Beatrice Schuler-Thurner

Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Mage-3 and Influenza-Matrix Peptide-Specific Cytotoxic T Cells Are Inducible in Terminal Stage HLA-A2.1+ Melanoma Patients by Mature Monocyte-Derived Dendritic Cells

Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 × 106 s.c. followed by two i.v. ones of 6 and 12 × 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2.1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-γ-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network

Background Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) blocking antibody, has been approved for the treatment of metastatic melanoma and induces adverse events (AE) in up to 64% of patients. Treatment algorithms for the management of common ipilimumab-induced AEs have lead to a reduction of morbidity, e.g. due to bowel perforations. However, the spectrum of less common AEs is expanding as ipilimumab is increasingly applied. Stringent recognition and management of AEs will reduce drug-induced morbidity and costs, and thus, positively impact the cost-benefit ratio of the drug. To facilitate timely identification and adequate management data on rare AEs were analyzed at 19 skin cancer centers. Methods and Findings Patient files (n = 752) were screened for rare ipilimumab-associated AEs. A total of 120 AEs, some of which were life-threatening or even fatal, were reported and summarized by organ system describing the most instructive cases in detail. Previously unreported AEs like drug rash with eosinophilia and systemic symptoms (DRESS), granulomatous inflammation of the central nervous system, and aseptic meningitis, were documented. Obstacles included patientś delay in reporting symptoms and the differentiation of steroid-induced from ipilimumab-induced AEs under steroid treatment. Importantly, response rate was high in this patient population with tumor regression in 30.9% and a tumor control rate of 61.8% in stage IV melanoma patients despite the fact that some patients received only two of four recommended ipilimumab infusions. This suggests that ipilimumab-induced antitumor responses can have an early onset and that severe autoimmune reactions may reflect overtreatment. Conclusion The wide spectrum of ipilimumab-induced AEs demands doctor and patient awareness to reduce morbidity and treatment costs and true ipilimumab success is dictated by both objective tumor responses and controlling severe side effects.

A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use.

Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a satisfactory cryopreservation of DC in the absence of FCS is not yet available, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required to date. We now aimed at developing an effective cryopreservation method, and by testing several variables found that it was crucial to combine the most advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF and IL-4 and showed that amongst several known maturation stimuli the cocktail consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survival of mature DC. We then systematically explored cryopreservation conditions, and found that freezing matured DC at 1 degrees C/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the best results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survival as well as functional properties (allogeneic mixed leukocyte reaction, induction of influenza matrix or melan A peptide-specific cytotoxic T cells) of these thawed DC were equivalent to freshly prepared ones. The addition of CD40L or TRANCE/RANKL further improved DC survival. Importantly, we demonstrate that DC can effectively be loaded with antigens (such as Tetanus Toxoid, influenza matrix and melan A peptides) before cryopreservation so that it is now possible to generate antigen-preloaded, frozen DC aliquots that after thawing can be used right away. This is an important advance as both the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated.

Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories™

Dendritic cells (DC) are increasingly used for the immunotherapy of cancer. Both the induction of tumor-specific T cells and some clinical regressions have been observed in early phase I/II trials by using either DC isolated from blood, DC generated from CD34+ precursors ex vivo, and most frequently, by employing monocyte-derived DC. As DC vaccination is now awaiting phase II/III trials with larger patient collectives, it becomes increasingly important to overcome prior limitations such as the repetitive, labor-intensive generation of DC in a large number of open culture vessels. We describe here as a result of several years of optimization, in detail, a procedure that uses the so-called Nunc cell factories to process a whole apheresis product, labor- and cost-effectively in a quasi-closed system to reproducibly generate (by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha +PGE(2)) large numbers (8.32+/-3.8% of input peripheral blood mononuclear cells (PBMC)) of mature (>85% CD83+), monocyte-derived DC that can be successfully cryopreserved. Our report is based on the processing of >100 aphereses including 52 unselected aphereses in advanced melanoma patients. This allows us also to suggest meaningful quality and validation criteria. The DC generation method appears particularly promising as respective DC vaccination proved to be immunogenic in cancer patients and cell factories can readily be converted to a fully closed system by using appropriate valves, tubings, and bags.

Polyclonal CTL Responses Observed in Melanoma Patients Vaccinated with Dendritic Cells Pulsed with a MAGE-3.A1 Peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3168–176 peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 × 10−6 to 9 × 10−4 of CD8+ blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.

Targeting of DEC-205 on human dendritic cells results in efficient MHC class II-restricted antigen presentation

The use of dendritic cells (DCs) in therapeutic cancer vaccination requires their loading with tumor-specific antigen(s). DEC-205, a phagocytosis receptor mediating antigen uptake, is associated with CD8(+) T-cell responses in mice. Here we fused an anti-DEC-205scFv to an HLA-DP4-restricted epitope from the tumor antigen MAGE-A3, and examined the suitability and efficacy of DEC-205 to deliver a helper epitope to human monocyte-derived DCs (moDCs). The construct specifically bound DEC-205 on human moDCs without negative impact on DC phenotype and function. We measured antigen presentation with specific autologous CD4(+) T cells, generated by TCR-RNA transfection. DEC-205 targeting resulted in significant major histocompatibility complex class II-restricted antigen presentation, and was superior to loading DCs by electroporation of mRNA encoding endosome-targeted MAGE-A3-DCLAMP or by direct peptide pulsing. Anti-DEC-205scFv-MAGE-A3 was presented 100 times more efficiently than the control constructs. DC maturation before or during incubation with anti-DEC-205scFv-MAGE-A3 reduced the interleukin-10/interleukin-2 ratio. Moreover, we successfully applied the DEC-205 targeting strategy to moDCs from malignant melanoma patients. Again, DEC-205-targeted mature DCs (mDCs) presented the antigen more efficiently than peptide-pulsed DCs and maintained their stimulatory capacity after cryoconservation. Thus, DEC-205 targeting represents a feasible and effective method to deliver helper epitopes to DCs in anticancer vaccine strategies, which may also be suitable for DC targeting in vivo.

Functional Analysis of Tumor-Specific Th Cell Responses Detected in Melanoma Patients after Dendritic Cell-Based Immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3243–258), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.

Frequency of Circulating Tregs with Demethylated FOXP3 Intron 1 in Melanoma Patients Receiving Tumor Vaccines and Potentially Treg-Depleting Agents

Purpose: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients. Experimental Design: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox. Results: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2. Conclusions: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs. Clin Cancer Res; 17(4); 1–8. ©2010 AACR.

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×108 IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.