Naoki Kojo

Erythropoietin-producing hepatocellular receptor A2 (EphA2) has emerged as a key mediator that promotes tumor malignant progression. EphA2 overexpression and its non-canonical signaling lead to oncogenic transformation, metabolic reprogramming, resistance to treatments, and metastasis. Therefore, strategies targeting EphA2 have been evaluated in clinical trials. However, the clinical effects were not sufficient. An anti-EphA2 monoclonal antibody (mAb), Ea₂Mab-7 (mouse IgG₁, κ), demonstrated high affinity and specificity among Eph receptors. In this study, we produced recombinant class-switched Ea₂Mab-7 variants, including Ea₂Mab-7-mG_2a (mouse IgG_2a) and Ea₂Mab-7-hG₁ (human IgG₁). Both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ recognized human triple-negative breast cancer MDA-MB-231, pancreatic cancer MIA PaCa-2, and colorectal cancer HCT-15 in flow cytometry. Furthermore, both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ exerted significant antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against these tumors. In mouse xenograft models of breast, pancreatic, and colorectal cancers, both mAbs demonstrated antitumor activity. These results indicate the potential of Ea₂Mab-7 variants for the treatment of EphA2-positive cancers.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) has emerged as a key mediator that promotes tumor malignant progression. EphA2 overexpression and its non-canonical signaling lead to oncogenic transformation, metabolic reprogramming, resistance to treatments, and metastasis. Therefore, strategies targeting EphA2 have been evaluated in clinical trials. However, the clinical effects were not sufficient. An anti-EphA2 monoclonal antibody (mAb), Ea2Mab-7 (mouse IgG1, κ), demonstrated high affinity and specificity among Eph receptors. In this study, we produced recombinant class-switched Ea2Mab-7 variants, including Ea2Mab-7-mG2a (mouse IgG2a) and Ea2Mab-7-hG1 (human IgG1). Both Ea2Mab-7-mG2a and Ea2Mab-7-hG1 recognized human triple-negative breast cancer MDA-MB-231, pancreatic cancer MIA PaCa-2, and colorectal cancer HCT-15 in flow cytometry. Furthermore, both Ea2Mab-7-mG2a and Ea2Mab-7-hG1 exerted significant antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against these tumors. In mouse xenograft models of breast, pancreatic, and colorectal cancers, both mAbs demonstrated antitumor activity. These results indicate the potential of Ea2Mab-7 variants for the treatment of EphA2-positive cancers.

CD44 variant (CD44v) isoforms are involved in promoting cancer metastasis, sustaining cancer stem cell (CSC) properties, and conferring resistance to therapeutic interventions. Consequently, the development of monoclonal antibodies (mAbs) targeting CD44v represents a crucial strategy for eliminating CD44v-positive cancer cells. Previously, an anti-CD44v6 mAb, C₄₄Mab-9 (mouse IgG₁, κ), was established. C₄₄Mab-9 recognizes explicitly the epitope encoded by the variant exon 6-encoded region of CD44 and applies to flow cytometry, western blotting, and immunohistochemistry. To assess the therapeutic potential, a mouse IgG_2a isotype of C₄₄Mab-9 (designated C₄₄Mab-9-mG_2a) was generated, and the in vitro and in vivo antitumor activities were evaluated using gastric and colorectal cancer cell lines. C₄₄Mab-9-mG_2a demonstrated specific binding to CD44v3–10-overexpressed Chinese hamster ovary cells (CHO/CD44v3–10), as well as gastric cancer (NUGC-4) and colorectal cancer (COLO201 and COLO205) in flow cytometry. C₄₄Mab-9-mG_2a exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10, NUGC-4, COLO201, and COLO205. Moreover, systemic administration of C₄₄Mab-9-mG_2a significantly inhibited tumor growth in CHO/CD44v3–10, NUGC-4, COLO201, and COLO205 xenografts compared with the control IgG_2a. These findings indicate that C₄₄Mab-9-mG_2a could be applied to the mAb-based therapy against CD44v6-positive tumors.

Background: CD44 variant (CD44v) isoforms are involved in promoting cancer metastasis, sustaining cancer stem cell (CSC) properties, and conferring resistance to therapeutic interventions. Consequently, the development of monoclonal antibodies (mAbs) targeting CD44v represents a crucial strategy for eliminating CD44v-positive cancer cells. Previously, an anti-CD44v6 mAb, C44Mab-9 (mouse IgG1, κ), was established. C44Mab-9 recognizes explicitly the epitope encoded by the variant exon 6-encoded region of CD44 and applies to multiple experimental techniques. Methods: To assess the therapeutic potential, a mouse IgG2a isotype of C44Mab-9 (designated C44Mab-9-mG2a) was generated, and the in vitro and in vivo antitumor activities were evaluated using gastric and colorectal cancer cell lines. Results: C44Mab-9-mG2a demonstrated specific binding to CD44v3–10-overexpressed Chinese hamster ovary cells (CHO/CD44v3–10), as well as gastric cancer (NUGC-4) and colorectal cancer (COLO201 and COLO205) in flow cytometry. C44Mab-9-mG2a exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10, NUGC-4, COLO201, and COLO205. Moreover, systemic administration of C44Mab-9-mG2a significantly inhibited tumor growth in CHO/CD44v3–10, NUGC-4, COLO201, and COLO205 xenografts compared with the control IgG2a. Conclusions: These findings indicate that C44Mab-9-mG2a could be applied to the mAb-based therapy against CD44v6-positive tumors.