Human Interleukin-1 Receptor Research Articles

Human group 2 innate lymphoid cells do not express the IL-5 receptor

This work was part funded by (for CEB) Airway Disease Predicting Outcomes through Patient Specific Computational Modelling (AirPROM) project (funded through FP7 EU grant), Wellcome Senior Fellowship, (for DJC) Medical Research Council (MRC) and Asthma UK (Centre Grant: G1000758), from the NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London, an equipment grant from the Midlands Asthma and Allergy Research Association (MAARA) and (for CEB and DJC) the National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Unit. BMJR was funded by an MRC and Asthma UK PhD studentship. This paper presents independent research funded by the NIHR.

Tocilizumab for giant cell arteritis with corticosteroid-resistant progressive anterior ischemic optic neuropathy

BackgroundGiant cell arteritis is an inflammatory disorder of the medium- and large-size arteries. Permanent visual loss related to arteritic anterior ischemic optic neuropathy is among the most serious complications of this disease and initial treatment usually consists of high dose corticosteroids. There is no consensus in the literature concerning the optimal therapeutic approach in giant cell arteritis patients with corticosteroid-resistant arteritic anterior ischemic optic neuropathy. Case reportA 73-year-old Caucasian female with biopsy-proven giant cell arteritis developed an acute visual loss of the right eye due to arteritic anterior ischemic optic neuropathy. Despite 5 daily methylprednisolone pulses, systemic symptoms persisted and rapid involvement of the controlateral eye was documented. Therefore, tocilizumab (humanised monoclonal antibody binding the human interleukin-6 receptor) was introduced as a potential salvage therapy with a swift consecutive resolution of the systemic symptoms and stabilization of the ophthalmic lesions. ConclusionsAlthough a late effect of steroids pulses cannot be formally ruled out in this dramatic situation, tocilizumab likely offered a decisive effect in preventing bilateral blindness and may have contributed to steroid tapering. Tocilizumab may represent a new early effective second-line treatment option in corticosteroid-resistant anterior ischemic optic neuropathy. More data are needed to confirm this observation and to evaluate the safety profile of this treatment.

Safety of tocilizumab in the treatment of juvenile idiopathic arthritis

ABSTRACTIntroduction: Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody and IL-6 receptor antagonist, currently approved for the treatment of systemic juvenile idiopathic arthritis (sJIA) and polyarticular juvenile idiopathic arthritis (pJIA) in children aged 2 years or older refractory to conventional treatment. The most common adverse events in patients treated with TCZ were infections, especially in the respiratory tract. The most frequent laboratory abnormalities were altered liver function, neutropenia and elevated cholesterol levels.Areas covered: The safety of TCZ in the treatment of children with JIA was determined based on a review of published clinical trials, including two multicenter studies of patients with sJIA and pJIA (the TENDER and CHERISH trials, respectively). The frequency of adverse events (AEs), serious adverse events (SAEs) and deaths reported in these studies was analyzed and discussed.Expert opinion: TCZ was effective and well tolerated in the treatment of severe forms of sJIA and pJIA, and can be considered a treatment of choice for these conditions. The risk of infections and laboratory abnormalities, such as neutropenia, should be constantly monitored. There is still a need for comparative studies of the risks and benefits of biological agents in patients with refractory JIA.

The Effects of Optic Atrophy Protein (OPA)-1 Deletion on Platelet Function Is Regulated By the Hormonal Milieu

Cardiovascular diseases (CVD) are the leading cause of death in the United States. Thrombosis represents one of the most common underlying pathologies in CVD and disproportionately plagues females. Analyses of transcripts from the Framingham offspring cohort, revealed enrichment in transcripts encoding for the mitochondrial dynamics protein: optic atrophy-1 in females that positively correlated with coronary heart disease and diabetes. Optic atrophy (OPA)-1 regulates mitochondrial fusion, electron transport chain (ETC), complex assembly and apoptosis. The correlation suggests that altered mitochondrial dynamics may contribute to platelet dysfunction. Thus, to determine whether the enrichment in OPA1 transcripts in females that correlates with increased CVD risk represents a physiological adaptation or a pathophysiological response, mice with platelet-specific deletion of OPA1 (pOPA-1 KO) were generated. Male pOPA-1 KO mice exhibited compromised cristae morphology and a 50% reduction in mitochondrial DNA. Additionally, mitochondrial function assessed by an extracellular flux analyzer (XF24 Seahorse biosciences) was reduced by 50%. Mitochondrial dysfunction in male pOPA-1 KO mice was associated with heightened agonist-mediated platelet activation (a hyperactive phenotype). Male pOPA-1 KO mice had a shortened time to stable occlusion of the carotid artery as assessed in vivo by (rose Bengal) photochemical injury (~25 min knockout vs ~ 35 min control), and were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava. In contrast, cristae morphology was normal and mitochondrial DNA unchanged in females. Females revealed no evidence of heightened agonist response or increased incidence of thrombus formation. In contrast, female pOPA-1 KO mice had increased time to stable occlusion of the carotid artery as assessed by photochemical injury (~75 min knockout vs ~35 min control). Additionally, when platelets from pOPA-1 KO or control males were transferred into female mice harboring the human interleukin 4 receptor (HIL4R) transgene that were depleted of their native platelets by HIL4R antibodies, the reconstituted male platelets acquired the phenotype of female pOPA-1 KO mice. Thus, the time to stable occlusion of the carotid artery following photochemical injury was increased. In parallel, gonadectomized pOPA-1 KO male mice were protected from the prothrombotic phenotype. Additionally, gonadectomized female pOPA-1 KO no longer exhibited increased time to stable carotid artery occlusion. These findings suggest a direct interaction between platelet genotype and the systemic milieu, which determines platelet function in pOPA-1 KO mice and the modulation by gonadectomy suggests a role for sex steroids. Thus, whereas preserving OPA-1 expression might be a protective adaptation in males, OPA-1 induction in platelets of females that correlates with CVD in humans, might promote platelet hyper-activation. DisclosuresNo relevant conflicts of interest to declare.

Expression, Purification, and Characterization of Interleukin-11 Orthologues.

Interleukin-11 (IL-11) is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11) production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.

Evaluating the autoinduction expression system and one-step purification for high-level expression and purification of gallbladder-derived rhIL-1Ra.

Recent advancement in fermentation technologies resulted in the increased yields of recombinant proteins of biopharmaceutical and medicinal importance. Consequently, there is an important task to develop simple and easily scalable methods that can facilitate the production of high-quality recombinant protein. Most of the recent reports described the expression of recombinant human IL-1 receptor antagonist (rhIL-1Ra) in Escherichia coli using isopropyl-β-d-thiogalacto pyranoside (IPTG), a nonmetabolizable and expensive compound, as an expression inducer. In this study, we describe the expression and one-step purification of gallbladder-derived rhIL-1Ra by autoinduction in E. coli. This method includes special media that automatically induce the target protein expression from T7 promoter and allow the production of the target protein in high yield than the conventional IPTG induction method. In addition to fermentation process improvements, one-step purification strategy is essential to make the process economical. We developed a single-step cation exchange chromatography and obtained 300 mg/L of rhIL-1Ra with 98% purity. Purified protein was characterized by SDS-PAGE and Ion exchange HPLC (IEX-HPLC). The described method can be used to scale up the production of rhIL-1Ra and other recombinant proteins.

Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model

We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3Δ741, Tac-β3Δ747, Tac-β3Δ754, Tac-β3Δ759, and Tac-β3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3ΔNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3Δ759, Tac-β3ΔNITY, and Tac-β3Δ754, but not Tac-β3Δ747 or Tac-β3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3Δ759 and 123/Tac-β3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3Δ747 and 123/Tac-β3Δ741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

Structure and target interaction of a G-quadruplex RNA-aptamer

ABSTRACTG-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A. These RNA aptamers are specific for human interleukin-6 receptor (hIL-6R), a key player in inflammatory diseases and cancer, and have recently been exploited for in vitro drug delivery studies. With the aim to resolve the RNA structure, global shape, RNA:protein interaction site and binding stoichiometry, we now investigated AIR-3 and AIR-3A by different methods including RNA structure probing, Small Angle X-ray scattering and microscale thermophoresis. Our findings suggest a broader spectrum of folding species than assumed so far and remarkable tolerance toward different modifications. Mass spectrometry based binding site analysis, supported by molecular modeling and docking studies propose a general Gquadruplex affinity for the target molecule hIL-6R.

Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells.

Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections.

Human and Murine Interleukin 23 Receptors Are Novel Substrates for A Disintegrin and Metalloproteases ADAM10 and ADAM17

IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor β1 (IL-12Rβ1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17.

Rationale and study design for a phase I/IIa trial of anakinra in children with Kawasaki disease and early coronary artery abnormalities (the ANAKID trial)

BackgroundAlthough Kawasaki disease (KD) is the most common cause of acquired heart disease in children and may result in coronary artery aneurysms (CAA) with an attendant risk of myocardial infarction, there is no recommended therapy to halt progression of arterial wall damage and prevent aneurysm formation in the acute phase of the vasculitis. While intravenous immunoglobulin (IVIG) reduces the risk of CAA, up to 20% of KD patients are IVIG resistant and have a higher risk for developing CAA. The IL-1 pro-inflammatory pathway is upregulated in children with acute KD and plays a critical role in the experimental animal model of KD. Thus, IL-1 is a logical therapeutic target. ObjectivesThe goal of this study is to determine the safety, tolerability, pharmacokinetics, and immunomodulatory effects of anakinra, a recombinant human IL-1 receptor antagonist, in acute KD patients with coronary artery abnormalities on the baseline echocardiogram. DesignThis is a two-center dose-escalation Phase I/IIa trial in 30 acute KD patients ≥8months old with a coronary artery Z score≥3.0 in the right coronary artery and/or left anterior descending artery or an aneurysm. Subjects will receive a 2- to 6-week course of anakinra by daily subcutaneous injection and will be assessed for resolution of inflammation and dose limiting toxicities (leukopenia, anaphylactoid reaction, or severe infection). ConclusionThe safety and tolerability of blocking both IL-1α and Il-1β by anakinra will be evaluated as a strategy to prevent or attenuate coronary artery damage in infants and children with acute KD.Trial registration: Clinical Trials.gov # NCT02179853, registered June 28, 2014

971f Discovery of Novel and Potent Orally Stable Peptide Antagonistsof the Human IL-23 Receptor and Their Pre-Clinical POC in Rat Colitis Models

971f Discovery of Novel and Potent Orally Stable Peptide Antagonistsof the Human IL-23 Receptor and Their Pre-Clinical POC in Rat Colitis Models

IL1 Receptor Antagonist Inhibits Pancreatic Cancer Growth by Abrogating NF-κB Activation

Constitutive NF-κB activation is identified in about 70% of pancreatic ductal adenocarcinoma (PDAC) cases and is required for oncogenic KRAS-induced PDAC development in mouse models. We sought to determine whether targeting IL-1α pathway would inhibit NF-κB activity and thus suppress PDAC cell growth. We determined whether anakinra, a human IL-1 receptor (rhIL-1R) antagonist, inhibited NF-κB activation. Assays for cell proliferation, migration, and invasion were performed with rhIL-1R antagonist using the human PDAC cell lines AsPc1, Colo357, MiaPaCa-2, and HPNE/K-ras(G12V)/p16sh. In vivo NF-κB activation-dependent tumorigenesis was assayed using an orthotopic nude mouse model (n = 20, 5 per group) treated with a combination of gemcitabine and rhIL-1RA. rhIL-1R antagonist treatment led to a significant decrease in NF-κB activity. PDAC cells treated with rhIL-1R antagonist plus gemcitabine reduced proliferation, migration, and invasion as compared with single gemcitabine treatment. In nude mice, rhIL-1R antagonist plus gemcitabine significantly reduced the tumor burden (gemcitabine plus rhIL-1RA vs. control, P = 0.014). We found that anakinra, an FDA-approved drug that inhibits IL-1 receptor (IL-1R), when given with or without gemcitabine, can reduce tumor growth by inhibiting IL1α-induced NF-κB activity; this result suggests that it is a useful therapeutic approach for PDAC.

IL-1 receptor antagonist (IL-1Ra)-Fc ameliorate autoimmune arthritis by regulation of the Th17 cells/Treg balance and arthrogenic cytokine activation

IntroductionIL-1β signalling has a critical role in the pathogenesis of various types of inflammatory arthritis including rheumatoid arthritis (RA). We aimed to investigate the therapeutic effects of human IL-1 receptor antagonist with Fc fragment (hIL-1Ra-Fc) on autoimmune arthritis and to identify the possible mechanisms by which hIL-1RA-Fc exerts anti-arthritic effects in a murine model of RA and patients with rheumatoid arthritis. MethodsCollagen-induced arthritis (CIA) murine model was established in DBA/1J mice. The levels of various cytokines were determined by using enzyme-linked immunosorbent assay. The mouse joints were assessed for clinical arthritis score and histologic features. Th17 cells and Treg cells were stained by using antibodies specific for CD4, IL-17, CD25, and FoxP3. Osteoclastogenesis was determined by TRAP staining and real-time PCR. ResultshIL-1RA-Fc reduced the arthritis incidence, histological inflammation and cartilage score in the CIA model. The expression of proinflammatory cytokines, VEGF and RANK, was reduced in the affected joint of hIL-1Ra-Fc-treated mice. hIL-1Ra-Fc-treated mice showed decreased number of Th17 cells with increased number of Treg cells in spleens. hIL-1Ra-Fc reduced Th17 cell differentiation by inactivation of STAT3 signalling, and reciprocally induced Treg cell differentiation through STAT5 signalling. In addition, the expression of IL-17, TNF-α, RANKL, and VEGF was decreased, while Foxp3 gene expression was increased in PBMCs of RA patients after administration of hIL-1Ra-Fc. ConclusionThe anti-arthritis effects of hIL-1RA-Fc are associated with regulation of balance between Th17 cells and Treg cells and suppression of osteoclastogenesis and angiogenesis in the affected joints.

Intractable Epilepsy (IE) and Responses to Anakinra, a Human Recombinant IL-1 Receptor Agonist (IL-1ra): Case Reports

Patients with intractable seizures (IE) who failed to respond currently available treatment measures are clinically challenging, causing significant morbidity and even mortality. Better understanding of contributing factors for IE will be helpful for exploring additional treatment options. A role of inflammation has long been suspected in pathogenesis of epilepsy, especially in idiopathic epilepsy with diffuse epileptic activity. In rodent models of epilepsy, blockage of inflammatory mediators such as interleukin 1-β (IL-1β) and IL-6 attenuates epileptic activities. However, therapeutic effects of blockers of such inflammatory mediators have not been addressed in patients with IE.This manuscript reports 4 IE cases in which anakira, a human recombinant IL-1 receptor antagonist (IL-1ra), was clinically effective in controlling their epileptic activity in a dose-dependent manner. In these patients, we also observed changes in ratios of IL-1β/IL-10 produced by peripheral blood monocytes, surrogates of bone marrow derived microglial cells. These findings could be associated with seizure control status.These presented cases indicate a possible therapeutic use of a commercially available IL-1β blocker (anakinra) and therapeutic utility of the IL-1β/IL-10 ratios produced by peripheral blood monocytes for assessing responses to this immune-modulating agent.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Patient-Derived Xenografts Are Faithful Genomic and Phenotypic Models of Primary Leukemia and Respond to the IL3 Receptor Targeting Agent SL-401 In Vivo

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive acute leukemia/lymphoma recently classified as a malignant transformation of plasmacytoid dendritic cells (pDCs) and a subtype of acute myeloid leukemia (AML). BPDCN has no standard treatment and a poor prognosis, with median survival <1 year. A significant roadblock to better understanding BPDCN is a lack of adequate model systems. We generated patient-derived xenografts (PDX) of BPDCN in NOD/Scid/IL2rgnull (NSG) mice. Bone marrow or peripheral blood cells involved by BPDCN blasts (CD45 low, CD123 high, HLA-DR high, CD3 neg) were transplanted into irradiated NSG recipients. Nine of 16 BPDCNs caused lethal leukemia involving blood, spleen, and bone marrow 2-6 months after transplantation. All nine BPDCN PDXs were serially transplantable. Flow characterization of each patient's BPDCN and corresponding xenograft revealed no major differences in BDCA2, BDCA4, FCeR1, ILT7, or cytoplasmic TCL1 staining. All samples maintained high expression of the human interleukin-3 (IL3) receptor (IL3Ralpha/CD123), a hallmark feature of BPDCN. To further characterize BPDCN pathogenesis we performed whole transcriptome sequencing (RNA-seq) on sorted blasts from 11 patients and on normal pDCs isolated from 4 healthy donors. These were compared to RNA-seq in six PDXs. The spectrum of mutations in BPDCN transcriptomes overlapped with that seen in other hematologic malignancies, particularly myeloid disorders, and was similar to reported DNA mutations in BPDCN, including in ASXL1, CTCF, IDH2, NRAS, RUNX1, STAG2, TET2, and TP53. Particularly striking was the presence of a canonical mutation in an RNA splicing factor in 7 of 11 cases (SRSF2 P95H/L/R in four, ZRSR2 R295* and gene locus deletion in two, and SF3B1 K666N in one). Known oncogenic mutations in the original disease were retained in the PDXs, including all splicing factor mutations, with the exception of an IDH2 R140Q that was lost in one PDX. BPDCN PDXs grouped together in unsupervised clustering of expression profiles, distinct from AML and ALL PDXs in an analysis of 134 models from the DFCI Public Repository of Xenografts (http://PRoXe.org). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways associated with differentially expressed genes between primary BPDCNs and non-malignant pDCs revealed signatures related to dendritic cell activation, cell cycle, and apoptosis. In addition, 3 of the top 11 sets were genes involved in mRNA processing, mRNA splicing, and processing capped intron-containing pre-mRNAs (all FDR<1e-6). To test the efficacy of BPDCN-targeted therapy using primary human leukemias in vivo, we performed a pre-clinical trial in NSG mice using SL-401, a recombinant biologic consisting of a fusion protein of IL3 and diphtheria toxin. Three independent BPDCN xenografts were injected into 20 NSG mice each, and followed by weekly peripheral blood monitoring for human CD45 and CD123. When leukemia burden reached >0.5% in at least half of the mice in the cohort, animals were randomized to receive SL-401 at 100 ug/kg or vehicle intraperitoneally daily for 5 days. Two mice in each group were sacrificed at day 7 for response assessment, and peripheral blood was followed weekly in the remaining mice for evidence of progression (>5% human CD45/CD123-positive cells). 7 days after treatment, mice receiving SL-401 had dramatic reductions in BPDCN in the peripheral blood, spleen, and bone marrow (0.31% vs 37.6% in marrow of SL-401 vs vehicle). SL-401 prolonged progression-free survival in all BPDCNs tested (12 vs 48 days, P<0.0001 by log-rank test). At the time of progression after SL-401, relapsing mice were re-randomized to receive a 2nd and in some cases 3rd cycle of SL-401 or vehicle. Repeated treatment in mice that progressed after SL-401 resulted in second and third peripheral blood remissions. All PDXs responded to SL-401 including those with and without splicing factor and TP53 mutations. CD123 expression was maintained at high levels on all SL-401 treated BPDCNs even after repeated cycles. Primary xenografts of BPDCN are faithful models of the human disease, maintain genetic and transcriptomic characteristics of the original tumor, and respond to multiple courses of IL3-DT in vivo, suggesting that they provide a valuable resource to study disease biology and response/resistance to targeted therapy. DisclosuresChen:Stemline Therapeutics, Inc.: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics, Inc.: Research Funding.

First Preclinical Report of the Efficacy and PD Results of KHK2823, a Non-Fucosylated Fully Human Monoclonal Antibody Against IL-3Rα

Human interleukin-3 receptor alpha (IL-3Ra, CD123), which promotes the proliferation and differentiation of hematopoietic cells, is highly expressed in myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).We newly generated KHK2823, a non-fucosylated fully human IgG1 monoclonal antibody against human IL-3Ra, by utilizing the POTELLIGENT® technology. Here, we describe the in vitro and in vivo preclinical efficacy and safety of KHK2823, as well as its pharmacodynamic (PD) profile.At first, we explored that KHK2823 bound to various hematological malignant cells and leukemic stem cells. The cells from AML and MDS bone marrows were found to be bound by KHK2823. A significant part of bone marrow cells derived from B-cell acute lymphoblastic leukemia (B-ALL) patients was also bound by KHK2823.KHK2823 bound to soluble human IL-3Ra protein with a sub-nanomolar dissociation constant (KD), and recognized CD34+ CD38+ (leukemic blast) and/or CD34+ CD38- (leukemic stem cell) cells in patients with AML/MDS, as well as AML cell lines, thereby obtaining a high antibody-dependent cellular cytotoxic activity without complement-dependent cytotoxicity. Interestingly, KHK2823 did not interfere with the binding of IL-3 to IL-3R. The lack of a receptor-ligand interaction may conserve the IL-3 signal, which plays an important role in normal hematopoiesis. In a tumor model xenografting the human AML cell line MOLM-13 on nude rats, KHK2823 significantly suppressed the tumor growth at doses of 0.1 and 1 mg/kg (Figure 1).The PD and toxicity profiles of KHK2823 were assessed in cynomolgus monkeys administered at doses ranging from 0.1 to 100 mg/kg by i.v. infusion, once weekly for 4 weeks. KHK2823 was generally well tolerated in monkeys, even at 100 mg/kg. The number of IL-3Ra-positive cells in the peripheral blood of cynomolgus monkeys decreased in all groups receiving KHK2823, which suggest KHK2823 could exert its depletion activity of IL-3Ra-positive cells in human (Figure 2). Currently, the safety and tolerability of KHK2823 is being investigated in patients with AML or MDS in a Phase 1 study (NCT02181699, https://clinicaltrials.gov/ct2/show/NCT02181699). This is the first non-randomized, open-label, dose escalation clinical study to investigate the safety, PK, immunogenicity and PD of repeated doses of KHK2823.In summary, KHK2823 was confirmed to bind to AML, MDS and B-ALL cells as the IL-3Ra in accordance with other publications. KHK2823 was also found to eliminate AML cells in vitro and also suppressed the AML tumor growth in the in vivo model. In addition, the number of IL-3Ra-positive cells in cynomolgus monkeys decreased following i.v. infusion of 0.1mg/kg KHK2823 with a tolerable safety profile, even at a dose of 100 mg/kg. Taken together, KHK2823 may therefore be a promising anti-IL-3Ra therapeutic drug for the treatment of AML. [Display omitted] [Display omitted] DisclosuresAkiyama:Kyowa Hakko Kirin Co., Ltd.: Employment. Takayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment. Maekawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Shimabe:Kyowa Hakko Kirin Co., Ltd.: Employment. Nishikawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Yamawaki:Kyowa Hakko Kirin Co., Ltd: Employment. Iijima:Kyowa Hakko Kirin Co., Ltd: Employment. Hiura:Kyowa Hakko Kirin Co., Ltd.: Employment. Takahashi:Kyowa Hakko Kirin Co., Ltd.: Employment. Akashi:Asahi Kasei: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau. Tawara:Kyowa Hakko Kirin Co., Ltd: Employment.

IL-1β, in contrast to TNFα, is pivotal in blood-induced cartilage damage and is a potential target for therapy

AbstractJoint bleeding after (sports) trauma, after major joint surgery, or as seen in hemophilia in general leads to arthropathy. Joint degeneration is considered to result from the direct effects of blood components on cartilage and indirectly from synovial inflammation. Blood-provided proinflammatory cytokines trigger chondrocytes and induce the production of cartilage-degrading proteases. In the presence of erythrocyte-derived iron, cytokines stimulate radical formation in the vicinity of chondrocytes inducing apoptosis. To unravel the role of interleukin (IL) 1β and tumor necrosis factor (TNF) α in the pathogenesis of this blood-induced cartilage damage, the effect of antagonizing these cytokines was examined in human in vitro cultures. Addition of recombinant human IL-1β monoclonal antibody or IL-1 receptor antagonist resulted in a dose- and time-dependent protection of cartilage from blood-induced damage. In higher concentrations, almost complete normalization of cartilage matrix proteoglycan turnover was achieved. This was accompanied by a reduction in IL-1β and IL-6 production in whole blood cultures, whereas TNFα production remained unaffected. Interestingly, addition of a TNFα monoclonal antibody, although demonstrated to inhibit the direct (transient) effects of TNFα on cartilage, exhibited no effect on blood-induced (prolonged) cartilage damage. It is demonstrated that IL-1β is crucial in the development of blood-induced joint damage, whereas TNFα is not. This hierarchical position of IL-1β in blood-induced joint damage warrants studies on targeting IL-1β to potentially prevent joint degeneration after a joint bleed.

Abstract 3605: Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment

Abstract Introduction: Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin (IL)-1, CXC receptors (CXCR)1/2 ligands, and Transforming Growth Factor β (TGFβ) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the anti-VEGF antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse the resistance to anti-VEGF treatment. Methods: Bevacizumab-resistant FGBR orthotopic tumor bearing mice received bevacizumab alone or in combination with a recombinant human IL-1 receptor antagonist, a monoclonal antibody against TGFβ Receptor type II, and a recombinant antibody binding CXCR1/2 ligands. Western blot analysis was performed to assess the activity of p65 NFB, Smad2 and on the expression of IL-6 in FGBR cells in culture. Immunohistochemical analysis of FFPE sections from FGBR tumors was performed to assess Smad2 phosphorylation and IL-6 expression. Wound healing assay was performed to assess cell motility in FGBR cells in culture. Immunohistochemical analysis was performed to assess the expression of E-Cadherin and Vimentin in FGBR tumors. Immunohistochemical analysis was performed to estimate the presence of Cd11b+ cells. Results: The combination of these agents with bevacizumab reduced the tumor burden and significantly prolonged mice survival if compared with single agent bevacizumab. Tumors from mice receiving the combination treatment demonstrated significantly lower expression of IL-6 and phosphorylation of p65 and Smad2 when compared with control. FGBR cells treated in vitro with the three agents plus bevacizumab had significantly higher levels of E-cadherin and lower levels of Vimentin, and exhibited significantly lower migration rates than did their bevacizumab- treated controls. Consistently, tumors from mice receiving the combination treatment demonstrated significantly higher expression of E-cadherin, lower levels of Vimentin, and a significantly lower infiltration by CD11b+ cells when compared with bevacizumab -treated controls. Conclusion: This study suggests that inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing EMT and inhibiting CD11b+ proangiogenic myeloid cells tumor infiltration. Citation Format: Carmine Carbone, Anna Tamburrino, Geny Piro, Marco Zanotto, Maria Mihaela Mina, Silvia Zanini, Federico Boschi, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3605. doi:10.1158/1538-7445.AM2015-3605

563. Rapid and Nuclease-Free Generation of Gene-Repaired Human iPS Cells from IL2RG-Defective Fibroblasts

Recent developments of artificial nucleases, such as TALENs and CRISPR/Cas9, have made gene repair therapy of inherited diseases more realistic. However, off-target chromosomal breaks resulting in imprecise DNA repair by the nucleases are still significant concerns from safety aspects, which is the most critical issue of clinical gene therapy. Furthermore, prolonged culture during induction and manipulation of induced pluripotent stem cells (iPSCs) has been shown to increase point mutations and copy number variations in the iPSC genome, raising additional safety concerns for therapeutic applications of iPSCs. In order to minimize cell culture for iPSC induction and achieve efficient gene repair without using nucleases, we examined a strategy of consecutive iPSC induction and gene repair without cell cloning between the two processes. For this purpose, we utilized Sendai virus vector (SeV)-mediated iPSC induction and helper-dependent adenoviral vector (HDAdV)-mediated gene targeting.To mimic somatic cells from patients of X-linked severe combined immunodeficiency, we firstly knocked out exon 1 of the common gamma chain of human interleukin-2 receptor (IL2RG) locus by utilizing TALENs, and subsequently derived fibroblast-like cells. Next, these cells were subject to iPSC induction by using the SeV expressing Yamanaka factors, SeVdp(KOSM). Instead of isolating and characterizing individual SeV-iPS colonies before gene targeting, iPSC-like colonies were grown as a cell population. Twenty-two to 26 days after SeV infection, the cells were transduced with a HDAdV targeting the IL2RG locus. This HDAdV was designed to introduce the PGK-neo cassette into intron 4 and to repair the mutation of exon 1. Cells were subject to G418/FIAU selection to isolate gene-targeted SeV-iPS colonies. Correct targeting at the IL2RG locus by the vector in G418/FIAU double-resistant colonies was verified by PCR analyses. From two independent experiments, the gene targeting frequencies were similar to those in authentic human iPSCs at 1.6 – 1.8 × 10-5 per cell and 60 – 75% of chromosomal integration (G418/FIAU-resistance), among them 60 – 80% of gene-targeted colonies had the exon 1 mutation repaired. At the time of HDAdV infection, the SeV-iPSCs already showed characteristics of pluripotency, revealed by bisulfite sequencing of the NANOG locus and TRA-1-60 immunostaining. The pluripotency was also maintained in gene-targeted iPSC colonies. From the start of iPSC induction (infection with SeV) to isolation of gene-repaired iPSC colonies, it took only 40 days. We are currently analyzing the expression of IL2RG in differentiated T cells derived from the gene-repaired iPSCs.These results suggest that, by combining efficient methods for iPSC induction and gene targeting, gene repair therapy using patients’ somatic cells becomes more realistic.