CONSTRUCTION AND IN-VITRO EVALUATION OF RFT5(SCFV)-ETA', A NEW RECOMBINANT SINGLE-CHAIN IMMUNOTOXIN WITH SPECIFIC CYTOTOXICITY TOWARD CD25(-DERIVED CELL-LINES() HODGKIN)

The data of a closed phase I/II trial in patients with resistant Hodgk in's lymphoma indicate promising results using a chemically linked ant i-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on t he high-affinity moab RFT5. Since recombinant DNA technology permits t he readier production of large amounts of ITs, we constructed a new RF T5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hyb ridoma cell line RFT5, synthesized first strand cDNA and performed RT- PCR. Amplified coding regions of the light and heavy chain variable do mains were joined together with a synthetic (Gly(4)-Ser)(3) linker. Th e resulting single chain variable fragment (scFv) was fused to a modif ied Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coil, the 70 kD a His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxi n was purified on a Ni2+-NTA chelating sepharose and eluted with 250 m M imidazole. Pooled protein was renatured, dialyzed and concentrated b y precipitation. Binding properties of RFT5(scFv)-ETA' were assessed o n the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FAGS analysis. CD25-specific binding was confirmed by immunoprecip itation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-deri ved cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Bu rkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosy nthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by c ompetitive toxicity assays. These data confirm for the first time bind ing specificity and toxicity of a recombinant anti-CD25 immunotoxin, a gainst Hodgkin-derived cell lines; its applicability on Hodgkin's lymp homa needs yet to be evaluated in vivo.