Proposed
Implementation

Discussions with relevant stakeholders such as University Medical Centers, the Catherina Hospital, Novartis, RIVM (National Institute for Public Health and the Environment) and MedTech company CiMaas, yielded some suggestions to further improve the design of !MPACT beyond the scope of this year's iGEM project. The complete input of these stakeholders in our project can be found on the Integrated Human Practices page. Their suggestions for future research make !MPACT potentially safer and more effective. The three suggestions we have investigated include:

1. The type of immune cells that should be harvested from the patient for our cell therapy against anti-neutrophil cytoplasmic antibody (ANCA)-associated Vasculitis (AAV).
2. The inhibition of our engineered cells.
3. The local production of interleukin 10 (IL-10).

For a complete understanding of our future research proposal, we advise you to read our Project description.

Cell type

To make a well-grounded decision about the immune cell type we should use for !MPACT, it is essential to understand the complete treatment procedure. The implemented treatment of !MPACT (Figure 2) starts by collecting immune cells from the patient by a clinician. The cells will be transported to the manufacturing laboratory, where the cells will be genetically engineered to incorporate the genes for the GEMS (Generalized extracellular molecule sensor platform), and IL-10 production.¹ The expansion of cells is followed by the injection of the modified cells into the patient. Lastly, the cells will interact with the pathogenic autoantibodies of AAV (ANCAs) and secrete locally and temporarily IL-10 to treat AAV symptoms. To learn more about the underlying mechanism of activation of our engineered cells and subsequent IL-10 production, you can read the our Project description.

The choice of which immune cell type to harvest from the patient is a difficult decision. From the interviews with Erasmus Medical Center and CiMaas two different options emerged; T cells or B cells. T cells are essential players in the adaptive immune system and are developed from stem cells in the bone marrow.² They express highly polymorphic antigen receptors through which they are able to recognize foreign body cell-associated antigens.² Recognition of antigens by T cells results in clonal amplification and the acquest of effector functions that range from producing soluble paracrine factors to "help" other immune cells to the capacity of killing pathogen-infected cells.² B cells are just like T cells, also crucial actors in the adaptive immune system.³ They are developed from lymphoid progenitors in the bone marrow and once matured they are able to travel through the bloodstream and the lymphatic system.³ They have the ability to differentiate in plasma cells once activated through antigen binding and subsequently produce and secrete millions of different antibodies against specific pathogens.³

Both B cells and T cells have advantages and disadvantages to be used for !MPACT. We made an appropriate assessment of which cells to use, combining the stakeholders' feedback and information from relevant literature. An overview of this assessment can be found in Table 1.

Table 1 | Cell type overview. Summary of the advantages and disadvantages of T and B cells as immune cells for !MPACT.

	T-cells	B-cells
Natural producer of IL-10	+	0
Mobility	+	0
Isolation	+	+
Activation and memory cell production	0	+
Current therapies	+	-

T helper 2 (Th2) cells are a subset of T cells that naturally produce IL-10.⁴ This means that after the isolation of Th2 cells from the patient, genes for IL-10 production are no longer required to be inserted in the genome of the cells. Moreover, Th2-engineered cells are able to natively fold and excrete IL-10, as was also confirmed by dr. Reno Debets from Erasmus Medical Center.⁴ Only a specific subset of B cells, regulatory B (Breg) cells are able to produce IL-10 in specific contexts and are thus not considered a major source of IL-10.⁵ Since T helper cells are considered better natural producers of IL-10, they are preferred for the design of !MPACT over B cells considering IL-10 production.

Secondly, T cells are much more used in cell therapies, such as Chimeric Antigen Receptor (CAR)-T cell therapy for various cancer types such as lymphomas and leukemias.⁶ Advances in the selection of optimal T-cell types, their genetic modification, and the underlying mechanisms of action of T cells therapies foster new applications for autoimmune therapies.⁷ For example engineered T cells are successfully used to dampen immune responses in mice models.⁸ Moreover, the RIVM and EMA pointed out that obtaining licenses for the use of T-cell therapies outside the lab is easier because environmental risk analyses of engineered T-cells are already available. On the contrary, B cell modulation as therapy for cancers and especially for autoimmune diseases is still in its infancy phase.^9,10 No B cell therapies are on the market and only a few early clinical trials for B cell therapies against cancers have been conducted.⁹ One of the main problems is the generation of good manufacturing practice (GMP) for B-cell immunotherapies, which still face major obstacles to overcome.⁹ In addition, targeting specific effector functions of B cells to treat auto-immune diseases requires further research.¹⁰ Considering the ability to genetically modify T cells by the generation of good manufacturing practice and because of a better understanding of the underlying mechanism of T cells damping immune responses compared to B cells, T cells have a significant advantage for implementation in !MPACT.

T cells as well as B cells are considered mobile since they must migrate throughout the body to recognize antigens.^11,12 They can easily shift from lymphoid organs to target organs.^11,12 Extensive migration is required for T cells responses as they should have the ability to detect antigens at the surface of infected cells in specific organs and because they are involved in interaction with other immune cells.¹¹ B cells also critically depend on mobility for their functional activity. For antigen recognition, B cells are required to travel from the blood to secondary lymphoid organs to "patrol" for antigens.¹² Large mobility of the engineered cells of !MPACT is a critical requirement to get the cells locally at the site of inflammation in the small blood vessels in case of AAV.¹³ The fact that both B and T cells are considered mobile and have the ability to circulate in the blood, does not result in a preference for one of these cells considering mobility.

Both B cells and T cells are isolated from patients via apheresis, a process in which blood from the patient is guided through a cell separator to extract the cells of interest.¹⁴ Since both cell types can be obtained in high purity using efficient protocols, the isolation procedure is not resulting in a preference for one cell type over the other.^15,16

For !MPACT to be commercially viable it is preferred that the therapy has high persistence (years) and preferable only one injection is required. Persistence is defined by Cremer et al. as "the extent to which a patient acts in accordance with the prescribed interval, and dose of a dosing regimen".¹⁷ Thereby, high persistence of !MPACT results in the engineered cells being long enough in the human body to prevent possible relapses of AAV through the reactivation of the engineered cells. For high persistence, the formation of memory cells is required, as was explained by MedTech company CiMaas. The formation of long-lasting T cells involves a complex set of events and requires multiple signaling pathways activated.¹⁸ The antigen interaction with the T cell receptor (TCR) is not sufficient because also inflammation and costimulation are necessary for T cell memory.¹⁹ After activation proliferation and massive apoptosis of T cells is required after which only the cells that survive form the memory T cells.¹⁸ Memory B cells, on the other hand, are relatively easily formed through binding T- dependent antigens.²⁰ T-dependent antigens are proteins with multiple epitopes.²⁰ An example of a protein with multiple epitopes is an antibody that is also required to activate !MPACT. Binding T-dependent antigens activate the germinal center (GC) pathway resulting in the activation of B cells (into antibody-producing plasma cells) and in parallel the formation of memory B cells.^20,21 The GC is a microstructure in the secondary lymphoid tissue where the antibody-secreting plasma cells and memory B cells are formed.22 After the formation of memory B cells, they have acquired migration properties to travel to the spleen and lymph nodes.²¹ They can get activated again by T-dependent antigens to form plasma cells.²¹ Because for memory B cell formation only T-dependent antigen binding is required, it is expected that it is easier to connect the binding of antibodies to the formation of memory B cells compared to memory T cells.

In conclusion, both cell types have advantages and disadvantages and follow-up in vitro research is necessary to determine which cell type should be harvested from the patient to produce !MPACT. Based on our literature studies, we would suggest first investigating the possibility of T-cells since GMP production of engineered T cells is optimized, T cell therapies are already on the market, they are major natural producers of IL-10, have high mobility, and there's much information available to genetically manipulate them to give them a required functionality. The only disadvantage of engineering T cells to produce !MPACT, is that formation of memory cells is more complicated than with B cells, so we will investigate whether sufficient T cell memory is formed when activating the engineered T cells with antibodies.

Inhibition system

Multiple academical stakeholders such as University Medical Centers, the Catherina Hospital, and pharmaceutical company Novartis pointed out that the immune system is tightly regulated and a small difference in IL-10 concentration could lead to dysregulation of the immune system or to cytokine release syndrome (CRS). CRS is a massive production of cytokines leading to systemic inflammation and is often encountered during the administration of CAR-T cell therapies.²³ The pathophysiology of CRS is only partly understood and is induced by massive T cell activation and subsequent activation of other immune cells and endothelial cells that produce large amounts of cytokines.²³ The academic hospitals, therefore, advised us to look for a way to inhibit T cell activation and subsequent IL-10 production. Moreover, the RIVM (National Institute for Public Health and the Environment) explained the possible consequences of GMOs on the environment or public health. As GMOs are living organisms, they have the potential to mutate and evolve undesirable traits such as increased stability, toxicity, uncontrollable cell division, and allergenicity over the course of the treatment.²⁴ This could disturb biodiversity or form a danger to human health.²⁴ Hence, also the RIVM advised us to design !MPACT in a way such that it can externally be inhibited (e.g. by adding a chemical) to stop the production of IL-10 and if necessary kill the engineered cells.

Our solution to inhibit IL-10 production is by killing engineered cells that produce it in a dose-dependent manner by adding a small molecule. This can be achieved by a so-called “suicide gene transfer”. Suicide gene transfer is a strategy in which apoptosis-inducing transgenes are introduced in our engineered cells.²⁵
For an appropriate suicide gene transfer that could be used in human cells, we looked at current methods to regulate and control CAR-T cells. There are three families of suicide gene technologies available in the medical industry, depending on their mode of action; metabolic, dimerization-inducing, and therapeutic monoclonal antibody-mediated.²⁶ A metabolic mode of action converts a non-toxic compound into a toxic one resulting in cell death. Dimerization-inducing is activating apoptosis in the cell by dimerizing proteins involved in apoptosis (e.g. Caspases), and monoclonal antibody-mediated uses protein expression in the plasma membrane of the engineered cells to remove the cells after administering a specific monoclonal antibody.²⁶ For use in the human body, the suicide gene activating agent should be biologically inert, have sufficient bio-availability and appropriate bio-distribution profiles and it should not be associated with toxicity or adverse events.²⁶ Dimerization-inducing apoptosis with the administration of AP1903 (a chemical protein dimerizer for FKBP protein systems) does fulfill all these requirements since AP1903 is found safe and well tolerated at high doses in healthy human subjects and has a favorable pharmacokinetic profile.²⁷
Moreover, this mechanism is compared to the other two mechanisms (metabolic and therapeutic monoclonal antibody-mediated) controllable in a dose-dependent manner, which makes this the best option to control IL-10 production of !MPACT.²⁸ The dimerization-inducing apoptosis approach works via apoptotic genes such as Caspases that eliminate cells by inducing apoptosis (“killswitch”) with the addition of the small dimerizing molecule AP1903.²⁶ The suicide gene for the dimerizing-inducing approach encodes for chimeric proteins that have an AP1903 domain (drug binding domain) linked in frame with Caspases of the apoptotic pathway. Upon dimerization of the drug binding by adding AP1903, apoptosis of the engineered cells is induced.

The literature describes the use of dimerization by iCasp9 which contains a FKBP12-F36V binding domain that dimerizes and activates the intrinsic mitochondrial apoptotic pathway upon administration of AP1903 (Figure 3).²⁶ Recently, this system is improved to a regulatable system where the T-cell survival can be controlled in a drug-dependent manner.²⁸ This allows controlling the rate of survival of our engineered cells administered and therewith the amount of IL-10 production. The method described serves as the solution for a regulatable killswitch for !MPACT to mitigate possible adverse advents (e.g. CRS) and to control IL-10 production.
In conclusion, we demonstrated the need for a killswitch in our system and we will perform future research to investigate the effect of the regulatable killswitch described on the IL-10 production and cell survival of !MPACT.

Localization

AAV is associated with inflammation and necrotizing of small blood vessels.¹³ Acute vascular inflammation is induced when ANCA antibodies bind the ANCA antigens on neutrophils. As a consequence, neutrophils get activated and adhere to blood vessel walls. They penetrate into the vascular wall where they release toxic oxygen radicals and enzymes that result in apoptosis of endothelial cells and vessel wall matrix.²⁹ To efficiently reduce inflammation with !MPACT, University Medical Centers explained that local production of IL-10 at the site of inflammation of the small blood vessels is preferred. Local production ensures that IL-10 is immediately at the site where it needs to have its effect. Subsequently, a lower amount of IL-10 needs to be excreted by our engineered cells, and the toxicity and adverse advents of the therapy decrease. To increase the localization of the engineered cells at the site of inflammation we will induce interaction between the engineered cells of !MPACT and the damaged endothelial cells. Literature states that during AAV flare-ups, major histocompatibility complex class I chain-related protein A (MICA) is upregulated at the cell membrane of vascular endothelial cells.³⁰ These recognition elements on the surface of endothelial cells can be used to stimulate interaction with a receptor on the engineered cells of !MPACT to upregulate the recruitment of our engineered cells to the site of inflammation (Figure 4).³⁰ Localization of our cell therapy is also an interesting topic we will optimize in future research.

To conclude, we aim to optimize the design of !MPACT to make it more effective, feasible, and responsibly based on the advice of the relevant stakeholders we approached. We will investigate the option of T cells as immune cells we will harvest from the patient in order to produce !MPACT. Moreover, we will implement a regulatable killswitch to control IL-10 production and the survival of the engineered cells, and lastly, we will improve the local production of IL-10 at the site of the inflammation. The optimized design will be legally protected as will be discussed in the next step of the drug development process.

The previous section described optimizations in the design of !MPACT for future research. As soon as the first results of the optimized therapy and technology are obtained, we will file a patent for the complete proof of concept of !MPACT. Discussions with stakeholders such as The Gate, made us realize the importance of legal protection of the technology underlying !MPACT our (Integrated Human Practices). They explained that protection is necessary to implement the therapy in the real world, as a patent makes it unlawful for others to develop, use, resell, rent out, or supply the patented cell therapy for a minimum of twenty years. Moreover, having legal protection on the technology is essential to defray the high development costs. Furthermore, the Gate stressed that patenting a system or technology requires that it should be new, not obvious, and capable of industrial applicability. Since !MPACT builds upon state-of-the-art research and applies a new conceptual cell therapy to a new application (disease) for industrial use, it is validated that !MPACT is patentable. Lastly, we learned that it is of crucial importance to not disclose the details of the technology that we aim to patent. Once disclosed to the general public without a non-disclosure agreement it becomes very hard to patent new technology. In this section, we will discuss the necessary steps to obtain a patent to protect the technology, as well as our plan for how we aim to tackle this.

The procedure of a patent application starts by filling out an application form. In addition, a patent document is required, including a description of the invention.³¹ As explained in our business plan which can be found on the Entrepreneurship wiki page, we will apply for a patent application in the United States of America and Europe. These applications will be filed after the Grand Jamboree as soon as possible and the patents should get extended each time new developments in the technology are made. Through the United States patent and trademark office (USPTO), we will apply for a utility patent which generally expires 20 years after the application filing date. Simultaneously, we will apply for a European patent because it is an easier and cheaper alternative for obtaining individual patents in countries that are members of the European Patent Convention (EPC).³² This patent is also valid for 20 years.³² The simplified timeline of a European patent application is shown in Figure 5.

The claims included in the patent application contain are based on the proof of concept of !MPACT extended with the proposed optimizations for !MPACT in technical detail. Since !MPACT is considered a modular platform technology, we aim to adjust !MPACT in the future to be applicable for other autoimmune diseases. For each new disease, the patent application will start immediately after the generation of the first results in the R&D phase.

DISCLAIMER

Although we have reached out to experts for legal protection of the technology of !MPACT, we did not manage to achieve a patent for !MPACT before the Grand Jamboree. We have decided to disclose the technology of !MPACT during the Grand Jamboree and with relevant stakeholders such that we can present our project in the best way possible. Moreover, we wanted to get the most relevant feedback on how to improve our design to make it more feasible, desirable, and responsible. On contrary, as discussed in Section 1. Optimizations and outlook, we aim to expand and optimize the mechanism underlying !MPACT after the Grand Jamboree even further. Through non-disclosure agreements, we will discuss with relevant stakeholders on the technical details of these optimizations and file a utility patent for the improved design through the process described. Our experience has taught us the value of intellectual property, and we would advise future iGEM teams to consider legal protection as early as possible in the project.

More information about legally protecting !MPACT and our business plan can be found on the Entrepreneurship wiki page.

After future in-vitro research to optimize !MPACT and after legal protection of the proof of concept through a utility patent, !MPACT will be subjected to (pre)-clinical trials. There are multiple (pre)-clinical trials that must be successfully conducted before the therapy is eligible for entering the market. The clinical trial process, including its permission and the different clinical trials, are defined through stakeholder interviews (Integrated Human Practices), complemented with literature, and will be discussed in this section.

Overview of clinical trials

Clinical trials are studies to investigate the safety and efficacy of a medicine as defined by the EMA (European Medicine Agency).³³ There are several phases of biomedical clinical trials:

• Pre-clinical trials: In vitro tests of the drug to find out whether the drug has the potential to cause serious harm, i.e. toxicity.³⁴
• Animal trials: Tests on animals to assess the safety and effectiveness of the drug in animals.³⁴

Above described trials test the safety of the drugs in general. Next, studies should be performed to investigate how !MPACT will interact with the human body (Figure 6):

• Phase I trials: To test the drugs for the first time in a small group (20-100) of often healthy people or people with the disease to evaluate a safe dosage range and to identify possible side effects.^35,36 The emphasis is on the safety of the drug and dosage.^36,37
• Phase II trials: These studies have a larger group of human subjects: up to several hundred people with the disease. They are focused on both the efficacy and side effects of the drug.³⁶ This phase is split into two phases: phase IIa and phase IIb. Phase IIa is specifically assigned to determine the dosing requirements and phase IIb is conducted to assess the efficacy of the drug at a certain dose.³⁸
• Phase III trials: Studies on a larger population, including different countries and regions, both focusing on safety and effectiveness. ^35,37 It includes 300 to 3000 participants who have the disease.³⁶ Approval of the drug is often done directly after this phase.^35,37
• Phase IV trials: These take place after approval by the FDA or EMA. The effectiveness and safety of the drug are monitored in large diverse populations (several thousand) for a longer period of time.^35,37

Required permissions

Clinical trials have strict regulations to ensure the protection of the rights, safety, and well-being of trial participants and to ensure that the results of the clinical trials are credible. Clinical trials conducted within the EU should comply with EU clinical trials legislation and have to receive permission from the CCMO (Central Committee on Research Involving Humans) to perform the studies.⁴⁰ In the case of Advanced therapy medicinal products (ATMPs), under which !MPACT is categorized, the minister of public health must give permission as well. To get permission, several dossiers have to be delivered, including, among others, the set-up of the studies, the protocols, and how safety is ensured.⁴¹ After submission of the application dossier, the dossier will be validated which takes a maximum of 25 days.⁴¹ After the validation phase, the assessment period starts which will take up to 76 days and in the case of ATMPs, it can be extended with an additional 50 days for consulting with experts.⁴² Afterwards, each member state draws separate conclusions and a decision is made about the application (see Figure 7).

Next to permission from the CCMO to perform clinical trials, a few other licenses are necessary. For activities with GMOs outside the laboratory, an additional license from the ministry of infrastructure and water management is required for which an environmental risk analysis has to be performed.⁴⁴ Furthermore, a license from the CCD (Central Commission of Animal studies in the Netherlands) is needed to execute animal studies.⁴⁵

In case of the clinical trial application is accepted, the study can be conducted. The conduct of the clinical trial should be monitored to verify the protection of the rights, safety, and well-being of the participants as well as to ensure reliable data.⁴⁶ In addition, intermediate data analysis should be submitted, and safety reports are required.^47,48 Within one year after the end of the clinical trial, a summary of the results should be submitted to the Clinical Trials Information System.⁴⁹ In case the clinical trial is intended to be used to obtain market authorization, also a clinical study report should be submitted.⁵⁰

Safety & challenges

Safety is extremely important during clinical trials. To ensure safety, we learned in the interview with the Centre for Human Drug Research (CHDR) that very low doses should be administered at first and several vital functions should be measured during the study, such as temperature, kidney function, liver function, and an ECG should be made. People are not only screened during the trials but also before and after the trials. Screening is especially important for medicines for autoimmune diseases, like !MPACT, because the CHDR explained that immune response in animal models differs from immune responses in humans. This could result in unexpected adverse events during clinical trials based on predictions of animal test results. The differences between animal and human immune responses oppose a challenge during clinical trials. However, extensive screening during clinical trials overcomes this challenge, as the study can immediately be stopped when unwanted changes are noticed during the screening procedure, the CHDR explained.

Conclusion

After successfully completing all clinical trials, upscaling of the production process is required and market authorization can be requested. However, our team will not perform all clinical trials for !MPACT ourselves. We will perform the clinical trials up and until phase IIa since a successful clinical IIa trial serves as a golden standard for licensing a proof of concept.⁵¹ So after clinical trial IIa, we will license !MPACT to a pharmaceutical company that performs the remaining the clinical trials. For more information on our business plan, you can visit the Entrepreneurship wiki page.

Previously, (pre)-clinical trials were discussed. To perform large clinical trials and to treat even larger populations of patients diagnosed with AAV, production of !MPACT should be scaled out. We discussed with Johnson & Johnson the production facilities for safe and efficient production of !MPACT. In this section, we will describe the production process of cell therapies and the corresponding challenges and safety issues.

Manufacturing cell therapies require multiple steps (Figure 8). Quality controls are performed during the entire process.⁵² The first step in the production of !MPACT is leukapheresis to remove blood from the patient’s body, the leukocytes are separated and the remaining blood is returned to the circulation of the patient.⁵² If a sufficient amount of leukocytes are isolated, the isolated mixture is enriched in the specific immune cell of interest (either B or T cells). This enrichment is accomplished by a multi-step process. First cells are washed out of the leukapheresis buffer. In the second step, counterflow centrifugal elutriation separates cells by size and density but ensures cell viability. By flowing the cells over bead conjugates with specific antibodies, a certain cell type can be isolated. If needed for the desired cell type, the cell can be activated by beads covered with monoclonal antibodies or Antigen Presenting Cells (APCs) together with feeder cells and growth factors.⁵² The Generalized Extracellular Molecule Sensor platform (GEMS) is inserted into the genome of the isolated immune cell during the activation procedure by a viral vector (e.g. a lentivirus). After a few days, the viral vectors are washed out of the medium.⁵² The lentivirus used viral machinery to bind the immune cells, enter the cell and introduce RNA genetic material that encodes for the GEMS platform, IL-10, and the JAK-STAT pathway. DNA is formed of the RNA by reverse transcription so that the DNA is permanently encoded into the genome of the patient cells.⁵² The cells are placed in a bioreactor where transcription and translation of the DNA that encodes for the GEMS platform, IL-10, and the JAK-STAT pathway takes place so that the GEMS platform is expressed on the cell surface. Another method that does not makes use of lentiviruses is mRNA transfection. mRNA is considered safer in its use since the risks of transgene insertion into host genomes are abolished.⁵³ Moreover, mRNA only needs to be transmitted in the cytoplasm whereas DNA of a lentiviral vector for example has to be transmitted towards the nucleus of the host cell.⁵³ To investigate the potential of mRNA in gene editing we performed experiments in which we used mRNA to express the GEMS receptor on the cell membrane (lab results).⁵² The bioreactor in which the cells are cultured is optimized and provides optimal gas exchange and growth medium so that the number of cells gets expanded sufficiently for clinical use. After a sufficient cell amount is reached they must be concentrated so that they can be infused back into the patient. Subsequently, the cells are washed and cryopreserved in an infusible medium and transported to the location the patient will receive the therapy.⁵² We learned that some steps such as culturing, medium replacement, and cell isolation are semi-automated. Optimizing these processes with more automation while retaining the same quality is essential to minimize the vein-to-vein time. The vein-to-vein time is of critical importance because, in the case of acute treatment, a shorter vein-to-vein time allows for more indications and a larger patient population that can be treated.⁵²

To get consistent cell processing one need Good Manufacturing Practise (GMP) facilities. The viral vector that encodes for the GEMS platform is considered a key raw material for GMP. It should be sterile because the final engineered cell that contains the GEMS platform cannot be sterilized anymore. So the viral vector manufacturing step should be controlled and performed in clean room conditions with minimal exposure to open air. Moreover, to ensure GMP, a variety of safety testing is required to maintain sterility. Last generation and a minimal amount of lentiviral vector should also be used to ensure maximal safety. Production of the viral vector is also an extensive process with numerous safety measures. To make sure this is a GMP process, multiple quality controls such as testing for safety, potency, purity, sterility, identity, and titer are performed to make sure all standards are met before the viral vector is used to transduce the immune cells. Optimization of the viral vector before the large-scale !MPACT manufacturing minimizes variability and increases the efficiency of the production process.⁵²

A significant challenge in upscaling the production of !MPACT is switching from a flexible academic setting to a controlled production process that contains multiple locations for manufacturing, collection, and treatment. Hence, effective logistics and coordination are required to make sure all manufacturing steps are handled safely and correctly and the patients are scheduled efficiently. To make this process as efficient as possible, knowledge about the production process itself and the necessary quality attributes of the product is essential. The product should have sufficient cell expansion, transduction efficiency, cellular phenotype, growth rate, and function. Quality controls are required to maintain a uniformly high-quality product.⁵²

Different steps of the production process, are executed in different biosafety-level rooms depending on the hazards of the vector, host, and insert you work with. A GMP facility for manufacturing cell therapies should fulfill all GMO laws and regulations and the organization should have all GMO licenses required to work with certain organisms, vectors and inserts. In the Netherlands, these laws and regulations are defined by "Besluit GGO" and "Regeling GGO", which contain the rules regarding the contained use and deliberate release into the environment of genetically modified organisms.⁵⁴ Permission requests for working with GMOs in the Netherlands should be filed to Bureau GGO. Bureau GGO assesses the notifications for contained use and provides permits for work with GMOs.⁵⁴

Production safety is not only restricted to the manufacturing facility. Johnson&Johnson stressed that we should also take into consideration the transport (packaging, conditions, way of transport) and the protocols by hospitals when they administer the therapy. GMOs should be transported such that they arrive at their destination in good conditions and with no hazards during transport.⁵⁵ Transport of GMOs is subject to strict national and international laws and regulations since they are potentially infectious. These regulations include rules for proper packaging, and also shipping requirements to minimize the chance of damage or leaks and thereby preventing unintended exposure in the environment during shipment.⁵⁵ A typical packaging system is triple packaging (three package layers) that are able to withstand shocks, vibrations, and changes in temperature, humidity or pressure during transport.⁵⁵ Moreover, the packages should be marked and labeled and every shipment should be documented.⁵⁵

For the production, distribution, market authorization, and market entrance of GMOs multiple licenses are required. Legal affairs are essential during the complete drug development process but in particular, after clinical trials, market authorization and assessment of the new therapy by the EMA (or FDA) is a critical step.

Previous sections about (pre)-clinical trials and production of !MPACT showed laws & legislation are inevitable in the entire drug development process. For market authorization of !MPACT, laws & legislation even play a larger role. To learn about legislation for market approval, we approached several stakeholders among which experts on the EMA (European Medicine Agency) and a life sciences lawyer. You can read more on how they contributed to our project on the Integrated Human Practices page.

Market authorization allows a medicine such as a cell therapy to be placed on the market for sale and supply.⁵⁶ Basically, there are two different ways for market authorization; a centralized and a national authorization procedure. Via the centralized procedure, the EMA gives advice on market approval for the complete European Union. The EMA reviews medicines and gives an opinion on the benefit-risk to the European Commission in Brussels which will make the final decision on market entry. For the US, the agency that reviews medicine is called the FDA (Food and Drug Administration).⁵⁶ For the national authorization procedure, different organizations and advisory bodies can be involved, and approval is restricted to the respective nation.⁵⁶ Drugs for rare diseases and Advanced Therapy Medical Products ATMPs such as !MPACT for ANCA-associated Vasculitis (AAV), must be approved via a centralized procedure by the EMA for market authorization. Drugs for rare diseases are also designated as orphan drugs. There are seven different scientific committees within the EMA that evaluate new medicine (Figure 9). For !MPACT the COMP and CAT will be the two most relevant committees.⁵⁶

The regulatory process of market authorization and the role of the different committees is visualized in Figure 10. Next, to committees, very often also certain external experts are consulted, called the Scientific Advice Working Party (SAWP) or Scientific Advisory Groups (SAG).⁵⁶

The evaluation phase itself can take up to 210 days during which multiple interruptions can take place to address questions and issues of the EMA to the applicant.⁵⁶ The standard market approval provides comprehensive data that includes a complete evaluation of the medicine's quality safety and efficacy. After the market authorization, the EMA keeps monitoring the safety of drugs for as long as they are on the market and they take regulatory actions if needed, to maintain public health in the EU.⁵⁶

To conclude there are tons of laws and regulations to be followed, documents to be filed, and licenses required to develop a new medicine and to get it to the market. The process of market entrance will be discussed in the next section.

The next step after successful market authorization is the market entrance. For a successful market entrance strategy, you need a solid and viable business plan. We would recommend future iGEM teams build a business plan at the very early start of new product development since a business plan can have significant consequences on the design of a new therapy. For building a viable business plan to successfully enter the market, we co-created our business plan together with companies such as Novartis, Organon, ThermoFisher, The Gate, Ambagon Therapeutics, and CiMaas and the National Healthcare Institute of the Netherlands (Zorginstituut Nederland). You can read more how they contributed on the Integrated Human Practices page. A solid business plan requires the following things (Figure 11);

We learned that to create a business plan that results in the successful implementation of a new therapy in the market, it is important you clearly state how the therapy fulfills the need of the end-users. Moreover, you should identify your assumptions, you should make it realistic, and specific (clear steps, deadlines, forecasts, and metrics).⁵⁸ In specific for our project !MPACT, we learned from the stakeholders we engaged, there are some challenges we should overcome. We had to think about which steps of the drug development process we aim to perform ourselves and which steps we want to outsource. We had to define who the important players are to bring !MPACT to the market and what partnerships we need for this. We had to look into reimbursement of our therapy, as this is of critical importance to reach the patient. And lastly, we had to create a financial forecast of when we expect to make a profit and which investors we need to ensure a positive cash flow. For market entrance, multiple actors play an essential role which is summarized in Figure 12.

These stakeholders are important members of the healthcare system in the Netherlands. The route to get !MPACT on the market can differ per country, but often similar stakeholders of the healthcare system are involved. In the center of the triangle (not shown in the figure), the government is positioned that tries to represent the interest of all involved parties. By engaging all these stakeholders we learned what the market for new therapies looks like, which parties are relevant and what the road is towards a successful market entrance for our new therapy.

You can read our business plan and how we aim to enter the market, on the Entrepreneurship page. If market entrance is achieved, !MPACT can be administered to the patient by clinicians.

After !MPACT enters the market, hospitals, and medical centers will apply this therapy to treat anti-neutrophil cytoplasmic antibody (ANCA)-associated Vasculitis (AAV) patients. Both the hospitals and AAV patients can be considered the end-users of !MPACT (Figure 13). During the meetings with clinicians/physicians of Maastricht University Medical Center, the Vasculitis Foundation, and AAV patients themselves, we obtained feedback on the feasibility and desirability of !MPACT. Moreover, we learned how cell therapies are administered to the patient and what the relevant safety issues are during this process (Integrated Human Practices). This feedback was used to suggest how !MPACT should be implemented by clinicians/physicians.

Patients

Currently, AAV is treated with immunosuppressive therapies, mostly rituximab or cyclophosphamide, in combination with glucocorticoids.⁵⁹ Although this treatment is often effective, the unspecific suppression of the immune system caused by this treatment can be extremely burdensome for many patients.^59,60 Peter Verhoeven, ex-chairman of the Vasculitis Foundation, and the AAV patients themselves confirmed these limitations of current treatments. From the patient interviews, we learned that patients are willing to receive !MPACT over the current treatments if it is proven effective and advised by clinicians.

Previous to the isolation of immune cells for the patient, the patients need to be screened by the physician to see if they are medically eligible for !MPACT. The specific eligibility criteria will depend on the results of the clinical trials but there are some general criteria. The patient should be diagnosed with the right subtype of AAV, the symptoms of AAV should be severe and the ANCA concentration should fluctuate with the activity of the disease. Besides, the patient should have sufficient organ function and performance status, the patient should not have active infections, and should not have cardiac, immune, or neurological dysfunction.⁶¹ If all criteria are met the patient can undergo leukapheresis.

Clinicians/physicians

After the leukapheresis and production of !MPACT, it needs to be administered to the AAV patients by clinicians (Figure 14). The hospital or treatment center should have the required licenses to administer advanced GMO-based cell therapies and the administration procedure should be carried out via standard protocols (as delivered by the manufacturer) by certified staff.

During the bridging phase (time between isolation and infusion of immune cells), the patients should be closely monitored for disease progression.⁶² In addition, lymphodepleting chemotherapy should be given to the patient. This treatment results in the depletion of specific lymphocytes. The purpose of this treatment is to prepare the patient for !MPACT and to create the ideal environment for the engineered immune cells to expand.⁶² The infusion and monitoring phase is the next. !MPACT is administered by infusion when thawed and at room temperature. The administration of !MPACT should be carried out in a sterile environment to protect the cells from contamination but also to protect the caregiver and the environment from unintended exposure.⁶³ Subsequently, the patient should be monitored to anticipate on possible adverse events.⁶² As the cells of !MPACT secrete the cytokine IL-10 upon sensing ANCAs, the most important challenge that needs to be considered when treating patients with !MPACT is the common arising side effect of cytokine release syndrome (CRS). CRS is an acute inflammatory response that can arise after massive cytokine release.⁶⁴ According to Dr. van der Poel from Maastricht UMC, CRS can occur in different gradations of severity. However, most patients almost always recover if patients are monitored closely. Severe CRS that would arise in treated AAV patients could be managed by intervening in the IL-6 signaling pathway. Tocilizumab, an IL-6-receptor inhibitor is acknowledged to be the best management strategy to treat CRS.⁶⁵ After administration of !MPACT patients should remain nearby of a certified treatment center for at least 4 weeks and should get a long-term follow-up.⁶⁶

In previous sections of the drug development process, we have touched upon safety issues for each respective step. There are however some more safety facets that should be considered during implementation of !MPACT into the real world. Interviews with the BioSafety Officer of our institute, experts in dual-use, Greenpeace, a worldwide public health organization, and the RIVM (National Institute for Public Health and the Environment) resulted in some key safety issues to consider (Integrated Human Pracitices). This section gives a clear overview of the remaining safety measures that are necessary for the successful implementation of !MPACT.

Future wet-lab research

For future wet-lab research to !MPACT, there are some important safety measures to consider. Since !MPACT is intended to be used in the human body and as it has an immune modulatory function, it could potentially harm human health by accidental exposure of the engineered cells of !MPACT. !MPACT has the potential to dysregulate the immune system of healthy individuals which could make the individual more prone to infectious diseases, auto-immunity, or systemic inflammation due to a cytokine storm.²³ Preparation of experiments is thus crucial and one should determine the experimental risks and take precautionary measures. For each experiment, one should think about what must be done when contaminated material is spilled and it should be checked whether all parts and cells used, are covered by the GMO license of the institution. More information on safety measures to manage risks in a laboratory can be found on our lab Safety page and our project safety form.

Environmental Risk Assessment

Risks of !MPACT to the environment and public health should also be considered. If you use GMOs for therapy, you use them outside the laboratory. This means they are introduced into the environment and a license is required by the Ministry of Infrastructure and Water Management in the Netherlands.⁴⁴ To apply for such a license, an Environmental Risk Assessment (ERA) must be conducted.⁴⁴ An ERA for a cell therapy has to be based on the probability of transmission of the cell therapy from patients to other persons, animals, other organisms, or the environment in general.⁶⁷ Clinical or experimental data is required and should contribute to the ERA. The ERA is based on 6 key principles according to the European Medical Agency (EMA):⁶⁷

Step 1: Identification of GMO characteristics that may cause adverse effects

Step 2: Evaluation of the potential consequences of each adverse effect, if it occurs

Step 3: Evaluation of the likelihood of the occurrence of each identified potential adverse effect

Step 4: Estimation of the risk posed by each identified characteristic of the GMO(s).

Step 5: Application of management strategies for risks arising from the deliberate release or marketing of the GMO(s).

Step 6: Determination of the overall risk of the GMO(s)

Chimeric Antigen Receptor (CAR)-T cell therapies for various cancer types such as lymphomas and leukemias are already available on the market and are approved for use in the environment (therapeutic purposes).⁶ Since !MPACT has a large resemblance to CAR-T cell therapy in terms of genetic engineering and functionality, the CAR-T cell environmental risk analysis could be used as starting point for the ERA.

It is hypothesized that !MPACT has minimal risks for the environment and public health based on discussions with the RIVM. Our engineered cells have minimal chance on survival outside the body and transmission of the engineered cell to a host is only possible via blood contact

Lentiviral vectors

As explained in 4. production process section, we need lentiviruses to implement genes into the genome of human immune cells. The risks of lentiviral vectors are already familiar, so they no longer require new risk analysis. Moreover measures to minimize risks are also available already.⁶⁸ The risks of lentiviral vectors (LVVs) include that they have oncogenic potential and recombination of the lentiviral vector could unintendedly lead to reconstitution of a replication-competent and pathogenic virus that results in infectious diseases.⁶⁹ The most important measure is to prevent exposure to LVVs by following strict safety protocols as delivered by the manufacturer. If exposure occurs, it is important to handle it by flushing the affected area with water or using specific medication to prevent infection by LLVs. Also, LLVs have been designed to minimize their oncogenic and viral potential.⁶⁹

Transgenerational transfer of engineered cells

A risk we were advised to delve into by the RIVM is the transfer of the engineered cells from a pregnant mother to the unborn child. Ideally !MPACT forms memory cells, which means the engineered T-cells will remain in the body long after treatment. When patients become parents, long after the treatment with !MPACT, literature states that engineered T-cells have the ability to transfer from mother to child by crossing the placenta, as well as via breastmilk.⁷⁰ Since matured T-cells could potentially influence the immune responses in the child, the possibility of transgenerational transfer of !MPACT and its consequences for the child should be investigated. We propose to test the transgenerational transfer of our engineered cells and its impact on the offspring in mice models. These studies are currently also performed for CAR T-cell therapy and investigate transgenic T-cell prevalence and location in the offspring, developmental abnormalities, and possible autoimmunity for the newborn.⁷⁰ This study could give insights in the long-term consequences of the transgenerational transfer of our engineered cells.⁷⁰ If risks for newborns were detected, measures on how to ablate these risks through a well-grounded research approach could be implemented in the design of !MPACT.⁷⁰ Next to research, also the women receiving !MPACT should be counseled appropriately about the risks and consequences for a newborn child. ⁷⁰

Precaution Principle

Greenpeace offered advice on a trustworthy risk assessment for new GMOs. New GMOs should be subject to the application of the Precautionary Principle (PP). The PP, correctly applied requires the following:

1. Preventive action;
2. prior to scientific proof regarding cause and effect (at which time it is too often too late);
3. a shift in the burden of proof onto the proponent of the activity to demonstrate/prove the activity is unlikely to cause harm;
4. implemented via an alternative, substitution.

Greenpeace believes that even if a risk is very low, but the consequences are potentially significant, a very strict risk assessment by an independent, objective third party is necessary. We propose to execute the environmental risk assessment according to the Precautionary Principle.

Global, national, and health security

Another important danger that we considered, is the dual-use of !MPACT. Seen the potential of !MPACT to dysregulate a person’s immune system, we had to think about the consequences of this technology if it falls into the wrong hands. We learned in the discussion with dr. Koos van der Bruggen (expert in biosecurity) that !MPACT could potentially be used as a bioweapon for individual or terrorist attacks. Visit our (Integrated Human Pracitices) page for more information on this interview. The possibility that !MPACT is used as a bioweapon in war, is however negligibly according to Koos because !MPACT does not survive outside the human body, does not transmit through the air, and is difficult to administer. We learned that complete prevention of dual-use is not possible but there are certain measures we take to minimize the risks.⁷¹ For example, we follow the guidelines for the Dutch Code of Conduct for Biosecurity, limit access to the lab, screen new lab members, and keep the trajectory of stocks and materials. These measures minimize the risks since it keeps bad people away from dangerous biological agents. We also learned that biosecurity is as important as biosafety since the chance of accidents is even larger than deliberate misuse of biological agents. Lastly we have a take-home message for future iGEM teams; we should increase awareness among life scientists of possible misuse of their research, however, awareness should not become so predominant that distrust is the default attitude in a laboratory.

The rules of conduct as literally stated by the Dutch Code of Conduct for Biosecurity can be found here:

Rules of conduct⁷¹

RESEARCH AND PUBLICATION POLICY ⁷¹

• Screen for possible dual-use aspects during the application and assessment procedure and during the execution of research projects.
• Weigh the anticipated results against the risks of the research if possible dualuse aspects are identified.
• Reduce the risk that the publication of the results of potential dual-use life sciences research in scientific publications will unintentionally contribute to misuse of that knowledge.

ACCOUNTABILITY AND OVERSIGHT ⁷¹

• Report any finding or suspicion of misuse of dual-use technology directly to the competent persons or commissions.
• Take whistleblowers seriously and ensure that they do not suffer any adverse effects from their actions.

INTERNAL AND EXTERNAL COMMUNICATION ⁷¹

• Provide (additional) security for internal and external e-mails, post, telephone calls and data storage concerning information about potential dual-use research or potential dual-use materials.

ACCESSIBILITY ⁷¹

• Carry out (additional) screening with attention to biosecurity aspects of staff and visitors to institutions and companies where potential dual-use life sciences research is performed or potential dual-use biological materials are stored.

SHIPMENT AND TRANSPORT ⁷¹

• Carry out (additional) screening with attention to biosecurity aspects of transporters and recipients of potential dual-use biological materials, in consultation with the competent authorities and other parties.

Creating or reinforcing social inequities

Since the production process of !MPACT is a long, labor-intensive, and complex procedure, we expect that !MPACT will be expensive. Reimbursement in the basic package of health insurance is required for !MPACT to reach the patient. Hence, we should consider creating or reinforcing social inequities. According to the public health organization we interviewed ( Integrated Human Pracitices ), differences in access to healthcare between certain countries are largely due to two things: First, some countries do not have the financial resources to offer certain treatments, and second, they often lack a well-educated health workforce. Therefore, it is expected that poor developing countries will not offer expensive ATMPs like !MPACT to their population. Therewith, the social inequities between countries increase. We learned from the public health organization that it is difficult to reduce the inequality in access to new ATMPs such as !MPACT. The responsibility lies with the manufacturer: the manufacturer should be willing to offer the therapy in certain countries for a lower price or even for free to help a larger population. On the other hand, we learned from the public health organization that innovation is necessary, even if the initial costs are very high. In the future, expensive technology can become the new standard in all countries. The public health organization named the example the CT scan which was very expensive at the beginning. Now costs of a CT scan are reduced, and CT scans are standard in the health systems in almost all countries.

We propose three actions to decrease social inequities:

1. We will work together with large public health organizations such as the World Health Organization (WHO) to determine the minimum facilities and workforce necessary for a country to offer !MPACT.
2. We will train healthcare staff in other countries to administer !MPACT. Moreover, we will make all required product information, production protocols, safety measures, etc. accessible.
3. We will negotiate with pharmaceutical companies to offer !MPACT for a lower price in developing countries.

Integer communication

Finally, communication is indispensable for the safe implementation of a new therapy. We will communicate about !MPACT in a transparent, honest, objective, and accessible way that is understandable for all different populations in society. We will communicate with patients, the general public, other scientists, clinicians, and pharmaceutical companies who have different educational backgrounds, levels of understanding, and preferred learning modes differ. We will listen to all stakeholders, make visualizations to get a message across and adjust your vocabulary and assumptions to the audience.⁷²