Jolanda de Vries

Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes

TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRαβ when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRαβ constructs enabled T lymphocytes to specifically kill and produce TNF-α when triggered by native gp100pos/HLA-A2pos tumor target cells as well as gp100 peptide-loaded HLA-A2pos tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial

BACKGROUND AIMS RNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients. METHODS CD14(+) cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilms tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients. RESULTS In a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14(+) cells (94.5+/-3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25+/-10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients. CONCLUSIONS Our method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.

A novel 19F agent for detection and quantification of human dendritic cells using magnetic resonance imaging

Monitoring of cell therapeutics in vivo is of major importance to estimate its efficacy. Here, we present a novel intracellular label for 19F magnetic resonance imaging (MRI)‐based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of 19F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The 19F label was tested in primary human monocyte‐derived dendritic cells. These cells took up label effectively, resulting in a labeling of 1.7 ± 0.1 × 1013 19F atoms per cell, with a viability of 80 ± 6%, without the need for electroporation or transfection agents. This results in a minimum detection sensitivity of about 2,000 cells/voxel at 7 T, comparable with gadolinium‐labeled cells. Comparison of the detection sensitivity of cells labeled with 19F, iron oxide and gadolinium over typical tissue background showed that unambiguous detection of the 19F‐labeled cells was simpler than with the contrast agents. The effect of the 19F agent on cell function was minimal in the context of cell‐based vaccines. From these data, we calculate that detection of 30,000 cells in vivo at 3 T with a reasonable signal to noise ratio for 19F images would require less than 30 min with a conventional fast spin echo sequence, given a coil similar to the one used in this study. This is well within acceptable limits for clinical studies, and thus, we conclude that 19F MRI for quantitative cell tracking in a clinical setting has great potential.

Immunotherapy: Cancer vaccine triggers antiviral-type defences

An immunotherapy approach targets nanoparticles to dendritic cells of the immune system, leading to an antitumour immune response with antiviral-like features. Initial clinical tests of this approach show promise. See Letter p.396

Immune infiltrates impact on the prediction of prognosis and response to immunotherapy of melanoma patients

Background Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite specific therapies have been explored for many years, no effective therapeutic options have been developed. Vaccination strategies, including Dendritic Cells (DC) based immunotherapy, are consistently increasing the proportion of cancer patients with anti-vaccine immune responses although the number of patients with increased overall survival is still limited. The efficacy of the immunotherapy is mainly dependent on tumor microenvironment –immune system interactions. Our aim is to evaluate the host immune response to melanoma by quantifying the density and location of T cells in primary tumors of patients treated with DC immunotherapy and correlate them with clinical variables such as overall survival (OS). Materials and methods We collected 60 FFPE primary tumors from melanoma patients treated with DC immunotherapy. Serial sections (4 micron in thickness) were stained with CD3, CD8 and CD45RO antibodies. Haematoxylin was used as a counterstain and Nova Red for the immunohistochemical stain. All the slides were digitalized and an automated quantitative analysis was performed in order to evaluate the density and location of two lymphocyte populations, cytotoxic (CD8) and memory (CD45RO) T cells. Of all samples the clinical outcome of the patient is known. Results The immunohistochemical analysis of primary melanoma using a small set of patients resulted in significant differences between short (OS 24 months). A high degree of T cells infiltration was seen in the tumor area of both patients, responding and non-responding to DC immunotherapy. However, the location but not the density of TILs was significantly different in the two cohorts of patients and showed a strong correlation with clinical response to DC vaccination. Conclusions Immune cells within melanoma tumors may have a prognostic value and clinical significance as a predictor of patient outcome and response to DC immunotherapy in melanoma patients.

Abstract A016: T cell landscape within primary melanoma as a predictive biomarker of survival after cancer vaccination in patients with metastatic disease

Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite different types of immunotherapy became available for the treatment of melanoma, including adoptive T cell transfer, immune checkpoint blockade, and vaccines such as dendritic cell vaccines, when applied to treat metastatic melanoma, only 10 to 40% of the patients have long term benefit. Furthermore, given the toxicity and high costs associated with immunotherapy there is a stringent need to identify biomarkers that may predict its potential efficacy. We have investigated whether the presence and distribution of T cells within the primary tumor of melanoma patients correlates with survival when treated for metastatic disease with dendritic cell based cancer vaccines. Multispectral imaging and quantitative immunohistochemistry have been used to compare the tumor microenvironment of responding patients, that survived longer than 20 months after immunotherapy, and non-responding patients that survived less than 12 months after immunotherapy. The density and location of T cell were initially assessed in primary melanomas of 19 metastatic melanoma patients treated with dendritic cell based immunotherapy (discovery set) and subsequently in an independent cohort of 39 patients (validation cohort). In the discovery cohort we observed a very high correlation between the ratios of peritumoral over intratumoral T cells in the primary tumor and overall survival; lower peri/intratumoral T cell ratios in primary tumors were associated with improved clinical outcome indicating that the intratumoral T cell component is more prominent in responding patients whereas T cells fail to infiltrate the tumor in non-responding patients. ROC curve and multivariate analysis were exploited to evaluate the predictive power of the T cell ratio compared and/or combined to established prognostic markers. The same statistical analyses in the validation cohort confirmed our findings showing that the peri/intratumoral T cell ratio correctly predicted long term survival after DC vaccination in 90% of the cases and was the strongest predictor of survival in the multivariate analysis. This study indicates that the ratio between CD3 positive peri/intra-tumoral T cells is a very strong predictor of clinical outcome and response to dendritic cells immunotherapy in melanoma patients. Citation Format: Angela Vasaturo, Altuna Halilovic, Kalijn Bol, Dagmar Verweij, Patricia Groenen, Han van Krieken, Willeke Blokx, Jolanda de Vries, Johannes Textor, Carl G. Figdor. T cell landscape within primary melanoma as a predictive biomarker of survival after cancer vaccination in patients with metastatic disease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A016.

Abstract ED01-01: Immunity against cancer: How to be enhanced?

Prevention of infectious diseases through immunization is one of the greatest achievements of modern medicine. Nonetheless, considerable challenges remain for improving the efficacy of vaccines currently employed to treat devastating diseases such as cancer. Dendritic cells (DC) are considered professional antigen-presenting cells of the immune system highly effective in initiating primary immune responses. Following infection or inflammation they undergo a complex process of maturation, and migrate to lymph nodes (LN) where they present antigens to T cells. Their decisive role in inducing immunity formed the rationale for DC immunotherapy: DC loaded with tumor antigens are injected into cancer patients to stimulate T cells to eradicate tumours. A major advantage of immunotherapy when compared to radio-, chemo- and targeted therapy is its low toxicity. To date, dendritic cell (DC)-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients. Although during the past 15 years the concept of DC vaccination has been clearly proven and found safe, the number of patients that have long-term benefit is still limited. Therefore the development of bioassays that predict clinical outcome are essential to optimize cellular anticancer immunotherapy. We have developed a robust and simple skin test to evaluate the capacity of tumor-specific T cells to migrate, recognize their targets, and exert effector functions. This bioassay detects skin infiltrating T lymphocytes (SKILs) with an elevated antineoplastic potential and hence identifies patients responding to immunotherapy once started. The identification of a biomarker already available before start of treatment would facilitate decision-making regarding (prophylactic) immunotherapy. We recently identified that the ratio between peri/intratumoral T cells in the primary tumor, is an excellent predictor of survival after DC-based immunotherapy in metastatic melanoma patients. Citation Format: Jolanda De Vries, Winald Gerritsen, Carl G. Figdor. Immunity against cancer: How to be enhanced?. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr ED01-01.

T cell landscape within primary melanoma as a biomarker of survival after cancer vaccination in patients with metastatic disease

Melanoma is a highly malignant melanocyte-derived tumor and its incidence is increasing at outstanding rate. Despite different types of immunotherapy became available for the treatment of melanoma, including adoptive T cell transfer, immune checkpoint blockade, and vaccines such as dendritic cell vaccines, when applied to treat metastatic melanoma, only 10 to 40% of the patients have long term benefit. Furthermore, given the toxicity and high costs associated with immunotherapy there is a stringent need to identify biomarkers that may predict its potential efficacy. We have investigated whether the presence and distribution of T cells within the primary tumor of melanoma patients correlates with survival when treated for metastatic disease with dendritic cell based cancer vaccines. Quantitative multispectral imaging has been used to compare the tumor microenvironment of responding patients that survived longer than 20 months after immunotherapy, and non-responding patients that survived less than 12 months after immunotherapy. T cell infiltrates have been initially assessed in primary melanomas of 19 metastatic melanoma patients treated with dendritic cell based immunotherapy (discovery set) and subsequently in an independent cohort of 39 patients (validation cohort). In the discovery cohort we observed a very high correlation between the ratio of peritumoral over intratumoral T cells in the primary tumor and overall survival. Lower peri/intratumoral T cell ratios in primary tumors were associated with improved clinical outcome. ROC curve and multivariate analysis were exploited to evaluate the predictive power of the T cell ratio and established prognostic markers. Statistical analyses in the validation cohort confirmed our findings showing that the peri/intratumoral T cell ratio correctly predicted long term survival after DC vaccination in 90% of the cases and was the strongest predictor of survival in the multivariate analysis. This study indicates that the ratio between CD3 positive peri/intra-tumoral T cells is a very strong predictor of patient outcome and response to dendritic cells immunotherapy in melanoma patients.

Abstract 2547: Peripheral immune signatures and survival in stage IV melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The presence of peripheral T-cells recognizing the melanoma-associated antigens NY-ESO-1 or Melan-A, as well as low frequencies of circulating CD14+ HLA-DR-/low myeloid-derived suppressor cells (MDSCs), is associated with prolonged survival in stage IV melanoma (n=94). In addition, the two of 12 patients with partial responses or stable disease in pilot studies of ipilimumab treatment both tended to have lower levels of MDSCs at baseline which decreased further on treatment. In contrast, two patients with progressive disease had high baseline MDSC levels, which increased further after treatment. These patients are being followed up to determine whether another mechanism of action of ipilimumab may be via an effect on MDSCs. A correlation was observed between the presence of antigen-reactive T cells and the frequency of MDSCs. Our results indicate that patients lacking antigen-reactive T cells had significantly higher levels of MDSCs. In addition, high frequencies of MDSCs correlated with lower frequencies of NY-ESO-1- and Melan-A-reactive T cells that also produced lower levels of IFN-gamma. NY-ESO-1 was recognized more frequently by CD4+ than by CD8+ T cells, whereas Melan-A more often stimulated CD8+ T cells. Possession of NY-ESO-1-reactive T cells conferred a survival advantage whether the response was mediated by CD4+ or CD8+ T cells or both. However, recognition of Melan-A by CD4+ T cells was associated with poorer clinical outcome even when the patient also possessed CD8+ reactive T cells. This negative effect was mediated by CD4+ T cells with a Th2- or Th17-type but not Th1-type cytokine response. Thus, tumor antigen-specificity and function of peripheral T cells and their interactions with MDSCs in advanced melanoma carries significant prognostic weight for predicting survival and may provide mechanistic insights into tumor immunosurveillance in such patients. Citation Format: Graham P. Pawelec, Alexander Martens, Claus Garbe, Benjamin Weide, Evelyna Derhovanessian, Dirk Schadendorf, Anna Maria Di Giacomo, Michele Maio, Jolanda de Vries, Henning Zelba. Peripheral immune signatures and survival in stage IV melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2547. doi:10.1158/1538-7445.AM2014-2547