Distinctive Low-Resolution Structural Features of Dimers of Antibody-Drug Conjugates and Parent Antibody Determined by Small-Angle X-ray Scattering.

Structural features of lysine-conjugated antibody-drug conjugate (ADC) from humanized IgG1 were studied by small-angle X-ray scattering (SAXS). As the physicochemical properties of the cytotoxic drug (payload) and linker may impact the conformational and colloidal stabilities of the conjugated monoclonal antibody (mAb), it is essential to characterize how the conjugation may affect the overall higher order structure and therefore the physical stability and integrity of the ADCs upon storage conditions. Here, the ADC monomer and aggregates generated upon thermal stress were analyzed by high performance liquid chromatography coupled to SAXS with a particular focus on the fraction of dimers (3-10% depending on the storage conditions at 25 and 40 °C). In addition to average parameters such as radius of gyration, molecular weight, and maximal end-to-end distance, the structural information obtained from SAXS patterns were visualized as a low-resolution average envelope of both monomers and dimers (implementation of two methods: ab initio reconstruction and modeling Fab and Fc as rigid bodies with a flexible hinge). We showed that the monomer envelope of the ADC was similar to the corresponding (nonconjugated) parent monoclonal antibody (mAb). ADC dimers appeared more compact and less polydisperse than the dimers of mAb, which was also confirmed by atomic force microscopy. The generated envelopes of the mAb dimers suggest elongated structures with one or few inter-mAb contacts at the outermost region of Fab or Fc domains. The structural features of ADC dimers are independent of the tested pH buffering system (pH 5.0/acetate and pH 6.0/histidine with or without NaCl) and characterized by multiple, tighter contacts between the Fab and Fc domains and distortion of the monomer native shape. Results from the SAXS structural study show in the present case that conjugation has favored innermost inter-ADC contacts in the dimer, which differ from the inter-mAb ones. In general, it is likely that many parameters affect inter-ADC association, including the chemical nature of linkers and drugs, degree of conjugation, conjugation sites, etc. Making a qualitative difference between mAb and ADC dimers as a function of these parameters can help point to the presence of tight associations that must be abolished in protein drug formulations.

Identification of a major intermediate along the self-assembly pathway of an icosahedral viral capsid by using an analytical model of a spherical patch.

Viruses are astonishing edifices in which hundreds of molecular building blocks fit into the final structure with pinpoint accuracy. We established a robust kinetic model accounting for the in vitro self-assembly of a capsid shell derived from an icosahedral plant virus by using time-resolved small-angle X-ray scattering (TR-SAXS) data at high spatiotemporal resolution. By implementing an analytical model of a spherical patch into a global fitting algorithm, we managed to identify a major intermediate species along the self-assembly pathway. With a series of data collected at different protein concentrations, we showed that free dimers self-assembled into a capsid through an intermediate resembling a half-capsid. The typical lifetime of the intermediate was a few seconds and yet the presence of so large an oligomer was not reported before. The progress in instrumental detection along with the development of powerful algorithms for data processing contribute to shedding light on nonequilibrium processes in highly complex systems such as viruses.

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Reconstruction of the Disassembly Pathway of an Icosahedral Viral Capsid and Shape Determination of Two Successive Intermediates.

Viral capsids derived from an icosahedral plant virus widely used in physical and nanotechnological investigations were fully dissociated into dimers by a rapid change of pH. The process was probed in vitro at high spatiotemporal resolution by time-resolved small-angle X-ray scattering using a high brilliance synchrotron source. A powerful custom-made global fitting algorithm allowed us to reconstruct the most likely pathway parametrized by a set of stoichiometric coefficients and to determine the shape of two successive intermediates by ab initio calculations. None of these two unexpected intermediates was previously identified in self-assembly experiments, which suggests that the disassembly pathway is not a mirror image of the assembly pathway. These findings shed new light on the mechanisms and the reversibility of the assembly/disassembly of natural and synthetic virus-based systems. They also demonstrate that both the structure and dynamics of an increasing number of intermediate species become accessible to experiments.