Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy.

Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3+ DC (cDC1) and CD1c/BDCA1+ DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy.

Adjuvant dendritic cell therapy in stage IIIB/C melanoma: the MIND-DC randomized phase III trial.

Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88-1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73-2.38; p = 0.44). Grade 3-4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p < 0.001). In conclusion, while adjuvant nDC treatment in stage IIIB/C melanoma patients generated specific immune responses and was well tolerated, no benefit in RFS was observed.

Adjuvant dendritic cell vaccination induces tumor-specific immune responses in the majority of stage III melanoma patients.

PURPOSE: To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients. EXPERIMENTAL DESIGN: Retrospective analysis of stage III melanoma patients, vaccinated with autologous monocyte-derived DC loaded with tumor-associated antigens (TAA) gp100 and tyrosinase after radical lymph node dissection. Skin-test infiltrating lymphocytes (SKILs) obtained from delayed-type hypersensitivity skin-test biopsies were analyzed for the presence of TAA-specific CD8(+) T cells by tetrameric MHC-peptide complexes and by functional TAA-specific T cell assays, defined by peptide-recognition (T2 cells) and/or tumor-recognition (BLM and/or MEL624) with specific production of Th1 cytokines and no Th2 cytokines. RESULTS: Ninety-seven patients were analyzed: 21 with stage IIIA, 34 with stage IIIB, and 42 had stage IIIC disease. Tetramer-positive CD8(+) T cells were present in 68 patients (70%), and 24 of them showed a response against all 3 epitopes tested (gp100:154-162, gp100:280-288, and tyrosinase:369-377) at any point during vaccinations. A functional T cell response was found in 62 patients (64%). Rates of peptide-recognition of gp100:154-162, gp100:280-288, and tyrosinase:369-377 were 40%, 29%, and 45%, respectively. Median recurrence-free survival and distant metastasis-free survival of the whole study population were 23.0 mo and 36.8 mo, respectively. CONCLUSIONS: DC vaccination induces a functional TAA-specific T cell response in the majority of stage III melanoma patients, indicating it is more effective in stage III than in stage IV melanoma patients. Furthermore, performing multiple cycles of vaccinations enhances the chance of a broader immune response.

Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity.

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktail.

Effective Clinical Responses in Metastatic Melanoma Patients after Vaccination with Primary Myeloid Dendritic Cells.

PURPOSE: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro-generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c(+) myeloid DCs, naturally circulating in the blood. EXPERIMENTAL DESIGN: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c(+) myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100. RESULTS: Our results show that therapeutic vaccination against melanoma with small amounts (3-10 × 10(6)) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12-35 months), which directly correlated with the development of multifunctional CD8(+) T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth. CONCLUSIONS: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155-66. ©2015 AACR.

Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients.

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×106 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100.

Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice.

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

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 (51)Cr 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.

The lymphoid chemokine CCL21 triggers LFA-1 adhesive properties on human dendritic cells.

Dendritic cells (DCs) are the most potent APCs, involved in the induction of immunity and tolerance. Recently we showed that during differentiation of human DCs from monocyte precursors, Lymphocyte function-associated antigen-1 (LFA-1)-binding capacity is lost, although integrin expression levels were maintained constant, suggesting a different regulation mechanism of this integrin on different cell types. However, the exact role of LFA-1 in DC adhesion and migration remains obscure. Chemokines are potent regulators of integrin function, influencing migratory and adhesive properties of leukocytes. Here, we show that upon vaccination of cancer patients with human DCs, cells that have migrated in vivo into the lymph nodes upregulated the active form of LFA-1. We further show that exposure of human DCs to the lymphoid chemokine CCL21 specifically restores the high-affinity form of LFA-1 and induces binding to its ligand ICAM-1 under low shear stress. Our data indicate that on DCs LFA-1 may function as an inducible anchor during lymphatic transmigration or within the lymph nodes. A thorough understanding of the adhesive events during the DC life cycle will help to improve the outcome of DC-based antitumor clinical trials.

Immunogenicity of dendritic cells pulsed with CEA peptide or transfected with CEA mRNA for vaccination of colorectal cancer patients.

BACKGROUND: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. PATIENTS AND METHODS: In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All DCs were loaded with keyhole limpet hemocyanin (KLH) as a control protein. Evaluation of vaccine-induced immune reactivity consisted of T-cell proliferative responses and B-cell antibody responses against KLH in peripheral blood. CEA reactivity was determined in T-cell cultures of biopsies of post-treatment delayed type hypersensitivity skin tests. RESULTS: Sixteen patients were included. All patients showed T-cell responses against KLH upon vaccination. CEA peptide-specific T-cells were detected in 8 out of 11 patients in the peptide group, but in none of the 5 patients in the RNA group. CONCLUSION: In our study, DC CEA mRNA transfection was not superior to DC CEA peptide pulsing in the induction of a tumor-specific immune response in colorectal cancer patients.

In situ expression of tumor antigens by messenger RNA-electroporated dendritic cells in lymph nodes of melanoma patients.

Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8(+) T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

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.

Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination.

UNLABELLED: Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. METHODS: The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. RESULTS: The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. CONCLUSION: When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.

Analysis of dendritic cell trafficking using EGFP-transgenic mice.

Dendritic cells (DCs) are professional antigen presenting cells, well equipped to initiate an immune response. For effective induction of an immune response, DC should migrate from the periphery to the lymph node to present the antigen to T lymphocytes. Currently, tumor-antigen loaded DCs are used in clinical vaccination trials in cancer patients. To investigate the migratory capacity of DC in vivo, a variety of fluorescent and radioactive labels have been used. Here we introduce a novel tool to study DC migration in vivo: DCs generated from enhanced green fluorescent protein (EGFP)-transgenic mice. DC from EGFP-transgenic mice display typical DC behavior and can be matured without affecting their autofluorescence in vitro. In addition, the continuously produced cytoplasmic EGFP in living cells functions as a viability marker, since EGFP released from dying cells does not stain DC from C57Bl/6 mice upon coculture. In vivo migration studies using EGFP-DC and indium-111-labeled DC were performed to determine the efficiency of i.d. versus s.c. administered DC to reach the draining lymph node. The analysis demonstrates that i.d. injection increases the amount of EGFP-DC/indium-111-labeled DC in the lymph node compared to s.c. injection. Subsequent quantitative, phenotypical and ultrastuctural analysis demonstrate that DC generated from EGFP-transgenic mice are well suited to study the migratory behavior, distribution and phenotype of DC in vivo.