Afatinib

C57BL/6J mice (stock #000664; Jackson labs, Bar Harbor, ME) were challenged subcutaneously with 500,000 MC38 colon cancer cells on their flanks and enrolled on-study when tumors reached 50 mm³. Mice were treated with vehicle plus IgG2a isotype control (10 mg/kg; Bio X Cell, West Lebanon, NH), anti–PD-1 (10 mg/kg; clone RMP1–14; Bio X Cell, West Lebanon, NH), afatinib (10 mg/kg; Selleck, Houston, TX), combination anti–PD-1 (10 mg/kg) and afatinib (10 mg/kg), or combination anti–PD-1 (10 mg/kg), afatinib (10 mg/kg), and anti-CD8α (200 μg; clone 53–6.7; Bio X Cell, West Lebanon, NH). Animals received intraperitoneal (IP) injections of anti–PD-1 on days 5, 8, and 12 and afatinib on days 6, 7, 8, 9, and 10 (as indicated). Depleting anti-CD8α was administered two days prior to first anti–PD-1 treatment. Mice used in experiments were 7–8 weeks of age at time of tumor challenge. Endpoint was considered to be when tumors reached a size of 2000 mm³ or as mandated by institutional guidelines due to development of necrotic lesions. Mice were monitored every 2–3 days and tumors measured with digital calipers. All animal studies were conducted in accordance with, as well as with the approval of, the Institutional Animal Care and Use Committee of Dana-Farber/Harvard Cancer Center.