Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Hobin Yang,Yong Jin Lee,Kyoung Song,Young Kee Shin,Sungyoul Hong,Saehyung Lee,Nirmal Rajasekaran,Soohyun Hwang,Young-Deug Kim,Jun Young Choi,Yoon-La Choi,Joon-Seok Choi,Hyunbo Shim,Hayeon Park,Taeeun Kim

doi:10.3390/biom10010051

Abstract

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell–cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.

Highlights

Malignant tumors, which account for approximately 90% of human cancers, arise from epithelial tissues
We demonstrated the antibody-dependent cellular cytotoxicity (ADCC) activity of h4G3 in many types of cancer cell lines according to CLDN3 expression levels
The scFv selection was monitored by measuring output-to-input ratios (Supplementary Materials Figure S1A) and by ELISA (Figure S1B) which showed the enrichment of scFv against CLDN3

Summary

Introduction

Malignant tumors, which account for approximately 90% of human cancers, arise from epithelial tissues. Tight junctions (TJs) regulate permeability across epithelial sheets and play a role in the development and maintenance of cell polarity [1]. Loss of TJ integrity leads to aberrant cell growth by increasing the influx of growth factor; it promotes the detachment of malignant cells from the primary tumor site by disrupting cell adhesion and polarity, resulting in the formation of distant metastasis [2,3]. Tight junction components are attractive targets, since they are accessible in cancer cells compared to normal cells. Cells grow parallel to the lumen maintaining their polarity, and the accessibility to the TJ components, located on the apical side of the epithelium, is limited. In the initial phase of epithelial tumorigenesis, the orientation of mitotic spindles becomes irregular, leading to out-of-plane division [4]

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Results

Discussion

Conclusion