title: CCL22-specific Antibodies Reveal That Engagement of Two Distinct Binding Domains on CCL22 Is Required for CCR4-mediated Function.
abstract: CCL22 inactivation in vivo occurs by cleavage at the N-terminus; however, it is unclear whether this encompasses the entire site of CCR4 interaction. CCL17 also binds CCR4 and its function requires binding via two discrete binding sites. Using monoclonal antibodies (MAbs), we report that there are two separate sites on CCL22 that are required for CCR4-mediated function. The CCL22-specific antibodies bind with affinities of 632 ± 297 pM (MC2B7) and 308 ± 43 pM (MAB4391) and neither exhibited detectable binding to CCL17. Both antibodies are comparable in their ability to inhibit CCL22-mediated calcium mobilization; however, competition binding studies demonstrate that MC2B7 and MAB4391 bind to distinct epitopes on CCL22. Both antibodies inhibit function through CCR4, which is demonstrated by loss of β-arrestin recruitment in a reporter cell line. In both assays, blocking either site independently abolished CCL22 function, suggesting that concurrent engagement of both sites with CCR4 is necessary for function. This is the first demonstration that CCL22 has two distinct binding sites that are required for CCR4 function. These antibodies are valuable tools for better understanding the interaction and function of CCL22 and CCR4 and will potentially help further understanding of the differential outcomes of CCL17 and CCL22 interaction with CCR4.
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Sandra
authors.initials: S
authors.lastname: Santulli-Marotto
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: John
authors.initials: J
authors.lastname: Wheeler
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Eilyn R
authors.initials: ER
authors.lastname: Lacy
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Ken
authors.initials: K
authors.lastname: Boakye
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Luongo
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Sheng-Jiun
authors.initials: SJ
authors.lastname: Wu
authors.affiliation: Janssen Research & Development , LLC, Spring House, Pennsylvania.
authors.forename: Mary
authors.initials: M
authors.lastname: Ryan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D019390:Arrestins / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D054414:Chemokine CCL17 / Q000235:genetics / Q000276:immunology; D054422:Chemokine CCL22 / Q000235:genetics / Q000276:immunology*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005786:Gene Expression Regulation; D006801:Humans; D007113:Immunity, Innate; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054398:Receptors, CCR4 / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology; D000071557:beta-Arrestins
chemical_list: D000911:Antibodies, Monoclonal; D019390:Arrestins; C499538:Ccl17 protein, mouse; C515483:Ccl22 protein, mouse; C515355:Ccr4 protein, mouse; D054414:Chemokine CCL17; D054422:Chemokine CCL22; D000939:Epitopes; D054398:Receptors, CCR4; D000071557:beta-Arrestins