Abstract
Neuregulin protein 1 (NRG1) is a large (> 60–amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.
Highlights
Neuregulin protein 1 (NRG1) is a large (> 60–amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4
The ligand conformation is sustained with three pairs of disulfide bonds and extensive hydrogen bonds, which can bind to the ECD of either HER4 or HER3 and convert the receptors from the unbound state through a major conformational change to the liganded state (Fig. 1A,B)
Using biolayer interferometry for direct binding to streptavidin-captured biotinylated recombinant HER3 and HER4 ECD proteins, we found that both NRG1-monomeric Fc (MFc) and NRG1-human serum albumin (HSA) bound to HER4 with similar equilibrium binding affinities, of 640 and 460 nM, respectively (Table 1)
Summary
Neuregulin protein 1 (NRG1) is a large (> 60–amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This challenge provides an important opportunity to advance novel protein designs and engineering platforms to expand the potential applications of agonistic antibodies One such example is the activation of the human epidermal growth factor receptor family member 4 (HER4) and its natural peptide agonist, neuregulin. We applied a monomeric Fc (MFc) fusion technology to carry out a selectivity engineering campaign to identify neuregulin variants with enhanced HER4 selectivity and diminished HER3 binding[22] By combining these protein engineering approaches, we were able to design ALM molecules with enhanced ligand specificity and potential to greatly improve the pharmacokinetics (PK), stability, and downstream developability profiles when compared with the natural ligand
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