Humanized mouse models

Two different methods of humanization were used. In one method, human cord blood CD34⁺ frozen cells from mixed donors were purchased from STEMCELL Technologies and engrafted intravenously into NSG-SGM3 mice (2 × 10⁴ cells per mouse). This method results in more complete engraftment of human immune cells, including myeloid cells. Humanization was determined after 12 weeks by flow cytometry on peripheral blood; mice with ≥25% of total CD45⁺ cells of human origin were considered humanized. These animals were inoculated with 5 × 10⁶ LG1306 or MDA-MB-231 cells subcutaneously on the right flank. Once tumors had reached ~75 mm³, mice were treated intraperitoneally 5× (for LG1306) or 3× (for MDA-MB-231) weekly with 300 μg of IgG1 isotype control, 300 μg of nivolumab, 300 μg of nivolumab +300 μg Ab1, or 300 μg of nivolumab +300 μg Ab2. In the second method, 1 × 10⁷ PBMCs were administered to NSG mice intravenously (a control group did not receive PBMCs), and on the same day, mice were inoculated with 3.5 × 10⁶ HT-29 cells subcutaneously on the right flank. In this model, only human T cells persist in the recipient mouse long term. One week later, when the tumors had reached ~90 mm³, mice were treated intraperitoneally with 300 μg of IgG1 isotype control, 300 μg of Ab1, or 300 μg of Ab2; dosing was repeated for a total of five weekly doses.