Reina Ito

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.

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-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.

Eph receptor A10 (EphA10) is undetectable in most normal tissues, and the increased expression has been shown to correlate with tumor progression and poor prognosis. Therefore, EphA10 is a promising therapeutic target with minimal adverse effects. The EphA10-targeting strategies, including monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs), bispecific Abs, and chimeric antigen receptor (CAR) T cells, have been developed in preclinical studies. Therefore, mAbs that specifically recognize cell sur-face-expressing EphA10 are essential for developing novel therapeutic modalities. In this study, we established novel anti-human EphA10 mAbs using the Cell-Based Immun-ization and Screening (CBIS) method. Among them, Ea₁₀Mab-3 reacted with EphA10-overexpressed Chinese hamster ovary-K1 (CHO/EphA10) and glioblastoma cell line (LN229/EphA10) in flow cytometry. The binding affinities (K_D values) were deter-mined as 2.6 × 10-10 M for CHO/EphA10 and 3.0 × 10-9 M for LN229/EphA10. Furthermore, Ea₁₀Mab-3 did not exhibit cross-reactivity with other Eph receptor-overexpressed CHO-K1. These results indicate that Ea10Mab-3 is a specific, high-affinity mAb that is expected to be used for mAb-based tumor diagnosis and therapy.

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 dissociation constant (KD) values of Ca17Mab-5 for CHO/CDH17 and COLO205 were estimated as 1.4 × 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 and colorectal cancers. Conclusion: Ca17Mab-5 is a versatile tool for detecting CDH17 and has potential for tumor diagnosis and therapy.

Glypican-3 (GPC3) belongs to the glypican family of heparan sulfate proteoglycans and is fre-quently overexpressed in hepatocellular carcinoma (HCC). The overexpression of GPC3 is associ-ated with the poor clinical outcomes, suggesting its potential as a clinically relevant biomarker and therapeutic target. Therefore, anti-GPC3 monoclonal antibodies (mAbs) have been developed in various modalities for tumor diagnosis and therapy. In this study, more than 80 clones of novel anti-GPC3 mAbs were established using a flow cytometry-based high-throughput screening, the Cell-Based Immunization and Screening (CBIS) method. Among them, a clone G3Mab-25 (IgG1, κ) recognized GPC3-overexpressed Chinese hamster ovary-K1 (CHO/GPC3) but not parental CHO-K1 in flow cytometry. Furthermore, G3Mab-25 recognizes endogenous GPC3 in GPC3-expressing HCC cell lines, including HepG2, HuH-7, and JHH-5. G3Mab-25 specifically recognized only CHO/GPC3, but not other GPC family-overexpressed CHO-K1. The dissociation constant values of G3Mab-25 for CHO/GPC3, HepG2, HuH-7, and JHH-5 were determined to be 1.8 × 10⁻8 M, 7.3 × 10⁻⁹ M, 3.9 × 10⁻⁹ M, and 1.4 × 10⁻⁹ M, respectively. Moreover, G3Mab-25 de-tects the N-terminal fragment of GPC3 in western blotting. In immunohistochemistry, G3Mab-25 showed a diverse staining pattern for GPC3 in HCC tissues. G3Mab-25, established by the CBIS method, is a versatile mAb for basic research and is expected to contribute to tumor diagnosis and therapy.