Mika K. Kaneko

Cadherin-4 (CDH4/R-cadherin) is a type I cadherin involved in diverse processes of neural development and cancer. Although several anti-CDH4 monoclonal antibodies (mAbs) have been established for Western blotting, mAbs suitable for flow cytometric analysis of CDH4 have not been described. To generate anti-CDH4 mAbs for flow cytometry, we used a flow cytometry-based high-throughput screening strategy. Among the generated mAbs, Ca₄Mab-3 (IgG₁, κ) selectively recognized CDH4-overexpressed Chinese hamster ovary-K1 (CHO/CDH4) cells by flow cytometry and showed no detectable cross-reactivity with 21 other CDHs, including both type I and type II CDHs. Ca4Mab-3 also detected endogenous CDH4 in human breast cancer (MDA-MB-231) and osteosarcoma (U2OS) cell lines. The apparent dissociation constants (KD) of Ca₄Mab-3 for CHO/CDH4 and U2OS cells were 1.4 × 10⁻⁹ M and 1.1 × 10⁻⁹ M, respectively. In addition, Ca4Mab-3 successfully detected endogenous CDH4 in Western blotting. These results indicate that Ca₄Mab-3 is a highly specific anti-CDH4 mAb that can be used in flow cytometry and Western blotting. Ca₄Mab-3 may serve as a useful tool for investigating CDH4 function and may have potential applications in the development of therapeutic strategies for CDH4-positive tumors.

Podocalyxin (PODXL) is a potential diagnostic biomarker and therapeutic target in various tumors. Development of anti-PODXL mAbs has been limited by concerns about potential reactivity with normal tissues. To minimize on-target, off-tumor toxicities and adverse effects, we developed cancer-specific mAbs (CasMabs) against PODXL and demonstrated antitumor efficacy in human tumor xenograft models. In this study, we evaluated humPcMab-60-DXd, an anti-PODXL CasMab conjugated with DXd, a deriv-ative of the topoisomerase I inhibitor exatecan. humPcMab-60-DXd retained reactivity with the pancreatic ductal adenocarcinoma cell line MIA PaCa-2 and inhibited cell pro-liferation in vitro. In the MIA PaCa-2 xenograft model, humPcMab-60-DXd showed su-perior antitumor efficacy compared with humPcMab-60. Furthermore, humP-cMab-60-DXd exhibited dose-dependent antitumor activity without reducing mice body weight. These results provide the first observation of antitumor efficacy for an an-ti-PODXL CasMab-drug conjugate, which could facilitate the clinical development of CasMabs for tumor therapy.

Background/Objectives: Cadherin-16 (CDH16, Ksp-cadherin) possesses unique seven extracellular cadherin repeats, and its expression is restricted to normal kidney epithelium. CDH16 is downregulated in renal cell carcinoma (RCC) and is associated with poor prognosis. Therefore, developing mAbs that specifically recognize cell-surface CDH16 is essential for tumor diagnosis and for isolating CDH16-positive renal epithelial cells. Methods: Anti-human CDH16 mAbs (designated as Ca₁₆Mabs) were developed by immunizing mice with CDH16-overexpressed tumor cells, followed by a high-throughput flow cytometry-based screening. Results: Among the 58 established Ca₁₆Mabs, a clone, Ca₁₆Mab-56 (IgG₁, κ), specifically recognized CDH16-overexpressed Chinese hamster ovary-K1 (CHO/CDH16) cells with no detectable cross-reactivity to 21 other CDHs in flow cytometry. Ca₁₆Mab-56 also detected endogenous CDH16 in human RCC cell lines (OS-RC-2 and KMRC-20) and normal kidney epithelial cell lines. The dissociation constant (K_D) values of Ca₁₆Mab-56 for CHO/CDH16 and OS-RC-2 were determined as 6.5 × 10⁻⁹ M and 1.2 × 10⁻⁹ M, respectively. Furthermore, Ca₁₆Mab-56 detected endogenous CDH16 by Western blotting and showed potent staining in normal kidney tubular epithelium and clear membranous staining in renal cell carcinoma in immunohistochemistry. Conclusions: Ca₁₆Mab-56 is a versatile tool for detecting CDH16 and has potential for tumor diagnosis.

The G protein-coupled receptor, CXC chemokine receptor 5 (CXCR5), is predominantly expressed on B cells located in the secondary lymphoid tissues, follicular helper T cells, and lymphoma cells. Binding to its ligand, CXCL13, mediates cell migration and regulates lymphocyte trafficking. Aberrant CXCL13/CXCR5 expression and signaling have been implicated in tumor progression, autoimmune diseases, and chronic inflammatory disorders. Therefore, specific mAbs against CXCR5 are expected to be useful for diagnosis and therapeutic applications. In this study, novel anti-human CXCR5 mAbs (Cx₅Mabs) were developed through flow cytometry-based high-throughput screening. One clone, Cx₅Mab-6 (IgG_2b, κ), recognized CXCR5-overexpressed Chinese hamster ovary (CHO)-K1 cells but did not react with the other five CXCR receptors-overexpressed CHO-K1 cells in flow cytometry. Additionally, Cx₅Mab-6 recognized endogenous CXCR5 in the human Burkitt lymphoma Raji cell line. The dissociation constant (K_D) values of Cx₅Mab-6 for CHO/CXCR5 and Raji were 3.4 × 10⁻⁹ M and 1.2 × 10⁻¹⁰ M, respectively. Furthermore, Cx₅Mab-6 is useful for Western blotting and can detect CXCR5 in human lymphoma tissue by immunohistochemistry. These findings suggest that Cx₅Mab-6 is versatile for basic research and has potential applications in clinical diagnosis.

Glypican-2 (GPC2) is a heparan sulfate proteoglycan that plays essential roles in enhancing receptor signaling by recruiting heparan sulfate-binding ligands. Overexpression of GPC2 is observed in pediatric cancers, including neuroblastoma and medulloblastoma, where it supports oncogenic signaling and tumor progression. High-affinity GPC2 binders have been developed as effective immunotherapies targeting tumors overexpressing GPC2. In this study, we generated novel anti-GPC2 monoclonal antibodies using a flow cytometry–based high-throughput screening. Among the clones, G2Mab-33 (IgG₁, κ) and G2Mab-7 (IgG₁, κ) selectively recognized GPC2-overexpressed Chinese hamster ovary cells (CHO/GPC2), with no reactivity against parental CHO-K1 cells in flow cytometry. G₂Mab-33 and G2Mab-7 recognized endogenous GPC2 in the human neuroblastoma cell line, IMR-32. Specificity testing showed that G₂Mab-33 and G₂Mab-7 reacted with CHO/GPC2 and did not cross-react with other GPCs overexpressed in CHO-K1. The dissociation constant (KD) values for G₂Mab-7 and G₂Mab-33 were 2.3 × 10⁻⁸ M and 3.5 × 10^⁻9 for CHO/GPC2, respectively. Additionally, G₂Mab-7 can be used in western blotting and immunohistochemistry. Overall, these findings show that G₂Mab-7 and G₂Mab-33 are versatile mAbs for basic research and promising candidates for antibody-based therapy.

CXCR4 (CD184) is a G protein-coupled receptor that binds CXCL12. Aberrant CXCR4 expression is associated with tumor progression, metastasis, therapeutic resistance, and poor prognosis in various hematological and solid malignancies, making it an attractive therapeutic target. Therefore, CXCR4-targeting monoclonal antibodies (mAbs) have advanced to clinical evaluation. In this study, we established a novel anti-human CXCR4 mAb, Cx4Mab-7 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Cx4Mab-7 specifically recognized CXCR4-overexpressed LN229 glioblastoma cells (LN229/CXCR4), CXCR4-overexpressed CHO-K1 cells (CHO/CXCR4), and endogenous CXCR4 on Jurkat cells without cross-reactivity to other C-X-C chemokine receptors. Flow cytometry demonstrated that Cx4Mab-7 exhibited high binding affinity for LN229/CXCR4 cells (KD: 7.4 ± 1.8 × 10−9 M) and Jurkat cells (KD: 3.5 ± 3.6 × 10−9 M). In addition, Cx4Mab-7 was suitable for immunohistochemistry using formalin-fixed paraffin-embedded CHO/CXCR4 cells, although it showed only weak reactivity in Western blot. Cx4Mab-7 may contribute to the future development of diagnostic applications targeting the CXCR4 axis.

The G protein-coupled seven-transmembrane receptor, CXC chemokine receptor 5 (CXCR5), is predominantly expressed on B cells located in the secondary lymphoid tissues, follicular helper T cells, and lymphoma cells. Binding to its ligand, CXCL13, mediates cell migration and regulates lymphocyte trafficking. Aberrant CXCL13/CXCR5 expression and signaling have been implicated in tumor progression, autoimmune diseases, and chronic inflammatory disorders. Therefore, specific mAbs against CXCR5 are expected to be useful for diagnosis and therapeutic applications. In this study, novel anti-human CXCR5 mAbs (Cx₅Mabs) were developed through flow cytometry-based high-throughput screening. One clone, Cx₅Mab-6 (IgG_2b, κ), recognized CXCR5-overexpressed Chinese hamster ovary (CHO)-K1 cells but did not react with the other 5 CXCR receptors-overexpressed CHO-K1 cells in flow cytometry. Additionally, Cx5Mab-6 recognized endogenous CXCR5 in the human Burkitt lymphoma Raji cell line. The dissociation constant (K_D) values of Cx₅Mab-6 for CHO/CXCR5 and Raji were 3.4 × 10⁻⁹ M and 1.2 × 10⁻10 M, respectively. Furthermore, Cx₅Mab-6 is useful for Western blotting and can detect CXCR5 in human lymphoma tissue by immunohistochemistry. These findings suggest that Cx₅Mab-6 is versatile for basic research and has potential applications in clinical diagnosis.

Background/Objectives: Cadherin-16 (CDH16, Ksp-cadherin) possesses unique seven extracellular cadherin repeats, and its expression is restricted to normal kidney epithelium. CDH16 is downregulated in renal cell carcinoma (RCC) and is associated with poor prognosis. Therefore, developing mAbs that specifically recognize cell-surface CDH16 is essential for tumor diagnosis and for isolating CDH16-positive renal epithelial cells. Methods: Anti-human CDH16 mAbs (designated as Ca₁₆Mabs) were developed by immunizing mice with CDH16-overexpressed tumor cells, followed by a high-throughput flow cytometry-based screening. Results: Among the 58 established Ca₁₆Mabs, a clone, Ca₁₆Mab-56 (IgG₁, κ), specifically recognized CDH16-overexpressed Chinese hamster ovary-K1 (CHO/CDH16) cells with no detectable cross-reactivity to 21 other CDHs in flow cytometry. Ca₁₆Mab-56 also detected endogenous CDH16 in human RCC cell lines (OS-RC-2 and KMRC-20) and normal kidney epithelial cell lines. The dissociation constant (K_D) values of Ca₁₆Mab-56 for CHO/CDH16 and OS-RC-2 were determined as 6.5 × 10⁻⁹ M and 1.2 × 10⁻⁹ M, respectively. Furthermore, Ca₁₆Mab-56 detected endogenous CDH16 by Western blotting and showed potent staining in normal kidney tubular epithelium and clear membranous staining in renal cell carcinoma in immunohistochemistry. Conclusion: Ca₁₆Mab-56 is a versatile tool for detecting CDH16 and has potential for tumor diagnosis.

Background/Objectives: Cadherin-17 (CDH17, LI-cadherin) is a non-classical cadherin with an atypical structure and unique functions. CDH17 expression is restricted to normal intestinal epithelium. Furthermore, CDH17 functions as an oncoprotein that promotes tumor migration and invasion in colorectal, gastric, and pancreatic cancers. Therefore, CDH17 is an important diagnostic marker and therapeutic target. The CDH17-directed strategies, including monoclonal antibodies (mAbs), bispecific Abs, antibody–drug conjugates (ADCs), and chimeric antigen receptor (CAR) T cells, have been evaluated in preclinical and clinical studies. Therefore, developing mAbs that specifically recognize cell surface-expressing CDH17 is essential for advancing both tumor diagnosis and therapy. Methods: Anti-human CDH17 mAbs (named Ca₁₇Mabs) were developed by immunizing a mouse with CDH17-overexpressed cells and a high-throughput screening using flow cytometry. Results: Among Ca₁₇Mabs, a clone, Ca₁₇Mab-5 (IgG₁, κ) specifically recognized CDH17-overexpressed Chinese hamster ovary-K1 (CHO/CDH17) cells with no detectable cross-reactivity to 21 other CDHs by flow cytometry. Ca₁₇Mab-5 also detected endogenous CDH17 in human colorectal cancer cell lines, COLO201 and COLO205. The apparent dissociation constant (K_D) values of Ca₁₇Mab-5 for CHO/CDH17 and COLO205 were estimated as 1.5 × 10⁻⁸ M and 1.3 × 10⁻⁸ M, respectively. Furthermore, Ca₁₇Mab-5 detected endogenous CDH17 by Western blotting. In immunohistochemistry, Ca₁₇Mab-5 exhibited clear membranous staining in normal colon epithelium, colorectal, gastric, and pancreatic cancers. Conclusions: Ca₁₇Mab-5 is a versatile tool for detecting CDH17 and has potential for tumor diagnosis.

Glypican-4 (GPC4) is a member of the heparan sulfate proteoglycan family and regulates a wide range of growth factor signaling through interactions with heparan sulfate-binding ligands. Pathogenic variants of GPC4 have been identified in human congenital disorders, and dysregula-tion of GPC4 has been reported in human cancer. Therefore, specific anti-GPC4 monoclonal anti-bodies (mAbs) are needed for both basic research and clinical applications. In this study, we estab-lished novel anti-glypican-4 (GPC4) mAbs using a flow cytometry–based high-throughput screening approach. Among the isolated clones, G₄Mab-9 (IgG_2b, κ) showed specific binding to Chinese hamster ovary-K1 (CHO/GPC4) cells overexpressed GPC4, with no reactivity against pa-rental CHO-K1. Furthermore, G₄Mab-9 detected endogenous GPC4 expression in human embry-onic kidney 293FT cells. Specificity analyses demonstrated that G₄Mab-9 selectively recognized GPC4 without cross-reactivity with other glypicans in CHO-K1 cells overexpressing them. The dissociation constant (K_D) of G₄Mab-9 was determined to be 1.4 × 10⁻7 M for CHO/GPC4, indicat-ing low binding affinity. Moreover, G₄Mab-9 detected a 33-kDa cleaved fragment of GPC4 by western blotting and stained CHO/GPC4 but not CHO-K1 in immunohistochemistry. Collectively, these results indicate that G₄Mab-9 is a valuable tool for GPC4-related basic research and a poten-tial candidate for the development of diagnostic and therapeutic approaches.