Bloconlugate Chem. 1993, 4, 581-585
581
Purification and Characterization of IL6-PE4E,a Recombinant Fusion of Interleukin 6 with Pseudomonas Exotoxin Robert J. Kreitman and Ira Pastan' Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 4E16, Bethesda, Maryland 20892. Received June 11,1993'
We have developed a procedure to purify the recombinant fusion toxin IL6-PE4E from Escherichia coli which results in a high yield of fully active monomeric protein of high purity and very low endotoxin content. The chimeric toxin is composed of human interleukin 6 (IL6) fused to a derivative of Pseudomonas exotoxin (PE) containing mutations in the binding domain which prevent binding to the PE receptor. In a typical preparation, 20 g of E. coli cells expressing the plasmid encoding IL6-PE4E were treated with lysozyme and washed repeatedly with detergent (Triton X-loo), to obtain 500 mg of inclusion bodies. The recombinant protein was denatured and reduced in guanidine hydrochloride solution containing dithioerythritol and refolded in a redox buffer containing oxidized glutathione and L-arginine. After purification of the dialyzed protein by anion-exchange, polymyxin B, and sizing chromatography, we obtained 100 mg (20% of recombinant protein) of purified monomer with 0.6-2.5 endotoxin units/mg of protein. Amino terminal sequencing confirmed the first 20 amino acids. IL6PE4Epurified in this manner was fully cytotoxic toward human multiple myeloma,hepatoma, epidermoid carcinoma, and prostate carcinoma cell lines. After intravenous injection into mice, we found the dose-limiting toxicity to be to the liver, by measurement of serum transaminases and histologic evaluation of the liver. The LD50 was 450 pg/kg. We conclude that IL6-PE4E can be purified efficiently for preclinical testing.
INTRODUCTION Interleukin 6 (IL6) is a multifunctional cytokine important for the differentiation of human B cells, but is also involved in several disease states, such as rheumatoid arthritis, human T cell leukemiavirus (HTLV-1)and HIV infection, and multiple myeloma ( I ) . IL6 receptors (IL6R's) have been found not only on myeloma cells (2) but on hepatocellular carcinoma cells as well (3). While hepatoma is an important cause of cancer throughout the world, prostate cancer incidence is increasing in the United States, and such cells also express IL6R ( 4 ) . Moreover, in a recent study 11of 13malignant prostates from patients contained high levels of IL6R mRNA (5). In 1989, it was shown that IL6R-bearing cells could be targeted using a recombinant fusion of IL6 with a derivative of Pseudomonus exotoxin (PE) (6). PE is a 613 amino acid protein which kills normal animal cells by binding to the PE receptor, internalizing, translocating to the cytosol, and ADP-ribosylating elongation factor 2. Of the three structural domains, domain Ia (amino acids 1-252) binds to the PE receptor, domain I1 (amino acids 253-364) mediates translocation of the C-terminal portion of the toxin (amino acids 280-613) to the cytosol, and domain I11(amino acids 400-613) contains the ADP-ribosylating activity (7, 8). In our most effective IL6-toxin, IL6-PE664G1U (IL6PE4E), human IL6 is fused to the amino terminus of full length PE (9),but in domain Ia of PE, four basic residues (Lys57, His246, Arg247, and His249) have been replaced with Glu to prevent the toxin from binding to the PE receptor. IL6-PE4E was selectively cytotoxic toward four groups of malignant cell lines: multiple myeloma, hepatoma, prostate carcinoma, and epidermoid carcinoma (3,4,
* Author to whom correspondence should be addressed. @
Abstract published in Advance ACS Abstracts, November
1, 1993.
9). IL6-PE4E caused regression of PLC/PRF/5 human hepatoma cells growing as xenografts in nude mice (IO). To assess its potential efficacy as a purging agent for autologous bone marrow transplantation in multiple myeloma, we tested fresh bone marrow specimens and over half of the patients had cells which were killed by the toxin (11). In contrast, normal hematopoietic precursor cells (BFU-E and CFU-GM) showed no sensitivity. For our previous tissue culture and murine studies, unwashed inclusion bodies of IL6-PE4E were dissolved in guanidine solution, diluted into PBS and purified by anion exchange and sizing chromatography (9). A typical yield was 1.5-7 mg of purified monomer from 5 g of cells producing -100 mg of total recombinant protein (1.57 % yield) (Siegall, Kreitman, and Pastan, unpublished data). For primate toxicity studies and clinical trials, we needed a more efficient protocol which would afford a higher yield and provide clinical-grade material with low endotoxin content. Currently, humans and other primates treated with recombinant protein should receive
