Trastuzumab herceptin

All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) or Thermo Fisher Scientific (Pittsburgh, PA, USA), unless otherwise specified. Aqueous solutions were prepared using ultrapure water (resistivity, 18 MΩ.cm) treated with Chelex resin purchased from Bio-Rad Laboratories, Inc. (Berkeley, CA, USA). p-SCN-Bn-DTPA was purchased from Macrocyclics, Inc. (Dallas, TX, USA). Indium-111 ([¹¹¹In] InCl₃) was purchased from MDS Nordion (Vancouver, BC, Canada). Trastuzumab (Herceptin), an anti-human HER-2 IgG monoclonal antibody was obtained from Genentech (San Francisco, CA, USA).

Study approval was obtained from the Institutional Review Board, and all patients signed consent prior to tissue collection according to the institutional guidelines. A total of 15 USC cell lines were established after the sterile processing of fresh tumor biopsy samples, as described previously 9 (link). All USC cell lines were established from biopsies taken from chemotherapy-naïve patients at the time of primary staging surgery (Table 1). Tumors were staged according to the International Federation of Gynecology and Obstetrics staging system. Patient characteristics are noted in Table 1. Primary USC cell lines with limited passages (i.e., <50) were analyzed by flow cytometry for HER2 expression both immediately after tumor processing as well as after being cultured in vitro for 1 week to 5 years. This was evaluated with Trastuzumab (Herceptin; Genentech, San Francisco, CA) as a humanized monoclonal antibody (mAb) of the IgG1 isotype and is further explained under the flow cytometry section of the Materials and Methods. The corresponding cell blocks were also analyzed for HER2 surface expression by IHC and c-erbB-2 gene amplification by fluorescent in situ hybridization (FISH).

The version of the anti-HER2 sdAb 5F7 bearing a C-terminus GGC tail (hereafter referred to as 5F7) was a gift from Dr. Hilde Revets, formerly of Ablynx NV (Ghent, Belgium). This anti-HER2 sdAb was selected from phage libraries derived from llamas immunized with SKBR-3 human breast carcinoma cells, and details for production, purification, and characterization have been reported previously [17 (link)].
All reagents used in cell culture studies were purchased from Invitrogen (Grand Island, NY, USA). HER2-expressing BT474M1 human breast carcinoma cells [47 (link)], a more tumorigenic version of the original BT474 line, were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum, streptomycin (100 μg/mL), and penicillin (100 IU/mL). HER2-expressing SKOV-3 human ovarian carcinoma cells, obtained from the Duke University Cell Culture Facility, were grown in McCoy’s 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37 °C in a 5% CO₂ humidified incubator, medium was changed every two days, and cells were passaged by trypsinization (0.05% Trypsin-EDTA) when they were about 80% confluent. Trastuzumab (Herceptin^®, Genentech, San Francisco, CA, USA) was obtained from the Duke Cancer Institute Pharmacy.