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: S Barth, M Huhn, W Wels, V Diehl, A Engert
• Journal: International journal of molecular medicine (Int J Mol Med) Vol. 1 Issue 1 Pg. 249-56 (Jan 1998) ISSN: 1107-3756 [Print] Greece
• PMID: 9852227 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Bacterial Toxins
• Exotoxins
• Immunoglobulin Variable Region
• Immunotoxins
• RFT5(scFv)-ETA'
• Single-Chain Antibodies
• Virulence Factors
• ADP Ribose Transferases
• Pseudomonas aeruginosa exotoxin A

Topics

• ADP Ribose Transferases
• Amino Acid Sequence
• Antibodies, Monoclonal (genetics, pharmacokinetics, pharmacology)
• Bacterial Toxins
• Base Sequence
• Evaluation Studies as Topic
• Exotoxins (genetics, pharmacology)
• Gene Expression
• Humans
• Immunoglobulin Variable Region (genetics, pharmacology)
• Immunotoxins (genetics, pharmacology)
• Molecular Sequence Data
• Single-Chain Antibodies
• Tumor Cells, Cultured
• U937 Cells
• Virulence Factors