Abstract
D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12 ± 0.1 mg/mL, the pH was at 7.4 ± 0.4, and endotoxin levels were below the detection limit of 10 EU/mL (1.26 EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42 months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72 h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov).
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Acknowledgements
We thank Jenna Lewis for the editorial assistance of the manuscript. The study was funded by the following grant from the National Institutes of Health (NIH) of the USA: P01-CA154291-03 (to D. D. Bigner). This research was also supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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Author 1 declares that she has no conflict of interest. Author 2 declares that he has no conflict of interest. Author 3 declares that she has no conflict of interest. Author 4 declares that he has no conflict of interest. Author 5 declares that he has no conflict of interest. Author 6 is inventor on immunotoxin patents, all of which have been assigned to NIH. Author 7 owns stock in Istari Oncology and is a consultant to Genetron Health.
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Chandramohan, V., Pegram, C.N., Piao, H. et al. Production and quality control assessment of a GLP-grade immunotoxin, D2C7-(scdsFv)-PE38KDEL, for a phase I/II clinical trial. Appl Microbiol Biotechnol 101, 2747–2766 (2017). https://doi.org/10.1007/s00253-016-8063-x
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DOI: https://doi.org/10.1007/s00253-016-8063-x