Freestyle 293 medium

Human prostate cancer cell line PacMetUT1 was isolated from the lymph node metastasis of a 57-year old prostate cancer patient at our university [49 (link)]. PC-3 and DU145 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). PacMetUT1 and DU145 cells were cultured in McCoy’s 5A medium supplemented with pyruvate, amino acids, nutrients and 10% fetal bovine serum (FBS), as described [50 (link)]. HEK293 FreeStyle cells (HEK293F, Invitrogen, Carlsbad, CA) were maintained in FreeStyle 293 Medium (Invitrogen, Carlsbad, CA) supplemented with 1% Penicillin/Streptomycin. Mink lung epithelial (Mv1Lu) cells were obtained from Prof. Dan Rifkin and were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS and 1% penicillin/streptomycin. PacMetUT1, DU145, and Mv1Lu cells were maintained at 37°C in a 5% CO₂ humidified incubator, while the HEK293F cells were maintained at 37°C in an 8% CO₂humidified incubator-shaker.

The mammalian cell line used in this work is a serum-free suspension-adapted HEK 293 cell line (HEK 293SF-3F6 from NRC, Montreal, QC, Canada) kindly provided by Dr. Amine Kamen from McGill University (Montreal, QC, Canada). Cells were cultured as previously described [36 (link)]. HIV-1 Gag-eGFP VLP production was achieved by transient transfection. Briefly, the pGag-eGFP plasmid encoding for the Gag-eGFP polyprotein, is diluted with FreeStyle 293 medium (Invitrogen, Carlsbad, CA, USA) and vortexed for 10 s, then polyethylenimine (PEI) is added at 1:2 DNA:PEI ratio (w/w) and vortexed three times, the mixture is incubated for 15 min at RT and is added to the cell culture, where a medium exchange has been already performed. HIV-1 Gag-eGFP VLPs were harvested at 72 h post transfection (hpt) by centrifugation at 1000× g during 15 min. Supernatants were stored at 4 °C until analysis. Non-transfected negative controls reproducing cell growth conditions were also produced, for comparison.

The HEK293 cell line provided by Dr. Amine Kamen from McGill University (Montreal, Canada) derives from a cGMP master cell bank that is available for manufacturing of clinical material. It is adapted to grow in suspension and in serum free medium. Cells were cultured in Freestyle 293 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.1% Pluronic® (Invitrogen, Carlsbad, CA, USA). Medium was also supplemented with 1.6 mg/L of r-transferrin (Merck Millipore, Kankakee, IL, USA), 19.8 mg/L of r-insulin (FeF Chemicals/Novo Nordisk, Køge, Denmark.), and 0.9X of an in-house lipid mixture [21 (link)]. Cells were routinely maintained in 125-mL disposable polycarbonate Erlenmeyer flasks (Corning, Steuben, NY, USA) in 20 mL of culture medium, shaken at 110 rpm using an orbital shaker (Stuart, Stone, UK) placed in an incubator maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. Cell count and viability were determined using NucleoCounter NC-3000 (ChemoMetec, Allerod, Denmark).

HEK 293T, HEK 293F and TF-1 cells were obtained from the American Type Culture Collection (ATCC). HEK 293T and HEK 293F cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) and FreeStyle™ 293 medium (Invitrogen, Carlsbad, CA, USA) respectively, supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (10,000 U/mL) (Gibco). TF-1 cells were cultured in RPMI 1640 containing 20% FBS and 50 U/mL GM-CSF (Thermo Fisher Scientific).

The cDNA sequences of anti-cMet variable domains were cloned from mouse hybridoma HB-11895 (5D5.11.6) (ATCC, Manassas, VA) with a mouse Ig primer set (Novagen, Cat # 69831-3). For cMet Halfbody molecules, mutations such as C226S, C229S, T366F, L368F, P395A, F405R, Y407R, K409D were introduced into the heavy chain hinge region using standard mutagenesis techniques. The variable domain sequences of the anti-CD3 antibody were derived from the OKT3 antibody and those of the anti-EGFR antibody were derived from cetuximab. Inner and outer domains were connected using a long-long linker (L-L): VH1-VH2 Linker, ASTKGPSVFPLAP (13a.a); VL1-VL2 Linker, TVAAPSVFIFPP (12a.a.). AbbVie-proprietary pHybE vectors with corresponding huIgG1 heavy chain and kappa light chain were used to transiently transfect 293-6E cells in Freestyle 293 medium (Invitrogen, Carlsbad, CA). Media containing the secreted protein was harvested 6–7 days post transfection and purified using protein A chromatography (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The proteins were then dialyzed into PBS buffer, pH 7.2 (Invitrogen, Carlsbad, CA). The monomer percentage of Half DVD-Ig proteins was analyzed by SDS-PAGE and size exclusion chromatography. Binding of anti-EGFR/anti-CD3 DVD-Ig and half DVD-Ig molecules to CD3 and EGFR was analyzed by FACS.