Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
Nucl Med Biol. 2011 Jan;38(1):129-36. doi: 10.1016/j.nucmedbio.2010.06.010. Epub 2010 Sep 1.
The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for (111)In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ((111)In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of (111)In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer.
Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37°C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (≥ 85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of (111)In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K(a) = 0.6-9.6 × 10(7) L/mol; B(max) = 0.6-10.4 × 10(6) sites/cell). (111)In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of (111)InCl(3) to a single kit vial and incubating for 30 min at room temperature. (111)In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility.
Pure and homogeneous Fab fragments were produced. Eleven lots of kits met established quality specifications. The labeling efficiency with (111)In was 90.6 ± 2.2%. (111)In-DTPA-trastuzumab Fab bound specifically to HER2 on SKBR-3 cells (K(a) = 4.8 ± 2.5 × 10(7) L/mol and B(max) = 1.6 ± 0.8 × 10(6) sites/cell). Thirteen lots of (111)In-DTPA-trastuzumab injection met all established specifications. Kits were stable for 90 days and (111)In-DTPA-trastuzumab Fab injection was stable for 24 h stored at 4 °C.
A kit was formulated under GMP conditions for the preparation of (111)In-DTPA-trastuzumab Fab injection suitable for human administration. The kits were approved by Health Canada.
人类表皮生长因子受体-2（HER2）基因在 25%的浸润性乳腺癌中扩增，在多达 60%的早期疾病[导管原位癌（DCIS）]中受体过表达。临床前研究表明，在荷有人乳腺癌异种移植的裸鼠中，HER2 单克隆抗体曲妥珠单抗（赫赛汀）（111）In-DTPA-曲妥珠单抗 Fab 的 Fab 片段在 72 小时时具有高肿瘤/血液比（>27:1）（111）In-DTPA-曲妥珠单抗 Fab）。本研究的目的是根据良好生产规范（GMP）条件制定试剂盒，以便在 HER2 阳性乳腺癌的成像和放射免疫引导手术（RIGS）的 I 期临床试验中对人进行（111）In-DTPA-曲妥珠单抗 Fab 注射。
使用固定化木瓜蛋白酶在 37°C 下对曲妥珠单抗 IgG（赫赛汀）进行 20 小时的酶解，产生 Fab 片段。通过超滤纯化 Fab 片段，然后用二乙三胺五乙酸（DTPA）二酐的 10 倍摩尔过量反应。DTPA-Fab 片段经过纯化，然后通过过滤无菌装入单位剂量玻璃小瓶（试剂盒）。试剂盒根据体积（0.9-1.1ml）、蛋白浓度（0.45-0.55mg/ml）、pH 值（5.5-6.5）、DTPA 取代度（0.5-4.0mol DTPA/mol Fab）、外观（透明、无色、无颗粒）、标记效率（≥85%）、无菌和无热源性（USP XXXII）进行测试。使用 SKBR-3 人乳腺癌细胞通过饱和放射性配体结合测定法测量（111）In-DTPA-曲妥珠单抗 Fab 对 HER2 的免疫反应性（规格：K(a)=0.6-9.6×10(7)L/mol；B(max)=0.6-10.4×10(6)个/细胞）。通过向单个试剂盒小瓶中添加 80-100MBq 的（111）InCl(3)，并在室温下孵育 30 分钟，制备（111）In-DTPA-曲妥珠单抗 Fab 注射液。（111）In-DTPA-曲妥珠单抗 Fab 的放射性活度和 pH 值、放射化学纯度（RCP）、外观和无菌性进行检测。
产生了纯净和均一的 Fab 片段。11 批试剂盒符合既定的质量规范。（111）In 的标记效率为 90.6±2.2%。（111）In-DTPA-曲妥珠单抗 Fab 特异性结合 SKBR-3 细胞上的 HER2（K(a)=4.8±2.5×10(7)L/mol 和 B(max)=1.6±0.8×10(6)个/细胞）。13 批（111）In-DTPA-曲妥珠单抗注射液符合所有既定规格。试剂盒在 90 天内稳定，（111）In-DTPA-曲妥珠单抗 Fab 注射液在 4°C 下储存 24 小时稳定。
根据 GMP 条件制定了试剂盒，用于制备适合人体给药的（111）In-DTPA-曲妥珠单抗 Fab 注射液。该试剂盒已获得加拿大卫生部的批准。
