A kit to prepare (111)In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer.

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.