4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines
Introduction
The magnitude of antigen specific T cell activation has been a focus of vaccine therapy for cancer. The interaction of the costimulatory molecule B7 with its ligand CD28 has been the most comprehensively investigated with several studies demonstrating potent anti-tumor immunity when B7.1 is co-expressed with tumor-associate antigens. This suggests that engagement of costimulatory molecules normally expressed on antigen-presenting cells (APCs) with cognate ligands on T cells could be an important factor in tumor eradication [1]. There have been few studies evaluating the effects of combining costimulatory molecules that target distinct signaling pathways within naïve T cells.
The TNF/TNFR family is a large group of related molecules that can act as costimulatory molecules for activation and maintenance of T cells [2]. In this family, 4-1BBL is a well-characterized costimulatory molecule expressed by APC, including B cells, macrophages, and DCs [3]. APC-expressed 4-1BBL binds to 4-1BB (CD137), a member of the TNFR superfamily expressed on monocytes, neutrophils, DC, NK cells and recently activated T cells. 4-1BB signaling has profound effects on T cells, including activation of both CD4+ and CD8+ T cells, increased levels of cytokine secretion [4], [5], enhanced clonal expansion [4], [6], [7] and increased survival [6], [7], [8]. Recent studies have noted strong anti-tumor effects mediated by 4-1BB stimulation using agonistic anti-4-1BB antibodies [9], [10], [11], [12] and adenoviruses expressing 4-1BBL [13], [14], [15]. Previous reports suggested that 4-1BB was superior to B7.1/CD28 signaling for stimulation of CD8+ T cell expansion [16] and that 4-1BB might activate T cells in the absence of B7.1/CD28 interactions [17], [18], [19] or APC stimulation [5]. Other reports, however, have demonstrated that combining B7.1/CD28 stimulation and 4-1BB signaling was required for optimal induction of CD25 and bcl-XL expression in CD8+ T cells [5], increased CTL activity [20] and protective anti-tumor immunity [21].
We have previously demonstrated that the addition of ICAM-1 and LFA-3 to replication-competent poxviruses expressing B7.1 improves antigen-specific therapeutic responses in murine models, especially when low levels of Signal 1 are provided [22]. In this study we describe the additional benefit of expressing 4-1BBL with a triad of costimulatory molecules, including B7.1, ICAM-1, and LFA-3 (designated TRICOM) in a carcinoembryonic antigen (CEA) transgenic mouse model. A recombinant vaccinia virus containing 4-1BBL transgene was generated (rV-4-1BBL) and resulted in enhanced T cell proliferation and survival in co-culture in vitro assays of virus-infected DC and naïve T cells. The rV-4-1BBL vaccine induced non-specific tumor growth arrest in an established murine tumor treatment model. When rV-4-1BBL was combined with a vaccinia virus expressing CEA and TRICOM a significant enhancement in responses were observed with a single immunization against both 4- and 7-day established CEA-bearing tumors in CEA-transgenic mice. This was associated with an increased level of CEA-specific CD4+ T cell proliferation, Th1 cytokine release, improved CD8+ T cell cytotoxicity and avidity, and induction of an antigen cascade against other tumor-associated antigens, i.e. p53 and gp70. In addition, the use of 4-1BBL with TRICOM resulted in increased expression of the anti-apoptotic proteins bcl-XL and bcl-2 in both CD4+ and CD8+ T cells and accumulation of CEA-specific T cells within the tumor microenvironment. These results have implications for the rational design of more potent vaccines in the treatment of established human tumors.
Section snippets
Animals and cell lines
Female C57BL/6 mice transgenic for human CEA were obtained from a breeding pair, provided by Dr. John Thompson (Institute of Immunobiology, University of Freiburg, Germany). The generation and characterization of the CEA-transgenic mouse has been previously described [23], [24]. Female C57BL/6 and BALB/c mice were obtained from the National Cancer Institute-Frederick Cancer Research Animal Facility (Frederick, MD). Mice were housed and maintained under pathogen-free conditions in microisolator
Characterization of rV-4-1BBL
We constructed a recombinant vaccinia virus expressing the full-length murine 4-1BBL gene. To confirm 4-1BBL protein expression, immature bone marrow-derived DCs were infected with rV-4-1BBL at MOI = 1 for 16 h. More than 30% of infected DCs expressed 4-1BBL on their surfaces by flow cytometry analysis (Fig. 1A). There was a dose-dependent increase in the percentage of 4-1BBL-positive cells with 60% of DCs expressing 4-1BBL at an MOI of 4 (data not shown). The expression of 4-1BBL was not noted on
Discussion
4-1BBL–4-1BB costimulation activates T cells through increased cytokine production [4], [5], enhanced clonal expansion [4], [6], [7] and induction of anti-apoptotic molecule expression [6], [7], [8]. Here, we extended these observations to a therapeutic poxvirus vaccine system in vitro utilizing DCs infected with rV-4-1BBL and in vivo utilizing T cells from tumor-bearing mice vaccinated with rV-4-1BBL. The results are important since poxviruses are currently in Phase III clinical trials and
Acknowledgments
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and in part by NIH grant K08 CA79881 (H. Kaufman).
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2014, Molecular Therapy OncolyticsCitation Excerpt :Combination of lentivirus-encoded tumor antigen vaccine with PD-1 or PD-L1 blocking antibodies has similarly been demonstrated to result in enhanced vaccination efficacy and improved tumor control.114 Expression of 4-1BB ligand (4-1BBL) by vaccinia virus has been shown to enhance the efficacy of vaccination of another vaccinia vector carrying the genes for CEA, B7.1, ICAM-1, and LFA-3 (rV-CEA-TRICOM).115 Similarly, combination of the recombinant vaccinia and fowlpox-based CEA-TRICOM vaccines with systemic CTLA-4 blockade led to enhanced antitumor immunity.116
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2014, Advances in Cancer ResearchCitation Excerpt :Initial studies demonstrated that the admixing of recombinant vaccinia (rV)-TAA with rV-B7.1 resulted in enhanced TAA-specific T-cell responses and antitumor immunity compared to either vector alone (Hodge et al., 1995; Kalus et al., 1999). Additional studies were conducted with recombinant vaccinia viruses containing other T-cell costimulatory molecules including LFA-3, CD70, ICAM-1, 4-1BBL, and OX-40L (Kudo-Saito et al., 2006; Lorenz, Kantor, Schlom, & Hodge, 1999a; Lorenz, Kantor, Schlom, & Hodge, 1999b; Uzendoski, Kantor, Abrams, Schlom, & Hodge, 1997). Each was shown to enhance antigen-specific T-cell responses, but the combined use of three specific costimulatory molecules (B7.1, ICAM-1, and LFA-3) acted synergistically to further enhance antigen-specific T-cell responses (Fig. 2.1).
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2013, Cancer Immunotherapy: Immune Suppression and Tumor Growth: Second EditionAntitumor immune response induced by DNA vaccine encoding human prostate-specific membrane antigen and mouse 4-1BBL
2010, UrologyCitation Excerpt :4-1BBL and its receptor 4-1BB induced on naive T cells after T cell receptor-major histocompatibility complex/peptide and CD80/86/CD28 ligation after interaction of cognate T cells with DCs are a pair of co-stimulatory molecules. Signaling through 4-1BB/4-1BBL enhances T cell expansion, augments T cell effector function (including cytokine release, CD4− mediated “help,” and CTL activity),27 prevents activation-induced apoptosis of CD8+ T cells,8 overcomes activation-induced nonresponsiveness in CD8+ T cells in vitro,28 and can break immunologic ignorance (promoting regression of poorly immunogenic tumors in vivo).29 Therefore, 4-1BBL could be a useful vaccine adjuvant.
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2010, Cancer LettersCitation Excerpt :Members of the TNFR superfamily such as CD27, CD30, CD40, 4-1BB (CD137) and OX40 have gained importance as costimulatory molecules delivering signals that prolong and propagate T-cell responses [12]. In this regard, engagement of CD137 can effect on T cells profoundly, including activation of both CD4+ and CD8+ T cells, enhanced expansion [13,14], increased long-term survival [15,16], and anti-apoptosis of activation-induced CD8+ T cells [17,18]. Costimulation through 4-1BB can also promote enhanced production of cytokines such as IL-2, IL-4 and IFN-γ [13,18].