In tumor-bearing hosts, myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) play important roles in immune suppression, the reversal of which is vitally important for the success of immune therapy. We have shown that ckit ligand is required for MDSC accumulation and Treg development. We hypothesized that sunitinib malate, a receptor tyrosine kinase inhibitor, could reverse MDSC-mediated immune suppression and modulate the tumor microenvironment, thereby improving the efficacy of immune-based therapies. Treatment with sunitinib decreased the number of MDSC and Treg in advanced tumor-bearing animals. Furthermore, it not only reduced the suppressive function of MDSCs but also prevented tumor-specific T-cell anergy and Treg development. Interestingly, sunitinib treatment resulted in reduced expression of interleukin (IL)-10, transforming growth factor-β, and Foxp3 but enhanced expression of Th1 cytokine IFN-γ and increased CTL responses in isolated tumor-infiltrating leukocytes. A significantly higher percentage and infiltration of CD8 and CD4 cells was detected in tumors of sunitinib-treated mice when compared with control-treated mice. More importantly, the expression of negative costimulatory molecules CTLA4 and PD-1 in both CD4 and CD8 T cells, and PDL-1 expression on MDSC and plasmacytoid dendritic cells, was also significantly decreased by sunitinib treatment. Finally, sunitinib in combination with our immune therapy protocol (IL-12 and 4-1BB activation) significantly improves the long-term survival rate of large tumor-bearing mice. These data suggest that sunitinib can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy for advanced malignancies.

IntroductionImmune-based therapy has achieved a certain level of success; however, the overall therapeutic effect has been much less promising due to the immune suppressive mechanisms associated with advanced malignancies. 1 To achieve a better therapeutic efficacy of immune activation therapy, the mechanism or mechanisms by which a large tumor burden prevents immune activation from inducing effective antitumor immunity needs to be elucidated.Tumor growth is accompanied by an increase in the number of Gr-1 ϩ Mac-1 ϩ myeloid-derived suppressor cells (MDSCs) 2-4 and tumor-specific T regulatory cells (Tregs) 5,6 with strong immune suppressive activity in cancer patients and in tumor-bearing mice. [7][8][9] Both MDSCs and Tregs may be directly involved in immune unresponsiveness in active immune therapy.It has been demonstrated that MDSCs are involved in T-cell hyporesponsiveness in tumor-bearing mice. Several mechanisms by which MDSCs regulate the tumor-specific T-cell response have recently been proposed and the in vivo immune regulatory effects of MDSCs on tumor-specific T-cell response have been identified. 7-12 T-cell inactivation can be mediated by MDSCs through IFN␥-dependent nitric oxide (NO) production [12][13][14][15][16] or the Th2-mediated IL-4/IL-13-dependent arginase 1 pathway. 14,[17][18][19][20][21][22] In addition, a mechanism of ROS-mediated cell killing has been proposed. 3,23,24 Furthermore, MDSCs can inhibit cytotoxic T lymphocyte (CTL) responses through NOdependent or -independent mechanisms. Cell-to-cell contact appeared to be crucial in these mechanisms. 25 Our laboratory has further identified a novel mechanism of MDSC-mediated immune suppression on activated T cells through the development of Foxp3 ϩ T regulatory cells (Tregs) and T-cell tolerance both in vitro and in tumor-bearing mice. The induction of Tregs by MDSCs requires IFN-␥ and IL-10 but is independent of the NO-mediated suppressive mechanism. 11 To overcome MDSCmediated immune suppression and prevent Treg induction, it is critical to identify the tumor factors that are required for MDSC accumulation in tumor-bearing animals.Several lines of evidence support the hypothesis that the development and expansion of MDSCs may be modulated by tumor-secreted factors. MDSCs in tumor-bearing animals can differentiate into mature dendritic cells or remain as MDSCs with inhibitory activities, depending on the local cytokine milieu. 26,27 Human renal cell carcinoma cell lines release soluble factors (IL-6, M-CSF) that inhibit the differentiation of CD34 ϩ cells into dendritic cells (DCs) and trigger their commitment toward monocytic cells. 28 In a transgenic mammary tumor, VEGF levels correlate with the MDSC number. 29 Moreover, the in vivo infusion of vascular endothelial growth factor (VEGF) can induce MDSC development in naive mice and impair DC function and differentiation. 30 Granulocyte macrophage-colony-stimulating factor (GM-CSF) secretion has correlated with the capacity of tumor metastases and the GM-CSF and IL-3 in conditioned mediu...

Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG−/− mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4+CD25+Foxp3+ Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases.

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Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2730.