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Immune checkpoint inhibitors can block inhibitory molecules on the surface of T cells, switching them from an exhausted to an active state. One of these inhibitory immune checkpoints, programmed cell death protein 1 (PD-1) is expressed on T cell subpopulations in acute myeloid leukemia (AML). PD-1 expression has been shown to increase with AML progression following allo-haematopoeitic stem cell transplantation, and therapy with hypomethylating agents. We have previously shown that anti-PD-1 can enhance the response of leukemia-associated antigen (LAA)-specific T cells against AML cells as well as leukemic stem and leukemic progenitor cells (LSC/LPCs) ex vivo. In concurrence, blocking of PD-1 with antibodies such as nivolumab has been shown to enhance response rates post-chemotherapy and stem cell transplant. The immune modulating drug lenalidomide has been shown to promote anti-tumour immunity including anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenicity. The effects of lenalidomide are distinct from chemotherapy, hypomethylating agents or kinase inhibitors, making lenalidomide an attractive agent for use in AML and in combination with existing active agents. To determine whether anti-PD-1 (nivolumab) and lenalidomide alone or in combination could enhance LAA-specific T cell immune responses, we used colony-forming immune and ELISpot assays. Combinations of immunotherapeutic approaches are believed to increase antigen-specific immune responses against leukemic cells including LPC/LSCs. In this study we used a combination of LAA-peptides with the immune checkpoint inhibitor anti-PD-1 and lenalidomide to enhance the killing of LSC/LPCs ex vivo. Our data offer a novel insight into how we could improve AML patient responses to treatment in future clinical studies.

XLSX file - 72K, RNAi PRAME

XLSX file - 51K, MSigDB RNAi PRAME

XLSX file - 102K, MSigDB RNAi ATRA

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XLSX file - 42K, HD98B sample overview.

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Purpose: In acute myeloid leukemia (AML) without retinoic acid receptor (RAR) rearrangement, the effect of all-trans-retinoic acid (ATRA) is still poorly understood despite an association of NPM1 mutation and ATRA response. Recently, preferentially expressed antigen in melanoma (PRAME) has been shown to be a dominant repressor of RAR signaling.Experimental Design: Thus, we further investigated ATRA response mechanisms, especially the impact of PRAME expression on ATRA responsiveness. We profiled gene expression in diagnostic samples derived from our AML HD98B trial, in which ATRA was administered in addition to intensive chemotherapy.Results: Our data revealed a PRAME expression-associated gene pattern to be significantly enriched for genes involved in the retinoic acid metabolic process. In leukemia cell line models, we could show that retinoic acid-regulated cell proliferation and differentiation are impacted by PRAME expression. In patients with primary AML, repressor activity of high-PRAME levels might be overcome by the addition of ATRA as indicated by better outcome in 2 independent studies (P = 0.029).Conclusions: PRAME seems to impair differentiation and to increase proliferation likely via blocking RAR signaling, which might be reversed by ATRA. PRAME therefore represents a promising target for both ATRA treatment and possibly future immunotherapeutic approaches in AML. Clin Cancer Res; 19(9); 2562–71. ©2013 AACR.

XLSX file - 52K, MSigDB RNAi PRAME C2-C4

PDF file - 110K, Supplementary figure 1 Apoptosis of PRAME-high AML cells in response to ATRA treatment. Supplementary figure 2 PRAME overexpression in PRAME-low KG1 cells impacts cell numbers following treatment with ATRA.

Críticas à crescente industrialização da agricultura têm incentivado sistemas alimentares diferenciados a partir de relações entre produtores e consumidores. Registra-se um aumento exponencial de Community Supported Agriculture (CSA) em todo o mundo. Na Alemanha, as CSAs expandiram-se mais lentamente e somente em anos recentes passaram a desfrutar de maior atenção do público. Neste artigo, objetivamos situar historicamente as CSAs, valorizando as suas especificidades e características e delineando tendências mais recentes. A metodologia constou de revisão de literatura e de entrevistas com atores-chave que participam da coordenação e com associados de duas experiências de CSA. Os principais resultados mostram iniciativas e tendências convergentes para a expansão de sistemas alimentares sustentáveis em novos estabelecimentos agrícolas na Europa. Influenciam nas iniciativas, a demanda das pessoas por produtos regionais, a possibilidade de rastreabilidade, o desejo de cooperação, e a consciência sobre os efeitos climáticos globais nos modos de vida, dentre outras razões.

Systemic administration of histone deacetylase inhibitors (HDACi), like valproic acid (VPA), is often associated with rapid drug metabolization and untargeted tissue distribution. This requires high-dose application that can lead to unintended side effects. Hence, drug carrier systems such as nanoparticles (NPs) are developed to circumvent these disadvantages by enhancing serum half-life as well as organ specificity.This chapter gives a summary of the biological characterization of HDACi-coupled NPs in vitro, including investigation of cellular uptake, biocompatibility, as well as intracellular drug release and activity. Suitable methods, opportunities, and challenges will be discussed to provide general guidelines for the analysis of HDACi drug carrier systems with a special focus on recently developed cellulose-based VPA-coupled NPs.

Nucleophosmin1 (NPM1) is one of the most commonly mutated genes in AML and is often associated with a favourable prognosis. Immune responses play an increasing role in AML treatment decisions; however, the role of immune checkpoint inhibition is still not clear. To address this, we investigated specific immune responses against NPM1, and three other leukaemia-associated antigens (LAA), PRAME, Wilms' tumour 1 and RHAMM in AML patients. We investigated T cell responses against leukaemic progenitor/stem cells (LPC/LSC) using colony-forming immunoassays and flow cytometry. We examined whether immune checkpoint inhibition with the anti-programmed death 1 antibody increases the immune response against stem cell-like cells, comparing cells from NPM1 mutated and NPM1 wild-type AML patients. We found that the anti-PD-1 antibody, nivolumab, increases LAA stimulated cytotoxic T lymphocytes and the cytotoxic effect against LPC/LSC. The effect was strongest against NPM1

The efficacy of immunotherapies in cancer treatment becomes more and more apparent not only in different solid tumors but also in hematological malignancies. However, in acute myeloid leukemia (AML), mechanisms to increase the efficacy of immunotherapeutic approaches have to be further elucidated. Targeting leukemic progenitor and stem cells (LPC/LSC) by specific CTL, for instance, in an adjuvant setting or in minimal residual disease, might be an option to prevent relapse of AML or to treat MRD. Therefore, we investigated the influence of immune checkpoint inhibitors on LAA-specific immune responses by CTL against leukemic myeloid blasts and colony-forming cells including leukemic progenitor cells (CFC/LPC). In functional immunoassays like CFU/CFI (colony-forming units/immunoassays) and ELISpot analysis, we detected specific LAA-directed immune responses against CFC/LPC that are postulated to be the source population of relapse of the disease. The addition of nivolumab (anti-PD-1) significantly increases LAA-directed immune responses against CFC/LPC, no effect is seen when ipilimumab (anti-CTLA-4) is added. The combination of ipilimumab and nivolumab does not improve the effect compared to nivolumab alone. The anti-PD1-directed immune response correlates to PD-L1 expression on progenitor cells. Our data suggest that immunotherapeutic approaches have the potential to target malignant CFC/LPC and anti-PD-1 antibodies could be an immunotherapeutic approach in AML. Moreover, combination with LAA-directed vaccination strategies might also open interesting application possibilities.

Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration.

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N’-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.

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.

Introduction Despite a large number of trials, the role of bevacizumab (BEV) in the treatment of recurrent high-grade gliomas is still controversial. Evidence regarding an effect on overall survival in this context is ultimately inconclusive. At the Department of Radiation Oncology at Erlangen, Germany we treated a large cohort of patients with recurrent gliomas where bevacizumab use was determined exclusively by the health care provider’s approval of reimbursement. Methods 61 patients (between 06/2008 and 01/2014) with recurrent high-grade gliomas had reimbursement requests for BEV sent to their health insurance. 37 patients out of 61 (60.7%) had their requests approved and therefore received bevacizumab (BEV-arm) as part of their treatment. The remaining 24 (39.3%) patients received standard therapy without bevacizumab (non-BEV-arm). Survival endpoints were defined with reference to the first BEV request to the health insurance provider. Results Median overall survival (OS) for the whole cohort was 7.0 months. OS was significantly better for BEV vs. Non-BEV patients (median, 10.3 vs. 4.2 months, logrank p = 0.023). There was an increased BEV benefit in cases of higher-order recurrences (first order recurrence BEV vs. Non-BEV, 12.5 vs. 10.2 months, p = 0.578) (second or higher order of recurrence, 9.9 vs. 2.6 months, p = 0.010). On multivariate analysis for overall survival the prognostic impact of bevacizumab (HR = 0.43, p = 0.034) remained significant. Conclusion Our results suggest an influence of BEV on overall survival in a heavily pretreated patient population suffering from high-grade gliomas with BEV benefit being greatest in case of second or later recurrence.

T cell responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI) due to graft-versus-leukemia effect. For better characterization of the T cell responses, we assessed frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells using ELISpot and pMHC multimer assays and analyzed the frequency of regulatory T cells (Treg) as well as cytokine profiles before/after DLI. The data were correlated to the clinical course of patients. Significantly more LAA-derived T cell epitopes (p = 0.02) were recognized in clinical responders (R) when compared to nonresponders (NR). In addition, pMHC multimer-based flow cytometry showed a significantly higher frequency of LAA-specific T cells in R versus NR. The frequency of Treg in R decreased significantly (p = 0.008) while keeping stable in NR. No differences in T cell subset analysis before/after DLI were revealed. Clinical responders were correlated to specific immune responses and all clinical responders showed an increase of specific immune responses after DLI. Cytokine assays using enzyme-linked immunosorbent assay showed a significant increase of IL-4 after DLI. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. Moreover, this study suggests that enhanced LAA diversity in T cell responses as well as decreasing numbers of Treg contribute to clinical outcome of patients treated with DLI.

Despite improved treatment options, multiple myeloma (MM) remains an incurable disease. The aim of this study was to investigate the prognostic value of positron emission tomography/computed tomography (PET/CT) using 18F-2’-deoxy-2’-fluorodeoxyglucose ([18F]FDG) in MM patients shortly before and ~100 days after allogeneic hematopoietic cell transplantation (allo-HCT). In this retrospective analysis, we evaluated [18F]FDG-PET/CT-scans of 45 heavily pre-treated MM patients before and 27 patients after scheduled allo-HCT. All scans were qualitatively and semi-quantitatively assessed for the presence of active disease. Serological response was recorded according to International Myeloma Working Group (IMWG) criteria. Progression-free (PFS) and overall survival (OS) were correlated with different PET/CT-derived parameters, such as presence, number and maximum standardized uptake value (SUVmax) of focal myeloma lesions. The impact of extramedullary disease on patient outcome was also assessed. PET/CT negativity -prior to or following allo-HCT- was a favorable prognostic factor for progression-free and overall survival (both, PFS and OS: pre-HSCT p 6.5) revealed a significantly shortened survival compared to patients with a lower SUVmax ( 3) of focal lesions (pre-HCT: both PFS and OS: p

Introduction Chimeric antigen receptor (CAR) T cell (CART) therapy has shown to be a new and very promising therapeutical method in patients with relapsed or refractory (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). The investigator-initiated Heidelberg-CAR-T-cell-trial 1 (HD-CAR-1) evaluates both efficacy and safety of escalating doses of 3 rd-generation CD19-directed CARTs comprising CD28 and 4-1BB as costimulatory molecules in patients with r/r ALL and NHL. Leukapheresis, manufacturing, administration, patient monitoring and follow-up were all conducted in-house at the Heidelberg University Hospital. Methods Treatment was conducted with escalating doses of autologous 3 rd-generation CARTs after lymphodepletion with fludarabine (90 mg/m 2) and cyclophosphamide (1,500 mg/m 2) in patients with r/r acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Non-Hodgkin's lymphoma (NHL), with subtype including diffuse large B-cell lymphoma (DLBCL), transformed follicular lymphoma (tFL) or mantle cell lymphoma (MCL). Treatment efficacy as well as occurrence of toxicities, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), infections or cytopenia, were evaluated. Following prior results regarding dose-dependent efficacy and excellent toxicity profile with dose levels (DL) I, II and III (10 6, 5×10 6 and 20×10 6 CARTs/m 2), a trial amendment was approved for treatment of patients with higher CART doses (DL IV, V and VI: 5x10 7, 10x10 7 and 20x10 7 CARTs/m 2). Results Overall, screening was performed for 32 patients. Two patients were considered screening failures due to rapidly progressive disease (PD) and uncontrolled hepatitis B infection, respectively; leading to 30 patients enrolled in the study. The HD-CAR-1 product was given to 27 patients (12 patients with ALL, four with CLL, four with MCL, five with DLBCL, and two with FL) in different dose levels (six patients with DL I, six patients with DL II, eight patients with DL III, 5 patients with DL IV, 2 patient with DL V). The CART product was not given to two patients due to PD and lethal septic complication, respectively. Severe CAR-T toxicity was rare, as only two patients developed CRS ≥ III°. Both patients received treatment with tocilizumab, while one was additionally treated with steroids. No ICANS ≥ III° were reported in the study. 54% of patients achieved a complete response (CR) with an overall response rate (ORR) of treated patients of 65%. In a subgroup response analysis, an ORR of 92% (83% CRs) with 50% of all patients achieving MRD-negative CR was seen in ALL patients. In the NHL/CLL cohort, an ORR of 43% and a CR rate of 29% were observed. Figure 1 displays OS and PFS results till the end of study (EOS, day +90). Conclusion Academic CART production was feasible for all enrolled patients. Patients responded clinically to treatment and CARTs displayed a highly favorable safety profile. Overall, HD-CAR-1 accounts for clinical evaluation of 3 rd generation CARTs. Figure 1 Figure 1. Disclosures Schubert: Gilead: Consultancy. Schmitt: TolerogenixX Ltd: Current Employment; Hexal: Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; Therakos/Mallinckrodt: Research Funding. Müller-Tidow: Pfizer: Research Funding; Janssen: Consultancy, Research Funding; Bioline: Research Funding. Dreger: Riemser: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; BMS: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Bluebird Bio: Consultancy; Roche: Consultancy, Speakers Bureau. Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees; TolerogenixX: Current holder of individual stocks in a privately-held company; Kite Gilead: Other: Travel grants; Bluebird Bio: Other: Travel grants; Novartis: Other: Travel grants, Research Funding; Hexal: Other: Travel grants, Research Funding; Apogenix: Research Funding.

Nucleophosmin1 (NPM1) is one of the most frequently mutated genes in AML, is often associated with a favorable prognosis and seems to be a suitable target structure for immunotherapeutic approaches. Other groups and ours described specific immune responses of CD8-positive T cells against immunogenic epitopes derived from the mutational region of NPM1 in AML patients (pts). In this extended immunological study, we investigated immune responses against the mutational epitope of NPM1 but also against other LAA in NPM1 mut compared to NPM1 wt pts. 30 AML pts were analyzed using FACS analysis, tetramer staining and colony forming immunoassays (CFI). 15 NPM1 mut and 15 NPM1 wt pts were investigated in CFI to detect CTL mediated immune responses against leukemic progenitor/stem cells (LPC/LSC). We also added immune checkpoint inhibitors to investigate whether these immune responses could be enhanced. Against the LAA PRAME-P3, WT1 and RHAMM-R3 we detected similar frequencies of T cell responses in CFI in NPM1 mut compared to NPM1 wt pts. Antigen specific immune responses were detected in CFI by comparing growth of patient cells alone with growth by addition of antigen specific CTL and calculating colony reduction. Comparing NPM1 mut/NPM1 wt pts many had an immune response to LAA, more than 50% of the pts in both cohorts exhibited an immune response against all epitopes. In NPM1 wt pts no responses were found against the NPM1 epitope as expected, whereas NPM1 mut patients showed a high frequency of immune responses in 10/15 NPM1 mut AML pts (67%) in CFI a reduction of colonies was detected. With the addition of anti-PD1 antibody to CFI we detected an increase of immune responses. For the LAA responses were similar comparing NPM1 mut/NPM1 wt. Compared to LAA, the epitope NPM1 showed a particularly strong immune response when the antibody anti-PD1 was added. All 15 NPM1 mut pts showed an immune response with anti-PD1, with a median reduction of colonies of 47%. 7 of 15 NPM1 mut pts showed a strong immune response against LPC/LSC in CFI with more than 50% reduction of colonies. The data suggest that especially NPM1 mut patients are suitable candidates for antibody therapy with the PD1 antibody. Combination with another immunotherapy such as an NPM1 specific vaccine would be a possibility. Even though no advantage in therapy with the anti-PD1 antibody has yet been shown in the overall AML collective, this therapy could be an option for patients with NPM1 mutated AML. Disclosures Greiner: Bristol Myers Squibb: Other: Unspecified Relationship. Schneider: AbbVie: Current Employment. Schrezenmeier: Novartis: Honoraria; Apellis: Honoraria; Sanofi: Honoraria; Alexion, AstraZeneca Rare Disease: Honoraria, Other: Travel support, Research Funding; Roche: Honoraria.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, 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.

Allogeneic stem cell transplantation (allo-SCT) is the preferred curative treatment for several hematological malignancies. The efficacy of allo-SCT depends on the graft-versus-leukemia (GvL) effect. However, the prognosis of patients with relapsed acute myeloid leukemia (AML) following allo-SCT is poor. Donor lymphocyte infusion (DLI) is utilized after allo-SCT in this setting to prevent relapse, to prolong progression free survival, to establish full donor chimerism and to restore the GvL effect in patients with hematological malignancies. Thus, there are different options for the administration of DLI in AML patients. DLI is currently used prophylactically and in the setting of an overt relapse. In addition, in the minimal residual disease (MRD) setting, DLI may be a possibility to improve overall survival. However, DLI might increase the risk of severe life-threatening complications such as graft-versus-host disease (GvHD) as well as severe infections. The transfusion of lymphocytes has been tested not only for the treatment of hematological malignancies but also chronic infections. In this context, manipulated DLI in a prophylactic or therapeutic approach are an option, e.g., virus-specific DLI using different selection methods or antigen-specific DLI such as peptide-specific CD8+ cytotoxic T lymphocytes (CTLs). In addition, T cells are also genetically engineered, using both chimeric antigen receptor (CAR) genetically modified T cells and T cell receptor (TCR) genetically modified T cells. T cell therapies in general have the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease after allo-SCT. The focus of this review is to discuss the different strategies to use donor lymphocytes after allo-SCT. Our objective is to give an insight into the functional effects of DLI on immunogenic antigen recognition for a better understanding of the mechanisms of DLI. To ultimately increase the GvL potency without raising the risk of GvHD at the same time.

IntroductionChimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin’s lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigator-initiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells.Methods and analysisAdult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1–20×106transduced cells/m2) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CAR-T-cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation ofin vivofunction and survival of CD19.CAR T cells and assessment of CD19.CAR T cell antitumour efficacy.HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique.Ethics and disseminationEthical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings.Trial registration numberEudra CT 2016-004808-60;NCT03676504; Pre-results.