Development of a Novel Anti−CD44 Monoclonal Antibody for Multiple Applications against Esophageal Squamous Cell Carcinomas

Abstract

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo−resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti−CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3−10) ectodomain and screening using enzyme−linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab−46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)−overexpressed Chinese hamster ovary−K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab−46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10−8 M, 4.9 × 10−8 M, and 4.1 × 10−8 M, respectively. C44Mab−46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab−46 strongly stained the formalin−fixed paraffin−embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab−46 is very useful for detecting CD44 in various applications.

Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody, C₄₄Mab-108

“…2A). Furthermore, C44Mab-108 never recognized CHO/CD44s cells, which was recognized by previously established pan-CD44 mAb, C44Mab-46 [16] at 10 µg/ml (Fig. 2B).…”

“…We previously established the novel anti-CD44 mAbs, C44Mab-5 (IgG1, kappa) [15] and C44Mab-46 (IgG1, kappa) [16] using Cell-Based Immunization and Screening (CBIS) method and immunization of CD44v3-10 ectodomain, respectively. Both C44Mab-5 and C44Mab-46 have epitopes in the first five exons-encoding sequences [17][18][19].…”

“…Therefore, they can recognize both CD44s and CD44v (pan-CD44). Furthermore, they showed high sensitivity for flow cytometry and immunohistochemical analysis in oral [15] and esophageal tumors [16]. We have also investigated the antitumor effects in mouse xenograft models of oral squamous cell carcinomas [20].…”

Development of a Novel Anti-CD44 variant 6 Monoclonal Antibody for Multiple Applications against Colorectal Carcinomas

“…Therefore, they can recognize both CD44s and CD44v (pan-CD44). Furthermore, C44Mab-5 and C44Mab-46 exhibited the high reactivity for flow cytometry and immunohistochemical analysis in oral [19] and esophageal [20] SCCs. We also examined the antitumor effects of C44Mab-5 in mouse xenograft models [24].…”

“…We established the novel anti-CD44 mAbs, C44Mab-5 (IgG1, kappa) [19] and C44Mab-46 (IgG1, kappa) [20] by Cell-Based Immunization and Screening (CBIS) method and immunization of CD44v3-10 ectodomain, respectively. Both C44Mab-5 and C44Mab-46 recognize the first five standard exons-encoding sequences [21][22][23].…”