Durable Clinical Responses and Long-Term Follow-Up of Stage III-IV Non-Small-Cell Lung Cancer (NSCLC) Patients Treated With IDO Peptide Vaccine in a Phase I Study-A Brief Research Report.

Background: Long-term follow-up on a clinical trial of 15 stage III-IV NSCLC patients treated with an Indoleamine 2,3-Dioxygenase (IDO) peptide vaccine (NCT01219348). Methods: Fifteen HLA-A2-positive patients with stable stage III-IV NSCLC after standard chemotherapy were treated with subcutaneous vaccinations (100 µg IDO5 peptide, sequence ALLEIASCL, formulated in 900 µl Montanide) biweekly for 2.5 months and thereafter monthly until progression or up to 5 years. Here we report long-term clinical follow-up, toxicity and immunity. Results: Three of 15 patients are still alive corresponding to a 6-year overall survival of 20 %. Two patients continued monthly vaccinations for 5 years (56 vaccines). One of the two patients developed a partial response (PR) of target lesions in the liver 15 months after the first vaccine and has remained in PR ever since. The other patient had a solitary distant metastasis in a lymph node in retroperitoneum at baseline which normalized during treatment. All following evaluation scans during the treatment have been tumor free. The vaccine was well tolerated for all 5 years with no long-term toxicities registered. The third long-term surviving patient discontinued vaccinations after 11 months due to disease progression. Flow cytometry analyses of PBMCs from the two long-term responders demonstrated stable CD8+ and CD4+ T-cell populations during treatment. In addition, presence of IDO-specific T-cells was detected by IFN-γ Elispot in both patients at several time points during treatment. Conclusion: IDO peptide vaccination was well tolerated for administration up to 5years. Two of 15 patients are long-term responders with ongoing clinical response 6 years after 1st vaccination.

[Multicentric reticulohistiocytosis is a rare form of paraneoplasia].

A 59-year-old woman developed a rash and severe arthralgia, which primarily affected her fingers. She displayed digital arthritis and nodules on the hands, chest, face, and oral cavity. Blood samples were normal. Skin biopsies revealed histiocytic proliferation. The surface marker profile and clinical findings were consistent with multicentric reticulohistiocytosis, which may occur as a paraneoplastic phenomenon. On workup, she was diagnosed with an otherwise asymptomatic stage IVC fallopian tube cancer. She experienced little effect of prednisolone, but her condition improved on antineoplastic treatment.

Indoleamine 2,3-dioxygenase and survivin peptide vaccine combined with temozolomide in metastatic melanoma.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) and survivin have been identified as potential targets for cancer vaccination. In this phase II study a vaccine using the peptides Sur1M2 and IDO5 was combined with the chemotherapy temozolomide (TMZ) for treatment of metastatic melanoma patients. The aim was to simultaneously target several immune inhibiting mechanisms and the highly malignant cells expressing survivin. METHODS: HLA-A2 positive patients with advanced malignant melanoma were treated biweekly with 150 mg/m2 TMZ daily for 7 days followed by subcutaneous vaccination with 250 µg of each peptide in 500 µL Montanide solution at day 8. Granulocyte-macrophage colony-stimulating factor was used as an adjuvant and topical imiquimod was applied prior to vaccination. Treatment was continued until disease progression. Clinical response was evaluated by PET-CT and immunological outcome was assessed by ELISPOT and flow cytometry. RESULTS: In total, 17 patients were treated with a clinical benefit rate of 18% including one patient with partial tumor regression. Immune analyses revealed a vaccine specific response in 8 (67%) of 12 patients tested, a significant decrease in the frequency of CD4+ T-cells during treatment, a tendency towards decreasing frequencies of naïve CD4+ and CD8+ T-cells, and increasing frequencies of memory CD4+ and CD8+ T-cells. CONCLUSIONS: These results demonstrate that vaccine-induced immunity towards survivin and IDO-derived peptides can be achieved in combination with TMZ in patients mainly suffering from grade M1c melanoma including patients with brain metastases. A significant clinical activity could not be proven in this small study and a larger setup is needed to properly assess clinical efficacy.

Dendritic cell vaccination in combination with docetaxel for patients with metastatic castration-resistant prostate cancer: A randomized phase II study.

BACKGROUND AIMS: We investigated whether the addition of an autologous dendritic cell-based cancer vaccine (DCvac) induces an immune response in patients with metastatic castration-resistant prostate cancer treated with docetaxel. METHODS: Forty-three patients were randomized 1:1 to receive up to 10 cycles of docetaxel alone, 75 mg/m2 every 3 weeks or in combination with DCvac. Monocytes were harvested following a leukapheresis procedure, matured ex vivo and subsequently transfected with messenger RNA encoding multiple tumor-associated antigens (TAAs). DCvac was administered intradermally twice through treatment cycles 1-4 and once through treatment cycles 5-10. Immune cell composition and antigen-specific responses were analyzed using flow cytometry, ELISpot and delayed type hypersensitivity (DTH) tests. Toxicity was graded according to Common Terminology Criteria for Adverse Events version 3.0. Progression-free survival (PFS) and disease-specific survival (DSS) was calculated using the Kaplan-Meier method. RESULTS: Prostate-specific antigen responses were similar in patients treated with docetaxel alone and combination therapy (58% versus 38%; P = 0.21). PFS and DSS were comparable: 5.5 versus 5.7 months (P = 0.62, log rank) and 21.9 versus 25.1 months (P = 0.60, log rank). Nine (50%) and 14 (78%) patients treated with docetaxel and DCvac had a TAA-specific or vaccine-specific immune response in the ELISpot and DTH analysis, respectively. Vaccine induced toxicity was limited to local reactions. Decline in myeloid-derived suppressor cells at the third treatment cycle was found to be an independent predictor of DSS. CONCLUSIONS: The addition of DCvac was safe. Immune responses were detected in approximately half of the patients investigated.

Safety, immune and clinical responses in metastatic melanoma patients vaccinated with a long peptide derived from indoleamine 2,3-dioxygenase in combination with ipilimumab.

BACKGROUND AIM: Indoleamine 2,3-dioxygenase (IDO) is an emerging new target in cancer therapy that can be targeted with active immunotherapy (e.g. through peptide vaccination). Furthermore, IDO has been identified as a key mechanism underlying resistance to treatment with the checkpoint blocking antibody ipilimumab (ipi). METHODS: Ten patients with metastatic melanoma participated in a phase I first-in-human clinical study assessing safety of combining ipi with a 21-mer synthetic peptide vaccine from IDO denoted IDOlong. Secondary and tertiary end points included vaccine and clinical response. RESULTS: Treatment was generally safe and well tolerated. Vaccine related adverse reactions included grade I and II erythema, oedema and pruritus at the vaccination site, which were manageable with mild topical corticosteroids. One patient developed presumed ipi-induced colitis. It initially responded to high-dose parenteral corticosteroids but later relapsed while the patient was admitted to a local hospital, where he died after receiving suboptimal therapy. Vaccine-specific T-cell responses were detectable ex vivo in three patients. At first evaluation, five of the 10 treated patients were in stable disease, one of whom had an unconfirmed partial response. CONCLUSIONS: Treatment with IDOlong synthetic peptide vaccine in combination with ipi was generally safe and without augmented toxicity. The vaccine induced readily detectable T-cell responses in a subset of patients. Treatment showed signs of clinical activity, although not exceeding efficacy of ipi alone. Results should be confirmed in a larger study.

Long-Lasting Complete Responses in Patients with Metastatic Melanoma after Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes and an Attenuated IL2 Regimen.

PURPOSE: Adoptive cell transfer therapy (ACT) based on autologous tumor-infiltrating lymphocytes (TIL) has achieved impressive clinical results in several phase I and II trials performed outside of Europe. Although transient, the toxicities associated with high-dose (HD) bolus IL2 classically administered together with TILs are severe. To further scrutinize whether similar results can be achieved with lower doses of IL2, we have carried out a phase I/II trial of TIL transfer after classical lymphodepleting chemotherapy followed by an attenuated IL2 regimen. EXPERIMENTAL DESIGN: Twenty-five patients with progressive treatment-refractory metastatic melanoma, good clinical performance, age < 70 years, and at least one resectable metastasis were eligible. TIL infusion was preceded by standard lymphodepleting chemotherapy and followed by attenuated doses of IL2 administered in an intravenous, continuous decrescendo regimen (ClinicalTrials.gov Identifier: NCT00937625). RESULTS: Classical IL2-related toxicities were observed but patients were manageable in a general oncology ward without the need for intervention from the intensive care unit. RECIST 1.0 evaluation displayed three complete responses and seven partial responses (ORR 42%). Median overall survival was 21.8 months. Tumor regression was associated with a higher absolute number of infused tumor-reactive T cells. Moreover, induction and persistence of antimelanoma T-cell responses in the peripheral blood was strongly correlated to clinical response to treatment. CONCLUSIONS: TIL-ACT with a reduced IL2 decrescendo regimen results in long-lasting complete responses in patients with treatment-refractory melanoma. Larger randomized trials are needed to elucidate whether clinical efficacy is comparable with TIL-ACT followed by HD bolus IL2. Clin Cancer Res; 22(15); 3734-45. ©2016 AACR.

Multinational study exploring patients' perceptions of side-effects induced by chemo-radiotherapy.

PURPOSE: We aimed to prospectively assess the incidence, severity and patients' perceptions of side-effects induced by radiotherapy and concomitant weekly cisplatin. PATIENTS AND METHODS: This multinational survey included patients with a diagnosis of gynaecological or head and neck cancer scheduled to receive radiotherapy and concomitant weekly cisplatin. Patients completed a questionnaire prior to anti-cancer treatment and after 3 weeks of treatment. Baseline frequency and severity of symptoms were compared to frequency and severity after 3 weeks of treatment, and patients were asked to rank the five most severe symptoms experienced. RESULTS: An increase in the severity as well as in the mean number of symptoms (18 compared to 24) was observed during treatment. Patients ranked 7 of the 10 most feared baseline symptoms as non-physical, whereas 8 of the 10 most feared symptoms after 3 weeks of treatment were physical. Nausea was ranked as the 5th most severe symptom during treatment, despite 98% of patients receiving antiemetic prophylaxis. CONCLUSION: Patients with head and neck cancer or gynaecological cancer suffer from a number of primarily non-physical symptoms before starting combined chemo-radiotherapy. After 3 weeks of treatment patients score 8 of the 10 most feared symptoms as physical. Future trials focusing on the prevention of side-effects in patients receiving radiotherapy and concomitant chemotherapy are highly warranted.

The targeting of indoleamine 2,3 dioxygenase -mediated immune escape in cancer.

The era of immunotherapies was unleashed in 2010 with the Food and Drug Administration (FDA) approval of the first therapeutic vaccine sipuleucel-T as a standard treatment for metastatic prostate cancer. Next, the first immune-activating anticytotoxic lymphocyte antigen-4 (CTLA-4) antibody ipilimumab exhibiting 'immune checkpoint blockade' was approved by FDA and European Medical Agency (EMA) for the treatment of patients with metastatic melanoma. New generations of immune checkpoint blockading antibodies targeting programmed cell death 1 (PD-1) and its ligand (PD-L1) are now under intense investigation in metastatic melanoma (MM) and non-small-cell lung cancer (NSCLC), and impressive clinical results are anticipated. Despite these successes, only a fraction of patients become clinical responders to therapy. Thus, to improve the selection of patients likely to respond, scrutinizing different immune parameters during treatment is essential. In the summary of this PhD thesis, we investigated changes in immune parameters and their possible correlation with clinical efficacy in patients with MM during treatments with the standard chemo- and immunotherapies, temozolomide (TMZ) and interferon-α2b/interleukin-2 (IFN-α/IL-2) immunotherapy. The overall aim was to assess changes in frequency and absolute counts of different immune cell subsets before and after treatment and correlate to clinical benefit. Furthermore, the thesis covers a finalized, clinical phase 1 study in patients with NSCLC testing a peptide vaccination with a HLA-A2-restricted epitope derived from indoleamine 2,3 dioxygenase (IDO). The overall aim in this trial was to evaluate safety and tolerability of IDO as an anticancer vaccine target in patients with NSCLC and to assess whether immunity correlated to clinical response.

Long-lasting disease stabilization in the absence of toxicity in metastatic lung cancer patients vaccinated with an epitope derived from indoleamine 2,3 dioxygenase.

PURPOSE: To investigate targeting of indoleamine 2,3 dioxygenase (IDO) enzyme using a synthetic peptide vaccine administered to patients with metastatic non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: In a clinical phase I study, we treated 15 HLA-A2-positive patients with stage III-IV NSCLC in disease stabilization after standard chemotherapy. Patients were treated with imiquimod ointment and subcutaneous vaccinations (100 µg IDO5 peptide, sequence ALLEIASCL, formulated in 900 µL Montanide). Primary endpoint was toxicity. Clinical benefit and immunity were assessed as secondary endpoints. RESULTS: No severe toxicity was observed. One patient developed a partial response (PR) after one year of vaccine treatment, whereas long-lasting stable disease (SD) ≥ 8.5 months was demonstrated in another six patients. The median overall survival (OS) was 25.9 months. Patients demonstrated significant improved OS (P = 0.03) when compared with the group of patients excluded because of HLA-A2 negativity. IDO-specific CD8(+) T-cell immunity was demonstrated by IFN-γ Elispot and Tetramer staining. Fluorescence-activated cell sorting analyses demonstrated a significant reduction of the Treg population (P = 0.03) after the sixth vaccine (2.5 months) compared with pretreatment levels. Furthermore, expression of IDO was detected in nine of ten tumor biopsies by immunohistochemistry. High-performance liquid chromatography analyses of kynurenine/tryptophan (Kyn/Trp) ratio in sera were performed. In long-term analyses of two clinical responding patients, the ratio of Kyn/Trp remained stable. CONCLUSIONS: The vaccine was well tolerated with no severe toxicity occurring. A median OS of 25.9 months was demonstrated and long-lasting PR+SD was seen in 47% of the patients.

Immune modulations during chemoimmunotherapy & novel vaccine strategies--in metastatic melanoma and non small-cell lung cancer.

This thesis describes the treatment of metastatic melanoma (MM) and non small-cell lung cancer (NSCLC) from an immunotherapeutic approach. The purpose of the first part of the thesis was to assess how treatment with Temozolomide (TMZ) chemotherapy affects the immune system in patients with metastatic MM. Our results showed that the number of T lymphocytes was significantly reduced after 3 treatment cycles. Furthermore, the induced lymphopenia was positive correlated to achievement of clinical benefit. We demonstrated that the proportion of CD4+ and Treg lymphocytes decreased whereas the CD8+ T cells increased. In particular, we demonstrated that mature CD8+ T cells increased during treatment. Analyses of peripheral blood before and after treatment showed that T cell responses against common viral epitopes were conserved despite chemotherapy. Surprisingly, we found a significant increase in T cell responses against well-known MM tumour specific antigens. Overall, we have verified that TMZ in addition to being an alkylating and cytotoxic chemotherapy, also possess immune modulatory effect in MM patients treated with standard dosage of TMZ. In the second part of the thesis we examined how treatment with Interferon alfa-2b and Interleukin 2 (IFNα/IL2) affects the immune system. We demonstrated a significant induced lymphocytosis during treatment. Furthermore, we showed that the percentage increase in lymphocytes was positively correlated to clinical outcome. Moreover, we have seen that IFNα/IL2 leads to significant increase in NK and Treg cells in both patients with and without clincal effect. In general, T cell responses against common viral epitopes and well-known melanoma tumour specific antigens were low. Furthermore, the study confirmed that elevated LDH is negatively correlated with both treatment response and median overall survival. Overall, we have characterized changes of immune cells and correlated them with clinical efficacy during the couse of IFNα/IL2 used in standard dosage. In the third part we investigated if vaccination with a peptide derived from IDO was feasible in patients with metastatic NSCLC. This "First in Man" trial was safe and showed modest side effects only. Since IDO was expressed in NSCLC tissues it was found to be a relevant target. One patient achieved significant regression of liver metastases (confirmed partial response) and another 6/15 patients achieved prolonged disease stabilization. Furthermore, median overall survival was 25.9 months demonstrating a better survival in vaccinated compared to non-vaccinated comparable NSCLC patients. The presence of IDO specific CD8+ T cells were detected by IFNy Elispot. In patients with clinical effect of the vaccine IDO-specific CD8+ T cells at pre-treatment was significanctly increased. Moreover, low-frequent IDO positive tetramer CD8+ T cells were detected and led to effective killing of an IDO+ HLA-A2 positive cancer cell line (SW480) in 1 patient. Moreover, flow cytometry was performed and in general no significant changes in CD8+ and CD4+ T cells were seen, although patients with clinical response showed a trend towards increased mature CD8+ T cells during treatment. In addition, we found lower levels of Tregs as well as an increased level of NK cells after 6 vaccinations. Elevated Kyn/Trp ratio is suggested to mirror IDO activity. In 8/11 patients the level after the 6th vaccine was stable compared to baseline. No differences between patients with clinical benefit (4/5) and patients with progressive disease (4/6) were demonstrated. Two patients had an increase in Kyn/Trp ration meanwhile demonstrating a high expression of IDO. In 2 patients with clinical response long-term stabilization of Kyn/Trp was observed. Overall, the vaccine was well tolerated with no adverse toxicity. Median overall survival was 25.9 months with long term disease stabilization achieved in 47% of the treated patients. Based on the promising clinical results achieved in the vaccine trial for NSCLC patients, we launched a new clinical trial for MM patients (ongoing patient recruitment) in June 2012. In order to enhance the immune response the vaccine comprises IDO plus Survivin peptide as well as the adjuvants Montanide, Aldara and GM-CSF. Finally, the vaccine is given in combination with TMZ. Patients are evaluated every 3rd month with PET-CT scan. Preliminary clinical data from the first 7/30 evaluable patients are presented. Two patients demonstrated a patial response with 57% and 45% tumour regression lasting for 10 months and 6+ months respectively, corresponding to a preliminary objective response rate of 29%. The vaccine has been manageable and without significant side effects.

Depletion of T lymphocytes is correlated with response to temozolomide in melanoma patients.

Therapeutic strategies to deplete lymphocytes, especially regulatory T cells, in cancer patients have been proposed to increase the benefits of (immuno)chemotherapy. In this study, we explored the influence of temozolomide (TMZ) on different T-cell populations and addressed if the depletion of CD4+ T cells would be associated to the clinical benefits of TMZ. Patients were treated with TMZ (150 mg/m2 daily, every two weeks on a four-week schedule) until disease progression. Changes in T-lymphocyte subsets were characterized by flow cytometry. All patients enrolled in this study had histologically verified unresectable stage IV melanoma. Objective responses were induced in 12.5% of the patients, while 42.5% of them obtained short-term disease stabilization. The median progression-free survival (PFS) of this patient cohort was 8.7 mo. Lymphopenia (< 0.7 × 109 cells/L, grade 2) developed in 71% of the patients after 3 treatment cycles (~100 d). The development of grade 2 lymphopenia after the 3rd cycle of therapy positively correlated with clinical outcome (p = 0.01), and was linked, though non-significantly, to prolonged median PFS (303 vs. 200 d). In addition, significant changes in CD8+ T-cell subgroups were observed, notably a shift from naïve T cells toward more differentiated memory T cells. Finally, we demonstrated that specific CD8+ T-cell responses against selected tumor associated antigens (TAAs) were enhanced by the administration of TMZ (p = 0.04), while virus-specific T-cell responses were stable. Thus, immunological monitoring in the course of TMZ treatment might become an important tool for clinical guidance in the future.

Methods to improve adoptive T-cell therapy for melanoma: IFN-γ enhances anticancer responses of cell products for infusion.

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term-cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8(+) and CD4(+) T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

Adoptive cell therapy with autologous tumor infiltrating lymphocytes and low-dose Interleukin-2 in metastatic melanoma patients.

BACKGROUND: Adoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion. Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed. Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections. METHODS: This is a pilot trial (ClinicalTrials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS ≤1, age <70, measurable and progressive disease and no involvement of the central nervous system. Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day. RESULTS: Low-dose IL-2 considerably decreased the treatment related toxicity with no grade 3-4 IL-2 related adverse events. Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment. Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed. Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission. CONCLUSION: Complete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy.

Natural CD4+ T-cell responses against indoleamine 2,3-dioxygenase.

BACKGROUND: The enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tumor antigens. Recently, we described cytotoxic CD8(+) T-cell reactivity towards IDO-derived peptides. METHODS AND FINDINGS: In the present study, we show that CD4(+) helper T cells additionally spontaneously recognize IDO. Hence, we scrutinized the vicinity of the previously described HLA-A*0201-restricted IDO-epitope for CD4(+) T-cell epitopes. We demonstrated the presence of naturally occurring IDO-specific CD4(+) T cells in cancer patients and to a lesser extent in healthy donors by cytokine release ELISPOT. IDO-reactive CD4(+) T cells released IFN-γ, TNF-α, as well as IL-17. We confirm HLA class II-restriction by the addition of HLA class II specific blocking antibodies. In addition, we detected a trend between class I- and class II-restricted IDO responses and detected an association between IDO-specific CD4(+) T cells and CD8(+) CMV-responses. Finally, we could detect IL-10 releasing IDO-reactive CD4(+) T cells. CONCLUSION: IDO is spontaneously recognized by HLA class II-restricted, CD4(+) T cells in cancer patients and in healthy individuals. IDO-specific T cells may participate in immune-regulatory networks where the activation of pro-inflammatory IDO-specific CD4(+) responses may well overcome or delay the immune suppressive actions of the IDO-protein, which are otherwise a consequence of the early expression of IDO in maturing antigen presenting cells. In contrast, IDO-specific regulatory T cells may enhance IDO-mediated immune suppression.

Metastatic melanoma patients treated with dendritic cell vaccination, Interleukin-2 and metronomic cyclophosphamide: results from a phase II trial.

Dendritic cells (DC) are the most potent antigen presenting cells and have proven effective in stimulation of specific immune responses in vivo. Competing immune inhibition could limit the clinical efficacy of DC vaccination. In this phase II trial, metronomic Cyclophosphamide and a Cox-2 inhibitor have been added to a DC vaccine with the intend to dampen immunosuppressive mechanisms. Twenty-eight patients with progressive metastatic melanoma were treated with autologous DCs pulsed with survivin, hTERT, and p53-derived peptides (HLA-A2(+)) or tumor lysate (HLA-A2(-)). Concomitantly the patients were treated with IL-2, Cyclophosphamide, and Celecoxib. The treatment was safe and tolerable. Sixteen patients (57 %) achieved stable disease (SD) at 1st evaluation and 8 patients had prolonged SD (7-13.7 months). The median OS was 9.4 months. Patients with SD had an OS of 10.5 months while patients with progressive disease (PD) had an OS of 6.0 months (p = 0.048) even though there were no differences in prognostic factors between the two groups. Despite the use of metronomic Cyclophosphamide, regulatory T cells did not decrease during treatment. Indirect IFN-γ ELISPOT assays showed a general increase in immune responses from baseline to the time of 4th vaccination. Induction of antigen-specific immune responses was seen in 9 out of 15 screened HLA-A2(+) patients. In conclusion, the number of patients obtaining SD more than doubled and 6-month survival significantly increased compared to a previous trial without Cyclophosphamide and Celecoxib. A general increase in immune responses against the tested peptides was observed.