Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome.

Striatal cholinergic interneurons are tonically active neurons and respond to sensory stimuli by transiently suppressing firing that is associated with sensorimotor learning. The pause in tonic firing is dependent on dopaminergic activity; however, its cellular mechanisms remain unclear. Here, we report evidence that dopaminergic inhibition of hyperpolarization-activated cation current (I h ) is involved in this process. In neurons exhibiting regular firing in vitro, exogenous application of dopamine caused a prolongation of the depolarization-induced pause and an increase in the duration of slow afterhyperpolarization (sAHP) after depolarization. Partially blocking I h with specific blocker ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride) reduced firing and mimicked the effects of dopamine on sAHP. The I h , being active at membrane potentials negative than Ϫ50 mV, was inhibited by dopamine via activation of the D 2 -like receptor, but not D 1 -like receptor. The inhibitory effects of the D 2 receptor activation on I h were mediated through a protein kinase A-independent cyclic AMP pathway. Consistently, D 2 -like receptor agonist quinpirole showed comparable effects on sAHP and firing rate as those induced by I h channel blocker. Moreover, dopamine was unable to further affect the sAHP duration in neurons when I h was blocked. These findings indicate that D 2 receptor-dependent inhibition of I h may be a novel mechanism for modulating the pause response in tonic firing in cholinergic interneurons.

Injection of NMDAR antagonist into the thalamus can produce delta frequency EEG oscillations in the thalamocortical system. It is surprising that an antagonist of an excitatory neurotransmitter should trigger such activity, and the mechanism is unknown. One hypothesis is that the antagonist blocks excitation of GABAergic cells, thus producing disinhibition. To test this hypothesis, we investigated the effect of NMDAR antagonist (APV) on cells of the nucleus reticularis (nRT) in rat brain slices, a thalamic nucleus that can serve as a pacemaker for thalamocortical delta oscillations and that is composed entirely of GABAergic neurons. We found, unexpectedly, that nRT cells are hyperpolarized by APV. This occurs because these cells have an unusual form of NMDAR (probably NR2C) that contributes inward current at resting potential in response to ambient glutamate. The hyperpolarization produced by APV is sufficient to deinactivate T-type calcium channels, and these trigger rhythmic bursting at delta frequency. The APV-induced delta frequency bursting is abolished by dopamine D2 receptor antagonist, indicating that dopamine and NMDAR antagonist work synergistically to stimulate delta frequency bursting. Our results have significant implications concerning the electrophysiological basis of schizophrenia and bring together the NMDAR hypofunction, dopamine, and GABA theories of the disease. Our results suggest that NMDAR hypofunction and dopamine work synergistically on the GABAergic cells of the nRT to generate the delta frequency EEG oscillations, a thalamocortical dysrhythmia (TCD) in the awake state that is an established abnormality in schizophrenia.

Eculizumab is a humanized mAb approved for treatment of patients with paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab binds complement component C5 and prevents its cleavage by C5 convertases, inhibiting release of both the proinflammatory metabolite C5a and formation of the membrane attack complex via C5b. In this study, we present the crystal structure of the complex between C5 and a Fab fragment with the same sequence as eculizumab at a resolution of 4.2 Å. Five CDRs contact the C5 macroglobulin 7 domain, which contains the entire epitope. A complete mutational scan of the 66 CDR residues identified 28 residues as important for the C5–eculizumab interaction, and the structure of the complex offered an explanation for the reduced C5 binding observed for these mutant Abs. Furthermore, the structural observations of the interaction are supported by the reduced ability of a subset of these mutated Abs to inhibit membrane attack complex formation as tested in a hemolysis assay. Our results suggest that eculizumab functions by sterically preventing C5 from binding to convertases and explain the exquisite selectivity of eculizumab for human C5 and how polymorphisms in C5 cause eculizumab-resistance in a small number of patients with paroxysmal nocturnal hemoglobinuria.

Postmortem studies have shown that schizophrenia produces a reduction in the 67-kilodalton isoform of glutamic acid decarboxylase (GAD67), a key enzyme for gamma-aminobutyric acid (GABA) synthesis. N-methyl-d-aspartate receptor (NMDAR) antagonists have been extensively used to study schizophrenia because they can induce many aspects of the disease, including the decrease in GAD67. It is generally thought that this reduction in GAD implies a reduction in functional inhibition, but direct evidence had been lacking. We have therefore performed physiological studies in slices of prefrontal cortex taken from rats treated with the NMDAR antagonist ketamine. Both frequency and amplitude of miniature inhibitory postsynaptic currents were reduced. Consistent with a reduction of inhibition, we observed an increase in postsynaptic excitability. The increased excitability is likely to result from disinhibition because miniature excitatory postsynaptic current properties and intrinsic excitability were not changed. Ketamine did not affect inhibition or GAD levels in young rats, indicating a developmental regulation that may be related to the developmental increase in ketamine sensitivity that occurs in humans. Our results show that NMDAR antagonist produces biochemical changes in the GABA system that lead to a functional disinhibition. Such disinhibition would be expected to decrease gamma oscillations, which are reduced in schizophrenia.