Weiyi Peng

BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. Results: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

Loss of PTEN promotes resistance to T cell–mediated immunotherapy

UNLABELLED T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.

Persistent antigen at vaccination sites induces tumor-specific CD8+ T cell sequestration, dysfunction and deletion

To understand why cancer vaccine–induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freunds adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8+ T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

BRAF Inhibition Increases Tumor Infiltration by T cells and Enhances the Antitumor Activity of Adoptive Immunotherapy in Mice

Purpose: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. Experimental Design: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen–independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. Results: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. Conclusion: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell–based immunotherapy for the treatment of patients with melanoma. Clin Cancer Res; 19(2); 393–403. ©2012 AACR.

PD-1 BLOCKADE ENHANCES T CELL MIGRATION TO TUMORS BY ELEVATING IFN-γ INDUCIBLE CHEMOKINES

Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements, many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor programmed cell death-1 (PD-1) expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. As PD-1 can attenuate T-cell-mediated antitumor responses, we tested whether its blockade with an anti-PD-1 antibody could enhance the antitumor activity of ACT in this model. Cotreatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared with treatments with either agent alone. While anti-PD-1 did not reduce the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow-transplant experiments using IFN-γR-/- mice implicated IFN-γ as a crucial nexus for controlling PD-1-mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites.

Transduction of Tumor-Specific T Cells with CXCR2 Chemokine Receptor Improves Migration to Tumor and Antitumor Immune Responses

Purpose: One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Because melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT by enhancing T-cell migration to tumor. Experimental Design: In this study, we used transgenic pmel-1 T cells, which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. To visualize luciferase-expressing T cells within a tumor, a nonpigmented tumor is required. Therefore, we used the MC38 tumor model, which naturally expresses CXCL1. Results: Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared with mice receiving control luciferase-transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared with mice treated with control pmel-1 T cells. Conclusions: These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors. Clin Cancer Res; 16(22); 5458–68. ©2010 AACR.

Antitumor T-cell responses contribute to the effects of dasatinib on c-KIT mutant murine mastocytoma and are potentiated by anti-OX40

Targeted and immune-based therapies are thought to eradicate cancer cells by different mechanisms, and these approaches could possibly complement each other when used in combination. In this study, we report that the in vivo antitumor effects of the c-KIT inhibitor, dasatinib, on the c-KIT mutant P815 mastocytoma tumor were substantially dependent on T cell-mediated immunity. We found that dasatinib treatment significantly decreased levels of Tregs while specifically enhancing tumor antigen-specific T-cell responses. We sought to further enhance this therapy with the addition of anti-OX40 antibody, which is known to provide a potent costimulatory signal to T cells. The combination of dasatinib and anti-OX40 antibody resulted in substantially better therapeutic efficacy compared with either drug alone, and this was associated with enhanced accumulation of tumor antigen-specific T cells in the tumor microenvironment. Furthermore, the combination regimen inhibited the function of Tregs and also resulted in significantly up-regulated expression of the IFN-γ-induced chemokines CXCL9, 10, and 11 in the tumor microenvironment, which provides a feasible mechanism for the enhanced intratumoral CTL infiltration. These studies delineate a strategy by which targeted therapy and immunotherapy may be combined to achieve superior antitumor responses in cancer patients.

Antitumor Activity Mediated by CpG: The Route of Administration is Critical

Unmethylated CpG oligodeoxynucleotides (CpG) are synthetic toll-like receptor 9 agonists that activate innate immune cells and which have been tested as an immune therapy in a number of cancer clinical trials. Although some antitumor immune responses have been reported, so far the majority of studies have failed to show significant clinical responses to CpG. Here we showed that the route of administration is critical to the antitumor activity of CpG. Although intravenous (i.v.) injection of CpG was capable of inducing the activation and expansion of tumor antigen-specific T cells, most of these activated T cells failed to migrate to tumor sites. By contrast, intratumoral (i.t.) injection of CpG led to extensive tumor infiltration of antigen-specific T cells and subsequent tumor suppression. We further showed that very high levels of inflammatory chemokines [regulated upon activation, normal T-cell expressed, and secreted (RANTES), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, monocyte chemotactic protein (MCP5), macrophage inflammatory proteins (MIP1&agr;, and MIP1&bgr;)] were induced in the tumor microenvironment after i.t. CpG injection, compared with administration by the i.v. route. It is interesting to note that, in vivo depletion of plasmacytoid dendritic cells greatly reduced the levels of chemokines induced; also, T-cell accumulation and antitumor effect were impaired. We also showed that i.t. but not i.v. CpG injection induced a broad antigen-specific T-cell response against tumor-derived antigens. Collectively, our data provides evidence that the route of CpG administration is a critical factor in mediating antitumor activity. By inducing localized inflammatory signals at tumor sites, i.t. CpG effectively promotes the migration, activation and function of immune cells, ultimately leading to improved tumor control.

Blockade of the PD-1 pathway enhances the efficacy of adoptive cell therapy against cancer

The blockade of the PD-1 pathway can increase the production of interferon γ by tumor-specific T cells located within or in the proximity of the malignant lesion, thereby increasing the chemokine-dependent trafficking of immune effector cells. This can boost the efficacy of adoptive T-cell therapy to achieve superior antitumor responses.

Agonistic antibody to CD40 boosts the antitumor activity of adoptively transferred T cells in vivo

CD40, a member of the tumor necrosis factor receptor superfamily, is broadly expressed on antigen-presenting cells and other cells, including fibroblasts and endothelial cells. Binding of CD40 and its natural ligand CD40L (CD154) triggers cytokine secretion, and increased expression of costimulatory molecules is required for T-cell activation and proliferation. However, to our knowledge, the use of agonistic antibodies to CD40 to boost adoptively transferred T cells in vivo has not been investigated. The purpose of this study was to determine whether anti-CD40 monoclonal antibody (mAb) in combination with interleukin (IL)-2 could improve the efficacy of in vitro-activated T cells to enhance antitumor activity. Mice bearing B16 melanoma tumors expressing the gp100 tumor antigen were treated with cultured, activated T cells transgenic for a T-cell receptor specifically recognizing gp100, with or without anti-CD40 mAb. In this model, the combination of anti-CD40 mAb with IL-2 led to expansion of adoptively transferred T cells and induced a more robust antitumor response. Furthermore, the expression of CD40 on bone marrow-derived cells and the presence of CD80/CD86 in the host were required for the expansion of adoptively transferred T cells. The use of neutralizing mAb to IL-12 provided direct evidence that enhanced IL-12 secretion induced by anti-CD40 mAb was crucial for the expansion of adoptively transferred T cells. Collectively, these findings provide a rationale to evaluate the potential application of anti-CD40 mAb in adoptive T-cell therapy for cancer.