Coronado Biosciences Research Articles

The efficacy and safety of lurasidone in adolescent patients with schizophrenia: Results of functional and quality of life measures from a 6-week, double-blind, placebo-controlled study

IntroductionLurasidone, an atypical antipsychotic, demonstrated efficacy and safety in adults with schizophrenia.Objective/AimsTo evaluate the efficacy and safety of lurasidone in adolescent patients with schizophrenia.MethodsAdolescents (13–17 years old) with schizophrenia were randomly assigned to six weeks of double-blind treatment with lurasidone 37 mg/day, 74 mg/day or placebo. An ANCOVA using an LOCF approach was performed to assess change from baseline on secondary study endpoints: Pediatric Quality of Life Enjoyment and Satisfaction Questionnaire (PQ-LES-Q) and Children's Global Assessment Scale (CGAS).ResultsPatients were randomized to lurasidone 37 mg/d (n = 108), 74 mg/day (n = 106), or placebo (n = 112). Placebo-adjusted LS mean improvement at week 6 on the PQ-LES-Q was 5.3 (P = 0.001) and 5.8 (P < 0.001) for the 37 mg/day and 74 mg/day groups, respectively; and, on the CGAS was 4.6 (P = 0.002) and 4.9 (P < 0.001) for the 37 mg/day and 74 mg/d groups, respectively. The most common adverse events occurring at ≥ 5% in either lurasidone group and at least twice the rate of placebo were: nausea, somnolence, akathisia, vomiting and sedation. Mean change in weight at week 6 for placebo, 37 mg/day, and 74 mg/day groups was 0.05 kg, 0.17 kg, and 0.49 kg, respectively. Lurasidone treated patients did not show clinically meaningful differences from placebo on laboratory measures of cholesterol, triglycerides, glucose, and prolactin.ConclusionsAdolescent patients with schizophrenia treated with lurasidone demonstrated significant improvement in quality of life and function. Lurasidone was generally well-tolerated and associated with minimal changes in weight and metabolic parameters. Sponsored by Sunovion Pharmaceuticals Inc. ClinicalTrials.gov identifier: NCT01911429.Disclosure of interestDr. Findling receives or has received research support, acted as a consultant and/or served on a speaker's bureau for Alcobra, American Academy of Child & Adolescent Psychiatry, American Physician Institute, American Psychiatric Press, Bracket, CogCubed, Cognition Group, Coronado Biosciences, Dana Foundation, Elsevier, Forest, Guilford Press, Ironshore, Johns Hopkins University Press, Jubilant Clinsys, KemPharm, Lundbeck, Merck, NIH, Neurim, Novartis, Otsuka, Oxford University Press, Pfizer, Physicians Postgraduate Press, Purdue, Rhodes Pharmaceuticals, Roche, Sage, Shire, Sunovion, Supernus Pharmaceuticals, Transcept Pharmaceuticals, Tris, Validus, and WebMD. Drs. Goldman, Cucchiaro, Deng, and Loebel are employees of Sunovion Pharmaceuticals Inc.

Pain Medicine Ethics Forum: Key Opinion Leaders with Industry Conflicts of Interest Should Be Able to Hold Leadership Positions in Professional Medical Associations.

Disclosure: Depomed, Advisory Board; Spinal Modulations, Data Safety and Monitoring Board; Boston Scientific, Consultant; Jazz Pharmaceutical, Advisory Board and Speaker; Jansen, Advisory Board; Zynerba, Advisory Board; Daiichi Sankyo, Advisory Board and Coronado Biosciences, Advisory Board. In 2009, the Journal of the American Medical Association published a proposal for controlling conflicts of interest between Professional Medical Associations (PMAs) and industry [1]. The proposal recommended $0 thresholds for presidents, officers, and board members. It also recommended $0 funding of any PMA activities. The premise for this recommendation was based on research showing the power of gifts to bias physician choices. However, it was not based on research on the effects of payments made to consulting physicians, those serving on scientific advisory boards for …

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Preliminary Results of a Phase 1/2 Clinical Trial of Cndo-109-Activated Allogeneic Natural Killer Cells in High Risk Acute Myelogenous Leukemia Patients in First Complete Remission

BACKGROUNDWe previously reported that resting human peripheral blood NK cells can be primed to kill NK-resistant tumor cells by co-incubation with a lysate of the leukemia cell line CTV-1. CNDO-109 is a clinical-grade CTV-1 lysate that primes NK cells ex-vivo to kill NK-resistant acute myeloid leukemia (AML) cells. CNDO-109-activated NK cells can be cryopreserved and remain primed when thawed. Incubation or treatment with IL-2 is not required. We report preliminary safety, outcome and NK chimerism data from an ongoing Phase 1/2 transitional clinical trial of CNDO-109-NK cells.METHODSA 3x3 dose escalation phase 1 trial was opened in 2013 for patients with AML in 1st CR with high-risk disease and no conventional treatment options. Patients were given preparative chemotherapy of cyclophosphamide and fludarabine on Study Days -6 to -2, followed on day 0 by a single dose of CNDO-109-activated NK cells at the following doses; cohort 1 = 3×105, cohort 2 = 1×106, cohort 3 = up to 3×106 cells/kg recipient body weight. The MTD will set the dose for the transitional phase 2 trial.CNDO-109-NK cells were generated from a single apheresis collection from HLA-haploidentical related donors. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) and co-incubated with CNDO-109 lysate (Coronado Biosciences) overnight under cGMP conditions. After lysate removal cells were cryopreserved in dosed aliquots and released for infusion. Exogenous IL-2 was not used. Quality control testing of the final product included NK purity (>50%), viability (> 70%), potency, sterility, mycoplasma and endotoxin. Residual T cell contamination (<104 cells/kg patient body weight) was a lot release safety criterion. Patients were assessed for safety and efficacy whilst expansion, proliferation and persistence of donor NK cells were assayed by molecular-based chimerism techniques, and by flow cytometry, where informative markers were available. Pre- and post-treatment samples from cohort 3 patients are being tested for flow cytometric and molecular MRD (University of Washington - Walter, R.B.2013 and NextGene Sequencing).RESULTSSeven eligible patients have been enrolled. All products met lot release and contained activated NK cells as determined by killing of NK resistant Raji cells as well as increased expression of CD69 & CD25: TableBaseline(Resting NK Cells)Activated NK CellsChangep-valueRaji Cell Lysis15.2%+/- 10.0%29.7% +/- 13.9%0.008CD69+ Expression5.9% +/- 2.9%42.6% +/- 12.5%0.008CD25+ Expression3.1% +/- 1.8%5.8% +/- 1.9%0.008No infusional toxicity, adverse events attributed to NK therapy, GvHD nor deaths have been reported. As expected, all patients experienced transient myelosuppression (approx. 2 weeks). 3 patients suffered early relapse post-treatment (2 in cohort 1, 1 in cohort 2; average time to relapse from CR1 for these 3 patients was 104 days).In 5 of 7 evaluable patients, persistence of donor activated NK cells was observed from Day +7 post-infusion (chimerism = 1%-84%) to as late as day +56 in one patient. Flow cytometric comparison of donor NK cells and patient NK cells in the same sample by selective gating on the mismatched HLA allele showed that circulating donor NK cells typically differed from recipient NK cell in higher expression of NKG2A (e.g. 63.5% vs 2.7%), CD57 (e.g. 80.5% vs 13.2%) and CD69 (e.g. 11.6% vs 2.52%) suggesting a more normal, mature and activated phenotype than the endogenous host NK population.Even after loss of circulating donor primed NK cells, 2 of the 3 patients tested showed persistence of low levels of activated autologous NK cells (~10-20% of circulating NK) exceeding the numbers circulating pre-CNDO-109 NK treatment, out to Day +56, suggesting that the therapy may induce endogenous NK activation to enhance the patients’ innate immunity to AML.CONCLUSIONSThis establishes proof of concept that CNDO-109-NK can persist transiently in patients with lasting microchimerism for > 1 month and can induce activation of endogenous NK cells in patients treated without cytokine administration. To date 4 of the 7 patients enrolled remain relapse free (max. = 410 days post CR1 date). These results highlight the potential of CNDO-109-NK cells in the treatment of AML and other malignancies. DisclosuresMiller:Coronado Biosciences: Consultancy. Hillman:Coronado Biosciences: Employment. Silver:Coronado Biosciences: Employment. Szarek:Coronado Biosciences: Consultancy. Lowdell:Coronado Biosciences: Consultancy, Patents & Royalties. Gorelik:Coronado Biosciences: Employment. Rowinsky:Coronado Biosciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Male Circumcision as a Religious Ritual

Incorrect Financial Disclosure: In the Original Investigation titled “Undertreatment, TreatmentTrends, andTreatmentDissatisfactionAmongPatientsWithPsoriasis and Psoriatic Arthritis in the United States: Findings From theNational Psoriasis Foundation Surveys, 2003-2011” publishedonlineAugust 14, 2013, and also in the October print issue of JAMA Dermatology (2013;149[10]:1180-1185. doi: 10.1001/jamadermatol.2013.5264), incorrect information appeared in the Conflict of Interest Disclosures section of the Article Information on page 1185. That section should have appeared as follows: “Dr Armstrong has received research grants or consultant honoraria from Abbott Laboratories, Amgen, Inc, and Janssen Biotech. Dr Lebwohl has been a consultant and/or investigator for Abbott Laboratories, Amgen, Inc, Anacor Pharmaceuticals, Inc, BioLine RX, Ltd, Celgene Corporation, Columbia Laboratories, Inc, Coronado Biosciences, Dermipsor, Ltd, Eli Lilly & Co, Galderma, GlaxoSmithKline-Stiefel, Janssen Ortho Biotech,LEOPharmaceuticals,MaruhoCo,Ltd,MedaPharmaceuticals,Novartis,Pfizer, Ranbaxy Laboratories, Ltd, Thesan Pharmaceuticals, and Valeant Pharmaceuticals. The other authors reported no disclosures.” This article was corrected online.

Death Receptor 3 (DR3) Is Expressed By Innate Lymphoid Cells (ILC) and Ligation By Tumor Like Antigen-1 (TL1A) Leads To Costimulation and Significant ILC Expansion

ROR-gt expressing innate lymphoid cells (ILCs) are unique lymphocytes that play important roles in the immune system and have applications to hematopoietic cell transplantation. In fetal life, ILCs orchestrate lymph node organogenesis, while in adults they facilitate the repair of damaged lymphoid structures and mediate mucosal immunity. ILCs function through the production of IL-22 and the expression of TNF-superfamily molecules (lymphotoxin, BAFF, and OX40L) that act on stroma or other lymphocytes, respectively. ILCs are almost exclusively found in the secondary lymphoid tissues (i.e., lymph nodes) and are essentially absent from the peripheral blood, making the study of these cells difficult and clinical application nearly impossible. To overcome these limitations, we devised a method to generate ILCs from hematopoietic stem cells (HSCs) cultured on irradiated stroma in the presence of cytokines (IL-7, IL-15, SCF and FLT3L) [Blood 11:4052-5, 2011]. After ∼21 days of culture, functional ILCs and conventional NK cells differentiate. While this system robustly leads to the ILCs differentiation, GMP-compatible methods of expansion will be required for clinical translation. We sought to identify factors involved in ILC growth and proliferation. After screening gene expression arrays on purified ILCs, the TNF superfamily receptor, known as Death Receptor 3 (DR3, TNFRSF25) was selected for further study. DR3 expression was confirmed using quantitative PCR. Compared to cNK cells, ILCs expressed significant quantities of mRNA for DR3 (p<0.001). While DR3 contains death receptor signaling domains, under some conditions it can also mediate T cell growth. We assessed DR3 functionality in purified ILCs by stimulating them with recombinant TL1A (TNSF15), the only reported ligand for DR3. Consistent with known effect of TL1A on NF-kB activation in T cells, NK-kB phosphorylation was also observed in ILCs. To determine the impact of TL1A on ILC function, IL-22 production was tested. When TL1A was added to ILCs, there was surprisingly no IL-22 production by intracellular cytokine staining or ELISA. We reasoned that TL1a may costimulate ILC activation and combined TL1A with IL-1b and IL-23, known to activate IL-22 production in ILCs. Compared to IL-1b and IL-23, the addition of TL1A led to a significantly higher percentage of IL-22 producing ILCs (9.3% vs. 23.3%, n=8, p<0.001) and more IL-22 production as determined by ELISA (3399 pg/ml vs. 8757 pg/ml, n=3, p<0.001). Nearly identical results were obtained for IL-8 production (p<0.001). Prior studies in T cells show that DR3 signaling increases the expression of the high affinity IL-2 receptor (CD25). While TL1A alone did not increase CD25 on resting ILCs and IL1b + IL-23 only marginally increased CD25 expression (∼1.7x increase from baseline), the combination of TL1A and IL1b + IL-23 led to significantly higher amounts of CD25 on the surface of ILCs (∼3.8x induction). We then tested whether IL-2 could be used to expand ILCs in vitro. Purified ILCs were treated in the following conditions: media (control), TL1A alone, IL1b + IL-23, or the combination of TL1A + IL-1b + IL-23 for 16 hours. Cells were then washed and cultured in media containing IL-2 (1,000U/ml). In short term (5 day) cultures, there was significantly more proliferation with TL1A + IL-1b + IL-23 (14.4% vs. 21.2% vs. 17.4% vs. 42%, n=7, p<0.001) as measured by CSFE dilution. When ILCs were cultured for 14 days, TL1A + IL-1b + IL-23 resulted in significantly greater expansion than IL1-b + IL23 cultured cells (39.3x vs. 14x, n=7, p=0.007). Since TL1A has been associated with skewing of T cells to IL-17 production and the onset of inflammatory conditions (Crohn's disease), we assessed the expanded ILCs for the loss of IL-22 and/or the acquisition of IL-17. ILCs expanded in the presence of TLA1 + IL-1b + IL-23 had no change in their surface phenotype or capacity to produce IL-22. Importantly, neither IL-17 nor changes in RORgt or AHR mRNA expression were detected after expansion. Collectively, these studies identify a novel axis where DR3/TL1A signaling costimulates IL1b and IL-23 induced production of IL-22 and results in the expression of IL-2R (CD25) along with the associated proliferative response to IL-2. These studies significantly advance our ability to devise GMP-compliant methods to generate ILCs and pave the way for adoptive transfer experiments using ILCs in humans. Disclosures:Miller:Coronado Biosciences: Scientific Advisory Board Other.

Characterization Of a Weakly Expressed KIR2DL1 Allele

The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR. Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of promoter elements revealed a cluster of three single nucleotide polymorphisms (SNPs) in the distal promoter approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a functional binding site for the ZEB1 transcription factor. Individuals possessing the ZEB1 site in their KIR2DL1 promoter produce high levels of a non-translatable distal KIR2DL1transcript that inhibits transcription from the proximal promoter, resulting in weak expression of this allele.This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract HHSN261200800001E. Disclosures:Miller:Coronado Biosciences: Scientific Advisory Board Other.

Early NK Cell Proliferation After Umbilical Cord Blood Transplantation Is Associated With Superior Disease-Free Survival Due To Reduced Leukemia Relapse

Compared to traditional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT) has the potential of triggering graft vs. leukemia (GVL) reactions that make this procedure uniquely curative. Despite this, relapse remains the most common cause of treatment failure. Early after allo-HCT the donor immune system reconstitutes within the host and natural killer (NK) cells are the earliest lymphocyte population to recover. Previous studies by us and other investigators have linked rapid lymphocyte recovery and/or high NK cell numbers early after transplant with less relapse. Mechanistically, the reasons for this are unknown. We hypothesized that NK cell proliferation would be associated with allo-HCT outcomes. In a large data set with short-term follow-up we compared stem cell source to NK cell proliferation at Day 28 after transplantation (using Ki67+ staining). In patients undergoing autologous (n=117), sibling (n=57), single umbilical cord blood (sUCB) (n=62) or double umbilical cord blood (dUCB) (n=50) transplantation there were significant differences in the median NK cell proliferation (2.1%, 3.3%, 3.8%, and 19.2%, respectively, p&lt;0.0001). These results suggest that NK cell proliferation is increased when patients are transplanted with stem cell sources that have an increased number of HLA mismatches (dUCB). We next examined factors extrinsic to the NK cells to determine whether differences in the four patient groups were due to these factors. Regulatory T cells (Tregs) are known to negatively regulate the proliferation and activation of a variety of cell types, including NK cells. We utilized the percentage and absolute number of Tregs (defined as CD4+CD25highFoxP3+CD127low) at Day 28 (available for a subset of the above patient samples) and correlated it with the percentage of proliferating NK cells at Day 28 after allogeneic transplant (n=212). The median percentage of Tregs within the lymphocyte fraction was 1.49%. In patients with higher than 1.49% Tregs, the mean NK cell proliferation was 10.8% +/-16.4. In contrast, patients with lower than 1.49% Tregs had a mean NK cell proliferation of 21.2% +/-24.3 (p=0.0004). A nearly identical trend was observed for the absolute numbers of Tregs, suggesting that Tregs may play an extrinsic role in NK cell proliferation after allo-HCT. To further address differences in lymphocyte proliferation and clinical outcomes, we used Ki67 staining to assess T cell (CD4+ and CD8+) and NK cell (CD3-CD56+) proliferation at Day 28 in a different subset of patients undergoing dUCB transplant with acute leukemia or MDS (80%) or other malignant disorders using either myeloablative (42%) or reduced intensity conditioning (58%) and a median of one year follow-up (n=53). There was no association of transplant outcomes with T cell (CD4 or CD8) proliferation. In the NK cell compartment there was a wide range in the percentage of proliferating NK cells (0-86%), with a median of 20%. Patients were segregated into high (&gt;20%) and low (&lt;20%) NK cell proliferating groups and assessed for cytokine levels and transplant outcomes. At Day 28 soluble IL15 levels were not different between high and low NK cell proliferating patients. There was no significant association between NK cell proliferation and the probability or time to neutrophil recovery (p=0.15) or treatment related mortality (p=0.88). Excluding the 14 patients who developed aGVHD prior to day of NK cell assessment (Day 28), the high NK proliferating group had a trend toward a higher cumulative incidence of aGVHD (46% vs. 25%, p=0.17). Using multivariable analysis to control for a variety of patient and disease-associated variables, patients with high NK cell proliferation had significantly better disease-free survival (HR=0.29, 95%CI=0.12-0.71, p=0.01) (Figure 1). Strikingly, patients with high NK cell proliferation were 4-times less likely to relapse (HR=0.24, 95%CI=0.08-0.77, p=0.02). Collectively, these studies show that early NK cell proliferation is associated with superior outcomes after dUCB transplantation, due to reduced relapse, and that this is partly modulated by Tregs. Prospective strategies to increase NK cell proliferation may be of therapeutic benefit to improve transplant outcomes.Figure 1Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant.Figure 1. Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant. Disclosures: Wagner: Novartis: Research Funding. Miller:Coronado Biosciences: Scientific Advisory Board Other.

Human IL-22 Producing CD56+ RORγt+ Innate Lymphoid Cells and Conventional NK Cells Have Distinctive Developmental Trajectories and Are Separate Cell Lineages

AbstractAbstract 1219Human IL-22 producing RORγt+ innate lymphoid cells (ILC22) and conventional NK (cNK) cells are present in secondary lymphoid tissues. Both cell types have an immunophenotype that correspond to stage III NK progenitors (CD56+/−CD117highCD94−), leading us and others to speculate that the IL-22 producing cells are part of the NK lineage and can give rise to cNK cells (Tang, Blood, 2010, Cupedo, Nat Imm, 2009 and Colona, Immunity, 2009). However, recent fate mapping studies in mice suggest that these cell types are separate lineages (Sawa, Science 2010). Given the significant phenotypic and functional differences between human and murine ILC22 cells, this issue is unresolved in humans. To address this, we used an established differentiation system where UCB-derived CD34+ cells are cultured on irradiated fetal liver stromal cells in the presence of IL-3 (5 ng/ml, for the first week), IL-7 (20 ng/ml), SCF (20 ng/ml), FLT3L (10 ng/ml) and IL-15 (10 ng/ml). We have previously demonstrated that this model precisely recapitulates NK cell developmental intermediates, as well as IL-22 producing ILCs (Gryzwacz, Blood, 2005 and Tang, Blood, 2011). We first set out to determine whether it was possible to distinguish IL-22 producing ILCs from cNK using intracellular cytokine staining and a panel of mAbs. Non-IL-22 producing cNK cells showed a CD56+CD117lo/-CD7+/−LFA-1high phenotype, while ILC22 cells were completely contained within the CD56+CD117highCD94−CD7−LFA-1− fraction. Purification of these two populations showed that ILC22 cells expressed high quantities of transcription factors associated with IL-22 production including AhR and RORγt, while these were absent or barely detectable in cNK cells (p<0.0001). Conversely, T-bet and Eomes were highly expressed in cNK progenitors, but not ILC22 cells. While cNK cells expressed granzyme and perforin, classical NK-associated receptors (NKp30, NKp46, NKG2A, NKG2D, CD8, CD16 and KIR) and showed degranulation (CD107a) and produced IFN-γ in response to K562 targets or IL-12+IL-18, ILC22 cells did not. Thus, ILC22 and cNK cells were distinguishable by transcription factor expression, surface receptor expression and function. To investigate the lineage relationship between ILC22 cells and cNK cells, stage III NK progenitors (defined as CD56+CD117+CD94−) were purified on the basis of LFA-1 expression and then further cultured. Cells that expressed LFA-1 (i.e., cNK progenitor cells) rapidly acquired CD94, and differentiated into stage IV and V cNK cells. Conversely, the vast majority of cells that lacked LFA-1 cells (i.e., ILC22 cells) acquired neither LFA-1 nor CD94, thus never differentiate into stage IV and V cNK cells. These results suggest that ILC22 cells represent a separated and stable cell lineage from cNK cells. To further address this and investigate the developmental requirements for cNK and ILC22 cells, CD34+ hematopoietic stem cells were cultured in the above conditions with or without IL-7 and SCF, which are known to be critical cytokines for lymphoid tissue inducer (LTi) cell generation in vivo (a population similar to ILC22 cells). In the absence of IL-7 and SCF, cNK cells developed normally while ILC22 cells did not develop. These results show that cNK cells differentiated even in the absence of ILC22 stage III cells, which require SCF and IL-7 for differentiation. Conversely, in the absence of IL-15, CD34+ cells showed a complete block in cNK differentiation and instead gave rise to a CD56+ILC22 cells, and their phenotype and function were normal. Thus, while human ILC22 cells and cNK progenitors have a phenotype that overlaps with stage III NK progenitors, these studies demonstrate that they are separate cell lineages, with differing phenotype, transcription factor expression, developmental requirements and functions. Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Bi-Specific Killer Cell Engagers (BiKEs) Signaling Through CD16 and Targeting CD33 (CD16 × CD33), Triggers Natural Killer (NK) Cell Cytotoxic and Cytokine Production Against Refractory Acute Myeloid Leukemia (AML)

AbstractAbstract 256AML accounts for a large number of annual deaths due to leukemia worldwide. NK cells are effectors of the innate immune system that mediate the graft versus leukemia (GVL) effect in patients with AML and other hematologic malignancies. The safety and success of using haploidentical NK cell infusions to treat patients with AML has been previously demonstrated, but this therapeutic approach has limitations of potency and lacks specificity for leukemic targets. NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) usually occurs trough the binding of the activating receptor FcγRIIIA (also known as CD16) to the Fc portion of antibodies. CD16 is expressed on most CD56dim NK cells and induces NK cell activation, leading to interferon (IFN-γ) and tumor necrosis factor (TNF-α) secretion. CD16 shedding occurs upon NK cell activation, an effect that we have shown is mediated by a specific metalloproteinase called a disintegrin and metalloproteinase-17 (ADAM-17). We hypothesized that a BiKE antibody molecule, developed specifically to signal through CD16 and targeting the myeloid differentiation antigen CD33 (i.e., CD16 × CD33 BiKE) would enhance NK cell function against AML. In addition, we predicted that selective inhibition of ADAM-17 activity would prevent CD16 shedding and enhance the activity of the CD16 × CD33 BiKE. Bone marrow (BM) samples from 10 patients with primary refractory AML were obtained from our leukemia tissue bank and used as targets. CD33 was expressed on 8 of the 10 BM samples. Purified NK cells from healthy donors were isolated and incubated overnight in the absence of cytokines. NK cells and AML BM samples were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE, scFv anti-CD16 (negative control) or DTCD33 × CD33 (anti-CD33 plus anti-CD33 spliced to truncated diphtheria toxin - negative control). NK cells under the above conditions were all treated with or without 5 uM of an ADAM17 inhibitor (Incyte). After 4 hours in culture, intracellular CD107a degranulation assay and intracellular TNF-α and IFN-γ assays were performed. A MTS survival assay was also performed on the target cells. In 8 of 10 AML samples, NK cells significantly degranulated and secreted cytokines (TNF-α and IFN-γ) only when treated with the CD16 × CD33 BiKE reagent. The MTS survival assay confirmed significant AML target cell death in the presence of the CD16 × CD33 BiKE. Combined treatment with the CD16 × CD33 BiKE and the ADAM17 inhibitor led to a significant increase in cytokine TNF-α and IFN-γ secretion by NK cells when compared to treatment with CD16 × CD33 BiKE alone. Phenotypic analysis of the NK cells after treatment with the ADAM17 inhibitor revealed a significant decrease in CD16 shedding as predicted. Of note, no NK cell activity was triggered by the 2 AML BM samples that lacked CD33 expression arguing in favor of the specificity of this molecule for CD33 positive AML. We then analyzed the ability of NK cells to kill multiple targets in the presence of the CD16 × CD33 BiKE over time. NK cells and HL60 cells (CD33 positive targets) were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE and incubated overnight in the presence or absence of the ADAM17 inhibitor. On the following day, a second target exposure with chromium (Cr-) labeled HL60 cells was added to the culture in order to visualize the effect of NK cells on second targets. After 4 hours, intracellular CD107a, TNF-α, IFN-γ and standard Cr- release assays were performed. In the presence of the CD16 × CD33 BiKE, NK cells showed significantly more degranulation killing and secreted more cytokines in response to secondary targets. Cytokine secretion was also enhanced by the addition of the ADAM17 inhibitor to the BiKE. Collectively, these findings support the ability of a CD16 × CD33 BiKE to trigger NK cell activation through direct signaling of CD16, inducing secretion of cytokines, lytic granules and causing target cell death in resistant AML BM samples and HL60 targets. BiKE enhanced AML killing occurs over a wide range of CD33 target cell density as long as some expression is present. In addition, targeting ADAM17 prevents activation induced CD16 shedding and enhances NK cell cytokine production when combine with therapeutic antibodies. NK cell directed therapy by these compounds could specifically enhance the anti-leukemic potency and efficacy of NK cell adoptive therapy against myeloid disorders. Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Optimal Xenogeneic Adoptive Transfer of Human NK Cells: Fresh NK Cells and IL-15 Administration Are Superior to Frozen NK Cells and IL-2

AbstractAbstract 346Expansion of NK cells after adoptive transfer is a major determinant of their anti-tumor efficacy but the mechanisms of their expansion are not completely understood. To study in vivo NK cell expansion and the relative merits of fresh versus frozen NK cell products, we used xenotransplantation of human GMP NK cell products (provided by two PACT centers funded by the NHLBI) into immune deficient mice. NOD/IL-2Rγc/Rag (NOG) that lack T-, B-, and NK cells and that have a macrophage defect that renders recipients highly amenable to human cell engraftment, were given 250 cGy total body irradiation, and infused with fresh or frozen NK cells. Two products were tested: 1) an enriched fresh NK cell product obtained by CD3 and CD19 depletion followed by overnight activation with IL-2 or IL-15 (U. of Minnesota) or 2) ex vivo expanded NK cells using membrane bound IL-15 and 41BB-L transduced K562 cells with 10 U/ml IL-2 in G-Rex devices for 10 days (Baylor). NK cells (1–2 million NK cells for each individual experiment) were given IV and mice were treated with no cytokines, 5 or 10 mg of IL-2 (Novartis), or 5 mg IL-15 (produced by the NCI for clinical use) as 3 injections per week for 2 weeks. Peripheral blood (Table), spleen and marrow were evaluated on days 7, 14, 21 and 28 after adoptive transfer. Counts were converted to absolute cell numbers per 100 μL of blood or the number of cells recovered per spleen or after flushing of a single femur. On day 7, the number of huNK cells in blood in the absence of cytokine administration was low with both cell products and numbers continued to diminish by day 14. In marked contrast, every other day IL-2 or IL-15 significantly increased huNK cells in murine blood. On day 7 and 14, ex vivo expanded NK cell products resulted in significantly higher numbers of huNK in mice receiving 5μg IL-2 compared to mice who received fresh NK cells activated overnight with IL-2. With 5μg IL-2, huNK number decreased between day 7 and 14 irrespective of cell product. In contrast, only IL-15 lead to increased numbers of huNK cells between days 7 and 14. Previously cryopreserved NK cells showed significantly worse survival for both cell products, with a larger fold decrease seen with ex vivo expanded cells. Blood collected from cytokine treated mice at day 14 after infusion of fresh products contained huNK cells that were fully functional as assessed by potent CD107a degranulation, TNF and IFN production after exposure to K562 target cells as well as augmented IFN production induced by IL-12 and IL-18. On day 8, Ki67+ (proliferating) huNK cells were significantly higher with IL-15 compared to IL-2 (both at 5μg) in marrow for fresh (76% vs 35%, p=<0.001) and ex vivo expanded (88% vs 53%, p=0.003) NK cells. Ki67+ proliferating NK cells had decreased by day 14 under all conditions. With fresh NK cells and IL-15 on day 8, there were 1371 huNK relative counts recovered from spleen compared to 450 recovered from marrow (p=0.01). In marked contrast, ex vivo expanded NK cells (that express a lower percentage of CD62L compared to fresh NK cells) had a reverse homing pattern with 874 huNK cells in spleen versus 2146 in marrow (p=0.07). There was a significant difference between the two cells products in terms of the absolute numbers of huNK cells found in marrow at day 8 (p=0.0007). We conclude that 1) ex vivo expanded NK cells home better to BM which may be useful in treating AML localized to BM, whereas fresh NK cells may be preferable for homing to lymphoid tissue in the periphery; 2) at early time points fresh huNK cells were less abundant than expanded cells but catch up at late time points; and 2) IL-15 is more effective than IL-2 on a mcg basis. Overall, fresh and ex vivo expanded NK cells have different properties and kinetics after infusion. This xenogeneic model results in definitive readouts to optimize the best characteristics of each cell product to tailor a product to have maximal efficacy for clinical testing.Results of experiments with fresh or ex vivo expanded NK cellsExp.Activated NK sourceIn vivo cytokine# miceAvg hu NK Day 7/100 μL muBloodAvg hu NK Day 14/100 μL muBlood1Fresh NKnone611724IL2-5 ug623853Ex vivo expandednone6281IL2-5 ug67602352Fresh NKnone421216IL2-5 ug446593IL15-5 ug45401151Ex vivo expandednone4639IL2-5 ug4888181IL15-5 ug4322129303Fresh NKnone12-1614155IL2-5 ug12-16331169IL2-10 ug12-16197182IL15-5 ug12-162661177Ex vivo expandednone12-16223IL2-5 ug12-16323106IL2-10 ug12-16372673IL15-5 ug12-167351955 Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Comparison of IPSS and IPSS-R Scoring in a Population Based Myelodysplastic Syndromes (MDS) Study

AbstractAbstract 3841 Introduction:The International Prognostic Scoring System (IPSS) has recently been revised (IPSS-R) to capture more inclusive cytogenetic characterization, more stringent blast description, and more detailed cytopenia assignments. To validate the IPSS-R in a prospective cohort of patients, we compared IPSS and IPSS-R categorization within a group of newly diagnosed MDS patients in a prospective population-based epidemiologic study in Minnesota. Methods:We identified all newly diagnosed MDS cases in Minnesota between the ages of 20 and 85 years from April 1, 2010 to April 2012. Our process includes rapid case identification by the Minnesota Cancer Surveillance System (MCSS), patient contact and enrollment, central medical review including: 1) independent review by two hematopathologists to confirm diagnosis and WHO subclassification; 2) independent cytogeneticist's review for classification as defined by IPSS and IPSS-R; and 3) medical chart review with prospective calculation of IPSS, IPSS-R, and WPSS by a an oncologist. Results:Classification of the 128 patients by IPSS versus IPSS-R is shown (Table)IPSS-R dxTotalVery LowLowIntermediateHighVery HighIPSS dxLow161910036INT-1410138035INT-2008172550High000077Total2029222532128Kendall's Tau = 0.77In this comparative classification, IPSS-R scoring refined the MDS prognostic risk groups. Low IPSS remained low risk in the IPSS-R spanning the Very Low and Low groups, while all high risk IPSS remained in the Very High risk group in the IPSS-R. However, those in the INT-1 and INT-2 IPSS categories were inconsistently reclassified in the IPSS-R. INT-1 IPSS spanned from Very Low to High and INT-2 IPSS spanned from Intermediate to Very High in the IPSS-R. While reclassification of this prospective cohort resembles results in the IPSS-R reports, ongoing follow-up will be needed to determine if the survival and AML progression measures are reproducible. Conclusion:Until molecular data are available to refine future prognostic scoring systems, the IPSS-R enhances our prognostic risk stratification abilities and is applicable to the general MDS population. As treatment decisions are based on this initial prognostic risk assessment, the more diverse differentiation of risk within the IPSS-R will directly refine prognosis and impact decisions on therapy. Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Education and Cytokine Receptor Balance Play a Critical Role in Long Term Survival of the Most Differentiated Natural Killer (NK) Cells.

AbstractAbstract 2148NK cells can effectively treat advanced acute myelogenous leukemia if they survive and maintain functional competency after adoptive transfer. In the peripheral blood of healthy human donors the balance between Killer-cell immunoglobulin-like receptor positive (KIR+) NK cells, which are more differentiated and functional in terms of tumor killing and cytokine production, and KIR− NK cells is maintained by proliferation, differentiation, and survival. The mechanism that governs this process is largely unknown. Given that we and others have shown that KIR− NK cells proliferate better than KIR+ NK cells with IL-15 in vitro, we chose to study whether the balance between these two subsets could be maintained by enhanced survival of KIR+ NK cells. To explore this further, peripheral blood mononuclear cells (PBMCs) from healthy human donors were put in serum free conditions overnight and NK cell survival was assessed. A significantly larger proportion of the KIR− NK cells died (33±2.41%) compared to well characterized KIR+ NK cells: KIR2DL1/DS1 (8.9±1.22%), KIR2DL2/DL3 (6.7±0.51%), and KIR3DL1 (6.1±0.95%) (all P < 0.0001). A similar effect was seen when looking at Annexin V+ cells that still had membrane integrity. In order to further understand why KIR+ NK cells exhibited superior survival, we quantified expression of anti-apoptotic markers and found that KIR-expressing NK cells express more Bcl-xL, by Q-PCR and Western blotting. They also express more Bcl-2, by Q-PCR and flow median fluorescence intensity (MFI), particularly when the cells are treated with IL-15 overnight (P ≤ 0.0005). We next investigated expression of cell death family receptors Fas and Fas-Ligand (FasL). Under every condition tested Fas was expressed at significantly higher levels in the KIR− NK cells (P ≤ 0.03). FasL on the other hand was only expressed at higher levels in KIR− NK cells post 60 hrs in culture alone or with IL-2 but not IL-15. Although NK cells can signal through IL-7 or IL-2, only IL-15 mouse knockouts demonstrate a complete deficit of NK cells. Both IL-15 and IL-2 signal through 2 common shared components, IL-2Rb and the common g chain, and an individual cytokine specific component, IL-2Ra for IL-2 and IL-15Ra for IL-15. Interestingly, although all the components for IL-2 signaling are present on the NK cell, IL-15Ra bound to IL-15 is presented in trans from other cells or in complexes in order to achieve IL-15 signaling in NK cells, thereby discarding the necessity for IL-15Ra expression on the NK cell itself. Based on this information, the expression of all of these components was evaluated on KIR+ and KIR− NK cells. We found that IL-2Ra is expressed, by MFI, at statistically significant lower levels (P ≤ 0.0023) on KIR+ NK cells when compared to KIR− NK cells. In contrast, the common g chain is expressed, by MFI, at statistically higher levels (P < 0.0001) on KIR+ NK cells. No statistical differences were seen in IL-15Ra and IL-2Rb expression. We hypothesized that competition for common components, IL-2Rb and the common g chain, between IL-2Ra and IL-15Ra (in trans) plays a role in regulating survival through IL-15. In agreement with this premise, IL-2Ralo NK cells died significantly less than IL-2Rahi NK cells with transpresentation of IL-15 (3.5±1.5% vs. 30.8±2.6%, P = 0.0025). Transient overexpression of IL-2Ra ablates this enhanced survival. Finally we wanted to see if the educational status of the NK cell has a role in its survival. Of the KIR+ NK cells the educated NK cells, which have KIRs corresponding to self-ligands, represent the most differentiated and functional subset. We found that educated NK cells consistently had more Bcl-2 than uneducated NK cells (P ≤ 0.0021). This was associated with enhanced common g chain expression in the educated NK cell population (P = 0.0001). Previous studies have shown that CD57+ NK cells are long lived. We show here that educated NK cells had a statistically larger proportion and density of CD57 expression on a per cell basis (P < 0.0001). These data indicate that educated NK cells could survive better, through enhanced Bcl-2 upregulation, and persist longer, as determined by CD57 expression, than their uneducated counterparts. In summary we find that the mechanism for maintenance of more differentiated NK cells involves enhanced cell survival and differential responses to cytokines limited by the alpha chains of IL-2 and IL-15, which may be manipulated for therapeutic purposes. Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Impact of Umbilical Cord Blood (UCB) T Regulatory Cells (Tregs) On Infection Risk Early After UCB Transplant

AbstractAbstract 4188We have previously shown that the infusion of ex vivo expanded UCB-derived Tregs at a dose of ≥3 × 106/kg immediately after UCB transplantation is associated with a reduced incidence of acute GVHD as compared to historical controls (p=0.05, Blood 2011; 117:1061). Together with a trend toward increased dual chimerism, these data support the conclusion that UCB Tregs are functional in vivo. We therefore asked the question whether Treg increased the increase risk of opportunistic infection (OI), particularly in the early transplant period when Treg are detectable in the peripheral blood. Opportunistic infections were defined as viral or fungal infections that were either documented or sufficiently supported by radiographic studies. For this analysis, patients receiving UCB-Tregs (n=24) were compared to historical controls (n=65) treated the same but without Tregs. All patients received a nonmyeloablative conditioning consisting of cyclophosphamide 50mg/kg/x1day, fludarabine 40 mg/m2/x5d, and total body irradiation 200 cGy/x1d and posttransplant immunosuppression consisting of mycophenate mofetil in combination with cyclosporine A (CsA, n=17 Treg patients and all controls) or sirolimus (n=7 Treg patients). Tregs were initially enriched by CD25+ selection (Miltenyi CliniMACS) from a partially HLA matched 3rd UCB unit Tregs were activated and expanded for 18+1 days in the presence of anti-CD3/CD28 mAb coated beads and rhIL-2. 90% of patients received one or two doses of 3 × 106/kg on d+1 (dose 1) and d+15 (dose 2) after UCB transplant. The median follow up of survivors is 2.6 (2.0–3.3) years and 4.8 (2.0–6.9) years for Treg and control patients, respectively. For non-Treg and Treg groups, the incidence of OI at day +100 was 58% (95%CI, 38–79%) and 57% (95%CI, 43–70%), respectively (p=0.74) at a median of 28 (21–87) and 41 (1–82) days after UCBT. Viral infections represented the majority of OI observed for Treg (58% [95%CI, 38–79%]) and non-Treg (55% [95%CI, 42–69%]) patient populations (p=.67). The overall day 0 to day +180 cumulative density of OI was 2.10 infections/1000 patient-days in Treg versus 1.52 infections/1000 patient-days in non-Treg patients (RR 1.39, p=.08). Notably, there was increased density of OI through day +30 with 18.06 infections/1000 patient-days for Treg patients versus 7.71 infections/1000 patient-days (RR 5.35, p=.02). In contrast, between day +31 and +180, the density of infection in Treg vs non-Treg patients was 4.22/1000 patient-days versus 7.22/1000 patient-days (RR 0.58, p=.07), respectively. The predominant viral infection observed in Treg patients through day +30 was HHSV-6 (n=9) with only two cases of CMV viremia and one case of parainflunenza upper respiratory infection). Despite the increased risk of early viral infections among Treg recipients, treatment related mortality at 1 year (18% vs. 10 %, p=0.36) and disease free survival at 2 years (33% vs. 34%, p=.95) were similar compared to historical controls. Interestingly, of the 7 recipients of Treg in the presence of sirolimus, none developed OI and 5/7 are alive. In conclusion, there is an increased risk of OI early after transplant in recipients of UCB Tregs and posttransplant pharmacological immunosuppression. Whether higher or multiple Treg doses may be more effective in suppressing acute GVHD, OI risk may be increased especially when used in combination with CsA. A dose escalation study UCB-derived Tregs expanded with KT86/64 artificial antigen presenting cells in the presence of sirolimus/MMF immune suppression is underway. Disclosures:Miller:Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

The CombiRx Trial: A Multi-Center, Double-Blind, Randomized Study Comparing the Combined Use of Interferon Beta-1a and Glatiramer Acetate to Either Agent Alone in Participants with Relapsing-Remitting Multiple Sclerosis - Clinical Outcomes (PL02.003)

Objective: To determine if combined use of interferon beta-1a (IFN) 30ug weekly and glatiramer acetate (GA) 20 mg daily is more efficacious than either alone in relapsing remitting multiple sclerosis (RRMS). Background This double-blind, multi-center, randomized RRMS clinical trial compared IFN plus GA versus single agent arms with matching placebo. Entry criteria included: EDSS ≤ 5.5, RRMS, age 18-60, ≥2 relapses in prior 3 years and no prior use of either medication. Design/Methods: 1,008 participants were randomized and underwent quarterly clinical assessments until the last subject enrolled completes 3 years in January 2012. Magnetic resonance imaging (MRI) was done at baseline and 6, 12, 24 and 36 months. The primary MRI variable was change in the Z4 composite MRI score (unweighted sum of the individual Z scores for enhanced tissue, T2-hyperintense and T1-hypointense lesion volumes, and normalized CSF - an inverse measure of atrophy) from baseline to 36 months by treatment cohort using general linear models. Assuming differences between groups on the Z4 composite, selected additional measures will be formally addressed as follows: cumulative sum of the number of enhanced and unique active lesions, differences in the cubed root of the T2 and T1 lesion volumes, and differences in brain atrophy analyzed over the 36 months on intent to treat basis. Results: Recruitment occurred from January 2005-April 2009. At baseline 39.7% of participants had enhancements, a median total T2 lesion volume of 7.48 ml (T1-hypointense component 0.84 ml), and mean Z4 composite = 0. On study MRI data will be provided, with clinical outcome data presented separately. Conclusions: CombiRx was a uniquely sponsored collaboration between NIH, academic institutions, practicing neurologists and pharma/biotech; the largest test thus far of the potential for combining immunotherapies in early RRMS. It provides the longest controlled clinical, MRI and biomarker dataset of any MS trial. Supported by: NIH/NINDS (5U01NS045719). Disclosure: Dr. Wolinsky has received personal compensation for activities with Astellas, Bayer Pharmaceuticals Corporation, Bayer Multiple Sclerosis Council, Celgene Corporation, Eli Lilly & Company, Roche Diagnostics Corporation, Novartis, Sanofi-Aventis Pharmaceuticals and Teva Neuroscience as consultant and participant on monitoring and advisory boards. Dr. Wolinsky has received (royalty or license fee or contractual rights) payments from University of Texas Health Science Center at Houston. Dr. Wolinsky has received research support from Sanofi-Aventis Pharmaceuticals, Inc. Dr. Narayana has received personal compensation for activities with Teva Neuroscience. Dr. Nelson has received personal compensation for activities with Bayer HealthCare, Biogen Idec, EMD Serono, National MS Society, MS Association of America, Novartis, Sanofi-Aventis Pharmaceuticals, Inc., Teva Neuroscience, and the University of Massachusetts Medical School. Dr. Nelson has received research support from Novartis and Sanofi-Aventis Pharmaceuticals, Inc. Dr. Datta has nothing to disclose. Dr. Cofield has received personal compensation for activities with GlaxoSmithKline, Inc., Teva Neuroscience, Orthotech Biotech, the Department of Defense, and the American Academy for Orthopedic Surgery. Dr. Cutter has received personal compensation for activities with Sanofi-Aventis Pharmaceuticals, Inc., Cleveland Clinic, Daiichi Pharmaceutical Corporation, GlaxoSmithKline, Inc., Genmab Biopharmaceuticals, Eli Lilly & Company, Medivation, Modigenetech, Ono Pharmaceutical, PTC Therapeutics, Teva Neuroscience, Vivus, University of Pennsylvania, NHLBI, NINDS, NMSS, Alexion, Bayhill Therapeutics, Bayer Pharmaceuticals Corporation, Celgene, Novartis, Consortium of MS Centers, Genzyme Corporation, Klein-Buendel Incorporated, Nuron Biotech, Peptimmune, Somnus Pharmaceuticals, Sandoz, University of Texas Southwestern and Visioneering Technologies, Inc. Dr. Cutter has received compensation for serving on the board of Pythagoras, INC. Dr. Cutter has received research support from various pharmaceutical corporations. Dr. Conwit has nothing to disclose. Dr. Gustafson has nothing to disclose. Dr. Lublin has received personal compensation for activities with Bayer HealthCare Pharmaceuticals, Biogen Idec, EMD Serono, Novartis, Pfizer, Teva Neuroscience, Genmab, Medicinova, Actelion, Allozyne, Sanofi, Acorda, Questcor, Avanir, Roche, Celgene, Abbott, MorphoSys, Johnson & Johnson, Revalesio, Coronado Bioscience, Genzyme, and MedImmune as a consultant.Dr. Lublin has received personal compensation in an editorial capacity for Elsevier: Multiple Sclerosis and Related Diseases. Dr. Lublin has received personal compensation for activities with Bayer HealthCare Pharmaceuticals, Biogen Idec, EMD Serono, Novartis, Pfizer, Teva Neuroscience, Genmab, Medicinova, Actelion, Allozyne, Sanofi, Acorda, Questcor, Avanir, Roche, Celgene, Abbott, MorphoSys, Johnson & Johnson, Revalesio, Coronado Bioscience, Genzyme, and MedImmune as a consultant.Dr. Lublin has received personal compensation in an editorial capacity for Elsevier: Multiple Sclerosis and Related Diseases.

Effect of Teriflunomide on Relapses with Sequelae and Relapse Leading to Hospitalization in a Population with Relapsing Forms of Multiple Sclerosis: Results from the TEMSO Study (S30.003)

Objective: To assess whether teriflunomide reduces the occurrence of relapses with sequelae and relapses leading to hospitalization. Background Incomplete recovery from relapses leads to some degree of disability. Teriflunomide is a novel oral disease-modifying therapy in development for the treatment of RMS. The TEriflunomide Multiple Sclerosis Oral (TEMSO) study showed that teriflunomide reduced the annualized relapse rate by over 30% (p Design/Methods: TEMSO was a 2-year, randomized, double-blind study that assessed the efficacy and safety of teriflunomide 7mg and 14mg in patients with relapsing forms of MS (RMS) (N=1088); a typical patient was 38 years old, had experienced MS symptoms for over 8 years and 2.2 relapses within the past 2 years. Relapse with sequelae (investigator-defined) and relapse leading to hospitalization were analyzed (post hoc) on the intention-to-treat population (N=1086). Annualized rates were derived using a Poisson model, with treatment, Expanded Disability Status Scale (EDSS) score strata at baseline and region as covariates. The risks of sequelae and of hospitalization per relapse were calculated as a raw percentage observed over the study and compared with a Chi-square test. Results: Teriflunomide reduced the annualized rate of relapses with sequelae by 25% (ns) with 7mg and 53% (p Conclusions: In addition to its efficacy on overall relapse rates, teriflunomide significantly reduced the occurrence of relapses leading to hospitalization. Teriflunomide 14mg also significantly reduced the occurrence of relapses with sequelae, the risks of sequelae and of hospitalization per relapse. These benefits may suggest an effect on relapse severity. Supported by: sanofi-aventis. Disclosure: Dr. Miller has received personal compensation for activities with Acordia Therapeutics, Avanir, Biogen Idec, Chelsea Therapeutics, EMD Serono, GlaxoSmithKline, La-Ser, Merck Serono, Novartis, Nuron BiotechONO, Pfizer, Sanofi-Aventis, and Teva Neuroscience. Dr. Miller has received personal compensation in an editorial capacity for Continuum and Real Living with Multiple Sclerosis. Dr. Miller has received research support from Acorda, Biogen Idec, Genentech, Genzyme, Novartis, Roche, Sanofi-Aventis, and Teva Neuroscience. Dr. Lublin has received personal compensation for activities with Bayer HealthCare Pharmaceuticals, Biogen Idec, EMD Serono, Novartis, Pfizer, Teva Neuroscience, Genmab, Medicinova, Actelion, Allozyne, Sanofi, Acorda, Questcor, Avanir, Roche, Celgene, Abbott, MorphoSys, Johnson & Johnson, Revalesio, Coronado Bioscience, Genzyme, and MedImmune as a consultant.Dr. Lublin has received personal compensation in an editorial capacity for Elsevier: Multiple Sclerosis and Related Diseases. Dr. O9Connor has received personal compensation for activities with Abbott Labratories, Inc., Bayer Pharmaceuticals Corporation, Biogen Idec, BioMS, Cognosci, Daiichi Pharmaceuticals Corporation, Serono, Inc., Genentech, Inc., Genmab, Novartis, Roche Diagnostics Corporation, Sanofi-Aventis Pharmaceuticals, Inc., Teva Neuroscience, Warburg Pincus and Wyeth Pharmaceuticals as a consultant. Dr. O9Connor has received research support from Abbott Labratories, Bayer Pharmaceuticals Corporation, Biogen Idec, BioMS, Cognosci, Daiichi Pharmaceutical Corporation, Serono, Inc., Genentech, Inc., Genmab, Novartis, Roche Diagnostics Corporation, Sanofi-Aventis Pharmaceuticals, Teva Neuroscience, Warburg Pincus and Wyeth Pharmaceuticals. Dr. Wolinsky has received personal compensation for activities with Astellas, Bayer Pharmaceuticals Corporation, Bayer Multiple Sclerosis Council, Celgene Corporation, Eli Lilly & Company, Roche Diagnostics Corporation, Novartis, Sanofi-Aventis Pharmaceuticals and Teva Neuroscience as consultant and participant on monitoring and advisory boards. Dr. Wolinsky has received (royalty or license fee or contractual rights) payments from University of Texas Health Science Center at Houston. Dr. Wolinsky has received research support from Sanofi-Aventis Pharmaceuticals, Inc. Dr. Comi has received personal compensation for activities with Novartis, Teva Neuroscience, Sanofi-Aventis Pharmaceuticals, Inc., Merck Serono, Bayer Schering, and Biogen Dompe. Dr. Kappos has received research support from Acorda Therapeutics, Actelion, Allozyne, BaroFold, Inc., Bayer Pharmaceuticals Corporation, Bayhill Therapeutics, Biogen Idec, Boehringer Ingelheim Pharmaceuticals, Inc, Elan Corporation, Genmab, GlaxoSmithKline, Inc., Glenmark Pharma, Merck Serono, MediciNova, Novartis, Sanofi-Aventis Pharmaceuticals, Santhera Pharmaceuticals, Shire, Roche Diagnostics, Teva Neuroscience, UCB Pharma, Pfizer Inc, Swiss MS Society, Swiss National Research Foundation, European Union, Gianni Rubatto Foundation, Novartis and Roche Research Foundations. Dr. Freedman has received personal compensation for activities with Bayer Healthcare, Genzyme Corporation, EMD Serono, Novartis, Sanofi-Aventis Pharmaceuticals, Inc., and Teva Neuroscience. Dr. Freedman has received research support from Genzyme Corporation and Bayer Healthcare. Dr. Olsson has received personal compensation for activities with Biogen Idec, Merck & Co., Inc., Sanofi-Aventis Pharmaceuticals, Inc., Novartis, and Bayer Healthcare. Dr. Olsson has received research support from Biogen Idec, Merck & Co., Inc., Sanofi-Aventis Pharmaceuticals, Inc., Novartis and Bayer Healthcare. Ms. Dive-Pouletty has received personal compensation for activities with Sanofi-Aventis. Dr. Bego-Le-Bagousse has received personal compensation for activities with Sanofi-Aventis Pharmaceuticals, Inc. as an employee. Dr. Confavruex has received personal compensation for activities with Biogen Dompe, Biogen Idec, Gemacbio, Genzyme Corporation, Hertie Foundation, Novartis, Sanofi-Aventis Pharmaceuticals, Inc., Teva Neuroscience, UCB Pharma, Bayer Schering, and Merck Serono. Dr. Confavruex has received research support from Bayer Schering, Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis Pharmaceuticals, and Teva Neuroscience.

Temporal Changes in MRI Activity, Inflammation, Immunomodulation, and Gene Expression in Relapsing-Remitting Multiple Sclerosis Subjects Treated with Helminth Probiotic Trichuris Suis (S30.004)

Objective: To define critical points at which immunological markers and gene expression patterns correlate with changes in brain MRI activity during treatment with Trichuris suis ova (TSO) in the HINT clinical trial for relapsing-remitting multiple sclerosis (RRMS). Background Despite extensive investigation in animal models and promising results in exploratory studies of helminth-induced immunomodulation for autoimmune diseases, including multiple sclerosis, at present the mechanism of action and key operative molecules are not well-understood. Design/Methods: Peripheral blood mononuclear cells (PBMC) and serum were obtained from RRMS subjects before, during, and after treatment with 2,500 live TSO given orally every 2 weeks. Serial determinations of inflammatory markers (CRP, eosinophils), immune responses (PBMC phenotypes),and gene expression (Affymetrix GeneChip 1.0 ST Array) were performed and compared with brain MRI scans read by masked neuroradiologists. Results: New enhancing MRI lesions (NEL) were significantly reduced in 5 of 6 subjects after TSO treatment. In individual subject 110, for whom frequent MRI studies at 2-4 week intervals were performed, a 91% relative reduction in NEL was observed after treatment. Markers of inflammation peaked at 1-2 months after treatment initiation and declined thereafter. CD8+/CD161hi T cells, which may be associated with IL-17 production, decreased in frequency in 2 out of 3 subjects. Gene expression tested in 3 subjects by enrichment analysis demonstrated significant changes (z-score > 4.0) in 17 Gene Ontology (GO) pathways after treatment, including GO:0034143, regulation of TLR-4 pathway; GO:0002684, positive regulation of immune system; GO:0002694, regulation of leukocyte activation; and GO:0004896, cytokine receptor activity. Conclusions: In exploratory studies of RRMS subjects treated with the probiotic helminth TSO, early inflammation, likely due to innate immunity, was followed by favorable adaptive immune responses temporally associated with reduction in brain MRI activity. Supported by: National Multiple Sclerosis Society research grant GR 3613A4/1 to JF. NIH research grant 1R56AI091462-01 to ZF. Disclosure: Dr. Fleming has received personal compensation for activities with Coronado Biosciences as a scientific advisory board member. Dr. Isaak has nothing to disclose. Dr. Hardin has nothing to disclose. Dr. Huston has nothing to disclose. Dr. Boland has nothing to disclose. Dr. Broman has nothing to disclose. Dr. Kendziorski has nothing to disclose. Dr. Field has nothing to disclose. Dr. Fabry has nothing to disclose.

Results of a Phase 1 Clinical Trial of Allogeneic Tumor-Primed Natural Killer Cells in AML

Abstract 946We have previously reported that resting human NK cells can be primed to lyse NK-resistant tumor cells, including AML, by co-incubation with membrane preparations from the leukemia cell line CTV-1. Tumor-primed NK cells can be cryopreserved and remain in a primed state when thawed. Following translation of this production method to GMP-compliance we designed a phase 1 trial for AML patients. PROTOCOL: AML patients meeting the inclusion criteria were enrolled and they and their children HLA-typed and screened for medical suitability for apheresis. KIR mismatched donors in the GvL direction were preferred when available. PATIENT INCLUSION CRITERIA: >60 years in PR (blasts >5<20% in BM) after 2nd course of induction chemotherapy; >60 years in CR2 after re-induction chemotherapy; >60 years in PR or CR after 2 courses of chemotherapy with poor risk disease by conventional cytogenetic criteria; <60 years beyond CR2 but unsuitable for transplantation. CONDITIONING: Fludarabine 25mg/m2/day for 3 days plus a single fraction (2Gy) TBI on day 4. MANUFACTURE OF Tumor-primed NK: T-pNK cells were generated from single 2hr apheresis from haploidentical related donors in 14 cases; 1 donor declined and the patient was not treated. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) with (n=2) or without (n=12) prior depletion of CD3+ cells. Purified NK cells were co-incubated with a lysate of CTV-1 cells (DSMZ repository) overnight. Lysate was removed by density gradient separation on day 2 and cells were cryopreserved in dosed aliquots and released for infusion within 15 days of manufacture. TREATMENT: Three groups of 5 patients were to receive 1×10^6/kg, 5×10^6/kg or 1×10^7/kg haploidentical related donor T-pNK as a single i.v. infusion 24 hours after completion of TBI. RESULTS: 15 patients were enrolled between July 2008 and Dec 2009 – Table 1. Mean donor T-pNK yield was 6.73×10^9 and CD56 purity 99.3%. T cell contamination was <10^4/kg. All products achieved QC release criteria. Median time from enrolment to product release was 46 days. 8/15 patients received the T-pNK therapy; 7 untreated due to relapse during product manufacture or failure to remit (6) and donor refusal (1). No infusional toxicity was observed. 7/8 patients experienced profound bone marrow suppression with median time of neutrophil recovery 55 days (19–101). Two patients who previously had an allogeneic transplant were salvaged with a CD34+ top up stem cell transfusion from their original donors. All experienced neutropenic fevers requiring antibiotic support with 2 of them requiring ICU admission. Due to the toxicities observed the dose escalation was abandoned and all treated patients received 1×10^6 cell/kg. The median duration of CR post T-pNK was 222 days (55–845) compared to previous CR median of 47 days (0–90) – Table 1. 7/8 patients experienced a longer CR post T-pNK than their previous CR; median increase in duration of CR was 242 days (range 54–815). CONCLUSIONS: T-pNK can be easily generated and administered with acceptable toxicity which was easily managed. In this non-controlled trial there was evidence of clinical efficacy but delays in donor screening and processing led to 40% attrition of enrolled patients prior to drug administration.Table 1:UPNSexAgeCytogeneticsAlloHSCTStatus at treatmentDuration of CR pre-TpNK (days)Duration CR post-TpNK01F54Trisomy 21NoCR33084503M72N/KNoPR1N/A25004M51t(9;11)YesCR33028407M71ComplexNoCR1605509F73Trisomy 13NoCR14714108M67normalNoCR26211611M61Trisomy 9YesCR39035212F67normalNoCR23098 Disclosures:Lowdell:Coronado Biosciences: Consultancy.

The Broad Spectrum Bcl-2 Inhibitor Apogossypol Induces Apoptosis and Differentiation of Blast Crisis Chronic Myeloid Leukemia Stem Cells.

Abstract 3275Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant. Introduction:Leukemia stem cells (LSC) are rare, self-renewing cells capable of proliferation and differentiation into the bulk of cells that make up a leukemia. LSC, like normal tissue-specific stem cells, can be highly quiescent and resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. While traditional chemotherapy may successfully eradicate the bulk of tumor cells, it often fails to kill LSC resulting in their reactivation and eventual relapse of disease.Apoptosis resistance in cancer often involves deregulation of Bcl-2 family proteins. Bcl-2 family expression also seems to have an important role in normal stem cell function as expression changes of Mcl-1, Bcl-2, and Bcl-XL in mouse hematopoietic stem and progenitors characterize the differentiation of hematopoietic cells to different lineages.Several studies have linked expression of Bcl-2 family members to the development of blast crisis chronic myeloid leukemia (BC CML) in vitro, however there has been little data on the role of Bcl-2 expression in apoptosis resistance in human LSC. Also, various data has shown that CML cells become increasingly resistant to BCR-ABL inhibition with progression to blast crisis. In BC CML, LSC are enriched in the progenitor population and can be serially transplanted in immunodeficient mice leading to BC CML in vivo. We hypothesize that human BC CML LSC may resist chemotherapy by overexpression of Bcl-2 family proteins and maintenance of quiescence. We analyzed Bcl-2 family protein expression in BC CML LSC and analyzed whether these cells were sensitive to chemotherapy treatment in vitro. We further analyzed whether BC CML LSC could maintain quiescence in vivo. Finally, we tested the efficacy of the broad spectrum Bcl-2 antagonist apogossypol on BC CML LSC in vitro and in vivo. Methods:Bcl-2 and Mcl-1 protein expression was measured in 1° BC CML LSC by intracellular FACS analysis and compared to expression in normal and chronic phase CML cells. CD34+ cells isolated from serially transplanted BC CML LSC were used for all subsequent studies. To assess whether BC CML cells maintained quiescence in vivo, they were stained with DiR, an infrared fluorescent cell membrane dye, transplanted into neonatal mice, and analyzed for DiR fluorescence 18 weeks later. Dividing cells will distribute DiR to all daughter cells leading to dilution of the dye and a decrease in fluorescent signal per cell. LSC drug resistance was tested in vitro by culturing the cells with etoposide, dasatinib, and apogossypol and by measuring apoptosis by FACS using annexin-V/7-AAD staining. Finally, apogossypol efficacy was tested in vivo in LSC transplanted mice. At 8 weeks post-transplantation, mice were treated for 3 weeks with apogossypol or vehicle and then analyzed for human hematopoietic cell engraftment and apoptosis by FACS. Results:1° BC CML progenitors expressed significantly higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. LSC cultured in vitro were also resistant to etoposide and dasatinib-induced apoptosis. Apogossypol treatment in vitro however led to a dose-dependent increase in cell death and apoptosis and resulted in a significant increase in the frequency of lin+ staining cells along with a significant shift in the frequency of the common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) populations compared to vehicle treated controls.Mice transplanted with BC CML LSC developed diffuse myeloid sarcomas and had high levels of human engraftment in the liver, spleen, and bone marrow. Human cells in tumors and the liver were uniformly DiR- while cells engrafted in the spleen and bone marrow retained DiR fluorescence. In vivo treatment with apogossypol led to a significant reduction in human cell engraftment in mouse bone marrow compared to vehicle controls. Consistent with the results in vitro, there was also a significant increase in the frequency of lin+ staining cells in engrafted mouse spleens as well as a significant decrease in engrafted GMP in the mouse bone marrow. Conclusions:Our results demonstrate that BC CML LSC are highly resistant to conventional chemotherapy but are sensitive to apogossypol in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC by inducing apoptosis as well as by inducing differentiation. Disclosures:Goff:Coronado Biosciences: Research Funding. Tesi:Coronado Biosciences: President and CEO. Jamieson:Coronado Biosciences: Research Funding. Jamieson:Coronado Biosciences: Research Funding.

Multiple Myeloma Cancer Stem Cell Animal Model.

Abstract 1848Poster Board I-874Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC.Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures:Goff:Coronado Biosciences: Research Funding.