Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics

Abstract

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aβ1–40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals.

De novodesign of high-affinity single-domain antibodies

“…In the end, the output phage libraries from desired rounds of selecVon were sub-cloned into the vector pLTDA118 (a modified pHEN6 vector with C-terminal 3xFlag/6xHis tag) and transformed into TG1 cells. Single colonies were randomly picked to grow in the 96-well deep-well plate for the preparaVon of periplasmic extracts, and Sanger sequenced as previously described (32).…”

Small Antibodies with Big Applications: Nanobody-Based Cancer Diagnostics and Therapeutics

“…In contrast, nanobodies present a great number of advantages that conventional antibodies do not. The mini size of nanobodies makes them have the ability to penetrate solid tumors due to exceptional permeability, as well as cross the blood-brain barrier (BBB)-which most of mAbs and drugs could not [7,8]. Nanobodies are highly thermally stable and tolerant to extreme pH values, allowing their structures and functions to remain intact under hostile conditions [9].…”

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery

“…We chose TfR1 as a target because of its high expression on the human BBB; its ability to mediate constitutive, ligand-independent receptor-mediated transcytosis (RMT) across the CNS vasculature (8)(9)(10)(11)(12); and its track record as a target to increase the delivery of biologics into the CNS of mice (13)(14)(15), nonhuman primates (NHPs) (16)(17)(18), and humans as investigational therapies (19)(20)(21)(22) and as an approved antibody-based therapeutic for Mucopolysaccharidosis type II (23). We first screened 7-mer-modified AAV9 capsid libraries for their ability to bind to human TfR1 in vitro.…”