Search Results (7)

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C₄₄Mab-9; IgG₁, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C₄₄Mab-9 recognizes CD44v6. Furthermore, C₄₄Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (K_D) of C₄₄Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10⁻⁹ M, 1.7 × 10⁻⁸ M, and 2.3 × 10⁻⁸ M, respectively. C₄₄Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C₄₄Mab-9 is useful for detecting CD44v6 in various applications. Full article

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C₉Mab-24 (rat IgG_2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1–19). C₉Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C₉Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C₉Mab-24 did not react with four peptides (M13A–F14A, F14A–D15A, D16A–F17A, and F17A–S18A), indicating that _13-MFDDFS_-18 is involved in C₉Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target–antibody interaction. Full article

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C₃Mab-3 (rat IgG_2a, kappa), and C₃Mab-4 (rat IgG_2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C₃Mab-3, C₃Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C₃Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C₃Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C₃Mab-3, C₃Mab-4, and J073E5. Full article

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG₁, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG_2a-type of EpMab-37 (EpMab-37-mG_2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG_2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (K_D): 2.9 × 10⁻⁸ M and 1.8 × 10⁻⁸ M, respectively] by flow cytometry. EpMab-37-mG_2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG_2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG_2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers. Full article

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (K_D: 1.3 × 10⁻⁹ M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors. Full article

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG₁, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The K_D of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10⁻⁸ M and 3.2 × 10⁻⁸ M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications. Full article

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo−resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti−CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3−10) ectodomain and screening using enzyme−linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C₄₄Mab−46; IgG₁, kappa) reacted with CD44 standard isoform (CD44s)−overexpressed Chinese hamster ovary−K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent K_D of C₄₄Mab−46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10⁻⁸ M, 4.9 × 10⁻⁸ M, and 4.1 × 10⁻⁸ M, respectively. C₄₄Mab−46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C₄₄Mab−46 strongly stained the formalin−fixed paraffin−embedded ESCC tissues in immunohistochemistry. Collectively, C₄₄Mab−46 is very useful for detecting CD44 in various applications. Full article