Cell-free production and streamlined assay of cytosol-penetrating antibodies.

Antibodies that target intracellular proteins hold great promise in the development of novel therapeutic interventions for various diseases. In particular, antibodies that can cross cellular membranes have potential applications in controlling disease-related intracellular protein-protein interactions. Given the large number of cytosolic proteins and complicated interactions that are potentially involved in disease development, discovery of antibodies targeting intracellular proteins requires iterative cycles of expression and assessment of candidate antibodies. Because current cell-based expression methods do not provide sufficient throughput for production and assay of cytosol-penetrating antibodies, we integrated a cell-free protein synthesis system designed to provide optimal conditions for production of functional antibodies with a cytosol-penetration assay. The proposed approach of consolidating cell-free synthesis and cell-based assay will substantially expand the capability of discovering and engineering antibodies that can cross the cell membrane and effectively control protein-mediated cellular functions. Biotechnol. Bioeng. 2016;113: 2107-2112. © 2016 Wiley Periodicals, Inc.

Evaluation of Intraoperative Near-Infrared Fluorescence Visualization of the Lung Tumor Margin With Indocyanine Green Inhalation.

Importance: Identification of the tumor margin during surgery is important for precise minimal resection of lung tumors. Intravenous injection of indocyanine green (ICG) has several limitations when used for intraoperative visualization of lung cancer. Objectives: To describe a technique for intraoperative visualization of lung tumor margin using ICG inhalation and evaluate the clinical applicability of the technique in mouse and rabbit lung tumor models as well as lung specimens of patients with lung tumors. Design, Setting, and Participants: In lung tumor models of both mice and rabbits, the distribution of inhaled ICG in the lung tumor margin was investigated in vivo and ex vivo using a near-infrared imaging system. Lung tumor margin detection via inhalation of ICG was evaluated by comparing the results obtained with those of the intravenous injection method (n = 32, each time point for 4 mice). Based on preclinical data, use of ICG inhalation to help detect the tumor margin in patients with lung cancer was also evaluated (n = 6). This diagnostic study was conducted from May 31, 2017, to March 30, 2019. Main Outcomes and Measures: The use of tumor margin detection by inhaled ICG was evaluated by comparing the inhaled formulation with intravenous administration of ICG. Results: From 10 minutes after inhalation of ICG to 24 hours, the distribution of ICG in the lungs was significantly higher than that in other organs (signal to noise ratio in the lungs: 39 486.4; interquartile range [IQR], 36 983.74-43 592.5). Ex vivo and histologic analysis showed that, in both lung tumor models, inhaled ICG was observed throughout the healthy lung tissue but was rarely found in tumor tissue. The difference in the fluorescent signal between healthy and tumor lung tissues was associated with the mechanical airway obstruction caused by the tumor and with alveolar macrophage uptake of the inhaled ICG in healthy tissues. Inhalation at a 20-fold lower dose of ICG had a 2-fold higher efficiency for tumor margin detection than did the intravenous injection (2.9; IQR, 2.7-3.2; P < .001). Conclusions and Relevance: The results of this study suggest that lung-specific inhalation delivery of ICG is feasible and may be useful for the intraoperative visualization of lung tumor margin in clinical practice.