The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression.Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5 -/-littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.

To create more effective T cells against human tumors, we have designed a strategy to allow T cells to recognize tumor cells using natural killer (NK) cell receptors but retain the effector responses of T lymphocytes. NKG2D is an activating cell surface receptor expressed on NK cells and on some T-cell subsets. Its ligands are primarily expressed on tumor cells. We have shown that by linking mouse NKG2D to the CD3Z chain, it was possible to generate a chimeric NKG2D (chNKG2D) receptor that allowed activation of murine T cells on engagement with NKG2D ligand-positive tumor cells leading to antitumor responses in mice. In this study, a human version of the chNKG2D receptor was expressed on primary human T cells, and antitumor responses were determined. Human peripheral blood mononuclear cell-derived T cells were retrovirally transduced with a human chNKG2D receptor gene. These chNKG2D-bearing human T cells responded to NKG2D ligand-positive tumor cells by producing T-helper 1 cytokines, proinflammatory chemokines, and significant cellular cytotoxicity. This response could be blocked by anti-NKG2D antibodies, and it was dependent on NKG2D ligand expression on the target cells but not on expression of MHC molecules. In addition, the activity of chNKG2D-bearing T cells remained unimpaired after exposure to a soluble NKG2D ligand, soluble MICA, at concentrations as high as 1.5 Mg/mL.

Adoptive transfer of T cells expressing chimeric NKG2D (chNKG2D) receptors, a fusion of NKG2D and CD3ζ, can lead to long-term, tumor-free survival in a murine model of ovarian cancer. To determine the mechanisms of chNKG2D T cell antitumor efficacy, we analyzed how chNKG2D T cells altered the tumor microenvironment, including the tumor-infiltrating leukocyte populations. chNKG2D T cell treatment of mice bearing ID8 tumor cells increased the number and activation of NK cells and increased the activation of host CD8+ T cells within the tumor. Foxp3+ regulatory T cells at the tumor site decreased more than 300-fold after chNKG2D T cell treatment. Tumor-associated regulatory T cells expressed cell surface NKG2D ligands and were killed by chNKG2D T cells in a perforin-dependent manner. chNKG2D T cells also altered the function of myeloid cells at the tumor site, changing these cells from being immunosuppressive to enhancing T cell responses. Cells isolated from the tumor produced elevated amounts of IFN-γ, NO, and other proinflammatory cytokines after chNKG2D T cell treatment. ChNKG2D T cells required perforin, IFN-γ, and GM-CSF to induce a full response at the tumor site. In addition, transfer of chNKG2D T cells into mice bearing tumors that were established for 5 weeks led to long-term survival of the mice. Thus, chNKG2D T cells altered the ovarian tumor microenvironment to eliminate immunosuppressive cells and induce infiltration and activation of antitumor immune cells and production of inflammatory cytokines. This induction of an immune response likely contributes to chNKG2D T cells’ ability to eliminate established tumors.

Despite advancements in the treatment of ovarian cancer, this disease continues to be a leading cause of cancer death in women. Adoptive transfer of tumor-reactive T cells is a promising antitumor therapy for many cancers. We designed a chimeric receptor linking NKG2D, a natural killer (NK) cellactivating receptor, to the CD3Z chain of the T-cell receptor to target ovarian tumor cells. Engagement of chimeric NKG2D receptors (chNKG2D) with ligands for NKG2D, which are commonly expressed on tumor cells, leads to T-cell secretion of proinflammatory cytokines and tumor cytotoxicity. In this study, we show that >80% of primary human ovarian cancer samples expressed ligands for NKG2D on the cell surface. The tumor samples expressed MHC class I-related protein A, MICB, and UL-16 binding proteins 1 and 3. ChNKG2D-expressing T cells lysed ovarian cancer cell lines. We show that T cells from ovarian cancer patients that express chNKG2D secreted proinflammatory cytokines when cultured with autologous tumor cells. In addition, we show that chNKG2D T cells can be used therapeutically in a murine model of ovarian cancer. These data indicate that treatment with chNKG2D-expressing T cells is a potential immunotherapy for ovarian cancer. [Cancer Res 2007;67(10):5003-8]

Ovarian cancer is one of the leading causes of cancer death in women and the development of novel therapies is needed to complement the standard treatment options such as chemotherapy and radiation. In this study, we show that treatment with T cells expressing a chimeric NKG2D receptor (chNKG2D) was able to lead to long-term, tumor-free survival in mice bearing established ovarian tumors. Tumor-free mice were able to reject a rechallenge with ovarian tumor cells 225 days after original tumor injection. In addition, chNKG2D T cell treatment induced specific host immune responses to ovarian tumor cells, including the development of both CD8+ and CD4+ T cell tumor-specific memory responses. The chNKG2D T cells reduced the ovarian tumor burden using both cytotoxic and cytokine-dependent pathways. Specifically, chNKG2D T cell expression of perforin, GM-CSF, and IFN-γ were essential for complete antitumor efficacy.