Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Clin Cancer Res. 2013 Sep 1;19(17):4717-27. doi: 10.1158/1078-0432.CCR-12-3891. Epub 2013 Jul 15.
The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.
The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.
The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6×10(-9) mol/L and 1.3×10(-9) mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P=0.006), 28% (P=0.002), and 166% (P=0.001), respectively.
In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both.
表皮生长因子受体（EGFR）基因在胶质母细胞瘤中最常被扩增和过表达，同时还有其缺失突变体 EGFRvIII。我们测试了重组免疫毒素 D2C7-(scdsFv)-PE38KDEL 的临床前疗效，该毒素与 EGFRwt（583-637 个氨基酸）和 EGFRvIII（292-346 个氨基酸）蛋白的细胞外结构域中存在的 55 个氨基酸（AA）区域反应。
通过表面等离子体共振和流式细胞术测量 D2C7-(scdsFv)-PE38KDEL 与 EGFRwt 和 EGFRvIII 的结合亲和力和特异性。通过抑制人 EGFRwt 转染 NR6（NR6W）、人 EGFRvIII 转染 NR6（NR6M）、EGFRwt 过表达 A431-表皮样癌和胶质母细胞瘤异种移植细胞（43、D08-0493MG、D2159MG 和 D270MG）中的蛋白质合成来测量 D2C7-(scdsFv)-PE38KDEL 的体外细胞毒性。使用 43、NR6M 和 D270MG 原位肿瘤模型评估 D2C7-(scdsFv)-PE38KDEL 的体内抗肿瘤疗效。
D2C7-(scdsFv)-PE38KDEL 对 EGFRwt 和 EGFRvIII 的 KD 分别为 1.6×10(-9) mol/L 和 1.3×10(-9) mol/L。用 NR6W 和 NR6M 细胞进行流式细胞术证实了 D2C7-(scdsFv)-PE38KDEL 对 EGFRwt 和 EGFRvIII 的特异性。D2C7-(scdsFv)-PE38KDEL 的 IC50 在表达 EGFRwt（NR6W、A431、43 和 D08-0493MG）的细胞上为 0.18 至 2.5 ng/mL。D2C7-(scdsFv)-PE38KDEL 的 IC50 约为 0.25 ng/mL，在 EGFRvIII 表达细胞（NR6M）和 EGFRwt 和 EGFRvIII 表达的胶质母细胞瘤异种移植细胞（D2159MG 和 D270MG）上。值得注意的是，在 43、NR6M 和 D270MG 的颅内肿瘤模型中，通过对流增强输送用 D2C7-(scdsFv)-PE38KDEL 治疗分别延长了 310%（P=0.006）、28%（P=0.002）和 166%（P=0.001）的存活时间。
在临床前研究中，D2C7-(scdsFv)-PE38KDEL 免疫毒素对表达 EGFRwt、EGFRvIII 或两者的脑肿瘤具有显著的治疗潜力。
