IL2 Fused to Lectin-Deficient Ricin Is Toxic to Human Leukemia Cells Expressing the IL2 Receptor
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Leukemia (1997) 11, 22–30 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 IL2 fused to lectin-deficient ricin is toxic to human leukemia cells expressing the IL2 receptor AE Frankel1,TFu1, C Burbage1, J Chandler2, MC Willingham3 and EP Tagge2 Departments of 1Medicine, 2Surgery and 3Pathology, Medical University of South Carolina, Charleston, SC, USA Interleukin-2 (IL2) fused to ricin B chain (RTB) with modifi- been fused to either IL2 or antibody Fv anti-IL2R peptides.3–10 cations of amino acid residues in each of three galactose-bind- All reagents showed potent selective cytotoxicities in vitro,in ing subdomains (1a,1band 2g) was expressed in insect cells, purified by immunoaffinity chromatography and reassociated some cases, in vivo. with ricin A chain (RTA). The fusion toxin-bound human leu- Ricin-based fusion proteins are attractive candidates for kemic cells with IL2 receptors and the binding was competed development for several reasons. (1) The toxin inactivates cell with IL2 but not asialofetuin. In contrast, binding was not protein synthesis by a mechanism independent of that used observed with receptor negative human cell lines, and the − by DT or PE. The RNA N-glycosidase activity of ricin cripples fusion molecule very weakly bound asialofetuin (K = 10 6M), d 1500 ribosomes/min and a single molecule of ricin in the indicating lectin-deficient RTB. The IL2–lectin-deficient RTB- 11,12 RTA intoxicated IL2 receptor bearing cells as well as ricin or cytosol can cause cell death. Thus, ricin fusion toxins may IL2–wild-type RTB-RTA. While ricin and IL2–wild-type RTB-RTA be used in combination with bacterial fusion toxins or when were equally toxic to receptor negative cell lines, the IL2–lectin- bacterial fusion toxin resistance is encountered. (2) Further- deficient RTB-RTA was two-two and one half logs less cyto- more, there is no immunologic cross-reactivity between ricin toxic to these cell lines. The sensitivity of receptor-positive and the bacterial toxins. Patients who have been immunized cells to the lectin-deficient fusion protein suggests that high avidity intracellular galactose binding may not be required for with diphtheria toxin or had previous exposure to PE do not 13 ricin intoxication, at least in the case of IL2 receptor-targeted show amnestic immune responses to ricin. (3) Finally, there molecules. Furthermore, the potent selective cytotoxicity of the is extensive clinical experience with RTA and blocked ricin fusion protein suggests that the IL2–lectin-deficient RTB-RTA immunotoxins suggesting safety in patients.14 However, con- and similar ricin fusion molecules directed against other leu- struction of ricin fusion toxins has been hampered by the kemic cell surface receptors provide a novel class of fusion toxins for therapy of human leukemias. requirement for a reducible disulfide between RTA and the 15 Keywords: ricin; fusion toxin; IL2 receptor ligand for cell intoxification. Initial efforts to produce IL2– RTA fusions yielded nontoxic molecules. Subsequent efforts to introduce a diphtheria toxin loop peptide or factor Xa Introduction recognition sequence between IL2 and RTA did not yield disulfide-linked molecules and were noncytotoxic to IL2R 16 Many patients with hematopoietic malignancies have incom- bearing cells. plete responses to chemoradiotherapy and die from progress- We chose an alternative strategy for producing biologically ive disease. Patients’ leukemic blasts may develop multiple active ricin fusion molecules. We fused oligohistidine tag or drug resistance phenotypes and normal tissue toxicities may IL2 to RTB and reassociated the fusion with RTA.17,18 Both + limit dose escalation. Novel therapeutic modalities with mini- ligand specificity (Ni2 or IL2R) and heterodimer cytotoxic mal toxicities and no cross-resistance with current cytotoxic potency were maintained. Because we used the holotoxin treatments are needed. in our construction, we needed to identify and modify One such class of drugs are fusion toxins which are hybrid normal tissue binding sites on ricin. Alterations were made proteins composed of peptide ligands reactive with malignant in amino acid residues in RTB subdomains 1a and 2g,but cells (antibody fragments or cytokines) fused to polypeptide persistent sugar and cell binding and cell cytotoxicity were toxins (diphtheria toxin, Pseudomonas exotoxin or ricin). The observed.19–21 When IL2 was fused to double lectin-site toxin–ligand–receptor complex internalizes into intracellular mutant RTB, the reassociated heterodimer displayed reduced compartments from which the catalytic domain of the toxin but continued normal tissue binding and toxicity.22 translocates to the cytosol and inactivates protein synthesis. We recently confirmed a third lectin site in RTB subdomain The target for several leukemia-directed fusion toxins has 1b and reduced its sugar binding affinity by the amino acid been the IL2 receptor (IL2R). IL2R is a heterotrimeric glyco- substitution Tyr-78 to histidine (unpublished data). The triple- protein complex on the cell membrane with a 55 kDa a sub- site mutant had 10- to 20-fold lower galactoside avidity and unit, a 75 kDa b subunit and a 64 kDa g subunit.1 The only showed similar reduction in cell sensitivity to heterodimer. normal human tissues expressing IL2Ra and IL2Rb are acti- Three groups of investigators have chemically or genetically vated T cells, B cells, LGL cells and monocytes and some liver modified lectin sites on ricin and used covalently attached Kupffer cells, lung macrophages and skin Langerhans’ cells. A ligands to study cell intoxication.23–25 In each case, reductions variety of hematologic neoplasms may show high affinity IL2R in lectin function led to profound decreases in cytotoxic expression including hairy cell leukemia, adult T cell leuke- potency. We chose to test the role of intracellular galactose mia, and a fraction of cutaneous T cell lymphomas and B cell binding in ricin intoxication by fusing IL2 to triple-site mutant 2 chronic lymphocytic leukemias. RTB. In this report, we describe the production, chemical Diphtheria toxin (DT) and Pseudomonas exotoxin (PE) have characterization of the fusion molecule and its reassociated heterodimer, and the biological activity of the ricin fusion pro- Corrrespondence: AE Frankel, Hollings Cancer Center Room 311, 86 tein. The results have pertinence both in understanding the Jonathan Lucas Street, Charleston, SC 29425, USA molecular mechanism of ricin cytotoxicity and in the design Received 9 July 1996; accepted 26 September 1996 of ricin-based fusion toxins. IL2–lectin-deficient ricin fusion protein AE Frankel et al 23 Materials and methods sodium azide, and 25 mM lactose (NTEAL), ultracentrifuged at 100 000 g for 1 h, and bound and eluted from a P2 mono- Construction of plasmid clonal antibody–acrylamide matrix as previously described.26 P2 is an anti-RTB monoclonal antibody. The affinity matrix Site-specific mutagenesis was performed on single-stranded was prepared using Ultralink azlactone functionality bis-acryl- pUC119-RTB[W37S/Y248H]DNA using the Sculptor in vitro amide following the recommendations of the manufacturer mutagenesis kit (Amersham, Arlington Heights, IL, USA) as (Pierce, Rockford, IL, USA). Recombinant protein was previously described.19 The aromatic ring residue Tyr-78 in absorbed to the column in NTEAL, washed with 0.5 M NaCl, the 1b subdomain was changed to histidine to reduce van der 25 mM Tris pH 9, 1 mM EDTA, 0.1% Tween 20, 0.02% Waals interactions between the protein and galactosides. The sodium azide, 25 mM lactose and eluted with 0.1 M triethyl- BamHI–EcoRI mutant RTB encoding DNA fragment was sub- amine hydrochloride pH 11. The eluant was neutralized with cloned into pAcGP67A plasmid (PharMingen, San Diego, CA, 1/10 volume 1 M sodium phosphate pH 4.25 and stored at USA) and used to transform INVaF′ E. coli cells (InVitrogen, −20°C until assayed. Three preparations were made. San Diego, CA, USA). Transfer vector with mutant RTB was then purified by cesium chloride density centrifugation, restricted with BamHI, bound and eluted from silica matrix Characterization of recombinant protein (Promega, Madison, WI, USA), digested with calf intestinal phosphatase (Boehringer-Mannheim, Indianapolis, IN, USA), Total protein concentration of the affinity column eluant was heat inactivated and repurified on silica matrix. Thre BamHI measured by absorbance at 280 nm. Since the optical den- fragment encoding IL2 prepared by polymerase chain reaction sities of a 1 mg/ml solution of RTB and IL2 were 1.4 and 0.7, of pDW27 plasmid DNA as previously described18,22 was iso- respectively, a 1 mg/ml solution of fusion protein should have lated from pUC119-IL2 by digestion of cesium chloride den- a mass average optical density of 1.16. Protein was also quan- sity gradient purified plasmid with BamHI, agarose electro- titated by BioRad (Hercules, CA, USA) protein assay as per phoresis and binding and elution from silica matrix. The 406- recommendations of the supplier. Aliquots of ADP-IL2-ADP- bp fragment was subcloned into pAcGP67A-ADP-RTB[W37S/ RTB[W37S/Y248H/Y78H], plant RTB and prestained low mol- Y248H/Y78H]. The expression vector was maintained in ecular weight standards were run on a reducing 15% SDS- INVaF′ E. coli using 100 mg/ml ampicillin. Plasmid isolated PAGE, stained with Coomassie Blue R-250 and scanned on by alkaline lysis followed by cesium chloride density gradient an IBAS automatic image analysis system (Kontron, Germany). centrifugation was double-stranded dideoxy