De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function.

Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.

Peptidomimetic-based antibody surrogate for HER2.

Inhibition of human epidermal growth factor receptor 2 mediated cell signaling pathway is an important therapeutic strategy for HER2-positive cancers. Although monoclonal antibodies are currently used as marketed drugs, their large molecular weight, high cost of production and susceptibility to proteolysis could be a hurdle for long-term application. In this study, we reported a strategy for the development of artificial antibody based on γ-AApeptides to target HER2 extracellular domain (ECD). To achieve this, we synthesized a one-bead-two-compound (OBTC) library containing 320,000 cyclic γ-AApeptides, from which we identified a γ-AApeptide, M-3-6, that tightly binds to HER2 selectively. Subsequently, we designed an antibody-like dimer of M-3-6, named M-3-6-D, which showed excellent binding affinity toward HER2 comparable to monoclonal antibodies. Intriguingly, M-3-6-D was completely resistant toward enzymatic degradation. In addition, it could effectively inhibit the phosphorylation of HER2, as well as the downstream signaling pathways of AKT and ERK. Furthermore, M-3-6-D also efficiently inhibited cell proliferation in vitro, and suppressed tumor growth in SKBR3 xenograft model in vivo, implying its therapeutic potential for the treatment of cancers. Its small molecular weight, antibody-like property, resistance to proteolysis, may enable it a new generation of artificial antibody surrogate. Furthermore, our strategy of artificial antibody surrogate based on dimers of cyclic γ-AApeptides could be applied to a myriad of disease-related receptor targets in future.

In vitro-engineered non-antibody protein therapeutics.

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, ß-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.