Latest revision as of 02:06, 2 November 2017

Integrated Human Practice

Science is often depicted in opposition to the general public. As scientists we are interested in building bridges between both groups. Our project is about the genetic modification of human immune cells for a therapeutic usage. Therefore it is especially important to take into account the opinion and concerns of the community and local experts. The engagement elaborated key factors of the projects implementation in a real life scenario.

Public Opinion

As a first step, a survey was conducted, where people had the chance to share their opinion towards our project and immune-based therapies in general. Respondents were asked whether they consider immunotherapies worthy of support, how they would feel about beeing treated with such a therapy and which aspects would be most important to them.
The majority would contemplate to undergo a therapy based on our approach in the case of cancer diagnosis. They consider research on immune-based cancer therapies important and worthy of support. Among efficacy, safety is regarded to be one of the most important aspects of our project.
Detailed information about the survey can be found below.

After a short introduction into the current status of CAR T cell therapies, and the improvement by our project, the participants were asked the following questions.

1. Do you consider research on immune based cancer therapy important and worthy of support?
2. In case of a cancer diagnosis: Would you consider undergoing a therapy based on our project, if clinical trials would have confirmed the security and compatibility? The cost of the treatment would be covered by the health insurance.
3. Which of the following aspects do you consider the most important regarding our therapy? (Safety, Compatibility, Efficiency; Two answers were possible)

Figure 1: Results of a small survey covering immunotherapy and our project. In total, 35 responses per question were collected. a) First two questions of this survey are about acceptance of immunotherapy and our project. b) Last question asking for aspects considered most important.

The responses show, that the two most important aspects are efficiency and safety.

Currently CAR T cell therapy development is challenged by overreaching efficacy rather than lack of potency, causing off-target effects. Since our project already aims to solve this issue we sought,inspired by these results, to address the second most frequent concern by improving safety aspects of the CARTEL^TM AND gate. We consulted experts to evaluate feasible safety implementations for our system.

Safety Improvement

At the science fair and the science days we had plenty of opportunities to give the community an understanding of CAR T cell therapies and our project. People recognized our efforts in improving the safety and the compatibility of a novel cancer therapy. Yet, they were concerned by the prospect of introducing genetically modified immune cells to specifically target and destroy cancer tissue.

In order to address these concerns we consulted the group of Prof. Dr. Toni Cathomen from the Center of Chronic Immunodeficiency (CCI). Being the director of the Institute for Transfusion Medicine and Gene Therapy in Freiburg, he is dedicated to research of the immune system and rare immune diseases. Based on this advice and contribution, we established a kill switch, which specifically eliminates our modified T cells in case of unexpected behaviour.

Kill Switch for Activated T cells

To improve the safety of the CARTEL^TM T cells a kill switch, which specifically eliminates CAR T cells that cause unexpected side effects, was implemented. A not negligible risk may occur due to lentiviral transduction of T cells, as it can integrate constructs near an oncogene. This favors the formation of cancer within the population of modified T cells themselves. The switch is controlled by a drug, that does not affect unmodified cells in moderate doses. This approach is frequently used in clinical studies of modified lymphocytes. It is based on the drug ganciclovir (GCV) and its effector, a thymidine kinase commonly expressed by cells infected with herpesviruses (Bondanza et al., 2006).

Mechanism

GCV is usually used as medication against human herpesviruses. It has a high affinity to viral kinases like the thymidine kinase expressed by infected cells. These kinases initiate metabolization of ganciclovir into a nucleotide analogue, which is subsequently integrated into the genome by the DNA polymerase. This results in disruption of DNA replication of proliferating cells leading to apoptosis (Fischer et al., 2005). Ganciclovir has a 1000-fold higher affinity to the herpes simplex virus 1 thymidine kinase (HSV-TK) than to native kinases and therefore exhibits excellent kinetics for a kill switch, as it leaves non-targeted cells unaffected (Dey & Evans, 2011).

Figure 2: Schematic representation of the functional mechanism of ganciclovir. a) Structures of ganciclovir, guanine nucleotide and ganciclovir triphosphate. b) Ganciclovir enters the cell (1) and is phosphorylated by the expressed HSV-TK (2). Cellular thymidine kinases lead to two additional phosphorylations of ganciclovir (3). The ganciclovir-triphosphate is a guanine analogue which inhibits the cellular DNA synthesis processes (4). This leads to apoptosis.

Based on the concerns of people and the advice of Prof. Dr. Cathomen to integrate an additional safety mechanism into our CAR T cells, we were able to introduce a drug based safety system. This will allow elimination of CARTEL^TM T cells in case of severe side effects and thus improve our system. Stable cell lines containing the suicide gene were established and used successfully to confirm its function. The experiment and the results are displayed in the results section.

Interviews with Experts

In order to learn more about safety aspects in CAR T cell therapy, we discussed our project with experts.

iGEM Freiburg members with Prof. Dr. Toni Cathomen

The estimated cost for the first approved CAR T cell therapy manufactured and marketed by Novartis (Tisagenleucel) in the United States is about 475 000 $ for 1 year (Hagen, 2017), an amount not every health insurance is willing to cover. The therapy is approved specifically against pediatric acute lymphoblastic leukemia. CAR T cell treatments for solid tumors are still in clinical trials since they are difficult to implement due to severe off-target effects.

Due to the early stage of research on this subject, we consulted experts about the prospects and future developments of CAR T cell therapy as an integral part of cancer therapies. We discussed how to make it suitable for the treatment of solid tumors, and how our CARTEL^TM system may impact this.

Prof. Dr. Cathomen informed us about possibilities for optimization and automation of manufacturing protocols. This is a critical step to make CAR T cell therapy affordable and ready to use at every treatment center.

I think that local automation will be the future key, meaning that every clinic will be able to produce its own CAR T cells in an automated process - Prof. Cathomen

Additionally he was enthusiastic about possible future applications of the CARTEL^TM system.

For prostate cancer you would for example choose PSMA (prostate-specific membrane antigen) as a target. These molecules are enriched in the tumor, but they also exist in healthy tissue, for example the brain.[...] in this situation your system is very interesting. The CAR is only expressed in a certain microenvironment and is further restricted by its specificity for the desired antigen - Prof. Cathomen

For the full interview see the dropdown below.

iGEM Freiburg: The newly approved CAR T cell therapy from Novartis costs 475 000 $. Is there a possibility of reducing these costs for example by using allogeneic T cells?
Prof. Cathomen: Allogeneic T cell therapy is already used in clinical trials. The procedure for this is as follows: The endogenous T cell receptor, which is responsible for allogenicity, is knocked out. This approach is combined with a very strong chemotherapy, eliminating the immune system and its blood stem cells. Afterwards, new stem cells along with CAR T cells are transplanted into the patient. The allogeneic T cells are present in the patient until the immune system is replenished by the engrafted stem cells. The CAR T cells are then eliminated by the newly developed immune cells. This means that the price will decrease as T cells can be produced in advance and can be derived from a healthy donor, enhancing the reproducibility of the whole process. The drawback, however, is that the T cells are only available for a limited timeframe of about twelve weeks before they are eliminated by the immune system. A clinical trial has already been published. It was successfully conducted on two children suffering from a type of T cell leukemia in London. The patients received a stem cell transplant along with allogeneic CAR T cells targeted against CD19 . Both patients were terminally diseased with no conventional therapies being available and both of them survived. This principally shows that the concept works.

iGEM Freiburg: Could this also be used against solid tumors? Or does that require more time?
Prof. Cathomen: Carl June (a pioneer of CAR T cell therapy) says that to prevent relapses it might be important to have memory CAR T cells. This is where the problem with “off-the-shelf CAR T cells” lies, which are completely eliminated. This raises the question if the tumor might reappear. This has not been answered yet. In solid tumors, a difficulty lies in the tumor microenvironment that develops some specific characteristics that limits the access of T cells and also CAR T cells to the tumor. We try to overcome this via a knockout of checkpoint inhibitors such as PD-1 (programmed cell death protein 1). If this approach is applicable has not been shown yet. There are many ongoing studies working in this field, but no data has been reported until now.

iGEM Freiburg: So the CAR T cell therapy cannot be applied without previously switching off the immune system?
Prof. Cathomen: I would not put it that way. In the case of allogeneic CAR T cells it is a must. Whether this approach is desirable for solid tumors is a different question. I would be more critical about this because the whole immune system needs to be eliminated. In the case of leukemia this is the desired effect chemotherapy. The situation is, however, completely different for solid tumors. The tumor cells cannot be completely eliminated with chemotherapy.

iGEM Freiburg: So allogeneic T cells are no option in fighting solid tumors?
Prof. Cathomen: It is not impossible but very difficult. The CAR T cells have to be modified, as not to be recognized by the immune system. By this modification, however, you endow the CAR T cells with the potential of developing into a tumor. You can equip CAR T cells with a safety switch such as TK (thymidine kinase), so you can eliminate them. But as tumor cells divide rapidly they accumulate a lot of mutations, and it has been shown that TK can lose its function due to mutations. Then these T cells cannot be killed anymore. This is a common problem in cancer treatment. 99.9 % of cells might be killed by a certain treatment, but the surviving 0.1 % of cells are resistant to all therapies previously used. They are positively selected.

iGEM Freiburg: Do you think that our approach of using the tumor microenvironment could contribute to this therapy?
Prof. Cathomen: I do find this idea very interesting. The CAR T cell being only active in the milieu created by the tumor. This could be possible, but still has to be tested in a mouse model, first. You would have to induce the formation of tumors in mice where such a micro milieu is formed. In this model, it could be tested whether the CAR T cells are activated and attack the tumor. Finding the right tumor model would be the next important step in this context. Because mouse models are usually based on xenografts, meaning that human tumor cells and human CAR T cells are transplanted into mice. This model is of course very artificial. The tumors grow, for example, much faster than in the natural human background. If the tumor grows too fast, the mouse has to be killed due to ethical reasons. So, creating the right mouse tumor model is always quite tricky.

iGEM Freiburg: Can you think of other factors defining the tumor microenvironment?
Prof. Cathomen: This is very specific for the type of tumor. The inputs you want to apply are very well chosen as they are pretty common for the most types of cancer. VEGF is necessary to stimulate tumor growth and hypoxia is also very common. The next level of specificity is achieved via the CAR. For prostate cancer, you could for example choose PSMA (prostate-specific membrane antigen) as a target. This antigen is enriched in the tumor, but it also exists in healthy tissue, for example the brain. The concentration of PSMA in the brain is far lower compared to the tumor. If a CAR T cell, however, reaches the brain and encounters its antigen severe problems could arise.

iGEM Freiburg: This would be the situation in which CARTEL could be beneficial?
Prof. Cathomen: Yes, in this situation your system is very interesting. The CAR is only expressed in a certain microenvironment and is further restricted by its specificity for the desired antigen. So, the concept is pretty good and interesting.

iGEM Freiburg: Back to allogeneic T cells. Do you think there is a possibility of using them for the treatment of solid tumors?
Prof. Cathomen: As I said before: For leukemia, the chances are pretty good that the concept will work out. A possibility for solid tumor could be the intratumoral application of allogeneic CAR T cells. But again: If the immune system cannot be easily eliminated – and this is the situation in solid tumors – allogeneic T cells have a low chance of survival.

iGEM Freiburg: A different problem is that metastases present different antigens than the primary tumor. Is there a chance for CAR T cell therapy to also eliminate metastases?
Prof. Cathomen: I do not think that CAR T cell therapy can ever be used as a single means, but has to be used in combination with other treatments such as kinase inhibitors, antibody based therapies, chemotherapy or surgery etc. I think it will always be a combinatorial therapy. Currently, CAR T cells are used as the ultima ratio. In the future, this will maybe change, if clinical trials confirm that CAR T cells become safer. For sure the therapy has a huge success, but fatal off-target effects are observed every now and then, so the risk has to be considered. We are talking about patients with a low chance of surviving the next two or three month. In these cases the risk is acceptable. In other cases, survival rates are higher, so one is going to be more reluctant in choosing relatively risky therapies.

iGEM Freiburg: So there are still no clinical trials on patients with early stage cancers?
Prof. Cathomen: No, this is at the moment not approved. Patients really have to be terminally diseased. CAR T cell therapy still is the ultima ratio.

iGEM Freiburg: Could this change in future?
Prof. Cathomen: I think so. At the moment, there are a lot of clinical trials concerning CAR T cells of the first generation. They do not yet include any safety aspects. Again, if this aspect of the therapy is further developed, CAR T cells could become more feasible for treating early stage cancers. For example, by introducing safety features to eliminate cells, via TK for example, or to use synthetic switches which allows us to switch them on and off again. In your case, this switch would be triggered by the microenvironment. But there are also other possibilities with which the CAR T cells can be externally switched on and off. If an overreaction of the immune system is observed CAR T cells could be inactivated and reactivated again after the symptoms have declined. Once CAR T cells have become safer, they will be used more commonly. This would really be meaningful in the context of metastases.

iGEM Freiburg: What are the potential long term consequences of CAR T cell therapy?
Prof. Cathomen: The only CAR of which we have sufficient data is directed against CD19. These CAR T cells persist in the patient. It was initially expected that these cells would disappear after some time, but apparently, memory T cells have been transduced with the CAR along with effector T cells and these memory cells persist for decades or even lifelong. This means that every B cell in the patient will be eliminated after the treatment. So, these patients will always lack B cells and never be able to produce antibodies anymore. This trade-off is less tragic as it seems as the patients have survived the tumor and the immune system can be stabilized by regularly injecting immunoglobulins, a mix of different antibodies.

iGEM Freiburg: But this would also mean that memory CARTEL T cells will also be able to fight the tumor if it reappears?

Prof. Cathomen: Right, your approach is really predestined for solid tumors, but not for leukemia.

iGEM Freiburg: Recently a therapy from Novartis against B-cell ALL was approved by the FDA. Can you imagine when the first therapy like this will be approved in Europe?
Prof. Cathomen: This is a somewhat difficult question, but I think that we will see an approval for a CAR T cell therapy next year or maybe one year later. The market is also present in Europe. But Novartis has to construct the infrastructure first. The concept of Novartis is centralized production in huge clean rooms. The isolated T cells have to be shipped to these centers for modification and afterwards back to the hospital for transplantation. The manufacturing process still has to be approved, but Novartis certainly has a big interest of an approval of this therapy in Europe.

iGEM Freiburg: The key to future centralized manufacturing?
Prof. Cathomen: Novartis (and other CAR T cell companies favor centralized manufacturing. I do not think that this is where we are heading in the future. I think that automation of the manufacturing processes will be the future, meaning that every clinic will be able to produce its own CAR T cells in an automated process. There is a German company, for example, that is about to produce such machines: you basically supply the device with the patient’s T cells and the desired CAR vector and after three weeks you end up with the ready to use CAR T cell product. This product, prepared on site can then be used directly to treat the patient. This is making the logistic process a lot easier, as you do not have to ship everything to one central location. Personally, I think that an automated local production is the way to go for the future.

iGEM Freiburg: But this is still some time ahead?
Prof. Cathomen: Well, these devices already exist.

iGEM Freiburg: Thank you very much for taking some time to discuss this project with us!

iGEM Freiburg members with PD Dr. Simone Hettmer

PD Dr. Simone Hettmer from the Comprehensive Cancer Centre Freiburg, is a practicing pediatric hematologist and oncologist, and shared her insights on how children with cancer and their parents perceive novel treatments. As one of 13 oncology centers of excellence in Germany we asked about her opinion concerning our project.

I think that your approach to CAR T cell therapies is very promising. I especially like that you prioritize the importance of the tumor microenvironment - PD Dr. Hettmer

When we asked about the possible acceptance of our CARTEL^TM system by future patients, she raised awareness for the individual situation of each future patient.

As far as patients and families are concerned, I believe that acceptance depends mostly on cultural and personal factors - PD Dr. Hettmer

For the full interview see the dropdown below.

PD Dr. Simone Hettmer from pediatric oncology at the university hospital of Freiburg

iGEM Freiburg: Dear PD Dr. Simone Hettmer, thank you for having this interview with us. First of all, how do you like our approach to this project?
PD Dr. Simone Hettmer: I think that your approach to CAR T cell therapies is very promising. I especially like that you prioritize the importance of the tumor microenvironment. At the moment, little is known about how tumor cells influence their niche and how healthy cells in their niche interact with neighbouring tumor cells.

iGEM Freiburg: Our project is not the universal solution to fight cancer, but we think it can contribute to the establishment of CAR T cell therapies against solid tumors. What obstacles do you see in our project or CAR T cell therapies in general?
PD Dr. Simone Hettmer: The key to developing a successful CAR T cell therapy is to identify a common antigen, that is expressed on all cells of an individual’s tumor, such as CD19 in B-lineage leukemias or CD30 in Hodgkin’s lymphoma. In case of a solid tumor, that includes the primary or relapsed tumor’s progeny at distant metastatic sites, too. At the same time, the antigen must not be expressed on other functionally relevant cells, in order to limit toxicity by off-target effects on healthy cells. There are practical issues as well. You need to be able to extract a sufficient number of functional T cells from the patient in order to be able to make CAR T cells to treat that particular patient’s tumor. Also, if you use lentiviruses to engineer cells, you need to very carefully control for off-target effects of lentiviral transduction that may promote malignant transformation after reinfusion into the patient.

iGEM Freiburg: Would people accept a project like ours which is based on genetic modification of immune cells?
PD Dr. Simone Hettmer: Well, you need to convince the medical community as well as the patients. For the latter, you need to provide sound safety and efficacy data in vitro and in preclinical animal studies, and initiate well-controlled clinical trials to prove that your approach is actually safe and effective in real patients. If well-established and highly successful treatments are available (as is the case for several pediatric cancers), you may also have to show that your treatment is better than what’s already sitting on the shelf. As far as patients and families are concerned, I believe that acceptance depends mostly on cultural and personal factors. Some families are very concerned about being part of an “experiment”. Others prioritize the quality of life at home at the end of life over hospital stays, logistical strains and side effects that may be associated with an experimental treatment. Still, losing a child is the worst thing that can happen to any parent, and most families are desperate to find a cure for their sick child.

iGEM Freiburg: The newly approved CAR T cell therapy in the United States has made a huge impact on the cancer research community. But there is barely any information about possible impending long term side effects…
PD Dr. Simone Hettmer: That’s not quite true… certain side effects are well-known, for example Cytokine Release Syndrome, which indicates a fulminant response to immune treatments. Other side effects will come to light as more and more patients undergo CAR T cell therapy. This is especially true for late effects of treatment, an important consideration for children, who may go on to live for decades after their cancer is cured.

iGEM Freiburg: Thank you very much for your time and your expertise!
PD Dr. Simone Hettmer: You’re welcome.

Human Practice

Collaborations

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-             <h1 align="center">Integrated Human Practce</h1>
+             <h1 align="center">Integrated Human Practice</h1>
              <div class="item">
-             <h2>Interview with Professor Cathomen</h2>
+             <p>Science is often depicted in opposition to the general public. As scientists we are interested in building bridges between both groups. Our project is about the genetic modification of human immune cells for a therapeutic usage. Therefore it is especially important to take into account the opinion and concerns of the community and local experts. The engagement elaborated key factors of the projects implementation in a real life scenario.</p>
-                <p>Toni Cathomen is Professor of Cell and Gene Therapy at the Center for Chronic Immunodeficiency and Director of the Institute for Transfusion Medicine and Gene Therapy in the University Medical Center Freiburg. He is leading the ’Cathomen research group’ which works with CAR T cells. Our team interviewed him to discuss  different aspects of our project such as the design, the application costs and the possible bottlenecks but also the potential of our CARTelTM system in modern cancer therapy.</p>
+</div>
-              <br />
-                <p><b>How are allogeneic CAR T cells used and what are the possible benefits and disadvantages?</b></p>
-                <p>Allogeneic CAR T cells are already in use for CAR therapy. The T cell receptor, which is responsible for the alloreactivity is knocked down because the used CAR T cells are not from the patient and would be seen as a threat. A strong chemotherapy is then used to destroy the immune system of the leukemic patient (like a classical leukemia treatment without T cells). Even the stem cells of the immune system have to be destroyed. A new immune system from a safe donator is inserted via bone marrow transplantation. Also allogeneic CAR T cells are then inserted. These allogeneic CAR T cells are active until the new immune system is up again and the CAR T cells are destroyed. This time span is about 8 to 12 weeks. <br />
-                This allogene therapy is cheaper because the allogeneic CAR T cells are produced for multiple patients and are kept in stocks, but also come from healthy donors and not from a weak patient. The production itself is then controllable. The disadvantages are that the allogeneic cells are only available for a short period of time. One publication (Waseem Quasim, et al.) mentioned two boys with acute lymphoblastic lymphoma (ALL) who got the allogeneic CAR T cells therapy after all other therapies had not worked. And both boys healed. This method with allogeneic cells was a success. <br />
-                If memory CAR T cells were produced by the body during the therapy, a possible relapse would be avoided. But all allogeneic CAR T cells are destroyed by the new immune system. Nevertheless, there is no known leukemia relapse after this allogenic CAR T cells therapy.
-                </p>
+<div class="item">
+<h2>Public Opinion</h2>
-                <p><b>Our project focuses on solid tumors and not on cancer like leukemia. How would these allogeneic CAR T cells be used against a solid tumor?</b></p>
-                <p>Solid tumors have the microenvironment which reduces the T cell activity. Researchers try to counter it with the so called «checkpoint inhibitors». Whether the CAR T cells would actually work against solid tumors is not known and research may not be far enough to allow a reliable answer. But there are many studies going on about this precise topic, but none are published yet. <br />
+<p>As a first step, a survey was conducted, where people had the chance to share their opinion towards our project and immune-based therapies in general. Respondents were asked whether they consider immunotherapies worthy of support, how they would feel about beeing treated with such a therapy and which aspects would be most important to them.<br>
-                For allogeneic CAR T cells against solid tumors, the immune system is not destroyed in opposition to the leukemia therapy. The allogeneic cells are then instantly recognized as foreign. A possible solution would be to transform the allogeneic T cells to hide them from the immune system. This method would be more complicated and dangerous: cells which are not recognized by the immune system are also not controlled. This is a great risk for cancer. CAR T cells can be modified to be deactivated from the outside (with drugs) but it has been shown that CAR T cells get many mutations and they become unresponsive to this deactivation.
+The majority would contemplate to undergo a therapy based on our approach in the case of cancer diagnosis. They consider research on immune-based cancer therapies important and worthy of support. Among efficacy, safety is regarded to be one of the most important aspects of our project.<br>
-                </p>
+Detailed information about the survey can be found below.</p>
-                <p><b>Do you think our project to activate T cells through the tumor microenvironment is a possible contribution to the current cancer research?</b></p>
-                <p>Your idea is very interesting and there is a chance that such a construct as yours will be used in the future. The next step would be to test the final CAR T cell construct within a mouse model with a solid tumor and its microenvironment. But then you should remember that mouse models react differently than  humans because the T cells and the tumor have human origin.
-                </p>
-                <p><b>Do you know other inputs for our AND gate which could be better activators?</b></p>
-                <p>Hypoxia, pH and growth factors like VEGF are well chosen and very tumor specific. Other inputs do not seem to be better. General inputs are very interesting because all tumors could be targeted and then the specific tumor could be targeted with the appropriate CAR. The general activation through the AND gate and the specific activation through the CAR is a good combination. This method promising.</p>
-                <p><b>Do you think that the CAR therapy will be an independent therapy one day?</b></p>
-                <p>I don’t think there will ever be a proper CAR therapy but rather a combination with other techniques like antibody therapy, of course chemotherapy, surgery, etc. Today, the CAR therapy is  only seen as the last option when other methods did not work, for patients that have a low chance to survive their illness and are ready to take the risks of an unknown therapy. With a solid tumor the patient will probably not do the therapy, because like all new therapy, all other resources have to fail before they do the CAR therapy.</p>
-                <p><b>Will this change?</b></p>
-                <p>Many studies are running but still without safety feature. When switches are developed (like AND gate, kill switches, activation/deactivation switch…) and CAR can be deactivated, the CAR therapy will be applicated sooner and not be considered as a last possibility.</p>
-                <p><b>The CAR Therapy has often been used now against minimal residual disease after classical leukemia therapy, mostly with big success. Do you know if there is any long term impact?</b></p>
-                <p>With the CAR therapy, there is a co-production of memory T cells which can last decades. Treated patients who had leukemia then never have B cells again and have to take antibody mixes to keep the immune system stable. There is no other known long term impact.</p>
-                <p><b>If our AND gate T cells also produce memory cells, any new tumor relapse or growing residue with a tumor microenvironment would then be destroyed?</b></p>
-                <p>Exactly. In this case this would be advantageous.</p>
-                <p><b>The first CAR therapy was accepted by the FDA in the USA last August. Do you have an idea when we will see the CAR therapy in Germany or in Europe?</b></p>
-                <p>There is no infrastructure yet in Europe that allows the production of T cells the way Novartis is doing it in the USA. Novartis production is central and all T cell samples are gathered there, modified and then sent back. Nevertheless, there is a big interest to expand in Europe and Novartis is certainly waiting for an approval. <br />
-                But in the future, the CAR T cells will be produced in the clinics itself and the production will be automated. There is already a machine which can modify T cells. The therapy will be cheaper and quicker. This leads me to believe that the CAR therapy will be present in Europe in the near future.</p>
-                <p><b></b></p>
-                <p></p>
-            <br />
              </div>
+<div class="item">
+                <button class="accordion">Survey</button>
+                    <div class="panel panel-default">
+                      <div class="item">
+<p>After a short introduction into the current status of CAR T cell therapies, and the improvement by our project, the participants were asked the following questions.<br><br>
+<i>1. Do you consider research on immune based cancer therapy important and worthy of support?<br>
+. In case of a cancer diagnosis: Would you consider undergoing a therapy based on our project, if clinical trials would have confirmed the security and compatibility? The cost of the treatment would be covered by the health insurance.<br>
+. Which of the following aspects do you consider the most important regarding our therapy? (Safety, Compatibility, Efficiency; Two answers were possible)<br></i>
+</div>
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+<div class="item">
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+       <div class="figure">
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+                   <div class="figurecaption">
+<p><strong>Figure 1: Results of a small survey covering immunotherapy and our project.</strong>
+In total, 35 responses per question were collected. <b>a)</b> First two questions of this survey are about acceptance of immunotherapy and our project. <b>b)</b> Last question asking for aspects considered most important.</p>
+                   </div>
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-             <div class="item">
-             <p>Waseem Quasim, et al. (2017): Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Science Translational Medicine,  25 Jan, Vol. 9, Issue 374.
+    <div class="mySlides">
-             </p>
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+      <img src="https://static.igem.org/mediawiki/2017/6/6b/T-FREIBURG-S2-2.png" style="width:100%">
+    </div>
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+             <div class="numbertext"> </div>
+      <img src="https://static.igem.org/mediawiki/2017/f/f3/T%E2%80%93FREIBURG%E2%80%93Suicide_gene_mechanism.png" style="width:100%">
+    </div>
+    <div class="mySlides">
+             <div class="numbertext"></div>
+      <img src="" style="width:100%">
+    </div>
+  </div>
+  </div>
+  </div>
+</div>
+</div>
+</div>
+<div class="item">
+<p>The responses show, that the two most important aspects are efficiency and safety.</p>
+</div>
+</div>
+</div>
+<div class="item">
+<p>Currently CAR T cell therapy development is challenged by overreaching efficacy rather than lack of potency, causing off-target effects. Since our project already aims to solve this issue we sought,inspired by these results, to address the second most frequent concern by improving safety aspects of the CARTEL<sup>TM</sup> AND gate. We consulted experts to evaluate feasible safety implementations for our system.</p>
+</div>
+<div class="item">
+<h2>Safety Improvement</h2>
+<p>
+At the <a href="https://2017.igem.org/Team:Freiburg/HP/Silver">science fair</a> and the <a href="https://2017.igem.org/Team:Freiburg/HP/Silver">science days</a> we had plenty of opportunities to give the community an understanding of CAR T cell therapies and our project. People recognized our efforts in improving the safety and the compatibility of a novel cancer therapy. Yet, they were concerned by the prospect of introducing genetically modified immune cells to specifically target and destroy cancer tissue.</p>
+<p>In order to address these concerns we consulted the group of <a href="https://www.uniklinik-freiburg.de/itg.html" target="_blank">Prof. Dr. Toni Cathomen</a> from the <a href="https://www.uniklinik-freiburg.de/cci.html" target="_blank">Center of Chronic Immunodeficiency (CCI)</a>. Being the director of the Institute for Transfusion Medicine and Gene Therapy in Freiburg, he is dedicated to research of the immune system and rare immune diseases. Based on this advice and contribution, we established a <a href="https://2017.igem.org/Team:Freiburg/Results">kill switch</a>, which specifically eliminates our modified T cells in case of unexpected behaviour.</p>
+</div>
+<div class="item">
+                <button class="accordion">Kill Switch</button>
+                    <div class="panel panel-default">
+<div class="item">
+<h2>Kill Switch for Activated T cells</h2>
+<p>To improve the safety of the CARTEL<sup>TM</sup> T cells a kill switch, which specifically eliminates CAR T cells that cause unexpected side effects, was implemented. A not negligible risk may occur due to lentiviral transduction of T cells, as it can integrate constructs near an oncogene. This favors the formation of cancer within the population of modified T cells themselves. The switch is controlled by a drug, that does not affect unmodified cells in moderate doses. This approach is frequently used in clinical studies of modified lymphocytes. It is based on the drug ganciclovir (GCV) and its effector, a thymidine kinase commonly expressed by cells infected with herpesviruses (Bondanza <i>et al.</i>, 2006).</p>
+</div>
+ <div class="item">
+<h2>Mechanism</h2>
+<p>GCV is usually used as medication against human herpesviruses. It has a high affinity to viral kinases like the thymidine kinase expressed by infected cells. These kinases initiate metabolization of ganciclovir into a nucleotide analogue, which is subsequently integrated into the genome by the DNA polymerase. This results in disruption of DNA replication of proliferating cells leading to apoptosis (Fischer <i>et al.</i>, 2005). Ganciclovir has a 1000-fold higher affinity to the herpes simplex virus 1 thymidine kinase (HSV-TK) than to native kinases and therefore exhibits excellent kinetics for a kill switch, as it leaves non-targeted cells unaffected (Dey & Evans, 2011).</p>
+</div>
+<div class="item">
+    <div class="image_box middle">
+       <div class="figure">
+           <div class="figureinner">
+               <img src="https://static.igem.org/mediawiki/2017/f/f3/T%E2%80%93FREIBURG%E2%80%93Suicide_gene_mechanism.png" onclick="openModal();currentSlide(2)" class="hover-shadow cursor" style="width:80%;height:80%">
+                   <div class="figurecaption">
+<p><strong>Figure 2: Schematic representation of the functional mechanism of ganciclovir. a)</strong> Structures of ganciclovir, guanine nucleotide and ganciclovir triphosphate. <b>b)</b> Ganciclovir enters the cell (1) and is phosphorylated by the expressed HSV-TK (2). Cellular thymidine kinases lead to two additional phosphorylations of ganciclovir (3). The ganciclovir-triphosphate is a guanine analogue which inhibits the cellular DNA synthesis processes (4). This leads to apoptosis.
+</p>
+                   </div>
+           </div>
+       </div>
+    </div>
+</div>
+<div class="item">
+<p>Based on the concerns of people and the advice of Prof. Dr. Cathomen to integrate an additional safety mechanism into our CAR T cells, we were able to introduce a drug based safety system. This will allow elimination of CARTEL<sup>TM</sup> T cells in case of severe side effects and thus improve our system. Stable cell lines containing the suicide gene were established and used successfully to confirm its function. The experiment and the results are displayed in the <a href="https://2017.igem.org/Team:Freiburg/Results">results section</a>.</p>
+</div>
+</div>
+</div>
+ <div class="item">
+<h2>Interviews with Experts</h2>
+<p>In order to learn more about safety aspects in CAR T cell therapy, we discussed our project with experts.</p>
+  <div class="image_box right">
+             <div class="figure">
+              <div class="figureinner" style="width:400px">
+                 <img src="https://static.igem.org/mediawiki/2017/a/a4/T-FREIBURG-Interview_Toni_Cathomen.png" height="100%" width="100%">
+                 <div class="figurecaption">
+                    <p style="text-align:center"><strong>iGEM Freiburg members with Prof. Dr. Toni Cathomen</strong><br>
+                       </p>
+                 </div>
+              </div>
              </div>
+            </div>
+<p>The estimated cost for the first approved CAR T cell therapy manufactured and marketed by Novartis (Tisagenleucel) in the United States is about 475 000 $ for 1 year (<a href="http://www.onclive.com/web-exclusives/novartis-sets-a-price-of-475000-for-car-tcell-therapy " target= " _blank">Hagen, 2017</a>), an amount not every health insurance is willing to cover. The therapy is approved specifically against pediatric acute lymphoblastic leukemia. CAR T cell treatments for solid tumors are still in clinical trials since they are difficult to implement due to severe off-target effects.</p>
+<p>Due to the early stage of research on this subject, we consulted experts about the prospects and future developments of CAR T cell therapy as an integral part of cancer therapies. We discussed how to make it suitable for the treatment of solid tumors, and how our CARTEL<sup>TM</sup> system may impact this.
+</p>
+<p><b>Prof. Dr. Cathomen</b> informed us about possibilities for optimization and automation of manufacturing protocols. This is a critical step to make CAR T cell therapy affordable and ready to use at every treatment center.</p>
+    </div>
+<div class="item">
+<p><i>I think that local automation will be the future key, meaning that every clinic will be able to produce its own CAR T cells in an automated process</i> - Prof. Cathomen<p>
+<p>Additionally he was enthusiastic about possible future applications of the CARTEL<sup>TM</sup> system.</p>
+<p><i>For prostate cancer you would for example choose PSMA (prostate-specific membrane antigen) as a target. These molecules are enriched in the tumor, but they also exist in healthy tissue, for example the brain.[...] in this situation your system is very interesting. The CAR is only expressed in a certain microenvironment and is further restricted by its specificity for the desired antigen</i> - Prof. Cathomen</p>
+<p>For the full interview see the dropdown below.</p>
+    </div>
+  <div class="item">
+                <button class="accordion">Interview with Prof. Dr. Toni Cathomen: Costs and Further Development</button>
+                    <div class="panel panel-default">
+                        <div class="item">
+                            <div class="item">
+                            <p>
+                            <b><i>iGEM Freiburg: The newly approved CAR T cell therapy from Novartis costs 475 000 $. Is there a possibility of reducing these costs for example by using allogeneic T cells?</i></b><br>
+                            <i>Prof. Cathomen:</i> Allogeneic T cell therapy is already used in clinical trials. The procedure for this is as follows: The endogenous T cell receptor, which is responsible for allogenicity, is knocked out. This approach is combined with a very strong chemotherapy, eliminating the immune system and its blood stem cells. Afterwards, new stem cells along with CAR T cells are transplanted into the patient. The allogeneic T cells are present in the patient until the immune system is replenished by the engrafted stem cells. The CAR T cells are then eliminated by the newly developed immune cells. This means that the price will decrease as T cells can be produced in advance and can be derived from a healthy donor, enhancing the reproducibility of the whole process. The drawback, however, is that the T cells are only available for a limited timeframe of about twelve weeks before they are eliminated by the immune system. A clinical trial has already been published. It was successfully conducted on two children suffering from a type of T cell leukemia in London. The patients received a stem cell transplant along with allogeneic CAR T cells targeted against CD19 . Both patients were terminally diseased with no conventional therapies being available and both of them survived. This principally shows that the concept works.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: Could this also be used against solid tumors? Or does that require more time?</i><br></b>
+                            <i>Prof. Cathomen:</i> Carl June (a pioneer of CAR T cell therapy) says that to prevent relapses it might be important to have memory CAR T cells. This is where the problem with “off-the-shelf CAR T cells” lies, which are completely eliminated. This raises the question if the tumor might reappear. This has not been answered yet.
+In solid tumors, a difficulty lies in the tumor microenvironment that develops some specific characteristics that limits the access of T cells and also CAR T cells to the tumor. We try to overcome this via a knockout of checkpoint inhibitors such as PD-1 (programmed cell death protein 1). If this approach is applicable has not been shown yet. There are many ongoing studies working in this field, but no data has been reported until now.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: So the CAR T cell therapy cannot be applied without previously switching off the immune system?<br></i></b>
+                            <i>Prof. Cathomen:</i>  I would not put it that way. In the case of allogeneic CAR T cells it is a must. Whether this approach is desirable for solid tumors is a different question. I would be more critical about this because the whole immune system needs to be eliminated. In the case of leukemia this is the desired effect chemotherapy. The situation is, however, completely different for solid tumors. The tumor cells cannot be completely eliminated with chemotherapy.
+                            </p>
+                            <p>
+                               <b> <i>iGEM Freiburg: So allogeneic T cells are no option in fighting solid tumors?<br></i></b>
+                            <i>Prof. Cathomen:</i> It is not impossible but very difficult. The CAR T cells have to be modified, as not to be recognized by the immune system. By this modification, however, you endow the CAR T cells with the potential of developing into a tumor. You can equip CAR T cells with a safety switch such as TK (thymidine kinase), so you can eliminate them. But as tumor cells divide rapidly they accumulate a lot of mutations, and it has been shown that  TK can lose its function due to mutations. Then these T cells cannot be killed anymore. This is a common problem in cancer treatment. 99.9 % of cells might be killed by a certain treatment, but the surviving 0.1 % of cells are resistant to all therapies previously used. They are positively selected.
+                            </p>
+                            <p>
+                           <b> <i>iGEM Freiburg: Do you think that our approach of using the tumor microenvironment could contribute to this therapy?<br></i></b>
+                            <i>Prof. Cathomen:</i> I do find this idea very interesting. The CAR T cell being only active in the milieu created by the tumor. This could be possible, but still has to be tested in a mouse model, first. You would have to induce the formation of tumors in mice where such a micro milieu is formed. In this model, it could be tested whether the CAR T cells are activated and attack the tumor. Finding the right tumor model would be the next important step in this context. Because mouse models are usually based on xenografts, meaning that human tumor cells and human CAR T cells are transplanted into mice. This model is of course very artificial. The tumors grow, for example, much faster than in the natural human background. If the tumor grows too fast, the mouse has to be killed due to ethical reasons. So, creating the right mouse tumor model is always quite tricky.
+                            </p>
+                            <p>
+                              <b>  <i>iGEM Freiburg: Can you think of other factors defining the tumor microenvironment?<br></i></b>
+                            <i>Prof. Cathomen:</i> This is very specific for the type of tumor. The inputs you want to apply are very well chosen as they are pretty common for the most types of cancer. VEGF is necessary to stimulate tumor growth and hypoxia is also very common.
+The next level of specificity is achieved via the CAR. For prostate cancer, you could for example choose PSMA (prostate-specific membrane antigen) as a target. This antigen is enriched in the tumor, but it also exists in healthy tissue, for example the brain. The concentration of PSMA in the brain is far lower compared to the tumor. If a CAR T cell, however, reaches the brain and encounters its antigen severe problems could arise.
+                            </p>
+                            <p>
+                               <b> <i>iGEM Freiburg: This would be the situation in which CARTEL could be beneficial?</i></b><br>
+                            <i>Prof. Cathomen:</i> Yes, in this situation your system is very interesting. The CAR is only expressed in a certain microenvironment and is further restricted by its specificity for the desired antigen. So, the concept is pretty good and interesting.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: Back to allogeneic T cells. Do you think there is a possibility of using them for the treatment of solid tumors?<br></i></b>
+                            <i>Prof. Cathomen:</i> As I said before: For leukemia, the chances are pretty good that the concept will work out. A possibility for solid tumor could be the intratumoral application of allogeneic CAR T cells. But again: If the immune system cannot be easily eliminated – and this is the situation in solid tumors – allogeneic T cells have a low chance of survival.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: A different problem is that metastases present different antigens than the primary tumor. Is there a chance for CAR T cell therapy to also eliminate metastases?<br></i></b>
+                            <i>Prof. Cathomen:</i> I do not think that CAR T cell therapy can ever be used as a single means, but has to be used in combination with other treatments such as kinase inhibitors, antibody based therapies, chemotherapy or surgery etc. I think it will always be a combinatorial therapy. Currently, CAR T cells are used as the ultima ratio. In the future, this will maybe change, if clinical trials confirm that CAR T cells become safer. For sure the therapy has a huge success, but fatal off-target effects are observed every now and then, so the risk has to be considered. We are talking about patients with a low chance of surviving the next two or three month. In these cases the risk is acceptable. In other cases, survival rates are higher, so one is going to be more reluctant in choosing relatively risky therapies.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: So there are still no clinical trials on patients with early stage cancers?<br></i></b>
+                            <i>Prof. Cathomen:</i> No, this is at the moment not approved. Patients really have to be terminally diseased. CAR T cell therapy still is the ultima ratio.
+                            </p>
+                            <p>
+                                <b><i>iGEM Freiburg: Could this change in future?<br></i></b>
+                            <i>Prof. Cathomen:</i> I think so. At the moment, there are a lot of clinical trials concerning CAR T cells of the first generation. They do not yet include any safety aspects. Again, if this aspect of the therapy is further developed, CAR T cells could become more feasible for treating early stage cancers. For example, by introducing safety features to eliminate cells, via TK for example, or to use synthetic switches which allows us to switch them on and off again. In your case, this switch would be triggered by the microenvironment. But there are also other possibilities with which the CAR T cells can be externally switched on and off. If an overreaction of the immune system is observed CAR T cells could  be inactivated and reactivated again after the symptoms have declined. Once CAR T cells have become safer, they will be used more commonly. This would really be meaningful in the context of metastases.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: What are the potential long term consequences of CAR T cell therapy?<br></i></b>
+                            <i>Prof. Cathomen:</i> The only CAR of which we have sufficient data is directed against CD19. These CAR T cells persist in the patient. It was initially expected that these cells would disappear after some time, but apparently, memory T cells have been transduced with the CAR along with effector T cells and these memory cells persist for decades or even lifelong. This means that every B cell in the patient will be eliminated after the treatment. So, these patients will always lack B cells and never be able to produce antibodies anymore. This trade-off is less tragic as it seems as the patients have survived the tumor and the immune system can be stabilized by regularly injecting immunoglobulins, a mix of different antibodies.
+                            </p>
+                            <p>
+                                <b>  <i>iGEM Freiburg: But this would also mean that memory CARTEL T cells will also be able to fight the tumor if it reappears?<br></i></b></p>
+                           <p> <i>Prof. Cathomen:</i> Right, your approach is really predestined for solid tumors, but not for leukemia.</p>
+                            <p>
+                            <b><i>iGEM Freiburg: Recently a therapy from Novartis against B-cell ALL was approved by the FDA. Can you imagine when the first therapy like this will be approved in Europe?<br></i></b>
+                            <i>Prof. Cathomen:</i> This is a somewhat difficult question, but I think that we will see an approval for a CAR T cell therapy next year or maybe one year later. The market is also present in Europe. But Novartis has to construct the infrastructure first. The concept of Novartis is centralized production in huge clean rooms. The isolated T cells have to be shipped to these centers for modification and afterwards back to the hospital for transplantation. The manufacturing process still has to be approved, but Novartis certainly has a big interest of an approval of this therapy in Europe.
+                            </p>
+                            <p>
+                            <b><i>iGEM Freiburg: The key to future centralized manufacturing?<br></i></b>
+                            <i>Prof. Cathomen:</i> Novartis (and other CAR T cell companies favor centralized manufacturing. I do not think that this is where we are heading in the future. I think that automation of the manufacturing processes will be the future, meaning that every clinic will be able to produce its own CAR T cells in an automated process. There is a German company, for example, that is about to produce such machines: you basically supply the device with the patient’s T cells and the desired CAR vector and after three weeks you end up with the ready to use CAR T cell product. This product, prepared on site can then be used directly to treat the patient. This is making the logistic process a lot easier, as you do not have to ship everything to one central location. Personally, I think that an automated local production is the way to go for the future.
+                            </p>
+                            <p>
+                                <b><i>iGEM Freiburg: But this is still some time ahead?<br></i></b>
+                            <i>Prof. Cathomen:</i> Well, these devices already exist.
+                            </p>
+                            <p>
+                            <b> <i>iGEM Freiburg: Thank you very much for taking some time to discuss this project with us!</i></b>
+                            </p>
+                            </div>
+                        </div>
+                    </div>
+                </div>
+<div class="item">
+ <div class="image_box right">
+            <div class="figure">
+              <div class="figureinner" style="width:400px">
+                 <img src="https://static.igem.org/mediawiki/2017/5/5e/T-FREIBURG-Interview_Simone_Hettmer.png" height="100%" width="100%">
+                 <div class="figurecaption">
+                    <p><strong>iGEM Freiburg members with PD Dr. Simone Hettmer</strong><br>
+                       </p>
+                 </div>
+              </div>
+            </div>
+           </div>
+<p><b>PD Dr. Simone Hettmer from the Comprehensive Cancer Centre Freiburg</b>, is a practicing pediatric hematologist and oncologist, and shared her insights on how children with cancer and their parents perceive novel treatments. As one of 13 oncology centers of excellence in Germany we asked about her opinion concerning our project.</p>
 </div>
+<div class="item">
+<p><i>I think that your approach to CAR T cell therapies is very promising. I especially like that you prioritize the importance of the tumor microenvironment</i> - PD Dr. Hettmer</p></div>
+<div class="item">
+<p>When we asked about the possible acceptance of our CARTEL<sup>TM</sup> system by future patients, she raised awareness for the individual situation of each future patient. </p></div>
+<div class="item">
+<p><i>As far as patients and families are concerned, I believe that acceptance depends mostly on cultural and personal factors</i> - PD Dr. Hettmer</p>
+<p style="text-align:left">For the full interview see the dropdown below.</p>
 </div>
+    <div class="item">
+                <button class="accordion">Interview with PD Dr. Simone Hettmer: The Patient/Therapy Relation</button>
+                    <div class="panel panel-default">
+                        <div class="item">
+                        <h3>PD Dr. Simone Hettmer from pediatric oncology at the university hospital of Freiburg</h3>
+                            <div class="item">
+                        <p>
+                            <b><i>iGEM Freiburg: Dear PD Dr. Simone Hettmer, thank you for having this interview with us.
+                                First of all, how do you like our approach to this project?<br></i></b>
+                        <i>PD Dr. Simone Hettmer:</i>
+                       I think that your approach to CAR T cell therapies is very promising. I especially like that you prioritize the importance of the tumor microenvironment. At the moment, little is known about how tumor cells influence their niche and how healthy cells in their niche interact with neighbouring tumor cells.
+                        </p>
+                        <p>
+                            <b><i>iGEM Freiburg:
+                        Our project is not the universal solution to fight cancer, but we think it can contribute to the establishment of CAR T cell therapies against solid tumors. What obstacles do you see in our project or CAR T cell therapies in general?<br></i></b>
+                        <i>PD Dr. Simone Hettmer:</i>
+                        The key to developing a successful CAR T cell therapy is to identify a common antigen, that is expressed on all cells of an individual’s tumor, such as CD19 in B-lineage leukemias or CD30 in Hodgkin’s lymphoma. In case of a solid tumor, that includes the primary or relapsed tumor’s progeny at distant metastatic sites, too. At the same time, the antigen must not be expressed on other functionally relevant cells, in order to limit toxicity by off-target effects on healthy cells. There are practical issues as well. You need to be able to extract a sufficient number of functional T cells from the patient in order to be able to make CAR T cells to treat that particular patient’s tumor. Also, if you use lentiviruses to engineer cells, you need to very carefully control for off-target effects of lentiviral transduction that may promote malignant transformation after reinfusion into the patient.
+                        </p>
+                        <p>
+                        <b><i>iGEM Freiburg:
+                        Would people accept a project like ours which is based on genetic modification of immune cells?<br></i></b>
+                        <i>PD Dr. Simone Hettmer:</i>
+                       Well, you need to convince the medical community as well as the patients. For the latter, you need to provide sound safety and efficacy data in vitro and in preclinical animal studies, and initiate well-controlled clinical trials to prove that your approach is actually safe and effective in real patients. If well-established and highly successful treatments are available (as is the case for several pediatric cancers), you may also have to show that your treatment is better than what’s already sitting on the shelf. As far as patients and families are concerned, I believe that acceptance depends mostly on cultural and personal factors. Some families are very concerned about being part of an “experiment”. Others prioritize the quality of life at home at the end of life over hospital stays, logistical strains and side effects that may be associated with an experimental treatment. Still, losing a child is the worst thing that can happen to any parent, and most families are desperate to find a cure for their sick child.
+                        </p>
+                        <p>
+                        <b><i>iGEM Freiburg:
+                        The newly approved CAR T cell therapy in the United States has made a huge impact on the cancer research community. But there is barely any information about possible impending long term side effects…</b><br></i>
+                        <i>PD Dr. Simone Hettmer:</i>
+                        That’s not quite true… certain side effects are well-known, for example Cytokine Release Syndrome, which indicates a fulminant response to immune treatments. Other side effects will come to light as more and more patients undergo CAR T cell therapy. This is especially true for late effects of treatment, an important consideration for children, who may go on to live for decades after their cancer is cured.
+                        </p>
+                        <p>
+                       <b> <i>iGEM Freiburg:
+                        Thank you very much for your time and your expertise!</b><br></i>
+                        <i>PD Dr. Simone Hettmer:</i>
+                        You’re welcome.
+                        </p>
+                            </div>
+                        </div>
+                    </div>
+                </div>
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+<div class="figure" style="width:195px">
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+                       	<p><strong>Human Practice</strong></p></a>
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+                        </div>
+                    <div class="figurecaption-car" style="float:left">
+                       	<p><strong>Collaborations</strong></p></a>
+        </div>
+        </div>
+        </div>
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