A Combination of the Immunotherapeutic Drug Anti-Programmed Death 1 with Lenalidomide Enhances Specific T Cell Immune Responses against Acute Myeloid Leukemia Cells.

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.

Analysis of HDACi-Coupled Nanoparticles: Opportunities and Challenges.

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.

Enhanced stimulation of antigen-specific immune responses against nucleophosmin 1 mutated acute myeloid leukaemia by an anti-programmed death 1 antibody.

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 NPM1mut cells when the immunogenic epitope was derived from the mutated region of NPM1 and these effects were enhanced through the addition of anti-PD-1. The data suggest that patients with NPM1 mutated AML could be treated with the immune checkpoint inhibitor anti-PD-1 and that this treatment combined with NPM1-mutation specific directed immunotherapy could be even more effective for this unique group of patients.

De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition.

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.

Stroke Angel: Effect of Telemedical Prenotification on In-Hospital Delays and Systemic Thrombolysis in Acute Stroke Patients.

INTRODUCTION: Door-to-CT scan time (DCT) and door-to-needle time (DNT) are important process measures in acute ischemic stroke (AIS) patients undergoing intravenous thrombolysis (IVT). We examined the impact of a telemedical prenotification by emergency medical service (EMS) (called the "Stroke Angel" program) on DCT and DNT and IVT rate compared to standard of care. PATIENTS AND METHODS: Two prospective observational studies including AIS patients admitted via EMS from 2011 to 2013 (cohort I; n = 496) and from January 1, 2015 to May 31, 2018 (cohort II; n = 349) were conducted. After cohort I, the 4-Item Stroke Scale and a digital thrombolysis protocol were added. Multivariable logistic and linear regression analysis was performed. RESULTS: In cohort I, DCT was lower in the intervention group (13 vs. 26 min using standard of care; p < 0.001), but no significant difference in median DNT (35 vs. 39 min; p = 0.24) was observed. In cohort II, a reduction of DCT (8 vs. 15 min; p < 0.001) and DNT (25 vs. 29 min p = 0.003) was observed in the intervention group. Compared to standard of care, the likelihood of DCT ≤10 min or DNT ≤20 min in the intervention group was 2.7 (adjusted odds ratio [aOR] 2.7; 95% CI: 2.1-3.5) and 1.8 (aOR 1.8; 95% CI: 1.1-2.9), respectively. In cohort II, IVT rate was higher (aOR 1.4; 95% CI: 1.1-1.9) in the intervention group. CONCLUSION: Although the positive effects of Stroke Angel in AIS provided a rationale for implementation in routine care, larger studies of practice implementation will be needed. Using Stroke Angel in the prehospital management of AIS impacts on important process measures of IVT delivery.

The N-terminal BRCT domain determines MCPH1 function in brain development and fertility.

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.

Evidence for improved survival with bevacizumab treatment in recurrent high-grade gliomas: a retrospective study with ("pseudo-randomized") treatment allocation by the health insurance provider.

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.

A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter.

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.

Polysaccharide Nanoparticles Bearing HDAC Inhibitor as Nontoxic Nanocarrier for Drug Delivery.

The histone deacetylase inhibitors (HDACi) are potent drugs in the treatment of inflammatory diseases and defined cancer types. However, major drawbacks of HDACi, such as valproic acid (VPA), are limited serum half-life, side effects and the short circulation time. Thus, the immobilization of VPA in a polysaccharide matrix is used to circumvent these problems and to design a suitable nanocarrier system. Therefore, VPA is covalently attached to cellulose and dextran via esterification with degree of substitution (DS) values of up to 2.20. The resulting hydrophobic polymers are shaped to spherical nanoparticles (NPs) with hydrodynamic diameter between 138 to 221 nm and polydispersity indices from 0.064 to 0.094 by nanoprecipitation and emulsification technique. Lipase treatment of the NPs leads to in vitro release of VPA and hence to an inhibition of HDAC2 activity in a HDAC2 assay. NPs are rapidly taken up by HeLa cells and mainly localize in the cytoplasm. The NPs are hemocompatible and nontoxic as revealed by the shell-less hen's egg model.

Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients.

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.

Specific T-cell immune responses against colony-forming cells including leukemic progenitor cells of AML patients were increased by immune checkpoint inhibition.

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.

Immunological and Clinical Impact of Manipulated and Unmanipulated DLI after Allogeneic Stem Cell Transplantation of AML Patients.

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.

Conformation space of a heterodimeric ABC exporter under turnover conditions.

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.

Treatment of patients with relapsed or refractory CD19+ lymphoid disease with T lymphocytes transduced by RV-SFG.CD19.CD28.4-1BBzeta retroviral vector: a unicentre phase I/II clinical trial protocol.

INTRODUCTION: Chimeric 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 ANALYSIS: Adult 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 (1-20×106 transduced 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 of in vivo function 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 DISSEMINATION: Ethical 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 NUMBER: Eudra CT 2016-004808-60; NCT03676504; Pre-results.

Chimeric Antigen Receptor (CAR) T Cell Therapy in Acute Myeloid Leukemia (AML).

Despite high response rates after initial chemotherapy in patients with acute myeloid leukemia (AML), relapses occur frequently, resulting in a five-year-survival by <30% of the patients. Hitherto, allogeneic hemotopoietic stem cell transplantation (allo-HSCT) is the best curative treatment option in intermediate and high risk AML. It is the proof-of-concept for T cell-based immunotherapies in AML based on the graft-versus-leukemia (GvL)-effect, but it also bears the risk of graft-versus-host disease. CD19-targeting therapies employing chimeric antigen receptor (CAR) T cells are a breakthrough in cancer therapy. A similar approach for myeloid malignancies is highly desirable. This article gives an overview on the state-of-the art of preclinical and clinical studies on suitable target antigens for CAR T cell therapy in AML patients.

Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders.

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.

Prognostic value of [18F]FDG-PET/CT in multiple myeloma patients before and after allogeneic hematopoietic cell transplantation.

PURPOSE: 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). METHODS: 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. RESULTS: 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 < 0.001, post-HCT p < 0.005). High FDG-uptake (SUVmax > 6.5) revealed a significantly shortened survival compared to patients with a lower SUVmax (<6.5) (OS, 5.0 ± 1.1 m vs. not reached - longest 122.0 m; p < 0.001). Moreover, our data prove that a higher number (>3) of focal lesions (pre-HCT: both PFS and OS: p < 0.001; post-HCT PFS: p < 0.001, OS: p = 0.139) as well as the presence of extramedullary disease serve as adverse prognostic factors prior to and after allo-HCT. At response assessment after allo-HCT, [18F]FDG-PET/CT had a complementary value in prognostication in addition to IMWG criteria alone. CONCLUSION: [18F]FDG-PET/CT before and shortly after allogeneic HCT is a powerful predictor for progression-free and overall survival in MM patients.

Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

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.

Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients.

INTRODUCTION: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (TN) vs. effector (TE) T cells, TN cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TN/TE ratio of CART cells. MATERIALS AND METHODS: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays. RESULTS: IL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (TSCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (TEM), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTN cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTN cells in untreated CLL patients. Final TN/TE ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion. CONCLUSION: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product.