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We have applied in vivo intracellular antibody capture (IAC) technology to isolate human intrabodies which bind to the oncogenic RAS protein. IAC facilitates the capture of antibody fragments, in this case single-chain Fvs (scFvs), which tolerate reducing environments, such as the cytoplasm of cancer cells. Three anti-RAS scFvs with different affinity, solubility and intracellular binding activity were characterized. The anti-RAS scFvs with highest affinity were expressed relatively poorly in mammalian cells, and greater soluble expression was achieved by mutating the antibody framework to canonical consensus scaffolds, previously derived from IAC, without losing antigen specificity. Mutagenesis experiments showed that the consensus scaffolds are functional as intrabody fragments without an intra-domain disulfide bond. Furthermore, we could convert an intrabody which does not bind RAS in mammalian cells into a high-affinity reagent capable of inhibiting RAS-mediated NIH 3T3 transformation by exchanging VH and VL complementarity-determining regions onto its consensus scaffold. These data show that the consensus scaffold is a robust framework by which to improve intrabody function.

Original publication

DOI

10.1093/emboj/cdg106

Type

Journal article

Journal

EMBO J

Publication Date

03/03/2003

Volume

22

Pages

1025 - 1035

Keywords

3T3 Cells, Amino Acid Sequence, Animals, Antibody Affinity, Antibody Specificity, COS Cells, Cell Transformation, Neoplastic, Cysteine, Humans, Immunoglobulin Variable Region, Immunosorbent Techniques, Mice, Molecular Sequence Data, Protein Binding, Sequence Alignment, Surface Plasmon Resonance, ras Proteins