Showing papers by "Takeo Yoshikawa published in 2023"

Antitumor activities of a defucosylated anti-EpCAM monoclonal antibody in colorectal carcinoma xenograft models

Abstract: 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-mG2a-f recognized EpCAM-overexpressing CHO-K1 (CHO/EpCAM) cells with a moderate binding-affinity [dissociation constant (KD)=2.2×10−8 M] using flow cytometry. EpMab-37-mG2a-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-mG2a-f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab-37-mG2a-f also exhibited a moderate binding-affinity (KD) =1.5×10−8 M] and high ADCC and CDC activities for a colorectal cancer cell line (Caco-2 cells). The administration of EpMab-37-mG2a-f to Caco-2 tumor-bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab-37-mG2a-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-mG2a-f may exert antitumor activities against EpCAM-positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

Epitope Mapping of the Novel Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by 2 × Alanine Scanning.

Abstract: We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb), C9Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA). This study aims to identify the binding epitope of C9Mab-11 by using 1 × and 2 × alanine (or glycine) substituted-hCCR9 peptides (1 × and 2 × Ala-scan) by ELISA. According to the 1 × Ala-scan analysis, the response of C9Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the 2 × Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, and M13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C9Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important. The successful identification of the C9Mab-11 epitope might be useful for the future pathophysiological analysis of hCCR9.

Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7).

Abstract: One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.

Epitope Mapping of an Anti-EpCAM Monoclonal Antibody (EpMab-37) Using the Alanine Scanning Method.

Abstract: The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.