Radiotherapy is a major part in the treatment of most common cancers, but many patients experience local recurrence with metastatic disease. In evaluating response biomarkers, we found that low doses of fractionated radiotherapy led to PD-L1 upregulation on tumor cells in a variety of syngeneic mouse models of cancer. Notably, fractionated radiotherapy delivered in combination with aPD-1 or aPD-L1 mAbs generated efficacious CD8 þ T-cell responses that improved local tumor control, long-term survival, and protection against tumor rechallenge. These favorable outcomes were associated with induction of a tumor antigen-specific memory immune response. Mechanistic investigations showed that IFNg produced by CD8 þ T cells was responsible for mediating PD-L1 upregulation on tumor cells after delivery of fractionated radiotherapy. Scheduling of anti-PD-L1 mAb was important for therapeutic outcome, with concomitant but not sequential administration with fractionated radiotherapy required to improve survival. Taken together, our results reveal the mechanistic basis for an adaptive response by tumor cells that mediates resistance to fractionated radiotherapy and its treatment failure. With attention to scheduling, combination immunoradiotherapy with radiotherapy and PD-1/PD-L1 signaling blockade may offer an immediate strategy for clinical evaluation to improve treatment outcomes. Cancer Res; 74(19); 5458-68. Ó2014 AACR.

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The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-Fc␥R interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo. IntroductionThe addition of the anti-CD20 mAb rituximab to chemotherapy has substantially improved the clinical outcome for many patients with a wide range of B-cell malignancies. 1-3 However, despite the unprecedented success of rituximab, a substantial proportion of patients with CD20-positive B-cell malignancies fail to achieve a complete remission or relapse after receiving rituximab-containing immunochemotherapy. 4 These areas of unmet clinical need highlight the requirement to develop improved treatments for these patients. Given both the success with rituximab and the rapid development of mAb engineering technology, there is currently intense investigation into the development of novel anti-CD20 mAbs aimed at improving therapeutic efficacy. Central to this challenge, is an enhanced understanding of the mechanism of action of anti-CD20 mAbs.Anti-CD20 mAbs can activate a range of potential tumor cell killing pathways (reviewed in Lim et al 5 ) including Fc-Fc␥ receptor (Fc␥R) interactions (namely Ab-dependent cellular cytotoxicity [ADCC] and phagocytosis mediated by Fc␥R-expressing immune effector cells such as macrophages and/or NK cells), complement-dependent cytotoxicity (CDC), or the direct induction of programmed cell death (PCD). Although it is well established that Fc-Fc␥R interactions are critical for the in vivo efficacy of anti-CD20 mAbs, 6-8 the role of complement remains disputed as to whether it is beneficial, 9,10 inconsequential, 7,11,12 or even detrimental to anti-CD20 mAb efficacy. 13,14 However, the potential importance of PCD in enhancing anti-CD20 mAb potency remains largely underinvestigated, perhaps because it does not appear to play a major role in the therapeutic efficacy of rituximab. 15 We have c...