Preclinical evidence suggests that high-dose hypofractionated ionizing radiation (IR) can enhance anti-tumor immunity and result in significant tumor control when combined with immune checkpoint blockade (ICB). However, low-dose daily fractioned IR used for many tumor types including head and neck squamous cell carcinoma results in lymphopenia and may be immunosuppressive. We compared immune correlates, primary tumor and abscopal tumor control rates following the addition of PD-1 mAb to either high-dose hypofractioned (8Gyx2) or low-dose daily fractionated (2Gyx10) IR in syngeneic models of cancer. When compared to 2Gyx10 IR, 8Gyx2 IR preserved peripheral and tumor-infiltrating CD8 T-lymphocyte accumulation and activation and reduced peripheral and tumor gMDSC accumulation. Regulatory T-lymphocytes were largely unaltered. Type I and I IFN levels and expression of IFN-responsive MHC class I and PD-L1 was enhanced in tumors treated with 8Gyx2 compared to 2Gyx10 IR. Functionally, tumor-specific CD8 T-lymphocyte IFN responses within tumor draining lymph nodes were enhanced following 8Gyx2 IR but suppressed following 2Gyx10 IR. When combined with PD-1 mAb, reversal of adaptive immune resistance and subsequent enhancement of CD8+ cell dependent primary and abscopal tumor control was observed following 8Gyx2 but not 2Gyx10 IR. These data strongly support that compared to daily fractionated low-dose IR, high-dose hypofractionated IR preserves or enhances anti-tumor immunity and, when combined with PD-1 mAb to reverse adaptive immune resistance, promotes anti-tumor immunity to control primary and distant tumors. These data critically inform the rational design of trials combining IR and ICB.

Purpose: Surgical resection of primary tumor with regional lymphadenectomy remains the treatment of choice for patients with advanced human papillomavirus-negative head and neck squamous cell carcinoma. However, even when pathologic disease-free margins can be achieved, locoregional and/or distant disease relapse remains high. Perioperative immunotherapy may improve outcomes, but mechanistic data supporting the use of neoadjuvant or adjuvant treatment clinically are sparse.Experimental Design: Two syngeneic models of oral cavity carcinoma with defined T-cell antigens were treated with programmed death receptor 1 (PD-1) mAb before or after surgical resection of primary tumors, and antigen-specific T-cell responses were explored with functional and in vivo challenge assays.Results: We demonstrated that functional immunodominance developed among T cells targeting multiple independent tumor antigens. T cells specific for subdominant antigens expressed greater levels of PD-1. Neoadjuvant, but not adjuvant, PD-1 immune checkpoint blockade broke immunodominance and induced Tcell responses to dominant and subdominant antigens. Using tumors lacking the immunodominant antigen as a model of antigen escape, neoadjuvant PD-1 immune checkpoint blockade induced effector T-cell immunity against tumor cells lacking immunodominant but retaining subdominant antigen. When combined with complete surgical excision, neoadjuvant PD-1 immune checkpoint blockade led to formation of immunologic memory capable of preventing engraftment of tumors lacking the immunodominant but retaining subdominant antigen.Conclusions: Together, these results implicate PD-1 expression by T cells in the mechanism of functional immunodominance among independent T-cell clones within a progressing tumor and support the use of neoadjuvant PD-1 immune checkpoint blockade in patients with surgically resectable carcinomas.

BackgroundNatural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined.MethodsTumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed.ResultsCultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab.ConclusionsTumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.