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Development of a Novel Anti-Mouse CCR4 Monoclonal Antibody (C4Mab-1) by N-Terminal Peptide Immunization
Junko Takei1, Hiroyuki Suzuki2, Teizo Asano1
1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Abstract:
The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10-9 M, 5.4 × 10-7 M, and 1.1 × 10-6 M, respectively. C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.
Insights
A novel anti-mouse CCR4 monoclonal antibody, C4Mab-1, was developed for research. This sensitive antibody can detect mouse CCR4 on various cell types, aiding in understanding T cell responses and potential therapeutic applications.
Area of Science:
- Immunology
- Molecular Biology
- Oncology
Background:
- CC chemokine receptor type-4 (CCR4) is a G-protein-coupled receptor on T cells, crucial for immune responses.
- CCR4 ligands CCL17 and CCL22 mediate T cell functions in health and disease.
- Anti-CCR4 antibody mogamulizumab is used in treating certain lymphomas and shows potential in solid tumors.
Purpose of the Study:
- To develop a sensitive and specific monoclonal antibody against mouse CCR4 (mCCR4).
- To establish a tool for basic research, diagnosis, and potential therapeutic development targeting mCCR4.
Main Methods:
- N-terminal peptide immunization was used to generate anti-mCCR4 antibodies.
- Flow cytometry was employed to assess antibody reactivity with mCCR4-expressing cells.
- Kinetic analysis determined the binding affinity (KD) of the developed antibody.
Main Results:
- A specific and sensitive anti-mCCR4 monoclonal antibody, C4Mab-1 (rat IgG1, kappa), was successfully established.
- C4Mab-1 demonstrated reactivity with mCCR4-overexpressed CHO-K1 cells, P388, and J774-1 cells via flow cytometry.
- Kinetic analysis revealed binding affinities (KD) in the nanomolar to low micromolar range for C4Mab-1.
Conclusions:
- C4Mab-1 is a valuable tool for detecting mouse CCR4.
- This antibody can aid in elucidating mCCR4-related biological responses in research settings.
