Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

Characterization and evaluation of the antitumour activity of a dual-targeting monoclonal antibody against claudin-3 and claudin-4.

BACKGROUND: Because human claudin-3 and claudin-4 (CLDN3 and CLDN4) are overexpressed in a variety of carcinomas, they are promising targets for cancer therapy. The aim of the present study was to generate a dual-targeting monoclonal antibody against CLDN3 and CLDN4 and evaluate its antitumour activity. MATERIALS AND METHODS: BALB/c mice were immunised with CLDN4-expressing Chinese hamster ovary cells and cell-based screening was performed. The antibody-binding epitope of CLDN3 and CLDN4 and the antitumour activity of the antibody were evaluated. RESULTS: A monoclonal antibody, KM3907 (IgG2a), which recognised CLDN3 and CLDN4, but not CLDN5, CLDN6 and CLDN9, was successfully isolated. The binding assay of KM3907 revealed that KM3907 recognised the extracellular loop 1 of CLDN3 and CLDN4. Mouse human chimeric IgG1 induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and treatment with murine KM3907 significantly inhibited tumour formation in SCID mice in vivo. CONCLUSION: A dual-targeting monoclonal antibody against CLDN3 and CLDN4 is a promising strategy for cancer immunotherapy.

Therapeutic antitumor efficacy of monoclonal antibody against Claudin-4 for pancreatic and ovarian cancers.

Claudin-4 (CLDN4) is a tetraspanin transmembrane protein of tight junction structure and is highly expressed in pancreatic and ovarian cancers. In this study, we aimed to generate an anti-Claudin-4 monoclonal antibody (mAb) and evaluate its antitumor efficacy in vitro and in vivo. To isolate specific mAb, we generated CLDN3, 4, 5, 6, and 9, expressing Chinese hamster ovary (CHO) cells, and then used them as positive and negative targets through cell-based screening. As a result, we succeeded in isolating KM3900 (IgG2a), which specifically bound to CLDN4, from BXSB mice immunized with pancreatic cancer cells. Immunoprecipitation and flow cytometry analysis revealed that KM3900 recognized the conformational structure and bound to extracellular loop 2 of CLDN4. Furthermore, binding of KM3900 was detected on CLDN4-expressing pancreatic and ovarian cancer cells, but not on negative cells. Next, we made the mouse-human chimeric IgG1 (KM3934) and evaluated its antitumor efficacy. KM3934 induced dose-dependent antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and significantly inhibited tumor growth in MCAS or CFPAC-1 xenograft SCID mice in vivo (P < 0.05). These results suggest that mAb therapy against CLDN4 is promising for pancreatic and ovarian cancers.

Alpha1-syntrophin modulates turnover of ABCA1.

ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein. Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h. To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ (PSD95-Discs large-ZO1) proteins, alpha1-syntrophin and Lin7, were found to interact with ABCA1. Immunoprecipitation revealed that alpha1-syntrophin interacted with ABCA1 strongly and that the interaction was via the C-terminal three amino acids SYV of ABCA1. Co-expression of alpha1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing alpha1-syntrophin. This effect is not common among PDZ-containing proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1. Co-expression of alpha1-syntrophin significantly increased the apoA-I-mediated release of cholesterol. ABCA1 was co-immunoprecipitated with alpha1-syntrophin from mouse brain. These results suggest that alpha1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release.

Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release.

ABCA7 is expressed predominantly in myelo-lymphatic tissues or reticuloendothelial cells. Physiological role and function of this protein are not fully understood. We isolated the full-length cDNA (type I) and a splicing variant cDNA (type II) of human ABCA7, and developed monoclonal antibodies against extracellular domain (ECD)1 of ABCA7. RT-PCR experiments suggested that human ABCA7 gene produced the type II mRNA in a tissue-specific manner. Immunostaining revealed that the type I ABCA7, expressed in HEK293 cells, was localized to the plasma membrane and ECD1 was exposed to the extracellular space as was the case for ABCA1. HEK293 cells expressing type I ABCA7 showed apoA-I-dependent cholesterol and phospholipid release. In contrast, type II ABCA7 appeared to be localized mainly in endoplasmic reticulum and did not show apoA-I-dependent cholesterol and phospholipid release. Alternative splicing could be involved in the post-transcriptional regulation of the expression and function of human ABCA7.