Preprint Communication Version 1 Preserved in Portico This version is not peer-reviewed

Development of Sensitive Anti-mouse CCR5 Monoclonal Antibodies Using the N-terminal Peptide Immunization

Rena Ubukata
,
Hiroyuki Suzuki
ORCID logo ,
Tomohiro Tanaka
,
Tsunenori Ouchida
ORCID logo ,
Guanjie Li
ORCID logo ,
Mika K Kaneko
,
Yukinari Kato
* ORCID logo
Version 1 : Received: 1 April 2024 / Approved: 16 April 2024 / Online: 18 April 2024 (09:29:28 CEST)

How to cite: Ubukata, R.; Suzuki, H.; Tanaka, T.; Ouchida, T.; Li, G.; Kaneko, M.K.; Kato, Y. Development of Sensitive Anti-mouse CCR5 Monoclonal Antibodies Using the N-terminal Peptide Immunization. Preprints 2024, 2024041066. https://doi.org/10.20944/preprints202404.1066.v1 Ubukata, R.; Suzuki, H.; Tanaka, T.; Ouchida, T.; Li, G.; Kaneko, M.K.; Kato, Y. Development of Sensitive Anti-mouse CCR5 Monoclonal Antibodies Using the N-terminal Peptide Immunization. Preprints 2024, 2024041066. https://doi.org/10.20944/preprints202404.1066.v1

Abstract

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to the ligands, CCR5 activates downstream signaling, which plays critical roles in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of pro-inflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10−8 M and 7.3 × 10−9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

Keywords

mouse CCR5; monoclonal antibody; peptide immunization; flow cytometry; western blotting

Subject

Medicine and Pharmacology, Oncology and Oncogenics

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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