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SM16 efficacy and radiosensitivity of CT26 and Panc02.

Supplemental Figure legends from TGFβ Inhibition Prior to Hypofractionated Radiation Enhances Efficacy in Preclinical Models

Quantified effect of tumor size and epithelial-to-mesenchymal transition.

The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial–mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy. Cancer Immunol Res; 2(10); 1011–22. ©2014 AACR.

Relapsed sarcomas remain essentially incurable for most children with cancer. Alpha-Tocopheryloxyacetic acid (αTEA) is a scalable semi-synthetic derivative of alpha-tocopherol that has shown promising activity preclinically and clinically in adult cancer, but has not yet been studied in pediatric sarcoma. We hypothesized that αTEA would mediate anti-tumor effects in rhabdomyosarcoma (RMS) by modulating the immunosuppressive milieu. Treatment of murine M3-9-M RMS in vitro with αTEA (2.5–100uM) resulted in increased apoptosis by flow cytometry (Annexin V+/7AAD+) and Incucyte analysis (Annexin V+) within 24 hours (p < 0.05). Using an established orthotopic luciferase+ M3-9-M in vivo RMS model, αTEA-supplemented chow administered for 21 days significantly reduced tumor volume (p < 0.001) and bioluminescence (p < 0.05) by 28 days, resulting in a significant prolongation of survival compared to matched control chow (p < 0.05), up to 4 weeks after the feed was stopped. Analysis of spleens from RMS mice fed αTEA-supplemented diets showed an increased percentage of Ki-67+ and IFNγ+CD4+ T cells (p < 0.01) at the end of treatment as compared to recipients of the control diet. In addition, a decrease in the percentage of CD11b+Arg-1+ (p < 0.01) and PD-L1+ (p < 0.05) cells within the spleen were observed in αTEA-treated RMS tumor-bearing mice. RNA-Seq analysis of RMS tumors is underway to explore changes in αTEA vs. control-treated mice. These results suggest that αTEA causes direct induction of apoptosis of RMS in vitro and in vivo, leading to prolonged survival through enhanced CD4+ T cell stimulation and reduced myeloid cell-mediated immunosuppression in RMS-bearing mice. Further investigation of αTEA as an immunomodulating agent is warranted.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

OX40 (CD134, TNFRSF4), a member of the tumor necrosis factor receptor (TNFR) superfamily is expressed on activated CD4+ and CD8+ T cells. Engagement of OX40 with cognate ligand or soluble agonist enhances T cell proliferation, survival and cytokine production by memory T cells. Although αOX40 therapy has been effective at controlling incipient tumors in a variety of mouse tumor models, it has been less effective at eradicating large established tumors. Pervasive immunosuppression in the tumor microenvironment has been implicated as a potential mechanism of immune evasion by tumor cells. Here we show that combining agonist antibody (αOX40) with an orally bioavailable small molecule inhibitor of TGF-β signaling, results in complete regression of well-established tumors in ~85% of treated mice. Tumor infiltrating lymphocytes (TIL) from these dual treated mice produce increased levels of Granzyme-B and IFNγ and higher levels of the proliferation marker Ki67. Differences seen in STAT3 expression by the TIL led us to investigate the role of STAT3 in this anti-tumor effect. Using an OX40 promoter-driven CRE model to knock out the STAT3 gene in activated T-lymphocytes, we demonstrated that STAT3 is essential for the therapeutic efficacy of the combination treatment as a 50% drop in tumor cure rate was observed in STAT-3 deficient mice compared to STAT3 sufficient mice. Together these results demonstrate the anti-tumor efficacy of combining OX40 therapy with TGF-β inhibition.

SM16 is an orally available small molecule TGF-b signaling inhibitor that binds to the ATP pocket of TGF-b receptor Type 1 and prevents phosphorylation by TGF-BRII following engagement of the ligand. OX40 (CD134) is a member of the TNF-a receptor superfamily and is upregulated on activated CD4 and CD8 T cells. OX40 ligation by its cognate ligand (OX40L) or agonistic antibody (anti-OX40) enhances effector function, expansion and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of SM16 plus anti-OX40 in a poorly immunogenic and highly metastatic 4T1 mammary tumor model. Our data show that SM16 synergizes with OX40 to elicit a potent anti-tumor effect against established primary tumors, with a 73% reduction in tumor size and a 92% reduction in the number of metastatic lung nodules; overall 71% of treated mice were either tumor free or showed stable disease by the end of the study. This positive treatment outcome is associated with a two-fold expansion of tumor-infiltrating effector CD8+ T cells and an accumulation of CD4+ and CD8+ cells overall, as well as a high tumor-specific IFN-g response. Depletion of both CD4+ and CD8+ T cells completely abrogates the effect of this therapy, indicating it is T cell dependent. Taken together, these data suggest that combining a TGF-b signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.

Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.