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

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 ).

• Authors: Vidyalakshmi Chandramohan, Charles N Pegram, Hailan Piao, Scott E Szafranski, Chien-Tsun Kuan, Ira H Pastan, Darell D Bigner
• Journal: Applied microbiology and biotechnology (Appl Microbiol Biotechnol) Vol. 101 Issue 7 Pg. 2747-2766 (Apr 2017) ISSN: 1432-0614 [Electronic] Germany
• PMID: 28013405 (Publication Type: Journal Article)

Chemical References

• Bacterial Toxins
• Exotoxins
• Immunotoxins
• Virulence Factors
• ADP Ribose Transferases
• Pseudomonas aeruginosa exotoxin A
• EGFR protein, human
• ErbB Receptors

Topics

• ADP Ribose Transferases (genetics)
• Adult
• Bacterial Toxins (genetics)
• Cell Line, Tumor
• Clinical Trials, Phase I as Topic
• Clinical Trials, Phase II as Topic
• Drug Stability
• Electrophoresis, Polyacrylamide Gel
• ErbB Receptors (genetics, metabolism)
• Escherichia coli (genetics)
• Exotoxins (genetics)
• Fermentation
• Glioblastoma (drug therapy)
• Humans
• Immunotoxins (chemistry, genetics, metabolism, therapeutic use)
• Quality Control
• Virulence Factors (genetics)