Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.

Schizophrenia is a severe mental disorder with an unclear pathophysiology. Increased expression of the immune gene C4 has been linked to a greater risk of developing schizophrenia; however, it is not known whether C4 plays a causative role in this brain disorder. Using confocal imaging and whole-cell electrophysiology, we demonstrate that overexpression of C4 in mouse prefrontal cortex neurons leads to perturbations in dendritic spine development and hypoconnectivity, which mirror neuropathologies found in schizophrenia patients. We find evidence that microglia-mediated synaptic engulfment is enhanced with increased expression of C4. We also show that C4-dependent circuit dysfunction in the frontal cortex leads to decreased social interactions in juvenile and adult mice. These results demonstrate that increased expression of the schizophrenia-associated gene C4 causes aberrant circuit wiring in the developing prefrontal cortex and leads to deficits in juvenile and adult social behavior, suggesting that altered C4 expression contributes directly to schizophrenia pathogenesis.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.

Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity.

How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization.

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

An antibody-cytotoxic conjugate, BIIB015, is a new targeted therapy for Cripto positive tumours.

BIIB015 is an immunoconjugate created for the treatment of solid tumours and is currently in Phase I of clinical evaluation. BIIB015 consists of a humanised monoclonal antibody against the Cripto protein carrying a payload, via a hindered disulphide linker, of the maytansinoid derivative, DM4. Cripto is a GPI-linked protein required for signal transduction of the TGF-beta ligand, Nodal. Cripto has been previously described as an oncogene and fits the classic pattern of an embryonic gene that is re-expressed in a transformed tumour cell. Cripto expression is highly prevalent on a number of solid tumours, including greater than 75% of breast, lung, and colorectal tumours. Our report documents for the first time that targeting the cell surface Cripto protein with an anti-Cripto antibody-cytotoxic conjugate is an effective means of inhibiting or regressing growth of Cripto positive tumours. BIIB015 which utilises a 'cleavable' linker containing a disulphide bond exhibits superior activity when compared to huB3F6 mAb conjugates with different linker systems, including one with a 'non-cleavable' linker. BIIB015 displays specificity for Cripto in both in vitro and in vivo experiments. In human xenograft models originating from lung (Calu-6), colon (CT-3), testicular (NCCIT) and breast (MDA-MB-231) tumour samples, BIIB015 shows robust activity with results ranging from >50% tumour inhibition to complete tumour regression. The efficacy seen in the MDA-MB-231 model, a triple negative (-HER2, -ER, and -PR) tumour, is particularly exciting since there is currently no approved therapy for this indication. In addition, BIIB015 can be combined with standard of care chemotherapeutics for enhanced efficacy.