Protocol for high-throughput cloning, expression, purification, and evaluation of bispecific antibodies.

Bispecific antibodies are a powerful new class of therapeutics, but their development often requires enormous amounts of time and resources. Here, we describe a high-throughput protocol for cloning, expressing, purifying, and evaluating bispecific antibodies. This protocol enables the rapid screening of large panels of bispecific molecules to identify top candidates for further development.

A biparatopic agonistic antibody that mimics fibroblast growth factor 21 ligand activity.

Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies ( those that activate receptor signaling) have been described.

Anti-PCSK9 antibody pharmacokinetics and low-density lipoprotein-cholesterol pharmacodynamics in nonhuman primates are antigen affinity-dependent and exhibit limited sensitivity to neonatal Fc receptor-binding enhancement.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect.

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes.

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation.

Targeted codon optimization improves translational fidelity for an Fc fusion protein.

High levels of translational errors, both truncation and misincorporation in an Fc-fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified.

Context-dependent role of angiopoietin-1 inhibition in the suppression of angiogenesis and tumor growth: implications for AMG 386, an angiopoietin-1/2-neutralizing peptibody.

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2.

FGF21 N- and C-termini play different roles in receptor interaction and activation.

Fibroblast growth factor-21 (FGF21) signaling requires the presence of beta-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF21 binds directly to beta-Klotho through its C-terminus. Serial C-terminal truncations of FGF21 weakened or even abrogated its interaction with beta-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response.