TOHOKU UNIVERSITY Researchers

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Mika Kaneko

Section

Graduate School of Medicine

Job title

Associate Professor

Degree

博士（理学）（総合研究大学院大学）
修士（工学）（創価大学）

researchmap

https://researchmap.jp/mkkaneko

J-GLOBAL ID

200901023890190012

Profile

学位
2000年3月総合研究大学院大学（生命科学研究科遺伝学専攻）博士（理学）取得

職歴
2000年4月〜2005年3月山形大学医学部法医学講座助手（2003年9月から助教）
2002年9月〜2003年8月文部科学省在外研究員としてアメリカ・ジョージア大学に研究留学
2005年4月〜2006年3月東京都神経科学総合研究所分子発生生物学部門流動研究員
2006年4月〜2008年3月産業技術総合研究所糖鎖医工学研究センター招聘研究員
2009年3月〜2010年3月アメリカ・デューク大学病理部上級研究員
2010年4月〜2013年3月山形大学医学部グローバルCOE プロジェクト教員（講師）
2013年４月〜2017年3月東北大学地域イノベーション分野准教授
2017年４月〜現在東北大学大学院医学系研究科抗体創薬共同研究講座准教授

Education 4

The Graduate University for Advanced Studies

- 2000
The Graduate University for Advanced Studies　Graduate School, Division of Life Science　Genetics

- 2000
Soka University

- 1997
Soka University　Faculty of Engineering　Department of Bioengineering

- 1997

Research Interests 8

Antibody Drug Development
Antibody engineering
分子進化学
糖鎖工学
糖鎖生物学
melecular evolution
glycoengineering
glycobiology

Research Areas 4

Life sciences / Evolutionary biology /
Life sciences / Functional biochemistry /
Life sciences / Structural biochemistry /
Life sciences / Genetics /

Papers 427

Development of an anti-human EphA2 monoclonal antibody Ea2Mab-7 for multiple applications

Hiroyuki Satofuka, Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Mika K. Kaneko, Yukinari Kato

Biochemistry and Biophysics Reports　2025/06

DOI： 10.1016/j.bbrep.2025.101998 　
A novel anti-mouse CXCR1 monoclonal antibody, Cx1Mab-8, demonstrates nanomolar affinity in flow cytometry. International-journal

Guanjie Li, Hiroyuki Suzuki, Tomohiro Tanaka, Hiroyuki Satofuka, Mika K Kaneko, Yukinari Kato

Biochemistry and biophysics reports　42　101965-101965　2025/06

DOI： 10.1016/j.bbrep.2025.101965 　

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CXC chemokine receptor 1 (CXCR1) is an important regulator for neutrophil granulocyte activation through binding to the ligand interleukin-8 (IL-8). Upon binding to IL-8, CXCR1 activates downstream signaling, critical for innate and adaptive immune responses. The IL-8-CXCR1 axis also plays an important role in tumor progression, especially in the tumor microenvironment. CXCR1 antagonists or anti-IL-8 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for inflammatory diseases and tumors. In this study, we developed novel mAbs for mouse CXCR1 (mCXCR1) using the N-terminal peptide immunization. Among the established anti-mCXCR1 mAbs, Cx1Mab-8 (rat IgG2b, kappa) recognized mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 (LN229/mCXCR1) by flow cytometry. The dissociation constant (K D) values of Cx1Mab-8 for CHO/mCXCR1 and LN229/mCXCR1 were determined as 4.1 × 10-10 M and 1.5 × 10-9 M, respectively. These results indicated that Cx1Mab-8 is useful for detecting mCXCR1 by flow cytometry with high affinity and could contribute to obtaining the proof of concept in preclinical studies.
Development of an Anti‐CDH15/M‐Cadherin Monoclonal Antibody Ca15Mab‐1 for Flow Cytometry, Immunoblotting, and Immunohistochemistry

Rena Ubukata, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

2025/04/08

DOI： 10.20944/preprints202504.0675.v1 　
PMab-322: A Novel Anti-Hippopotamus Podoplanin Monoclonal Antibody for Multiple Applications

Haruto Yamamoto, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

2025/04/08

DOI： 10.20944/preprints202504.0646.v1 　
Ea₃Mab-20: A Specific Anti-Human EphA3 Monoclonal Antibody for Flow Cytometry

Hiroyuki Satofuka, Hiroyuki Suzuki, Miu Hirose, Keisuke Shinoda, Takuya Nakamura, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

2025/04/07

DOI： 10.20944/preprints202504.0524.v1 　
Antitumor Activities by a Humanized Cancer-Specific Anti-Podoplanin Monoclonal Antibody humPMab-117 Against Human Tumors

Tomohiro Tanaka, Hiroyuki Suzuki, Tomokazu Ohishi, Manabu Kawada, Mika K. Kaneko, Yukinari Kato

2025/03/28

DOI： 10.20944/preprints202503.2150.v1 　
Development of a Novel Anti-Mouse CCR7 Monoclonal Antibody C7Mab-2 by Immunization of the Extracellular Loop Domain

Haruto Yamamoto, Hiroyuki Suzuki, Tomohiro Tanaka, Hiroyuki Satofuka, Mika K. Kaneko, Yukinari Kato

2025/03/10

DOI： 10.20944/preprints202503.0581.v1 　
E_a1Mab-30: A Novel Monoclonal Antibody Against Erythropoietin-Producing Hepatocellular Receptor A1 for Versatile Applications

Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Shiori Fujisawa, Mika K. Kaneko, Yukinari Kato

2025/03/07

DOI： 10.20944/preprints202503.0525.v1 　
A novel anti-mouse CCR7 monoclonal antibody, C7Mab-7, demonstrates high sensitivity in flow cytometry, western blot, and immunohistochemistry. International-journal

Hiroyuki Satofuka, Hiroyuki Suzuki, Tomohiro Tanaka, Rena Ubukata, Miu Hirose, Haruto Yamamoto, Yu Kaneko, Shiori Fujisawa, Guanjie Li, Mika K Kaneko, Yukinari Kato

Biochemistry and biophysics reports　41　101948-101948　2025/03

DOI： 10.1016/j.bbrep.2025.101948 　

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C-C chemokine receptor type 7 (CCR7) is a member of the G protein-coupled receptor family and functions as a lymph node-homing receptor for immune cells. Upon ligand binding, CCR7 promotes the migration of immune cells to secondary lymphoid organs. In cancers, CCR7 has been revealed as a critical molecule in lymph node metastasis. Consequently, anti-CCR7 monoclonal antibodies (mAbs) have been developed as cancer therapeutic agents. In this study, we established an anti-mouse CCR7 (mCCR7) mAb, C7Mab-7 (rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. C7Mab-7 demonstrated high sensitivity in flow cytometry. The dissociation constant (K D) value of C7Mab-7 was determined to be 2.5 × 10⁻⁹ M for mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells. Furthermore, C7Mab-7 detected mCCR7 with high sensitivity in western blot and immunohistochemistry. C7Mab-7, developed by the CBIS method, accelerates the development of CCR7-targeted antibody therapies and cancer diagnostics.
Development of a novel anti-erythropoietin-producing hepatocellular receptor B6 monoclonal antibody Eb6Mab-3 for flow cytometry. International-journal

Tomohiro Tanaka, Yu Kaneko, Haruto Yamamoto, Guanjie Li, Shiori Fujisawa, Hiroyuki Satofuka, Keisuke Shinoda, Takuya Nakamura, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Biochemistry and biophysics reports　41　101960-101960　2025/03

DOI： 10.1016/j.bbrep.2025.101960 　

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Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the largest Eph subfamily of receptor tyrosine kinases. EphB6 is widely expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphB6 is involved in cancer pathology despite lacking kinase activity. Developing sensitive monoclonal antibodies (mAbs) for EphB6 has been desired for treatment, diagnosis, and further analysis of EphB6. This study established a novel specific and sensitive anti-human EphB6 mAb clone Eb6Mab-3 (mouse IgG1, kappa) by the Cell-Based Immunization and Screening (CBIS) method. In flow cytometry, Eb6Mab-3 demonstrated reactivity with EphB6-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB6) and endogenously EphB6-expressing DLD-1 colorectal cancer cells. Cross-reactivity of Eb6Mab-3 was not observed. Eb6Mab-3 demonstrated a moderate binding affinity (dissociation constant; K D) for CHO/EphB6 (K D: 2.6 ± 1.0 × 10-8 M) and a high binding affinity for DLD-1 (K D: 3.4 ± 1.3 × 10-9 M). Eb6Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in Western blot. Eb6Mab-3, established by the CBIS method, could be valuable for analyzing the EphB6-associated cellular functions and has potential applications in diagnosis and treatment with specificity and high affinity for cancer cells.
Cryo-EM structure of the bacterial intramembrane metalloprotease RseP in the substrate-bound state. International-journal

Kikuko Asahi, Mika Hirose, Rie Aruga, Yosuke Shimizu, Michiko Tajiri, Tsubasa Tanaka, Yuriko Adachi, Yukari Tanaka, Mika K Kaneko, Yukinari Kato, Satoko Akashi, Yoshinori Akiyama, Yohei Hizukuri, Takayuki Kato, Terukazu Nogi

Science advances　11　(9)　eadu0925　2025/02/28

DOI： 10.1126/sciadv.adu0925 　

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Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in bacterial pathogens. A structural model of how S2Ps discriminate and accommodate substrates could help us develop selective antimicrobial agents. We previously proposed that the Escherichia coli S2P RseP unwinds helical substrate segments before cleavage, but the mechanism for accommodating a full-length membrane-spanning substrate remained unclear. Our present cryo-EM analysis of Aquifex aeolicus RseP (AaRseP) revealed that a substrate-like membrane protein fragment from the expression host occupied the active site while spanning a transmembrane cavity that is inaccessible via lateral diffusion. Furthermore, in vivo photocrosslinking supported that this substrate accommodation mode is recapitulated on the cell membrane. Our results suggest that the substrate accommodation by threading through a conserved membrane-associated region stabilizes the substrate-complex and contributes to substrate discrimination on the membrane.
Development of a Novel Anti-Human Glypican 5 Monoclonal Antibody (G5Mab-1) for Multiple Applications

Yu Kaneko, Tomohiro Tanaka, Shiori Fujisawa, Guanjie Li, Hiroyuki Satofuka, Mika K. Kaneko, Hiroyuki Suzuki, Yukinari Kato

2025/01/28

DOI： 10.20944/preprints202501.2117.v1 　
Antitumor Activities of a Humanized Cancer-Specific Anti-HER2 Monoclonal Antibody, humH2Mab-250 in Human Breast Cancer Xenografts. International-journal

Mika K Kaneko, Hiroyuki Suzuki, Tomokazu Ohishi, Takuro Nakamura, Miyuki Yanaka, Tomohiro Tanaka, Yukinari Kato

International journal of molecular sciences　26　(3)　2025/01/26

DOI： 10.3390/ijms26031079 　

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Monoclonal antibody (mAb) and cell-based immunotherapies represent cutting-edge strategies for cancer treatment. However, safety concerns persist due to the potential targeting of normal cells that express reactive antigens. Therefore, it is crucial to develop cancer-specific mAbs (CasMabs) that can bind to cancer-specific antigens and exhibit antitumor activity in vivo, thereby reducing the risk of adverse effects. We previously screened mAbs targeting human epidermal growth factor receptor 2 (HER2) and successfully developed a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (mouse IgG1, kappa). In this study, we assessed both the in vitro and in vivo antitumor efficacy of the humanized H2Mab-250 (humH2Mab-250). Although humH2Mab-250 showed lower reactivity to HER2-overexpressed Chinese hamster ovary-K1 (CHO/HER2) and breast cancer cell lines (BT-474 and SK-BR-3) than trastuzumab in flow cytometry, both humH2Mab-250 and trastuzumab showed similar antibody-dependent cellular cytotoxicity (ADCC) against CHO/HER2 and the breast cancer cell lines in the presence of effector splenocytes. In addition, humH2Mab-250 exhibited significant complement-dependent cellular cytotoxicity (CDC) in CHO/HER2 and the breast cancer cell lines compared to trastuzumab. Furthermore, humH2Mab-250 possesses compatible in vivo antitumor effects against CHO/HER2 and breast cancer xenografts with trastuzumab. These findings highlight the distinct roles of ADCC and CDC in the antitumor effects of humH2Mab-250 and trastuzumab and suggest a potential direction for the clinical development of humH2Mab-250 for HER2-positive tumors.
Establishment of a Highly-Sensitive and Specific anti-EphB2 Monoclonal Antibody Eb₂Mab-12 for Flow Cytometry

Rena Ubukata, Hiroyuki Suzuki, Miu Hirose, Hiroyuki Satofuka, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

2025/01/07

DOI： 10.20944/preprints202406.0704.v2 　
Establishment of a high-affinity anti-mouse CXCR5 monoclonal antibody for flow cytometry

Kenichiro Ishikawa, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

Microbes & Immunity　2　(1)　101-101　2024/12/26
Publisher: AccScience Publishing
DOI： 10.36922/mi.5664 　

ISSN： 3041-0886

eISSN： 3029-2883

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The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx5Mab-3; rat IgG2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx5Mab-3 for CHO/mCXCR5 cell is 7.2 &times; 10&minus;10 M. Furthermore, Cx5Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx5Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.
Development of specific anti-mouse atypical chemokine receptor 4 monoclonal antibodies. International-journal

Miu Hirose, Hiroyuki Suzuki, Rena Ubukata, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Biochemistry and biophysics reports　40　101824-101824　2024/12

DOI： 10.1016/j.bbrep.2024.101824 　

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Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K D) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10-9 M, 1.3 × 10-8 M, and 1.7 × 10-9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.
A Cancer-Specific Anti-Podoplanin Monoclonal Antibody, PMab-117-mG2a Exerts Antitumor Activities in Human Tumor Xenograft Models

Tomohiro Tanaka, Hiroyuki Suzuki, Tomokazu Ohishi, Mika K. Kaneko, Yukinari Kato

Cells　13　(22)　1833-1833　2024/11/06
Publisher: MDPI AG
DOI： 10.3390/cells13221833 　

eISSN： 2073-4409

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Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.
A Cancer-Specific Anti-Podoplanin Monoclonal Antibody, PMab-117-mG2a Exerts Antitumor Activities in Human Tumor Xenograft Models. International-journal

Tomohiro Tanaka, Hiroyuki Suzuki, Tomokazu Ohishi, Mika K Kaneko, Yukinari Kato

Cells　13　(22)　2024/11/06

DOI： 10.3390/cells13221833 　

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Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.
An Anti-Human EphA2 Monoclonal Antibody Ea₂Mab-7 Shows High Sensitivity for Flow Cytometry, Western Blot, and Immunohistochemical Analyses

Hiroyuki Satofuka, Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Mika K Kaneko, Yukinari Kato

2024/10/09

DOI： 10.20944/preprints202410.0572.v1 　
An Anti-Mouse CXCR5 Monoclonal Antibody (Cx₅Mab-3) Shows High Affinity for Flow Cytometry

Kenichiro Ishikawa, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

2024/10/08

DOI： 10.20944/preprints202410.0497.v1 　
Antitumor activities of anti‑CD44 monoclonal antibodies in mouse xenograft models of esophageal cancer

Kenichiro Ishikawa, Hiroyuki Suzuki, Tomokazu Ohishi, Takuro Nakamura, Miyuki Yanaka, Guanjie Li, Tomohiro Tanaka, Akira Ohkoshi, Manabu Kawada, Mika Kaneko, Yukio Katori, Yukinari Kato

Oncology Reports　52　(5)　2024/08/29
Publisher: Spandidos Publications
DOI： 10.3892/or.2024.8806 　

ISSN： 1021-335X

eISSN： 1791-2431
Anti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor Activity in Mouse Xenograft Models of Oral Squamous Cell Carcinomas

Kenichiro Ishikawa, Hiroyuki Suzuki, Tomokazu Ohishi, Guanjie Li, Tomohiro Tanaka, Manabu Kawada, Akira Ohkoshi, Mika K. Kaneko, Yukio Katori, Yukinari Kato

International Journal of Molecular Sciences　25　(17)　9190-9190　2024/08/24
Publisher: MDPI AG
DOI： 10.3390/ijms25179190 　

eISSN： 1422-0067

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CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3–10-overexpressed CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3–10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.
Anti-HER2 Cancer-Specific mAb, H2Mab-250-hG1, Possesses Higher Complement-Dependent Cytotoxicity than Trastuzumab. International-journal

Hiroyuki Suzuki, Tomokazu Ohishi, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

International journal of molecular sciences　25　(15)　2024/08/01

DOI： 10.3390/ijms25158386 　

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Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.
Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization. International-journal

Rena Ubukata, Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2024/06/13

DOI： 10.1089/mab.2024.0009 　

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One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.
Development of a Sensitive Anti-Mouse CCR5 Monoclonal Antibody for Flow Cytometry. International-journal

Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Tsunenori Ouchida, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2024/06/05

DOI： 10.1089/mab.2024.0004 　

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C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.
Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody (C₄₄Mab-108) Using Enzyme-Linked Immunosorbent Assay

Hiroyuki Suzuki, Mayuki Tawara, Aoi Hirayama, Nohara Goto, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　43　(3)　85-89　2024/06/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2023.0022 　

eISSN： 2167-9436
Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies Peer-reviewed

Tetsuya Inoue, Yuichiro Yamamoto, Kaoru Sato, Yuko Okemoto-Nakamura, Yoshimi Shimizu, Motohiko Ogawa, Taishi Onodera, Yoshimasa Takahashi, Takaji Wakita, Mika K. Kaneko, Masayoshi Fukasawa, Yukinari Kato, Kohji Noguchi

iScience　27　(4)　109363-109363　2024/04
Publisher: Elsevier BV
DOI： 10.1016/j.isci.2024.109363 　

ISSN： 2589-0042
Cx1Mab-1: A Novel Anti-mouse CXCR1 Monoclonal Antibody for Flow Cytometry. International-journal

Guanjie Li, Tomohiro Tanaka, Tsunenori Ouchida, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　43　(2)　59-66　2024/04

DOI： 10.1089/mab.2023.0031 　

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The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.
PMab-314: An Anti-Giant Panda Podoplanin Monoclonal Antibody. International-journal

Tsunenori Ouchida, Guanjie Li, Hiroyuki Suzuki, Miyuki Yanaka, Takuro Nakamura, Saori Handa, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　43　(2)　53-58　2024/04

DOI： 10.1089/mab.2024.0003 　

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The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.
Cx3Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal Antibody for Flow Cytometry. International-journal

Tsunenori Ouchida, Yu Isoda, Tomohiro Tanaka, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2024/03/20

DOI： 10.1089/mab.2023.0024 　

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C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.
Epitope Mapping of an Anti-Mouse CD39 Monoclonal Antibody Using PA Scanning and RIEDL Scanning. International-journal

Yuki Okada, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2024/03/20

DOI： 10.1089/mab.2023.0029 　

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A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.
C₈Mab-21: A Novel Anti-human CCR8 Monoclonal Antibody for Flow Cytometry

Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Mika K Kaneko, Yukinari Kato

2024/03/19

DOI： 10.20944/preprints202403.1166.v1 　
Locally misfolded HER2 expressed on cancer cells is a promising target for development of cancer-specific antibodies

Takao Arimori, Emiko Mihara, Hiroyuki Suzuki, Tomokazu Ohishi, Tomohiro Tanaka, Mika K. Kaneko, Junichi Takagi, Yukinari Kato

Structure　2024/03
Publisher: Elsevier BV
DOI： 10.1016/j.str.2024.02.007 　

ISSN： 0969-2126
A Cancer-Specific Monoclonal Antibody against HER2 Exerts Antitumor Activities in Human Breast Cancer Xenograft Models

Mika K. Kaneko, Hiroyuki Suzuki, Tomokazu Ohishi, Takuro Nakamura, Tomohiro Tanaka, Yukinari Kato

International Journal of Molecular Sciences　2024/02

DOI： 10.3390/ijms25031941 　
Development of a Sensitive Anti-Mouse CD39 Monoclonal Antibody (C39Mab-1) for Flow Cytometry and Western Blot Analyses. International-journal

Yuki Okada, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　43　(1)　24-31　2024/02

DOI： 10.1089/mab.2023.0016 　

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CD39 is involved in adenosine metabolism by converting extracellular ATP to adenosine. As extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. This study developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening method. The established anti-mCD39 mAb, C39Mab-1 (rat IgG2a, kappa), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant of C39Mab-1 for CHO/mCD39 was 7.3 × 10-9 M. Furthermore, C39Mab-1 detected the lysate of CHO/mCD39 by western blot analysis. These results indicated that C39Mab-1 is useful for the detection of mCD39 in many functional studies.
Establishment of Anti-Dog Programmed Cell Death Ligand 1 Monoclonal Antibodies for Immunohistochemistry. International-journal

Tsunenori Ouchida, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2024/01/18

DOI： 10.1089/mab.2023.0014 　

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Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa), and L1Mab-354 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed high-binding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 × 10-10 M and 7.2 × 10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-overexpressed cells. These results indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.
Establishment of a Novel Cancer-Specific Anti-HER2 Monoclonal Antibody H2Mab-250/H2CasMab-2 for Breast Cancers

Mika K Kaneko, Hiroyuki Suzuki, Tomokazu Ohishi, Tomohiro Tanaka, Yukinari Kato

2023/12/28

DOI： 10.20944/preprints202309.0906.v4 　
Establishment of a Novel Anti-mouse CCR1 Monoclonal Antibody C₁Mab-6

Tsunenori Ouchida, Yu Isoda, Tomohiro Tanaka, Mika K. Kaneko, Hiroyuki Suzuki, Yukinari Kato

2023/12/27

DOI： 10.20944/preprints202312.2059.v1 　
Antipodoplanin antibody enhances the antitumor effects of CTLA-4 blockade against malignant mesothelioma by natural killer cells. International-journal

Hiroto Yoneda, Atsushi Mitsuhashi, Aito Yoshida, Hirokazu Ogino, Satoshi Itakura, Na Thi Nguyen, Hiroshi Nokihara, Seidai Sato, Tsutomu Shinohara, Masaki Hanibuchi, Shinji Abe, Mika K Kaneko, Yukinari Kato, Yasuhiko Nishioka

Cancer science　2023/12/26

DOI： 10.1111/cas.16046 　

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Combination immunotherapy with multiple immune checkpoint inhibitors (ICIs) has been approved for various types of malignancies, including malignant pleural mesothelioma (MPM). Podoplanin (PDPN), a transmembrane sialomucin-like glycoprotein, has been investigated as a diagnostic marker and therapeutic target for MPM. We previously generated and developed a PDPN-targeting Ab reagent with high Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, the effects of anti-PDPN Abs on various tumor-infiltrating immune cells and their synergistic effects with ICIs have remained unclear. In the present study, we established a novel rat-mouse chimeric anti-mouse PDPN IgG2a mAb (PMab-1-mG2a ) and its core-fucose-deficient Ab (PMab-1-mG2a -f) to address these limitations. We identified the ADCC and CDC activity of PMab-1-mG2a -f against the PDPN-expressing mesothelioma cell line AB1-HA. The antitumor effect of monotherapy with PMab-1-mG2a -f was not sufficient to overcome tumor progression in AB1-HA-bearing immunocompetent mice. However, PMab-1-mG2a -f enhanced the antitumor effects of CTLA-4 blockade. Combination therapy with anti-PDPN Ab and anti-CTLA-4 Ab increased tumor-infiltrating natural killer (NK) cells. The depletion of NK cells inhibited the synergistic effects of PMab-1-mG2a -f and CTLA-4 blockade in vivo. These findings indicated the essential role of NK cells in novel combination immunotherapy targeting PDPN and shed light on the therapeutic strategy in advanced MPM.
Cx4Mab-1: A Novel Anti-Mouse CXCR4 Monoclonal Antibody for Flow Cytometry. International-journal

Tsunenori Ouchida, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2023/12/21

DOI： 10.1089/mab.2023.0023 　

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The CXC chemokine receptor 4 (CXCR4, CD184) is a member of the G protein-coupled receptor family that is expressed in most leukocytes. Overexpression of CXCR4 is associated with poor prognosis in not only hematopoietic malignancy but also solid tumors. Because CXCR4 is an attractive target for tumor therapy, reliable preclinical murine models using anti-CXCR4 monoclonal antibodies (mAbs) have been warranted. This study established a novel anti-mouse CXCR4 (mCXCR4) mAb using the Cell-Based Immunization and Screening method. Flow cytometric analysis showed that an anti-mCXCR4 mAb, Cx4Mab-1 (rat IgG2a, kappa), recognized mCXCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR4) cells and endogenously mCXCR4-expressing mouse myeloma P3X63Ag8U.1 (P3U1) cells. Furthermore, Cx4Mab-1 did not recognize mCXCR4-knockout P3U1 cells. The dissociation constants of Cx4Mab-1 for CHO/mCXCR4 and P3U1 were determined as 6.4 × 10-9 M and 2.3 × 10-9 M, respectively, indicating that Cx4Mab-1 possesses a high affinity to both endogenous and exogenous mCXCR4-expressing cells. These results indicate that Cx4Mab-1 could be a useful tool for preclinical mouse models.
Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding Peer-reviewed

Kohdai Yamada, Ryouhei Shioya, Kohei Nishino, Hirotake Furihata, Atsushi Hijikata, Mika K. Kaneko, Yukinari Kato, Tsuyoshi Shirai, Hidetaka Kosako, Tatsuya Sawasaki

Nature Communications　14　(1)　2023/12/14
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-023-43931-7 　

eISSN： 2041-1723

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Abstract Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment against EGFR (EGFR-FabID), which could then biotinylate the extracellular region of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors were identified as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were influenced by treatment with EGF ligand and its specific kinase inhibitor, gefitinib. These results indicate that FabID provides accurate proximity exPPI analysis of target receptor proteins on cell membranes with ligand and drug responses.
A Rat Anti-Mouse CD39 Monoclonal Antibody for Flow Cytometry. International-journal

Hiroyuki Suzuki, Tomohiro Tanaka, Yuma Kudo, Mayuki Tawara, Aoi Hirayama, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(6)　203-208　2023/12

DOI： 10.1089/mab.2023.0018 　

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By converting extracellular adenosine triphosphate to adenosine, CD39 is involved in adenosine metabolism. The extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment. Therefore, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is thought to be one of the important strategies for tumor therapy. In this study, we developed novel mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCD39 mAbs, C39Mab-2 (rat IgG2a, lambda), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) and an endogenously mCD39-expressed cell line (SN36) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of C39Mab-2 for CHO/mCD39 and SN36 were 5.5 × 10-9 M and 4.9 × 10-9 M, respectively. These results indicated that C39Mab-2 is useful for the detection of mCD39 in flow cytometry.
Epitope Mapping of an Anti-ferret Podoplanin Monoclonal Antibody Using the PA Tag-Substituted Analysis. International-journal

Yu Isoda, Mika K Kaneko, Tomohiro Tanaka, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(6)　189-193　2023/12

DOI： 10.1089/mab.2023.0026 　

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In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (VRPEDD) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292 recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.
Antitumor activities against breast cancers by an afucosylated <scp>anti‐HER2</scp> monoclonal antibody <scp>H₂Mab‐77‐mG_2a</scp>‐f

Tomohiro Tanaka, Hiroyuki Suzuki, Tomokazu Ohishi, Mika K. Kaneko, Yukinari Kato

Cancer Science　2023/11/09
Publisher: Wiley
DOI： 10.1111/cas.16008 　

ISSN： 1347-9032

eISSN： 1349-7006

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Abstract Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti‐HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2‐positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti‐HER2 mAb, H₂Mab‐77 (mouse IgG₁, kappa). This was then altered to produce H₂Mab‐77‐mG_2a‐f, an afucosylated mouse IgG_2a. In the present work, we examined the reactivity of H₂Mab‐77‐mG_2a‐f and antitumor effects against breast cancers in vitro and in vivo. BT‐474, an endogenously HER2‐expressing breast cancer cell line, was identified by H₂Mab‐77‐mG_2a‐f with a strong binding affinity (a dissociation constant [K_D]: 5.0 × 10⁻⁹ M). H₂Mab‐77‐mG_2a‐f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H₂Mab‐77‐mG_2a‐f demonstrated strong antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) for BT‐474 cells. MDA‐MB‐468, a HER2‐negative breast cancer cell line, was unaffected by H₂Mab‐77‐mG_2a‐f. Additionally, in the BT‐474‐bearing tumor xenograft model, H₂Mab‐77‐mG_2a‐f substantially suppressed tumor development when compared with the control mouse IgG_2a mAb. In contrast, the HER2‐negative MDA‐MB‐468‐bearing tumor xenograft model showed no response to H₂Mab‐77‐mG_2a‐f. These findings point to the possibility of H₂Mab‐77‐mG_2a‐f as a treatment regimen by showing that it has antitumor effects on HER2‐positive breast tumors.
A Cancer-Specific Monoclonal Antibody against Podocalyxin Exerted Antitumor Activities in Pancreatic Cancer Xenografts

Hiroyuki Suzuki, Tomokazu Ohishi, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

2023/10/20

DOI： 10.20944/preprints202310.1348.v1 　
A Humanized and Defucosylated Antibody against Podoplanin (humLpMab-23-f) Exerts Antitumor Activities in Human Lung Cancer and Glioblastoma Xenograft Models. International-journal

Hiroyuki Suzuki, Tomokazu Ohishi, Mika K Kaneko, Yukinari Kato

Cancers　15　(20)　2023/10/20

DOI： 10.3390/cancers15205080 　

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A cancer-specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa), was established in our previous study. We herein produced a humanized IgG1 version (humLpMab-23) and defucosylated form (humLpMab-23-f) of an anti-PDPN mAb to increase ADCC activity. humLpMab-23 recognized PDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN), PDPN-positive PC-10 (human lung squamous cell carcinoma), and LN319 (human glioblastoma) cells via flow cytometry. We then demonstrated that humLpMab-23-f induced ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10, and LN319 cells in vitro and exerted high antitumor activity in mouse xenograft models, indicating that humLpMab-23-f could be useful as an antibody therapy against PDPN-positive lung squamous cell carcinomas and glioblastomas.
Development of an Anti-EphB4 Monoclonal Antibody for Multiple Applications Against Breast Cancers

Ren Nanamiya, Hiroyuki Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2023/10/12

DOI： 10.1089/mab.2023.0015 　
PMab-301: An Anti-Giraffe Podoplanin Monoclonal Antibody for Immunohistochemistry

Tsunenori Ouchida, Tomohiro Tanaka, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

2023/10/09

DOI： 10.20944/preprints202310.0453.v1 　
Defucosylated Monoclonal Antibody (H2Mab-139-mG2a-f) Exerted Antitumor Activities in Mouse Xenograft Models of Breast Cancers against Human Epidermal Growth Factor Receptor 2. International-journal

Hiroyuki Suzuki, Tomokazu Ohishi, Ren Nanamiya, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Current issues in molecular biology　45　(10)　7734-7748　2023/09/23

DOI： 10.3390/cimb45100488 　

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The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot be recognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab-139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10-9 M and 7.7 × 10-9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities in mouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.
A Cancer-Specific Monoclonal Antibody against HER2 for Breast Cancers

Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

2023/09/14
Publisher: {MDPI} {AG}
DOI： 10.20944/preprints202309.0906.v1 　

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<jats:p>Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy such as trastuzumab. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs have been desired to reduce adverse effects. In this study, we provide a strategy for the selection of cancer-specific mAb against HER2. We screened more than 200 of anti-HER2 mAbs obtained by our laboratory and established a novel cancer-specific anti-HER2 antibody, H2Mab-250 (IgG1, kappa). H2Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 never showed reactivity to non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H2Mab-119 (IgG1, kappa) reacted with both cancer and normal epithelial cells. The epitope mapping revealed that H2Mab-250 recognized the domain VI of HER2 and the Trp614 mainly contributes to the recognition by H2Mab-250. In immunohistochemical analysis, H2Mab-250 exhibited a superior reactivity to HER2-positive breast cancer section compared to H2Mab-119. Importantly, H2Mab-250 never showed any reactivity to normal tissues by immunohistochemical analysis. The strategy to select cancer-specific mAbs would contribute to the development of novel antibodies and modalities for cancer therapy.</jats:p>
Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

Tsunenori Ouchida, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

International Journal of Translational Medicine　3　(3)　310-320　2023/08/10
Publisher: MDPI AG
DOI： 10.3390/ijtm3030022 　

eISSN： 2673-8937

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Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.
Establishment of a Novel Anti-Human CCR6 Monoclonal Antibody C₆Mab-19 with the High Binding Affinity in Flow Cytometry

Tomohiro Tanaka, Kaishi Kitamura, Hiroyuki Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2023/07/10
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2023.0004 　

eISSN： 2167-9436
Development of a Novel Anti-CD44 Variant 8 Monoclonal Antibody C44Mab-94 against Gastric Carcinomas

Hiroyuki Suzuki, Nohara Goto, Tomohiro Tanaka, Tsunenori Ouchida, Mika K. Kaneko, Yukinari Kato

Antibodies　12　(3)　45-45　2023/07/04
Publisher: MDPI AG
DOI： 10.3390/antib12030045 　

eISSN： 2073-4468

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Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy.
EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry

Nohara Goto, Hiroyuki Suzuki, Tomohiro Tanaka, Kenichiro Ishikawa, Tsunenori Ouchida, Mika K. Kaneko, Yukinari Kato

Antibodies　12　(3)　42-42　2023/06/21
Publisher: MDPI AG
DOI： 10.3390/antib12030042 　

eISSN： 2073-4468

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Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.
Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas

Kenichiro Ishikawa, Hiroyuki Suzuki, Mika K. Kaneko, Yukinari Kato

Current Issues in Molecular Biology　45　(7)　5248-5262　2023/06/21
Publisher: MDPI AG
DOI： 10.3390/cimb45070333 　

eISSN： 1467-3045

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Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3–10 and HSC-3 was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. Furthermore, C44Mab-18 detected CD44v3–10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.
Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells. International-journal

Motohide Murate, Noriko Yokoyama, Nario Tomishige, Ludovic Richert, Nicolas Humbert, Brigitte Pollet, Asami Makino, Nozomu Kono, Laura Mauri, Junken Aoki, Yasushi Sako, Sandro Sonnino, Naoko Komura, Hiromune Ando, Mika K Kaneko, Yukinari Kato, Kei-Ichiro Inamori, Jin-Ichi Inokuchi, Yves Mély, Kazuhisa Iwabuchi, Toshihide Kobayashi

Cellular and molecular life sciences : CMLS　80　(6)　167-167　2023/05/30

DOI： 10.1007/s00018-023-04813-9 　

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Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.
Recombinant production of antibody antigen-binding fragments with an N-terminal human growth hormone tag in mammalian cells. International-journal

Yuriko Adachi, Mika K Kaneko, Yukinari Kato, Terukazu Nogi

Protein expression and purification　208-209　106289-106289　2023/05/07

DOI： 10.1016/j.pep.2023.106289 　

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Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at ∼4 μg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.
A Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6 Was Established for Multiple Applications. International-journal

Hiroyuki Suzuki, Kaishi Kitamura, Nohara Goto, Kenichiro Ishikawa, Tsunenori Ouchida, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

International journal of molecular sciences　24　(9)　2023/05/07

DOI： 10.3390/ijms24098411 　

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Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.
Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C44Mab-3 for Multiple Applications against Pancreatic Carcinomas. International-journal

Yuma Kudo, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Antibodies (Basel, Switzerland)　12　(2)　2023/04/28

DOI： 10.3390/antib12020031 　

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Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant's (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant-5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3-10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3-10 and PK-1 was 1.3 × 10-9 M and 2.6 × 10-9 M, respectively. C44Mab-3 could detect the exogenous CD44v3-10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.
Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning. International-journal

Tomohiro Tanaka, Mayuki Tawara, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Antibodies (Basel, Switzerland)　12　(2)　2023/04/28

DOI： 10.3390/antib12020032 　

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CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1-20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.
Development of anti-feline PD-1 antibody and its functional analysis. International-journal

Shoma Nishibori, Mika K Kaneko, Takayuki Nakagawa, Kazuo Nishigaki, Yukinari Kato, Masaya Igase, Takuya Mizuno

Scientific reports　13　(1)　6420-6420　2023/04/24

DOI： 10.1038/s41598-023-31543-6 　

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Antibodies against immune checkpoint molecules restore T-cell function by inhibiting the binding of PD-1 and PD-L1 and have been shown to exert therapeutic effects in various human cancers. However, to date, no monoclonal antibody that recognizes feline PD-1 or PD-L1 has been reported, and there are many unknowns regarding the expression of immune checkpoint molecules and their potential as therapeutic targets in cats. Here we developed anti-feline PD-1 monoclonal antibody (1A1-2), and found that the monoclonal antibody against anti-canine PD-L1 (G11-6), which was previously developed in our laboratory, cross-reacted with feline PD-L1. Both antibodies inhibited the interaction of feline PD-1 and feline PD-L1 in vitro. These inhibitory monoclonal antibodies augmented the interferon-gamma (IFN-γ) production in activated feline peripheral blood lymphocytes (PBLs). Furthermore, for clinical application in cats, we generated a mouse-feline chimeric mAb by fusing the variable region of clone 1A1-2 with the constant region of feline IgG1 (ch-1A1-2). Ch-1A1-2 also augmented the IFN-γ production in activated feline PBLs. From this study, 1A1-2 is first anti-feline PD-1 monoclonal antibody with the ability to inhibit the interaction of feline PD-1 and PD-L1, and the chimeric antibody, ch-1A1-2 will be a beneficial therapeutic antibody for feline tumors.
A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers. International-journal

Mayuki Tawara, Hiroyuki Suzuki, Nohara Goto, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Current issues in molecular biology　45　(4)　3658-3673　2023/04/20

DOI： 10.3390/cimb45040238 　

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Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.
Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7). International-journal

Nami Tateyama, Teizo Asano, Tomohiro Tanaka, Yu Isoda, Yuki Okada, Hiyori Kobayashi, Guanjie Li, Ren Nanamiya, Takeo Yoshikawa, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(2)　68-72　2023/04/18

DOI： 10.1089/mab.2022.0034 　

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One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.
Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas International-journal

Hiroyuki Suzuki, Kazuki Ozawa, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

Biomedicines　11　(4)　2023/04/05

DOI： 10.3390/biomedicines11041099 　

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Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.
Development of Highly Sensitive Anti-Mouse CD39 Monoclonal Antibodies C39Mab-1 and C39Mab-2 for flow cytometry

Hiroyuki Suzuki, Yuma Kudo, Mayuki Tawara, Nohara Goto, Kenichiro Ishikawa, Tsunenori Ouchida, Tomohiro Tanaka, TEIZO ASANO, Takuro Nakamura, Miyuki Yanaka, Mika K. Kaneko, Yukinari Kato

2023/04/04
Publisher: {MDPI} {AG}
DOI： 10.20944/preprints202304.0032.v1 　

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<jats:p>CD39 is involved in adenosine metabolism through conversion of extracellular ATP to adenosine. Because extracellular adenosine plays a critical role in the immune suppression of tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. In this study, we developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCD39 mAbs, which were established by the CBIS method including C39Mab-1 (rat IgG2a, kappa) and C39Mab-2 (rat IgG2a, lambda), reacted with not only mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) but also endogenously mCD39-expressed cell lines, such as L1210 (mouse lymphocytic leukemia) and J774-1 (mouse macrophage-like) cell lines through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constant (KD) of C39Mab-1 and C39Mab-2 for CHO/mCD39 was 7.3 &times; 10&minus;9 M and 5.5 &times; 10&minus;9 M, respectively. KD of C39Mab-1 and C39Mab-2 for L1210 was 3.3 &times; 10&minus;9 M and 3.6 &times; 10&minus;10 M, respectively. Furthermore, C39Mab-1 could detect the lysate of CHO/mCD39 by western blot analysis. These results indicate that C39Mab-1 and C39Mab-2 are useful for the detection of mCD39 in many functional studies.</jats:p>
Epitope Mapping of the Novel Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by 2 × Alanine Scanning. International-journal

Yu Isoda, Tomohiro Tanaka, Hiroyuki Suzuki, Teizo Asano, Kaishi Kitamura, Yuma Kudo, Ryo Ejima, Kazuki Ozawa, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(2)　73-76　2023/04

DOI： 10.1089/mab.2022.0035 　

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We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb), C9Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA). This study aims to identify the binding epitope of C9Mab-11 by using 1 × and 2 × alanine (or glycine) substituted-hCCR9 peptides (1 × and 2 × Ala-scan) by ELISA. According to the 1 × Ala-scan analysis, the response of C9Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the 2 × Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, and M13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C9Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important. The successful identification of the C9Mab-11 epitope might be useful for the future pathophysiological analysis of hCCR9.
Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody C44Mab-108 for Immunohistochemistry. International-journal

Hiroyuki Suzuki, Tomohiro Tanaka, Nohara Goto, Mika K Kaneko, Yukinari Kato

Current issues in molecular biology　45　(3)　1875-1888　2023/02/25

DOI： 10.3390/cimb45030121 　

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CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10-7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.
Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas. International-journal

Ryo Ejima, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

International journal of molecular sciences　24　(4)　2023/02/16

DOI： 10.3390/ijms24044007 　

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CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.
Epitope Mapping of an Anti-EpCAM Monoclonal Antibody (EpMab-37) Using the Alanine Scanning Method. International-journal

Guanjie Li, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(1)　41-47　2023/02

DOI： 10.1089/mab.2022.0031 　

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The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.
Antitumor activities of a defucosylated anti‑EpCAM monoclonal antibody in colorectal carcinoma xenograft models. International-journal

Guanjie Li, Hiroyuki Suzuki, Tomokazu Ohishi, Teizo Asano, Tomohiro Tanaka, Miyuki Yanaka, Takuro Nakamura, Takeo Yoshikawa, Manabu Kawada, Mika K Kaneko, Yukinari Kato

International journal of molecular medicine　51　(2)　2023/02

DOI： 10.3892/ijmm.2023.5221 　

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Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.
Determination of the Binding Epitope of an Anti-Mouse CCR9 Monoclonal Antibody (C9Mab-24) Using the 1× Alanine and 2× Alanine-Substitution Method. International-journal

Hiyori Kobayashi, Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Antibodies (Basel, Switzerland)　12　(1)　2023/01/31

DOI： 10.3390/antib12010011 　

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C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A-F14A, F14A-D15A, D16A-F17A, and F17A-S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target-antibody interaction.
Current Targeted Therapy for Metastatic Colorectal Cancer. International-journal

Tomokazu Ohishi, Mika K Kaneko, Yukihiro Yoshida, Atsuo Takashima, Yukinari Kato, Manabu Kawada

International journal of molecular sciences　24　(2)　2023/01/15

DOI： 10.3390/ijms24021702 　

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Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer deaths worldwide. Surgery or surgery plus radiotherapy and/or chemotherapy for patients with metastatic CRC (mCRC) were accepted as the main therapeutic strategies until the early 2000s, when targeted drugs, like cetuximab and bevacizumab, were developed. The use of targeted drugs in clinical practice has significantly increased patients' overall survival. To date, the emergence of several types of targeted drugs has opened new possibilities and revealed new prospects for mCRC treatment. Therapeutic strategies are continually being updated to select the most suitable targeted drugs based on the results of clinical trials that are currently underway. This review discusses the up-to date molecular evidence of targeted therapy for mCRC and summarizes the Food and Drug Administration-approved targeted drugs including the results of clinical trials. We also explain their mechanisms of action and how these affect the choice of a suitable targeted therapy.
Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors. International-journal

Naoya Maekawa, Satoru Konnai, Yumie Asano, Takumi Otsuka, Eri Aoki, Hiroto Takeuchi, Yukinari Kato, Mika K Kaneko, Shinji Yamada, Yumiko Kagawa, Maki Nishimura, Satoshi Takagi, Tatsuya Deguchi, Hiroshi Ohta, Takayuki Nakagawa, Yasuhiko Suzuki, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi

PloS one　18　(1)　e0281143　2023

DOI： 10.1371/journal.pone.0281143 　

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Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.
SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing. International-journal

Yuichiro Yamamoto, Tetsuya Inoue, Miyu Inoue, Mana Murae, Masayoshi Fukasawa, Mika K Kaneko, Yukinari Kato, Kohji Noguchi

International journal of molecular sciences　23　(24)　2022/12/13

DOI： 10.3390/ijms232415834 　

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.
Establishment of a Sensitive Monoclonal Antibody Against Mouse CCR9 (C₉Mab-24) for Flow Cytometry

Hiyori Kobayashi, Teizo Asano, Hiroyuki Suzuki, Tomohiro Tanaka, Takeo Yoshikawa, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2022/12/13
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2022.0032 　

eISSN： 2167-9436
Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2. International-journal

Mitsuhiro Machitani, Junko Takei, Mika K Kaneko, Saori Ueki, Hirofumi Ohashi, Koichi Watashi, Yukinari Kato, Kenkichi Masutomi

Virology journal　19　(1)　213-213　2022/12/10

DOI： 10.1186/s12985-022-01948-2 　

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A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.
Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

Nami Tateyama, Teizo Asano, Hiroyuki Suzuki, Guanjie Li, Takeo Yoshikawa, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

Antibodies　11　(4)　75-75　2022/12/02
Publisher: MDPI AG
DOI： 10.3390/antib11040075 　

eISSN： 2073-4468

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The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.
A Defucosylated Anti-EpCAM Monoclonal Antibody (EpMab-37-mG2a-f) Exerts Antitumor Activity in Xenograft Model

Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Tomokazu Ohishi, Manabu Kawada, Takeo Yoshikawa, Mika K. Kaneko, Yukinari Kato

Antibodies　11　(4)　74-74　2022/11/24
Publisher: MDPI AG
DOI： 10.3390/antib11040074 　

eISSN： 2073-4468

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The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (KD): 2.9 × 10−8 M and 1.8 × 10−8 M, respectively] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers.
Defucosylated Mouse-Dog Chimeric Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody (H77Bf) Exerts Antitumor Activities in Mouse Xenograft Models of Canine Osteosarcoma. International-journal

Ren Nanamiya, Tomokazu Ohishi, Hiroyuki Suzuki, Takuya Mizuno, Takeo Yoshikawa, Teizo Asano, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　42　(1)　27-33　2022/11/18

DOI： 10.1089/mab.2022.0022 　

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Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an important therapeutic target in various cancer types, including breast and gastric cancers. We previously developed an anti-HER2 monoclonal antibody (mAb), H2Mab-77 (mouse IgG1, kappa), which detects HER2 and dog HER2 (dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors and could be valuable as part of an antibody treatment regimen for them.
Antitumor Activity of an Anti-EGFR/HER2 Bispecific Antibody in a Mouse Xenograft Model of Canine Osteosarcoma

Nami Tateyama, Hiroyuki Suzuki, Tomokazu Ohishi, Teizo Asano, Tomohiro Tanaka, Takuya Mizuno, Takeo Yoshikawa, Manabu Kawada, Mika K. Kaneko, Yukinari Kato

Pharmaceutics　14　(11)　2494-2494　2022/11/17
Publisher: MDPI AG
DOI： 10.3390/pharmaceutics14112494 　

eISSN： 1999-4923

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The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3 × 10−9 M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.
Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization. International-journal

Tomohiro Tanaka, Hiroyuki Suzuki, Yu Isoda, Teizo Asano, Takuro Nakamura, Miyuki Yanaka, Saori Handa, Nozomi Takahashi, Saori Okuno, Takeo Yoshikawa, Guanjie Li, Ren Nanamiya, Nohara Goto, Nami Tateyama, Yuki Okada, Hiyori Kobayashi, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(6)　303-310　2022/11/16

DOI： 10.1089/mab.2022.0027 　

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The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.
Development of a Novel Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) by N-Terminal Peptide Immunization. International-journal

Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Ren Nanamiya, Nami Tateyama, Yu Isoda, Yuki Okada, Hiyori Kobayashi, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(6)　343-349　2022/11/16

DOI： 10.1089/mab.2022.0021 　

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The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.
Epitope Mapping Using the Cell-Based 2 × Alanine Substitution Method About the Anti-mouse CXCR6 Monoclonal Antibody, Cx₆Mab-1

Yu Isoda, Tomohiro Tanaka, Hiroyuki Suzuki, Teizo Asano, Takeo Yoshikawa, Kaishi Kitamura, Yuma Kudo, Ryo Ejima, Kazuki Ozawa, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2022/11/16
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2022.0029 　

eISSN： 2167-9436
Epitope Mapping of an Anti-Mouse CCR2 Monoclonal Antibody (C2Mab-6) Using Enzyme-Linked Immunosorbent Assay. International-journal

Tomohiro Tanaka, Hiroyuki Suzuki, Teizo Asano, Guanjie Li, Ren Nanamiya, Nami Tateyama, Yu Isoda, Yuki Okada, Hiyori Kobayashi, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(6)　339-342　2022/11/07

DOI： 10.1089/mab.2022.0020 　

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CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C2Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C2Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C2Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C2Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.
近接ビオチン化酵素AirID融合抗体を用いた膜タンパク質細胞外ドメイン相互作用解析

山田航大, 塩屋亮平, 金子美華, 西野耕平, 加藤幸成, 小迫英尊, 澤崎達也

日本生化学会大会プログラム・講演要旨集　95回　1P-209　2022/11
Publisher: (公社)日本生化学会
CBIS法を用いた新規抗EpCAM抗体の樹立

浅野禎三, 李冠傑, 鈴木裕之, 田中智大, 金子美華, 加藤幸成

日本生化学会大会プログラム・講演要旨集　95回　1P-393　2022/11
Publisher: (公社)日本生化学会
Epitope Mapping of the Anti-Human CC Chemokine Receptor Type-2 Monoclonal Antibody (K036C2). International-journal

Tomohiro Tanaka, Hiroyuki Suzuki, Guanjie Li, Ren Nanamiya, Yu Isoda, Yuki Okada, Hiyori Kobayashi, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(5)　285-289　2022/10

DOI： 10.1089/mab.2022.0018 　

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CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, and is localized on cell surface of tumor cells and some immune cells, including monocytes and macrophages. CCR2 is a receptor for monocyte chemoattractant protein-1/C-C motif chemokine 2, and is involved in the progression of various diseases such as cancers. Therefore, the development of CCR2-targeted monoclonal antibody (mAb) is desired. Its characterization, including epitope of mAb, is very important for antibody applications. In this study, we investigated the critical epitope of K036C2, which is a commercially available anti-human CCR2 (hCCR2) mAb. We conducted enzyme-linked immunosorbent assay (ELISA) using three N-terminal peptides of hCCR2 and demonstrated that K036C2 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. K036C2 lost the reaction to the alanine-substituted peptides of D25A, Y26A, D27A, G29A, and A30G. These results indicate that the critical binding epitope of K036C2 includes Asp25, Tyr26, Asp27, Gly29, and Ala30 of hCCR2.
Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6Mab-1) Using the 2 × Alanine Scanning Method. International-journal

Yu Isoda, Tomohiro Tanaka, Hiroyuki Suzuki, Teizo Asano, Takuro Nakamura, Miyuki Yanaka, Saori Handa, Yu Komatsu, Saori Okuno, Nozomi Takahashi, Yuki Okada, Hiyori Kobayashi, Guanjie Li, Ren Nanamiya, Nohara Goto, Nami Tateyama, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(5)　275-278　2022/10

DOI： 10.1089/mab.2022.0019 　

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The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.
KLMab-1: An Anti-human KLRG1 Monoclonal Antibody for Immunocytochemistry. International-journal

Masaki Saito, Hiroyuki Suzuki, Teizo Asano, Tomohiro Tanaka, Takeo Yoshikawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(5)　279-284　2022/10

DOI： 10.1089/mab.2022.0016 　

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Immune checkpoint molecules have received attention as targets of cancer immunotherapy. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is one of the immune checkpoint molecules expressed in CD4+ T, CD8+ T, and natural killer (NK) cells. KLRG1 exhibits antiviral and antitumor immunity, and its expression in T and NK cells is upregulated by viral infectious diseases and some tumors. Thus, monoclonal antibodies (mAbs) for KLRG1 would be useful tools for the diagnosis and immunotherapy against viral infectious diseases and cancers. We have developed anti-human KLRG1 (hKLRG1) mAb (clone KLMab-1, mouse IgG1, kappa) by the Cell-Based Immunization and Screening method. We have also demonstrated that KLMab-1 recognizes both exogenous and endogenous hKLRG1 in flow cytometry. In this study, we first showed that KLMab-1 and its recombinant mAb (recKLMab-1) bound to exogenous hKLRG1 overexpressed in Chinese hamster ovary (CHO)-K1 cells, but not in parental CHO-K1 cells, in immunocytochemistry. We next showed that both mAbs detected endogenous hKLRG1 expressed in human NK cells. These results demonstrate that KLMab-1 and recKLMab-1 are available for immunocytochemistry.
脱フコシル化キメラ抗EGFR抗体の犬がんモデルに対する抗腫瘍効果(Defucosylated mouse-dog chimeric anti-EGFR antibody exerts antitumor activity in canine cancer models)

田中智大, 大石智一, 鈴木裕之, 金子美華, 川田学, 加藤幸成

日本癌学会総会記事　81回　P-3047　2022/09
Publisher: (一社)日本癌学会
ISSN： 0546-0476
Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors. International-journal

Hiroyuki Suzuki, Tomokazu Ohishi, Teizo Asano, Tomohiro Tanaka, Masaki Saito, Takuya Mizuno, Takeo Yoshikawa, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Oncology reports　48　(3)　2022/09

DOI： 10.3892/or.2022.8366 　

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Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti‑HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2‑positive breast and gastric cancer. An anti‑HER2 mAb, H2Mab‑77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse‑dog chimeric anti‑HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding‑affinity [a dissociation constant (KD): 7.5x10‑10 M, as determined by flow cytometric analysis] for dog HER2‑overexpressed CHO‑K1 (CHO/dHER2) cells. H77Bf highly exerted antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 xenograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding‑affinity (KD: 7.2x10‑10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2‑positive canine cancers, and could be valuable treatment regimen for canine cancers.
Epitope Mapping of an Anti-elephant Podoplanin Monoclonal Antibody (PMab-295) Using Enzyme-Linked Immunosorbent Assay. International-journal

Yuki Okada, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(4)　221-227　2022/08/02

DOI： 10.1089/mab.2022.0017 　

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Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.
Development of a Monoclonal Antibody PMab-295 Against Elephant Podoplanin. International-journal

Yuma Kudo, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(4)　194-201　2022/08/02

DOI： 10.1089/mab.2022.0007 　

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Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.
Development of an Anti-human CCR2 Monoclonal Antibody (C2Mab-9) by N-Terminal Peptide Immunization. International-journal

Tomohiro Tanaka, Guanjie Li, Masaki Saito, Hiroyuki Suzuki, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(4)　188-193　2022/08/02

DOI： 10.1089/mab.2022.0001 　

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The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells, including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), using the synthetic peptide immunization method. Flow cytometric and immunocytochemical results showed that C2Mab-9 reacted with hCCR2-expressing U937 (human histiocytic lymphoma) and natural killer cells. Furthermore, C2Mab-9 showed the moderate binding affinity for both cells. Conclusively, C2Mab-9 can be a useful tool for analyzing hCCR2-related biological responses.
Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal Antibody, C4Mab-1. International-journal

Teizo Asano, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(4)　214-220　2022/08/02

DOI： 10.1089/mab.2022.0015 　

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C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.
Improved anti-solid tumor response by humanized anti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model. International-journal

Akihiro Ishikawa, Masazumi Waseda, Tomoko Ishii, Mika K Kaneko, Yukinari Kato, Shin Kaneko

Genes to cells : devoted to molecular & cellular mechanisms　27　(9)　549-558　2022/07/05

DOI： 10.1111/gtc.12972 　

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Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional non-humanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in non-humanized NZ-1 CAR-T cells. This article is protected by copyright. All rights reserved.
Development of a Novel Anti-EpCAM Monoclonal Antibody for Various Applications. International-journal

Guanjie Li, Hiroyuki Suzuki, Teizo Asano, Tomohiro Tanaka, Hiroyoshi Suzuki, Mika K Kaneko, Yukinari Kato

Antibodies (Basel, Switzerland)　11　(2)　2022/06/08

DOI： 10.3390/antib11020041 　

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The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10-8 M and 3.2 × 10-8 M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications.
Epitope Mapping of the Anti-Human CCR2 Monoclonal Antibody C2Mab-9. International-journal Peer-reviewed

Tomohiro Tanaka, Guanjie Li, Teizo Asano, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(3)　150-156　2022/06/06

DOI： 10.1089/mab.2022.0012 　

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CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, localized on cell surface of some immune-related cells, including monocytes and macrophages. CCR2 and its ligand CCL2 are involved in the progression of various diseases such as cancers. Therefore, CCR2-targeted monoclonal antibodies (mAbs) are needed for treatment and diagnosis. Previously, we successfully developed an anti-human CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C2Mab-9. We conducted enzyme-linked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C2Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C2Mab-9 lost the reaction to the alanine-substituted peptides of F23A, F24A, D25A, Y26A, and D27A. Among them, F23A, F24A, D25A, and Y26A did not block the C2Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C2Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.
Epitope Mapping of an Anti-Chinese/Golden Hamster Podoplanin Monoclonal Antibody. International-journal Peer-reviewed

Nohara Goto, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(3)　163-169　2022/06/06

DOI： 10.1089/mab.2022.0014 　

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Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.
Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Exerted Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor. International-journal Peer-reviewed

Tomohiro Tanaka, Tomokazu Ohishi, Masaki Saito, Hiroyuki Suzuki, Mika K Kaneko, Manabu Kawada, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(3)　142-149　2022/06/06

DOI： 10.1089/mab.2022.0009 　

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The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors, and could be valuable as part of an antibody treatment regimen for them.
Cx6Mab-1: A Novel Anti-Mouse CXCR6 Monoclonal Antibody Established by N-Terminal Peptide Immunization. International-journal

Kaishi Kitamura, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(3)　133-141　2022/06

DOI： 10.1089/mab.2022.0010 　

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The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells in tumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx6Mab-1 (rat IgG1, kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx6Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 × 10-9 M, 3.4 × 10-7 M, and 3.8 × 10-7 M, respectively. Furthermore, Cx6Mab-1 could detect endogenous mCXCR6 in P388 and J774-1 cells by Western blotting. These results indicated that Cx6Mab-1 is useful for detecting mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing cells in vivo experiments.
TgMab-2: An Anti-human T Cell Immunoglobulin and Immunoreceptor Tyrosine-Based Inhibitory Motif Domain Monoclonal Antibody for Immunocytochemistry

Masaki Saito, Hiroyuki Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　41　(3)　157-162　2022/06/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2022.0013 　

eISSN： 2167-9436
Phosphorylation of hTERT at threonine 249 is a novel tumor biomarker of aggressive cancer with poor prognosis in multiple organs. International-journal

Yoko Matsuda, Taro Yamashita, Juanjuan Ye, Mami Yasukawa, Keiko Yamakawa, Yuri Mukai, Mitsuhiro Machitani, Yataro Daigo, Yohei Miyagi, Tomoyuki Yokose, Takashi Oshima, Hiroyuki Ito, Soichiro Morinaga, Takeshi Kishida, Toshinari Minamoto, Shinji Yamada, Junko Takei, Mika K Kaneko, Motohiro Kojima, Shuichi Kaneko, Tsutomu Masaki, Masahiro Hirata, Reiji Haba, Keiichi Kontani, Nobuhiro Kanaji, Nobuyuki Miyatake, Keiichi Okano, Yukinari Kato, Kenkichi Masutomi

The Journal of pathology　257　(2)　172-185　2022/06

DOI： 10.1002/path.5876 　

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Recent evidence indicates that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates expression of target genes and is directly involved in tumor formation in a telomere-independent manner. Non-canonical function of hTERT has been considered as a therapeutic target for cancer therapy. We have previously shown that hTERT phosphorylation at threonine 249 (p-hTERT), which promotes RdRP activity, is an indicator of an aggressive phenotype and poor prognosis in liver and pancreatic cancers, using two cohorts with small sample sizes with polyclonal p-hTERT antibody. To clarify the clinical relevance of p-hTERT, we developed a specific monoclonal antibody and determined the diagnostic and prognostic value of p-hTERT in cancer specimens using a large cohort. A monoclonal antibody for phosphorylated hTERT (p-hTERT) at threonine 249 was developed and validated. The antibody was used for the immunohistochemical staining of formalin-fixed, paraffin-embedded specimens from 1523 cases of lung, colon, stomach, pancreatic, liver, breast, and kidney cancers. We detected elevated p-hTERT expression levels in cases with a high mitotic activity, high pathological grade, and high nuclear pleomorphism. Elevated p-hTERT expression was an independent prognostic factor for lung, pancreatic, and liver cancers. Furthermore, p-hTERT expression was associated with immature and aggressive features, such as adenosquamous carcinoma (lung and pancreas), invasive type of cancer (lung), high serum alpha-fetoprotein level (liver), and triple-negative status (breast). In conclusion, RdRP activity indicated by p-hTERT expression predicts aggressive cancer phenotypes in various types of cancer. Thus, p-hTERT is a novel biomarker for the diagnosis of aggressive cancers with a poor prognosis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine. International-journal

Mahabub Alam, Hiroki Shima, Yoshitaka Matsuo, Nguyen Chi Long, Mitsuyo Matsumoto, Yusho Ishii, Nichika Sato, Takato Sugiyama, Risa Nobuta, Satoshi Hashimoto, Liang Liu, Mika K Kaneko, Yukinari Kato, Toshifumi Inada, Kazuhiko Igarashi

The Journal of biological chemistry　298　(7)　102084-102084　2022/05/27

DOI： 10.1016/j.jbc.2022.102084 　

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Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.
Development of a Novel Anti-CD44 Monoclonal Antibody for Multiple Applications against Esophageal Squamous Cell Carcinomas. International-journal Peer-reviewed

Nohara Goto, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

International journal of molecular sciences　23　(10)　2022/05/16

DOI： 10.3390/ijms23105535 　

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CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo-resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3-10) ectodomain and screening using enzyme-linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab-46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab-46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10-8 M, 4.9 × 10-8 M, and 4.1 × 10-8 M, respectively. C44Mab-46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab-46 strongly stained the formalin-fixed paraffin-embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab-46 is very useful for detecting CD44 in various applications.
Development of Monoclonal Antibody 281-mG2a-f Against Golden Hamster Podoplanin. International-journal Peer-reviewed

Ren Nanamiya, Hiroyuki Suzuki, Junko Takei, Guanjie Li, Nohara Goto, Hiroyuki Harada, Masaki Saito, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2022/04/27

DOI： 10.1089/mab.2021.0058 　

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Golden (Syrian) hamster (Mesocricetus auratus) is a small animal model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Pathological analyses of the tissues are required to understand the pathogenesis of SARS-CoV-2 and the evaluation of therapeutic modalities, including neutralizing monoclonal antibodies (mAbs). However, mAbs that recognize the golden hamster-derived antigens and distinguish specific cell types, such as the pneumocytes, are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-Chinese hamster (Cricetulus griseus) PDPN mAb PMab-281 (IgG3, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. A defucosylated mouse IgG2a version of PMab-281 (281-mG2a-f) was also developed. The 281-mG2a-f strongly recognized both the Chinese hamster and the golden hamster PDPN using flow cytometry and could detect lung type I alveolar epithelial cells, lymphatic endothelial cells, and Bowman's capsules in the kidney from the golden hamster using immunohistochemistry. These results suggest the usefulness of 281-mG2a-f for analyzing the golden hamster-derived tissues and cells for SARS-CoV-2 research.
Development of an Anti-Mouse CCR8 Monoclonal Antibody (C8Mab-1) for Flow Cytometry and Immunocytochemistry. International-journal Peer-reviewed

Masaki Saito, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2022/04/27

DOI： 10.1089/mab.2021.0069 　

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It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells and many cancers and is associated with the progression of the cancers. However, its role in cancer progression remains unclear. Thus, the development of mAbs for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-1, rat IgG2a, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C8Mab-1 and its recombinant antibody (recC8Mab-1) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry and immunofluorescence. Moreover, C8Mab-1 and recC8Mab-1 specifically reacted to P388 (a mouse lymphocyte-like cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C8Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.
A Defucosylated Mouse Anti-CD10 Monoclonal Antibody (31-mG2a-f) Exerts Antitumor Activity in a Mouse Xenograft Model of Renal Cell Cancers. International-journal Peer-reviewed

Hiroki Kawabata, Tomokazu Ohishi, Hiroyuki Suzuki, Teizo Asano, Manabu Kawada, Hiroyoshi Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　2022/04/27

DOI： 10.1089/mab.2021.0049 　

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CD10 is a cell surface metalloendopeptidase that cleaves and degrades many secreted physiologically active peptides by its enzymatic activity. Although CD10 expression has been found in various types of cells, its expression is increased in several cancers, including renal cancer. In this study, the antitumor activity of a novel anti-human CD10 monoclonal antibody (mAb) was investigated. A defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) was created from an anti-CD10 mAb, C10Mab-31 (IgG1, kappa). Both C10Mab-31 and 31-mG2a-f specifically reacted with endogenous CD10 in renal cancer cells, VMRC-RCW, with the dissociation constant (KD) values of 6.3 × 10-9 M and 1.1 × 10-9 M, respectively, indicating high binding affinity. To further examine the anti-CD10 mAb-mediated effector functions, the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were examined. The 31-mG2a-f significantly exhibited ADCC and CDC against VMRC-RCW cells in vitro. Furthermore, 31-mG2a-f exhibited antitumor activities in mouse xenografts of VMRC-RCW cells. These results suggest that 31-mG2a-f exerts antitumor activities against CD10-expressing renal cancers and could be a valuable therapeutic candidate for treating them.
Development of a Novel Anti-Mouse CCR2 Monoclonal Antibody (C2Mab-6) by N-Terminal Peptide Immunization. International-journal Peer-reviewed

Tomohiro Tanaka, Guanjie Li, Teizo Asano, Masaki Saito, Mika K Kaneko, Hiroyuki Suzuki, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　80-86　2022/04/04

DOI： 10.1089/mab.2021.0063 　

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The CC chemokine receptor type-2 (CCR2) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of immune cells and tumors. CCR2 binds to the CC motif chemokine 2/monocyte chemoattractant protein-1, a CC chemokine, which is produced by various cells, including immune-related cells and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. This study established a novel, specific, and sensitive anti-mouse CCR2 (mCCR2) mAb; C2Mab-6 (rat IgG1, kappa), using the mCCR2 synthetic peptide immunization method. C2Mab-6 reacted with mCCR2-overexpressed Chinese hamster ovary-K1 cells and L1210 (murine leukemia) cells, which express endogenous mCCR2 in flow cytometry. Furthermore, C2Mab-6 showed a high binding affinity for both cells. Hence, C2Mab-6 can be a useful tool for analyzing mCCR2-related biological responses, using flow cytometry.
Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody. International-journal Peer-reviewed

Nohara Goto, Hiroyuki Suzuki, Tomokazu Ohishi, Akiko Harakawa, Guanjie Li, Masaki Saito, Junko Takei, Tomohiro Tanaka, Teizo Asano, Masato Sano, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　67-73　2022/04/04

DOI： 10.1089/mab.2021.0059 　

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The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFR-expressing canine fibroblastic tumors.
C8Mab-3: An Anti-Mouse CCR8 Monoclonal Antibody for Immunocytochemistry. International-journal Peer-reviewed

Hiroyuki Suzuki, Masaki Saito, Teizo Asano, Tomohiro Tanaka, Kaishi Kitamura, Yuma Kudo, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　110-114　2022/04/04

DOI： 10.1089/mab.2022.0002 　

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The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also expressed in many cancers and is associated with those progression. The development of monoclonal antibodies (mAbs) for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-3, rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C8Mab-3 and its recombinant antibody (recC8Mab-3) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry. In addition, C8Mab-3 and recC8Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C8Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C8Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.
Establishment of Novel Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7) by N-terminal Peptide Immunization. International-journal Peer-reviewed

Teizo Asano, Hiroyuki Suzuki, Nohara Goto, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　94-100　2022/04

DOI： 10.1089/mab.2021.0065 　

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The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 × 10-9 M, 1.4 × 10-7 M, and 1.7 × 10-7 M, respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 × 10-9 M, 5.1 × 10-7 M, and 3.1 × 10-7 M, respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3 through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.
Development of a Monoclonal Antibody PMab-292 Against Ferret Podoplanin. International-journal Peer-reviewed

Nohara Goto, Hiroyuki Suzuki, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　101-109　2022/04

DOI： 10.1089/mab.2021.0067 　

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Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.
C8Mab-2: An Anti-Mouse C-C Motif Chemokine Receptor 8 Monoclonal Antibody for Immunocytochemistry. International-journal Peer-reviewed

Masaki Saito, Tomohiro Tanaka, Teizo Asano, Takuro Nakamura, Miyuki Yanaka, Saori Handa, Yu Komatsu, Yasuhiro Harigae, Nami Tateyama, Ren Nanamiya, Guanjie Li, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　115-119　2022/04

DOI： 10.1089/mab.2021.0045 　

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C-C motif chemokine receptor 8 (CCR8) is a G protein-coupled receptor predominantly expressed in regulatory T (Treg) and T helper 2 cells. The evidence that CCR8 expression in Treg is increased in cancers, CCR8 increases migration activity of Treg, and CCR8 induces the anti-apoptotic activity in T cell leukemia and lymphoma suggests that CCR8 is associated with cancer development. Thus, developing a specific monoclonal antibody (mAb) for CCR8 is useful for diagnostic and therapeutic purposes and the anti-CCR8 mAb becomes a remarkable experimental tool for basic research. We previously developed an anti-mouse CCR8 (mCCR8) mAb called C8Mab-2 (rat IgG2b, kappa) that was applicable to flow cytometric analysis for both endogenous and exogenous mCCR8. This study showed that C8Mab-2 and recombinant C8Mab-2 (recC8Mab-2) were specifically bound to exogenously expressed mCCR8 in mCCR8-overexpressed Chinese hamster ovary-K1 cells. In addition, we found that C8Mab-2 and recC8Mab-2 recognized endogenous mCCR8 in P388 (a mouse lymphocyte-like cell line) and J774-1 cells (a mouse macrophage-like cell line). These data demonstrate that C8Mab-2 and recC8Mab-2 are useful for immunocytochemical analysis.
A Defucosylated Mouse Anti-CD10 Monoclonal Antibody (31-mG2a-f) Exerts Antitumor Activity in a Mouse Xenograft Model of CD10-Overexpressed Tumors. International-journal Peer-reviewed

Hiroki Kawabata, Hiroyuki Suzuki, Tomokazu Ohishi, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　59-66　2022/04

DOI： 10.1089/mab.2021.0048 　

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CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Therefore, antibodies against CD10 are used for diagnosing follicular lymphoma and solid tumors, including renal carcinomas. In this study, we developed an anti-human CD10 monoclonal antibody, clone C10Mab-31 (IgG1, kappa), which detects CD10 by flow cytometry and shows high affinity for CD10-overexpressed CHO-K1 (CHO/CD10) cells. Furthermore, the defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) exhibits antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antitumor activities in mouse xenografts of CHO/CD10 cells. These results indicate that 31-mG2a-f exerts antitumor effects against CD10-expressing tumors and could be valuable as part of an antibody treatment regimen for them.
C3Mab-3: A Monoclonal Antibody for Mouse CC Chemokine Receptor 3 for Flow Cytometry. International-journal Peer-reviewed

Teizo Asano, Hiroyuki Suzuki, Tomohiro Tanaka, Masaki Saito, Guanjie Li, Nohara Goto, Ren Nanamiya, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　74-79　2022/04

DOI： 10.1089/mab.2021.0062 　

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CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed in eosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 was found in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be a useful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the Cell-Based Immunization and Screening method. The established anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry. C3Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells, which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that KDs of C3Mab-3 for CHO/mCCR3, P388, and J774-1 cells were 4.3 × 10-8 M, 2.6 × 10-7 M, and 2.4 × 10-7 M, respectively. C3Mab-3 could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.
Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor by Defucosylated Mouse-Dog Chimeric Anti-Epidermal Growth Factor Receptor Antibody (E134Bf). International-journal Peer-reviewed

Guanjie Li, Hiroyuki Suzuki, Junko Takei, Teizo Asano, Masato Sano, Tomohiro Tanaka, Hiroyuki Harada, Takuya Mizuno, Tomokazu Ohishi, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　53-58　2022/04

DOI： 10.1089/mab.2021.0040 　

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The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.
C9Mab-1: An Anti-Mouse CCR9 Monoclonal Antibody for Immunocytochemistry. International-journal Peer-reviewed

Masaki Saito, Hiroyuki Suzuki, Yasuhiro Harigae, Guanjie Li, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　120-124　2022/04

DOI： 10.1089/mab.2021.0052 　

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C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.
Development of a Novel Anti-Mouse CCR4 Monoclonal Antibody (C4Mab-1) by N-Terminal Peptide Immunization. International-journal Peer-reviewed

Junko Takei, Hiroyuki Suzuki, Teizo Asano, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(2)　87-93　2022/04

DOI： 10.1089/mab.2021.0064 　

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The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10-9 M, 5.4 × 10-7 M, and 1.1 × 10-6 M, respectively. C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.
Roles of Podoplanin in Malignant Progression of Tumor. International-journal

Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Cells　11　(3)　2022/02/07

DOI： 10.3390/cells11030575 　

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Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial-to-mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is upregulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating its involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in the malignant progression of tumors and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.
Epitope Mapping of the Anti-CD20 Monoclonal Antibodies (C20Mab-11 and 2H7) Using HisMAP Method. International-journal

Hiroyuki Suzuki, Teizo Asano, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　20-26　2022/02

DOI： 10.1089/mab.2021.0051 　

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CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.
C3Mab-2: An Anti-Mouse CCR3 Monoclonal Antibody for Immunocytochemistry. International-journal

Masaki Saito, Yasuhiro Harigae, Guanjie Li, Teizo Asano, Tomohiro Tanaka, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　45-49　2022/02

DOI： 10.1089/mab.2021.0050 　

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The C-C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4, and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressed in eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic and therapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognized endogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cell line) cells and are usable in immunocytochemistry.
Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody (134-mG2a-f) Exerts Antitumor Activities in Mouse Xenograft Models of Canine Osteosarcoma. International-journal

Ren Nanamiya, Junko Takei, Tomokazu Ohishi, Teizo Asano, Tomohiro Tanaka, Masato Sano, Takuro Nakamura, Miyuki Yanaka, Saori Handa, Nami Tateyama, Yasuhiro Harigae, Masaki Saito, Hiroyuki Suzuki, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　1-7　2022/02

DOI： 10.1089/mab.2021.0036 　

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore, 134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry. In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cell line, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly suppressed the development of D-17 compared with the results in response to control mouse IgG. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could be valuable as part of an antibody treatment regimen for them.
Epitope Mapping of an Anti-CD10 Monoclonal Antibody (MME/1870) Using Enzyme-Linked Immunosorbent Assay. International-journal

Hiroki Kawabata, Hiroyuki Suzuki, Junko Takei, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　15-19　2022/02

DOI： 10.1089/mab.2021.0046 　

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CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. CD10 is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Antibodies against CD10 are used for the diagnosis of follicular lymphoma. Anti-human CD10 monoclonal antibody (clone MME/1870) can be used for Western blotting and immunohistochemical analyses. This study examined the critical epitope of MME/1870 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and MME/1870 reacted to the 501-520 amino acid sequence of CD10. Next, we analyzed the reaction to 20 point mutants, and MME/1870 did not recognize the alanine-substituted peptides of Y507A, I511A, I512A, and L515A. These results indicate that the binding epitope of MME/1870 includes Tyr507, Ile511, Ile512, and Leu515 of CD10.
TrMab-6 Exerts Antitumor Activity in Mouse Xenograft Models of Breast Cancers. International-journal

Tomohiro Tanaka, Tomokazu Ohishi, Masaki Saito, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　32-38　2022/02

DOI： 10.1089/mab.2021.0056 　

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Trophoblast cell surface antigen 2 (TROP2) has been reported to be overexpressed in many cancers, and is involved in cancer cell proliferation, invasion, and metastasis. We previously developed a highly sensitive anti-TROP2 monoclonal antibody (mAb) (clone TrMab-6; mouse IgG2b, kappa) using a Cell-Based Immunization and Screening method. TrMab-6 is useful for investigations using flow cytometry, Western blotting, and immunohistochemistry and possesses antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against TROP2-expressing triple-negative breast cancer (TNBC) cell lines, such as MDA-MB-231 and MDA-MB-468. This study investigated whether TrMab-6 possesses in vivo antitumor activities via ADCC/CDC activities using mouse xenograft models of TNBC cell lines. In vivo experiments on MDA-MB-231 and MDA-MB-468 xenografts revealed that TrMab-6 significantly reduced tumor growth compared with normal mouse IgG treatment. The findings of this study suggest that TrMab-6 is a promising treatment option for TROP2-expressing TNBC.
Immunohistochemical Analysis Using Monoclonal Antibody PMab-269 Against Steller Sea Lion Podoplanin. International-journal

Guanjie Li, Hiroyuki Suzuki, Junko Takei, Masaki Saito, Nohara Goto, Kazuyuki Uchida, Takayuki Nakagawa, Hiroyuki Harada, Tomohiro Tanaka, Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　39-44　2022/02

DOI： 10.1089/mab.2021.0055 　

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Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.
Epitope Mapping of Rituximab Using HisMAP Method. International-journal

Teizo Asano, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　41　(1)　8-14　2022/02

DOI： 10.1089/mab.2021.0044 　

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CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by mAbs could contribute to understanding the function of mAbs and could be useful for the development of vaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved for the treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has been studied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also important for rituximab binding to CD20. In this study, we tried to determine the binding epitope of rituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximab-binding for CD20.
Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Yuki Imaizumi, Kazunori Takanuki, Takuya Miyake, Mizuki Takemoto, Kunio Hirata, Mika Hirose, Rika Oi, Tatsuya Kobayashi, Kenichi Miyoshi, Rie Aruga, Tatsuhiko Yokoyama, Shizuka Katagiri, Hiroaki Matsuura, Kenji Iwasaki, Takayuki Kato, Mika K. Kaneko, Yukinari Kato, Michiko Tajiri, Satoko Akashi, Osamu Nureki, Yohei Hizukuri, Yoshinori Akiyama, Terukazu Nogi

2022/01/31
Publisher: Cold Spring Harbor Laboratory
DOI： 10.1101/2022.01.31.478169 　

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Abstract Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.
Antitumor activity of a novel anti‑HER3 monoclonal antibody in mouse xenografts of colorectal cancer

Asano Teizo, Ohishi Tomokazu, Suzuki Hiroyuki, Kawada Manabu, Kaneko Mika K., Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　95　2-O-056　2022
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.95.0_2-o-056 　

eISSN： 2435-4953

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Background: Human epidermal growth factor receptor 3 (HER3) has been found to be overexpressed in many cancers such as lung, breast, and colon cancers. High expression of HER3 is thought to be a negative prognostic factor in several solid tumors including colorectal cancer. Moreover, HER3 is associated with drug resistance to EGFR- and HER2-targeted therapy in some cancers. Therefore, HER3 is thought to be an important therapeutic target. Purpose: We aimed to develop a novel anti-HER3 monoclonal antibody (mAb), and investigated its antitumor activities against colorectal cancer. Methods: We employed the Cell-Based Immunization and Screening (CBIS) method using HER3-overexpressed CHO-K1 cells for producing anti-HER3 mAbs. Anti-HER3 mAbs were screened using flow cytometry (FCM). Then, we examined the antibody‑dependent cellular cytotoxicity (ADCC) and the complement‑dependent cytotoxicity (CDC) activities of anti-HER3 mAbs for Caco‑2 (a human colorectal adenocarcinoma cell line). Moreover, a mouse xenograft model of Caco-2 was used for examining the antitumor activity. Results: We developed an anti-HER3 mAbs, H₃Mab-17 (IgG_2a, kappa) using CBIS method. H₃Mab-17 reacted with HER3-expressing cells in FCM. In vitro analysis demonstrated that H₃Mab-17 showed the ADCC and CDC activities against Caco-2. H₃Mab-17 significantly reduced tumor development in Caco-2 xenograft compared with control mouse IgG. Conclusion: We have successfully established a novel anti-HER3 mAb (H₃Mab-17), which could be a useful antibody-based therapy for patients with HER3-expressing colorectal cancers.
Development of anti-mouse CC chemokine receptor 8 monoclonal antibody C₈Mab-2

Saito Masaki, Tanaka Tomohiro, Asano Teizo, Suzuki Hiroyuki, K. Kaneko Mika, Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　95　1-P-095　2022
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.95.0_1-p-095 　

eISSN： 2435-4953

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CC motif chemokine receptor 8 (CCR8), a G protein-coupled receptor (GPCR), is highly expressed in regulatory T cells, T helper 2 cells, and cancer cells. It plays important role in allergic inflammation and cancer development. Therefore, specific monoclonal antibodies (mAbs) for CCR8 would be useful for diagnostic and therapeutic purposes of the diseases. However, the production of mAbs for GPCRs has remained very difficult. We have developed a novel method for the development of mAbs, named the Cell-Based Immunization and Screening (CBIS) method. In the present study, an SD rat was immunized with mouse CCR8-overexpressed CHO-K1 cells (CHO/mCCR8). The hybridomas expressing anti-mCCR8 mAbs were screened by using CHO/mCCR8 and CHO-K1 cells. We obtained 73 strongly anti-mCCR8 mAb-expressing hybridomas from 1,916 hybridomas, and we finally established C₈Mab-2 (IgG_2b, kappa). C₈Mab-2 selectively reacted to CHO/mCCR8 cells in a dose-dependent manner, but not to CHO-K1 cells, in flow cytometry. C₈Mab-2 also recognized endogenous mCCR8 in a mouse lymphocyte-like cell line (P388) and a mouse macrophage-like cell line (J774-1). Furthermore, C₈Mab-2 visualized mCCR8 in CHO/mCCR8, P388, and J774-1 cells in immunocytochemistry. In conclusion, we developed the anti-mCCR8 mAb, C₈Mab-2, which is available for detecting endogenous and exogenous mCCR8 in flow cytometry and immunocytochemistry. C₈Mab-2 would be usable for diagnosis and medical treatment for allergic inflammation and cancer in mouse models.
Antitumor Activities by a Defucosylated Mouse–Dog Chimeric Anti-EGFR Antibody in Canine Tumor Xenograft Models

Hiroyuki Suzuki, Li Guanjie, Asano Teizo, Tanaka Tomohiro, Kaneko Mika, Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　96　4-B-O12-5　2022
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.96.0_4-b-o12-5 　

eISSN： 2435-4953

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The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity by flow cytometry, western blotting and immunohistochemistry. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, E134Bf. Kinetic analysis of the interactions of E134Bf with the canine osteosarcoma cell line (D-17) and canine fibroblastic cell line (A-72) cells was conducted by flow cytometry. The K_Dfor the interaction of E134Bf with the D-17 and A-72 cells was 5.5 × 10⁻¹⁰ M and 6.0 × 10⁻¹⁰M, respectively, indicating that E134Bf exhibits high affinity for D-17 and A-72 cells. Furthermore, E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 and A-72 cells. In vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.
A novel anti-TROP2 monoclonal antibody TrMab-6 possesses antitumor activity in breast cancer models

Tanaka Tomohiro, Suzuki Hiroyuki, Ohishi Tomokazu, Kawada Manabu, K.Kaneko Mika, Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　95　2-O-055　2022
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.95.0_2-o-055 　

eISSN： 2435-4953

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Trophoblast cell surface antigen 2 (TROP2) has become one of the effective therapeutic targets by antibody-drug conjugates (ADCs) such as sacituzumab govitecan which US FDA approved in 2020. TROP2 is reported to be overexpressed, and involved in tumor cell proliferation, invasion, metastasis, and poor prognosis in several types of solid tumor. Several clinical trials of anti-TROP2 ADCs are ongoing worldwide in patients with breast and lung cancer. Here, using a Cell-Based Immunization and Screening (CBIS) method, we developed a novel anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG_2b, kappa). TrMab-6 was found to be applicable for many experiments, including flow cytometry, Western blotting, and immunohistochemistry. Furthermore, we investigated the potential of TrMab-6 for in vitro antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activities, and in vivo antitumor activities. TrMab-6 strongly induced anti-tumor effects against breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468 in vitro. In vivo experiments revealed that TrMab-6 significantly reduced tumor growth on breast cancer xenograft models. These results indicated that TrMab-6 could be a promising treatment option for TROP2-expressing breast cancers.
Defucosylated Mouse-Dog Chimeric Anti-EGFR Antibody Exerts Antitumor Activities in Mouse Xenograft Models of Canine Tumors. International-journal

Guanjie Li, Tomokazu Ohishi, Mika K Kaneko, Junko Takei, Takuya Mizuno, Manabu Kawada, Masaki Saito, Hiroyuki Suzuki, Yukinari Kato

Cells　10　(12)　2021/12/20

DOI： 10.3390/cells10123599 　

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The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.
Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C20Mab-60) Using Enzyme-Linked Immunosorbent Assay. International-journal

Junko Takei, Hiroyuki Suzuki, Teizo Asano, Guanjie Li, Masaki Saito, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(6)　250-254　2021/12

DOI： 10.1089/mab.2021.0042 　

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CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies. Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody-antigen binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed an anti-human CD20 monoclonal antibody, C20Mab-60 (IgG2a, kappa), using the Cell-Based Immunization and Screening (CBIS). C20Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses. In this study, we examined the critical epitope of C20Mab-60 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. We performed ELISA with deletion mutants, and C20Mab-60 reacted to the 160-179 amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C20Mab-60 did not recognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that the binding epitope of C20Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.
Epitope Mapping of an Anti-HER2 Monoclonal Antibody (H2Mab-181) Using Enzyme-Linked Immunosorbent Assay. International-journal

Teizo Asano, Junko Takei, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(6)　255-260　2021/12

DOI： 10.1089/mab.2021.0029 　

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Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein expressed in various types of normal or cancer cells. Overexpression of HER2 is found in many cancers and is related to cell proliferation, differentiation, and migration. We recently developed a novel anti-HER2 monoclonal antibody, H2Mab-181, by immunizing mice with the purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 not only in flow cytometry and Western blotting for gastric cancer cell lines, but also in immunohistochemical analyses for gastric cancer tissues. In this study, we analyzed the binding epitope of H2Mab-181 to HER2 using enzyme-linked immunosorbent assay (ELISA). Results showed that the H2Mab-181 epitope was determined to be Gly383, Asp384, Ala386, Asn388, and Pro391 by ELISA.
Epitope Mapping of an Anti-Human CCR9 Monoclonal Antibody (C9Mab-1) Using Enzyme-Linked Immunosorbent Assay. International-journal

Junko Takei, Teizo Asano, Guanjie Li, Masaki Saito, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(6)　239-242　2021/12

DOI： 10.1089/mab.2021.0037 　

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One of G protein-coupled receptors, CCR9, is mainly expressed in the thymocytes and the small bowel. The ligand of CCR9 is CCL25 (TECK), and the CCR9-CCL25 axis controls T cell maturation and intestinal immune response. CCR9 is related to graft-versus-host disease and autoimmune diseases. Recent studies have been reported that CCR9 is also associated with tumor proliferation, apoptosis, migration, and drug resistance. Therefore, CCR9-targeting therapy is receiving a lot of attention. Previously, we developed an anti-human CCR9 (hCCR9) monoclonal antibody, C9Mab-1 (IgG1, kappa), which can be used for flow cytometry, by immunizing mice with hCCR9-overexpressed Chinese hamster ovary-K1 cells. In this study, we examined the critical epitope of C9Mab-1, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and C9Mab-1 reacted to the 1-20 amino acids sequence of hCCR9. Next, we analyzed the reaction to 20 point mutants, and C9Mab-1 did not recognize the alanine-substituted peptides of I10A, P11A, N12A, M13A, A14G, D16A, and Y17A. The results indicate that the binding epitope of C9Mab-1 includes Ile10, Pro11, Asn12, Met13, Ala14, Asp16, and Tyr17 of hCCR9.
Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C20Mab-60) Using the HisMAP Method. International-journal

Teizo Asano, Junko Takei, Yoshikazu Furusawa, Masaki Saito, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(6)　243-249　2021/12

DOI： 10.1089/mab.2021.0035 　

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CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, we developed a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also for Western blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb, HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag. Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag was selected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa), C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicate that the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.
Immunocytochemical Analysis of DGKη in Cultured Cells Using a Monoclonal Antibody DhMab-4. International-journal

Tomoyuki Nakano, Toshiaki Tanaka, Fumio Sakane, Mika K Kaneko, Yukinari Kato, Kaoru Goto

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(6)　261-265　2021/12

DOI： 10.1089/mab.2021.0034 　

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Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) to phosphatidic acid (PA). Since both DG and PA serve as intracellular second messenger molecules, DGK plays a pivotal role in balancing these two signaling pathways. Of the DGK family, DGKη is classified as a type II DGK. Reportedly, DGKη is expressed ubiquitously through mammalian tissues and cells. Previous studies using cDNA transfection methods reported cytoplasmic localization of DGKη in cultured human cells. However, subcellular localization of native protein is still unknown. Recently, we established a human DGKη-specific monoclonal antibody, DhMab-4. In this study, we examined subcellular localization of native protein of DGKη using DhMab-4 by immunocytochemistry in human cultured cells.
Expression of podoplanin in various types of feline tumor tissues.

Satoshi Kamoto, Masahiro Shinada, Daiki Kato, Masaya Tsuboi, Sho Yoshimoto, Ryohei Yoshitake, Shotaro Eto, Namiko Ikeda, Yosuke Takahashi, Yuko Hashimoto, James Chambers, Kazuyuki Uchida, Shinji Yamada, Mika K Kaneko, Ryohei Nishimura, Yukinari Kato, Takayuki Nakagawa

The Journal of veterinary medical science　83　(11)　1795-1799　2021/11/24

DOI： 10.1292/jvms.20-0608 　

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Podoplanin is expressed in various human tumors where it promotes tumor progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of this study was to evaluate podoplanin expression in various types of feline tumor tissues. Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%) squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin, indicating the importance of the comparative aspects of podoplanin expression, which may be used as a novel research model for podoplanin biology.
Epitope Mapping of a Cancer-Specific Anti-Podocalyxin Monoclonal Antibody (PcMab-60) Using Enzyme-Linked Immunosorbent Assay and Surface Plasmon Resonance. International-journal

Teizo Asano, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(5)　227-232　2021/10

DOI： 10.1089/mab.2021.0030 　

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Podocalyxin (PODXL) is a type I transmembrane sialoglycoprotein that is overexpressed in human cancers, including breast, oral, and lung. PODXL promotes tumor progression, and its expression is associated with poor prognosis. Since PODXL is expressed in normal cells, including kidney podocytes and vascular endothelial cells (VECs), cancer-specific monoclonal antibodies (mAbs) are necessary to reduce the adverse effects of antibody therapy on PODXL-expressing cancers. Previously, we established a cancer-specific mAb against PODXL, PcMab-60 (mouse IgM, kappa), by immunizing mice with soluble PODXL produced by LN229 glioblastoma cells. PcMab-60 reacted with PODXL-expressing cancer cells, but did not react with VECs. In this study, we investigated an epitope of PcMab-60 using flow cytometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). The results of SPR revealed that the PcMab-60 epitope consisted of Thr105, Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117. In contrast, the results of ELISA revealed that the PcMab-60 epitope consisted of Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116. These results demonstrate the cancer-specific epitope, which was recognized by PcMab-60.
Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46) Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance. International-journal

Junko Takei, Teizo Asano, Hiroyuki Suzuki, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(5)　219-226　2021/10

DOI： 10.1089/mab.2021.0028 　

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CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the C44Mab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to be located in exon 5 of CD44.
Preclinical Evaluation of Podoplanin-Targeted Alpha-Radioimmunotherapy with the Novel Antibody NZ-16 for Malignant Mesothelioma. International-journal

Hitomi Sudo, Atsushi B Tsuji, Aya Sugyo, Mika K Kaneko, Yukinari Kato, Kotaro Nagatsu, Hisashi Suzuki, Tatsuya Higashi

Cells　10　(10)　2021/09/22

DOI： 10.3390/cells10102503 　

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The prognosis of advanced mesothelioma is poor. Podoplanin (PDPN) is highly expressed in most malignant mesothelioma. This study aimed to evaluate the potential alpha-radioimmunotherapy (RIT) with a newly developed anti-PDPN antibody, NZ-16, compared with a previous antibody, NZ-12. METHODS: The in vitro properties of radiolabeled antibodies were evaluated by cell binding and competitive inhibition assays using PDPN-expressing H226 mesothelioma cells. The biodistribution of 111In-labeled antibodies was studied in tumor-bearing mice. The absorbed doses were estimated based on biodistribution data. Tumor volumes and body weights of mice treated with 90Y- and 225Ac-labeled NZ-16 were measured for 56 days. Histologic analysis was conducted. RESULTS: The radiolabeled NZ-16 specifically bound to H226 cells with higher affinity than NZ-12. The biodistribution studies showed higher tumor uptake of radiolabeled NZ-16 compared with NZ-12, providing higher absorbed doses to tumors. RIT with 225Ac- and 90Y-labeled NZ-16 had a significantly higher antitumor effect than RIT with 90Y-labeled NZ-12. 225Ac-labeled NZ-16 induced a larger amount of necrotic change and showed a tendency to suppress tumor volumes and prolonged survival than 90Y-labeled NZ-16. There is no obvious adverse effect. CONCLUSIONS: Alpha-RIT with the newly developed NZ-16 is a promising therapeutic option for malignant mesothelioma.
Development of a Novel Epitope Mapping System: RIEDL Insertion for Epitope Mapping Method. International-journal

Teizo Asano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(4)　162-167　2021/08

DOI： 10.1089/mab.2021.0023 　

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To clarify the binding region of monoclonal antibodies (mAbs) to target molecules, it is very essential to understand the pharmacological function of each mAb. Although deletion mutants and point mutants are usefully utilized for epitope mapping, we often experience the difficulty of determining the mAb epitope against membrane proteins. We aimed to develop a novel method to determine the binding region of mAbs using epitope tag system. We first checked the reactivity of an anti-CD44 mAb (C44Mab-5) to several deletion mutants of CD44. We then employed the RIEDL tag system ("RIEDL" peptide and LpMab-7 mAb). We inserted the "RIEDL" peptide into the CD44 protein from the 21st to 41st amino acid (AA). The transfectants produced were stained by LpMab-7 and C44Mab-5 in flow cytometry. C44Mab-5 did not react with 30th-361st AA of the deletion mutant of CD44. Furthermore, the reaction of C44Mab-5 to RIEDL tag-inserted CD44 from 25th to 36th AA was lost, although LpMab-7 detected most of the RIEDL tag-inserted CD44 from 21st to 41st AA. The epitope of C44Mab-5 for CD44 was determined to be the peptide from 25th to 36th AA of CD44 using RIEDL insertion for epitope mapping (REMAP) method. The REMAP method might be useful for determining the critical epitope of functional mAbs against many target molecules.
Epitope Mapping of an Anti-Human Epidermal Growth Factor Receptor Monoclonal Antibody (EMab-51) Using the RIEDL Insertion for Epitope Mapping Method. International-journal

Ren Nanamiya, Masato Sano, Teizo Asano, Miyuki Yanaka, Takuro Nakamura, Masaki Saito, Tomohiro Tanaka, Hideki Hosono, Nami Tateyama, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(4)　149-155　2021/08

DOI： 10.1089/mab.2021.0010 　

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The classic method for identifying the epitope that monoclonal antibodies (mAbs) bind uses deletion mutants and point mutants of the target protein. However, determining the epitope of mAbs-reactive membrane proteins is often challenging. We recently developed the RIEDL insertion for epitope mapping (REMAP) method to identify mAb-binding epitopes. Herein, we first checked the reactivity of an anti-epidermal growth factor receptor (EGFR) mAb (EMab-51) to several EGFR deletion mutants such as EGFR/dN152, EGFR/dN313, EGFR/dN370, EGFR/dN375, EGFR/dN380, and EGFR/dN482. We found the N-terminus of the EMab-51-binding epitope between residues 375 and 380 of EGFR. We next produced EGFR/dN313 mutants with the RIEDL peptide tag inserted at each possible position of 375-AFRGDSFTHTPPLDP-389. EMab-51 lost its reactivity with the mutants having a RIEDL tag inserted at each position of 377-RGDSFTHTPP-386, whereas LpMab-7 (an anti-RIEDL mAb) detected every mutant. Thus, using the REMAP method, we identified the EMab-51-binding epitope of EGFR as 377-RGDSFTHTPP-386.
Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 134-mG2a-f Exerts Antitumor Activities in Mouse Xenograft Models of Dog Epidermal Growth Factor Receptor-Overexpressed Cells

Nami Tateyama, Ren Nanamiya, Tomokazu Ohishi, Junko Takei, Takuro Nakamura, Miyuki Yanaka, Hideki Hosono, Masaki Saito, Teizo Asano, Tomohiro Tanaka, Masato Sano, Manabu Kawada, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　40　(4)　177-183　2021/08/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0022 　

eISSN： 2167-9436
An Anti-HER2 Monoclonal Antibody H2Mab-41 Exerts Antitumor Activities in Mouse Xenograft Model Using Dog HER2-Overexpressed Cells

Nami Tateyama, Teizo Asano, Tomokazu Ohishi, Junko Takei, Hideki Hosono, Ren Nanamiya, Tomohiro Tanaka, Masato Sano, Masaki Saito, Manabu Kawada, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　40　(4)　184-190　2021/08/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0025 　

eISSN： 2167-9436
Development of a Novel Anti-HER2 Monoclonal Antibody H2Mab-181 for Gastric Cancer

Junko Takei, Teizo Asano, Tomohiro Tanaka, Masato Sano, Hideki Hosono, Ren Nanamiya, Nami Tateyama, Masaki Saito, Hiroyoshi Suzuki, Hiroyuki Harada, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　40　(4)　168-176　2021/08/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0021 　

eISSN： 2167-9436
Epitope Mapping of the Anti-California Sea Lion Podoplanin Monoclonal Antibody PMab-269 Using Alanine-Scanning Mutagenesis and ELISA

Tomohiro Tanaka, Teizo Asano, Masato Sano, Junko Takei, Hideki Hosono, Ren Nanamiya, Nami Tateyama, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2021/07/19
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0017 　

eISSN： 2167-9436
Epitope Mapping of an Antihuman EGFR Monoclonal Antibody (EMab-134) Using the REMAP Method. International-journal

Masato Sano, Mika K Kaneko, Teizo Aasano, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(4)　191-195　2021/07/19

DOI： 10.1089/mab.2021.0014 　

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The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays an important role in normal epidermal cell physiology. EGFR is overexpressed in cancer cells and has a number of mutations that implicate tumor malignancy, development, and poor patient prognosis; thus, EGFR is an attractive target for cancer therapy. At present, anti-EGFR monoclonal antibodies (mAbs) have been approved and are used for treating patients with a variety of EGFR-expressing cancers. Epitope mapping is important in identifying the therapeutic mechanism of anti-EGFR mAbs; however, the development of epitope mapping techniques lags behind the development of antimolecular target mAbs, including anti-EGFR mAbs. Hence, in this study, a novel epitope mapping method, RIEDL insertion for epitope mapping (REMAP) method, was developed. The results of this study demonstrated that the critical epitope of anti-EGFR mAb EMab-134 is Gly378, Asp379, Ser380, Phe381, Thr382, His383, Thr384, Pro385, and Pro386 of EGFR. The REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.
Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46) Using the REMAP Method. International-journal

Teizo Asano, Mika K Kaneko, Junko Takei, Nami Tateyama, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(4)　156-161　2021/07/19

DOI： 10.1089/mab.2021.0012 　

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CD44 functions as a major hyaluronan receptor on most cell types, with roles in cell adhesion, migration, proliferation, differentiation, and survival. The CD44 gene comprises 20 exons, with alternative splicing producing many different isoforms. CD44 variant isoforms exhibit tissue-specific expression patterns and have been studied as therapeutic targets for several cancers; therefore, anti-CD44 monoclonal antibodies (mAbs) are useful for investigating CD44 expression in various cancers. Previously, we established an anti-CD44 mAb, C44Mab-46 (IgG1, κ), by immunizing mice with the CD44v3-10 ectodomain. Although C44Mab-46 recognized all CD44 isoforms, the binding epitope of C44Mab-46 has not been determined. In this study, we first checked the reactivity of C44Mab-46 to several CD44v3-10 deletion mutants such as dN79, dN124, dN147, and dN224. We found the N-terminus of the C44Mab-46-binding epitope between residues 147 and 224 of CD44v3-10. We next investigated this epitope using a novel mapping system: RIEDL insertion for epitope mapping (REMAP) method. We constructed 31 CD44 standard (CD44s) mutants where the RIEDL tag was inserted into the expected epitope region in CD44s. We observed that the C44Mab-46 epitope constituted five amino acids: 174-TDDDV-178 of CD44s. Thus, the REMAP method could be used to determine mAb binding epitopes for membrane proteins.
An anti‑TROP2 monoclonal antibody TrMab‑6 exerts antitumor activity in breast cancer mouse xenograft models. International-journal

Tomohiro Tanaka, Tomokazu Ohishi, Teizo Asano, Junko Takei, Ren Nanamiya, Hideki Hosono, Masato Sano, Hiroyuki Harada, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Oncology reports　46　(1)　2021/07

DOI： 10.3892/or.2021.8083 　

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Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive anti‑TROP2 monoclonal antibody (clone TrMab‑6; mouse IgG2b, κ) was developed using a Cell‑Based Immunization and Screening (CBIS) method. TrMab‑6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab‑6 possesses in vitro antibody‑dependent cellular cytotoxicity (ADCC) or complement‑dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2‑overexpressed CHO‑K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA‑MB‑231, and MDA‑MB‑468. In vitro experiments revealed that TrMab‑6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHO‑K1 and MCF7/TROP2‑knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 xenografts revealed that TrMab‑6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO‑K1 and MCF7/TROP2‑knockout xenografts. The findings suggest that TrMab‑6 is a promising treatment option for TROP2‑expressing breast cancers.
Anti‑HER3 monoclonal antibody exerts antitumor activity in a mouse model of colorectal adenocarcinoma

Teizo Asano, Tomokazu Ohishi, Junko Takei, Takuro Nakamura, Ren Nanamiya, Hideki Hosono, Tomohiro Tanaka, Masato Sano, Hiroyuki Harada, Manabu Kawada, Mika Kaneko, Yukinari Kato

Oncology Reports　46　(2)　2021/06/28
Publisher: Spandidos Publications
DOI： 10.3892/or.2021.8124 　

ISSN： 1021-335X

eISSN： 1791-2431
Ferret Podoplanin Is Detected by PMab-241 in Immunohistochemistry

Ren Nanamiya, Junko Takei, Teizo Asano, Masato Sano, Tomohiro Tanaka, Hideki Hosono, Hiroyuki Harada, Yusuke Sakai, Takuya Mizuno, Hiroyoshi Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　40　(3)　134-140　2021/06/01
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0016 　

eISSN： 2167-9436
Development of an Anti-Elephant Podoplanin Monoclonal Antibody PMab-265 for Flow Cytometry

Hideki Hosono, Teizo Asano, Junko Takei, Masato Sano, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2021/05/26
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0015 　

eISSN： 2167-9436
Development of Monoclonal Antibody PMab-269 Against California Sea Lion Podoplanin

Tomohiro Tanaka, Teizo Asano, Masato Sano, Junko Takei, Hideki Hosono, Ren Nanamiya, Takuro Nakamura, Miyuki Yanaka, Hiroyuki Harada, Masato Fukui, Hiroyoshi Suzuki, Kazuyuki Uchida, Takayuki Nakagawa, Yukinari Kato, Mika K. Kaneko

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2021/05/26
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0011 　

eISSN： 2167-9436
Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion. International-journal

Risako Tamura-Sakaguchi, Rie Aruga, Mika Hirose, Toru Ekimoto, Takuya Miyake, Yohei Hizukuri, Rika Oi, Mika K Kaneko, Yukinari Kato, Yoshinori Akiyama, Mitsunori Ikeguchi, Kenji Iwasaki, Terukazu Nogi

Acta crystallographica. Section D, Structural biology　77　(Pt 5)　645-662　2021/05/01

DOI： 10.1107/S2059798321002527 　

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Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.
Development of Anti-Mouse CC Chemokine Receptor 3 Monoclonal Antibodies for Flow Cytometry

Teizo Asano, Ren Nanamiya, Junko Takei, Takuro Nakamura, Miyuki Yanaka, Hideki Hosono, Tomohiro Tanaka, Masato Sano, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　2021/04/20
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2021.0009 　

eISSN： 2167-9436
Development of Antihuman Killer Cell Lectin-Like Receptor Subfamily G Member 1 Monoclonal Antibodies for Flow Cytometry. International-journal

Teizo Asano, Ren Nanamiya, Tomohiro Tanaka, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(2)　76-80　2021/04

DOI： 10.1089/mab.2021.0008 　

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Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, was identified as an inhibitory receptor expressed on natural killer (NK) cells and certain T cells. The protein regulates effector functions and developmental processes in these cells. In this study, we established a specific and sensitive monoclonal antibody (mAb) for human KLRG1 (hKLRG1), which is useful for flow cytometry, using a Cell-Based Immunization and Screening (CBIS) method. The established anti-hKLRG1 mAb, KLMab-1 (mouse IgG1, kappa), reacted with overexpressed hKLRG1 in Chinese hamster ovary-K1 (CHO/hKLRG1) and human NK cells, which also expressed endogenous hKLRG1 as confirmed by flow cytometry. KLMab-1, which was established by the CBIS method, could be useful for elucidating the hKLRG1-related biological response by flow cytometry.
Development of Anti-human T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) Monoclonal Antibodies for Flow Cytometry. International-journal

Junko Takei, Teizo Asano, Ren Nanamiya, Takuro Nakamura, Miyuki Yanaka, Hideki Hosono, Tomohiro Tanaka, Masato Sano, Mika K Kaneko, Hiroyuki Harada, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　40　(2)　71-75　2021/04

DOI： 10.1089/mab.2021.0006 　

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Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1 (PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2, resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than their single use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new anti-human TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG1, kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (KD) of TgMab-2 for CHO/hTIGIT cells were determined to be 3.5 × 10-9 M. These results suggest that TgMab-2, which was developed by CBIS method, is useful for analyzing the function of hTIGIT by flow cytometry.
Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis. International-journal

Yusuke Sayama, Mika K Kaneko, Junko Takei, Hideki Hosono, Masato Sano, Teizo Asano, Yukinari Kato

Biochemistry and biophysics reports　25　100902-100902　2021/03

DOI： 10.1016/j.bbrep.2020.100902 　

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TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.
Prevention of necrosis caused by transient expression in Nicotiana benthamiana by application of ascorbic acid

Shohei Nosaki, Mika K Kaneko, Fuminori Tsuruta, Hideki Yoshida, Yukinari Kato, Kenji Miura

Plant Physiology　186　(2)　832-835　2021/02/26
Publisher: Oxford University Press (OUP)
DOI： 10.1093/plphys/kiab102 　

ISSN： 0032-0889

eISSN： 1532-2548

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Application of high concentrations of sodium ascorbate suppresses necrosis caused by the expression of recombinant proteins in Nicotiana benthamiana, resulting in an increase in protein accumulation.
Development and characterization of TrMab‑6, a novel anti‑TROP2 monoclonal antibody for antigen detection in breast cancer. International-journal

Yusuke Sayama, Mika K Kaneko, Yukinari Kato

Molecular medicine reports　23　(2)　2021/02

DOI： 10.3892/mmr.2020.11731 　

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Trophoblast cell‑surface antigen 2 (TROP2) is a type I transmembrane glycoprotein that is overexpressed in a number of cancer types, including triple‑negative breast cancer. The current study aimed to develop a highly sensitive and specific monoclonal antibody (mAb) targeting TROP2, which could be used to evaluate TROP2 expression using flow cytometry, western blot analysis and immunohistochemistry by employing the Cell‑Based Immunization and Screening (CBIS) method. The established anti‑TROP2 mAb, TrMab‑6 (mouse IgG2b, κ), detected TROP2 on PA‑tagged TROP2‑overexpressing Chinese hamster ovary‑K1 (CHO/TROP2‑PA) and breast cancer cell lines, including MCF7 and BT‑474 using flow cytometry. Western blot analysis indicated a 40 kDa band in lysates prepared from CHO/TROP2‑PA, MCF7 and BT‑474 cells. Furthermore, TROP2 in 57/61 (93.4%) of the breast cancer specimens was strongly detected using immunohistochemical analysis with TrMab‑6. In conclusion, the current study demonstrated that TrMab‑6 may be a valuable tool for the detection of TROP2 in a wide variety of breast cancer types.
Development of a monoclonal antibody for CD44-expressing oral cancers

Takei Junko, K. Kaneko Mika, Ohishi Tomokazu, Asano Teizo, Kawada Manabu, Harada Hiroyuki, Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　94　2-O-D1-4　2021
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.94.0_2-o-d1-4 　

eISSN： 2435-4953

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Purpose: CD44 is widely expressed on the surface of most tissues and all hematopoietic cells. CD44 plays important roles in cell proliferation, adhesion, and migration. Previously, we developed an anti-CD44 monoclonal antibody, C₄₄Mab-5 (IgG₁, kappa) by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. In this study, we converted the mouse IgG₁ subclass antibody C₄₄Mab‑5 into an IgG_2a subclass antibody, 5‑mG_2a, and further produced a defucosylated version, 5‑mG_2a‑f and investigate its antitumor activities against oral cancers. Methods: To generate 5‑mG_2a‑f, appropriate V_H cDNA of mouse C₄₄Mab‑5 and C_H of mouse IgG_2a were subcloned into pCAG‑Neo vector, and light chain of C₄₄Mab‑5 was subcloned into pCAG‑Ble vector. Vectors were transfected into BINDS‑09 (FUT8‑deficient ExpiCHO‑S cells) using the ExpiCHO Expression System. Mouse xenograft models of HSC-2 and SAS (human oral cancer cell lines) were used for examining the antitumor activity. Results: 5-mG_2a-f demonstrated a sensitive and specific reaction against oral cancer cells in flow cytometry and immunohistochemical analyses. The sensitivity of 5‑mG_2a‑f was similar with that of C₄₄Mab‑5. In vitro analysis demonstrated that 5‑mG_2a‑f showed moderate ADCC and CDC activities against HSC-2 and SAS. 5-mG_2a-f significantly reduced tumor development in HSC-2 and SAS xenografts in comparison to control mouse IgG. Conclusion: 5‑mG_2a‑f may be a useful antibody-based therapy for patients with CD44-expressing oral cancers.
The anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody EpMab-16 exerts antitumor activity in a mouse model of colorectal adenocarcinoma. International-journal

Hideki Hosono, Tomokazu Ohishi, Junko Takei, Teizo Asano, Yusuke Sayama, Manabu Kawada, Mika K Kaneko, Yukinari Kato

Oncology letters　20　(6)　383-383　2020/12

DOI： 10.3892/ol.2020.12246 　

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The epithelial cell adhesion molecule (EpCAM), which is a calcium-independent homophilic intercellular adhesion factor, contributes to cell signaling, differentiation, proliferation and migration. EpCAM is essential for carcinogenesis in numerous types of human cancer. The purpose of the present study was to establish an anti-EpCAM monoclonal antibody (mAb) for targeting colorectal adenocarcinomas. Thus, an anti-EpCAM mAb, EpMab-16 (IgG2a, κ), was established by immunizing mice with EpCAM-overexpressing CHO-K1 cells, and validated using flow cytometry, western blot, and immunohistochemical analyses. EpMab-16 reacted with endogenous EpCAM specifically in a colorectal adenocarcinoma cell line as determined by flow cytometry and western blot analyses. Immunohistochemical analysis demonstrated that EpMab-16 stained a plasma membrane-like pattern in clinical colorectal adenocarcinoma tissues. The dissociation constant (K D) for EpMab-16 in a Caco-2 colorectal adenocarcinoma cell line determined by flow cytometry was 1.8×10-8 M, suggesting moderate binding affinity of EpMab-16 for EpCAM. Whether the EpMab-16 induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Caco-2 or antitumor activity was then assessed in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC induction in Caco-2 cells by EpMab-16 treatment. In vivo experiments in a Caco-2 ×enograft model demonstrated that EpMab-16 treatment significantly reduced tumor growth compared with that in mice treated with the control mouse IgG. These results suggested that EpMab-16 may be a promising treatment option for EpCAM-expressing colorectal adenocarcinomas.
Anti‑EpCAM monoclonal antibody exerts antitumor activity against oral squamous cell carcinomas. International-journal

Mika K Kaneko, Tomokazu Ohishi, Junko Takei, Masato Sano, Takuro Nakamura, Hideki Hosono, Miyuki Yanaka, Teizo Asano, Yusuke Sayama, Hiroyuki Harada, Manabu Kawada, Yukinari Kato

Oncology reports　44　(6)　2517-2526　2020/12

DOI： 10.3892/or.2020.7808 　

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The epithelial cell adhesion molecule (EpCAM) is a calcium‑independent, homophilic, intercellular adhesion factor classified as a transmembrane glycoprotein. In addition to cell adhesion, EpCAM also contributes to cell signaling, differentiation, proliferation, and migration. EpCAM is an essential factor in the carcinogenesis of numerous human cancers. In the present study, we developed and validated an anti‑EpCAM monoclonal antibody (mAb), EpMab‑16 (IgG2a, kappa), by immunizing mice with EpCAM‑overexpressing CHO‑K1 cells. EpMab‑16 specifically reacted with endogenous EpCAM in oral squamous cell carcinoma (OSCC) cell lines in flow cytometry and Western blot analyses. It exhibited a plasma membrane‑like stain pattern in OSCC tissues upon immunohistochemical analysis. The KD for EpMab‑16 in SAS and HSC‑2 OSCC cells were assessed via flow cytometry at 1.1x10‑8 and 1.9x10‑8 M, respectively, suggesting moderate binding affinity of EpMab‑16 for EpCAM. We then assessed whether the EpMab‑16 induced antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) against OSCC cell lines, and antitumor capacity in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC inducement against OSCC cells treated with EpMab‑16. In vivo experiments on OSCC xenografts revealed that EpMab‑16 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that EpMab‑16 could be a promising treatment option for EpCAM‑expressing OSCCs.
A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts antitumor effects in mouse xenograft models of pancreatic carcinoma. International-journal

Mika K Kaneko, Tomokazu Ohishi, Manabu Kawada, Yukinari Kato

Biochemistry and biophysics reports　24　100826-100826　2020/12

DOI： 10.1016/j.bbrep.2020.100826 　

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Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG2a-type mAb, named as 60-mG2a, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG2a, named as 60-mG2a-f, to augment its ADCC activity. In vivo analysis revealed that 60-mG2a-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG2a-f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.
Epitope mapping of an anti-diacylglycerol kinase delta monoclonal antibody DdMab-1. International-journal

Masato Sano, Teizo Asano, Mika K Kaneko, Yukinari Kato

Biochemistry and biophysics reports　24　100808-100808　2020/12

DOI： 10.1016/j.bbrep.2020.100808 　

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Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG2a, kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, and performed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670 mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly located between amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A, R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1, indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGKδ.
A defucosylated anti-PD-L1 monoclonal antibody 13-mG<inf>2a</inf>-f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Junko Takei, Tomokazu Ohishi, Mika K. Kaneko, Hiroyuki Harada, Manabu Kawada, Yukinari Kato

Biochemistry and Biophysics Reports　24　2020/12

DOI： 10.1016/j.bbrep.2020.100801 　

eISSN： 2405-5808
Phase I/II Clinical Trial of the Anti-Podoplanin Monoclonal Antibody Therapy in Dogs with Malignant Melanoma. International-journal

Satoshi Kamoto, Masahiro Shinada, Daiki Kato, Sho Yoshimoto, Namiko Ikeda, Masaya Tsuboi, Ryohei Yoshitake, Shotaro Eto, Yuko Hashimoto, Yosuke Takahashi, James Chambers, Kazuyuki Uchida, Mika K Kaneko, Naoki Fujita, Ryohei Nishimura, Yukinari Kato, Takayuki Nakagawa

Cells　9　(11)　2020/11/23

DOI： 10.3390/cells9112529 　

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Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed on tumor cells. PDPN is known to be linked with several aspects of tumor malignancies in certain types of human and canine tumors. Therefore, it is considered to be a novel therapeutic target. Monoclonal antibodies targeting PDPN expressed in human tumor cells showed obvious anti-tumor effects in preclinical studies using mouse models. Previously, we generated a cancer-specific mouse-dog chimeric anti-PDPN antibody, P38Bf, which specifically recognizes PDPN expressed in canine tumor cells. In this study, we investigated the safety and anti-tumor effects of P38Bf in preclinical and clinical trials. P38Bf showed dose-dependent antibody-dependent cellular cytotoxicity against canine malignant melanoma cells. In a preclinical trial with one healthy dog, P38Bf administration did not induce adverse effects over approximately 2 months. In phase I/II clinical trials of three dogs with malignant melanoma, one dog vomited, and all dogs had increased serum levels of C-reactive protein, although all adverse effects were grade 1 or 2. Severe adverse effects leading to withdrawal of the clinical trial were not observed. Furthermore, one dog had stable disease with P38Bf injections. This is the first reported clinical trial of anti-PDPN antibody therapy using spontaneously occurring canine tumor models.
A defucosylated anti‑CD44 monoclonal antibody 5‑mG2a‑f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma. International-journal

Junko Takei, Mika K Kaneko, Tomokazu Ohishi, Hideki Hosono, Takuro Nakamura, Miyuki Yanaka, Masato Sano, Teizo Asano, Yusuke Sayama, Manabu Kawada, Hiroyuki Harada, Yukinari Kato

Oncology reports　44　(5)　1949-1960　2020/11

DOI： 10.3892/or.2020.7735 　

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CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti‑CD44 monoclonal antibody (mAb), C44Mab‑5 (IgG1, kappa) was established by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab‑5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab‑5 lacks antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC), the antitumor activity of C44Mab‑5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab‑5 into an IgG2a subclass antibody, 5‑mG2a, and further produced a defucosylated version, 5‑mG2a‑f, using FUT8‑deficient ExpiCHO‑S (BINDS‑09) cells. Defucosylation of 5‑mG2a‑f was confirmed using fucose‑binding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5‑mG2a‑f against SAS and HSC‑2 oral cancer cells were determined through flow cytometry to be 2.8x10‑10 M and 2.6x10‑9 M, respectively, indicating that 5‑mG2a‑f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5‑mG2a‑f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5‑mG2a‑f showed moderate ADCC and CDC activities against SAS and HSC‑2 oral cancer cells. In vivo analysis revealed that 5‑mG2a‑f significantly reduced tumor development in SAS and HSC‑2 xenografts in comparison to control mouse IgG, even after injection seven days post‑tumor inoculation. Collectively, these results suggest that treatment with 5‑mG2a‑f may represent a useful therapy for patients with CD44‑expressing oral cancers.
Crystal structure of an anti-podoplanin antibody bound to a disialylated O-linked glycopeptide

Satoshi Ogasawara, Kano Suzuki, Kentaro Naruchi, Seiwa Nakamura, Junpei Shimabukuro, Nanase Tsukahara, Mika K. Kaneko, Yukinari Kato, Takeshi Murata

Biochemical and Biophysical Research Communications　533　(1)　57-63　2020/11
Publisher: Elsevier BV
DOI： 10.1016/j.bbrc.2020.08.103 　

ISSN： 0006-291X
新規抗TROP2抗体を用いた乳がんに対する抗腫瘍効果の検討

佐山勇輔, 武井潤子, 大石智一, 金子美華, 川田学, 加藤幸成

日本癌学会総会記事　79回　OJ15-3　2020/10
Publisher: (一社)日本癌学会
ISSN： 0546-0476
HER2を標的とした抗体医薬開発

武井潤子, 金子美華, 大石智一, 佐山勇輔, 川田学, 原田浩之, 加藤幸成

日本癌学会総会記事　79回　OJ16-7　2020/10
Publisher: (一社)日本癌学会
ISSN： 0546-0476
Development of Core-Fucose-Deficient Humanized and Chimeric Anti-Human Podoplanin Antibodies. International-journal

Mika K Kaneko, Tomokazu Ohishi, Takuro Nakamura, Hiroyuki Inoue, Junko Takei, Masato Sano, Teizo Asano, Yusuke Sayama, Hideki Hosono, Hiroyoshi Suzuki, Manabu Kawada, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(5)　167-174　2020/10

DOI： 10.1089/mab.2020.0019 　

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Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2 interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanized anti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27, named as P1027 and a core-fucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f against LN319 (a human glioblastoma cell line) are 1.1 × 10-8 and 3.9 × 10-9 M, respectively. ADCC reporter assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f may be a useful therapy for patients with PDPN-expressing cancers.
Anti-EGFR monoclonal antibody 134-mg<inf>2a</inf> exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Hideki Hosono, Junko Takei, Tomokazu Ohishi, Masato Sano, Teizo Asano, Yusuke Sayama, Takuro Nakamura, Miyuki Yanaka, Manabu Kawada, Hiroyuki Harada, Mika Kato Kaneko, Yukinari Kato

International Journal of Molecular Medicine　46　(4)　1443-1452　2020/10

DOI： 10.3892/ijmm.2020.4700 　

ISSN： 1107-3756

eISSN： 1791-244X
RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

Kenji Miura, Hideki Yoshida, Shohei Nosaki, Mika K. Kaneko, Yukinari Kato

Frontiers in Plant Science　11　2020/09/10
Publisher: Frontiers Media SA
DOI： 10.3389/fpls.2020.510444 　

eISSN： 1664-462X
DGKzetaに対する新規抗体作製とそのエピトープ解析

佐野雅人, 淺野禎三, 佐山勇輔, 武井潤子, 細野秀樹, 金子美華, 加藤幸成

日本生化学会大会プログラム・講演要旨集　93回　[2Z11-435)]　2020/09
Publisher: (公社)日本生化学会
新規ペプチドタグシステムであるRIEDLタグはタンパク質精製に有用である(RIEDL tag, a novel peptide tag system is useful for protein purification)

淺野禎三, 佐山勇輔, 佐野雅人, 武井潤子, 細野秀樹, 金子美華, 加藤幸成

日本生化学会大会プログラム・講演要旨集　93回　[3Z12-702)]　2020/09
Publisher: (公社)日本生化学会
Anti-metastatic activity of an anti-egfr monoclonal antibody against metastatic colorectal cancer with kras p.G13d mutation

Tomokazu Ohishi, Yukinari Kato, Mika K. Kaneko, Shun Ichi Ohba, Hiroyuki Inoue, Akiko Harakawa, Manabu Kawada

International Journal of Molecular Sciences　21　(17)　1-16　2020/09/01

DOI： 10.3390/ijms21176037 　

ISSN： 1661-6596

eISSN： 1422-0067
RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification. International-journal Peer-reviewed

Teizo Asano, Mika K Kaneko, Yukinari Kato

Biochemistry and biophysics reports　23　100780-100780　2020/09

DOI： 10.1016/j.bbrep.2020.100780 　

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Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide "tag" and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel "RIEDL tag system," which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.
Epitope Mapping of DhMab-1: An Antidiacylglycerol Kinase Monoclonal Antibody. International-journal

Masato Sano, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　140-143　2020/08

DOI： 10.1089/mab.2020.0014 　

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Diacylglycerol kinase (DGK) η is classified as a type II DGK and catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKη has been reported to be highly expressed in the hippocampus and cerebellum. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not been developed. Recently, we established a specific antihuman DGKη (hDGKη) mAb, DhMab-1 (mouse IgG2a, kappa). For epitope mapping of DhMab-1, here we produced deletion or point mutants of hDGKη and performed Western blotting to determine the binding epitope of DhMab-1. DhMab-1 reacted with the dN755 mutant, but not with the dN760 mutant, indicating that the N-terminus of the DhMab-1 epitope is mainly located between amino acids 755 and 760 of the protein. A more detailed analysis using point mutants demonstrated that seven mutants, that is, A751G, I755A, D756A, P757A, D758A, L759A, and D760A, were not detected by DhMab-1. These results indicate that Ala751, Ile755, Asp756, Pro757, Asp758, Leu759, and Asp760 are important for DhMab-1 binding to hDGKη.
An Antihuman Epidermal Growth Factor Receptor 2 Monoclonal Antibody (H2Mab-19) Exerts Antitumor Activity in Glioblastoma Xenograft Models. International-journal

Yukinari Kato, Tomokazu Ohishi, Junko Takei, Takuro Nakamura, Manabu Kawada, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　135-139　2020/08

DOI： 10.1089/mab.2020.0013 　

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Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in glioblastoma as well as breast, gastric, lung, colorectal, and pancreatic cancers. Its expression is associated with poor clinical outcomes. Anti-HER2 antibodies have provided significant survival benefits to patients with HER2-overexpressing breast and gastric cancers. We recently developed an anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa), by immunizing mice with the extracellular domain of HER2, which is expressed in LN229 glioblastoma cells. In this study, we investigated the antitumor activity of H2Mab-19 in an LN229 glioblastoma xenograft model. H2Mab-19 showed high binding affinity (KD: 1.1 × 10-8 M) against LN229 cells. Furthermore, H2Mab-19 significantly reduced tumor development in an LN229 xenograft. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing glioblastomas.
Establishment of an Anti-CD20 Monoclonal Antibody (C20Mab-60) for Immunohistochemical Analyses. International-journal

Yoshikazu Furusawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　112-116　2020/08

DOI： 10.1089/mab.2020.0015 　

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Sensitive and specific monoclonal antibodies (mAbs) are needed for detecting CD20. This antigen, one of several B lymphocyte antigens, is a target for every application used in the diagnosis of B cell lymphoma. Many anti-CD20 mAbs have been established, although applications of these antibodies are limited. This study aims to establish sensitive and specific anti-CD20 mAbs suitable for broad application, such as flow cytometry, Western blotting, and immunohistochemical analyses. Using the Cell-Based Immunization and Screening (CBIS) method, all procedures were performed by utilizing CD20-stable transfectants, and a clone, C20Mab-60 (IgG2a, kappa), was developed. In flow cytometry, C20Mab-60 detected overexpression of CD20 in LN229 cell and endogenous CD20 in BALL-1 (a human B cell leukemia cell line) but did not react with CD20-knockout BALL-1 (BINDS-24), indicating specificity for CD20. In Western blotting, C20Mab-60 detected CD20-overexpressing Chinese hamster ovary-K1, BALL-1, and Raji (a human Burkitt's lymphoma cell line) displaying both sensitivity and specificity. Furthermore, B cell but not T cell lymphomas were strongly stained with C20Mab-60 in immunohistochemical analyses. C20Mab-60, which was developed by CBIS method, is shown to be useful for the detection of cells expressing CD20 in lymphoma tissues by flow cytometry, Western blotting, and immunohistochemical analyses.
H2Mab-19, an anti-human epidermal growth factor receptor 2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts. International-journal

Junko Takei, Mika Kato Kaneko, Tomokazu Ohishi, Manabu Kawada, Hiroyuki Harada, Yukinari Kato

Experimental and therapeutic medicine　20　(2)　846-853　2020/08

DOI： 10.3892/etm.2020.8765 　

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Human epidermal growth factor receptor 2 (HER2) is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanized anti-HER2 antibody that offers significant survival benefits to patients with HER2-overexpressing breast cancer. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa) was developed. Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of H2Mab-19 were investigated using both breast cancer and oral cancer cell lines. H2Mab-19 demonstrated cytotoxicity in BT-474 (a human breast cancer cell line) and HSC-2 or SAS (human oral cancer cell lines). H2Mab-19 also possessed both ADCC and CDC activity against BT-474, HSC-2, and SAS cell lines. In comparison to control mouse IgG, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2, and SAS xenografts. Collectively, these results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing breast and oral cancers.
Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260. International-journal Peer-reviewed

Mika K Kaneko, Masato Sano, Junko Takei, Teizo Asano, Yusuke Sayama, Hideki Hosono, Atsushi Kobayashi, Satoru Konnai, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　144-155　2020/07/16

DOI： 10.1089/mab.2020.0018 　

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Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
Generation of a canine anti-canine CD20 antibody for canine lymphoma treatment. International-journal Peer-reviewed

Takuya Mizuno, Yukinari Kato, Mika K Kaneko, Yusuke Sakai, Toshinori Shiga, Masahiro Kato, Toshihiro Tsukui, Hirofumi Takemoto, Akio Tokimasa, Kenji Baba, Yuki Nemoto, Osamu Sakai, Masaya Igase

Scientific reports　10　(1)　11476-11476　2020/07/10

DOI： 10.1038/s41598-020-68470-9 　

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Lymphoma is the most common hematological cancer in dogs. Canine diffuse large B cell lymphoma shows a relatively good response to treatment with multi-agent cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy; however, the 2-year survival rate is as low as 20%. For human B cell type lymphoma, the anti-CD20 chimeric antibody, rituximab, was developed two decades ago. The combination of rituximab and CHOP chemotherapy was highly successful in improving patient prognosis. However, no anti-canine CD20 antibody is available for the treatment of canine lymphoma. During this study, a rat anti-canine CD20 monoclonal antibody was established. We also generated a rat-canine chimeric antibody against canine CD20 designed for clinical application. This chimeric antibody (4E1-7-B) showed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the canine B cell lymphoma cell line CLBL-1. Moreover, to obtain stronger ADCC activity, a defucosylated 4E1-7-B antibody (4E1-7-B_f) was also generated, and it showed tenfold stronger ADCC activity compared with 4E1-7-B. 4E1-7-B_f as well as 4E1-7-B suppressed the growth of CLBL-1 tumors in an immunodeficient xenotransplant mouse model. Finally, a single administration of 4E1-7-B_f induced considerable peripheral B cell depletion in healthy beagles. Thus, 4E1-7-B_f is a good antibody drug candidate for canine B cell type lymphoma.
Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs. International-journal

Goichi Akiyama, Sindhu Saraswathy, Thania Bogarin, Xiaojing Pan, Ernesto Barron, Tina T Wong, Mika K Kaneko, Yukinari Kato, Young Hong, Alex S Huang

Experimental eye research　196　108049-108049　2020/07

DOI： 10.1016/j.exer.2020.108049 　

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The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.
An Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody H2Mab-19 Exerts Antitumor Activity in Mouse Colon Cancer Xenografts. International-journal Peer-reviewed

Yukinari Kato, Tomokazu Ohishi, Junko Takei, Takuro Nakamura, Masato Sano, Teizo Asano, Yusuke Sayama, Hideki Hosono, Manabu Kawada, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　123-128　2020/06/18

DOI： 10.1089/mab.2020.0009 　

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Trastuzumab is a humanized antibody against human epidermal growth factor receptor 2 (HER2) that offers significant survival benefits to patients with HER2-overexpressing breast or gastric cancer. HER2 is also known to be overexpressed in colon cancers. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, κ) was characterized for its anticancer activity in colon cancers. H2Mab-19 showed both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against Caco-2, a colon cancer cell line. Furthermore, H2Mab-19 significantly reduced tumor development in a Caco-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing colon cancers.
Establishment of C20Mab‑11, a novel anti‑CD20 monoclonal antibody, for the detection of B cells International-journal Peer-reviewed

Yoshikazu Furusawa, Mika Kaneko, Yukinari Kato

Oncology Letters　20　(2)　1961-1967　2020/06/17

DOI： 10.3892/ol.2020.11753 　

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CD20 is one of several B-lymphocyte antigens that has been shown to be an effective target for the detection and treatment of B-cell lymphomas. Sensitive and specific monoclonal antibodies (mAbs) are required for every application used for the diagnosis of B-cell lymphoma. Although many anti-CD20 mAbs have been established, the types of applications, those anti-CD20 can be used in, are limited. In this study, we aimed to establish novel anti-CD20 mAbs to be used for broad applications, such as flow cytometry, western blot, and immunohistochemical analyses, using the Cell-Based Immunization and Screening (CBIS) method. One of the established mAbs, C20Mab-11 (IgM, kappa), detected overexpression of CD20 in CHO-K1 or LN229 cell lines, indicating that C20Mab-11 is specific for CD20. In western blot analyses, C20Mab-11 detected not only overexpression of CD20 in CHO-K1 or LN229 cell lines, but also CD20 of BALL-1 and Raji cells with both sensitivity and specificity. Furthermore, C20Mab-11 strongly stained B cells of the lymph follicle and B cell lymphomas in immunohistochemical analyses. These results indicate that C20Mab-11 develped by CBIS method, is useful for the detection of CD20 in lymphoma tissues by flow cytometry, western blot, and immunohistochemical analyses and potentially could be beneficial for the treatment of B cell lymphomas.
Epitope Mapping of the Anti-Diacylglycerol Kinase Monoclonal Antibody DhMab-4 for Immunohistochemical Analysis. International-journal Peer-reviewed

Teizo Asano, Masato Sano, Junko Takei, Yusuke Sayama, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(4)　117-122　2020/06/02

DOI： 10.1089/mab.2020.0012 　

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Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKη is classified as a type II DGK. Reports have shown that DGKη is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG2b, kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKη and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKη.
Epitope Mapping of PMab-241, a Lymphatic Endothelial Cell-Specific Anti-Bear Podoplanin Monoclonal Antibody. International-journal

Yusuke Sayama, Masato Sano, Teizo Asano, Yoshikazu Furusawa, Junko Takei, Takuro Nakamura, Miyuki Yanaka, Saki Okamoto, Saori Handa, Yu Komatsu, Yoshimi Nakamura, Mikiko Yanagawa, Mika K Kaneko, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(3)　77-81　2020/06

DOI： 10.1089/mab.2020.0004 　

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Anti-bear podoplanin (bPDPN) monoclonal antibodies (mAbs), including PMab-247 and PMab-241, have been previously established. Although PMab-247 has shown positive immunostaining for lymphatic endothelial cells (LECs), type I alveolar cells of the lung, and podocytes of the kidney, PMab-241 stains LECs but does not react with lung type I alveolar cells. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and the PLAG-like domain (PLD). The binding epitope of PMab-247 was previously determined to include bPDPN residues Asp76, Arg78, Glu80, and Arg82. Among these, Glu80 and Arg82 are included in PLD of bPDPN. The purpose of this study is to determine the binding epitope of PMab-241 and to clarify the difference between these two anti-bPDPN mAbs. Analysis of bPDPN deletion mutants revealed that the N-terminus of the PMab-241 epitope exists between amino acids (aa) 75 and 80 of bPDPN. In addition, analysis of bPDPN point mutants demonstrated that the critical epitope of PMab-241 includes Thr75, Asp76, and Arg78 of bPDPN. The binding epitopes of PMab-241 and PMab-247 seem to overlap, but this slight difference may be sufficient to provide the specificity of PMab-241 to discriminate LECs from type I alveolar cells of the lung.
H2Mab-19 Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody Therapy Exerts Antitumor Activity in Pancreatic Cancer Xenograft Models. International-journal Peer-reviewed

Yukinari Kato, Tomokazu Ohishi, Masato Sano, Teizo Asano, Yusuke Sayama, Junko Takei, Manabu Kawada, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(3)　61-65　2020/05/18

DOI： 10.1089/mab.2020.0011 　

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Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast cancer, gastric, lung, colorectal, oral, and pancreatic cancers. HER2 expression is associated with poor clinical outcomes. An anti-HER2 humanized antibody, trastuzumab, has improved survival rates in patients with HER2-overexpressing breast and gastric cancers. Previously, we established a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa). It has also been characterized for breast, oral, and colon cancers. In this study, we investigated the antitumor activities of H2Mab-19 in pancreatic cancer xenograft models. We selected MIA PaCa-2, a pancreatic cancer cell line which expresses HER2. H2Mab-19 showed high binding affinity (KD: 1.2 × 10-8 M) against MIA PaCa-2 cells. Furthermore, H2Mab-19 significantly reduced tumor development in a MIA PaCa-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing pancreatic cancers.
Thr80 of Sheep Podoplanin Is a Critical Epitope of the Antisheep Podoplanin Monoclonal Antibody: PMab-256. International-journal Peer-reviewed

Yukinari Kato, Masato Sano, Teizo Asano, Yusuke Sayama, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(3)　95-100　2020/05/18

DOI： 10.1089/mab.2020.0010 　

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An antisheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established. PMab-256 shows positive immunostaining for lymphatic endothelial cells, lung type I alveolar cells, and kidney podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG domains or PLD. The purpose of this study is to determine the binding epitope of PMab-256. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids 75 and 80 of sPDPN. Furthermore, analysis of sPDPN point mutations demonstrated that the critical epitope includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.
Site-specific epitope insertion into recombinant proteins using the MAP tag system. International-journal Peer-reviewed

Ayami Wakasa, Mika K Kaneko, Yukinari Kato, Junichi Takagi, Takao Arimori

Journal of biochemistry　168　(4)　375-384　2020/05/09

DOI： 10.1093/jb/mvaa054 　

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The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8-residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted β-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in "site-specific epitope insertion" application.
PDPN Is Expressed in Various Types of Canine Tumors and Its Silencing Induces Apoptosis and Cell Cycle Arrest in Canine Malignant Melanoma. International-journal Peer-reviewed

Masahiro Shinada, Daiki Kato, Satoshi Kamoto, Sho Yoshimoto, Masaya Tsuboi, Ryohei Yoshitake, Shotaro Eto, Namiko Ikeda, Kohei Saeki, Yuko Hashimoto, Yosuke Takahashi, James Chambers, Kazuyuki Uchida, Mika K Kaneko, Naoki Fujita, Ryohei Nishimura, Yukinari Kato, Takayuki Nakagawa

Cells　9　(5)　2020/05/05

DOI： 10.3390/cells9051136 　

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Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed. It is also known to be linked with several aspects of tumor malignancy in some types of human tumors, including invasion, metastasis, and cancer stemness. However, there are few reports on the expression of dog PDPN (dPDPN) in canine tumors, and the association between dPDPN and tumor malignancy has not been elucidated. We identified that 11 out of 18 types of canine tumors expressed dPDPN. Furthermore, 80% of canine malignant melanoma (MM), squamous cell carcinoma, and meningioma expressed dPDPN. Moreover, the expression density of dPDPN was positively associated with the expression of the Ki67 proliferation marker. The silencing of dPDPN by siRNAs resulted in the suppression of cell migration, invasion, stem cell-like characteristics, and cell viability in canine MM cell lines. The suppression of cell viability was caused by the induction of apoptosis and G2/M phase cell cycle arrest. Overall, this study demonstrates that dPDPN is expressed in various types of canine tumors and that dPDPN silencing suppresses cell viability through apoptosis and cell cycle arrest, thus providing a novel biological role for PDPN in tumor progression.
Targeted Phototherapy for Malignant Pleural Mesothelioma: Near-Infrared Photoimmunotherapy Targeting Podoplanin. International-journal Peer-reviewed

Yuko Nishinaga, Kazuhide Sato, Hirotoshi Yasui, Shunichi Taki, Kazuomi Takahashi, Misae Shimizu, Rena Endo, Chiaki Koike, Noriko Kuramoto, Shota Nakamura, Takayuki Fukui, Hiroshi Yukawa, Yoshinobu Baba, Mika K Kaneko, Toyofumi F Chen-Yoshikawa, Hisataka Kobayashi, Yukinari Kato, Yoshinori Hasegawa

Cells　9　(4)　2020/04/20

DOI： 10.3390/cells9041019 　

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Malignant pleural mesothelioma (MPM) has extremely limited treatment despite a poor prognosis. Moreover, molecular targeted therapy for MPM has not yet been implemented; thus, a new targeted therapy is highly desirable. Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer therapy that combines the specificity of antibodies for targeting tumors with toxicity induced by the photoabsorber after exposure to NIR-light. In this study, we developed a new phototherapy targeting podoplanin (PDPN) for MPM with the use of both NIR-PIT and an anti-PDPN antibody, NZ-1. An antibody-photosensitizer conjugate consisting of NZ-1 and phthalocyanine dye was synthesized. In vitro NIR-PIT-induced cytotoxicity was measured with both dead cell staining and luciferase activity on various MPM cell lines. In vivo NIR-PIT was examined in both the flank tumor and orthotopic mouse model with in vivo real-time imaging. In vitro NIR-PIT-induced cytotoxicity was NIR-light dose dependent. In vivo NIR-PIT led to significant reduction in both tumor volume and luciferase activity in a flank model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). The PDPN-targeted NIR-PIT resulted in a significant antitumor effect in an MPM orthotopic mouse model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). This study suggests that PDPN-targeted NIR-PIT could be a new promising treatment for MPM.
Detection of Circulating Tumor Cells (CTCs) in Malignant Pleural Mesothelioma (MPM) with the "Universal" CTC-Chip and An Anti-Podoplanin Antibody NZ-1.2. International-journal Peer-reviewed

Taiji Kuwata, Kazue Yoneda, Masataka Mori, Masatoshi Kanayama, Koji Kuroda, Mika K Kaneko, Yukinari Kato, Fumihiro Tanaka

Cells　9　(4)　2020/04/05

DOI： 10.3390/cells9040888 　

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Circulating tumor cell (CTC) is a potentially useful surrogate of micro-metastasis, but detection of rare tumor cells contaminated in a vast majority of normal hematologic cells remains technical challenges. To achieve effective detection of a variety of CTCs, we have developed a novel microfluidic system (CTC-chip) in which any antibody to capture CTCs is easily conjugated. In previous studies, we employed an antibody (clone E-1) against podoplanin that was strongly expressed on mesothelioma cells. The CTC-chip coated by the E-1 antibody (E1-chip) provided a modest sensitivity in detection of CTCs in malignant pleural mesothelioma (MPM). Here, to achieve a higher sensitivity, we employed a novel anti-podoplanin antibody (clone NZ-1.2). In an experimental model, MPM cells with high podoplanin expression were effectively captured with the CTC-chip coated by the NZ-1.2 antibody (NZ1.2-chip). Next, we evaluated CTCs in the peripheral blood sampled from 22 MPM patients using the NZ1.2-chip and the E1-chip. One or more CTCs were detected in 15 patients (68.2%) with the NZ1.2-chip, whereas only in 10 patients (45.5%) with the E1-chip. Of noted, in most (92.3%, 12/13) patients with epithelioid MPM subtype, CTCs were positive with the NZ1.2-chip. The CTC-count detected with the NZ1.2-chip was significantly higher than that with the E1-chip (p = 0.034). The clinical implications of CTCs detected with the NZ1.2-chip will be examined in a future study.
Antibody-Drug Conjugates Using Mouse-Canine Chimeric Anti-Dog Podoplanin Antibody Exerts Antitumor Activity in a Mouse Xenograft Model. International-journal Peer-reviewed

Yukinari Kato, Yuji Ito, Tomokazu Ohishi, Manabu Kawada, Takuro Nakamura, Yusuke Sayama, Masato Sano, Teizo Asano, Miyuki Yanaka, Saki Okamoto, Saori Handa, Yu Komatsu, Junko Takei, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(2)　37-44　2020/04

DOI： 10.1089/mab.2020.0001 　

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Antibody-drug conjugates (ADCs), which consist of a monoclonal antibody (mAb), a linker, and a payload, can deliver a drug to cancer tissues. We previously produced an anti-dog podoplanin (dPDPN) mAb, PMab-38, which reacts with dPDPN-expressing canine melanomas and squamous cell carcinomas (SCCs), but not with dPDPN-expressing canine type I alveolar cells or lymphatic endothelial cells, indicating that PMab-38 possesses cancer specificity. In this study, we developed an ADC, P38B-DM1, using the mouse-canine chimeric anti-dPDPN antibody, P38B as the antibody, a peptide linker, and emtansine as the payload using the chemical conjugation by affinity peptide (CCAP) method. We investigated its cytotoxicity against dPDPN-overexpressed Chinese hamster ovary (CHO/dPDPN) cells in vitro and its antitumor activity using a mouse xenograft model of CHO/dPDPN cells. P38B-DM1 showed cytotoxicity to CHO/dPDPN cells in a dose-dependent manner in vitro. Furthermore, P38B-DM1 exhibited higher antitumor activity than P38B in the mouse xenograft model. These results suggest that P38B-DM1, developed using the CCAP method, is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.
A novel anti-EGFR monoclonal antibody (EMab-17) exerts antitumor activity against oral squamous cell carcinomas via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. International-journal Peer-reviewed

Junko Takei, Mika Kato Kaneko, Tomokazu Ohishi, Manabu Kawada, Hiroyuki Harada, Yukinari Kato

Oncology letters　19　(4)　2809-2816　2020/04

DOI： 10.3892/ol.2020.11384 　

ISSN： 1792-1082

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The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG2a, kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.
Development of Novel Mouse Monoclonal Antibodies Against Human CD19. International-journal Peer-reviewed

Shinji Yamada, Mika K Kaneko, Yusuke Sayama, Teizo Asano, Masato Sano, Miyuki Yanaka, Takuro Nakamura, Saki Okamoto, Saori Handa, Yu Komatsu, Yoshimi Nakamura, Yoshikazu Furusawa, Junko Takei, Yukinari Kato

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(2)　45-50　2020/04

DOI： 10.1089/mab.2020.0003 　

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CD19 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is expressed in normal and neoplastic B cells, and it modulates the threshold of B cell activation for amplifying B cell receptor signaling. Blinatumomab (a CD3-CD19-bispecific T cell-engaging antibody) and tisagenlecleucel (genetically modified T cells that express a CD19 chimeric antigen receptor [CART-19]) provide significant benefits for patients with CD19-positive relapsed or refractory B cell malignancies. In this study, we first employed the Cell-Based Immunization and Screening (CBIS) method to produce anti-CD19 monoclonal antibodies using CD19-overexpressing cells for both immunization and screening. One established clone-C19Mab-1-proved to be useful in flow cytometry assays against lymphoma cell lines, such as BALL-1, P30/OHK, and Raji. Second, the extracellular domain of CD19 was immunized into mice, and enzyme-linked immunosorbent assays were performed for the first screening. One established clone-C19Mab-3-was determined to be useful for Western blotting and immunohistochemical analysis. Due to their complementary utility, a combination of C19Mab-1 (established using CBIS) and C19Mab-3 (established using conventional method) could be useful for the pathological analysis of CD19.
CDK1 dependent phosphorylation of hTERT contributes to cancer progression. International-journal Peer-reviewed

Mami Yasukawa, Yoshinari Ando, Taro Yamashita, Yoko Matsuda, Shisako Shoji, Masaki Suimye Morioka, Hideya Kawaji, Kumiko Shiozawa, Mitsuhiro Machitani, Takaya Abe, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Yasuhide Furuta, Tadashi Kondo, Mikako Shirouzu, Yoshihide Hayashizaki, Shuichi Kaneko, Kenkichi Masutomi

Nature communications　11　(1)　1557-1557　2020/03/25

DOI： 10.1038/s41467-020-15289-7 　

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The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.
Establishment of anti-horse podoplanin monoclonal antibody using Cell-Based Immunization and Screening (CBIS) method

Sayama Yusuke, K. Kaneko Mika, Kato Yukinari

Proceedings for Annual Meeting of The Japanese Pharmacological Society　93　3-O-127　2020
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.93.0_3-o-127 　

eISSN： 2435-4953

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Purpose: Podoplanin is expressed in normal tissues including renal podocytes and lymphatic endothelial cells. To investigate the expression and function of horse podoplanin (horPDPN), sensitive and specific mAbs against horPDPN are necessary. In this study, we aimed to develop useful anti-horPDPN mAbs for many applications such as flow cytometry (FCM), western blot (WB), and immunohistochemistry (IHC). Methods: We employed a conventional immunization method using synthetic peptides or Cell-Based Immunization and Screening (CBIS) method using horPDPN-expressed mammalian cells for producing anti-horPDPN mAbs. Anti-horPDPN mAbs were screened using enzyme-linked immunosorbent assay or FCM. Established anti-horPDPN mAbs were characterized using FCM, WB, and IHC. Results: We developed two anti-horPDPN mAbs, PMab-202 using the peptide immunization and PMab-219 using CBIS method. PMab-202 reacted with horPDPN in FCM and WB, but did not stain horPDPN in IHC. In contrast, PMab-219 detected horPDPN in not only FCM and WB, but also IHC. Conclusion: We have successfully established mouse anti-horPDPN mAbs, PMab-202 and PMab-219. PMab-219 is applicable for FCM, WB, and IHC analyses. CBIS method could be more advantageous to establish immunohistochemistry-applicable mAbs for elucidating the pathophysiological function of horPDPN.
An anti-CD133 monoclonal antibody CMab-43 exerts anti-tumor and anti-metastasis activities for colon cancers

Kato Yukinari, Kaneko Mika, Ohishi Tomokazu, Kawada Manabu

Proceedings for Annual Meeting of The Japanese Pharmacological Society　93　(0)　3-O-111　2020
Publisher: Japanese Pharmacological Society
DOI： 10.1254/jpssuppl.93.0_3-O-111 　

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Background: Cancer stem cells contribute to tumorigenesis, metastasis, and chemoresistance. A pentaspan membrane glycoprotein CD133 has been used for the isolation of stem-like cells from several cancers.Purpose: In this study, we aimed to develop sensitive and specific anti-CD133 mAbs, which exerts anti-tumor and anti-metastasis activities.Methods: Cell-Based Immunization and Screening (CBIS) method was employed for the development of anti-CD133 mAbs. LN229/CD133 glioblastoma cells were immunized into mice, and FCM was used for the first screening. WB and IHC screenings were further performed. Human colon cancer cell lines were used for examining the anti-tumor and the anti-metastasis activities of anti-CD133 mAbs.Results: We established a novel anti-CD133 mAb, CMab-43 (IgG_2a, kappa), which demonstrated a sensitive and specific reaction against colon cancer cells in FCM, WB, and IHC analyses. CMab-43 showed cancer-specific staining patterns in colon cancer tissues. Furthermore, CMab-43 significantly reduced tumor development of colon cancer cell xenografts, and inhibited experimental metastasis of colon cancer cells.Conclusion: CMab-43 is useful for many applications and exerts anti-tumor or anti-metastasis activities. CMab-43 could be advantageous for antibody therapy against CD133-expressing colon cancers.
Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry. International-journal Peer-reviewed

Sayama Y, Sano M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(1)　17-22　2020/01

DOI： 10.1089/mab.2019.0045 　

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Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregation-stimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.
BACH1 promotes pancreatic cancer metastasis by repressing epithelial genes and enhancing epithelial-mesenchymal transition. International-journal Peer-reviewed

Sato M, Matsumoto M, Saiki Y, Alam M, Nishizawa H, Rokugo M, Brydun A, Yamada S, Kaneko MK, Funayama R, Ito M, Kato Y, Nakayama K, Unno M, Igarashi K

Cancer research　80　(6)　1279-1292　2020/01

DOI： 10.1158/0008-5472.CAN-18-4099 　

ISSN： 0008-5472

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Pancreatic ductal adenocarcinoma (PDAC) is among the cancers with the poorest prognoses due to its highly malignant features. BTB and CNC homology 1 (BACH1) has been implicated in RAS-driven tumor formation. We focused on the role of BACH1 in PDAC, more than 90% of which have KRAS mutation. Knockdown of BACH1 in PDAC cell lines reduced cell migration and invasion, in part, by increasing E-cadherin expression, whereas its overexpression showed opposite effects. BACH1 directly repressed the expression of FOXA1 that is known to activate the expression of CDH1 encoding E-cadherin and to inhibit epithelial-to-mesenchymal transition. BACH1 also directly repressed the expression of genes important for epithelial cell adhesion including CLDN3 and CLDN4. In a mouse orthotopic implantation model, BACH1 was required for the high metastatic ability of AsPC-1 cells. IHC analysis of clinical specimens with a newly developed anti-BACH1 mAb revealed that high expression of BACH1 is a poor prognostic factor. These results suggest that the gene regulatory network of BACH1 and downstream genes including CDH1 contribute to the malignant features of PDAC by regulating epithelial-to-mesenchymal transition. SIGNIFICANCE: Greater understanding of the gene regulatory network involved in epithelial-to-mesenchymal transition of pancreatic cancer cells will provide novel therapeutic targets and diagnostic markers.
Upregulation of PD-L1 expression by prostaglandin E2 and the enhancement of IFN-γ by anti-PD-L1 antibody combined with a COX-2 inhibitor in Mycoplasma bovis infection International-journal Peer-reviewed

Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

Front Vet Sci　7　12-12　2020

DOI： 10.3389/fvets.2020.00012 　

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Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.
Clinical efficacy of the combined treatment of anti-PD-L1 rat-bovine chimeric antibody with a COX-2 inhibitor in calves infected with Mycoplasma bovis Peer-reviewed

Goto S, Konnai S, Hirano Y, Kohara J, Okagawa T, Maekawa N, Sajiki Y, Watari K, Minato E, Kobayashi A, Gondaira S, Higuchi H, Koiwa M, Tajima M, Taguchi E, Ishida M, Uemura R, Yamada S, Kaneko M, Kato Y, Yamamoto K, Toda M, Suzuki Y, Murata S, Ohashi K

Jpn J Vet Res　68　(2)　77-90　2020

DOI： 10.14943/jjvr.68.2.77 　

ISSN： 0047-1917
Epitope Mapping of PMab-225 an Anti-Alpaca Podoplanin Monoclonal Antibody Using Flow Cytometry. International-journal Peer-reviewed

Sayama Y, Sano M, Furusawa Y, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(6)　255-260　2019/12

DOI： 10.1089/mab.2019.0033 　

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Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.
Epitope Mapping of Monoclonal Antibody PMab-233 Against Tasmanian Devil Podoplanin. International-journal Peer-reviewed

Sano M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(6)　261-265　2019/12

DOI： 10.1089/mab.2019.0032 　

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The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses three tandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAG-like domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to be clarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34 of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.
Epitope Analysis of an Antihorse Podoplanin Monoclonal Antibody PMab-219. International-journal Peer-reviewed

Kato Y, Sayama Y, Sano M, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(6)　266-270　2019/12

DOI： 10.1089/mab.2019.0034 　

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Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression is associated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. In addition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In this study, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between 55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 of horPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.
The Epitope of PMab-210 Is Located in Platelet Aggregation-Stimulating Domain-3 of Pig Podoplanin. International-journal Peer-reviewed

Kaneko MK, Sayama Y, Sano M, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(6)　271-276　2019/12

DOI： 10.1089/mab.2019.0037 　

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Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.
PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear International-journal Peer-reviewed

Takei J, Yamada S, Konnai S, Ishinazaka T, Shimozuru M, Kaneko MK, Kato Y

Monoclon Antib Immunodiagn Immunother.　38　(6)　282-284　2019/12

DOI： 10.1089/mab.2019.0038 　

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Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.
Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy. International-journal Peer-reviewed

Sakamoto S, Inoue H, Kaneko MK, Ogasawara S, Kajikawa M, Urano S, Ohba SI, Kato Y, Kawada M

Cancer science　110　(11)　3595-3602　2019/11

DOI： 10.1111/cas.14196 　

ISSN： 1347-9032

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Coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein that is associated with adenoviral infection. CAR is involved in the formation of epithelial tight junctions and promotes tumor growth in some cancers. Previously, we developed mouse monoclonal antibodies against human CAR and found that one, mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells. Herein, we generated and characterized a mouse-human chimeric anti-CAR antibody (ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and in vivo anti-tumor activity against CAR-expressing prostate cancer DU-145 cells. In addition, cancer tissue array analysis confirmed that CAR is highly expressed in neuroendocrine lung cancers including small cell lung cancer, and treatment with ch6G10A effectively inhibited in vivo subcutaneous tumor growth of NCI-H69 small cell lung cancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibited both in vivo orthotopic tumor growth and distant metastatic formation in mouse xenograft models of a highly metastatic subline of human small cell lung cancer DMS273 cells. These results suggest that targeting therapy to CAR with a therapeutic antibody might be effective against several cancer types including small cell lung cancer.
Characterization of Anti-Goat Podoplanin Monoclonal Antibody PMab-235 Using Immunohistochemistry Against Goat Tissues. International-journal Peer-reviewed

Takei J, Itai S, Harada H, Furusawa Y, Miwa T, Fukui M, Nakamura T, Sano M, Sayama Y, Yanaka M, Handa S, Hisamatsu K, Nakamura Y, Yamada S, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(5)　213-219　2019/10

DOI： 10.1089/mab.2019.0022 　

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Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.
Epitope Mapping of Antipig Podoplanin Monoclonal Antibody PMab-213. International-journal Peer-reviewed

Yamada S, Itai S, Furusawa Y, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(5)　224-229　2019/10

DOI： 10.1089/mab.2019.0023 　

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Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on lymphatic endothelial cells, podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor, comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported a mouse antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developed by CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213 using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN.
Epitope mapping of anti-bear podoplanin monoclonal antibody PMab-247 International-journal Peer-reviewed

Kato Y, Takei J, Sayama Y, Sano M, Konnai S, Kobayashi A, Harada H, Yamada S, Kaneko MK

Monoclon Antib Immunodiagn Immunother.　38　(5)　230-233　2019/10

DOI： 10.1089/mab.2019.0025 　

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Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs.
新規HER2モノクローナル抗体の大腸がんxenograftモデルにおける抗腫瘍効果(Antitumor activity of novel anti-HER2 monoclonal antibodies in a mouse xenograft model of colon cancer)

山田慎二, 大石智一, 金子美華, 川田学, 加藤幸成

日本癌学会総会記事　78回　P-1096　2019/09
Publisher: 日本癌学会
ISSN： 0546-0476
がん幹細胞マーカーCD133を標的とした抗体医薬開発

武井潤子, 金子美華, 山田慎二, 大石智一, 川田学, 原田浩之, 加藤幸成

日本生化学会大会プログラム・講演要旨集　92回　[3T17m-03]　2019/09
Publisher: (公社)日本生化学会
Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection. International-journal Peer-reviewed

Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Kei Watari, Erina Minato, Atsushi Kobayashi, Junko Kohara, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Hirofumi Takahashi, Nobuhiro Terasaki, Akira Takeda, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi

Journal of immunology (Baltimore, Md. : 1950)　203　(5)　1313-1324　2019/09/01

DOI： 10.4049/jimmunol.1900342 　

ISSN： 0022-1767
イヌ腫瘍組織におけるPodoplanin発現の解析 Peer-reviewed

品田真央, 嘉本諭, 加藤大貴, 坪井誠也, 吉本翔, Chambers James, 内田和幸, 池田凡子, 佐伯亘平, 山田慎二, 金子美華, 西村亮平, 加藤幸成, 中川貴之

日本獣医学会学術集会講演要旨集　162回　459-459　2019/08
Publisher: (公社)日本獣医学会
ISSN： 1347-8621
Epitope Mapping of Anti-Diacylglycerol Kinase Zeta Monoclonal Antibody DzMab-1 for Immunohistochemical Analyses. International-journal Peer-reviewed

Sano M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(4)　175-178　2019/08

DOI： 10.1089/mab.2019.0020 　

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The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has been reported to be terminated by DGKζ, 1 of 10 DGK isoforms in most cases. We previously established an anti-DGKζ monoclonal antibody (mAb) DzMab-1 (rat IgG1, kappa), which reacts with both mouse DGKζ and human DGKζ (hDGKζ). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues of hDGKζ are important for facilitating DzMab-1 binding to hDGKζ. Furthermore, DzMab-1 was shown to be useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings could be applied for the production of more functional anti-hDGKζ mAbs.
PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. International-journal Peer-reviewed

Furusawa Y, Yamada S, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, Kaneko MK, Kato Y

Biochemistry and biophysics reports　18　100616-100616　2019/07

DOI： 10.1016/j.bbrep.2019.01.009 　

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Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.
Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin. International-journal Peer-reviewed

Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano M, Harada H, Fukui M, Kaneko MK, Kato Y

Biochemistry and biophysics reports　18　100631-100631　2019/07

DOI： 10.1016/j.bbrep.2019.100631 　

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Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.
Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses. International-journal Peer-reviewed

Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M, Kaneko MK

Biochemistry and biophysics reports　18　100633-100633　2019/07

DOI： 10.1016/j.bbrep.2019.100633 　

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Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.
Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis. Peer-reviewed

Furusawa Y, Takei J, Sayama Y, Yamada S, Kaneko MK, Kato Y

Biochemistry and biophysics reports　18　100644　2019/07

DOI： 10.1016/j.bbrep.2019.100644 　
PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin International-journal Peer-reviewed

Furusawa Yoshikazu, Yamada Shinji, Nakamura Takuro, Sano Masato, Sayama Yusuke, Itai Shunsuke, Takei Junko, Harada Hiroyuki, Fukui Masato, Kaneko Mika K, Kato Yukinari

HELIYON　5　(7)　e02063　2019/07

DOI： 10.1016/j.heliyon.2019.e02063 　

ISSN： 2405-8440
Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody H₂Mab-41 Exerts Antitumor Activity in a Mouse Xenograft Model of Colon Cancer. International-journal Peer-reviewed

Kato Y, Ohishi T, Yamada S, Itai S, Takei J, Sano M, Nakamura T, Harada H, Kawada M, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(4)　157-161　2019/06

DOI： 10.1089/mab.2019.0017 　

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The expression of human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed in several cancers, such as breast, lung, gastric, pancreatic, and colorectal cancers, and be associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H2Mab-41 (IgG2b, kappa), and the antitumor activity of H2Mab-41 was investigated using mouse xenograft models. Caco-2 cells (human colon cancer cell line), which expresses HER2, were subcutaneously implanted into the flanks of nude mice. H2Mab-41 and control mouse IgG were injected three times into the peritoneal cavity of mice. H2Mab-41 significantly reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 5, 8, 11, 15, and 19. Taken together, these results suggest that H2Mab-41 is useful for antibody therapy against HER2-expressing colon cancers.
Establishment of an Anticetacean Podoplanin Monoclonal Antibody PMab-237 for Immunohistochemical Analysis. International-journal Peer-reviewed

Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M, Harada H, Yamada S, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(3)　108-113　2019/06

DOI： 10.1089/mab.2019.0013 　

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Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.
PMab-247 Detects Bear Podoplanin in Immunohistochemical Analysis International-journal Peer-reviewed

Takei J, Furusawa Y, Yamada S, Nakamura T, SayamaY, Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK, Kato Y

Monoclon Antib Immunodiagn Immunother.　38　(4)　171-174　2019/06

DOI： 10.1089/mab.2019.0019 　

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Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.
Immunohistochemical analysis of harbor porpoise using anti-podoplanin antibody PMab-237 International-journal Peer-reviewed

Yamada S, Itai S, Takei J, Konnai S, Kobayashi A, Nakagun S, Kobayashi Y, Kaneko MK, Kato Y

Monoclon Antib Immunodiagn Immunother.　38　(3)　104-107　2019/06

DOI： 10.1089/mab.2019.0014 　

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Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.
Establishment and Epitope Mapping of Anti-Diacylglycerol Kinase α Monoclonal Antibody DaMab-8 for Immunohistochemical Analyses. International-journal Peer-reviewed

Sano M, Kaneko MK, Suzuki H, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(1)　1-5　2019/05

DOI： 10.1089/mab.2019.0004 　

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Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). DGKα, 1 of the 10 DGK isozymes, is involved in T cell function. In the present study, we describe a specific monoclonal antibody DaMab-8 (mouse IgG1, kappa) against DGKα, which is extremely useful for performing immunohistochemical analysis for T cells in oropharyngeal squamous cell carcinomas. Furthermore, we characterized the binding epitope of DaMab-8 using Western blotting and found that the sites Asn610, Leu611, Trp612, Gly613, Asp614, His619, Tyr623, and Gly624 of DGKα are important for facilitating the DaMab-8 binding to the DGKα protein. Thus, DaMab-8 could be advantageous for immunohistochemical analyses toward clarifying the distribution of DGKα-expressing T cells in every pathophysiological tissue.
Epitope Mapping of Anti-Diacylglycerol Kinase ζ Monoclonal Antibody for the Detection of T Cells by Immunohistochemical Analyses. Peer-reviewed

Kato Y, Itai S, Yamada S, Suzuki H, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(3)　124-128　2019/05

DOI： 10.1089/mab.2019.0005 　
Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231. International-journal Peer-reviewed

Takei J, Itai S, Furusawa Y, Yamada S, Nakamura T, Sano M, Harada H, Fukui M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(3)　129-132　2019/05

DOI： 10.1089/mab.2019.0012 　

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Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.
Epitope Mapping of Antihuman Diacylglycerol Kinase Gamma Monoclonal Antibody DgMab-6. International-journal Peer-reviewed

Sano M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(3)　120-123　2019/05

DOI： 10.1089/mab.2019.0011 　

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Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). Both DG and PA serve as lipidic second messengers; therefore, DGK plays a critical role in regulating the balance of two signaling pathways mediated by DG and PA in cellular functions. DGK gamma (DGKγ), 1 of the 10 mammalian DGK isozymes, has been reported to be involved in membrane traffic, mast cell function, and leukemic cell differentiation. We previously developed a novel anti-DGKγ monoclonal antibody (mAb), DgMab-6, which is very useful in immunocytochemistry of human cultured cells. In this study, we characterized the binding epitope of DgMab-6 using Western blotting, and Glu12 is important for facilitating the DgMab-6 binding to the DGKγ protein. These results might lead to further development for sensitive and specific mAbs against DGKγ.
Therapeutic efficacy evaluation of radioimmunotherapy with Y-90-labeled anti-podoplanin antibody NZ-12 for mesothelioma International-journal Peer-reviewed

Sudo Hitomi, Tsuji Atsushi B, Sugyo Aya, Saga Tsuneo, Kaneko Mika K, Kato Yukinari, Higashi Tatsuya

CANCER SCIENCE　110　(5)　1653-1664　2019/05

DOI： 10.1111/cas.13979 　

ISSN： 1347-9032
Epitope Mapping of the Antihorse Podoplanin Monoclonal Antibody PMab-202. International-journal Peer-reviewed

Kaneko MK, Furusawa Y, Sano M, Itai S, Takei J, Harada H, Fukui M, Yamada S, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(2)　79-84　2019/04

DOI： 10.1089/mab.2019.0001 　

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Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.
Anti-CD133 Monoclonal Antibody CMab-43 Exerts Antitumor Activity in a Mouse Xenograft Model of Colon Cancer. International-journal Peer-reviewed

Kato Y, Ohishi T, Yamada S, Itai S, Furusawa Y, Sano M, Nakamura T, Kawada M, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(2)　75-78　2019/04

DOI： 10.1089/mab.2019.0002 　

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Cancer stem cells contribute to tumorigenesis, metastasis, recurrence, and chemoresistance. CD133/prominin-1-a pentaspan membrane glycoprotein-has been used as a stem cell biomarker for the isolation of stem-like cells from a variety of normal and pathological tissues. In our previous studies, we developed several anti-CD133 monoclonal antibodies using Cell-Based Immunization and Screening (CBIS) methods, followed by characterization of their efficacy by flow cytometry, western blotting, and immunohistochemical analyses. One of the 100 clones, CMab-43 (IgG2a, kappa), demonstrated a sensitive and specific reaction against colon cancer cells. This study aimed to investigate the antitumor activity of CMab-43. Caco-2 cells (human colon cancer cell line) were subcutaneously implanted into the flanks of nude mice. CMab-43 and control mouse IgG were injected three times into the peritoneal cavity of mice. Tumor formation was observed in the control and CMab-43-treated mice of Caco-2 xenograft models. CMab-43 significantly reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 12, 14, and 17. Our results cumulatively suggest that CMab-43 is useful for antibody therapy against CD133-expressing colon cancers.
Establishment of a Monoclonal Antibody PMab-231 for Tiger Podoplanin. Peer-reviewed

Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M, Harada H, Yamada S, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(2)　89-95　2019/04

DOI： 10.1089/mab.2019.0003 　
Anti-Bovine Podoplanin Monoclonal Antibody PMab-44 Detects Goat Podoplanin in Immunohistochemistry. International-journal Peer-reviewed

Yamada S, Kaneko MK, Furusawa Y, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Hisamatsu K, Nakamura Y, Koyanagi M, Fukui M, Harada H, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(2)　96-99　2019/04

DOI： 10.1089/mab.2018.0031 　

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Podoplanin (PDPN) is expressed in type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. We have characterized the PDPNs of various animal species using specific anti-PDPN monoclonal antibodies (mAbs). In this study, we investigated whether these anti-PDPN mAbs cross-react with goat PDPN (gPDPN). Flow cytometry demonstrated that the anti-bovine PDPN mAb PMab-44 (IgG1, kappa) reacts with gPDPN, which is overexpressed in CHO-K1 cells. Using immunohistochemical analysis, type I alveolar cells of goat lung were strongly detected by PMab-44. These results indicate that PMab-44 is useful for investigating gPDPN.
DzMab-1: Anti-Human Diacylglycerol Kinaseζ Monoclonal Antibody for Immunocytochemistry. Peer-reviewed

Nakano T, Ogasawara S, Tanaka T, Hozumi Y, Sano M, Sayama Y, Yamada S, Kaneko MK, Kato Y, Goto K

Monoclon Antib Immunodiagn Immunother.　38　(4)　179-182　2019/04

DOI： 10.1089/mab.2019.0024. 　
Application of the NZ-1 Fab as a crystallization chaperone for PA tag-inserted target proteins International-journal Peer-reviewed

Tamura Risako, Oi Rika, Akashi Satoko, Kaneko Mika K, Kato Yukinari, Nogi Terukazu

PROTEIN SCIENCE　28　(4)　823-836　2019/04

DOI： 10.1002/pro.3580 　

ISSN： 0961-8368
Epitope Mapping of Antidiacylglycerol Kinase α Monoclonal Antibody DaMab-2. International-journal Peer-reviewed

Sano M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(1)　8-11　2019/02

DOI： 10.1089/mab.2018.0047 　

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Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.
PMab-213: A Monoclonal Antibody for Immunohistochemical Analysis Against Pig Podoplanin. International-journal Peer-reviewed

Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(1)　18-24　2019/02

DOI： 10.1089/mab.2018.0048 　

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Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.
PMab-210: A Monoclonal Antibody Against Pig Podoplanin. International-journal Peer-reviewed

Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu M, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　38　(1)　30-36　2019/02

DOI： 10.1089/mab.2018.0038 　

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Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.
Evaluation of immunohistochemical staining with PMab-38, an anti-dog podoplanin monoclonal antibody, in various canine tumor tissues Peer-reviewed

Kiname Kohei, Yoshimoto Sho, Kato Daiki, Tsuboi Masaya, Tanaka Yuiko, Yoshitake Ryohei, Eto Shotaro, Shinada Masahiro, Chamberes James, Saeki Kohei, Kinoshita Ryohei, Yamada Shinji, Uchida Kazuyuki, Kaneko Mika K, Nishimura Ryohei, Kato Yukinari, Nakagawa Takayuki

JAPANESE JOURNAL OF VETERINARY RESEARCH　67　(1)　25-34　2019/02

DOI： 10.14943/jjvr.67.1.25 　

ISSN： 0047-1917
Immunohistochemical Detection of Sheep Podoplanin Using an Antibovine Podoplanin Monoclonal Antibody PMab-44 Peer-reviewed

Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK

Monoclon Antib Immunodiagn Immunother.　37　(6)　265-268　2018/12

DOI： 10.1089/mab.2018.0036 　
Detection of Alpaca Podoplanin by Immunohistochemistry Using the Antibovine Podoplanin Monoclonal Antibody PMab-44 Peer-reviewed

Kato Y, Yamada S, Itai S, Konnai S, Kobayashi A, Kaneko MK

Monoclon Antib Immunodiagn Immunother.　37　(6)　269-271　2018/12

DOI： 10.1089/mab.2018.0037 　
Anti-Horse Podoplanin Monoclonal Antibody PMab-219 is Useful for Detecting Lymphatic Endothelial Cells by Immunohistochemical Analysis Peer-reviewed

Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, Kaneko MK

Monoclon Antib Immunodiagn Immunother.　37　(6)　272-274　2018/12

DOI： 10.1089/mab.2018.0044 　
Antitumor activity by ADCC against oral squamous cell carcinomas by anti-podocalyxin antibody Peer-reviewed

Itai Shunsuke, Ohishi Tomokazu, Kaneko Mika, Yamada Shinji, Abe Shinji, Nishioka Yasuhiko, Kawada Manabu, Harada Hiroyuki, Kato Yukinari

CANCER SCIENCE　109　269　2018/12

ISSN： 1349-7006
Efficacy and safety study of anti-podoplanin cancer-specific monoclonal antibody, chLpMab-23 Peer-reviewed

Yamada Shinji, Kaneko Mika, Kunita Akiko, Abe Shinji, Itai Shunsuke, Fukayama Masashi, Nishioka Yasuhiko, Kato Yukinari

CANCER SCIENCE　109　465　2018/12

ISSN： 1349-7006
Establishment of P38Bf, a Core-Fucose-Deficient Mouse-Canine Chimeric Antibody Against Dog Podoplanin. International-journal Peer-reviewed

Kato Y, Mizuno T, Yamada S, Nakamura T, Itai S, Yanaka M, Sano M, Kaneko MK

Monoclonal antibodies in immunodiagnosis and immunotherapy　37　(5)　218-223　2018/11

DOI： 10.1089/mab.2018.0035 　

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Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10-7, 5.2 × 10-9, and 6.5 × 10-9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.
Detection of Tiger Podoplanin Using the Anti-Cat Podoplanin Monoclonal Antibody PMab-52. International-journal Peer-reviewed

Yamada S, Itai S, Furusawa Y, Sano M, Nakamura T, Yanaka M, Handa S, Hisamatsu K, Nakamura Y, Fukui M, Harada H, Mizuno T, Sakai Y, Ogasawara S, Murata T, Uchida H, Tahara H, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　37　(5)　224-228　2018/11

DOI： 10.1089/mab.2018.0033 　

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Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies (mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, and cat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab-52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These results indicate that PMab-52 may be useful for the detection of tiger PDPN.
Establishment of Monoclonal Antibody PMab-202 Against Horse Podoplanin. International-journal Peer-reviewed

Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　37　(5)　233-237　2018/11

DOI： 10.1089/mab.2018.0030 　

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Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs, PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN (CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses. PMab-202 is expected to prove useful in investigating the function of horse PDPN.
DgMab-6: Antihuman DGKγ Monoclonal Antibody for Immunocytochemistry. Peer-reviewed

Nakano T, Ogasawara S, Tanaka T, Hozumi Y, Yamaki A, Sakane F, Shirai Y, Nakamura T, Yanaka M, Yamada S, Kaneko MK, Kato Y, Goto K

Monoclon Antib Immunodiagn Immunother.　37　(5)　229-232　2018/11
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2018.0026 　

eISSN： 2167-9436
The mouse-canine chimeric anti-dog podoplanin antibody P38B exerts antitumor activity in mouse xenograft models Peer-reviewed

Kato Y, Ohishi T, Kawada M, Maekawa N, Konnai S, Itai S, Yamada S, Kaneko MK

Biochem Biophys Rep.　17　23-26　2018/11
がん特異的podoplaninを認識するヒトキメラ改変モノクローナル抗体(chLpMab-23)の抗腫瘍効果および安全性(Efficacy and safety study of anti-podoplanin cancer-specific monoclonal antibody, chLpMab-23)

山田慎二, 金子美華, 国田朱子, 阿部真治, 板井俊介, 深山正久, 西岡安彦, 加藤幸成

日本癌学会総会記事　77回　703-703　2018/09
Publisher: (一社)日本癌学会
ISSN： 0546-0476
口腔扁平上皮癌における抗ポドカリキシン抗体のADCCによる抗腫瘍効果(Antitumor activity by ADCC against oral squamous cell carcinomas by anti-podocalyxin antibody)

板井俊介, 大石智一, 金子美華, 山田慎二, 阿部真治, 西岡安彦, 川田学, 原田浩之, 加藤幸成

日本癌学会総会記事　77回　47-47　2018/09
Publisher: 日本癌学会
ISSN： 0546-0476
Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1. Peer-reviewed

Yamada S, Itai S, Kaneko MK, Konnai S, Kato Y

Biochemistry and biophysics reports　15　52-56　2018/09

DOI： 10.1016/j.bbrep.2018.07.002 　
Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6. Peer-reviewed

Kaneko MK, Yamada S, Itai S, Furusawa Y, Nakamura T, Yanaka M, Handa S, Hisamatsu K, Nakamura Y, Fukui M, Harada H, Kato Y

Biochemistry and biophysics reports　15　76-80　2018/09

DOI： 10.1016/j.bbrep.2018.07.003 　
Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2. International-journal Peer-reviewed

Itai S, Yamada S, Kaneko MK, Sano M, Nakamura T, Yanaka M, Handa S, Hisamatsu K, Nakamura Y, Furusawa Y, Fukui M, Ohishi T, Kawada M, Harada H, Kato Y

Biochemistry and biophysics reports　15　93-96　2018/09

DOI： 10.1016/j.bbrep.2018.07.008 　

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Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG2a-type, 47-mG2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.
猫腫瘍組織におけるPodoplaninの発現分析 Peer-reviewed

嘉本諭, 加藤大貴, 吉本翔, 坪井誠也, 池田凡子, 品田真央, Chambers James, 内田和幸, 佐伯亘平, 山田慎二, 金子美華, 西村亮平, 加藤幸成, 中川貴之

日本獣医学会学術集会講演要旨集　161回　426-426　2018/08
Publisher: (公社)日本獣医学会
ISSN： 1347-8621
Epitope Mapping of Anti-Telomerase Reverse Transcriptase Monoclonal Antibodies. Peer-reviewed

Furusawa Y, Itai S, Yamada S, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　37　(4)　185-187　2018/08

DOI： 10.1089/mab.2018.0019 　
Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2. Peer-reviewed

Furusawa Y, Yamada S, Itai S, Nakamura T, Fukui M, Harada H, Kaneko MK, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　37　(4)　188-193　2018/08

DOI： 10.1089/mab.2018.0025 　
A core fucose-deficient anti-podocalyxin antibody exhibits antitumor activity via augmented antibody-dependent cellular cytotoxicity in oral squamous cell carcinoma Peer-reviewed

Itai Shunsuke, Kaneko Mika K, Yamada Shinji, Kato Yukinari

CANCER RESEARCH　78　(13)　2018/07

DOI： 10.1158/1538-7445.AM2018-5617 　

ISSN： 0008-5472
An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system Peer-reviewed

Sanae Tabata, Yu Kitago, Yuki Fujii, Emiko Mihara, Keiko Tamura-Kawakami, Naoko Norioka, Katsu Takahashi, Mika K. Kaneko, Yukinari Kato, Junichi Takagi

Protein Expression and Purification　147　94-99　2018/07/01

DOI： 10.1016/j.pep.2018.03.004 　

ISSN： 1046-5928
Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134 Peer-reviewed

Mika K. Kaneko, Shinji Yamada, Shunsuke Itai, Yao-Wen Chang, Takuro Nakamura, Miyuki Yanaka, Yukinari Kato

Biochemistry and Biophysics Reports　14　54-57　2018/07/01
Publisher: Elsevier B.V.
DOI： 10.1016/j.bbrep.2018.03.010 　

ISSN： 2405-5808
Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5 Peer-reviewed

Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Mika K. Kaneko, Yukinari Kato

Biochemistry and Biophysics Reports　14　64-68　2018/07/01
Publisher: Elsevier B.V.
DOI： 10.1016/j.bbrep.2018.03.007 　

ISSN： 2405-5808
Determination of critical epitope of PcMab-47 against human podocalyxin. Peer-reviewed

Itai S, Yamada S, Kaneko MK, Kato Y

Biochemistry and biophysics reports　14　78-82　2018/07

DOI： 10.1016/j.bbrep.2018.04.003 　
Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin Peer-reviewed

Shinji Yamada, Mika K. Kaneko, Shunsuke Itai, Yao-Wen Chang, Takuro Nakamura, Miyuki Yanaka, Satoshi Ogasawara, Takeshi Murata, Hiroaki Uchida, Hideaki Tahara, Hiroyuki Harada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(3)　162-165　2018/06
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2018.0006 　

eISSN： 2167-9436
47-mG2a: A Mouse IgG2a-Type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry Peer-reviewed

Mika K. Kaneko, Shunsuke Itai, Shinji Yamada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(3)　158-161　2018/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2018.0003 　

ISSN： 2167-9436
Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase. Peer-reviewed

Kaneko MK, Yamada S, Itai S, Chang YW, Nakamura T, Yanaka M, Harada H, Suzuki H, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　2018/05

DOI： 10.1089/mab.2018.0007 　
Anti-podocalyxin antibody exerts antitumor effects via antibodydependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma Peer-reviewed

Shunsuke Itai, Tomokazu Ohishi, Mika K. Kaneko, Shinji Yamada, Shinji Abe, Takuro Nakamura, Miyuki Yanaka, Yao-Wen Chang, Shun-Ichi Ohba, Yasuhiko Nishioka, Manabu Kawada, Hiroyuki Harada, Yukinari Kato

Oncotarget　9　(32)　22480-22497　2018/04/27
Publisher: Impact Journals LLC
DOI： 10.18632/oncotarget.25132 　

ISSN： 1949-2553

eISSN： 1949-2553
LpMab-23-recognizing cancer-type podoplanin is a novel predictor for a poor prognosis of early stage tongue cancer Peer-reviewed

Akihiro Miyazaki, Hiromi Nakai, Tomoko Sonoda, Yoshihiko Hirohashi, Mika K. Kaneko, Yukinari Kato, Yoshihiko Sawa, Hiroyoshi Hiratsuka

Oncotarget　9　(30)　21156-21165　2018/04/20
Publisher: Impact Journals LLC
DOI： 10.18632/oncotarget.24986 　

ISSN： 1949-2553
Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin Peer-reviewed

Yao-Wen Chang, Mika K. Kaneko, Shinji Yamada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(2)　95-99　2018/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0067 　

ISSN： 2167-9436
Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers Peer-reviewed

Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Yao-Wen Chang, Hiroyoshi Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(2)　110-115　2018/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2018.0004 　

ISSN： 2167-9436
Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects Lung Cancers by Immunohistochemistry Peer-reviewed

Shinji Yamada, Shunsuke Itai, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(2)　91-94　2018/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2018.0002 　

ISSN： 2167-9436
Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4 Peer-reviewed

Shinji Yamada, Shunsuke Itai, Mika K. Kaneko, Yukinari Kato

Biochemistry and Biophysics Reports　13　123-128　2018/03/01
Publisher: Elsevier B.V.
DOI： 10.1016/j.bbrep.2018.01.009 　

ISSN： 2405-5808
PMab-48 Recognizes Dog Podoplanin of Lymphatic Endothelial Cells Peer-reviewed

Shinji Yamada, Shunsuke Itai, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(1)　63-66　2018/02/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0053 　

ISSN： 2167-9436
Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer Peer-reviewed

Mika K. Kaneko, Shinji Yamada, Shunsuke Itai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(1)　59-62　2018/02/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0052 　

ISSN： 2167-9436
Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel Peer-reviewed

Tomoto Suzuki, Yuya Takakubo, Hiroharu Oki, Xing Liu, Ryusuke Honma, Yasushi Naganuma, Stuart B. Goodman, Mika K. Kaneko, Yukinari Kato, Michiaki Takagi

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　37　(1)　12-19　2018/02/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0047 　

ISSN： 2167-9436
47-mG2a: A Mouse IgG2a-type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry Peer-reviewed

Mika K. Kaneko, Shunsuke Itai, Shinji Yamada, Yukinari Kato

Monoclon. Antib. Immunodiagn. Immunother.　37　(2)　91-94　2018
Anti-podocalyxin chimeric antibody chPcMab-47 targeting colorectal adenocarcinomas Peer-reviewed

Chang Yao-Wen, Kunita Akiko, Kaneko Mika K, Itai Shunsuke, Yamada Shinji, Ohishi Tomokazu, Abe Shinji, Nishioka Yasuhiko, Kawada Manabu, Fukayama Masashi, Kato Yukinari

CANCER SCIENCE　109　840　2018/01

ISSN： 1349-7006
Efficacy and safety study of anti-podocalyxin cancer-specific monoclonal antibody, chPcMab-6 Peer-reviewed

Yamada Shinji, Kaneko Mika K, Kunita Akiko, Ohishi Tomokazu, Abe Shinji, Chang Yao-Wen, Itai Shunsuke, Kawada Manabu, Fukayama Masashi, Nishioka Yasuhiko, Kato Yukinari

CANCER SCIENCE　109　848　2018/01

ISSN： 1349-7006
PcMab-47: a novel anti-human podocalyxin monoclonal antibody for immunohistochemistry against oral cancers Peer-reviewed

Itai Shunsuke, Yamada Shinji, Kaneko Mika K, Chang Yao-Wen, Kunita Akiko, Fukayama Masashi, Harada Hiroyuki, Kato Yukinari

CANCER SCIENCE　109　1052　2018/01

ISSN： 1349-7006
Motion analysis of furniture under seismic excitation using the finite element method Peer-reviewed

Isobe Daigoro, Yamashita Takuzo, Tagawa Hiroyuki, Kaneko Mika, Takahashi Toru, Motoyui Shojiro

JAPAN ARCHITECTURAL REVIEW　1　(1)　44-55　2018/01

DOI： 10.1002/2475-8876.1015 　

ISSN： 2475-8876
Elevated TERT Expression in TERT-Wildtype Adult Diffuse Gliomas: Histological Evaluation with a Novel TERT-Specific Antibody. International-journal Peer-reviewed

Kenta Masui, Takashi Komori, Yukinari Kato, Kenkichi Masutomi, Koichi Ichimura, Satoshi Ogasawara, Mika K Kaneko, Hiroharu Oki, Hiroyoshi Suzuki, Masayuki Nitta, Takashi Maruyama, Yoshihiro Muragaki, Takakazu Kawamata, Tatsuo Sawada, Noriyuki Shibata

BioMed research international　2018　7945845-7945845　2018

DOI： 10.1155/2018/7945845 　

ISSN： 2314-6133
Expression of Cat Podoplanin in Feline Squamous Cell Carcinomas Peer-reviewed

Shunsuke Itai, Shinji Yamada, Mika K. Kaneko, Hiroyuki Harada, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(6)　243-250　2017/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0046 　

ISSN： 2167-9436
Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity Peer-reviewed

Shunsuke Itai, Shinji Yamada, Mika K. Kaneko, Yao-Wen Chang, Hiroyuki Harada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(6)　272-281　2017/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0042 　

ISSN： 2167-9436
Epitope Mapping of Monoclonal Antibody PMab-38 Against Dog Podoplanin Peer-reviewed

Yao-Wen Chang, Shinji Yamada, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(6)　291-295　2017/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0048 　

ISSN： 2167-9436
Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer Peer-reviewed

Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Yao-Wen Chang, Hiroyuki Harada, Hiroyoshi Suzuki, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(6)　287-290　2017/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0050 　

ISSN： 2167-9436
PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry Peer-reviewed

Shinji Yamada, Shunsuke Itai, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Yao-Wen Chang, Saori Handa, Hiroyuki Harada, Yumiko Kagawa, Osamu Ichii, Satoru Konnai, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(5)　224-230　2017/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0027 　

ISSN： 2167-9436
Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas Peer-reviewed

Shunsuke Itai, Shinji Yamada, Mika K. Kaneko, Hiroyuki Harada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(5)　220-223　2017/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0030 　

ISSN： 2167-9436
Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry Peer-reviewed

Shunsuke Itai, Mika K. Kaneko, Yuki Fujii, Shinji Yamada, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Saori Handa, Yao-Wen Chang, Hiroyoshi Suzuki, Hiroyuki Harada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(5)　214-219　2017/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0028 　

ISSN： 2167-9436
Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry Peer-reviewed

Shunsuke Itai, Yuki Fujii, Takuro Nakamura, Yao-Wen Chang, Miyuki Yanaka, Noriko Saidoh, Saori Handa, Hiroyoshi Suzuki, Hiroyuki Harada, Shinji Yamada, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(5)　231-235　2017/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0031 　

ISSN： 2167-9436
口腔がんに対する免疫染色に有用な抗ポドカリキシン抗体PcMab-47の樹立

板井俊介, 山田慎二, 金子美華, 張耀文, 国田朱子, 深山正久, 原田浩之, 加藤幸成

日本癌学会総会記事　76回　P-3166　2017/09
Publisher: (一社)日本癌学会
ISSN： 0546-0476
ポドカリキシンを標的としたヒトキメラ型抗体chPcMab-47によるヒト大腸がん移植片モデルでの抗腫瘍効果の検討

張耀文, 国田朱子, 金子美華, 板井俊介, 山田慎二, 大石智一, 阿部真治, 西岡安彦, 川田学, 深山正久, 加藤幸成

日本癌学会総会記事　76回　E-3008　2017/09
Publisher: 日本癌学会
ISSN： 0546-0476
がん特異的podocalyxinを認識するヒトキメラ改変モノクローナル抗体(chPcMab-6)の抗腫瘍効果および安全性

山田慎二, 金子美華, 国田朱子, 大石智一, 阿部真治, 張耀文, 板井俊介, 川田学, 深山正久, 西岡安彦, 加藤幸成

日本癌学会総会記事　76回　J-3083　2017/09
Publisher: 日本癌学会
ISSN： 0546-0476
Antipodocalyxin Antibody chPcMab-47 Exerts Antitumor Activity in Mouse Xenograft Models of Colorectal Adenocarcinomas Peer-reviewed

Mika K. Kaneko, Akiko Kunita, Shinji Yamada, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Yao-Wen Chang, Saori Handa, Satoshi Ogasawara, Tomokazu Ohishi, Shinji Abe, Shunsuke Itai, Hiroyuki Harada, Manabu Kawada, Yasuhiko Nishioka, Masashi Fukayama, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(4)　157-162　2017/08/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0020 　

ISSN： 2167-9436

eISSN： 2167-9436
H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer Peer-reviewed

Shunsuke Itai, Yuki Fujii, Mika K. Kaneko, Shinji Yamada, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Yao-Wen Chang, Saori Handa, Maki Takahashi, Hiroyoshi Suzuki, Hiroyuki Harada, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(4)　143-148　2017/08/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0026 　

ISSN： 2167-9436
DaMab-2: Anti-Human DGKα Monoclonal Antibody for Immunocytochemistry Peer-reviewed

Tomoyuki Nakano, Satoshi Ogasawara, Toshiaki Tanaka, Yasukazu Hozumi, Satoru Mizuno, Eri Satoh, Fumio Sakane, Naoki Okada, Akinobu Taketomi, Ryusuke Honma, Takuro Nakamura, Noriko Saidoh, Miyuki Yanaka, Shunsuke Itai, Saori Handa, Yao-Wen Chang, Shinji Yamada, Mika K. Kaneko, Yukinari Kato, Kaoru Goto

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(4)　181-184　2017/08/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0023 　

ISSN： 2167-9436
Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44 Peer-reviewed

Shinji Yamada, Ryusuke Honma, Mika K. Kaneko, Takuro Nakamura, Miyuki Yanaka, Noriko Saidoh, Michiaki Takagi, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(3)　129-134　2017/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0016 　

ISSN： 2167-9436
ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity Peer-reviewed

Mika K. Kaneko, Takuro Nakamura, Akiko Kunita, Masashi Fukayama, Shinji Abe, Yasuhiko Nishioka, Shinji Yamada, Miyuki Yanaka, Noriko Saidoh, Kanae Yoshida, Yuki Fujii, Satoshi Ogasawara, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(3)　104-112　2017/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0014 　

ISSN： 2167-9436
Structural basis for perception of diverse chemical substances by T1r taste receptors Peer-reviewed

Nipawan Nuemket, Norihisa Yasui, Yuko Kusakabe, Yukiyo Nomura, Nanako Atsumi, Shuji Akiyama, Eriko Nango, Yukinari Kato, Mika K. Kaneko, Junichi Takagi, Maiko Hosotani, Atsuko Yamashita

NATURE COMMUNICATIONS　8　15530　2017/05

DOI： 10.1038/ncomms15530 　

ISSN： 2041-1723
Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity Peer-reviewed

Mika K. Kaneko, Shinji Yamada, Takuro Nakamura, Shinji Abe, Yasuhiko Nishioka, Akiko Kunita, Masashi Fukayama, Yuki Fujii, Satoshi Ogasawara, Yukinari Kato

CANCER MEDICINE　6　(4)　768-777　2017/04

DOI： 10.1002/cam4.1049 　

ISSN： 2045-7634
Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification Peer-reviewed

Yuki Fujii, Mika K. Kaneko, Satoshi Ogasawara, Shinji Yamada, Miyuki Yanaka, Takuro Nakamura, Noriko Saidoh, Kanae Yoshida, Ryusuke Honma, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(2)　68-71　2017/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0052 　

ISSN： 2167-9436
PcMab-47: Novel Antihuman Podocalyxin Monoclonal Antibody for Immunohistochemistry Peer-reviewed

Satoshi Ogasawara, Mika K. Kaneko, Shinji Yamada, Ryusuke Honma, Takuro Nakamura, Noriko Saidoh, Miyuki Yanaka, Kanae Yoshida, Yuki Fujii, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(2)　50-56　2017/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0008 　

ISSN： 2167-9436
LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin Peer-reviewed

Shinji Yamada, Satoshi Ogasawara, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(2)　72-76　2017/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0001 　

ISSN： 2167-9436
Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin Peer-reviewed

Shinji Yamada, Mika K. Kaneko, Takuro Nakamura, Osamu Ichii, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(2)　77-79　2017/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2017.0002 　

ISSN： 2167-9436
The chimeric antibody NZ-12 or CAR T cells targeting human podoplanin possesses antitumor activity against orthotopic xenograft models Peer-reviewed

Kato Yukinari, Shiina Satoshi, Natsume Atsushi, Abe Shinji, Nishioka Yasuhiko, Ogasawara Satoshi, Kaneko Mika K

CANCER IMMUNOLOGY RESEARCH　5　(3)　2017/03

DOI： 10.1158/2326-6074.TUMIMM16-B89 　

ISSN： 2326-6066
Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN Peer-reviewed

Mika K. Kaneko, Takuro Nakamura, Ryusuke Honma, Satoshi Ogasawara, Yuki Fujii, Shinji Abe, Michiaki Takagi, Hiroyuki Harada, Hiroyoshi Suzuki, Yasuhiko Nishioka, Yukinari Kato

CANCER MEDICINE　6　(2)　382-396　2017/02

DOI： 10.1002/cam4.954 　

ISSN： 2045-7634
Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain Peer-reviewed

Mika K. Kaneko, Shinji Abe, Satoshi Ogasawara, Yuki Fujii, Shinji Yamada, Takeshi Murata, Hiroaki Uchida, Hideaki Tahara, Yasuhiko Nishioka, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(1)　25-29　2017/02
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2016.0047 　

eISSN： 2167-9436
Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Peer-reviewed

Yukinari Kato, Akiko Kunita, Masashi Fukayama, Shinji Abe, Yasuhiko Nishioka, Hiroaki Uchida, Hideaki Tahara, Shinji Yamada, Miyuki Yanaka, Takuro Nakamura, Noriko Saidoh, Kanae Yoshida, Yuki Fujii, Ryusuke Honma, Michiaki Takagi, Satoshi Ogasawara, Takeshi Murata, Mika K. Kaneko

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　36　(1)　20-24　2017/02
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2016.0045 　

eISSN： 2167-9436
MAP Tag: A Novel Tagging System for Protein Purification and Detection Peer-reviewed

Yuki Fujii, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(6)　293-299　2016/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0039 　

ISSN： 2167-9436
Podoplanin Expression in Canine Melanoma Peer-reviewed

Satoshi Ogasawara, Ryusuke Honma, Mika K. Kaneko, Yuki Fujii, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(6)　304-306　2016/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0040 　

ISSN： 2167-9436
A platelet aggregation-inducing factor podoplanin is highly expressed in metastatic lesions of osteosarcoma

Hiroharu Oki, Mika Kaneko, Satoshi Ogasawara, Yuta Tujimoto, Xing Liu, Masato Sugawara, Yuya Takakubo, Takashi Tuchiya, Yukinari Kato, Michiaki Takagi

Journal of Orthopaedic Translation　7　119-120　2016/10
Publisher: Elsevier BV
DOI： 10.1016/j.jot.2016.06.144 　

ISSN： 2214-031X
LpMab-19 Recognizes Sialylated O-Glycan on Thr76 of Human Podoplanin Peer-reviewed

Satoshi Ogasawara, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(5)　245-253　2016/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0031 　

ISSN： 2167-9436
Establishment of Anti-Human ATRX Monoclonal Antibody AMab-6 Peer-reviewed

Satoshi Ogasawara, Yuki Fujii, Mika K. Kaneko, Hiroharu Oki, Hemragul Sabit, Mitsutoshi Nakada, Hiroyoshi Suzuki, Koichi Ichimura, Takashi Komori, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(5)　254-258　2016/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0037 　

ISSN： 2167-9436
PMab-38 Recognizes Canine Podoplanin of Squamous Cell Carcinomas Peer-reviewed

Mika K. Kaneko, Ryusuke Honma, Satoshi Ogasawara, Yuki Fujii, Takuro Nakamura, Noriko Saidoh, Michiaki Takagi, Yumiko Kagawa, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(5)　263-266　2016/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0036 　

ISSN： 2167-9436
Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies. International-journal Peer-reviewed

Masataka Umitsu, Katsuya Sakai, Satoshi Ogasawara, Mika K Kaneko, Ryoko Asaki, Keiko Tamura-Kawakami, Yukinari Kato, Kunio Matsumoto, Junichi Takagi

Scientific reports　6　33149-33149　2016/09/09

DOI： 10.1038/srep33149 　

ISSN： 2045-2322
Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model Peer-reviewed

Shinji Abe, Mika Kato Kaneko, Yuki Tsuchihashi, Toshihiro Izumi, Satoshi Ogasawara, Naoto Okada, Chiemi Sato, Makoto Tobiume, Kenji Otsuka, Licht Miyamoto, Koichiro Tsuchiya, Kazuyoshi Kawazoe, Yukinari Kato, Yasuhiko Nishioka

CANCER SCIENCE　107　(9)　1198-1205　2016/09

DOI： 10.1111/cas.12985 　

ISSN： 1347-9032

eISSN： 1349-7006
Structural basis for multi-specific peptide recognition by the anti-IDH1/2 monoclonal antibody, MsMab-1 Peer-reviewed

Yu Kitago, Mika K. Kaneko, Satoshi Ogasawara, Yukinari Kato, Junichi Takagi

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　478　(3)　1274-1279　2016/09

DOI： 10.1016/j.bbrc.2016.08.110 　

ISSN： 0006-291X

eISSN： 1090-2104
PMab-44 Detects Bovine Podoplanin in Immunohistochemistry Peer-reviewed

Ryusuke Honma, Satoshi Ogasawara, Mika K. Kaneko, Yuki Fujii, Hiroharu Oki, Takuro Nakamura, Michiaki Takagi, Satoru Konnai, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(4)　186-190　2016/08/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0016 　

ISSN： 2167-9436
Specific Detection of Dog Podoplanin Expressed in Renal Glomerulus by a Novel Monoclonal Antibody PMab-38 in Immunohistochemistry Peer-reviewed

Ryusuke Honma, Mika K. Kaneko, Satoshi Ogasawara, Yuki Fujii, Satoru Konnai, Michiaki Takagi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(4)　212-216　2016/08/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0022 　

ISSN： 2167-9436
A cancer-specific monoclonal antibody against podocalyxin developed by CasMab technology inhibited the tumor growth by antibody dependent cellular cytotoxicity Peer-reviewed

Yukinari Kato, Satoshi Ogasawara, Akiko Kunita, Yuki Fujii, Mika K. Kaneko

CANCER RESEARCH　76　2016/07

DOI： 10.1158/1538-7445.AM2016-2958 　

ISSN： 0008-5472

eISSN： 1538-7445
Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin Peer-reviewed

Satoshi Ogasawara, Mika K. Kaneko, Ryusuke Honma, Hiroharu Oki, Yuki Fujii, Michiaki Takagi, Hiroyoshi Suzuki, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(3)　155-162　2016/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2016.0006 　

ISSN： 2167-9436
Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin. Peer-reviewed

Ogasawara S, Kaneko MK, Honma R, Oki H, Fujii Y, Takagi M, Suzuki H, Kato Y

Monoclonal antibodies in immunodiagnosis and immunotherapy　2016/05
Publisher: Mary Ann Liebert Inc
DOI： 10.1089/mab.2016.0006.rev 　

ISSN： 2167-9436
Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state Peer-reviewed

Yuki Fujii, Yukiko Matsunaga, Takao Arimori, Yu Kitago, Satoshi Ogasawara, Mika K. Kaneko, Yukinari Kato, Junichi Takagi

JOURNAL OF CELL SCIENCE　129　(7)　1512-1522　2016/04

DOI： 10.1242/jcs.176685 　

ISSN： 0021-9533

eISSN： 1477-9137
Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin Peer-reviewed

Yukinari Kato, Satoshi Ogasawara, Hiroharu Oki, Ryusuke Honma, Michiaki Takagi, Yuki Fujii, Takuro Nakamura, Noriko Saidoh, Hazuki Kanno, Mitsuo Umetsu, Satoshi Kamata, Hiroshi Kubo, Mitsuhiro Yamada, Yoshihiko Sawa, Kei Ichi Morita, Hiroyuki Harada, Hiroyoshi Suzuki, Mika Kato Kaneko

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(2)　109-116　2016/04

DOI： 10.1089/mab.2015.0077 　

eISSN： 2167-9436
Critical Epitope of Anti-Rabbit Podoplanin Monoclonal Antibodies for Immunohistochemical Analysis Peer-reviewed

Ryusuke Honma, Yuki Fujii, Satoshi Ogasawara, Hiroharu Oki, Satoru Konnai, Yumiko Kagawa, Michiaki Takagi, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(2)　65-72　2016/04/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2015.0078 　

ISSN： 2167-9436
CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains Peer-reviewed

Satoshi Shiina, Masasuke Ohno, Fumiharu Ohka, Shunichiro Kuramitsu, Akane Yamamichi, Akira Kato, Kazuya Motomura, Kuniaki Tanahashi, Takashi Yamamoto, Reiko Watanabe, Ichiro Ito, Takeshi Senga, Michinari Hamaguchi, Toshihiko Wakabayashi, Mika K. Kaneko, Yukinari Kato, Vidyalakshmi Chandramohan, Darell D. Bigner, Atsushi Natsume

CANCER IMMUNOLOGY RESEARCH　4　(3)　259-268　2016/03

DOI： 10.1158/2326-6066.CIR-15-0060 　

ISSN： 2326-6066

eISSN： 2326-6074
LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin Peer-reviewed

Yukinari Kato, Satoshi Ogasawara, Hiroharu Oki, Polina Goichberg, Ryusuke Honma, Yuki Fujii, Mika K. Kaneko

PLOS ONE　11　(3)　e0152912　2016/03

DOI： 10.1371/journal.pone.0152912 　

ISSN： 1932-6203
Establishment of Novel Monoclonal Antibody PMab-32 Against Rabbit Podoplanin Peer-reviewed

Ryusuke Honma, Yuki Fujii, Satoshi Ogasawara, Hiroharu Oki, Xing Liu, Takuro Nakamura, Mika K. Kaneko, Michiaki Takagi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　35　(1)　41-47　2016/02/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2015.0066 　

ISSN： 2167-9436
An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas Peer-reviewed

Akane Yamamichi, Toshihiro Kasama, Fumiharu Ohka, Hiromichi Suzuki, Akira Kato, Kazuya Motomura, Masaki Hirano, Melissa Ranjit, Lushun Chalise, Michihiro Kurimoto, Goro Kondo, Kosuke Aoki, Noritada Kaji, Manabu Tokeshi, Toshio Matsubara, Takeshi Senga, Mika K. Kaneko, Hidenori Suzuki, Masahito Hara, Toshihiko Wakabayashi, Yoshinobu Baba, Yukinari Kato, Atsushi Natsume

SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS　17　(1)　618-625　2016

DOI： 10.1080/14686996.2016.1227222 　

ISSN： 1468-6996

eISSN： 1878-5514
新規抗ポドプラニンキメラ抗体による抗腫瘍効果の検討

国田朱子, 小笠原諭, 金子美華, 加藤幸成

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[3T18p-10(3P1057)]　2015/12
Publisher: (公社)日本生化学会
Development of sensitive monoclonal antibody PMab-2 against rat podoplanin Peer-reviewed

Hiroharu Oki, Ryusuke Honma, Satoshi Ogasawara, Yuki Fujii, Xing Liu, Michiaki Takagi, Mika K. Kaneko, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(6)　396-403　2015/12/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2015.0041 　

ISSN： 2167-9436
The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity Peer-reviewed

Yukinari Kato, Akiko Kunita, Shinji Abe, Satoshi Ogasawara, Yuki Fujii, Hiroharu Oki, Masashi Fukayama, Yasuhiko Nishioka, Mika K. Kaneko

ONCOTARGET　6　(34)　36003-36018　2015/11

DOI： 10.18632/oncotarget.5339 　

ISSN： 1949-2553
A high-sensitive HMab-2 specifically detects IDH1-R132H, the most common IDH mutation in gliomas Peer-reviewed

Yuki Fujii, Satoshi Ogasawara, Hiroharu Oki, Xing Liu, Mika K. Kaneko, Shingo Takano, Yukinari Kato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　466　(4)　733-739　2015/10

DOI： 10.1016/j.bbrc.2015.09.070 　

ISSN： 0006-291X

eISSN： 1090-2104
Monoclonal Antibody LpMab-9 Recognizes O-glycosylated N-Terminus of Human Podoplanin Peer-reviewed

Mika K. Kaneko, Hiroharu Oki, Yasukazu Hozumi, Xing Liu, Satoshi Ogasawara, Michiaki Takagi, Kaoru Goto, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(5)　310-317　2015/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2015.0022 　

ISSN： 2167-9436
Development of Monoclonal Antibody LpMab-10 Recognizing Non-glycosylated PLAG1/2 Domain Including Thr34 of Human Podoplanin Peer-reviewed

Satoshi Ogasawara, Hiroharu Oki, Mika K. Kaneko, Yasukazu Hozumi, Xing Liu, Ryusuke Honma, Yuki Fujii, Takuro Nakamura, Kaoru Goto, Michiaki Takagi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(5)　318-326　2015/10/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2015.0018 　

ISSN： 2167-9436
Diagnostic advantage of double immunohistochemistry using two mutation-specific anti-IDH antibodies (HMab-1 and MsMab-1) in gliomas Peer-reviewed

Shingo Takano, Yukinari Kato, Tetsuya Yamamoto, Xing Liu, Eiichi Ishikawa, Mika K. Kaneko, Satoshi Ogasawara, Masahide Matsuda, Masayuki Noguchi, Akira Matsumura

BRAIN TUMOR PATHOLOGY　32　(3)　169-175　2015/07

DOI： 10.1007/s10014-015-0214-8 　

ISSN： 1433-7398

eISSN： 1861-387X
Anti-podoplanin Monoclonal Antibody LpMab-7 Detects Metastatic Lesions of Osteosarcoma Peer-reviewed

Mika K. Kaneko, Hiroharu Oki, Satoshi Ogasawara, Michiaki Takagi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(3)　154-161　2015/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2014.0091 　

ISSN： 2167-9436
Characterization of Monoclonal Antibody LpMab-7 Recognizing Non-PLAG Domain of Podoplanin Peer-reviewed

Hiroharu Oki, Mika K. Kaneko, Satoshi Ogasawara, Yuta Tsujimoto, Xing Liu, Masato Sugawara, Yuya Takakubo, Michiaki Takagi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(3)　174-180　2015/06/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2014.0090 　

ISSN： 2167-9436
A novel monoclonal antibody SMab-2 recognizes endogenous IDH2-R172S of chondrosarcoma Peer-reviewed

Xing Liu, Satoshi Ogasawara, Mika K. Kaneko, Hiroharu Oki, Yasukazu Hozumi, Kaoru Goto, Michiaki Takagi, Yukinari Kato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　459　(4)　636-642　2015/04

DOI： 10.1016/j.bbrc.2015.02.162 　

ISSN： 0006-291X

eISSN： 1090-2104
Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms Peer-reviewed

Jhon A. Ochoa-Alvarez, Harini Krishnan, John G. Pastorino, Evan Nevel, David Kephart, Joseph J. Lee, Edward P. Retzbach, Yongquan Shen, Mahnaz Fatahzadeh, Soly Baredes, Evelyne Kalyoussef, Masaru Honma, Martin E. Adelson, Mika K. Kaneko, Yukinari Kato, Mary Ann Young, Lisa Deluca-Rapone, Alan J. Shienbaum, Kingsley Yin, Lasse D. Jensen, Gary S. Goldberg

ONCOTARGET　6　(11)　9045-9060　2015/04

DOI： 10.18632/oncotarget.3515 　

ISSN： 1949-2553
Characterization of Monoclonal Antibody LpMab-3 Recognizing Sialylated Glycopeptide of Podoplanin Peer-reviewed

Hiroharu Oki, Satoshi Ogasawara, Mika Kato Kaneko, Michiaki Takagi, Masanori Yamauchi, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　34　(1)　44-50　2015/02/01
Publisher: Mary Ann Liebert Inc.
DOI： 10.1089/mab.2014.0087 　

ISSN： 2167-9436
Determination of O-glycosylation sites around PLAG domains of human podoplanin using CasMab method

Kato Yukinari, Ogasawara Satoshi, Fujii Yuki, K. Kaneko Mika

Japan Journal of Molecular Tumor Marker Research　31　64-65　2015
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.31.64 　
Development of high-sensitive anti-podoplanin mAbs against osteosarcoma

Oki Hiroharu, Kato Yukinari Kato, Kaneko Mika, Ogasawara Satoshi, Tsujimoto Yuta, Liu Xing, Sugawara Masato, Takakubo Yuya, Tsuchiya Takashi, Takagi Michiaki

Japan Journal of Molecular Tumor Marker Research　30　(0)　70-71　2015
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.30.70 　
A Platform of C-type Lectin-like Receptor CLEC-2 for Binding O-Glycosylated Podoplanin and Nonglycosylated Rhodocytin Peer-reviewed

Masamichi Nagae, Kana Morita-Matsumoto, Masaki Kato, Mika Kato Kaneko, Yukinari Kato, Yoshiki Yamaguchi

STRUCTURE　22　(12)　1711-1721　2014/12

DOI： 10.1016/j.str.2014.09.009 　

ISSN： 0969-2126

eISSN： 1878-4186
Atomic details of the glycosylation-dependent and independent ligand interactions of C- type lectin-like receptor-2 (CLEC-2) Peer-reviewed

Masamichi Nagae, Kana Morita-Matsumoto, Masaki Kato, Mika Kaneko, Yukinari Kato, Yoshiki Yamaguchi

GLYCOBIOLOGY　24　(11)　1131-1131　2014/11

ISSN： 0959-6658

eISSN： 1460-2423
IDH2とTP53変異はMaffucci症候群患者における神経膠腫発症と関連している(IDH2 and TP53 mutations are correlated with gliomagenesis in a patient with Maffucci syndrome)

Moriya Kunihiko, Kaneko Mika, Hosaka Masami, Watanabe Mika, Sakuma Jun, Sasahara Yoji, Kure Shigeo, Kato Yukinari

日本小児血液・がん学会雑誌　51　(4)　198-198　2014/10
Publisher: (一社)日本小児血液・がん学会
ISSN： 2187-011X

eISSN： 2189-5384
Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin Peer-reviewed

Yukinari Kato, Mika K. Kaneko

CANCER RESEARCH　74　(19)　2014/10

DOI： 10.1158/1538-7445.AM2014-663 　

ISSN： 0008-5472

eISSN： 1538-7445
A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin Peer-reviewed

Yukinari Kato, Mika Kato Kaneko

SCIENTIFIC REPORTS　4　5924　2014/08

DOI： 10.1038/srep05924 　

ISSN： 2045-2322
Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone Peer-reviewed

Mika Kato Kaneko, Xing Liu, Hiroharu Oki, Satoshi Ogasawara, Takuro Nakamura, Noriko Saidoh, Yuta Tsujimoto, Yuka Matsuyama, Akira Uruno, Masato Sugawara, Takashi Tsuchiya, Mitsunori Yamakawa, Masayuki Yamamoto, Michiaki Takagi, Yukinari Kato

CANCER SCIENCE　105　(6)　744-748　2014/06

DOI： 10.1111/cas.12413 　

ISSN： 1349-7006
IDH2 and TP53 mutations are correlated with gliomagenesis in a patient with Maffucci syndrome Peer-reviewed

Kunihiko Moriya, Mika K. Kaneko, Xing Liu, Masami Hosaka, Fumiyoshi Fujishima, Jun Sakuma, Satoshi Ogasawara, Mika Watanabe, Yoji Sasahara, Shigeo Kure, Yukinari Kato

CANCER SCIENCE　105　(3)　359-362　2014/03

DOI： 10.1111/cas.12337 　

ISSN： 1349-7006
PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin Peer-reviewed

Yuki Fujii, Mika Kaneko, Makiko Neyazaki, Terukazu Nogi, Yukinari Kato, Junichi Takagi

PROTEIN EXPRESSION AND PURIFICATION　95　240-247　2014/03

DOI： 10.1016/j.pep.2014.01.009 　

ISSN： 1046-5928

eISSN： 1096-0279
Establishment of a multi-specific antibody against IDH1 and IDH2 mutations

Tsujimoto Yuta, Kaneko Mika Kato, Ogasawara Satoshi, Kato Yukinari

Japan Journal of Molecular Tumor Marker Research　29　56-57　2014
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.29.56 　
Expression analysis of isocitrate dehydrogenase mutations in osteosarcoma

Liu Xing, Sugawara Masato, Kaneko Mika Kato, Ogasawara Satoshi, Tsujimoto Yuta, Naganuma Yasushi, Tsuchiya Takashi, Takagi Michiaki, Kato Yukinari

Japan Journal of Molecular Tumor Marker Research　29　(0)　54-55　2014
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.29.54 　
IDH2 and TP53 mutations are correlated with gliomagenesis in a patient with Maffucci syndrome Peer-reviewed

Kunihiko Moriya, Mika K. Kaneko, Xing Liu, Masami Hosaka, Fumiyoshi Fujishima, Jun Sakuma, Satoshi Ogasawara, Mika Watanabe, Yoji Sasahara, Shigeo Kure, Yukinari Kato

Cancer Science　105　(3)　359-362　2014

DOI： 10.1111/cas.12337 　

ISSN： 1349-7006 1347-9032
Multi-specific monoclonal antibody MsMab-2 recognizes IDH1-R132L and IDH2-R172M mutations Peer-reviewed

Satoshi Ogasawara, Mika Kato Kaneko, Yuta Tsujimoto, Xing Liu, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　32　(6)　377-381　2013/12/01

DOI： 10.1089/mab.2013.0050 　

ISSN： 2167-9436
Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1 Peer-reviewed

Xing Liu, Yukinari Kato, Mika Kato Kaneko, Masato Sugawara, Satoshi Ogasawara, Yuta Tsujimoto, Yasushi Naganuma, Mitsunori Yamakawa, Takashi Tsuchiya, Michiaki Takagi

CANCER MEDICINE　2　(6)　803-814　2013/12

DOI： 10.1002/cam4.149 　

ISSN： 2045-7634
Establishment of a cancer-specific anti-podoplanin antibody not reacting with lymphatic endothelial cells. Peer-reviewed

Kato Yukinari, Kaneko Mika K

MOLECULAR CANCER THERAPEUTICS　12　(11)　2013/11

DOI： 10.1158/1535-7163.TARG-13-C167 　

ISSN： 1535-7163
A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody Peer-reviewed

Shinji Abe, Yuki Morita, Mika Kato Kaneko, Masaki Hanibuchi, Yuta Tsujimoto, Hisatsugu Goto, Souji Kakiuchi, Yoshinori Aono, Jun Huang, Seidai Sato, Masatoshi Kishuku, Yuki Taniguchi, Mami Azuma, Kazuyoshi Kawazoe, Yoshitaka Sekido, Seiji Yano, Shin-ichi Akiyama, Saburo Sone, Kazuo Minakuchi, Yukinari Kato, Yasuhiko Nishioka

The Journal of Immunology　Vol.190　(No.12)　6239-6249　2013/06/15

DOI： 10.4049/jimmunol.1300448 　

ISSN： 1550-6606

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Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.
A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody. International-journal Peer-reviewed

Shinji Abe, Yuki Morita, Mika Kato Kaneko, Masaki Hanibuchi, Yuta Tsujimoto, Hisatsugu Goto, Soji Kakiuchi, Yoshinori Aono, Jun Huang, Seidai Sato, Masatoshi Kishuku, Yuki Taniguchi, Mami Azuma, Kazuyoshi Kawazoe, Yoshitaka Sekido, Seiji Yano, Shin-ichi Akiyama, Saburo Sone, Kazuo Minakuchi, Yukinari Kato, Yasuhiko Nishioka

Journal of immunology (Baltimore, Md. : 1950)　190　(12)　6239-49　2013/06/15

DOI： 10.4049/jimmunol.1300448 　

ISSN： 0022-1767
Establishment of a Multi-Specific Monoclonal Antibody MsMab-1 Recognizing Both IDH1 and IDH2 Mutations Peer-reviewed

Mika Kato Kaneko, Satoshi Ogasawara, Yukinari Kato

TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE　230　(2)　103-109　2013/06

DOI： 10.1620/tjem.230.103 　

ISSN： 0040-8727

eISSN： 1349-3329
Novel monoclonal antibodies GMab-r1 and LMab-1 specifically recognize IDH1-R132G and IDH1-R132L mutations Peer-reviewed

Mika Kato Kaneko, Yuta Tsujimoto, Yasukazu Hozumi, Kaoru Goto, Yukinari Kato

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy　32　(3)　224-228　2013/06/01

DOI： 10.1089/mab.2013.0006 　

ISSN： 2167-9436
Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors Peer-reviewed

Vidyalakshmi Chandramohan, Xuhui Bao, Mika Kato Kaneko, Yukinari Kato, Stephen T. Keir, Scott E. Szafranski, Chien-Tsun Kuan, Ira H. Pastan, Darell D. Bigner

INTERNATIONAL JOURNAL OF CANCER　132　(10)　2339-2348　2013/05

DOI： 10.1002/ijc.27919 　

ISSN： 0020-7136
Generation of a novel monoclonal antibody WMab-1 specific for IDH2-R172W mutation Peer-reviewed

Yukinari Kato, Mika Kato Kaneko

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　433　(4)　374-378　2013/04

DOI： 10.1016/j.bbrc.2013.02.106 　

ISSN： 0006-291X
Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations Peer-reviewed

Mika Kato Kaneko, Shunpei Morita, Yuta Tsujimoto, Ryo Yanagiya, Kana Nasu, Hiroko Sasaki, Yasukazu Hozumi, Kaoru Goto, Atsushi Natsume, Mika Watanabe, Toshihiro Kumabe, Shingo Takano, Yukinari Kato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　432　(1)　40-45　2013/03

DOI： 10.1016/j.bbrc.2013.01.088 　

ISSN： 0006-291X
A novel monoclonal antibody GMab-m1 specifically recognizes IDH1-R132G mutation Peer-reviewed

Yukinari Kato, Atsushi Natsume, Mika Kato Kaneko

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　432　(4)　564-567　2013/03

DOI： 10.1016/j.bbrc.2013.02.049 　

ISSN： 0006-291X
Development of specific monoclonal antibodies against IDH1/2 mutation

Kato Yukinari, Kaneko Mika

Japan Journal of Molecular Tumor Marker Research　28　21-22　2013
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.28.21 　
Chimeric anti-podoplanin antibody suppresses tumor metastasis through neutralization and antibody-dependent cellular cytotoxicity Peer-reviewed

Mika Kato Kaneko, Akiko Kunita, Shinji Abe, Yuta Tsujimoto, Masashi Fukayama, Kaoru Goto, Yoshihiko Sawa, Yasuhiko Nishioka, Yukinari Kato

CANCER SCIENCE　103　(11)　1913-1919　2012/11

DOI： 10.1111/j.1349-7006.2012.02385.x 　

ISSN： 1347-9032
Immunohistochemical detection of IDH1 mutation, p53, and internexin as prognostic factors of glial tumors Peer-reviewed

Shingo Takano, Yukinari Kato, Tetsuya Yamamoto, Mika Kato Kaneko, Eiichi Ishikawa, Yuta Tsujimoto, Masahide Matsuda, Kei Nakai, Ryo Yanagiya, Shunpei Morita, Koji Tsuboi, Akira Matsumura

JOURNAL OF NEURO-ONCOLOGY　108　(3)　361-373　2012/07

DOI： 10.1007/s11060-012-0837-0 　

ISSN： 0167-594X

eISSN： 1573-7373
Podoplanin expression in advanced atherosclerotic lesions of human aortas Peer-reviewed

Kinta Hatakeyama, Mika Kato Kaneko, Yukinari Kato, Tetsunori Ishikawa, Kensaku Nishihira, Yuta Tsujimoto, Yoshisato Shibata, Yukio Ozaki, Yujiro Asada

THROMBOSIS RESEARCH　129　(4)　E70-E76　2012/04

DOI： 10.1016/j.thromres.2012.01.003 　

ISSN： 0049-3848
Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin Peer-reviewed

Chiaki Kaji, Yuta Tsujimoto, Mika Kato Kaneko, Yukinari Kato, Yoshihiko Sawa

ACTA HISTOCHEMICA ET CYTOCHEMICA　45　(4)　227-237　2012

DOI： 10.1267/ahc.12008 　

ISSN： 0044-5991

eISSN： 1347-5800
CONSTRUCTION OF A NOVEL ANTIPODOPLANIN (NZ-1) IMMUNOTOXIN FOR BRAIN TUMOR THERAPY Peer-reviewed

Vidyalakshmi Chandramohan, Mika Kato Kaneko, Yukinari Kato, Charles N. Pegram, Stephen T. Keir, Ira H. Pastan, Chien-Tsun Kuan, Darell D. Bigner

NEURO-ONCOLOGY　13　I26-I26　2011/05

ISSN： 1522-8517
Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing (vol 28, pg 115, 2011) Peer-reviewed

Shingo Takano, Wei Tian, Masahide Matsuda, Tetsuya Yamamoto, Eiichi Ishikawa, Mika Kato Kaneko, Kentaro Yamazaki, Yukinari Kato, Akira Matsumura

BRAIN TUMOR PATHOLOGY　28　(2)　125-125　2011/04

DOI： 10.1007/s10014-011-0028-2 　

ISSN： 1433-7398

eISSN： 1861-387X
Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing Peer-reviewed

Shingo Takano, Wei Tian, Masahide Matsuda, Tetsuya Yamamoto, Eiichi Ishikawa, Mika Kato Kaneko, Kentaro Yamazaki, Yukinari Kato, Akira Matsumura

BRAIN TUMOR PATHOLOGY　28　(2)　115-123　2011/04

DOI： 10.1007/s10014-011-0023-7 　

ISSN： 1433-7398

eISSN： 1861-387X
Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation. International-journal

Kaneko Mika Kato, Tian Wei, Takano Shingo, Suzuki Hiroyuki, Sawa Yoshihiko, Hozumi Yasukazu, Goto Kaoru, Yamazaki Kentaro, Kitanaka Chifumi, Kato Yukinari

Biochem Biophys Res Commun　406　(4)　608-613　2011/03/25

DOI： 10.1016/j.bbrc.2011.02.102 　

ISSN： 1090-2104

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Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.
Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation Peer-reviewed

Mika Kato Kaneko, Wei Tian, Shingo Takano, Hiroyuki Suzuki, Yoshihiko Sawa, Yasukazu Hozumi, Kaoru Goto, Kentaro Yamazaki, Chifumi Kitanaka, Yukinari Kato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　406　(4)　608-613　2011/03

DOI： 10.1016/j.bbrc.2011.02.102 　

ISSN： 0006-291X
Neutralization of blood group A-antigen by anti-A antibody

Hasegawa Yasushi, Tanabe Minoru, Kato Yukinari, Kaneko Mika, Shimazu Motohide, Wakabayashi Go, Kawachi Shigeyuki, Obara Hideaki, Shinoda Masahiro, Narimatsu Hisashi, Kitajima Masaki, Kitagawa Yuko

Organ Biology　18　(1)　112-115　2011
Publisher: The Japan Society for Organ Preservation and Biology
DOI： 10.11378/organbio.18.112 　

ISSN： 1340-5152

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Background. The major barrier to ABO-incompatible solid organ transplantation is acute humoral rejection. Various strategies to reduce anti-blood group antibodybyovercoming ABO incompatible transplantation have been tried. However, antigen-suppressing procedures have not been performed. Methods. We investigated whether an anti-A antibody(K7508)can mask A-antigen on the cells in vitro. Next, we immunized mice with A-antigen-expressing cells coated with K7508 or its Fab fragment, and measured anti-A antibodyproduction in the mice. Results. Blood group A-antigen-expressing cells coated with K7508 were not recognized by another anti-A antibodyin flow cytometry, indicating that A-antigen was masked byK7508 in vitro. The A-antigen on the paraffin-embedded liver tissue was also masked byK7508. Furthermore, the production of anti-A antibodyin mice immunized with A-antigen-expressing cells coated with K7508 or its Fab fragment was significantlysuppressed compared to that in mice immunized with non-coated cells alone, indicating that A-antigen was neutralized byK7508 in vivo. Conclusions. The neutralization of blood group antigen byanti-blood group antibodyand especiallyits Fab fragment might represent one strategyto overcome ABO-incompatible organ transplantation
Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas Peer-reviewed

Yukinari Kato, Ganesan Vaidyanathan, Mika Kato Kaneko, Kazuhiko Mishima, Nidhi Srivastava, Vidyalakshmi Chandramohan, Charles Pegram, Stephen T. Keir, Chien-Tsun Kuan, Darell D. Bigner, Michael R. Zalutsky

NUCLEAR MEDICINE AND BIOLOGY　37　(7)　785-794　2010/10

DOI： 10.1016/j.nucmedbio.2010.03.010 　

ISSN： 0969-8051
GMab-1, a high-affinity anti-3 '-isoLM1/3 ',6 '-isoLD1 IgG monoclonal antibody, raised in lacto-series ganglioside-defective knockout mice Peer-reviewed

Yukinari Kato, Chien-Tsun Kuan, Jinli Chang, Mika Kato Kaneko, Joanne Ayriss, Hailan Piao, Vidyalakshmi Chandramohan, Charles Pegram, Roger E. McLendon, Pam Fredman, Jan-Eric Mansson, Darell D. Bigner

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　391　(1)　750-755　2010/01

DOI： 10.1016/j.bbrc.2009.11.132 　

ISSN： 0006-291X
Podoplanin is a novel antibody-based therapeutic target for glioblastoma Peer-reviewed

Yukinari Kato, Kazuhiko Mishima, Mika Kaneko, Michael Zalutsky, Chien-Tsun Kuan, Darell Bigner

CANCER RESEARCH　69　2009/05

ISSN： 0008-5472

eISSN： 1538-7445
Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification Peer-reviewed

Atsushi Kuno, Yukinari Kato, Atsushi Matsuda, Mika Kato Kaneko, Hiromi Ito, Koh Amano, Yasunori Chiba, Hisashi Narimatsu, Jun Hirabayashi

MOLECULAR & CELLULAR PROTEOMICS　8　(1)　99-108　2009/01

DOI： 10.1074/mcp.M800308-MCP200 　

ISSN： 1535-9476

eISSN： 1535-9484
Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties. Peer-reviewed

Nemoto-Sasaki Y, Hayama K, Ohya H, Arata Y, Kaneko MK, Saitou N, Hirabayashi J, Kasai K

Biochimica et biophysica acta　1780　(10)　1131-1142　2008/10

DOI： 10.1016/j.bbagen.2008.07.003 　

ISSN： 0006-3002
Podocalyxin expression in malignant astrocytic tumors Peer-reviewed

Norihito Hayatsu, Mika Kato Kaneko, Kazuhiko Mishima, Ryo Nishikawa, Masao Matsutani, Janet E. Price, Yukinari Kato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　374　(2)　394-398　2008/09

DOI： 10.1016/j.bbrc.2008.07.049 　

ISSN： 0006-291X
Characterization of anti-podoplanin monoclonal antibodies: Critical epitopes for neutralizing the interaction between podoplanin and CLEC-2 Peer-reviewed

Satoshi Ogasawara, Mika Kato Kaneko, Janet E. Price, Yukinari Kato

HYBRIDOMA　27　(4)　259-267　2008/08

DOI： 10.1089/hyb.2008.0017 　

ISSN： 1554-0014
Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors Peer-reviewed

Yukinari Kato, Norihito Hayatsu, Mika Kato Kaneko, Satoshi Ogasawara, Tetsutaro Hamano, Satoru Takahashi, Ryo Nishikawa, Masao Matsutani, Kazuhiko Mishima, Hisashi Narimatsu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　369　(4)　1041-1046　2008/05

DOI： 10.1016/j.bbrc.2008.02.130 　

ISSN： 0006-291X
Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells Peer-reviewed

Norihito Hayatsu, Satoshi Ogasawara, Mika Kato Kaneko, Yukinari Kato, Hisashi Narimatsu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　368　(2)　217-222　2008/04

DOI： 10.1016/j.bbrc.2008.01.058 　

ISSN： 0006-291X
Engineering of mucin-type human glycoproteins in yeast cells Peer-reviewed

Koh Amano, Yasunori Chiba, Yoshiko Kasahara, Yukinari Kato, Mika Kato Kaneko, Atsushi Kuno, Hiromi Ito, Kazuo Kobayashi, Jun Hirabayashi, Yoshifumi Jigami, Hisashi Narimatsu

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　105　(9)　3232-3237　2008/03

DOI： 10.1073/pnas.0710412105 　

ISSN： 0027-8424
Neutralization of blood group A-antigen by a novel anti-A antibody: Overcoming ABO-incompatible solid-organ transplantation Peer-reviewed

Yasushi Hasegawa, Yukinari Kato, Mika Kato Kaneko, Satoshi Ogasawara, Motohide Shimazu, Minoru Tanabe, Shigeyuki Kawachi, Hideki Obara, Masahiro Shinoda, Yuko Kitagawa, Hisashi Narimatsu, Masaki Kitajima

TRANSPLANTATION　85　(3)　378-385　2008/02

DOI： 10.1097/TP.0b013e3181612f84 　

ISSN： 0041-1337
Podoplanin expression in human tumors: glycan structure and immunohistochemical analysis

Kato Yukinari, Kato Kaneko Mika, Ogasawara Satoshi, Ito Hiromi, Kameyama Akihiko, Narimatsu Hisashi

Japan Journal of Molecular Tumor Marker Research　23　33-34　2008
Publisher: Japanese Society for Molecular Tumor Marker Research
DOI： 10.11241/jsmtmr.23.33 　
Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2 Peer-reviewed

Yukinari Kato, Mika Kato Kaneko, Akiko Kunita, Hiromi Ito, Akihiko Kameyama, Satoshi Ogasawara, Nana Matsuura, Yasushi Hasegawa, Katsue Suzuki-Inoue, Osamu Inoue, Yukio Ozaki, Hisashi Narimatsu

CANCER SCIENCE　99　(1)　54-61　2008/01

DOI： 10.1111/j.1349-7006.2007.00634.x 　

ISSN： 1347-9032
Induction of podoplanin by transforming growth factor-beta in human fibrosarcoma Peer-reviewed

Hiroyuki Suzuki, Yukinari Kato, Mika Kato Kaneko, Yukari Okita, Hisashi Narimatsu, Mitsuyasu Kato

FEBS LETTERS　582　(2)　341-345　2008/01

DOI： 10.1016/j.febslet.2007.12.028 　

ISSN： 0014-5793
Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells Peer-reviewed

Katsue Suzuki-Inoue, Yukinari Kato, Osamu Inoue, Mika Kato Kaneko, Kazuhiko Mishima, Yutaka Yatomi, Yasuo Yamazaki, Hisashi Narimatsu, Yukio Ozaki

JOURNAL OF BIOLOGICAL CHEMISTRY　282　(36)　25993-26001　2007/09

DOI： 10.1074/jbc.M702327200 　

ISSN： 0021-9258
Functional glycosylation of human podoplanin: Glycan structure of platelet aggregation-inducing factor Peer-reviewed

Mika Kato Kaneko, Yukinari Kato, Akihiko Kameyama, Hiromi Ito, Atsushi Kuno, Jun Hirabayashi, Tomomi Kubota, Koh Amano, Yasunori Chiba, Yasushi Hasegawa, Isoji Sasagawa, Kazuhiko Mishima, Hisashi Narimatsu

FEBS LETTERS　581　(2)　331-336　2007/01

DOI： 10.1016/j.febslet.2006.12.044 　

ISSN： 0014-5793
Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain Peer-reviewed

Yukinari Kato, Mika Kato Kaneko, Atsushi Kuno, Noboru Uchiyama, Koh Amano, Yasunori Chiba, Yasushi Hasegawa, Jun Hirabayashi, Hisashi Narimatsu, Kazuhiko Mishima, Motoki Osawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　349　(4)　1301-1307　2006/11

DOI： 10.1016/j.bbrc.2006.08.171 　

ISSN： 0006-291X
Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor Peer-reviewed

Mika Kato Kaneko, Yukinari Kato, Takashi Kitano, Motoki Osawa

GENE　378　52-57　2006/08

DOI： 10.1016/j.gene.2006.04.023 　

ISSN： 0378-1119
Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma. International-journal Peer-reviewed

Kazuhiko Mishima, Yukinari Kato, Mika K Kaneko, Youya Nakazawa, Akiko Kunita, Naoya Fujita, Takashi Tsuruo, Ryo Nishikawa, Takanori Hirose, Masao Matsutani

Acta neuropathologica　111　(6)　563-8　2006/06

DOI： 10.1007/s00401-006-0033-4 　

ISSN： 0001-6322
Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression Peer-reviewed

K Mishima, Y Kato, MK Kaneko, R Nishikawa, T Hirose, M Matsutani

ACTA NEUROPATHOLOGICA　111　(5)　483-488　2006/05

DOI： 10.1007/s00401-006-0063-y 　

ISSN： 0001-6322
Functional glycosylation and platelet-aggregation stimulating activity of Aggrus Peer-reviewed

M Kaneko

SEIKAGAKU　77　(4)　332-335　2005/04

ISSN： 0037-1017
Association of alpha 2-HS glycoprotein (AHSG, fetuin-A) polymorphism with AHSG and phosphate serum levels Peer-reviewed

M Osawa, W Tian, H Horiuchi, M Kaneko, K Umetsu

HUMAN GENETICS　116　(3)　146-151　2005/02

DOI： 10.1007/s00439-004-1222-7 　

ISSN： 0340-6717
Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma Peer-reviewed

Y Kato, M Kaneko, M Sata, N Fujita, T Tsuruo, M Osawa

TUMOR BIOLOGY　26　(4)　195-200　2005

DOI： 10.1159/000086952 　

ISSN： 1010-4283
Aggrus: a diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors. International-journal Peer-reviewed

Yukinari Kato, Isoji Sasagawa, Mika Kaneko, Motoki Osawa, Naoya Fujita, Takashi Tsuruo

Oncogene　23　(52)　8552-6　2004/11/04

DOI： 10.1038/sj.onc.1207869 　

ISSN： 0950-9232

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Aggrus (also known as T1alpha/podoplanin) is a membrane sialoglycoprotein whose function in tumors is unknown. We recently determined that Aggrus possessed the ability of inducing platelet aggregation and that its expression was frequently upregulated in colorectal tumors. Thus, Aggrus expression might be associated with tumor-induced platelet aggregation and tumor metastasis. Here we show, by means of cancer profiling array and real-time PCR, that aggrus mRNA expression is frequently upregulated in testicular germ cell tumors when compared with the surrounding normal tissue. Immunohistochemical staining revealed that Aggrus protein expression was detected in 10 of 11 seminomas (90.9%), but its expression was not observed in embryonal carcinomas (0/4; 0%). Specific markers for seminomas have not been reported, and Aggrus is a potential diagnostic marker for seminomas and may be associated with malignancies of the testis.
Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells. International-journal Peer-reviewed

Mika Kaneko, Yukinari Kato, Akiko Kunita, Naoya Fujita, Takashi Tsuruo, Motoki Osawa

The Journal of biological chemistry　279　(37)　38838-43　2004/09/10

DOI： 10.1074/jbc.M407210200 　

ISSN： 0021-9258

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Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.
Normal embryonic and germ cell development in mice lacking alpha 1,3-fucosyltransferase IX (Fut9) which show disappearance of stage-specific embryonic antigen 1 Peer-reviewed

T Kudo, M Kaneko, H Iwasaki, A Togayachi, S Nishihara, K Abe, H Narimatsu

MOLECULAR AND CELLULAR BIOLOGY　24　(10)　4221-4228　2004/05

DOI： 10.1128/MCB.24.10.4221-4228.2004 　

ISSN： 0270-7306
Divergent evolution of the prolactin-inducible protein gene and related genes in the mouse genome Peer-reviewed

M Osawa, H Horiuchi, W Tian, M Kaneko

GENE　325　179-186　2004/01

DOI： 10.1016/j.gene.2003.10.013 　

ISSN： 0378-1119
Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. International-journal

Yukinari Kato, Naoya Fujita, Akiko Kunita, Shigeo Sato, Mika Kaneko, Motoki Osawa, Takashi Tsuruo

The Journal of biological chemistry　278　(51)　51599-605　2003/12/19

ISSN： 0021-9258

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Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.
Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. International-journal Peer-reviewed

Yukinari Kato, Naoya Fujita, Akiko Kunita, Shigeo Sato, Mika Kaneko, Motoki Osawa, Takashi Tsuruo

The Journal of biological chemistry　278　(51)　51599-605　2003/12/19

DOI： 10.1074/jbc.M309935200 　

ISSN： 0021-9258

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Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.
A novel beta(1,6)-N-acetylglucosaminyltransferase V (GnT-VB)

M Kaneko, G Alvarez-Manilla, M Kamar, Lee, I, JK Lee, K Troupe, WJ Zhang, M Osawa, M Pierce

FEBS LETTERS　554　(3)　515-519　2003/11

DOI： 10.1016/S0014-5793(03)01234-1 　

ISSN： 0014-5793
Molecular characterization of a human monoclonal antibody to B antigen in ABO blood type

M Kaneko, Y Kato, H Horiuchi, M Osawa

IMMUNOLOGY LETTERS　86　(1)　45-51　2003/03

DOI： 10.1016/S0165-2478(02)00294-8 　

ISSN： 0165-2478
Intragenic haplotypes and molecular evolution of the human α2-HS glycoprotein (AHSG/fetuin) gene Peer-reviewed

M. Osawa, I. Yuasa, J. Henke, M. Kaneko, K. Umetsu

International Congress Series　1239　(C)　765-767　2003/01/01

DOI： 10.1016/S0531-5131(02)00557-5 　

ISSN： 0531-5131
Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans Peer-reviewed

M Osawa, M Kaneko, H Horiuchi, T Kitano, Y Kawamoto, N Saitou, K Umetsu

GENOMICS　81　(1)　78-84　2003/01

DOI： 10.1016/S0888-7543(02)00010-1 　

ISSN： 0888-7543
Increased urine phenylethylamine after methylphenidate treatment in children with ADHD Peer-reviewed

A Kusaga, Y Yamashita, T Koeda, M Hiratani, M Kaneko, S Yamada, T Matsuishi

ANNALS OF NEUROLOGY　52　(3)　371-374　2002/09

DOI： 10.1002/ana.10302 　

ISSN： 0364-5134
CD15 expression in mature granulocytes is determined by alpha 1,3-fucosyltransferase IX,but in promyelocytes and monocytes by alpha 1,3-fucosyltransferase IV

F Nakayama, S Nishihara, H Iwasaki, T Kudo, R Okubo, M Kaneko, M Nakamura, M Karube, K Sasaki, H Narimatsu

JOURNAL OF BIOLOGICAL CHEMISTRY　276　(19)　16100-16106　2001/05

DOI： 10.1074/jbc.M007272200 　

ISSN： 0021-9258
The evolutionary history of glycosyltransferase genes

M Kaneko, S Nishihara, H Narimatsu, N Saitou

TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY　13　(70)　147-155　2001/03

DOI： 10.4052/tigg.13.147 　

ISSN： 0915-7352
Identification of two rodent genes encoding homologues to seminal vesicle autoantigen: A gene family including the gene for prolactin-inducible protein

M Yoshida, M Kaneko, H Kurachi, M Osawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　281　(1)　94-100　2001/02

DOI： 10.1006/bbrc.2001.4311 　

ISSN： 0006-291X
Haplotype analysis of the human alpha 2-HS glycoprotein (fetuin) gene

M Osawa, Yuasa, I, T Kitano, J Henke, M Kaneko, T Udono, N Saitou, K Umetsu

ANNALS OF HUMAN GENETICS　65　27-34　2001/01

ISSN： 0003-4800
The beta-1,3-galactosyltransferase-4 (B3GALT4) gene is located in the centromeric segment of the human MHC class II region

T Shiina, E Kikkawa, H Iwasaki, M Kaneko, H Narimatsu, K Sasaki, S Bahram, H Inoko

IMMUNOGENETICS　51　(1)　75-78　2000/01

ISSN： 0093-7711
alpha 1,3-fucosyltransferase 9 (FUT9; Fuc-TIX) preferentially fucosylates the distal GlcNAc residue of polylactosamine chain while the other four alpha 1,3FUT members preferentially fucosylate the inner GlcNAc residue

S Nishihara, H Iwasaki, M Kaneko, A Tawada, M Ito, H Narimatsu

FEBS LETTERS　462　(3)　289-294　1999/12

DOI： 10.1016/S0014-5793(99)01549-5 　

ISSN： 0014-5793
HLA領域6p21.3上に存在するbeta1,3ガラクトース転移酵素遺伝子シークエンシング解析

吉川枝里, 椎名隆, 金子美華, 成松久, 猪子英俊

MHC: Major Histocompatibility Complex　6　(1)　86-86　1999/06
Publisher: 日本組織適合性学会
ISSN： 2186-9995

eISSN： 2187-4239
alpha 1,3-fucoslytransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX

M Kaneko, T Kudo, H Iwasaki, Y Ikehara, S Nishihara, S Nakagawa, K Sasaki, T Shiina, H Inoko, N Saitou, H Narimatsu

FEBS LETTERS　452　(3)　237-242　1999/06

DOI： 10.1016/S0014-5793(99)00640-7 　

ISSN： 0014-5793
Assignment of the human alpha 1,3-fucosyltransferase IX gene (FUT9) to chromosome band 6q16 by in situ hybridization

M Kaneko, T Kudo, H Iwasaki, T Shiina, H Inoko, T Kozaki, N Saitou, H Narimatsu

CYTOGENETICS AND CELL GENETICS　86　(3-4)　329-330　1999

ISSN： 0301-0171
HLA領域6p21.3上に存在するbeta1,3ガラクトース転移酵素遺伝子のマッピング並びにシークエンシング解析

吉川枝里, 椎名隆, 金子美華, 成松久, 猪子英俊

日本臨床免疫学会会誌　(26回抄録集)　121-121　1998/10
Publisher: 日本臨床免疫学会
ISSN： 0911-4300

eISSN： 1349-7413
Expression cloning and characterization of a novel murine alpha 1,3-fucosyltransferase, mFuc-TIX, that synthesizes the Lewis x (CD15) epitope in brain and kidney

T Kudo, Y Ikehara, A Togayachi, M Kaneko, T Hiraga, K Sasaki, H Narimatsu

JOURNAL OF BIOLOGICAL CHEMISTRY　273　(41)　26729-26738　1998/10

DOI： 10.1074/jbc.273.41.26729 　

ISSN： 0021-9258
Distinct substrate specificities of five human alpha-1,3-fucosyltransferases for in vivo synthesis of the sialyl Lewis x and Lewis x epitopes

H Kimura, N Shinya, S Nishihara, M Kaneko, T Irimura, H Narimatsu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　237　(1)　131-137　1997/08

DOI： 10.1006/bbrc.1997.7100 　

ISSN： 0006-291X
Wide variety of point mutations in the H gene of Bombay and para-Bombay individuals that inactivate H enzyme

M Kaneko, S Nishihara, N Shinya, T Kudo, H Iwasaki, T Seno, Y Okubo, H Narimatsu

BLOOD　90　(2)　839-849　1997/07

ISSN： 0006-4971

Show all ︎Show first 5

Misc. 53

近接ビオチン化酵素AirID融合抗体を用いた膜タンパク質細胞外ドメイン相互作用解析

山田航大, 塩屋亮平, 金子美華, 西野耕平, 加藤幸成, 小迫英尊, 澤崎達也

日本生化学会大会プログラム・講演要旨集　95回　1P-209　2022/11
Publisher: (公社)日本生化学会
抗TROP2モノクローナル抗体TrMab-6の乳がんモデルに対する抗腫瘍効果

田中智大, 大石智一, 川田学, 金子美華, 加藤幸成

日本癌学会学術総会抄録集(Web)　80th　2021
膵がんを標的としたコアフコース欠損型がん特異的抗ポドカリキシン抗体の開発

金子美華, 大石智一, 川田学, 加藤幸成, 加藤幸成

日本生化学会大会(Web)　94th　2021
新規抗EpCAM抗体(EpMab-16)の大腸がんに対する抗腫瘍活性の評価

細野ヒデキ, 大石智一, 淺野禎三, 金子美華, 川田学, 加藤幸成, 加藤幸成

日本生化学会大会(Web)　94th　2021
新規抗EGFRモノクローナル抗体134-mG2aの口腔扁平上皮がん移植片モデルにおける抗腫瘍効果の検討

七宮蓮, 武井潤子, 武井潤子, 大石智一, 川田学, 原田浩之, 金子美華, 加藤幸成, 加藤幸成

日本癌学会学術総会抄録集(Web)　80th　2021
抗体ラベリング法を用いた膜内切断プロテアーゼの電子顕微鏡単粒子解析

有賀理江, 廣瀬未果, 坂口(田村)梨沙子, 金子美華, 加藤幸成, 加藤貴之, 岩崎憲治, 禾晃和

日本蛋白質科学会年会プログラム・要旨集　21st　2021
Detection of Lion Podoplanin Using the Antitiger Podoplanin Monoclonal Antibody PMab-231. International-journal

Yukinari Kato, Junko Takei, Masato Sano, Teizo Asano, Yusuke Sayama, Kazuyuki Uchida, Takayuki Nakagawa, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(3)　91-94　2020/05/14

DOI： 10.1089/mab.2020.0008 　

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Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.
Development of an Anti-Sheep Podoplanin Monoclonal Antibody PMab-256 for Immunohistochemical Analysis of Lymphatic Endothelial Cells. International-journal

Yukinari Kato, Yoshikazu Furusawa, Masato Sano, Junko Takei, Takuro Nakamura, Miyuki Yanaka, Saki Okamoto, Saori Handa, Yu Komatsu, Teizo Asano, Yusuke Sayama, Mika K Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy　39　(3)　82-90　2020/04/28

DOI： 10.1089/mab.2020.0005 　

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Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the Cell-Based Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1, kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256 stained type I alveolar cells of lung, renal glomerulus and Bowman's capsule, and lymphatic endothelial cells of lung and colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.
新規抗EGFR抗体EMab-17はKRAS(G13D)変異型大腸がん細胞に抗腫瘍効果を示す

大石智一, 加藤幸成, 金子美華, 大庭俊一, 井上裕幸, 原川晃子, 川田学, 川田学

臨床薬理　51　(Supplement)　2020

ISSN： 0388-1601
NZ-1ラベリングによる立体構造解析の一般化に向けたPAタグ挿入方法の最適化

有賀理江, 田村梨沙子, 浴本亨, 廣瀬未果, 大井里香, 金子美華, 加藤幸成, 岩崎憲治, 池口満徳, 池口満徳, 禾晃和

日本結晶学会年会講演要旨集　2020 (CD-ROM)　2020
CBIS法を用いた抗ブタポドプラニン抗体の樹立

古澤慶一, 古澤慶一, 古澤慶一, 山田慎二, 金子美華, 武井潤子, 佐野雅人, 佐山勇輔, 三輪崇志, 福井真人, 加藤幸成, 加藤幸成

日本生化学会大会(Web)　92nd　2019
Cell-Based Immunization and Screening(CBIS)法を用いた抗ヤギポドプラニンモノクローナル抗体の樹立

佐山勇輔, 山田慎二, 佐野雅人, 古澤慶一, 古澤慶一, 古澤慶一, 三輪崇志, 福井真人, 金子美華, 加藤幸成, 加藤幸成

日本生化学会大会(Web)　92nd　2019
Establishment of a novel rat anti-podoplanin antibody detecting a cancer-specific glycopeptide

加藤幸成, 加藤幸成, 金子美華, 山田慎二

日本癌学会学術総会抄録集(Web)　78th　2019
ポドプラニンに対するがん特異的認識抗体の抗腫瘍効果および安全性評価

加藤幸成, 加藤幸成, 金子美華, 山田慎二

日本薬理学雑誌　153　(Supplement)　2019

ISSN： 0015-5691
Development of monoclonal antibodies against a cancer stem cell marker CD133

加藤幸成, 加藤幸成, 山田慎二, 大石智一, 川田学, 金子美華

日本がん転移学会学術集会・総会プログラム抄録集　28th　2019
電子顕微鏡単粒子解析に向けた抗体断片の結合による標的タンパク質のサイズと形状の最適化

三好賢一, 田村梨沙子, 高貫一徳, 廣瀬未果, 大井里香, 金子美華, 加藤幸成, 岩崎憲治, 禾晃和

日本細胞生物学会大会(Web)　71st　2019
親水性領域部分断片とFab断片の共結晶構造解析による膜内切断酵素特異的抗体のエピトープ領域の探索

廣瀬建, 金子美華, 加藤幸成, 禾晃和

日本結晶学会年会講演要旨集　2019　2019
ネコpodoplaninに対するモノクローナル抗体の樹立とネコ扁平上皮がんにおけるpodoplanin発現解析

山田慎二, 板井俊介, 賀川由美子, 今内覚, 金子美華, 加藤幸成, 加藤幸成

日本獣医学会学術集会講演要旨集　161st　2018

ISSN： 1347-8621
ポドプラニンに対するがん特異的抗体の抗腫瘍効果と安全性評価

金子美華, 山田慎二, 加藤幸成, 加藤幸成

日本糖質学会年会要旨集　37th　2018
イヌポドプラニンに対するがん特異的認識抗体の樹立およびエピトープ解析

古澤慶一, 古澤慶一, 板井俊介, 板井俊介, 山田慎二, 金子美華, 加藤幸成

日本生化学会大会(Web)　91st　2018
構造情報に基づいたエピトープ挿入部位の最適化による抗体断片との安定な複合体形成

田村梨沙子, 大井里香, 金子美華, 加藤幸成, 禾晃和

日本結晶学会年会講演要旨集　2018　2018
脳腫瘍IDH1遺伝子変異の新しい迅速診断技術

山道茜, 大岡史治, 笠間敏博, 鈴木啓道, 青木恒介, 本村和也, 千賀威, 金子美華, 若林俊彦, 馬場嘉信, 加藤幸成, 木村晋也, 夏目敦至

日本癌学会総会記事　75回　E-2012　2016/10
Publisher: 日本癌学会
ISSN： 0546-0476
PAタグのループへの挿入による蛋白質の構造変化評価システム

藤井勇樹, 藤井勇樹, 松永幸子, 有森貴夫, 北郷悠, 小笠原諭, 金子美華, 加藤幸成, 高木淳一

日本蛋白質科学会年会プログラム・要旨集　16th　154　2016/05/19
抗変異型IDH抗体MsMab‐1の多重認識機構についての構造基盤

北郷悠, 金子美華, 加藤幸成, 高木淳一

日本蛋白質科学会年会プログラム・要旨集　15th　121　2015/05/26
超高親和性を有する新規アフィニティータグシステム“PAタグシステム”

藤井勇樹, 高木淳一, 金子美華, 加藤幸成

和光純薬時報　82　(4)　2-4　2014/10/15

ISSN： 1347-4804
Maffucci症候群においてIDH2とTP53の変異が脳腫瘍発生に関わる

森谷邦彦, 金子美華, 保坂正美, 渡辺みか, 佐久間潤, 笹原洋二, 呉繁夫, 加藤幸成

日本整形外科学会雑誌　88　(6)　S1249-S1249　2014/06
Publisher: (公社)日本整形外科学会
ISSN： 0021-5325
抗PAタグペプチド抗体NZ‐1の特異性の改変

藤井勇樹, 山下和男, 金子美華, 北郷悠, 加藤幸成, STANDLEY Daron M, 高木淳一

日本蛋白質科学会年会プログラム・要旨集　14th　79　2014/05/26
Maffucci症候群においてIDH2とTP53の変異が脳腫瘍発生に関わる

森谷邦彦, 金子美華, 保坂正美, 渡辺みか, 佐久間潤, 笹原洋二, 呉繁夫, 加藤幸成

小児の脳神経　39　(1)　91-91　2014/04
Publisher: (一社)日本小児神経外科学会
ISSN： 0387-8023
血小板凝集因子受容体CLEC-2のリガンド認識機構の結晶学的研究

長江雅倫, 森田加奈, 加藤雅樹, 金子美華, 加藤幸成, 山口芳樹

日本結晶学会年会講演要旨集　2014　2014
新規アフィニティータグ「PA tag」のループへの挿入による効果的な利用法

藤井勇樹, 金子美華, 北郷悠, 加藤幸成, 高木淳一

日本生化学会大会(Web)　87th　2P-108 (WEB ONLY)　2014
ヒトNK細胞を介した抗ポドプラニン抗体NZ-8の悪性胸膜中皮腫に対する抗腫瘍効果

阿部真治, 加藤幸成, 金子美華, 木宿昌俊, 川添和義, 東満美, 埴淵昌毅, 水口和生, 西岡安彦

日本呼吸器学会誌　2　(増刊)　331-331　2013/03
Publisher: (一社)日本呼吸器学会
ISSN： 2186-5876
抗ポドプラニン抗体NZ-1の悪性胸膜中皮腫に対する抗体依存性細胞障害活性と抗腫瘍効果

埴淵昌毅, 阿部真治, 加藤幸成, 金子美華, 木宿昌俊, 川添和義, 黄俊, 三橋惇志, 水口和生, 西岡安彦

肺癌　52　(5)　769-769　2012/10
Publisher: (NPO)日本肺癌学会
ISSN： 0386-9628

eISSN： 1348-9992
抗eTEVペプチド抗体2H5の結晶構造解析による抗原認識機構

北郷悠, 金子美華, 加藤幸成, 高木淳一

日本蛋白質科学会年会プログラム・要旨集　12th　82　2012/05/31
悪性胸膜中皮腫に対する抗ポドプラニン抗体NZ-1の抗腫瘍効果に関する検討

阿部真治, 加藤幸成, 金子美華, 木宿昌俊, 川添和義, 東満美, 水口和生, 曽根三郎, 西岡安彦

日本呼吸器学会誌　1　(増刊)　166-166　2012/03
Publisher: (一社)日本呼吸器学会
ISSN： 2186-5876
Caenorhabditis elegans galectins LEC-1-LEC-11: Structural features and sugar-binding properties

Yoko Nemoto-Sasaki, Ko Hayama, Hiroyuki Ohya, Yoichiro Arata, Mika Kato Kaneko, Naruya Saitou, Jun Hirabayashi, Ken-ichi Kasai

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS　1780　(10)　1131-1142　2008/10

DOI： 10.1016/j.bbagen.2008.07.003 　

ISSN： 0304-4165

eISSN： 1872-8006
血小板凝集因子Podoplaninの機能性糖鎖の解析

金子美華, 加藤幸成, 亀山昭彦, 伊藤浩美, 久野敦, 平林淳, 久保田智巳, 天野仰, 千葉靖典, 成松久

日本糖質学会年会要旨集　28th　33　2008/07/29
血小板凝集因子Podoplaninの糖鎖構造解析

金子美華, 加藤幸成, 亀山昭彦, 伊藤浩美, 久野敦, 平林淳, 久保田智巳, 天野仰, 千葉靖典, 成松久

バイオテクノロジーシンポジウム予稿集　25th　12-15　2007/11/06
酵母を活用した糖鎖認識プローブの作製技術開発

千葉靖典, 天野仰, 加藤幸成, 金子美華, 久野敦, 伊藤浩美, 平林淳, 成松久

バイオテクノロジーシンポジウム予稿集　25th　63-65　2007/11/06
比較糖鎖プロファイリングシステム応用技術開発

久野敦, 関矢聡, 松田厚志, 加藤行成, 金子美華, 成松久, 平林淳

バイオテクノロジーシンポジウム予稿集　25th　74-75　2007/11/06
ムチン型糖鎖生産酵母株での糖タンパク質の発現と機能解析

天野仰, 笠原由子, 久野敦, 平林淳, 加藤幸成, 金子美華, 成松久, 千葉靖典, 地神芳文

日本農芸化学会大会講演要旨集　2007　149　2007/03/05
血小板凝集因子Podoplaninの糖鎖構造解析

金子美華, 加藤幸成, 亀山昭彦, 伊藤浩美, 久野敦, 平林淳, 久保田智己, 天野仰, 千葉靖典, 成松久

生化学　2P-0001　2007

ISSN： 0037-1017
A homolog of N-acetylglucosaminyltransferase V (GnT-V) shows high expression levels in mouse and human brain

JK Lee, K Abbott, K Troupe, M Kaneko, R Matthews, M Pierce

GLYCOBIOLOGY　14　(11)　1149-1149　2004/11

ISSN： 0959-6658
A Novel beta(1,6) N-acetylglucosaminyltransferase V (GnT-VB) transcript is expressed in high levels in mouse and human brain.

M Kaneko, G Alvarez-Manilla, M Kamar, Lee, I, JK Lee, K Troupe, M Pierce

GLYCOBIOLOGY　13　(11)　877-877　2003/11

ISSN： 0959-6658
α1,3‐フコース転移酵素遺伝子XI(Fut9)‐KOマウスの機能解析

成松久, 工藤崇, 西原祥子, 池原譲, 金子美華, 阿部訓也, 池上司郎, 井ノ口馨, 末松誠

生化学　74　(8)　856-856　2002/08/25
Publisher: (公社)日本生化学会
ISSN： 0037-1017

eISSN： 2189-0544
Molecular analysis of the dodecamer repeat and intragenic haplotypes in the human cystatin B gene

OSAWA Motoki, UMETSU Kazuo, KANEKO Mika, HORIUCHI Hidekazu

10　6-8　2002/06/15
Intragenic haplotypes of the human α 2-HS glycoprotein (AHSG) gene

OSAWA M., UMETSU K., KANEKO M., YUASA I., KITANO T., SAITOU N.

9　216-219　2001/06/30
FUT9の組織分布,基質特異性とFut9‐KOマウスの解析

成松久, 工藤崇, 西原祥子, 岩崎裕子, 池原譲, 金子美華, 阿部訓也

日本分子生物学会年会プログラム・講演要旨集　23rd　235　2000/11/25
α1,3‐フコース転移酵素(Fut9)KOマウスでのLewis x抗原の発現

工藤崇, 池原譲, 金子美華, 西原祥子, 阿部訓也, 成松久

生化学　72　(8)　855-855　2000/08/25
Publisher: (公社)日本生化学会
ISSN： 0037-1017
糖鎖発現制御と機能・病態解析 α1,3-フコース転移酵素遺伝子IX(Fuc-TIX)の発現と機能

工藤崇, 金子美華, 栂谷内晶, 岩崎裕子, 西原祥子, 阿部訓也, 石井泰雄, 大隅典子, 佐々木克敏, 成松久

生化学　71　(8)　633-633　1999/08
Publisher: (公社)日本生化学会
ISSN： 0037-1017
Mapping and nucleotide sequence determination of the beta 1.3 galactose transferage gene located on the 6p21.3 region

KIKKAWA Eri, SHIINA Takashi, KANEKO Mika, NARIMATSU Hisashi, INOKO Hidetoshi

21　240-240　1998/12/01
Cloning and expression of a novel α1 1,3-fucosyltransferase (FUT9) from mouse and human.

NARIMATSU H, KUDO T, KANEKO M, TOGAYACHI A, NAKAGAWA S, SASAKI K, SAITO N, SHIINA T, INOKO H

日本分子生物学会年会プログラム・講演要旨集　21　1998/12/01
糖鎖の機能解析新規ヒトα1,3フコース転移酵素遺伝子(hFuc-TIX)のクローニング

金子美華, 工藤崇, 池原譲, 新屋直子, 岩崎裕子, 西原祥子, 中川智, 佐々木克敏, 椎名隆, 猪子英俊

生化学　70　(8)　760-760　1998/08
Publisher: (公社)日本生化学会
ISSN： 0037-1017
(翻訳)「DNAから見た生物進化」第5章を担当

別冊日経サイエンス　1997

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Research Projects 16

Development and characterization of cancer-specific monoclonal antibodies against tumor-associated glycoproteins.

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2021/04/01 - 2024/03/31
Development and characterization of a cancer-specific monoclonal antibody against a tumor-associated sialoglycoprotein.

Kaneko Mika

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2017/04/01 - 2020/03/31

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Podocalyxin (PODXL), a CD34-related sialomucin, is expressed not only in many tumors but also in normal cells such as epithelial cells and endothelial cells; therefore, PODXL could not be a target of antibody therapy. We recently established cancer-specific anti-PODXL mAb (ex.clone: PcMab-6), which could target only cancer cells although PODXL is highly expressed in both cancer and normal cells. PcMab-6 reacted with PODXL-expressing many cancer cell lines whereas it did not bind to vascular endothelial cells (VECs) in flow cytometry. Furthermore, PcMab-6 reacted only with PODXL-expressing cancer cells, not with VECs in breast and oral cancer tissues using immunohistochemistry. We have further constructed chPcMab-6, a mouse-human chimeric mAb from the PcMab-6, which also responds with only podocalyxin-expressing cancer cells. chPcMab-6 has ADCC activity in vitro and anti-tumor effect in vivo.
Development and characterization of a cancer-specific monoclonal antibody (CasMab) against podoplanin

Kaneko Mika, KUNITA Akiko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tohoku University

2014/04/01 - 2017/03/31

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Podoplanin (PDPN) is a platelet aggregation-inducing mucin-like sialoglycoprotein that is highly expressed in many cancers and normal tissues. PDPN has also been reported to be a marker to enrich tumor-initiating cells. Recently, we established the platform to produce a cancer-specific mAb (CasMab) by combining glycobiology and cell engineering technologies. In this study, we also established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas or TALEN. We investigate several novel cancer-specific anti-PDPN or anti-glycopeptide mAbs. Antitumor activity of chimeric anti-human PDPN antibodies were validated. The CasMab-producing platform could lead to establishment of the side effect-reducing mAbs.
Regulatory mechanisms of glycosyltransferase genes which synthesizetumor specific glycan on PDPN.

MIKA Kaneko

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Young Scientists (B)

Institution: Yamagata University

2011 - 2012

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Podoplanin (PDPN) is a mucin-type sialoglycoprotein associatedwith tumor metastasis, malignant progression, and cancer stem cells. Recently, werevealed the existence of tumor specific glycan on PDPN. In this study, we showed thatseveral glycosyltransferase genes are involved in the tumor specific glycan on PDPN.Furthermore, we investigated the potential role of noncoding RNA such as microRNAs inglycosyltransferase gene regulation.
ヘパラン硫酸の糖鎖構造多様性による神経回路形成の制御

金子美華

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 若手研究(B)

Institution: (財)東京都医学研究機構

2005 - 2006

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本研究課題において、ヘパラン硫酸糖鎖構造の多様性の神経回路形成の制御機構の解明を目指し、マウス子宮内胎仔脳へのエレクトロポレーション法による遺伝子導入により糖鎖構造を改変するため、その実験系の確立に着手した。プロテオグリカン糖鎖を改変するため、基本骨格であるEXT1遺伝子のノックダウンsiRNAを検討行い、ノックダウン効果を示す、有効な配列を特定した。さらに、shRNA発現用プラスミドの構築を行った。また、基本骨格糖鎖合成遺伝子、また、グリコサミノグリカン鎖修飾遺伝子に関して、遺伝子改変後の成果を判定する手段として、real-time PCR法によるmRNA量の定量法を確立した。本研究成果により、in vitro培養細胞系において、shRNA発現ベクターを用いたEXT1遺伝子のノックダウンに成功した。また、ひとつの遺伝子をノックダウンしたとき、他の糖鎖修飾関連酵素の遺伝子発現量の変化も観察することが可能となった。今回、EXT1遺伝子のみを行ったベクターを用いたノックダウンの実験系を、同様にして、他のグリコサミノグリカン鎖修飾遺伝子にも適応することで、さらに詳細な糖鎖構造の改変を可能にし、ヘパラン硫酸糖鎖構造の多様性の機能を解析することが可能となった。
Signal transduction of chondroitin sulfate proteoglycans and neuronal network formation

MAEDA Nobuaki, ISHII Maki, HATA Toshihiro, KANEKO Mika

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Tokyo Metropolitan Organization for Medical Research

2005 - 2006

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PTPζ, a receptor-type protein tyrosine phosphatase, uses a heparin-binding growth factor pleiotrophin as a ligand and play important roles in the morphogenesis of cerebellar Purkinje cells. In this study, we studied functional interaction between PTPζ and DNER, a Delta/Notch-like receptor highly expressed in Purkinje cells. PTPζ and DNER displayed patchy co-localization in the dendritic shafts of Purkinje cells and in the COS-7 transfectants expressing both proteins. Immunoprecipitation experiments indicated that PTPζ and DNER formed complex both in vivo and in vitro. Several tyrosine residues in the intracellular region of DNER including the ones in and adjacent to the tyrosine-based sorting motif were phosphorylated, and were easily dephosphorylated by the PTPζ catalytic domain. The tyrosine-based sorting motif played critical roles in the endocytosis and intracellular transport of DNER, and tyrosine phosphorylation and destruction of this motif resulted in the accumulation of DNER on the plasma membrane of the PTPζ-expressing cells. Accumulation of DNER on the plasma membrane of Neuro-2A cells led to the morphological changes including extension of processes. Pleiotrophin suppressed the endocytosis of DNER and DNER-induced inhibition of neurite outgrowth of Neuro-2A cells. These observations suggests that pleiotrophin-PTPζ signaling regulates the morphogenesis of Purkinje cells by modifying the intracellular transport of DNER.
コンドロイチン硫酸プロテオグリカンPTPζの情報伝達と小脳皮質の形成機構

前田信明, 石井万幾, 秦敏宏, 金子美華

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 特定領域研究

Institution: (財)東京都医学研究機構

2005 - 2006

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PTPζは、コンドロイチン硫酸プロテオグリカンとして生合成される脳特異的受容体型チロシンホスファターゼである。昨年度我々は、本分子がDelta様の細胞外領域を持つ膜蛋白質、DNERと相互作用することを見い出した。本年度は、PTPζの情報伝達がDNERとどのような共役関係にあるかを検討した。我々は既に、PTPζがヘパリン結合性成長因子プレイオトロフィンをリガンドとして情報伝達することを明らかにしている。そこで、PTPζ陽性のNeuro-2A神経芽細胞にDNERを強制発現させ、プレイオトロフィン刺激の効果を解析した。その結果、プレイオトロフィンは、DNERのエンドサイトーシスを抑制し、本分子の細胞膜への蓄積を促進することが明らかになった。また、DNERはNeuro-2A細胞のレチノイン酸依存的な細胞分化を抑制したが、プレイオトロフィン刺激は、これを脱抑制し、細胞分化を促進した。DNERはNotchのリガンドとして機能し、神経細胞の分化に重要な役割を果たしていることが知られている。PTPζ-プレイオトロフィン情報伝達は、DNERのチロシン燐酸化状態を規定することによって、本分子の細胞膜への提示とエンドサイトーシスを調節し、DNER-Notch情報伝達の強度を制御している可能性が考えられる。また、PTPζのコンドロイチン硫酸部分は、PTPζ-プレイオトロフィン情報伝達の必須構成要素となっており、今後、コンドロイチン硫酸の除去あるいはその構造改変が、DNERの情報伝達にどのような影響を及ぼすかを解析する予定である。
原因不明の乳幼児突然死例における遺伝的背景の検討

大澤資樹, 金子美華

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 萌芽研究

Institution: 山形大学

2004 - 2005

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乳幼児突然死症候群(sudden infant death syndrome ; SIDS)は、剖検と諸検査においても死因が不明のものとして除外診断されている。国内では1歳未満の死因の第三位を占め、病態の解明が重要な課題となっている。原因としては、うつ伏せ寝に伴う神経系の機能異常が推定されているが、1〜2割程度の症例では呼吸器系・循環系・脳神経系等の先天性異常が原因と考えられている。先天性異常としては、睡眠中の無呼吸発作を主症状とする先天性中枢性肺胞低換気症候群(CCHS、Ondineの呪い)が病状の類似性から最も可能性のある候補となる。しかし、これを解剖から診断することは難しく、原因遺伝子についてDNA解析を行うことにより遺伝性疾患を判断できる可能性がある。実験計画について学内倫理委員会の承認を得た上で、実験目的での使用について遺族から承諾を得た48例につき、血液よりDNAを抽出し遺伝子解析を行った。現在までに、RET proto-oncogene、Phox2b(paired mesoderm homeobox 2b)、Phox2a、EDNRB(endothelin receptor type B)の解析を終了した。その結果、非同義置換をいくつか検出したが、それが直接的に発現や機能の異常に結びついているとは考えにくかった。特に、Phox2b遺伝子内のポリアラニン異常伸長はCCHSの10例中9例に検出されたのに対し、SIDS例では全く検出できなかった。すなわち、意外なことにSIDSにこのCCHSが遺伝的背景として関与している可能性は低いと推定された。現在は、心臓刺激伝導系の機能異常であるQT延長症やBurgata症候群の原因遺伝子であるSCN5A・KCAQ1・KCNE1・KCNH2等の遺伝子解析を進行中である。
Molecular analysis of congenital central hypoventilation syndrome in infant sudden death cases

OSAWA Motoki, KANEKO Mika

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (C)

Institution: Yamagata University

2002 - 2003

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Concerning sudden infant death syndrome, the unknown causes have been suspected to be not only physical factors such as asphyxia, but also congenital disorders involving the respiratory and circulation systems. However ; it is difficult for the inherited diseases, in particular congenital central hypoventilation syndrome (CCHS, Ondine's curse), to be diagnosed by post-mortem examinations of autopsy and histology. We performed molecular analysis of the candidate genes to DNA specimens from SIDS victims. In the analysis of RET proto-oncogene, Phox2b (paired mesoderm homeobox 2b), ZFHX1B (zinc finger homeobox 1B), CSTB (cystatin B), EDNRB (endothelin receptor type B) genes, no remarkable mutations were evident, indicating that CCHS is not closely related to SIDS. However, methodological improvements were obtained during the analysis, which has been published as research articles. In another aspect, mitochondrial disorder of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) was detected in a case. Although progressing slowly as a chronic disorder, MELAS is potentially involved in some of SIDS cases.
ガン転移に関与する血小板凝集因子の作用機序 Competitive

2003 -
臓器特異的たんぱく発現の研究 Competitive

2003 -
Aggrus, a platelet aggregation-inducing fuctor associated with tumor metastasis Competitive

2003 -
Tissue specific protein expression Competitive

2003 -
糖鎖抗原に対するヒト抗体の研究 Competitive

System: 科学研究費補助金

2001 - 2002
Study on natural antibodies for sugar antigens Competitive

2001 - 2002
法医学実務に利用するヒトABH抗体のin vitro発現系の開発

金子美華

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 若手研究(B)

Institution: 山形大学

2001 - 2002

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本研究では、従来、法医実務においてABO式血液型の型判定に利用されてきたヒト抗血清と同等の力価を持つ代替品、すなわち多価ないし高親和性ヒト抗体を、分子生物学的手法を用いて作製することを目的とし、抗体活性と抗体の塩基配列の相関性を調べた。本研究の最も基礎となる部分、全リンパ球から目的の抗体産生Bリンパ球を、少なくともある一定期間培養出来る株として単離・樹立する方法として、細胞融合法を用いたヒトイムノグロブリンの産生方法を検討した。まだ実験に改善の余地はあるが、ほぼ確実に目的のクローンが得られる方法を樹立した。得られたクローンより抗ABH抗体の塩基配列を決定し、抗体配列の特性に関して詳細な解析を行った。その結果、ヒト抗ABH自然抗体配列は、生殖細胞(germline cell)の配列と比べ、数少ない変異しか持たなかった。また、我々の取得した抗B自然抗体と他の研究グループから報告された抗B抗体を比較すると、いずれも同じ配列グループに所属する事などから、特定の生殖細胞系列の抗体配列がほとんど変異を持たずに抗ABH抗体になり得る可能性が示唆された。さらに、抗B、抗H自然抗体のH鎖が第3相補性決定領域(CDR3)のみが非常に異なり、その他の領域においては相同であるという結果を得て、H鎖のCDR3が抗体の特異性決定に重要な役割を持つことも示唆された(投稿中)。我々は、細胞融合法を用いることで、目的のヒト抗体産生細胞を分離・取得し、その抗体の塩基配列を解析する事が容易な方法を樹立した。抗体特異性と塩基配列に関する基礎的な研究に非常に有用な方法である。こうした配列の情報を蓄積することで、目的に合った抗体を人工的に設計し作製する事が可能となると考えている。

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