Development of a High-Affinity Anti-Human CXCR4 Monoclonal Antibody, Cx₄Mab-7, for Flow Cytometry

CXCR4 (CD184) is a G protein-coupled receptor that binds CXCL12. Aberrant CXCR4 expression is associated with tumor progression, metastasis, therapeutic resistance, and poor prognosis in various hematological and solid malignancies, making it an attractive therapeutic target. Therefore, CXCR4-targeting monoclonal antibodies (mAbs) have advanced to clinical evaluation. In this study, we established a novel anti-human CXCR4 mAb, Cx4Mab-7 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Cx4Mab-7 specifically recognized CXCR4-overexpressed LN229 glioblastoma cells (LN229/CXCR4), CXCR4-overexpressed CHO-K1 cells (CHO/CXCR4), and endogenous CXCR4 on Jurkat cells without cross-reactivity to other C-X-C chemokine receptors. Flow cytometry demonstrated that Cx4Mab-7 exhibited high binding affinity for LN229/CXCR4 cells (KD: 7.4 ± 1.8 × 10−9 M) and Jurkat cells (KD: 3.5 ± 3.6 × 10−9 M). In addition, Cx4Mab-7 was suitable for immunohistochemistry using formalin-fixed paraffin-embedded CHO/CXCR4 cells, although it showed only weak reactivity in Western blot. Cx4Mab-7 may contribute to the future development of diagnostic applications targeting the CXCR4 axis.