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Abstract Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cryo-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Abstract Multi-specific antibodies (msAbs) are being developed as next generation antibody-based therapeutics. Knowledge of the three-dimensional structures, in the full antibody context, of their fragment antigen-binding (Fab) moieties with or without bound antigens is key to elucidating their therapeutic efficiency and stability. However, the flexibility of msAbs, a feature essential for their multi specificity, has hindered efforts in this direction. Cross-Over Dual Variable immunoglobulin (CODVIg) is a promising bispecific antibody format, designed to simultaneously target the interleukins IL4 and IL13. In this work we present the biophysical and structural characterisation of a CODVFab:IL13 complex in the full antibody context, using cryo-electron microscopy at an overall resolution of 4.2 Å. Unlike the 1:2 stoichiometry previously observed for CODVIg:IL4, CODVIg:IL13 shows a 1:1 stoichiometry. As well as providing details of the IL13-CODV binding interface, including the residues involved in the epitope-paratope region, the structure of CODVFab:IL13 also validates the use of labelling antibody as a new strategy for the single particle cryo-EM study of msAbs in complex with one, or more, antigens. This strategy reduced the inherent flexibility of the IL13 binding domain of CODV without inducing either structural changes at the epitope level or steric hindrance between the IL4 and IL13 binding regions of CODVIg. The work presented here thus also contributes to the development of methodology for the structural study of msAbs, a promising platform for cancer immunotherapy.

Background: Multispecific antibodies are artificially engineered molecules designed to bind simultaneously to several (different) antigens. Potential advantages of generating viable multispecific antibodies include the identification of malignant cells coupled with the concurrent recruitment of immune cells and the blocking of complex viral escape mechanisms. The cross-over dual-variable immunoglobulin (CODV-Ig) has been proposed as a universal bispecific therapeutic format. Its unique antigen-binding fragment (Fab) architecture provides pM affinities for ligands, no positional effect in target binding and a stable self-supporting structure. However, the three-dimensional arrangement of the constant and antigen-binding fragments in the CODV-Ig format may play a role in its in vivo effects. To further understand the structure and function of multispecific antibodies based on the CODV-Ig format high-resolution structural information is required. Towards this, we use cryo-electron microscopy (cryo-EM). Methods: We purified CODV-Ig both in an unbound state and in complex with a single antigen and validated sample quality using SDS-PAGE, Small Angle X-Ray Scattering (SAXS) and negative-stain electron microscopy (NSEM; Tecnai T12 and F20 microscopes at IBS, Grenoble). Data of sufficient quality for image analysis was obtained using a Titan Krios microscope (ESRF, Grenoble) equipped with a Quantum LS energy filter and K2 Summit direct electron detector. Results: NSEM of CODV-Ig resulted in low-resolution structural models and suggested a preferential orientation of the antibody under negative-stain conditions. Close-to-optimal vitrification conditions for CODV-Ig and antibody-antigen complexes have been identified. Efforts are in progress to reduce the antibody’s propensity to aggregation and aversion to conventional cryo-EM supports. Nevertheless, image processing of both CODV-Ig alone and in complex with antigens suggests very high flexibility and conformational heterogeneity. Conclusion: Particle heterogeneity may require additional data to be collected, in order to have sufficient signal that will lead to well-defined classes. An additional strategy may involve efforts to immobilize the molecules as to obtain fewer and better-defined classes.