Inhibiting the programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) axis by monoclonal antibodies (mAbs) is a successful cancer immunotherapy. However, mAbbased drugs have various disadvantages including high production costs and large molecular sizes, which motivated us to develop a smaller alternative drug. Since PD-L1 binds PD-1 with moderate affinity, a higher affinity PD-1 variant should serve as a competitive inhibitor of the wild-type PD-1/PD-L1 interaction. In this report, we conducted in silico point mutagenesis of PD-1 to identify potent PD-1 variants with a higher affinity toward PD-L1 and refined the in silico results using a luciferase-based in-cell protein−protein interaction (PPI) assay. As a result, a PD-1 variant was developed that had two mutated amino acids (T76Y, A132V), termed 2-PD-1. 2-PD-1 could bind with PD-L1 at a dissociation constant of 12.74 nM. Moreover, 2-PD-1 successfully inhibited the PD-1/PD-L1 interaction with a half maximal inhibitory concentration of 19.15 nM and reactivated the T cell with a half maximal effective concentration of 136.1 nM. These results show that in silico mutagenesis combined with an in-cell PPI assay verification strategy successfully prepared a non-IgG inhibitor of the PD-1/PD-L1 interaction.

BackgroundNerve regeneration is important for the treatment of degenerative diseases and neurons injured by accidents. Nerve growth factor (NGF) has been previously conjugated to materials for promotion of neurogenesis.Materials and methodsPhotoreactive gelatin was prepared by chemical coupling of gelatin with azidobenzoic acid (P-gel), and then NGF was immobilized on substrates in the presence or absence of micropatterned photomasks. UV irradiation induced crosslinking reactions of P-gel with itself, NGF, and the plate for immobilization.ResultsBy adjustment of the P-gel concentration, the nanometer-order height of micropatterns was controlled. NGF was quantitatively immobilized with increasing amounts of P-gel. Immobilized NGF induced neurite outgrowth of PC12 cells, a cell line derived from a pheochromocytoma of the rat adrenal medulla, at the same level as soluble NGF. The immobilized NGF showed higher thermal stability than the soluble NGF and was repeatedly used without loss of biological activity. The 3D structure (height of the formed micropattern) regulated the behavior of neurite guidance. As a result, the orientation of neurites was regulated by the stripe pattern width.ConclusionThe micropattern-immobilized NGF nanolayer biochemically and topologically regulated neurite formation.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation‐inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin‐like growth factor with a key amino acid (3,4‐dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid‐phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long‐lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.