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.

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.

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.

Providing cytokine signaling is a key strategy to boost the efficacy of chimeric antigen receptor (CAR) T-cell therapy. However, the individual roles of key downstream mediators, STAT3 and STAT5, remain incompletely understood. In this study, we engineered CAR T cells to express constitutively active mutants of STAT3 (Y640F; caSTAT3) and STAT5 (N642H; caSTAT5) to investigate their individual functions. In vitro, caSTAT3 CAR T cells exhibited enhanced effector function and a robust memory phenotype, with broader transcriptional changes involving both effector- and memory-associated genes compared with caSTAT5 CAR T cells. However, caSTAT3 CAR T cells failed to expand because of activation of apoptosis-related gene programs. In contrast, caSTAT5 CAR T cells demonstrated sustained proliferation over time. Despite the limited in vitro expansion, caSTAT3 CAR T cells exhibited reduced transgene toxicity in vivo and exerted durable antitumor activity in both leukemia and solid tumor models without significant off-tumor toxicity. Titrated expression of caSTAT3 maintained enhanced effector function without inducing apoptosis. On the other hand, caSTAT5 CAR T cells efficiently accumulated in tumors but also infiltrated nontumor tissues, causing lethal systemic toxicity. Co-expression of caSTAT3 and caSTAT5 markedly enhanced the long-term proliferative capacity of CAR T cells, even in the absence of antigen stimulation or cytokine supplementation. These findings elucidate the distinct impacts of STAT3 and STAT5 activation on CAR T-cell behavior and suggest that selective activation of STAT3 at optimal levels may improve CAR T-cell efficacy while minimizing off-tumor toxicities.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H₂Mab-300 (rat IgG_2b, kappa) and H₂Mab-304 (rat IgG₁, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (K_D) values of H₂Mab-300 and H₂Mab-304 for CHO/mHER2 were 1.2 × 10⁻⁹ M and 1.7 × 10⁻⁹ M, respectively. The K_D values of H₂Mab-300 and H₂Mab-304 for NMuMG were 4.9 × 10⁻¹⁰ M and 9.0 × 10⁻¹⁰ M, respectively. These results indicated that H₂Mab-300 and H₂Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.