Development of a Novel Anti-Mouse CCR7 Monoclonal Antibody C7Mab-2 by Immunization of the Extracellular Loop Domain

The chemokine receptors possess seven‐transmembrane helices connected by an extracellular N‐terminal region, three extracellular loops (ECL1‐3), three intracellular loops, and an intracellular C‐terminal region. We have developed specific monoclonal antibodies (mAbs) against chemokine receptors for flow cytometry using the Cell-Based Immunization and Screening (CBIS) and the N-terminal peptide immunization methods. However, there are few reports for establishing anti-chemokine receptor mAbs by immunization of ECL peptides. Here, we established an anti-mouse CCR7 (mCCR7) mAb, C7Mab-2 (rat IgG2b, kappa) by immunization of the ECL3 peptide. C7Mab-2 demonstrated the reactivity to mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells in flow cytometry, which was blocked by the ECL3 peptide. C7Mab-2 did not show the cross-reactivity to other mouse CC, CXC, CX3C, and XC chemokine receptors. The dissociation constant (KD) value of C7Mab-2 was determined to be 2.8 ± 0.3 × 10⁻⁹ M for CHO/mCCR7 cells. Furthermore, C7Mab-2 detected mCCR7 in immunohistochemistry. The strategy could accelerate the development of novel chemokine receptor mAbs with high affinity and specificity.