INDIVIDUAL PROJECTS – WORK PACKAGE 2: IMMUNO-ONCOLOGY

INTRODUCTION 

Today it is well accepted that immune cells play an important role in the context of tumour development. In regard to brain tumours, immune cells of the myeloid compartment are of special importance as they can build up to 20 % of the tumour mass. Moreover, it is well known that within the tumour microenvironment, tumour associated macrophages become reprogrammed towards a cancer cell supportive rather than anti-cancer phenotype. However, the intrinsic and extrinsic factors conferring such reprogramming and especially the role of metabolism need to be specified. Especially, our understanding of the role of mitochondrial folate metabolism in myeloid cells is still rudimentary. Yet, this pathway is a topic of intense research in the field of cancer metabolism and it is targeted in the clinic by the use of antifolates. Moreover, small molecules are being developed to target specific enzymes of mitochondrial folate metabolism. 

AIM AND HYPOTHESIS 

As immune cells residing in the tumour microenvironment can play an important role in the clinical outcome of intervention strategies, it is important to study folate metabolism not only in cancer cells but also in immune cells of the tumour microenvironment.  

Therefore, we are currently developing different in vitro and in vivo tools to study the role of folate mediated 1C metabolism in macrophages/microglia. Using these models, we will be able to study (i) tumour development in vivo using mouse derived cancer cell lines and (ii) study macrophage metabolism ex vivo using bone-marrow derived macrophages (BMDMs). 

METHODS 

Analysis of metabolism using GC-MS and LC-MS, BMDMs, ex vivo brain slice cultures, orthotopic brain tumour models, mouse models for melanoma, in vitro invasion and migration assays, co-culture system (macrophages and cancer cells) 

INTRODUCTION 

Immune checkpoint blockade is currently one of the most attractive therapeutic approaches to treat cancer. Inhibitors of the PD-1/PD-L1 pathway are increasingly used in the clinic and have become part of the standard therapy for multiple malignancies. The most commonly used (FDA-approved) biomarker to predict success of immune check point therapy efficacy is PD-L1 protein in the tumor microenvironment. However, there is growing evidence that patients with low/no PD-L1 expression before treatment might also benefit from immune check point therapy. 

AIM AND HYPOTHESIS 

In positive clinical samples, PD-L1 protein frequently shows strongest expression in tumour areas with prominent infiltration of lymphocytes, suggesting that short distance interaction with lymphocytes induces PD-L1 upregulation in tumour cells (hypothesis 1). Very interestingly, at higher magnification, IHC staining reveals asymmetrical distribution of PD-L1 in individual tumour cells with PD-L1 polarisation toward the inflammatory stroma. Such an observation is consistent with our recent in vitro investigations showing that, in resistant tumour cells engaged in an immunological synapse with cytotoxic lymphocytes, PD-L1 molecules cluster in the region of the immunological synapse. We hypothesise that PD-L1 recruitment to the synapse, which is driven by actin cytoskeleton remodeling in tumour cells, provides a potent inhibitory signal to cytotoxic lymphocytes. The present project aims at investigating the significance of PD-L1 polarisation at both tissue and cell levels. More specifically, the project will investigate the following questions. 1) Is PDL1 expression in tumour cells induced by lymphocyte interaction, and if so by which mechanism? 2) Is PD-L1 clustering in the immunological synapse involved in tumour immune evasion? 

METHODS 

The project is largely based on tissue and cell imaging methods, including light microscopy (mostly for IHC analyses of tumor tissue), fluorescence (confocal) microscopy, correlative light and electron microscopy and imaging flow cytometry. In vitro models that recapitulate PD-L1 upregulation at the tumour/lymphocyte border will be established (e.g. using 3D tumour spheroids co-cultured with cytotoxic lymphocytes) and used in functional studies. The signal driving PD-L1 upregulation (soluble vs. direct mechanical trigger) and the molecular link(s) between PD-L1 and the actin cytoskeleton (ongoing BioID analysis) will be characterised. Finally, the findings will be validated in both primary and metastatic patient samples and correlated with clinico-epidemiologic data. 

INTRODUCTION 

Chronic Lymphocytic leukemia (CLL) represents the most frequent leukemia in adults. Despite new targeted therapies, CLL remains an incurable disorder and a high burden for the healthcare system. Therefore alternative therapies should be developed. CLL cells survival, proliferation and resistance to therapies are highly dependent on the microenvironment in lymphoid organs. Interactions with stromal cells and the immune compartment by inducing a strong immuno-suppression and various leukemic promoting immune cells is key to leukemia progression. In recent years, immunotherapy has revolutionised the management of cancer patients. However, only a limited number of patients respond to these new therapies (30-40% response to single-agent therapy) and these are not approved yet for all cancers. It is therefore necessary to further optimise the benefits of these therapies aimed at reactivating the immune system to fight against the tumour. Based on deep immune-phenotyping results, our group recently demonstrated that dual immune checkpoint blockade-based immunotherapy was efficient to treat a murine pre-clinical model of CLL (Wierz et al, 2018). However, direct translation of these type of findings in the clinic is difficult due to the lack of human CLL-based model.   

AIM AND HYPOTHESIS 

This project will aim at developing a PDX model of CLL in humanised NSG mice to test several immunotherapy combinations aiming at reactivating the exhausted immune system. Artificial lymph nodes and other 3D co-culture systems will be used to screen new therapies in complex models mimicking the tumor and its microenvironment.  

METHODS 

(i) establish a humanised mouse and set up the PDX of CLL cells, (ii) test immunotherapies in vivo and analyse the immune system (CyTOF, Hyperion), (iii) develop an artificial lymph node / 3D model with CLL and normal bystander cells (air-liquid interface, cell printing), (iv) perform high-throughput screening of drugs and immunotherapies on 3D organoids, and (v) validate hits in vivo in Eµ-TCL1 and CLL-PDX mice. 

INTRODUCTION 

Ulcerative colitis (UC) represents a major form of Inflammatory Bowel Diseases (IBD), which are chronic relapsing disorders affecting the gastrointestinal tract. There is growing evidence that changes in nutritional habits and microbiota-associated alterations contribute to increasing UC incidences, especially in industrialized countries, where diets characterised by insufficient fiber intake are common^(1,2).

We have previously shown that a diet devoid of fiber led to overgrowth of mucin glycan-degrading bacteria in mice, resulting in decreased colonic mucus thickness,  heightened susceptibility to enteropathogenic infection, and increased mucosal immune responses^(3–5). Additionally, we have demonstrated that genetically susceptible mice develop severe forms of IBD in the face of increased microbial mucin foraging⁽⁶⁾.

AIM AND HYPOTHESIS 

In line with our published work and given that UC patients typically demonstrate defects in mucus layer integrity, we hypothesize that mucin-degrading commensals play a pivotal role not only in the pathogenesis of UC, but also in the success of biological therapies targeting the immune responses of the host.

METHODS 

The proposed project will make use of already collected samples (between 2020 to 2022) from one cross-sectional and one longitudinal IBD cohort study involving UC patients. Both studies have been conducted in collaboration with the CHL, Luxembourg. The cross-sectional study targeted UC patients with currently active, moderate disease irrespective of medical treatment. Meanwhile, the longitudinal study targeted patients that were newly diagnosed with UC and were to be treated with antibodies targeting the host immune response (Infliximab, interfering with TNF response; Vedolizumab interfering with T cell homing to the gut).

Using these patient cohorts, we will test the hypothesis that dysregulated mucin glycan degradation by commensal gut bacteria is associated with acute inflammation in UC patients and impacts success of antibody-based therapies. Dietary therapeutics could be employed in the mid-term to reduce mucin-degrading activity by the gut microbiota and improve the success of IBD therapies.

This translational PhD project will involve clinical samples, cutting-edge microbiome-related omics analyses, broad immune cell profiling from patient blood samples and high-throughput culturing of patients’ mucin-degrading intestinal microbial populations followed by in vitro phenotyping. This interdisciplinary project will prepare the PhD student for a career in academia or industry or clinical settings.

REFERENCES

1. Wolter, M. et al. Leveraging diet to engineer the gut microbiome. Nat. Rev. Gastroenterol. Hepatol. 0123456789, (2021).
2. Miyauchi, E., Shimokawa, C., Steimle, A., Desai, M. S. & Ohno, H. The impact of the gut microbiome on extra-intestinal autoimmune diseases. Nat. Rev. Immunol. 23, 9–23 (2023).
3. Desai, M. S. et al. A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility. Cell 167, 1339-1353.e21 (2016).
4. Martens, E. C., Neumann, M. & Desai, M. S. Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier. Nat. Rev. Microbiol. 16, 457–470 (2018).
5. Neumann, M. et al.  Deprivation of dietary fiber in specific-pathogen-free mice promotes susceptibility to the intestinal mucosal pathogen Citrobacter rodentium . Gut Microbes 13, (2021).
6. Pereira, G. et al. Unravelling specific diet and gut microbial contributions to inflammatory bowel disease. BioRxiv https://doi.org/10.1101/2022.04.03.486886 (2022).

INTRODUCTION 

ICB-based therapies have emerged as promising treatment for aggressive cancers, but their success requires efficient infiltration of cytotoxic immune cells into the tumour. Therefore, a major challenge in the field of immuno-oncology is to develop strategies, to be combined with ICB, promoting tumor infiltration by these cells. IPI and TIME jointly identified one chemokine receptor as a key receptor involved in preventing the recruitment of cytotoxic immune cells to tumours. 

AIM AND HYPOTHESIS 

Targeting this chemokine receptor using neutralising monoclonal antibodies (mAbs), antibody fragments such as Nanobodies (Nbs) or small molecules represents an innovative strategy to induce infiltration of immune cells into tumours, rendering them eligible for ICB-based therapy. This project aims at (I) developing receptor modulators (mAbs, Nbs or small molecules) and (II) validating their pharmacological/functional properties in cellular assays using the know-how of IPI group and (III) evaluating their benefit for combined ICB therapy in relevant cancer models using the expertise of TIME group. 

METHODS 

mAbs and Nbs against mouse and human receptors will be elicited using the magnetic GPCRliposome-based technology developed by IPI group, allowing to purify and display a high density of properly folded receptors for mouse or llama immunisation. Mabs and Nbs will be selected by hybridoma and phage-display technologies, respectively. The validation of the binders and the characterisation of their activity towards the receptor will be performed using tailor made cellular assays based on the NanoBiT/NanoBRET technologies at the DII. These assays will also be adapted for screening of small receptor modulators using the drug-screening platform of Dr. Yong-Jun Kwon. Finally, selected modulators will be evaluated alone or in combination with anti-PD-1 therapy using mouse melanoma and colon cancer models. 

INTRODUCTION 

As a novel cancer immunotherapeutic approach, we generated two types of immunoconjugates, called CoMiX, that enhanced complement directed activity and cytotoxicity at the surface of HER2-tumour cells or CD20-overexpressing lymphomas (Seguin-Devaux et al., 2019). Based on the CoMix technology (patent PCT/EP2017/062283), we have generated recombinant molecules targeting and activating NK cells against different cell types infected with various pathogens. These molecules also increase the cytotoxic activity of NK cells against K562 and Raji cancer cells through IFN-y release and cytotoxic protein degranulation.  

Pancreatic cancer is a cancer with high unmet medical needs. Pancreatic cancer has a catastrophic prognosis (recent five year survival in the USA: 9.3%) and moreover is expected to become the second most frequent cancer in 2030. Treatment is difficult, as in most cases diagnosis is made at late stages when tumour cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients due to therapy resistance and a limited response to checkpoint inhibitors. The CEA expression is generally very high in pancreatic adenocarcinoma, Many pancreatic cancers are resistant to immunotherapeutic approaches due to their immune-hostile microenvironment  

AIM AND HYPOTHESIS 

We propose here to evaluate novel immunoconjugates activating NK cell function in different models of cancer to evaluate their potency as compared to current therapies and in combination with checkpoint inhibitors. Based on our preliminary results, we will evaluate our therapeutic molecules against a hematopoietic cancer (B cell lymphoma), and pancreatic tumours. These immunoconjugates will allow investigation of the therapeutic efficacy of innate immunity and whether our recombinant molecules can overcome the current resistance to the chemo- and targeted therapies in a model of pancreatic cancer. This PhD project may provide additional applications for CoMIX in cancer therapies and consolidate the potential of CoMIX against other solid tumours. 

METHODS 

The PhD student will develop different in vitro (2D and 3D cellular models) and in vivo (mouse models of lymphoma and pancreatic cancer) cancer models, evaluate several molecules activating NK cells against tumour cells, and investigate their mode of action using different immunoassays. 

INTRODUCTION 

Targeted therapy can enhance the antitumour immune responses by releasing new antigens; this provides a theoretical basis for immunotherapy combined with targeted therapy as recently observed in lung cancer (Akbay et al., 2013; Liang et al., 2018). Along this line, the small REGONIVO phase Ib clinical trial (n=48 patients) very recently showed promising results on the combination of the tyrosine inhibitor Regorafenib and the anti-PD1 therapy Nivolumab in gastric and CRC patients (ASCO 2019). These initial results are promising and encourage the testing of more combinations of different tyrosine kinase inhibitors and immune-based therapies.  

AIM AND HYPOTHESIS 

We hypothesise that the screening of single drugs and the combination thereof directly on patient material will lead to the development of new personalised therapy options. In addition, patient-derived models composed of tumour and stromal cells (note that stromal cells, among which fibroblasts, are known to be responsible for immune-suppression), as established over the past years within the MDM group, will help to understand the underlying mechanisms of innate/acquired resistance and thereby prevent therapy resistance in long-term treatments.  

METHODS 

Fresh biopsies from stage III and stage IV CRC patients will be collected at the different involved hospitals and directly transferred to LIH and the MDM group at UL. Screening of therapy options (including different combinations of targeted and immune-based therapies) directly on patient material will be done by applying the high throughput drug-screening library from LIH. Using in silico modelling, as previously described by our group in collaboration with the Sauter group at UL (Pacheco et al., 2019), we will further refine the list of tested drugs/drug combinations. Additionally, patient- derived models from the same patients (composed of tumour and stromal cells as well as cells from the normal counterpart tissue) will be used to (i) understand the mechanisms of innate and acquired resistance, (ii) to address the efficiency of the identified therapy options (iii) as well as study their potential to induce resistance in long-term treatments, by the means of molecular biology (i.e. bulk and single-cell transcriptomics combined with signaling network analysis) and functional assays (Cell Titer Glo viability assays). Finally, most promising pre-clinical candidates will be validated in our in house orthotopic mouse CRC models.  

INTRODUCTION 

Precision medicine provides a promising avenue for cancer patients, however robust preclinical models to predict sensitivity of novel treatments are key for advancing clinical oncology (Byrne et al., 2017; Gao et al., 2015). Based on a large platform of Glioblastoma patient-derived orthotopic xenografts (PDOXs), which retain histopathological, genetic and epigenetic features of patient tumours superior to in vitro cultures, we have successfully applied biomarker-driven drug treatment in PDOX in vivo and in PDOX-derived 3D tumour organoids ex vivo (Bougnaud et al., 2016; Abdul Rahim et al., 2017; Golebiewska et al., BioRvix, 2020). Both approaches, however, suffer from the reduction and/or absence of immune system components, limiting the opportunity to test novel immune modulating therapies. 

AIM AND HYPOTHESIS 

In this project we plan to expand our GBM PDOX platform to humanised mouse models. The immune component, i.e. peripheral blood mononuclear cells and/or patient-derived microglia/macrophages will also be combined with ex vivo 3D primary tumour organoids. 

METHODS 

Relevant tumour-stroma interactions will be investigated at the single cell level (single cell RNA-seq, Flow and mass cytometry) to monitor tumour progression and treatment response. Standardised short-term tumour-immune co-cultures will be applied for high-throughput drug screening and successful compounds will be validated in humanised PDOX models in vivo. 

INTRODUCTION 

The treatment of brain tumours remains challenging. Whereas the large majority of benign tumours can be treated surgically or by radiosurgery, a large majority of malignant brain tumours are still not curable. Whereas neuronavigation technologies have been coming up in Neurosurgery as a standard of care within the last two decades, innovative technologies, such as spectroscopic imaging (RS and MRS), as well as precision MRI, offer new possibilities for targeted treatment approaches. However, despite the development of many new and promising drugs, delivery through the Blood Brain Barrier (BBB) remains challenging. 

AIM AND HYPOTHESIS 

The aim of the project is it, to develop heat maps of the spatial distribution of biochemical tumour properties by a combination of navigated RS, MRS and diffusional MRI and to combine those with models of drug distribution for CED by computer modelling of diffusional MRI images. 

METHODS 

We will analyse RS with navigated RS fiber probes and integrate those into data sets of multiple 3D-MRI datasets. Therefore, we will develop heat maps to depict the biochemical tumour property distribution. By integration of diffusability (diffusion weighted MRI), we will develop models for fluid (drug) distribution within the individual patients’ brains. Those models will be tested by labelled Gadolinium particles. 

INTRODUCTION 

Precision oncology, guided by next-generation sequencing of tumour DNA and RNA, implies the ability to predict which patients will likely respond to specific cancer therapies (Horak et al., 2017). Recent studies demonstrated the utility of tumour mutational burden (TMB) measured by whole exome sequencing (WES) as a promising biomarker to identify patients most likely to benefit from immunotherapy across a wide range of cancer types (Chan et al., 2019). In the clinical setting, where WES is not routinely available, panel sequencing is emerging as the technology for TMB assessment. However, lack of harmonisation between platforms and of robust predictive cut-offs are major limitations of panel-based TMB quantification (Fancello et al., 2019). Furthermore, there is an unmet clinical need for the assessment of TMB in liquid biopsies, since measuring TMB in circulating tumour DNA (ctDNA) presents a major detection challenge (Gandara et al., 2018). 

AIM AND HYPOTHESIS 

This project aims to evaluate and validate different strategies for reliable detection of TMB in solid tumour tissues and blood-derived ctDNA to identify patients eligible for immunotherapy. Implementation of TMB-assessment in translational oncology programs will improve personalised therapeutic decision-making and patient management in the future. 

METHODS 

We will perform WES and panel sequencing on tumour tissues and corresponding blood samples as well as ctDNA to identify actionable somatic and hereditary alterations, including TBM. Bioinformatics pipelines to determine TBM from different data sets will be developed utilising own and public available data sets. Results will be correlated with clinical data. We will integrate our approaches in translational research programs such as PFP and in multidisciplinary tumour boards to enable fast translation into the clinics.