An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFV)-ETA ') is a potent immunotoxin against a Hodgkin-derived cell line

The human CD30 receptor is highly overexpressed on the surface of Hodgkin R eed-Sternberg cells and has been shown to be an excellent target for select ive immunotherapy using monoclonal antibody-based agents such as immunotoxi ns. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybr idoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembl ed, cloned and expressed as a mini-library for display on filamentous phage . Functional Ki-4 scFv were obtained by selection of binding phage on the H odgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected rec ombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pse udomonas exotoxin A (ETA'). The resulting immunotoxin Ki-4(scFv)-ETA' speci fically binds to CD30(+) L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgki n's lymphoma or other CD30(+) malignancies.