Correction to: Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

In the original publication of the article the following abstract and keywords were inadvertently omitted.

Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7­1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

EGFR signaling suppresses type 1 cytokine-induced T-cell attracting chemokine secretion in head and neck cancer.

Resistance to antitumor immunity can be promoted by the oncogenic pathways operational in human cancers, including the epidermal growth factor receptor (EGFR) pathway. Here we studied if and how EGFR downstream signaling in head and neck squamous cell carcinoma (HNSCC) can affect the attraction of immune cells. HPV-negative and HPV-positive HNSCC cell lines were analyzed in vitro for CCL2, CCL5, CXCL9, CXCL10, IL-6 and IL-1ß expression and the attraction of T cells under different conditions, including cetuximab treatment and stimulation with IFNγ and TNFα using qPCR, ELISA and migration experiments. Biochemical analyses with chemical inhibitors and siRNA transfection were used to pinpoint the underlying mechanisms. Stimulation of HNSCC cells with IFNγ and TNFα triggered the production of T-cell attracting chemokines and required c-RAF activation. Blocking of the EGFR with cetuximab during this stimulation increased chemokine production in vitro, and augmented the attraction of T cells. Mechanistically, cetuximab decreased the phosphorylation of MEK1, ERK1/2, AKT, mTOR, JNK, p38 and ERK5. Chemical inhibition of EGFR signaling showed a consistent and pronounced chemokine production with MEK1/2 inhibitor PD98059 and JNK inhibitor SP600125, but not with inhibitors of p38, PI3K or mTOR. Combination treatment with cetuximab and a MEK1/2 or JNK inhibitor induced the highest chemokine expression. In conclusion, overexpression of EGFR results in the activation of multiple downstream signaling pathways that act simultaneously to suppress type 1 cytokine stimulated production of chemokines required to amplify the attraction of T cells.

A novel allogeneic off-the-shelf dendritic cell vaccine for post-remission treatment of elderly patients with acute myeloid leukemia.

In elderly acute myeloid leukemia (AML) patients post-remission treatment options are associated with high comorbidity rates and poor survival. Dendritic cell (DC)-based immunotherapy is a promising alternative treatment strategy. A novel allogeneic DC vaccine, DCP-001, was developed from an AML-derived cell line that uniquely combines the positive features of allogeneic DC vaccines and expression of multi-leukemia-associated antigens. Here, we present data from a phase I study conducted with DCP-001 in 12 advanced-stage elderly AML patients. Patients enrolled were in complete remission (CR1/CR2) (n = 5) or had smoldering disease (n = 7). All patients were at high risk of relapse and ineligible for post-remission intensification therapies. A standard 3 + 3 dose escalation design with extension to six patients in the highest dose was performed. Patients received four biweekly intradermal DCP-001 injections at different dose levels (10, 25, and 50 million cells DCP-001) and were monitored for clinical and immunological responses. Primary objectives of the study (feasibility and safety) were achieved with 10/12 patients completing the vaccination program. Treatment was well tolerated. A clear-cut distinction between patients with and without detectable circulating leukemic blasts during the vaccination period was noted. Patients with no circulating blasts showed an unusually prolonged survival [median overall survival 36 months (range 7-63) from the start of vaccination] whereas patients with circulating blasts, died within 6 months. Long-term survival was correlated with maintained T cell levels and induction of multi-functional immune responses. It is concluded that DCP-001 in elderly AML patients is safe, feasible and generates both cellular and humoral immune responses.

Intratumoral HPV16-Specific T Cells Constitute a Type I-Oriented Tumor Microenvironment to Improve Survival in HPV16-Driven Oropharyngeal Cancer.

Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome.Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database.Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages.Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACRSee related commentary by Laban and Hoffmann, p. 505.

Monitoring of the Immune Dysfunction in Cancer Patients.

Immunotherapy shows promising clinical results in patients with different types of cancer, but its full potential is not reached due to immune dysfunction as a result of several suppressive mechanisms that play a role in cancer development and progression. Monitoring of immune dysfunction is a prerequisite for the development of strategies aiming to alleviate cancer-induced immune suppression. At this point, the level at which immune dysfunction occurs has to be established, the underlying mechanism(s) need to be known, as well as the techniques to assess this. While it is relatively easy to measure general signs of immune suppression, it turns out that accurate monitoring of the frequency and function of immune-suppressive cells is still difficult. A lack of truly specific markers, the phenotypic complexity among suppressive cells of the same lineage, but potentially with different functions and functional assays that may not cover every mechanistic aspect of immune suppression are among the reasons complicating proper assessments. Technical innovations in flow and mass cytometry will allow for more complete sets of markers to precisely determine phenotype and associated function. There is, however, a clear need for functional assays that recapitulate more of the mechanisms employed to suppress the immune system.

Local delivery of CpG-B and GM-CSF induces concerted activation of effector and regulatory T cells in the human melanoma sentinel lymph node.

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I-II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3(+) DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8(+) T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs.

A phase 1/2 study combining gemcitabine, Pegintron and p53 SLP vaccine in patients with platinum-resistant ovarian cancer.

PURPOSE: Preclinical tumor models show that chemotherapy has immune modulatory properties which can be exploited in the context of immunotherapy. The purpose of this study was to determine the feasibility and immunogenicity of combinations of such an immunomodulatory chemotherapeutic agent with immunotherapy, p53 synthetic long peptide (SLP) vaccine and Pegintron (IFN-α) in patients with platinum-resistant p53-positive epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: This is a phase 1/2 trial in which patients sequential 6 cycles of gemcitabine (1000 mg/kg2 iv; n = 3), gemcitabine with Pegintron before and after the first gemcitabine cycle (Pegintron 1 µg/kg sc; n = 6), and gemcitabine and Pegintron combined with p53 SLP vaccine (0.3 mg/peptide, 9 peptides; n = 6). At baseline, 22 days after the 2nd and 6th cycle, blood was collected for immunomonitoring. Toxicity, CA-125, and radiologic response were evaluated after 3 and 6 cycles of chemotherapy. RESULTS: None of the patients enrolled experienced dose-limiting toxicity. Predominant grade 3/4 toxicities were nausea/vomiting and dyspnea. Grade 1/2 toxicities consisted of fatigue (78%) and Pegintron-related flu-like symptoms (72%). Gemcitabine reduced myeloid-derived suppressor cells (p = 0.0005) and increased immune-supportive M1 macrophages (p = 0.04). Combination of gemcitabine and Pegintron stimulated higher frequencies of circulating proliferating CD4+ and CD8+ T-cells but not regulatory T-cells. All vaccinated patients showed strong vaccine-induced p53-specific T-cell responses. CONCLUSION: Combination of gemcitabine, the immune modulator Pegintron and therapeutic peptide vaccination is a viable approach in the development of combined chemo-immunotherapeutic regimens to treat cancer.

Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

Development of thyroglobulin antibodies after GVAX immunotherapy is associated with prolonged survival.

Cancer immunotherapy induces a variety of autoinflammatory responses, including those against the thyroid gland, which can be exploited to predict clinical outcomes. Considering the paucity of information about thyroid autoimmunity in patients receiving cancer vaccines, we designed our study to assess the development of thyroglobulin antibodies (TgAbs) in patients treated with GVAX (vaccine made of a tumor cell type transfected with GM-CSF) and/or ipilimumab and correlated seroconversion with survival. Using both in house and commercial ELISA assays, we measured TgAbs in patients with pancreatic (No. = 53), prostate (No. = 35) or colon (No. = 8) cancer, before and after treatment with GVAX only (No. = 34), GVAX plus ipilimumab (No. = 42) or ipilimumab (No. = 20), and correlated their levels with patient's survival, disease status and T-cell surface markers. Antibodies to thyroperoxidase, myeloperoxidase, proteinase 3, insulin and actin were also measured. TgAbs specifically developed after GVAX, independent of the underlying cancer (81% in prostate, 75% colon cancer and 76% pancreatic cancer) and co-administration of ipilimumab (75% in GVAX only and 78% in GVAX plus ipilimumab). This TgAbs seroconversion could be detected mainly by the in house assay, suggesting that the thyroglobulin epitopes recognized by the antibodies induced by GVAX are different from the epitopes seen in the classic form of Hashimoto thyroiditis. Notably, TgAbs seroconversion was associated with significantly prolonged survival (p = 0.01 for pancreas and p = 0.005 for prostate cancer). In conclusion, GVAX immunotherapy induces the appearance of TgAbs that recognize a unique antigenic repertoire and associate with prolonged survival.

IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14(+)CD141(+)DC-SIGN(+) DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a(+) subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8(+) T cells, migration of immature CD14(+) DDC was accompanied by increased release of IL-10, poor expansion of CD4(+) and CD8(+) T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.

Functional characterization of a STAT3-dependent dendritic cell-derived CD14+ cell population arising upon IL-10-driven maturation.

Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a+ DCs found in the human skin to CD14+CD141+ macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14- and CD14+ DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14+ DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14+ cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14+ DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8+ T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14+ counterparts, CD14- monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14+ DCs as compared with DCs matured in standard conditions or CD14- DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14+ phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.

Priming of PRAME- and WT1-specific CD8+ T cells in healthy donors but not in AML patients in complete remission: Implications for immunotherapy.

Active immunotherapy may prevent the relapse of acute myeloid leukemia (AML) by inducing leukemia-specific T cells. Here, we investigated whether Wilms' tumor 1 (WT1) and preferentially expressed antigen in melanoma (PRAME)-specific T cells could be induced upon the priming of healthy donor- and AML patient-derived T cells with HLA-A2-matched, peptide-loaded allogeneic dendritic cells. AML-reactive, tetramer (Tm)-binding and interferon-producing, cytotoxic T lymphocytes specific for PRAME could readily be isolated from healthy individuals and maintained in culture. In this setting, priming efficacy was significantly higher for PRAME than for WT1. The priming of T cells from patient-derived material proved to be near-to-impossible: No leukemia-associated antigen (LAA)-specific T cell could be primed in 4 patients that had recently achieved a complete response (CR), and in only 1 out of 3 patients exhibiting a sustained CR we did observe WT1-specific T cells, though with a low frequency. These findings suggest that the functionality and/or repertoire of T cells differ in healthy subjects and AML patients in CR, and may have repercussions for the implementation of active vaccination approaches against AML.

Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients.

BACKGROUND: New treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells. METHODS: Tetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells. RESULTS: Ex vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein. CONCLUSIONS: Here we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells.

BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.

T cell profiling reveals high CD4+CTLA-4 + T cell frequency as dominant predictor for survival after prostate GVAX/ipilimumab treatment.

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.

Combined immunotherapy with granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells and ipilimumab in patients with metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial.

BACKGROUND: The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5×10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3×10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0·3, 1·0, 3·0, or 5·0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288. FINDINGS: We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3·0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5·0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3·0 mg/kg dose level, rather than enrol a further three patients at the 5·0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3·0 mg/kg or 5·0 mg/kg ipilimumab. INTERPRETATION: GVAX combined with 3·0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted. FUNDING: Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam.