title: Construction and in vitro evaluation of RFT5(scFv)-ETA', a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines.
abstract: The data of a closed phase I/II trial in patients with resistant Hodgkin's lymphoma indicate promising results using a chemically linked anti-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on the high-affinity moab RFT5. Since recombinant DNA technology permits the readier production of large amounts of ITs, we constructed a new RFT5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hybridoma cell line RFT5, synthesized first strand cDNA and performed RT-PCR. Amplified coding regions of the light and heavy chain variable domains were joined together with a synthetic (Gly4-Ser)3 linker. The resulting single chain variable fragment (scFv) was fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coli, the 70 kDa His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxin was purified on a Ni2+-NTA chelating sepharose and eluted with 250 mM imidazole. Pooled protein was renatured, dialyzed and concentrated by precipitation. Binding properties of RFT5(scFv)-ETA' were assessed on the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FACS analysis. CD25-specific binding was confirmed by immunoprecipitation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-derived cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Burkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosynthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by competitive toxicity assays. These data confirm for the first time binding specificity and toxicity of a recombinant anti-CD25 immunotoxin, against Hodgkin-derived cell lines; its applicability on Hodgkin's lymphoma needs yet to be evaluated in vivo.
authors.affiliation: Medizinische Klinik I der Universitat zu Koln, Labor fur Immunotherapie, D-50924 Koln, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Barth
authors.forename: M
authors.initials: M
authors.lastname: Huhn
authors.forename: W
authors.initials: W
authors.lastname: Wels
authors.forename: V
authors.initials: V
authors.lastname: Diehl
authors.forename: A
authors.initials: A
authors.lastname: Engert
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology*; D001427:Bacterial Toxins; D001483:Base Sequence; D005069:Evaluation Studies as Topic; D005098:Exotoxins / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000494:pharmacology*; D000922:Immunotoxins / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D057127:Single-Chain Antibodies; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C117703:RFT5(scFv)-ETA'