drug. Prof Perkins and his team have now shown the structural reasons behind this inactivity.
Prof Perkins explains “First we purify the protein, and put it through various data collections to figure out whether it’s a monomer or a mixture. Then we do the X-ray and neutron scattering and plot out all the data. Then with computer modelling you get a nice curve fit that proves that we know what the antibody structure looks like. By looking at these structures we found out the Fab arms of the IgG4 antibodies block the binding sites in the Fc part and so now we have an explanation into why human IgG4 does not interact with the immune system.”
Antibody stability is also a major concern for the pharmaceutical Industry as manufacturing, shipping and storage may compromise the stability of the antibody.
“When you work with antibodies you also need to work out how they behave in solution”, explains Dr Perkins. “So in order to be able to manufacture these antibodies, we also need to know the stability of the antibody in standard bioprocessing conditions. The classic example of that is, when you make the antibody, you have to take it to pH 3 to kill off any viruses in the prep, yet pH 3 destabilises the antibody. So we want to try and stop this.”
“So here we can use the neutron scattering approach together with X-ray scattering, and then back in London we use the ultracentrifuge to figure out the antibody state and stability. We have already done this with rabbit antibodies, but now we have looked at the human antibody IgG4”
Research date: June 2014
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