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BACKGROUND: Cytomegalovirus (CMV) disease represents a serious complication after allogeneic peripheral blood stem cell (PBSC) transplantation. If possible, stem cell donors for transplantation are selected on the basis of their CMV serostatus. However, the cytomegalovirus-specific immune status can be further characterized by measuring CMV phosphoprotein 65-specific CD8(+) T cell frequencies using tetramers, pentamers, and streptamers. We therefore investigated the specificity and sensitivity of all 3 methods and compared the results to patient serostatus. METHODS: Twenty-three samples from CMV-seropositive healthy volunteers and 15 samples from CMV-seropositive patients before and after allogeneic PBSC transplantation were stained with tetramers, pentamers, or streptamers and analyzed by flow cytometry. RESULTS: Similar frequencies of CD8(+) and multimer(+) T cells could be measured by all 3 multimer technologies. The lowest background signals (< or =0.02%) were obtained using tetramer technology. Frequencies of 0.19%-2.48% of CMV phosphoprotein 65 495-503-specific CD8(+) T cells were detected in healthy volunteers. Antigen-specific T cells were detected in only 11 (48%) of 23 seropositive healthy volunteers. CMV antigenemia before day 100 after allogeneic PBSC transplantation occurred in 2 of 3 patients without any specific T cells. CONCLUSION: These findings demonstrate the power of multimer staining and a certain limitation of serologic testing to define appropriate donors for transplantation. Therefore, whenever possible, CMV-seropositive donors of transplants to seropositive recipients should be screened for their CD8(+) T cell frequency. All 3 multimer technologies can be used, yielding similar results. The streptamer technology additionally offers the advantage of selecting CMV phosphoprotein 65-specific CD8(+) T cells at the good manufacturing practice level for adoptive T cell transfer.

Objective To investigate the inhibitory effects of dasatinib on proliferation, function, and signaling events on CD8 + T cells. Materials and Methods Carboxyfluorescein diacetate succinimidyl ester and 5-bromo-2-deoxyuridine were used to detect proliferation and cell cycle of CD8 + T cells treated with dasatinib, respectively. Frequency and function of viral and leukemia-antigen–specific CD8 + T cells from healthy donors were measured by tetramer staining and ELISPOT assay. Western blotting analysis was performed to detect T-cell receptor (TCR), nuclear factor k appa B (NF-κB) and Src signaling events in T cells treated with dasatinib or imatinib. Results Dasatinib inhibited proliferation of CD8 + T cells in a dose-dependent manner, which was associated with lower secretion of interferon-gamma and granzyme B, as well as with arrest of CD8 + T cells in the G 0 /G 1 phase of cell cycle. Inhibition of CD8 + T cells was proven for blood samples from a patient under dasatinib medication when compared with their T-cell status without dasatinib. Western blotting confirmed that these effects were mediated through downregulation of the phosphorylation level of molecules from the TCR and the NF-κB signaling transduction cascade. Dasatinib proved to be more potent than imatinib on Src and TCR signaling events in Jurkat T cells. Conclusion Our study demonstrated that dasatinib impaired proliferation and function of CD8 + T cells via TCR and NF-κB signaling events without inducing apoptosis. Therefore, dasatinib might alter the graft-vs-leukemia effect and the graft-vs-host disease after allogeneic stem cell transplantation sustained by CD8 + T cells. Dasatinib might also be used as a novel immunosuppressant agent.

BACKGROUND: Dendritic cells (DCs) generated from leukemic blasts constitute a promising tool in immunotherapy for acute myeloid leukemia patients (AML-DCs), because AML-DCs express human leukocyte antigens and costimulatory molecules such as CD40, CD80, and CD86 at a higher level than leukemic blasts. Potentiation of AML-DC vaccine might become feasible by the addition of adjuvants such as lipopolysaccharides (LPS) or CPG-rich oligodeoxyribonucleotides binding to Toll-like receptors (TLR) and inducing a stronger Type 1 T-cell response. STUDY DESIGN AND METHODS: mRNA and protein expression of TLR-2, -4, and -9 were analyzed with quantitative real-time polymerase chain reaction, Western blot, and flow cytometry for mature monocyte-derived DCs generated from 14 AML patients versus 14 healthy volunteers (HV-DCs), and the response of the AML- and HV-DCs to different microbial TLR ligands was determined by enzyme-linked immunosorbent assay for the proinflammatory cytokines tumor necrosis factor (TNF)-α, inducible protein (Ip)-10, and interleukin (IL)-6. RESULTS: AML-DCs and HV-DCs strongly expressed TLR-2 and TLR-4, while TLR-9 was expressed at a lower level in both groups. There was no significant difference in TLR expression between the two groups of AML-DCs and HV-DCs. In accordance with the TLR expression levels, DCs generated from both AML patients and HVs responded to the known microbial ligands peptidoglycan (PGN) and lipoteichoic acid for TLR-2 and LPS as ligand for TLR-4, by producing TNF-α and IL-6. A response to the ODNs 2006 and 2216 binding to TLR-9 was only detected in AML-DCs. CONCLUSION: Microbial ligands like ODNs and LPS constitute promising adjuvants for enhancing (AML-) DC vaccines.

The novel selective BCR-ABL Breakpoint cluster region--Abelson murine leukemia viral oncogene homolog 1 (BCR-AML) inhibitor nilotinib (AMN107) is a tyrosine kinase inhibitor that is more potent against leukaemia cells in vitro than imatinib. As nilotinib might be used in the context of allogeneic stem cell transplantation where CD8+ T lymphocytes play a pivotal role in the graft-versus-leukaemia (GVL) effect, we investigated effects of nilotinib on this lymphocyte subpopulation. Nilotinib inhibits phytohemagglutinin (PHA)-induced proliferation of CD8+T lymphocytes in vitro at therapeutically relevant concentrations (0.5-4 microM). The inhibition of CD8+ T lymphocytes specific for leukaemia or viral antigens through nilotinib was associated with a reduced expansion of antigen peptide specific CD8+ T lymphocytes and with a decreased release of interferon-gamma and granzyme B by these cells as analysed by flow cytometry and enzyme-linked immunospot (ELISPOT) assays. The inhibitory effect caused by nilotinib was two times stronger than by imatinib. These effects were mediated through the inhibition of the phosphorylation of ZAP-70, Lck and ERK 1/2 and the NF-kappaB signalling transduction pathway. Taken together, we observed a strong suppressive impact of nilotinib on the CD8+ T lymphocyte function which should be considered carefully in the framework of allogeneic stem cell transplantation or other T cell based immunotherapies.

The receptor for hyaluronic acid–mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8+ T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in accordance with an increase of R3-specific CD8+ T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.

The tyrosine kinase inhibitor imatinib has been reported to inhibit CD8+ T lymphocytes. Little is known about its effects on CD4+CD25+ regulatory T cells (T(reg) cells) which might regulate the graft-vs.-leukemia (GVL) reaction after allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion (DLI). This is of particular interest in patients with relapse of chronic myeloid leukemia (CML) after allo-SCT, as the two therapeutical options DLI and imatinib might interact reversely. Here, we demonstrate that the proliferation of CD4+CD25+ T(reg) cells and their production of IL-10, TGF-beta1 and granzyme B as markers of activation were significantly down-regulated by imatinib in a dose-dependent manner. In addition, the expression of surface CD69, both surface and intracellular GITR, FoxP3, CD152 (CTLA) of activated CD4+CD25+ T(reg) cells were inhibited by imatinib in a dose-dependent manner. In light of these findings, clinical administration of imatinib might not result in a reduction of the GVL effect on CML patients receiving imatinib after allo-SCT and/or DLI or other CD8+ T lymphocyte based immunotherapies as the function of CD8+ cytotoxic T lymphocytes and CD4+CD25(hi) Treg cells is hampered in a similar way by imatinib.

Recently, we described the receptor for hyaluronic acid-mediated motility (RHAMM) as a leukemia-associated antigen and characterized the RHAMM-derived peptide R3 (pos. 165-173: ILSLELMKL) as a CD8+ T cell epitope. Directing CD8+ T lymphocytes specifically to R3 might help to shape the graft-versus-leukemia effect observed after allogeneic stem cell transplantation (allo-SCT). To detect the potential induction of R3-specific cytotoxic T lymphocytes in chronic myeloid leukemia (CML) patients after allo-SCT and healthy donors, we used mixed lymphocyte peptide culture, enzyme-linked immunospot (ELISPOT) release assays for interferon (IFN)-gamma and granzyme B, tetramer staining and 51Cr release cytotoxicity assays. The R3 peptide showed the capacity to elicit specific CD8+ T cell responses characterized by the release of IFN-gamma and granzyme B upon stimulation with R3-pulsed T2 cells. Responses to R3 peptide were detected in 67% (6/9) of the CML patients after allo-SCT and 24% (8/34) of healthy donors in ELISPOT assays for IFN-gamma and granzyme B. These R3-specific CD8+ T cells comprised predominantly effector cells (CCR7-CD45RA+ or CD27-CD45RA+) in patients with CML after allo-SCT or healthy donors respectively. Cytotoxicity assays demonstrated effective lysis of CML progenitor cells by R3-primed CD8+ T lymphocytes. Imatinib inhibited the functional activation of R3-specific CD8+ T lymphocytes. In summary, we demonstrated R3-specific CD8+ effector T lymphocytes after allo-SCT in CML patients which might have been augumented by R3 peptide vaccination and hampered at least partially by imatinib in this particular patient cohort.

Dendritic cells are the most professional antigen-presenting cells to elicit T-cellular responses toward microbial agents and cancer cells. The graft-versus-leukemia effect observed after allogeneic stem cell transplantation strongly suggests that T lymphocytes play a major role in the rejection of leukemic cells. This graft-versus-leukemia effect might be enhanced through dendritic cell vaccination. The characterization of leukemia-specific antigens eliciting immune responses in the autologous host has prompted researchers and clinicians to broaden the spectrum of dendritic cell vaccines to hematological malignancies. Recently, the focus is on acute myeloid leukemia and chronic lymphocytic leukemia. This review summarizes data on the administration of autologous and allogeneic dendritic cells to leukemia patients as an interesting approach in cellular therapy of leukemias.

Aim: Recently, extracorporeal photopheresis (ECP) has become a promising therapeutic approach for steroid-refractory chronic graft-versus-host disease (cGVHD) because of its advantage over other therapies. The mechanism of action in ECP still remains to be elucidated. To investigate the evidence for clinical efficacy of ECP in the treatment of steroid-refractory cGVHD, we conducted a systematic search and meta-analysis comprising several databases and abstracts presented at recent annual meetings in the field. Methods: A systematic review of publications indexed in the PubMed, Embase, the Cochrane-controlled trials registry, the Cochrane Library, and ISI Web of knowledge were performed on January 10, 2015. Eight studies including 176 patients were identified. Meta-analyses were carried out to calculate the overall response rate (ORR) and complete response rate (CRR) of cGVHD and the organ-specific responses. Results: In patients with cGVHD, pooled ORR and CRR were 0.66 and 0.46, respectively, with organ-specific responses of 0.80 for skin, 0.79 for gut, 0.57 for oral mucosa, 0.57 for liver, 0.50 for eye, and 0.46 for joint involvement. Conclusion: The present analysis revealed a promising clinical benefit of ECP for patients with steroid-refractory cGVHD. Further prospective trials with larger cohorts are mandatory.

Catumaxomab is an antibody that binds with one arm epithelial cell adhesion molecule (EpCAM) positive tumors and with the other arm CD3+ T cells. Intravenous application of therapeutic antibodies may result in intravascular cytokine release.In this pilot trial we assessed whether cytokine release can be controlled by ex vivo cell opsonization and cytokine wash-out before administration of catumaxomab, preserving its anti-cancer activity. In addition, preliminary data on safety of and clinical response to catumaxomab coated autologous immune cells were acquired.Peripheral blood mononuclear cells (PBMNC) of four patients with recurrent head and neck carcinoma were collected by leukapheresis, incubated ex vivo with catumaxomab for 24 h and cleared from released cytokines. Each patient received an escalated number of antibody-coated PBMNC equivalent to 1 x 10(4), 1 x 10(5), 1 x 10(6) and 1 x 10(7) CD3(+) cells/kgBW intravenously at bi-weekly intervals.After opsonization, PBMNC released substantial amounts of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in vitro, which were removed before administration. Catumaxomab up-regulated CD25, CD69, and CD83 on PBMNC, and catumaxomab loaded PBMNC released IFNgamma and granzyme B when coincubated with EpCAM(+) BHY cells, suggesting cell activation and target directed biological activity. During the study period, one patient died of aspiration pneumonia and one patient needed a tracheotomy. Treatment related adverse events (AE) occurred at the highest cell dose in two patients, whereas 1 x 10(6) loaded CD3(+) cells/kgBW were well tolerated by all patients. One patient showed stable disease for 6 months and one patient is in complete remission for 27 months.Ex vivo opsonization of PBMNC with catumaxomab provided biologically active, tumor targeting cells. Extracorporeal PBMNC coating may be an option to control intravascular cytokine release induced by therapeutic antibodies.

The Bcr-Abl tyrosine kinase inhibitor imatinib mesylate is highly effective in the front-line treatment of chronic myeloid leukemia (CML) and is increasingly used in patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT). Since an impairment of anti-viral CD8+ T-lymphocyte function by imatinib has been described, we question whether imatinib also affects specific anti-leukemic CD8+ T lymphocytes generated from the peripheral blood of healthy donors, and of CML patients after allo-SCT. Here, we assessed CD8+ T-cell expansion and function from healthy donors and patients with CML. The release of IFN-gamma and granzyme B by CD8+ T-lymphocytes specific for R3, a recently described T-cell epitope peptide derived from a leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility), was inhibited by imatinib in a dose-dependent fashion (range: 1-25 microM). These T cells were able to lyse cognate peptide labeled T2 cells and CD34+ CML progenitor cells. This lysis was inhibited by imatinib. The inhibitory effect was not associated with an increased rate of apoptosis of T cells and reversible after removal of imatinib. In the light of these findings, clinical administration of imatinib might result in the reduction of efficacy of the graft-versus-leukemia effect or other T-cell-based immunotherapies.

Specific immunotherapies for patients with chronic myeloid leukemia (CML) might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft-versus-leukemia effect after allogeneic stem cell transplantation. Here, we investigated the mRNA expression and T-cell recognition of tumor-associated antigens or leukemia-associated antigens (LAAs) in 34 patients with CML. Several LAAs are expressed in CML and therefore are candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%), and WT1 (53%), but not BAGE, MAGE-A1, SSX2, or NY-ESO-1. The frequency of mRNA expression of RHAMM/CD168, Proteinase3, and PRAME was higher in acceleration phase and blast crisis. In flow cytometry, CD34 + progenitor cells typed positive for HLA molecules but were deficient for CD40, CD80, CD83, and CD86. However, RHAMM/CD168 R3-peptide (ILSLELMKL)-specific T-cell responses in CML patients were demonstrated by ELISPOT analysis and specific lysis of RHAMM/CD168 R3-pulsed T2 cells and CD34 + CML cells in chromium-51 release assays. RHAMM-R3-specific T cells could be phenotyped as CD8 + R3 ∗ tetramer + CD45RA + CCR7 − CD27 − early effector T cells by tetramer staining. Therefore, vaccination strategies inducing such RHAMM-R3-directed effector T cells might be a promising approach to enhance specific immune responses against CML cells.

The present study investigated the effects of CD34(+) cell selection in 102 patients using the CliniMACS device. Patients were at high risk for the development of graft versus host disease (GvHD) because of age, or the use of a haploidentical, mismatched or unrelated donor (UD). The median age of the patients was 44 years. The CliniMACS procedure yielded 8.0 x 10(6) CD34(+) cells/kg and the number of residual T cells was 1.3 x 10(4)/kg (median). The median follow up was 20.6 months. The probability of graft failure was 7%. The rate of acute GvHD was low (compatible family donors 10%, UDs 17%, and haploidentical donors 26%) with no patient enduring more than grade II disease. The cumulative incidence of chronic GvHD at the median follow up after transplant was 15% for the compatible family donor group, 40% for the UD group and 78% in the group transplanted from a haploidentical donor Treatment failure was mainly because of transplant-related mortality, especially aspergillus infection, and not due to relapse. The probability of disease-free survival, stratified for the risk of treatment failure, was 27% for the high risk, 46% for the intermediate risk and 83% for the low risk group.

Specific immunotherapies for patients with acute myeloid leukemia (AML) using leukemia-associated antigens (LAA) as target structures might be a therapeutic option to enhance the graft-vs.-leukemia effect observed after allogeneic stem cell transplantation or to prolong a complete remission (CR) achieved by chemotherapy. Significant mRNA expression of LAA is a prerequisite for such immunotherapies. Here, previously characterized antigens associated with solid tumors (TAA) and newly characterized LAA were investigated for their expression in up to 60 AML patients and in leukemia cell lines. To investigate their specificity for leukemic blasts, the mRNA expression was also characterized in PBMN and CD34 positive cells of healthy volunteers and in a panel of normal tissues. The following antigens showed high mRNA expression in AML patients: MPP11 was detected in 43/50 (86%), RHAMM in 35/50 (70%), WT1 in 40/60 (67%), PRAME in 32/50 (64%), G250 in 18/35 (51%), hTERT in 7/25 (28%) and BAGE in 8/30 (27%) of AML patients. Real-time RT-PCR showed a tumor-specific expression of the antigens BAGE, G250 and hTERT, as well as highly tumor-restricted expression for RHAMM, PRAME and WT1. The antigen MPP11 was overexpressed. These antigens might be candidates for immunotherapies of leukemia patients and, because of their simultaneous expression, also for polyvalent vaccines.

To design a specific immunotherapy for leukemia patients, the identification of leukemia-associated antigens (LAAs) is a pivotal step. Antileukemic effects after hematopoetic stem cell transplantation for myeloid leukemias are observed and might be related to the recognition of LAAs. Using the serological screening of an expression library (SEREX) of K562 cells, we identified 16 different clones encoding LAAs eliciting a humoral immune response, among them the heat shock proteins HSJ2 and HSP70, the M-phase phosphoprotein 11 (MPP11), the BRCA1-associated protein (BRAP), the Jkappa recombination binding protein (RBPJkappa) and the receptor for hyaluronic acid mediated motility (RHAMM). Serological responses to MPP11 were observed in 7/19 (37%) of patients with acute myeloid leukemia (AML) and 6/16 (38%) of patients with chronic myeloid leukemia (CML), but not in healthy volunteers (0/20). IgG antibodies directed against MPP11 were also detected in 25-50% of the sera of patients with solid tumors such as melanoma, renal cell, ovarian and breast carcinoma. mRNA expression of MPP11 was detected in 20/20 AML patients and 7/10 patients with CML. In normal tissues, strong mRNA expression of MPP11 was only detected in testis. By real-time PCR, we detected upregulation of MPP11 in leukemic blasts. Simultaneous humoral immune responses to 2 or more of the 16 LAAs identified here was observed, suggesting the feasibility of a polyvalent vaccination as an option for immunotherapies in leukemia patients.

Acute myeloid leukemia (AML) is a clonal disease of hematopoiesis with poor clinical outcome despite recent improvements in chemotherapy and stem cell transplantation regimens. Immunotherapy with dendritic cells (DCs) eliciting specific T cell responses to leukemia-associated antigens (LAAs) might be a therapeutic option. DCs must express HLA class I/II molecules and the costimulatory molecules CD40, CD80 and CD86 to effectively activate T cells for the subsequent lysis of leukemic blasts. The expression of these antigens on DCs generated from 15 AML patients (AML-DCs) and on DCs generated from 15 healthy volunteers (HV-DCs) was analyzed by FACS. All DCs displayed the typical morphology and tested negative for B, T and NK cell markers. The sustained mRNA expression of LAAs such as PRAME, RHAMM or WT-1 proved that the AML-DCs originated from AML blasts. Compared with AML blasts, the expression of CD40, CD80, CD86 and HLA-DR was upregulated during DC culture to a median of 80-98% on AML-DCs. HLA-ABC was preserved on AML-DCs (median 95%). Expression of CD40, CD80 and CD83 remained lower on AML-DCs than on HV-DCs. AML-DCs express at least one LAA and strongly express HLA and costimulatory molecules, the prerequisites for eliciting T cell responses. AML-DCs may play a role in vaccine-based immunotherapies for AML patients.

Introduction Hematopoietic growth factors including G-CSF are manufactured by the use of recombinant technology. When biologically equivalent agents are manufactured they are termed “biosimilars”. The use of biosimilars in general and in particular for peripheral blood hematopoietic stem cell (PBSC) mobilization has stimulated an ongoing debate regarding their efficacy and safety. The use of biosimilar G-CSF (Ratiograstim®, Tevagrastim®, Biograstim®, Zarzio®, Nivestim®) was approved by the European Medicines Agency (EMA) for all the registered indications of the originator (Neupogen®) including mobilization of stem cells. Methods We performed a comprehensive review of published reports on the use of biosimilar G-CSF covering patients with hematological malignancies as well as healthy donors that underwent stem cell mobilization with a biosimilar G-CSF for autologous and allogeneic stem cell transplantation, evaluating mobilization yield and safety profile as well as engraftment and transplantation outcome. Results An extensive literature review produced 903 patients mostly with hematological malignancies as well as healthy donors that underwent successful autologous or allogeneic stem cell mobilization respectively using a biosimilar G-CSF (Ratiograstim®/Tevagrastim® or Zarzio®). A total of 519 patients or donors underwent stem cell mobilization with Ratiograstim®/Tevagrastim®, while 384 patients or donors underwent stem cell mobilization with Zarzio®. The indication for stem cell mobilization in hematology patients included 326 with multiple myeloma, 273 with Non-Hodgkin’s lymphoma (NHL), 79 with Hodgkin’s lymphoma (HL), and others. 155 sibling or volunteer unrelated donors were mobilized using either Ratiograstim®/Tevagrastim® or Zarzio®. Biosimilar G-CSF based stem cell mobilization for both autologous and allogeneic transplantation resulted in good mobilization of CD34+ stem cells with side effects similar to reference G-CSF. Post transplantation engraftment did not significantly differ from results previously documented with the originator filgrastim (Neupogen®) in historical controls. The side effects experienced by the patients or donors mobilized by biosimilar G-CSF were minimal and were comparable to those of originator G-CSF. (More detailed results regarding yield, engraftment and side effects will be disclosed in the conference.) Conclusions In summary, we present the published experience for the use of biosimilar G-CSFs in more than 900 patients and normal family related and volunteer unrelated donors. Both the toxicity profile PBSC yield and efficacy seem equivalent to historical data with the reference G-CSF. Until results from multi-center randomized clinical trials that directly compare biosimilar G-CSF with the originator G-CSF are reported, it is important to collect and summarize all of the available clinical experience in order to allow the transplant community to make informed decisions regarding the choice of G-CSF. Disclosures: Schmitt: AMGEN: Honoraria; Teva: Consultancy, Honoraria, Research Funding, Travel Grant, Research Grants Other. Publicover:Teva: Travel Grants, unrestricted educational grant Other. Orchard:Teva: Honoraria, Travel Grants, research grants Other. Schmitt:TEVA: Travel grant Other. Nagler:Teva: Honoraria, Travel grants, research grants Other.

Abstract 3592 Nucleophosmin gene 1 mutations (NPM1mut) are one of the most frequent molecular alterations in AML and distinct immune responses might contribute to the favorable prognosis of AML patients with NPM1mut. Recently, we showed specific T cell responses of CD4+ and CD8+ T cells against epitopes derived from mutated regions of NPM1 (Greiner et al., Blood. 2012 May 16, Epub). In the present study, we investigated clinical parameters and the clinical outcome of NPM1mut AML patients in accordance to their immune responses against different NPM1 epitopes. Moreover, we examined the quantitative expression of different leukemia-associated antigens (LAAs) in NPM1mutAML patients. In ELISpot analysis of 33 healthy volunteers and 27 AML patients, we detected T cell responses of CD4+ and CD8+ T cells against epitopes derived from the mutated region of NPM1. We performed further tetramer assays against the most interesting epitopes and chromium release assays to show the cytotoxicity of peptide-specific T cells. Microarray analysis was performed to analyze the expression of different LAAs in NPM1mut and NPM1wtAML patients. Two epitopes (peptide #1 and #3) derived from NPM1mut induced CD8+ T cell responses. 33% of the NPM1mut AML patients showed immune responses against peptide #1 and 44% against peptide #3. NPM1mut AML patients showed a significantly higher frequency of T cell responses against peptide #3 in contrast to HVs (p=0.046), whereas for peptide #1 the frequency of T cell responses of AML NPM1mut patients and HVs was not significantly different. Specific lysis of pulsed T2 cells but also NPM1mut leukemic blasts was detected in chromium release assays. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and the peptide called OL8 showed favorable results to activate both CD8+ and CD4+ T cells. We performed survival analysis for these 33 NPM1mut patients analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a trend to a better overall survival (OS) for patients with specific T cell responses against peptide #1 or #3. However, the patient numbers are small and the data have to be interpreted carefully. Analyses with material from larger controlled clinical trials with a high number of patients with NPM1mut AML have to be performed. Our microarray analysis of 30 AML patients showed a high expression of different LAAs like RHAMM, WT-1 and BCL-2 in all subtypes of cells of NPM1mutAML patients, also in leukemic progenitor cells. This demonstrates that NPM1 is an AML subtype suitable for poly-targeted immunotherapeutic trials. Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses to NPM1 mutation may contribute to the favorable prognosis. Disclosures: No relevant conflicts of interest to declare.

6567 Background: Mutations of the nucleophosmin gene (NPM1mut) are one of the most frequent molecular alterations in AML and constitute an important prognostic marker. The impact of NPM1mut on leukemogenesis and progression remains to be elucidated. Immune responses against NPM1mut might contribute to the favourable prognosis of AML patients with NPM1mut. Therefore, we examined T cell responses against NPM1mut. Methods: NPM1 wildtype as well as NPM1mut were screened for HLA-A*0201 binding T cell epitopes with the help of different algorithm programs. Ten peptides with most favourable characteristics were tested with ELISpot analysis for interferon-γ and granzyme B in 33 healthy volunteers and 30 AML patients. Tetramer assays against most interesting epitopes were performed and chromium release assays were used to show the cytotoxicity of peptide-specific CD8+ T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Results: Two epitopes (#1 and #3) derived from NPM1mut induced CD8+ T cell responses in a high frequency. In healthy volunteers, immune responses were detected in 39%/18% against #1 and #3, and in 33%/44% of NPM1mut AML patients against #1 and #3. NPM1-peptide primed effector T cells showed specific lysis of pulsed T2 cells as well as leukemic blasts in chromium release assays. In tetramer assays a significant CD8+ T cell population could be detected. To obtain a robust and continuous T cell reaction, the help of CD4+ T cells is indispensable. Therefore, we investigated the increase of CD8+ T cell responses by the activation of CD4+ T cells stimulated with longer peptides called overlapping peptides (OL). Potent HLA-DR epitopes were predicted and several favourable peptides (OL 1 to 8) were synthesized. OL8 showed favourable results to activate both CD8+ and CD4+ T cells. Conclusions: Taken together, NPM1mut represents a candidate for immunotherapeutic approaches and we hypothesize that it is also potentially involved in immunogenic rejection of NPM1mut leukemic blasts. Therefore, NPM1mut is a promising target structure for specific immunotherapies in AML patients.

Abstract 1928 INTRODUCTION: There is limited experience in the application of the biosimilar Ratiograstim® for mobilization of peripheral stem cells and especially in healthy donors (HD). The G-CSF biosimilar Ratiograstim® was approved by the EMA for mobilization of peripheral stem cells. Here, we investigated on two cohorts the efficacy and safety of peripheral stem cell mobilization by either the biosimilar or the reference G-CSF. MATERIAL AND METHODS: 22 patients and their related donors were investigated for: 1. leukocyte-count in the peripheral blood of healthy donors, 2. number of collected CD34+ stem cells, 3. number of apheresis procedures, 4. CD34+ cell count per kg body weight of the patients, 5. time till regeneration, 6. side effects. RESULTS: In HD receiving subcutaneously Ratiograstim®, WBC counts in the peripheral blood ranged from 29.9 G/L to 64.6 G/L (mean 48.9 G/L) and the CD34 cell counts from 19.3/μl to 114.6/μl (mean 67.6/μl). In a mean of 1.45 apheresis procedures 172.5×106 up to 494×106 CD34+ cells (mean 354.7×106) were collected. This resulted in CD34+ cell numbers of 2.28×106 up to 1×107 (mean 5.17×106) per kg body weight of the patients. In HD receiving subcutaneously the reference G-CSF, WBC counts ranged from 27.1 G/L up to 62.5 G/L (mean 43.3 G/L) and the CD34+ cell counts from 13.55/μl up to 122.4/μl (mean 57.3/μl). A mean of 1.27 apheresis procedures were necessary to collect 181.74×106 up to 598.00×106 CD34+ cells (mean 370.49×106) absolutely. This translates into 2.33×106 up to 7.9×106 (mean 4.95×106) CD34+ cells per kg body weight of the patients. Patients regenerated >0.5 G/L neutrophils/μl within 15 days in the Ratiograstim® cohort versus 17 days in the reference group. SUMMARY: In total we analyzed two cohorts, one cohort of 22 patients and donors receiving the biosimilar Ratiograstim® versus another cohort receiving reference G-CSF. No unexpected side effects and no increased frequencies in side effects as flu-like symptoms, allergic reactions and alterations in kidney and liver function were observed. In comparison to the reference group we did not see significant differences in 1. WBC count in the peripheral blood after mobilisation, 2. CD34+ cell count after mobilisation, 3. CD34+ cell count absolute numbers, 4. CD34+ cells per kg body weight of the patients, 5. engraftment, 6. regeneration of WBC, neutrophils and platelets. All patients engrafted. Therefore, the efficacy of the biosimilar Ratiograstim® when compared to the reference G-CSF was comparable in our cohort of patients in the context of allogeneic stem cell transplantation. Disclosures: Schmitt: ratiopharm: Honoraria, Research Funding. Freund:Medac: Honoraria, Research Funding.

Abstract 2569 Introduction In AML, mutations in the nucleophosmin (NPM1) gene are one of the most frequent molecular alterations and predominantly occur in AML with normal cytogenetics. Patients with NPM1 mutation without FLT3-ITD mutation show a favourable prognosis of their disease. The functional role of mutated NPM1 for the improved clinical outcome is under evaluation. Immune responses might be involved in the clinical outcome of the disease. In this work, we demonstrate both CD4+ and CD8+ T cell responses against the mutated region of NPM1. Methods The entire amino acid sequences of the NPM1 wild type protein as well as of the mutated cytoplasmic NPM1 types A, B, C and D were screened for HLA-A*0201 binding T cell epitopes using the algorithms of the SYFPEITHI, the Rankpep and the HLA-Bind software programs. Ten peptides with most favourable characteristics were subjected to ELISpot analysis for interferon-γ and granzyme B in 22 healthy volunteers and 27 AML patients to test specific T cell responses of CD8+ T cells. Tetramer assays against the two most interesting epitopes have been performed and chromium release assays have been used to show the cytotoxicity of peptide-specific T cells to lyse T2 cells and leukemic blasts. Moreover, HLA-DR binding epitopes were screened in algorithmic analysis and HLA-DR*0701 binding peptides were exploited to stimulate CD4+ T cells. In the presence of overlapping peptide stimulated CD4+ T cells, NPM1-A specific CD8+ T cells revealed augmented interferon-γ and granzyme B secretion and up-regulation of intracellular interferon-γ. CD4+, CD4-CD8+, CD4-CD8- cell fractions were separated from PBMCs of HLA-A2+DR*0701+ healthy volunteers using a combination of CD4 and CD8 MicroBeads. Results Two epitopes (P3 and P9) derived from the NPM1-mutated protein showed specific T cell responses in healthy volunteers and AML patients. In NPM1-mutated AML patients 33% showed immune responses of CD8+ T cells against peptide P3 and 42% against peptide P9. Specific lysis was detected in chromium release assays NPM1 peptide-primed effector T cells generated from NPM1-mutated AML patients. Tetramer assays showed peptide-specific T cells. To obtain a robust and effective immune response against tumor cells, the activation of CD4 + helper T cells is crucial. Thus NPM1-peptide-A overlapping MHC class II epitopes were searched by primary structure analysis program. Based on plenary search, eight favourable overlapping peptides OL 1–8 were synthesized and exploited for CD4+ T cell stimulation. In granzyme B ELISPOT assay, OL8 co-pulsed NPM1-A CD8+ T cells indicated notable S.I., in contrast other OL1-7 disabled to increase granzyme B secretion. To ensure that Th1 cytokine secretion, under the condition of CD8+ and CD4+ T cells mixed culture, was resulted from NPM1-A CD8+ T cells but not HLA-DR epitope stimulated CD4+ T cells activation, HLA-A2 blocking effect was confirmed in ELISPOT assay. NPM1-A CD8+ T cells co-pulsed with OL6, 7 and 8 showed lesser interferon-γ secretion after HLA-A2 blocking antibody exposure as 73, 35 and 57%. Of note, 83–94% of granzyme B secretion levels were reduced by HLA-A2 blockade administration, and by which NPM1-A CD8+ T cells seemed to be the most probable IFN-gamma and granzyme B producers and CD4+ T cells to interfere with CD8+ T cells. Conclusion Taken together, mutated NPM1 is a promising target structure for specific immunotherapies in AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4951 Objective To investigate the relevance of Inducing apoptosis and reversal effect of nilotinib in combination with Tet on multidrug reststahoe of K562/A02 cell line and its mechanism. Methods Methyl-thiazol tetrazolium(MTT) assay is employed to examine the pharmacological effect of Nilotinib or Tet alone on K562/A02 cell line, to calculate the inhibitor concerntration 50(IC50) of daunorubicin(DNR) in K562/A02 cell line and the resistance multiple of DNR to K562/A02 cell line. Flow cytometry(FCM) was employed to comple the effect on the inducing apoptosis of K562/A02. The expression of bax/survivin mRNA was determined by RT-PCR, and the expression of bax/survivin protein was assessed by Western blot. Results After 48 h 5 nmol/L nilotinib or 1.0μmoL/L Tet treatment, IC50 of DNR to K562/A02 was 5.71mg/L or 6.52 mg/L respectively;While on combinative treatment, its IC50 decreased to 3.12 mg/L. Nilotinib or Tet alone was able to increase the DNR induced apoptosis rate of K562/A02 cell (P Conclusion Nilotinib or Tet alone can pattially reverse drug resistance of K562/A02 cells. There is resistance phenomena of DNR to K562/A02 cells, and Nilotinib or Tet alone alone is able to reserve the resistance of DNR and enhance the inducing apoptasis effect of DNR. The mechanism may be associated with the increase of bax mRNA and protein expression and decrease of survivin mRNA and protein expression and increase of the apoptosis rate. And there is a synergistic action with these two agants in combination. Disclosures: No relevant conflicts of interest to declare.

Abstract 3671 Poster Board III-607 For effective elimination of malignant cells by specific T cells a co-activation of CD4- and CD8-positive T cells might be important. We performed two RHAMM-R3 peptide vaccination trials using 300μg and 1000μg for patients with AML, MDS and multiple myeloma overexpressing RHAMM. Similar mild toxicity of both cohorts was found, only mild drug-related adverse events were observed such as erythema and induration of the skin. In the 300μg cohort we detected in 7/10 (70 %) patients specific immune responses and also positive clinical effects in 5/10 (50 %) patients. In the high dose peptide vaccination trial (1000μg peptide) 4/9 (44 %) patients showed positive immune responses. These patients showed an increase of CD8+RHAMM-R3 tetramer+/CD45RA+/CCR7-/CD27-/CD28- effector T cells and an increase of R3-specific CD8+ T cells. In the higher peptide dose cohort three patients showed positive clinical effects. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial and might induce immune tolerance. In this work, we investigated the co-existence of serological immune responses against RHAMM detected by a RHAMM-specific ELISA of patients with AML, MDS and multiple myeloma treated in these two peptide vaccination trials. We correlated these results to specific T cell responses of CD8-positive T cells measured by ELISpot assays for interferon gamma and Granzyme B, tetramer staining and chromium release assays. Moreover, these results were compared to the frequency of regulatory T cells. 4/19 patients have a positive serological immune response in ELISA assay, all of these patients developed also strong specific CD8-positive T cell responses during peptide vaccination detected by ELISpot assays and tetramer staining. As expected, peptide vaccination did not result in the induction of humoral immune responses. In further ELISA assays we measured IL-2 and IL-10 levels in the sera of the patients before and three weeks after four vaccinations. While IL-10 levels remained at a rather low level over the time of vaccination, we detected an increase of IL-2 up to the five-fold of the initial levels in four of ten patients. Moreover, we performed a proteome array to detect cytokine and chemokine regulation in sera of patients vaccinated in these two trials during and after RHAMM-R3 peptide vaccination. 36 cytokines, chemokines and acute phase proteins were measured and both cohorts vaccinated with different peptide doses were compared. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Co-existence of immune responses of CD4-positive T cells against the target RHAMM seems to be important for an induction of strong immune responses of CD8-positive T cells. Disclosures: No relevant conflicts of interest to declare.

The leukemia-associated antigen PRAME (preferentially expressed antigen of melanoma) is frequently expressed in several solid tumors. Earlier, we reported (Greiner et al., Blood 2006) that co-expression of leukemia-associated antigens including PRAME constituted a favorable prognostic parameter for AML patients. By microassay analysis and conventional RT-PCR, we detected over-expression of PRAME in more than 60% of AML and of CML patients. In contrast, PRAME is expressed neither in normal CD34-positive hematopoietic stem cells nor in normal tissues (other than testis and placenta). Here, we describe for the first time that retinoic acid-induced cell proliferation and differentiation of several AML cell lines is dependent on PRAME expression: PRAME negative cell lines treated with all-trans retinoic acid (ATRA) in cell culture showed lower cell proliferation than PRAME positive cells as assessed by cell counts and FACS analysis for BrdU, but an increase of cell differentiation detected by FACS analysis for CD66b in contrast with PRAME positive leukemia cell lines. The leukemia-associated antigen PRAME seems to be responsible for the resistance of PRAME-positive AML to ATRA treatment. We detected no differences in induction of apoptosis in PRAME-positive or PRAME-negative cell lines treated with ATRA in FACS analysis for annexin. Over-expression of the antigen and this critical role of PRAME in cell differentiation might be the reason for specific T cell induction against PRAME-derived peptides. To detect specific T cell responses against PRAME-derived peptides we examined samples from healthy volunteers and AML patients. Positive results in ELISPOT assays for IFN-g and Granzyme B secretion were observed in 70% of AML cases analyzed for the peptide PRAME-P3. In chromium release assays, we found a PRAME-specific cell lysis of PRAME-positive AML blasts. Taken together, PRAME is involved in a crucial mechanism for cell growth of leukemic cells, induces specific T cell responses in a high frequency of AML patients, and this constitutes an appropriate target structure for specific immunotherapies or other targeted therapies in AML.

Background: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). For the clearance of CMV, CD8+ T cells are pivotal. Patients after allo-PBSCT with recurrent CMV reactivation usually lack such CMV specific T cells. Conventional antiviral therapy of CMV reactivation characteristically results in myelosuppression and further suppression of CMV specific T cells. Adoptive transfer of CMV specific T cells may help to overcome this problem. A novel technology designated “streptamers” allows the selection of CMVpp65 specific CD8+ T cell up to 98% purity without altering the functional properties of the selected T cells and without requiring cumbersome and time consuming T cell cultures. Materials and Methods: Here, the novel streptamer technology was used for adoptive transfer of CMV specific T cells into two acute leukemia patients with recurrent high CMV antigenemia after allo-PBSCT. Standard peripheral blood mononuclear cell apheresis was performed on the former stem cell donors of two patients with acute leukemia. Isolation of CMV specific donor lymphocytes was performed using a Good Manufacturing Product (GMP)-grade Streptamer selection kit on a CliniMacs™ device. Briefly, MHC-Streptamers (CMVpp65/HLA-B7 for patient 1; CMVpp65/HLA-A2 for patient 2) were labeled with beads overnight to obtain MHC-streptamer-bead complexes. Subsequently CMV specific T-lymphocytes were immunomagnetically labeled by incubating mononuclear cells with MHC-Streptamer-bead complexes. Cells were run on a CliniMacs™ device. The positive fraction was then incubated with biotin to detach the steptamers from the T cells. Results: A single specific donor lymphocyte infusion (sDLI) of 0.4 or 2.2 ×105 CMVpp65 specific T cells per kg body weight was performed in an AML or ALL patient respectively, after allogeneic PBSCT developing a CMVpp65 antigenemia with a maximum of 959 or 716 CMVpp65 positive/500,000 cells and treatment with foscarnet, ganciclovir and valganciclovir. After sDLI, the CMV antigenemia was cleared and remained persistently controlled even after discontinuation of valganciclovir therapy in both patients. No acute or chronic toxic side effect, particularly no aggravation of graft-versus-host disease (GvHD) was observed. A strong and sustained increase of the absolute count of CMV-specific CD8+ T cells in concordance with the increase of CD3+CD8+ T cells up to 440/μl was detected. CMV-specific CD8+ T cells showed no significant expression of CCR7, CD62L or CD107, but stained increasingly positive for CD45RA, indicating a preferential effector T cell phenotype. Results from stimulation experiments of CD3+ T cells with HLA-B7 versus HLA-A2 restricted CMVpp65 derived peptides demonstrate late reconstitution of HLA-A2-restricted CMV-specific T cells, whereas the adoptively transferred HLA-B7-restricted CMV-specific T-cell response augmented very early und was maintained over time. The chimerism analysis of the in vivo expanded CMV-specific CD8+ T cells demonstrated a 100% donor chimerism. T cell receptor excision circle (sjTRECs) analysis revealed a frequency of sjTRECs two logs lower than expected, indicating peripheral expansion rather than thymic proliferation of CMV specific CD8+ T cells. cDNA generated from FACS-purified donor-derived CMV B7 pp65-specific CD8+ T cells was probed with the indicated 5′ Vß14-specific and 3′ CDR3-specific primers for the presence of clonotypic T cells. The respective CDR3 region sequence was identical for both donor T cells and CMVpp65 specific T cells in the patients at different time points after the adoptive T cell transfer, thus clearly indicating that the expanded CMV specific T cell were of clonogenic donor origin. Conclusion: Streptamer technology offers the advantage of selecting CMV specific CD8+ T cells at GMP level for adoptive T cell transfer. Two CMVpp65 specific T cell transfers resulted in a marked increase of CMV-specific CD8+ T cells and induced long-lasting CD8+ T cell responses, which allowed the patients to discontinue toxic antiviral drug therapy without further high level reactivation of CMV.

Objective: BK polyoma virus (BKV) can cause hemorrhagic cystitis (BKV-HC) in patients after allogeneic stem cell transplantation (allo-SCT). Levofloxacine can hamper the proliferation of BKV in patients after kidney transplantation even resulting in less rejections of transplants. We therefore wondered whether the prophylactic use of levofloxacine might also reduce the incidence of BKV-HC in patients after allo-SCT. Methods: 178 patients undergoing SCT at our institution were included in this study. 72 patients received a BKV-HC prophylaxis with 500 mg/d levofloxacine administered orally till d+50 while 102 patients did not. Patients developing clinical symptoms of cystitis were screened for BKV in the urine by PCR. Results: 31/102 (30%) patients from the non-prophylaxis group tested positive for BKV after developing clinical symptoms of cystitis. The severity of BKV-HC was classified clinically as follows: grade I (no microhematuria) = 10, grade II (micro-, but no macrohematuria) = 11, grade III (macrohematuria) = 9, and grade IV (intravesicular blood clots) = 1. Patients with BKV-HC grade I were treated symptomatically by oxybutynin. Grade II patients received levofloxacin alone, grade III patients additionally leflunomide. The patient with grade IV BKV-HC received in addition bladder instillations with prostaglandin. BKV-HC was not associated with higher mortality (p=0.77). Differences according to the conditioning regime could be observed: 29 % of patients receiving a radio immune therapy (RIT) developed BKV-HC, vs. 26 % of patients after reduced intensity conditioning (RIC), vs. 19 % of patients after standard or FLAMSA regimen, vs. 7 % of patients after mini-RIT. In contrast, only 12 of 72 patients (17%) with levofloxacin tested positive for BKV in the urine. These patients showed mild or moderate clinical symptoms with microhematuria. Oxybutinin and leflunomide were added and the patients recovered. As for the conditioning, BKV-HC occurred in 4/20 patients after FLAMSA conditioning and 8/36 patients after standard conditioning, but not patients after (mini-)RIT. Of note, the difference between the prophylactic group with a BKV-HC incidence of 17% vs. 30% in the non-prophylaxis group tested significant (p Summary: Incidence of BKV-HC depends on the type of conditioning, and can be significantly reduced by levofloxacin prophylaxis. The role of T cell immune response to BKV remains to be elucidated.

We have demonstrated immunological responses and positive clinical effects of a peptide vaccination for patients with AML, MDS, MM and CLL with a limited tumor load or a minimal residual disease over-expressing RHAMM using 300 μg RHAMM-R3 peptide (Schmitt et al., Blood 2008; Giannopoulos et al., abstract submitted). To date, 26 patients were enrolled in this clinical peptide vaccination trial. Here, we report on the second cohort of nine patients with AML, MDS and MM vaccinated with a higher peptide dose (1000 μg RHAMM-R3 peptide). The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Similar to the patients vaccinated with 300 μg peptide only mild drug-related adverse events were observed such as erythema and induration of the skin. Immunomonitoring was performed using ELISpot assays for Interferon gamma and Granzyme B, tetramer-based flow cytometry and chromium release assays. Moreover, the frequency of regulatory T cells was quantified at different time points of vaccination. In this second cohort of patients treated with 1,000 μg peptide we detected specific immune responses in a lower frequency (4/9 patients) in contrast to patients in the 300 μg cohort (7/10 patients). In these patients with immune responses we found an increase of CD8+/HLA-A2/RHAMM-R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. Two patients with positive immune responses showed a significant decrease of regulatory T cells. One patient without positive immune and clinical effects showed an increase of the frequency of regulatory T cells (5.03% to 15.9%). Three out of nine patients treated with 1,000 μg showed positive clinical effects: One patient with MDS RAEB-2 showed a reduction of leukemic blasts in the bone morrow to lower than 5%, one MDS patient achieved a normalization of the peripheral blood counts and one patient with multiple myeloma experienced a reduction of light chain in serum. The patients in the 300 μg cohort showed also a higher frequency of positive clinical effects (5 out of 10 patients). Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial.

Introduction: Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity. Methods: As a paradigm for peptide vaccine preparation the immunogenic CMV peptide (pp65, pos.: 495–503 NLVPMVATV) lyophilisate was resolubilized in dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and admixed with Montanide™ (ISA51: incomplete Freund’s adjuvant [IFA]). This mixture freshly prepared, stored at +4°C and cryopreserved at −20°C was subjected to lymphocyte peptide cultures (MLPC) followed by enzyme linked immunospot (ELISPOT) assays and flow cytometry (FACS) as bio-assay. Results: Addition of different amounts of peptide (10–80 μg) to a MLPC resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8+ CMV tetramer+ T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at ±4°C over 24 hrs did not result in a significant change of the immunogenicity of the vaccine, as assessed by ELISPOT and FACS. In contrast, cryopreservation of the vaccine at −20°C resulted in a loss of immunogenicity. Conclusion: Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as IFA, is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.

B-cell chronic lymphocytic leukemia (B-CLL) is a very attractive disease to be targeted by immunotherapy due to its rather low progression and due to the availability of cancer cells in the peripheral blood. Dendritic cells (DCs) are the most professional antigen presenting cells, but are deficient in B-CLL. Therefore administration of ex vivo generated DCs might improve the anti-leukemic T cell response in patients with B-CLL. Our group reported the first clinical trial with allogeneic DCs stimulated with leukemic cells lysates in B-CLL patients. To answer the question whether autologous DCs might cause a similar immunotherapeutical effect we conducted the present study assessing the safety and feasibility as well as the clinical and immunological responses to autologous monocyte-derived DC immunotherapy in B-CLL patients. Precursor cells for DC generation were obtained using two methods: by plastic adherence of PBMC from leukapheresis or CD14 positive separation through magnetic beads of PBMC from 150mL of peripheral blood. Twelve patients at clinical stage 0 and 2 according to Rai were vaccinated intradermally up to eight times with a mean number of 7.4×106 DCs pulsed with B-CLL cell lysates. Specific CD8+ T cell responses to RHAMM and fibromodulin - recently described as tumor specific antigens (TAA) in B-CLL were evaluated by multi-parametric tetramer-based flow cytometry and ELISPOT assays. Furthermore, the percentage of CD4+CD25hiFoxP3+ T regulatory cells in the peripheral blood over the time of vaccination was assessed. We observed a decrease of peripheral blood leukocytes and B CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. The best clinical response was observed in the patients treated with DCs generated from adherent fraction of PBMC obtained by leukapheresis. No severe toxicities were noted. After the end of therapy the in vitro proliferation of autologous T cells stimulated by DC pulsed with lysates, as well as by lysates alone increased significantly comparing to the values before treatment (1391±2170, p

Cytomegalovirus (CMV) reactivation constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (PBSCT). The frequency of CMVpp65 specific CD8+ T cells is pivotal for the clearance of CMV. CMVpp65 specific CD8+ T cell frequencies can be measured using tetra-, penta- and streptamer technologies, streptamers can also be applied therapeutically. In donors, these frequencies might allow us to define the best available donor in addition to the mere serostatus. In the present study we investigated the specificity and sensitivity of all three methods and compared the results to the serostatus. A therapeutical application, i.e. an adoptive transfer of CMV specific CD8+ T cells selected by streptamer technology to a patient with acute lymphatic leukemia suffering from life-threatening CMV antigenemia after allogeneic PBSCT was performed. 23 samples from CMV seropositive healthy volunteers (HV) and 10 samples from CMV seropositive patients before and after allogeneic stem cell transplantation (all HLA-A2 or -B7 positive) were analyzed with tetra-, penta- or streptamer conjugated to PE by flow cytometry. Our lab took part in an inter-lab CMV multimer assay including 20 European countries in the framework of www.kimt.de. For the adoptive T cell transfer a donor leukapheresis was performed followed by an HLA-B7 CMVpp65 streptamer positive selection. The patient received 2×10E5 CMV specific CD8+ T cells per kg body weight as a single transfusion. Optimal amounts of HLA-A2 multimers to stain a pellet of 10E6 cells were 0.44 mcg tetramer, 0.15 mcg pentamer and 0.2 mcg MHC/0.3 mcg streptactin complex. Surprisingly, only in 48% (11/23) seropositive HV CD8+ multimer+ T cells could be detected. The ALL patient developed a foscarvir resistant CMV antigenemia with a maximum of 959/500,000 CMVpp65 positive cells. After a switch to ganciclovir/valganciclovir and an adoptive transfer of CMV specific T cells, the antigenemia was cleared. Valganciclovir was discontinued, but CMV antigenemia remained controlled. The frequency of CMVpp65 specific CD8+ T cells increased dramatically from 0.0% till 19.8%. All of these T cells were donor derived as demonstrated by small tandem repeat (STR) analysis. The patient did not develop signs of CMV disease at any time point. This study demonstrates the power of multimer staining to define appropriate donors for transplantation. Donors should be screened for their CMVpp65 specific CD8+ T cell frequency. All three multimer technologies can be used yielding similar results. The streptamer technology additionally offers the advantage to select CMVpp65 specific CD8+ T cells at the GMP level for adoptive T cell transfer and can induce long-lasting CD8+ T cell responses effectively clearing even a high virus load.

Background: For CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT), one might administer tyrosine kinase inhibitors (TKIs) like imatinib (Glivec), nilotinib (Tasigna) and dasatinib (Sprycel), or donor lymphocyte infusions (DLIs). TKIs will inhibit the proliferation of CML progenitor cells, but might also hamper the graft-versus-leukemia (GVL) effect considered to be crucial for the eradication of the disease. Moreover, CD8+ T cells specific for cytomegalovirus (CMVpp65) might be impaired. Methods: Imatinib, nilotinib and dasatinib were added at concentrations of 0–20 μM, 0–4 μM and 0–50 nM respectively to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived from influenza matrix protein (IMP), CMVpp65 as a viral antigen and the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemia-associated antigen. CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PSCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as Tregs after 3 days of culture with anti-CD3 and anti-CD28. Activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed. Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2/tetramer+effector T cells specific for IMP, CMVpp65 and RHAMM-R3 was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibition was reversible after removal of the drugs from the MLPC. The proliferation and function of Tregs were also significantly inhibited by TKIs in a dose-related fashion. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NFκB p65/p100/p105 and c-Rel. T cell activity was impaired when TKIs were clinically administered. The potency of T cell inhibition was imatinib: nilotinib: dasatinib = 1:2:40 under therapeutical serum respectively culture medium levels (2 μM:1 μM: 25 nM). Conclusion: When administering TKIs to patients after allo-PBSCT, the inhibition of both CD8+ and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. Inhibition of CD8+ T lymphocytes was partially reversible after removal of TKIs from the cultures. Using western blotting analysis, we found that these effects are mediated at least in part by down-regulating the levels of phosphorylation of TCR and NF-κB family members.

Most patients with AML, MDS, MM and chronic lymphatic leukemia (CLL) express the receptor for hyaluronic acid mediated motility (RHAMM/CD168) on their tumor cells. We characterized RHAMM/CD168 as a leukemia-associated antigen (LAA) eliciting both humoral and cellular immune responses in patients with different hematological malignancies. CD8 positive T cells primed with the RHAMM/CD168-derived peptide R3 (ILSLELMKL) were able to lyse autologous AML blasts expressing this LAA. Therefore, we initiated a phase I/II R3 peptide vaccination to induce immunological and hematological responses for patients with AML, MDS or MM overexpressing RHAMM/CD168. Patients were included with positive RHAMM/CD168 expression but with a limited tumor load. At a biweekly interval, RHAMM R3 peptide (300 mcg for the first 12 patients and 1000 mcg for patients 13–24) emulsified with the incomplete Freund’s adjuvant (day 3) and GM-CSF (100 mcg, days 1–5) was administrated four times subcutaneously. The primary aim of the study is safety and feasibility of this peptide vaccination, secondary aims the evaluation of a specific T cell immune response to RHAMM/CD168 R3 peptide and the assessment of the influence of the R3 peptide vaccination on the remission status. Since January 2005, 14 patients have been enrolled in the study. The first ten patients (2 AML, 4 MDS, 4 MM) have completed the course of four vaccinations and have been completely evaluated. Therapy related adverse events observed under R3-peptide vaccination were erythema and induration of the skin at the site of injection (CTC I°). In 5/10 patients, we detected in the peripheral blood a significant increase of specific CD8+ T cells recognizing the R3 peptide in ELISPOT analysis and seven-color flow cytometry including tetramer staining. As expected, after vaccination with the HLA class I peptide R3 no significant increase of IgG titers against the antigen RHAMM could be detected. Clinical responses have been assessed by the examination of peripheral blood and bone-marrow samples before and after vaccination. Patients showed a reduction of the tumor-specific expressed antigen RHAMM/CD168 in real-time RT-PCR analysis after vaccination. 3/6 patients with myeloid disorders (1 AML, 2 MDS/RAEB1) showed a reduction of CD33+ cells in FACS analysis of the bone-marrow after four vaccinations from 10 and 7 % to 1–2 and

Several Tumor-associated antigens (TAAs) are expressed in acute myeloid leukemia (AML) and elicit specific immune responses of CD8 positive T cells. These specific T cell responses against leukemic blasts expressing TAAs might play a critical role in the control of minimal residual disease (MRD) in AML. Therefore, we investigated whether TAAs inducing specific immune responses in AML patients were associated with the clinical outcome. A DNA-microarray analysis of 116 AML samples was performed to correlate expression of TAAs to the clinical outcome. In these AML patients specific T cell responses to TAAs were assessed by ELISPOT analysis, tetramer staining and chromium release assays. Quantitative RT-PCR based validation of our results demonstrated the power of DNA microarray technology. We found a significant correlation of high mRNA expression of the TAA G250/CA9 with a longer overall survival (P=0.022), a trend for better outcome in patients with high expression levels of PRAME (P=0.103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1 we found no correlation with clinical outcome of AML patients. Moreover, co-expression of RHAMM/HMMR, PRAME and G250/CA9 provided a favorable prognostic effect (P=0.005). We found specific T cell responses at high frequency for these three antigens in AML patients. Positive immune reactions were detected in 8/17 (47%) AML patients for RHAMM/HMMR-R3-derived, in 7/10 (70%) for PRAME-P3-derived, and in 6/10 (60%) for newly characterized G250/CA9-G2-derived peptides. We detected a significant increased immune response of AML patients in complete remission compared to AML patients with refractory disease (P

Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.

Background/Purpose Cytomegalovirus (CMV) disease constitutes a serious complication after stem cell transplantation and has been treated by adoptive transfer of donor-derived CMV-specific CD8+ T cells. CMV-specific CD8+ T cells were selected by multimers, and the technologies may alter the function of these T cells. Therefore, here we evaluated the impact of multimer reagents on the function of CD8+ T lymphocytes. Methods CMV-specific CD8+ T cells were purified from the peripheral blood of donors using tetra- and streptamer technologies. The functional status of purified CMV-specific CD8+ T cells was assessed by multiparametric immunophenotyping and carboxyfluorescein succinimidyl ester proliferation assays as well as by enzyme-linked immunospot assays. Results A similar percentage of CMV-specific CD8+ T cells could be purified by both tetra- (90%) and streptamer (92%) technologies. That constitutes a 30- to 50-fold concentration of CMV-specific CD8+CD45RA+CCR7−effector T cells. Selected cells secreted interferon-gamma and granzyme B upon stimulation with CMVpp65 peptide, thus demonstrating their functionality. Conclusion Our study demonstrated that both tetra- and streptamer technologies can be used to purify CMV-specific cytotoxic CD8+ T cells for adoptive T-cell transfer. Both multimer technologies did not have any negative influence on the proliferation of selected T cells. Importantly, streptamer technology is available at good manufacturing practice level.