[Show abstract][Hide abstract] ABSTRACT: Background:
Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity.
We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD).
A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)(+) and CCR4(+) T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively.
rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1-specific proliferation of CLA(+) and CCR4(+) T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1-specific CLA(+) and CCR4(+) proliferation and cytokine secretion in patients with and without APT reactions.
Hypoallergenic rBet v 1 fragments induce T cell-dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.
The Journal of allergy and clinical immunology 10/2015; DOI:10.1016/j.jaci.2015.08.042 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin's lymphoma and anaplastic large cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MC) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MC in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MC in 3/25 patients (12%) with indolent SM (ISM), 4/7 patients (57%) with aggressive SM (ASM) and 4/7 patients (57%) with MC leukemia (MCL). The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MC, with lower IC50-values obtained in CD30(+) MCPV-1.1 cells (10 µg/ml) compared to CD30(-) HMC-1.2 cells (>50 µg/ml). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC cell lines tested as well as in primary neoplastic MC in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MC in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MC. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug-target for patients with CD30(+) advanced SM.
[Show abstract][Hide abstract] ABSTRACT: Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multi-kinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction-profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin-metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC50 values. Midostaurin and CGP62221 also produced growth-inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, that accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug-competition experiments revealed that midostaurin interacts with KIT and several additional kinase-targets. The key downstream-regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE-receptor-downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.Leukemia accepted article preview online, 09 September 2015. doi:10.1038/leu.2015.242.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2015; DOI:10.1038/leu.2015.242 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT).
We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were done with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof was studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments.
Allergens from Timothy grass (Phl p 7), Alder (Aln g 4), Birch (Bet v 4), Turnip rape (Bra r 1), Lamb's quarter (Che a 3) and Olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3 whereas binding and affinity to the other allergens was low. mAb102.1F10 showed limited inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity.
Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Advanced systemic mastocytosis (SM) is a life-threatening neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MCs) in various organs and a poor survival. So far, no curative treatment concept has been developed for these patients. We identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as novel drug target in aggressive SM (ASM) and MC leukemia (MCL). As assessed by immunohistochemistry and PCR, neoplastic MCs expressed substantial amounts of BRD4 in ASM and MCL. The human MCL lines HMC-1 and ROSA also expressed BRD4, and their proliferation was blocked by a BRD4-specific shRNA. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition and apoptosis in HMC-1 cells and ROSA cells, regardless of the presence or absence of KIT D816V. In addition, JQ1 suppressed the proliferation of primary neoplastic MCs obtained from patients with ASM or MCL (IC50: 100-500 nM). In drug combination experiments, midostaurin (PKC412) and all-trans retinoic acid (ATRA) were found to cooperate with JQ1 in producing synergistic effects on survival in HMC-1 and ROSA cells. Together, we have identified BRD4 as a promising drug target in advanced SM. Whether JQ1 or other BET-bromodomain inhibitors are effective in vivo in patients with advanced SM remains to be elucidated.Leukemia accepted article preview online, 09 June 2015. doi:10.1038/leu.2015.138.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 06/2015; DOI:10.1038/leu.2015.138 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called ‘stem cell niche.’ Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.
[Show abstract][Hide abstract] ABSTRACT: The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease‐specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34+/CD38− LSCs in BCR/ABL1+ CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210+ acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine‐targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.
European Journal of Clinical Investigation 12/2014; 44(12). DOI:10.1111/eci.12368 · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: House dust mites belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high molecular weight allergen, Der p 11, in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in faeces. IgE reactivity of rDer p 11 was tested with sera from HDM allergic patients from Europe and Africa in RAST-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM allergic patients suffering from HDM-associated AD.Journal of Investigative Dermatology accepted article preview online, 07 July 2014; doi:10.1038/jid.2014.271.
[Show abstract][Hide abstract] ABSTRACT: The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown.
We asked whether neoplastic stem cells (NSC) in patients with MDS (n=29) or acute myeloid leukemia, AML (n=62) express CD52.
As assessed by flow cytometry, CD52 was found to be expressed on NSC-enriched CD34+/CD38- cells in 8/11 patients with MDS and isolated del(5q). In most other MDS patients, CD52 was weakly expressed or not detectable on NSC. In AML, CD34+/CD38- cells displayed CD52 in 23/62 patients, including 4 with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In qPCR analyses, purified NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab was found to induce complement-dependent lysis of CD34+/CD38-/CD52+ NSC, but did not induce lysis in CD52- NSC. Alemtuzumab also suppressed engraftment of CD52+ NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML.
The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC-marker and a potential NSC-target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients.
Clinical Cancer Research 05/2014; 20(13). DOI:10.1158/1078-0432.CCR-13-2811 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV=CD26) as a novel specific and pathogenetically relevant biomarker of CD34(+)/CD38(-) CML LSC. In functional assays, CD26 was identified as target-enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1+ cells, whereas CD26(-) SC from the same patients produced multilineage BCR/ABL1-negative engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.
[Show abstract][Hide abstract] ABSTRACT: Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH-1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV-1, by lentiviral immortalization of cord blood-derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52-targeting antibody alemtuzumab induced cell death in CD52(+) primary neoplastic MCs obtained from patients with SM as well as in MCPV-1 cells. NSG mice xenotransplanted with MCPV-1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.-Hoermann, G., Blatt, K., Greiner, G. Putz, E. M., Berger, A., Herrmann, H., Cerny-Reiterer, S., Gleixner, K. V., Walz, C., Hoetzenecker, K., Müllauer, L., Reiter, A., Sotlar, K., Sexl, V., Valent, P., Mayerhofer, M. CD52 is a molecular target in advanced systemic mastocytosis.
The FASEB Journal 04/2014; 28(8). DOI:10.1096/fj.14-250894 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus-derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23-specific IgG Abs that inhibited binding of patients' IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23-induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-γ in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.
The Journal of Immunology 04/2014; 192(10). DOI:10.4049/jimmunol.1400064 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs. However, no human cell line model useful for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF)-dependent human MC line, ROSA(KIT WT), expressing a fully functional IgE-receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into a SCF-independent clone, ROSA(KIT D816V) which produced a mastocytosis-like disease in NSG mice. However, although several signaling pathways are activated, ROSA(KIT D816V) did not exhibit an increased, but even a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V+ MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 up-regulation. In conclusion, our data show that KIT D816V is a disease-propagating oncoprotein but does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur only in the context of a co-existing allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs used to treat SM patients.
[Show abstract][Hide abstract] ABSTRACT: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was the rational design of recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction.
Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in E. coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients` basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization.
Although each of the mosaic proteins had lost the alpha helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunisation of rabbits with Fel d 1 MB, MC, MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat allergic patients to Fel d 1 in a comparable manner as IgG induced with the wildtype allergen.
We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat allergic patients. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Mast cell leukemia (MCL) is a life-threatening disease associated with high mortality and drug-resistance. Only few patients survive more than 12 months. We report on a 55-year-old female patient with acute MCL diagnosed in May 2012. The disease was characterized by a rapid increase in white blood cells and mast cells (MC) in the peripheral blood, and a rapid increase of serum tryptase levels. The KIT D816H mutation was detected in the blood and bone marrow (BM). Induction chemotherapy with high-dose ARA-C and fludarabine (FLAG) was administered. Unexpectedly, the patient entered a hematologic remission with almost complete disappearance of neoplastic MC and a decrease of serum tryptase levels to normal range after 2 cycles of FLAG. Consecutively, the patient was prepared for allogeneic stem cell transplantation. However, shortly after the third cycle of FLAG, tryptase levels increased again, immature MC appeared in the blood, and the patient died from cerebral bleeding. Together, this case shows that intensive chemotherapy regimens, like FLAG, may induce remission in acute MCL. However, treatment responses are short-lived and the overall outcome remains dismal in these patients. We propose to separate this acute type of MCL from more subacute or chronic variants of MCL.
Leukemia Research Reports 11/2013; 3(1). DOI:10.1016/j.lrr.2013.11.001
[Show abstract][Hide abstract] ABSTRACT: The induction of blocking IgG antibodies that compete with IgE for allergen binding is one important mechanism of allergen-specific immunotherapy. The application of blocking antibodies may be an alternative treatment strategy. A synthetic gene coding for a single-chain fragment (ScFv) specific for the major timothy grass pollen allergen Phl p 2 was inserted into plasmid pCANTAB 5 E, and the recombinant ScFv was expressed in Escherichia coli and purified by affinity chromatography. The ScFv was tested for allergen binding by ELISA, and its association and dissociation were measured by surface plasmon resonance (Biacore) technology. The ability of the ScFv to inhibit allergic patients' IgE binding to Phl p 2 and Phl p 2-induced basophil degranulation was studied by ELISA competition and basophil activation (CD203c) assays. We report the expression, purification, biochemical and immunological characterization of a monomeric single-chain fragment (ScFv) of human origin specific for the major timothy grass pollen allergen, Phl p 2. The Phl p 2-ScFv showed high affinity binding to the allergen and blocked the binding of allergic patients' polyclonal IgE to Phl p 2 up to 98%. Furthermore, it inhibited allergen-induced basophil activation. The Phl p 2-ScFv inhibited allergic patients' IgE binding to Phl p 2 as well as Phl p 2-induced basophil activation and might be useful for passive immunotherapy of grass pollen allergy.