Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin

Abstract

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.

Detection and destruction of HER2‐positive cancer cells by Ultra Quenchbody‐siRNA complex

“…It affords simpler and faster detection than previous heterogeneous immunoassays, such as ELISA and immunofluorescence, yet with sufficient sensitivity and selectivity. Although Q‐bodies specific to small molecules (peptides and haptens) were relatively well characterized, successful detection of proteins including serum albumin and claudin 4 have been reported (Jeong et al, 2017). Especially, Fab‐based, double‐labeled UQ‐bodies showed higher responses than scFv‐based, single‐labeled Q‐bodies, and double‐labeled UQ‐bodies were successfully used to image overexpressed claudin 4 on LoVo colon cancer cells by wash‐free fluorescence imaging.…”

“…In this study, we demonstrated that UQ‐bodies could be used to stain cancer cells without any washing steps. In our previous study (Jeong et al, 2017), by using UQ‐bodies that show 2.5‐3‐fold antigen‐dependent dequenching response, we have successfully imaged claudin‐positive cancer cells without any washing steps. When we performed the same assay using randomly AF488‐labeled UQ‐body, no clear cellular image was observed with high background fluorescence.…”

“…We have found that Q‐body made of antibody Fab fragment (Ultra Q‐body [UQ‐body]) is more stable, and generally gives a higher antigen‐dependent signal than the corresponding single‐chain Fv‐based Q‐body. UQ‐bodies have been successfully constructed for the detection of various substances, including small molecules, such as illegal drugs (Abe et al, 2014), pesticides (Zhao, Dong, Jeong, Okumura, & Ueda, 2018), and peptides, as well as large proteins, such as influenza virus hemagglutinin (Jeong, Dong, & Ueda, 2018) and the tight junction‐associated membrane protein, claudin (Jeong et al, 2017). We have successfully imaged claudin‐positive cancer cells by fluorescence microscopy without any washing steps, which could potentially be harnessed for use in cancer diagnostics.…”

Synthesis of Quenchbodies for One-Pot Detection of Stimulant Drug Methamphetamine

“…Since the fluorescence of the Q-body is quenched in the absence of antigen, its signal-to-background ratio is higher than that of other conventional dye-conjugated antibodies. We have utilized Q-bodies as biosensors for the rapid detection of various targets in a solution, and in some cases on cells, without the need for washing steps [13,14]. Moreover, Q-body assay for detecting BGP peptide was successfully performed not only in the biochemical buffer-based samples but also in 50% plasma with no apparent loss of response [8].…”

Single-Step Detection of the Influenza Virus Hemagglutinin Using Bacterially-Produced Quenchbodies

“…Therefore, adding a Quenchbody to a target sample and measuring its fluorescence intensity, allow for the antigen to be quantified, without the need for additional reagents or experimental steps. To date, we have applied Quenchbodies as biosensors, for the rapid detection of various targets in a solution, and in some cases on the cells, without performing any washing steps [11,12,13,14,15,16,17,18]. Among these studies, we have developed a Fab-type Quenchbody that recognizes the HAs of the influenza virus H1N1 and H5N1, via a cell-free transcription and translation system [13].…”