Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG₁, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG_2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (K_D)=2.2x10^‑8 M] using flow cytometry. EpMab‑37‑mG_2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG_2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG_2a‑f also exhibited a moderate binding‑affinity (K_D=1.5x10^‑8 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG_2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG_2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG_2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death.

CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed in eosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 was found in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be a useful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the Cell-Based Immunization and Screening method. The established anti-mCCR3 mAb, C ₃ Mab-3 (rat IgG _2a , kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry. C ₃ Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells, which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that K _D s of C ₃ Mab-3 for CHO/mCCR3, P388, and J774-1 cells were 4.3 × 10 ⁻⁸ M, 2.6 × 10 ⁻⁷ M, and 2.4 × 10 ⁻⁷ M, respectively. C ₃ Mab-3 could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C ₅ Mab-2 (rat IgG _2b , kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant ( K _D ) of C ₅ Mab-2 for CHO/mCCR5 was determined as 4.3 × 10 ⁻⁸ M. These results indicated that C ₅ Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.

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.

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.