The Eph receptor-ephrin system is essential for embryonic, neural, and vascular development and contributes to tumor promotion or suppression. Ephrin type-A receptor 7 (EphA7) has been identified as a tumor suppressor often lost or downregulated in lymphomas. In contrast, EPHA7 mutations have been suggested as potential driver genes in other cancers. However, the detailed molecular function has not been clarified. Therefore, a monoclonal antibody (mAb) against EphA7 is essential for basic research and clinical applications. In this study, we developed a novel anti-human EphA7 mAb, Ea7Mab-10, using the Cell-Based Immunization and Screening (CBIS) method. Ea7Mab-10 reacted with EphA7-overexpressed Chinese hamster ovary-K1 (CHO/EphA7) and EphA7-overexpressed LN229 (LN229/EphA7) in flow cytometry. The binding affinities were determined as 3.1 × 10−9 M for CHO/EphA7 and 6.9 × 10−9 M for LN229/EphA7. Furthermore, Ea7Mab-10 did not show cross-reactivity with other Eph family members and was suitable for Western blotting and immunohistochemistry. These results demonstrate that Ea7Mab-10, developed using the CBIS method, supports basic research on EphA7 and could be used for mAb-based targeted therapies.

Eph receptor-ephrin system is essential for embryonic, neural, and vascular development, and also plays important roles in tumor promotion or suppression. EphA7 was described as a tumor suppressor that is frequently lost in lymphomas. In contrast, EphA7 mutations have been suggested to be a driver gene in several cancers. However, the detailed molecular function has not been clarified. Therefore, monoclonal antibody (mAb) against EphA7 is essential for basic research and clinical applications. In this study, we developed a novel anti-human EphA7 mAb, Ea7Mab-10, using the Cell-Based Immunization and Screening (CBIS) method. Ea7Mab-10 reacted with EphA7-overexpressed Chinese hamster ovary-K1 (CHO/EphA7) and EphA7-overexpressed LN229 (LN229/EphA7) in flow cytometry. The binding affinities were determined as 3.1×10-9 M for CHO/EphA7 and 6.9×10-9 M for LN229/EphA7. Furthermore, Ea7Mab-10 did not show cross-reactivity with other Eph family members. Furthermore, Ea7Mab-10 is suitable for western blotting and immunohistochemistry. These results indicate that Ea7Mab-10, established by the CBIS method, facilitates basic studies of EphA7 and is expected for future therapeutic applications, including mAb-based targeted therapy.

Glypican-1 (GPC1) is a heparan sulfate proteoglycan that plays a critical role in regulating various signaling pathways and tumor development. Overexpression of GPC1 promotes tumor cell proliferation and invasiveness, and is associated with poor clinical outcomes. Therefore, anti-GPC1 monoclonal antibodies (mAbs) have been developed in various modalities for tumor therapy. Here, we developed novel anti-GPC1 mAbs using a flow cytometry-based high-throughput screening approach, the Cell-Based Immunization and Screening (CBIS) method. A clone G1Mab-28 (IgG1, κ) reacted with GPC1-overexpressed Chinese hamster ovary-K1 (CHO/GPC1), but not parental CHO-K1, in flow cytometry. Furthermore, G1Mab-28 recognizes endogenous GPC1-expressing human esophageal squamous cell carcinoma KYSE770 cell line. Furthermore, G1Mab-28 specifically recognized only CHO/GPC1, but not other GPC family-overexpressed CHO-K1. The dissociation constant values of G1Mab-28 for CHO/GPC1 and KYSE770 were determined to be 3.3 × 10⁻8 M and 4.6 × 10⁻⁹ M, respectively. Moreover, G1Mab-28 is suitable for western blotting and immunohistochemistry. G1Mab-28, established by the CBIS method, is versatile for basic research and is expected to contribute to the antibody-based tumor therapy.

Background/Objectives: Glypican-1 (GPC1) is a heparan sulfate proteoglycan that plays a critical role in regulating various signaling pathways and tumor development. Overexpression of GPC1 promotes tumor cell proliferation and invasiveness, and is associated with poor clinical outcomes. Therefore, anti-GPC1 monoclonal antibodies (mAbs) have been developed in various modalities for tumor therapy. Methods: We developed novel anti-GPC1 mAbs using a flow cytometry-based high-throughput screening approach, the Cell-Based Immunization and Screening (CBIS) method. Results: A clone G₁Mab-28 (IgG₁, κ) reacted with GPC1-overexpressed Chinese hamster ovary-K1 (CHO/GPC1), but not parental CHO-K1, in flow cytometry. Furthermore, G₁Mab-28 recognizes the endogenous GPC1-expressing human esophageal squamous cell carcinoma KYSE770 cell line. Furthermore, G₁Mab-28 specifically recognized only CHO/GPC1, but not the other GPC family-overexpressed CHO-K1. The dissociation constant values of G₁Mab-28 for CHO/GPC1 and KYSE770 were determined to be 3.3 × 10⁻⁸ M and 4.6 × 10⁻⁹ M, respectively. Moreover, G₁Mab-28 is suitable for Western blotting and immunohistochemistry. Conclusions: G₁Mab-28, established by the CBIS method, is versatile for basic research and is expected to contribute to antibody-based tumor therapy.

Glypican-1 (GPC1) has emerged as a critical mediator of malignant tumor progression. GPC1 plays essential roles in regulating various signaling pathways involved in tumor cell proliferation, invasiveness, and tumorigenesis. Overexpression of GPC1 in tumors mediates oncogenic transformation, epithelial-to-mesenchymal transition, metastatic dissemination, and therapeutic resistance. Accordingly, GPC1-targeted therapeutic strategies have been investigated in clinical and preclinical studies. However, clinical efficacy has been limited. We previously developed an anti-GPC1 monoclonal antibody (mAb), G₁Mab-28 (mouse IgG₁, κ), which exhibits high affinity and specificity for GPC1. In the present study, we generated recombinant isotype-converted G₁Mab-28, including G₁Mab-28-mG_2a (mouse IgG_2a) and G₁Mab-28-hG₁ (human IgG₁). Both mAbs recognized GPC1-expressing human tumor cell lines, including lung squamous cell carcinoma PC-10 and pancreatic ductal adenocarcinoma PK-45H, by flow cytometry. Moreover, both mAbs exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against those cell lines. In mouse xenograft models, treatment with the mAbs resulted in potent antitumor efficacy against PC-10 and PK-45H tumors. Collectively, these findings support the therapeutic potential of G₁Mab-28 for the treatment of GPC1-positive tumors.

Erythropoietin-producing hepatocellular receptor A8 (EphA8) is a type I transmembrane protein that belongs to the largest erythropoietin-producing hepatocellular (Eph) family among receptor tyrosine kinases. By binding to its membrane-bound ephrin-A or ephrin-B ligands on adjacent cells, Eph receptors form complexes and mediate bidirectional signaling activities, triggering cell-cell adhesion and repulsion. Increased expression of EphA8 correlates with poor prognosis in some types of cancer. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphA8 has been desired for treatment, diagnosis, and further basic research. In particular, there are no anti-EphA8 mAbs that can be used for flow cytometry. A novel, specific, and sensitive anti-human EphA8 mAb, which applies to flow cytometry, clone Ea8Mab-9 (mouse immunoglobulin G1, kappa), was established using the Cell-Based Immunization and Screening method. Ea8Mab-9 reacted with EphA8-overexpressed Chinese hamster ovary-K1 cells (CHO/EphA8) and EphA8-overexpressed LN229 glioblastoma cells (LN229/EphA8) in flow cytometry. Notably, Ea8Mab-9 did not recognize other members of the Eph receptor family. Furthermore, Ea8Mab-9 demonstrated a high binding affinity for CHO/EphA8 and LN229/EphA8, with dissociation constants of 1.3 × 10-9 M and 1.6 × 10-9 M, respectively. The reaction of Ea8Mab-9 with CHO/EphA8 was completely blocked by a recombinant EphA8 protein. Ea8Mab-9 could be useful for analyzing the EphA8-related biological responses using flow cytometry, owing to its high affinity and specificity.