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The benefit of combining immunotherapy with photon irradiation has been shown pre-clinically and clinically. This current pre-clinical study was designed to investigate the anti-tumour action of combining immunotherapy with protons. Male CDF1 mice, with a C3H mammary carcinoma inoculated on the right rear foot, were locally irradiated with single radiation doses when tumours reached 200mm 3 . Radiation was delivered with an 83–107MeV pencil scanning proton beam in the centre of a 3 cm spread out Bragg peak. Following irradiation (day 0), mice were injected intraperitoneal with anti-CTLA-4, anti-PD-1, or anti-PD-L1 (10 mg/kg) twice weekly for two weeks. Endpoints were tumour growth time (TGT3; time to reach 3 times treatment volume) or local tumour control (percent of mice showing tumour control at 90 days). A Student’s T-test (tumour growth) or Chi-squared test (tumour control) were used for statistical analysis; significance levels of  p  &lt; 0.05. Untreated tumours had a mean (± 1 S.E.) TGT3 of 4.6 days (± 0.4). None of the checkpoint inhibitors changed this TGT3. A linear increase in TGT3 was seen with increasing radiation doses (5–20 Gy), reaching 17.2 days (± 0.7) with 20 Gy. Anti-CTLA-4 had no effect on radiation doses up to 15 Gy, but significantly enhanced 20 Gy; the TGT3 being 23.0 days (± 1.3). Higher radiation doses (35–60 Gy) were investigated using a tumour control assay. Logit analysis of the dose response curve, resulted in a TCD50 value (radiation dose causing 50% tumour control; with 95% confidence intervals) of 48 Gy (44–53) for radiation only. This significantly decreased to 43 Gy (38–49) when mice were treated with anti-CTLA-4. Neither anti-PD-1 nor anti-PD-L1 significantly affected tumour control. Checkpoint inhibitors enhanced the response of this C3H mammary carcinoma to proton irradiation. However, this enhancement depended on the checkpoint inhibitor and radiation dose.

Using immunotherapy to enhance the response of a C3H mammary carcinoma to proton radiation