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 SDS-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 immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296EU/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 degrees C in the plastic syringe and administration tubing for 48 h.
Cancer Research Institute, Scott & White Memorial Hospital, Temple, TX, USA. ysu@swmail.sw.org
Yunpeng
Y
Su
Shi-Yan
SY
Li
Sunil
S
Ghosh
Janelle
J
Ortiz
Donna E
DE
Hogge
Arthur E
AE
Frankel
D019943:Amino Acid Substitution; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D002478:Cells, Cultured; D017321:Clinical Trials, Phase I as Topic / Q000379:methods*; D004167:Diphtheria Toxin / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004339:Drug Compounding / Q000379:methods; D004340:Drug Contamination / Q000517:prevention & control; D004353:Drug Evaluation, Preclinical; D004355:Drug Stability; D014644:Genetic Variation / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000473:pathology; D008239:Lysine / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D013242:Sterilization; D018675:Toxicity Tests; D014364:Tryptophan / Q000235:genetics
D004167:Diphtheria Toxin; D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D008239:Lysine