C₇Mab-2: A novel monoclonal antibody against mouse CCR7 established by immunization of the extracellular loop domain

Abstract

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. Specific monoclonal antibodies (mAbs) against chemokine receptors for flow cytometry have been developed using Cell-Based Immunization and Screening, and the N-terminal peptide immunization methods. However, there are few reports on the establishment of anti-chemokine receptor mAbs through immunization with ECL peptides. Here, an anti-mouse C-C chemokine receptor type 7 (mCCR7) mAb, C₇Mab-2 (rat immunoglobulin G_2b, kappa), was established through immunization with the ECL3 peptide. C₇Mab-2 demonstrated reactivity to mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells in flow cytometry, which was inhibited by the ECL3 peptide. C₇Mab-2 did not show cross-reactivity with other mouse CC, CXC, CX3C, and XC chemokine receptors. The dissociation constant value of C₇Mab-2 was determined to be 2.8 × 10⁻⁹ M for CHO/mCCR7 cells. Furthermore, C₇Mab-2 detected mCCR7 in immunohistochemistry. This strategy could accelerate the development of novel chemokine receptor mAbs with high affinity and specificity.

Keywords

Mouse C-C chemokine receptor type 7 / Monoclonal antibody / Extracellular loop / Peptide immunization / Flow cytometry / Immunohistochemistry

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