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

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.


Introduction
CD44 is a transmembrane glycoprotein expressed on many cell types, including endothelial cells, epithelial cells, fibroblasts, keratinocytes, and leukocytes [1]. CD44 is believed to play important roles in physiological processes such as cell proliferation, adhesion, migration, and lymphocyte activation [2]. The CD44 gene consists of 20 exons [3], with ten constitutively expressed to produce the smallest isoform, which is the standard form of CD44 (CD44s). The other possible isoforms are categorized as CD44 variants (CD44v) and are generated by alternatively spliced transcripts [4]. The diversity of the CD44 protein is also augmented by post-translational modifications such as N-and Oglycosylation and heparan sulfate [5,6].
Cancer cells often express a large variety of CD44 variants, particularly in the advanced stages. Indeed, the CD44v6 isoform was first identified as one of the metastatic determinants in cancer. CD44v6specific monoclonal antibodies (mAbs) have been shown to inhibit the metastasis of rat pancreatic tumor cells [7,8]. Furthermore, the transfection of CD44v4-7 cDNA has been shown to confer a metastatic phenotype to non-metastatic cells [9]. In addition, CD44v6 isoforms have been reported to act as co-receptors for receptor tyrosine kinases (RTKs), such as Met and VEGFR-2 [10][11][12]. Another variant isoform, CD44v3, is able to bind several heparan sulfate-binding growth factors, such as FGFs and HB-EGF [13,14]. These studies suggest that the coreceptor function of CD44v for RTKs is required for tumor progression. Several reports have also indicated that CD44v likely functions as a prognostic marker in many tumors, including lung, colorectal, breast, hepatocellular, and head and neck cancers [15][16][17][18][19]. Considering that CD44 has multifunctional roles and a promising prognostic value in various cancers, targeting CD44 for cancer therapy may prove to be a promising approach. MAbs, which neutralize the binding of hyaluronic acid to CD44, have been shown to inhibit anchorage-independent growth of murine mammary carcinoma cells and human colon carcinoma cells, and to induce apoptosis in vitro [20]. In addition, mAbs against CD44 or CD44v have been demonstrated to exert significant antitumor activity in in vivo animal models of human xenograft tumors [21,22]. Although a number of studies have been conducted on CD44, CD44v isoforms have not been fully characterized. This is, in part, due to the lack of critical probes needed for the specific detection of CD44v isoforms, as limited antibodies against CD44 variant exons are commercially available. Therefore, sensitive antibodies to CD44v-specific isoforms are necessary.
Recently, we immunized mice with cat podoplanin-expressed CHO-K1 cells (CHO/catPDPN) and performed screening using CHO/catPDPN in flow cytometry [23]. This method was named as the Cell-Based Immunization and Screening (CBIS) method. Using this CBIS method, we obtained highly sensitive mAbs against various membrane proteins. In this study, we aimed to develop a novel anti-CD44 mAb using the CBIS method.

Production of novel anti-CD44 mAbs using the Cell-Based Immunization and Screening (CBIS) method
We immunized two mice with CHO/CD44v3-10 cells (Fig. 1A), and performed flow cytometric screening. We previously named this cellbased strategy as the CBIS method, as shown in Fig. 1B. Culture supernatants of 1008 wells were mixed with CHO/CD44v3-10 and CHO-K1 cells, and 15 wells (15/1008; 1.5%) showing a stronger reaction against CHO/CD44v3-10 cells than CHO-K1 cells were ultimately selected. After limiting dilution, 7 clones were established, all of which were classified as IgG 1 subclass. Clone C 44 Mab-5 (IgG 1 , kappa) reacted with oral cancer tissues in further immunohistochemical analyses (data not shown).
In conclusion, we successfully produced C 44 Mab-5, a novel anti-CD44s mAb, using the CBIS method. This method is advantageous because it does not require the membrane protein to be purified in all steps of mAb production. C 44 Mab-5 appears to be promising for the detection of CD44s, as well as many CD44 variants using flow cytometry and immunohistochemical analysis. Future studies investigating the reactivity of C 44 Mab-5 towards other human cancers are warranted.