Nicole Scharenborg - Academia.edu (original) (raw)

Papers by Nicole Scharenborg

Research paper thumbnail of Maturation State Lymph Nodes in Melanoma Patients Is Determined by Their Effective Migration of Antigen-pulsed Dendritic Cells to Updated

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an ... more Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naı̈ve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed byin vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in vivo.

Research paper thumbnail of Intranodal vaccination with mRNA-optimised dendritic cells in metastatic melanoma patients

OncoImmunology, 2015

y These authors equally contributed to this work.

Research paper thumbnail of Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Nuclear Medicine and Biology, 2006

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune ... more Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.

Research paper thumbnail of 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

Cancer Immunology, Immunotherapy, 2011

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients. Several strateg... more 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 9 10 6 DC) and intradermally (on average 11 9 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. Antigenspecific 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.

Research paper thumbnail of In situ Expression of Tumor Antigens by Messenger RNA-Electroporated Dendritic Cells in Lymph Nodes of Melanoma Patients

Cancer Research, 2009

Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for ca... more Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8(+) T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

Research paper thumbnail of Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skin tests

Annals of Oncology, 2006

Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune sys... more Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. As such they are currently used in clinical vaccination protocols in cancer patients.

Research paper thumbnail of Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skin tests

Annals of Oncology, 2006

Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune sys... more Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. As such they are currently used in clinical vaccination protocols in cancer patients.

Research paper thumbnail of Adjuvant dendritic cell vaccination induces tumor-specific immune responses in the majority of stage III melanoma patients

OncoImmunology, 2016

To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specif... more To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients. Retrospective analysis of stage III melanoma patients, vaccinated with autologous monocyte-derived DC loaded with tumor-associated antigens (TAA) gp100 and tyrosinase after radical lymph node dissection. Skin-test infiltrating lymphocytes (SKILs) obtained from delayed-type hypersensitivity skin-test biopsies were analyzed for the presence of TAA-specific CD8(+) T cells by tetrameric MHC-peptide complexes and by functional TAA-specific T cell assays, defined by peptide-recognition (T2 cells) and/or tumor-recognition (BLM and/or MEL624) with specific production of Th1 cytokines and no Th2 cytokines. Ninety-seven patients were analyzed: 21 with stage IIIA, 34 with stage IIIB, and 42 had stage IIIC disease. Tetramer-positive CD8(+) T cells were present in 68 patients (70%), and 24 of them showed a response against all 3 epitopes tested (gp100:154-162, gp100:280-288, and tyrosinase:369-377) at any point during vaccinations. A functional T cell response was found in 62 patients (64%). Rates of peptide-recognition of gp100:154-162, gp100:280-288, and tyrosinase:369-377 were 40%, 29%, and 45%, respectively. Median recurrence-free survival and distant metastasis-free survival of the whole study population were 23.0 mo and 36.8 mo, respectively. DC vaccination induces a functional TAA-specific T cell response in the majority of stage III melanoma patients, indicating it is more effective in stage III than in stage IV melanoma patients. Furthermore, performing multiple cycles of vaccinations enhances the chance of a broader immune response.

Research paper thumbnail of Adherence and Proliferation of Endothelial Cells on Surface-Immobilized Albumin-Heparin Conjugate

Tissue Engineering, Feb 1, 1998

Research paper thumbnail of Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

Journal of Biomedical Materials Research, Mar 5, 1999

Research paper thumbnail of Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity

Cancer immunology, immunotherapy : CII, Jan 10, 2016

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly w... more 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 coi...

Research paper thumbnail of Effective clinical responses in metastatic melanoma patients after vaccination with primary myeloid dendritic cells

Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 28, 2015

PURPOSE Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vit... more PURPOSE 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. EXPERIMENTAL DESIGN Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16h) ex vivo culture and loaded with tumor associated antigens of tyrosinase and gp100. RESULTS Our results show that therapeutic vaccination against melanoma with small amounts (3-10x106) of myeloid DCs is feasible and without substantial toxicity. Four out of fourteen patients showed long-term progression-free survival (12-35 months), which directly correlated with the development of multifunctional CD8+ T cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity...

Research paper thumbnail of Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion

J Biomed Mater Res, 1999

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfac... more Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.

Research paper thumbnail of Maturation of Dendritic Cells Is a Prerequisite for Inducing Immune Responses in Advanced Melanoma Patients

Clinical Cancer Research

Experimental Design: Advanced HLA-A2.1 ؉ melanoma patients were vaccinated with peptide-and keyho... more Experimental Design: Advanced HLA-A2.1 ؉ melanoma patients were vaccinated with peptide-and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor ␣/prostaglandin E 2 (10 patients).

Research paper thumbnail of Immunogenicity of dendritic cells pulsed with CEA peptide or transfected with CEA mRNA for vaccination of colorectal cancer patients

Anticancer research, 2010

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have... more Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All D...

Research paper thumbnail of Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients

Clinical cancer research : an official journal of the American Association for Cancer Research, 2003

We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanom... more We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. I...

Research paper thumbnail of Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state

Cancer research, 2003

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an ... more Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naïve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed by in vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in vivo.

Research paper thumbnail of A pilot study on the immunogenicity of dendritic cell vaccination during adjuvant oxaliplatin/capecitabine chemotherapy in colon cancer patients

BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses... more BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system. METHODS: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccineinduced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)peptide pulsed DCs. RESULTS: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished. CONCLUSION: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatinbased chemotherapy.

Research paper thumbnail of Adherence and Proliferation of Endothelial Cells on Surface-Immobilized Albumin-Heparin Conjugate

Research paper thumbnail of Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Nuclear Medicine and Biology, 2006

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune ... more Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.

Research paper thumbnail of Maturation State Lymph Nodes in Melanoma Patients Is Determined by Their Effective Migration of Antigen-pulsed Dendritic Cells to Updated

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an ... more Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naı̈ve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed byin vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in vivo.

Research paper thumbnail of Intranodal vaccination with mRNA-optimised dendritic cells in metastatic melanoma patients

OncoImmunology, 2015

y These authors equally contributed to this work.

Research paper thumbnail of Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Nuclear Medicine and Biology, 2006

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune ... more Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.

Research paper thumbnail of 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

Cancer Immunology, Immunotherapy, 2011

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients. Several strateg... more 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 9 10 6 DC) and intradermally (on average 11 9 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. Antigenspecific 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.

Research paper thumbnail of In situ Expression of Tumor Antigens by Messenger RNA-Electroporated Dendritic Cells in Lymph Nodes of Melanoma Patients

Cancer Research, 2009

Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for ca... more Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8(+) T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

Research paper thumbnail of Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skin tests

Annals of Oncology, 2006

Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune sys... more Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. As such they are currently used in clinical vaccination protocols in cancer patients.

Research paper thumbnail of Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skin tests

Annals of Oncology, 2006

Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune sys... more Background: Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. As such they are currently used in clinical vaccination protocols in cancer patients.

Research paper thumbnail of Adjuvant dendritic cell vaccination induces tumor-specific immune responses in the majority of stage III melanoma patients

OncoImmunology, 2016

To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specif... more To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients. Retrospective analysis of stage III melanoma patients, vaccinated with autologous monocyte-derived DC loaded with tumor-associated antigens (TAA) gp100 and tyrosinase after radical lymph node dissection. Skin-test infiltrating lymphocytes (SKILs) obtained from delayed-type hypersensitivity skin-test biopsies were analyzed for the presence of TAA-specific CD8(+) T cells by tetrameric MHC-peptide complexes and by functional TAA-specific T cell assays, defined by peptide-recognition (T2 cells) and/or tumor-recognition (BLM and/or MEL624) with specific production of Th1 cytokines and no Th2 cytokines. Ninety-seven patients were analyzed: 21 with stage IIIA, 34 with stage IIIB, and 42 had stage IIIC disease. Tetramer-positive CD8(+) T cells were present in 68 patients (70%), and 24 of them showed a response against all 3 epitopes tested (gp100:154-162, gp100:280-288, and tyrosinase:369-377) at any point during vaccinations. A functional T cell response was found in 62 patients (64%). Rates of peptide-recognition of gp100:154-162, gp100:280-288, and tyrosinase:369-377 were 40%, 29%, and 45%, respectively. Median recurrence-free survival and distant metastasis-free survival of the whole study population were 23.0 mo and 36.8 mo, respectively. DC vaccination induces a functional TAA-specific T cell response in the majority of stage III melanoma patients, indicating it is more effective in stage III than in stage IV melanoma patients. Furthermore, performing multiple cycles of vaccinations enhances the chance of a broader immune response.

Research paper thumbnail of Adherence and Proliferation of Endothelial Cells on Surface-Immobilized Albumin-Heparin Conjugate

Tissue Engineering, Feb 1, 1998

Research paper thumbnail of Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

Journal of Biomedical Materials Research, Mar 5, 1999

Research paper thumbnail of Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity

Cancer immunology, immunotherapy : CII, Jan 10, 2016

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly w... more 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 coi...

Research paper thumbnail of Effective clinical responses in metastatic melanoma patients after vaccination with primary myeloid dendritic cells

Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 28, 2015

PURPOSE Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vit... more PURPOSE 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. EXPERIMENTAL DESIGN Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16h) ex vivo culture and loaded with tumor associated antigens of tyrosinase and gp100. RESULTS Our results show that therapeutic vaccination against melanoma with small amounts (3-10x106) of myeloid DCs is feasible and without substantial toxicity. Four out of fourteen patients showed long-term progression-free survival (12-35 months), which directly correlated with the development of multifunctional CD8+ T cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity...

Research paper thumbnail of Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion

J Biomed Mater Res, 1999

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfac... more Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.

Research paper thumbnail of Maturation of Dendritic Cells Is a Prerequisite for Inducing Immune Responses in Advanced Melanoma Patients

Clinical Cancer Research

Experimental Design: Advanced HLA-A2.1 ؉ melanoma patients were vaccinated with peptide-and keyho... more Experimental Design: Advanced HLA-A2.1 ؉ melanoma patients were vaccinated with peptide-and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor ␣/prostaglandin E 2 (10 patients).

Research paper thumbnail of Immunogenicity of dendritic cells pulsed with CEA peptide or transfected with CEA mRNA for vaccination of colorectal cancer patients

Anticancer research, 2010

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have... more Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. We have demonstrated that vaccination of autologous ex vivo cultured DCs results in the induction of tumor-specific immune responses in cancer patients, which correlates with clinical response. Optimization of antigen loading is one of the possibilities for further improving the efficacy of DC vaccination. Theoretically, transfection of DCs with RNA encoding a tumor-specific antigen may induce a broader immune response as compared to the most widely used technique of peptide pulsing. In this clinical study, RNA transfection was compared with peptide pulsing as an antigen loading strategy for DC vaccination. Patients with resectable liver metastases of colorectal cancer were vaccinated intravenously and intradermally 3 times weekly with either carcinoembryogenic antigen (CEA)-derived HLA-A2 binding peptide-loaded or CEA mRNA electroporated DCs prior to surgical resection of the metastases. All D...

Research paper thumbnail of Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients

Clinical cancer research : an official journal of the American Association for Cancer Research, 2003

We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanom... more We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. I...

Research paper thumbnail of Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state

Cancer research, 2003

Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an ... more Dendritic cells are the professional antigen-presenting cells of the immune system. To induce an effective immune response, these cells should not only express high levels of MHC and costimulatory molecules but also migrate into the lymph nodes to interact with naïve T cells. Here, we demonstrate that in vitro-generated mature, but not immature dendritic cells, efficiently migrate into the T-cell areas of lymph nodes of melanoma patients. This difference is confirmed by in vitro studies, in which immature dendritic cells are strongly adherent, whereas mature dendritic cells remain highly motile. Our present findings demonstrate that the ability of dendritic cells to mount a proper immune response correlates with their ability to migrate both in vitro and in vivo.

Research paper thumbnail of A pilot study on the immunogenicity of dendritic cell vaccination during adjuvant oxaliplatin/capecitabine chemotherapy in colon cancer patients

BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses... more BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system. METHODS: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccineinduced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)peptide pulsed DCs. RESULTS: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished. CONCLUSION: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatinbased chemotherapy.

Research paper thumbnail of Adherence and Proliferation of Endothelial Cells on Surface-Immobilized Albumin-Heparin Conjugate

Research paper thumbnail of Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Nuclear Medicine and Biology, 2006

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune ... more Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.