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Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88-1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73-2.38; p = 0.44). Grade 3-4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p < 0.001). In conclusion, while adjuvant nDC treatment in stage IIIB/C melanoma patients generated specific immune responses and was well tolerated, no benefit in RFS was observed., (© 2024. The Author(s).)

Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3 + DC (cDC1) and CD1c/BDCA1 + DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy., Competing Interests: Conflict of interest K.P., M.B., A.D., C.A. and C.S. are employees of Miltenyi Biomedicine or Biotec. All other authors declare not conflict of interest., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34 + hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27)., (© 2023. The Author(s).)

Purpose: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma., Patients and Methods: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding., Results: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed., Conclusions: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses., (©2022 The Authors; Published by the American Association for Cancer Research.)

We evaluated the immunological responses of lymph-node involved (stage III) melanoma patients to adjuvant dendritic cell vaccination with subsets of naturally occurring dendritic cells (nDCs). Fifteen patients with completely resected stage III melanoma were randomized to receive adjuvant dendritic cell vaccination with CD1c + myeloid dendritic cells (cDC2s), plasmacytoid dendritic cells (pDCs) or the combination. Immunological response was the primary endpoint and secondary endpoints included safety and survival. In 80% of the patients, antigen-specific CD8 + T cells were detected in skin test-derived T cells and in 55% of patients, antigen-specific CD8 + T cells were detectable in peripheral blood. Functional interferon-γ-producing T cells were found in the skin test of 64% of the patients. Production of nDC vaccines meeting release criteria was feasible for all patients. Vaccination only induced grade 1-2 adverse events, mainly consisting of fatigue. In conclusion, adjuvant dendritic cell vaccination with cDC2s and/or pDCs is feasible, safe and induced immunological responses in the majority of stage III melanoma patients., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background Aims: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies., Methods: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€)., Results: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices., Conclusions: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

Background: Clinical benefit of cellular immunotherapy has been shown in patients with castration-resistant prostate cancer (CRPC). We investigated the immunological response and clinical outcome of vaccination with blood-derived CD1c + myeloid dendritic cells (mDCs; cDC2) and plasmacytoid DCs (pDCs)., Methods: In this randomized phase IIa trial, 21 chemo-naive CRPC patients received maximally 9 vaccinations with mature mDCs, pDCs or a combination of mDCs plus pDCs. DCs were stimulated with protamine/mRNA and loaded with tumor-associated antigens NY-ESO-1, MAGE-C2 and MUC1. Primary endpoint was the immunological response after DC vaccination, which was monitored in peripheral blood and in T cell cultures of biopsies of post-treatment delayed-type hypersensitivity-skin tests. Main secondary endpoints were safety, feasibility, radiological PFS (rPFS) and overall survival. Radiological responses were assessed by MRIs and contrast-enhanced 68 Ga-prostate-specific membrane antigen PET/CT, according to RECIST 1.1, PCWG2 criteria and immune-related response criteria., Results: Both tetramer/dextramer-positive (dm + ) and IFN-γ-producing (IFN-γ + ) antigen specific T cells were detected more frequently in skin biopsies of patients with radiological non-progressive disease (5/13 patients; 38%) compared to patients with progressive disease (0/8 patients; 0%). In these patients with vaccination enhanced dm + and IFN-γ + antigen-specific T cells median rPFS was 18.8 months (n = 5) vs. 5.1 months (n = 16) in patients without IFN-γ-producing antigen-specific T cells (p = 0.02). The overall median rPFS was 9.5 months. All DC vaccines were well tolerated with grade 1-2 toxicity., Conclusions: Immunotherapy with blood-derived DC subsets was feasible and safe and induced functional antigen-specific T cells. The presence of functional antigen-specific T cells correlated with an improved clinical outcome., Trial Registration: ClinicalTrials.gov identifier NCT02692976, registered 26 February 2016, retrospectively registered.

Objective: This study aimed to evaluate the effect of dendritic cell (DC) vaccination against HIV-1 on host gene expression profiles., Design: Longitudinal PBMC samples were collected from participants of the DC-TRN trial for immunotherapy against HIV. Microarray-assisted gene expression profiling was performed to evaluate the effects of vaccination and subsequent interruption of antiretroviral therapy on host genome expression. Data from the DC-TRN trial were compared with results from other vaccination trials., Methods: We used Affymetrix GeneChips for microarray gene expression analysis. Data were analyzed by principal component analysis and differential gene expression was assessed using linear modeling. Gene ontology enrichment and gene set analysis were used to characterize differentially expressed genes. Transcriptome analysis included comparison with PBMCs obtained from DC-vaccinated melanoma patients and of healthy individuals who received seasonal influenza vaccination., Results: DC-TRN immunotherapy in HIV-infected individuals resulted in a major shift in the transcriptome. Longitudinal analysis demonstrated that changes in the transcriptome sustained also during interruption of antiretroviral therapy. After DC-vaccination, the transcriptome was enriched for cellular immunity associated genes that were also induced in healthy adults who received live attenuated influenza virus vaccination. These beneficial responses were accompanied by detrimental signals of general immune activation., Conclusions: The DC-TRN induced changes in the transcriptome were profound, lasting, and consisted of both protective signals and signatures of inflammation and immune exhaustion, with a net result of decreased viral load, without clinical benefit. Thus transcriptome analysis provides useful information, dissecting both positive and negative effects, for the evaluation of safety and efficacy of immunotherapeutic strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

HIV infection leads to a gradual loss CD4(+) T lymphocytes comprising immune competence and progression to AIDS. Effective treatment with combined antiretroviral drugs (cART) decreases viral load below detectable levels but is not able to eliminate the virus from the body. The success of cART is frustrated by the requirement of expensive lifelong adherence, accumulating drug toxicities and chronic immune activation resulting in increased risk of several non-AIDS disorders, even when viral replication is suppressed. Therefore, there is a strong need for therapeutic strategies as an alternative to cART. Immunotherapy, or therapeutic vaccination, aims to increase existing immune responses against HIV or induce de novo immune responses. These immune responses should provide a functional cure by controlling viral replication and preventing disease progression in the absence of cART. The key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control of viral replication, and thus how these responses can be elicited and how they can be monitored. Part one of this review provides an extensive overview of the (patho-) physiology of HIV infection. It describes the structure and replication cycle of HIV, the epidemiology and pathogenesis of HIV infection and the innate and adaptive immune responses against HIV. Part two of this review discusses therapeutic options for HIV. Prevention modalities and antiretroviral therapy are briefly touched upon, after which an extensive overview on vaccination strategies for HIV is provided, including the choice of immunogens and delivery strategies., (Copyright © 2014. Published by Elsevier Masson SAS.)

HIV infection leads to a gradual loss CD4+ T lymphocytes comprising immune competence and progression to AIDS. Effective treatment with combined antiretroviral drugs (cART) decreases viral load below detectable levels but is not able to eliminate the virus from the body. The success of cART is frustrated by the requirement of expensive life-long adherence, accumulating drug toxicities and chronic immune activation resulting in increased risk of several non-AIDS disorders, even when viral replication is suppressed. Therefore there is a strong need for therapeutic strategies as an alternative to cART. Immunotherapy, or therapeutic vaccination, aims to increase existing immune responses against HIV or induce de novo immune responses. These immune responses should provide a functional cure by controlling viral replication and preventing disease progression in the absence of cART. The key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control of viral replication, and thus how these responses can be elicited and how they can be monitored. Part one of this review provides an extensive overview of the (patho-) physiology of HIV infection. It describes the structure and replication cycle of HIV, the epidemiology and pathogenesis of HIV infection and the innate and adaptive immune responses against HIV. Part two of this review discusses therapeutic options for HIV. Prevention modalities and antiretroviral therapy are briefly touched upon, after which an extensive overview on vaccination strategies for HIV is provided, including the choice of immunogens and delivery strategies., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objectives: This study aimed to evaluate HIV sequence evolution in whole genes and in CD8 T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates., Design: HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI., Methods: HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8 T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution., Results: Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8 T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes., Conclusion: Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8 T-cell epitope level.

Recently we have shown that liposomes can be used as artificial microbes for the production and delivery of DNA-encoded antigens. These so-called antigen-expressing immunostimulatory liposomes (AnExILs) were superior in inducing antigen-specific antibodies compared to conventional liposomal protein or DNA vaccines when tested in mice after i.m. immunization. In this study, we investigated the capacity of AnExILs to induce T-cell responses. By using a plasmid vector encoding a model antigen under control of both the prokaryotic T7 and the eukaryotic CMV promoter we hypothesized that antigen production could lead to CTL activation via two distinct routes: i. production of antigens inside the AnExILs with subsequent cross-presentation after processing by APCs and ii. endogenous production of antigens after AnExIL-mediated transfection of the pDNA. Although we were not able to demonstrate transfection-mediated expression of luc-NP in mice, i.m. injection of AnExILs producing luc-NP resulted in T-cell responses against the encoded NP epitope, as determined by tetramer staining. T-cell responses were comparable to the responses obtained after i.m. injection of naked pDNA. In order to find out whether CTL activation was caused by cross-presentation of the exogenous antigens produced inside AnExILs or by endogenous antigen production from transfection with the same pDNA source a second study was initiated in which the contribution of each of these effects could be separately determined. These results demonstrate that the observed T-cell responses were not exclusively caused by cross-presentation of the AnExIL-produced antigens alone, but were rather a combination of dose-dependent antigen cross-presentation and low levels of endogenous antigen production., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Many drugs are unavailable in suitable paediatric dosage forms. We describe the development and validation of a stable paediatric oral formulation of clonidine hydrochloride 50 μg/ml, allowing individualised paediatric dosing and easy administration. Stability of the extemporaneously compounded formulation of clonidine hydrochloride was assessed using a validated HPLC method. Clonidine hydrochloride was stable in the buffered aqueous solution at room temperature for up to 9 months. The described formulation is chemically stable for at least 9 months when stored in brown 100 ml PET bottles at room temperature, enabling adequate oral treatment in paediatric patients., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition., (© 2012 John Wiley & Sons A/S.)

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have generated a recombinant influenza A virus with the HIV-1 p17(Gag) (rFlu-p17) gene inserted into the influenza virus neuraminidase (NA) gene. Expression of HIV-1 p17 protein was detected by conventional Western blot analysis and also by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of rFlu-p17 infected cells. The latter method does not depend on protein-specific antibody preparations and proved to be a powerful tool to detect proteins of interest. Next, antigen presentation of p17 expressed after infection of antigen-presenting cells was determined. Cloned p17-specific CD8+ T-cells were co-cultured with rFlu-p17 infected B-cells and produced IFN-gamma upon stimulation. Furthermore, we showed that immunization with rFlu-p17 elicited a humoral immune response in mice. This study shows that replication-deficient rFlu-p17 is antigenic in vitro and immunogenic in vivo and warrants further development as a candidate vaccine vector.

Despite initial enthusiasm, the use of pharmacogenetics has remained limited to investigation in only a few clinical fields such as oncology and psychiatry. The main reason is the paucity of scientific evidence to show that pharmacogenetic testing leads to improved clinical outcomes. Moreover, for most pharmacogenetic tests (such as tests for genetic variants of cytochrome P450 enzymes) a detailed knowledge of pharmacology is a prerequisite for application in clinical practice, and both physicians and pharmacists might find it difficult to interpret the clinical value of pharmacogenetic test results. Guidelines that link the result of a pharmacogenetic test to therapeutic recommendations might help to overcome these problems, but such guidelines are only sparsely available. In 2001, an early step was taken to develop such guidelines for the therapeutic use of antidepressants, and these included CYP2D6-related dose recommendations drawn from pharmacokinetic study data. However, the use of such recommendations in routine clinical practice remains difficult, because they are currently outside the ambit of the clinical environment and are not accessible during the decision-making process by physicians and pharmacists, namely the prescription and dispensing of drugs.