INDUSTRY

23June 2021 ESRFnews

A Sanofi ESRF PhD student uses cryo-EM to see the bigger picture.

Many diseases, including those deriving from viruses, involve more than one alien protein, which is why scientists are interested in engineering multispecific antibodies that can target two or more targets. Now, a PhD student working for the multinational pharmaceutical company Sanofi, and backed by the French Industrial Agreement of Training through Research (CIFRE) programme, has assisted the study of multispecific antibodies by showing that full reconstructions of their structures can be obtained at the ESRF s CM01 cryo-EM facility. Sanofi has already developed

multispecific antibody formats. One of them is cross-over dual-variable immunoglobulin (CODV-Ig), which has two domains to bind to antigens, interconnected by glycine links of various lengths. Sanofi researchers have already found that, in vitro, the specific architecture that results from these links allows the domains to bind to any antigens, together or individually, regardless of their ordering. However, in vivo experiments have not always found the same benefit, prompting the researchers to hypothesise that the 3D position and orientation of the individual domains relative to the overall architecture play a crucial role. CIFRE, a programme of the French

Ministry of Higher Education and Research, has been running for 30 years, linking 20,000 PhD students with France-based companies and research institutions. It has financed David Fernandez, a student structural biologist from Spain, for a PhD co-supervised between Sanofi and the ESRF, with the aim of developing a pipeline for full-antibody studies using cryo-EM sample tube to structure and to try to understand

why CODV-Ig is sometimes not as effective in vivo as in vitro. As a person who was already determined to follow a career in industry, being able to pursue a PhD in the field has helped me understand how research in industry works and given me the experience required for future opportunities, he says. Sanofi has amazing scientists that do not hesitate to provide the help or financial means necessary in order to make a project successful. At the same time, I have spent most of my time at the ESRF, which has an environment more akin to academia. Because the ESRF is part of the Partnership for Structural Biology (PSB) in Grenoble, this has allowed me to exchange ideas and ask for help or tools at the other institutes partnered through the PSB the European Molecular Biology Laboratory, the Institut de Biologie Structurale and the Institut Laue- Langevin. Thanks to this, my project has been able to gradually advance. Scientifically, Fernandez s project

has not been straightforward: for

Honing multi-specific antibodies

Fernandez (right) at work at the ESRF with Magali Mathieu, his supervisor at Sanofi who is a long-term ESRF user.

E TI E N N E B O U Y

instance, he found a high level of positional heterogeneity in the domains and fragments of his samples, vastly increasing the amount of data and time necessary to obtain high- resolution structures. Nevertheless, using the Titan Krios microscope at CM01 currently the highest-end cryo-electron microscope available he managed to obtain the first structure of CODV-Ig bound to an antigen, IL13, which plays a part in various diseases such as allergies and cancer, at 3.9 Å resolution. The results show that there is indeed a way of obtaining full antibody maps by cryo-EM, even though it requires a large amount of data, and that one can observe small antigens bound to antibodies at high resolution within the full-antibody context, he says.

Jon Cartwright

The aim was to develop a pipeline for full-antibody studies using cryo-EM