High-level expression and purification of the recombinant diphtheria fusion toxin DTGM for PHASE I clinical trials

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. Inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride with dithioerythritol. Recombinant protein was refolded by diluting 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymixin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at -80 degrees 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 lysate endotoxin assay, human AML HL60 cell cytotoxicity assay, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL6 mouse toxicity, and immunohistochemistry. Yields were 23 mg/liter bacterial 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 IC50 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 LD10 in mice was 110 microg/kg/day x5. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 3 months at -80 and -20 degrees C. Further, the drug was stable at 4, 25, and 37 degrees C in human serum for 48 h. Drug reacted only with human monocytes, granulocytes, and myeloid precursors in frozen human tissue sections by immunohistochemistry. The synthesis of this protein drug should be useful for production for clinical 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.