ALBERT

Description: Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P

Description: Anti-HLA donor-specific Abs (DSAs) have been reported to be associated with graft failure in mismatched hematopoietic stem cell transplantation; however, their role in the development of graft failure in matched unrelated donor (MUD) transplantation remains unclear. We hypothesize that DSAs against a mismatched HLA-DPB1 locus is associated with graft failure in this setting. The presence of anti-HLA Abs before transplantation was determined prospectively in 592 MUD transplantation recipients using mixed-screen beads in a solid-phase fluorescent assay. DSA identification was performed using single-Ag beads containing the corresponding donor's HLA-mismatched Ags. Anti-HLA Abs were detected in 116 patients (19.6%), including 20 patients (3.4%) with anti-DPB1 Abs. Overall, graft failure occurred in 19 of 592 patients (3.2%), including 16 of 584 (2.7%) patients without anti-HLA Abs compared with 3 of 8 (37.5%) patients with DSA (P = .0014). In multivariate analysis, DSAs were the only factor highly associated with graft failure (P = .0001; odds ratio = 21.3). Anti-HLA allosensitization was higher overall in women than in men (30.8% vs 12.1%; P 〈 .0001) and higher in women with 1 (P = .008) and 2 or more pregnancies (P = .0003) than in men. We conclude that the presence of anti-DPB1 DSAs is associated with graft failure in MUD hematopoietic stem cell transplantation.

Description: Minor histocompatibility antigens (mHA) mediate immune responses that can cause both graft vs. host disease (GvHD) and the graft vs. leukemia (GvL) effect observed in allogeneic stem cell transplantation (SCT). These responses result from donor T-cells being exposed to non-self peptides that are presented by HLA molecules on recipient cells. In contrast, cancer testis antigens (CTA) are peptide antigens derived from proteins, normally expressed only in male germ cells, but also aberrantly expressed in some tumors. These antigens can induce T-cell responses in an autologous setting without using T-cells from an allogeneic donor. Because, mHA that are expressed by hematopoietic tissue or leukemia could be therapeutic targets, which would enhance GVL without increasing the risk of GVHD we performed a genomics based mHA screen to identify high-value mHA. However, one of our predicted antigens, UNC-GRK4-1, was only expressed in testis and human AML, and has properties more consistent with a CTA. We used the Illumina NS-12 microarray platform to genotype 99 HLA-A0201 expressing myeloid leukemia patients and their HLA-matched donors at 13,917 non-synonymous coding single nucleotide polymorphisms (cSNPs). For both alleles in each of the 13,917 cSNPs we identified genetically predicted donor-recipient mHA responses, which were defined as the donor being homozygous for 1 allele and the recipient being either heterozygous or homozygous for the alternate allele. Using Fisher's exact test for each allele, we tested for the association between genetically predicted mHA responses and the clinical outcomes of remission vs. relapse over all 99 patients. Using the BioGPS database, we determined the tissue expression for the 261 alleles associated with remission to a p-value of

Description: PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)–restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)–like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [KD] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSCs) but not normal leukocytes (P 〈 .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P 〈 .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.

Description: Abstract 2090 We have shown that cytotoxic T lymphocytes (CTL) with specificity for the cyclin E (CCNE) derived HLA-A2-restricted peptide CCNE144-152 (ILLDWLMEV) specifically lyse myeloid and lymphoid leukemia in proportion to CCNE overexpression. Full length (FL) CCNE is also overexpressed in most solid tumors, including breast cancer where it is a poor prognostic factor. In addition, leukemia and many breast cancers express tumor-specific low molecular weight (LMW) isoforms of CCNE that result from post-translational processing of the FL protein. Neutrophil elastase (NE), derived from the primary granules of neutrophils, cleaves FL CCNE into LMW forms and NE has been identified in breast cancer tissue. Therefore, we hypothesized that CCNE may also be a breast cancer tumor antigen because the CCNE144-152 peptide is contained within the overexpressed FL and LMW forms, and that effective T cell immunity could be amplified by increased availability of LMW forms within tumor cells exposed to NE. The link between innate immunity, inflammation, and tumor immunity is poorly understood, and this mechanism could explain a role for tumor-infiltrating inflammatory cells in breast cancer. To test this, we elicited CCNE-CTL from peripheral blood lymphocytes of HLA-A2+ healthy donors by weekly stimulation with CCNE-pulsed T2 cells and low-dose IL-2. After 21 days, cytotoxicity of target cells by the lymphocytes was tested with a standard 4-hour calcein AM-based assay. The CCNE-CTL specifically lysed T2 cells pulsed with CCNE (30%) but not non-pulsed T2 cells (0%) at an effector:target (E:T) ratio of 20:1 (p 〈 0.01). Next, we tested whether CCNE-CTL killed HLA-A2+ MDA-MB-231 (231) breast cancer cells. First, we confirmed that FL CCNE and LMW forms were expressed in 231 cells, while low expression of FL CCNE and no expression of the LMW forms was observed in benign epithelial cells by Western blot. Next, we showed that CCNE-CTL mediated 49% lysis of 231 breast cancer cells at an E:T ratio of 20:1. To look for in vivo evidence of CCNE recognition, we studied peripheral blood lymphocytes from breast cancer patients by flow cytometry with CCNE/HLA-A2 tetramers and anti-CD8 antibodies. In 3 of 4 breast cancer patients we identified CCNE-CTL, with no detectable CCNE-CTL in healthy controls. Together, these results confirm that CCNE is also a tumor antigen in breast cancer. NE, which cleaves CCNE and is expressed in breast cancer tissue, may be produced endogenously by breast cancer cells, or exogenously by inflammatory cells in the tumor microenvironment. Therefore, we studied 231 cells and three other breast cancer cell lines (MDA-MB-453, MCF-7, and HER18) for NE expression. RT-PCR performed with NE-specific primers showed that none of the cells expressed NE mRNA and Western blot showed no NE protein expression, suggesting that NE in tumor tissue derives from neutrophils or other inflammatory cells. To determine whether NE is taken up by breast cancer cells, we used flow cytometry to show that 231 cells pulsed with soluble NE took up an increasing amount of NE and was maximal by 24 hours when intracellular NE expression in 231 cells was comparable to that of HL60 leukemia cells that express high levels of NE. In addition, LMW isoforms of CCNE were increased in NE-pulsed 231 cells, by Western blot. Importantly, CCNE-CTL specific lysis of NE-pulsed 231 cells was 2-fold higher compared to nonpulsed 231 (46% versus 22% at E:T 10:1, p = 0.01). Taken together, these data show that overexpressed CCNE144-152 is a novel breast cancer peptide antigen. Furthermore, exogenous NE is taken up by breast cancer cells, increasing LMW forms of CCNE and enhancing the susceptibility of breast cancer cells to CCNE-CTL-mediated cytolysis. This study links a specific enzyme secreted by neutrophils in the innate immune response to tumors to a specific adaptive immune response against breast cancer, and it suggests that immunotherapy targeting CCNE is warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4301 The leukemia associated antigens (LAA) proteinase-3 (P3) and neutrophil elastase (NE) are serine proteases found within neutrophil azurophil granules and are aberrantly expressed by leukemia. The human leukocyte antigen (HLA)-A2 restricted nonomeric peptide PR1, derived from P3 and NE, has been detected on leukemia cell surface in association with HLA-A2. PR1-specific cytotoxic T lymphocytes (CTL) were detected in patients following hematopoietic stem cell transplantation and were associated with clinical remission. Furthermore, in a phase I/II clinical trial, PR1-vaccine showed immunologic and clinical responses in patients with MDS and AML. Antigen cross presentation is a mechanism by which exogenous antigens are taken up by antigen presenting cells (APC) and presented in association with major histocompatibility (MHC)-I molecule. Cross presentation is thought to be the primary mechanism by which tumor antigens are presented to elicit tumor specific immune responses. Cross presentation by dendritic cells (DC), which express co-stimulatory molecules, leads to immune priming, while antigen presentation by B cells is associated with immune tolerance. Since cross presentation of leukemia antigens has been associated with anti-leukemia immune responses, we investigated whether P3 and NE are cross presented by DCs and B cells, and whether the source of P3 and NE (soluble vs. leukemia-cell associated) determines priming of the anti-PR1 immune response. We have previously reported that serum P3 levels are significantly higher in leukemia patients, and we now also report higher serum NE levels in patients with AML (median±standard deviation (SD)= 1236 ±572 ng/ml; n=15) and CML (median ± SD = 941 ± 494 ng/ml; n=15), in comparison with healthy donor blood (median ± SD = 65 ± 177 ng/ml; n=5) (p

Description: Abstract 2502 Leukemia stem cells (LSCs) in acute myeloid leukemia (AML) are resistant to conventional chemotherapy, able to sustain leukemia through treatment, and may regenerate disease after treatment completion. Understanding the biological differences between LSCs and non-LSC leukemic blasts will be important for developing improved and targeted AML therapies. We report here the results of an RNA-Seq analysis of differential gene and splice variant expression in LSC-enriched vs. non-enriched leukemia fractions in 8 AML patients. Using fluorescence activated cell sorting (FACS) we divided 8 adult AML patient blast samples into LSC-enriched (“LSC” = Lin-, CD34+, CD38-) and “non-LSC” fractions. We extracted total RNA from each sample, purified mRNA, and performed massively parallel RNA sequencing using the Illumina HiSeq platform. Hierarchical clustering based on RPKM gene expression values showed greater similarity between LSC and non-LSC fractions within each patient's leukemia than similarity of LSC and non-LSC fractions among patients. Three hundred seventy-one genes were differentially expressed between the LSC and non-LSC fractions, with the most prominent public differences being increased expression of myeloid differentiation markers (e.g. LYZ, MPO, HBA/B) in the non-LSC samples. The LSC fractions displayed increased expression of CD34, FLT3, N-ras, K-ras, AML1, cyclin D1, CD61, and M-SCF. Pathway analysis revealed statistically significant differences in gene expression in the KEGG-defined Acute Myeloid Leukemia, Cell Cycle, and Hematopoietic Cell Lineage pathways between the LSC and non-LSC fractions. Using the MapSplice software package we identified multiple novel splice variants present in ≥ 6 of the 8 AML patient samples. These splices were found in genes known to be associated with leukemogenesis including MYC, MTOR, and FLT3. In addition to the identification of novel splices in AML associated genes, we used the FDM software package to measure differential splice utilization between the LSC vs. non-LSC fractions. Using FDM, we measured statistically significant splice utilization between LSCs and non-LSCs in 87 genes including ZNF34, NUMA1, NBEAL2, BAT2D1, FGR, MAP3K6, RFX5, NFATC2IP, and LRMP. In addition to these globally differentially spliced genes, several splice variants were highly differentially expressed between LSC and non-LSC fractions in individual patients (e.g. MAGED4, CYBB, IL17RA). Somewhat unexpectedly, no genes were both significantly differentially expressed and differentially spliced in the LSC vs. non-LSC fractions. To our knowledge, the data reported here represent the first RNA-Seq analysis of LSC-enriched vs. non-enriched AML fractions. Our results confirm prior gene-expression microarray studies that showed differential gene expression in LSC-enriched vs. non-enriched fractions. We extend this work by reporting the discovery of novel splice variants in AML associated genes and observe, for the first time differential splice utilization between LSC and non-LSC fractions. These results should prove valuable for both understanding LSC biology and guiding development of LSC-specific therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2089 The human leukocyte antigen (HLA)-A2 restricted nonapeptide PR1 (VLQELNVTV) was shown to be immunogenic in leukemia. A phase I/II clinical trial has been initiated with PR1 peptide vaccine and to date has demonstrated clinical efficacy, including complete remission and immunologic responses in patients with acute (AML) and chronic (CML) myeloid leukemia, as well as myelodysplastic syndrome. PR1 is derived from the serine proteases proteinase-3 (P3) and neutrophil elastase (NE), which are normally found within neutrophil azurophil granules and are released into the inflammatory milieu. We have shown that P3 and NE are taken up and cross presented by antigen presenting cells and that their cross presentation elicits PR1 immunity. Because P3 and NE are present in breast cancer biopsies, we hypothesized that P3/NE may be taken up by breast cancer cells and cross presented to PR1-CTL. We recently demonstrated that the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 and HER18 do not endogenously express NE and that NE is taken up by these cell lines. In this report, using PCR, western blot and flow cytometry, we show that P3 also is NOT endogenously expressed by the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 or HER18. Using confocal microscopy, we demonstrate that P3 is taken up by these breast cancer cell lines within 10 minutes of pulsing and localizes to LAMP-2 containing lysosomal vesicles by 4 hours, suggesting its processing for presentation by (HLA)-I (i.e. HLA-A2). Using 8F4, the novel PR1-HLA-A2 monoclonal antibody, we show that PR1 is cross presented from P3 by 3 of 4 HLA-A2+ breast cancer cell lines (MDA-MB-231, MDA-MB-453-A2+, MCF-7), and from NE by 1 of 4 breast cancer cell lines (MDA-MB-231). Next, we studied whether PR1 presentation made cells susceptible to PR1-specific killing by PR1-CTL and the 8F4 monoclonal antibody. We show that following 12-hour pulsing of the MDA-MB-231 cell line with NE or P3, PR1 CTLs killed up to 31% and 38% of the NE- or P3-pulsed breast cancer cells respectively, vs.

Description: Background: Noncoding RNAs play an important role in the pathogenesis of CLL. Recent publications suggested that higher miR-155 plasma levels (above 56th percentile) were associated with a significantly lower survival rate (P= 0.0122) in CLL patients treated with conventional treatments (Ferrajoli et al. Blood 2013;122:1891). Other studies revealed that expression of miR-29b or miR-181b significantly inhibits T cell leukemia/lymphoma 1(Tcl1) oncogene (Pekarsky et al. Cancer Res 2006;66:11590) whereas high Tcl1 expression correlates with aggressive CLL phenotype showing unmutated immunoglobulin variable region genes and ZAP70 positivity. The impact of these miR expressions in CLL patients undertaking allogeneic stem cell transplantation (alloSCT) is unknown. Methods and Patients: We identified 41 patients with relapsed/refractory CLL who had received a non-myeloablative alloSCT at our center and in whom pre-transplant serum samples collected before initiating the patients’ allogeneic conditioning were available. We measured the serum expression of miR-155 as well as miR-15a/16-1 cluster, miR-29b and miR-181b. Total RNA was isolated from 100 µL of serum using the Total RNA Purification Kit. Serum miRNA levels were measured by qRT-PCR (TaqMan MicroRNA assays). The relative serum levels of miR-155, miR -15, miR-29b, miR-181b, and miR-16-1 were calculated using the equation 2−ΔCt, where ΔCt = mean CtmiRNA – mean Ctcel-miR-39, and Ct = threshold cycle. Twenty fmol of synthetic C. elegans miRNA (cel-miR-39) was spiked into each serum samples to normalize the experimental qRT-PCR data (cel-miR-39 Ct mean ± SD = 17.777± 0.628). Forty one patients were initially evaluated. Thirteen of these 41 patients were later excluded as they received alemtuzumab for graft-versus-host disease (GVHD) prophylaxis. Therefore, our analysis was limited to 28 patients who received their alloSCT between 2000-2010. Median age (range) was 59 (45-70) years. Median number of prior therapies was 3 (range, 2-8). Eleven (39%) had a beta-2 microglobulin level of 〉3 mg/L. Ten of 12 (83%) patients with data that could be evaluated had unmutated immunoglobulin variable-region heavy-chain gene, and 4/19 (21%) had 17p13.1 deletion. 46% of patients had refractory disease at transplantation. The proportion of patients who received a matched related and a matched unrelated donor was 68% and 32%, respectively. All patients received non-myeloablative conditioning with fludarabine, cyclophosphamide, and rituximab as previously published (Khouri et al. Cancer 2011;117:4679). GVHD prophylaxis consisted of tacrolimus and methotrexate. Results: Median (range) follow-up months was 68 (43-141). OS from the time of alloSCT was studied according to the relative expressions of miR under investigation (low, below median; high, above median). The 5-year OS rates of patients with low and high mir-155 were 63% and 50%, respectively (HR=1.6, P=0.4; Figure). No statistically significant differences were found in the other miRs studied. The 5-year OS was 57% for both the low and high miR-15a expression. The 5-year OS in low and high miR-29b and miR-181b were 52% and 61% (HR=0.8; P=0.7), and 60% and 53% (HR=1.1, P=0.9), respectively. The 5-year OS in low and high miR-16 were 43% and 71% (HR=0.4, P=0.2). Conclusions: Our preliminary results suggest that serum levels of miR-155, miR -15, miR-29b, miR-181b, and miR-16 are not a statistically significant prognostic biomarker for survival in relapsed/refractory CLL undertaking alloSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.