Joannes F.M. Jacobs

Dendritic Cell Vaccination in Combination with Anti-CD25 Monoclonal Antibody Treatment: A Phase I/II Study in Metastatic Melanoma Patients

Purpose: The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody–mediated depletion of CD25high regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. Experimental Design: Thirty HLA-A2.1+ metastatic melanoma patients were vaccinated with mature dendritic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patients were pretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor α-chain (CD25), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parameters were determined. Results: Daclizumab efficiently depleted all CD25high immune cells, including CD4+FoxP3+CD25high cells, from the peripheral blood within four days of administration. Thirty days after administration, daclizumab was cleared from the circulation and all CD25+ cells reappeared. The presence of daclizumab during dendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the presence of antigen-specific T cells. Daclizumab, however, did prevent these CD25+ T cells from acquiring effector functions. Consequently, significantly less patients pretreated with daclizumab developed functional, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatment had no significant effect on progression-free survival compared with the control group. Conclusions: Although daclizumab depleted the CD4+FoxP3+CD25high Tregs from the peripheral circulation, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionally suppressed de novo induced CD25+ effector cells during dendritic cell vaccinations. Our results indicate that for immunotherapeutic benefit of transient Treg depletion, timing and dosing as well as Treg specificity are extremely important. Clin Cancer Res; 16(20); 5067–78. ©2010 AACR.

Limited amounts of dendritic cells migrate into the T-cell area of lymph nodes but have high immune activating potential in melanoma patients.

Purpose: The success of immunotherapy with dendritic cells (DC) to treat cancer is dependent on effective migration to the lymph nodes and subsequent activation of antigen-specific T cells. In this study, we investigated the fate of DC after intradermal (i.d.) or intranodal (i.n.) administration and the consequences for the immune activating potential of DC vaccines in melanoma patients. Experimental Design: DC were i.d. or i.n. administered to 25 patients with metastatic melanoma scheduled for regional lymph node resection. To track DC in vivo with scintigraphic imaging and in lymph nodes by immunohistochemistry, cells were labeled with both [111In]-indium and superparamagnetic iron oxide. Results: After i.d. injection, maximally 4% of the DC reached the draining lymph nodes. When correctly delivered, all DC were delivered to one or more lymph nodes after i.n. injection. Independent of the route of administration, large numbers of DC remained at the injection site, lost viability, and were cleared by infiltrating CD163+ macrophages within 48 hours. Interestingly, 87 ± 10% of the surviving DC preferentially migrated into the T-cell areas, where they induced antigen-specific T-cell responses. Even though more DC reached the T-cell areas, i.n. injection of DC induced similar antigen-specific immune responses as i.d. injection. Immune responses were already induced with <5 × 105 DC migrating into the T-cell areas. Conclusions: Monocyte-derived DC have high immune activating potential irrespective of the route of vaccination. Limited numbers of DC in the draining lymph nodes are sufficient to induce antigen-specific immunologic responses.

Prognostic significance and mechanism of Treg infiltration in human brain tumors.

Regulatory T cells (Tregs) accumulate in tumors and can contribute to the dismal immune responses observed in these tumors. We reported that the percentage of tumor infiltrating Tregs is strongly correlated with the WHO grade of the brain tumor. We now report on the clinical follow-up of this patient cohort (n=83). Subgroup analyses in patients with glioblastomas (n=29) showed a moderate, not significant, inverted association between Tregs and survival. We further show that Tregs in glioblastomas, in contrast to other tumor infiltrating effector lymphocytes, highly express the CCR4 chemokine receptor. Moreover, the CCR4 ligand CCL22 is secreted by glioblastomas and may explain the specific Treg accumulation seen in these tumors.

Regulatory T cells in melanoma: the final hurdle towards effective immunotherapy?

Immunotherapy studies in patients with melanoma have reported success in the expansion of tumour-specific effector T cells in vivo, but even in the presence of substantial numbers of functional T cells circulating in the blood, favourable clinical outcomes are scarce. This failure to induce robust clinical responses might be related to tumour-induced immune evasion, rendering the host tolerant to melanoma antigens. Immunosuppression in the tumour microenvironment mediated by regulatory T cells (Treg) is a dominant mechanism of tumour immune escape and is a major hurdle for tumour immunotherapy. Accumulation of Treg in melanoma is frequently recorded and the ratio of CD8-positive T cells versus Treg in the tumour microenvironment is predictive for survival of patients with melanoma. Hence, depletion of Treg seems to be a promising strategy for the enhancement of melanoma-specific immunity. Indeed, murine studies have shown that Treg depletion greatly increases the efficacy of immunotherapy. But despite the success of some strategies in depletion of Treg in patients, overall clinical efficacy has been disappointing. The lack of Treg specificity of the Treg depleting strategies applied so far imply that well-designed studies into dosage, timing, and administration regimens with more specific agents are urgently needed. Depletion of functional Treg from the tumour microenvironment as part of multifaceted immunotherapeutic treatments is a major challenge to induce clinically relevant immune responses against melanomas.

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.

Regulatory T cells and the PD-L1/PD-1 pathway mediate immune suppression in malignant human brain tumors

The brain is a specialized immune site representing a unique tumor microenvironment. The availability of fresh brain tumor material for ex vivo analysis is often limited because large parts of many brain tumors are resected using ultrasonic aspiration. We analyzed ultrasonic tumor aspirates as a biosource to study immune suppressive mechanisms in 83 human brain tumors. Lymphocyte infiltrates in brain tumor tissues and ultrasonic aspirates were comparable with respect to lymphocyte content and viability. Applying ultrasonic aspirates, we detected massive infiltration of CD4+FoxP3+CD25(high) CD127(low) regulatory T cells (Tregs) in glioblastomas (n = 29) and metastatic brain tumors (n = 20). No Treg accumulation was observed in benign tumors such as meningiomas (n = 10) and pituitary adenomas (n = 5). A significant Treg increase in blood was seen only in patients with metastatic brain tumors. Tregs in high-grade tumors exhibited an activated phenotype as indicated by decreased proliferation and elevated CTLA-4 and FoxP3 expression relative to blood Tregs. Functional analysis showed that the tumor-derived Tregs efficiently suppressed cytokine secretion and proliferation of autologous intratumoral lymphocytes. Most tumor-infiltrating Tregs were localized in close proximity to effector T cells, as visualized by immunohistochemistry. Furthermore, 61% of the malignant brain tumors expressed programmed death ligand-1 (PD-L1), while the inhibitory PD-1 receptor was expressed on CD4+ effector cells present in 26% of tumors. In conclusion, using ultrasonic tumor aspirates as a biosource we identified Tregs and the PD-L1/PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumors.

Route of Administration Modulates the Induction of Dendritic Cell Vaccine–Induced Antigen-Specific T Cells in Advanced Melanoma Patients

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

Targeting CD4+ T-Helper Cells Improves the Induction of Antitumor Responses in Dendritic Cell―Based Vaccination

To evaluate the relevance of directing antigen-specific CD4(+) T helper cells as part of effective anticancer immunotherapy, we investigated the immunologic and clinical responses to vaccination with dendritic cells (DC) pulsed with either MHC class I (MHC-I)-restricted epitopes alone or both MHC class I and II (MHC-I/II)-restricted epitopes. We enrolled 33 stage III and IV HLA-A*02:01-positive patients with melanoma in this study, of whom 29 were evaluable for immunologic response. Patients received intranodal vaccinations with cytokine-matured DCs loaded with keyhole limpet hemocyanin and MHC-I alone or MHC-I/II-restricted tumor-associated antigens (TAA) of tyrosinase and gp100, depending on their HLA-DR4 status. In 4 of 15 patients vaccinated with MHC-I/II-loaded DCs and 1 of 14 patients vaccinated with MHC-I-loaded DCs, we detected TAA-specific CD8(+) T cells with maintained IFN-γ production in skin test infiltrating lymphocyte (SKIL) cultures and circulating TAA-specific CD8(+) T cells. If TAA-specific CD4(+) T-cell responses were detected in SKIL cultures, it coincided with TAA-specific CD8(+) T-cell responses. In 3 of 13 patients tested, we detected TAA-specific CD4(+)CD25(+)FoxP3(-) T cells with high proliferative capacity and IFN-γ production, indicating that these were not regulatory T cells. Vaccination with MHC-I/II-loaded DCs resulted in improved clinical outcome compared with matched control patients treated with dacarbazine (DTIC), median overall survival of 15.0 versus 8.3 months (P = 0.089), and median progression-free survival of 5.0 versus 2.8 months (P = 0.0089). In conclusion, coactivating TAA-specific CD4(+) T-helper cells with DCs pulsed with both MHC class I and II-restricted epitopes augments TAA-specific CD8(+) T-cell responses, contributing to improved clinical responses.

PD-1 Blockade Augments Th1 and Th17 and Suppresses Th2 Responses in Peripheral Blood From Patients With Prostate and Advanced Melanoma Cancer

Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon &ggr;, interleukin (IL)-2, tumor necrosis factor &agr;, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.

Sorafenib reduces the percentage of tumour infiltrating regulatory T cells in renal cell carcinoma patients

Tyrosine kinase inhibitors (TKIs) as sorafenib are known to reduce the number of immunosuppressive regulatory T cells (Tregs) in the peripheral blood and thereby shifting the immune balance to a more stimulating setting. The effect of sorafenib on intratumoural Tregs is unclear but important for future combinations of TKIs and immunotherapy. We, therefore, evaluated the accumulation of regulatory T cells (Tregs, defined as, CD4+FoxP3+CD25highCD127low‐cells) in blood, ascites, metastases and primary tumours of patients with renal cell carcinoma (RCC), and we explored the effect of neoadjuvant treatment with sorafenib 400 mg bid on intratumoural Tregs in 11 patients with RCC in comparison to 15 nontreated RCC patients. We found that immunosuppressive Tregs specifically accumulate in primary tumour, metastases and ascites of RCC‐patients. Tumour infiltrating Tregs were functional. Neoadjuvant sorafenib treatment significantly reduced the percentage of tumour‐infiltrating Tregs (mean 17.3% vs. 28.1%, p = 0.046). Diminished Treg accumulation at the tumour site adds to explain the clinical effectiveness of sorafenib treatment. This observation may have important implications for the use of sorafenib in combination with immunotherapy in patients with RCC, since the depletion of Tregs has been associated with enhanced responses on vaccine mediated immunotherapy.