Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

Rational design of a humanized glucagon-like peptide-1 receptor agonist antibody.

Bovine antibody BLV1H12 possesses a unique "stalk-knob" architecture in its ultralong heavy chain CDR3, allowing substitutions of the "knob" domain with protein agonists to generate functional antibody chimeras. We have generated a humanized glucagon-like peptide-1 (GLP-1) receptor agonist antibody by first introducing a coiled-coil "stalk" into CDR3H of the antibody herceptin. Exendin-4 (Ex-4), a GLP-1 receptor agonist, was then fused to the engineered stalk with flexible linkers, and a Factor Xa cleavage site was inserted immediately in front of Ex-4 to allow release of the N-terminus of the fused peptide. The resulting clipped herceptin-Ex-4 fusion protein is more potent in vitro in activating GLP-1 receptors than the Ex-4 peptide. The clipped herceptin-Ex-4 has an extended plasma half-life of approximately four days and sustained control of blood glucose levels for more than a week in mice. This work provides a novel approach to the development of human or humanized agonist antibodies as therapeutics.

An antibody CDR3-erythropoietin fusion protein.

X-ray crystallographic analysis of a bovine antibody (BLV1H12) revealed a unique scaffold in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent exposed, antiparallel ß-stranded "stalk" fused with a disulfide cross-linked "knob" domain. This unusual variable region motif provides a novel approach for generating chimeric antibodies with novel activities. Toward this end, human erythropoietin (hEPO) was substituted for the "knob" domain in this antibody to afford an antibody-hEPO (Ab-hEPO) fusion protein that efficiently expresses in mammalian cells. Ab-hEPO proliferated TF-1 cells with a potency comparable to that of hEPO (EC50 ∼ 0.03 nM) and exhibits a significantly extended plasma half-life (>6 days) in mice relative to hEPO (∼4 h). Mice treated with the Ab-hEPO fusion protein show sustained elevated hematocrit for more than two weeks. This work demonstrates the utility of BLV1H12 CDR3 fusions as a novel approach for generating potent polypeptides with enhanced pharmacological properties.

Enhanced proteolytic clearance of plasma Aß by peripherally administered neprilysin does not result in reduced levels of brain Aß in mice.

Accumulation of ß-amyloid (Aß) in the brain is believed to contribute to the pathology of Alzheimer's Disease (AD). Aß levels are controlled by the production of Aß from amyloid precursor protein, degradation by proteases, and peripheral clearance. In this study we sought to determine whether enhancing clearance of plasma Aß with a peripherally administered Aß-degrading protease would reduce brain Aß levels through a peripheral sink. Neprilysin (NEP) is a zinc-dependent metalloprotease that is one of the key Aß-degrading enzymes in the brain. We developed a NEP fusion protein with in vitro degradation of Aß and a 10 day plasma half-life in mouse. Intravenous administration of NEP to wild-type and APP23 transgenic mice resulted in dose-dependent clearance of plasma Aß. However, this did not correspond to reduced levels of soluble brain Aß with treatment up to 5 weeks in WT mice or formic acid-extractable brain Aß with 3 month treatment in aged APP23. In contrast, intracranial injection of NEP resulted in an acute decrease in soluble brain Aß. We found no change in amyloid precursor protein gene expression in mice treated with intravenous NEP, suggesting that the lack of effects in the brain following this route of administration was not caused by compensatory upregulation of Aß production. Taken together, these results suggest a lack of a robust peripheral Aß efflux sink through which brain amyloid burdens can be therapeutically reduced.