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 ).

Keywords: Epidermal growth factor receptor; Good laboratory practice; Malignant glioma; Mutant EGFR variant III; Recombinant immunotoxin.

Figures

Fig. 1

D2C7-IT fermentation profile. A representative 8L D2C7-IT fermentation profile is shown. The X-axis represents fermentation time in minutes and the Y-axis represents OD600 values.

Fig. 2

pRB199-D2C7-(scdsFv)-PE38KDEL plasmid map with key features.

Fig. 3

Representative coomassie-stained SDS-PAGE gel of crude D2C7-IT bacterial lysate and purified D2C7-IT inclusion body. aE. coli BLR (λ DE3) cells transformed with pRB199-D2C7-(scdsFv)-PE38KDEL were grown in fermentor until OD600 reached around 6–8 and the D2C7-IT expression was induced with 1 mM IPTG for 2 h. Samples were harvested at the end of the induction period and different volumes (5 and 15μl) of crude bacterial lysate were analyzed in a SDS-PAGE gel. M: molecular weight standard. b 25 μl of the D2C7-IT inclusion body was analyzed in a SDS-PAGE gel during the final TE 50/20 buffer wash. M: molecular weight standard and IB: inclusion body.

Fig. 4

Representative chromatographic profile and coomassie-stained SDS-PAGE gel of D2C7-IT Q-Sepharose fractions. a Chromatographic profile of D2C7-IT eluate from the Q-Sepharose FF column. 30 μl of the D2C7-IT eluate fractions 25-42 from the Q-Sepharose FF column was analyzed in a SDS-PAGE gel. M: molecular weight standard. b 20 μl of the pooled D2C7-IT Q-Sepharose fractions 29-32 was analyzed in a SDS-PAGE gel. M: molecular weight standard and MQ: merged Q-Sepharose fractions 29-32.

Fig. 5

Representative chromatographic profile and coomassie-stained SDS-PAGE gel of D2C7-IT TSK SEC fractions. A chromatographic profile of D2C7-IT eluate from the TSKgel SuperSW3000 column. A D2C7-IT sample was injected into the TSK column and 30-second fractions (2 mL/min, total runtime 60 min) were collected and 30 μl of the D2C7-IT eluate fractions 81-88 from the TSK SEC column was analyzed in a SDS-PAGE gel. M: molecular weight standard.

Fig. 6

Purified bulk D2C7-IT Lot 212041 analytical release testing summary. The purified bulk reference standard, D2C7-IT Lot 212041 was subjected to analytical release testing including product inspection, identity, content, potency, purity, and safety.

Fig. 7

Final vialed D2C7-IT Lot 211131 analytical release testing summary. The final vialed product, D2C7-IT Lot 211131, was subjected to analytical release testing including product inspection, identity, content, potency, purity, and safety.

Fig. 8

Flow cytometric analysis to determine the specificity of D2C7-IT Lot 211131. a Parental NR6 cells were used as control. Indirect flow cytometry analysis demonstrated the specificity of D2C7-IT Lot 211131 against b A431p cells expressing EGFRwt or c NR6M cells transfected with human EGFRvIII. Cells were stained with D2C7-IT Lot 211131 (black open peaks) or a non-specific scFv derived immunotoxin (P588-IT) control (filled grey peaks).

Fig. 9

Forty two month stability data including a protein concentration, b IEF gel, c SDS-PAGE analysis, d HPLC-SEC analysis, and Potency-Cytotoxicity of D2C7-IT Lot 212041 () and D2C7-IT Lot 211131 () against e A431p and f NR6M cells.

Fig. 9

Forty two month stability data including a protein concentration, b IEF gel, c SDS-PAGE analysis, d HPLC-SEC analysis, and Potency-Cytotoxicity of D2C7-IT Lot 212041 () and D2C7-IT Lot 211131 () against e A431p and f NR6M cells.

Fig. 9

Forty two month stability data including a protein concentration, b IEF gel, c SDS-PAGE analysis, d HPLC-SEC analysis, and Potency-Cytotoxicity of D2C7-IT Lot 212041 () and D2C7-IT Lot 211131 () against e A431p and f NR6M cells.

Fig. 9

Forty two month stability data including a protein concentration, b IEF gel, c SDS-PAGE analysis, d HPLC-SEC analysis, and Potency-Cytotoxicity of D2C7-IT Lot 212041 () and D2C7-IT Lot 211131 () against e A431p and f NR6M cells.