Natural killer (NK) cells are innate immune effectors capable of broad cytotoxicity via germline-encoded receptors and can have conferred cytotoxic potential via the addition of chimeric antigen ...receptors. Combined with their reduced risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), NK cells are an attractive therapeutic platform. While significant progress has been made in treating hematological malignancies, challenges remain in using NK cell-based therapy to combat solid tumors due to their immunosuppressive tumor microenvironments (TMEs). The development of novel strategies enabling NK cells to resist the deleterious effects of the TME is critical to their therapeutic success against solid tumors. In this review, we discuss strategies that apply various genetic and non-genetic engineering approaches to enhance receptor-mediated NK cell cytotoxicity, improve NK cell resistance to TME effects, and enhance persistence in the TME. The successful design and application of these strategies will ultimately lead to more efficacious NK cell therapies to treat patients with solid tumors. This review outlines the mechanisms by which TME components suppress the anti-tumor activity of endogenous and adoptively transferred NK cells while also describing various approaches whose implementation in NK cells may lead to a more robust therapeutic platform against solid tumors.
Myeloid-derived suppressor cells (MDSCs) represent a heterogenous population of immature myeloid cells capable of modulating immune responses. In the context of cancer, MDSCs are abnormally produced ...and recruited to the tumor microenvironment (TME) to aid in the establishment of an immunosuppressive TME that facilitates tumor escape. Additionally, MDSCs contribute to non-immunologic aspects of tumor biology, including tumor angiogenesis and metastasis. The clinical significance of MDSCs has recently been appreciated as numerous studies have suggested a correlation between circulating and intratumoral MDSC frequencies and tumor stage, progression, and treatment resistance. In this chapter, we review MDSC characterization, development, expansion, and mechanisms that facilitate immunosuppression and tumor progression. Furthermore, we highlight studies demonstrating the clinical significance of MDSCs in various disease states in addition to strategies that modulate various aspects of MDSC biology for therapeutic gain.
BackgroundSuccessful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor ...microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor–related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs.MethodsThe function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB.ResultsNuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model.ConclusionsOur findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.
Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion. MDSC programming or polarization has been proposed as a strategy for leveraging the ...developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-β1 (TGFβ-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. TGFβ-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFβ-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFβ-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Thus, TGFβ1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.
...γδ-T cells can discriminate tumor cells with dysregulated metabolism, which express these danger signals, from healthy cells, which do not. γδ-T cells are thought to help bridge the innate and ...adaptive immune systems, and, thus, functionally and phenotypically share components of both.6 For example, γδ-T cells express numerous receptors typically found on innate immune effectors, such as natural killer (NK) cells, which play crucial roles in anti-tumor responses. ...the authors compared the expression of exhaustion markers on γδ-T cells expressing the traditional CAR versus the “co-stimulation only” CAR. ...as the authors themselves concede, the concept of a co-stimulatory CAR approach in which signals 1 and 2 of T cell activation are provided by separate receptors has been proposed and investigated previously in αβ-T cells.
High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting ...immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients.
The anti-tumor activity of recombinant mAb's directed against tumor cell growth receptors has generally been considered to result from direct antiproliferative effects, the induction of apoptosis, or ...possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in vivo. We show here that NK cells secrete a distinct profile of potent immunostimulatory cytokines in response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be duplicated by costimulation with other ILs and was significantly enhanced in the presence of monocytes. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIII) and the IL-12 receptor expressed on NK cells. Coadministration of Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK cell-derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAb's might also result from activation of a potent cytokine secretion program within immune effectors capable of recognizing mAb-coated targets.
Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), ...but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.
The Her2 / neu (c‐erbB‐2) oncogene encodes a 185‐kDa protein tyrosine kinase which is overexpressed in 20 % of breast adenocarcinomas and is recognized by a humanized anti‐Her2 / neu monoclonal ...antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody‐dependent cell cytotoxicity (ADCC) against antibody‐coated targets via their expression of a low‐affinity receptor for IgG (FcγRIII or CD16). NK cells can be expanded in cancer patients via the administration of low‐dose interleukin‐2 (IL‐2) and become potent cytotoxic effectors following exposure to high doses of IL‐2. We tested IL‐2‐activated NK cells against Her2 / neu+ (MCF‐7Her2 / neu) and Her2 / neu– (MDA‐468) breast cancer cell lines in a 4‐h 51Cr‐release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5‐coated MCF‐7Her2 / neu and MDA‐468 target cells by IL‐2‐activated NK cells was 35 % and 3 %, respectively (p < 0.05). Lysis was less than 5 % when targets were treated with either the non‐humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5‐coated MCF‐7Her2 / neu cells was inhibited by 80 % when NK cells were pre‐treated with an anti‐Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF‐7Her2 / neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low‐dose IL‐2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5‐coated tumor cells in the presence of IL‐2 also produced large amounts of IFN‐γ with concomitant up‐regulation of cell‐surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL‐2 therapy in patients with cancers that express the Her2 / neu oncogene.
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