Leopold Sellner

Aminoferrocene-based prodrugs and their effects on human normal and cancer cells as well as bacterial cells.

Aminoferrocene-based prodrugs are activated under cancer-specific conditions (high concentration of reactive oxygen species, ROS) with the formation of glutathione scavengers (p-quinone methide) and ROS-generating iron complexes. Herein, we explored three structural modifications of these prodrugs in an attempt to improve their properties: (a) the attachment of a -COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro but also decreases cell-membrane permeability and biological activity, (b) the alkylation of the N-benzyl residue does not show any significant affect, and (c) the attachment of the second arylboronic acid fragment improves the toxicity (IC50) of the prodrugs toward human promyelocytic leukemia cells (HL-60) from 52 to 12 μM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphocytic leukemia (CLL) cells, with the best compounds exhibiting an IC50 value of 1.5 μM. The most active compounds were found to not affect mononuclear cells and representative bacterial cells.

Improved synthesis of N-Benzylaminoferrocene-based prodrugs and evaluation of their toxicity and antileukemic activity

We report on an improved method of synthesis of N-benzylaminoferrocene-based prodrugs and demonstrate its applicability by preparing nine new aminoferrocenes. Their effect on the viability of selected cancer cells having different p53 status was studied. The obtained data are in agreement with the hypothesis that the toxicity of aminoferrocenes is not dependent upon p53 status. Subsequently the toxicity of a selected prodrug (4) was investigated ex vivo using rat precision cut liver slices and in vivo on hybrid male mice BDF1. In both experiments no toxicity was observed: ex vivo, up to 10 μM; in vivo, up to 6 mg/kg. Finally, prodrug 4 was shown to extend the survival of BDF1 mice carrying L1210 leukemia from 13.7 ± 0.6 days to 17.5 ± 0.7 days when injected daily 6 times at a dose of 26 μg/kg starting from the second day after injection of L1210 cells.

Generation of efficient human blood progenitor–targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells

OBJECTIVEnCurrently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC).nnnMATERIALS AND METHODSnAn advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells.nnnRESULTSnOn a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors.nnnCONCLUSIONnThese novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.

Normal-Tissue Radioprotection by Overexpression of the Copper-Zinc and Manganese Superoxide Dismutase Genes

Background and Purpose:Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation.Material and Methods:HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1–6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined.Results:After transduction, 90.0% ± 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 ± 12.6, CuZnSOD: 95.1 ± 17.1, MnSOD: 108.5 ± 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06–1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07–1.43; p = 0.01).Conclusion:Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.Hintergrund und Ziel:Der Ansatz, Normalgewebszellen gegen bestrahlungsinduzierte Schäden zu schützen, kann möglicherweise die therapeutische Breite strahlentherapeutischer Ansätze erhöhen. Ein potentieller Ansatz wäre die gentherapeutische Überexpression der Kupfer-Zink-(CuZnSOD) oder Mangan-Superoxiddismutase (MnSOD) mit rekombinanten Adeno-assoziierten viralen (rAAV2) Vektoren. Das Ziel dieser Studie war die Bestimmung der bestrahlungsmodulierenden Effekte der SOD-Gene auf humane primäre Lungenfibroblasten (HPLF).Material und Methodik:HPLF wurden mit rAAV2-Vektoren transduziert, die die cDNA für CuZnSOD, MnSOD oder ein Kontrollgen enthielten. Die Zellen wurden mit 1–6 Gy bestrahlt und deren Gentransfereffizienz, Apoptoserate, Proteinexpression/-aktivität und Strahlensensitivität mit einem Koloniebildungsassay bestimmt.Ergebnisse:Nach Transduktion exprimierten 90,0% ± 6,4% der Zellen das entsprechende Transgen. Für beide SOD konnte mittels Immunhistochemie sowie SOD-Aktivitätsassay eine signifikante Überexpression (fünffach; Kontrolle: 21,1 ± 12,6, CuZnSOD: 95,1 ± 17,1, MnSOD: 108,5 ± 36,0 U SOD/mg Gesamtprotein) gezeigt werden. Diese Überexpression führte zu einer signifikanten Radioprotektion der HPLF im Vergleich zu den Kontrollen (Überlebensfraktion-[SF-]Quotient SOD/Kontrolle > 1): CuZnSOD: 1,18-fach (95%-Konfidenzintervall [CI]: 1,06–1,32; p = 0,005), MnSOD: 1,23-fach (95%-CI: 1,07–1,43; p = 0,01).Schlussfolgerung:Im Vergleich zu den Kontrollen führte die Überexpression von CuZnSOD oder MnSOD in HPLF zu einem erhöhten klonogenen Überleben nach Bestrahlung. Da in vorherigen Arbeiten eine mangelnde Radioprotektion nach Überexpression der SOD-Gene in Tumorzellen beobachtet werden konnte, sind rAAV2-Vektoren erfolgversprechend für die Übertragung von radioprotektiven Genen in Normalgewebszellen.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

BackgroundFor many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants.MethodsTo determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls.ResultsTransduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed.ConclusionUsing an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors.

Neutralization of membrane complement regulators improves complement-dependent effector functions of therapeutic anticancer antibodies targeting leukemic cells

Complement-dependent cytotoxicity (CDC) is one of the effector mechanisms mediated by therapeutic anticancer monoclonal antibodies (mAbs). However, the efficacy of antibodies is limited by the resistance of malignant cells to complement attack, primarily due to the over-expression of one or more membrane complement regulatory proteins (mCRPs) CD46, CD55, and CD59. CD20-positive Burkitt lymphoma Raji cells and primary CLL cells are resistant to rituximab (RTX)-induced CDC whereas ofatumumab (OFA) proved to be more efficient in cell killing. Primary CLL cells but not CD52-positive acute lymphoblastic leukemia (ALL) REH cells were sensitive to alemtuzumab (ALM)-induced CDC. Upon combined inhibition on Raji and CLL cells by mCRPs-specific siRNAs or neutralizing antibodies, CDC induced by RTX and by OFA was augmented. Similarly, CDC of REH cells was enhanced after mCRPs were inhibited upon treatment with ALM. All mAbs induced C3 opsonization, which was significantly augmented upon blocking mCRPs. C3 opsonization led to enhanced cell-mediated cytotoxicity of leukemia cells exposed to PBLs or macrophages. Furthermore, opsonized CLL cells were efficiently phagocytized by macrophages. Our results provide conclusive evidence that inhibition of mCRPs expression sensitizes leukemic cells to complement attack thereby enhancing the therapeutic effect of mAbs targeting leukemic cells.

Chimeric Antigen Receptor T Cell Therapy Targeting CD19-Positive Leukemia and Lymphoma in the Context of Stem Cell Transplantation

Novel therapies with chimeric antigen receptor (CAR)-transduced T cells (TCs) sparked new hope for patients with relapsed or refractory CD19-positive leukemia or lymphoma even after stem cell therapies. This review focuses on CARs recognizing the B cell antigen CD19. Both retroviral and lentiviral vectors are used, encoding various anti-CD19 CAR constructs comprising costimulatory molecules such as CD28, CD137/4-1BB, and OX40 either alone (second-generation CARs) or in combination (third-generation CARs). Current, up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 58 ongoing trials is given. Clinical responses were achieved in up to 81% of ALL, 50% of CLL, and 40% of NHL patients. Factors with potential influence on the clinical outcome might be the design of the vector, the preconditioning regimen, and the number and quality of transfused CAR TCs. The applicability of clinical CAR TC therapy might include relapse after allogeneic stem cell transplantation (alloSCT), and ineligibility for or bridging until alloSCT. In summary, CAR therapy represents a highly promising treatment option even in heavily pretreated patients.

Next-generation sequencing of cancer consensus genes in lymphoma

Abstract Sensitive identification of mutations in genes related to the pathogenesis of cancer is a prerequisite for risk-stratified therapies. Next-generation sequencing (NGS) in lymphoma has revealed genetic heterogeneity which makes clinical translation challenging. We established a 454-based targeted resequencing platform for robust high-throughput sequencing from limited material of patients with lymphoma. Hotspot mutations in the most frequently mutated cancer consensus genes were amplified in a two-step multiplex-polymerase chain reation (PCR) which was optimized for homogeneous coverage of all regions of interest. We show that targeted resequencing based on NGS technologies allows highly sensitive detection of mutations and assessment of clone size. The application of this or similar techniques will help the development of genotype-specific treatment approaches in lymphoma.

Modulation of B Cells and Homing Marker on NK Cells Through Extracorporeal Photopheresis in Patients With Steroid-Refractory/Resistant Graft-Vs.-Host Disease Without Hampering Anti-viral/Anti-leukemic Effects

Graft-vs.-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation, significantly affects the post-transplant morbidity and mortality. Systemic steroids remain the gold standard for the initial management of GvHD. However, up to 60% of patients will not sufficiently respond to steroids. Extracorporeal photopheresis (ECP), a cell-based immunotherapy, has shown good clinical results in such steroid-refractory/resistant GvHD patients. Given its immunomodulatory, but not global immunosuppressive and steroid-sparing capacity, ECP constitutes an attractive option. In the case of GvHD, the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells. ECP therapy may restore this balance. However, the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear. In this study, 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD). A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining, LuminexTM-based cytokine, interferon-γ enzyme-linked immunospot, and chromium-51 release assays. Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis. Immunologically, modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L+ NK cells and newly defined CD19hiCD20hi B cells were modulated, but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved. (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33−CD11b+) to an activated subset (CD33+CD11b+). (4) The frequency of Foxp3+CD4+ regulatory T cells (Tregs) and CD24+CD38hi regulatory B cells was considerably increased in aGvHD patients, and Foxp3+CD8+ Tregs in cGvHD patients. (5) Proinflammatory cytokines like IL-1β, IL-6, IL-8, and TNF-α were significantly reduced. In summary, ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects.

Abstract 5557: Systematic mapping of drug sensitivity in hematological malignancies identifies vulnerability of chronic lymphocytic leukemia with mutant p53

Introduction: The impact of mutations and pathway deregulation for drug sensitivity is only partly understood. Here we systematically investigated heterogeneity of drug response and genetic lesions in leukemia and lymphoma using primary tumor cells in order to identify pathway dependencies which can be exploited by rational treatment approaches. Methods: Primary leukemia / lymphoma cells obtained from peripheral blood were characterized with an ex vivo high-throughput drug screening platform in two steps: In an initial screen 2221 different compounds were tested for cytotoxicity on a limited cohort (n=20). 67 promising compounds targeting different pathways were selected for further validation on a larger cohort (n=111; 97 chronic lymphocytic leukemia (CLL), 5 T-prolymphocytic leukemia (T-PLL), 6 non-CLL B-Non-Hodgkin-Lymphoma (B-NHL), mononuclear cells of 3 healthy donors). Heat inactivated human serum was supplemented to mimic micro-environmental conditions. Cell viability was assessed by quantification of ATP (CellTiterGlo®) 48 and 72 hours after drug application. To understand heterogeneous pathway dependencies, drug sensitivity was regressed on genetic profiles. Genetic characterization was performed by FISH, targeted sequencing of recurrent aberrations (BRAF, MYD88, NOTCH1, SF3B1, TP53) as well as whole exome sequencing. Results: We initially focused on the impact of compounds interfering with or dependent on the p53 pathway. Nutlin-3 and fludarabine induced cell death more efficiently in cells with wild-type (n=80) than in CLL with mutated p53 (n=17; Nutlin-3 10µM: 51±18 vs 80±18; fludarabine 10µM: 40±36 vs 67±23 [% viability of untreated control], both p Citation Format: Leopold Sellner, Malgorzata Oles, Mikolaj Slabicki, Carolin Blume, Jennifer Hullein, Tatjana Stolz, Marina Lukas, Martin Sill, Christopher C. Oakes, Sascha Dietrich, Olaf Merkel, Anna Jauch, Manfred Hensel, Davide Rossi, Katja Zirlik, Jan Durig, Ingo Ringshausen, Marco Herling, Martina Seiffert, Peter Dreger, Christof von Kalle, Anthony D. Ho, Hanno Glimm, Wolfgang Huber, Thorsten Zenz. Systematic mapping of drug sensitivity in hematological malignancies identifies vulnerability of chronic lymphocytic leukemia with mutant p53. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5557. doi:10.1158/1538-7445.AM2014-5557