Due to limitations in concentrating bsAb, the bsAb + IVIg dose was separated into two injections separated by 4 h (1st injection: 30 g Fab-IgG1 + 10 mg IVIg; 2nd injection: 20 mg IVIg). Forty-eight hours after PLD dose, mice were sacrificed, and tissues (heart, liver, kidneys, spleen, lungs, tumor) and blood via cardiac puncture were collected. IgG1HER2, tandem FabHER2PEG, Fab-IgG1HER2PEG, and non-specific IVIg. C) Binding affinity of IgG1HER2, tandem FabHER2PEG, and Fab-IgG1HER2PEG to HER2-Fc chimera analyzed by ELISA (n=2). D) Binding affinity of IgG1PEG (left), tandem FabHER2PEG (middle), and Fab-IgG1HER2PEG (right) to DSPE-PEG5k in the presence and absence of free PEG8K Goat polyclonal to IgG (H+L)(HRPO) competition analyzed by ELISA (n=2). Methods Cell lines and animals Human SKBR3 (HER2+), A2780 (HER2-), and BT474 (HER2+) were purchased from the UNC-CH Tissue Culture Facility. SKBR3 cells were cultured in McCoys medium containing 15% fetal bovine serum, and A2780 cells were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% L-glutamine. BT474 cells were maintained in RPMI 1640 media with 2 g/L sodium bicarbonate and 2 mM L-glutamine, and supplemented with 10% fetal bovine serum, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 0.01 mg/ml human insulin. All cells were maintained at 37C and 5% CO2. Female athymic nude (6C8 week old) mice were obtained from Charles River Laboratories (Wilmington, MA, USA) or bred in-house by UNC Animal Services Core (Chapel Hill, NC), and maintained in a sterile housing suite. Keap1?CNrf2-IN-1 All animal experiments were carried out in accordance with an animal use protocol (16C333.0) approved by the University of North Carolina Animal Care and Use Committee. Mice were randomly assigned to treatment groups and investigators were blinded to Keap1?CNrf2-IN-1 the treatments. Cell uptake assay Cells were seeded at 5104 cells/well into 96-well plates. Next day, the cells were incubated with 10 nM mAb controls or bsAb for 4 h at 37C. After washing to remove unbound Ab, the cells were then incubated with fluorescent, PEGylated polystyrene beads (1:104 cell:bead ratio) for 12 h at 37C. Cells were washed to remove unbound beads and flow cytometry was performed using iQue Screener PLUS (Intellicyt, Albuquerque, NM). Data were analyzed using ForeCyt and BD FACSDiva software. To determine if bsAb remained on the surface of cells 24 h after bsAb incubation, cells were treated using extended uptake conditions. After cells were treated with 10 nM mAb or bsAb for 4 h at 37C, cells were washed and incubated in fresh media for 24 h at 37C. Then, cells were washed and incubated with fluorescent, PEGylated polystyrene beads (1:104 cell:bead ratio) for 4 h at 37C. Unbound beads were removed through washing, and cell-associated fluorescence was analyzed via flow cytometry. Pharmacokinetics of bispecific antibody in the presence and absence of high dose IVIg Female athymic nude mice (6C8 weeks old) either received a single intravenous injection of 30 g bispecific Fab-IgG1 or two intravenous injections separated by 15 minutes of 30 g bispecific Fab-IgG1 and 30 mg human intravenous Keap1?CNrf2-IN-1 immune globulin (IVIg, Provigen) via tail vein. Blood was collected from mice at different time points (5 min, 1 h, 3 h, 5 h, 7 h, 24 h, 48 h, 72 h; n = 8 mice per treatment group, n = 4 mice per time point). Whole blood was stored undisturbed at room temperature for 20 min to allow clotting. Samples were then centrifuged at 2,000 xg in a refrigerated centrifuge for 15 min to isolate serum. PEG-specific ELISAs33 were used to quantify the serum concentration of bispecific Fab-IgG1HER2xPEG at various time points by detecting antibody with goat anti-human IgG F(ab)2 (Rockland Immunochemicals, cat no. 209C1304, 1:10,000 dilution). PK analysis of the blood concentration of bsAb was conducted using PKSolver with a two-compartment model.34 Biodistribution of pretargeted PLD in tumor-bearing mice Female athymic nude (nu/nu) mice received a subcutaneous implantation of a single 60day, 0.36 mg 17-estradiol pellet six days prior to left mammary pad injection of BT474 cells; estradiol supplement was used to support growth of HER2+ BT474 cells. BT474 cells were resuspended in a 1:1 Matrigel/PBS solution to a final concentration of 4107 cells/ml. Each mouse received a 100 l injection of BT474 (4106) cells into the left mammary pad. Once tumors were 100 mm3, mice were randomized into antibody treatment groups (n Keap1?CNrf2-IN-1 = 4 mice per group). PBS, bsAb (30 g Fab-IgG1), and bsAb + IVIg (30 g Fab-IgG1 with 30 mg IVIg) were administered i.v., followed by generic PEGylated liposomal doxorubicin (PLD, 3 mg/kg.