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Abstract Background: Conflicting data exists on whether T-cell dose of allogeneic stem cell product influences transplant outcome. Methods: Using CIBMTR database, we identified 2,736 adult patients who underwent first allogeneic HCT for AML/ALL/MDS (between 2008-2014) with PBSC from an HLA-identical sibling donor (MSD) or 8/8-matched unrelated donor (MUD). This cohort excluded ex-vivo and in-vivo T-cell depletion transplants. Correlative analysis was done between CD3 cell dose and risk of GVHD, relapse, NRM, DFS and OS. Results: T-cell dose cutoff values were identified (using maximum likelihood estimation method) to identify differential risk of aGVHD (grade II-IV) in both MSD and MUD groups. A CD3 cell dose cutoff of 14 x107 cells/kg identified MSD/low CD3 (n=223) and MSD/high CD3 (n=1214) and a dose of 15 x107 cells/kg identified MUD/low CD3 (n=197) and MUD/high CD3 (n=1102). CD3 and CD34 cell doses did not correlate. Median CD3 cell dose were 11 and 29 in the MSD/Low and MSD/High groups, respectively, and 10 and 28 in the MUD/Low and MUD/High groups, respectively. In MSD/high CD3, cumulative incidence (CI) of D100-aGVHD (GII-IV) was higher (33% vs 25%) (P value =0.009) without influence on engraftment, severe aGVHD (GIII-IV), cGVHD, NRM, relapse, DFS, or OS. In MUD/high CD3, CI of D100-aGVHD (GII-IV) was higher (50% vs 40%) (p value=0.009) and so was the CI of 6-month-cGVHD (31% vs23%) (p value = 0.02) with no influence on engraftment, severe aGVHD, 2-year-GVHD, NRM, relapse, DFS, or OS. On multivariate analysis, both MSD (table 1) and MUD (table 2) groups failed to show a correlation between CD3 cell dose and aGVHD (GII-IV) (p value =0.1 and 0.07), cGVHD (p value=0.8 and 0.3) for both groups respectviely. Sub-analysis of CD4, CD8 and CD4/CD8 ratio failed to identify cutoff values predictive of transplant outcome. Using log-rank test, the sample size was, however, suboptimal to identify difference (with 80% power at 0.05% significant level) at these cutoff cell dose. Conclusion: In this relatively small registry study, the CD3 cell dose in the PBSCT product did not influence the risk of acute or chronic GVHD or other transplant outcome when using matched sibling or 8/8 unrelated donors. Disclosures Saad: Actinium: Consultancy. Lamb:Incysus Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Eukaryotic translation initiation factor 5A (eIF5A) has been implicated in the regulation of cell proliferation, apoptosis, and inflammation, and is the only known protein to be modified by hypusination. Hypusinated eIF5A, the predominant form of eIF5A in cancer cells, is involved in cell survival and activation of inflammatory pathways. In contrast, accumulation of the unhypusinated form of eIF5A is associated with apoptosis and mutants of eIF5A that cannot be hypusinated (e.g. eIF5AK50R) are pro-apoptotic. SNS01-T was designed to treat B-cell cancers and consists of two active components: a plasmid DNA expressing the pro-apoptotic eIF5AK50R under the control of a B cell-specific promoter, and an siRNA against an untranslated region of native eIF5A mRNA. When these two components are combined with linear polyethyleneimine (PEI), the nucleic acids are condensed into nanoparticles for protection from degradation in the blood and enhanced cellular delivery. The mode of action of SNS01-T is siRNA-mediated inhibition of hypusinated eIF5A and simultaneous over-expression of pro-apoptotic eIF5AK50R to induce cell death. In vitro cell studies and in vivo xenograft studies have demonstrated the efficacy of this approach. The safety and tolerability of intravenous administration of SNS01-T is being investigated in a first-in-human Phase1b/2a study in patients with relapsed or refractory multiple myeloma (MM), mantle cell lymphoma (MCL) or diffuse large B cell lymphoma (DLBCL). Eligible patients are being enrolled sequentially into four cohorts at increasing doses. Each patient receives an intravenous infusion of SNS01-T twice weekly for 6 consecutive weeks. Eligible patients must have been diagnosed with MM according to IMWG criteria, or with MCL or DLBCL with histologic confirmation. Patients also must have measurable disease, have relapsed or refractory disease after two or more prior treatment regimens, have a life expectancy of at least 3 months, and not be eligible to receive any other standard therapy known to extend life expectancy. The primary objective is to evaluate the safety and tolerability of multiple escalating doses of SNS01-T. Secondary objectives include analysis of pharmacokinetics, immunogenicity, pro-inflammatory cytokines, and therapeutic efficacy. The required 3 patients per cohort have completed the dosing schedule in cohorts 1 and 2 from a total of 10 patients enrolled (9 patients with MM and 1 with DLBCL). Of the ten patients enrolled, four completed the full treatment period, two did not complete dosing but were evaluable for safety, and four (three in cohort 1 and one in cohort 2) discontinued treatment after fewer than 8 doses and were not evaluable. There were no drug-related serious adverse events or dose limiting toxicities in either cohort 1 or 2. In cohort 1 (0.0125 mg/kg SNS01-T), two of three evaluable patients did not progress on treatment and were considered stable at week 3 and week 6, the end of the dosing regimen. The third patient progressed after receiving 10 of the 12 doses and was evaluable for safety. In cohort 2 (0.05 mg/kg), 3 patients (2 with MM and 1 with DLBCL) were evaluable for safety. Stabilization of serum monoclonal protein levels was observed in one MM patient of cohort 2. Two patients (1 with MM and 1 with DLBCL) progressed after receiving 8 of the 12 doses and were evaluable for safety. Results from ongoing pharmacokinetic studies, immunogenicity studies, and quantification of pro-inflammatory cytokines will be discussed. The planned dose levels for the third and fourth groups are 0.2 and 0.375 mg/kg, respectively. The results to date of this first-in-human clinical trial indicate that SNS01-T can be administered safely and the MTD has not yet been reached (Clinical Trials.gov Identifier: NCT01435720). Disclosures: Barranco: Senesco Technologies: Consultancy. Usmani:Celgene, Onyx, Millenium: Consultancy, Research Funding, Speakers Bureau. van Rhee:Jansen&Jansen: Research Funding. Thompson:Senesco Technologies: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Taylor:Senesco Technologies: stock options Other. Dondero:Senesco Technologies: Employment. Browne:Senesco Technologies Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Siegel:Celgene, Millenium, Onyx (same for all): Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Abstract Background Allogeneic stem cell transplantation (allo-SCT) is a potentially curative option for patients with myelodysplastic syndromes (MDS). However, allo-SCT can be associated with significant morbidity and mortality along with a risk for post-transplant disease relapse. While many MDS prognostic models exist, a prognostic model specifically designed to predict post-transplant disease relapse is yet to be developed. Therefore, we carried out this single institution study. Methods After due IRB approval, the Mayo Clinic MDS database (1995-2012) was retrospectively reviewed. This database contains comprehensive information on MDS clinical and pathological characteristics, treatments and SCT. Primary outcomes analyzed included disease free survival (DFS), overall survival (OS) and relapse rates at five years post allo-SCT. In addition, we evaluated transplant related mortality (TRM), non-relapse mortality (NRM), and rates of acute and chronic graft versus host disease (GVHD). DFS and OS were calculated from the time of transplantation. The prognostic relevance of several disease and transplant specific characteristics were analyzed, including: age, gender, World Health Organization (WHO) categories, therapy related MDS, cytogenetics, International Prognostic Scoring System (IPSS) and IPSS-Revised (IPSS-R) at the time of MDS diagnosis, transfusion dependence, bone marrow and circulating blast count at transplant, serum ferritin levels, SCT Co-morbidity Index, donor source, HLA matching, CMV serostatus, and conditioning regimen. Multivariate analysis was performed using the Cox Proportional Hazard Model. Survival was estimated and compared using the Kaplan Meier and Log Rank tests, respectively. Results From 1995 through 2012, 96 consecutive patients with MDS underwent allo-SCT at the Mayo Clinic, Rochester. Of these, 62 were male (65%) with a median age of 54 (24-68 years). The median follow up was 31 (1-181) months. Twenty seven patients (28%) had therapy related MDS and 39 (40%) had MDS with a monosomal karyotype. IPSS-R risk stratification identified 55 (57%) patients with very high, 20 (21%) with high and 21 (22%) with intermediate/good risk disease. Fifty (52%) patients received a reduced intensity conditioning. The TRM was 8% and NRM at 5 years was 22%. Acute (grade 2 to 4) GVHD developed in 57 (59%) patients and chronic extensive GVHD was noted in 52 (54%) patients. Five-year relapse, DFS and OS rates were 24%, 42%, and 44%, respectively. In a univariate analysis, IPSS-R (high/very high, P=0.005), therapy related MDS (P=0.009), bone marrow blast count at transplant (≥10%, P=0.01), and the presence of a monosomal karyotype (P=0.05) were significantly associated with worse DFS. On a multivariable analysis, only high or very high risk IPSS-R category (HR 7.8, P=0.005) and therapy related MDS (HR 3.9, P=0.04) retained their negative prognostic impact. Based on this finding, we developed a prognostic model that assigned one point to each of these two factors (low risk-0, intermediate-1 and high risk-2). This model successfully stratified patients based on DFS, with a 5-year DFS of 67%, 40% and 23% for low, intermediate and high risk categories respectively, P=0.0012. This model was also successful in risk stratifying patients with regards to OS (table 1, figure 1). The presence of chronic GVHD was associated with a significantly improved DFS (62% vs 24%, P<0.0001) and OS (78% vs 19%, P<0.0001), however, as this is a post-transplant outcome, it was not included in the analysis/model. Conclusions Although the IPSS-R has not been validated in therapy related MDS or in patients who have received MDS directed therapy, the high/very high risk categories along with therapy related MDS, serve as independent predictors of OS and disease relapse after allo-SCT. Using these two variables, our prognostic model efficiently stratified patients with regards to DFS, relapse rates, and OS. Given the smaller sample size validation in a larger patient cohort will be needed. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 504 Background: There is limited data on the outcomes of AHCT for MM as a salvage approach following relapse after an initial (upfront) AHCT. We analyzed the outcomes of second salvage AHCT for relapsed MM. Methods: From the CIBMTR database we identified 187 patients (63% male) who had second AHCT (AHCT2) as salvage for MM relapsing after a prior AHCT (AHCT1). Planned tandem AHCT and AHCT2 within 6 months after AHCT1 were excluded. Cumulative incidence of relapse, non-relapse mortality (NRM) and the probabilities of progression-free (PFS) and overall survival (OS) were analyzed and multivariate analysis performed using proportional hazards models. Results: Median age at transplant was 57 (range 28–72) and 59 years (range 28–74) respectively at AHCT1 and AHCT2 respectively. Median follow up after AHCT2 was 47 months (range: 3–97). The proportion of IgG, IgA and free light chain subtypes was 48, 20 and 18% respectively. Durie Salmon stage at diagnosis was stage I (6%), II (21%), III (59%) respectively. Seven and 13% had serum creatinine > 1.5 mg/dl at AHCT1 and AHCT2 respectively. Conditioning regimen was single agent melphalan was in 78% and 84% for AHCT1 and AHCT2 respectively. At the time of AHCT2, majority (60%) were chemotherapy resistant compared with 80% patients with sensitive disease prior to AHCT1. Median time from AHCT1 to relapse was 18 months, with 32% relapsing within the first year, 29% in the second year, 19% in the third year and 19% beyond the third year after AHCT1. There was heterogeneity in the time to AHCT2 after relapse although 67% had AHCT2 within a year after relapse (median 7 months). Median between AHCT1 and AHCT2 was 32 months with the majority (69%) beyond 24 months. After AHCT2, day 28 engraftment rates of neutrophils and platelets were 96% (95% CI: 93–98) and 88% (95% CI: 83–92). Complete remission (CR) rates after AHCT1 were 43% whereas it was only 25% following AHCT2. After AHCT2, cumulative incidence of relapse at 1, 3 and 5 years was 51, 82, and 91% respectively. After AHCT2, one year NRM was 2%. PFS was 47%, 13% and 5% and OS was 83%, 46% and 29% at 1, 3 and 5 years respectively. In multivariate analysis, those relapsing within 36 months of AHCT1 had a higher risk of relapse (HR 1.58, p = 0.036) and higher risk of treatment failure and inferior PFS (HR1.52, p= 0.04). Mortality was higher for those relapsing early after AHCT1 (<36 months, HR 1.91, p = 0.02). OS was superior and mortality lower for AHCT2 performed after 2004-08 (HR 0.61, p=0.02). Conclusion: AHCT2 can be safely performed in relapsed MM with the best outcomes observed in later relapses (>36 months from AHCT1). OS has improved since 2004, but NRM, relapse and PFS after AHCT2 were not impacted by year of AHCT2. Thus the improved OS likely reflects greater availability of novel agents for post AHCT progression. Disclosures: No relevant conflicts of interest to declare.

Abstract Chimeric antigen receptor T cell (CART) therapy has developed as a powerful and potentially curative therapy in hematological malignancies over the last few years. CD19 directed CART cells have resulted in impressive complete response rates of ~90% in acute lymphoblastic leukemia that are durable for the majority of patients. However, the overall response rates in other malignancies such as chronic lymphocytic leukemia are around 50%. This could be partially related to CART exhaustion and dysfunction induced by leukemia cells. In this study, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction and exhaustion in hematological malignancies. As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 directed CART cells (second generation CARs using 4-1BB and CD3z signaling domains and a lentiviral vector). Incubation of primary AML samples or MOLM14 cell line with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PD-L1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P <0.001), and up-regulation of PD-1 and TIM-3 on T cells 3-7 days post co-culture (8% of T cells expressed PD-1 on day 0 vs 43% on day 3, P=0.03 and 13% of T cells expressed TIM-3 on day 0 vs 71% on day 3, p=0.001, Figure 1). For in vivo experiments, we used NSG (NOD-SCID-g-/-) mice and engrafted them with the MOLM14 cell line. Treatment of these AML xenografts with suboptimal doses of CD33 or CD123 CARTs resulted in initial anti-tumor responses, followed by disease relapses in 40-60% of the mice (Figure 2A). T cells were isolated from the bone marrow of these mice and analyzed for differential expression of inhibitory receptors. There was a significantly increased up-regulation of TIM-3 receptors on T cells isolated from mice with relapsed disease compared with T cells isolated from mice in remission after CART cell therapy (Figure 2B). Next, we investigated the role of adding checkpoint blockade to improve T cell function ex vivo after CART cell therapy. Marrows of mice that relapsed after CART cell therapy contained both residual CART cells and leukemia and were used to model the administration of checkpoint blockade in the setting of CART cell exhaustion. Cells were cultured with PD-1 or TIM-3 blocking antibodies or the combination of both (10 ug/ml for 72 hours). CART cell effector functions such as cytokine production and Ki-67 proliferation marker improved in the presence of checkpoint antagonists especially when both PD-1 and TIM-3 blocking antibodies were combined (figure 2C). Finally, we tested the combination of checkpoint blockade with CARTs in AML xenografts. In this approach, we treated MOLM14 xenografts with suboptimal doses of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD), with or without checkpoint blocking antibodies. NSG mice bearing MOLM14 AML xenografts were established. Engraftment was confirmed by bioluminescent imaging. The tumor bearing mice were then treated with suboptimal doses (0.25-0.5 x106 total T cells I.V) of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD). Mice also received PD-1 blockade, TIM-3 blockade or the combination of both on days 3, 6, 9 and 12 post T cell therapy, with the rationale for early checkpoint blockade being based on our in vitro observations of early upregulation of inhibitory ligands on AML after exposure to CART cells. Mice were then followed with serial imaging to assess disease burden. The addition of checkpoint antagonists to untransduced T cells, in particular anti-TIM3, did not lead to an anti-leukemic effect. However, the addition of PD-1 or TIM-3 blockade to CART cell therapy resulted in a synergistic anti-tumor activity as shown in Figure 3. The durable complete response rate was: 45% for treatment with CART123 alone, 80% for treatment with CART123+PD-1 blockade, 100% for treatment with CART123+TIM-3 blockade, and 80% for treatment with CART123+ both PD-1 and TIM-3 blockade). Our preclinical results indicate that PD-1 and TIM-3 pathways are involved in CART exhaustion and dysfunction in AML. Combination of checkpoint inhibitors with CART cells may lead to enhanced efficacy in AML and other hematological malignancies. Current studies are investigating mechanisms of synergy and the role of these combinations in other hematological malignancies. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision. Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision. Results : 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified. Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1). Conclusion : The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding.

Abstract Patients with Hodgkin lymphoma (HL) who relapse or are refractory to first-line therapy have a poor prognosis and some of these patients require innovative therapeutic approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor T cells (CART19) for B cell malignancies. However, B cell antigens such as CD19 are typically absent from Hodgkin Reed-Sternberg (HRS) cells. Targeting the CD30 antigen using CART cells has met with limited clinical success to date. In HL the neoplastic HRS cells often represent ~1% of the tumor mass and the remainder is comprised of a highly immunosuppressive tumor microenvironment (TME). Within the TME, tumor-associated macrophages (TAM) have a key role in promoting tumor growth and inhibiting the anti-tumor immune response, and the presence of TAM in HL is associated with a poor prognosis. HL therefore represents an excellent model of tumor-induced immunosuppression that can potentially represent a barrier to successful CART therapy. The objective of this study was therefore to develop an anti-HL CAR T cell immunotherapy that would also overcome the immunosuppression of the HL microenvironment. We sought to define a tumor antigen that was co-expressed on HRS and on TAM. Using immunohistochemistry and flow cytometry we confirmed previous reports that CD123 (the IL-3Rα chain) is expressed on HRS in 50-60% of HL patients and in 100% HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). Importantly, CD123 was also seen on several HL-associated immune cells of the TME, including TAM. Exposure of macrophages to HDLM-2 cells polarized them to an M2-like phenotype (Figure 1 A) with expression of CD123. Luminex analysis of HDLM-2/TAM co-culture supernatants revealed high levels of IL-13, and blockade of IL-13 partially reversed the immunosuppressive phenotype. We then developed a model of TAM-mediated CAR T cell inhibition, using immunosuppressive M2-like macrophages derived by exposure to IL-4 or to HDLM-2 cells. These macrophages could suppress “third party” CART19 proliferation in response to B-acute lymphoblastic leukemia. We then used anti-CD123 CART cells (CART123) to target TAM. Importantly, CART123 actively recognized and killed TAM, thereby resisting TAM-mediated inhibition (as opposed to control CART19 cells) (Figure 1 B). We therefore sought to investigate the utility of anti-CD123 CART for the treatment of HL (initial results for this part presented at ASH 2014). In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells. A clinically-relevant xenograft model of HL was generated using luciferase+ HDLM-2 cells in NSG mice. In this model mice showed progressive increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day ~43 mice were randomized to receive 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 8 months while mice treated with control T cells had a median survival of 128 days (Figure 2 A). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In order to prove the formation of an immunological memory, at day 250 CART123 treated mice were re-challenged with the HDLM-2 cell line. The tumor was promptly rejected, associated with a re-expansion of CART123 cells in the peripheral blood (~10 months after T cell injection) leading to an overall survival advantage as compared to controls (Figure 2 B). In summary, we showed that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. In Hodgkin Lymphoma (HL) CD123 is expressed on malignant Reed-Sternberg cells as well as in M2-polarized tumor associated macrophages (TAMs): importantly CART123 can target simultaneously both the HRS and the TAM without being resistant to immunosuppression. We have previously demonstrated that CART123 can lead to myelosuppression in preclinical models, suggesting that our findings could be translated to treat patients with refractory HL in the context of stem cell transplantation using “short-acting” RNA-CAR123 T cells or depletable lentivirally-transduced CART123. Disclosures Ruella: Novartis: Patents & Royalties. Kenderian:Novartis: Patents & Royalties, Research Funding. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies. Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%). Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Objectives: Infection with the causative agent of visceral leishmaniasis (VL) may be either symptomatic or asymptomatic. In this study we aimed at investigating the prevalence of asymptomatic infections of leishmania in non-endemic villages in Gedaref state, Sudan. A descriptive cross-sectional study conducted during September and October 2014. Blood samples were collected for serological and molecular analysis. Sticky-traps, knockdown spray and CDC-miniature light traps were used for the collection of sandflies. Results: Ninety-Five participants were included; 52 from Abukishma, 15 Algadamblia Tirfa, 25 Abualnaja and 3 were from Algadamblia Aljabal . Females constituted 56 (58.9%) of the study participants while males were 39 (41.1%). The most frequent age group was >40-years (54.7%). Balanites/Acacia trees were the most planted tree inside the houses; 78 (82.1%). Also, 85 (89.5%) of the participants breed animals inside the house. DAT test revealed 5 positive participants (5.2%). 4/5 DAT positive were past VL infection. PCR detected 35 (36.8%) positive participants. A total of 31/35 was considered asymptomatic infections based on PCR. Households planted Balanites/Acacia trees or breed domestic animals were found in high percentages with L. donovani PCR positive participants (60.1%, 91.4%). No statistically significant was found for VL associated risk factors and VL asymptomatic participants.

Abstract Background. Many children and adults with acute myeloid leukemia (AML) relapse or are incurable with current treatment modalities, highlighting a need for alternative therapies. Chimeric antigen receptor (CAR) T cells targeting CD123 have demonstrated potent anti-leukemia activity in murine xenograft models of human AML. However, CD123-redirected T cell treatment of mice engrafted with normal human hematopoietic cells resulted in profound myeloablation, raising concerns for hematologic toxicity in patients with AML who may be treated with such therapies. We hypothesized that T cell deletion after CD123-redirected T cell-induced eradication of AML could minimize this bystander toxicity without impairing leukemia control, thereby increasing the therapeutic window of anti-AML CAR T cell immunotherapy. Methods. To test this hypothesis, we compared three termination strategies in human AML xenograft models: (1) T cell ablation with the anti-CD52 antibody alemtuzumab after treatment with T cells lentivirally-transduced with anti-CD123-41BB-CD3ζ (CART123), (2) T cell ablation with the anti-CD20 antibody rituximab after treatment with CART123 engineered to co-express CD20 (CART123/CD20), and (3) treatment with "biodegradable" anti-CD123 mRNA-electroporated CAR T cells (RNA-CART123). Mice engrafted with luciferase-expressing human AML cell lines (MOLM14, MOLM13, U937) or primary AML specimens (n=3) were treated with CD123-redirected CAR T cells as above. For T cell depletion studies, alemtuzumab 1 or 5 mg/kg was injected intraperitoneally (IP) at 1-4 weeks after 1x105-106 CART123 to determine optimal dosing and timing of T cell ablation. In subsequent studies, rituximab 10 mg/kg was injected IP 4 weeks after 1x105 -106 CART123/CD20, or 1x107 RNA-CART123 were injected intravenously at 5, 9, and 16 days after AML engraftment. Mice were followed by weekly bioluminescent imaging and/or quantitative flow cytometry analyses of blood, spleen, and/or bone marrow. Results. CART123 treatment of CD123+ AML xenografts induced marked T cell expansion and leukemia eradication in vivo, resulting in long-term animal survival (p<0.0001 vs untransduced T cell-treated controls). Minimal xenogeneic graft-versus-host effects were observed. One dose of alemtuzumab rapidly eliminated T cells in all tested models with best efficacy of 5 mg/kg dosing at 4 weeks post-CART123. CART123/CD20 inhibited AML proliferation with similar kinetics to those of CART123, and 1 dose of rituximab at 4 weeks post-CART123/CD20 infusion rapidly eliminated T cells while preserving leukemia remission. Alemtuzumab or rituximab alone did not inhibit AML proliferation in non-CART123-treated xenograft models vs AML-only controls (p=1.00). Mice with CART123- or CART123/CD20-induced AML remission at time of T cell ablation remained leukemia-free for ≥12 weeks, and animal survival did not differ from that of CD123-redirected CAR T cell-treated mice that did not undergo T cell depletion (p=1.00). In contrast, early depletion of T cells with alemtuzumab at 1, 2, or 3 weeks post-CART123 resulted in failure to eliminate AML with subsequent leukemia progression and animal death. Furthermore, AML rechallenge of animals with alemtuzumab- or rituximab-ablated T cells resulted in rapid leukemia proliferation without T cell re-expansion, confirming completeness of T cell depletion. Non-ablated mice demonstrated CAR T cell re-expansion with rejection of CD123+ leukemia rechallenge (p<0.0001). RNA-CART123 rapidly eliminated AML and facilitated long-term animal survival, although RNA-CART123 had expectedly shorter persistence in vivo than did CART123 or CART123/CD20. Conclusions. Alemtuzumab and rituximab completely eliminated CD123-redirected CAR T cells in human AML xenograft models. Sustained leukemia remission required CART123 or CART123/CD20 persistence for 4 weeks prior to T cell termination via alemtuzumab or rituximab post-CART123 or CART123/CD20, respectively. Ongoing studies are investigating efficacy of T cell elimination in additional xenograft models and against other anti-AML CAR T cell immunotherapies. Results from these studies may help to identify promising T cell termination strategies that will augment efficacy of CAR T cell therapy in patients with AML, particularly prior to stem cell transplantation. RNA-CART123 trials in patients with relapsed/refractory AML will open soon. Disclosures Off Label Use: depletion of CAR T cells with alemtuzumab or rituximab. Kenderian:Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Aplenc:Sigma Tau: Consultancy. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.