Yoram Reiter

Construction of a functional disulfide-stabilized TCR Fv indicates that antibody and tcr fv frameworks are very similar in structure

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions. We now design and produce a disulfide-stabilized Fv of a T cell receptor. It is composed of V alpha and V beta variable domains of the 2B4 TCR stabilized by a disulfide bond between framework residues of the TCR Fv at a site corresponding to that used for disulfide stabilization of antibody Fvs. For ease of production and detection, the TCRdsFv was fused to a truncated form of Pseudomonas exotoxin (PE38). The TCR(dsFv) retains its native conformation and is much more stable than a TCR scFv. Moreover, it is functional in biological assays. Because successful disulfide stabilization of the TCR Fv by the positions used for antibody Fv stabilization would not occur unless the mutated residues in TCR Fv are at positions closely similar to those in antibody Fvs, most likely within less than 1.5 A, these results provide very strong experimental evidence for the structural similarity between immunoglobulin and TCR antigen-binding variable domains.

Recombinant single-chain and disulfide-stabilized Fv-immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice.

Monoclonal antibody (MAb) 55.1 specifically recognizes an antigen on the surface of human colon adenocarcinoma cells. We constructed recombinant immunotoxins composed of the heavy‐ and light‐chain variable regions of MAb 55.1 fused to a recombinant form of Pseudomonas exotoxin (PE). The heavy‐ and light‐chain variable regions are stabilized by 2 means. One is by a flexible peptide linker to form a single‐chain antigen binding protein (scFv) and the second by an interchain disulfide bond engineered between structurally conserved framework regions. These are termed disulfide stabilized Fvs (dsFv). The 2 Fv forms are fused to truncated forms of PE lacking the cell binding domain. The recombinant scFv‐ and dsFv‐immunotoxins were expressed in E. coli and purified to near homogeneity. The scFv‐ and dsFv‐immunotoxins were shown to be specifically cytotoxic to human colon adenocarcinoma cell lines. The scFv‐immunotoxin containing PE38KDEL was more active than the immunotoxin containing PE38 with the native carboxyl terminus (REDLK). However, the PE38KDEL immunotoxin is about 2‐fold more toxic in mice, and therefore it does not appreciably increase the therapeutic window in mice. Intravenous administration of the scFv‐ and dsFv‐ recombinant immunotoxins caused complete regression of a human colon carcinoma (Colo205) growing subcutaneously in immunodeficient mice. The dsFv‐immunotoxin has better antitumor activity compared with its scFv‐immunotoxin counterpart.