A randomized controlled phase II clinical trial on mRNA electroporated autologous monocyte-derived dendritic cells (TriMixDC-MEL) as adjuvant treatment for stage III/IV melanoma patients who are disease-free following the resection of macrometastases.

BACKGROUND: Autologous monocyte-derived mRNA co-electroporated dendritic cells with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively activated TLR4 (caTLR4) (referred to as TriMixDC-MEL) have anti-tumor activity in advanced melanoma patients. We investigated the safety and activity of adjuvant TriMixDC-MEL in stage III/IV melanoma patients. MATERIALS AND METHODS: Forty-one patients were randomly assigned to treatment with TriMixDC-MEL (n = 21) and standard follow-up (n = 20). "Cross-over" was allowed at the time of non-salvageable recurrence. The primary endpoint was the percentage of patients alive and disease-free at 1-year. For a subset of patients, (formalin-fixed paraffin-embedded), tumor tissue samples were available for mRNA expression profiling and PD-L1 immunohistochemical staining. RESULTS: Baseline characteristics were well balanced. One-year after randomization, 71% of patients in the study arm were alive and free of disease compared to 35% in the control arm. After a median follow-up of 53 months (range 3-67), 23 patients experienced a non-salvageable melanoma recurrence (TriMixDC-Mel arm n = 9 and control arm n = 14).The median time to non-salvageable recurrence was superior in the TriMixDC-MEL arm (median 8 months (range 1-6) vs. not reached; log-rank p 0.044). TriMixDC-MEL-related adverse events (AE) consisted of transient local skin reactions, flu-like symptoms and post-infusion chills. No grade ≥ 3 AE's occurred. The mRNA expression profiling revealed four genes (STAT2, TPSAB1, CD9 and CSF2) as potential predictive biomarkers. CONCLUSION: TriMixDC-MEL id/iv as adjuvant therapy is tolerable and may improve the 1-year disease-free survival rate. Combination of optimized autologous monocyte-derived DC-formulations warrants further investigation in combination with currently approved adjuvant therapy options.

TriMix and tumor antigen mRNA electroporated dendritic cell vaccination plus ipilimumab: link between T-cell activation and clinical responses in advanced melanoma.

BACKGROUND: We previously reported that dendritic cell-based mRNA vaccination plus ipilimumab (TriMixDC-MEL IPI) results in an encouraging rate of tumor responses in patients with pretreated advanced melanoma. Here, we report the TriMixDC-MEL IPI-induced T-cell responses detected in the peripheral blood. METHODS: Monocyte-derived dendritic cells electroporated with mRNA encoding CD70, CD40 ligand, and constitutively active TLR4 (TriMix) as well as the tumor-associated antigens tyrosinase, gp100, MAGE-A3, or MAGE-C2 were administered together with IPI for four cycles. For 18/39 patients, an additional vaccine was administered before the first IPI administration. We evaluated tumor-associated antigen specific T-cell responses in previously collected peripheral blood mononuclear cells, available from 15 patients. RESULTS: Vaccine-induced enzyme-linked immunospot assay responses detected after in vitro T-cell stimulation were shown in 12/15 patients. Immune responses detected in patients with a complete or partial response were significantly stronger and broader, and exhibited a higher degree of multifunctionality compared with responses in patients with stable or progressive disease. CD8+ T-cell responses from patients with an ongoing clinical response, either elicited by TriMixDC-MEL IPI or on subsequent pembrolizumab treatment, exhibited the highest degree of multifunctionality. CONCLUSIONS: TriMixDC-MEL IPI treatment results in robust CD8+ T-cell responses in a meaningful portion of stage III or IV melanoma patients, and obviously in patients with a clinical response. The levels of polyfunctional and multiantigen T-cell responses measured in patients with a complete response, particularly in patients evidently cured after 5+ years of follow-up, may provide a benchmark for the level of immune stimulation needed to achieve a durable clinical remission. TRIAL REGISTRATION NUMBER: NCT01302496.

Single Domain Antibody-Mediated Blockade of Programmed Death-Ligand 1 on Dendritic Cells Enhances CD8 T-cell Activation and Cytokine Production.

Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1pos) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8pos) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.

Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent.

The PD-1:PD-L1 immune checkpoint axis is central in the escape of cancer cells from anticancer immune responses. Monoclonal antibodies (mAbs) specific for PD-L1 have been approved for treatment of various cancer types. Although PD-L1 blockade has proven its merit, there are still several aspects that require further attention to fully capitalize on its potential. One of these is the development of antigen-binding moieties that enable PD-L1 diagnosis and therapy. We generated human PD-L1 binding single domain antibodies (sdAbs) and selected sdAb K2, a sdAb with a high affinity for PD-L1, as a lead compound. SPECT/CT imaging in mice following intravenous injection of Technetium-99m (99mTc)-labeled sdAb K2 revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and relatively low kidney retention, which is a unique property for radiolabeled sdAbs. We further showed using surface plasmon resonance that sdAb K2 binds to the same epitope on PD-L1 as the mAb avelumab, and antagonizes PD-1:PD-L1 interactions. Different human cell-based assays corroborated the PD-1:PD-L1 blocking activity, showing enhanced T-cell receptor signaling and tumor cell killing when PD-1POS T cells interacted with PD-L1POS tumor cells. Taken together, we present sdAb K2, which specifically binds to human PD-L1, as a new diagnostic and therapeutic agent in cancer management.

Impact of lenalidomide maintenance on the immune environment of multiple myeloma patients with low tumor burden after autologous stem cell transplantation.

Lenalidomide is a potent anti-myeloma drug with immunomodulatory properties. It is increasingly used in a low-dose maintenance setting to prolong remission duration after standard treatment. Data on the in vivo effects of lenalidomide are scarce and sometimes different from the well-described in vitro effects. We therefore evaluated the numerical, phenotypical and functional impact of lenalidomide maintenance on several immune cell types in a cohort of seventeen homogeneously treated myeloma patients achieving a low residual myeloma burden after a bortezomib based-induction followed by autologous stem cell transplantation. Lenalidomide maintenance: 1) increased the fraction of naïve CD8+ T cells and several memory T-cell subsets, 2) reduced the numbers of terminal effector CD8+ T cells, 3) resulted in a higher expression of co-stimulatory molecules on resting T cells and of the inhibitory checkpoint molecules LAG-3 on CD4+ T cells and TIM-3 on CD4+ and CD8+ T cells, 4) reduced the number of TIGIT+ CD8+ T cells, 5) increased the number of regulatory T cells with a phenotype associated with strong suppressive capacity. Purified CD8+ T cells showed increased and more polyfunctional recall viral responses. However, PBMC responses were not enhanced during lenalidomide maintenance and CD4+ T-cell responses specific for the myeloma-associated antigen MAGE-C1 even tended to become lower. We conclude that lenalidomide maintenance after autologous stem cell transplantation has complex pleotropic effects on the immune environment. Immune interventions such as anti-myeloma vaccination should include measures to tackle an expanded inhibitory Treg compartment.

mRNA Electroporation of Dendritic Cells with WT1, Survivin, and TriMix (a Mixture of caTLR4, CD40L, and CD70).

The immune system is a crucial player in the development of cancer. Once it is in imbalance and immunosuppressive mechanisms supporting tumor growth take over control, dendritic cell immunotherapy might offer a solution to restore the balance. There are several methods to manufacture dendritic cells but none of them has yet proven to be superior to others. In this chapter, we discuss the methodology using electroporation of mRNA encoding Wilms' tumor gene 1, survivin, and TriMix (mixture of caTLR4, CD40L, and CD70) to simultaneously load and mature dendritic cells.

Phase II Study of Autologous Monocyte-Derived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma.

PURPOSE: Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic mRNA (TriMixDC-MEL) are immunogenic and have antitumor activity as a monotherapy in patients with pretreated advanced melanoma. Ipilimumab, an immunoglobulin G1 monoclonal antibody directed against the cytotoxic T-lymphocyte-associated protein 4 receptor that counteracts physiologic suppression of T-cell function, improves the overall survival of patients with advanced melanoma. This phase II study investigated the combination of TriMixDC-MEL and ipilimumab in patients with pretreated advanced melanoma. PATIENTS AND METHODS: Thirty-nine patients were treated with TriMixDC-MEL (4 × 10(6) cells administered intradermally and 20 × 10(6) cells administered intravenously) plus ipilimumab (10 mg/kg every 3 weeks for a total of four administrations, followed by maintenance therapy every 12 weeks in patients who remained progression free). Six-month disease control rate according to the immune-related response criteria served as the primary end point. RESULTS: The 6-month disease control rate was 51% (95% CI, 36% to 67%), and the overall tumor response rate was 38% (including eight complete and seven partial responses). Seven complete responses and one partial tumor response are ongoing after a median follow-up time of 36 months (range, 22 to 43 months). The most common treatment-related adverse events (all grades) consisted of local DC injection site skin reactions (100%), transient post-DC infusion chills (38%) and flu-like symptoms (84%), dermatitis (64%), hepatitis (13%), hypophysitis (15%), and diarrhea/colitis (15%). Grade 3 or 4 immune-related adverse events occurred in 36% of patients. There was no grade 5 adverse event. CONCLUSION: The combination of TriMixDC-MEL and ipilimumab is tolerable and results in an encouraging rate of highly durable tumor responses in patients with pretreated advanced melanoma.

Long-term clinical outcome of melanoma patients treated with messenger RNA-electroporated dendritic cell therapy following complete resection of metastases.

PURPOSE: Melanoma patients with a high risk of recurrence may benefit from immunotherapy with mRNA-electroporated autologous monocyte-derived dendritic cells (DCs). Further benefit may be found in combining DC-therapy with interferon alfa-2b. PATIENTS AND METHODS: The long-term clinical outcome of AJCC stage III/IV melanoma patients who had no evidence of disease at the time of treatment with autologous mRNA-electroporated DCs in a single-center pilot clinical trial was analyzed. Antigen loading was accomplished by co-electroporation of mRNA encoding a fusion protein between MAGE-A1, -A3, -C2, Tyrosinase, MelanA/MART-1, or gp100, and an HLA class II-targeting sequence. DCs were administered by 4-6 bi-weekly intradermal injections. IFN-α-2b (5 MIU TIW) was initiated either at recurrence (cohort 1), concomitant with DCs (cohorts 2 and 3), or following the fourth DC administration (cohort 4). RESULTS: Thirty melanoma patients were recruited between April 2006 and June 2009. DC-related adverse events included grade 2 local injection site reactions in all patients, grade 2 fever and flu-like symptoms in one patient, and skin depigmentation in seven patients. After a median follow-up of over 6 years, the median relapse-free survival is 22 months (95% CI 12-32 months). Twelve patients have died. The median overall survival has not been reached; the 2-year and 4-year survival rates are 93 and 70%, respectively. CONCLUSIONS: Adjuvant therapy following the resection of melanoma metastases with autologous mRNA-electroporated DCs, combined with interferon alfa-2b, is tolerable and results in encouraging long-term overall survival rates justifying further evaluation in a randomized clinical trial.

AZD1480 delays tumor growth in a melanoma model while enhancing the suppressive activity of myeloid-derived suppressor cells.

AZD1480 is a potent, competitive small-molecule inhibitor of JAK1/2 kinase which inhibits STAT3 phosphorylation and tumor growth. Here we investigated the effects of AZD1480 on the function of different immune cell populations in a melanoma model. When MO4 tumor-bearing mice were treated with AZD1480 we observed a strong inhibition of tumor growth as well as a prolonged survival. Moreover, a significant decrease in the percentage of myeloid-derived suppressor cells (MDSCs) was observed after treatment with AZD1480. However, AZD1480 enhanced the suppressive capacity of murine MDSCs while at the same time impairing the proliferative as well as the IFN-γ secretion capacity of murine T cells. The addition of AZD1480 to co-cultures of human MDSCs and T cells does not affect the suppressive activity of MDSCs but it does reduce the IFN-γ secretion and the proliferative capacity of T cells. We showed that although AZD1480 has the ability to delay the tumor growth of MO4 tumor-bearing mice, this drug has detrimental effects on several aspects of the immune system. These data indicate that systemic targeting of the JAK/STAT pathway by JAK1/2 inhibition can have divergent effects on tumor growth and anti-tumor immune responses.

Immunomodulatory drugs improve the immune environment for dendritic cell-based immunotherapy in multiple myeloma patients after autologous stem cell transplantation.

Multiple myeloma (MM) is characterized by a malignant proliferation of plasma cells in the bone marrow with associated organ damage. Although the prognosis of MM has improved recently, the disease remains incurable for the large majority of patients. The eradication of residual disease in the bone marrow is a main target on the road toward cure. Immune cells play a role in the control of cancer and can be tools to attack residual MM cells. However, the myeloma-associated immune deficiency is a major hurdle to immunotherapy. We evaluated ex vivo the effects of low doses of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide on several immune cell types from MM patients after autologous stem cell transplantation and with low tumor burden. We observed that these drugs increased CD4(+) and CD8(+) T-cell proliferation and cytokine production, enhanced the lytic capacity of cytotoxic T lymphocytes and reduced the suppressive effects of regulatory T cells on CD8(+) T-cell responses. In addition, we found that functional dendritic cells (DCs) can be generated from mononuclear cells from MM patients. The presence of IMiDs improved the quality of antigen-specific T cells induced or expanded by these DCs as evidenced by a higher degree of T-cell polyfunctionality. Our results provide a rationale for the design of early phase clinical studies to assess the efficacy of DC-based immunotherapy in combination with posttransplant maintenance treatment with IMiDs in MM.

Optimized dendritic cell-based immunotherapy for melanoma: the TriMix-formula.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. In this regard, one of the major focuses of cancer immunotherapy has been the design of vaccines promoting strong tumor-specific cytotoxic T lymphocyte responses in cancer patients. Here, dendritic cells (DCs) play a pivotal role as they are regarded as nature's adjuvant and as such have become the natural agents for antigen delivery in order to finally elicit strong T cell responses (Villadangos and Schnorrer in Nat Rev Immunol 7:543-555, 2007; Melief in Immunity 29:372-383, 2008; Palucka and Banchereau in Nat Rev Cancer 12:265-277, 2012; Vacchelli et al. in Oncoimmunology 2:e25771, 2013; Galluzzi et al. in Oncoimmunology 1:1111-1134, 2012). Therefore, many investigators are actively pursuing the use of DCs as an efficient way of inducing anticancer immune responses. Nowadays, DCs can be generated at a large scale in closed systems, yielding sufficient numbers of cells for clinical application. In addition, with the identification of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, a whole range of strategies using DCs for immunotherapy have been designed and tested in clinical studies. Despite the evidence that DCs loaded with tumor-associated antigens can elicit immune responses in vivo, clinical responses remained disappointingly low. Therefore, optimization of the cellular product and route of administration was urgently needed. Here, we review the path we have followed in the development of TriMixDC-MEL, a potent DC-based cellular therapy, discussing its development as well as further modifications and applications.

Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.

Modulation of regulatory T cell function by monocyte-derived dendritic cells matured through electroporation with mRNA encoding CD40 ligand, constitutively active TLR4, and CD70.

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1ß, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

Characterization of CD8+ T-cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL).

Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL) stimulates T-cell responses against the presented tumor-associated antigens (TAAs). In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8(+) T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs) and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71%) patients screened, CD8(+) T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8(+) T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8(+) T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8(+) T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

Dendritic cells loaded with mRNA encoding full-length tumor antigens prime CD4+ and CD8+ T cells in melanoma patients.

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

Epitope and HLA-type independent monitoring of antigen-specific T-cells after treatment with dendritic cells presenting full-length tumor antigens.

The efficacy of cancer immunotherapy can be improved by treatment with full-length tumor antigen and by combining several antigens. This approach allows the induction of a broad immune response irrespective of the patient's HLA type which at the same time challenges immune monitoring. Also, the number of available lymphocytes is most often limited and minimal in vitro restimulations of the lymphocytes should maintain information about the actual in vivo situation. To overcome these hurdles, we developed a method to measure the CD8(+) and CD4(+) T-cell responses directly ex vivo. Skin biopsies taken from dendritic cell (DC)-induced DTH reactions from melanoma patients participating in a DC-clinical trial served as lymphocyte source. Antigen-specificity of skin infiltrating lymphocytes was investigated by coculture with antigen-presenting autologous B cells and assessed for CD137 upregulation and enhanced cytokine secretion. Using this approach we could detect treatment-specific CD8(+) T-cells without restimulation in vitro. Upregulation of the activation marker CD137 correlated with the upregulation of the lytic marker CD107a. CD137 upregulation by treatment-specific CD4(+) lymphocytes however was more pronounced after antigen-specific in vitro restimulation. Both CD8(+) and CD4(+) lymphocytes could be further expanded using the same B cells as for screening allowing characterization of the recognized antigenic region. In addition, this technique can be extended to detect a broader array of T-cell functions and to monitor a large cohort of patients. We believe that this approach of direct ex vivo monitoring, irrespective of the patient's HLA-type or the recognized peptide, and using a limited number of lymphocytes is a valuable tool in the immune monitoring of current cellular immunotherapies.

A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption.

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.

Intravenous and intradermal TriMix-dendritic cell therapy results in a broad T-cell response and durable tumor response in a chemorefractory stage IV-M1c melanoma patient.

Dendritic cells (DCs) electroporated with mRNA encoding CD70, CD40L and a constitutively active toll-like receptor 4 (TriMix-DC) have an increased T-cell stimulatory capacity. In a prospective phase IB clinical trial, we treated melanoma patients with intradermal and intravenous injections of autologous TriMix-DC co-electroporated with mRNA encoding full-length MAGE-A3, MAGE-C2, tyrosinase and gp100. We report here the immunological and clinical results obtained in one patient with a particularly favorable outcome. This patient had stage IV-M1c melanoma with documented progression during dacarbazine chemotherapy and received 5 TriMix-DC injections. Following DC therapy, a broad CD8(+) T-cell response against multiple epitopes derived from all four treatment antigens was found in the blood and among T cells derived from DTH biopsy. In addition, CD4(+) T cells recognizing different MAGE-A3-derived epitopes were detected in DTH-derived cells. A spontaneous anti-MAGE-C2 CD8(+) T-cell response was present prior to TriMix-DC therapy and increased during treatment. The tumor response was assessed with 18-fluorodeoxyglucose-positron emission/computed tomography. We documented a partial tumor response according to RECIST criteria with a marked reduction in (18)F-FDG-uptake by lung, lymph node and bone metastases. The patient remains free from progression after 12 months of follow-up. This case report indicates that administration of autologous TriMix-DC by the combined intradermal and intravenous route can mediate a durable objective tumor response accompanied by a broad T-cell response in a chemorefractory stage IV-M1c melanoma patient.

Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma.

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-α-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-α-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-α-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-α-2b.

The combination of 4-1BBL and CD40L strongly enhances the capacity of dendritic cells to stimulate HIV-specific T cell responses.

One of the consequences of HIV infection is a progressive loss of T cell functions, resulting in decreased cytokine secretion and proliferation and an increased sensitivity to apoptosis. Therefore, successful therapeutic vaccination approaches should aim at restoring the functionality of existing HIV-specific T cells, as well as to efficiently induce potent, HIV-specific T cells from naïve T cells. In this study, we wanted to determine the stimulatory capacity of DCs coelectroporated with mRNA encoding for different costimulatory molecules of the TNFSF, together with HIV antigen-encoding mRNA. We show that DCs electroporated with 4-1BBL can enhance the proliferation, functionality, cytokine production, and survival of HIV-specific CD8(+) T cells. Furthermore, we are the first to show that a combination of 4-1BBL and CD40L overexpression on DCs dramatically enhances CD4(+) and CD8(+) T cell responses. Finally, we demonstrate that signaling through 4-1BB, but not through CD40, can alleviate the suppressive effect of Tregs on CD8(+) T cell proliferation. Thus, the combination of 4-1BBL and CD40L enhances HIV-specific CD8(+) T cell responses in a synergistic way, resulting in enhanced proliferation of CD4(+) and CD8(+) T cell subsets, an increased cytokine secretion, and a reduced sensitivity to Treg-mediated immune suppression.