Clinical Trial of SBRT and Systemic Pembrolizumab With or Without Avelumab/Ipilimumab+ Dendritic Cells in Solid Tumors

Cancer cells can be recognized by the patient's own immune system, a process that is referred to as the "cancer immunity cycle" (Chen \& Mellman 2013; Mellman 2013; Chen \& Mellman 2017).

Remarkable anti-tumor activity has been achieved by blocking the inhibitory T-cell receptor CTLA-4 and/or the PD-1/-L1 axis. Immune checkpoint inhibition by monoclonal antibody (mAb) therapy has become a standard of care in patients with advanced melanoma, renal cell carcinoma, non-small cell lung carcinoma, Hodgkin's lymphoma and bladder cancer. Indications are continuously expanding. Activity of PD-1/-L1 and CTLA-4 inhibition has been correlated with hallmarks of pre-existing anti-tumor T-cell response (presence of activated cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) as evident from transcription profiles) and PD-L1 expression by tumor cells (in response to T-cell secreted IFN-). Mutational load of the cancer cells and presence of highly immunogenic neo-epitopes in the cancer cell genome underlies the capacity for cancer cells to elicit an immune response (Tumeh, Harview et al. 2014). Responsiveness to treatment with CTLA-4 has been correlated with the expression of HLA class I molecules by the cancer cells (Tumeh, Harview et al. 2014).

In immune-evasive tumors a pivotal role has been attributed to the elimination of myeloid dendritic cells (myDC) from the TME. myDC play a pivotal role in the initiation and coordination of the activity of anti-tumor CTL activity within the TME (Rodig, Gusenleitner et al. 2017). In animal models, myDC have been demonstrated to play an essential role in "licensing" anti-tumor CTLs to eradicate tumor cells. Activation of oncogenic signaling pathways such as the WNT/Catenin pathway can lead to the exclusion of myeloid DC from the TME (Broz et al. 2014; Spranger \& Gajewski 2016). Absence of myDCs at the invasive margin and within metastases has been correlated with defective CTL activation allowing the metastasis to escape the anti-tumor immune response (Salmon, Idoyaga et al. 2016). These myDC also migrate to tumor-draining lymph nodes and present tumor antigens to T-cells in these secondary lymphoid organs (Salmon, Idoyaga et al. 2016). In mouse models, tumor-residing Batf3 dendritic cells were shown to be required for effector T Cell trafficking and success of adoptive T-cell therapy (Roberts et al. 2016). Presence of myeloid DC's was more strongly correlated with a "T-cell inflamed TME signature" as compared to neo-antigen load in 266 melanomas from The Cancer Genome Atlas (Spranger, Luke et al. 2016).

Two important human myDCs subsets exist that are differentiated by expression of either the BDCA-1 or BDCA-3 surface marker. The CD1c (BDCA-1)+ antigen is specifically expressed on human dendritic cells, which are CD11chighCD123low and represent the major subset of myDCs in human blood (about 0.6 % of all peripheral blood mononuclear cells (PBMCs)). CD1c (BDCA-1)+ myDC have a monocytoid morphology and express myeloid markers such as CD13 and CD33 as well as Fc receptors such as CD32, CD64, and FceRI. Furthermore, myDC are determined to be CD4+, Lin (CD3, CD16, CD19, CD20, CD56)-, CD2+, CD45RO+, CD141 (BDCA-3)-, CD303 (BDCA-2)-, and CD304 (BDCA-4/Neuropilin-1)-.

A proportion of CD1c (BDCA-1)+ myDC co-expresses CD14 and CD11b. These dual positive cells for CD14 and CD1c (BDCA-1) have immunosuppressive capacity and inhibit T-cell proliferation in vitro. Depletion of this cell type is preferred prior to using CD1c (BDCA-1)+ cells for immune-stimulatory purposes (Bakdash, Buschow et al. 2016).

CD1c (BDCA-1)+ myDC play an important role in the cross-presentation of tumor antigens following immunogenic cell death (Di Blasio, Wortel et al. 2016). Under conditions of tumor growth, myDC will be poorly recruited to the tumor microenvironment, do not get activated and thereby fail to efficiently coordinate anti-tumor immunity within the tumor micro-environment and present tumor associated antigens within tumor-draining lymph nodes. When activated appropriately, human CD1c (BDCA-1)+ dendritic cells secrete high levels of IL-12 and potently prime CTL responses (Di Blasio, Wortel et al. 2016). In vitro, IL-12 production by CD1c (BDCA-1)+ myDC can be boosted by exogenous IFN-(Nizzoli, Krietsch et al. 2013) CD1c (BDCA-1)+ myDC spontaneously "partially mature" within 12 hours following their isolation. Optimal maturation with secretion of IFN- as well as the orientation of stimulated T-lymphocytes towards a Th1 phenotype is only achieved following Toll-like receptor stimulation (Nizzoli, Krietsch et al. 2013).

Animal models have established the safety and efficacy of intra-tumoral administration of ipilimumab. An intratumoral dose of CTLA-4 blocking mAb administered at a ratio of [1:100] compared to intravenous dosing was found to result in equivalent anti-tumor effect and was associated with less systemic toxicity (Skold, van Beek et al. 2015) (Marabelle, Kohrt et al. 2013). In an ongoing clinical trial for patients with advanced melanoma, conducted by our research group, intratumoral injection of CD1c (BDCA-1)+ myDC together with the CTLA-4 blocking mAb ipilimumab plus intravenous administration of the PD-1 blocking mAb nivolumab have been proven feasible and safe. Intratumoral administration of an anti-PD-L1 IgG1 mAb may increase the potential for antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

Additionally, the investigators have previously investigated the utility of SBRT to primary tumor and metastatic locations in oligometastatic non-small cell lung carcinoma (NSCLC) patients and found that this treatment was feasible, safe and resulted in an overall metabolic response rate in 60% of the treated population, with 30% of patients achieving a complete metabolic remission (Collen, Christian et al. 2014). Radiation therapy (RT) has been recognized as potentially synergistic with immune checkpoint blockade. Tumor cell killing by radiation results in release of tumor antigens, reduces the immunosuppression within the TME and can reinvigorate the cancer-immunity cycle by upregulation of immunogenic cell surface markers and inducing immunogenic cell death (Dewan, Galloway et al. 2009; Kulzer, Rubner et al. 2014; Frey, Ruckert et al. 2017). Moreover, it has been shown that RT-induced immunogenic cell death can trigger DC maturation and activation in vitro (Kulzer et al. 2014), providing a clear rationale for combining DC-therapy together with RT.

In this randomized phase II clinical trial, the investigators propose to conduct hypofractionated SBRT in combination with systemic PD-1 immune checkpoint blockade (pembrolizumab) with or without intratumoral PD-L1/CTLA-4 inhibition plus intratumoral administration of CD1c (BDCA-1)+ / CD141 (BDCA-3)+ myDC.