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 K_D 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.

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 (K_D) 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.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3–10-overexpressed cells. In this study, we established C₄₄Mab-6 (IgG₁, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C₄₄Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent K_D of C₄₄Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10⁻⁹ M, 6.3 × 10⁻⁹ M, and 1.9 × 10⁻⁹ M, respectively. C₄₄Mab-6 could detect the CD44v3–10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C₄₄Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C₄₄Mab-108; IgG₁, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The K_D of C₄₄Mab-108 for CHO/CD44 v3-10 was 3.4 × 10⁻⁷ M. In western blot analysis, C₄₄Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C₄₄Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C₄₄Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG₁, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the K_D of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10⁻⁸ M and 1.9 × 10⁻⁸ M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C₄₄Mab-94; IgG₁, kappa) recognized the variant-8-encoded region and peptide, indicating that C₄₄Mab-94 is a specific mAb for CD44v8. Furthermore, C₄₄Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C₄₄Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C₄₄Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy.