PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemiaassociated 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)- IntroductionCD8 T cells specific for the human leukocyte antigen-A2 (HLA-A2)-restricted peptides WT1 and PR1, which are derived from the endogenous leukemia-associated antigens Wilms' tumor antigen [1][2][3] and proteinase 3 (P3), respectively, mediate cytotoxicity against acute myeloid leukemia (AML). PR1-specific T cells also contribute to cytogenetic remission of chronic myeloid leukemia (CML) in patients treated with interferon, 4,5 and vaccination with WT1 and PR1 6,7 can induce specific CD8 immunity in patients with myeloid malignancies. These results validate endogenous self-peptides as targets for immunotherapy, including vaccination, adoptive cell therapy, or antibodies that bind peptide/MHC. Such T-cell receptor (TCR)-like monoclonal antibodies (mAbs) may have selective activity against leukemia if target peptide/MHC complexes are aberrantly expressed on leukemia. Furthermore, mAbs are easy to administer and can be dosed frequently, which may increase their effectiveness against high leukemia burdens.Eliciting TCR-like mAbs has been technically challenging, 8 primarily because of the high immunogenicity of HLA molecules in mice. Phage-display libraries, 9 peptide/MHC immunization, 10,11 and the combination of both strategies 8,12 have been used to produce TCR-like mAbs targeting peptides derived from solid-tumor antigens (eg, MAGE, ␤-HCG, TARP, and NY-ESO-1) in the context of HLA-A1 or HLA-A2. [9][10][11]13,14 Although antibody activity against primary tumors has not been well studied, complement-dependent cytotoxicity (CDC) against tumor cell lines has been reported. 11 Some toxin-conjugated antibodies also show activity against tumor cells. 14-16 However, to eradicate cancer, these antibodies must be active against cancerinitiating cells, and TCR-like mAb-induced cytolysis of cancer stem cells has not been reported. Nevertheless, because PR1-specific CTLs suppress leukemia progenitor cells in vitro 17 and because Lin Ϫ CD34 ϩ CD38 Ϫ cells are enriched for leukemia stem cells (LSCs) 18 and can be easily studied, we hypothesized that if an anti-PR1/HLA-A2 antibody could be produced, it may be active against blasts and LSCs from HLA-A2 ϩ AML patients.We report the discovery of 8F4, a novel mAb that binds with high affinity to a conformational epitope of PR1/HLA-A2 and induces dose-dependent cytolysis of myeloid leukemia cells but not normal hematopoietic cells. 8F4 mediates CDC against Lin Ϫ CD34 ϩ CD38 Ϫ LSCs and preferentially inhibits the growth of leukemia progenitor cells. These results justify further study of TCR-like antibodies to verify the differential effects against normal stem cells and LSCs. Biologically significant differences may justify further study of a humanized form o...

BackgroundEmerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity.DesignMouse GL261-glioma model was set up in immunocompetent C57BL/6 mouse for Delta-24-RGD treatment. The changes of the immune cell populations were analyzed by immunohistochemistry and flow cytometry. The anti-glioma immunity was evaluated with functional study of the splenocytes isolated from the mice. The efficacy of the virotherapy was assessed with animal survival analysis. The direct effect of the virus on the tumor-associated antigen presentation to CD8+ T cells was analyzed with an in vitro ovalbumin (OVA) modeling system.ResultsDelta-24-RGD induced cytotoxic effect in mouse glioma cells. Viral treatment in GL261-glioma bearing mice caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, viral infection and IFNγ increased the presentation of OVA antigen in OVA-expressing cells to CD8+ T-cell hybridoma B3Z cells, which is blocked by brefeldin A and proteasome inhibitors, indicating the activity is through the biosynthesis and proteasome pathway.ConclusionsOur results demonstrate that Delta-24-RGD induces anti-glioma immunity and offers the first evidence that viral infection directly enhances presentation of tumor-associated antigens to immune cells.

SUMMARY Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including srsf2. RNA-Seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling and histone modification, which are also enriched in mouse CMP haploinsufficient for srsf2 and in CD34+ CMML patient cells harboring srsf2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing and myeloid progenitor differentiation.

There is little understanding of the impact of tumor-associated neutrophils (TAN) on adaptive immunity to tumors. In this study, we report the results of an investigation of the pathobiological basis for the prognostic significance of neutrophil elastase (NE), a serine protease found in neutrophil granules, in a model of cyclin E-overexpressing breast cancer. We established that NE was expressed by TAN within breast cancer tissues but not by breast cancer cells. NE modulated killing of breast cancer cells by cytotoxic T lymphocytes (CTL) specific for cyclin E-derived HLA-A2 restricted peptide (ILLDWLMEV). Breast cancer cells exhibited striking antigen-specific uptake of NE from the microenvironment that was independent of NE enzymatic activity. Further, NE uptake increased expression of low molecular weight forms of cyclin E and enhanced susceptibility to peptide-specific CTL lysis, suggesting that cyclin E peptides are naturally presented on breast cancer cells. Taken together, our findings reveal a previously unknown mechanism of antitumor adaptive immunity that links cancer cell uptake of an inflammatory mediator to an effective cytolytic response against an important breast cancer antigen.

PR1 is a human leukocyte antigen (HLA)-A2 restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived antigen presenting cells, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific cytotoxic T lymphocytes (PR1-CTL) and the anti-PR1/HLA-A2 antibody 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an antigen become susceptible to therapies that target cross-presented antigens and suggest PR1 as a broadly expressed tumor antigen.

PR1, an HLA-A2-restricted peptide derived from both proteinase 3 and neutrophil elastase, is recognized on myeloid leukemia cells by cytotoxic T lymphocytes (CTL) that preferentially kill leukemia and contribute to cytogenetic remission. To evaluate safety, immunogenicity and clinical activity of PR1 vaccination, a phase I/II trial was conducted. Sixty-six HLA-A2+ patients with acute myeloid leukemia (AML: 42), chronic myeloid leukemia (CML: 13) or myelodysplastic syndrome (MDS: 11) received three to six PR1 peptide vaccinations, administered subcutaneously every 3 weeks at dose levels of 0.25, 0.5 or 1.0 mg. Patients were randomized to the 3 dose levels after establishing the safety of the highest dose level. Primary endpoints were safety and immune response, assessed by doubling of PR1/HLA-A2 tetramer-specific CTL, and the secondary endpoint was clinical response. Immune responses were noted in 35 of 66 (53%) patients. Of the 53 evaluable patients with active disease, 12 (24%) had objective clinical responses (complete: 8, partial: 1 and hematological improvement: 3). PR1-specific immune response was seen in 9 of 25 clinical responders vs. 3 of 28 clinical non-responders (p=0.03). In conclusion, PR1 peptide vaccine induces specific immunity that correlates with clinical responses, including molecular remission, in AML, CML and MDS patients.

Background aims The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T-cells to mediate anti-leukemia activity. Methods Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endodomain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

Purpose Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. Experimental Design We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were performed to characterize the immune response to CG in patients. Results CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and demonstrated immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL mediated cytotoxicity, further confirming HLA-A*0201 dependent killing. Finally, we demonstrated functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. Conclusion CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.