High-Level Expression and Purification of the Recombinant Diphtheria Fusion Toxin DTGM for PHASE I Clinical Trials Arthur E. Frankel,* ,1 Jason Ramage,* Amy Latimer,* Theodore Feely,* Stephen Delatte,† Philip Hall,‡ Edward Tagge,† Robert Kreitman,§ and Mark Willingham ¶ *Department of Cancer Biology and ¶ Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; ‡Department of Pharmacy and †Department of Surgery, Medical University of South Carolina, Charleston, South Carolina 29425; and §Laboratory of Molecular Biology, NCI/NIH, Bethesda, Maryland 20892 Received January 12, 1999, and in revised form February 24, 1999 A genetically engineered fusion toxin targeted to acute myeloid leukemic (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human granulocyte– macrophage colony-stimulating factor. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BL21 (DE3) Escherichia coli harboring pUBS500 plasmid. Transformants were grown in Superbroth and induced with IPTG. Inclu- sion bodies were isolated, washed, and denatured in guanidine hydrochloride with dithioerythritol. Re- combinant protein was refolded by diluting 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after an- ion-exchange, size exclusion on FPLC, and polymixin B affinity chromatography. The final material was fil- ter sterilized, aseptically vialed, and stored at 80°C. Fifty-four 3-liter bacterial culture preparations were made and pooled into 27 batches. The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS–PAGE, limulus amebocyte ly- sate endotoxin assay, human AML HL60 cell cytotox- icity assay, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL6 mouse toxicity, and immunohistochemistry. Yields were 23 mg/liter bacte- rial culture of denatured fusion toxin. After refolding and chromatography, final yields were 24  4% or 5 mg/liter. Vialed product was sterile and 1.7  0.4 mg/ml in PBS. Purity by SDS–PAGE was 99  1%. Aggregates by HPLC were < 1%. Potency revealed a 24-h IC 50 of 2.7  0.5 pM on HL60 cells. Endotoxin levels were 1 eu/mg. The N-terminal sequence was confirmed, and E. coli DNA was < 113 pg/mg. The LD 10 in mice was 110 g/kg/day 5. There was no evidence of loss of solubil- ity, proteolysis, aggregation, or loss of potency over 3 months at 80 and 20°C. Further, the drug was stable at 4, 25, and 37°C in human serum for 48 h. Drug reacted only with human monocytes, granulocytes, and myeloid precursors in frozen human tissue sec- tions by immunohistochemistry. The synthesis of this protein drug should be useful for production for clin- ical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development. This is the first report of the scaleup of a recombinant fusion toxin for clinical trials. © 1999 Academic Press Acute myeloid leukemia (AML) is the most common leukemia in adults and the second most common leu- kemia in children (1). With 10,000 estimated cases/ year in the United States and the prolonged hospital- izations associated with treatment and complications, the disease represents a significant share of health care costs. Types of drugs that have shown significant activity in AML include cytosine arabinoside and the topoisomerase-II inhibitors—anthracyclines, amsa- crine, and etoposide. With combination induction and consolidation chemotherapy, complete remission rates of about 70% have been achieved (2). However, most patients ultimately relapse and die from the disease or complications of treatment. Even with allogeneic bone marrow transplantation (3), modulators of drug efflux transporters such as quinine and cyclosporine (4), and several different antileukemic cytotoxic drugs, includ- ing topoisomerase-I inhibitors, nucleoside analogs, and hypomethylating agents (5), the prognosis remains dis- mal for relapsed or refractory AML patients. Median survival is measured in weeks to months. Radiochemo- 1 To whom correspondence should be addressed at Hanes 4046, Wake Forest University School of Medicine, Medical Center Boule- vard, Winston-Salem, NC 27157. Fax: 336-716-0255. E-mail: afrankel@wfubmc.edu. Protein Expression and Purification 16, 190 –201 (1999) Article ID prep.1999.1071, available online at http://www.idealibrary.com on 190 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.