IFN-gamma responses in peptide-treated melanoma patients measured by an ELISPOT assay using allogeneic dendritic cells

BACKGROUND: Several melanoma-specific peptides are currently used in clinical trials. However, the monitoring of the T cell response remains non-standardised and is often limited by shortage of cells. MATERIALS AND METHODS: We established an IFN-gamma ELISPOT assay to detect the CD8+ T cell response in HLA-A2-positive melanoma patients using pre-frozen, peptide-loaded HLA-A2-positive but otherwise allogeneic, monocyte-derived dendritic cells (DC) as antigen-presenting cells. We tested HLA-A2-positive stage III or IV melanoma patients before and after peptide immunotherapy. RESULTS: The number of EBV and influenza-specific IFN-gamma-spots were comparable irrespective of the use of autologous or allogeneic HLA-A2 immature DCs when using purified CD8+ cells as responder cells, but a high allogeneic background was seen when using PBMC. We observed modifications of the in vitro response to the melanoma peptides in three out of four responding patients, while virus responses remained constant; however, similar results were seen in the group with progressive disease. CONCLUSION: This demonstrates the possibility of monitoring an immune response by using allogeneic DCs, reducing the consumption of patient cells. The in vitro IFN-gamma responses increased in response to the peptide therapy, however this could not be correlated to clinical outcome.     

Melanoma immunology: past, present and future

PURPOSE OF REVIEW: Metastatic melanoma is a disease for which no effective therapeutic options have been developed during the last 30 years with the possible exception of high dose interferon-alpha in the adjuvant setting of stage III patients. The immunotherapy approach was initiated decades ago using cell-based vaccines and adoptive immunotherapy with functionally ill-defined lymphocytes. This paper aims to evaluate the last three decades of research in melanoma immunotherapy and to provide insights in the future of this strategy. RECENT FINDINGS: Thanks to the development of knowledge in basic and applied immunology, clinical studies of immunotherapy have been followed by trials based on molecular characterization of melanoma antigens and availability of ex-vivo assays allowing the quantitative assessment of the immune response against the given vaccine and against patient tumor cells. This second generation of immunotherapy trials, along with additional preclinical studies, while not yet resulting in a convincing clinical outcome, provided a wealth of data on immunogenicity of different melanoma antigens, mechanism of antigen presentation and factors that impair immune recognition of melanoma cells. SUMMARY: We discuss how this information will be exploited for designing new and more successful clinical trials of both active and adoptive antigen-specific immunotherapy of metastatic melanoma patients.     

Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2. Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-alpha to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 107 mDCs for 10 weeks and given 350-700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0-1). Median survival from the first vaccination was 240 days (range 31-735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocyte-derived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.     

Antigen-based immunotherapy of melanoma: Canvaxin therapeutic polyvalent cancer vaccine

As yet there are no FDA-approved cancer vaccines for malignant melanoma, but encouraging response rates and low toxicities reported in phase I/II trials suggest that antigen-based active immunotherapy may complement current treatment modalities. The cumulative data for Canvaxin therapeutic polyvalent cancer vaccine represent the largest phase II clinical trial of any cancer vaccine. Univariate and multivariate analyses of these data have demonstrated the prognostic significance of this allogeneic whole-cell preparation as a postoperative adjuvant treatment for patients with stage III and IV melanoma. The vaccine has also been shown promising results after resection of stage II melanoma and in patients with regional in-transit disease. The consistent correlation between immune and clinical responses to the vaccine suggests that immune parameters may be used to monitor a patient's response to vaccine therapy.     

Monitoring of circulating angiogenic factors in dendritic cell-based cancer immunotherapy

BACKGROUND: A promising treatment approach for patients with malignant disease that recently has found its way into clinical trials is based on vaccination with autologous dendritic cells loaded with tumor antigens. However, adequate assays for monitoring clinical and immunologic responses still are under debate. In recent years, the determination of angiogenic markers has shown considerable potential in the diagnosis and prognosis of patients with malignant disease, because tumor growth and spread are promoted by angiogenesis, the formation of new blood vessels. METHODS: The authors established a method for measuring the plasma levels of three modulators of angiogenesis: vascular endothelial growth factor, platelet-derived endothelial cell growth factor, and thrombospondin-1. The angiogenic blood profile of a healthy control group was characterized and compared with a group of patients with malignant disease. Ultimately, levels of circulating angiogenic factors were monitored in the course of dendritic cell-based cancer immunotherapy. RESULTS: Baseline levels of angiogenic mediators varied substantially among healthy individuals but showed consistent values for each individual. Blood levels of circulating angiogenic factors were elevated significantly in patients with advanced disease and were highly sensitive to dendritic cell-based immunotherapy. CONCLUSIONS: To our knowledge, the current report was the first to analyze circulating levels of angiogenic factors during dendritic cell-based immunotherapy. The authors observed a noteworthy change in the angiogenic blood profile with treatment, and this change was correlated with the induction of an immunologic response.     

Dendritic cell-based glioma immunotherapy (review)

Despite advances in radiation and chemotherapy along with surgical resectioning, the prognosis of patients with malignant glioma is poor. Among the new treatments currently being investigated for malignant glioma, immunotherapy is theoretically very attractive, since it offers the potential for high tumor-specific cytotoxicity. There are increasing reports demonstrating that systemic immunotherapy using dendritic cells is capable of inducing an antiglioma response. Therefore, dendritic cell-based immunotherapy could be a new treatment modality for patients with glioma. In this review, we will discuss the implications of these findings for glioma therapy. A literature review of dendritic cell-based glioma immunotherapy was used to overview the dendritic cell in immunobiology, in the central nervous system and in tumor immunology, glioma-associated antigens, dendritic cell therapy in animal glioma model, dendritic cell therapy in clinical trials and future directions in dendritic cell therapy. Dendritic cell-based immunotherapy strategies appear promising as an approach to successfully induce an antitumor immune response and increase survival in patients with glioma. Dendritic cell therapy of glioma seems to be safe and without major side effects. Its efficacy should be further determined in randomized, controlled clinical trials. The development of methods for manipulating dendritic cells for the purpose of vaccination will enhance the clinical usefulness of these cells for biotherapy for malignant glioma.     

Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy

Regional lymph nodes are the first site for melanoma metastases. The sentinel node (SN), on the direct lymphatic drainage pathway, which usually harbors first metastases, demonstrates significant suppression in its ability to respond to antigenic stimulation. This down-regulation of SN immunity is likely the basis of its susceptibility to tumor metastases, suggesting a potential role of the immune system in the control of malignant tumors. Despite immune dysfunction in the SN, phase II trials of systemic post-operative immunotherapy with a polyvalent melanoma vaccine developed at the John Wayne Cancer Institute showed improved 5-year overall survival in patients with melanoma metastatic to regional nodes. However, most immunotherapy clinical trials have failed to demonstrate a significant clinical response, and analyses of immune responses to tumor-associated antigens that correlate clinical responses have not been established. Therefore, refinements in assay methodologies and improvements in vaccine designs are critical to the success of cancer immunotherapy. Antigen presentation by dendritic cells (DCs) is the most potent means to initiate a T cell immunity. Dendritic cell-based immunotherapies have been vigorously attempted in the past decade. To improve the immunogenicity of cancer vaccines, we recently generated heterokaryons of DCs and tumor cells by electrofusion. The fusion hybrids retained their full antigen-presenting capacity and all natural tumor antigens. In pre-clinical animal experiments, a single injection of the DC-tumor fusion hybrids was sufficient to mediate the regression of tumors established in the lung, skin and brain. Most interestingly, successful therapy required the delivery of fusion hybrids directly into lymphoid organs such as lymph nodes. A clinical trial is now being carried out to test the immunogenicity and therapeutic effects of fusion hybrids for the treatment of metastatic melanoma. Presented as Session V of the First International Symposium on Cancer Metastasis and the Lymphovascular System. April 28–30, 2005, San Francisco, CA.     

Durable benefit and the potential for long-term survival with immunotherapy in advanced melanoma

Historically, the median overall survival for patients with stage IV melanoma was less than 1 year and the 5-year survival rate was ∼10%. Recent advances in therapy have raised 5-year survival expectations to ∼20%. Notably, a subset of melanoma patients who receive immunotherapy with high-dose interleukin-2, and now ipilimumab, can achieve long-term survival of at least 5 years. A major goal in melanoma research is to increase the number of patients who experience this overall survival benefit. In this review, we discuss the attributes of immunotherapy and newer targeted agents, and consider how combination strategies might improve the chances of achieving durable benefit and long-term survival. We also discuss three areas that we believe will be critical to making further advances in melanoma treatment. To better understand the clinical profile of patients who achieve long-term survival with immunotherapy, we first present data from ipilimumab clinical trials in which a subset of patients experienced durable responses. Second, we discuss the limitations of traditional metrics used to evaluate the benefits of immunotherapies. Third, we consider emerging issues that clinicians are currently facing when making treatment decisions regarding immunotherapy. A better understanding of these novel treatments may improve survival outcomes in melanoma, increase the number of patients who experience this overall survival benefit, and inform the future use of these agents in the treatment of other cancer types.     

Combined treatment of dendritoma vaccine and low-dose interleukin-2 in stage IV renal cell carcinoma patients induced clinical response: A pilot study

Vaccination using dendritic/tumor cell hybrids represents a novel and promising cancer immunotherapy. We have developed a technology that can instantly purify the hybrids (dendritomas) from the fusion mixture of dendritic cells (DCs) and tumor cells. Our animal studies and a phase I study of stage IV melanoma patients demonstrated that dendritoma vaccination could be conducted without major toxicity and induced tumor cell-specific immunological and clinical responses. In this pilot study, ten stage IV renal cell carcinoma patients were studied. Dendritomas were made from autologous DCs and tumor cells and administered by subcutaneous injection. After initial vaccination, three escalating doses of IL-2 (3, 6, and 9 million units each) were followed within five days. This treatment regimen was tolerated well without severe adverse events directly related to the dendritoma vaccine. Most adverse events were related to IL-2 administration or pre-existing disease. Patient-specific immune responses were evaluated by flow cytometric measurement of interferon-gamma-producing T-cells before and after vaccination in response to stimulation with tumor antigens. Nine out of nine patients eligible for the analysis showed an increase of IFN-gamma-expressing CD4+ T cells after vaccination(s); while five out of eight patients eligible for the analysis showed an increase of IFN-gamma-expressing CD8+ T cells. Clinical responses were documented in 40% of the patients, three with stabilization of disease and one with a partial response documented by a reduction in tumor size. This pilot study demonstrated that dendritoma vaccines could be administered safely to patients with metastatic renal cell carcinoma, while producing both clinical and immunologic evidence of response.     

Vaccination with hybrids of tumor and dendritic cells induces tumor-specific T-cell and clinical responses in melanoma stage III and IV patients

Hybrid cell vaccination was developed as therapeutic approach that aims at stimulating tumor-specific cytotoxic T-cell responses in cancer patients using hybrids of autologous tumor and allogeneic dendritic cells. We tested this concept and the efficacy of the vaccines in inducing clinical and immunologic responses in a clinical trial with melanoma stage III and IV patients. Of the 17 patients evaluated, 1 experienced a complete response, 1 a partial response and 6 stable disease with remarkably long survival times. In 11 of 14 patients analyzed, high-frequency T-cell responses to various tumor-associated T-cell epitope were induced and detectable in the peripheral blood. These immune responses were detected in clinical response patients as well as nonresponders. Failures of clinical responses in all the cases investigated correlated with loss of antigen expression and presentation. Hybrid cell vaccination thus proves effective in inducing tumor-specific T-cell responses in cancer patients.     

Biochemotherapy for advanced melanoma

The outcome of chemotherapy for patients with stage IV melanoma is unsatisfactory, since durable responses are rarely achieved. More experimental treatments, such as vaccine approaches, antibody treatments, and gene therapy are being developed and are of high scientific interest; however, their efficacy in advanced melanoma patients has so far been very limited. Based on the observation of a small proportion of long-term responses, the use of biotherapy or biochemotherapy is currently preferred in many institutions as first-line treatment in stage IV melanoma. Various interleukin-2 (IL-2) dosing schedules and combinations with interferon alpha (IFN-alpha) have been tested in patients with advanced melanoma during the past decade. The response rates reported with IL-2 as a single agent or in combination with IFN-alpha varies from 10% to 41%, with a small, but remarkable proportion of durable responses. Subsequently, biochemotherapy regimens combining IL-2, IFN-alpha, and chemotherapy have been evaluated in phase II trials, which have suggested improved response rates. Recent randomized trials have investigated the role of biochemotherapy as compared to biotherapy alone or as compared to chemotherapy for the treatment of advanced melanoma. So far, none of the approaches has been proven to confer a survival benefit and thus the uniform desire is to include as many patients as possible in controlled clinical trials.     

Role of bacillus Calmette–Guérin in the treatment of advanced melanoma

Early trials of Bacillus Calmette–Guérin (BCG)-based immunotherapy for melanoma consistently show a trend toward improved clinical outcomes in patients treated with BCG compared with observation alone. As an extension of these findings, investigators have initiated the Malignant Melanoma Active Immunotherapy (MMAIT) trials in patients with stage III (MMAIT–III) and stage IV (MMAIT–IV) disease. The overall survival of the patients receiving BCG plus placebo was much better than expected in both studies, thus suggesting a potential for BCG as an adjuvant after the resection of advanced disease. The work contained herein will explore the clinical rationale for adjuvant BCG in future trials focused on the treatment of patients with advanced malignant melanoma.     

Immunotherapy of melanoma

Melanoma is considered one of the immunogenic – if not the most immunogenic – malignancies. This is based on several observations.

• 1.Spontaneous remissions occur occasionally.
• 2.In about 5% of melanomas no primary tumour is found. The genetic aberrations of these tumours closely resemble those of cutaneous melanomas, and therefore are suggestive of spontaneous regressions of the primary tumours.
• 3.Both primary tumours and metastases often have brisk lymphocytic infiltrates, a phenomenon that is correlated with better outcome.
• 4.Studies of isolates of these tumour-infiltrating T lymphocytes have revealed that a proportion of these cells recognise melanoma antigens.
• 5.Melanomas respond to immunotherapy.

These observations have led to over 30 years of research on immunotherapy for melanoma; many of these efforts have failed, with only a few exceptions: interleukin-2 (IL-2) and to a lesser degree interferon-a (IFN-〈). Recently, new developments in immunotherapy have revolutionised this treatment modality. Anti-CTLA4 has received approval from the Food and Drugs Administration (FDA) and the European Medicines Agency (EMA) for the treatment of stage IV melanomas based on the improvement in overall survival in phase III trials, and more recently blockade of PD1/PDL1 interactions has shown objective clinical responses in a stage IV melanoma in early-phase clinical trials. In addition, several independent single-institution phase I/II trials using adoptive cell therapy have shown a consistently high response rate, including durable complete remissions in a substantial percentage of treated patients.Now, for the first time, immunotherapy has moved beyond the treatment of melanoma as both CTLA4 and PD1 blockade have been shown to induce objective responses in other tumour types as well.This chapter will discuss the mechanism of action, clinical efficacy and side effects of IL-2, the novel treatments consisting of the immune checkpoint blockade drugs anti-CTLA4 and anti-PD1 and adoptive cell therapy.     

Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous dendritic cells.

PURPOSE: We wanted to evaluate feasibility and safety of dendritic cell-based immunotherapy in patients with metastatic renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: Patients with metastatic RCC (n = 35) received vaccinations (i.v. or i.d.) of CD83+ autologous monocyte-derived dendritic cells (moDCs). MoDCs were loaded with lysate of cultured autologous or allogeneic permanent tumor cells (A-498) as well as keyhole limpet hemocyanin as control and helper antigen. Maturation of moDCs was induced by a combination of tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and prostaglandin E2. RESULTS: Treatment was associated with transient flu-like symptoms. In 2 of 27 evaluable patients, any evidence of disease disappeared (complete response). In both cases, metastatic tissue had been the source of tumor antigen. One patient had an objective partial response. Seven patients had stable disease, the remaining 17 patients had progressive disease. In 11 of 11 patients evaluated, moDCs induced strong immune responses against keyhole limpet hemocyanin. In 5 of 6 patients tested, enhanced immune responses against oncofetal antigen (immature laminin receptor; OFA/LRP) could also be detected. The strongest responses against OFA/LRP were detectable in 2 patients with complete response and partial response, respectively. At the time of submission, mean follow up is 32 months and 8 patients are currently alive. CONCLUSIONS: Our data indicate that moDC-based vaccination is well tolerated and has immunological as well as clinical effects in patients with metastatic RCC. OFA/LRP might be an attractive candidate antigen for DC-based immunotherapy of RCC.     

Dendritic cell vaccination in glioblastoma after fluorescence-guided resection

AIM: To assess whether the addition of a customized, active immunotherapy to standard of care including fluorescence-guided surgery, may provide hints of an improved survival for patients with poor-prognosis, incurable glioblastoma multiform.METHODS: Preliminary to our ongoing, phase-II clinical trial, we conducted a small pilot study enrolling five consecutive patients with resectable glioblastoma. In terms of Recursive Partitioning Analysis, four patients were class V and one was class IV. In all five cases, fluorescence-guided surgery was employed, followed by rapid steroid discontinuation. Patients were then treated with a combination of standard radio-chemotherapy with temozolomide and tumor lysate-pulsed, mature dendritic cell-based vaccinations.RESULTS: Though all five patients ultimately progressed, with any further treatment left to the sole decision of the treating oncologist, active immunotherapy was very well tolerated and induced specific immune responses in all three patients for whom enough material was available for such an assessment. Median progression-free survival was 16.1 mo. Even more important, median and mean overall survival were 27 mo and 26 mo, respectively. Three patients have died with an overall survival of 9 mo, 27 mo and 27.4 mo, while the other two are still alive at 32 mo and 36 mo, the former receiving treatment with bevacizumab, while the latter has now been off therapy for 12 mo. Four of five patients were alive at two years.CONCLUSION: Active immunotherapy with tumor lysate-pulsed, autologous dendritic cells is feasible, safe, well tolerated and biologically efficacious. A phase-II study is ongoing to possibly improve further on our very encouraging clinical results.     

Immunotherapies for non-small-cell lung cancer and mesothelioma

Non-small-cell lung cancer and mesothelioma are thoracic malignancies, which in their advanced stages are incurable and have poor prognosis. Advances in our understanding of immune responses to tumours, tumour immunosuppression mechanisms, and tumour-specific shared antigens enabled successful early clinical trials of several specific and non-specific immunotherapies. For non-small-cell lung cancer, phase 3 clinical trial results of Toll-like receptor agonists show little promise. However, ongoing phase 3 trials are assessing melanoma-associated antigen A3 vaccine, liposomal BLP25, belagenpumatucel-L, and talactoferrin. In mesothelioma, immunotherapies being investigated include dendritic cell-based and Listeria-based vaccines, and allogeneic tumour cell and WT1 analogue peptide vaccines. Selection of appropriate patients and disease stages for immunotherapy, measurement of tumour-specific immune responses, and understanding the association between immune and clinical responses are some of the major challenges for the development of immunotherapies for these malignancies.     

Increasing the efficacy of tumor cell vaccines by enhancing cross priming

Cancer immunotherapy has been attempted for more than a century, and investment has intensified in the last 20years. The complexity of the immune system is exemplified by the myriad of immunotherapeutic approaches under investigation. While anti-tumor immunity has been achieved experimentally with multiple effector cells and molecules, particular promise is shown for harnessing the CD8 T cell response. Tumor cell-based vaccines have been employed in hundreds of clinical trials to date and offer several advantages over subunit and peptide vaccines. However, tumor cell-based vaccines, often aimed at cross priming tumor-reactive CD8 T cells, have shown modest success in clinical trials. Here we review the mechanisms of cross priming and discuss strategies to increase the efficacy of tumor cell-based vaccines. A synthesis of recent findings on tissue culture conditions, cell death, and dendritic cell activation reveals promising new avenues for clinical investigation.     

Role of bacillus Calmette-Guérin in the treatment of advanced melanoma

Early trials of Bacillus Calmette-Guérin (BCG)-based immunotherapy for melanoma consistently show a trend toward improved clinical outcomes in patients treated with BCG compared with observation alone. As an extension of these findings, investigators have initiated the Malignant Melanoma Active Immunotherapy (MMAIT) trials in patients with stage III (MMAIT-III) and stage IV (MMAIT-IV) disease. The overall survival of the patients receiving BCG plus placebo was much better than expected in both studies, thus suggesting a potential for BCG as an adjuvant after the resection of advanced disease. The work contained herein will explore the clinical rationale for adjuvant BCG in future trials focused on the treatment of patients with advanced malignant melanoma.     

Dendritic cell vaccination as a treatment modality for melanoma

As melanoma is an immunogenic tumor, immunotherapy has been investigated as a possible treatment modality for melanoma patients at high risk of relapse and those with metastatic disease. In the past decade progress has been made, ranging from rather nonspecific stimulations of the immune system with IL-2 and IFN-α to more specific approaches based on vaccination with tumor antigens. Owing to their unique features, dendritic cells (DCs) represent an important tool for tumor antigen-specific immunotherapy. However, clinical vaccination trials with DCs showed sobering results with respect to objective responses and improvement of overall survival. In this review, principles and methods of DC-based vaccination are presented. Mechanisms impairing clinically successful vaccination strategies are described. Finally, we will discuss perspectives for future developments of DC-based vaccines that might lead melanoma treatment to a new era.     

Immunization of NSCLC patients with antigen-pulsed immature autologous dendritic cells

Only a handful of NSCLC patients have been included in dendritic cell (DC) vaccine clinical trials. We had previously reported a series of 16 individuals with stages IA-IIIB NSCLC who received autologous DC vaccines matured with dendritic cell/T cell-derived maturation factor (DCTCMF). Here we report the results of a continuation study with similar inclusion criteria, immunization protocol, and analysis, using an immature DC vaccine. Of the 14 participants, 7 had undergone surgical resection (stage I/II), with or without adjuvant therapy, and 7 with unresectable stage III had been treated with chemo-radiation alone. Autologous DCs were pulsed with apoptotic bodies derived from an allogeneic NSCLC cell line that over-expresses Her2/neu, CEA, WT1, Mage2, and survivin. DCs were not exposed to any maturation stimulus. Individuals received two intradermal vaccines (average 8.1x10(7) DC per immunization) 1 month apart. Immune responses were measured by IFN-gamma ELISPOT, comparing relative number of antigen-reactive T-cells from pre-vaccine to timepoints post-immunization. Immunologic responses were seen in 4/7 stage III unresectable, and 6/7 stage I/II surgically resected patients, including 3/3 resected patients who had also received adjuvant chemo-radiation. There were no related adverse events. One of seven surgically resected patients recurred and 4/7 stage III patients progressed. Three of five patients with progressive disease showed no immunologic response. Data indicate that immature DC pulsed with apoptotic tumour cells have similar biologic activity to a DCTCMF-matured DC preparation delivered in a similar clinical protocol. Therapeutic efficacy is unknown and clinical outcomes are anecdotal.