作者: Mu, Jingxi ; Murate, Kentaro ; Kawashima, Hiroki ; Yamamoto, Kenta ; Hirose, Takashi ; Nakamura, Masanao ; Urano, Takeshi ; Nariai, Yuko ; Sawada, Tsunaki ; Maeda, Keiko ; Furukawa, Kazuhiro ; Kamino, Hiroki ; Ishikawa, Eri ; Yamamura, Takeshi ; Ohashi, Ayako ; Fujishiro, Mitsuhiro

BACKGROUNDNumerous biological interventions and small molecules are used to treat Crohn's disease; however, the effectiveness of these treatments varies largely. Non-responsiveness to biological therapies is associated with interleukin (IL)-18 gene polymorphisms and high IL-18 expression has been implicated in the pathogenesis of Crohn's disease.AIMSThe aim of this study was to elucidate the expression of precursor and mature IL-18 in patients with Crohn's disease who exhibited varied responses to cytokine-targeted treatments and determine whether selective inhibition of mature IL-18 offers a novel therapeutic avenue.METHODSWe generated a monoclonal antibody that specifically recognizes the neoepitope of caspase-cleaved mature IL-18. Expression of precursor and mature IL-18 was analyzed in patients with Crohn's disease. Anti-mature IL-18 monoclonal antibodies were intraperitoneally administered in an acute colitis mouse model, and the disease activity index, body weight loss, tissue pathology, proinflammatory cytokine expression, goblet cell function, and microbiota composition were assessed.RESULTSPrecursor and mature IL-18 expression was upregulated and goblet cell function was impaired in patients with Crohn's disease who were unresponsive to biological therapies. Administration of anti-mature IL-18 antibodies ameliorated induced colitis by repairing goblet cell function and restoring the mucus layer.CONCLUSIONSThe newly developed monoclonal antibody holds promise as a therapeutic alternative for Crohn's disease.

2023-05-01·Biochimie

Generation of the novel anti-FXYD5 monoclonal antibody and its application to the diagnosis of pancreatic and lung cancer

Article

作者: Maruyama, Riruke ; Tajima, Yoshitsugu ; Hotta, Takamasa ; Kamino, Hiroki ; Kajitani, Naoyo ; Kadota, Kyuichi ; Isobe, Takeshi ; Nariai, Yuko ; Urano, Takeshi

Despite recent advances in cancer treatments, pancreatic cancer has a dismal prognosis globally. Early detection of cancer cells and effective treatments for recalcitrant tumors are required, but the innovative therapeutic tools remain in development. Cancer-specific antigens expressed only on cancer cells may help resolve these problems, and antibodies to such antigens have potential in basic research and clinical applications. To generate specific antibodies that bind to proteins expressed on the surface of pancreatic cancer cells, we immunized mice with human pancreatic cancer MIA PaCa-2 cells, and isolated a hybridoma that produces a monoclonal antibody (mAb), named 12-13.8. This antibody was applied to molecular biological experiments such as immunocytochemistry, immunoblotting, flow cytometry, and immunoprecipitation. In addition, we showed that mAb 12-13.8 could accumulate in tumors, through in vivo experiments using cancer-bearing mice. Immunohistochemical staining of pancreatic and lung tumor tissues indicated that the increase of the staining strength by mAb 12-13.8 positively and inversely correlated with the patients' cancer recurrence and survival rate, respectively. We identified the FXYD5 protein as the target protein of mAb 12-13.8, by a human protein array screening system. The FXYD5 protein is overexpressed in various types of cancer and is modified by O-linked glycosylation. We confirmed the binding of the FXYD5 protein to mAb 12-13.8 by using FXYD5-knockout MIA PaCa-2 cells, and detailed epitope mapping identified amino acid residues 45-52 as the minimal peptide sequence. Our results indicate that mAb 12-13.8 could be a valuable tool for FXYD5 studies, and useful in diagnostic and drug delivery applications for cancer patients.

2022-09-01·Archives of Biochemistry and Biophysics

Generation of antagonistic monoclonal antibodies against the neoepitope of active mouse interleukin (IL)-18 cleaved by inflammatory caspases

Article

作者: Uchida, Yuki ; Urano, Takeshi ; Obayashi, Eiji ; Koga, Tomohiro ; Nariai, Yuko ; Tajima, Yoshitsugu ; Kamino, Hiroki ; Kawakami, Atsushi

Interleukin 18 (IL-18) is a member of the IL-1 family and plays an important role in both the innate and acquired immune systems. It is constitutively expressed as an inactive precursor (24 kDa) in various cell types, and the mature IL-18 (18 kDa) cleaved by inflammatory caspase-1/4 binds to the interleukin-18 receptor, thereby activating downstream signaling pathways. We previously generated anti-human IL-18 antibodies that specifically recognize the human IL-18 neoepitope cleaved by inflammatory caspase-1/4. Because the N-terminal amino acid sequences of the neoepitopes are different between human IL-18 and mouse IL-18, the anti-human IL-18 neoepitope antibodies do not recognize mouse mature IL-18. We have now generated novel anti-mouse IL-18 neoepitope antibodies. We also confirmed CXCL2 secretion from P-815 mouse cells by mouse IL-18 stimulation, and established a simple assay to evaluate the activity of mouse IL-18. Using this evaluation system, we confirmed that the anti-mouse IL-18 neoepitope antibodies could inhibit mouse IL-18. By demonstrating the therapeutic efficacy of the anti-mouse IL-18 neoepitope and function-blocking mAbs established in the present study in mouse models, corresponding to human inflammatory diseases in which IL-18 may be involved, such as inflammatory bowel diseases, we can provide the proof-of-concept that the previously established anti-human IL-18 neoepitope and function-blocking mAbs work in human inflammatory disorders corresponding to mouse models.

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