A Novel Combination of Immunotherapy and Radiation Therapy in a Primary Mouse Model of Soft Tissue Sarcoma With High Mutational Load.

Purpose/objective(s) Preclinical studies using transplant tumor models have shown promising results for immunotherapy combined with radiotherapy (RT). However, research using primary tumor models is limited, and results from tumors using transplanted cell lines that do not develop in a native microenvironment under immunosurveillance often do not translate to the clinic. We previously found that tumors generated by transplanting cell lines from primary mouse sarcomas into syngeneic mice were cured by anti-PD-1 and RT. However, identical treatment failed to achieve local control in autochthonous sarcomas from the same model. Recent data has shown agonistic anti-OX40 immunotherapy combined with CpG oligonucleotide, a TLR9 agonist, eradicates tumors in a spontaneous mouse model of mammary cancer. Other studies have shown synergistic effects of anti-OX40 with anti-PD-1 or RT in transplant models. We hypothesize that the combination of anti-OX40, CpG, anti-PD-1, and RT will improve local control compared to RT or immunotherapy alone in a primary mouse model of soft tissue sarcoma with high mutational load. Materials/methods We generated autochthonous soft tissue sarcomas in 129/SvJ mice by intramuscular injection of adenovirus expressing Cas9 and a gRNA targeting Trp53 followed by exposure to the carcinogen 3-methylcholanthrene. Once tumors reached 70 mm3, mice were treated with RT (20 Gy, day 0), anti-OX40 (20 µg intraperitoneal (IP), randomized to one of two schedules: days 1 & 8 or days 3 & 10), CpG (50 µg intratumor, days 1 & 8 or 3 & 10), and anti-PD-1 (200 µg IP, days 1, 4, 7 or 3, 7, 10). Tumors were measured 3x/week. Time to tumor quintupling was compared to historical control mice treated with RT and isotype controls for anti-PD-1 and/or anti-CTLA-4 using the Mann-Whitney test. Results Sarcomas from this model are histologically similar to undifferentiated pleomorphic sarcoma. For mice treated with anti-OX40, CpG, and anti-PD-1 beginning day 1 (n = 17) compared to day 3 after RT (n = 13), median time to tumor quintupling was 36.2 vs. 44.3 days, respectively (P = 0.09). Time to tumor quintupling for mice treated with RT, anti-OX40, CpG, and anti-PD-1 (pooled groups with immunotherapy-initiated day 1 or 3) was significantly delayed compared to historical control mice treated with RT and isotype controls (n = 52) (median 38.7 vs. 30.5 days, respectively, P = 0.0009). Studies comparing combined RT, anti-OX40, CpG, and anti-PD-1 to contemporaneous controls are ongoing. Conclusion In a high mutational load primary mouse model of soft tissue sarcoma that is resistant to RT and anti-PD-1, RT combined with anti-OX40, CpG, and anti-PD-1 immunotherapy significantly delays tumor growth compared to historical controls. Results using this novel treatment combination with contemporaneous control groups are pending to determine if this regimen warrants further evaluation in a phase I clinical trial.