Delineation of target expression profiles in CD34+/CD38- and CD34+/CD38+ stem and progenitor cells in AML and CML.

In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38- and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38- and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38- LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38- cells variably expressed "aberrant" membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication-mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38- LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

BACKGROUND: In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects. METHODS: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively. RESULTS: House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10. CONCLUSION: IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.

Identification of a leukemia-initiating stem cell in human mast cell leukemia.

Mast cell leukemia (MCL) is a highly fatal malignancy characterized by devastating expansion of immature mast cells in various organs. Although considered a stem cell disease, little is known about MCL-propagating neoplastic stem cells. We here describe that leukemic stem cells (LSCs) in MCL reside within a CD34+/CD38- fraction of the clone. Whereas highly purified CD34+/CD38─ cells engrafted NSGhSCF mice with fully manifesting MCL, no MCL was produced by CD34+/CD38+ progenitors or the bulk of KIT+/CD34- mast cells. CD34+/CD38- MCL cells invariably expressed CD13 and CD133, and often also IL-1RAP, but did not express CD25, CD26 or CLL-1. CD34+/CD38- MCL cells also displayed several surface targets, including CD33, which was homogenously expressed on MCL LSCs in all cases, and the D816V mutant form of KIT. Although CD34+/CD38- cells were resistant against single drugs, exposure to combinations of CD33-targeting and KIT-targeting drugs resulted in LSC-depletion and markedly reduced engraftment in NSGhSCF mice. Together, MCL LSCs are CD34+/CD38- cells that express distinct profiles of markers and target antigens. Characterization of MCL LSCs should facilitate their purification and should support the development of LSC-eradicating curative treatment approaches in this fatal type of leukemia.

Phenotyping and Target Expression Profiling of CD34+/CD38- and CD34+/CD38+ Stem- and Progenitor cells in Acute Lymphoblastic Leukemia.

Leukemic stem cells (LSCs) are an emerging target of curative anti-leukemia therapy. In acute lymphoblastic leukemia (ALL), LSCs frequently express CD34 and often lack CD38. However, little is known about markers and targets expressed in ALL LSCs. We have examined marker- and target expression profiles in CD34+/CD38- LSCs in patients with Ph+ ALL (n = 22) and Ph- ALL (n = 27) by multi-color flow cytometry and qPCR. ALL LSCs expressed CD19 (B4), CD44 (Pgp-1), CD123 (IL-3RA), and CD184 (CXCR4) in all patients tested. Moreover, in various subgroups of patients, LSCs also displayed CD20 (MS4A1) (10/41 = 24%), CD22 (12/20 = 60%), CD33 (Siglec-3) (20/48 = 42%), CD52 (CAMPATH-1) (17/40 = 43%), IL-1RAP (13/29 = 45%), and/or CD135 (FLT3) (4/20 = 20%). CD25 (IL-2RA) and CD26 (DPPIV) were expressed on LSCs in Ph+ ALL exhibiting BCR/ABL1p210, whereas in Ph+ ALL with BCR/ABL1p190, LSCs variably expressed CD25 but did not express CD26. In Ph- ALL, CD34+/CD38- LSCs expressed IL-1RAP in 6/18 patients (33%), but did not express CD25 or CD26. Normal stem cells stained negative for CD25, CD26 and IL-1RAP, and expressed only low amounts of CD52. In xenotransplantation experiments, CD34+/CD38- and CD34+/CD38+ cells engrafted NSG mice after 12-20 weeks, and targeting with antibodies against CD33 and CD52 resulted in reduced engraftment. Together, LSCs in Ph+ and Ph- ALL display unique marker- and target expression profiles. In Ph+ ALL with BCR/ABL1p210, the LSC-phenotype closely resembles the marker-profile of CD34+/CD38- LSCs in chronic myeloid leukemia, confirming the close biologic relationship of these neoplasms. Targeting of LSCs with specific antibodies or related immunotherapies may facilitate LSC eradication in ALL.

The KIT and PDGFRA switch-control inhibitor DCC-2618 blocks growth and survival of multiple neoplastic cell types in advanced mastocytosis.

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of KIT, which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC50 <1 µM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.

Variability of PD-L1 expression in mastocytosis.

Mastocytosis is a rare disease with heterogeneous clinical manifestations and few effective therapies. Programmed death-1 (PD-1) and its ligands (PD-L1 and PD-L2) protect tissues from immune-mediated damage and permit tumors to evade immune destruction. Therapeutic antibodies against PD-1 and PD-L1 are effective in the treatment of a variety of neoplasms. In the present study, we sought to systematically analyze expression of PD-1 and PD-L1 in a large number of patients with mastocytosis using immunohistochemistry and multiplex fluorescence staining. PD-L1 showed membrane staining of neoplastic mast cells (MCs) in 77% of systemic mastocytosis (SM) cases including 3 of 3 patients with MC leukemia, 2 of 2 with aggressive SM, 1 of 2 with smoldering SM, 3 of 4 with indolent SM, and 9 of 12 with SM with an associated hematologic neoplasm (SM component only). Ninety-two percent (23 of 25) of cutaneous mastocytosis (CM) cases and 1 of 2 with myelomastocytic leukemia expressed PD-L1, with no expression found in 15 healthy/reactive marrows, 18 myelodysplastic syndromes (MDSs), 16 myeloproliferative neoplasms (MPNs), 5 MDS/MPNs, and 3 monoclonal MC activation syndromes. Variable PD-L1 expression was observed between and within samples, with PD-L1 staining of MCs ranging from 10% to 100% (mean, 50%). PD-1 dimly stained 4 of 27 CM cases (15%), with no expression in SM or other neoplasms tested; PD-1 staining of MCs ranged from 20% to 50% (mean, 27%). These results provide support for the expression of PD-L1 in SM and CM, and PD-1 expression in CM. These data support the exploration of agents with anti-PD-L1 activity in patients with advanced mastocytosis.

The pan-BCL-2-blocker obatoclax (GX15-070) and the PI3-kinase/mTOR-inhibitor BEZ235 produce cooperative growth-inhibitory effects in ALL cells.

Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph+) ALL and Ph- ALL (obatoclax IC50: 0.01-5 µM; BEZ235, IC50: 0.01-1 µM). Both drugs were also found to produce growth-inhibitory effects in all Ph+ and all Ph- cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34+/CD38- stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.

Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia.

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of BCR-ABL1 mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant BCR-ABL1+ cell lines and primary leukemic cells, including cells harboring BCR-ABL1T315I or T315I+ compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34+/CD38- leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize BCR-ABL1+ cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in BCR-ABL1+ cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1T315I or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.

Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.

Chronic myeloid leukemia (CML) is a stem cell-derived leukemia in which neoplastic cells exhibit the Philadelphia chromosome and the related oncoprotein BCR-ABL1. The disease is characterized by an accumulation of myeloid precursor cells in the peripheral blood and bone marrow (BM). A small fraction of neoplastic cells in the CML clone supposedly exhibits self-renewal and thus long-term disease-propagating ability. However, so far, little is known about the phenotype, function, and target expression profiles of these leukemic stem cells (LSCs). Recent data suggest that CML LSCs aberrantly express the interleukin-2 receptor alpha chain CD25. Whereas normal CD34+/CD38- BM stem cells display only low amounts of CD25 or lack CD25 altogether, CD34+/CD38- LSCs express CD25 strongly in more than 90% of all patients with untreated CML. As a result, CD25 can be used to identify and quantify CML LSCs. In addition, it has been shown that CD25 serves as a negative growth regulator of CML LSCs. Here, we review the value of CD25 as a novel marker and potential drug target in CML LSCs.

Extracorporeal IgE Immunoadsorption in Allergic Asthma: Safety and Efficacy.

BACKGROUND: Prevention of IgE-binding to cellular IgE-receptors by anti-IgE (Omalizumab) is clinically effective in allergic asthma, but limited by IgE threshold-levels. To overcome this limitation, we developed a single-use IgE immunoadsorber column (IgEnio). IgEnio is based on a recombinant, IgE-specific antibody fragment and can be used for the specific extracorporeal desorption of IgE. OBJECTIVE: To study safety and efficacy of IgEnio regarding the selective depletion of IgE in a randomized, open-label, controlled pilot trial in patients with allergic asthma and to investigate if IgEnio can bind IgE-Omalizumab immune complexes. METHODS: Fifteen subjects were enrolled and randomly assigned to the treatment group (n=10) or to the control group (n=5). Immunoadsorption was done by veno-venous approach, processing the twofold calculated plasma volume during each treatment. A minimum average IgE-depletion of 50% after the last cycle in the intention-to-treat population was defined as primary endpoint. Safety of the treatment was studied as secondary endpoint. In addition, possible changes in allergen-specific sensitivity were investigated, as well as clinical effects by peak flow measurement and symptom-recording. The depletion of IgE-Omalizumab immune complexes was studied in vitro. The study was registered at clinicaltrials.gov (NCT02096237) and conducted from December 2013 to July 2014. RESULTS: IgE immunoadsorption with IgEnio selectively depleted 86.2% (±5.1% SD) of IgE until the end of the last cycle (p<0.0001). Removal of pollen allergen-specific IgE was associated with a reduction of allergen-specific basophil-sensitivity and prevented increases of allergen-specific skin-sensitivity and clinical symptoms during pollen seasons. IgEnio also depleted IgE-Omalizumab immune complexes in vitro. The therapy under investigation was safe and well-tolerated. During a total of 81 aphereses, 2 severe adverse events (SAE) were recorded, one of which, an episode of acute dyspnea, possibly was related to the treatment and resolved after administration of antihistamines and corticosteroids. CONCLUSIONS: This pilot study indicates that IgE immunoadsorption with IgEnio may be used to treat patients with pollen-induced allergic asthma. Furthermore, the treatment could render allergic patients with highly elevated IgE-levels eligible for the administration of Omalizumab and facilitate the desorption of IgE-Omalizumab complexes. This study was funded by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.

Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy.

BACKGROUND: We have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier. METHODS: We conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n=70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment. RESULTS: Sixty-eight patients completed the trial. TNSS significantly decreased with mean changes of -1.41 (BM32/20µg) (P=0.03) and -1.34 (BM32/40µg) (P=0.003) whereas mean changes in the BM32/10µg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P<0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P=0.0063). CONCLUSION: The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.).

Evaluation of in vitro effects of various targeted drugs on plasma cells and putative neoplastic stem cells in patients with multiple myeloma.

Multiple myeloma (MM) is a malignancy characterized by monoclonal paraproteinemia and tissue plasmocytosis. In advanced MM cytopenia and osteopathy may occur. Although several effective treatment strategies have been developed in recent years, there is still a need to identify new drug targets and to develop more effective therapies for patients with advanced MM. We examined the effects of 15 targeted drugs on growth and survival of primary MM cells and 5 MM cell lines (MM.1S, NCI-H929, OPM-2, RPMI-8226, U-266). The PI3-kinase blocker BEZ235, the pan-BCL-2 inhibitor obatoclax, the Hsp90-targeting drug 17AAG, and the Polo-like kinase-1 inhibitor BI2536, were found to exert major growth-inhibitory effects in all 5 MM cell lines tested. Moreover, these drugs suppressed the in vitro proliferation of primary bone marrow-derived MM cells and induced apoptosis at pharmacologic drug concentrations. Apoptosis-inducing effects were not only seen in the bulk of MM cells but also in MM stem cell-containing CD138-/CD20+/CD27+ memory B-cell fractions. Synergistic growth-inhibitory effects were observed in MM cell lines using various drug combinations, including 17AAG+BI2536 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+BEZ235 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+obatoclax in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+BEZ235 in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+obatoclax in MM.1S, OPM-2 and RPMI-8226 cells, and BEZ235+obatoclax in MM.1S and RPMI-8226 cells. Together, our data show that various targeted drugs induce profound and often synergistic anti-neoplastic effects in MM cells which may have clinical implications and may contribute to the development of novel treatment strategies in advanced MM.

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

BACKGROUND: The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated. OBJECTIVE: To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level. METHODS: Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments. RESULTS: Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients. CONCLUSION: Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.

Frequent occurrence of T cell-mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments.

BACKGROUND: Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity. OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

PURPOSE: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. EXPERIMENTAL DESIGN: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812. RESULTS: In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. CONCLUSIONS: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.

Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis.

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs 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 MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. 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 MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with 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 lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. 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.

Cancer stem cells in basic science and in translational oncology: can we translate into clinical application?

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.

Molecular evolution of hypoallergenic hybrid proteins for vaccination against grass pollen allergy.

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy.

BACKGROUND: Grass pollen is one of the most important sources of respiratory allergies worldwide. OBJECTIVE: This study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach. METHODS: Fusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity. RESULTS: Ten hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation. CONCLUSION: A recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.