Fewer burdensome side effects, by a patient-specific treatment that acts locally in the body
"Description begins in the writer's imagination, but should finish in the reader's" - Stephen King
Design of !MPACT
Currently, 3-5% of the world population is affected by autoimmune diseases and this number is rising.1,2 Autoimmune diseases are caused by a breach of immunologic tolerance leading to an immune response against molecules of the own body.3
The iGEM TU Eindhoven 2022 team focuses on a group of rare, but life-threatening autoimmune diseases named antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).4 This is a collection of severe chronic disorders characterized by granulomatous and neutrophilic tissue inflammation causing necrosis of blood vessels.5,6 The necrosis of vessels leads to insufficient oxygen supply to the organs connected to these vessels. As a consequence, a decrease in tissue functioning or tissue death occurs.7
Three types of AAV are classified, namely granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic GPA (EGPA). As GPA is the most common type of AAV, our project focuses on the chronic, systemic, and relapsing disease GPA.8 GPA predominantly involves the nose and sinuses, lungs, kidneys, joints, and eyes.5
AAV is associated with the production of pathogenic antineutrophil cytoplasmic antibodies (ANCAs). The ANCA concentration in the body is correlated with the disease activity. These antibodies target two major antigens: leukocyte proteinase 3 (PR3) and myeloperoxidase (MPO).5 The majority of the GPA cases have ANCA autoantibodies targeting PR3.8 These PR3-ANCA-positive patients are at great risk of getting relapses of the disease.7 That is why our project specifically focuses on GPA cases that are PR3-ANCA positive.
In conclusion, there is a high unmet need in the treatment of AAV (Figure 1):
To meet the shortcomings of the current therapies against ANCA-associated vasculitis, we designed !MPACT: a Modular and Personalized Autoimmune Cell Therapy. This cell-based therapy detects the ANCA autoantibodies associated with ANCA-associated vasculitis. In the presence of ANCAs, interleukin-10 (IL-10), a natural anti-inflammatory cytokine, gets produced resulting in a specific suppression of the autoimmune response.
!MPACT is based on the Generalized Extracellular Molecule Sensor (GEMS) platform described in Scheller et al. (2018). This modular synthetic receptor allows the coupling of an extracellular input to an intracellular signaling pathway.14 In our case, binding of the pathogenic ANCA autoantibodies to the receptor leads to receptor dimerization and activation, starting a signaling pathway that results in the production and local release of anti-inflammatory cytokine IL-10 (Figure 2). Local release of IL-10 is suggested to have great potential in treating autoimmune diseases according to both literature and stakeholder interviews (see IL-10 in autoimmune diseases and Human Practices).
The activity of !MPACT is dependent on the concentration of ANCAs present. Higher ANCA concentrations, i.e. higher disease activity, will lead to more IL-10 production, resulting in stronger suppression of the inflammation. Furthermore, the presence of ANCAs is strictly necessary for IL-10 production. When no ANCAs are present, i.e. no disease activity, the cells are “sleeping” and thus will not release IL-10. The production of IL-10 is only desired when ANCAs are present, as elevated IL-10 levels could lead to side effects in the absence of disease.15 In case of a relapse of the disease, the increase in ANCA concentration again activates the !MPACT cells. This way, the relapse of the disease is prevented before it gets problematic. !MPACT is temporarily active, adjusted to the disease activity, and can intervene early on in relapses.
The implementation of !MPACT is similar to CAR T-cells: harvested immune cells from a patient will be genetically engineered with the synthetic receptor and multiplied, after which the cells will be injected back into the patient's body (see Proposed Implementation). In the body, the engineered cells will function as described above to fight the inflammation.
!MPACT is innovative and powerful since its therapeutic effect is personalized to the patient, i.e. the amount of IL-10 production adjusts to the disease activity. Consequently, fewer burdensome side effects are expected compared to current treatments. In addition, !MPACT can detect and intervene relapses early on, which is not possible with the current treatments. The prevention of relapses and fewer side effects will reduce organ damage and will thereby reduce both healthcare costs and workload. This is confirmed by the Maastricht University Medical Center (see Human Practices). Therefore, we are convinced that our design will greatly contribute to a better treatment of ANCA-associated vasculitis.
The GEMS platform used in our proof of concept cell therapy consists of a mutated erythropoietin receptor (EpoR) fused to an extracellular domain containing proteinase 3 (PR3), to which ANCAs can bind. In addition, the GEMS platform contains signal transduction domains of interleukin 6 receptor B (IL-6RB) that can activate the JAK/STAT signaling pathway.
Upon binding of the ANCAs to the PR3 domain, the extracellular affinity domains dimerize, causing a rotation of each receptor subunit around its axis. This results in a changed orientation of the intracellular IL-6RB domains, which in turn induces the JAK/STAT pathway. The JAK/STAT pathway results in downstream formation of STAT3 dimers which induce transcription of an IL-10 gene regulated by a STAT3 promoter. After translation of IL-10, the IL-10 is automatically secreted by the cell due to the signal sequence Igk. The mechanism is shown in Figure 3.
Interleukin-10 (IL-10) is a cytokine involved in regulating the immune response. Its primary role is to deactivate or dampen the immune response without damaging surrounding tissue.19 This is done by suppressing antigen-specific immunity and thus allowing for T-cell tolerance.20 Since IL-10 lowers the inflammatory host response, it is co-responsible for preventing inflammation in autoimmune pathologies, hence why IL-10 deficiency or dysregulation is also known to be the cause of several autoimmune diseases. The powerful anti-inflammatory characteristics of IL-10 suggest the therapeutic potential of (artificial) IL-10 administration in autoimmune diseases.15 Also interviews with Utrecht University Medical Center, Novartis, and Catherina Hospital showed that IL-10 could have a promising therapeutic effect in autoimmune diseases due to its anti-inflammatory properties (see Human Practices). Administration of IL-10 is especially promising in AAV where the IL-10 production is decreased in case of the development of a relapse.21 This supports the power of !MPACT to prevent relapses of AAV.
Although the potential of IL-10 in a clinical application has been demonstrated, many challenges still remain. A significant drawback, which currently withholds clinicians from using IL-10, is that the biologically active form of such a molecule is an unstable homodimer. As a result, IL-10 has a short half-life and is easily degraded in vivo.22 To use IL-10 as a potential therapeutic agent, it is, therefore, necessary to continually administer small doses to maintain the therapeutic concentration. Furthermore, research has shown that, although small concentrations are safe and overall well tolerated, high doses of IL-10 are associated with systemic side effects like fever, headache, and malaise.23 Most of these side effects however are temporary, mild to moderate, and disappear after the cancelation of the treatment.24 The last difficulty that presents itself is the complexity of the IL-10 mechanism. New research suggests that IL-10 is not solely immune inhibitory, but can also stimulate the immune response in some immune diseases and cancers.24,25 A proposed explanation is that IL-10 also induces pro-inflammatory cytokine production, which may be heightened in presence of other proteins, like disease-specific antibodies.26 This is an unwanted effect when using IL-10 as a therapeutic agent. However, much remains uncertain about this effect of IL-10.27
Despite these disadvantages, local administration of IL-10 has proven to be effective in multiple studies.28-30 Small IL-10 concentrations are shown to be safe, well-tolerated, and effective in the treatment of several autoimmune diseases. Only too high IL-10 concentrations should be prevented since that is associated with pro-inflammatory and systemic side effects.23 To prevent too high IL-10 concentrations, we suggested an inhibition system that can be implemented in !MPACT to ensure safe IL-10 concentrations (see Proposed Implementation).
The Generalized Extracellular Molecule Sensor (GEMS) platform is a novel synthetic receptor with great potential for many applications in synthetic biology and for developing cell-based diagnostics and therapeutics. The GEMS platform senses extracellular molecules by modified erythropoietin receptor (EpoR) dimers that are inert for erythropoietin but are fused to extracellular affinity domains which consist of single-chain variable fragments (scFvs) of antibodies, enabling the receptor to sense soluble molecules.14 These EpoR dimers are also linked to different intracellular domains, which transmit the signal and activate distinct natural signaling pathways (JAK/STAT, MAPK, PLCG, and PI3K/Akt).17 Upon ligand binding, the extracellular affinity domains dimerize, causing a rotation of each receptor subunit around its own axis. This results in a changed orientation of the intracellular domains, which in turn activates the downstream signaling pathways.14
The modular structure of the GEMS platform allows combinations of different intra- and extracellular domains, making it a customizable epitope sensor. As a result, GEMS are able to detect a large variety of target molecules.14 Until now, the GEMS platform is constructed for the detection of rapamycin, an azo dye (RR120), caffeine, nicotine, a peptide tag fused to mCherry (SunTag), prostate-specific antigen (PSA), and a de novo designed protein displaying two viral epitopes.18 Thereby, the validity of the GEMS platform is confirmed for signaling molecules in a wide range of molecular weights.14 However, it is not yet validated whether the GEMS platform is able to detect antibodies.
The activation of the receptor is usually quantified by the reporter protein SEAP (human placental secreted alkaline phosphatase), making it suitable for detection purposes. Exchanging the reporter protein SEAP for the expression of a therapeutic agent could give rise to multiple other therapeutic applications for the GEMS platform.14
During the iGEM project, we designed, built, and tested the proof of concept of !MPACT. Together with stakeholders we validated the problem, designed the therapy, and validated this solution to make the therapy desirable, feasible, safe, and responsible for the world (see Human Practices). The proof of concept is developed and tested in vitro in the lab (see Experiments, Results, and Proof of Concept) Our goal was to show that the designed synthetic receptor could be activated through the binding of antibodies and could subsequently produce IL-10. In addition, we built a kinetic model of the synthetic receptor to support future lab work by simulating the behavior of the system and by predicting sensitive parameters that can be adjusted in the lab to optimize the system and the experimental conditions. This allows the lab work to be used to its full potential (see Model). Furthermore, together with relevant stakeholders in the industry, we created a business plan for !MPACT that allows translation of the project idea directly into a new venture (see Entrepreneurship).
Fewer burdensome side effects, by a patient-specific treatment that acts locally in the body
Reduce healthcare workloads
Enlarge public outreach
As iGEM TU Eindhoven team with many Biomedical Engineering students, we were interested in tackling a global healthcare problem where we could make a real societal impact. First, we started brainstorming about present-day healthcare-related problems and about novel technologies in synthetic biology. We also performed literature research on these topics. Papers suggesting that Long Covid is triggered by an autoimmune response and that the Epstein-Barr virus is the trigger for Multiple Sclerosis sparked our interest. We dived deeper into the topic of autoimmunity. By brainstorming, also together with our PIs, we developed a first concept for our project: designing a synthetic receptor as a treatment for an autoimmune disease. We reached out to several stakeholders, including University Medical Centers, hospitals, and experts in the field of autoimmune diseases, to brainstorm with them about our project idea and to validate the need for new treatments for several autoimmune diseases. All stakeholder interviews can be found on the Human Practices page. Based on the obtained insights from our stakeholders, literature research, and discussions within our team we specified our design. As the involvement of stakeholders in our project was a high priority in our project, we kept validating our project with them throughout the entire journey. Consequently, we had to make several pivots in our project design, but this resulted in a better validated and more desirable design of our project that can eventually make a stronger societal impact.
The proof of concept of our modular and personalized autoimmune cell therapy serves as a foundation for a new cell therapy against ANCA-associated vasculitis and for research in synthetic biology. Considering our promising results (see Results), future research should optimize the design of the cell therapy, especially by focusing on enhancing the binding and activation of the GEMS platform by autoantibodies (see Experimental Outlook on the Results page). It is suggested to use a large linker length library to optimize the binding of antibodies and activation of the receptor. Besides, the design could be optimized in order to make the therapy act more locally in the body and the inhibition system for the receptor we envisioned could be implemented to prevent too high IL-10 concentrations (see Proposed Implementation). Next to optimizing the proof of concept, the technology should be patented to protect the IP for future business opportunities (see Entrepreneurship).
The proposed implementation of !MPACT is shown in Figure 5: immune cells will be harvested from the patient, will be genetically engineered with the GEMS platform, and multiplied, after which the cells will be injected back into the patient's body. Before !MPACT can be implemented in the real world, the current proof of concept could be optimized and the treatment should be developed. The development process of the therapy includes performing animal and clinical trials, large-scale production, market authorization, and ensuring reimbursement of the therapy in the basic package of the health insurance (see Proposed Implementation).
!MPACT is currently designed for ANCA-associated vasculitis, however, the modularity of the innovative platform technology offers great potential for this treatment to be applicable to multiple autoimmune diseases. With 400 million people suffering from autoimmune diseases, our technology can eventually help many people and it, therefore, contributes to a healthier future and a reduction in healthcare workloads.
As the activation of the synthetic receptor depends on the autoantibody concentration, the therapeutic action will be adjusted to the disease activity. Besides, the goal is that the therapeutic agent IL-10 will be produced locally at the site of inflammation. Consequently, it is expected that fewer side effects will occur compared to the current non-specific immunosuppressive treatments. On the Proposed Implementation page, suggestions are made to achieve the local action of !MPACT.
Ideally, memory cells of the engineered therapeutic cells will be formed to increase the persistence of the therapy in the body. These memory cells can remain in a resting state for years in the human body.16 The memory cells can be activated when a relapse of the disease occurs. This way, the relapse of the disease is prevented before it gets problematic. More information about the memory cells and the implementation of !MPACT can be found on the Proposed Implementation wiki page.
Since !MPACT will prevent relapses of the disease, it will prevent further health damage and multimorbidity. Consequently, less hospitalization is needed, resulting in lower healthcare expenditures and workloads.
In order to be able to make a societal impact, it is important to validate the problem and the designed solution with stakeholders. Our goal was therefore to involve many relevant stakeholders, including patients, patient foundations, University Medical Centers, hospitals, and pharmaceutical companies throughout the entire project. All input from stakeholders and how we implemented their feedback in our project can be found on the Human Practices wiki page.
Since synthetic biology is not yet very well-known at our university and beyond, our goal was to create more awareness for iGEM and synthetic biology in general. Therefore, we focused on public outreach both within our university and outside. This goal is realized by presenting iGEM and the field of synthetic biology in several presentations, social media posts, and publications (see Communication). Besides, we engaged with many stakeholders as well as the general public in our project (see Human Practices, Education, and Partnership). Furthermore, we organized three educational activities about synthetic biology: lessons at elementary schools, a grand Challenge Day for more than 100 High School students, and a workshop and lecture for students at our university (see Education).
The iGEM Competition is growing and getting more professional, as well as the student teams at the Eindhoven University of Technology. We aimed to follow that trend and wanted to professionalize in order to be able to address and inspire a larger audience to make synthetic biology more well-known. To achieve this goal, we improved the branding of our team, including the use of high-quality media in our outreach (see Communication). Furthermore, the clear organization within our team allowed us to work efficiently and goal-driven. Moreover, we involved relevant experts in our project that together cover all aspects of our project (see Human Practices).
Collaborating with others can give new ideas and insights which can benefit the project. Our goal was to collaborate with other iGEM teams, our university, and stakeholders, as well as to stimulate others to collaborate. For example, we did not only organize two iGEM meetups to stimulate collaborations between iGEM teams, but we also stimulated collaborations between our partner companies, such as RiboPro, and the university (see Collaborations and Communication).
This year’s iGEM TU Eindhoven team focused a lot on getting new sponsorships in order to have enough financial resources to realize all aspects of our project. Finding companies that sponsor our team increases public awareness of the potential of synthetic biology and it indicates that people have faith in the team and project to be successful. All sponsoring collaborations can be found on the page Sponsors.
To make a real societal impact, our goal is to create a startup out of !MPACT after the iGEM competition. This way, the innovation will be further developed and !MPACT may get on the market eventually. We can thereby help patients suffering from AAV as well as reduce healthcare expenditures and workloads. Therefore, we developed a valid business plan for !MPACT (see Entrepreneurship).