The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.
A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.
Biogen Inc., Cambridge, Massachusetts 02142, USA. darren_baker@biogen.com
D P
DP
Baker
A
A
Whitty
M R
MR
Zafari
D L
DL
Olson
C A
CA
Hession
K
K
Miatkowski
L S
LS
Avedissian
S F
SF
Foley
M L
ML
McKay
C D
CD
Benjamin
L C
LC
Burkly
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015374:Biosensing Techniques; D002850:Chromatography, Gel; D006639:Histidine / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007376:Interleukin-2 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D015375:Receptors, Interleukin-2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014162:Transfection
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007376:Interleukin-2; D046911:Macromolecular Substances; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D006639:Histidine