Tomohiro Tanaka

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.

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.

Leukocyte migration is a fundamental process in both innate and adaptive immune responses. This process is tightly regulated by chemokines and their cognate receptors. The bioavailability of chemokines is further modulated by atypical chemokine receptors (ACKRs), a subset of chemokine receptor–like molecules that lack coupling to canonical G protein–mediated signaling pathways. Among these, ACKR4 regulates dendritic cell migration through ligand scavenging and has been implicated in tumor progression in murine models. We previously established anti-mouse ACKR4 (mACKR4) mAbs, A4Mab-1, A4Mab-2, and A4Mab-3, by N-terminal peptide immunization. This study examined the binding epitopes of A4Mabs. Alanine (or glycine) scanning within the N-terminal region (amino acids 2–19) was performed using flow cytometry and Western blotting. The results demonstrated that Tyr11, Tyr12, Glu14, Glu15, and Glu17 are critical for recognition by A4Mab-1, while Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are essential for recognition by A4Mab-2 in flow cytometry and Western blotting. Furthermore, Glu14, Asn16, and Glu17 are essential for recognition by A4Mab-3 in flow cytometry. These findings contribute to the understanding of mACKR4 recognition by A4Mabs.

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.

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.

Cadherin 13 (CDH13), also known as T-cadherin or H-cadherin, is a member of the cadherin superfamily. CDH13 is anchored to the plasma membrane via glycosylphosphatidylinositol. CDH13 plays an essential role in the development of the heart and nervous systems, including the brain. Many reports have identified CDH13 as a risk factor for neurodevelopmental disorders. Furthermore, CDH13 has been shown to be expressed in numerous cancers, but its role as a cancer-promoting or -suppressing factor remains unclear. Therefore, the development of highly sensitive and specific anti-CDH13 monoclonal antibodies (mAbs) is necessary to elucidate the biological and pathological functions of CDH13. In this study, we established a novel anti-human CDH13 mAb (clone Ca₁₃Mab-4) using the Cell-Based Immunization and Screening (CBIS) method. Ca₁₃Mab-4 can be used for flow cytometric analysis. Ca₁₃Mab-4 binds specifically to CDH13 and not to other cadherin family members. The dissociation constant values of Ca₁₃Mab-4 for CDH13-overexpressed CHO-K1 and U87MG glioma cells were determined as 2.5 (± 0.6) x 10^-8 M and 8.9 (± 2.1) x 10^-9 M, respectively. Furthermore, Ca₁₃Mab-4 clearly detected CDH13 in the western blot and immunohistochemistry of cell sections. Therefore, the Ca₁₃Mab-4, established by CBIS method, could be a valuable tool for basic research and is expected to contribute to elucidating the relationship between CDH13 and diseases, including neurodevelopmental disorders and cancer.

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.

The CXC chemokine receptor 2 (CXCR2) is a member of the G-protein-coupled receptor superfamily and regulates a diverse range of immune responses and tumor progression. CXCR2 is expressed on immune cells, especially neutrophils, and is involved in various immune responses by interacting with its chemokine ligands. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR2 has been desired for treatment and diagnosis. This study established a novel sensitive anti-mouse CXCR2 (mCXCR2) mAb; Cx2Mab-5 (rat IgG2a, kappa), using the mCXCR2 synthetic N-terminus peptide immunization method. In flow cytometry, Cx2Mab-5 recognized mCXCR2-overexpressed Chinese hamster ovary-K1 cells (CHO/mCXCR2) and WEHI-3B (murine myelomonocytic leukemia cell) cells, which express endogenous mCXCR2. Cx2Mab-5 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. Cx2Mab-5 showed a moderate binding affinity for both CHO/mCXCR2 and WEHI-3B. Further-more, Cx2Mab-5 detected mCXCR2 in western blot and immunohistochemistry in CHO/mCXCR2 cells. Hence, Cx2Mab-5 can be a valuable tool for analyzing mCXCR2-expressing cells, such as immune cells and 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.