S96 Abstracts

of BMT: patients transplanted during the years 1990-2000(n=16) and 2001-2009 (n=31). Results: Myeloablativeconditioning with Busulfan/Cytoxan/ATG was well tolerat-ed in both cohorts. The number of matched sibling donortransplants did not differ, however MUD BMT numbersincreased significantly from 9/16 during 1990-2000 com-pared to 23/31 during 2001-2009. Transplant relatedmortality within the first 100 days was significantly greaterfor patients transplanted during1990-2000 (3/16 patients)compared to patients transplanted in 2001-2009 (0/31patients) (p=0.03, Fischer's exact test). The overallincidence of GVHD was similar in both cohorts. Long term5 year OS was 62.5% (95% CI: 34.5% to 81.1%) and 91.2%(95% CI: 69% to 97.7%) in the 1990-2000 and 2001-2009 timeperiods, respectively. Conclusions: MUD BMT in excellentoutcomes in the treatment of WAS. Overall survival hasimproved significantly over the last decade with MUD BMToutcomes now equivalent to or better than prior reportswith MSD donors. MUD BMT at an early age is recommendedand allows many patients without a MSD to receive curativetherapy without increased risk of morbidity or mortalityfrom transplantation.

doi:10.1016/j.clim.2010.03.287

F.66. Characteristics and Development of TRX518,an Aglycosyl Humanized Monoclonal Antibody (Mab)Agonist of huGITRJoe Ponte, Daniel Doty, Rudy Christmas, Paul Ponath, LouVaickus, Michael Rosenzweig. Tolerx, Cambridge, MA

Glucocorticoid-induced tumor necrosis factor receptor(GITR) is an immunomodulator found on many cell types,including regulatory and effector T cells. In murine estab-lished tumor models, combination therapy with Tolerx' anti-mouse GITR Mab (2F8) significantly reduced tumor burdenand prolonged survival compared with antibody or chemo-therapy alone. Cumulative data with 2 chemotherapeuticagents revealed N60% complete remissions with combinationtherapy compared with b10% with chemotherapy. Incomplete responders, combination therapy elicited specificand robust memory responses. Combination therapy en-hanced CD8+ T cell number and reduced the number of Tregulatory cells (Tregs), altering the Treg/Teff ratio in tumordraining lymph nodes. CD8+ or CD4+ T cell depletion ablatedefficacy. A fully humanized aglycosylated IgG1 mAb, TRX518,was constructed based on our parental murine 6C8 anti-huGITR. Aglycosylation abrogates antibody-dependent cel-lular cytotoxicity and complement-mediated lysis. ThoughTRX518 co-stimulates sub-optimally activated peripheralblood lymphocytes, it is not a superagonist. Plate-boundTRX518 did not induce appreciable cytokine release (IL-2, IL6, IL-10, TNF-alpha, IFN-gamma). TRX518 blocked theinteraction of GITR with its ligand and enhanced thecytotoxicity of NK cells. TRX518 did not block an allogeneicmixed lymphocyte reaction (MLR) or enhance an autologousMLR. In vivo, TRX518 was not depleting, but bound to GITR onCD4+ and CD8+ naïve and memory T cells, B cells, NK cells,NKT cells, monocytes, and macrophages. Cynomolgous

macaque and human tissue cross studies and an IND-enablingGMP toxicology study are complete. Based on thesepromising results, TRX518 will be evaluated in a Phase 1clinical oncology trial.

doi:10.1016/j.clim.2010.03.288

F.67. Assessment of Adalimumab's ComplementActivity with Human Cells Expressing ComplementRegulatory ProteinsKristie Grebe, Jochen Salfeld, Zehra Kaymakcalan. AbbottLaboratories, Worcester, MA

Complement-dependent cell cytotoxicity (CDC) has beena topic of interest since the advent of biologic therapeuticagents similar or identical to human antibodies. The CDCactivity of adalimumab with human cells either transfectedwith tumor necrosis factor (TNF) or activated by lipopoly-saccharide (LPS) was assessed and compared with a TNF-transfected mouse cell line that has previously shown CDCactivity. CDC was examined in three different cell types:human MonoMax-6 (MM6) cell line and THP-1 cell line (bothhuman monocytic cell lines), and freshly isolated, humanperipheral blood mononuclear cells (PBMC). Calcein-labeledcells were cultured with test antibodies plus complementand released Calcein from dying cells was measured. HumanPBMC and THP-1 cell line were activated by LPS. Adalimumabwas added to human PBMC, MM6 and THP-1 cell line cultures,in the presence of complement. Neither transfected MM6cells nor LPS-activated human THP-1 cells and PBMC werekilled by adalimumab and C'. Antibodies for MHC moleculeswere used as positive controls, and CDC did take place inthese same cells in the presence of complement and controlantibodies. Surface expression of mTNF was demonstratedby binding of adalimumab and another control anti-TNFantibody. Expression of complement regulatory proteins,which inhibit complement activation, was detected with theactivated PBMC, the THP-1, and the mTNF-transduced MM6cell lines, but not the transfected mouse cell line. Inconclusion, despite adalimumab's ability to bind to mTNFexpressed on the surface of activated human PBMC, THP-1cells, and a mTNF-transfected cell line, CDC was notobserved.

doi:10.1016/j.clim.2010.03.289

F.68. Developing a Topical HIV Microbicide:CD4-targeting Aptamers Inhibit HIV Infection inPrimary Cells in vitro and in Polarized HumanCervical ExplantsAdam Wheeler, Judy Lieberman, Radiana Trifonova, EmreBasar, Derek Dykxhoorn. Harvard Medical School, Boston,MA

The therapeutic use of small interfering RNAs (siRNA) toprevent or treat HIV infection requires an effective means

S97Abstracts

for in vivo delivery into susceptible target cells. Aptamers,small structured nucleic acid sequences that bind with highspecificity to individual proteins, provide an attractiveapproach for cell-specific targeting. We designed a chimericRNA, which was transcribed in vitro and complexed with thecomplementary 21-nucleotide active siRNA strand. Wehypothesized that the partially-duplexed RNA would beselectively internalized into CD4+ cells, and subsequentlyprocessed into an active siRNA capable of silencing targetgenes. Internalization of Cy3-labeled aptamer-siRNA chi-meras was first demonstrated by fluorescence microscopyand flow cytometry. Delivery was restricted to those cellsexpressing CD4. Functional silencing was verified by showingrobust knockdown of host genes (e.g. CCR5, CD45 and lamin),at both the protein and mRNA level. To investigate whetherthis system could be used to suppress HIV infection, CD4-chimeras were designed to encode siRNAs targeting the viralgenes gag, vif and the HIV co-receptor, CCR5. Theseinhibited HIV infection in both macrophages and CD4+ T-cells by 60–90%. Efficient inhibition of HIV replication wasalso demonstrated in polarized human cervical explants.Importantly, chimera treatment was non-cytotoxic to tissuesand did not induce an interferon-mediated inflammatoryresponse. These data suggest that aptamer-siRNA chimerascould be an effective, cell-type specific therapeutic genesilencing approach to topically prevent sexual transmissionof HIV, providing the framework for an HIV microbicide thatis economical, easily produced, and amenable to use inresource-poor settings.

doi:10.1016/j.clim.2010.03.290

F.69. Rituximab Transiently Affects Islet-specific TCell Responses in Type 1 Diabetes PatientsBarbara Brooks-Worrell1, Craig Beam2, Jessica Reichow3,Jerry Palmer1TrialNet Study Group. 1University ofWashington/DVA Puget Sound Health Care System, Seattle,WA; 2University of South Florida, Gainsville, FL; 3SeattleInstitute for Biomedical Research, Seattle, WA

Rituximab is a human/murine chimeric monoclonalantibody that targets the transmembrane protein CD20 of Bcells. The binding of rituximab to CD20 leads to significantdepletion of peripheral B cells. Although type 1 diabetes is aT cell mediated autoimmune disease, recent evidence alsopoints to a potential role of B cells in the pathogenesis oftype 1 diabetes. We have evaluated whether rituximabaffects the islet specific T cell responses in newly diagnosedtype 1 diabetes patients who participated in a randomized,double-blind study. Multiple infusions (days 1, 8, 15 and 22)of either rituximab or placebo were given to the studyparticipants. T cell responses were available from 25rituximab-treated type 1 diabetes patients and 21 type 1diabetes patients receiving placebo. Islet specific T cellresponses were evaluated using cellular immunoblotting, atechnique demonstrated to have excellent sensitivity andspecificity for studying T cell responses to islet proteins fromtype 1 diabetes patients. The median baseline T cellproliferative responses to the islet proteins were not

significantly different between the rituximab-treated andthe placebo-treated groups (p=0.12, one-sided ExactWilcoxon test). However, at 6 months post-infusions, themedian T cell responses to islet proteins in the rituximab-treated group was significantly (pb0.04) lower comparedto the median T cell responses to islet proteins from theplacebo-treated group. The significant differences be-tween the groups was lost by 12 months post-infusion(p=0.29). We conclude that rituximab appears to down-regulate the T cell responses to islet proteins in type 1diabetes patients, however this downregulation appears tobe short lived.

doi:10.1016/j.clim.2010.03.291

F.70. The Role of Ly6C+ InflammatorySpleen-derived Monocytes in an Animal Modelof Brain IschemiaLaurel Yong-Hwa Lee1, Oleg Butovsky1, Ying Wei2, MakotoHayase2, Christian Waeber2, Michael Moskowitz2, HowardWeiner1. 1Brigham and Women's Hospital, Boston, MA;2Massachusetts General Hospital, Boston, MA

Stroke is a leading cause of mortality and disabilityworldwide. Upon ischemic injury, local activation oftranscription factors within the brain tissue leads toexpression of pro-inflammatory factors within hours.There is increasing evidence suggesting that post-ischemicimmune responses, particularly peripheral monocytes, playa crucial role in determining clinical outcome of stroke.While therapeutic strategies selectively targeting mono-cytes may prove to be effective, the exact mechanism ofmonocytes-mediated post-stroke brain injury remains un-clear. The heterogeneity of monocytes and residentmicroglia and their unknown identity further complicatesthis challenge. CD11b+Ly6C+ cells are a subset of murinemonocytes and are functionally equivalent to humanCD14highCD16- monocyte subtype. They are readilyrecruited to inflamed tissues and lymph nodes in vivo andplay an important role in acute inflammatory response.Using a mouse model of brain ischemia, we evaluated thekinetics of post-stroke CD11b+Ly6C+ response in peripheryand in ischemic brain. Following 60-min right-sided occlu-sion of the middle cerebral artery (MCAO), the animalswere reperfused for 1, 3, 7, 14, 21, and 28 d before FACSanalysis. CD11b+Ly6C+ monocytes were rapidly recruited tothe ischemic brain from the periphery as early as 1d postMCAO in a biphasic manner. Gene expression study of thesorted CD11b+Ly6C+ infiltrates suggested that they differfrom the CD11b+Ly6C+ splenic reservoir in functionalprofiles and express high levels of pro-inflammatorycytokines, including IL-1β and TNF-α. Therapeutic strate-gies that selectively block or immunomodulate the pro-inflammatory CD11b+Ly6C+ monocyte infiltrates may helpreduce infarct size and improve clinical outcome followingstroke.

doi:10.1016/j.clim.2010.03.292