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Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy.
Mai 2018 | González, Fermín E; Chernobrovkin, Alexey; Pereda, Cristián; García-Salum, Tamara; Tittarelli, Andrés; López, Mercedes N; Salazar-Onfray, Flavio; Zubarev, Roman A
Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions. PMID 29744371

Molecular signatures associated with tumor-specific immune response in melanoma patients treated with dendritic cell-based immunotherapy.
Apr. 2018 | García-Salum, Tamara; Villablanca, Andrea; Matthäus, Franziska; Tittarelli, Andrés; Baeza, Mauricio; Pereda, Cristián; Gleisner, M Alejandra; González, Fermín E; López, Mercedes N; Hoheisel, Jörg D; Norgauer, Johannes; Gebicke-Haerter, Peter J; Salazar-Onfray, Flavio
We previously showed that autologous dendritic cells (DCs) loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination, correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers. PMID 29682201

Randomized phase II trial of autologous dendritic cell vaccines versus autologous tumor cell vaccines in metastatic melanoma: 5-year follow up and additional analyses.
März 2018 | Dillman, Robert O; Cornforth, Andrew N; Nistor, Gabriel I; McClay, Edward F; Amatruda, Thomas T; Depriest, Carol
Despite improved survival following checkpoint inhibitors, there is still a potential role for anti-cancer therapeutic vaccines. Because of biological heterogeneity and neoantigens resulting from each patient's mutanome, autologous tumor may be the best source of tumor-associated antigens (TAA) for vaccines. Ex vivo loading of autologous dendritic cells with TAA may be associated with superior clinical outcome compared to injecting irradiated autologous tumor cells. We conducted a randomized phase II trial to compare autologous tumor cell vaccines (TCV) and autologous dendritic cell vaccines (DCV) loaded with autologous TAA. PMID 29510745

Dendritic cell therapy in melanoma.
Nov. 2017 | Alvarez-Dominguez, Carmen; Calderón-Gonzalez, Ricardo; Terán-Navarro, Hector; Salcines-Cuevas, David; Garcia-Castaño, Almudena; Freire, Javier; Gomez-Roman, Javier; Rivera, Fernando
Dendritic cell (DC) vaccines are cancer vaccines used currently as melanoma therapies. They act as adjuvants initiating the immune responses, but not only as they can also have effector activities redirecting cytotoxic CD8 T cells against melanoma. Ex vivo preparation of monocyte derived DCs has been implemented to produce large numbers of DCs for clinical therapy, highlighting the necessity of activate DC s to produce Th1 cytokines, especially TNF-a and IL-12 with potent anti-tumour actions. Several clinical trials both in the European Union and USA are open currently using DC vaccines, alone or in combination with other immunotherapies. The type of antigen is also an active area of investigation involving tumour antigens and bacterial epitopes, both providing good responses. Bacterial epitopes presented the advantage versus tumour antigens that they can prepare the vaccination site as they induce innate and specific immune responses as they are potent recall antigens that expand cytotoxic responses. PMID 29114544

A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and α-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients.
Nov. 2017 | Gasser, Olivier; Sharples, Katrina J; Barrow, Catherine; Williams, Geoffrey M; Bauer, Evelyn; Wood, Catherine E; Mester, Brigitta; Dzhelali, Marina; Caygill, Graham; Jones, Jeremy; Hayman, Colin M; Hinder, Victoria A; Macapagal, Jerome; McCusker, Monica; Weinkove, Robert; Painter, Gavin F; Brimble, Margaret A; Findlay, Michael P; Dunbar, P Rod; Hermans, Ian F
Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 10 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4 and CD8 T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation. PMID 29094183

Twelve-year survival and immune correlates in dendritic cell-vaccinated melanoma patients.
Apr. 2017 | Gross, Stefanie; Erdmann, Michael; Haendle, Ina; Voland, Steve; Berger, Thomas; Schultz, Erwin; Strasser, Erwin; Dankerl, Peter; Janka, Rolf; Schliep, Stefan; Heinzerling, Lucie; Sotlar, Karl; Coulie, Pierre; Schuler, Gerold; Schuler-Thurner, Beatrice
Reports on long-term (≥10 years) effects of cancer vaccines are missing. Therefore, in 2002, we initiated a phase I/II trial in cutaneous melanoma patients to further explore the immunogenicity of our DC vaccine and to establish its long-term toxicity and clinical benefit after a planned 10-year followup. PMID 28422751

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells.
Dez. 2016 | Kyte, Jon Amund; Aamdal, Steinar; Dueland, Svein; Sæbøe-Larsen, Stein; Inderberg, Else Marit; Madsbu, Ulf Erik; Skovlund, Eva; Gaudernack, Gustav; Kvalheim, Gunnar
The most effective anticancer immune responses are probably directed against patient-specific neoantigens. We have developed a melanoma vaccine targeting this individual mutanome based on dendritic cells (DCs) loaded with autologous tumor-mRNA. Here, we report a phase I/II trial evaluating toxicity, immune response and clinical outcome in 31 metastatic melanoma patients. The first cohort (n = 22) received the vaccine without any adjuvant; the next cohort (n = 9) received adjuvant IL2. Each subject received four weekly intranodal or intradermal injections, followed by optional monthly vaccines. Immune response was evaluated by delayed-type hypersensitivity (DTH), T cell proliferation and cytokine assays. Data were collected for 10 y after inclusion of the last patient. No serious adverse events were detected. In the intention-to-treat-cohort, we demonstrated significantly superior survival compared to matched controls from a benchmark meta-analysis (1 y survival 43% vs. 24%, 2 y 23% vs. 6.6%). A tumor-specific immune response was demonstrated in 16/31 patients. The response rate was higher after intradermal than intranodal vaccination (80% vs. 38%). Immune responders had improved survival compared to non-responders (median 14 mo vs. 6 mo; p = 0.030), and all eight patients surviving >20 mo were immune responders. In addition to the tumor-specific response, most patients developed a response against autologous DC antigens. The cytokine profile was polyfunctional and did not follow a Th1/Th2 dichotomy. We conclude that the favorable safety profile and evidence of a possible survival benefit warrant further studies of the RNA/DC vaccine. The vaccine appears insufficient as monotherapy, but there is a strong rationale for combination with checkpoint modulators. PMID 27999747

Adjuvant Dendritic Cell Vaccination in High-Risk Uveal Melanoma.
Sep. 2016 | Bol, Kalijn F; van den Bosch, Thomas; Schreibelt, Gerty; Mensink, Hanneke W; Keunen, Jan E E; Kiliç, Emine; Japing, Wouter J; Geul, Kaspar W; Westdorp, Harm; Boudewijns, Steve; Croockewit, Sandra A J; van Rossum, Michelle M; de Goede, Anna L; Naus, Nicole C; van der Graaf, Winette T A; Gerritsen, Winald R; de Klein, Annelies; Punt, Cornelis J A; Figdor, Carl G; Cohen, Victoria M; Paridaens, Dion; de Vries, I Jolanda M
PMID 27476772

Adjuvant dendritic cell vaccination induces tumor-specific immune responses in the majority of stage III melanoma patients.
Sep. 2016 | Boudewijns, Steve; Bol, Kalijn F; Schreibelt, Gerty; Westdorp, Harm; Textor, Johannes C; van Rossum, Michelle M; Scharenborg, Nicole M; de Boer, Annemiek J; van de Rakt, Mandy W M M; Pots, Jeanne M; van Oorschot, Tom G M; Duiveman-de Boer, Tjitske; Olde Nordkamp, Michel A; van Meeteren, Wilmy S E C; van der Graaf, Winette T A; Bonenkamp, Johannes J; de Wilt, Johannes H W; Aarntzen, Erik H J G; Punt, Cornelis J A; Gerritsen, Winald R; Figdor, Carl G; de Vries, I Jolanda M
To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients. PMID 27622047

Diversification of Antitumour Immunity in a Patient with Metastatic Melanoma Treated with Ipilimumab and an IDO-Silenced Dendritic Cell Vaccine.
Aug. 2016 | Sioud, Mouldy; Nyakas, Marta; Sæbøe-Larssen, Stein; Mobergslien, Anne; Aamdal, Steinar; Kvalheim, Gunnar
Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) inhibits T-cell activation and promotes T-cell differentiation into regulatory T-cells. Moreover, IDO expression promotes resistance to immunotherapies targeting immune checkpoints such as the cytotoxic T lymphocyte antigen-4 (CTLA-4). Here, a patient with metastatic melanoma pretreated with ipilimumab, an anti-CTLA-4 blocking antibody, was vaccinated with IDO-silenced DCs cotransfected with mRNA for survivin or hTERT tumour antigens. During vaccination, T-cell responses to survivin and hTERT tumour antigens were generated, and a certain degree of clinical benefit was achieved, with a significant reduction in lung, liver, and skin metastases, along with a better performance status. T-cell responses against MART-1 and NY-ESO-1 tumour antigens were also detected in the peripheral blood. The patient also mounted an antibody response to several melanoma proteins, indicating diversification of the antitumour immunity in this patient. The identification of such serum antibody-reacting proteins could facilitate the discovery of tumour neoantigens. PMID 27504122

Dendritic cell vaccination in melanoma patients: From promising results to future perspectives.
Juli 2016 | Boudewijns, Steve; Bloemendal, Martine; Gerritsen, Winald R; de Vries, I Jolanda M; Schreibelt, Gerty
Dendritic cells (DCs) play an important role in the induction of antitumor immunity. Therefore, they are used as anti-cancer vaccines in clinical studies in various types of cancer. DC vaccines are generally well tolerated and able to induce antigen-specific T cell responses in melanoma patients. After DC vaccinations, functional tumor-specific T cells are more frequently detected in stage III melanoma patients, as compared to patients with advanced melanoma, indicating that the tumor load influences immunological responses. Furthermore, long-lasting clinical responses were rarely seen in metastatic melanoma patients after DC vaccination. Since more potent treatment options are available, e.g. immune checkpoint inhibitors and targeted therapy, DC vaccination as monotherapy may not be preferred in the treatment of advanced melanoma. However, encouraging results of DC vaccines combined with ipilimumab have been reported in advanced melanoma patients with an objective response rate of 38%. DC vaccines show promising clinical results in stage III patients, although clinical efficacy still needs to be proven in a phase 3 trial. The clinical and immunological results of DC vaccination in stage III melanoma patients might be further improved by using naturally circulating DCs (myeloid DCs and plasmacytoid DCs) and neoantigens to load DCs. PMID 27322496

T cell landscape in a primary melanoma predicts the survival of patients with metastatic disease after their treatment with dendritic cell vaccines.
Mai 2016 | Vasaturo, Angela; Halilovic, Altuna; Bol, Kalijn F; Verweij, Dagmar I; Blokx, Willeke A M; Punt, Cornelis J A; Groenen, Patricia J T A; van Krieken, Han J H J M; Textor, Johannes; de Vries, I Jolanda M; Figdor, Carl G
Tumor infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3+ T cells infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (p=0.000026) and a validation cohort of 39 patients (p=0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum LDH level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared to chemotherapy-treated patients was observed for high (p=0.000566), but not low (p=0.154) I/P ratios. In conclusion, T cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. PMID 27197179

Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity.
März 2016 | Bol, Kalijn F; Aarntzen, Erik H J G; Pots, Jeanette M; Olde Nordkamp, Michel A M; van de Rakt, Mandy W M M; Scharenborg, Nicole M; de Boer, Annemiek J; van Oorschot, Tom G M; Croockewit, Sandra A J; Blokx, Willeke A M; Oyen, Wim J G; Boerman, Otto C; Mus, Roel D M; van Rossum, Michelle M; van der Graaf, Chantal A A; Punt, Cornelis J A; Adema, Gosse J; Figdor, Carl G; de Vries, I Jolanda M; Schreibelt, Gerty
Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktail. PMID 26861670

Effective Clinical Responses in Metastatic Melanoma Patients after Vaccination with Primary Myeloid Dendritic Cells.
Feb. 2016 | Schreibelt, Gerty; Bol, Kalijn F; Westdorp, Harm; Wimmers, Florian; Aarntzen, Erik H J G; Duiveman-de Boer, Tjitske; van de Rakt, Mandy W M M; Scharenborg, Nicole M; de Boer, Annemiek J; Pots, Jeanette M; Olde Nordkamp, Michel A M; van Oorschot, Tom G M; Tel, Jurjen; Winkels, Gregor; Petry, Katja; Blokx, Willeke A M; van Rossum, Michelle M; Welzen, Marieke E B; Mus, Roel D M; Croockewit, Sandra A J; Koornstra, Rutger H T; Jacobs, Joannes F M; Kelderman, Sander; Blank, Christian U; Gerritsen, Winald R; Punt, Cornelis J A; Figdor, Carl G; de Vries, I Jolanda M
Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro-generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c(+) myeloid DCs, naturally circulating in the blood. PMID 26712687

Type I-polarized BRAF-pulsed dendritic cells induce antigen-specific CD8+ T cells that impact BRAF-mutant murine melanoma.
Okt. 2015 | Cintolo, Jessica A; Datta, Jashodeep; Xu, Shuwen; Gupta, Meera; Somasundaram, Rajasekharan; Czerniecki, Brian J
Existing therapies targeting the mutated BRAF oncodriver (BRAF) successfully treat melanoma but are susceptible to resistance. This study assessed the potential of a dendritic cell-based BRAF vaccine for the treatment of BRAF-mutant melanoma. Type 1-polarized dendritic cells (DC1) pulsed with affinity-modified BRAF peptide were administered to C57Bl/6 mice both before (prevention) and twice weekly after (treatment) the development of established tumor with B16 melanoma transfected to express BRAF (B16). The efficacy of the BRAF-pulsed DC1 vaccine was corroborated in a novel transplantable BRAF-mutant murine melanoma model (BRAF; PTEN; CDK2NA). Three-dimensional tumor measurements and survival were determined. Induction of BRAF-specific CD8 T-cell responses after brief in-vitro sensitization was assessed by interferon-γ enzyme-linked immunosorbent assay and/or enzyme-linked immunospot. Mice receiving BRAF-pulsed DC1 vaccines before B16 tumor challenge demonstrated increased tumor-doubling times (P<0.001) and improved survival (P=0.0186) compared with those that received ovalbumin (control)-pulsed DC1 vaccines. In mice bearing established B16 tumors (mean 32 mm), BRAF-pulsed DC1 vaccines delayed tumor growth (P<0.001) and improved survival (P=0.0008), compared with untreated mice. Likewise, in mice bearing BRAF; PTEN; CDK2NA tumors, compared with controls, BRAF-DC1 vaccination recapitulated these effects by delaying tumor growth (P<0.001) and improving survival (P=0.002). Vaccination elicited specific CD8 T-cell recognition of BRAF-pulsed antigen-presenting cells (P<0.05), as well as BRAF-expressing cancer cells (P<0.001), measured by interferon-γ release in vitro. BRAF-pulsed DC1 vaccines induce oncogene-specific CD8 T-cell immune responses that impact tumor growth and survival in preclinical models of BRAF-mutant melanoma. Exploration of BRAF-targeted vaccines, in combination with BRAF-targeted therapies and checkpoint inhibitors, is warranted. PMID 26451873

Sensitivity of Human Malignant Melanoma Cell Lines to Newcastle Disease Virus.
Sep. 2015 | Pap, Marianna; Bátor, Judit; Szeberényi, József
Virotherapy may be a promising alternative to chemotherapy of malignant melanoma. In clinical trials using strains of Newcastle disease virus (NDV), only a fraction of patients with cancer responded to virotherapy. In the present study, we tried to find a correlation between the susceptibility of human melanoma cell lines to NDV and growth factor signaling pathways. PMID 26408702

Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model.
Sep. 2015 | Niu, Zeshan; Bai, Fuliang; Sun, Tian; Tian, Hui; Yu, Dan; Yin, Jiechao; Li, Siming; Li, Tianhe; Cao, Hongwei; Yu, Qingzhong; Wu, Yunzhou; Ren, Guiping; Li, Deshan
Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma. PMID 24645750

Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma.
Sep. 2015 | Markov, Oleg V; Mironova, Nadezhda L; Sennikov, Sergey V; Vlassov, Valentin V; Zenkova, Marina A
Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless the antimetastatic effect was less effective in comparison with prophylactic DC vaccine. PMID 26325576

Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells.
Mai 2015 | Carreno, Beatriz M; Magrini, Vincent; Becker-Hapak, Michelle; Kaabinejadian, Saghar; Hundal, Jasreet; Petti, Allegra A; Ly, Amy; Lie, Wen-Rong; Hildebrand, William H; Mardis, Elaine R; Linette, Gerald P
T cell immunity directed against tumor-encoded amino acid substitutions occurs in some melanoma patients. This implicates missense mutations as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as targets of antitumor immunity is lacking. Moreover, it remains unknown whether vaccination can augment such responses. We found that a dendritic cell vaccine led to an increase in naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-β usage and clonal composition. Our results demonstrate that vaccination directed at tumor-encoded amino acid substitutions broadens the antigenic breadth and clonal diversity of antitumor immunity. PMID 25837513

Novel dendritic cell-based vaccination in late stage melanoma.
Jan. 2015 | Schneble, Erika J; Yu, Xianzhong; Wagner, T E; Peoples, George E
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play an important role in stimulating an immune response of both CD4(+) T helper cells and CD8(+) cytotoxic T lymphocytes (CTLs). As such, DCs have been studied extensively in cancer immunotherapy for their capability to induce a specific anti-tumor response when loaded with tumor antigens. However, when the most relevant antigens of a tumor remain to be identified, alternative approaches are required. Formation of a dentritoma, a fused DC and tumor cells hybrid, is one strategy. Although initial studies of these hybrid cells are promising, several limitations interfere with its clinical and commercial application. Here we present early experience in clinical trials and an alternative approach to manufacturing this DC/tumor cell hybrid for use in the treatment of late stage and metastatic melanoma. PMID 25483650

Radiotherapy as an immunological booster in patients with metastatic melanoma or renal cell carcinoma treated with high-dose Interleukin-2: evaluation of biomarkers of immunologic and therapeutic response.
Okt. 2014 | Ridolfi, Laura; de Rosa, Francesco; Ridolfi, Ruggero; Gentili, Giorgia; Valmorri, Linda; Scarpi, Emanuela; Parisi, Elisabetta; Romeo, Antonino; Guidoboni, Massimo
Tumor cells killed by radiation therapy (RT) are a potentially good source of antigens for dendritic cell (DC) uptake and presentation to T-cells. RT upregulates cell death receptors such as Fas/CD95 and MHC-I, induces the expression of co-stimulatory molecules on tumor cells, and promotes production of pro-inflammatory cytokines. High-dose interleukin-2 (HD-IL-2) bolus has been shown to obtain objective response rates ranging from 15% to 17% in patients with metastatic melanoma or renal cell carcinoma (RCC), with 6% to 8% of cases experiencing a durable complete response. However, HD-IL-2 is also associated with severe side-effects; if it is to remain a component of the curative treatment strategy in patients with metastatic melanoma or RCC, its therapeutic efficacy must be improved and patients who are most likely to benefit from treatment must be identified a priori. We designed a clinical study combining immunomodulating RT and HD-IL-2 to evaluate their clinical and immunological efficacy and to explore the predictive and prognostic value of 1) tumor-specific immune response and 2) serum levels of proangiogenic cytokines. PMID 25245327

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.
Sep. 2014 | González, Fermín E; Ortiz, Carolina; Reyes, Montserrat; Dutzan, Nicolás; Patel, Vyomesh; Pereda, Cristián; Gleisner, Maria A; López, Mercedes N; Gutkind, J Silvio; Salazar-Onfray, Flavio
We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas. PMID 24673602

Vaccination with autologous dendritic cells loaded with autologous tumor lysate or homogenate combined with immunomodulating radiotherapy and/or preleukapheresis IFN-α in patients with metastatic melanoma: a randomised "proof-of-principle" phase II study.
Aug. 2014 | de Rosa, Francesco; Ridolfi, Laura; Ridolfi, Ruggero; Gentili, Giorgia; Valmorri, Linda; Nanni, Oriana; Petrini, Massimiliano; Fiammenghi, Laura; Granato, Anna Maria; Ancarani, Valentina; Pancisi, Elena; Soldati, Valentina; Cassan, Serena; Riccobon, Angela; Parisi, Elisabetta; Romeo, Antonino; Turci, Livia; Guidoboni, Massimo
Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. PMID 25053129

Efficiency of dendritic cell vaccination against B16 melanoma depends on the immunization route.
Aug. 2014 | Edele, Fanny; Dudda, Jan C; Bachtanian, Eva; Jakob, Thilo; Pircher, Hanspeter; Martin, Stefan F
Dendritic cells (DC) presenting tumor antigens are crucial to induce potent T cell-mediated anti-tumor immune responses. Therefore DC-based cancer vaccines have been established for therapy, however clinical outcomes are often poor and need improvement. Using a mouse model of B16 melanoma, we found that the route of preventive DC vaccination critically determined tumor control. While repeated DC vaccination did not show an impact of the route of DC application on the prevention of tumor growth, a single DC vaccination revealed that both the imprinting of skin homing receptors and an enhanced proliferation state of effector T cells was seen only upon intracutaneous but not intravenous or intraperitoneal immunization. Tumor growth was prevented only by intracutaneous DC vaccination. Our results indicate that under suboptimal conditions the route of DC vaccination crucially determines the efficiency of tumor defense. DC-based strategies for immunotherapy of cancer should take into account the immunization route in order to optimize tissue targeting of tumor antigen specific T cells. PMID 25121970

Long-term complete remission following radiosurgery and immunotherapy in a melanoma patient with brain metastasis: immunologic correlates.
Mai 2014 | Karbach, Julia; Gnjatic, Sacha; Biskamp, Melina; Atmaca, Akin; Weidmann, Eckhart; Brandt, Kathrin; Wahle, Claudia; Bernhard, Helga; Knuth, Alexander; Jäger, Elke
A melanoma patient with brain metastases was treated by gamma-knife radiosurgery and immunotherapy with autologous tumor-lysate-loaded dendritic cells (DC). Ten years after the combined treatment, the patient remains in complete remission. Remarkable immunologic correlates to the clinical development were the transient induction of NY-ESO-1 antibody and the durable expansion of MAGE-A1p161-169 EADPTGHSY-specific CD8+ T cells. Although the induction of NY-ESO-1 antibody most likely resulted from gamma-knife-mediated "auto-vaccination," the persistence of circulating MAGE-A1-specific T cells, which are still detectable ex vivo in the absence of any tumor manifestation, coincides with DC-based vaccination administered monthly until today. PMID 24795353

Dasatinib promotes the expansion of a therapeutically superior T-cell repertoire in response to dendritic cell vaccination against melanoma.
Apr. 2014 | Lowe, Devin B; Bose, Anamika; Taylor, Jennifer L; Tawbi, Hussein; Lin, Yan; Kirkwood, John M; Storkus, Walter J
Dasatinib (DAS) is a potent inhibitor of the BCR-ABL, SRC, c-KIT, PDGFR, and ephrin tyrosine kinases that has demonstrated only modest clinical efficacy in melanoma patients. Given reports suggesting that DAS enhances T cell infiltration into the tumor microenvironment, we analyzed whether therapy employing the combination of DAS plus dendritic cell (DC) vaccination would promote superior immunotherapeutic benefit against melanoma. Using a M05 (B16.OVA) melanoma mouse model, we observed that a 7-day course of orally-administered DAS (0.1 mg/day) combined with a DC-based vaccine (VAC) against the OVA257-264 peptide epitope more potently inhibited tumor growth and extended overall survival as compared with treatment with either single modality. The superior efficacy of the combinatorial treatment regimen included a reduction in hypoxic-signaling associated with reduced levels of immunosuppressive CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSC) and CD4(+)Foxp3(+) regulatory T (Treg) populations in the melanoma microenvironment. Furthermore, DAS + VAC combined therapy upregulated expression of Type-1 T cell recruiting CXCR3 ligand chemokines in the tumor stroma correlating with activation and recruitment of Type-1, vaccine-induced CXCR3(+)CD8(+) tumor-infiltrating lymphocytes (TILs) and CD11c(+) DC into the tumor microenvironment. The culmination of this bimodal approach was a profound "spreading" in the repertoire of tumor-associated antigens recognized by CD8(+) TILs, in support of the therapeutic superiority of combined DAS + VAC immunotherapy in the melanoma setting. PMID 24734217

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome.
Aug. 2013 | Dannull, Jens; Haley, N Rebecca; Archer, Gary; Nair, Smita; Boczkowski, David; Harper, Mark; De Rosa, Nicole; Pickett, Nancy; Mosca, Paul J; Burchette, James; Selim, Maria A; Mitchell, Duane A; Sampson, John; Tyler, Douglas S; Pruitt, Scott K
Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. PMID 23934126

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses.
Juni 2013 | Durán-Aniotz, Claudia; Segal, Gabriela; Salazar, Lorena; Pereda, Cristián; Falcón, Cristián; Tempio, Fabián; Aguilera, Raquel; González, Rodrigo; Pérez, Claudio; Tittarelli, Andrés; Catalán, Diego; Nervi, Bruno; Larrondo, Milton; Salazar-Onfray, Flavio; López, Mercedes N
Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH(+) with respect to DTH(-) unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood. PMID 23242374

Metastatic melanoma patients treated with dendritic cell vaccination, Interleukin-2 and metronomic cyclophosphamide: results from a phase II trial.
Sep. 2012 | Ellebaek, Eva; Engell-Noerregaard, Lotte; Iversen, Trine Zeeberg; Froesig, Thomas Moerch; Munir, Shamaila; Hadrup, Sine Reker; Andersen, Mads Hald; Svane, Inge Marie
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. PMID 22426890

Dendritic cell-based vaccination in metastatic melanoma patients: phase II clinical trial.
Sep. 2012 | Oshita, Chie; Takikawa, Masako; Kume, Akiko; Miyata, Haruo; Ashizawa, Tadashi; Iizuka, Akira; Kiyohara, Yoshio; Yoshikawa, Shusuke; Tanosaki, Ryuji; Yamazaki, Naoya; Yamamoto, Akifumi; Takesako, Kazutoh; Yamaguchi, Ken; Akiyama, Yasuto
Metastatic and chemoresistant melanoma can be a good target of immunotherapy because it is an intractable cancer with a very poor prognosis. Previously, we tested a dendritic cell (DC)-based phase I vaccine, and confirmed that it was safe. In the present study, we performed a phase II trial of a DC vaccine for metastatic melanoma patients with mainly the HLA-A24 genotype, and investigated the efficacy of the vaccine. Twenty-four patients with metastatic melanoma were enrolled into a phase II study of DC-based immunotherapy. The group included 19 HLA-A24-positive (A*2402) patients and 3 HLA-A2-positive (A*0201) patients. The protocol for DC production was similar to that in the phase I trial. Briefly, a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-A2, MAGE-A3 and MART-1 or MAGE‑A1) restricted to HLA-A2 or A24 and KLH were used for DC pulsing. Finally, DCs were injected subcutaneously (s.c.) into the inguinal region in the dose range of 1-5x107 per shot. The DC ratio (lin-HLA-DR+) of the vaccine was 38.1±13.3% and the frequency of CD83+ DCs was 25.7±20.8%. Other parameters regarding DC processing were not different from phase I. Immune response-related parameters including the ELISPOT assay, DTH reaction to peptide or KLH, DC injection numbers were shown to be related to a good prognosis. The ELISPOT reaction was positive in 75% of the patients vaccinated. The increase of anti-melanoma antigen antibody titer before vaccination was also shown to be a prognosis factor, but that post-vaccination was not. Based on immunohistochemical analysis, CD8 and IL-17 were not involved in the prognosis. Adverse effects of more than grade III were not seen. Overall survival analysis revealed a significant survival prolongation effect in DC-given melanoma patients. These results suggest that peptide cocktail-treated DC vaccines may be a safe and effective therapy against metastatic melanoma in terms of prolongation of overall survival time. PMID 22895835

The dermis as a portal for dendritic cell-targeted immunotherapy of cutaneous melanoma.
Sep. 2011 | Oosterhoff, D; Sluijter, B J R; Hangalapura, B N; de Gruijl, T D
Complete surgical excision at an early stage remains the only curative treatment for cutaneous melanoma with few available adjuvant therapy options. Nevertheless, melanoma is a relatively immunogenic tumor type and particularly amenable to immunotherapeutic approaches. A dense network of cutaneous dendritic cells (DC) may account for the reported efficacy of vaccination through the skin and provide an attractive target for the immunotherapy of melanoma. Several phenotypically distinct DC subsets are discernable in the skin, among others, epidermal Langerhans cells and dermal DC. Upon appropriate activation both subsets can efficiently migrate to melanoma-draining lymph nodes (LN) to prime T cell-mediated responses. Unfortunately, from an early stage, melanoma development is characterized by strong immune suppression, facilitating unchecked tumor growth and spread. Particularly the primary tumor site and the first-line tumor-draining LN, the so-called sentinel LN, bear the brunt of this melanoma-induced immune suppression-and these are exactly the sites where anti-melanoma effector T cell responses should be primed by DC in order to prevent early metastasis. Through local immunopotentiation or through DC-targeted vaccination, the dermis may be utilized as a portal to activate DC and kick-start or boost effective T cell-mediated anti-melanoma immunity, even in the face of this immune suppression. PMID 21681685

Route of administration modulates the induction of dendritic cell vaccine-induced antigen-specific T cells in advanced melanoma patients.
Sep. 2011 | Lesterhuis, W Joost; de Vries, I Jolanda M; Schreibelt, Gerty; Lambeck, Annechien J A; Aarntzen, Erik H J G; Jacobs, Joannes F M; Scharenborg, Nicole M; van de Rakt, Mandy W M M; de Boer, Annemiek J; Croockewit, Sandra; van Rossum, Michelle M; Mus, Roel; Oyen, Wim J G; Boerman, Otto C; Lucas, Sophie; Adema, Gosse J; Punt, Cornelis J A; Figdor, Carl G
It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. PMID 21771874

Phase I/II trial of a dendritic cell vaccine transfected with DNA encoding melan A and gp100 for patients with metastatic melanoma.
Juni 2011 | Steele, J C; Rao, A; Marsden, J R; Armstrong, C J; Berhane, S; Billingham, L J; Graham, N; Roberts, C; Ryan, G; Uppal, H; Walker, C; Young, L S; Steven, N M
This trial tested a dendritic cell (DC) therapeutic cancer vaccine in which antigen is loaded using a novel non-viral transfection method enabling the uptake of plasmid DNA condensed with a cationic peptide. Proof of principle required the demonstration of diverse T lymphocyte responses following vaccination, including multiple reactivities restricted through both major histocompatibility complex (MHC) class I and II. Patients with advanced melanoma were offered four cycles of vaccination with autologous DC expressing melan A and gp100. Disease response was measured using Response Evaluation Criteria in Solid Tumours. Circulating MHC class I- and II-restricted responses were measured against peptide and whole antigen targets using interferon-γ ELIspot and enzyme-linked immunosorbent assay assays, respectively. Responses were analyzed across the trial population and presented descriptively for some individuals. Twenty-five patients received at least one cycle. Vaccination was well tolerated. Three patients had reduction in disease volume. Across the trial population, vaccination resulted in an expansion of effector responses to both antigens, to the human leukocyte antigen A2-restricted modified epitope, melan A ELAGIGILTV, and to a panel of MHC class I- and II-restricted epitopes. Vaccination with mature DC non-virally transfected with DNA encoding antigen had biological effect causing tumour regression and inducing diverse T lymphocyte responses. PMID 21307889

Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2.
Feb. 2011 | Bjoern, J; Brimnes, M K; Andersen, M H; Thor Straten, P; Svane, I M
In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospectively analysed in fresh blood, and treatment-associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4+ CD25(high) T cell subset from 6% to 22% (P<0.001). At the 6th vaccine, a general decline was observed and a significantly (P=0.01) lower level of CD4+ CD25(high) Treg cells was reached in the group of patients who attained disease stabilization (9.5%) compared to patients with continued progressive disease (14.5%). However, when FoxP3 was employed for retrospective analysis of Tregs on frozen blood, this difference did not reach significance (P=0.09). The vast majority of the Treg produced IL-10 and, to a varying extent, TGF-β. In addition, sorted CD4+ CD25(high) CD127⁻ Tregs were able to suppress proliferation of peripheral blood mononuclear cells in a dose-dependent manner, thus suggesting a regulatory functionality. These findings emphasize the need for strategies to effectively eliminate Treg cells to optimize the clinical effectiveness of cancer immunotherapy. PMID 21204893

Resistance to the proapoptotic effects of interferon-gamma on melanoma cells used in patient-specific dendritic cell immunotherapy is associated with improved overall survival.
Jan. 2011 | Cornforth, A N; Fowler, A W; Carbonell, D J; Dillman, R O
The use of whole cell tumor vaccines and various means of loading antigen onto dendritic cells have been under investigation for over a decade. Induction of apoptosis and the exposure of immune-stimulating proteins are thought to be beneficial for the use in immunotherapy protocols, but conclusive evidence in the clinical setting has been lacking. Incubation of melanoma cell lines with interferon-gamma (IFN-γ) increased phosphatidylserine and calreticulin exposure, but not in the IFN-γ-resistant cell line Lu-1205. Short-term autologous melanoma cell lines used for loading dendritic cells for immunotherapy showed differential response to the pro-apoptotic effects of IFN-γ. These IFN-γ-treated tumor cells (TCs) were irradiated and used for loading antigen for dendritic cell therapy. A log-rank comparison of survival for patients whose TCs were found to be either sensitive (upregulated phosphatidylserine and calreticulin) or insensitive to IFN-γ revealed a strongly significant correlation to progression-free (p = 0.003) and overall survival (p = 0.002) favorably in those patients whose cell lines were resistant to the proapoptotic effect of IFN-γ. These results suggest that the use of IFN-γ in anti-melanoma dendritic cell-based immunotherapy may only be beneficial when the cells do not undergo apoptosis in response to IFN-γ and support the contention that the use of some apoptotic cells in vaccines may be detrimental. PMID 20960187

Wild-type and modified gp100 peptide-pulsed dendritic cell vaccination of advanced melanoma patients can lead to long-term clinical responses independent of the peptide used.
Jan. 2011 | Lesterhuis, W Joost; Schreibelt, Gerty; Scharenborg, Nicole M; Brouwer, H Mary-lène H; Gerritsen, Marie-Jeanne P; Croockewit, Sandra; Coulie, Pierre G; Torensma, Ruurd; Adema, Gosse J; Figdor, Carl G; de Vries, I Jolanda M; Punt, Cornelis J A
Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients. Several strategies have been employed to load DC with antigen, including peptide loading. To increase immunogenicity of peptides, major histocompatibility complex (MHC) class I binding affinity and stability of peptide-MHC complexes at the cell surface may be improved by modification of the amino acid sequence. In this study, we compared the capacity of DC loaded with wild-type versus modified gp100 peptides with higher binding affinities to induce an immune and clinical response in advanced melanoma patients. Metastatic HLA-A2.1(+) melanoma patients were vaccinated intravenously (on average 25 × 10(6) DC) and intradermally (on average 11 × 10(6) DC) with mature DC loaded with keyhole limpet hemocyanin (KLH) together with tyrosinase peptide and either wild-type (15 patients) or modified (12 patients) gp100 peptides. All vaccinated patients showed a pronounced proliferative T cell or humoral response against KLH. Gp100-specific T cell responses were monitored in post-treatment delayed type hypersensitivity (DTH) skin biopsies by tetramer and functional analysis. Antigen-specific T cells were found in 2 of 15 patients vaccinated with wild-type gp100-loaded DC, versus 1 of 12 patients vaccinated with modified peptide-loaded DC. These three patients also had the best clinical response, with long-term (>8 years) complete responses in two patients, one in each group. We conclude that vaccination with peptide-loaded DC can result in long-term clinical responses in a minority of metastatic melanoma patients, and that the use of modified as compared to wild-type gp100 peptides for DC loading does not result in a relevant enhanced immune responses. PMID 21069321

New approaches to the development of adenoviral dendritic cell vaccines in melanoma.
Dez. 2010 | Butterfield, Lisa H; Vujanovic, Lazar
Considerable research in the field of immunotherapy for melanoma has demonstrated that this tumor type can be responsive to therapeutic immune activation strategies. In early clinical trials, vaccine strategies using dendritic cells (DCs) and adenovirus (Ad) vectors (AdVs) were safe and immunogenic, and induced clinical responses in a minority of patients. Research from the past several years has yielded an improved mechanistic understanding of DC biology, AdV effects on DCs and the crosstalk that occurs between antigen-loaded DCs and specific lymphocyte subsets. This knowledge base is being combined with technological advances in cytokine delivery, AdV design and in vivo DC targeting. These developments are leading to novel AdV-transduced DC-based therapeutic modalities that may further advance melanoma immunotherapy. Interactions between AdVs and DCs, initial clinical trial results, and new developments in DC engineering and in AdV biology are reviewed. PMID 21154122

Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients.
Okt. 2010 | Jacobs, Joannes F M; Punt, Cornelis J A; Lesterhuis, W Joost; Sutmuller, Roger P M; Brouwer, H Mary-Lène H; Scharenborg, Nicole M; Klasen, Ina S; Hilbrands, Luuk B; Figdor, Carl G; de Vries, I Jolanda M; Adema, Gosse J
The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody-mediated depletion of CD25(high) regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. PMID 20736326

Multicenter phase II study of matured dendritic cells pulsed with melanoma cell line lysates in patients with advanced melanoma.
Okt. 2010 | Ribas, Antoni; Camacho, Luis H; Lee, Sun Min; Hersh, Evan M; Brown, Charles K; Richards, Jon M; Rodriguez, Maria Jovie; Prieto, Victor G; Glaspy, John A; Oseguera, Denise K; Hernandez, Jackie; Villanueva, Arturo; Chmielowski, Bartosz; Mitsky, Peggie; Bercovici, Nadège; Wasserman, Ernesto; Landais, Didier; Ross, Merrick I
Several single center studies have provided evidence of immune activation and antitumor activity of therapeutic vaccination with dendritic cells (DC) in patients with metastatic melanoma. The efficacy of this approach in patients with favorable prognosis metastatic melanoma limited to the skin, subcutaneous tissues and lung (stages IIIc, M1a, M1b) was tested in a multicenter two stage phase 2 study with centralized DC manufacturing. PMID 20875102

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial.
Sep. 2010 | Trepiakas, Redas; Berntsen, Annika; Hadrup, Sine Reker; Bjørn, Jon; Geertsen, Poul F; Straten, Per Thor; Andersen, Mads H; Pedersen, Anders E; Soleimani, Amir; Lorentzen, Torben; Johansen, Julia S; Svane, Inge Marie
Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2+ patients) or with autologous/allogeneic tumor lysate (HLA-A2(−) patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-alpha2b. PMID 20429791

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.
Dez. 2009 | Lazar, Itay; Yaacov, Barak; Shiloach, Tamar; Eliahoo, Elad; Kadouri, Luna; Lotem, Michal; Perlman, Riki; Zakay-Rones, Zichria; Panet, Amos; Ben-Yehuda, Dina
Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma. PMID 19864394

Dendritic cell vaccination combined with CTLA4 blockade in patients with metastatic melanoma.
Okt. 2009 | Ribas, Antoni; Comin-Anduix, Begoña; Chmielowski, Bartosz; Jalil, Jason; de la Rocha, Pilar; McCannel, Tara A; Ochoa, Maria Teresa; Seja, Elizabeth; Villanueva, Arturo; Oseguera, Denise K; Straatsma, Bradley R; Cochran, Alistair J; Glaspy, John A; Hui, Liu; Marincola, Francesco M; Wang, Ena; Economou, James S; Gomez-Navarro, Jesus
Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma. PMID 19789309

Mechanisms of murine dendritic cell antitumor dysfunction in aging.
Sep. 2009 | Grolleau-Julius, Annabelle; Abernathy, Lisa; Harning, Erin; Yung, Raymond L
Effective cancer immunotherapy depends on the body's ability to generate tumor antigen-presenting cells and tumor-reactive effector lymphocytes. As the most potent antigen presenting cells (APCs), dendritic cells (DCs) are capable of sensitizing T cells to new and recall antigens. Clinical trials of antigen-pulsed autologous DCs have been conducted in patients with a number of hematological and solid cancers, including malignant melanoma, lymphoma, myeloma, and non-small cell lung cancer. These studies suggest that antigen-loaded DC vaccination is a potentially safe and effective cancer therapy. However, the clinical results have been variable. Since the elderly are preferentially affected by diseases targeted by DC-directed immunotherapy, it is quite striking that few studies to date have focused on the effect of aging on DC function, a key aspect of optimal immunotherapy design in an aging population. In the present paper, we will discuss the consequences of aging on murine bone marrow-derived DC function and their use in cancer immunotherapy. PMID 19082999

Immunotherapy with dendritic cells pulsed by autologous dactinomycin-induced melanoma apoptotic bodies for patients with malignant melanoma.
Sep. 2009 | Chang, John W C; Hsieh, Jia-Juan; Shen, Yung-Chi; Ho, E; Chuang, Cheng-Keng; Chen, Yu-Ray; Liao, Shuen-Kuei; Chen, Jen-Shi; Leong, Stanley P L; Hou, Ming-Mo; Chang, Nai-Jen; Wang, Cheng-Hsu
The primary goal of this study was to evaluate the efficacy of immunotherapy for patients with metastatic melanoma with autologous melanoma apoptotic bodies (MAB)-pulsed dendritic cells (DCs). Accessible tumors from eligible patients with refractory metastatic melanoma were surgically removed and processed for primary culture. The autologous tumor cells were treated with dactinomycin to obtain MAB. To generate DCs, adherent peripheral blood mononuclear cells were cultured in complete medium containing granulocyte macrophage-colony stimulating factor and interleukin-4. MAB-pulsed DCs were given either intradermally (i.d.) or intravenously. Patients were immunized at monthly intervals and boosted with keyhole limpet hemocyanin (KLH) and MAB 2 weeks post-vaccination, with a maximum of four cycles. Of the 10 patients enrolled in this trial, nine were treated with MAB-pulsed DCs; two were given intravenous vaccinations and the other seven were i.d. injected. Mild tenderness in the draining lymph nodes lasting for less than 48 h and enlargement of the draining lymph nodes were noted in all seven i.d. cases. Treatment-related grade 3-4 toxicity, neutropenia, skin ulceration, tumor growth at the injection site, and sepsis were not observed in any of the patients. Delayed-type hypersensitivity to KLH was observed in all patients, whereas no delayed-type hypersensitivity to autologous tumor antigens was observed. One patient achieved partial response with reduction in lung metastatic tumor mass, and a presence of vesicles in the post-vaccination KLH response. Two patients had stable disease for more than 24 months; one was still alive at the time of submission of this report, the other eventually developed multiple metastases. MAB-pulsed DC immunotherapy is well tolerated in patients with malignant melanoma; however, its efficacy is only modest. Combination with other modalities is required to enhance DC-based immunotherapy. PMID 19750589

Increases in serum TARC/CCL17 levels are associated with progression-free survival in advanced melanoma patients in response to dendritic cell-based immunotherapy.
Aug. 2009 | Cornforth, Andrew N; Lee, Gregory J; Fowler, Abner W; Carbonell, Denysha J; Dillman, Robert O
Changes in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy. PMID 19421847

Genetically engineered Newcastle disease virus for malignant melanoma therapy.
Juni 2009 | Zamarin, D; Vigil, A; Kelly, K; García-Sastre, A; Fong, Y
Despite the advances in cancer therapies in the past century, malignant melanoma continues to present a significant clinical challenge due to lack of chemotherapeutic response. Systemic therapy with immunostimulatory agents such as interferon and interleukin-2 (IL-2) has shown some promise, though each is associated with significant side effects. Over the past 50 years, oncolytic Newcastle disease virus (NDV) has emerged as an alternative candidate for cancer therapy. The establishment of reverse-genetics systems for the virus has allowed us to further manipulate the virus to enhance its oncolytic activity. Introduction of immunomodulatory molecules, especially IL-2, into the NDV genome was shown to enhance the oncolytic potential of the virus in a murine syngeneic colon carcinoma model. We hypothesize that a recombinant NDV expressing IL-2 would be an effective agent for therapy of malignant melanoma. We show that recombinant NDV possesses a strong cytolytic activity against multiple melanoma cell lines, and is effective in clearing established syngeneic melanoma tumors in mice. Moreover, introduction of murine IL-2 into NDV significantly enhanced its activity against syngeneic melanomas, resulting in increased overall animal survival and generation of antitumor immunity. These findings warrant further investigations of IL-2-expressing NDV as an antimelanoma agent in humans. PMID 19242529

Vaccination with recombinant adenoviruses and dendritic cells expressing prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate cancer.
Mai 2009 | Kim, Sol; Lee, Jee-Boong; Lee, Geon Kook; Chang, Jun
Prostate cancer is currently the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men in the US. Immunological approaches may provide an alternative option for prevention and treatment of prostate cancer. PMID 19267351

Single-step antigen loading and activation of dendritic cells by mRNA electroporation for the purpose of therapeutic vaccination in melanoma patients.
Mai 2009 | Bonehill, Aude; Van Nuffel, An M T; Corthals, Jurgen; Tuyaerts, Sandra; Heirman, Carlo; François, Violaine; Colau, Didier; van der Bruggen, Pierre; Neyns, Bart; Thielemans, Kris
A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen-derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen-encoding mRNA. PMID 19417017

Melanoma vaccines: The problems of local immunosuppression.
Apr. 2009 | Polak, Marta E; Borthwick, Nicola J; Jager, Martine J; Cree, Ian A
The incidence of cutaneous melanoma in Europe is rising, and the disease is incurable once metastases occur. Because melanoma expresses antigens that can be specifically recognized by the immune system, and because this disease occasionally undergoes spontaneous regression mediated by anti-tumor immunity, a number of different melanoma vaccines have been developed and tested clinically. Although most such vaccines show efficacy in vitro and an ability to stimulate anti-melanoma immune responses in blood, they have proved disappointing in clinical practice. It has become increasingly clear that the interaction between melanoma and the immune system is determined locally, within the tumor or draining lymph nodes. It is now clear that melanoma cells have the ability to anergize the immune system by inducing an immunosuppressive microenvironment that may explain the inability of systemic vaccines to alter patient outcomes. This subversion of the immune system involves alteration of dendritic cell (DC) function by tumor-derived cytokines, leading to the generation of suppressive and regulatory T lymphocytes. Successful melanoma vaccination probably requires therapeutic neutralization of the immunosuppressive microenvironment, which will require greater understanding of the molecular mechanisms used by the tumor to promote immunosuppression. Nevertheless, if these problems can be overcome, it seems likely that the efficacy of melanoma vaccines could be greatly enhanced. PMID 19405172

Limited amounts of dendritic cells migrate into the T-cell area of lymph nodes but have high immune activating potential in melanoma patients.
Apr. 2009 | Verdijk, Pauline; Aarntzen, Erik H J G; Lesterhuis, W Joost; Boullart, A C Inge; Kok, Ellemieke; van Rossum, Michelle M; Strijk, Simon; Eijckeler, Femke; Bonenkamp, Johannes J; Jacobs, Joannes F M; Blokx, Willeke; Vankrieken, J Han J M; Joosten, Irma; Boerman, Otto C; Oyen, Wim J G; Adema, Gosse; Punt, Cornelis J A; Figdor, Carl G; de Vries, I Jolanda M
The success of immunotherapy with dendritic cells (DC) to treat cancer is dependent on effective migration to the lymph nodes and subsequent activation of antigen-specific T cells. In this study, we investigated the fate of DC after intradermal (i.d.) or intranodal (i.n.) administration and the consequences for the immune activating potential of DC vaccines in melanoma patients. PMID 19318472

The induction of tumor-specific CD4+ T cells via major histocompatibility complex class II is required to gain optimal anti-tumor immunity against B16 melanoma cell line in tumor immunotherapy using dendritic cells.
Apr. 2009 | Fujisawa, Yasuhiro; Nabekura, Tsukasa; Nakao, Tomohei; Nakamura, Yasuhiro; Takahashi, Takenori; Kawachi, Yasuhiro; Otsuka, Fujio; Onodera, Masafumi
We have demonstrated that dendritic cells (DCs) genetically modified to express tumor-associated antigens (TAAs) with retroviral vectors elicit more potential anti-tumor effect than those loaded with peptides because they can prime antigen-specific CD4+ T cells resulting in production of tumor-specific antibody. In this study, we showed the importance of antigen presentation via a major histocompatibility complex (MHC) class II molecule in cancer immunity against non-membrane bound TAAs such as the melanoma antigen gp100 by using DCs derived from MHC class II-deficient mice (C2KO). DCs were prepared by transduction of gp100 cDNA into haematopoietic progenitor cells obtained from C2KO followed by differentiation with cytokines (C2KO-gp/DCs). When C2KO-gp/DCs were inoculated into immunocompetent mice, the mice scarcely primed the antigen-specific Th1 cells and developed fewer CD8 T cells than did those inoculated with transduced DCs prepared from normal mice. The attenuated anti-tumor effect was also confirmed in a postimmunization setting where, while two of eight control mice eradicated the pre-existing melanoma cell line B16 (25%), no mice inoculated with C2KO-gp/DCs did. These results suggested not only the limitation of current protocols using MHC class I-restricted tumor peptides but also the usefulness of DCs expressing gp100 in vaccine therapy against melanoma. PMID 19054057

Prolonged survival of dendritic cell-vaccinated melanoma patients correlates with tumor-specific delayed type IV hypersensitivity response and reduction of tumor growth factor beta-expressing T cells.
März 2009 | López, Mercedes N; Pereda, Cristian; Segal, Gabriela; Muñoz, Leonel; Aguilera, Raquel; González, Fermín E; Escobar, Alejandro; Ginesta, Alexandra; Reyes, Diego; González, Rodrigo; Mendoza-Naranjo, Ariadna; Larrondo, Milton; Compán, Alvaro; Ferrada, Carlos; Salazar-Onfray, Flavio
The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. PMID 19139436

Immunoregulatory T cells in the peripheral blood of melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.
März 2009 | Nakai, Noriaki; Katoh, Norito; Kitagawa, Tomoko; Ueda, Eiichiro; Takenaka, Hideya; Kishimoto, Saburo
Regulatory T cells (Treg) may inhibit monocyte-derived melanoma antigen-pulsed dendritic cells (DC) vaccination in treatment of melanoma. However, the Treg level in peripheral blood mononuclear cells (PBMCs) following DC vaccination has not been examined in melanoma patients in Japan. PMID 19157789

Dendritic cells in the skin--potential use for melanoma treatment.
Jan. 2009 | El Marsafy, Sanaa; Bagot, Martine; Bensussan, Armand; Mauviel, Alain
Melanoma is an aggressive malignancy with poor prognosis. Eradication of tumor cells requires an effective interaction between melanoma cells and different players of the immune system. As the most potent professional antigen-presenting cells, dendritic cells (DCs) play a pivotal role in mounting a specific immune response where their intratumoral and peritumoral density as well as their functional status are correlated with clinical staging of the disease and with patients' survival. Under steady-state conditions, internalization of apoptotic cells by immature DCs designates a state of tolerance to self-antigens. Nevertheless, pathogens and necrotic cells interacting with pattern recognition receptors trigger downstream signaling pathways that evoke maturation of DCs, leading to the production of pro-inflammatory cytokines. These mature DCs are essential for T-cell priming and subsequent development of a specific immune response. Altered functions of DCs have an impact on the development of various disorders including autoimmune diseases and cancers. Herein, we focus on the checkpoints created throughout DCs antigen capturing and presentation to T cells, with subsequent development of either tolerance or immune response, with an emphasis on the role played by DCs in melanoma tumorigenesis and their therapeutic potential. PMID 19040502

TIMP-1-GPI in combination with hyperthermic treatment of melanoma increases sensitivity to FAS-mediated apoptosis.
Jan. 2009 | Djafarzadeh, Roghieh; Milani, Valeria; Rieth, Nicole; von Luettichau, Irene; Skrablin, Petra S; Hofstetter, Monika; Noessner, Elfriede; Nelson, Peter J
Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8 degrees C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes. PMID 18618109

Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.
Dez. 2008 | Nakai, Noriaki; Katoh, Norito; Germeraad, Wilfred T V; Kishida, Tsunao; Ueda, Eiichiro; Takenaka, Hideya; Mazda, Osam; Kishimoto, Saburo
In melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH. PMID 18804963

Tumor infection by oncolytic reovirus primes adaptive antitumor immunity.
Nov. 2008 | Prestwich, Robin J; Errington, Fiona; Ilett, Elizabeth J; Morgan, Ruth S M; Scott, Karen J; Kottke, Timothy; Thompson, Jill; Morrison, Ewan E; Harrington, Kevin J; Pandha, Hardev S; Selby, Peter J; Vile, Richard G; Melcher, Alan A
Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. PMID 19010851

Type 2 Bias of T cells expanded from the blood of melanoma patients switched to type 1 by IL-12p70 mRNA-transfected dendritic cells.
Nov. 2008 | Minkis, Kira; Kavanagh, Daniel G; Alter, Galit; Bogunovic, Dusan; O'Neill, David; Adams, Sylvia; Pavlick, Anna; Walker, Bruce D; Brockman, Mark A; Gandhi, Rajesh T; Bhardwaj, Nina
Melanoma patients may exhibit a T(H)2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial T(H)1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1beta, tumor necrosis factor alpha, and IL-6] and prostaglandin E(2) (PGE(2)), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE(2) suppresses the production of IL-12p70, a cytokine essential for differentiation of T(H)1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the T(H)2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew T(H)1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8(+) T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy. PMID 19010919

Dendritic cells pulsed with keyhole limpet hemocyanin and cryopreserved maintain anti-tumor activity in a murine melanoma model.
Nov. 2008 | Teitz-Tennenbaum, Seagal; Li, Qiao; Davis, Mary A; Chang, Alfred E
We compared viability, phenotype, in vitro function and therapeutic efficacy of murine unpulsed-dendritic cells (-DC), DC pulsed with keyhole limpet hemocyanin (KLH-DC) and cryopreserved KLH-DC (C-KLH-DC). Mean viability (%+/-SE) of unpulsed-DC, KLH-DC and C-KLH-DC was 93.6+/-0.9, 93.9+/-0.8 and 87.4+/-1.6, respectively. Pulsing DC with KLH did not induce maturation or affect in vitro function. Cryopreservation of KLH-DC reduced MHC I, CD80 and CD86 expression, endocytic capacity and allogeneic splenocyte stimulatory capacity. Intratumoral (i.t.) vaccination of mice bearing s.c. D5 melanoma with unpulsed-DC, KLH-DC or C-KLH-DC elicited comparable anti-tumor immune responses and inhibited tumor growth to the same extent. Combining radiotherapy with i.t. unpulsed-DC, KLH-DC or C-KLH-DC administration enhanced induction of anti-tumor immune responses and inhibition of tumor growth to a similar degree. Cryopreservation of KLH-DC slightly reduces viability, expression of co-stimulatory cell surface markers and in vitro function; however, in vivo anti-tumor activity is fully maintained with or without radiotherapy. PMID 18845485

Review of clinical studies on dendritic cell-based vaccination of patients with malignant melanoma: assessment of correlation between clinical response and vaccine parameters.
Okt. 2008 | Engell-Noerregaard, Lotte; Hansen, Troels Holz; Andersen, Mads Hald; Thor Straten, Per; Svane, Inge Marie
During the past years numerous clinical trials have been carried out to assess the ability of dendritic cell (DC) based immunotherapy to induce clinically relevant immune responses in patients with malignant diseases. A broad range of cancer types have been targeted including malignant melanoma which in the disseminated stage have a very poor prognosis and only limited treatment options with moderate effectiveness. Herein we describe the results of a focused search of recently published clinical studies on dendritic cell vaccination in melanoma and review different vaccine parameters which are frequently claimed to have a possible influence on clinical response. These parameters include performance status, type of antigen, DC maturation status, route of vaccine administration, use of adjuvant, and vaccine induced immune response. In total, 38 articles found through Medline search, have been included for analysis covering a total of 626 patients with malignant melanoma treated with DC based therapy. Clinical response (CR, PR and SD) were found to be significantly correlated with the use of peptide antigens (p = 0.03), the use of any helper antigen/adjuvant (p = 0.002), and induction of antigen specific T cells (p = 0.0004). No significant correlations between objective response (CR and PR) and the tested parameters were found. However, a few non-significant trends were demonstrated; these included an association between objective response and use of immature DCs (p = 0.08), use of adjuvant (p = 0.09), and use of autologous antigen preparation (p = 0.12). The categorisation of SD in the response group is debatable. Nevertheless, when the SD group were analysed separately we found that SD was significantly associated with use of peptide antigens (p = 0.0004), use of adjuvant (p = 0.01), and induction of antigen specific T cells (p = 0.0003). No specific route of vaccine administration showed superiority. Important lessons can be learned from previous studies, interpretation of these findings should, however, be done with reservation for the many minor deviations in the different treatment schedules among the published studies, which were not considered in order to be able to process and group the data. PMID 18719915

Immunotherapy as an adjuvant therapy in the management of advanced, surgically resected, melanoma.
Okt. 2008 | Kalani, A D; Jack, A; Montenegro, G; Degliuomini, J; Wallack, M K
Metastatic melanoma continues to be one of the most devastating of all cancers. It is a heterogeneous solid tumor whose treatment is challenging and difficult. It afflicts thousands of otherwise healthy patients annually, and clinicians have yet to discover an effective treatment for locally advanced disease. Over the years, much attention has been devoted to the development of an effective adjuvant treatment for patients with resected melanoma who remain at high risk for recurrence. The new advances in the understanding of melanoma's microenvironment and the complexity of its disease process, makes it clear that the treatment approach to this disease needs to be multi-directional. Numerous studies have tested various immunotherapeutic strategies in the treatment of advanced melanoma, in particular. These strategies include melanoma vaccines, interferon-alpha, interleukin-2 (IL-2), and dendritic cell vaccines. The Dr. Wallack's Surgery Research Laboratory has been studying melanoma vaccines for the past three decades. The first generation melanoma vaccine proposed by the Laboratory showed promising results in a subset of patients. Recently, the same Laboratory has produced a second generation melanoma vaccine (DC-Melvac) that consists of five human melanoma cell lines, a recombinant vaccinia virus that encodes for IL-2, as well as dendritic cells that have been programmed to recognize certain melanoma associated antigens. DC-MelVac was recently approved by the Food and Drug Administration for its use in Phase I clinical trials. These trials are expected to be underway in the near future. The ensuing review discusses many of the immunotherapeutic strategies that have been studied in the treatment of melanoma, including DC-MelVac. PMID 18833052

Strategies to overcome obstacles to successful immunotherapy of melanoma.
Okt. 2008 | Pandolfi, F; Cianci, R; Lolli, S; Dunn, I S; Newton, E E; Haggerty, T J; Boyle, L A; Kurnick, J T
The immunogenicity of malignant melanomas has been recognized by the observed recruitment of tumor-specific cytotoxic T-cells (CTL), leading to the identification of several melanoma associated antigen (MAA). However, numerous strategies to treat melanoma with immunotherapy have resulted in only partial success. In this editorial, we discuss recent data related to the ability of tumors to elude immune responses. We therefore discuss different strategies to induce a clinically effective immune response. These approaches include 1) immunostimulation: including peptide/protein based vaccines, dendritic cell vaccines, and adoptive cell transfer; and 2) overcoming immunosuppression, including targeting of checkpoint molecules such as CTLA-4, circumventing the activity of Tregs, and assuring antigen expression by tumor cells (thwarting antigen silencing). Finally, we discuss recent advances in gene therapy, including adoptive therapy with engineered T cell receptors (TCRs). These issues lead to the conclusion that successful immunotherapy in malignant melanoma requires a combination of strategies aimed at both inducing immunostimulation and blocking immunosuppression. PMID 18831916

Dendritic cell vaccines in metastasized malignant melanoma.
Okt. 2008 | Erdmann, M; Schuler-Thurner, B
Dendritic cells as immunotherapeutic agents against malignancies have been applied for over ten years. Proof of principle studies demonstrated immunogenicity of dendritic cells even in patients suffering from advanced malignancies. Clinicians and immunologists early focused on this innovative immunotherapeutic approach in metastasized malignant melanoma--a malignancy so far resisting most traditional oncologic treatment modalities. In this review we summarize the experience obtained of dendritic cell therapy in patients with malignant melanoma and state past, present and future obstacles. So far over 850 melanoma patients in 51 trials have been reported since 1998. Within these trials there exists a vast heterogeneity concerning type of dendritic cell applied, differentiation and maturation of dendritic cells, type of antigen target and nature, application mode, number of cells applied, vaccination intervals in addition to patients treated at various stages of melanoma. A minority of patients developed anticipated autoimmune adverse events in addition to expected immune system activation symptoms such as fever and local site reaction. As only solitary World Health Organization (WHO) grade III or IV adverse events were reported one can state that dendritic cell therapy is safe. Objective clinical responses have repeatedly been observed in a minority of heavily pretreated and far advanced melanoma patients. Future challenges include optimization and standardization of dendritic cell generation and application, addition of synergistic immunomodulatory agents to enhance immunogenicity and block tumor escape and treatment of patients at earlier stages of disease who will benefit from this innovative therapy. PMID 18833080

Engineering of highly immunogenic long-lived DC vaccines by antiapoptotic protein gene transfer to enhance cancer vaccine potency.
Sep. 2008 | Yoshikawa, T; Niwa, T; Mizuguchi, H; Okada, N; Nakagawa, S
Dendritic cells (DCs) have a critical role in the induction of antigen-specific immune responses, transporting antigens from peripheral tissue to regional lymph nodes where they interact with antigen-specific T lymphocytes. Recent studies revealed that the efficacy of the T cell-dependent immune response depends on the lifespan of the antigen-presenting DCs in the lymph nodes. Here, we succeeded in engineering long-lived antigen-presenting DCs via Bcl-XL-derived hyperactive mutant antiapoptotic protein (Bcl-X FNK) gene transfer. In a B16BL6 melanoma model, these long-lived DCs exerted potent antitumor immunity that depended mainly on antigen-specific cytotoxic T lymphocytes. Furthermore, in vivo longevity of the long-lived DC vaccine led to antigen-specific activation of interferon-gamma-producing CD4+ and CD8+ T cells. Thus, the long-lived DC vaccine strategy is highly useful for constructing DC vaccines, as well as other cell-based medicines, such as stem cell therapy. PMID 18480845

Melanoma vaccines.
Sep. 2008 | Riley, Lee B; Agarwala, Sanjiv S
Over the last century, vaccine studies have demonstrated that the human immune system, with appropriate help, can limit or prevent infection against otherwise lethal pathogens. Encouraged by these results, success in animal models and numerous well-documented reports of immune-mediated melanoma regression in humans, investigators developed melanoma vaccines. However, despite considerable laboratory evidence for vaccine-induced immune responses, clinical responses remain poor. Recent studies have elucidated several mechanisms that hinder or prevent the creation of successful vaccines and suggest novel approaches to overcome these barriers. Unraveling the mechanisms of autoimmunity, dendritic cell activation, regulatory T cells and Toll-like receptors will generate novel vaccines that, when used in conjunction with standard adjuvant therapies, may result in improved clinical outcomes. The objective of this review is to provide an overall summary of recent clinical trials with melanoma vaccines and highlight novel vaccine strategies to evaluate in the near future. PMID 18767944

Dendritic cell based genetic immunization stimulates potent tumor protection dependent on CD8 CTL cells in the absence of autoimmunity.
Aug. 2008 | Zhang, Sheng; Huang, Weiyi
Although antibodies (Abs) produced by B cells can treat cancer in certain models, T cells have been accountable for the major effector to control cancer. Immune recognition toward tyrosinase-related protein-1 (TRP-1), a melanoma associated antigen up-regulated on the surface of B16F10 melanomas, generally leads to tumor protection mediated by Abs. In this study, immunization with dendritic cells ex vivo transduced with adenovirus encoding TRP-1 stimulates immune activation and potent tumor protection mediated by CD8 T cells in the absence of autoimmune consequence. Transfer of CD8 T cells from immunized mice also leads to tumor protection. The immune activation and CD8 T cell mediated tumor protection rely on the CD4 T cell help. Thus DC based genetic immunization targeting TRP-1, an antigen usually causes Ab predominant immune recognition, is capable of stimulating potent tumor protection dependent on CD8 T cells in the absence of autoimmunity. PMID 18299892

Immunological response in the mouse melanoma model after local hyperthermia.
Juli 2008 | Kubes, J; Svoboda, J; Rosina, J; Starec, M; Fiserová, A
Our study was aimed to characterize the phenotype and functional endpoints of local microwave hyperthermia (LHT, 42 degrees C) on tumor infiltrating and spleen leukocytes. The effectiveness of LHT applied into the tumor of B16F10 melanoma-bearing C57/BL6 mice was compared with anesthetized and non-treated animals. Subpopulations of leukocytes were analyzed using the flow cytometry, and the cytotoxic activity of splenocytes against syngeneic B16F10 melanoma and NK-sensitive YAC-1 tumor cell lines was evaluated in (51)Cr-release assay. Similarly, the in vitro modification of the heat treatment was performed using healthy and melanoma-bearing splenocytes. We found a 40 % increase of activated monocytes (CD11b+CD69+) infiltration into the tumor microenvironment. In the spleen of experimental animals, the numbers of cytotoxic T lymphocytes (CTLs-CD3+CD8+) and NK cell (CD49b+NK1.1+) raised by 22 % and 14 %, respectively, while the NK1.1+ monocytes decreases by 37 %. This was accompanied by an enhancement of cytotoxic effector function against B16F10 and YAC-1 targets in both in vivo and in vitro conditions. These results demonstrate that LHT induces better killing of syngeneic melanoma targets. Furthermore, LHT evokes the homing of activated monocytes into the tumor microenvironment and increases the counts of NK cells and CTL in the spleen. PMID 17552874

Phenotypic and functional analysis of dendritic cells and clinical outcome in patients with high-risk melanoma treated with adjuvant granulocyte macrophage colony-stimulating factor.
Juli 2008 | Daud, Adil I; Mirza, Noweeda; Lenox, Brianna; Andrews, Stephanie; Urbas, Patricia; Gao, Gui X; Lee, Ji-Hyun; Sondak, Vernon K; Riker, Adam I; Deconti, Ronald C; Gabrilovich, Dmitry
Granulocyte macrophage colony-stimulating factor (GM-CSF) can induce differentiation of dendritic cells (DCs) in preclinical models. We hypothesized that GM-CSF-stimulated DC differentiation may result in clinical benefit in patients with high-risk melanoma. PMID 18591558

IFN-producing killer dendritic cells contribute to the inhibitory effect of poly I:C on the progression of murine melanoma.
Juni 2008 | Jiang, Qun; Wei, Haiming; Tian, Zhigang
Toll-like receptor 3 agonist polyinosinic-polycytidilic acid (poly I:C) has been widely used as a potent adjuvant in tumor immunotherapy. In the present study, it was demonstrated that intraperitoneal injection of poly I:C could inhibit lung and liver metastasis of B16 melanoma cells in C57BL/6 mice in natural killer (NK) cells and interferon (IFN)-gamma dependent manner, leading to prolonged survival of the mice. B220 CD11c NK1.1 cells, recently defined as IFN-producing killer dendritic cells (IKDCs) were markedly increased in the spleen, lung, and liver of poly I:C-treated tumor bearing mice, compared with the control group. IFN-gamma induction by poly I:C in this unique NK cell subset indicated its critical contribution in tumor suppression in this model. Meanwhile, results of in vitro culture assay showed that poly I:C synergized with B16 cells could significantly promote IKDCs expansion in lymphocytes from different organs along with IFN-gamma production. Moreover, these ex vivo expanded IKDCs also exerted cytolytic activities against B16 cells and YAC-1 cells as conventional NK cells did. In conclusion, the findings of this study provide new insights into the role of IFN-gamma and IKDCs in the antitumor effect of poly I:C, and will possibly be helpful to explain why poly I:C may work as an adjucant to improve the antitumor effects of innate cells. PMID 18528299

Phase Ib trial assessing autologous, tumor-pulsed dendritic cells as a vaccine administered with or without IL-2 in patients with metastatic melanoma.
Juni 2008 | Redman, Bruce G; Chang, Alfred E; Whitfield, Joel; Esper, Peg; Jiang, Guihua; Braun, Thomas; Roessler, Blake; Mulé, James J
Twenty-four subjects with metastatic melanoma were treated on a randomized Phase Ib trial evaluating an autologous tumor lysate-pulsed dendritic cell (DC) vaccine with or without interleukin (IL)-2. The vaccine consisted of autologous DCs obtained from peripheral blood mononuclear cells (PBMCs) cultured in granulocyte macrophage-colony stimulating factor and IL-4 then pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH). The primary end points of the trial were safety and immune response to vaccine. Subjects were randomized to vaccine administered every other week times 3, vaccine x 3 followed by low-dose IL-2, or vaccine x 3 followed by high-dose IL-2. Immune response was monitored pretreatment and at 2 and 4 weeks after the third vaccine administration. Disease evaluation was performed at 4 weeks after the third vaccination. Therapy was well tolerated with no local vaccine toxicity greater than grade 1 in any arm. IL-2 toxicity was as expected without additional toxicity from the addition of IL-2 to vaccine. Immune response defined as delayed-type hypersensitivity, PBMC interferon-gamma enzyme-linked immunosorbent spot, and PBMC proliferation, to both autologous tumor and KLH were detected in all arms. Interferon-gamma enzyme-linked immunosorbent spot response to KLH (7 of 10 patients) and autologous tumor (4 of 10 patients) were also detected in subjects with available vaccine draining lymph node cells. There were no differences in immune response between treatment arms. No clinical responses were seen. Autologous tumor lysate-pulsed DC vaccine with or without IL-2 was well tolerated and immunogenic but failed to induce clinical response in patients with advanced melanoma. PMID 18528294

Therapeutic vaccination with dendritic cells pulsed with tumor-derived Hsp70 and a COX-2 inhibitor induces protective immunity against B16 melanoma.
Juni 2008 | Toomey, Deirdre; Conroy, Helen; Jarnicki, Andrew G; Higgins, Sarah C; Sutton, Caroline; Mills, Kingston H G
Prophylactic immunization of mice with autologous tumor-derived heat shock proteins (Hsp) generates effective anti-tumor immunity. However, this approach is ineffective when used therapeutically, partly due to the immunosuppressive effects of the growing tumor. Here we sought to overcome this problem by therapeutic vaccination with dendritic cells (DC) pulsed with Hsp70 and a COX-2 inhibitor. We found that Hsp70 induces IL-6 and IL-10 production and suppressed expression of CD40 on DC. Incubation of DC with tumor-conditioned medium attenuated Hsp70-induced expression of CD80 and induced expression of COX-2. Inhibition of COX-2 partially reversed the stimulatory effect of Hsp70 on DC IL-6 and IL-10 production and enhanced expression of CD80 and MHC classes I and II. Therapeutic administration of DC pulsed in vitro with Hsp70 in the presence of a COX-2 inhibitor significantly reduced progression of B16 tumors in mice and significantly enhanced survival. This was associated with a reduction in the frequency of IL-10-producing CD4(+) T cells and enhancement of IFN-gamma-producing CD8(+) T cells. Our findings provide a novel immunotherapeutic approach against cancer based on attenuation of COX-2-mediated immunosuppression using in vitro modulated DC. PMID 18479787

Melanoma and lymphoma rejection associated with eosinophil infiltration upon intratumoral injection of dendritic and NK/LAK cells.
Juni 2008 | Capobianco, Annalisa; Manfredi, Angelo A; Monno, Antonella; Rovere-Querini, Patrizia; Rugarli, Claudio
Dendritic cells (DCs) are promising tools for tumor immunotherapy. Their efficacy in the tumor environment increases when tumor cells die as a consequence of chemo/radiotherapy or when local stimuli promoting DC maturation and function are available. Dying tumor cells could represent a source of tumor antigens, which DCs cross-present to tumor-specific T cells. The outcome of cross presentation is in turn determined by the maturation state of DCs. Natural killer (NK)/lymphokine-activated killer (LAK) cells injected into growing tumors could both provide a source of dying cells for cross-presentation and deliver stimuli for DC maturation. Here, we report that NK/LAK cells recognized and killed in vivo major histocompatibility complex class I(low) highly tumorigenic, nonimmunogenic B16F1 melanoma cells when injected into exponentially growing neoplastic lesions. The simultaneous injection of immature DCs was required to heal animals. Similar results were obtained injecting NK/LAK cells and DC into growing Raucher leukaemia virus induced cell line lymphomas. Cured mice failed to reject other implantable tumors, and developed a specific cytotoxic response against the original neoplasm; moreover, they developed a long-lasting memory, and were protected against further challenges with living tumor cells only when both cell populations were introduced. The response associated to the preferential recruitment within tumors of eosinophils. The simultaneous injection in solid tumors of DCs and NK/LAK cells represents an attractive approach for antineoplastic immunotherapeutic strategies. PMID 18463539

Dendritic cell immunotherapy for melanoma.
Mai 2008 | Peng, Judy C; Thomas, Ranjeny; Dredge, Keith
Dendritic cells (DC) are the most potent antigen-presenting cells that initiate T cell-mediated immune responses against cancer. It has been almost a decade since the first trial of DC-based cancer immunotherapy was published. Despite the many clinical trials conducted since, few solid conclusions have been reached, and no specific-immunotherapy has routinely demonstrated meaningful anti-tumour responses. Clinical-grade DC can be obtained from three distinct cell populations in the blood - monocytes, CD34(+) progenitors or direct isolation of circulating blood DC. This review discusses the science behind DC-based cancer immunotherapy, with a particular emphasis on the use of monocyte-derived DC in melanoma clinical trials, and the various potential avenues for improvement of patient clinical response rates. PMID 18473960

Lack of elevation of serum S100B in patients with metastatic melanoma as a predictor of outcome after induction with an autologous vaccine of proliferating tumor cells and dendritic cells.
Mai 2008 | Schiltz, Patric M; Dillman, Robert O; Korse, Catharina M; Cubellis, James M; Lee, Gregory J; De Gast, Gijsbert C
Serum levels of S100B and/or lactate dehydrogenase (LDH) are putative tumor markers in melanoma. Early changes in such markers may correlate with a positive immune response to vaccine therapy. In patients with metastatic melanoma, S100B and LDH serum levels were measured at baseline, and 1 week after 3 weekly subcutaneous injections of investigational, patient-specific vaccines consisting of autologous dendritic cells loaded with antigens from irradiated proliferating autologous tumor cells, and suspended in granulocyte macrophage colony-stimulating factor. There was a poor correlation between S100B and LDH levels at baseline (p = 0.324). Fourteen (14) patients with measurable disease had higher S100B (p = 0.0456) and LDH (p = 0.0013) levels than 31 patients who lacked measurable disease at that time. Fourteen (14) deceased patients (median survival, 13 months) had a mean baseline S100B of 0.62 mug/L (95% confidence interval [CI] 0.00-1.66) and LDH of 815 U/L (95% CI 222-1408); 31 surviving patients (median follow-up, 35.4 months) had mean S100B of 0.07 mug/L (95% CI 0.00-0.23; p = 0.006) and LDH of 442 U/L (95% CI 296-588; p = 0.002). Elevated baseline levels of LDH and S100B were each predictive of inferior progression-free survival (PFS) and overall survival (OS) (both p < 0.0001), but S100B was a better predictor for PFS than LDH. Changes in LDH between baseline and week 4 were not predictive of survival, but an increase in S100B predicted for inferior OS ( p = 0.039). Both LDH and S100B are predictive tumor markers in patients with metastatic melanoma. This is the first study to examine the changes in serum levels of LDH and S100B in response to an autologous tumor-cell vaccine. PMID 18454690

Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy.
Apr. 2008 | Teitz-Tennenbaum, Seagal; Li, Qiao; Okuyama, Ryuji; Davis, Mary A; Sun, Rong; Whitfield, Joel; Knibbs, Randall N; Stoolman, Lloyd M; Chang, Alfred E
We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4 or CD8 effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy. PMID 18391761

Nonmyeloablative chemotherapy followed by T-cell adoptive transfer and dendritic cell-based vaccination results in rejection of established melanoma.
Apr. 2008 | Koike, Nobusada; Pilon-Thomas, Shari; Mulé, James J
We demonstrated previously that dendritic cell (DC)-based vaccines could mediate a specific and long-lasting antitumor immune response during early lymphoid reconstitution after lethal irradiation and bone marrow transplant. The purpose of this current study was to examine the potential therapeutic efficacy of DC-based vaccines in combination with sublethal lymphodepletion and T-cell transfer. In an aggressive model of melanoma, treatment with the combination of 200 mg/kg cyclophosphamide (Cy) and 100 mg/kg fludarabine (Flu) led to a lymphopenic state lasting approximately 14 days, but had no effect on the growth of an established M05 melanoma. Addition of ovalbumin (OVA) peptide-pulsed DC-based immunization resulted in a delay in tumor growth but did not enhance overall survival in this model. To improve treatment, adoptively transferred naive T cells were added. After induction of lymphopenia with Cy and Flu, transferred T cells demonstrated an activated memory phenotype including high expression of CD44 and low expression of CD62L. Induction of lymphopenia with Cy and Flu in combination with adoptive transfer of naive T cells and OVA peptide-pulsed DCs immunization led to an enhancement in the number of OVA specific, CD8 T cells that demonstrated specific cytotoxic activity, proliferation, and interferon-gamma production in response to the OVA expressing M05 melanoma. This combination therapy also led to tumor regression and enhanced survival in mice bearing M05 melanoma. PMID 18391755

Dendritic cell vaccines in melanoma: from promise to proof?
Apr. 2008 | Lesterhuis, W J; Aarntzen, E H J G; De Vries, I J M; Schuurhuis, D H; Figdor, C G; Adema, G J; Punt, C J A
Dendritic cells (DC) are the directors of the immune system, capable of inducing tumour antigen-specific T- and B-cell responses. As such, they are currently applied in clinical studies in cancer patients. Early small clinical trials showed promising results, with frequent induction of anti-cancer immune reactivity and clinical responses. In recent years, additional trials have been carried out in melanoma patients, and although immunological responses are often reported, objective clinical responses remain anecdotal with objective response rates not exceeding 5-10%. Thus, DC vaccination research has now entered a stage in between 'proof of principle' and 'proof of efficacy' trials. Crucial questions to answer at this moment are why the clinical responses remain scarce and what can be done to improve the efficacy of vaccination. The answers to these questions probably lie in the preparation and administration of the DC vaccines. Predominantly, cytokine-matured DC are used in clinical studies, while from preclinical studies it is evident that DC that are activated by pathogen-associated molecules are much more potent T cell activators. For sake of easy accessibility monocyte-derived DC are often used, but are these cells also the most potent type of DC? Other yet unsettled issues include the optimal antigen-loading strategy and route of administration. In addition, trials are needed to investigate the value of manipulating tolerizing mechanisms, such as depletion of regulatory T cells or blockade of the inhibitory T cell molecule CTLA-4. These issues need to be addressed in well-designed comparative clinical studies with biological endpoints in order to determine the optimal vaccine characteristics. DC vaccination can then be put to the ultimate test of randomized clinical trials. Here, we review the immunobiology of DC with emphasis on the different aspects that are most relevant for the induction of anti-tumour responses in vivo. The different variables in preparing and administering DC vaccines are discussed in this context and the immunological and clinical results of studies with DC vaccines in melanoma patients are summarized. PMID 18262431

Strategies and challenges in eliciting immunity to melanoma.
März 2008 | Ferguson, Andrew R; Nichols, Lisa A; Zarling, Angela L; Thompson, Elizabeth D; Brinkman, C Colin; Hargadon, Kristian M; Bullock, Timothy N; Engelhard, Victor H
The ability of CD8+ T cells to recognize melanoma tumors has led to the development of immunotherapeutic approaches that use the antigens CD8+ T cells recognize. However, clinical response rates have been disappointing. Here we summarize our work to understand the mechanisms of self-tolerance that limit responses to currently utilized antigens and our approach to identify new antigens directly tied to malignancy. We also explore several aspects of the anti-tumor immune response induced by peptide-pulsed dendritic cells (DCs). DCs differentially augment the avidity of recall T cells specific for self-antigens and overcome a process of aberrant CD8+ T-cell differentiation that occurs in tumor-draining lymph nodes. DC migration is constrained by injection route, resulting in immune responses in localized lymphoid tissue, and differential control of tumors depending on their location in the body. We demonstrate that CD8+ T-cell differentiation in different lymphoid compartments alters the expression of homing receptor molecules and leads to the presence of systemic central memory cells. Our studies highlight several issues that must be addressed to improve the efficacy of tumor immunotherapy. PMID 18363993

Cancer vaccines for established cancer: how to make them better?
März 2008 | Andrews, Daniel M; Maraskovsky, Eugene; Smyth, Mark J
If one envisions dendritic cells (DCs) as nature's adjuvant, then it is easy to predict that they would be advantageous for cancer immunotherapy. Advances in culture processes that generate large numbers of purified and functionally mature DCs raised the possibility that DCs might be promising clinical agents to generate effective immune responses against cancer. The use of mature DCs as cellular vaccines was proposed to be superior to conventional strategies aimed at treating cancer, yet a phase III clinical trial in patients with melanoma demonstrated no increased benefit of DCs over standard therapy. Despite this and other apparent failures, we propose that DC-based therapy should not be discarded but rather reassessed. The heterogeneity of DCs and their interaction with other innate cells and regulatory and effector pathways must be clearly understood before the full therapeutic benefit of DCs are recognized. Several aspects of DC vaccination require optimization including the following: effective delivery of vaccines to DCs in lymphoid tissues; incorporation of components that induce appropriate DC activation; and facilitation of innate and adaptive interactions and reduction of regulatory T-cell networks or suppressive microenvironments that hinder the function of immune effectors. Application of this knowledge is resulting in encouraging new data in pre-clinical settings, where multiple arms of the immune system are targeted for cancer therapy. PMID 18364006

Hyperthermia induces endoplasmic reticulum-mediated apoptosis in melanoma and non-melanoma skin cancer cells.
März 2008 | Shellman, Yiqun G; Howe, William R; Miller, Leslie A; Goldstein, Nathaniel B; Pacheco, Theresa R; Mahajan, Roop L; LaRue, Susan M; Norris, David A
Hyperthermia has been revived as a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, we examined the cytotoxic effects of hyperthermia on various skin cancer cell lines using cell viability, morphological analyses, and caspase activation assays. Hyperthermia induced cytotoxicity in a time- and temperature-dependent manner. At middle dose/time combinations, heat-induced apoptosis, whereas at higher doses, necrosis was the mechanism of cell death. To investigate the mechanisms of hyperthermia-induced apoptosis, we examined the activation of extrinsic (Caspase 8) and intrinsic (Caspase 9) apoptotic pathways. Hyperthermia did not activate Caspases 8 or 9, but did activate Caspase 3/7, suggesting a non-conventional apoptotic pathway. Last, analysis of Grp78 expression and Caspase 12 or 4 activation indicated that hyperthermia induced endoplasmic reticulum-mediated apoptosis. Thus, hyperthermia induced apoptosis in two types of skin cancer cells through endoplasmic reticulum-mediated apoptosis and not through the classical intrinsic or extrinsic apoptosis pathways. Hyperthermia may be a promising treatment for basal cell carcinoma and melanoma, bypassing the antiapoptotic defenses concentrated in the intrinsic and extrinsic apoptosis pathways. These results also raise the possibility that heat may be combined with other approaches for induction of apoptosis to achieve synergistic killing of skin cancers. PMID 17989736

A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression.
März 2008 | von Euw, Erika M; Barrio, María M; Furman, David; Levy, Estrella M; Bianchini, Michele; Peguillet, Isabelle; Lantz, Olivier; Vellice, Alejandra; Kohan, Abraham; Chacón, Matías; Yee, Cassian; Wainstok, Rosa; Mordoh, José
Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. PMID 18221542

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells.
Feb. 2008 | Tamura, Kyoichi; Yamada, Misato; Isotani, Mayu; Arai, Hiroyoshi; Yagihara, Hiroko; Ono, Kenichiro; Washizu, Tsukimi; Bonkobara, Makoto
To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma. PMID 17208475

Analysis and characterization of antitumor T-cell response after administration of dendritic cells loaded with allogeneic tumor lysate to metastatic melanoma patients.
Dez. 2007 | Bercovici, Nadege; Haicheur, Nacilla; Massicard, Severine; Vernel-Pauillac, Frederique; Adotevi, Olivier; Landais, Didier; Gorin, Isabelle; Robert, Caroline; Prince, H Miles; Grob, Jean-Jacques; Leccia, Marie Thérèse; Lesimple, Thierry; Wijdenes, John; Bartholeyns, Jacques; Fridman, Wolf H; Salcedo, Margarita; Ferries, Estelle; Tartour, Eric
The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-gamma enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-gamma in enzyme-linked immunospot. Differential expression of IFN-gamma and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-gamma only after restimulation. The differential expression of perforin and IFN-gamma by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-gamma production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines. PMID 18157017

Modulation of viability and maturation of human monocyte-derived dendritic cells by oncolytic adenoviruses.
Nov. 2007 | Schierer, Stephan; Hesse, Andrea; Müller, Ina; Kämpgen, Eckhart; Curiel, David T; Schuler, Gerold; Steinkasserer, Alexander; Nettelbeck, Dirk M
Adenoviral oncolysis is a promising new modality for treatment of cancer based on selective viral replication in tumor cells. However, tumor cell killing by adenoviral oncolysis needs to be improved to achieve therapeutic benefit in the clinic. Towards this end, the activation of anti-tumor immunity by adenoviral oncolysis might constitute a potent mechanism for systemic killing of uninfected tumor cells, thereby effectively complementing direct tumor cell killing by the virus. Knowledge of anti-tumor immune induction by adenoviral oncolysis, however, is lacking mostly due to species-specificity of adenovirus replication, which has hampered studies of human oncolytic adenoviruses in animals. We suggest the analysis of interactions of oncolytic adenoviruses with human immune cells as rational basis for the implementation of adenoviral oncolysis-induced anti-tumor immune activation. The goal of our study was to investigate how oncolytic adenoviruses affect human dendritic cells (DCs), key regulators of innate and adoptive immunity that are widely investigated as tumor vaccines. We report that melanoma-directed oncolytic adenoviruses, like replication-deficient adenoviruses but unlike adenoviruses with unrestricted replication potential, are not toxic to monocyte-derived immature DCs and do not block DC maturation by external stimuli. Of note, this is in contrast to reports for other viruses/viral vectors and represents a prerequisite for anti-tumor immune activation by adenoviral oncolysis. Furthermore, we show that these oncolytic adenoviruses alone do not or only partially induce DC maturation. Thus additional signals are required for optimal immune activation. These could be delivered, for example, by inserting immunoregulatory transgenes into the oncolytic adenovirus genome. PMID 17764070

Plasmacytoid dendritic cells represent a major dendritic cell subset in sentinel lymph nodes of melanoma patients and accumulate in metastatic nodes.
Okt. 2007 | Gerlini, Gianni; Urso, Carmelo; Mariotti, Giulia; Di Gennaro, Paola; Palli, Domenico; Brandani, Paola; Salvadori, Adriana; Pimpinelli, Nicola; Reali, Umberto Maria; Borgognoni, Lorenzo
Plasmacytoid dendritic cells (pDC) represent the main source of interferon-alpha, a cytokine with antitumor activity. However, in vitro studies point to pDC as a key subset for induction of tolerance. Herein, we investigated pDC in sentinel lymph nodes (SLN) of melanoma patients. We report that pDC were constantly found in SLN and represented, with Langerhans cells, the most frequent dendritic cell subset. Their frequency in positive (with metastasis) SLN was significantly higher than in negative (without metastasis) SLN. PDC were observed in the T cell-rich areas of lymph nodes, particularly around high endothelial venules and, in metastatic nodes, they accumulated in close vicinity with melanoma nests. Finally, pDC capability to produce interferon-alpha in situ was impaired. Consistently, pDC expressed CD86, but neither CD80 nor CD83, suggesting a not complete activation in melanoma-draining lymph nodes. These results are consistent with the hypothesis of a tolerogenic role played by pDC in tumor immunology. PMID 17827069

Dendritic cell immunotherapy for stage IV melanoma.
Sep. 2007 | O'Rourke, Michael G E; Johnson, Maree K; Lanagan, Catherine M; See, Janet L; O'Connor, Linda E; Slater, Gregory J; Thomas, David; Lopez, José Alejandro; Martinez, Nathan R; Ellem, Kay A O; Schmidt, Christopher W
Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings. PMID 17885587

Immunity of unloaded dendritic cells in lung melanoma of mice.
Sep. 2007 | Liu, Hongju; Xin, Jianbao; Tao, Xiaonan; Shang, Dan; Zhou, Qiong
In order to investigate the immunity of unloaded dendritic cells (DCs) derived from murine bone marrow to preexisting lung melanoma metastases of mice, MO5 were intravenously injected to induce lung metastases in syngeneic C57BL/6 mice. Unloaded GM-CSF DCs, PBS and DCs+SIINFEKEL were subcutaneously injected into the mice, which were divided as experimental group, negative control group and positive control group respectively. Monoclonal antibody was used to deplete NK or T cells separately. The immunity-inhibitory effects on the lung melanoma were observed and the corresponding effector cells were examined. It was found that in the experimental and positive groups, the regression was induced in metastatic nodules in the lungs of tumor-bearing mice, but abrogated by treatment with anti-asialo-GM1 but not anti-CD8. It was concluded that the unloaded DCs could suppress the lung melanoma metastases to some extent, which was mediated by NK cells, and could be used as a potent therapeutic agents for lung tumor. PMID 17828491

Review: dendritic cell-based vaccine in the treatment of patients with advanced melanoma.
Sep. 2007 | Zhang, Sheng; Wang, Qing; Miao, Beiping
Traditionally, immunology is mainly about the study of the immune response against foreign antigens, such as bacteria and viruses. Accordingly, tumor cells expressing alien or altered antigens make the attractive targets against which cancer immunology is initiated. However, recent comprehensive studies demonstrated that most prevalent antigens recognized by our immune system in cancer are those shared, nonmutated self-antigens expressed also by normal tissue cells. Thus, how to break the self-tolerance and avoid the concomitant autoimmunity remain the two challenges in cancer immunology. Dendritic cells (DCs) are the most effective antigen-presenting cells. They are capable of capturing, processing, and presenting antigens to T- and B-cells. This feature targets dendritic cells as the ideal candidates for breaking self-tolerance in cancer immunology. Clinical trials have demonstrated that dendritic cells are effective messengers. They circulate around the body and stimulate cytotoxic T-lymphocytes to clear tumor cells. Vaccination with DCs led to a clinical response in patients with melanoma, specifically those without significant autoimmunity. In this paper, we will examine the strategies and efficacies of DC-based vaccinations in the treatment of patients with melanoma. PMID 17803444

Lymphocyte subpopulations in melanoma patients treated with dendritic cell vaccines.
Aug. 2007 | Kadagidze, Zaira G; Borunova, Anna A; Zabotina, Tatiana N
The main goal of cancer immunotherapy is to induce or boost tumor-specific effector cells able to eliminate or reduce tumor progression. In this study, we characterized lymphocyte phenotypes in melanoma patients receiving dendritic cell (DC)-based vaccinotherapy. We found that several biological markers served as unfavorable prognostic factors for patients' response to therapy. This included decrease of CD4+ and CD8+ lymphocyte levels, 10% and higher increase of CD16+CD3+CD8+ lymphocyte population, and increase of CD16+CD8+perforin+ T lymphocytes, especially in combination with decreased levels of CDI6+CD8(-)perforin+ and CD8+CD16(-)perforin+ cells. Increase in CD8+CD16(-)perforin+ T lymphocytes with normal levels of CD16+CD8(-)perforin+ cells and the absence of CD16+CD8+perforin+ and regulatory lymphocytes were shown to be the positive prognostic markers for patients' response to DC vaccines. PMID 17713027

Dendritic cell vaccination.
Aug. 2007 | Proudfoot, Owen; Pouniotis, Dodie; Sheng, Kuo-Ching; Loveland, Bruce E; Pietersz, Geoffrey A
There has been a surge of interest in the use of dendritic cell (DC) vaccination as cellular immunotherapy for numerous cancers. Despite some encouraging results, this therapeutic modality is far from being considered as a therapy for cancer. This review will first discuss preclinical DC vaccination in murine models of cancer, with an emphasis on comparative studies investigating different methods of antigen priming. We will then comment on the various murine DC subsets and how these relate to human DC preparations used for clinical studies. Finally, the methodology used to generate human DCs and some recent clinical trials in several cancers are reviewed. PMID 17669014

Melanoma-infiltrating dendritic cells induce protective antitumor responses mediated by T cells.
Mai 2007 | Preynat-Seauve, Olivier; Contassot, Emmanuel; Schuler, Prisca; French, Lars E; Huard, Bertrand
Dendritic cells are the most potent antigen-presenting cells inducing innate and adaptive immune response. Dendritic cells infiltrate melanomas, but their ability to induce host antitumor immunity remains obscure. In a previous study, we have observed that melanoma-infiltrating dendritic cells have the capacity to process antigens and migrate to lymph nodes to prime T lymphocytes. Here, we observed that melanoma-infiltrating dendritic cells extracted from melanoma without any additional manipulations were able to protect naive mice against a lethal challenge with the tumor. Remarkably, this was achieved with reinjection of 10(5) melanoma-infiltrating dendritic cells, a number that did not exceed the total number of melanoma-infiltrating dendritic cells recovered from one single tumor. Three observations indicate that protection was due to the natural loading of melanoma-infiltrating dendritic cells with tumor antigens. First, the protective effect was not observed with equivalent numbers of bone marrow-derived dendritic cells. Second, the protection induced was specific for the tumor from which the tumor-infiltrating dendritic cells were isolated. Third, depletion experiments indicate that both CD4+ and CD8+ T lymphocytes were required during the effector phase of the antitumor response. Hence, designing strategies aimed at rendering melanoma-infiltrating dendritic cells visible to host T cells may boost spontaneous antitumor immunity. PMID 17505262

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.
Mai 2007 | von Euw, Erika M; Barrio, María M; Furman, David; Bianchini, Michele; Levy, Estrella M; Yee, Cassian; Li, Yongqing; Wainstok, Rosa; Mordoh, José
In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells). PMID 17448240

Results of a pilot trial of immunotherapy with dendritic cells pulsed with autologous tumor lysates in patients with advanced cancer.
Apr. 2007 | Mayordomo, Jose Ignacio; Andres, Raquel; Isla, Maria Dolores; Murillo, Laura; Cajal, Rosana; Yubero, Alfonso; Blasco, Carmen; Lasierra, Pilar; Palomera, Luis; Fuertes, Miguel Angel; Güemes, Antonio; Sousa, Ramon; Garcia-Prats, Maria Dolores; Escudero, Pilar; Saenz, Alberto; Godino, Javier; Marco, Ivan; Saez, Berta; Visus, Carmen; Asin, Laura; Valdivia, Gabriel; Larrad, Luis; Tres, Alejandro
The purpose of the study was to test the immunological and clinical effects of infusions of dendritic cells pulsed with autologous tumor lysate in patients with advanced cancer. PMID 17455868

Intratumoral injection of dendritic cells in combination with local hyperthermia induces systemic antitumor effect in patients with advanced melanoma.
Apr. 2007 | Guo, Jun; Zhu, Jun; Sheng, Xinan; Wang, Xiaopei; Qu, Li; Han, Yan; Liu, Yuexiang; Zhang, Hui; Huo, Ling; Zhang, Shuhui; Lin, Baohe; Yang, Zhi
Dendritic cells (DC) are potent antigen-presenting cells that can present tumor antigens chaperoned by heat shock proteins (HSPs), while local hyperthermia (LHT) can increase the expression of HSPs. In this study, we determine if intratumoral injection of immature DC after LHT (LHT+IT-DC) induces systemic antitumor immunity in patients with advanced melanoma, and investigate the potential immunological mechanisms involved in the treatments. Patients were randomly assigned to intratumoral administration of autologous immature DC triweekly, with (LHT+IT-DC, arm A, n = 9) or without (IT-DC, arm B, n = 9) LHT. Our results showed that there were no grade 3/4 toxicities. The time to progress (TTP) of arm A was 5 months, significantly longer than that in arm B (2 months, p < 0.05). However, the overall survival time had no statistical difference (13 months vs. 6 months, p > 0.05) between the 2 groups. Our ELISPOT assay showed a significantly increased melanoma-specific IFN-gamma production in arm A, suggesting that LHT+IT-DC was more effective in the induction of cytotoxic T lymphocytes (CTL) than IT-DC alone. Furthermore, we detected an increased HSPs expression 4 hr after the first LHT, an enhanced Th1/Th2 chemokines production 24 hr after the first LHT+IT-DC treatment, a promoted migration of DC to afferent lymph nodes, and a decreased infiltration of regulatory T cells (CD4(+)CD25(+)) and an increased infiltration of active CTL (CD8(+)CD28(+)) 48 hr after the third DC injection in arm A patients. Therefore, LHT+IT-DC can induce effective specific antitumor immunity and facilitate a Th1-polarized immune response in patients with advanced melanoma. PMID 17294445

Dendritic cell-based immunotherapy of malignant melanoma: success and limitations.
März 2007 | Tuettenberg, Andrea; Schmitt, Edgar; Knop, Jürgen; Jonuleit, Helmut
Dendritic cells (DC) are professional antigen-presenting cells in the immune system which are able to induce primary T-cell responses. Because of their central role in the initiation of immune responses, DC are an important tool for tumor-antigen-specific immunotherapy of cancer. DC vaccination using tumor-antigen-loaded DC has led to tumor regression in individual advanced-stage cancer patients. However, there is a discrepancy between strong and antigen-specific T cell responses in vaccinated cancer patients detectable ex vivo and only weak clinical responses. In most cases the immune system of advanced stage IV cancer patients allows only a temporary anti-tumor response and increasing evidence exists that active suppressive mechanisms of the immune system as well as of the tumor itself ultimately prevent "autoaggressive" immune reactions against the tumor. Active counter-regulation of effector T cells by tumor-antigen-specific regulatory T-cell (Treg) populations play a central role in limiting the efficacy of the vaccines. Nevertheless, recent studies have shown that DC,additionally activated byToll-Like-receptor ligands (TLRL) can neutralize these suppressive effects of Treg and facilitate the induction of long-lasting effector T cell responses even in the presence of activated Treg. These studies open a new way for "conditioning" of DC by TLRL and might significantly enhance the efficiency of DC-based melanoma vaccines in the future. PMID 17338793

Larger numbers of immature dendritic cells augment an anti-tumor effect against established murine melanoma cells.
Feb. 2007 | Lee, Tae-Hyung; Cho, Young-Hun; Lee, Min-Geol
The dendritic cell (DC) is a potentially promising tool for cancer immunotherapy. To date, however, DC-based immunotherapy has not yielded data with which firm conclusions can be drawn. In the present study, we tested the dose-dependant enhancement of the anti-tumor effect induced by DCs. When large numbers of DCs were used, tumor growth was suppressed up to 41% when compared to control mice. Survival of the animals was prolonged to 54 days compared to the 33-day survival the control mice. The delayed-type hypersensitivity (DTH) response induced was 26-fold higher than in the controls. Larger numbers of DCs also led to higher expansion of IFN-gamma-secreting-CD8(+) T cells. Furthermore, the secretion of IL-12p70 and IFN-gamma by spleen cells were enhanced in proportion to the dosage. However, the level of IL-4 secreted from spleen cells was negligible compared to the level of IFN-gamma that was released. These results indicate that DCs induce Th1-dominant immune response and that more DCs could lead to better immunological results, a finding which was consistent with our therapeutic results. PMID 17180546

Hyperthermia and immunity. A brief overview.
Jan. 2007 | Baronzio, Gianfranco; Gramaglia, Alberto; Fiorentini, Gianmaria
After many years, hyperthermia (HT) is experiencing a new resurgence as seen by the positive results of many randomized trials all over the world. Tumour immunity similarly is suggested as the fourth modality of therapy for metastatic tumours from renal carcinoma and melanoma. An overwhelming amount of data from animal models and human patients indicate that whole body and locoregional hyperthermia exerts many biological and therapeutic effects on immune competent cells and cytokines. Among these effects, hyperthermia has recently been demonstrated to enhance the antigen presentation and consequently the activity of dendritic cells. This improvement is obtained through several mechanisms: a) increased lymphocyte recruitment and trafficking into the tumour area; b) increased immunogenicity of heat treated tumour cells; and c) increased production of the heat-shock proteins and costimulatory molecules. The effects and mechanisms of HT on immunity, lymphocyte recruitment and dendritic cell stimulation by heat shock proteins are reviewed here. Moreover the use of HT as an innate immunity booster in association with biological response modifiers is suggested. PMID 17203747

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity.
Sep. 2006 | Palucka, Anna K; Ueno, Hideki; Connolly, John; Kerneis-Norvell, Fabienne; Blanck, Jean-Philippe; Johnston, Dennis A; Fay, Joseph; Banchereau, Jacques
Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma. PMID 16971810

Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous tumor-mRNA.
Sep. 2006 | Kyte, J A; Mu, L; Aamdal, S; Kvalheim, G; Dueland, S; Hauser, M; Gullestad, H P; Ryder, T; Lislerud, K; Hammerstad, H; Gaudernack, G
We have developed an individualized melanoma vaccine based on transfection of autologous dendritic cells (DCs) with autologous tumor-mRNA. Dendritic cells loaded with complete tumor-mRNA may generate an immune response against a broad repertoire of antigens, including unique patient-specific antigens. The purpose of the present phase I/II trial was to evaluate the feasibility and safety of the vaccine, and the ability of the DCs to elicit T-cell responses in melanoma patients. Further, we compared intradermal (i.d.) and intranodal (i.n.) vaccine administration. Twenty-two patients with advanced malignant melanoma were included, each receiving four weekly vaccines. Monocyte-derived DCs were transfected with tumor-mRNA by electroporation, matured and cryopreserved. We obtained successful vaccine production for all patients elected. No serious adverse effects were observed. A vaccine-specific immune response was demonstrated in 9/19 patients evaluable by T-cell assays (T-cell proliferation/interferon-gamma ELISPOT) and in 8/18 patients evaluable by delayed-type hypersensitivity (DTH) reaction. The response was demonstrated in 7/10 patients vaccinated intradermally and in 3/12 patients vaccinated intranodally. We conclude that immuno-gene-therapy with the described DC-vaccine is feasible and safe, and that the vaccine can elicit in vivo T-cell responses against antigens encoded by the transfected tumor-mRNA. The response rates do not suggest an advantage in applying i.n. vaccination. PMID 16710345

Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34(+) progenitor-derived dendritic cells.
Juni 2006 | Fay, Joseph W; Palucka, A Karolina; Paczesny, Sophie; Dhodapkar, Madhav; Johnston, Dennis A; Burkeholder, Susan; Ueno, Hideki; Banchereau, Jacques
Between March 1999 and May 2000, 18 HLA-A*0201(+) patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34(+) hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-gamma production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2-83), and the median event-free survival was 7 months (range: 2-83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs. PMID 16331519

Induction of anti-tumor immunity by vaccination with dendritic cells pulsed with anti-CD44 IgG opsonized tumor cells.
Juni 2006 | Pilon-Thomas, Shari; Verhaegen, Monique; Kuhn, Lisa; Riker, Adam; Mulé, James J
Due to the pivotal role that dendritic cells (DC) play in eliciting and maintaining functional anti-tumor T cell responses, these APC have been exploited against tumors. DC express several receptors for the Fc portion of IgG (Fcgamma receptors) that mediate the internalization of antigen-IgG complexes and promote efficient MHC class I and II restricted antigen presentation. In this study, the efficacy of vaccination with DC pulsed with apoptotic B16 melanoma cells opsonized with an anti-CD44 IgG (B16-CD44) was explored. Immature bone marrow derived DC grown in vitro with IL-4 and GM-CSF were pulsed with B16-CD44. After 48 h of pulsing, maturation of DC was demonstrated by production of IL-12 and upregulation of CD80 and CD40 expression. To test the efficacy of vaccination with DC+B16-CD44, mice were vaccinated subcutaneously Lymphocytes from mice vaccinated with DC+B16-CD44 produced IFN-gamma in response to B16 melanoma lysates as well as an MHC class I restricted B16 melanoma-associated peptide, indicating B16 specific CD8 T cell activation. Upon challenge with viable B16 cells, all mice vaccinated with DC alone developed tumor compared to 40% of mice vaccinated with DC+B16-CD44; 60% of the latter mice remained tumor free for at least 8 months. In addition, established lung tumors and distant metastases were significantly reduced in mice treated with DC+B16-CD44. Lastly, delayed growth of established subcutaneous tumors was induced by combination therapy with anti-CD44 antibodies followed by DC injection. This study demonstrates the efficacy of targeting tumor antigens to DC via Fcgamma receptors. PMID 16315029

Immunity to melanoma antigens: from self-tolerance to immunotherapy.
Mai 2006 | Slingluff, Craig L; Chianese-Bullock, Kimberly A; Bullock, Timothy N J; Grosh, William W; Mullins, David W; Nichols, Lisa; Olson, Walter; Petroni, Gina; Smolkin, Mark; Engelhard, Victor H
The development of effective immune therapy for cancer is a central goal of immunologists in the 21st century. Our laboratories have been deeply involved in characterization of the immune response to melanoma and translation of laboratory discoveries into clinical trials. We have identified a cohort of peptide antigens presented by Major Histocompatibility Complex (MHC) molecules on melanoma cells and widely recognized by T cells from melanoma patients. These have been incorporated into peptide-based vaccines that induce CD8(+) and CD4(+) T-cell responses in 80-100% of patients. Major objective clinical tumor regressions have been observed in some patients, and overall survival in vaccinated patients exceeds expected stage-specific survival. New clinical trials will determine the value of combination of melanoma helper peptides (MHP) into multipeptide vaccines targeting CD8 cells. New trials will also evaluate new approaches to modulating the host-tumor relationship and will develop new combination therapies. Parallel investigations in murine models are elucidating the immunobiology of the melanoma-host relationship and addressing issues that are not feasible to approach in human trials. Based on the fact that the largest cohort of melanoma antigens are derived from normal proteins concerned with pigment production, we have evaluated the mechanisms of self-tolerance to tyrosinase (Tyr) and have determined how T cells in an environment of self-tolerance are impacted by immunization. Using peptide-pulsed dendritic cells as immunogens, we have also used the mouse model to establish strategies for quantitative and qualitative enhancement of antitumor immunity. This information creates opportunities for a new generation of therapeutic interventions using cancer vaccines. PMID 16730266

Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy.
Apr. 2006 | Lopes, Luciene; Fletcher, Kate; Ikeda, Yasuhiro; Collins, Mary
Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A(27-35) epitope. The expanded cells are functional in that they release IFN-gamma upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A(27-35) peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial. PMID 16311731

Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG.
März 2006 | Schadendorf, D; Ugurel, S; Schuler-Thurner, B; Nestle, F O; Enk, A; Bröcker, E-B; Grabbe, S; Rittgen, W; Edler, L; Sucker, A; Zimpfer-Rechner, C; Berger, T; Kamarashev, J; Burg, G; Jonuleit, H; Tüttenberg, A; Becker, J C; Keikavoussi, P; Kämpgen, E; Schuler, G; ,
This randomized phase III trial was designed to demonstrate the superiority of autologous peptide-loaded dendritic cell (DC) vaccination over standard dacarbazine (DTIC) chemotherapy in stage IV melanoma patients. PMID 16418308

Induction of strong and persistent MelanA/MART-1-specific immune responses by adjuvant dendritic cell-based vaccination of stage II melanoma patients.
März 2006 | Tuettenberg, Andrea; Becker, Christian; Huter, Eva; Knop, Jürgen; Enk, Alexander H; Jonuleit, Helmut
A significant percentage of stage II melanoma patients (tumor thickness>1 mm) remain at risk of tumor recurrence after primary tumor excision. In this study, we used tumor antigen-pulsed dendritic cells as an adjuvant for immunization of these "high-risk" melanoma patients after resection of the primary tumor. A total of 13 patients were included and vaccinated 6 times every 14 days with autologous dendritic cells pulsed with a MelanA/MART-1 peptide in combination with a recall antigen. Antigen-specific immune responses were monitored before, during and up to 1 year after the last vaccination. The majority of patients exhibited increased recall antigen-specific CD4+ T cell responses upon vaccination. MelanA/MART-1-specific CD8+ T cells were expanded in 9/13 patients resulting in increased frequencies of memory cells in these patients. CD8+ T cells acquired the capacity to secrete IFN-gamma, to proliferate in culture in response to the tumor antigen used for vaccination and postvaccine samples contained MelanA/MART-1-specific T cells that recognized also the natural MelanA/MART-1-antigen expressed by tumor cells. Moreover, vaccination induced a long-lived tumor antigen-specific DTH-reactivity in the majority of the patients, detectable even 12 months after the last immunization. These data demonstrate for the first time that vaccination with tumor antigen-pulsed dendritic cells in a clinically adjuvant setting induces strong and persistent antigen-specific T-cell responses in tumor-free stage II melanoma patients, suggesting that tumor protective T cell immunity can be achieved. PMID 16353138

Dendritoma vaccination combined with low dose interleukin-2 in metastatic melanoma patients induced immunological and clinical responses.
Feb. 2006 | Wei, Yanzhang; Sticca, Robert P; Holmes, Lillia M; Burgin, Kelly E; Li, Jinhua; Williamson, Jane; Evans, Lyndon; Smith, Samuel J; Stephenson, Joseph J; Wagner, Thomas E
A pilot clinical trial using dendritomas, purified hybrids from the fusion of dendritic/tumor cells combined with a low dose of IL-2, in metastatic melanoma patients was conducted in order to determine its safety and potential immunological and clinical responses. Ten metastatic melanoma patients were enrolled into this study. Dendritoma vaccines were created by fusing dendritic cells stained with green fluorescent dye with irradiated autologous tumor cells stained with red fluorescent dye and purifying the hybrids using immediate fluorescent-activated cell sorting. Initial vaccine was given subcutaneously and followed by IL-2 in serially elevated doses from 3-9 million units/m2 for 5 days. Repeated vaccinations were administered without IL-2, at 3-month intervals for a maximum of 5 times. Immune reactions were measured by the increase of interferon-gamma (IFN-gamma) expressing T cells. Vaccine doses ranged from 250,000 to 1,000,000 dendritomas. There was no grade 2 or higher toxicity directly attributable to the vaccine. All patients experienced toxicity due to IL-2 administration (9-grade 2, 3-grade 3, 1-grade 4). Eight of nine evaluable patients demonstrated immunologic reactions by increased IFN-gamma expressing T cells. One patient developed partial response at 12 weeks after the first vaccine. Nine months later, this patient achieved a complete response. In addition, two patients had stable disease for 9 and 4 months, respectively; one patient had a mixed response. Our findings demonstrated that dendritoma vaccines with a low dose of IL-2 can be safely administered to patients with metastatic melanoma and induce immunological and clinical responses. PMID 16465362

Vaccine efficacy of fusogenic liposomes containing tumor cell-lysate against murine B16BL6 melanoma.
Jan. 2006 | Yoshikawa, Tomoaki; Okada, Naoki; Tsujino, Masaki; Gao, Jian-Qing; Hayashi, Akira; Tsutsumi, Yasuo; Mayumi, Tadanori; Yamamoto, Akira; Nakagawa, Shinsaku
Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes. PMID 16394519

Combined immunocell therapy using activated lymphocytes and monocyte-derived dendritic cells for malignant melanoma.
Nov. 2005 | Goto, Shigenori; Kaneko, Toru; Miyamoto, Yoju; Eriguchi, Masazumi; Kato, Akira; Akeyama, Teruhisa; Fujimoto, Katsuhiro; Tomonaga, Masamichi; Egawa, Koji
The beneficial effects of immunocell therapy, using either activated lymphocytes (ALs) or dendritic cells (DCs), in the treatment of melanoma has been demonstrated. DCs are professional antigen-presenting cells that induce cytotoxic T lymphocytes against tumor cells. DC therapy may be promising when combined with ALs. PMID 16302734

Dendritic cell immunizations alone or combined with low doses of interleukin-2 induce specific immune responses in melanoma patients.
Nov. 2005 | Escobar, A; López, M; Serrano, A; Ramirez, M; Pérez, C; Aguirre, A; González, R; Alfaro, J; Larrondo, M; Fodor, M; Ferrada, C; Salazar-Onfray, F
Dendritic cell (DC)-based therapy has proved to be effective in patients with a variety of malignancies. However, an optimal immunization protocol using DCs and the best means for delivering antigens has not yet been described. In this study, 20 patients with malignant melanoma in stages III or IV were vaccinated with autologous DCs pulsed with a melanoma cell lysate, alone (n = 13) or in combination with low doses of subcutaneous (s.c.) interleukin (IL)-2 injections (n = 7), to assess toxicity, immunological and clinical responses. Monocyte-derived DCs were morphological, phenotypic and functionally characterized in vitro. Peripheral blood mononuclear cells (PBMC), harvested from patients either prior to and after the treatment, were analysed using enzyme-linked immunosorbent spot (ELISPOT). After vaccination, 50% of the patients tested (seven of 13) from the first group and (three of seven) from the second, showed an increase in interferon (IFN)-gamma production in response to allogeneic melanoma cell lines but not to controls. Four of five tested human leucocyte antigen (HLA)-A2(+) patients with anti-melanoma activity also showed specific T cell responses against peptides derived from melanoma-associated antigens. Delayed type IV hypersensitivity reaction (DTH) against melanoma cell lysate was observed in six of 13 patients from the group treated with DC vaccines only and four of seven from the group treated with the combination of DCs and IL-2. Significant correlations were found between DTH-positive responses against tumour lysate and both disease stability and post-vaccination survival on the stage IV patients. There were no toxicities associated with the vaccines or evidence of autoimmunity including vitiligo. Furthermore, no significant enhancement was observed as a result of combining DC vaccination with IL-2. Our data suggest that autologous DCs pulsed with tumour lysate may provide a standardized and widely applicable source of melanoma specific antigens for clinical use. It is safe and causes no significant side effects and has been demonstrated to be partially efficient at triggering effective anti-melanoma immunity. PMID 16297169

Efficacy of superficial and deep regional hyperthermia combined with systemic chemotherapy and radiotherapy in metastatic melanoma.
Nov. 2005 | Richtig, E; Hoff, M; Rehak, P; Kapp, K; Hofmann-Wellenhof, R; Zalaudek, I; Poschauko, J; Uggowitzer, M; Kohek, P; Smolle, J
Response rates of cutaneous-subcutaneous or lymph node metastases of melanoma to systemic chemotherapy are rather low. We report our clinical experience with superficial and deep regional hyperthermia in combination with radiotherapy and/or chemotherapy with carboplatin. PMID 16296154

Intradermal injection of Newcastle disease virus-modified autologous melanoma cell lysate and interleukin-2 for adjuvant treatment of melanoma patients with resectable stage III disease.
Nov. 2005 | Voit, Christiane; Kron, Martina; Schwürzer-Voit, Markus; Sterry, Wolfram
The value of active specific immunotherapy (ASI) for the treatment of solid tumours still has to be assessed. The objective was to test an autologous tumour cell vaccine for adjuvant treatment of stage III melanoma patients. PMID 16285179

Immunization using autologous dendritic cells pulsed with the melanoma-associated antigen gp100-derived G280-9V peptide elicits CD8+ immunity.
Nov. 2005 | Linette, Gerald P; Zhang, Dongsheng; Hodi, F Stephen; Jonasch, Eric P; Longerich, Simonne; Stowell, Christopher P; Webb, Iain J; Daley, Heather; Soiffer, Robert J; Cheung, Amy M; Eapen, Sara G; Fee, Sharon V; Rubin, Krista M; Sober, Arthur J; Haluska, Frank G
To determine the toxicity, maximal tolerated dose, and clinical and immunologic response to autologous dendritic cells pulsed with melanoma-associated antigen gp100-derived G280-9V peptide. PMID 16278389

Melanoma immunotherapy: past, present, and future.
Okt. 2005 | Saleh, Farid; Renno, Waleed; Klepacek, Ivo; Ibrahim, Ghada; Asfar, Sami; Dashti, Hussein; Romero, Pedro; Dashti, Ali; Behbehani, Abdullah
The incidence of cancer and its related morbidity and mortality remain on the increase in both developing and developed countries. Cancer remains a huge burden on the health and social welfare sectors worldwide and its prevention and cure remain two golden goals that science strives to achieve. Among the treatment options for cancer that have emerged in the past 100 years, cancer vaccine immunotherapy seems to present a promising and relatively safer approach as compared to chemotherapy and radiotherapy. The identification of different tumour antigens in the last fifteen years using a variety of techniques, together with the molecular cloning of cytotoxic T lymphocytes (CTLs)- and tumour infiltrating lymphocytes (TILs)-defined tumour antigens allowed more refining of the cancer vaccines that are currently used in different clinical trials. In a proportion of treated patients, some of these vaccines have resulted in partial or complete tumour regression, while they have increased the disease-free survival rate in others. These outcomes are more evident now in patients suffering from melanoma. This review provides an update on melanoma vaccine immunotherapy. Different cancer antigens are reviewed with a detailed description of the melanoma antigens discovered so far. The review also summarises clinical trials and individual clinical cases in which some of the old and current methods to vaccinate against or treat melanoma were used. These include vaccines made of autologous or allogenic melanoma tumour cells, melanoma peptides, recombinant bacterial or viral vectors, or dendritic cells. PMID 16248801

Comparison of the anti-tumor effects of various whole-body hyperthermia protocols: correlation with HSP 70 expression and composition of splenic lymphocytes.
Sep. 2005 | Zhang, Honghai; Wang, Weirong; Zhang, Shuhong; Huang, Weida
Whole-body hyperthermia (WBH) has been used as an adjunct approach to radio-/ chemotherapy for tumor therapy for many years. However, the molecular mechanism underlying the enhancement of tumor control is not clearly understood. It has been hypothesized that WBH might activate immune system by inducing the expression of heat shock proteins (HSPs), which are thought to facilitate the presentation of tumor-specific antigens. In the present work, we examined the effects of various thermal doses of WBH on tumor growth delay and HSP70 levels in tumors on C57BL/6 mice, as well as on splenic lymphocyte subpopulations. The maximal WBH effect (about 40% decrease in tumor weight) was achieved by a 2-hour WBH treatment everyday at 40.0 degrees C. By using this treatment schedule, the populations of CD3+/CD4+ T cells and CD3+/CD8+ T cells increased by 4 and 3 times, respectively, at the end of WBH treatment period. When the length of day-by-day WBH treatment was longer than 2 hours or the frequency of WBH treatment was lower than once a day, the effect of tumor growth delay and the population of CD3+ T lymphocyte in spleen increase were discounted. On the other hand, the HSP70 levels in tumor nodules rose continuously as the WBH treating time increased, but the populations of NK cells in spleen did not change significantly. The results suggest that an increased CD3+ T lymphocyte population is closely related to the anti-tumor effect of WBH, which might be a useful marker for effectiveness of hyperthermia. However, neither the levels of HSP70 nor the NK cell populations in spleen appear to correlate to tumor control. PMID 16136780

Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendritic cells derived from CD34+ progenitors and activated with type I interferon.
Aug. 2005 | Banchereau, Jacques; Ueno, Hideki; Dhodapkar, Madhav; Connolly, John; Finholt, Jennifer P; Klechevsky, Eynav; Blanck, Jean-Philippe; Johnston, Dennis A; Palucka, A Karolina; Fay, Joseph
Twenty-two HLA A*0201 patients with stage IV melanoma were enrolled in a phase 1 safety and feasibility trial using a composite dendritic cell (DC) vaccine generated by culturing CD34 hematopoietic progenitors and activated with IFN-alpha. The DC vaccine was loaded with peptides derived from four melanoma tissue differentiation antigens (MART-1, tyrosinase, MAGE-3, and gp100) and influenza matrix peptide (Flu-MP). Twenty patients were evaluable, 14 of whom received vaccination with peptide-pulsed DCs without keyhole limpet hemocyanin (KLH) and 6 of whom received vaccination with KLH-loaded DCs. Patients were vaccinated until disease progression or until they had received eight vaccinations. None of the analyzed patients showed the expansion of melanoma-peptide-specific circulating effector memory T cells that secrete IFN-gamma in direct ELISPOT. Melanoma-peptide-specific recall memory CD8 T cells able to secrete IFN-gamma and to proliferate could be detected in six of the seven analyzed patients. There were no objective clinical responses. The estimated median overall survival was 12 months (range 2-38), and the median event-free survival was 4 months (range 1-12). There was no statistically significant survival advantage in patients who received KLH-loaded vaccines. As of March 2005, four patients remained alive, 26+, 28+, 28+, and 36+ months. Three of them had received KLH-loaded vaccines and all of them had had additional therapy. Overall, these results suggest that IFN-alpha-activated CD34-DCs are safe but elicit only limited immune responses, underscoring the need to test different DC maturation factors. PMID 16113607

Randomized trial of hyperthermia and radiation for superficial tumors.
Apr. 2005 | Jones, Ellen L; Oleson, James R; Prosnitz, Leonard R; Samulski, Thaddeus V; Vujaskovic, Zeljko; Yu, Daohai; Sanders, Linda L; Dewhirst, Mark W
Randomized clinical trials have demonstrated hyperthermia (HT) enhances radiation response. These trials, however, generally lacked rigorous thermal dose prescription and administration. We report the final results of a prospective randomized trial of superficial tumors (

Tumour-dendritic hybrid cell vaccination for the treatment of patients with malignant melanoma: immunological effects and clinical results.
März 2005 | Trefzer, Uwe; Herberth, Gunda; Wohlan, Karolina; Milling, Annett; Thiemann, Max; Sharav, Tumenjargal; Sparbier, Katrin; Sterry, Wolfram; Walden, Peter
Hybrid cell vaccines of autologous tumour cells fused with allogenic dendritic cells (DC) combine the tumour's antigenicity with the immune-stimulatory capacity of mature dendritic cells and allogenic MHC class II molecules to activate T cell help and induce tumour-specific cytotoxic T cells. This concept was tested in a clinical trial with melanoma stage III and IV patients. Seventeen patients were evaluated: one experienced complete, one partial response and six stable disease with long survival times. Eleven of fourteen patients, clinical responders and non-responders alike, mounted high-frequency T cell responses to various tumour-associated antigens. Failing clinical responses correlated with loss of antigenicity. PMID 15755630

Phase I/II trial of melanoma patient-specific vaccine of proliferating autologous tumor cells, dendritic cells, and GM-CSF: planned interim analysis.
Jan. 2005 | Dillman, Robert; Selvan, Senthamil; Schiltz, Patric; Peterson, Cheryl; Allen, Kanoe; Depriest, Carol; McClay, Edward; Barth, Neil; Sheehy, Patric; de Leon, Cristina; Beutel, Linda
The aim of this study was to investigate the feasibility, safety, and clinical efficacy of patient-specific dendritic cell vaccines in patients with metastatic melanoma. A planned interim analysis was conducted on the first 20 patients. PMID 15650459

[Antitumor research on mouse melanoma with combined application of Newcastle disease virus and its HN gene].
Aug. 2004 | Mi, Zhi-Qiang; Jin, Ning-Yi; Sun, Ying-Chun; Li, Xiao; Lian, Hai; Li, Jie; Guan, Guo-Fang
Although Newcastle disease virus (NDV) shows antitumor effect on many tumors, its mechanism is unclear. Hemagglutinin-neuraminidase (HN) gene was found to play an important role in NDV antitumor effect and HN protein located on tumor cell surface. This research was to evaluate the possibility of HN protein as a foreign antigen of tumor cell and the antitumor effect of the combined application of HN gene and NDV. PMID 15301713

Vaccination with hybrids of tumor and dendritic cells induces tumor-specific T-cell and clinical responses in melanoma stage III and IV patients.
Mai 2004 | Trefzer, Uwe; Herberth, Gunda; Wohlan, Karolina; Milling, Annett; Thiemann, Max; Sherev, Tumenjargal; Sparbier, Katrin; Sterry, Wolfram; Walden, Peter
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. PMID 15146563

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.
Sep. 2003 | Nagayama, Hitomi; Sato, Katsuaki; Morishita, Mariko; Uchimaru, Kaoru; Oyaizu, Naoki; Inazawa, Takeshi; Yamasaki, Tomoko; Enomoto, Makoto; Nakaoka, Takashi; Nakamura, Tetsuya; Maekawa, Taira; Yamamoto, Akifumi; Shimada, Shinji; Saida, Toshiaki; Kawakami, Yutaka; Asano, Shigetaka; Tani, Kenzaburo; Takahashi, Tsuneo A; Yamashita, Naohide
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. PMID 14512794

A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire.
Okt. 1999 | Batliwalla, F M; Bateman, B A; Serrano, D; Murray, D; Macphail, S; Maino, V C; Ansel, J C; Gregersen, P K; Armstrong, C A
The development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future. PMID 9990864

Hyperthermia as an adjuvant to radiation therapy of recurrent or metastatic malignant melanoma. A multicentre randomized trial by the European Society for Hyperthermic Oncology.
Aug. 1996 | Overgaard, J; Gonzalez Gonzalez, D; Hulshof, M C; Arcangeli, G; Dahl, O; Mella, O; Bentzen, S M
The ESHO protocol 3-85 is a multicentre randomized trial investigating the value of hyperthermia as an adjuvant to radiotherapy in treatment of malignant melanoma. A total of 134 metastatic of recurrent malignant melanoma lesions in 70 patients were randomized to receive radiotherapy alone (3 fractions in 8 days) or each fraction followed by hyperthermia (aimed for 43 degrees C for 60 min). Radiation was given with high voltage photons or electrons. Tumours were stratified according to institution and size (above or below 4 cm) and randomly assigned to a total radiation dose of either 24 or 27 Gy to be given with or without hyperthermia. The endpoint was persistent complete response in the treated area. A number of 128 tumours in 68 patients were evaluable, with an observation time between 3 and 72 months. Sixty-five tumours were randomized to radiation alone and 63 to radiation + heat. Sixty received 24 Gy and 68 tumours received 27 Gy, respectively. Size was < or = 4 cm in 81 and > 4 cm in 47 tumours. Overall the 2-year actuarial local tumour control was 37%. Univariate analysis showed prognostic influence of hyperthermia (rad alone 28% versus rad + heat 46%, p = 0.008) and radiation dose (24 Gy 25% versus 27 Gy 56%, p = 0.02), but not of tumour size (small 42% versus large 29%, p = 0.21). A Cox multivariate regression analysis showed the most important prognostic parameters to be: hyperthermia (odds ratio: 1.73 (1.07-2.78), p = 0.02), tumour size (odds ratio: 0.91 (0.85-0.99), p = 0.05) and radiation dose (odds ratio: 1.17 (1.01-1.36), p = 0.05). Analysis of the heating quality showed a significant relationship between the extent of heating and local tumour response. Addition of heat did not significantly increase the acute or late radiation reactions. The overall 5-year survival rate of the patients was 19%, but 38% in patients if all known disease was controlled, compared to 8% in the patients with persistent active disease. PMID 8676005