Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C 8 Mab-2 Using Flow Cytometry.
Hiyori KobayashiHiroyuki SuzukiTomohiro TanakaMika K KanekoYukinari KatoPublished in: Monoclonal antibodies in immunodiagnosis and immunotherapy (2024)
The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8 + cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C 8 Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C 8 Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C 8 Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17- DFFTAP -22 sequence is important for the recognition by C 8 Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C 8 Mab-2.
Keyphrases
- monoclonal antibody
- induced apoptosis
- flow cytometry
- amino acid
- cell cycle arrest
- dendritic cells
- regulatory t cells
- immune response
- high resolution
- signaling pathway
- cell death
- oxidative stress
- poor prognosis
- toll like receptor
- pi k akt
- inflammatory response
- mass spectrometry
- dna binding
- type iii
- high density
- electron microscopy
- wild type
- high speed