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Applied Microbiology and Biotechnology

, Volume 101, Issue 7, pp 2747–2766 | Cite as

Production and quality control assessment of a GLP-grade immunotoxin, D2C7-(scdsFv)-PE38KDEL, for a phase I/II clinical trial

  • Vidyalakshmi ChandramohanEmail author
  • Charles N. Pegram
  • Hailan Piao
  • Scott E. Szafranski
  • Chien-Tsun Kuan
  • Ira H. Pastan
  • Darell D. Bigner
Biotechnological products and process engineering

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).

Keywords

Good laboratory practice Recombinant immunotoxin Malignant glioma Epidermal growth factor receptor Mutant EGFR variant III 

Notes

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.

Compliance with ethical standards

Conflict of interest

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.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

For this type of study, formal consent is not required.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Preston Robert Tisch Brain Tumor Center at Duke and Department of PathologyDuke University Medical CenterDurhamUSA
  2. 2.Duke University Medical CenterDurhamUSA
  3. 3.Duke University Medical CenterDurhamUSA
  4. 4.Duke University Medical CenterDurhamUSA
  5. 5.Center for Cancer ResearchNational Cancer InstituteBethesdaUSA
  6. 6.Laboratory of Molecular Biology, Center for Cancer Research, National Cancer InstituteNational Institutes of HealthBethesdaUSA
  7. 7.Duke University Medical CenterDurhamUSA

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