Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis.

Complement component 5 (C5), an important molecule in the complement cascade, blockade by antibodies shows clinical efficacy in treating complement-mediated disorders. However, insufficient blockading induced by single-nucleotide polymorphisms in the C5 protein or frequent development of "breakthrough" intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab have been reported. Herein, we developed a lipid nanoparticle (LNP)-formulated siRNA targeting C5 that was efficiently delivered to the liver and silenced C5 expression. We identified a potent C5-siRNA with an in vitro IC50 of 420 pM and in vivo ED50 of 0.017 mg/kg following a single administration. Single or repeated administrations of the LNP-formulated C5-siRNA allowed robust and durable suppression of liver C5 expression in mice. Complement C5 silencing ameliorated C5b-dependent anti-acetylcholine receptor antibody-induced myasthenia gravis and C5a-dependent collagen-induced arthritis symptoms. Similarly, in nonhuman primates, a single administration of C5-siRNA/LNP-induced dose-dependent plasma C5 suppression and concomitantly inhibited serum complement activity; complement activity recovered to the pre-treatment levels at 65 days post administration, thus indicating that the complement activity can be controlled for a specific period. Our findings provide the foundation for further developing C5-siRNA delivered via LNPs as a potential therapeutic for complement-mediated diseases.

Prediction of relaxin-3-induced downstream pathway resulting in anxiolytic-like behaviors in rats based on a microarray and peptidome analysis.

The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.