Characterization Of Variant Diphtheria Toxin-Interleukin-3 Fusion Protein, Dtil3k116w, For Phase I Clinical Trials

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin interleukin-3 fusion protein, for treatment of acute myeloid leukemia The product was filter sterilized, aseptically vialed, and stored at -80 degrees C. It was characterized by Coomassie-stained SIDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy. receptor binding affinity. ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography HPLC, sequencing, and immunolustochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 79, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99% Aggregates by HPLC were <1% Endotoxin levels were 0.296 EU/mg Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies Potency study revealed a 48-h EC(50) of 0.5 pM on TF1/H-ras cell Its binding properties were confirmed by competitive experiments showing IC(50) of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 degrees C. Further, the drug was stable at 4 and 25 C in the plastic syringe and administration tubing for 48 h. (C) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.