Open-label, multicenter, single-arm phase II DeCOG-study of ipilimumab in pretreated patients with different subtypes of metastatic melanoma.

BACKGROUND: Ipilimumab is an approved immunotherapy that has shown an overall survival benefit in patients with cutaneous metastatic melanoma in two phase III trials. As results of registrational trials might not answer all questions regarding safety and efficacy of ipilimumab in patients with advanced melanoma seen in daily clinical practice, the Dermatologic Cooperative Oncology Group conducted a phase II study to assess the efficacy and safety of ipilimumab in patients with different subtypes of metastatic melanoma. PATIENTS AND METHODS: We undertook a multicenter phase II study in melanoma patients irrespective of location of the primary melanoma. Here we present data on patients with pretreated metastatic cutaneous, mucosal and occult melanoma who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals. Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria. Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0. Primary endpoint was the OS rate at 12 months. RESULTS: 103 pretreated patients received at least one dose of ipilimumab, including 83 cutaneous, seven mucosal and 13 occult melanomas. 1-year OS rates for cutaneous, mucosal and occult melanoma were 38 %, 14 % and 27 %, respectively. Median OS was 6.8 months (95 % CI 5.3-9.9) for cutaneous, 9.6 months (95 % CI 1.6-11.1) for mucosal, and 9.9 months (lower 95 % CI 2.3, upper 95 % CI non-existent) for occult melanoma. Overall response rates for cutaneous, mucosal and occult melanoma were 16 %, 17 % and 11 %, respectively. Eleven patients had partial response (16 %) and ten patients experienced stable disease (14 %), none achieved a complete response. Treatment-related AEs were observed in 71 patients (69 %), including 20 grade 3-4 events (19 %). No new and unexpected safety findings were noted. CONCLUSIONS: Ipilimumab is a treatment option for pretreated patients with advanced cutaneous melanoma seen in daily routine. Toxicity was manageable when treated as per protocol-specific guidelines. TRIAL REGISTRATION: Clinical Trials.gov NCT01355120.

Phase II DeCOG-study of ipilimumab in pretreated and treatment-naïve patients with metastatic uveal melanoma.

PURPOSE: Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options. The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials. As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM. PATIENTS AND METHODS: We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma. Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals. Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria. Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0. Primary endpoint was the OS rate at 12 months. RESULTS: Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively. Median OS was 6.8 months (95% CI 3.7-8.1), median progression-free survival 2.8 months (95% CI 2.5-2.9). The disease control rate at weeks 12 and 24 was 47% and 21%, respectively. Sixteen patients had stable disease (47%), none experienced partial or complete response. Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3-4 events (36%). One drug-related death due to pancytopenia was observed. CONCLUSIONS: Ipilimumab has very limited clinical activity in patients with metastatic UM. Toxicity was manageable when treated as per protocol-specific guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT01355120.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg.

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, ß-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma.

BACKGROUND: Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. PATIENTS AND METHODS: This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). RESULTS: Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. CONCLUSION: Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.

Autocrine TNF is critical for the survival of human dendritic cells by regulating BAK, BCL-2, and FLIPL.

The life span of dendritic cells (DCs) is determined by the balance of pro- and antiapoptotic proteins. In this study, we report that serum-free cultured human monocyte-derived DCs after TLR stimulation with polyinosinic acid-polycytidylic acid or LPS underwent apoptosis, which was correlated with low TNF production. Apoptosis was prevented by the addition of exogenous TNF or by concomitant stimulation with R-848, which strongly amplified endogenous TNF production. Neutralization of TNF confirmed that DC survival was mediated by autocrine TNF induced either by stimulation with R-848 or by ligation of CD40. DCs stimulated by polyinosinic acid-polycytidylic acid or IFN-ß, another known inducer of DC apoptosis, were characterized by high levels and activation of the proapoptotic protein BAK. The ratio of antiapoptotic BCL-2 to BAK correlated best with the survival of activated DCs. Addition of TNF increased this ratio but had little effect on BAX and XIAP. Knockdown experiments using small interfering RNAs confirmed that the survival of activated and also of immature DCs was regulated by BAK and showed that TNF was protective only in the presence of FLIP(L). Together, our data demonstrate that the survival of DCs during differentiation and activation depends on autocrine TNF and that the inhibition of BAK plays an important role in this process.

Generation of human dendritic cells that simultaneously secrete IL-12 and have migratory capacity by adenoviral gene transfer of hCD40L in combination with IFN-gamma.

Dendritic cells (DCs) are professional antigen presenting cells and have key functions in the initiation of immune responses. Hence, antigen-loaded DCs have become important tools for active-specific immunotherapy. In addition to defining strategies for antigen loading, effective T-cell activation by DCs will depend on vaccination protocols that facilitate DC migration to secondary lymphoid tissues and expression of costimulatory molecules and cytokines. Adenoviral gene transfer has been successfully implemented for genetic antigen loading of DCs. In this study, we exploit an adenoviral vector encoding human CD40 ligand (CD40L), Ad5hCD40L, to establish DCs that feature both migration potential and prolonged secretion of the key T-helper 1 cytokine interleukin-12p70 (IL-12p70). Transduction of human monocyte-derived DCs with Ad5hCD40L resulted in efficient CD40L expression, which was detected only intracellularly, and in secretion of IL-12p70. Addition of recombinant interferon (IFN)-gamma shortly after DC transduction substantially increased IL-12p70 secretion. Maturation of DCs was achieved with a standard cytokine maturation cocktail (MC) containing prostaglandin E2 which, however, abolished IL-12p70 secretion by Ad5hCD40L-transduced cells in the absence of IFN-gamma. Only DCs treated with Ad5hCD40L, MC, and IFN-gamma migrated efficiently towards CCL19 and continued to secrete IL-12p70. Finally, DCs transduced with both Ad5hCD40L and an adenoviral vector encoding the melanoma antigen MelanA/MART-1 and treated with MC and IFN-gamma efficiently primed naive autologous CD8+ T cells into antigen-specific cytotoxic T lymphocyte. This strategy to generate DCs that exert both migration capacity and prolonged IL-12p70 secretion after intracellular CD40L expression and IFN-gamma treatment has the potential to further improve current DC vaccination protocols.

A fast and robust method to clone and functionally validate T-cell receptors.

Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 5' end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research.

A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells.

A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA'), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA' was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 mug/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA' induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.

Targeting HIV-1 Gag into the defective ribosomal product pathway enhances MHC class I antigen presentation and CD8+ T cell activation.

The main source for endogenous peptides presented by the MHC class I (MHC-I) pathway are de novo-synthesized proteins which are degraded via the ubiquitin proteasome pathway. Different MHC-I Ag pools can be distinguished: first, short-lived defective ribosomal products, which are degraded in concert with or shortly after their synthesis, and, second, functional proteins that enter the standard protein life cycle. To compare the contribution of these two Ag sources to the generation of MHC-I-presented peptides, we established murine cell lines which express as a model Ag the HIV-1 Gag polyprotein fused to ubiquitin (Ub) carrying the epitope SIINFEKL (SL). Gag was expressed either in its wild-type form (UbMGagSL) or as a variant UbRGagSL harboring an N-end rule degron signal. Although UbRGagSL displayed wild-type protein stability, its inherent defective ribosomal products rate observed after proteasome shutdown was increased concomitant with enhanced presentation of the SL epitope. In addition, UbRGagSL induces enhanced T cell stimulation of SL-specific B3Z hybridoma cells as measured in vitro and of adoptively transferred TCR-transgenic OT-1 T cells in vivo. Furthermore, an elevated frequency of SL-specific T cells was detected by IFN-gamma ELISPOT after immunization of naive C57BL/6 mice with UbRGagSL/EL4 cells. These results further underline the role of the defective ribosomal product pathway in adaptive immunity.

Introduction of functional chimeric E/L-selectin by RNA electroporation to target dendritic cells from blood to lymph nodes.

BACKGROUND: Inefficient migration of dendritic cells (DC) to regional lymph nodes (LN) upon intracutaneous injection is a major obstacle for effective DC vaccination. Intravenous vaccination is unfavorable, because DC cannot migrate directly from the blood into LN. METHODS: To enable human monocyte-derived (mo)DC to enter LN directly from the blood, we manipulated them by RNA electroporation to express a human chimeric E/L-selectin (CD62E/CD62L) protein, which binds to peripheral node addressin expressed on high endothelial venules. RESULTS: Transfection efficiency exceeded 95%, and high E/L-selectin surface expression was detected for >48 h. E/L-selectin RNA-transfected DC displayed an identical mature DC phenotype as mock-transfected DC. Furthermore, E/L-selectin-transfected DC maintained their normal CCR7-mediated migration capacity, and their ability to prime and expand functional cytotoxic T cells recognizing MelanA. Most importantly, E/L-selectin-RNA-transfected DC gained the capability to attach to and roll on sialyl-Lewis(X) in vitro. OUTLOOK: The presented strategy can be readily translated into the clinic, as it involves no stable genetic manipulation or viral transformation, and allows targeting of a large number of LN.

Modulation of viability and maturation of human monocyte-derived dendritic cells by oncolytic adenoviruses.

Adenoviral oncolysis is a promising new modality for treatment of cancer based on selective viral replication in tumor cells. However, tumor cell killing by adenoviral oncolysis needs to be improved to achieve therapeutic benefit in the clinic. Towards this end, the activation of anti-tumor immunity by adenoviral oncolysis might constitute a potent mechanism for systemic killing of uninfected tumor cells, thereby effectively complementing direct tumor cell killing by the virus. Knowledge of anti-tumor immune induction by adenoviral oncolysis, however, is lacking mostly due to species-specificity of adenovirus replication, which has hampered studies of human oncolytic adenoviruses in animals. We suggest the analysis of interactions of oncolytic adenoviruses with human immune cells as rational basis for the implementation of adenoviral oncolysis-induced anti-tumor immune activation. The goal of our study was to investigate how oncolytic adenoviruses affect human dendritic cells (DCs), key regulators of innate and adoptive immunity that are widely investigated as tumor vaccines. We report that melanoma-directed oncolytic adenoviruses, like replication-deficient adenoviruses but unlike adenoviruses with unrestricted replication potential, are not toxic to monocyte-derived immature DCs and do not block DC maturation by external stimuli. Of note, this is in contrast to reports for other viruses/viral vectors and represents a prerequisite for anti-tumor immune activation by adenoviral oncolysis. Furthermore, we show that these oncolytic adenoviruses alone do not or only partially induce DC maturation. Thus additional signals are required for optimal immune activation. These could be delivered, for example, by inserting immunoregulatory transgenes into the oncolytic adenovirus genome.

Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection.

Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is the current lack of a simple functionally "closed" system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized the use of the Elutra counterflow elutriation system to enrich monocytic DC precursors by (1) developing an algorithm to avoid red blood cell debulking and associated monocyte loss before elutriation, and (2) by elutriation directly in culture medium rather than phosphate-buffered saline. Upon elutriation the bags containing the collected monocytes are simply transferred into the incubator to generate DC progeny as the final "open" washing step is no longer required. Elutriation resulted in significantly more (> or = 2-fold) and purer DC than the standard gradient centrifugation/adherence-based monocyte enrichment, whereas morphology, maturation markers, viability, migratory capacity, and T cell stimulatory capacity were identical. Subsequently, we compared RNA transfection, as this is an increasingly used approach to load DC with antigen. Elutra-derived and adherence-derived DC could be electroporated with similar, high efficiency (on average >85% green fluorescence protein positive), and appeared also equal in antigen expression kinetics. Both Elutra-derived and adherence-derived DC, when loaded with the MelanA peptide or electroporated with MelanA RNA, showed a high T cell stimulation capacity, that is, priming of MelanA-specific CD8+ T cells. Our optimized Elutra-based procedure is straightforward, clearly superior to the standard gradient centrifugation/plastic adherence protocol, and now allows the generation of large numbers of peptide-loaded or RNA-transfected DC in a functionally closed system.

Vaccinia virus impairs directional migration and chemokine receptor switch of human dendritic cells.

A crucial event for the induction of an anti-viral immune response is the coordinated, phenotype-dependent migration of dendritic cells (DC) to sites of infection and secondary lymphoid organs. Here we show that the vaccinia virus (VV) strains Western Reserve (WR) and modified virus Ankara (MVA) inhibit directional migration of mature DC toward the lymphoid chemokines CCL19 and CXCL12 without affecting surface expression of the respective chemokine receptors or impairing undirected cellular locomotion. Instead, infection with VV results in a deficiency of extracellular signal-regulated kinase-1 and a disturbance of intracellular calcium mobilization, indicating a viral interference with signaling events downstream of the surface chemokine receptors. In immature DC, apart from inhibiting chemokine-induced migration of infected DC, infection with both VV strains increases expression of the inflammatory chemokine receptors CCR1 and CXCR1 on non-infected bystander DC, which depends on the activity of IFN-alpha. Although functional, these chemokine receptors are resistant to lipopolysaccharide-induced down-regulation. In addition, VV-infected and non-infected bystander DC fail to up-regulate the lymphoid chemokine receptor CCR7 upon activation, together pointing to a disability to undergo the chemokine receptor switch. This study shows that VV targets directional migration of professional antigen-presenting cells at multiple functional levels, revealing a potent viral strategy of immune escape.

An improved method for RNA isolation and removal of melanin contamination from melanoma tissue: implications for tumor antigen detection and amplification.

Several kits are available to isolate RNA(1) from tissues. However, melanoma tissue is often rich in melanin that co-purifies with DNA/RNA and inhibits subsequent PCR reactions, hampering tumor antigen detection and amplification. This problem has not yet been addressed systematically. Here we generated a photometric protocol to determine both the melanin and RNA concentration by correcting the latter for the absorption coefficient of melanin at 260 nm. Subsequently, different combinations of silica-based RNA-binding, size-exclusion, and ion-exchange columns were used in 8 protocols for isolation of RNA from melanoma tissue to compare efficacy of melanin removal and yield of RNA. Furthermore, the capability of the different RNA preparations to function as template in RT-PCRs with products of different length, i.e. GAP-DH, tyrosinase, and gp100, was tested. We found that the combination of silica-based RNA-binding and size-exclusion columns was not sufficient to remove melanin from highly contaminated tumor samples, and subsequent RT-PCR failed to give larger products. However, protocols including ion-exchange columns resulted in efficient removal of melanin, while retaining reasonable RNA yields in samples from highly pigmented melanomas. Efficient RT-PCR of larger products turned out to be inversely correlated to the melanin contamination. This RNA-purification method will help scientists to isolate polynucleotides from melanin-containing tumor samples, which subsequently can be used in antigen detection assays and vaccination strategies using amplified total tumor RNA.

Mature monocyte-derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration.

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long-lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra- and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

Lack of toxicity of therapy-induced T cell responses against the universal tumour antigen survivin.

Prognosis of disseminated melanoma remains gloomy as neither chemotherapeutic nor unspecific immune modulatory approaches were able to improve the overall survival of these patients. Hence, specific immunotherapy has received increasing attention. Disappointing clinical results, however, indicate that the choice of suitable antigens is of special importance. To this end, the inhibitor of apoptosis (IAP) protein survivin, which is over-expressed in several tumours but is largely undetectable in adult tissues, appears to be a promising target for vaccination purposes, since down-regulation or loss of expression is associated with impaired tumour progression. Consequently, five heavily pretreated stage IV melanoma patients were vaccinated with the HLA-A2 restricted survivin(96-104) epitope presented by autologous dendritic cells (DCs) in a compassionate use setting. Four of these patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining confirmed that these survivin reactive cells infiltrated both visceral and soft tissue metastases.

Shift from systemic to site-specific memory by tumor-targeted IL-2.

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.

MHC class II-mediated apoptosis in dendritic cells: a role for membrane-associated and mitochondrial signaling pathways.

Cytotoxic elimination of dendritic cells (DC) in lymphoid tissue represents an important pathway of immune regulation. However, the mechanism of DC removal is still controversial since mature DC are insensitive to death receptor-mediated killing and other surface or soluble molecules mediating DC death in vivo have yet to be characterized. Class II ligation is the only known signal that induces rapid cell death in mature DC, thus our studies have now focused on the requirements for this cell death using the advantages of tools available for both the mouse and human systems. Anti-class II mAb could be grouped into (i) mAb that both bound to class II and caused class II-mediated cell death as well as (ii) those that bound to class II, but did not cause apoptosis. mAb binding stable class II dimers as well as those mAb recognizing either the alpha or beta chains of class II were found in both groups. Whereas class II-mediated death was enhanced by DC-DC homotypic interactions, DC clustering itself was insufficient to induce apoptosis. Although DC death could be inhibited by uncoupling actin filament bundling, the inhibition of various proteases, including the caspases, and protein transport mediators failed to inhibit class II-mediated cell death. Neither Bid, poly-ADP-ribose polymerase, caspases-3, -7 and -8 nor FLICE-inhibitory protein were found to be cleaved during class II apoptosis. Lastly, although class II mAb induced a rapid mitochondrial membrane depolarization in DC, cell death was not inhibited by Bcl-2 over-expression in DC. The independence of this form of apoptosis from protein or RNA synthesis, coupled to the rapidity of the mitochondrial depolarization and the lack of protection by Bcl-2, suggests that mature DC express pre-formed pro-apoptotic molecules that are involved in class II-mediated death.