Showing papers by "Shinae Kizaka-Kondoh published in 2020"

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

Abstract: 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.

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

Abstract: 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

Abstract: 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 (KD) 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.

Slicing Spheroids in Microfluidic Devices for Morphological and Immunohistochemical Analysis

Abstract: Microfluidic devices utilizing spheroids play important roles in in vitro experimental systems to closely simulate morphological and biochemical characteristics of the in vivo tumor microenvironment. For the observation and analysis of the inner structure of spheroids, sectioning is an efficient approach. However, conventional microfluidic devices are difficult for sectioning, and therefore, spheroids inside the microfluidic channels have not been sliced well. We proposed a microfluidic device created from embedding resin for sectioning. Spheroids were cultured, embedded by resin, and sectioned in the microfluidic device. Slices of the sectioned spheroids yielded clear images at the cellular level. According to morphological and immunohistochemical analyses of the slices of the spheroid, specific protein distribution was observed.

ROR2 regulates the survival of murine osteosarcoma cells in lung capillaries

Abstract: Aim: Lung metastasis is a leading cause of death in patients with osteosarcoma (OS). No effective therapy exists that improves the five-year overall survival rate of OS patients with metastasis. Therefore, finding novel therapeutic targets will help develop new treatment strategies for OS patients with lung metastasis. Methods: Based on analysis of gene expression profiles between sublines of the Dunn OS LM8 cell line with high (LM8-H) and low (LM8-L) metastatic ability, we have identified Wnt signal-related genes that play an important role in lung metastasis of OS. Function of the genes was investigated by establishing sublines of gene knockout and assessing their metastatic ability using a mouse lung metastasis model. The molecular mechanism underlying the function of the genes was further investigated by in vitro experiments. Results: We have identified that receptor tyrosine kinase-like orphan receptor 2 (ROR2), a receptor of the noncanonical Wnt signaling pathway, was involved in OS cell survival in lung capillaries during metastasis. LM8-H knocked out of Ror2 (H/Ror2-KO) significantly reduced lung metastasis by decreasing the viability in lung capillaries 48 h after intravenous injection. In vitro study revealed that ROR2 increased anoikis resistance through AKT activation. Reconstitution of ROR2 expression in H/Ror2-KO cells restored their metastatic ability and viability in

Development of near-infrared fluorescent probes with large stokes shifts for non-invasive imaging of tumor hypoxia

Abstract: A series of near-infrared (NIR) fluorochromes with large Stokes shifts was designed, synthesized, and evaluated for application in non-invasive imaging of tumor hypoxia. Each NIR fluorescent hypoxia probe comprised a tricarbocyanine dye and a 2-nitroimidazole-containing moiety as a hypoxia marker that binds to cellular nucleophiles via bioreductive activation under hypoxic conditions. Nucleophilic displacement of the amino-nucleophilic linker moiety of heptamethine cyanine dyes having a 2-chloro-1-cyclohexenyl ring and a 2-nitroimidazole moiety yielded various fluorochromes with different hydrophilicity. These exhibited long emission wavelengths (747–758 nm) with large Stokes shifts (111–125 nm) and high quantum yield (0.04–0.34). GPU-210, 297, and 316 showed significantly higher levels of fluorescence under hypoxic than under normoxic conditions on treating SUIT-2/HRE-Luc pancreatic cancer cells. Among these, only GPU-316 showed significant fluorescence intensity in tumor tissue in in vivo fluorescence imaging of mouse xenograft models. INTRODUCTION Tumor hypoxia is now known as an important factor that negatively influences the prognosis of cancer patients. Hypoxic tumor cells are not only resistant to ionizing radiation and many chemotherapeutic agents but also metastasize to distant organs. Therefore, there has been a growing impetus to develop non-invasive imaging methods to detect and assess tumor hypoxia. Nitroimidazole compounds conjugated with a radionuclide function as hypoxia markers and have been used for non-invasive imaging of tumor hypoxia. For example, pimonidazole is widely used as a hypoxia marker for immunohistochemical analysis (Figure 1). These compounds are known to be selectively trapped in hypoxic tissue via enzymatic reduction of the nitro group to form anion radicals that are further reduced in the absence of oxygen. Although several radiopharmaceuticals with a nitroimidazole group are in clinical use, current radionuclide imaging methods have poor spatial and temporal resolution and are subject to stringent safety regulations that limit their repeated use. Optical imaging, however, has comparable sensitivity to that of radionuclide imaging and can be employed if the emitting fluorophore is conjugated to a targeting ligand. It is safer and easier to perform than nuclear imaging. Optical imaging in the NIR region (650–900 nm) involves low absorption by intrinsic photoactive biomolecules and allows light to penetrate a few centimeters into the tissue, a sufficient depth for imaging practically all small animal models. Therefore, NIR fluorescence imaging, a less expensive and non-invasive technique, may be a powerful tool for imaging tumor hypoxia. Motivated by the aforementioned facts, we developed a NIR hypoxia probe, GPU-167 (Figure 1), comprising two pimonidazole moieties and a tricarbocyanine dye, for in vivo imaging of tumor hypoxia. This is the first chemical probe to visualize tumor hypoxia by NIR fluorescence with a significant tumor-to-background (T/B) ratio (muscle as the reference tissue) in a mouse xenograft model. However, the presence of diastereomers of GPU-167, due to its two chiral carbons, hinder its synthesis and further elaboration and its pharmacokinetics and tumor distribution require improvement. We also synthesized GPU-311 as another achiral candidate for a hypoxia-selective NIR fluorochrome (Figure 1). The heptamethine cyanine dye used has a cyclohexenyl ring moiety that increases its photostability and provides a chemically reactive site for nucleophilic substitution at the center methine carbon of the dye. A sulfur nucleophile was utilized to couple tricarbocyanine and a 2-nitroimidazole moiety with a hydrophilic tetraethylene glycol linker. Evaluation of its hypoxia selectivity in vitro was promising; however, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination by the liver. Another potential drawback of GPU-167 and 311 was their small Stokes shifts (25 and 17 nm, respectively), which can cause self-quenching and measurement errors due to excitation light and scattered light. Therefore, we designed a new molecule, inspired by the fact that direct attachment of the alkylamine to the cyclohexene ring of heptamethine cyanine dramatically increases the Stokes shift. Here we report on the design, synthesis, and functional evaluation of hypoxia-selective NIR probes with large Stokes shifts for non-invasive imaging of tumor hypoxia.