Cancer Research 64, , , . doi:
© 2009 American Association for Cancer Research
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Fig. 1. Immunotoxin (IT) construction and composition. A, construction of recombinant single-chain Fv (scFv) and disulfide-linked Fv (dsFv) ITs. DNA encoding the 8H9 Fv in a single-chain form has been described previously (12)
. To make psc8H9, which is the expression vector for sc8H9(Fv)-PE38, a VH 5' primer introduced a NdeI restriction site, and a VL 3' primer introduced a HindIII restriction site to facilitate cloning of the scFv into the expression vector by PCR. Construction of the dsFv involves the generation of two expression plasmids (p8H9-VH-PE38 and p8H9-VL) that encode the two components of the dsFv: VH-44cys and VL-100cys. The cysteines are introduced into position 44 in framework region 2 of VH and position 100 of FR4 of VL (Kabat number) by PCR using primers containing the restriction sites. In addition to the cysteine, cloning sites, ATG translation initiation codons, and stop codons are introduced at the 5' end and 3' end of the VH and VL genes by PCR. The VH-44cys gene is subcloned into an expression vector that contains the gene for a truncated form of Pseudomonas exotoxin, PE38. The expression vector is controlled by the T7 promoter. On induction of the T7 RNA polymerase by isopropyl-1-thio-ß-D-galactopyranoside, which is under control of the lacUV5 promoter in Escherichia coli BL21 
DE3, large amounts of recombinant protein are produced. B, composition of recombinant IT with PE38. The Fv region of the recombinant IT is fused to the NH2 terminus of PE38. In the scFv IT, the scFv fragment is stabilized by a peptide linker [(G4S)3] that connects the COOH terminus of VH with the NH2 terminus of the VL domain. In the dsFv IT, there are two components. The VH domain is fused to the NH2 terminus of PE38, and the VL domain is covalently linked to VH by a disulfide bond.
