Anders Elm Pedersen

Seromic profiling of colorectal cancer patients with novel glycopeptide microarray

Cancer‐associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post‐translational variants of proteins are likely to induce autoantibodies, and changes in O‐linked glycosylation represent one of the most important cancer‐associated post‐translational modifications (PTMs). Short aberrant O‐glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide‐specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer‐associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM‐peptide arrays in diagnostic measures.

Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study.

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.

Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a clinical phase 1/2 trial.

Therapeutic dendritic cell (DC) vaccination against cancer is a strategy aimed at activating the immune system to recognize and destroy tumor cells. In this nonrandomized phase 1/2 trial, we investigated the safety, feasibility, induction of T-cell response, and clinical response after treatment with a DC-based vaccine in patients with metastatic renal cell carcinoma. Twenty-seven patients with progressive cytokine-refractory metastatic renal cell carcinoma were vaccinated with DCs loaded with either a cocktail of survivin and telomerase peptides or tumor lysate depending on their HLA-A2 haplotype, and low-dose IL-2 was administered concomitantly. Tumor response, immune response, and serum IL-6 and YKL-40 were measured during treatment. Vaccine generation was successful in all patients and no serious adverse events were observed. None of the patients had an objective response but 13/27 patients obtained disease stabilization (SD) for more than 8 weeks. An antigen-specific immune response was demonstrated in 6/6 patients tested. Furthermore, significant alterations in serum YKL-40 and IL-6 were found during treatment. In conclusion, DC vaccination in our setting is feasible and without severe toxicity. Almost half of the patients obtained SD, and in more than 1/3 of the patients, SD persisted for more than 6 months. However, the evaluation of SD is difficult to interpret in the absence of a randomized trial and, therefore, these results should be interpreted with caution. Antigen-specific immune responses were observed in a subset of the treated patients.

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

BACKGROUND AND AIM 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. RESULTS Of 46 patients who initiated treatment, 10 stopped treatment within 1-4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2+ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25(high) CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD. CONCLUSIONS Vaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.

Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes.

Objective:To investigate the potential to induce additional cytotoxic T-lymphocyte (CTL) immunity during chronic HIV-1 infection. Design:We selected infrequently targeted or subdominant but conserved HLA-A*0201-binding epitopes in Gag, Pol, Env, Vpu and Vif. These relatively immune silent epitopes were modified as anchor-optimized peptides to improve immunogenicity and delivered on autologous monocyte-derived dendritic cells (MDDCs). Methods:Twelve treatment-naïve HLA-A*0201 HIV-1-infected Danish individuals received 1 × 107 MDDCs subcutaneously (s.c.) (weeks 0, 2, 4 and 8), pulsed with seven CD8+ T-cell epitopes and three CD4+ T-cell epitopes. Epitope-specific responses were evaluated by intracellular cytokine staining for interferon-γ, tumor necrosis factor α and interleukin-2 and/or pentamer labeling 3 weeks prior to, 10 weeks after and 32 weeks after the first immunization. Results:Previously undetected T-cell responses specific for one or more epitopes were induced in all 12 individuals. Half of the participants had sustained CD4+ T-cell responses 32 weeks after immunization. No severe adverse effects were observed. No overall or sustained change in viral load or CD4+ T-cell counts was observed. Conclusion:These data show that it is possible to generate new T-cell responses in treatment-naive HIV-1-infected individuals despite high viral loads, and thereby redirect immunity to target new multiple and rationally selected subdominant CTL epitopes. Further optimization could lead to stronger and more durable cellular responses to selected epitopes with the potential to control viral replication and prevent disease in HIV-1-infected individuals.

Dendritic cells in peripheral tolerance and immunity

Dendritic cells capable of influencing immunity exist as functionally distinct subsets, T cell‐tolerizing and T cell‐immunizing subsets. The present paper reviews how these subsets of DCs develop, differentiate and function in vivo and in vitro at the cellular and molecular level. In particular, the role of DCs in the generation of regulatory T cells is highlighted.

Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials

Abstract The costimulatory molecule CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on various antigen presenting cells (APCs) as well as some tumor cells. The binding to the natural ligand CD40L, which is expressed on T helper cells, leads to APC activation and thus enhancement of immune responses. Treatment with anti-CD40 monoclonal antibodies has been exploited in several cancer immunotherapy studies in mice and led to the development of anti-CD40 antibodies for clinical use. Here, Dacetuzumab and Lucatumumab are in the most advanced stage and are being tested as treatment for malignancies such as chronic lymphatic leukemia (CLL), Multiple Myeloma (MM), and non-Hodgkin’s lymphoma (NHL). The promising results from these early clinical trials have encouraged clinical drug development in order to investigate the effect of CD40 mAbs in combination with other cancer immunotherapies, in particular interleukin (IL)-2. An in-depth analysis of this immunotherapy is provided elsewhere. In the present review, we provide an update of the most recent clinical trials with anti-CD40 antibodies. We present and discuss recent and ongoing clinical trials in this field, including clinical studies which combine anti-CD40 treatment with other cancer-treatments, such as Rituximab and Tremelimumab.

Phenotypic and Functional Characterization of Clinical Grade Dendritic Cells Generated from Patients with Advanced Breast Cancer for Therapeutic Vaccination

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro‐generated monocyte‐derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte‐derived DC were generated in vitro utilizing granulocyte macrophage colony‐stimulating factor and rh‐interleukin‐4 (IL‐4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor‐alpha (TNF‐α) induced significant IL‐12 p70 secretion, which was increased after addition of a decoy IL‐10 receptor. The lymph node homing chemokine receptor CCR‐7 expression was induced by TNF‐α + IL‐1β + IL‐6 + prostaglandin E2 but was not induced by Poly I:C + TNF‐α. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient‐derived DC for antigen‐specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T‐cell stimulation, whereas the allostimulatory capacity of patient‐derived DC was significantly decreased. These data suggest that patient‐derived DC are more differentiated but are less sensitive to maturation‐inducing agents than DC obtained from healthy individuals.

Early detection of cancer in the general population: a blinded case-control study of p53 autoantibodies in colorectal cancer.

Background:Recent reports from cancer screening trials in high-risk populations suggest that autoantibodies can be detected before clinical diagnosis. However, there is minimal data on the role of autoantibody signatures in cancer screening in the general population.Methods:Informative p53 peptides were identified in sera from patients with colorectal cancer using an autoantibody microarray with 15-mer overlapping peptides covering the complete p53 sequence. The selected peptides were evaluated in a blinded case–control study using stored serum from the multimodal arm of the United Kingdom Collaborative Trial of Ovarian Cancer Screening where women gave annual blood samples. Cases were postmenopausal women who developed colorectal cancer following recruitment, with 2 or more serum samples preceding diagnosis. Controls were age-matched women with no history of cancer.Results:The 50 640 women randomised to the multimodal group were followed up for a median of 6.8 (inter-quartile range 5.9–8.4) years. Colorectal cancer notification was received in 101 women with serial samples of whom 97 (297 samples) had given consent for secondary studies. They were matched 1 : 1 with 97 controls (296 serial samples). The four most informative peptides identified 25.8% of colorectal cancer patients with a specificity of 95%. The median lead time was 1.4 (range 0.12–3.8) years before clinical diagnosis.Conclusion:Our findings suggest that in the general population, autoantibody signatures are detectable during preclinical disease and may be of value in cancer screening. In colorectal cancer screening in particular, where the current need is to improve compliance, it suggests that p53 autoantibodies may contribute towards risk stratification.

Modelling diverse root density dynamics and deep nitrogen uptake—A simple approach

We present a 2-D model for simulation of root density and plant nitrogen (N) uptake for crops grown in agricultural systems, based on a modification of the root density equation originally proposed by Gerwitz and Page in J Appl Ecol 11:773–781, (1974). A root system form parameter was introduced to describe the distribution of root length vertically and horizontally in the soil profile. The form parameter can vary from 0 where root density is evenly distributed through the soil profile, to 8 where practically all roots are found near the surface. The root model has other components describing root features, such as specific root length and plant N uptake kinetics. The same approach is used to distribute root length horizontally, allowing simulation of root growth and plant N uptake in row crops. The rooting depth penetration rate and depth distribution of root density were found to be the most important parameters controlling crop N uptake from deeper soil layers. The validity of the root distribution model was tested with field data for white cabbage, red beet, and leek. The model was able to simulate very different root distributions, but it was not able to simulate increasing root density with depth as seen in the experimental results for white cabbage. The model was able to simulate N depletion in different soil layers in two field studies. One included vegetable crops with very different rooting depths and the other compared effects of spring wheat and winter wheat. In both experiments variation in spring soil N availability and depth distribution was varied by the use of cover crops. This shows the model sensitivity to the form parameter value and the ability of the model to reproduce N depletion in soil layers. This work shows that the relatively simple root model developed, driven by degree days and simulated crop growth, can be used to simulate crop soil N uptake and depletion appropriately in low N input crop production systems, with a requirement of few measured parameters.