RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Claudina-3/imunologia , Estrutura Terciária de Proteína , Animais , Anticorpos Monoclonais/química , Células CHO , Claudina-3/química , Cricetinae , Cricetulus , Humanos , CamundongosRESUMO
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.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/terapia , Citotoxicidade Imunológica/imunologia , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/terapia , Sequência de Aminoácidos , Animais , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Células CHO , Linhagem Celular , Proliferação de Células , Claudina-3 , Claudina-4 , Cricetinae , Cricetulus , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos SCID , Dados de Sequência Molecular , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Homologia de Sequência de Aminoácidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.