Department of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Neurosurg. 2011 Jun;114(6):1662-71. doi: 10.3171/2010.11.JNS101214. Epub 2011 Feb 4.
The authors of this study aimed to genetically design a bispecific targeted toxin that would simultaneously target overexpressed markers on glioma as well as the tumor vasculature, to mutate certain amino acids to reduce the immunogenicity of this new drug, and to determine whether the drug was able to effectively reduce aggressive human brain tumors in a rat xenograft model via a novel hollow fiber (HF) catheter delivery system.
A new bispecific ligand-directed toxin (BLT) was created in which 2 human cytokines-epidermal growth factor ([EGF], targeting overexpressed EGF receptor) and amino acid terminal fragment ([ATF], targeting urokinase plasminogen activator receptor)-were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin with a terminal lysyl-aspartyl-glutamyl-leucine (KDEL) sequence. Site-specific mutagenesis was used to mutate amino acids in 7 key epitopic toxin regions that dictate the B cell generation of neutralizing antitoxin antibodies to deimmunize the drug, now called "EGFATFKDEL 7mut." Bioassays were used to determine whether mutation reduced the drug's potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were decreased. Aggressive brain tumors were intracranially established in nude rats by using human U87 glioma genetically marked with a firefly luciferase reporter gene (U87-luc), and the rats were stereotactically treated with 2 intracranial injections of deimmunized EGFATFKDEL via convection-enhanced delivery (CED). Drug was administered through a novel HF catheter to reduce drug backflow upon delivery.
In vitro, EGFATFKDEL 7mut selectively killed the human glioblastoma cell line U87-luc as well as cultured human endothelial cells in the form of the human umbilical vein endothelial cells. Deimmunization did not reduce drug activity. In vivo, when rats with brain tumors were intracranially treated with drug via CED and a novel HF catheter to reduce backflow, there were significant tumor reductions in 2 experiments (p < 0.01). Some rats survived with a tumor-free status until 130 days post-tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The maximal tolerated dose of EGFATFKDEL 7mut was established at 2 μg/injection or 8.0 μg/kg, and data indicated that this dose was nontoxic. Antitoxin antibodies were reduced by at least 90%.
First, data indicated that the BLT framework is effective for simultaneously targeting glioma and its neovasculature. Second, in the rodent CED studies, newly developed HF catheters that limit backflow are effective for drug delivery. Third, by mutating critical amino acids, the authors reduced the threat of the interference of neutralizing antibodies that are generated against the drug. The authors' experiments addressed some of the most urgent limitations in the targeted toxin field.
本研究的作者旨在通过基因工程设计一种双特异性靶向毒素，该毒素能同时靶向神经胶质瘤过度表达的标志物和肿瘤血管，通过突变某些氨基酸来降低这种新药的免疫原性，并通过新型中空纤维（HF）导管输送系统，确定该药物是否能有效减少大鼠异种移植模型中侵袭性人脑肿瘤。
作者构建了一种新的双特异性配体导向毒素（BLT），其中 2 个人类细胞因子 - 表皮生长因子（EGF，靶向过度表达的表皮生长因子受体）和氨基酸末端片段（ATF，靶向尿激酶纤溶酶原激活物受体） - 被克隆到同一个带有末端赖氨酰-天冬氨酰-谷氨酸-亮氨酸（KDEL）序列的截断假单胞菌外毒素的单链分子上。使用定点突变技术突变决定 B 细胞产生中和抗毒素抗体的 7 个关键表位毒素区域的氨基酸，使药物去免疫化，现称为“EGFATFKDEL 7mut”。生物测定用于确定突变是否降低了药物的效力，酶联免疫吸附测定研究用于确定是否减少了抗毒素抗体。通过使用基因标记有萤火虫荧光素酶报告基因的人 U87 神经胶质瘤（U87-luc），在裸鼠中建立侵袭性脑肿瘤，通过对流增强输送（CED）对大鼠进行 2 次颅内免疫原性 EGFATFKDEL 注射治疗。通过新型 HF 导管给药以减少给药后的药物回流。
在体外，EGFATFKDEL 7mut 选择性地杀死了人神经母细胞瘤细胞系 U87-luc 以及人脐静脉内皮细胞形式的培养人内皮细胞。去免疫化并没有降低药物的活性。在体内，当颅内用 CED 和新型 HF 导管给药治疗携带脑肿瘤的大鼠以减少回流时，在 2 项实验中均观察到肿瘤显著缩小（p<0.01）。一些大鼠在肿瘤接种后 130 天仍保持无肿瘤状态。无关的 BLT 对照不能保护建立特异性。EGFATFKDEL 7mut 的最大耐受剂量确定为 2μg/注射或 8.0μg/kg，数据表明该剂量无毒性。抗毒素抗体减少至少 90%。
首先，数据表明 BLT 框架可有效同时靶向神经胶质瘤及其新生血管。其次，在啮齿动物 CED 研究中，新开发的限制回流的新型 HF 导管可有效输送药物。第三，通过突变关键氨基酸，作者降低了针对药物产生的中和抗体的干扰威胁。作者的实验解决了靶向毒素领域最紧迫的一些限制。
