# Development of specific anti-mouse atypical chemokine receptor 4 monoclonal antibodies

## TL;DR

Researchers developed new monoclonal antibodies to detect a mouse protein involved in immune cell migration and cancer.

## Contribution

Novel monoclonal antibodies against mouse ACKR4 were developed for flow cytometry and western blotting.

## Key findings

- Three monoclonal antibodies (A4Mab-1, A4Mab-2, A4Mab-3) specifically recognize mouse ACKR4.
- The antibodies have measurable binding affinities and can detect ACKR4 in western blotting.
- These antibodies may aid preclinical tumor immunotherapy studies in mouse models.

## Abstract

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (KD) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10−9 M, 1.3 × 10−8 M, and 1.7 × 10−9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

•Novel anti-mACKR4 mAbs were developed by the N-terminal peptide immunization.•Three anti-mACKR4 mAbs are useful for flow cytometry or western blotting.•Anti-mACKR4 mAbs could contribute to the preclinical studies for tumor immunotherapy.

Novel anti-mACKR4 mAbs were developed by the N-terminal peptide immunization.

Three anti-mACKR4 mAbs are useful for flow cytometry or western blotting.

Anti-mACKR4 mAbs could contribute to the preclinical studies for tumor immunotherapy.