A single molecular descriptor to predict solution behavior of therapeutic antibodies.

Despite the therapeutic success of monoclonal antibodies (mAbs), early identification of developable mAb drug candidates with optimal manufacturability, stability, and delivery attributes remains elusive. Poor solution behavior, which manifests as high solution viscosity or opalescence, profoundly affects the developability of mAb drugs. Using a diverse dataset of 59 mAbs, including 43 approved products, and an array of molecular descriptors spanning colloidal, conformational, charge-based, hydrodynamic, and hydrophobic properties, we show that poor solution behavior is prevalent (>30%) in mAbs and is singularly predicted (>90%) by the diffusion interaction parameter (k D), a dilute-solution measure of colloidal self-interaction. No other descriptor, individually or in combination, was found to be as effective as k D. We also show that well-behaved mAbs, a substantial subset of which bear high positive charge and pI, present no disadvantages with respect to pharmacokinetics in humans. Here, we provide a systematic framework with quantitative thresholds for selecting well-behaved therapeutic mAbs during drug discovery.

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (OtofAla515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Dilation of fusion pores by crowding of SNARE proteins.

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).