• Applying MyD88 signaling in CD8+ T cells to enhance anti-tumor immunity

      Kaczanowska, Sabina; Davila, Eduardo, Ph.D.; 0000-0003-0191-6371 (2016)
      T cell immunotherapy is a promising strategy for the treatment of patients with advanced cancers. Despite promising results in patients with hematological malignancies, response rates to solid tumors remain low. One of the biggest challenges is effectively activating and sustaining anti-tumor T cell responses in the context of an immunosuppressive tumor environment. Several approaches are currently being explored to improve the efficacy of adoptive T cell transfer, including the activation of co-stimulatory signaling pathways in T cells. Toll-like receptor (TLR) engagement on T cells is a potent co-stimulatory signal that increases anti-tumor activity by enhancing T cell proliferation, effector function, and T cell survival. We developed and tested two genetic engineering strategies to exploit the co-stimulatory effects of TLR signaling in CD8+ T cells. First, CD8+ T cells were modified to express and secrete the TLR5 ligand (TLR5L), flagellin, as a means to deliver this immune adjuvant to the tumor for enhanced anti-tumor activity. TLR5L-secreting T cells exhibited improved proliferation, cytokine secretion, and anti-tumor activity in both xenogeneic and syngeneic models of melanoma. The anti-tumor activity of TLR5L-secreting T cells was associated with decreased numbers of phenotypically exhausted T cells and fewer myeloid-derived suppressor cells. Second, we designed and characterized a synthetic fusion protein composed of the T cell co-receptor CD8α and the TLR adaptor protein MyD88, termed CD8α:MyD88. The expression of CD8α:MyD88 on T cells increased T cell responses to low concentrations of tumor antigens as well as augmented the expression of effector molecules and co-stimulatory proteins. These effects were antigen-dependent and accompanied by elevated levels of TLR signaling-related proteins. The enhanced anti-tumor activity of CD8α:MyD88-expressing T cells in tumor-bearing mice was associated with a unique tumor cytokine/chemokine signature, improved T cell infiltration, elevated levels of antigen presentation, and fewer macrophages with an immunosuppressive phenotype. The use of T cells as vehicle to deliver TLR5L to the tumor site and the co-stimulation of T cells through a synthetic CD8α:MyD88 receptor represent two novel and versatile approaches for modulating the tumor microenvironment to enhance anti-tumor immunity.
    • Costimulatory Effects of TLR1-TLR2 signaling in CD8+ T Cells

      Joseph, Ann Mary; Davila, Eduardo, Ph.D. (2017)
      The future of T cell-based immunotherapy for cancer currently faces two predominant challenges; the development of an effective strategy to enhance the effector function of anti-tumor T cells and a method to overcome the barrage of suppressive mechanisms mediated by suppressive cells (such as MDSCs) within the immunosuppressive tumor microenvironment. The activation of the TLR-MyD88 signaling pathway in CD8+ T cells augments their proliferation, cytokine production and anti-tumor potency in vivo. Our studies explored the molecular pathways activated by TLR stimulation with a focus on mechanisms that enhance anti-tumor properties. Transcriptomic analysis of TLR-stimulated T cells revealed an enhanced expression of several TNFRSF members including 4-1BB. Blocking or deleting 4-1BB diminished the effects of TLR1-TLR2 stimulation. Furthermore, combined administration of a TLR1-TLR2 ligand and an agonistic 4-1BB antibody augmented T cell proliferation and cytokine production more than either therapeutic agent alone. This combination therapy strategy also induced significant tumor regression in mice bearing an established B16 melanoma. We also tested the ability of TLR1-TLR2-stimulated T cells to withstand MDSC-mediated suppression. TLR1-TLR2-treated T cells exhibited greater proliferation and cytokine production than non-treated T cells in the presence of tumor-derived MDSCs. This phenomenon was recapitulated in T cells transduced to express a synthetic CD8α:MyD88 coreceptor, which activates the MyD88 pathway specifically upon TCR engagement. The results presented in this study emphasize the strength and potential clinical use of activating the TLR signaling pathway in the development of immunotherapy strategies against cancer.
    • Developing Therapies to Overcome Immunosuppressive Myeloid Cells in the Tumor Microenvironment

      Ceradoy, Justine Anne; Davila, Eduardo, Ph.D. (2018)
      Myeloid cells in the tumor microenvironment represent significant barriers to the development of successful cancer immunotherapies. A multi-kinase inhibitor, Regorafenib (Reg), and a DNA-PK inhibitor, NU7441 (NU) were shown in a previous study to reduce expression of immunoinhibitory proteins in adaptive immune cells while increasing stimulatory MHC-I on cancer cells. In this study, we explored whether these drugs could reverse the suppressive activity of myeloid-derived suppressor cells (MDSCs) and alternatively activated macrophages. To test this idea, we used splenocytes from tumor-bearing mice and a human monocytic cell line differentiated into suppressive macrophages and assessed Arginase activity, their ability to suppress effector T cells, and mRNA expression of immunosuppressive and activating markers. We showed that Reg/NU decrease arginase activity and increase immunoactivating markers. These data demonstrate that treatment of suppressive myeloid cells with Reg/NU confers a less suppressive phenotype and leads to the generation of a more activating phenotype.
    • Identification and Characterization of Regorafenib and NU7441 as Targeted Immunotherapies for Melanoma

      Tsai, Alexander; Davila, Eduardo, Ph.D. (2017)
      The emergence of new therapeutics, including targeted therapies and immunotherapies, has improved prognoses for melanoma patients. Yet, each class of therapy suffers from unique shortcomings. Targeted therapies are only approved for approximately half of melanoma patients that carry a certain mutation in the BRAF gene. Additionally, responses to targeted therapies are almost always transient, as resistance arises rapidly. Immunotherapies offer, for the first time, the potential for long-term responses and complete regressions. However, these remarkable responses only occur in a small fraction of patients. The use of combination therapies is therefore emerging as a popular strategy to further improve outcomes in melanoma patients. The challenge is to identify appropriate agents that can be rationally combined to improve patient responses. We hypothesized that a targeted therapeutic that also altered the immunoregulatory milieu in a tumor might best synergize with an immunotherapy. Toward this end, high-throughput flow cytometry-based screening was performed with two large and diverse drug libraries to identify candidate therapies that augment T cell immunotherapy efficacy. Two lead therapies, regorafenib and NU7441, were selected based on their ability to alter a variety of immunomodulatory proteins, including CD55, CD73, CD155, programmed death ligand 1 (PD-L1), nerve growth factor receptor (NGFR), and human leukocyte antigen (HLA) class I in a heterogeneous panel of melanomas. The therapies were also found to upregulate several melanoma antigens, inhibit proliferation, and perturb commonly activated oncogenic signaling pathways in melanomas. T cells treated with the therapies proliferated normally and expressed increased CD25, CD28, inducible T cell costimulator (ICOS), and reduced co-inhibitory receptors. These phenotypic changes were associated with improved T cell function. In murine models, the compounds suppressed melanoma progression and altered various T cell subsets in the tumor microenvironment when used with and without various immunotherapies. Finally, regorafenib skewed intratumoral macrophages away from a phenotype associated with pro-tumor function. Collectively, these studies demonstrate for the first time that regorafenib and NU7441 influence the expression of immunomodulatory proteins on both tumor cells and T cells, and enhance the efficacy of various immunotherapies.
    • MyD88 Co-stimulation in CD8+ T Cells Improves Tumor Immunotherapy in Allogeneic and Syngeneic Models

      Ciavattone, Nicholas; Cao, Xuefang; Davila, Eduardo, Ph.D.; 0000-0001-9265-8202 (2021)
      Improving the efficacy of T cell therapies for solid tumors and leukemias could improve clinical outcomes. In leukemia, allogeneic hematopoietic cell transplantation and donor lymphocyte infusions can be potentially curative, however, tumor evasion of the graft-versus-leukemia effects still limit their efficacy. In the solid tumor microenvironment, immune suppressive myeloid cells inhibit T cell activation which can be detrimental to anti-tumor T cell responses. Toll-like Receptor/MyD88 signaling in cytotoxic T cells can provide a strong co-stimulation signal to improve T cell activation, function, and efficacy to counteract evasion mechanisms in hematopoietic and solid tumors. Unfortunately, TLR agonists lack specificity to T cells and may induce hyper inflammation or induce pro-tumor effects. Rather than provide TLR agonist therapy in leukemia and solid tumors, there exists potential to modify or engineer T cells to provide a direct MyD88 co-stimulus. Our research group developed a CD8α:MyD88 T cell co-receptor that mimics TLR co-stimulation in conjunction with T cell receptor activation. Our goal was to first determine whether this engineered T cell co-receptor could enhance graft-versus-leukemia responses in allogeneic hematopoietic cell transplantation and donor lymphocyte infusion therapies. In a suppressive solid tumor, we asked if MyD88 co-receptor could improve T cell activation and function in a suppressive tumor microenvironment. Using multiple experimental transplant models, we found that MyD88 co-stimulation in donor CD8+ T cells could improve the graft versus tumor response with some non-lethal increases in graft-versus-host disease. Looking further, we found that the CD8α:MyD88 co-receptor increased donor cytotoxic T cell proliferation, survival, and function in vivo. Donor CD8α:MyD88 T cells were able to directly kill tumor better than transduced controls. In the second part of this project we found that cytotoxic T cells receiving a synthetic MyD88 co-stimulation maintained strong basal activation in the presence of myeloid suppression. In the suppressive B16-GMCSF melanoma model, CD8α:MyD88 T cell were able to control tumor growth and reduce populations of suppressive myeloid cells in the tumor. These data show that augmented MyD88 co-stimulation in cytotoxic T cells could benefit patients undergoing both autologous and allogeneic T cell therapies.