Combination CTLA-4 Blockade and 4-1BB Activation Enhances Tumor Rejection by Increasing T-Cell Infiltration, Proliferation, and Cytokine Production

Howard Hughes Medical Institute, Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
PLoS ONE (Impact Factor: 3.23). 04/2011; 6(4):e19499. DOI: 10.1371/journal.pone.0019499
Source: PubMed


The co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.
We find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.
This study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

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    • "Adding anti–CTLA-4 further increased the potency of this approach, resulting in ~80% tumor rejection of established mammary 4 T1 tumors in mice [46]. Combining CTLA-4 blockade and 4-1BB co-stimulation with a granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting melanoma vaccine greatly improved tumor eradication and promoted survival compared with vaccine plus either agent [47]. Combination treatment increased proliferation and tumor infiltration by both CD4+ and CD8+ T cells, and intratumor inflammatory cytokine production. "
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    ABSTRACT: With the success of ipilimumab and promise of programmed death-1 pathway-targeted agents, the field of tumor immunotherapy is expanding rapidly. Newer targets for clinical development include select members of the tumor necrosis factor receptor (TNFR) family. Agonist antibodies to these co-stimulatory molecules target both T and B cells, modulating T-cell activation and enhancing immune responses. In vitro and in vivo preclinical data have provided the basis for continued development of 4-1BB, OX40, glucocorticoid-induced TNFR-related gene, herpes virus entry mediator, and CD27 as potential therapies for patients with cancer. In this review, we summarize the immune response to tumors, consider preclinical and early clinical data on select TNFR family members, discuss potential translational challenges and suggest possible combination therapies with the aim of inducing durable antitumor responses.
    04/2014; 2(1):7. DOI:10.1186/2051-1426-2-7
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    • "However, CD8+ T cells do not always traffic into tissues, which has been a special problem when CD8+ T cells are used for tumor immunotherapy [68]. While BAFF has not been studied in this context, it is interesting that 4-1BB stimulation has been shown to promote CD8+ T cell entry into the tumor microenvironment [69], [70]. While these studies used an agonistic anti-4-1BB antibody, the present report shows how a vaccine strategy using SP-D-4-1BBL can promote lymphocyte migration into diseased tissues. "
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    PLoS ONE 02/2014; 9(2):e90100. DOI:10.1371/journal.pone.0090100 · 3.23 Impact Factor
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    • "Consistent with previous studies [30], [39], [40], single OX40 triggering significantly promoted the accumulation of CD4+FoxP3+ Treg and mildly increased the frequencies of peritoneal CD4+ and CD8+ T cells. Incorporating the decreasing effect of PD-1 blockade on the immunosuppressive Treg and MDSC and promoting effect of OX40 triggering on the effector CD4+ and CD8+ T cells, combined anti-PD-1/OX40 mAb treatment significantly enhanced the accumulation of peritoneal effector CD4+ and CD8+ T cells with concomitantly attenuating Treg and MDSC, giving rise to a favorable ratio of the effector T cells to the immunosuppressive cells which is closely related with the effective immunotherapy as previously stated [41], [42]. Consistent with the roles of OX40 and PD-1 in memory T-cell formation [23], [43], individual PD-1 blockade or OX40 triggering modestly increased the percentage of CD44+CD62L− effector/memory and/or CD44+CD62L+ central memory T cells; importantly, combined anti-PD-1/OX40 mAb synergistically promoted the development of effector/memory and central memory T cells in peritoneal cavity, which constitutes the basis for the resistance of long-term surviving mice from 2 mAb treated group to the same tumor rechallenge. "
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    ABSTRACT: The co-inhibitory receptor Programmed Death-1 (PD-1) curtails immune responses and prevent autoimmunity, however, tumors exploit this pathway to escape from immune destruction. The co-stimulatory receptor OX40 is upregulated on T cells following activation and increases their clonal expansion, survival and cytokine production when engaged. Although antagonistic anti-PD-1 or agonistic anti-OX40 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In the present study, we evaluated the antitumor effects and mechanisms of combinatorial PD-1 blockade and OX40 triggering in a murine ID8 ovarian cancer model. Although individual anti-PD-1 or OX40 mAb treatment was ineffective in tumor protection against 10-day established ID8 tumor, combined anti-PD-1/OX40 mAb treatment markedly inhibited tumor outgrowth with 60% of mice tumor free 90 days after tumor inoculation. Tumor protection was associated with a systemic immune response with memory and antigen specificity and required CD4(+) cells and CD8(+) T cells. The anti-PD-1/OX40 mAb treatment increased CD4(+) and CD8(+) cells and decreased immunosuppressive CD4(+)FoxP3(+) regulatory T (Treg) cells and CD11b(+)Gr-1(+) myeloid suppressor cells (MDSC), giving rise to significantly higher ratios of both effector CD4(+) and CD8(+) cells to Treg and MDSC in peritoneal cavity; Quantitative RT-PCR data further demonstrated the induction of a local immunostimulatory milieu by anti-PD-1/OX40 mAb treatment. The splenic CD8(+) T cells from combined mAb treated mice produced high levels of IFN-γ upon tumor antigen stimulation and exhibited antigen-specific cytolytic activity. To our knowledge, this is the first study testing the antitumor effects of combined anti-PD-1/OX40 mAb in a murine ovarian cancer model, and our results provide a rationale for clinical trials evaluating ovarian cancer immunotherapy using this combination of mAb.
    PLoS ONE 02/2014; 9(2):e89350. DOI:10.1371/journal.pone.0089350 · 3.23 Impact Factor
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