Functional expression and characterization of macaque C-C chemokine receptor 3 (CCR3) and generation of potent antagonistic anti-macaque CCR3 monoclonal antibodies.
Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The beta-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop in vivo models of inflammatory diseases, it is essential to identify and characterize the homologues of human eotaxin (C-C chemokine ligand 11) and CCR3 from other species, such as non-human primates. Accordingly, we cloned the macaque eotaxin and CCR3 genes and revealed that they were 91 and 92% identical at the amino acid level to their human homologues, respectively. Macaque CCR3 expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (K(d) = 0.1 nm) and exhibited a robust eotaxin-induced Ca(2+) flux and chemotaxis. Characterization of beta-chemokines on native macaque CCR3 on eosinophils was performed by means of eotaxin-induced shape change in whole blood using a novel signaling assay known as gated autofluorescence forward scatter. Additionally, mAbs were raised against macaque CCR3 using two different immunogens: a 30-amino acid synthetic peptide derived from the predicted NH(2) terminus of macaque CCR3 and intact macaque CCR3-transfected cells. These anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist activity in receptor binding and functional assays. The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of ameliorating chronic inflammatory diseases in humans.
Zhang
Liwen
L
Department of Immunology and Rheumatology, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ 07065, USA.
Soares
Marco P
MP
Guan
Yanfen
Y
Matheravidathu
Stephen
S
Wnek
Richard
R
Johnson
Kristine E
KE
Meisher
Anna
A
Iliff
Susan A
SA
Mudgett
John S
JS
Springer
Martin S
MS
Daugherty
Bruce L
BL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000627:therapeutic use; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D054413:Chemokine CCL11; D018946:Chemokine CCL5 / Q000502:physiology; D019742:Chemokines, CC / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis; D003001:Cloning, Molecular; D004804:Eosinophils / Q000502:physiology; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*
D000911:Antibodies, Monoclonal; C515456:CCL11 protein, human; C515350:CCR3 protein, human; C515457:Ccl11 protein, mouse; C515351:Ccr3 protein, mouse; D054413:Chemokine CCL11; D018946:Chemokine CCL5; D019742:Chemokines, CC; D054397:Receptors, CCR3; D019707:Receptors, Chemokine; D002118:Calcium