ALBERT

Description: The need for safer gene therapy vectors was highlighted by the occurrence of three cases of retroviral vector-induced leukemia in children after the cure of severe combined immunodeficiency by gene therapy. These severe adverse events enhanced the development of new gene therapy vector systems, which aim to reduce influence of insertions on integrity and expression of genomic host DNA. Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) have no LTR enhancer activity and are not expected to produce insertional gene activation. In our study we analyzed the integrations sites of three different lentiviral SIN-HIV-based- vectors. These vectors differed in their internal elements (Promotor, Transgene, WPRE) which allowed us to investigate whether internal elements are influencing target site selection and clone survival. A total of 1422 integration sites were analyzed at three different time points (1 day, 30 days, 60 days) after transduction of identical HeLa cells by LAM- PCR. Our results showed similar gene involvement and chromosomal distribution of integration sites for all vector types analyzed, independent of vector composition. 62–63% of the integrations were detected in Refseq genes, 82–86% were found within Refseq genes and their surrounding 10kb. Surprisingly, 271 of 1422 integration sites were clustered as common integration sites (CIS). Computer simulations allowed us to show that this high number of CIS was significantly different from a modeled random distribution of integration sites. Gene ontology analysis showed no difference in significantly overrepresented gene categories between the distinct vector types. Interestingly, we detected a time dependent increase or decrease in significance. Specific gene categories like phosphorylation, protein kinase or ATP binding activity increased in significance from freshly transduced cells (1 day) to 30 days. This observation was even more pronounced at the latest time point analyzed (60 days). Other gene categories showed the exactly opposite effect. Gene ontology analysis of freshly transduced cells showed a significant overrepresentation of genes involved in cell cycle regulation, whereas the analysis of the later time points did not show overrepresentation of this gene category. These results suggest that lentiviral SIN-HIV-based vectors may induce clonal selection in vitro independently of internal vector elements. The character of such effects as well as any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations.

Description: Successful gene therapy trials of ADA-SCID and SCID-X1 demonstrated the curative potential of oncoretroviral gene transfer. Integration of the retroviral vectors used in these studies has been thought to be a random process but severe side effects in gene therapy and in vitro studies revealed preferred insertion of these vectors mainly around transcription start sites. In SCID patients proliferation advantage of gene corrected cells was one reason for the success of the trials, whereas in the most recent chronic granulomatous disease (CGD) gene therapy trial corrected cells do not have any selective advantage therefore the two patients received mild busulfan treatment before transplantation. High efficiency transduction and conditioning have helped in the successful correction of the patients. Peripheral blood granulocytes show a stable expression (〉10%) of the transgene (gp91phox) in patient 1 (15 months post treatment) as well as in patient 2 (11 months post treatment). We reasoned that, unlike T cells, which have the capability to proliferate independent of their bone marrow progenitors, granulocytes more directly reflect the influence of retrovirus insertion, and should therefore allow to closely monitor clonal fate in vivo and its potential relation to vector insertion. To study the clonality of the corrected myelopoiesis, the long term activity of individual cell clones, and the distribution of integration sites in active cells we carried out high sensitive LAM-PCR. The highly polyclonal composition of transduced cells forming myelopoiesis caused the sustained expression of gp91phox. Individual clones carrying the transgene could be detected at multiple time points. To define whether corrected cells have a proliferation advantage due to their vector integration we started large-scale sequencing and mapping of involved insertion sites. We here present 〉700 unique mappable integration sites of the two treated patients. The distribution of the SFFV based retroviral vector integration sites in this trial turned non random 5 months after transplantation. Corrected long-term myelopoiesis expanded 3- to 5- fold in the two patients due to activating common integration sites (CIS) in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1, suggesting that these genes influence regulation of normal long-term hematopoiesis in humans. Our data indicate that the therapeutic benefit in this trial was activated through insertional side effects, therefore our findings have important implications in novel gene therapy approaches.

Description: GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Description: Down-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4-CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide–HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2–restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A–HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag–specific T cells after transplantation.

Description: Background The prophylactic use of systemic antifungal agents reduces morbidity and mortality after allogeneic hemopoietic stem cell transplantation (HSCT). However, the efficacy of this approach in pts receiving intensive chemotherapy or autologous HSCT is yet unproven. This trial was designed to evaluate the efficacy of L-AmB prophylaxis in high-risk neutropenic pts. Methods: 231 pts with hematological malignancies and expected neutropenia (N) of more than 10 days (d) following intensive chemotherapy or autologous HSCT were enrolled, 219 pts became neutropenic and were randomized to receive either 50 mg L-AmB i.v. every second d (arm A) or no systemic antifungal prophylaxis (arm B). Treatment with L-AmB started 1–3 d prior onset of N and was continued until neutrophil recovery, breakthrough IFI, intolerable toxicity or death. The level of significance was 0.05 (two-sided) for all statistical analyses. Calculations were performed using commercially (SPSSWIN 12.0) software, the calculations for GEE were performed using the software MAREG. Results Pt. characteristics: Eligible pts 219; arm A: 110; arm B: 109. Reasons for exclusion were: Absence of N (8), infection prior N (3) and pts decision (1). Baseline characteristics were balanced for age (mean 53.8 years), underlying disease (119 AML, 27 ALL, 64 NHL, 9 other), duration of N (mean 14.8 D) and treatment modality (primary 149, secondary 42, transplant 28). Primary endpoint: The incidence of proven and probable IFI was 5 of 110 pts (4.6%) in arm A and 22 of 109 pts (20.2%) in arm B (p = 0.001, RR = 2.9, CI 1.3 – 6.5). Key secondary endpoints: Pneumonia of unknown origin occurred in 6 pts (5.5%) vs. 28 pts (25.7%) (p 〈 0.001), the incidence of possible, probable and proven IFI was 11 pts (10.2%) vs. 42 pts (39.6%) (p 〈 0.001), systemic antifungals were used in 24 pts (22%) vs. 64 pts (59%) (p 〈 0.001), and death occurred in 4 pts (3.7%) vs. 9 pts (8.2%) (p = 0.16) in arm A vs. arm B. Toxicity: No grade 3 or 4 toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not different between the treatment groups. Conclusion: Intermittent application of low dose L-AmB is save. The significant lower incidence of IFI in pts treated with L-AmB prophylaxis supports its use in prolonged N.

Description: Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. Cdkn2a−/ − mice are defective for p53 and Rb pathways, and are susceptible to a broad range of cancer-triggering genetic lesions. We developed an in-vivo genotoxicity assay, based on transplantation of Cdkn2a−/ − hematopoietic stem cells (HSCs), treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. In our rationale if RV or LV treatment is genotoxic, transplanted mice will show a significantly earlier tumor onset. Using this approach, we detected a dose-dependent acceleration in tumor onset in the mice transplanted with RV treated cells. We compared the RV and LV integration site distribution in pre-transplant cells and tumors from the transplanted mice. As expected, RV integrates close to gene promoters in pre-transplant cells and tumors. Moreover, we found that RV preferentially targeted genes encoding for transcription factors, kinases and genomic positions previously described as Common Integration Sites (CIS). CIS are genomic regions targeted at high frequency in tumors by retroviral integrations and probably map within or close to proto-oncogenes activated upon integration. Interestingly, in tumors, RV insertions at CIS and cell cycle genes were further enriched and associated to early lymphomagenesis. Remarkably, LV tested in the same conditions, did not show any tumor acceleration, and targeted CIS much less frequently than RV in pre-transplant cells or tumors, and did not show selection for integrations at any specific gene class. This is the first evidence that prototypic LV have low oncogenic potential, and provides a major rationale for their application to HSC gene therapy. To dissect the role in lymphomagenesis of the strong enhancers in the RV LTRs and the different integration site selection of each vector, we tested an RV with enhancer deleted LTRs (SIN RV) and a moderate promoter in internal position, an LV with a strong RV-like enhancer-promoter into the LTRs or in internal position. Our results show that: SIN RV shows reduced effect on lymphomagenesis acceleration with respect the conventional RV; LV with RV like LTRs treatment significantly accelerates lymphomagenesis, underlining important role of RV-LTRs in oncogenesis; LV with RV-like enhancer in internal position show a reduced genotoxicity. These data show that the type of genetic elements used (strong enhancers or moderate promoter) and their position in the vector genome (LTR or internal positions) significantly influence the vector safety profile. We are currently mapping the integration sites in pre-transplant cells and tumors marked by each vector to identify the genes targeted by the integrations and elucidate the potential mechanism of deregulation. The described model provide an important tool to compare the risk of insertional mutagenesis of different integrating vectors and provides a platform to test different vector types, designs and safety improvements.

Description: As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that inhibit signaling pathways driving cyclin D2 promoter transactivation, assaying more than 4,000 compounds. The top-ranked compound from these studies was a natural triterpenoid, pristimerin. Pristimerin markedly suppressed cyclin D2 promoter activity (〉90%) in 3T3 fibroblast cells and inhibited cyclin D1, D2 and D3 protein expression in myeloma tumor cells. Strikingly, the early (4 hour) transcriptional response of myeloma cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors (P90-fold), activating transcription factor (ATF) 3 and CHOP. Enzymatic assays performed with purified 20S proteosome, or with total cellular extract, confirm that pristimerin rapidly and specifically inhibits chymotrypsin-like 20S proteosome activity at low concentration (6 hours. Consistent with inhibition of proteosome function, pristimerin causes rapid and sustained accumulation of high molecular weight poly-ubiquitinated protein in myeloma cell lines. Notably, related cytotoxic triterpenoid drugs, such as the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me, RTA 402) or betulinic acid or the ginsenosides - all of which show promising anti-cancer activities and are currently in clinical trials for advanced lymphoma, leukemia or solid malignancies - commonly inhibit NF-kB activation via direct inhibition of IKKα or IKKβ. In contrast drugs that function as proteosome inhibitors also commonly suppress NF-kB function instead by impairing degradation of ubiquitinated IkB. Immunoblotting for phosphorylated IkB confirms that pristimerin, like other triterpenoids, acts upstream of IkB to inhibit its phosphorylation, although pristimerin simultaneously inhibits proteosome activity with marked potency to diminish the clearance of ubiquitinated IkB. As a result of this two-fold stabilization of IkB, pristimerin causes overt and specific suppression of NF-kB mediated transcription, measured by a panel of transcriptional reporters with synthetic promoters containing 5x repeats of generic binding sites for NF-kB, AP-1, CREB or TCF4. Importantly, specific suppression of constitutive NF-kB transcriptional activity was pronounced in myeloma cells with inherent NF-kB pathway activation resulting from bi-allelic deletion of the TRAF3 tumor suppressor. Constitutive activation of the NF-kB pathway occurs in a significant proportion of primary myeloma tumors, most commonly via inactivation of TRAF3. Selective silencing of NF-kB driven transcription in myeloma cells may mediate the potent suppression of cyclin D proteins induced by this compound. Significantly, multiple myeloma cells are exquisitely sensitive to both proteosome inhibition or NFkB pathway inhibition. Consistent with these twin vulnerabilities, pristimerin is potently and selectively lethal to primary myeloma cells from patients (IC50

Description: Developmental intermediates of human natural killer (NK) cells are found within secondary lymphoid tissue (SLT), and five distinct stages of these intermediates have been identified. While it is well documented that developing NK cells are reliant on interleukin (IL)-15 as a survival factor, it is likely that additional cytokines and growth factors are required for complete NK cell differentiation. Microarray transcriptional profiling of purified stage 1–4 cells from human tonsil and stage 4 and 5 cells from peripheral blood (PB) identified a developmental window of interleukin-1 receptor 1 (IL-1R1) messenger RNA (mRNA) expression restricted to stages 2 and 3. We confirmed this finding by quantitative RT-PCR, and analysis of IL-1R1 surface protein expression revealed that, on average, 81% of stage 3 immature NK cells are IL-1R1(+), whereas the majority of cells from stages 1, 2, and 4 are IL-1R1(−). When cultured in vitro with IL-1β, a physiologic ligand for IL-1R1, cells from all four stages died within 48 hours, consistent with an absolute requirement for IL-15 as a survival factor. However, the combination of IL-1β and IL-15 led to a significant and reproducible 4.64±−0.68–fold increase in stage 3 cell number over that seen with IL-15 alone (p 〈 0.0005). This phenomenon was completely restricted to stage 3 immature NK cells, and is attributed to increased proliferation. The effects of IL-1β were abrogated by a molar excess of IL-1 receptor antagonist (IL-1RA), a physiologic competitor for IL-1R1 binding. Collectively, our data indicate that IL-1R1 expression fluctuates dramatically during NK cell development, and that unique responses of IL-1R1(+) stage 3 cells to IL-1β and IL-15 govern the expansion of these immature NK cells. Our findings support a model in which IL-1β promotes stage 3 proliferation and survival in vivo, driving stage 3 cells to be the most prevalent NK cell intermediates within SLT.

Description: Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)–mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3−/− mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

Description: The HOVON-65/GMMG-HD4 trial is a prospective, randomized phase III trial to evaluate the efficacy of bortezomib prior to high-dose melphalan (200 mg/m2, HDM) on response and progression-free survival (PFS) in patients with newly diagnosed MM stage II or III according to Salmon & Durie (SD). Until May 2008 in total 833 patients aged 18–65 years were included in the trial. Patients were randomized to receive three cycles of VAD (arm A; vincristin 0,4mg, days 1–4, adriamycin 9 mg/m2, days 1–4, dexamethasone 40 mg, days 1–4, 9–12, and 17–20) or PAD (arm B; bortezomib 1.3 mg/m2, days 1,4,8,11, adriamycin 9 mg/m2, days 1–4, dexamethasone 40 mg, days 1–4, 9–12, and 17–20). Hematopoietic stem cells were mobilized in patients using the CAD regimen (cyclophosphamide 1000 mg/m2 iv day 1, adriamycin 15mg/m2, days 1–4, dexamethasone 40mg, days 1–4) and G-CSF. After stem cell harvesting all patients received one or two cycles of HDM with autologous stem cell transplantation followed by maintenance therapy with thalidomide 50 mg daily (arm A) and bortezomib 1.3 mg/m2 once every 2 weeks (arm B), respectively. As of August 2008 stem cell harvesting data from the first consecutively enrolled 150 patients (75 per arm) were analyzed. The data of the initial 300 patients (150 per arm) will be available by November 2008. Both treatment arms did not differ in age, SD stage of disease, ISS stage, and FISH abnormalities. 132 patients (88%) were treated with CAD plus G-CSF. Dosing and type of G-CSF treatment were comparable in both arms. In all patients stem cell collection was successful and at least two autografts could be harvested (minimal number of stem cells permitted per autograft was 2.0 ×106 CD 34+ cells per kg BW). Induction treatment Days until first leukapheresis Median (range) Number of leukaphereses Median (range) Number of harvested CD34+ stem cells (× 106) per kg BW Median (range) PAD 110 (96–149) 1 (1–4) 10.5 (4.1–37.6) VAD 111 (95–156) 1 (1–5) 9.3 (4.0–37.0) In 64% of the patients in arm A (VAD) and 79% of the patients in arm B (PAD) only one leukapheresis was sufficient for stem cell harvesting. In all patients hematopoietic reconstitution was achieved after HDM followed by autografts harvested after PAD or VAD. We conclude that stem cell harvesting after PAD is very well feasible.