Ensuring Safe and Responsible Development of Our Synthetic Immune Cell Platform
We aim to develop not only a cell therapy, but a modular toolkit to treat a wide variety of cancers. The sensing of specific molecular markers and the release of therapeutically active proteins requires high safety standards for potential clinical applications while also presenting challenges in preventing system misuse by malicious actors. Below, we explore strategies we have adopted or plan to incorporate to ensure the safe use of our technology.
It is important to note that our system remains in early development stages, utilizing only transient transfection methods. We have not yet produced stable cell lines that would pose heightened safety concerns. Additionally, the HEK293T cells used in our current validation studies have limited viability outside controlled laboratory conditions.
Identifying and Mitigating Potential Risks
Risk 1: Systemic Overactivation and Cytokine Release Syndrome
Cell therapies can trigger severe immune overreactions, particularly cytokine release syndrome (CRS), a potentially life-threatening condition and immune effector cell-associated neurotoxicity syndrome (ICANS). These conditions occur in a large fraction of patients with significant toxicities (Morris et al., 2022), the reason being that activated therapeutic cells lead to uncontrolled cytokine release and systemic inflammation.
Mitigation Strategy
Our dual-input system addresses this fundamental limitation of currently approved cell therapies. By implementing an inhibitory receptor which counteracts premature system activation, we prevent unchecked responses in non-target tissues. Unlike conventional cell therapies, our system can deactivate following initial activation, providing an additional safety layer. Additionally, we are exploring another safety mechanism: administering a systemic negative ligand that binds our inhibitory receptor throughout the body. By fine-tuning the dosage, positive signals within the tumor microenvironment (TME) would still outweigh this baseline inhibition, ensuring activation occurs only where intended.
To provide a fail-safe mechanism complementing these regulatory features, we plan to incorporate an inducible safety switch based on Caspase-9 activation: The kill-switch consists of inducible Caspase9 (iCasp9) linked to the DmrB protein. Upon administration of a Chemical Inducer of Dimerization (CID) (AP20187 or AP1903), DmrB dimerization brings two Casp9 molecules into proximity, triggering activation. Once activated, iCasp9 initiates Caspase-3, which subsequently cleaves a broad spectrum of cellular proteins, driving cells into irreversible apoptosis (Yuan et al., 2021). While our dual-input architecture is designed to prevent uncontrolled activation, this inducible kill-switch provides an orthogonal safety layer—enabling rapid termination of all engineered cells via CID administration in case unexpected systemic toxicity occurs, despite the primary regulatory controls.
Figure 1: Rapalog-inducible cellular kill switch. To ensure cell therapy safety, an orthogonal rapalog-inducible kill switch can be used to induce apoptosis in administered cellular therapeutics in case of adverse events. Rapalog AP20187 induces is engaged by DmrB-fused Casp9, which dimerizes and activates Caspase3. This initiates apoptotic signaling in cellular vehicles.
Risk 2: Dual-Use Potential and Malicious Reprogramming
Our system functions as a precise biosensor capable of targeting diverse biomarkers. This versatility, while therapeutically valuable, could theoretically be exploited to target healthy cells, essential tissue markers, or specific genetic polymorphisms. The modular design that enables therapeutic flexibility could facilitate harmful reconfiguration or payload substitution with toxic agents.
Mitigation Strategy
We maintain close collaboration with regulatory bodies and biosafety oversight committees to ensure responsible development. Importantly, we utilize primarily published, well-characterized components rather than developing novel elements with unknown safety profiles. The few novel sequences we generate (such as GDF-15 binding domains) undergo rigorous biosafety screening.
Our safety architecture integrates the kill switch as an essential system component which is coupled to our synthetic substrate, making it technically challenging to remove without disrupting core functionality. Future iterations of our design software will implement strict whitelisting protocols, restricting target selection to validated therapeutic markers and preventing potentially harmful configurations (Hoffmann et al., 2023).
Risk 3: Environmental Release and Containment Breach
Concerns about engineered cells escaping clinical settings or deliberate environmental release require careful consideration.
Mitigation Strategy
Our therapeutic cells are inherently self-limiting. We employ exclusively ex vivo expansion protocols without in vivo amplification mechanisms such as viral vectors. The cells require specific laboratory conditions for survival and expansion, making environmental persistence highly unlikely. Clinical application involves careful expansion under controlled conditions before patient administration, eliminating risks associated with self-replicating biological agents (Wang et al., 2025)
Addressing Healthcare Equity
Current cell therapies impose significant financial burdens, with CAR-T treatments often exceeding $400,000 per patient (Di et al., 2024).
Our Approach: By developing a modular platform adaptable to multiple cancer types, we aim to reduce development costs and streamline manufacturing processes. This standardization could substantially lower treatment costs, improving accessibility to advanced cell therapies across diverse patient populations.
Ethical Engagement and Informed Consent
Throughout our project, we prioritized ethical responsibility and safety in all our human practices activities. Recognizing the sensitive nature of cancer research, we made a deliberate choice to avoid direct engagement with cancer patients. Without professional training in counseling or patient support, we understood the potential risks of inadvertently causing emotional harm or creating unrealistic expectations. To ensure meaningful and responsible stakeholder engagement, we focused our conversations on cancer patient advocates and healthcare professionals who possess the expertise and experience to represent patient perspectives appropriately.
Informed consent formed a cornerstone of our ethical framework. We ensured that all interview participants fully understood how their contributions would be used before any information appeared on our wiki or in project materials. Each participant provided explicit agreement for the publication of their insights. This careful approach reflects our commitment to upholding the dignity, autonomy, and well-being of every individual involved in our research, ensuring that our outreach efforts remained both impactful and ethically sound.
Continuous Safety Evaluation
As our project progresses from proof-of-concept to potential therapeutic application, we remain committed to ongoing safety assessment and stakeholder engagement. Regular consultation with biosafety experts, ethicists, and regulatory authorities, as displayed on our Human Practices page, ensures our development pathway prioritizes both innovation and responsible implementation.
Safety in Our Laboratory
Facility and Regulatory Compliance
Our laboratory work was conducted at the Students' Laboratory at BioQuant in Heidelberg, with all activities strictly adhering to German national biosafety regulations and European Union standards. The laboratory maintains BSL-1 classification according to the German Biological Agents Ordinance (Biostoffverordnung).
Safety Training and Personnel Requirements
Prior to commencing any laboratory work, all team members completed mandatory biosafety training conducted by Prof. Stefan Wölfl and technical assistant Nina Beil. This comprehensive training covered emergency response procedures and fire safety protocols, proper use of safety equipment and protective gear, handling procedures for biological materials and hazardous chemicals, as well as waste management and decontamination protocols. To ensure continuous safety oversight, we implemented a buddy system requiring at least two team members to be present during all experimental procedures.
Personal Protective Equipment and Laboratory Conduct
Our safety protocols mandated wearing laboratory coats, gloves, and safety glasses at all times during laboratory work. Additionally, closed-toe footwear and full-length clothing were required, and personnel with long hair were required to keep it secured. We maintained a strict prohibition of food, beverages, and cosmetics in all laboratory spaces. The facility features proper ventilation systems meeting regulatory requirements, ensuring a safe working environment for all personnel.
Equipment Safety Measures
We implemented several equipment-specific safety protocols throughout our work. For cell counting, we utilized an automated cell counter instead of traditional hemocytometers, eliminating the risk of glass slide breakage and potential exposure to biological materials. We hope that this automated cell counter will serve as a lasting contribution that enables future iGEM teams in Heidelberg to work not only more efficiently but also more safely. Team members operating larger volume centrifuges, high-speed centrifuges, and ultracentrifuges received specialized training to ensure proper balancing, secure closure, and safe operation of these instruments.
Chemical Safety and Hazard Mitigation
Our approach to chemical safety included several important substitutions and precautions to minimize risks. We chose AP21976 over traditional rapamycin due to its lack of mTOR inhibitory activity, thereby reducing immunosuppressive risks to laboratory personnel. Following biosafety committee recommendations regarding TNF-alpha handling, we were advised that pregnant individuals should not handle this cytokine. Though no team members were pregnant during our project, we maintained awareness of this important safety consideration.
In our desinfection protocols, we transitioned from Antifect N to Bacillol following university hygiene guidelines, using pour methods rather than spray applications to prevent harmful aerosol formation that could damage lung tissue. For DNA visualization, we replaced ethidium bromide with the safer SYBR Safe alternative and established a dedicated gel electrophoresis station to contain any potential contamination.
For experiments involving hypoxia induction using cobalt chloride, we implemented enhanced safety measures including performing all work under fume hoods with respiratory protection, utilizing a double-gloving protocol with glove changes after stock solution preparation, and ensuring careful waste segregation and disposal. Similarly, microscopy preparations involving paraformaldehyde fixation were conducted exclusively under fume hoods with multiple PBS washing steps to minimize exposure risks.
Mammalian Cell Culture Safety
All team members working with mammalian cell cultures received appropriate training specific to cell culture techniques and safety requirements. Cell culture work was performed in the BSL-1 facility at BioQuant under vertical laminar flow hoods to maintain sterility and prevent contamination. As we exclusively worked with the well-characterized HEK293T cell line, we avoided the additional S2-level requirements associated with primary human cells, allowing us to maintain standard S1 safety protocols throughout our experiments.
Waste Management
Our waste disposal procedures complied with German and EU regulations through systematic segregation and treatment of different waste types. Biological waste was segregated and autoclaved before disposal, while chemical waste was collected in appropriate containers according to hazard classification. Sharp objects, including syringes and glass shards, were collected in specialized puncture-resistant containers to prevent cutting hazards. Trained BioQuant personnel supervised all waste management processes, and special procedures were implemented for cytotoxic and mutagenic substances to ensure safe disposal.
Continuous Safety Improvement
Throughout our project, we maintained regular communication with the university biosafety committee and our principal investigator to ensure our protocols remained current and appropriate for our experimental needs. This collaborative approach allowed us to adapt our safety measures as our project evolved while maintaining the highest safety standards.
- Di, M., Potnis, K. C., Long, J. B., Isufi, I., Foss, F., Seropian, S., Gross, C. P., & Huntington, S. F. (2024). Costs of care during chimeric antigen receptor T-cell therapy in relapsed or refractory B-cell lymphomas. JNCI Cancer Spectrum, 8(4), pkae059. https://doi.org/10.1093/jncics/pkae059
- Hoffmann, S. A., Diggans, J., Densmore, D., Dai, J., Knight, T., Leproust, E., Boeke, J. D., Wheeler, N., & Cai, Y. (2023). Safety by design: Biosafety and biosecurity in the age of synthetic genomics. iScience, 26(3), 106165. https://doi.org/10.1016/j.isci.2023.106165
- Morris, E. C., Neelapu, S. S., Giavridis, T., & Sadelain, M. (2022). Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy. Nature Reviews Immunology, 22(2), 85–96. https://doi.org/10.1038/s41577-021-00547-6
- Wang, S., Li, X., Ning, H., & Wu, Y. (2025). Dual-target CAR-T cell therapy: A new strategy to improve the therapeutic effect of hematological malignancies. Critical Reviews in Oncology/Hematology, 214, 104824. https://doi.org/10.1016/j.critrevonc.2025.104824
- Yuan, Y., Ren, H., Li, Y., Qin, S., Yang, X., & Tang, C. (2022). Cell-to-cell variability in inducible Caspase9-mediated cell death. Cell Death & Disease, 13(1), 34. https://doi.org/10.1038/s41419-021-04468-z