In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody.
The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable "kemptide" (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of 32P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate.
Patrick
M R
MR
Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.
Chester
K A
KA
Pietersz
G A
GA
D001665:Binding Sites; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000922:Immunotoxins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D010761:Phosphorus Radioisotopes / Q000737:chemistry*; D010766:Phosphorylation; D016499:Radioimmunotherapy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014407:Tumor Cells, Cultured
D002272:Carcinoembryonic Antigen; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D009842:Oligopeptides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins