Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5

Abstract

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa), recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C44Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C44Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231

“…We have previously produced mAbs against HER2 (22) or EGFR (23) using CasMab technology and mAbs against CD44, PD-L1, or CD133 using the CBIS method. (24)(25)(26)(27)(28) However, the binding epitopes of these mAbs (H 2 Mab-77 against HER2, EMab-51 against EGFR, C 44 Mab-5 against CD44, L 1 Mab-13 against PD-L1, and CMab-43 against CD133) remain to be determined because these mAbs did not react with synthesized peptides. Conversely, the binding epitopes of mAbs produced by immunizing with synthesized peptides have been easily determined using deletion mutants and point mutants of synthesized peptides.…”

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

“…Furthermore, LpMab-23-recognizing cancer-type podoplanin could be a novel predictor for a poor prognosis of early stage tongue cancer [ 40 ]. Recently, we also utilized a Cell-Based Immunization and Screening (CBIS) method to establish mAbs against various membrane proteins, such as CD133 [ 41 ], CD44 [ 42 ], PD-L1 [ 43 ], pig PDPN [ 34 ], horse PDPN [ 44 ], and cat PDPN [ 32 ]. Importantly, those mAbs are very useful for flow cytometry, Western blot, and immunohistochemistry.…”

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

“…XM_012545641.2) bearing an N-terminal PA16 tag (PA16-tasPDPN) was inserted into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [ 32 ]. The PA16 tag comprises 16 amino acids (GLEGGVAMPGAEDDVV) [ 33 ]. The CHO-K1 cells were transfected with pCAG-Ble vector containing PA16-tasPDPN using the Lipofectamine ® LTX and Plus™ reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA).…”

“…We employed a Cell-Based Immunization and Screening (CBIS) method [ 25 , 33 , 35 , 36 ] to develop sensitive and specific mAbs against tasPDPN. Briefly, two BALB/c mice were immunized with CHO/tasPDPN cells (1 × 10 8 ) intraperitoneally (i.p.)…”