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

Ibrutinib-based therapy reinvigorates CD8 T cells compared to chemoimmunotherapy: immune-monitoring from the E1912 trial

Bruton's tyrosine kinase Inhibitors (BTKis) that target B cell receptor signaling have led to a paradigm shift in CLL treatment. BTKis have been shown to reduce abnormally high CLL-associated T cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pre- and on-treatment (6- and 12-months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab to fludarabine, cyclophosphamide and rituximab (FCR). Intriguingly, we report that despite reduced overall T cell counts, higher numbers of T cells including effector CD8+ subsets at baseline and at the 6-month time-point associated with no infections and favorable progression-free survival (PFS) in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T cell killing function during ibrutinib-rituximab, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T cell cytotoxicity during ibrutinib-rituximab using the bispecific antibody glofitamab - supporting combination immunotherapy approaches

Heat shock proteins as indicators for wound healing and coordination of maturation and antigen presentation of murine dendritic cells

Teil 1: Hitzeschockproteine als Indikatoren der Wundheilung Im ersten Teil der Arbeit wurden Zusammenhänge zwischen Anwesenheit und Konzentrationen der Hitzeschockproteine (Hsps) Gp96 und Hsp70 in humanen Wundflüssigkeiten (WF) und Wundheilungsverläufen untersucht. Während Hsp70 durch Verwendung eines ELISA-Kits in WF und Seren nachgewiesen werden konnte, musste für Gp96 ein geeignetes immunologisches Detektionssystem entwickelt werden. Hierfür wurde eine Gp96-Immunpräzipitation mit anschließendem Western Blot zur Detektion und Semiquantifizierung des Hsps etabliert. Um mögliche Korrelationen zwischen Konzentrationen der Hsps in WF und der Wundheilung aufzudecken, wurden WF und Seren von Patienten verschiedener Kategorien über einen Zeitraum von mehreren Tagen beginnend nach der OP gesammelt und analysiert. Verglichen wurden die Hsp-Verläufe von elektiven Eingriffen (Endo), sowie von traumatischen und chronisch/septischen Wunden. Während in allen Patientenseren nur geringe Mengen Gp96 und Hsp70 ohne erkennbaren Verlauf detektiert wurden, fanden sich interessante Konzentrationsverläufe beider Hsps in den WF. In den WF von Traumapatienten nahm sowohl die Konzentration von Hsp70 wie auch Gp96 im Laufe der Wundheilung ab, gleiches galt für Gp96 in WF nach elektiven Eingriffen. Hier gab es deutliche Unterschiede zum Verhalten von Hsp70, dass eine starke Zunahme erfuhr. Im Gegensatz dazu konnten keine Änderungen der Hsp-Konzentrationen in chronisch/septischen Wunden beobachtet werden. Hier blieben sowohl Gp96 als auch Hsp70 bis zur Abheilung nahezu konstant. Diese Beobachtungen wiesen darauf hin, dass Anwesenheit und Konzentrationsverläufe von Hsps Rückschlüsse auf die Wundheilung zu lassen. Das z. T. unterschiedliche Verhalten der beiden verschiedenen Hsps könnte auf ihre unterschiedliche Herkunft zurückzuführen sein. Während es sich bei Gp96 um ein ER lokalisiertes Glykoprotein handelt, dass hauptsächlich durch Nekrose in den extrazellulären Raum gelangt, gehört Hsp70 zu den Stress-induzierten Hsps. Teil 2: Koordination von Reifung und Antigenpräsentation bei Dendritischen Zellen der Maus Der zweite Teil der Arbeit beschäftigte sich mit den Vorgängen während der Reifung von BMDCs in Zusammenhang mit der Antigenpräsentation. Im Gegensatz zu nicht-professionellen antigenpräsentierenden Zellen (APCs), bei denen eine frühe Herstellung und Präsentation von CTL-Epitopen wichtig für eine schnelle Viruselimination ist, benötigen DCs Zeit, einen reifen Phänotyp zu entwickeln, um ihre Aufgabe als T-Zell-Aktivatoren erfüllen zu können. Tatsächlich wiesen die Ergebnisse dieser Arbeit auf eine verzögerte Antigenpräsentation von BMDCs nach Influenz-Infektion im Vergleich zu nicht-professionellen APCs hin. Diese koinzidierte mit dem Auftreten von so genannten DALIS (dendritic cell aggresome-like induced structures). Hierbei handelt es sich um transiente Anhäufungen ubiquitinylierter Proteine (DRiPs) im Zytoplasma, die während der Reifung von DCs und Makrophagen auftreten. Durch ihre Zwischenspeicherung in DALIS wird der Abbau der DRiPs verzögert, daher wurde in dieser Arbeit postuliert, dass es sich bei DALIS um Antigenspeicher handeln könnte, die es den DCs ermöglichen, die Vorgänge der Reifung und Antigenpräsentation zu koordinieren. Hierfür sprach auch die Entdeckung des Influenza Nukleoproteins in DALIS infizierter BMDCs und das zeitliche Zusammenfallen von DALIS-Entwicklung und Expression von kostimulatorischen Molekülen auf der Oberfläche der DCs sowie anderen Reifungsmarkern. Ferner konnte durch Inhibition der DALIS-Entstehung die Antigenerkennung auf BMDCs beschleunigt werden. Im Vergleich zu Influenza-infizierten BMDCs wiesen mCMV-infizierte Zellen keine DALIS Bildung auf. Der Vergleich von Influenza und mCMV Infektion war von besonderem Interesse, da Zytomegaloviren (mCMV) die Reifung von DCs und somit ihre Fähigkeit, T Zellen zu aktivieren, beeinträchtigten. Hier zeigten BMDCs dieselbe Präsentationskinetik wie infizierte Fibroblasten, die ebenfalls nicht zur DALIS-Bildung fähig waren. Neben der Aufgabe der DALIS sind auch die Mechanismen, die zu ihrer Entstehung führen, bislang ungeklärt. In dieser Arbeit konnte durch den Einsatz von Inhibitoren gezeigt werden, dass die Aktivierung der MAPK-Wege ERK1/2 und p38 ebenso eine wichtige Rolle spielen wie der PI3K/Akt-Weg. Desweiteren scheint die Aktivierung des Translationsfaktors eIF4E für die DALIS-Bildung und IL-6 Produktion von BMDCs von Bedeutung zu sein.Part 1: Heat shock proteins as indicators for wound healing The first part of this thesis is about the coherence of concentrations of the heat shock proteins (hsps) gp96 and hsp70 in human wound fluids (WFs) and the process of wound healing. Whereas detection of hsp70 in WFs and serum was performed with the help of an special ELISA-kit, an adequate and specific immunological detection method for gp96 had to be developed. This method includes gp96 immunoprecipitation and western blotting for detection and semiquantification of the hsp. To detect possible correlations of hsp concentrations in WFs and wound healing, WFs and sera of different patients were collected over a few days beginning one day after surgery and analysed. Hsp concentrations in WFs of trauma, chronic/septic and elective surgery wounds were compared. In contrast to WFs, patients sera contained only small amounts of hsps. In the WFs of trauma patients were high levels of both hsps at the beginning which decreased during wound healing. The same could be observed for gp96 in WFs after elective surgery in contrast to hsp70, which increased over the time. WFs of chronic and septic wounds showed constant hsp levels. These results suggested the possibility to draw conclusions about the process of wound healing by monitoring the hsp-levels of WFs. The partly different behaviour of gp96 and hsp70 can be explained by their different backgrounds. Gp96 is an ER-localised glycoprotein, which can be found in the extracellular space mainly after necrosis, whereas hsp70 belongs to the famliy of stress-induced proteins. Part 2: Coordination of maturation and antigen presentation of murine dendritic cells The second part of the dissertation dealt with the procedures during BMDC maturation in context with antigen presentation. In contrast to nonprofessional antigen presenting cells (nonprofessional APCs), in which early generation and presentation of CTL epitopes is required to allow fast elimination of infected cells by activated cytotoxic T lymphocytes (CTLs), the situation for dendritic cells (DCs) is different. Although viral infection and expression of virus-derived proteins are very fast, DCs need time to acquire an activated phenotype that allows optimal activation of naïve T cells. Actually, the results of this thesis revealed that the presentation of the immunodominant influenza nucleoprotein (NP)-derived CTL epitope is delayed in BMDCs compared to nonprofessional APCs. This delay coincided with the so called DALIS formation (dendritic cell aggresome-like induced structures). DALIS are transient aggregations of ubiquitinated proteins (DRiPs = defective ribosomal proteins) which appear in the cytosol of maturing DCs and macrophages. Normally, DRiPs are degraded rapidly by proteasomes. However, their storage in DALIS delay their degradation and stabilize them. So, it is hypothesized that DALIS can function as antigen depots, allowing DCs to coordinate regulation of DC maturation and antigen presentation. This assumption is supported by the finding of influenza NP in DALIS of infected cells. Besides, DALIS appearance and upregulation of MHC class I molecules as well as costimulatory molecules coincided during DC maturation. Furthermore, inhibition of DALIS formation led to an earlier recognition of influenza infected BMDCs by CTLs. In comparison to influenza-infected cells, cytomegalovirus (mCMV)-infected BMDCs showed no DALIS formation. In contrast to influenza, mCMV affects maturation and antigen presentation of BMDCs and therefore their ability to elicit a T cell response. In this case, mCMV-infected BMDCs showed the same antigen presentation kinetics as infected fibroblasts, which were also unable of DALIS formation. Apart from the exact functions of DALIS the mechansim leading to DALIS formation are also still unknown. Using different inhibitors of the toll like receptor (TLR) signaling pathways, this study show an important role of the MAPK pathways ERK1/2 and p38 as well as the PI3K/Akt pathway in DALIS formation. Furthermore, the activation of the translation initiation factor eIF4E seemed significant for IL-6 production and DALIS formation in BMDCs

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism