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 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C₄₄Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG_2a version of C₄₄Mab-18 (C₄₄Mab-18-mG_2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C₄₄Mab-46-mG_2a. C₄₄Mab-18-mG_2a exhibited higher reactivity compared with C₄₄Mab-46-mG_2a to CD44v3–10-overexpressed CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C₄₄Mab-18-mG_2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3–10. In contrast, C₄₄Mab-46-mG_2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C₄₄Mab-18-mG_2a or C₄₄Mab-46-mG_2a significantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG_2a. These results indicate that C₄₄Mab-18-mG_2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed PANC-1 cells. Among the established clones, C₄₄Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C₄₄Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C₄₄Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C₄₄Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (K_D) of C₄₄Mab-18 for CHO/CD44v3–10 and HSC-3 was 1.6 × 10⁻⁷ M and 1.7 × 10⁻⁷ M, respectively. Furthermore, C₄₄Mab-18 detected CD44v3–10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C₄₄Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

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.

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, the elucidation of each CD44 isoform’s function in tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3) determined by 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) by flow cytometry. The apparent KD 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 expected for the application of tumor diagnosis and therapy.

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx5Mab-3; rat IgG2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx5Mab-3 for CHO/mCXCR5 cell is 7.2 × 10−10 M. Furthermore, Cx5Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx5Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.