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Posttransplant cyclophosphamide (PTCy) platform has shown low rates of graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) after haploidentical hematopoietic cell transplantation (HaploHCT). However, because of the limited disease control, relapse rate remains a major cause of treatment failure in high-risk patients. Total marrow and lymphoid irradiation (TMLI) allows for delivery of high radiation to bone marrow and other targeted structures, without increasing off-target radiation exposure and toxicity to end organs. In this phase 1 trial, 31 patients with high-risk and/or active primary refractory leukemias or myelodysplastic syndrome underwent peripheral blood stem cell HaploHCT with TMLI, fludarabine, and cyclophosphamide as the conditioning regimen. Radiation dose was escalated in increments of 200 cGy (1200-2000 cGy). GVHD prophylaxis was PTCy with tacrolimus/mycophenolate mofetil. Grade 2 toxicities by the Bearman scale were mucositis (n = 1), hepatic (n = 3), gastrointestinal (n = 5), and cardiac (n = 2). One patient (1800 cGy) experienced grade 3 pulmonary toxicity (dose-limiting toxicity). At a follow-up duration of 23.9 months for the whole cohort; 2-year NRM was 13%. Cumulative incidence of day 100 grade 2 to 4 and 3 to 4 acute GVHD was 52% and 6%, respectively. Chronic GVHD at 2 years was 35%. For patients treated with 2000 cGy, with a median follow-up duration of 12.3 months, 1-year relapse/progression, progression-free survival, and overall survival rates were 17%, 74%, and 83%, respectively. In conclusion, HaploHCT-TMLI with PTCy was safe and feasible in our high-risk patient population with promising outcomes.

Purpose: To conduct a phase I trial of a Modified Vaccinia Ankara vaccine delivering wild-type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer.Experimental Design: Patients received gemcitabine on days 1 and 8 and p53MVA vaccine on day 15, during the first 3 cycles of chemotherapy. Toxicity was classified using the NCI Common Toxicity Criteria and clinical response assessed by CT scan. Peripheral blood samples were collected for immunophenotyping and monitoring of anti-p53 immune responses.Results: Eleven patients were evaluated for p53MVA/gemcitabine toxicity, clinical outcome, and immunologic response. Toxicity: there were no DLTs, but 3 of 11 patients came off study early due to gemcitabine-attributed adverse events (AE). Minimal AEs were attributed to p53MVA vaccination. Immunologic and clinical response: enhanced in vitro recognition of p53 peptides was detectable after immunization in both the CD4+ and CD8+ T-cell compartments in 5 of 11 and 6 of 11 patients, respectively. Changes in peripheral T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC) did not correlate significantly with vaccine response or progression-free survival (PFS). Patients with the greatest expansion of p53-reactive T cells had significantly longer PFS than patients with lower p53-reactivity after therapy. Tumor shrinkage or disease stabilization occurred in 4 patients.Conclusions: p53MVA was well tolerated, but gemcitabine without steroid pretreatment was intolerable in some patients. However, elevated p53-reactive CD4+ and CD8+ T-cell responses after therapy correlated with longer PFS. Therefore, if responses to p53MVA can be enhanced with alternative agents, superior clinical responses may be achievable. Clin Cancer Res; 24(6); 1315–25. ©2018 AACR.

Figure S1: Frequency of Tregs does not significantly decline post treatment and does not correlate with p53-reactivity or PFS.; Figure S2: Frequency of MDSC does not significantly differ to healthy controls, does not decline post treatment and does not correlate with p53-reactivity or PFS.; Supplementary Table T1: Multi variation analysis* shows an association between progression free survival and frequency of p53-reactive T cells, disease stage and baseline MDSC. Supplementary Table T2: Correlations (Pearson) of variables included in multi variation analysis.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome. CXCR5-PD-1hi peripheral T-helper (Tph) cells have an important pathogenic role in autoimmune diseases, but the role of Tph cells in cGVHD remains unknown. We show that in patients with cGVHD, expansion of Tph cells among blood CD4+ T cells was associated with cGVHD severity. These cells augmented memory B-cell differentiation and production of immunoglobulin G via interleukin 21 (IL-21). Tph cell expansion was also observed in a murine model of cGVHD. This Tph cell expansion in the blood is associated with the expansion of pathogenic tissue-resident T-helper (Trh) cells that form lymphoid aggregates surrounded by collagen in graft-versus-host disease (GVHD) target tissues. Adoptive transfer experiments showed that Trh cells from GVHD target tissues give rise to Tph cells in the blood, and conversely, Tph cells from the blood give rise to Trh cells in GVHD target tissues. Tph cells in the blood and Trh cells in GVHD target tissues had highly overlapping T-cell receptor α and β repertoires. Deficiency of IL-21R, B-cell lymphoma 6 (BCL6), or T-bet in donor T cells markedly reduced the proportions of Tph cells in the blood and Trh cells in GVHD target tissues and reduced T-B interaction in the lymphoid aggregates. These results indicate that clonally related pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues, and that IL-21R-BCL6 signaling and T-bet are required for the development and expansion of Tph and Trh cells in the pathogenesis of cGVHD.

Patients with colon cancer remain largely refractory to current immunotherapeutic strategies. This is, in part, due to the overexpression of the immune checkpoint protein indoleamine 2,3-dioxygenase 1 (IDO). IDO is an important enzyme contributing to tumor-mediated immunosuppression and also correlates with poor prognosis in colon cancer patients. The aim of this study was to assess the therapeutic efficacy of attenuated Salmonella typhimurium delivering an shRNA plasmid targeting IDO (shIDO-ST) in two mouse models of colorectal cancer. In vitro, the CT26 and MC38 murine colon cancer cell lines were shown to upregulate IDO expression following stimulation with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Transfection of both cell lines with shIDO plasmid reduced IDO protein expression and function. In vivo, shIDO-ST treatment significantly delayed CT26 and MC38 tumor progression compared to mice treated with scrambled shRNA control (shScr-ST) or the clinically-tested IDO inhibitor epacadostat. Increased tumor infiltration of neutrophils was found to be the primary immune cell population associated with shIDO-ST treatment, suggesting robust activation of innate immunity. Although increased tumor expression of IDO is associated with resistance to antibody therapy against programed cell death-1 (anti-PD1), co-administration of anti-PD1 with shIDO-ST did not provide additional tumor growth control in either model of colorectal cancer. Altogether, we demonstrate that treatment with shIDO-ST markedly delays tumor growth in two immunocompetent colorectal mouse models and this appears to be a superior therapeutic strategy compared to epacadostat or blocking anti-PD1 antibody therapy in colon cancer.

Efficacy of PTCy after mismatched unrelated donor (MMUD) HCT is unknown. In this pilot clinical trial, we enrolled 38 patients with hematologic malignancies scheduled to undergo MMUD-HCT (≥6/8 HLA-matched donors) onto 1 of 2 conditioning strata: myeloablative using fludarabine and fractionated total body irradiation (n = 19) or reduced intensity with fludarabine/melphalan (n = 19). Graft source was peripheral blood stem cells (PBSCs), and GVHD prophylaxis was PTCy, tacrolimus, and mycophenolate mofetil. Patients’ median age was 53 years (range, 21-72 years). Median number of HLA mismatches was 2 (range, 1-4) of 12 loci. Twenty-three patients (61%) were considered racial (n = 12) or ethnic (n = 11) minorities. Median time to neutrophil engraftment was 16 days (range, 13-35 days). With a median follow-up of 18.3 months (range, 4.3-25.0 months) for surviving patients, 1-year overall survival (OS) and GVHD-free/relapse-free survival (GRFS) were 87% (95% confidence interval [CI]: 71-94) and 68% (95% CI: 51-81), respectively. Cumulative incidence of nonrelapse mortality at 100 days and 1 year were 0% and 11% (95% CI: 4-27), respectively, whereas relapse/progression was 11% (95% CI: 4-27). Cumulative incidence of 100-day acute GVHD grades 2-4 and 3-4 and 1-year chronic GVHD were 50% (95% CI: 36-69), 18% (95% CI: 9-36), and 48% (95% CI: 34-68), respectively. The rate of moderate/severe chronic GVHD was 3% in the entire cohort. We showed highly promising OS/GRFS rates with an acceptable risk profile after PBSC-MMUD-HCT with PTCy. This trial was registered at www.clinicaltrials.gov as #NCT03128359.

Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933.

Chronic graft-versus-host disease (cGVHD) is a late-occurring inflammatory condition post- allogeneic hematopoietic cell transplantation (HCT), which involves multiple distinct interactions among alloreactive dysregulated T and B cells. Prior studies have shown that autoantibodies to platelet-derived growth factor receptor (PDGFR) induce tyrosine phosphorylation and accumulation of Reactive Oxygen Species (ROS), causing expression of collagen type-1 gene through the Ha-Ras-Erk1/2–ROS signaling pathway. We hypothesize that antioxidant markers of inflammation (8-OHdG, total antioxidant levels, CXCL4 and pH2Ax) will be elevated in patients with scleroderma cGVHD compared to patients who do not develop cGVHD or have non-scleroderma cGVHD. Patients (n=36) were consented on an IRB-approved protocol for a cross-sectional single time point biospecimen collection. Patients with established cGVHD (n=29) or ≥2 years post-HCT without clinical manifestations of cGVHD (n=7) were included. Patients with cGVHD were divided into Scleroderma cGVHD (n=14) and Non-scleroderma cGVHD (n=15). Median age was 49 (range: 21-69) and 56 years (range: 18-72) for patients with scleroderma (group 1) and without scleroderma or no GVHD (group 2), respectively. Median prior lines of therapy was 3.2 (range: 2-5) for patients in group 1. All patients is group 1 and 77% of patients in group 2 received PBCSs as the graft source. Transplant was from a matched unrelated (group 1: 75%, group 2: 73%) or a matched sibling donor (group 1: 25%, group 2: 23%). Tacrolimus/sirolimus was the most commonly used GVHD prophylactic regimen in both groups (group 1: 83% and group 2: 64%). Peripheral blood samples were drawn at a single time point post-HCT, and serum samples were used for ELISA assays for levels of total antioxidants, 8-OHdG, and CXCL4. Flow cytometric analysis was performed to investigate percentage of Tregs and pH2AX+ cells. There were no differences in the total antioxidant levels, CXCL4 and Tregs between groups 1 and 2. Using a cutoff threshold of 10 ng/ml of 8-OHdG, 11 patients (78%) in group 1 and 9 patients (40.9%) in groups 2 had elevated levels of 8-OHdG (p=0.029, Fisher's exact). Percentage of pH2AX expressing cells was lower in group 2. At the cutoff threshold of 37%, only one patient (7%) in group 1 and 13 patients (59%) in group 2 had more than 37% pH2AX-expressing CD45+ cells in their peripheral blood (p=0.002, Fisher's exact). In conclusion, our pilot cross-sectional study showed that scleroderma cGVHD is associated with increased ROS levels (8-OHdG) without elevation of pH2AX cells in peripheral blood T cells. Our results indicate that ROS elevation in scleroderma patients may be a stress response to inflammatory milieu and is independent of DNA damage. Our data justify further prospective cohort studies to define the role of ROS markers in scleroderma cGVHD and to develop novel strategies to treat/prevent cGVHD.

Chronic GVHD remains as the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Systemic corticosteroids are the first line of therapy, but outcomes are poor in steroid refractory (SR) patients. Previous reports show that in SR patients, IL-2 therapy results in partial response (PR) associated with increase in regulatory T cells (Tregs). ECP has been used for cGVHD with skin response rates of 40-100%. Based on non-overlapping toxicities and, we prospectively studied a combination of low dose IL-2 + ECP to treat SR-cGVHD. Patients with SR-cGVHD, were treated with low dose IL-2 (1 × 106 U/m2, daily) + ECP for 12 weeks. IL-2 was provided by Prometheus Laboratories. ECP was administered 2 times/week for 4 weeks followed by 2 treatments every 2 weeks for the next 8 weeks. The NIH consensus criteria was used to assess cGVHD severity. PBMC and plasma samples were banked at baseline and on 3-week intervals to assess regulatory T cell (Tregs) and reactive oxygen species (ROS). Of the 12 consented patients, 9 completed the study (2 were not treated due to rapid disease progression before enrollment and 1 died from sepsis at week 11). All enrolled patients (n=10) had scleroderma as the predominant cGVHD manifestation. Median age was 45.6 years (range: 23-66). Median prior lines of therapy were 4.2(range 3-6), 5 patients had prior exposure to ruxolitinib and 4 were on ECP at the enrollment. Donors were fully matched unrelated (n=8), matched sibling (n=1) or mismatched unrelated (n=1). Conditioning regimen was myeloablative (n=5) or reduced intensity (n=4). GVHD prophylaxis was tacrolimus/Sirolimus (tac/sir) (n=6), tac/methotrexate (MTX) (n=2), or Tac/sir/MTX (n=1). Of the 10 enrolled patients, 9 responded to IL-2 + ECP combination (all classified as PR). A 37% dose reduction in steroid dose was noted among 7 patients. The remaining 3 patients were on stable steroid dose at end of study. Only 1 patient had progressive cGVHD in form of scleroderma. ECP+IL-2 therapy was well tolerated with no drug related serious adverse events (SAE). Grade 3-4 AEs included grade 3 metabolic and nutrition (n=8), grade 3 anemia (n=3), and grade 4 atrial flutter (n=1). Infectious complications were noted in 3 patients (one grade 4 [sepsis]). There was a robust increase in Tregs from the baseline mean of 0.755% (range 0.08-1.88) to end of treatment 6.999% (range 0.78-17.13) [p=0.02] Fig 1. Increment in Tregs was seen in patients who were on ECP therapy prior to enrollment (n=4) and in one patient who progressed while on therapy. No correlation was found between the treatment response and ROS levels. In conclusion, the combination of ECP +IL-2 is well tolerated in patients with SR-cGVHD with no apparent increase in infectious complications or other toxicities. Clinical response correlates with a robust increase in Tregs, but no correlation was found with ROS levels.

Purpose: To conduct a phase I trial of a Modified Vaccinia Ankara vaccine delivering wild-type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer. Experimental Design: Patients received gemcitabine on days 1 and 8 and p53MVA vaccine on day 15, during the first 3 cycles of chemotherapy. Toxicity was classified using the NCI Common Toxicity Criteria and clinical response assessed by CT scan. Peripheral blood samples were collected for immunophenotyping and monitoring of anti-p53 immune responses. Results: Eleven patients were evaluated for p53MVA/gemcitabine toxicity, clinical outcome, and immunologic response. Toxicity: there were no DLTs, but 3 of 11 patients came off study early due to gemcitabine-attributed adverse events (AE). Minimal AEs were attributed to p53MVA vaccination. Immunologic and clinical response: enhanced in vitro recognition of p53 peptides was detectable after immunization in both the CD4+ and CD8+ T-cell compartments in 5 of 11 and 6 of 11 patients, respectively. Changes in peripheral T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC) did not correlate significantly with vaccine response or progression-free survival (PFS). Patients with the greatest expansion of p53-reactive T cells had significantly longer PFS than patients with lower p53-reactivity after therapy. Tumor shrinkage or disease stabilization occurred in 4 patients. Conclusions: p53MVA was well tolerated, but gemcitabine without steroid pretreatment was intolerable in some patients. However, elevated p53-reactive CD4+ and CD8+ T-cell responses after therapy correlated with longer PFS. Therefore, if responses to p53MVA can be enhanced with alternative agents, superior clinical responses may be achievable. Clin Cancer Res; 24(6); 1315–25. ©2018 AACR.

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT) for acute leukemia and myelodysplastic syndrome (MDS). Wilms' tumor antigen (WT1) is overexpressed in the majority of acute leukemia and MDS patients and has been proposed as a universal diagnostic marker for detection of impending relapse. Comprehensive studies have shown that WT1 transcript levels have predictive value in acute leukemia patients in complete remission after chemotherapy. However, the focus of this study is the period after alloHCT for predicting relapse onset. We analyzed the accumulation of WT1 mRNA transcripts in peripheral blood of 82 leukemia and MDS patients and defined specific molecular ratios for relapse prediction. The extensively validated WT1/c-ABL ratio was used to normalize increases in WT1 transcript levels. The observed lead time of crossing or exceeding set WT1 levels is presented along with linear interpolation to estimate the calculated day the WT1 thresholds were crossed. The WT1/c-ABL transcript ratio of 50 or above yielded 100% specificity and 75% sensitivity reliably predicting future relapse with an observed average of 29.4 days (SD=19.8) and a calculated average of 63 days (SD=29.3) lead time before morphologic confirmation. A lower ratio of 20 or above gave lower specificity but higher sensitivity (84.8 and 87.5%, respectively) identified more patients that relapsed, at earlier times, providing an earlier warning with actual average lead time of 49.1 days (SD=30.8) and calculated average of 78 days (SD=28.8). WT1 transcript levels serve as a diagnostic relapse test with greater sensitivity than the morphologic approach used in the clinic as a readout.

Wilms' tumor antigen (WT1) is overexpressed in many different solid tumors and hematologic malignancies. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). In a cross-sectional survey study, we investigated the immune recognition of WT1 by patients with NHL. Utilizing a WT1 overlapping peptide library, we discovered that a large percentage of patients with NHL of all grades maintain WT1-specific T cells. Ex vivo frequencies of these T cells measured from unfractionated samples by the CD137 activation marker assay were high in many patients (some > 1% CD8+). Using standard in vitro techniques we discovered that they were cytotoxic to WT1 peptide library-loaded T2 cells and WT1 antigen-primed autologous Epstein-Barr virus-transformed B cell lines (EBV-LCLs) and expressed interferon gamma (IFN-γ). In addition, we detected WT1 mRNA transcripts in diseased lymph node tissues of patients with NHL utilizing real-time quantitative polymerase chain reaction (RT-qPCR) technology. These results are the first example of strong T cell reactivity against WT1 in patients with NHL which also demonstrate strong cytotoxicity against peptide-loaded tumor cells. The potential for developing WT1 as a target for immunotherapy in NHL deserves further exploration.

Despite the clinical success of anti-PD1 antibody (α-PD1) therapy, the immune mechanisms contributing to the antineoplastic response remain unclear. Here, we describe novel aspects of the immune response involved in α-PD1-induced antitumor effects using an orthotopic Kras (G12D)/p53(R172H)/Pdx1-Cre (KPC) model of pancreatic ductal adenocarcinoma (PDA). We found that positive therapeutic outcome involved both the innate and adaptive arms of the immune system. Adoptive transfer of total splenocytes after short-term (3 d) but not long-term (28 d) PD1 blockade significantly extended survival of non-treated tumor-bearing recipient mice. This protective effect appeared to be mostly mediated by T cells, as adoptive transfer of purified natural killer (NK) cells and/or granulocyte receptor 1 (Gr1)(+) cells or splenocytes depleted of Gr1(+) cells and NK cells did not exhibit transferrable antitumor activity following short-term PD1 blockade. Nevertheless, splenic and tumor-derived CD11b(+)Gr1(+) cells and NK cells showed significant persistence of α-PD1 bound to these cells in the treated primary recipient mice. We observed that short-term inhibition of PD1 signaling modulated the profiles of multifunctional cytokines in the tumor immune-infiltrate, including downregulation of vascular endothelial growth factor A (VEGF-A). Altogether, the data suggest that systemic blockade of PD1 results in rapid modulation of antitumor immunity that differs in the tumor microenvironment (TME) when compared to the spleen. These results demonstrate a key role for early immune-mediated events in controlling tumor progression in response to α-PD1 treatment and warrant further investigation into the mechanisms governing responses to the therapy at the innate-adaptive immune interface.

The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis. Previous studies have shown that the 2C polypeptide specifically interacts with the 3′-terminal sequence of poliovirus negative-strand RNA. This interaction is facilitated by the presence of the conserved sequence UGUUUU in the “stem a” within the 3′-terminal cloverleaf structure. We show that the 2C precursor, 2BC, also interacts with the 3′-terminal cloverleaf of the negative-strand RNA. We also demonstrate here that interaction of 2C/2BC with the negative strand 3′-terminal sequence not only depends on an intact “stem a” containing the UGUUUU sequence but is equally influenced by the presence of an intact “stem b” within the negative-strand cloverleaf. The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3′-terminal negative-strand cloverleaf structure.

Despite the growing understanding of how PD1 signaling contributes to the immunosuppressed tumor microenvironment (TME), the mechanisms underlying tumor regression by antibody blockade remain unclear. In this study, we sought to identify the early events involved in mediating the anti-tumor effects of anti-PD1 monoclonal antibody (α-PD1 mAb) using an orthotopic mutant KrasG12D/p53R172H Pdx-Cre (KPC) pancreatic ductal adenocarcinoma (PDA) model. This model is clinically relevant because in addition to KPC cells constitutively expressing the programmed death 1 ligand 1 (PD-L1), it represents advanced tumors due to its fast growth and drug resistant phenotype. When compared to hamster IgG isotype, single dose treatment with α-PD1 mAb (clone J43, 250µg/mouse) prolonged survival in C57BL6 mice bearing well established tumors (32 vs 76 days median survival time, respectively, p=0.0003). Mice that received α-PD1 mAb therapy presented transient moderate splenomegaly with increased spleen cellularity 72 hours following mAb administration. To determine if the transient splenomegaly represented a correlate of immune protection against tumor challenge, splenocytes from tumor bearing mice treated with IgG isotype or α-PD1 mAb for 72 hours, 7 days or 4 weeks were transferred into recipient tumor bearing mice that were pre-conditioned with cyclophosphamide (100mg/kg). Surprisingly, transferable protective immunity was only observed in the group that received splenocytes from donor mice treated with α-PD1 mAb for 72 hours (39.5 vs. 63 days median survival time for the IgG isotype vs. α-PD1 mAb treated group, p=0.042). Analysis by real time qPCR of tumor-infiltrating lymphocytes (TIL) obtained by selection of CD45+ cells using magnetic beads showed an increase in mRNA expression of IL-6 (4.6 fold), IL-8 (2.7 fold), IL-10 (6.2 fold), IFN-γ (3.8 fold) and TNF-α (4 fold) at 7 days, but not at 72 hours after PD1 blockade, suggesting that alterations in the splenic microenvironment influenced subsequent immune activation in the TME. Furthermore, individual depletion of immune cell subsets generally regarded as involved with the innate (Gr1+ cells, NK cells) or adaptive arms of the immune response (CD4 or CD8 T cells) were all capable of blocking the effect of α-PD1 mAb therapy in donor mice (p≤0.001). These results corroborate the importance of tumor recognition by the innate immune system in order to activate T cell-mediated killing, showing that stimulation of innate and/or adaptive immunity may play a direct role in controlling tumor progression. Alternatively, gene expression analysis using the non-TIL enriched fraction (remaining CD45- cells) extracted from tumors 7 days after PD1 inhibition showed downregulation of vascular endothelial growth factor A (VEGF-A) and G1/S related cyclin mRNA, such as cyclin-A, -D2 and E, suggesting that inhibition of PD1 induced tumor cell cycle arrest occurred concomitantly with upregulation of cytokine expression. Downregulation of VEGF-A was confirmed by ELISA using whole tumor extracts (p=0.03). Even though IL-10 is an inhibitor of VEGF-A, in vitro experiments using KPC cells failed to demonstrate a direct connection. However, TME conditions are complex, and often non-reproducible in vitro. Based on our results, we challenge the notion that PD1 blockade inhibits tumor progression mainly as a result of activating canonical adaptive immune responses. In addition, induction of G1/S tumor cell cycle arrest could increase sensitivity to cytotoxic therapies, thus providing a rationale for drug combinations. Given the broad role of VEGF-A in fostering an immunosuppressive TME and as an endothelial/epithelial growth factor, further studies are underway to investigate the role of VEGF-A inhibition in α-PD1 mAb-mediated tumor regression. Citation Format: Marcela d'Alincourt Salazar, Edwin Manuel, Weimin Tsai, Massimo D'Apuzzo, Leanne Goldstein, Bruce R. Blazar, Don J. Diamond. Blockade of checkpoint programmed death 1 (PD1) delays tumor growth and induces early transferable protective immunity in a murine orthotopic pancreatic ductal adenocarcinoma model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B170.

Poliovirus (PV) is the prototype virus of a large group of medically important viruses (picornaviruses) that include those inducing poliomyelitis (polioviruses), infectious hepatitis (hepatitis A virus), the common cold (rhinoviruses), and encephalitis and myocarditis (coxsackie viruses) (31). The single-stranded, plus-polarity RNA genome (∼7,500 nucleotides) of PV (18, 30) is translated into one large polyprotein, which is cotranslationally processed by virally encoded proteases 2Apro, 3Cpro, and 3CDpro into mature viral structural and nonstructural proteins (22). The viral proteases have been studied extensively and found to be very specific in polyprotein cleavage; 3Cpro and 3CDpro cleave the polyprotein at glutamine-glycine (Q-G) bonds while the 2Apro cleaves only at tyrosine-glycine (Y-G) bonds (20). The proteases do not cleave every potential cleavage site within the polyprotein; other determinants such as accessibility and context of the cleavage site are also important. Accurate initiation of transcription by RNA polymerase (Pol) II requires the assembly of a multiprotein complex on the core promoter around the mRNA start site (13). The multiprotein complex, consisting of at least seven to eight general transcription factors (GTFs) and RNA Pol II form a preinitiation complex at specific cis-acting elements (promoters) on the DNA template. The most common cis-acting element, the TATA box, is situated approximately 25 nucleotides upstream of the transcription start site. Among the GTFs in the complex, the TATA-binding protein (TBP) has been studied extensively (29). The TBP was first identified through its role in Pol II transcription, where it associates with 13 or 14 TBP-associated factors to form the TFIID complex (34). The unique aspect of TBP is its additional presence in complexes required for RNA Pol I (SL1) and RNA Pol III (TFIIIB) transcription. In both SL1 and TFIIIB complexes, TBP associates with a distinct set of TBP-associated factors (7, 33). Thus, TBP appears to be involved in all three RNA polymerase-mediated transcriptions. TBP has a bipartite structure with a highly conserved C-terminal core domain (amino acids 159 to 339), which folds as a molecular saddle, and is responsible for DNA binding via the concave underside. Additionally, the core also interacts with GTFs and other regulatory proteins via the solvent-exposed convex side. In contrast, the N-terminal region (amino acids 1 to 141) is variable in both length and sequence but is well conserved among vertebrates. It modulates DNA binding by interacting with the core domain and is characterized by a glutamine repeat region (Fig. ​(Fig.1).1). Recent studies have underscored the importance of the TBP N-terminal domain in transcriptional regulation both in yeast and mammalian systems (15, 21). Moreover, deletion of 55 and 96 amino acids from the N-terminal domain of TBP leads to inactivation of TATA-mediated transcription from the U6 small nuclear RNA promoter (24). FIG. 1. Shutoff of RNA polymerase II-mediated transcription does not correlate with 3Cpro-induced cleavage at the 18th glutamine-glycine site in TBP. (A) The domain structure of TBP consisting of the core, N-terminal with the glutamine stretch (Q), and acidic ... Infection of HeLa cells with PV causes a severe decrease in cellular transcription catalyzed by all three cellular RNA polymerases (10). Transcription mediated by RNA polymerase I (Pol I) is inhibited first, at 1 to 2 h postinfection, followed by inhibition of Pol II and Pol III transcription at approximately 3 and 4 h postinfection, respectively. Crawford et al. first showed that PV-induced inhibition of transcription observed in vivo could be recapitulated in vitro (8). Further studies showed that the viral protease 3Cpro alone was responsible for the shutoff of transcription by all three cellular RNA polymerases (36). Recent studies have shown that 3Cpro enters the nucleus in the form of its precursor 3CD (32), which presumably undergoes autocatalysis to generate 3Cpro in the nucleus of infected cells. A primary target of 3Cpro in PV-infected cells was previously identified to be the TATA-binding protein (6). Consistent with this observation, the TFIID complex isolated from PV-infected HeLa cells was transcriptionally inactive in an in vitro reconstituted transcription assay compared to the TFIID isolated from uninfected cells (19). Moreover, purified TBP could be directly cleaved by the purified 3Cpro in vitro, and the addition of purified TBP could completely restore both basal and activated transcription from the TATA and initiator promoters in HeLa cell extracts from PV-infected cells (37). Examination of the human TBP sequence revealed three Q-G sites at positions 12, 18, and 112 (Fig. ​(Fig.1).1). Subsequent in vitro studies using purified components showed that only the 18th Q-G site in TBP could be efficiently cleaved by 3Cpro both in vitro and in vivo (9). To determine if cleavage of TBP at the 18th Q-G bond is responsible for the shutoff of transcription in PV-infected cells, we expressed and purified a recombinant TBP that lacks the N-terminal 18 amino acids (called ΔN18 TBP). If cleavage at the 18th Q-G bond is the primary cause of transcriptional inactivation of TBP, then ΔN18 TBP should not be able to restore transcription in PV-infected cell extracts. We found that ΔN18 TBP was as active as the wild-type (wt) TBP in fully restoring Pol II transcription in PV-infected cell extracts from the adenovirus major late promoter (Ad MLP). We also found that the transcriptional activity of ΔN18 TBP was comparable to that of wt TBP in an in vitro transcription reconstitution assay. Since both genetic and biochemical evidence suggested that 3Cpro was involved in the shutoff of transcription and that TBP was the primary target of 3Cpro, we set out to determine if TBP was being cleaved by 3Cpro at sites other than the 18th Q-G bond. We report here identification of a Q-S site at position 104 to 105 of TBP, which appears to be cleaved by the viral protease 3Cpro. We also demonstrate that an alanine residue at P4 is critical for 3C-mediated cleavage of the 104th Q-S bond. A truncated form of TBP lacking the first 100 amino acids is significantly less active transcriptionally than the wt TBP in vitro. Finally, we demonstrate that a stable HeLa cell line expressing a recombinant TBP (rTBP) resistant to cleavage by the viral proteases is significantly refractive to the shutoff of transcription by poliovirus compared to the control cell line expressing wt TBP. Infection with poliovirus of the HeLa cell line expressing the noncleavable form of TBP shows small plaques and significantly reduced viral RNA synthesis compared to the control TBP cell line, suggesting that a defect in the shutoff of transcription can lead to inefficient replication of the virus.

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3C(pro), is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICalpha, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2A(pro), also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.

Infection of HeLa cells with poliovirus leads to rapid shut-off of host cell transcription by RNA polymerase II. Previous results have suggested that both the basal transcription factor TBP (TATA-binding protein) and transcription activator proteins such as CREB (cyclic AMP-responsive element-binding protein) and Oct-1 (the octamer-binding factor) are cleaved by the viral-encoded protease, 3C Pro . Here we demonstrate that the transcriptional activator (and tumor suppressor) p53 is degraded by the viral protease 3C both in vivo and in vitro. Unlike other transcription factors that are directly cleaved by 3C pro , degradation of p53 requires a HeLa cell activity in addition to 3C Pro . The degradation of p53 by 3C Pro does not appear to involve the ubiquitin pathway of protein degradation. Vaccinia virus infection of HeLa cells leads to inactivation of the cellular activity required for 3C Pro -mediated degradation of p53. The vaccinia-encoded protein (CrmA) is known to inhibit caspase I (ICE protease) that converts inactive IL-1β to an active secreted form. Incubation of HeLa cells with caspase I inhibitor Z-VAD-fmk does not interfere with 3C Pro -mediated degradation of p53. The cellular activity present in extracts of HeLa cells can be fractionated through phosphocellulose. A partially purified fraction that elutes at 0.6 M KCl from phosphocellulose contains the activity that degrades p53 in a 3C Pro -dependent manner. These results suggest that both poliovirus-encoded protease 3C Pro and a cellular activity are required for the degradation of p53 observed in cells infected with poliovirus.

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (HCT) for leukemia and myelodysplastic syndrome (MDS). In order to improve the outcome of leukemia patients after allogeneic HCT, it is imperative to identify reliable markers to predict impending relapse. Wilms’ tumor antigen (WT1) is overexpressed in the majority of leukemia and MDS patients and is being considered as a possible universal diagnostic marker for minimal residual disease (MRD), especially since no chromosomal translocation has nearly the frequency of association as WT1 does with leukemia. In this study we prospectively evaluated the prognostic value of MRD monitoring by qRT-PCR for WT1 transcripts. WT1 transcript levels in peripheral blood mononuclear cells (PBMC) were measured utilizing SYBR-Green qRT-PCR on the ABI7300 instrument (Applied Biosystems, Carlsbad, CA) and results were expressed as a ratio of WT1/c-ABL transcript copies normalized by 104 (WT1 ratio: WT1/c-ABLx104). PBMC samples were obtained monthly for 6 months post-HCT, then on alternating monthly schedule until 3 years post-HCT. Patients >18y.o. with confirmed diagnosis of MDS with A total of 83 patients (median age: 54, range: 19-74) with AML (n=39), ALL (n=24), MDS (n=17), or CML (n=3) received allogeneic HCT after fully ablative (n=39) or reduced-intensity (n=44) conditioning. Donor sources were matched related (n=33), unrelated (n=50), or umbilical cord blood (n=2). Fifty-one patients were considered low-risk (AML/ALL in CR1, CML in 1stchronic phase, or MDS-RA/RARS subtypes) while the remaining 32 patients were considered high risk. Sixteen of 83 patients relapsed with a median time of 238 days post-HCT (range: 76-747). The minimum WT1 ratio that gave specificity of 100% in predicting relapse was 50 (95% binomial exact CI: 92.5-100%), as none of the non-relapsed patients crossed this level. Of 16 patients who relapsed, 12 crossed the WT1 ratio of 50, providing a sensitivity of 75% (95% binomial exact CI: 48- 93%). The positive predictive value (PPV) and the negative predictive value (NPV) performance parameters for the WT1 ratio of 50 were 100% and 94.4%, respectively (Table 1). There was an average number of 63 days (SD=29.3) from crossing the WT1 ratio of 50 to hematologic relapse for the 12 relapsed patients. Since PBMC samples from healthy donors consistently demonstrated a WT1 ratio In summary, our data demonstrate that the quantitative measurement of WT1 transcripts is a reliable marker to assess MRD post-HCT for leukemia patients, and its real-time prospective monitoring provides a 2-3 month window of opportunity to introduce medical/immunologic interventions prior to overt hematologic relapse.Table 2WT1 ratio Specificity Sensitivity Positive Predictive Value Negative Predictive Value Days to Relapse (SD)50100%75%100%94.4%63 days (29)4095.5%75%80%94.1%66.9 days (29)3094%75%75%94%71.5 days (30)2085%87.5%58.3%96.6%78 days (28)1056.7%93.8%34.1%97.4%107.5 days (58) Disclosures: No relevant conflicts of interest to declare.

Abstract 953 Wilms' tumor antigen (WT1) is expressed in many solid tumors and hematologic malignancies and serves a physiological role in the maintenance of the tumorigenic phenotype. Immunologic recognition of WT1 has been observed in patients with myeloid and lymphoid leukemia, solid tumors, and even in healthy adults. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). Current therapeutic modalities for NHL patients are predominantly based on the highly successful application of the anti-CD20 mAb (Rituximab™) or radioactive derivatives (Zevalin™ or Bexxar™) combined with standard chemotherapy. Recent approaches have focused on generating NHL-specific idiotypes as an example of personalized medicine. However, harnessing a T cell response to malignant B or T cells in NHL patients has not succeeded, despite its attractiveness as a universal therapeutic option. We developed a detection system for WT1-specific T cells that is HLA independent by constructing a peptide library of 15mers (11 residues overlapping) corresponding to full length WT1 antigen. We analyzed peripheral blood mononuclear cells (PBMC) after priming with the peptide library for immune responses to the WT1 antigen detectable by flow cytometry based on the CD137 activation marker. Sixty-three NHL patients and 30 healthy donors (HD) were recruited under IRB-approved protocols. The cohort included patients who received allogeneic stem cell transplantation (alloHCT: n=42) or chemotherapy/autologous HCT (n=21). We observed that WT1-specific ex vivo CD8+ T cell responses were significantly greater in our NHL patient cohort (median 0.035%) compared to our randomly chosen HD cohort (0%, p=0.0013)(Figure 1). These WT1-specific CD8+CD137+ T cells co-expressed CD45RA and CD57 surface markers, consistent with them being a TEMRA (Terminal Effector Memory, RA+) population of highly differentiated T cells. Twenty-four of 43 patients (55.8%) with high/intermediate-grade NHL had a positive CD8+ T cell response (CD8+CD137+ ≥0.05%) to the WT1 antigen compared to four of 20 patients (20%) with low-grade NHL (p=0.011). None of the other clinical variables such as patient age, treatment modality (alloHCT vs. autoHCT or chemotherapy), disease status (CR vs. non-CR), prior acute GVHD (only in alloHCT, grade 0-I vs. II–IV) were significantly associated (p≤0.05) with WT1-specific CD8+ T-cell response in NHL patients when we conducted a multivariate analysis. PBMC from one HD and 12 NHL patients were expanded using in vitro stimulation (IVS) with the WT1 peptide library. All PBMC samples expanded in culture after 10–14 days by single IVS (median 7-fold expansion). We observed significantly higher IFN-γ responses in CD8+ T cells when IVS cells were re-stimulated with the WT1 library (median=3.0%, range: 0.08–5.5%) compared with the medium control (median=0.39%, range: 0.05–1.7%, p=0.002). Remarkably, using standard in vitro techniques, the expanded PBMC were cytotoxic to WT1-peptide library-loaded T2 cells and autologous EBV-transformed B-cell lines derived from patients enrolled in the study. These results challenge the notion that central tolerance mechanisms eliminate high affinity WT1-specific T cells that kill tumor cells. In order to distinguish whether WT1-specific T cells are maintained in NHL patients because of an aberration of tolerance mechanisms or the presence of WT1 antigen as a stimulator, we used sensitive real time quantitative PCR (RT-qPCR) methods to detect transcripts in LN from NHL patients and HD. We succeeded in detecting WT1 mRNA transcripts in diseased lymph node (LN) tissues of NHL patients utilizing RT-qPCR technology. There was significantly greater WT1 mRNA in NHL LN (n=6) with an average copy number ratio of 39 (WT1/c-ABL × 104) compared with 0.66 in non-disease LN obtained from individuals without a cancer diagnosis (n=7, p Disclosures: No relevant conflicts of interest to declare.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.