iGEM Munich: How do you go about when setting out to design a brand-new CAR T-cell? For our team it was quite hard when we tried to first search for CAR sequences, maybe you could share some of the tools you use?
Daniel Nixdorf: A lot of times we already have the sequences from the industry or prior works at our professorship. Designing a brand-new CAR or just starting out in this field can be discouraging because that there is nothing like a database for CARs or a tool that specifically supports CAR design, at least that I know of. It is in general also difficult to access the DNA sequences due to patents. The ones that are publicly accessible have to be searched for in papers and supporting material which can be time-consuming. However, there are data banks for Antibody sequences already.
iGEM Munich: Do you think that this non-existent database for CARs that you talked about, would be possible to develop and would help scientists in the field? Daniel Nixdorf: There is definitely room to improve on for CARs in the public domain. I also think it is possible to develop at least a prototype in a reasonable timeframe. iGEM Munich: So of course we would want to store original CAR designs in our database. Would you say a modular approach would be interesting to consider with the CARs?
Daniel Nixdorf: In CAR design one often does mix and match of domains but one has to be careful because there is no real validation tool to check the integrity of the new structure. This is also not quite possible because CAR T-cells is a very noisy field so results have to be very well experimentally verified before even going into animal trials.
iGEM Munich: Yes, we of course should make sure that newly assembled CARs are clearly labeled as such. Thank you for your insight into the field and the idea abou improving the availability of databases.

iGEM Munich: We are planning to develop a database in combination with a web-tool to help scientists to engineer their CAR T-cells faster and with fewer mistakes. In our own work we had problems finding any database collecting specifically CARs or even simply ScFvs, so we thought this was a gap worth filling. Would a database of CARs be helpful in your field?
PD. Dr. Kobold: Resources like sequences and building blocks are not readily available and in silico assembly of the different domains is not trivial and error-prone. This is why I think modularity is an idea worth trying, das one would be able to select sequences for the different domains and assemble them in place and overcome copy-paste issues.
iGEM Munich: How big of a problem are these assembly errors?
PD. Dr. Kobold: Many projects and publications in places around the world never go anywhere, in part likely due to such errors. Helping these researchers who are just starting out in this field would be a great deed. So even a non-modular database with only finished sequences could already be helpful.
PD. Dr. Kobold:These concepts are not exclusive. Even in our modular database you can select finished, already published sequences that will be clearly labeled as such.

Epilogue: With PD Dr. Kobold we did not only discuss our Database idea but we also asked for feedback for our research (SpecifiCAR) project. Due to this not really fitting within the theme of HP. Here we only present the part where we discussed our database plans.

iGEM Munich: [explanation of our database concept, see Daniel Nixdorf discussion]. Do you feel that a central information storage like that is missing in your field?
Nina Barceló: Yes, and I would love to see something like that. We currently use Benchling for our design workflow but of course we have to search the sequences before importing them to Benchling, which can be time-consuming. We do this about 10 times a year, so having a central place to look up some sequences would be a time saver. It also happens that we only change parts of the CAR so an implemented tool to assemble domains would be even more helpful.
iGEM Munich: [showing her the DB Schema] what do you think about our Database design. Is this what you imagined the modularity of the CAR would look like? Is there anything missing in your opinion?
Nina Barceló: I would need to of course use and test it, but in these first moments I don’t see anything that would be missing.
iGEM Munich: Would you be ready to publish and share your sequences in the Database eventually?
Nina Barceló: Of course some of the CAR sequences we are using are private and not meant for the public. But in general I think if it is possible, scientists should share this information in a central place for others to find.

Introduction: Dr. Ayuk is a specialist in internal medicine with a focus on hematology and internal oncology. He currently works at Universitätsklinikum Hamburg-Eppendorf at the Department of Stem Cell Transplantation where he practices CAR T-cell therapy, among others. He is also associated with the The German Cancer Society where he does public communication for CAR T-cell therapy. For more information about him and a list of his publications please visit his profile.

Doctor and Patient interaction

iGEM Munich: What are the criteria for choosing CAR T-cell therapy, i.e. is it a first-line therapy or must other therapies be tried beforehand? (Please be careful with this information, it can be quickly outdated and might be mostly Germany specific)
Dr. Ayuk: Currently CAR T-Cell therapy is available in the context of approval (standard treatment) or in the context of studies.
In the context of approval: it is only meant to be used after at least two previous therapies (in America possible after 1 prior therapy, EU approval expected at the end of this year).
In the context of studies: CAR T-Cell Therapy is on the way to be used as a first-line therapy (complementary) in high-risk cases of multiple myeloma.
There is however rapid development in this field.
iGEM Munich: How do patients react to the suggestion of CAR T-cell therapy, especially talking about the introduction of living, genetically modified cells.
Dr. Ayuk: I am surprised at how understanding almost all patients are. What startles them more is if they hear the price. However due to the fact that it is a one time cost, the price is actually comparable with other therapies. Few people reject the treatment and ask for a conventional alternative.
iGEM Munich: When talking to patients, which information do you try to give before the therapy?
Dr. Ayuk: There is no grander pattern in the conversations with the patients. As a doctor I have to be ready to adapt to the individual interest and knowledge level of the patients. It is important to give them all the necessary information while not overloading the patients with details they might not care about. Some also inform themselves via the internet, where one can find a lot of valuable information about this therapy.
iGEM Munich: How long do those initial conversations take?
Dr. Ayuk: For the first interaction I normally plan about an hour to discuss all the necessary details with the patient and answer all potential questions. Then we discuss the treatment at a tumorboard with other doctors. Following that we submit a claim to the insurance company. Then there are more conversations before and during the whole process of treatment (consisting of extraction of T cells, processing in the lab and finally reintroduction into the patient). It is important to inform the patient well in the beginning, as canceling during the treatment is very costly.
iGEM Munich: Are there many patients that stop after starting the treatment?
Dr. Ayuk: No, that I think shows the importance of taking these initial conversations seriously.

Interaction between doctors and scientists

iGEM Munich: In your opinion what is currently the biggest limitation of CAR T-cell therapy?
Dr. Ayuk: The bureaucracy. Applying for funding from insurance companies is difficult and due to the possible expenses for the hospital if the insurance company declines, the responsibility of the doctors then is not only to the patient but also to be mindful about the usage of funds. As a doctor it is therefore very important but sometimes difficult not to lose the focus on the patient in-between all the paperwork and disagreements.
The problem in the system is, therefore, that if the insurance company decides that the treatment could have been done in a less expensive way, the hospital can lose a lot of money, which in my opinion should not be the main concern of the practitioner.
iGEM Munich: How is the interaction between scientists, the industry and doctors?
Dr. Ayuk: In our hospital we have a lab, with which we stand in close interaction, focussing on research, especially side effects but also genetic modification of t cells, so we can produce CAR-T cells ourselves, but currently lack approved facilities. With the industry we are generally in that close contact, but we make sure to keep our treatment decisions and are research independent. Everything currently on the market is mainly from university research, however industries have high hopes for new developments with these therapies. With central processing of the T cells, one could cut down on the three-month processing time that we currently have. Currently, such companies exist in foreign countries but with the bureaucracy for transporting genetically modified cells, the waiting time is not really shortened. There is also the wait time between registering and getting a slot for processing, which adds two more weeks and probably will not change any time soon. So much shorter than 1 month is currently not very realistic.

Introduction: D'Ann S. was diagnosed with multiple myeloma at the age of 22 years. She has been fighting cancer for 10 years now. Her disease burden is low at the moment which is why she was a good candidate for giving us a look into the life of a patient.

iGEM Munich: What kind of treatment have you received before CAR-T therapy?
D'Ann: In the last 10 years we tried a lot of different standard treatment starting with Velcade, Revlimid [Lenalidomide] and Dexamethasone as well as radiation therapy. Because I didn’t respond so well to these first tries we switched to a harsher treatment: VDR PACE. Then I had a stem cell transplant and finally a bone marrow transplant from a related donor. For eight and a half years I was in remission, and was only on Revlimid maintenance. After I relapsed, I had to do more radiation and started two new chemo drugs (Carib & DAR Tova). Following that I received three donor lymphocyte infusions from the same related donor. So those have been my past treatments and now I’m prepping for CAR T.
iGEM Munich: How did you first learn about CAR T? From your doctor or did you know about it before?
D'Ann: I knew about it before. I’m pretty diligent with doing my own research on the latest trends for treatments and had already looked into CAR T-cell therapy. But it was still new and I asked my doctor about it a few times, but I was responding so well to the previous treatments that they never felt it was necessary. My oncologist then proposed it to me some time ago but he wanted to wait for the donor lymphocyte fusion before because he said the quality of the cells going into the growth of the CAR T correlates to how they turn out.
iGEM Munich: Is your case still a clinical trial?
D'Ann: I know that there are a couple of CAR T-cell therapies that are already approved and the one that I am getting (CARVYKTI™) was just approved earlier this year.
iGEM Munich: Would you still consider that treatment if it was still in trials?
D'Ann: I guess I kind of feel neutral about it. I’m ready to give it a good go and I feel pretty confident going into it.
iGEM Munich: You are currently waiting for the T-cells to go through the lab right? How long is this waiting time and do you find it acceptable?
D'Ann: They took the T-cells about a month ago, and I am set to get those back in a month. For me this waiting time is acceptable because my disease burden is low, but it could be a problem for somebody for whom this is their last resort.
iGEM Munich: How well were you informed about the therapy by medical staff & doctors?
D'Ann: I do feel like my questions were answered thoroughly. I was given a lot of good information from the transplant coordinators. My doctor was very open about possible side effects, but then he was also hopeful about what it could mean for my future, about potentially curing me and giving me a really long life, so for me that outweighs a lot of the risks.
iGEM Munich: How long was this process of consulting & decision time?
D'Ann: I had three or four meetings just with my doctor and I never felt rushed in our discussions and he always was really good about listening to all my concerns and then also giving me feedback.
iGEM Munich: Does the label of genetically modified cells being injected into your body make you worry more than with previous therapies?
D'Ann: Of course it does raise a bit of concern because it is something new and you think “this is crazy”, but in this case the hope of what it can bring for a lot of cancer patients outweighs that. The fear of losing the battle to cancer is greater than the concern I have for receiving genetically modified cells.
iGEM Munich: What if the illness we are trying to cure with genetically modified cells would be non deadly but it would mean improvements of the quality of life for the person?
D'Ann: I know this is very controversial, but I think when it comes to something that can improve someone's life I think I at least would give it a try.
iGEM Munich: Do you have anything to say to other patients that are considering CAR T-cell therapy?
D'Ann: Do your own research, ask the questions that you have, tell your doctor if you are not understanding something. Think about and understand the side effects but also think about the end result. For me it is the hope of a cure to live a long life with my two little kids and my husband.
iGEM Munich: After your therapy and hospital phase, could we come back and get an update on how the whole treatment went?
D'Ann: Yes, sometime around November would be great.

Introduction: Michelle R. from the UK was diagnosed with DLBCL (Diffuse large B cell lymphoma) in 2019 and since went through multiple treatments of which CAR T-cell therapy was one.

iGEM Munich: Did you receive CAR T-cell therapy as part of a clinical trial or was it approved by the NHS before?
Michelle: My treatment was approved by the NHS. Before I received it there were about 70 people who went through the trials.
iGEM Munich: Before receiving CAR T-cell therapy, which form of treatment had you tried?
Michelle: Before, I had Atrox Chemotherapy, intrathecal Chemotherapy and Radiotherapy. Then I received CAR T-cell therapy. Since then, I have also been on a clinical trial, more chemotherapy, and received a stem cell transplant.
iGEM Munich: How did you learn about this form of therapy?
Michelle: I was very lucky here, when I was first diagnosed in 2019. My consultant is quite renowned for treating lymphoma and he knew that this CAR T cell therapy just came on the NHS. Obviously, it had to be approved by a board of people at that time, but because he was high up it just got put through, and I was accepted for it straight away.
iGEM Munich: Did you have any concerns about having genetically modified cells in your body?
Michelle: Only because of the side effects that they told me, obviously, the neurotoxicity things like seizures and the risk of being in a coma was unlike any treatment that I had before. The statistics were like 1 in 3 people had a seizure and so it was really scary. Just before that I was on another form of chemotherapy that I forgot about, and it failed. So, I didn't have another choice, I had to have this.
iGEM Munich: Did you experience any (long-term) side effects?
Michelle: I had a high fever, chills, nausea, vomiting, and diarrhea, all that and headaches and a fast heartbeat,which then turned into neurotoxicity. I was in intensive care for only a day or two, but it basically felt like I was paralyzed. I could move my mouth, but I was not speaking English. I was just stuttering and even the thoughts that were in my head, I couldn't verbalize them. People were asking me, “what's your name,” and I just hummed. I couldn't move my body. They rushed me back into intensive care and my speech came back, but it took longer than my body took to come back. Months afterwards it still felt like my brain wasn't working in the same way it did before. When I went back to university, I used to be really good at writing and things like that, and after I found it incredibly difficult, and I couldn't comprehend things in the same way then I did before. Obviously, it is hard to know, but I imagine it was neurotoxicity. However, no one really told me it was.
iGEM Munich: How many days were you stationary during the therapy?
Michelle: I was in hospital for two weeks, then I had to stay at a hotel next to the hospital for another two weeks and I had to go in for daily check ups, sometimes multiple times a day.
iGEM Munich: Do you think CAR T-cell therapy had more severe side effects than the other form of treatments you have received?
Michelle: They were a lot more intense, but they were short-lived. So, like with chemo, I had it like once every three weeks and for two of these weeks I felt really ill, like standard chemo side effects. Then I would be okay for one week. With CAR T, I only had one week of really intense side effects, and then I was pretty much fine other than the feeling that my mental capacity was reduced for a few months afterwards. I feel like I’m back to normal now. I had a stem cell transplant most recently, and with that I was in the hospital for like ten weeks. That was more difficult because I had so many different prolonged side effects going on for ages, I couldn't leave the hospital full stop for these ten weeks, so honestly CAR T was probably better than stem cell, even though the neurotoxicity was the scariest side effect that I have ever had.
iGEM Munich: How long were you in remission after receiving CAR T-cell therapy?
Michelle: So, I was in remission for 8 months with CAR T. Which was the longest that I have been in remission for. With the stem cell transplant that I currently have, I've been in remission for like 7 or 8 months. Hopefully, it will take over the record with CAR T.
iGEM Munich: Do you find it important that people get to know about other forms of cancer therapy other than chemotherapy?
Michelle: Chemo was the only form of treatment I knew about, and for me it was the least effective. And I think it has the most long-lasting and the biggest range of side effects, and we all accept that you are going to be unwell for a long period of time. It was really crazy when I had CAR T therapy. Even though it was intense, I knew that I'm going to be really sick for a week or so. Compared to six months or for however long I thought that it was really good.
iGEM Munich: Do you have any recommendations for other people who are considering CAR T-cell therapy?
Michelle: I did have someone ask me whether they should do CAR T. So, I think, it is a very scary treatment, so don’t underestimate what your body will go through, but at the end of the day I wouldn't be here if I didn't have it. So comparatively, if you already put yourself through chemo, you're willing to put yourself through other treatments. I would personally say, do it. It is scary, but they have gotten a lot better to reverse my symptoms very quickly with steroids. It's been like a year and a half since I had it, so I imagine there now has been more research done, and they know how to manage all of the side effects. They told me that they have come a long way learning how to treat neurotoxicity.
iGEM Munich: How well were you informed about the therapy before you received the treatment?
Michelle: I think I have always been very well-informed. They gave me a contract which says exactly which side effects I can expect, what I would experience, how they would fix those and what the chances are of getting those. So I definitely felt like I was informed enough to make a choice.
iGEM Munich: Have there been any kinds of special monitoring for the CAR T-cells, are they still in your body?
Michelle: No, they told me that they would probably go away after a few months. They can last anywhere from a few months and in very rare cases up to a year. I don't think I have them in my body now. They never really confirmed that with me though. When I relapsed, they rushed me through different kinds of treatments, so I've been passed around between teams a lot.
iGEM Munich: What is your final conclusion about CAR T therapy? Whether it would be worth it?
Michelle: I still think that CAR T therapy is going to be revolutionary for cancer treatment. I think I was in the very early phases of it, for it to work so well for me,considering they had only just come on the NHS. I was really impressed with it; beforehand I went in remission for one month with chemo and then the other chemotherapy didn't work for me at all, and the tumor progressed. CAR T was really impressive, to bring me into remission for 8 months, and it is still the longest that I have been in remission. I think it has a huge amount of potential. You know it is so early in the phases compared to how long chemotherapy has been running. I think it is going to be probably one of the leading ways to treat cancer.