Charakterisierung der Regulation und Funktion von PPARbeta

Im Zusammenhang mit Tumorerkrankungen kristallisierte sich in den letzten Jahren eine Gruppe von Kernrezeptoren heraus, die zunehmend mit Prozessen wie dem Zellzyklus, der Apoptose oder der Angiogenese in Verbindung gebracht wurden - die Familie der PPARs. Zusammengesetzt ist sie aus den drei Liganden-induzierbaren Transkriptionsfaktoren PPARalpha, PPARbeta und PPARgamma. Ihre transkriptionelle Aktivität wird durch Fettsäuren und ihre Derivate, sowie verschiedene onkogene Signalwege, wie die Ras-Raf-ERK-Kaskade, reguliert. Der Arachidonsäure-Metabolit Prostazyklin wurde in der Literatur häufig als Agonist von PPARbeta beschrieben. In der vorliegenden Arbeit konnte jedoch nachgewiesen werden, dass Prostazyklin zumindest in den verschiedenen eingesetzten Testsystemen keine signifikante Bedeutung bei der Regulation von PPARbeta besitzt. So führte beispielsweise die Aktivierung eines cRaf-Östrogen-Rezeptorfusionsproteins durch 4-OH-Tamoxifen zwar zu einer Induktion von Cox-2 und PPARbeta, sowie zu einem deutlichen Anstieg der Prostazyklinsynthese, die erwartete Zunahme der transkriptionellen Aktivität von PPARbeta blieb hingegen aus. Weiterhin beschäftigte sich die vorliegende Arbeit mit der Identifizierung von Zielgenen von PPARbeta.Anhand von siRNA-Experimenten, sowie Microarray-Studien und Real-Time PCR konnten in diesem Zusammenhang zwei interessante, potentielle Zielgene von PPARbeta charakterisiert werden: CXCR-4 und Angiopoietin-1, die durch Beeinflussung der Angiogenese eine wichtige Funktion von PPARbeta bei der Tumorigenese vermitteln könnten

A Novel Approach for Quantifying the Pharmacological Activity of T-Cell Engagers Utilizing In Vitro Time Course Experiments and Streamlined Data Analysis

CD3-bispecifc antibodies are a new class of immunotherapeutic drugs against cancer. The pharmacological activity of CD3-bispecifcs is typically assessed through in vitro assays of cancer cell lines co-cultured with human peripheral blood mononuclear cells (PBMCs). Assay results depend on experimental conditions such as incubation time and the efector-to-target cell ratio, which can hinder robust quantifcation of pharmacological activity. In order to overcome these limitations, we developed a new, holistic approach for quantifcation of the in vitro dose–response relationship. Our experimental design integrates a time-independent analysis of the dose–response across diferent time points as an alternative to the static, “snap-shot” analysis based on a single time point commonly used in dose–response assays. We show that the potency values derived from staticin vitro experiments depend on the incubation time, which leads to inconsistent results across multiple assays and compounds. We compared the potency values from the time-independent analysis with a model-based approach. We fnd comparably accurate potency estimates from the model-based and time-independent analyses and that the timeindependent analysis provides a robust quantifcation of pharmacological activity. This approach may allow for an improved head-to-head comparison of diferent compounds and test systems and may prove useful for supporting frst-in-human dose selection

Pharmacokinetics and pharmacodynamics of t-cell bispecifics in the tumour interstitial fluid

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.publishedVersio

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration

Charakterisierung der Regulation und Funktion von PPARbeta

Im Zusammenhang mit Tumorerkrankungen kristallisierte sich in den letzten Jahren eine Gruppe von Kernrezeptoren heraus, die zunehmend mit Prozessen wie dem Zellzyklus, der Apoptose oder der Angiogenese in Verbindung gebracht wurden - die Familie der PPARs. Zusammengesetzt ist sie aus den drei Liganden-induzierbaren Transkriptionsfaktoren PPARalpha, PPARbeta und PPARgamma. Ihre transkriptionelle Aktivität wird durch Fettsäuren und ihre Derivate, sowie verschiedene onkogene Signalwege, wie die Ras-Raf-ERK-Kaskade, reguliert. Der Arachidonsäure-Metabolit Prostazyklin wurde in der Literatur häufig als Agonist von PPARbeta beschrieben. In der vorliegenden Arbeit konnte jedoch nachgewiesen werden, dass Prostazyklin zumindest in den verschiedenen eingesetzten Testsystemen keine signifikante Bedeutung bei der Regulation von PPARbeta besitzt. So führte beispielsweise die Aktivierung eines cRaf-Östrogen-Rezeptorfusionsproteins durch 4-OH-Tamoxifen zwar zu einer Induktion von Cox-2 und PPARbeta, sowie zu einem deutlichen Anstieg der Prostazyklinsynthese, die erwartete Zunahme der transkriptionellen Aktivität von PPARbeta blieb hingegen aus. Weiterhin beschäftigte sich die vorliegende Arbeit mit der Identifizierung von Zielgenen von PPARbeta.Anhand von siRNA-Experimenten, sowie Microarray-Studien und Real-Time PCR konnten in diesem Zusammenhang zwei interessante, potentielle Zielgene von PPARbeta charakterisiert werden: CXCR-4 und Angiopoietin-1, die durch Beeinflussung der Angiogenese eine wichtige Funktion von PPARbeta bei der Tumorigenese vermitteln könnten

Pharmacokinetics and pharmacodynamics of t-cell bispecifics in the tumour interstitial fluid

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies

miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

BackgroundIn humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.Principal findingsDNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.ConclusionInvestigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression