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.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 10 9 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin's enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.Peptides are increasingly attractive as therapeutics given their diverse biological functions and potentially high potency and target specificity (1). Yet the development of therapeutic peptides remains challenging given their typically short in vivo half-life that results from rapid renal clearance and from degradation by peptidases in the blood, liver, and kidneys (2). Susceptibility to both exo-and endopeptidases can contribute substantially to the clearance of peptides in circulation, with slow renal filtration increasing exposure to protease degradation (3). Furthermore, peptidase susceptibility may increase the risk of immunogenicity, mediated by antigen-processing proteases (4). Consequently, several strategies to enhance peptide resistance to proteases have been developed including Nterminal acetylation and C-terminal amidation (5), the use of backbone cyclization (6), incorporation of non-canonical or D-amino acids (7)(8), and PEGylation (9). Although these approaches have proven useful alone or in combination, there remains a need for general strategies to improve half-life that are compatible with recombinant protein synthesis and preserve the benefits of small size.Address correspondence to: Patrick S. Daugherty, Department of Chemical Engineering, Engineering II Room 3357, University of California, Santa Barbara, 893-2610; Fax: (805) 893-4731; psd@engineering.ucsb An alternative strategy to enhance the intrinsic stability of peptides is the use of naturally occurring disulfide-constrained peptide scaffolds that are re...

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid β (Aβ) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aβ pathology in mice producing both human Aβ and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aβ deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aβ pathology and may represent a selective approach to treat AD.

Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40-60 nM and slow dissociation rate constants (∼1 × 10(-3) s(-1)). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ≈ 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties.