Selection of phage-displayed Fab antibodies on the active conformation of Ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effecter s like c-Raf kinase, leading to cell grow th. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecule s that prevent downstream signaling may be of interest. Here, me show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al, (1999) J, Biol, Chem, 274, 18218-18230], a Fab antibody (Fab H2) was identified that exclusively bind s to active Ras, and not to inactive Ras, Using surface plasmon resonance ( SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to t he homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilize d Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC50 value of 135 nM. This value compares well to the KD of active Ras-binding to immobilized Raf -RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, w hich does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping st udies using SPR analysis demonstrate that the binding sites for the antibod ies are non-identical. This antibody could be used for dissecting functiona l differences between Ras effecters. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies , and thus could be used for gene therapy of cancer with intracellular anti bodies. (C) 1999 Federation of European Biochemical Societies.