The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Edited by Peter CresswellThe antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-␥ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-␥-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti–β-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood–brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.