Fig. 3. Sequence of anti-RAS intracellular scFvs. The nucleotide sequences were obtained and the derived protein translations (shown as the single letter code) were aligned. Dashes in the framework regions (FRs) represent identities with the consensus (Con) sequence (derived from anti-BCR and anti-ABL scFvs isolated by the IAC method; Tse et al., 2002). The numbers indicate the reference positions of the residues, according to the system of Lefranc and Lefranc (2001) (top column number, indicated as IMGT) and Kabat et al. (1991) (second column, Kabat). The 15 residues of the linker, (GGGGS)₃, between the heavy chain domain (VH) and light chain variable domain (VL) are not shown. The complementarity-determining regions (CDRs) are highlighted on a grey background and demarcated from FRs. The three anti-RAS intracellular scFvs are designated 33, J48 and I21. All anti-RAS scFvs belong to the VH3 subgroup of heavy chains and Vκ1 subgroup of light chains shown in the middle (designed VH3 or Vκ1) from the Kabat database (Kabat et al., 1991) or IGHV3 and IGKV1 from the Lefranc database (Lefranc and Lefranc, 2001). The mutated anti-RAS scFvs are shown designated as I21K33, I21R33, I21R33VHI21VL, con33 and I21R33VH (VHC22S;C92S) or (VHC23S;C104S) (Kabat or IMGT nomenclatures, respectively). I21K33 comprises the six CDRs of scFv33 in the I21 framework, and I21R33 is identical except for a mutation Lys94(106)Arg; I21R33VHI21VL comprises the VH domain of I21R33 fused to the VL domain of I21; con33 has all six CDRs of scFv33 in the canonical consensus framework (Tse et al., 2002b); I21R33 (VHC22S;C92S) or (VHC23S;C104S) is a mutant of clone I21R33 with the mutations Cys22(23)Ser and Cys92(104)Ser of the VH domain. There are only four amino acid differences (at positions H1, H5, L0 and L3) between consensus and I21R framework regions. ScFv 33 and J48 belong to IGHV3-48 with IGHJ4 and IGHD1-26 (VH domain), and to IGKV1-39 with IGKJ4 (VL domain) according to the IMGT database (Lefranc and Lefranc, 2001). ScFvI21 belongs to IGHV3-23 with IGHJ4 and IGHD1-8 (VH domain) and IGKV1D-39 with IGKJ1 (VL domain).