Category: Preclinical Development
Purpose: The regulatory success rate for central nervous system (CNS) drugs is less than half that of non-CNS drugs. A current bottleneck in the development of CNS drugs is the lack of drug delivery systems to the brain. The blood-brain barrier (BBB) is a membrane barrier formed by brain capillary endothelial cells and protects the brain from harmful materials circulating in the blood. Most small and all large drugs do not readily cross the BBB. The intercellular space between endothelial cells of the BBB is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein, is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. However, it remains unclear whether CLDN-5 can be a target for CNS delivery of drugs. In the present study, we generated anti-CLDN-5 monoclonal antibodies (mAbs) and examined the effects of the mAbs on the integrity of TJ seal and the permeation of solutes.
Methods: mAbs against the extracellular loop domains (ECLs) of CLDN-5 were generated by DNA immunization with male BXSB mice and female Wistar rats. A mutant of the C-terminal half of a Clostridium perfringens enterotoxin (C-CPEY306W/S313H, C-CPEmt) that recognizes a broad range of claudins, including CLDN-5, was used as a positive control.1) The effects of these mAbs on the integrity of the TJ seal were investigated by measurement of trans-epithelial/endothelial electrical resistance (TEER) in cell monolayers of CLDN-5-expressing MDCKII cells and of cynomolgus monkey brain microvasculature cells (a commercial available in vitro BBB model). The permeability of the monolayers to sodium fluorescein (FNa; 376 Da) or fluorescein-labeled dextran (FD-4; 4 kDa) was also determined in the cell monolayers.
Results: Previously, we generated anti-CLDN-1 and anti-CLDN-4 mAbs by using DNA immunization methods.2, 3) In the present study, we used these techniques to generate anti-CLDN-5 mAbs by immunizing BXSB mice and Wistar rats with a plasmid encoding human CLDN-5. Four mAbs targeting the ECLs of human CLDN-5 were isolated: M11 and M48 from the mice and R2 and R9 from the rats. All four mAbs specifically bound to CLDN-5 but not to CLDN-1 to -4, -6, or -7. A cross-species reactivity analysis of the mAbs revealed that all four mAbs bound to human and cynomolgus monkey CLDN-5. Next, to investigate whether the anti-CLDN-5 mAbs decreased TJ integrity in a CLDN-5-dependent manner, MDCKII transfectants expressing human (h), cynomolgus monkey (c), or mouse (m) CLDN-5 were established. Treatment of all transfectants with C-CPEmt decreased TJ integrity (Figure 1). All four of the anti-CLDN-5 mAbs dose- and time-dependently decreased TEER in the hCLDN-5- and cCLDN-5-expressing transfectants. In contrast, none of the anti-CLDN-5 mAbs affected TJ integrity in the mCLDN-5-expressing transfectant. These findings suggest that M11, M48, R2, and R9 decreased TJ integrity by interacting with CLDN-5. TEER values returned to baseline 24 h after the anti-CLDN-5 mAbs were removed. Next, we investigated the effects of the anti-CLDN-5 mAbs on the BBB in a commercially available BBB model. Treatment of the BBB model with the anti-CLDN-5 mAbs dose- and time-dependently reduced TEER (Figure 2). In addition, no cytotoxicity was observed after treatment with the anti-CLDN-5 mAbs. Control IgG did not increase the permeability of the BBB model to the fluorescein dye or FD-4. However, treatment with the anti-CLDN-5 mAbs significantly and dose-dependently increased the permeability of the model BBB to the fluorescein dye and FD-4 compared with vehicle.
Conclusion: CLDN-5 is a potential target for the development of drug delivery systems to the brain. The anti-CLDN-5 mAbs are potential leads for development of drug delivery to the brains.
1) Protze J, Eichner M, Piontek A, Dinter S, Rossa J, Blecharz KGZ, Vajkoczy P, Piontek J and Krause G (2015) Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5. Cell Mol Life Sci 72: 1417-1432.
2) Fukasawa M, Nagase S, Shirasago Y, Iida M, Yamashita M, Endo K, Yagi K, Suzuki T, Wakita T, Hanada K, Kuniyasu H and Kondoh M (2015) Monoclonal antibodies against extracellular domains of claudin-1 block hepatitis C virus infection in a mouse model. J Virol 89: 4866-4879.
3) Hashimoto Y, Kawahigashi Y, Hata T, Li X, Watari A, Tada M, Ishii-Watabe A, Okada Y, Doi T, Fukasawa M, Kuniyasu H, Yagi K and Kondoh M (2016) Efficacy and safety evaluation of claudin-4-targeted antitumor therapy using a human and mouse cross-reactive monoclonal antibody. Pharmacol Res Perspect 4: e00266.
Masuo Kondoh– Suita, Osaka, Japan
Yosuke Hashimoto– Osaka, Osaka, Japan
Yosuke Hashimoto– Osaka, Osaka, Japan
Keisuke Shirakura– Osaka, Osaka, Japan
Yoshiaki Okada– Osaka University, Osaka, Osaka, Japan
Hiroyuki Takeda– Ehime, Ehime, Japan
Akihiro Watari– Osaka, Osaka, Japan
Tatsuya Sawasaki– Ehime, Ehime, Japan
Keisuke Tachibana– Suita, Osaka, Japan
Takefumi Doi– Suita, Osaka, Japan
Kiyohito Yagi– Osaka, Osaka, Japan
Masuo Kondoh– Professor, Osaka University, Suita, Osaka, Japan