title: 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.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA. vidyalakshmi.chandramohan@duke.edu.|Duke University Medical Center, Box 3156, 181 MSRB-1, 203 Research Drive, Durham, NC, 27710, USA. vidyalakshmi.chandramohan@duke.edu.
authors.forename: Vidyalakshmi
authors.identifier: http://orcid.org/0000-0002-0653-3014
authors.initials: V
authors.lastname: Chandramohan
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA.|Duke University Medical Center, Box 3156, 203 Research Drive, Durham, NC, 27710, USA.
authors.forename: Charles N
authors.initials: CN
authors.lastname: Pegram
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA.|Duke University Medical Center, Box 3156, 181 MSRB-1, 203 Research Drive, Durham, NC, 27710, USA.
authors.forename: Hailan
authors.initials: H
authors.lastname: Piao
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA.|Duke University Medical Center, Box 3156, 163 MSRB-1, 203 Research Drive, Durham, NC, 27710, USA.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Szafranski
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA.|Duke University Medical Center, Box 3156, 203 Research Drive, Durham, NC, 27710, USA.
authors.forename: Chien-Tsun
authors.initials: CT
authors.lastname: Kuan
authors.affiliation: Center for Cancer Research, National Cancer Institute, Building 37, Room 5106, Bethesda, MD, 20892, USA.|Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Ira H
authors.initials: IH
authors.lastname: Pastan
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC, USA.|Duke University Medical Center, Box 3156, 177 MSRB-1, 203 Research Drive, Durham, NC, 27710, USA.
authors.forename: Darell D
authors.initials: DD
authors.lastname: Bigner
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D000328:Adult; D001427:Bacterial Toxins / Q000235:genetics; D045744:Cell Line, Tumor; D017321:Clinical Trials, Phase I as Topic; D017322:Clinical Trials, Phase II as Topic; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics; D005285:Fermentation; D005909:Glioblastoma / Q000188:drug therapy; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D011786:Quality Control; D037521:Virulence Factors / Q000235:genetics; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D066246:ErbB Receptors; D005098:Exotoxins; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C512478:EGFR protein, human
keywords: Epidermal growth factor receptor; Good laboratory practice; Malignant glioma; Mutant EGFR variant III; Recombinant immunotoxin