A key advantage of our B cell engineering approach is its prophylactic use: Memory B cells lie dormant in the body and respond promptly to infection or disease onset. Given the impending pandemic of neurodegenerative diseases, we propose B-cell therapy as a prophylactic treatment for Alzheimer’s disease - an idea we have discussed extensively with Alzheimer’s expert Prof. Oliver Wirths. We want to introduce the genes of novel, recently FDA-approved antibodies, Aducanemab or Lecanemab, into the B-cell genome. These antibodies are designed to bind to the pathological Aβ plaques and attract microglia, which in turn clear the plaques. However, as Professor Wirths pointed out, the efficacy of these antibodies is highly controversial. Even if the Aβ plaque burden is significantly reduced, this does not necessarily lead to an decrease in cognitive decline, he explained. However, it is important to remember that the patients in the study were already severely affected by Alzheimer’s disease. Since the actual onset of the disease, and thus neuronal damage, usually begins years before diagnosis, the disease may have already progressed too far. Although the antibody treatment removed the plaques, the neuronal damage was too severe. This could be a major advantage of a B-cell-based prophylactic treatment, Professor Wirths said: Antibodies are produced directly at the onset of the disease, preventing cognitive decline in the first place. Especially high-risk patients, for example those with a genetic predisposition, could benefit immensely from such a therapeutic approach. He also emphasized that outpatient, long-term antibody treatment is very costly for our healthcare system, whereas B-cell engineering can be seen as an expensive but one-time investment.

After discussing Alzheimer’s disease (AD) as a promising target for B-cell engineering with Prof. Wirths, another challenge emerged: less than 1% of antibodies actually cross the blood-brain barrier after treatment, making therapy highly inefficient. To overcome this problem in our B cells, we collaborated with Lis de Weerd, a scientist testing novel Alzheimer’s antibodies in animal models. She explained how antibodies can be engineered to include transferrin receptor binding sites, allowing them to bind to the transferrin receptor (TfR) on the blood-brain barrier. Association of these binding sites with the TfR then facilitates transcytosis. Lis de Weerd’s valuable insights prompted us to incorporate a platform into our antibody constructs that enhances transport across the blood-brain barrier. Building upon her feedback, we have extended this concept by attaching single chain variable fragments (scFvs) with TfR binding sites to our antibodies. This not only increases the valency but also has the potential to optimize and enhance transport into the brain.

B cell therapy can be applied to combat diverse pathogens and diseases. A major challenge these days is the increase in multi-resistant pathogens that do not respond to conventional antibiotic therapy. This is why our iGEM Team discussed tackling multi-resistant pathogens, such as MRSA, through B-cell engineering with Prof. Dr. Sieber – a renowned expert in this field. He shared our opinion that long-term B-cell therapy might provide a key advantage in comparison to common antibody therapy: when applied prophylactically, memory B cells are able to constantly produce antibodies and thereby prevent the spread of MRSA from the start. Referring to this, he pointed us to a specific target group for clinical implementation: high-risk, possibly immune-suppressed patients that are facing several hospitalizations – at constant risk to suffer from a life-threatening S. aureus infection. Due to his expertise in the field, Prof. Sieber guided us towards α-hemolysin, a very conserved S. aureus exotoxin, as a crucial antibody target for our therapeutic strategy. α-hemolysin plays a pivotal role in inducing cellular damage by creating pores in cell membranes and targeting it with antibodies would substantially diminish the infection’s impact. Lastly, Prof. Sieber emphasized the importance of developing treatment against multi-resistant pathogens, since pharma companies begin to withdraw from sponsoring research for novel antibiotics.

As an expert in microbiology and its clinical applications, Prof. Hall confirmed our idea to use Staphylococcus aureus’ a-Hemolysin as a target for bacterial toxins. Due to its highly conserved structure, published antibody structures already under clinical examination were available for us integrate into our model. However, she also advised us to expand our list of targets to other bacterial proteins and pathogens such as Enterococcus faecium, which are gaining in importance due to increasing antibiotic resistances in ICUs. Our B cell therapy would also be suitable as a supplementary treatment option for the decolonization of patients before admission to the ICU to prevent transmission. Another key point we took home was to search for dedicated experts through scientific networks focusing on specific topics or diseases, like the BactiVac consortium that Prof. Hall recommended to us.

As one of the pioneers of B cell engineering, Prof Barzel provided valuable feedback on potential targets for our project, as well as on genomic engineering and our antibody designs. First of all, Prof. Barzel highlighted chronic infections as optimal targets for B cell therapy, as the cost and inconvenience of long-term outpatient antibody treatment far exceeds that of cell therapy. Following his advice, we also adapted our therapeutic approach to HBV - hepatitis B virus. The big advantage: Antibodies are produced continuously, keeping viral replication in check. In addition, Prof. Barzel emphasized that the engineered B cells are still capable of class-switching, which allows them to respond more effectively to multiple viral targets than conventional antibody therapies. In addition to IgG, which provides humoral protection, significant amounts of IgA are produced to enhance the mucosal response to the invading virus. We also discussed with Prof. Barzel the challenges of engineering B cells according to his designs: By expressing the endogenous light chain alongside the integrated target light chain, mispairing reduces the number of functional antibodies. Therefore, our team decided to integrate linkers into our antibody constructs that fuse the light and heavy chains together to prevent inefficient mispairing. In future approaches, we also plan to generate light chain knock-outs to circumvent this problem.

Together with Prof. Protzer and her lab, we have established chronic HBV infection as one of our targets, specifically developing antibodies against the surface protein HBsAg to facilitate protection against viral entry. HBV utilizes HBsAg for cell entry and infection - by blocking its functionality via antibodies produced by our B cells, viral particles are bound and prevented from infecting healthy cells. Chronic HBV infection poses a serious health risk for affected patients, who may potentially develop live-threatening diseases such as liver cirrhosis and hepatocellular carcinoma among other liver-related diseases. For infected individuals, B cell therapy may offer an additional method of treatment to control and suppress HBV spread in order to preserve liver function and viability. Suggestions from Prof. Protzer furthermore included a refinement of our Transferrin-dependent system towards a more organ-specific system depending on the construct, as Transferrin receptors exists in many places throughout the body and may therefore cause the uptake of antibody into unrelated compartments which may contribute to a decrease in serological antibody concentration. Looking towards combination approaches as the future of cell therapy and our 2022 iGEM project, her suggestion of including T-Cell-Engager antibodies into our repertoire of constrcuts especially peaked our interest.

In addition to focusing on the scientific feasibility of our approach to B cell therapy, our project was designed to gather physicians’ insights into clinical translation. How do we take B cell therapy from bench to bedside? What would the extraction and reinsertion of B-cells into the germinal center look like? In order to answer these questions, we consulted one of the most renowned experts in CAR-T-cell therapy in Germany: Prof. Dr. Uwe Platzbecker, who came up with a brilliant idea. He suggested combining a patient’s personal B-cell therapy directly with surgical procedures in clinical practice. In breast cancer treatment, for example, not only the tumor but also the local lymph nodes are removed and tested for malignant cells. The introduction of an engineered B-cell into the germinal center could be easily implemented. A major advantage of this approach is that it minimizes invasive procedures and provides a rapid, local response to recurrent malignancy. In addition, the potential side effects of antibody treatment could be reduced since the levels of secreted antibodies are regulated by the body’s own immune system and mainly exert this local effect. In particular, Professor Platzbecker’s proposal to combine B-cell therapy with surgery led us to one of our most important targets: Renal cell carcinoma (RCC). In addition to removing the tumour, we propose to place B cells that produce antibodies against CAIX, or carbonic anhydrase IX, in the local lymph node. As CAIX is also a unique marker that is upregulated only in RCC, this will specifically eliminate cancer cells while allowing healthy kidney cells to persist. We also discussed the possibility of offering a B-cell therapy to improve secondary acquired immunodeficiency - to protect the elderly from infection. This could be achieved by replenishing their immune systems with specific B cells that target multiple pathogens, providing broad protection.

Unlimited opportunities in synthetic biology are accompanied by great dangers. Novel CRISPR/Cas technology allows us to arbitrarily modify and adapt organisms to our desires and requirements. But only because it is possible, does not mean it is ethically justifiable – especially when it comes to treatment of human disease. For which purposes should we use genetical engineering approaches? Which ethical standards should apply to biosciences? All these concerns, our iGEM Team discussed in an extensive workshop together with Dr. Mirko Himmel and Dr. Maria Riedner. With a special focus on biosafety, we first evaluated possible risks and unintended consequences that could result from clinical B-cell therapy implementation. Together, we came to the conclusion that ensuring correct engineering of the patient’s B-cell itself before reinsertion is crucial and easily feasible. Furthermore, safety measures need to be integrated to prevent unpredicted side-effects such as autoimmune reactions. This can for example be achieved by our proposed kill-switch, CD20 overexpression and targeting or scFv-mediated control over the cellular metabolism. We set a special focus on ensuring the safety of our technology – taking responsibility for our research. Nevertheless, it is essential to anticipate the potential long-term consequences of B-cell engineering and the ethical considerations that may arise as a result. Will there be a future where individuals seek to engineer their entire immune systems? Could this lead to a two-class society, with some possessing the financial means to enhance their immune defenses while others lack the opportunity to do so? Answering these questions, we clearly came to the conclusion that it is our legislator’s task to ensure equality of opportunity as well as defining ethical principles for the use of genetic engineering.

Unlimited opportunities in synthetic biology are accompanied by great dangers. Novel CRISPR/Cas technology allows us to arbitrarily modify and adapt organisms to our desires and requirements. But only because it is possible, does not mean it is ethically justifiable – especially when it comes to treatment of human disease. For which purposes should we use genetical engineering approaches? Which ethical standards should apply to biosciences? All these concerns, our iGEM Team discussed in an extensive workshop together with Dr. Mirko Himmel and Dr. Maria Riedner. With a special focus on biosafety, we first evaluated possible risks and unintended consequences that could result from clinical B-cell therapy implementation. Together, we came to the conclusion that ensuring correct engineering of the patient’s B-cell itself before reinsertion is crucial and easily feasible. Furthermore, safety measures need to be integrated to prevent unpredicted side-effects such as autoimmune reactions. This can for example be achieved by our proposed kill-switch, CD20 overexpression and targeting or scFv-mediated control over the cellular metabolism. We set a special focus on ensuring the safety of our technology – taking responsibility for our research. Nevertheless, it is essential to anticipate the potential long-term consequences of B-cell engineering and the ethical considerations that may arise as a result. Will there be a future where individuals seek to engineer their entire immune systems? Could this lead to a two-class society, with some possessing the financial means to enhance their immune defenses while others lack the opportunity to do so? Answering these questions, we clearly came to the conclusion that it is our legislator’s task to ensure equality of opportunity as well as defining ethical principles for the use of genetic engineering.

Prof. Schmidt and his team have advised us to enter strong collaborations with professionals and companies active in the medical field to qualify for an application of our research at the ethics committee - only extensive preclinical research and thorough investigation under GMPs should be admitted to further continue in clinical studies. Furthermore, they were able to raise our awareness for the quality and sources of our research material such as publications, cell lines and more, as ethical research can only be based on previous findings which also uphold the necessary ethical standards. In order to conduct clinical studies, the informed consent of all patients and participants is required to fulfill the underlying ethical guidelines. To fulfill these criteria, the ethics committee recommended us to utilize tools specifically designed to generate viable documents to confirm participant information and consent, such as their “eTIC” tool. Another point we were guided to think about was the medical necessity of experimental treatments such as B Cell therapy - for diseases with extensive treatment options and good survival prognoses, experimental therapies will not fulfill the necessary ethical criteria. However, ethical applications can be successful for diseases lacking viable treatment options, such as many forms of cancer or chronic HBV infection.

Main points in our discussion with Prof. Buxy were the considerations of medical need and the conduction of clinical trials regarding our therapy. She advised us to focus on currently challenging diseases, as only positive outcomes from a “risk-reward”-framework for the evaluation of novel therapeutics may be approved by the corresponding ethics committee. For our studies, an extensive risk evaluation for potential participants would be necessary, as well as a carefully selected group of patients to fit the study’s design. It is important to note that only licensed medical professionals specialized in conducting medical studies are approved to facilitate studies at the clinical stage and are essential for the tasks of informing patients, gathering the necessary consent, administering the therapeutic agent and evaluating the resulting data.

In our collaboration with iGEM Team from McGill university, we contributed a chapter focusing on B cell therapy to their bioethics report, which aims to build an ethical framework and guide for future iGEM Teams, who will potentially investigate similar projects. By utilizing the Bioethics Report’s content, future teams will be made aware of and benefit from preestablished ethical guidlines for their desired research project, enabling them to target their Human Practices work and research towards the right direction. You can find the bioethics report on their wiki. (2023.igem.wiki/mcgill/bioethics/)