Publications by authors named "Achim Schneeberger"

23 Publications

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3D screen printing - An innovative technology for large-scale manufacturing of pharmaceutical dosage forms.

Int J Pharm 2021 Jan 18;592:120096. Epub 2020 Nov 18.

Laxxon Medical GmbH, Hans-Knöll-Str. 6, 07745 Jena, Germany. Electronic address:

Three-dimensional (3D) screen printing was used to fabricate oral dosage forms of different geometry and size. The paste required as starting material for the 3D screen printing process was designed for delayed release and contained the model drug paracetamol (acetaminophen). A prototype screen printing unit was used to fabricate different tablets in a single production process. The resulting tablets were produced with three different sizes and designed geometries (disk, donut, cuboid, oval and grid). Investigation of size and mass of the individual tablets demonstrated high uniformity within the various groups of tablets. Further characterization of their physical properties, such as breaking force and friability, yielded results comparing favorably to conventionally produced tablets. Finally, drug release tests in artificial gastric media showed paracetamol release to depend on the surface-area-to-volume ratio. In conclusion, the study shows the potential of 3D screen printing to fabricate more complex oral dosage forms in the setting of mass production with high reproducibility.
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http://dx.doi.org/10.1016/j.ijpharm.2020.120096DOI Listing
January 2021

A Phase 1 Randomized Trial of Specific Active α-Synuclein Immunotherapies PD01A and PD03A in Multiple System Atrophy.

Mov Disord 2020 11 3;35(11):1957-1965. Epub 2020 Sep 3.

Université de Toulouse 3, CHU de Toulouse, INSERM, Centre de Reference AMS, Service de Neurologie et de Pharmacologie Clinique, Centre d'Investigation Clinique CIC1436, Réseau NS-Park/FCRIN et Centre of Excellence for Neurodegenerative Disorders (COEN) de Toulouse, Toulouse, France.

Multiple system atrophy (MSA) is a rare and fatal neurodegenerative disease with limited symptomatic treatment options. Aggregation of α-synuclein in oligodendrocytes is believed to be a central mechanism of the neurodegenerative process. PD01A and PD03A are 2 novel therapeutic vaccine candidates containing short peptides as antigenic moieties that are designed to induce a sustained antibody response, specifically targeting pathogenic assemblies of α-synuclein. The objectives of the current study were to evaluate primarily the safety and tolerability of PD01A and PD03A in patients with early MSA. Thirty patients (11 women) were randomized to receive 5 subcutaneous injections of either PD01A (n = 12), PD03A (n = 12), or placebo (n = 6) in this patient- and examiner-blinded, placebo-controlled, 52-week phase 1 clinical trial (ClinicalTrial.gov identifier: NCT02270489). Immunogenicity and clinical scores were assessed as secondary objectives. Twenty-nine patients reported a total of 595 treatment-emergent adverse events (mild or moderate, n = 555; severe, n = 40). Treatment-related adverse events included 190 injection-site reactions typically observed in vaccination trials with similar per-subject incidence in the treatment groups over time. Sustained IgG titers were observed in the PD01A-treated group, and 89% of treated patients developed a PD01-specific antibody response after receiving all injections. Induced antibodies displayed clear reactivity to the α-synuclein target epitope. Titers and antibody responder rate (58%) were lower in the PD03A-treated group. In conclusion, both PD01A and PD03A were safe and well tolerated. PD01A triggered a rapid and long-lasting antibody response that specifically targeted the α-synuclein epitope. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754431PMC
November 2020

Safety and immunogenicity of the α-synuclein active immunotherapeutic PD01A in patients with Parkinson's disease: a randomised, single-blinded, phase 1 trial.

Lancet Neurol 2020 07;19(7):591-600

AFFiRiS, Vienna, Austria.

Background: Robust evidence supports the role of α-synuclein pathology as a driver of neuronal dysfunction in Parkinson's disease. PD01A is a specific active immunotherapy with a short peptide formulation targeted against oligomeric α-synuclein. This phase 1 study assessed the safety and tolerability of the PD01A immunotherapeutic in patients with Parkinson's disease.

Methods: We did a first-in-human, randomised, phase 1 study of immunisations with PD01A, followed by three consecutive study extensions. Patients aged 45-65 years with a clinical diagnosis of Parkinson's disease (≤4 years since diagnosis and Hoehn and Yahr Stage 1 to 2), imaging results (dopamine transporter single photon emission CT and MRI) consistent with their Parkinson's disease diagnosis, and on stable doses of Parkinson's disease medications for at least 3 months were recruited at a single private clinic in Vienna, Austria. Patients were randomly assigned (1:1), using a computer-generated sequence with varying block size, to receive four subcutaneous immunisations with either 15 μg or 75 μg PD01A injected into the upper arms and followed up initially for 52 weeks, followed by a further 39 weeks' follow-up. Patients were then randomly assigned (1:1) again to receive the first booster immunisation at 15 μg or 75 μg and were followed up for 24 weeks. All patients received a second booster immunisation of 75 μg and were followed up for an additional 52 weeks. Patients were masked to dose allocation. Primary (safety) analyses included all treated patients. These four studies were registered with EU Clinical Trials Register, EudraCT numbers 2011-002650-31, 2013-001774-20, 2014-002489-54, and 2015-004854-16.

Findings: 32 patients were recruited between Feb 14, 2012, and Feb 6, 2013, and 24 were deemed eligible and randomly assigned to receive four PD01A priming immunisations. One patient had a diagnosis change to multiple system atrophy and was withdrawn and two patients withdrew consent during the studies. 21 (87%) of 24 patients received all six immunisations and completed 221-259 weeks in-study (two patients in the 15 μg dose group and one patient in the 75 μg dose group discontinued). All patients experienced at least one adverse event, but most of them were considered unrelated to study treatment (except for transient local injection site reactions, which affected all but one patient). Serial MRI assessments also ruled out inflammatory processes. Systemic treatment-related adverse events were fatigue (n=4), headache (n=3), myalgia (n=3), muscle rigidity (n=2), and tremor (n=2). The geometric group mean titre of antibodies against the immunising peptide PD01 increased from 1:46 at baseline to 1:3580 at week 12 in the 15 μg dose group, and from 1:76 to 1:2462 at week 12 in the 75 μg dose group. Antibody titres returned to baseline over 2 years, but could be rapidly reactivated after booster immunisation from week 116 onwards, reaching geometric group mean titres up to 1:20218.

Interpretation: Repeated administrations of PD01A were safe and well tolerated over an extended period. Specific active immunotherapy resulted in a substantial humoral immune response with target engagement. Phase 2 studies are needed to further assess the safety and efficacy of PD01A for the treatment of Parkinson's disease.

Funding: AFFiRiS, Michael J Fox Foundation.
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http://dx.doi.org/10.1016/S1474-4422(20)30136-8DOI Listing
July 2020

Effects of single and combined immunotherapy approach targeting amyloid β protein and α-synuclein in a dementia with Lewy bodies-like model.

Alzheimers Dement 2019 09 1;15(9):1133-1148. Epub 2019 Aug 1.

Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA; VA San Diego Healthcare System, La Jolla, CA, USA. Electronic address:

Introduction: Immunotherapeutic approaches targeting amyloid β (Aβ) protein and tau in Alzheimer's disease and α-synuclein (α-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting Aβ and/or α-syn may be effective.

Methods: Amyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aβ (AD02) or α-syn (PD-AFF1) and the combination.

Results: AD02 more effectively reduced Aβ and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced α-syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects.

Discussion: Specific active immunotherapy targeting Aβ and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies.
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http://dx.doi.org/10.1016/j.jalz.2019.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503212PMC
September 2019

Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer.

Sci Rep 2018 08 28;8(1):12954. Epub 2018 Aug 28.

Cell Therapy Institute, Paracelsus Medical University, Salzburg, Austria.

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-α) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN-α mRNA variants resulted in significantly improved IFN-α protein expression in human skin compared to native IFN-α mRNA transfection. IFN-α secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN-α mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN-α protein therapy has previously shown a strong therapeutic effect.
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http://dx.doi.org/10.1038/s41598-018-31061-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113332PMC
August 2018

Active immunization therapies for Parkinson's disease and multiple system atrophy.

Mov Disord 2016 Feb 11;31(2):214-24. Epub 2015 Aug 11.

AFFiRiS AG, Vienna, Austria.

Vaccination is increasingly being investigated as a potential treatment for synucleinopathies, a group of neurodegenerative diseases including Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies associated with α-synuclein pathology. All lack a causal therapy. Development of novel, disease-altering treatment strategies is urgently needed. Vaccination has positioned itself as a prime strategy for addressing these diseases because it is broadly applicable, requires infrequent administration, and maintains low production costs for treating a large population or as a preventive measure. Current evidence points to a causal role of misfolded α-synuclein in the development and progression of synucleinopathies. In the past decade, significant progress in active immunization against α-synuclein has been shown both in preclinical animal models and in early clinical development. In this review, we describe the state-of-the-art in active immunization approaches to synucleinopathies, with a focus on advances in Parkinson's disease (PD) and multiple-system atrophy (MSA). We first review preclinical animal models, highlighting their progress in translation to the clinical setting. We then discuss current clinical applications, stressing different approaches taken to address α-synuclein pathology. Finally, we address challenges, trends, and future perspectives of current vaccination programs.
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http://dx.doi.org/10.1002/mds.26377DOI Listing
February 2016

Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy.

Mol Neurodegener 2015 Mar 19;10:10. Epub 2015 Mar 19.

Department of Neurosciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA.

Background: Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation.

Results: In this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells.

Conclusions: This study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies.
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http://dx.doi.org/10.1186/s13024-015-0008-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411775PMC
March 2015

Tailoring the antibody response to aggregated Aß using novel Alzheimer-vaccines.

PLoS One 2015 22;10(1):e0115237. Epub 2015 Jan 22.

AFFiRiS AG, Karl-Farkas-Gasse 22, A-1030, Vienna, Austria.

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115237PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303436PMC
September 2015

Peptide-based anti-PCSK9 vaccines - an approach for long-term LDLc management.

PLoS One 2014 4;9(12):e114469. Epub 2014 Dec 4.

AFFiRiS AG, Karl-Farkas-Gasse 22, Vienna, 1030, Austria.

Background: Low Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models.

Methods And Finding: PCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr+/- mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested.

Conclusions: Peptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256444PMC
December 2015

Pyroglutamylated amyloid-β is associated with hyperphosphorylated tau and severity of Alzheimer's disease.

Acta Neuropathol 2014 Jul 27;128(1):67-79. Epub 2014 May 27.

AFFiRiS AG, Vienna Biocenter, Karl-Farkas-Gasse 22, Vienna, Austria.

Pyroglutamylated amyloid-β (pE(3)-Aβ) has been suggested to play a major role in Alzheimer's disease (AD) pathogenesis as amyloid-β (Aβ) oligomers containing pE(3)-Aβ might initiate tau-dependent cytotoxicity. We aimed to further elucidate the associations among pE(3)-Aβ, full-length Aβ and hyperphosphorylated tau (HP-τ) in human brain tissue. We examined 41 post mortem brains of both AD (n = 18) and controls. Sections from frontal and entorhinal cortices were stained with pE(3)-Aβ, HP-τ and full-length Aβ antibodies. The respective loads were assessed using image analysis and western blot analysis was performed in a subset of cases. All loads were significantly higher in AD, but when using Aβ loads as independent variables only frontal pE(3)-Aβ load predicted AD. In frontal and entorhinal cortices pE(3)-Aβ load independently predicted HP-τ load while non-pE(3)-Aβ failed to do so. All loads correlated with the severity of AD neuropathology. However, partial correlation analysis revealed respective correlations in the frontal cortex only for pE(3)-Aβ load only while in the entorhinal cortex respective correlations were seen for both HP-τ and non-pE(3)-Aβ loads. Mini Mental State Examination scores were independently predicted by entorhinal HP-τ load and by frontal pE(3)-Aβ load. Here, we report an association between pE(3)-Aβ and HP-τ in human brain tissue and an influence of frontal pE(3)-Aβ on both the severity of AD neuropathology and clinical dementia. Our findings further support the notion that pE(3)-Aβ may represent an important link between Aβ and HP-τ, and investigations into its role as diagnostic and therapeutic target in AD are warranted.
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http://dx.doi.org/10.1007/s00401-014-1296-9DOI Listing
July 2014

Next-generation active immunization approach for synucleinopathies: implications for Parkinson's disease clinical trials.

Acta Neuropathol 2014 14;127(6):861-79. Epub 2014 Feb 14.

AFFiRiS AG, Vienna Biocenter, 1030, Vienna, Austria,

Immunotherapeutic approaches are currently in the spotlight for their potential as disease-modifying treatments for neurodegenerative disorders. The discovery that α-synuclein (α-syn) can transmit from cell to cell in a prion-like fashion suggests that immunization might be a viable option for the treatment of synucleinopathies. This possibility has been bolstered by the development of next-generation active vaccination technology with short peptides-AFFITOPEs(®) (AFF)- that do not elicit an α-syn-specific T cell response. This approach allows for the production of long term, sustained, more specific, non-cross reacting antibodies suitable for the treatment of synucleinopathies, such as Parkinson's disease (PD). In this context, we screened a large library of peptides that mimic the C-terminus region of α-syn and discovered a novel set of AFF that identified α-syn oligomers. Next, the peptide that elicited the most specific response against α-syn (AFF 1) was selected for immunizing two different transgenic (tg) mouse models of PD and Dementia with Lewy bodies, the PDGF- and the mThy1-α-syn tg mice. Vaccination with AFF 1 resulted in high antibody titers in CSF and plasma, which crossed into the CNS and recognized α-syn aggregates. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn oligomers in axons and synapses, accompanied by reduced degeneration of TH fibers in the caudo-putamen nucleus and by improvements in motor and memory deficits in both in vivo models. Clearance of α-syn involved activation of microglia and increased anti-inflammatory cytokine expression, further supporting the efficacy of this novel active vaccination approach for synucleinopathies.
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http://dx.doi.org/10.1007/s00401-014-1256-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034750PMC
August 2015

AFFITOME® technology in neurodegenerative diseases: the doubling advantage.

Hum Vaccin 2010 Nov 1;6(11):948-52. Epub 2010 Nov 1.

AFFiRiS AG,Vienna, Austria.

Neurodegenerative diseases are still an area of unmet medical need. This is in contrast to our increasing knowledge on their pathology (e.g., Alzheimer's- (AD), Parkinson's (PD) disease). They are driven by the cerebral accumulation and aggregation of specific proteins (e.g., β-amyloid and hyperphosphorylated tau in the case of AD) in defined brain regions and, as a consequence, death of neurons. Accordingly, removal of given protein aggregates is expected to modify the course of the respective neurodegenerative disease. This has been convincingly demonstrated in animal models of human diseases. However, not every technology that can be used and proves successful in animal models can be translated to the human situation. As highlighted by recent progress in the field of AD research, specific immunotherapy is a viable option in this regard. Given the fact that the aggregates are composed of self-proteins, immunotherapeutic approaches have to consider the issue of potential autoimmunity. This is especially true in case of vaccines. An innovative solution to this problem is offered by the so called AFFITOME® technology, which relies on the use of "doubles" of native molecules, functionally mimotopes or AFFITOPES® if identified by AFFiRiS, as the antigenic vaccine component.
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http://dx.doi.org/10.4161/hv.6.11.13217DOI Listing
November 2010

Perforating folliculitis, angioedema, hand-foot syndrome--multiple cutaneous side effects in a patient treated with sorafenib.

J Dtsch Dermatol Ges 2009 May 29;7(5):449-52. Epub 2009 Jan 29.

Donauspital SMZ Ost, Department of Dermatology and Venereology, Vienna, Austria.

A patient with clear cell renal cell carcinoma was treated with sorafenib, a multikinase inhibitor, which induced a variety of cutaneous side effects. In addition to xerosis, he developed angioedema (AE), hand-foot syndrome (HFS) and perforating folliculitis (PF). The latter three occurred in a dose-dependent manner. AE was observed at the recommended daily dose of 800 mg. Dose reduction to 400 mg prevented its recurrence. At this dose level, the patient exhibited HFS, which cleared upon further reduction of the dose. While receiving 200 mg, the patient developed PF. To the best of our knowledge, this is the first description of a case of PF during treatment with sorafenib.
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http://dx.doi.org/10.1111/j.1610-0387.2009.07017.xDOI Listing
May 2009

Continuous engagement of a self-specific activation receptor induces NK cell tolerance.

J Exp Med 2008 Aug 7;205(8):1829-41. Epub 2008 Jul 7.

Gastroenterology Division, Department of Medicine, Washington University School of Medicine and Howard Hughes Medical Institute, St. Louis, MO 63110, USA.

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self-major histocompatability complex (MHC)-specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)-encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H(+) NK cells. There was a reversible hyporesponsiveness of Ly49H(+) NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H-m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H-m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC-specific inhibitory receptors.
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http://dx.doi.org/10.1084/jem.20072446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525593PMC
August 2008

Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma.

J Clin Oncol 2006 Dec;24(36):5716-24

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Purpose: The recent identification of toll-like receptors (TLRs) and respective ligands allows the evaluation of novel dendritic cell (DC) -activating strategies. Stimulation of TLR9 directly activates human plasmacytoid DCs (PDCs) and indirectly induces potent innate immune responses in preclinical tumor models. We performed an open-label, multicenter, single-arm, phase II pilot trial with a TLR9-stimulating oligodeoxynucleotide in melanoma patients.

Patients And Methods: Patients with unresectable stage IIIb/c or stage IV melanoma received 6 mg PF-3512676 weekly by subcutaneous injection for 24 weeks or until disease progression to evaluate safety as well as clinical and immunologic activity. Clinical and laboratory safety assessments were performed weekly; blood samples for immunological measurements were taken every 8 weeks. Tumor measurements were performed according to Response Evaluation Criteria in Solid Tumors.

Results: Twenty patients received PF-3512676 for a mean of 10.9 weeks with a mean of 10.7 injections. Laboratory and nonlaboratory adverse events were limited, transient, and did not result in any withdrawals. Two patients experienced a confirmed partial response; one response is ongoing for 140+ weeks. Three patients experienced stable disease. Immunologic measurements revealed induction of an activated phenotype of PDC, elevation of serum levels of 2',5'-oligoadenylate, a surrogate marker of type I interferon production, and significant stimulation of natural killer cell cytotoxicity (the latter was associated with clinical benefit).

Conclusion: These results indicate that TLR9-targeted therapy can stimulate innate immune responses in cancer patients, identify biomarkers that may be associated with TLR9-induced tumor regression, and encourage the design of follow-up studies to evaluate the ability of this therapeutic approach to target human cancer.
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http://dx.doi.org/10.1200/JCO.2006.07.9129DOI Listing
December 2006

IgA pemphigus--occurrence of anti-desmocollin 1 and anti-desmoglein 1 antibody reactivity in an individual patient.

J Dtsch Dermatol Ges 2006 Dec;4(12):1045-50

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna General Hospital, Austria.

Background: IgA pemphigus is a rare pustular autoimmune disease with exclusive IgA anti-keratinocyte cell surface antibody reactivity. Two subtypes have been discerned: in the subcorneal pustular dermatosis type, desmocollin 1 has been identified as a targeted autoantigen, while in few cases of the intraepidermal neutrophilic type, IgA anti-desmoglein 1 or IgA anti-desmoglein 3 reactivity has been demonstrated.

Patients And Methods: A 48-year-old white male presented with generalized large confluent pustules. Skin pathology was assessed by histology and direct immunofluorescence analysis. IgG/IgA autoantibodies against desmoglein 1/3 and desmocollin 1 were measured by ELISA and indirect immunofluorescence using desmocollin 1 cDNA-transfected COS7 cells, respectively.

Results: Histopathology revealed subcorneal pustules and direct immunofluorescence microscopy exclusively showed in vivo bound IgA with an intercellular pattern in the epidermis. Desmocollin 1 was identified as a target of IgA autoantibodies by indirect immunofluorescence microscopy utilizing desmocollin 1 cDNA-transfected COS7 cells. In addition, IgA anti-desmoglein 1 reactivity was demonstrated by ELISA. Neither IgA anti-desmoglein 3 nor IgG anti-desmoglein 1/3 autoantibodies were present.

Conclusions: Both desmocollin 1 and desmoglein 1 were autoantigens in this patient with IgA pemphigus and a distinct clinical presentation. To our knowledge, this is the first IgA pemphigus case with dual autoantibody reactivity.
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http://dx.doi.org/10.1111/j.1610-0387.2006.06166.xDOI Listing
December 2006

The effect of intensified extracorporeal photochemotherapy on long-term survival in patients with severe acute graft-versus-host disease.

Haematologica 2006 Mar;91(3):405-8

Medical University of Vienna, Department of Internal Medicine I, Bone Marrow Transplantation, Austria.

Acute graft-versus-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation. We performed a phase II study on patients with acute steroid-refractory GVHD grades II to IV given extracorporeal photochemotherapy (ECP) weekly and analyzed response and long-term survival. Complete resolution of GVHD was achieved in 82% of patients with cutaneous involvement, 61% with liver involvement, and 61% with gut involvement. The probability of survival was 59% among patients who responded completely to ECP compared to 11% in patients not responding completely. We conclude that intensified ECP is highly effective in acute GVHD and that sustained responses are associated with over 50% long-term survival.
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March 2006

Somatostatin receptor scintigraphy with 111In-DOTA-lanreotide and 111In-DOTA-Tyr3-octreotide in patients with stage IV melanoma: in-vitro and in-vivo results.

Melanoma Res 2005 Dec;15(6):523-9

Department of Dermatology, Medical University of Vienna, Vienna, Austria.

The overexpression of somatostatin receptors (SST-Rs) on various tumour cells provides the molecular basis for the successful use of radiolabelled SST analogues in clinical oncology. The objective of the study was to evaluate the tumour binding of In-1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetic acid-lanreotide (In-DOTA-LAN) and In-DOTA-tyrosine-octreotide (In-DOTA-Tyr-OCT) in patients with stage IV melanoma. In addition, we evaluated the potential antiproliferative effect of SST analogues, together with an assessment of the functionality of SST-Rs, on four melanoma cell lines. Twenty-three patients with advanced metastatic melanoma underwent scintigraphy. Thirty-eight of 61 lesions (62%) were positively imaged with In-DOTA-LAN, whereas 23 (37%) were negative. With In-DOTA-Tyr-OCT, 10 of the 23 documented lesions (43%) were positive and 13 (56%) were negative. In vitro, cell lines showed no growth inhibition in the presence of SST analogues and no influence on cell cycle distribution was found with the addition of SST analogues to cultured cells. In addition, no functional surface SST-Rs could be demonstrated on these cell lines. Taken together, our results demonstrate the visualization of metastatic melanoma in a high percentage of patients, probably due to binding of SST analogues to SST-Rs on tumour vessels or infiltrating immune cells. Judging from our data, however, there is no evidence of functional SST-R expression on melanoma cells.
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http://dx.doi.org/10.1097/00008390-200512000-00007DOI Listing
December 2005

CpG motifs are efficient adjuvants for DNA cancer vaccines.

J Invest Dermatol 2004 Aug;123(2):371-9

Division of Immnunology, Allergy, and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

DNA vaccines can induce impressive specific cellular immune response (IR) when taking advantage of their recognition as pathogen-associated molecular patterns (PAMP) through Toll-like receptors (TLR) expressed on/in cells of the innate immune system. Among the many types of PAMP, immunostimulatory DNA, so-called CpG motifs, was shown to interact specifically with TLR9, which is expressed in plasmacytoid dendritic cells (pDC), a key regulatory cell for the activation of innate and adaptive IR. We now report that CpG motifs, when introduced into the backbone, are a useful adjuvant for plasmid-based DNA (pDNA) vaccines to induce melanoma antigen-specific protective T cell responses in the Cloudman M3/DBA/2 model. The CpG-enriched pDNA vaccine induced protection against subsequent challenge with melanoma cells at significantly higher levels than its parental unmodified vector. Preferential induction of an antigen-specific, protective T cell response could be demonstrated by (i) induction of antigen-dependent tumor cell protection, (ii) complete loss of protection by in vivo CD4+/CD8+T cell- but not NK cell-depletion, and (iii) the detection of antigen-specific T cell responses but not of relevant NK cell activity in vitro. These results demonstrate that employing PAMP in pDNA vaccines improves the induction of protective, antigen-specific, T cell-mediated IR.
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http://dx.doi.org/10.1111/j.0022-202X.2004.23208.xDOI Listing
August 2004

Intraperitoneal administration of pMP6/liposome complexes inhibits the growth of co-localized colon-26 adenocarcinoma cells by inducing a tumor-specific immune response.

Anticancer Res 2003 Nov-Dec;23(6C):4843-51

Department of General Surgery, University of Vienna Medical School, Vienna, Austria.

Background: In our previous study, we found complexes of the non-coding pMP6 plasmid and cationic liposomes which exerted antitumor activity in the C26 model. We now sought to unravel the underlying protective effector mechanism(s).

Materials And Methods: C26 recipients (i.p.; day 0) were injected once (day -2) or twice (days -2 and 2) with pMP6/liposome complexes. Thus treated mice were evaluated for tumor growth and the occurrence of innate and specific immune responses.

Results: A single pMP6/liposome injection prolonged the survival of the animals as compared to non-treated C26 recipients (median survival 28 vs. 19 days). Two injections not only prolonged the survival time (median survival 55 days) but completely prevented tumor development in 50% of C26 recipients. I.p. administration of pMP6/liposome complexes resulted in the expression of the proinflammatory cytokines IL-6, IFN-gamma and TNF-alpha. This was followed by the appearance of activated NK cells within the peritoneal cavity and, somewhat later, by the induction of C26-specific CTLs. T cell depletion studies demonstrated the latter to be critical for the protective effect to occur.

Conclusion: Our data suggests that co-application of tumor cells and immunostimulatory pDNA/liposome complexes induces a protective and tumor-specific T cell response. Therefore these complexes may be considered promising agents in the immunological gene therapy of cancer.
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April 2004

Granulocyte-macrophage colony-stimulating factor-based melanoma cell vaccines immunize syngeneic and allogeneic recipients via host dendritic cells.

J Immunol 2003 Nov;171(10):5180-7

Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, University of Vienna Medical School, Vienna, Austria.

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the beta-galactosidase (betagal) gene into M3-GM cells. Their administration activated betagal-specific, L(d)-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-betagal injection sites revealed the presence of cells presenting the respective L(d)-binding betagal peptide epitope. Based on their capacity to activate betagal-specific CTL, they were identified as being CD11c(+) dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.
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http://dx.doi.org/10.4049/jimmunol.171.10.5180DOI Listing
November 2003

Induction of specific immune responses by polycation-based vaccines.

J Immunol 2002 Nov;169(9):5217-26

Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Germany.

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.
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http://dx.doi.org/10.4049/jimmunol.169.9.5217DOI Listing
November 2002