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Cryo-EM insights into the structural flexibility of bi-specific antibodies

Antibodies are vital for targeting disease-related proteins and cells, thus aiding in immune responses. Understanding their structure can inform therapeutic advances. French pharmaceutical company Sanofi teamed up with the ESRF to study bi-specific antibodies using cryo-electron microscopy.

Antibodies are Y-shaped proteins composed of variable and constant regions. Variable regions are crucial for recognising and binding to targets. While medicine has long benefited from antibodies that target single disease- causing factors, there s a growing need for multi-specific antibodies that can simultaneously target multiple factors in complex diseases, improving treatment efficacy, particularly in cancer and autoimmune diseases.

Sanofi and the ESRF investigated bi-specific antibodies, specifically CODVIg, which has a distinctive architecture enabling it to bind independently to two antigens in any sequence with strong affinity. CODVIg binds to IL4 and IL13 key cytokines essential in immune function and pertinent to conditions like asthma and allergies, and which play crucial roles in cancer immunotherapy treatments. Understanding the 3D structure of the versatile antibody CODVIg is therefore crucial for therapeutic applications.

Cryo-electron microscopy (cryo-EM) at beamline CM01 was used to investigate the interaction between CODVIg and the IL13 antigen. The flexibility of CODVIg presented challenges, even in cryo-EM, necessitating extensive data collection or the presence of a stabilising partner antibody. Both approaches were integrated, and a 4Å-resolution structure of the CODVFab:IL13 complex was thus obtained (Figure 128), revealing for the first time the CODV-IL13 interface and the flexibility of the Fc region of CODVIg.

Cryo-EM unveiled insights into the binding interface, stoichiometry, and structural changes upon IL13 binding, highlighting cryo-EM s potential for visualising complex antibody-antigen interactions. Understanding structures like CODVIg informs effective therapy and cancer immunotherapy development.

PRINCIPAL PUBLICATION AND AUTHORS

Structural insights into the bi-specific cross-over dual variable antibody architecture by cryo-EM, D. Fernandez-Martinez (a,b), M. Tully (a), G. Leonard (a), M. Mathieu (b), E. Kandiah (a), Sci. Rep. 13, 8694 (2023); https:/doi.org/10.1038/s41598-023-35678-4 (a) ESRF (b) Sanofi R&D, Bio Structure and Biophysics, Centre de Recherche Vitry-sur-Seine (France)

Fig. 128: 3D Cryo-EM reconstruction of CODVFab:IL13:RefAb (TC) and its CODVFab:IL13 interface. Constant domains: orange; IL4 binding domains: purple; IL13 binding domains: green. RefAb is in orange (heavy chain) and yellow (light chain).