Epiphycan is specifically expressed in cochlear supporting cells and is necessary for normal hearing.

The study of inner ear specific transcripts has revealed novel information about hereditary hearing loss and a mechanism of normal hearing. In this study, by analyzing a published cDNA library, we focused on Epiphycan (Epyc), a member of the small leucine-rich repeat proteoglycan family, whose transcript is enriched in the inner ear. Epyc mRNA was expressed abundantly and specifically in adult mice cochleae and was localized in supporting cells within the organ of Corti of both neonatal and adult mice. To examine the function of Epyc, we generated Epyc knockout (KO) mice using the CRISPR/Cas9 system. Epyc KO mice cochleae exhibited normal morphology. However, measurement of the auditory brain-stem response in Epyc KO mice revealed an elevated hearing threshold above 16 kHz frequency. This study suggests that Epyc is necessary for normal auditory function.

Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display.

Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.

Reconstitution of an Anti-HER2 Antibody Paratope by Grafting Dual CDR-Derived Peptides onto a Small Protein Scaffold.

Target-binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design strategy to create small proteins called fluctuation-regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity-determining region (CDR) loops of mAbs (CDR-derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target-binding capability. Here, a strategy to create FLAPs bearing dual CDR-derived peptides (D-FLAPs) using the anti-human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR-derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D-FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D-FLAP with HYTTPP and GDGFYA peptides from CDR-L3 and CDR-H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold.

Tumor-binding peptides such as human epidermal growth factor receptor 2 (HER2)-binding peptides are attractive therapeutic and diagnostic options for cancer. However, the HER2-binding peptides (HBPs) developed thus far are susceptible to proteolysis and lose their affinity to HER2 in vivo. In this report, a method to create a HER2-binding fluctuation-regulated affinity protein (HBP-FLAP) consisting of a fibronectin type III domain (FN3) scaffold with a structurally immobilized HBP is presented. HBPs were selected by phage-library screening and grafted onto FN3 to create FN3-HBPs, and the HBP-FLAP with the highest affinity (HBP sequence: YCAHNM) was identified after affinity maturation of the grafted HBP. HBP-FLAP containing the YCAHNM peptide showed increased proteolysis-resistance, binding to HER2 with a dissociation constant (K D) of 58 nM in ELISA and 287 nM in biolayer interferometry and specifically detects HER2-expressing cancer cells. In addition, HBP-FLAP clearly delineated HER2-expressing tumors with a half-life of 6 h after intravenous injection into tumor-bearing mice. FN3-based FLAP is an excellent platform for developing target-binding small proteins for clinical applications.

Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use.

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270-350 nM in biolayer interferometry and 24-65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

Catheter-related fungemia due to fluconazole-resistant Candida nivariensis.

We report on a case of fungemia due to fluconazole-resistant Candida nivariensis (MIC, > or =128 microg/ml). Internal transcribed spacer PCR followed by microchip gel electrophoresis with a blood culture that tested positive revealed a unique pattern different from those of other pathogenic yeasts.