Fraunhofer Institute for Cell Therapy and Immunology IZIInflammation and Tumor Models
Inflammatory diseases
Preclinical animal models for the development of new IBD therapies
Inflammatory bowel diseases (IBDs) are multifactorial diseases of the gastrointestinal tract, the incidence and prevalence of which have been rising constantly in industrial and newly industrialized countries. The etiology of IBD is unknown but is thought to be a combination of genetic predisposition, environmental factors and a dysregulated immune response to the gut microbiota. Current therapeutic approaches, such as immunosuppressives and biologics, often show severe side effects. Thus, there is an urgent need for the development of new therapies based on improved knowledge of the etiological factors of IBDs.
The goal of the in-house research project is to develop and characterize different IBD animal models for testing new therapies and elucidating mechanisms of pathogenesis. Thus, a model of chronic DSS colitis was established which shows typical symptoms such as weight loss and chronic bloody diarrhea. Moreover, the colon tissue of the animals features a continuous immune reaction with resulting ulcerations. In addition, the bacteria-induced chronic colitis has been established as an alternative animal model for IBDs which mainly focuses on the role of the microbiota in disease pathogenesis and / or as a target for novel treatment strategies. Both models can be used for preclinical studies as well as elucidating IBD pathogenesis and developing new therapies. In the latter case, the focus is currently on evaluating the therapeutic potential of phytochemicals.
We are also performing a functional analysis of the gut microbiota in the chronic DSS colitis model using BALB/c mice, together with the Helmholtz Centre for Environmental Research UFZ. The goal of this project is to better understand the interactions of the microbiota and the mucosal immune system in IBDs.
Project management
Dr. Nadine Vollack-Hesse
Development of a novel therapeutic concept for chronic inflammatory bowel diseases (IBD) via non-toxic ligands of the aryl hydrocarbon receptor
The umbrella term chronic inflammatory bowel disease (IBD) covers disease patterns that are characterized by recurring or continuously emerging inflammatory changes to the bowels. The two main types of IBD are Crohn's disease and ulcerative colitis. In Germany, around 300,000 people currently suffer from these two disorders, which are associated with long-term abdominal pain and diarrhea as well as high levels of fatigue.
Conventional IBD therapies focus on repressing the inflammation using anti-inflammatory drugs. A cure is yet to be found for these diseases, which means any drug therapy generally has to be administered for a person’s entire lifetime. This is first and foremost due to the fact that these therapies are systemic treatments. A therapeutic approach is yet to be found which has a causal and sustainable impact on the dysregulation of the intestinal immune system associated with IBD. Surgical measures often give rise to complications and impact on quality of life. The gut microbiome, which is significantly influenced not only by nutrition but also by the use of antibiotics, plays an important role in the emergence of IBD. This is why nutrition and alternatives to antibiotics are playing an increasing role in the development of new approaches to treating these diseases. More recent studies attest to the fact that the aryl hydrocarbon receptor (AhR) presents a highly promising, novel therapeutic target in the case of IBD.
Furthermore, numerous research papers have shown that the AhR plays a significant role in both the innate and the adaptive immune system in terms of maintaining immune homeostasis and controlling inflammatory responses in the intestines. The AhR is central to the communication between immune cells and enterocytes. AhR ligands found in food or the microbiota activate the AhR, promoting the survival and proliferation of immune cells and making a substantial contribution to immune homeostasis. Based on the recently published AhR structure, a highly diagnostic pharmacophore model is to be developed which will be used to select new, previously unknown AhR ligands as candidates for future therapeutic application in IBD cases using structure- and ligand-based methods. Moreover, a drug repositioning approach is to be taken, i.e. drugs which have already been approved or drug candidates currently undergoing clinical testing are to be identified as potential AhR ligands for other indications.
This work was supported by the Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD.
Project management
Dr. Nadine Vollack-Hesse / Dr. Jörg Lehmann
Plant extracts as active agents for the treatment of chronic inflammatory bowel diseases
Inflammatory bowel diseases (IBD), such as Crohn’s disease and Ulcerative Colitis, are multifactorial disorders of the gastrointestinal tract. The incidence and prevalence of these diseases have been rising constantly in industrial and newly industrialized countries for several decades. Although the diseases show a low mortality rate, patients suffer lifelong from episodes of severe pain and bloody diarrhea. The etiology of IBD is unknown but is assumed to be a combination of genetic predisposition, environmental factors and a dysregulated immune response to the gut microbiota. Hence, current therapies mainly focus on inhibiting the chronic immune response in the gut using immunosuppressive drugs and biologics. Since these therapies are often associated with strong side effects, pharmaceutical companies are pursuing the development of new IBD therapies with less side effects. The model often used for such developments - the acute dextran sulfate sodium (DSS)-induced colitis - cannot adequately represent the chronic course of the diseases. Therefore, the Preclinical Models Unit has developed a chronic model that better represents the course of disease and reduces animal burden. Due to its high reproducibility, the model is very well suited for the preclinical evaluation of therapeutic agents. The model of chronic DSS colitis has already been successfully applied in several industrial projects. Moreover, the therapeutic effect of a plant extract based on sage and bitter apple could be demonstrated in the frame of an internal research project. As a next step, the active components of the extract will be identified and tested for their therapeutic efficacy. The use of phytopharmaceuticals could result in a reduction of the dosis and hence the side effects of classical therapeutics. Furthermore, such therapies could be used in phases of remission and thereby prevent the development of resistance to classical therapeutics. The goal is to make the lifelong therapy needed by IBD patients as effective and tolerable as possible and thus improve the quality of life for these patients.
Project management
Dr. Nadine Vollack-Hesse
Press releases
Immunomodulatory role of the arylhydrocarbon receptor (AhR)
Within the frame of this project, the immunomodulatory effect of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) was characterized in collaboration with the Federal Institute for Risk Assessment (BfR). While the carcinogenic effect of BaP has been known for decades, little attention has been paid so far to the immunomodulatory effect of BaP, particularly of concentrations classified as uncritical. The immunomodulatory effects as well as the decomposition of BaP are mainly regulated by its binding to the arylhydrocarbon receptor (AhR) with subsequent receptor activation. A murine salmonella infection model is used to investigate the impact of BaP on an ongoing immune response. The analysis of the underlying mechanisms is carried out using suitable in vitro models. Previous investigations have mainly been restricted to the maturation and functional activation of murine macrophages. By using AhR-deficient mice or cells from these animals, the AhR dependence of the observed effects could be demonstrated. Interestingly, the initial findings in our model showed an AhR-dependent immunomodulatory effect of BaP which had a positive impact on the course of disease. In further experiments, selected non-toxic AhR ligands are being analyzed for their therapeutic potential in different disease models.
Project management
Sina Riemschneider
Completed projects
- Study of the therapeutic efficacy of an ion-based immunomodulator in a chronic DSS colitis model
- Study of the therapeutic efficacy of a transglutaminase inhibitor in an acute DSS colitis model
Tumor diseases
Preclinical and clinical development of a novel CAR-T cell therapy for the treatment of multiple myeloma and clear cell renal cell carcinoma
CAR-T cell therapy is based on the principle of equipping immune cells (T cells) with an artificial chimeric antigen receptor (CAR) by genetic modification. This enables the immune cells to identify specific surface structures (antigens) on cancer or other target cells and to activate a corresponding immune response.
In the therapies approved to date, the T cells are modified using viral vectors and in most cases address the cell surface molecule CD19, which is expressed by the target cells in particular in certain types of blood cancer and lymphomas.
With the ROR2-CAR-T cell therapy, scientists at the University Hospital of Würzburg have developed an immunotherapy that differs from previously approved therapies both in the type of genetic modification and the target antigen addressed. This is now to be transferred to clinical application as part of a project funded by the German Federal Ministry of Education and Research.
The ROR2 protein is a transmembrane receptor that plays an important role especially during embryonic development. It is normally not expressed, or only very slightly expressed, in normal, healthy cells and tissues. However, in some cancers, including multiple myeloma and clear cell renal cell carcinoma, it is highly expressed on the cancer cells in question. This makes the antigen a suitable target for appropriately targeted CAR-T cells.
In this project, a new method for the production of autologous CAR-T cells, which is still being tested, is used. The genetic modification of the patient's own T cells is carried out via a non-viral gene transfer, which, compared to viral gene transfer, will enable a simpler, more scalable and thus less expensive production process. The chimeric antigen receptor was designed to initiate overexpression of the transcription factor Batf3 in addition to T cell activation to improve T cell persistence and tumoricidal activity.
Fraunhofer IZI is responsible for two main areas within the project. On the one hand, the preclinical testing of the safety and efficacy of the novel CAR-T cell product within the scope of a GLP study, and on the other hand, the pharmaceutical manufacturing of the investigational medicinal products for the clinical study, including the prior establishment and validation of the manufacturing process as well as the safety-relevant quality controls.
The multicenter clinical study (phase I, first-in-human) will be realized at the University Hospitals of Würzburg (coordination, Prof. Dr. M. Hudecek), Regensburg and Leipzig.
According to international (OECD framework) and national guidelines and laws for the approval of new drugs, non-clinical safety testing of newly developed active substances and therapies must be carried out under conditions of Good Laboratory Practice (GLP). The results of such tests are an essential prerequisite for applying for and conducting clinical trials. Safety and efficacy studies on ROR2-CAR-T cells are performed at the Fraunhofer IZI GLP testing facility. For this purpose, humanized mouse models for multiple myeloma and clear cell renal cell carcinoma are established and applied under GLP. The aim of these non-clinical studies is to detect and evaluate adverse effects that can be derived from the activity of CAR-T cells specifically directed against the tumor, such as cytokine release syndrome or tumor lysis syndrome. In addition, special attention will be paid to the tumoricidal effect and persistence of CAR-T cells.
In vitro models for investigating new targets against tumor antigens
The Inflammation and Tumor Models Unit not only offers scientific expertise on preclinical tumor models; it is also highly experienced in analyzing potential tumor targets in appropriate in vitro models. Both 2D and 3D cell culture systems can be drawn on here, allowing the behavior of tumor cells to be examined upon introducing potential anti-tumor agents. Depending on the type of agent, tests can be conducted that look at internalization and invasion behavior, potential cell activation / inhibition and also the effect on proliferation. These tests can be carried out with real-time analyses.
Project management
Claudia Müller
Humanized mice
Preclinical testing of a CAR-T cell therapy in the NSG mouse model
The use of CAR-T cell therapy may be associated with considerable adverse effects that fall into three categories:
- on-target/on-tumor toxicity,
- on-target/off-tumor toxicity, and
- off-target/off-tumor toxicity.
Tumor lysis syndrome is just one of the critical on-target/on-tumor adverse effects (Bonifant et al., 2016). In the case of tumor lysis syndrome, a high number of cancer cells are rapidly destroyed, leading to a massive, abrupt release of intracellular ions, nucleic acids, proteins and their metabolites into the extracellular space. These metabolites can disrupt the body’s normal homeostatic mechanisms and cause, among other things, hyperuricemia, hyperkalemia, hyperphosphatemia and hypocalcemia. As a result, tumor lysis syndrome can lead to acute renal failure (Cairo and Bishop, 2004).
It is therefore important to document and evaluate possible side effects of CAR-T cell therapy in correlation with the therapeutic effect achieved using suitable in vivo models. For this kind of preclinical testing, we use humanized tumor mice that develop tumors by being injected with either tumor cell lines (CDX – cell-line derived) or patient material (PDX – patient derived). Besides assessing the animals’ clinical behavior, the effect of CAR-T cells can also be evaluated by means of bioluminescence imaging. Once the test has ended, the on-target/off-tumor and also the off-target/off-tumor toxicities can be examined through histological analyses.
Preclinical trials of a CAR-T cell therapy in the NSG mouse model have already been conducted in our department as a GLP study in accordance with guideline EMA/CAT/GTWP/671639/2008 in conjunction with EMA/CAT/852602/2018.
Project management
Claudia Müller
Development and production of human monoclonal antibodies in a humanized mouse model
This project focuses on developing and producing new kinds of fully humanized monoclonal antibodies for the treatment of tumour diseases (pilot project: triple-negative breast cancer). The development and production of such antibodies can be divided into several steps: The first step entails establishing a humanized mouse model which can be used to generate human monoclonal antibodies against known and yet unknown tumour antigens. A variety of immunization strategies have been created for this purpose in immune-deficient NSG or BRGS mice, which have developed a humanized immune system after the transfusion of human haematopoietic stem cells from cord blood. Tumour-specific human monoclonal antibodies are generated by fusing tumour-specific human B cells from these humanized mice with human plasmocytoma cells, and selected using suitable strategies. Selected candidates are finally tested in an established tumour mouse model in preclinical studies and modified if required (e.g. antibody-drug conjugates), in order to continue improving therapeutic efficacy.
As part of a cooperation project with the University of Regensburg (Dr. Anja Wege), it could be shown that antibodies were detectable in the serum following the co-transplantation of the human immune system and the tumor cells.
Further analysis of the IgG-bound proteins demonstrated that the detected antibodies were directed against target structures of the tumor.
Project management
Claudia Müller
In vivo model development
It is important that new therapeutic options are first established before being tested in suitable animal models. Small animal models such as the mouse model are widely used to investigate newly developed drugs or methods at the preclinical stage. However, the mouse model has to be adapted again and again in response to new questions and findings. It takes a lot of time and money to develop a genetically modified mouse strain. Mouse strains that do not yet have a transgenic mouse model can be established through the transduction or transfection modes of gene transfer into the respective organs.
Mice that have been genetically modified in this way can then be used for the preclinical testing of new drugs or methods and can even be transferred to immunodeficient mice with human immune systems. This is the approach currently being taken by the Inflammation and Tumor Models Unit as part of research into the coronavirus in order to establish a humanized NSG mouse model intended to replicate hACE2 in the lung tissue via AAV-based transduction or lipofection.