ALBERT

Description: Abstract 3283 Donor-derived regulatory T cells (Treg) and natural killer (NK) cells can respectively improve stem cell transplant (SCT) outcome by reducing graft versus host disease (GVHD) severity and exerting a graft-versus-leukemia effect. High frequencies of donor Treg are associated with less GVHD, and low doses of interleukin-2 (IL-2) can expand both NK and Treg after allogeneic SCT. To explore the feasibility of improving the quality of peripheral blood SCT donations, we evaluated the safety and the tolerability of ultra-low dose IL-2 administration to volunteers with the aim of preferentially expanding Treg and NK cells. Twelve healthy volunteers (mean age 34 years; range 22–57) received 0.1 or 0.2 million U/m2/day IL-2 subcutaneously for 5 days (NIH protocol 11-H-0268). Blood samples were collected before and 1, 2, 3, 4, 7 and 28 days after IL-2 injection. Samples were analyzed by multiplex techniques including whole transcriptome gene expression with HumanGene 1.0ST microarrays; serum levels of 69 cytokines and chemokines by Luminex assay; and lymphocyte phenotyping by flow cytometry, to comprehensively characterize the cellular and molecular immune response to IL-2 (“IL-2 immunome”). Treg subsets were determined within the CD4+ T cell population using FoxP3, Helios, CD45RA and CD31 to identify thymus-derived natural Treg (nTreg), induced Tregs (iTreg) and their recent thymic emigrants (RTE). NK cell subsets were determined within CD56+CD3- population using NKG2A, KIR2DL1, KIR2DL2/3, KIR3DL1 and CD57 to identify CD56bright, CD56dim NKG2A+KIR-, and CD56dim KIR+CD57+ cells. All subjects tolerated ultra-low dose IL-2 with minimal adverse events (mainly grade 1–2 injection site reactions). The fraction of FoxP3+Treg in CD4 rose significantly above baseline peaking at 4 days (3.7% vs 5.8%; p=0.0004) after the first dose of IL-2. Treg subset analysis demonstrated that the fraction of nTreg and RTE nTreg in CD4 expanded significantly in the lower dose cohort compared to the higher dose cohort (p=0.004 and p=0.005 respectively). %CD56bright NK significantly increased at 7 days (p=0.008), whereas CD56dimNKG2A+KIR-, and CD56dimKIR+CD57+ NK cells remained at baseline. The Ki67 proliferation marker further verified a significant in vivo expansion of CD56bright NK cells with ultra-low dose IL-2. Cytokine and chemokine profiling demonstrated significant increase circulating level of IP-10 (P=0.0018) through day 2 to 4 after IL-2 injections. In contrast, circulating levels of IL-2, IL-6, IL-10, IL-15 and IL-17 remained unchanged after IL-2 injection. Gene expression microarray studies revealed significant changes in 24 genes (P value 〈 0.1 corrected by false discovery rate (FDR) for multiple testing), including up-regulation of IL-2RA and FOXP3 as early as 2 days after IL-2 injections. Gene Set Analysis (GSA) revealed significant changes (P value 〈 0.1 after FDR) in innate immune response pathways, including Toll-like receptor signaling and interferon signaling. This is the first study to show that ultra-low dose IL-2 could be safely administrated to healthy volunteers to expand thymic-derived natural Treg and CD56bright NK cells. These results raise the possibility of using ultra-low dose IL-2 to boost Treg and NK cells in stem cell donors. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Imatinib achieves deep and durable remissions in patients with myeloid neoplasms bearing PDGFRB. Allogeneic stem cell transplantation is no longer indicated for patients with chronic myeloproliferative neoplasm bearing PDGFRB who respond to imatinib.

Description: Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity. VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST’s were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro. We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free 〉3 and 〉9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease. In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Description: Bone marrow stromal cells (BMSC, also known as bone marrow-derived “mesenchymal stem cells”) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage, BMSC for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following BMSC infusion. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were monitored for plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and after BMSC infusion. BMSCs were prepared from marrow aspirates from healthy volunteers with the expansion of 3 passages. Ten subjects were infused a fixed dose of 2 x 106 BMSCs /kg weekly for 3 doses. There was no treatment related toxicity (primary endpoint). Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first BMSC infusion (fig1). Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for tissue injury. The GVHD complete responders survived significantly longer (〉300 days vs a median of 33 days), had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts but there was no clear difference in natural or induced Tregs. Cytokine changes also segregated with survival. These results confirm that BMSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. However BMSC were ineffective in patients with more aggressive GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSC, requires a relatively intact immune system. Early detection and BMSC treatment appear important in patients with refractory GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Background T-large granular lymphocytosis (T-LGL) is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes, presenting with immune mediated cytopenias and associated with autoimmune syndromes. Immunosuppressive therapy (IST) with methotrexate, cyclosporine, or cyclophosphamide can improve the cytopenias in about half the patients but can lead to significant toxicity in older patients. The anti CD52 antibody alemtuzumab is a potent immunosuppressive agent with a good safety profile. We therefore initiated a pilot phase II study to evaluate alemtuzumab as a treatment for T-LGL. Methods 20 consecutive patients with T-LGL were enrolled from October 2006 to August 2012 at National Institutes of Health (www.clinicaltrials.gov - NCT00345345). After a 1 mg test dose, alemtuzumab was administered at 10 mg/dose/day intravenously for 10 days. Peripheral blood, bone marrow, and plasma samples were collected from patients before and at 3 or 6 months after treatment. Blood was analyzed for 1) lymphocytes subsets, T-cell receptor V-beta repertoire and CD57 and CD52 expression by flow cytometry (LSR II, BD, San Jose, California), 2) plasma cytokines using a a magnetic bead based Luminex assay (Affymetrix, CA, USA), 3) STAT3 mutation by direct Sanger sequencing and 4) expression level of 84 genes of the JAK-STAT signaling pathway quantified by PCR array 384 well from SABiosciences (Frederick, MD, USA). Results We report here the results of treatment with alemtuzumab in 20 T-LGL patients enrolled in the first stage of the protocol. Three had associated myelodysplasia (MDS) and two had T-LGL following hematopoietic stem cell transplantation (HSCT). The median age was 61 years (range, 26-82). The median number of prior therapeutic interventions for T-LGL leukemia was 3 (range, 0-7) and the median time from diagnosis to alemtuzumab therapy was 1096 days (range, 18-6054). The median follow-up for all patients is 508 days (range, 99-1481) and for surviving patients 650 days (range, 120-1481). One patient was lost to follow-up 4 months after alemtuzumab therapy. Alemtuzumab was generally well tolerated. Labeled infusion related reactions were common and managed symptomatically. Prolonged and subclinical EBV and CMV reactivations were common but there were no cases of EBV or CMV disease without instituting prophylactic or pre-emptive therapy. Hematological response as defined by protocol was observed in 11 of 20 patients by 3 months after treatment. No patient with MDS or post HSCT responded to alemtuzumab. Four patients relapsed and received a second round of immunosuppression. One patient achieved stable blood counts on cyclosporine, three received alemtuzumab with one patient responding but relapsing 1 year later. STAT3 mutations in the SH2 domain identified in 10 of 20 patients did not correlate with response to alemtuzumab (5 responders and 5 non-responders). Treatment with alemtuzumab reduced significantly the absolute clonal population of T-cytotoxic lymphocytes, as identified by their V-beta receptor phenotype, but they tended to persist in frequency in the peripheral blood of responders. The expanded V-beta clone expressed both CD52 positive and negative cells and both compartments reduced in size after the treatment. When compared with healthy volunteers T-LGL patients had a distinct plasma cytokine signature (IL-12p40, TRAIL, IL22, IP10, MCP1, M-CSF, PDGF-AA, LIF, SCF) as well as JAK-STAT pathway activation prior to treatment but neither was correlated to clinical responses to alemtuzumab, likely due to the various prior IST regimens. Conclusion This is the largest cohort of T-LGL patients treated with alemtuzumab yet reported. Treatment was well tolerated and at this dose minimal side effects were observed. Alemtuzumab treatment in previously heavily treated T-LGL patients results in over 50% response rate and represents a good treatment option for this condition. Disclosures: Off Label Use: Alemtuzumab for T-LGL.

Description: The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p

Description: Abstract 2528 The quantitative lymphocyte recovery after remission induction is known to correlate with the outcome of the patients with acute myeloid leukemia (AML), suggesting the immune-surveillance is at least partly responsible for maintained remissions. However defects in lymphocyte function and increased regulatory T cell numbers suggest a permissive immune environment for leukemia cell growth. Understanding the interplay between the leukemia and the immune system may provide insight into the mechanisms governing leukemia cure or relapse after induction chemotherapy and lead to better remission treatments to prevent relapse. To look for evidence of immune escape and possible immunoediting of lymphocyte function by the leukemia, we undertook an extensive phenotypic characterization of lymphocytes and AML blasts in blood and marrow at presentation, through remission to full hematological recovery. Twenty patients (mean age 57 years; range 31–69) with newly diagnosed AML were enrolled in a protocol permitting blood and marrow collection for investigational purposes (VICCHEM 1073). Mononuclear cells were obtained from blood and marrow samples collected at onset, 14–21 days and 28–35 days after start of induction chemotherapy. Mononuclear cells were analyzed for lymphocyte phenotyping by flow cytometry and subset frequencies were compared with 5 healthy controls (mean age 31). Leukemic blast cells (Lineage CD2/3/19 negative, CD45+ cells) were characterized in blood and marrow. T cells showed several, largely persisting, abnormalities: (1) A decrease in central memory CD197+, CD27+, CD45RO+ CD8+ (p=0.03) and CD4+ T cells (p=0.1) in the blood at presentation with a corresponding decrease in CD197+CD27+ CD45RO-naïve subsets (2) A significant increase in PD1+ high expression suppressor phenotype for CD8+ (p=0.001) and CD4+ (p=0.01) cells at recovery (days 28–35). (3) At presentation FoxP3+ CD4+ regulatory T cells (Treg) were increased significantly at presentation (p=0.03) and remained elevated. This increase was due mainly to augmentation in the HeliosloFoxP3hi induced Treg (p=0.04) and CD45RAloFoxP3hi effector Treg fraction (p=0.002). These changes persisted into remission. The leukemic blast population included a variable population of cells with the CD11b+CD124+HLA-DRlo myeloid derived suppressor (MDSC) phenotype. The frequency of MDSC was higher in bone marrow (median 6.1% vs 3.1% in blood; p=0.44) and a significantly higher proportion of marrow MDSC expressed the lymphocyte suppressor molecule PDL-1 (54.5% vs 28.5%; p=0.0189). NK cell subsets showed significantly lower frequencies of immature CD56bright NK cells (p=0.02), and correspondingly decreased more mature CD56dim CD57-KIR-NKG2A+ subsets at presentation (p=0.039). NK cell subsets trend to recover during remission but with reduced frequency of mature CD57+, KIR+ CD56dim NK cells in recovery (p=0.018). This study confirms the occurrence of significant alterations in T cell phenotype associated with increased regulatory and suppressor phenotype. The occurrence of the MDSC phenotype within the leukemia blast population suggests that myeloid leukemia cells promote a permissive immune environment for leukemia growth and persistence through immunoediting. These changes as well as abnormalities in NK cells tend to persist into remission and may contribute to leukemic relapse. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Little is known about the lethality of acute GVHD (aGVHD) in T -cell depleted (TCD) allogeneic hematopoietic stem cell transplantation (HSCT). We examined the incidence of aGVHD and its relative contribution to non relapse mortality (NRM) in a cohort of consecutive TCD HSCT at a single institute. Methods: We report 132 consecutive subjects who had undergone TCD HLA-identical sibling HSCT between 2006 and 2016 for hematologic malignancies. All subjects received conditioning with Fludarabine 125mg/m2, Cytoxan 120 mg/kg and 1200 cGy total body irradiation (TBI) ( 31 days) achievement of complete donor lymphoid chimerism. Significantly improved outcomes were noted for those transplanted beyond 2012: OS, NRM and CIR being 82%,6.2% and 20% at one year, and 68%, 6.2% and 41% at 3 years. Cox proportionate hazard modeling identified steroid refractory aGVHD (HR 4.0, P=0.007) and transplant prior to 2012 (HR 6.7, P=0.001) as significant factors impacting NRM. Conclusions: T cell addback after ex vivo TCD HSCT was associated with a significant burden of aGVHD. Steroid refractory aGVHD impacted NRM, but slow lymphoid engraftment, disease risk, CMV reactivation and age did not. Significant improvements in NRM in the current era suggest greatly improved salvage of steroid refractory aGVHD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.