The Murine Anti-Human Common Chain Monoclonal Antibody CP.B8 Blocks the Second Step in the Formation of the Intermediate Affinity I
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- 作者：Darren P. Baker ; Adrian Whitty ; Mohammad R. Zafari ; Dian L. Olson ; Catherine A. Hession ; Konrad Miatkowski ; Lena S. Avedissian ; Susan F. Foley ; Michele L. McKay ; Christopher D. Benjamin ; and Linda C.
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 13, 1998
- 年：1998
- 卷：37
- 期：41
- 页码：14337 - 14349
- 全文大小：359K
- 年卷期：v.37,no.41(October 13, 1998)
- ISSN：1520-4995
文摘
A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the humancommon 
(c) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfectedwith the cDNA for the full-length IL-2 receptor 
(IL-2R) and c chains, components which togethercomprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NKcells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, theextracellular portions of the IL-2R and c chains were expressed and purified, and their interactionswith 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 nostable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2was shown to associate rapidly with IL-2R, forming a binary complex with an equilibrium dissociationconstant (Kd) of 800 nM, which permitted subsequent association of the c chain. Dissociation of theIL-2/IL-2R/c chain complex was significantly slower than dissociation of the IL-2/IL-2R complex.Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the c chainwith IL-2 and IL-2R. By gel filtration, mAb CP.B8 formed a stable complex with the c chain, preventingits association with IL-2 and IL-2R. MAb CP.B8 was also capable of dissociating the c chain alreadycomplexed with IL-2 and IL-2R. SPR analysis confirmed these findings and showed, in addition, thatthe Fab fragment of CP.B8 was also capable of inhibiting the association of the c chain with the IL-2/IL-2R complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediateaffinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the c chainthat is involved in contact with IL-2 and/or IL-2R.
(c) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfectedwith the cDNA for the full-length IL-2 receptor (IL-2R) and c chains, components which togethercomprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NKcells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, theextracellular portions of the IL-2R and c chains were expressed and purified, and their interactionswith 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 nostable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2was shown to associate rapidly with IL-2R, forming a binary complex with an equilibrium dissociationconstant (Kd) of 800 nM, which permitted subsequent association of the c chain. Dissociation of theIL-2/IL-2R/c chain complex was significantly slower than dissociation of the IL-2/IL-2R complex.Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the c chainwith IL-2 and IL-2R. By gel filtration, mAb CP.B8 formed a stable complex with the c chain, preventingits association with IL-2 and IL-2R. MAb CP.B8 was also capable of dissociating the c chain alreadycomplexed with IL-2 and IL-2R. SPR analysis confirmed these findings and showed, in addition, thatthe Fab fragment of CP.B8 was also capable of inhibiting the association of the c chain with the IL-2/IL-2R complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediateaffinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the c chainthat is involved in contact with IL-2 and/or IL-2R.