Chandramohan Vidyalakshmi, Pegram Charles N, Piao Hailan, Szafranski Scott E, Kuan Chien-Tsun, Pastan Ira H, Bigner Darell D
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.
Appl Microbiol Biotechnol. 2017 Apr;101(7):2747-2766. doi: 10.1007/s00253-016-8063-x. Epub 2016 Dec 24.
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 ).
D2C7-(scdsFv)-PE38KDEL（D2C7-IT）是一种新型的基于重组绿脓杆菌外毒素A的免疫毒素（IT），靶向在胶质母细胞瘤中过表达的野生型表皮生长因子受体（EGFRwt）和突变型EGFR变体III（EGFRvIII）蛋白。初步临床前试验证明了D2C7-IT对表达EGFRwt、EGFRvIII或同时表达EGFRwt和EGFRvIII的原位胶质母细胞瘤异种移植模型具有抗肿瘤疗效。已开发出一种良好实验室规范（GLP）生产工艺，以生产足够的材料用于I/II期临床试验。D2C7-IT在大肠杆菌BLR（λ DE3）中T7启动子的控制下表达。D2C7-IT通过10升分批发酵工艺生产，然后从包涵体中通过阴离子交换、尺寸排阻和内毒素去除工艺进行纯化，获得了超过300毫克的纯化蛋白产量。用于临床测试的最终瓶装批次的D2C7-IT浓度为0.12±0.1毫克/毫升，pH值为7.4±0.4，内毒素水平低于10 EU/毫升的检测限（1.26 EU/毫升）。通过蛋白质浓度、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、等电聚焦、尺寸排阻色谱、细胞毒性、无菌性和pH值测量，对瓶装D2C7-IT的稳定性进行了42个月的监测。目前，瓶装D2C7-IT正在通过瘤内对流增强给药对复发性胶质母细胞瘤患者进行72小时的I/II期临床试验（NCT02303678，D2C7用于复发性恶性胶质瘤成年患者；clinicaltrials.gov）。
