Cloning, expression and characterization of the Fv fragments of the anti-carbohydrate mAbs Bl and B5 as single-chain immunotoxins

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Abstract
The mAbs Bl (IgGlK) and B5 (IgMK) recognize carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were separately cloned from hybridoma RNA using reverse transcription and PCR with oligonucleo-tide primers designed according to the amino acid sequences of the N-termini. The Fv regions also provide sequences for translation initiation in Escherichia coli (Frl oligos) and sequences of the constant region of the heavy and light domains (CHI or C-k oligos). Following the determination of the DNA sequence of the Fvs, primers were designed according to the 3' ends of the VH and VL domains. These also provided for a peptide linker at the C-terminus of the VH and a short connector at the C-terminus of the VL (Fr4 oligos). The VH and VL were then each PCR-amplified using their corresponding Frl and phosphorylated Fr4 oligos. The resulting PCR products were annealed as 'mutagenic primers' to a uracil-containing single-stranded template obtained from an expression plasmid encoding a single-chain immunotoxin in which the B3 single-chain Fv is fused to a truncated form of Pseudomonas exotoxin (PE). Thus, the Bl and B5 variable domains replaced their corresponding B3 domains in the expression plasmid by ‘variable domain shuffling’ without subcloning. The resulting Bl(Fv)-PE38 and B5(Fv)-PE38 were expressed in E.coli and purified to near homogeneity. Both show specific cytotoxidtles to human carcinoma cell lines, but Bl(Fv)-PE38 is much more active, reflecting its higher affinity to the target cells.

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