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

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemistry and Biophysics Reports Pub Date : 2024-09-07 DOI:10.1016/j.bbrep.2024.101824

Miu Hirose, Hiroyuki Suzuki, Rena Ubukata, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

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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 IgG_2b, kappa), A₄Mab-2 (rat IgG_2b, kappa), and A₄Mab-3 (rat IgG_2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K_D) 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.

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开发特异性抗小鼠非典型趋化因子受体 4 单克隆抗体

白细胞迁移是先天性和适应性免疫反应的一项基本功能。趋化因子及其受体控制着迁移系统。非典型趋化因子受体（ACKRs）是不与 G 蛋白信号通路耦合的趋化因子受体样分子，它控制着趋化因子的丰度。其中，ACKR4 通过控制配体来调节树突状细胞的迁移，并参与小鼠模型的肿瘤发生。由于还没有用于流式细胞术的抗小鼠 ACKR4（mACKR4）单克隆抗体（mAb）的报道，本研究旨在开发一种新型的 mACKR4 mAb。在已建立的抗 mACKR4 mAb 中，A4Mab-1（大鼠 IgG2b，kappa）、A4Mab-2（大鼠 IgG2b，kappa）和 A4Mab-3（大鼠 IgG2b，kappa）通过流式细胞术识别了过表达的中国仓鼠卵巢-K1（CHO/mACKR4）。经测定，A4Mab-1、A4Mab-2 和 A4Mab-3 对 CHO/mACKR4 的解离常数（KD）分别为 6.0 × 10-9 M、1.3 × 10-8 M 和 1.7 × 10-9 M。此外，A4Mab-1 和 A4Mab-2 还能通过免疫印迹检测到 mACKR4。这些结果表明，A4Mab-1、A4Mab-2和A4Mab-3有助于通过流式细胞术和Western印迹检测mACKR4，并在临床前模型中获得了概念验证。

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来源期刊

Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics

CiteScore

4.60

自引率

0.00%

发文量

191

审稿时长

59 days

期刊介绍： Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.