Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody (C44Mab-108) Using Enzyme-Linked Immunosorbent Assay.

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C44Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10) and found that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.

A Rat Anti-Mouse CD39 Monoclonal Antibody for Flow Cytometry.

By converting extracellular adenosine triphosphate to adenosine, CD39 is involved in adenosine metabolism. The extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment. Therefore, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is thought to be one of the important strategies for tumor therapy. In this study, we developed novel mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCD39 mAbs, C39Mab-2 (rat IgG2a, lambda), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) and an endogenously mCD39-expressed cell line (SN36) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of C39Mab-2 for CHO/mCD39 and SN36 were 5.5 × 10-9 M and 4.9 × 10-9 M, respectively. These results indicated that C39Mab-2 is useful for the detection of mCD39 in flow cytometry.

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning.

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1-20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.