Cancer is among the leading causes of death worldwide. In 2022, there were almost 20 million new cases and 9.7 million cancer-related deaths worldwide. By 2040, the number of new cases is expected to rise to 29.9 million, while the number of related deaths is expected to rise to 15.3 million . Approximately 40.5% of men and women is diagnosed with cancer at some point during their lifetimes .

Lung cancer is responsible for the highest number of cancer-related deaths among both men and women, accounting for approximately 28% of all cancer fatalities non-small cell lung cancer (NSCLC) comprises about 85% of all lung cancer cases. This type of cancer can be divided into two main subtypes: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUAD accounts for approximately 50-60% of NSCLC cases, while LUSC represents 20-30% of cases . iGEM Eindhoven 2024 focuses on both subtypes within the spectrum of NSCLC. NSCLC is often diagnosed after the cancer has spread to other areas of the body. Early-stage diagnosis is challenging due to the lack of specific symptoms. While smoking is the leading cause of lung cancers, NSCLC is also common in non-smokers, women, and younger patients .

The facts about NSCLC are summarized in Figure 1.

Surgery is the first-line treatment for NSCLC. However, by the time symptoms appear and diagnosis occurs, treatment options are more limited and less effective . In the advanced stages, NSCLC can be treated using a combination of chemotherapy, radiation therapy, immunotherapy, or targeted therapy. The five-year survival rate for lung cancer is only 26.6%, suggesting a lack of targeted and effective therapies . Current treatments can severely impact patients' quality of life and limit their ability to withstand the full course of therapy. The Disability Adjusted Life Years (DALY) lung cancer is 5, meaning that on average, each person affected by the disease losing the equivalent of 5 healthy years . Moreover, the costs for NSCLC patients are notably high, with total mean hospital expenses reaching €33,143 per patient over the course of the disease, contributing to an estimated €69.6 billion global economic burden annually . New therapies have the potential to reduce these costs by improving treatment efficiency, reducing the need for prolonged hospitalizations, and minimizing complications and side effects.

Herein, we provide an overview of the current therapies and shortcomings, highlighting the need for innovative therapies to improve lung cancer prognosis and patient quality of life.

Chemotherapy and radiation therapy, while sometimes effective, do not differentiate between cancerous and normal cells, resulting in widespread toxicity and a host of adverse reactions such as nausea, fatigue, and increased susceptibility to infections . Consequently, there has been a pressing need for more targeted and less harmful treatment approaches.

Targeted therapy works by acting on unique proteins on the cancer cells. However, the high mutation rate in lung cancer cells leads to significant genetic diversity within tumors . This diversity makes it difficult to develop treatments that can effectively target all cancer cells, as some patients demonstrate inherent resistance while others acquire it during therapy.

Immune checkpoint inhibition (ICI) has revolutionized treatment for a wide variety of cancer types, including lung cancer . Checkpoint inhibitors function by blocking specific proteins found on cancer or immune cells that usually signal the immune system to avoid attacking the cancer cells. These proteins act like “do not kill me” signals, which shield the cancer cells from immune destruction. When the checkpoint inhibitor binds to these proteins, it disables this protective signal, allowing the immune system to recognize and attack the cancer cells, as can be seen in Figure 2. Despite the success of these therapies in some patients, recent clinical trials have shown that many cancer patients remain resistant to ICI, even when their cancer cells express high levels of these inhibitory proteins . Certain patients are inherently resistant to ICI, while others develop it as they undergo immunotherapy. The failure of checkpoint inhibition is caused by various factors within the tumor microenvironment (TME), characterized by an immunosuppressive milieu. Antitumor responses of T-cells are hindered by immune inhibitory cells, suppressive cytokines, and metabolic changes . Additionally, the TME often lacks or has dysfunctional key innate immune cells like dendritic cells (DCs) and natural killer (NK) cells . These factors contribute to tumor immune evasion and ineffectiveness of immune checkpoint inhibitors. Besides, checkpoint inhibitors are associated with a wide range of immune-related adverse events because they are not specific for cancer cells. As oncological indications widen, their rare side effects become increasingly visible in clinical practice and impact therapy decisions . Consequently, there is an urgent need for novel therapeutic strategies aimed at activating key innate immune cells, enhancing the overall antitumor response, while minimizing harm to healthy cells .

To overcome the resistance towards current therapies, the iGEM TU Eindhoven team is developing a cancer vaccine called PROMISE: PRecision Oncology - Modular Immunotherapy using Surface-Engineered vesicles. A major challenge in cancer treatment is that there are typically not enough activated immune cells within the tumor microenvironment to effectively target cancer cells, as shown in Figure 3. This weak immune response allows tumors to evade detection and grow unchecked. PROMISE addresses this by leveraging trained immunity, a concept in which the immune system is trained to recognize cancer cells as foreign invaders. By enhancing immune cell activation through this approach, PROMISE empowers the immune system to launch a stronger, more targeted attack on cancer cells. This not only eliminates the cancer but also provides long-term protection against recurrence or relapse. Additionally, because the vaccine precisely targets cancer cells, damage to healthy cells is minimal, resulting in fewer side effects.

Modular Platform and Personalization

Our approach is modular, allowing for customization and versatility across various cancer types and infectious diseases. We have chosen NSCLC to validate our proof of concept due to its high mutational burden, which provides an abundance of neoantigens that can be targeted. This cancer type also has significant unmet therapeutic needs, especially in advanced stages where current treatments show limited effectiveness. NSCLC prevalence and high mortality rates further justify our focus, aiming to impact patient's life. For more detailed information, please refer to our market research on the entrepreneurship page.

Our modular cancer vaccine offers two strategies to maximize efficacy across NSCLC patients: the general and the personalized approach (Figure 7). We can identify a cocktail of antigens prevalent in a large percentage of NSCLC cases to develop a general vaccine. This vaccine can be widely administered, reducing costs and expediting treatment. However, for patients with unique expression profiles and specific mutations, we will employ a personalized pipeline, involving detailed tumor profiling, to target the individual’s unique cancer antigens. This combined strategy ensures personalized care, optimizing treatment outcomes for each NSCLC patient.

Our technology, PROMISE, uses BMVs to display neoantigens on their surface. Each individual has a different expression profile, suggesting that each type of cancer is different. Therefore, we created a modular platform consists of engineered BMVs, including a Spycatcher functionalization and a clickable neoantigen functionalized with a Spytag (Figure 9). With this system, we aim at facilitating the platform's functionalization with different antigens for personalized medicine.

1. Overcoming therapy resistance: PROMISE is composed of antigen-functionalized BMVs derived from BCG. This vaccine stimulates innate, adaptive and trained immunity, effectively priming immune cells to recognize and eliminate cancer cells.
2. Reduced side-effects: PROMISE aims to specifically target cancer cells, which reduces the likelihood of collateral damage and the associated side effects commonly seen with traditional cancer therapies.
3. Long-lasting protection:The antigen-functionalized BMVs derived from BCG create memory in both innate and adaptive immune-cells.
4. Modular platform: PROMISE is highly customizable to other cancer types or immune-mediated diseases because of the modular system used to display disease-specific antigens.
5. Personalization: PROMISE provides flexibility in treatment: we develop a general vaccine platform, while also generating a personalized pipeline tailored to the individual patient profile.

The mentioned benefits are validated using expert opinions from different stakeholders from companies like Johnson & Johnson, MSD (Merck), and ThermoFisher. More about this can be found on our Human Practices page.

Our team used a comprehensive and iterative brainstorming process, exploring a wide range of ideas from sustainability to diagnostics and therapeutics. We utilized diverse brainstorming techniques, prioritizing quantity over quality through methods such as mind mapping, brainwriting, literature research, and examining past iGEM team projects. This process allowed us to generate numerous potential project ideas.

Our brainstorming sessions adopted two fundamental approaches: ‘technology push’ and ‘market pull’. In the ‘technology push’ approach, we identified existing technologies and explored ways to enhance them, subsequently defining applications and markets for their use. Conversely, the ‘market pull’ approach started with identifying market needs or problems, such as diseases lacking effective treatments, and then searching for suitable technological solutions. For this process, we received help from experts within the TU/e contest. More information can be found on the communication page under events. After generating and refining our ideas, we engaged with professors and researchers at TU/e to assess the feasibility and potential impact of each concept. Through these discussions, we narrowed our focus to three leading ideas related to diagnostics and therapeutics: bacterial components for tumor therapy, intracellular protein delivery to tumor cells, and antibiotic resistance in bacteria. Each idea was inspired by recent literature articles and aligned with the expertise of some of our team members.

We ultimately chose the bacterial components for tumor therapy project due to its innovative nature and the alignment with our team's interests and the university's resources. This idea stood out as the most promising and feasible within the competition's timeframe. We validated our project choice through further consultations with research experts and business advisors, ensuring it had strong potential for scientific and entrepreneurial success. These conversations can be found on our Human Practices page.

In developing our trained immunity vaccine, PROMISE, we have made significant progress while also identifying key areas for further improvement. In the wet lab, we successfully isolated BMVs from both E. coli BL21 and M. smegmatis. We confirmed the presence of the membrane protein OmpA(N21) fused to GFP in BL21 cells using antibody staining and FACS analysis, proving successful plasmid transformation and fusion protein expression on the surface of the bacterial cell. More information can be found on our Lab page. In the future, we aim to show the same results for functionalizing M. smegmatis using genetic engineering. After this, we will focus on characterizing the presence of these fusion protein in BMVs post-isolation. In parallel, we are still exploring the post-insertion as functionalization method, for which we have successfully developed the necessary (fusion) proteins, and are currently optimizing micelle-stabilization processes. In the future, we aim to refine these protocols and scale them up for use with M. bovis BCG, advancing towards a functional trained immunity vaccine.

On the computational side, our dry lab developed an end-to-end pipeline that predicts tumor neoantigens from DNA sequence data, informing the design of our vaccine. More information can be found on our Modeling page. While the current model focuses on single nucleotide mutatons, we plan to incorporate structural variations, which often generate highly immunogenic neoantigens. Furthermore, we want to give more attention to the user-friendliness of the software As we progress, experimental validation of these predictions will be crucial. Additionally, we see potential in using our platform to develop a broader-spectrum vaccine by identifying antigens common across multiple patient profiles.

Looking ahead, our long-term goal is to advance PROMISE from the lab to clinical application. We are laying the groundwork for preclinical and clinical trials. More information can be found on our Human Practices page. We have secured our intellectual property through a provisional patent, ensuring a pathway for commercialization. The full business plan can be seen on our Entrepreneurship page. Our modular platform is designed to be adaptable, meaning once approved for one type of cancer, it can be expanded to treat others and potentially even other immune-mediated diseases.

In conclusion, while we have made exciting progress, there is much more to explore. We are committed to continuing this journey, collaborating with other iGEM teams, and pushing the boundaries of immunotherapy. The future of PROMISE is bright, and we are eager to see where it leads!