NN-800s: Brain-penetrant trimeric nanobodies enable potent TNFα inhibition via TfR1-mediated transcytosis

Abstract

Tumor necrosis factor alpha (TNF) is a central mediator of neuroinflammation and synaptic dysfunction in multiple central nervous system (CNS) disorders, including Alzheimers disease. Although clinically approved TNF inhibitors are highly effective in peripheral inflammatory diseases, their therapeutic application in CNS disorders is severely limited by poor blood-brain barrier (BBB) penetration. Here, we report the development of NN-800s, a class of heterotrimeric nanobody-based biologics engineered to achieve both high TNF neutralization potency and efficient BBB transcytosis. NN-800s consist of two humanized anti-TNF nanobodies flanking a humanized anti-transferrin receptor 1 (TfR1) nanobody that mediates receptor-dependent transport across the BBB. These constructs exhibit picomolar TNF inhibitory activity and achieve cerebrospinal fluid (CSF)-to-serum ratios of up to ~0.4 following systemic administration. Importantly, NN-800s do not disrupt transferrin-TfR1 interactions and do not induce hematological toxicity in vivo in humanized Tf/TfR1 rat models, supporting a favorable safety profile. The constructs are efficiently produced in CHO cells with high purity, low endotoxin levels, and strong scalability, supporting their developability as therapeutic biologics. Together, these data establish NN-800s as a promising platform for CNS-targeted TNF inhibition and demonstrate a generalizable strategy for delivering biologics across the BBB with therapeutic-level exposure.