Abstract PO-049: TGFβ and PD-L1 inhibition in combination with radiotherapy can overcome glioblastoma resistance to immunotherapies
Authors: Reiners Oliver , Lazar Ann , Chou William , Zhang Hui , Jones Trevor , Hoffelt Dixon , Barcellos-Hoff Mary Helen 
Published: April 2021

Abstract
Glioblastoma multiforme (GBM) is treated by surgical excision followed by radiotherapy (RT) and concomitant temozolomide. Despite intense effort, patients still have a median survival of 15 months. Immunotherapies, in particular immune checkpoint inhibitors (ICI), have provided durable benefit in many solid tumors but clinical trials in GBM have not shown any survival benefit. However, the potential for combinations of RT and ICI have garnered significant interest. Here, we investigated the response to RT and ICI using syngeneic GBM cell lines GL261 or SB28 orthotopically implanted in C57Bl6/J mice. Most GL261 tumor-bearing mice (80%) were cured by treatment with anti-PD-L1 (clone 6E11, Genentech, 10 mg/kg, weekly). Combination of anti-PD-L1 and focal irradiation with 10 Gy cured all mice, accompanied by immune memory defined as rejection of subsequent tumor rechallenge. In sharp contrast, mice bearing SB28 tumors were highly resistant. Anti-PD-L1 alone provided no survival benefit. RT treatment alone increased survival by 12 days and combination with anti-PD-L1 increased the median survival by 19 days, but none survived. Thus, the highly immunogenic GL261 demonstrate that brain tumors can respond to ICI and is augmented by RT, whereas SB28 brain tumors more accurately reflect the resistant nature of human GBM. To investigate the mechanism of SB28 resistance, we analyzed the expression of PD-L1 and CD8 lymphocyte infiltrate in tumor sections. As expected, based on treatment response, GL261 tumors are PD-L1 positive and highly infiltrated by CD8 lymphocytes, whereas SB28 tumors had little PD-L1 expression and no lymphocyte infiltration. Failure of ICI treatment in other cancers is associated with lymphocyte exclusion attributed to activated fibroblasts that produce extracellular matrix (10.1038/nature25501). The brain does not contain fibroblasts, but we found immunosuppressive microglia surrounding SB28 tumors, which were embedded in tenascin C (TNC) accompanied by dense hyaluronic acid, which we speculate constitute a tissue-intrinsic, immunosuppressive barrier. Transforming growth factor β (TGFβ) is abundant in GBM, is immunosuppressive, and elicits microglia polarization. Blocking TGFβ in irradiated SB28 brain tumors reduces TNC and prolongs survival (10.1016/j.ijrobp.2020.09.043). Hence, we tested whether reducing TGFβ activity could promote immunological memory. Monotherapy with 10 Gy focal RT, anti-PD-L1 or TGFβ inhibitor IPW-5371 (20 mg/kg, daily) had no effect on immunological memory (0/27). Combinations of 2 of the 3 treatments had negligible effect on immunological memory (5%, 2/43). However, 13% (2/15) of mice treated with 10 Gy, anti-PD-L1 and IPW-5371 rejected subsequent SB28 intracranial rechallenge. Taken together these preclinical data suggest that an optimal combination of RT, ICI and TGFβ inhibitor could overcome intrinsic GBM barriers to immunotherapy.
Citation Format: Oliver Reiners, Ann Lazar, William Chou, Hui Zhang, Trevor Jones, Dixon Hoffelt, Mary Helen Barcellos-Hoff. TGFβ and PD-L1 inhibition in combination with radiotherapy can overcome glioblastoma resistance to immunotherapies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-049.