Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Abstract

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3–10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 and 3.2 × 10−9 M, respectively. C44Mab-34 could detect CD44v3–10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

Development of a Novel Anti-CD44 Variant 8 Monoclonal Antibody C44Mab-94 against Gastric Carcinomas

“…We determined the epitope as a v8-encoded region using deletion mutants of CD44 ( Figure 2 ), and synthetic peptides ( Figure 3 ). We have established anti-CD44 mAbs using CHO/CD44v3–10 [ 29 , 35 , 36 , 38 ], PANC-1/CD44v3–10 (in this study), or purified CD44v3–10 ectodomain [ 30 , 37 ] as antigens. We listed them in our original “Antibody Bank” (see Supplementary Materials ).…”

“…Furthermore, we produced a class-switched and a defucosylated version of C 44 Mab-5 (5-mG 2a -f) using fucosyltransferase 8-deficient ExpiCHO-S cells and evaluated the antitumor effects of 5-mG 2a -f in OSCC xenograft-bearing mice [ 33 ]. We have developed various anti-CD44v mAbs, including anti-CD44v4 (C 44 Mab-108) [ 34 ], anti-CD44v5 (C 44 Mab-3) [ 35 ], anti-CD44v6 (C 44 Mab-9) [ 36 ], anti-CD44v7/8 (C 44 Mab-34) [ 37 ], and anti-CD44v9 (C 44 Mab-1) [ 38 ].…”

Editorial of Special Issue “Oral Cancer: From Pathophysiology to Novel Therapeutic Approaches”

“…Therefore, non-invasive biomarkers such as tumor oxygenation and signals from autofluorescence imaging, intensity signals from optical coherence tomography, and ultrasound wave signals from surface tumor tissues have been shown to provide accurate predictions for early detection and prognosis assessments in OSCC [30]. To overcome these challenges, this Special Issue has accepted a few publications that highlighted the potential biomarkers that can be used for diagnostic and therapeutic purposes [31][32][33][34][35][36][37][38][39][40]. The diagnostic section can be divided into biomarkers for OSCC and prognosis.…”

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

“…Furthermore, we produced a defucosylated version (5-mG 2a -f) using FUT8-deficient ExpiCHO-S cells (BINDS-09) and investigated the antitumor effects of 5-mG 2a -f in mouse xenograft models of oral SCC [ 29 ]. Recently, we have established various CD44v mAbs, including C 44 Mab-108 (v4) [ 30 ], C 44 Mab-3 (v5) [ 31 ], C 44 Mab-9 (v6) [ 32 ], and C 44 Mab-34 (v7/8) [ 33 ].…”