Targeting PD-L1/ PD-1-mediated inhibitory signaling with BTK inhibitors in Chronic Lymphocytic Leukemia (CLL).

PhD ThesisChronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults in the West. An unmet need for equally curative and tolerable treatment approaches exists. Our group has previously shown that the PD-1/PD-L1 immune checkpoint pathway is pivotal in mediating CLL-associated T-cell dysfunction. Bruton’s tyrosine kinase inhibitors such as Ibrutinib have been shown to be able to modulate the function of T-cells and myeloid cells. Using the Eμ-TCL1 mouse model of CLL, we have aimed to analyse the effect of BTK inhibitors on expression of immune checkpoint molecules, immune phenotype and T-cell function as well as develop a combination approach of BTK inhibitors and anti-PD-L1 immune checkpoint blockade. We detected a modest increase in PD-L1 expression among CLL B-cells and a decrease among myelomonocytic cells with both Ibrutinib and Acalabrutinib treatment. We have demonstrated an amelioration of the exhaustion phenotype of CD4+ and CD8+ T-cells with BTK-inhibitor treatment with downregulation of CD69, PD-1, LAG-3 and KLRG-1. We also found downregulation of inhibitory receptor 2B4, LAG-3 and KLRG-1 on NK cells. On myeloid cells we observed downregulation of PD-1 and 2B4 as well as a differential effect on expression of TIM-3 with upregulation among myelomonocytic cells and downregulation among classical dendritic cells. Immunophenotypes of BTK inhibitor and BTK inhibitor/anti-PD-L1 combination treated animals were similar with a slightly higher expression level of PD-1 among combination treated animals. Both substances improved helper cell cytokine profiles, degranulation capacity of cytotoxic T-cells and T-cell synapse formation to a similar extent. The combination of BTK inhibitor treatment and PD-L1 blockade failed to achieve improved correction of CLL-associated T-cell exhaustion phenotype and Ibrutinib/anti-PD-L1 combination treatment achieved only a very modest improvement of T-cell function over single agent treatments. Suprisingly, the combination of Acalabrutinib and anti-PD-L1 immune checkpoint blockade was detrimental regarding both helper cell and cytotoxic T-cell function. These findings would caution against the use of Acalabrutinib/anti-PD1 or anti-PD-L1 combinations in the clinical setting

Minimal renal toxicity after Rituximab DHAP with a modified cisplatin application scheme in patients with relapsed or refractory diffuse large B-cell lymphoma

Background: Rituximab (R) in combination with DHAP is a widely accepted salvage regimen for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). A common adverse effect of this protocol is renal toxicity which may result in treatment discontinuation. Assuming that a lower single dose of cisplatin over several days would reduce renal toxicity, our institution has chosen to administer cisplatin in a dosage of 25 mg/m2 per day as a 3-h infusion over 4 consecutive days. Methods: In this study, we analyzed the renal function of 122 patients with relapsed or refractory DLBCL treated with R-DHAP at our institution. Overall, 256 R-DHAP cycles were administered. 31 (25 %), 61 (50 %), 14 (12 %) and 16 (13 %) patients received one, two, three or four R-DHAP courses, respectively. Results: A glomerular filtration rate (GFR) decrease was observed after each R-DHAP cycle. However, in none of the subgroups the median GFR was lower than 60 ml/min/1.73 m2. In most patients, only renal impairment stage I and II was observed. Renal impairment stage III was seen in 10 % and stage IV only in 1 % of patients. Conclusion: We conclude that a modified R-DHAP regimen with administration of cisplatin 25 mg/m2 over 4 consecutive cycles leads only to minimal renal toxicity

Targeted Overexpression of Osteoactivin in Cells of Osteoclastic Lineage Promotes Osteoclastic Resorption and Bone Loss in Mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption

Analysis of the expressional pattern of the gene Osteoactivin (Dc-Hil) differentially expressed in dendritic cells

Dendritische Zellen sind die effektivsten Antigen präsentierenden Zellen des Organismus und nehmen in dieser Funktion eine zentrale Rolle bei der Induktion von spezifischen Immunantworten ein. Diese besondere Stellung im Immunsystem macht sie wichtig für das Verständnis von Reaktionen des adaptiven Immunsystems aber auch interessant für die Entwicklung therapeutischer Ansätze, insbesondere in der Onkologie. In der vorliegenden Arbeit wurde die differentielle Expression und das Expressionsmuster des Oberflächenproteins Osteoactivin (GPNMB) in menschlichen dendritischen Zellen untersucht. Es konnte gezeigt werden, dass Osteoactivin differentiell in dendritischen Zellen des Menschen exprimiert wird. Auch wurde festgestellt, dass Behandlung mit TLR-Liganden zu einer verminderten Expression, Behandlung mit STI571 oder IL10 jedoch zu einer gesteigerten Expression führt. Diese Ergebnisse stützen die These von Osteoactivin (DC-HIL) als coinihibitorisch wirkendem Protein in dendritischen Zellen.Dendritic cells are the most powerful antigen presenting cells of the human body and play a central role in induction of specific immune responses. This outstanding position in the immune system make them especially important in understanding the adaptive immune system but also in developing novel therapeutic strategies, most prominently in oncology. In this thesis the differential expression and expressional pattern of the surface protein Osteoactivin (GPNMB) in human dendritic cells was studied. It could be demonstrated that Osteoactivin is differentially expressed in human dendritic cells. Furthermore, treatment with TLR-ligands resulted in a decrease in expression while treatment with STI571 or IL10 resulted in an increase in expression. These results support the hypothesis of Osteoactivin (DC-Hil) as a coinhibitory protein in dendritic cells

Supplementary Material for: Cellular Immunotherapy in B-Cell Malignancy

<div>In recent years, cellular immunotherapy in B-cell malignancies has been driven by adoptive transfer of genetically engineered T cells expressing chimeric antigen receptors (CARs). CARs consist of a single chain variable </div><div>fragment (scFv) of a monoclonal antibody, a spacer domain, a transmembrane domain, an intracellular signaling domain, and additional costimulatory domains. The bulk of clinical data available is on CD19-targeting CAR T cells for the treatment of B-cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia, and B-cell non-Hodgkin lymphoma. Results so far have been promising with impressive rates and depth of remission especially among B-ALL patients. However, CAR T-cell therapy is a complex multi-step process, and clinical trials so far differ profoundly in CAR construct used, gene transfer method, composition of the cellular product, lymphodepletion, and CAR T-cell dose used. Randomized trials will be needed to conclusively evaluate the implications of these differences. The treatment concept is associated with significant neurotoxicity and potentially lethal cytokine release syndrome, both of which require specific management. Improvements in CAR design may help to overcome toxicity, the effects of an immunosuppressive microenvironment, and tumor escape by development of antigen-negative clones. This review will explain the mechanism of action, summarize the clinical experience with this treatment modality so far, and explore future developments in the field.</div

Cationic, Methylene-Bridged, Intramolecular Donor-Acceptor Systems Based on Zirconium and Hafnium and Phosphino-methanides

Pieper M, Auer P, Schwarzbich S, et al. Cationic, Methylene-Bridged, Intramolecular Donor-Acceptor Systems Based on Zirconium and Hafnium and Phosphino-methanides. ZEITSCHRIFT FUER ANORGANISCHE UND ALLGEMEINE CHEMIE. 2017;643(14):909-915.A new access to cationic zirconium and hafnium compounds [L2MCH2PR2][MeB(C6F5)(3)] (L = Cp, Ind; R = iso-Pr, tert-Bu; M = Zr, Hf) exhibiting an intramolecular donor-acceptor system was established by treating the precursors L2M(Me)CH2PR2 with B(C6F5)(3) (BCF). Precursors 1-6 [L2M(Me)CH2PR2 with L = Cp, Ind; R = iso-Pr, tert-Bu; M = Zr, Hf] were fully characterized. The crystal structures of these compounds revealed large M-CH2-P bond angles with values of about 134 degrees indicating the absence of interactions between the Lewis-acid and Lewis-base. The cationic compounds [L2MCH2PR2][MeB(C6F5)(3)] (7-12) were obtained by treatment of 1-6 with BCF. They were characterized by NMR spectroscopy, mass spectrometry, and elemental analyses; in H/D-scrambling experiments with H-2/D-2 mixtures 7-12 disclosed their reactivity towards cleavage of hydrogen

Dinuclear complex-induced DNA melting

Biere N, Kreft D, Walhorn V, Schwarzbich S, Glaser T, Anselmetti D. Dinuclear complex-induced DNA melting. Journal of Nanobiotechnology. 2023;21(1): 26.Dinuclear copper complexes have been designed for molecular recognition in order to selectively bind to two neighboring phosphate moieties in the backbone of double strand DNA. Associated biophysical, biochemical and cytotoxic effects on DNA were investigated in previous works, where atomic force microscopy (AFM) in ambient conditions turned out to be a particular valuable asset, since the complexes influence the macromechanical properties and configurations of the strands. To investigate and scrutinize these effects in more depth from a structural point of view, cutting-edge preparation methods and scanning force microscopy under ultra-high vacuum (UHV) conditions were employed to yield submolecular resolution images. DNA strand mechanics and interactions could be resolved on the single base pair level, including the amplified formation of melting bubbles. Even the interaction of singular complex molecules could be observed. To better assess the results, the appearance of treated DNA is also compared to the behavior of untreated DNA in UHV on different substrates. Finally, we present data from a statistical simulation reasoning about the nanomechanics of strand dissociation. This sort of quantitative experimental insights paralleled by statistical simulations impressively shade light on the rationale for strand dissociations of this novel DNA interaction process, that is an important nanomechanistic key and novel approach for the development of new chemotherapeutic agents. © 2023. The Author(s)

Pre-Transplant Serum Leptin Levels and Relapse of Acute Myeloid Leukemia after Allogeneic Transplantation

Weight loss and metabolic activity influence outcome after allogeneic stem cell transplantation (alloSCT). This study evaluates pre-conditioning Leptin, a peptide hormone involved in metabolism and immune homeostasis, as a prognostic factor for survival, relapse and non-relapse mortality (NRM) following alloSCT. Leptin serum levels prior to conditioning were determined in a cohort of patients transplanted for various hematologic malignancies (n = 524) and correlated retrospectively with clinical outcome. Findings related to patients with acute leukemia (AL) from this sample were validated in an independent cohort. Low pre-conditioning serum Leptin was an independent prognostic marker for increased risk of relapse (but not of NRM and overall mortality) following alloSCT for AL of intermediate and advanced stage (beyond first complete remission). Multivariate analysis revealed a hazard ratio (HR) for relapse of 0.75 per log2 increase (0.59–0.96, p = 0.020). This effect was similar in an independent validation cohort. Pre-conditioning serum Leptin was validated as a prognostic marker for early relapse by fitting the multivariate Cox model to the validation data. Pre-conditioning serum Leptin levels may serve as an independent prognostic marker for relapse following alloSCT in intermediate and advanced stage AL patients. Prospective studies are required to prove whether serum Leptin could be used for guiding nutritional intervention in patients with AL undergoing alloSCT