ALBERT

Description: Journal of the American Chemical Society DOI: 10.1021/jacs.6b06615

Description: ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

Description: Introduction: Patients receiving an allogeneic stem cell graft from a cytomegalovirus (CMV) seronegative donor are prone to CMV reactivation with high risk of disease and mortality. Therefore we initiated a clinical phase I trial with a novel vaccine designed by our group: a CMV phosphoprotein 65 (CMVpp65)-derived peptide in water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor. Material and Methods: 10 patients after allogeneic stem cell transplantation received four vaccines s.c. at a biweekly interval. Patients were monitored for clinical course and CMVpp65 antigenemia. CMV-specific CD8+ and gamma/delta T cells were analyzed by multi-color flow cytometry. Neutralizing anti-CMV antibody assays were established and correlated to clinical parameters. Results: Peptide vaccination was well tolerated, no drug-related serious adverse events were detected. 7 of 9 patients with CMVpp65 antigenemia cleared the CMV with enduring response 〉 1.5 years after 4 vaccinations and are still free from antigenemia until present. 2 patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An up to 6-fold increase in frequency of both CMV-specific CD8+ T cells or Vdelta2- gamma/delta T cells with a maximum of 1.32% CMV-specific CD8+ T cells ex vivo without priming and 13.7% Vdelta2- gamma/delta T cells of all CD8+ T cells was detected in five patients. Also titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load. Conclusion: Administration of CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing. Disclosures Kuball: Gadeta B.V.: Membership on an entity's Board of Directors or advisory committees. Wuchter:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy. Greiner:BMS: Research Funding.

Description: Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Treatment for acute myeloid leukemia (AML) patients became more effective, yet relapse rates are still high. Immunotherapeutic approaches might overcome the immune escape mechanisms of LSC and might be a therapeutic option. Leukemia-associated antigens (LAA) represent targets that are recognized by cytotoxic T-lymphocytes (CTL) and might be useful for specific immunotherapy. We enriched the LSC containing fraction as CD34+CD38- cells (called LSC-EF) of 20 AML patients using FACS, analyzed expression patterns of LAA in LSC-EF and compared them to patterns in enriched HSC und bulk AML cells by microarray expression analysis and qRT-PCR. In immunoassays, we analysed the functional inhibition of LCS in colony forming assays (CFU) of AML patients by stimulated T cells. We detected a high expression of several LAA in LSC-EF. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC-EF compared to HSC. Survivin (p = 0.7), HAGE (p = 0.5), SSXIP2 (p = 0.4) and Aurorakinase A (p = 0.7) did not indicate a significant differential expression. BAGE does not seem to be an ideal candidate as it is significantly higher expressed on normal HSC (p = 0.0056). Further, we evaluated the expression differences of LSC-EF compared to leukemic bulk. PRAME, RHAMM and WT1 qualified as interesting potential targets as they are lower expressed on leukemic bulk than on LSC-EF. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk but lower in LSC-EF. qRT-PCR confirmed our array data. In immunoassays, T cells stimulated against LAA indicated a significant inhibition of CFUs in AML patient samples. PRAME, RHAMM and WT1 showed good immunogenic responses with CTL recognition rates in a range of 58-83%. PRAME hereby proved as most effective using two different patient samples, with a reduction of 83% colonies (p = 0.004). WT1 shows a significant reduction of 74% (p = 0.05) and RHAMM of 58% colonies (p = 0.2). Taken together, several LAA are significantly expressed on the LSC-EF, whereas PRAME, RHAMM and WT1 seemed most promising as these LAA are differentially expressed compared to normal stem cells and stimulation of cytotoxic T cells against these LAAs inhibit LSC in CFU assays. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The role of allogeneic stem cell transplantation (allo-HSCT) in the treatment algorithms for patients with multiple myeloma remains controversial although it is the only potentially curative approach currently available. Here we present the retrospective analysis of 95 allo-HSCT performed between 1994 and 2013 at Ulm University Hospital. We focused on the impact of cytogenetics, graft-versus-host disease (GvHD) and intensity of conditioning on overall survival (OS), progression- free survival (PFS), relapse and non-relapse mortality (NRM). Study population Median age at initial diagnosis was 49 years (range 25-64), median age at time of allo-HSCT was 51 years (range 26-65). Median time from initial diagnosis to allo-HSCT was 13 months (range 3-106). Indications for allo-HSCT were 1) primary allo-HSCT after induction therapy (11 pts), 2) planned tandem auto-allo-HSCT (44 pts), 3) relapse after single allo-HSCT (25 pts), 4) relapse after tandem-auto-HSCT (15 pts). The conditioning regimen in 60 pts was a reduced intensity conditioning (RIC), in 35 pts myeloablative conditioning (MAC). In 68 pts cytogenetic data were available: 13 pts were stratified into standard-, 39 pts into the intermediate- and 16 pts into the high-risk group according to the mSMART recommendations. Results: Median follow-up was 70 months (95% CI, 64,5-75,5). The estimate 1-, 2- and 5-year OS was 87,4 %, 74,7 % and 46,4 % with a median OS of 36 months (95 % CI, 19,7 -52,2). For both, RIC and MAC median OS was 36 months (95% CI, 24,4–47,6 versus 95% CI, 0-108,4). The cumulative incidence of TRM was not different for RIC and MAC but there was a trend for lower relapse in patients receiving MAC (p=0,0612). With respect to the indication for allo–HSCT outcomes were as follows: Median OS was 89 months for primary allo-HSCT, 47 months for tandem auto-allo-HSCT, 20 months for relapse after auto-HSCT and 26 months for relapse after double auto-HSCT. OS did not differ significantly. Median OS in the standard risk group was 20 months (95% CI, 6,7-33,3), in the intermediate group 41 months (95% CI, 17,3-64,7) and in the high-risk group 7 months (95% CI, 0-14,8), showing no statistical significance. 1-year OS was 61,5% vs 66,7% vs 43,8%, 2-year OS was 35,9% vs 66,7% vs 43,8% and 5-year OS was 35,9% vs 43,2% vs 11,7% (standard vs intermediate vs high risk). The median PFS was 12 months (95% CI 8,4-15,6). 1-year PFS was 49,2%, 2-year PFS 32,9% and 5-year PFS was 21,8%. PFS according to the cytogenetic aberration showed a median PFS of 20 vs 14 vs 5 months, 1-year PFS was 53,8% vs 51,3 % vs 30 %, 2-year PFS with 35,9% vs 30,8% vs 7,5%, and 5-year PFS with 26,9% vs 11,5 % vs 7,5% (standard vs intermediate vs high risk). These differences are not statistically significant. Considering the impact acute GvHD, OS significantly differed between the groups with no aGvHD or aGvHD grade I and aGvHD grade II-IV with inferior survival for patients suffering from aGvHD grade II-IV. Chronic GvHD had no impact on outcome. Conclusion Our data of a 19 year experience in treatment of patients with advanced multiple myeloma with allo-HSCT showed an effective treatment option with a curative potential even for patients after intensive pretreatment including autologous stem cell transplantation. Patients who received MAC had a trend for lower cumulative incidence of relapse compared to RIC without increasing TRM in our study. Disclosures No relevant conflicts of interest to declare.

Description: In order to maintain remission and to prolong overall-survival after allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion (DLI), cytotoxic T-cell responses against malignant cells play a pivotal role, however they are not well characterized so far. In this study, we focused on the detection of immune responses in patients before and after DLI. A broader epitope-specific T cell activity is associated with clinical response of patients treated with DLI and a concurrent reduction of regulatory T cell frequency may contribute to clinical response in patients after DLI. For a better characterization of the T cell responses, frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells was assessed using ELISpot and pMHC multimer assays. Furthermore, the frequency of regulatory T cells (Treg) before and after DLI was analyzed. Results were correlated to the clinical course of the patients. Independently of their clinical response, 7/11 patients (63.6%) showed an immunological response through an increase in the number of recognized epitopes over the course of DLI. There was a significant increase (p=0.02) in epitope recognition comparing early screening and maximum response after DLI and a significant increase in the mean augmentation from 1 to 4 of the spotted epitopes in the course of DLI was detected in the cohort of clinical responders (R) compared to non-responders (NR) who did not show any dynamics in epitope recognition with a median of 3 to 4 recognized LAA. The proportion of the CD4+CD25highFoxP3+ Treg within the CD4+CD25high T cell fraction decreased significantly in R from a median of 72.9% to 54.6% when comparing the variation at the time points before and after DLI (p=0.04). In NR there was no significant change in the Treg fraction in the course of DLI. In general, significantly more LAA-derived T cell epitopes (p=0.02) were recognized in R when compared to NR. Moreover, the frequency of Treg in R decreased significantly (p=0.008) while remaining stable in NR. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. This study suggests that decreasing numbers of Treg as well as enhanced LAA diversity in T cell responses contribute to clinical outcome of patients treated with DLI. Disclosures Döhner: AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding.

Description: Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies. Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays. Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction. CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays. In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. Disclosures Schrezenmeier: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

Description: Mutations in the nucleophosmin gene (NPM1mut) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1mut. In the present study, we were able to demonstrate both CD4+ and CD8+ T-cell responses against NPM1mut. Ten peptides derived from wild-type NPM1 and NPM1mut were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr51-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR–binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1mut induced CD8+ T-cell responses. A total of 33% of the NPM1mut AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4+ T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8+ and CD4+ T cells. The results of the present study show that NPM1mut induces specific T-cell responses of CD4+ and CD8+ T cells and therefore is a promising target for specific immunotherapies in AML.

Description: Introduction The advent of T cells expressing chimeric antigen receptors (CART) for the treatment of cancer patients has been a milestone in the field of immunotherapy. For clinical application, reproducibility and safe generation of CART must be guaranteed. We investigated how different culture conditions of human peripheral blood mononuclear cells (PBMCs) influence the phenotype, i.e. naïve (N), central / effector memory (CM/EM), effector (E) cells, and the cellular composition of the resulting CART preparation, among others: the percentage of CD56+, γ/δ TCR+ and CD4+CD8+ double positive cells. Eventually, freezing/thawing of CART might also have an impact on their number, phenotype and function. Methods CART were generated by transducing human PBMCs from healthy donors with a CD19.CAR-CD28-CD137zeta vector (kindly provided by the Center for Cell and Gene Therapy, Houston) under two different stimulating culture conditions - anti-CD3/anti-CD28 antibodies with the addition of either IL-7/IL-15 or IL-2. CART generation lasted for 15 days. Cytotoxic ability of the generated CART was assessed by a standard chromium (Cr-51) release assay. CD19+ (Daudi) and CD19- (K562) cells were labeled for 2h with Cr-51 and coincubated with CART for 4h. Cr-51 release was measured in a liquid scintillation counter. In addition, multi-parametric flow cytometry was performed using a FACS LSR device. Results Overall, the transduction rate for both cytokine cocktails was around 50% of all CD3+ T cells. Freezing/thawing of CART reduced their cytotoxic activity: Lysis of the CD19+ target cell line (Daudi) reached a maximum of 80% for fresh cells and 50% for freshly thawed cells, both stimulated with IL-7/IL-15. FACS analysis revealed that freshly prepared cells showed an increased percentage of activated lymphocytes compared to cryopreserved cells. The majority of these activated lymphocytes were CD4+CD8+ double positive. Using freshly thawed cells, CART stimulated with IL-7/IL-15 showed higher cytotoxic activity than CART stimulated with IL-2: 50% vs. 38% (Daudi) and 7% vs. 5% (K562). Stimulation with IL-7/IL-15 led to an increase in the CD56+ CAR NK T cell population (20%) compared to IL-2 (8%). γ/δ TCR+ CART differed as well (3% for IL-7/IL-15, 8% for IL-2). The percentage of CD8+ and CD4+ CART was similar for both cytokine cocktails with around 60% and 30%, respectively. However, stimulation with IL-7/IL-15 showed a trend towards generation of a lower percentage of CD4+CD8+ double positive CART than stimulation with IL-2 (2% vs. 6%). The analysis of the different CART phenotypes showed significant differences: N 24% vs. 3%, CM 21% vs. 13%, EM 24% vs. 77%, E 31% vs. 7%, for IL-7/IL-15 vs. IL-2, respectively. A general phenomenon observed for both cytokine cocktails, was the difference in phenotype for CD4+ vs. CD8+ CART: CD4+ CART showed a more distinct memory phenotype, i.e. central and effector memory cells. Correspondingly, the CD8+ CART shifted more towards the naïve and effector phenotype. The percentage of highly effective stem cell memory CART (TSCM: naïve CD27+CD95+) was assessed. Using IL-7/IL-15, CAR TSCM cells accounted for 4.8%, whereas for IL-2, 2.7% had a TSCM phenotype. 75% of the CART were HLA-DR positive (activation marker) and 100% were positive for CD95 (FAS receptor, inhibition marker). Using PBMCs from healthy donor samples, the generated CART products did not contain any contamination of either B (CD19+) or stem cells (CD34+). Conclusions We have established a combination of immunophenotyping and cytotoxicity assays for CART as a preclinical potency assay. Stimulation with IL-7/IL-15 led to a balanced phenotype expression, with a similar number of N, CM, EM and E CART, whereas the majority of CART under IL-2 stimulation adopted an EM phenotype. Correlation of these data with the clinical outcome of patients receiving the corresponding cell products will allow optimization and standardization of CART therapy. Disclosures Wuchter: Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy.