Abstract 4068: Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors
Authors: Newton Jared M. , Hanoteau Aurelie , Liu Hsuan-Chen , Gaspero Angelina , Gartrell Robyn D. , Hart Thomas D. , Laoui Damya , Parikh Falguni , Saenger Yvonne M. , Sikora Andrew G. 
Published: August 2019

Abstract
Immune checkpoint inhibitors (ICI), including those targeting cytotoxic T-lymphocyte-associated-antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), have shown tremendous potential against solid tumor malignancies; however, response to ICI remains unpredictable with 60-90% of patients receiving minimal to no benefit. Lack of efficacy is commonly attributed to inadequate tumor-specific T cell generation and the immunosuppressive effects of the tumor immune microenvironment (TIME). Thus, we hypothesized that a combinatory treatment strategy aiming to enhance antigen presentation and revert the immunosuppressive TIME could improve response rates of ICI in established solid tumors. Using a syngeneic tumor model of HPV-associated head and neck cancer (mEER) established to 60-75 mm2 in size, we found that CTLA-4 and/or PD-1 inhibition only minorly delayed tumor growth and flow cytometry profiling revealed that the TIME maintained a “cold” or immunosuppressed state similar to untreated tumors. When PD-1/CTLA-4 inhibition was combined with a weekly dose of tumor-directed radiation (10 Gy x 2), we observed upregulation of antigen presentation molecules in the draining lymph node but the combination remained incapable of generating long-term survival benefit. This lack of efficacy was attributed to the immunosuppressed and lymphodepleted TIME, a common consequence of radiation. Thus, to improve the TIME, we optimized an immune-stimulating drug combination previously developed in our lab combining cyclophosphamide (CTX) and a small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). When we combined CTX/L-NIL immunomodulation, PD-1/CTLA-4 checkpoint inhibition, and radiation (collectively called the “CPR” regimen), we observed complete rejection of approximately 70% of established tumors in a CD8 T-cell dependent manner and potent development of immunologic memory against tumor-associated antigens. Tumor immune profiling after treatment revealed a “cold to hot” transition of the TIME, including increased levels of myeloid and lymphoid subsets associated with anti-tumoral immune responses (i.e. CD8 T cells, dendritic cells, M1 macrophages) and downregulation of immunosuppressive cellular subsets (i.e. T regulatory cells, granulocytic myeloid derived suppressor cells). We observed strong lymphoproliferation effects in the tumor-draining lymph node which resulted in significant TIME improvements including a 15-fold increase in the CD8 to regulatory T cell ratio. Thus, we have demonstrated that the rational combination of TIME immunomodulation, localized radiation to enhance antigen presentation, and immune checkpoint inhibitors to prevent T-cell exhaustion can promote the immunologic rejection of established solid tumors.
Citation Format: Jared M. Newton, Aurelie Hanoteau, Hsuan-Chen Liu, Angelina Gaspero, Robyn D. Gartrell, Thomas D. Hart, Damya Laoui, Falguni Parikh, Yvonne M. Saenger, Andrew G. Sikora. Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4068.