Made during the past year and a half, we are super excited to share this with everyone. I would like to say that we have some Q&A sessions organized for each session here. We will have 3 of those, and we really encourage people also online to ask their questions. Please write them. We will not do it with audio, please write in the messaging device in there, and we will read them out loud and try and answer them the best possible. Also for the crowd here, feel free to ask questions. We would like to have a nice discussion. At Evaxion, what are we doing? We are doing immunotherapy, and we really aspire to lead the exploration of AI in developing new vaccines. That goes for both immuno-oncology and also for the infectious disease area. Today we have a few examples in the oncology arm of the company, and then we also have a few examples in the infectious disease area. What will we be talking about today? The highlight of today is our platform, which is a vaccine platform developed in-house, and it's a DNA platform that has been optimized to target antigen-presenting cells. Why target antigen-presenting cells? These are our main stakeholders in the immune systems, where we need to engage those to get our vaccines to work. That is why we really want to target the antigen-presenting cells. Today, we hope to convince you that we have developed a platform that can deliver both cancer antig ens and also infectious disease antigens, so both for bacteria and virus. That will come as we get the speakers on stage. What is this platform? Again, it's a DNA plasmid. It's depicted up here. Let me see if I can get my little pointer to work. It is not happy with that. Good. On the right side of the screen, we have this round circle, which is a DNA plasmid. It is mainly backbone, which is the bright blue part of it, but the coding frame is where the action happens. That part is consisting of three different parts. We have what we call an APC targeting unit or antigen-presenting cell targeting unit. That's the green part. We have a multimerization unit, and then we also have a cassette for an antigen of choice. What we're going to try and convince you today is that you can put any antigen you desire, especially AI-predicted antigens, into this cassette that is depicted in the red. Today, we'll show you a lot of examples where we use the chemokine CCL19 as antigen-presenting cell unit. We have seen a lot of examples of how CCL19 can enhance the immune response we get for both cancer antigens and bacterial antigens. This DNA plasmid is what we give to the patient. We immunize with a DNA plasmid, insert it into the muscle or inject it into the muscle, and this plasmid will encode a protein product. The muscle cells will generate this product on its own. This protein product has been depicted up here. Again, it consists of this antigen-presenting cell targeting unit, depicted in green. When we have a dimerization unit, this is to stabilize the protein product, and then we have an antigen of choice, which is in the red. We will have three cases presented today by some of our top scientists. We will have cancer to begin with, then we will show some viral examples and end with the bacterial proof of concept. Yes. Without further ado, I will invite Marina to speak a little bit about the mechanism of action for our DNA vaccines. Thank you, Stine, for a really nice introduction. To understand how this APC targeting molecule can actually enhance the immunogenicity and the efficacy of DNA vaccines harboring so many different antigens, it's important that we look at what is the mechanism of action of DNA vaccines. DNA vaccines are typically injected in the muscle. The pointer doesn't work, but you will inject your vaccine in the muscle, and here the DNA can be either uptake by patrolling antigen-presenting cells, or it can be taken up by the non-immune cells, such as muscle cells. The muscle cells will produce the antigens that we have delivered in our vaccine and will release them in a non-specific manner to the environment. Those antigens can be engulfed by the patrolling antigen-presenting cells, which become loaded with the antigens. If the DNA were to go to the antigen-presenting cell, then the antigens will be processed inside the cells, and they are also loaded. These cells now that carry the antigens of the vaccine will travel to the lymph nodes, where they will meet naive immune cells, and can train these cells to recognize and get activated to fight either cancer cells or cells infected with virus or bacteria, which present the antigens that we have delivered in the vaccine. You may know that DNA cancer vaccines historically have not been really efficient in the clinic, and that is because this step of loading the antigen-presenting cells with the antigens is really inefficient. Targeting and then focusing on this specific step, we can actually enhance the efficacy of any DNA vaccine. That is what we aim to do with our APC-targeting technology. Yeah, we can see how that goes. The first step is the DNA uptake, and that happens exactly the same as it would in any DNA vaccine. The DNA will go into the patrolling APCs or into the muscle cells. Now the muscle cells can actively secrete the antigens, and that increases the amount of vaccine antigens that is available in the immunization site. The APC-targeting unit is also responsible of mediating the interaction between the antigen and the APC, and allowing the internalization in a much more efficient model. Furthermore, our APC-targeting unit also can recruit additional APCs to the immunization site. Summarizing, the APC-targeting unit enhances the amount of antigen that we find in the immunization site, it mediates the interaction via receptor internalization, and it also recruits additional APCs. All of this results in large amounts of APCs loaded with the antigens that enhance the immunogenicity and the efficacy of our DNA vaccines. This is how a vaccine can train the immune system to just fight cancer cells or infections, either from virus or from bacteria, and it can be applied to any disease that we may target. Yes. As Stine mentioned, we have CCL19 as our APC-targeting unit, and I would like to show a bit what is this and how can it mediate all these steps that we have seen so far. CCL19 is a small globular protein from the family of cytokines, as you can see here in the cartoon. It has a secretion signal that carries the protein outside the cell. It interacts with the receptor CCL7, which sits in the surface of the APCs. The interaction with the receptor mediates the internalization of the CCL19, and furthermore, and very importantly, it's a very potent cue for migration of APCs. When fusing an antigen to this molecule, we can benefit of all these characteristics and use it to deliver our antigens very specifically to these kinds of cells. Now I would like to show you a bit with some data, how we can investigate this in the lab and how it looks. Here I would like to illustrate the recruitment of the APCs by our APC-targeting vaccine. We have here this small plastic cassette that has 3 different compartments. In the middle one, we can place or seed our APCs embedded in a 3D matrix, and then on the right or on the left, we can add different solutions. In this case, we will place in the left our APC-targeting protein, and then we can look in the microscope and see how the cells move and whether they will migrate and get directed towards our proteins or not. Here in this little picture, each of the dots that you're seeing is a cell, that like an APC, and they are sensing our APC-targeted protein on the left. When you look... Sorry, there is a video that needs to play now. Meanwhile, we will see the cells moving in a second, but you will see that it's not that evident to figure out in which direction the cells are moving when you just look in the microscope. We can actually use a software to place those cells in a coordinate system, and then generate these spaghettis that allow us to follow the speed, the direction, and, like, where in the space the cells are going. If you see here on the very right, when the cells see some antigens, in this case, it's some cancer antigens, but without the APC-targeting unit, the cells move around, but they don't have a specific direction. You can see that by this very round cloud that is forming. that the cells are forming. Now I would really like to play some videos, and you could see how it looks when the cell sends the APC-targeting unit, CCL19. Otherwise, you will have to believe me. Oh, here we go. Maybe. I'm really sorry for the Well, maybe, we will take it after, and we can play the movies. You get more or less how the setup is looking and what is the idea, and you saw how those spaghetti looks around the center of the coordinate axis. When we have in the left, in this case, an APC-targeting unit CCL19, it is very clear that the cells are very directed into the left, and they cannot extend and form a non-center axis. Well, here in the right, we have a graph that is just an example to show how a molecule containing both an APC-targeting unit, CCL19, and an antigen, in this case, it's cancer antigens, can recruit APCs, while the antigens alone don't have that characteristic. We can say that APC-targeting vaccines containing CCL19 recruits APCs, and it's something that is derived by the APC-targeting molecule, in this case, CCL19, as the antigens alone cannot drive this process. Now we have almost seen an introduction of what is the mechanism of action of an APC-targeting vaccine and some data on this. I would like to move into the first practical case, that is, the delivery of cancer-specific antigens using this DNA platform. I would like to introduce our product, EVX-03. It's a DNA vaccine, targeting cancer-specific antigens, both neoepitopes and ERVs, and it follows the structure that Stine has already described. We have a DNA plasmid encoding CCL19, a dimerization domain, and then some silico-predicted cancer epitopes. We saw that this platform is really good at priming and loading APCs, and then the APC will go in the lymph node and train some immune cells. In this case, we are interested in training T cells. It's a kind of immune cell that has the ability to recognize and eliminate the cancer cells. We have them here in this cartoon. In yellow and in blue, we have two types of T cells, CD8 and CD4 T cells, and they are one of the main readouts that we will see through the presentation of today as how we determine that our product is working. Induction of these cancer-specific T cells, together with tumor elimination, is how we are evaluating our product. A really brief introduction of what are neoepitopes and ERVs, the cancer-specific antigens that are contained in our EVX-03 product. Neoepitopes are these small mutated peptides that we can see here in red, display on the surface of cancer cells, and that can be recognized by the immune system, by the T cells, and allows the immune system to eliminate the cells. Neoepitopes are the result of mutations acquired in the genome of cancer cells that translate into these small mutated peptides. On the other hand, ERVs, which are also cancer-specific antigens, have a different source. In this case, we are looking at the expression of ancient viral DNA that we all carry in our genomes, that is silenced and repressed in healthy tissue. However, cancer cells lose the ability to silence this type of DNA and results in the expression of protein and peptide products that are specific for the cancer. Therefore, it's also a really good target to train the immune system to identify the cancer cells. Yeah. How has been the process of developing EVX-03? I think we are pioneering the implementation of APC-targeting delivery for both neoepitopes and ERVs. I will show some data using a mouse models. Here we have taken the tumor that comes from a mouse and sequenced the genome to identify, using our AI platforms, both some neoepitopes and some ERVs. We have done extensive research into selecting what is the best APC-targeting molecule for delivering cancer antigens. Finally, that has resulted in the generation of a DNA vaccine with clinical applicability that we are happy to take into clinical trials. Yeah. Let's look into some of the early data where we did our screening for the most optimal APC-targeting unit. Here we are looking at a animal model where we have a prophylactic vaccination setup. That means that we are first immunizing with the DNA for the animal to mount an immune response, and then after, we are challenging them with some tumor cells. We will follow the development of these tumors and use tumor model, tumor volume as the main measure to determine the efficacy of our vaccines. Here you will see how the tumors grow when you immunize just with the neoepitopes without a targeting unit. How does it look when we immunize with CCL19 as an APC targeting unit, which is our selected product after, and other examples of other APC targeting molecules. Here is how the graphs look. In black, you see a negative control, where the tumors grow really big and really fast. You can see that when we immunize with cancer antigens without the APC targeting unit, we still achieve some tumor control, and that would be a regular DNA vaccine. When we include CCL19 as an APC targeting unit, the tumors are much more smaller, and actually, the majority of the animals do not develop a tumor at all. The efficacy of the vaccine is really improved. It's important to say that there are other APC targeting units that also have some good effect, and that can improve the efficacy of cancer neoepitopes in this case. We select CCL19 to move forward. Next, I would like to show some data on how efficient is actually our EVX-03 product containing the neoepitopes. Here we have done a titration study, where we have reduced the dose of DNA that we immunize the animals with, to see how until which dose we retain some both antitumor effect, but also some nice immune responses. The setup is the same, but here we have added an additional readout, that is, whether we can measure neoepitope-specific CD8 T cells. That is, cells in the immune system that have the ability to recognize the antigens that we have delivered with our vaccine. Looking at the tumor data, we are here seeing a bit of a different plot. Each line represents the development of a tumor of an individual mouse. The first one on the left is our negative control, where all of our animals develop tumor, and none of them are tumor-free at the end of the study. Moving to the right, we can see with 5 micrograms of EVX-03, the animals do not develop any tumors, and most of them, 12 out of 13, remain tumor-free at the end. That effect is maintained when we reduce the DNA dose down to even 0.5 micrograms of DNA, where we can see 5 out of 13 animals still remain tumor-free. The graph on the right then represents the T cells that have the ability to recognize some of these neoepitopes that we deliver in our work. You can see that they are also present even when we reduce the dose of the DNA quite significantly. Finally, I would like to show the therapeutic effect of EVX-03, and that means the effect of the vaccine when we are first challenging the animals with the tumors and then dosing them with our vaccine. This data represents much more the reality of a cancer patient, really. Here we will show as readout, both the tumor growth inhibition, but also cytotoxic T cells. That means cells that are not only able to recognize neoepitopes, but that they also secrete the cytokines that mediate tumor cells killing. You can see here that, compared to the negative control, EVX-03 is able to induce therapeutic antitumor effect as a monotherapy, just delivering EVX-03. That is also able to induce very potent, both CD8 and CD4 T cell responses, the two types of immune cells that mediate tumor killing. As a summary, APC targeting of cancer neoepitopes is a really potent way of improving the efficacy of DNA vaccine. I would also like to show some early data of our work with the ERVs, the second source of cancer-specific antigens that we have here, and the vaccine construct is the same. Now, instead of neoepitopes, we have some ERVs. We have here some prophylactic experiment, where we first vaccinate the animals, then challenge with the tumors, and follow the tumor volume and the induction of ERVs-specific T cells. As you can see here, ERVs are also capable, when delivered through this mechanism, to control tumor growth very efficiently. In the same way, they are able to induce T cells that recognize the ERVs, and therefore have the ability to kill cancer cells. Yeah. CCL19 is a very potent APC targeting unit for the delivery of cancer-specific epitopes in general, and can significantly enhance the immunogenicity of DNA vaccines. I think that summarizes the first part. I don't know if we could maybe try again to look at some videos of migrating cells, if the IT allows? She's try. Yeah. I think, while we are waiting, because it's a very important video, it is a human- ...antigen-presenting cells moving towards actually the human backbone we have that we will use in our patients in our clinical trial. We are preparing that to start in the end of this year. We have tested that compound that will go into people. I think it's a very important movement to see that the human antigen-presenting cells move towards our product. Also now we have a Q&A session, so we can maybe load up for that. Think about some questions, if you have any. Also, I will encourage online. If you have any, please write them. Don't hold back. We will be happy to answer any questions you might have. Yes, yeah. Your CD8 and CD4 response in mice is nicely correlating with your tumor take. Are you also expecting to use this as a marker in patients later, CD8 and CD4, as a response and as a prognostic marker or responder marker? Yes, that is part of the evaluation that the patients in that clinical trial will go through on that. Since the vaccine is personalized, actually, every patient will be probed and evaluated for the induction of CD4 and CD8 towards the specific antigens that arise from the mutations in their specific cancer. Yeah. Yeah. I was just wondering, is there a need also to use an adjuvant? Do you use an adjuvant also in your experimental setting with the mice? We have explored different adjuvants or delivery methods, we could say, in our in vivo models. I think in the clinic we have selected a jet injection that suits with pressure, with air pressure, the DNA molecules into the muscle. It's a way of enhancing the transfection of the muscle cells with the DNA. Yes, maybe. The expression is always in the muscle cells, or do you also expecting some expression of the DNA construct directly in the dendritic cells, or, and what is the percentage? You have a feeling that everything goes through muscles first? No. Of course, the DNA will go into the cells that are present on the injection point. It is very likely that it will get into the APCs, the antigen-presenting cells, that are patrolling around. The amount of antigen-presenting cells that we find in healthy muscle is very low compared to the muscle cells that we have present. Just statistically, we would expect to have more DNA going into the muscle. However, we have not analyzed what is the relevance of these two types of cells in the efficacy of the vaccine. Yeah. Any questions online? No. Thank you very much. I think we will transition into our break. Tina is. Play a video. Playing, wanted to play. Of course, it worked fantastically when we did the test today just before the presentation. Working. Just give it a go. Okay, great. If you remember now here we have our cells sitting in this little yellow somewhere in the middle, and they are sensing on the left the APC targeting molecule, CCL19. Let's see if we get some movement now. Yes. Each one of these is an APC that is moving around. This is a video that when you follow over time, this is over 12 to 16 hours, and you can visualize as these little movies. It's a bit difficult, though, to see what is exactly the direction, and is the movement directed towards a specific side or it is stochastic and random? Yes. Okay, great. We came to this little diagram where we could see that when you have only the antigens, then the cells move all over, they are alive, they are having a good time. They create this very round cloud center in the middle. Yes, there we go. When they sense the APC targeting unit, CCL19, then I hope it's obvious that they are very biased towards where they should go, and they are being recruited to the source of the APC targeting element. Yeah. As you see here now, we have probed only the first part of our molecule that contains the CCL19 and the dimer, right? Also, to make it evident that this would be the case to whatever antigen comes to use after. When adding something real, some neoepitopes, in this case, they, this molecule still has the ability to recruit, the human APCs. As Stine said, these ones are APCs coming from a human donor, and they are sensing a human construct, that resembles what, could be, for example, an EVX-03 final product. Yeah, some questions? For how long do the muscle cells express this construct? They should express it for at least, 10 days after a single immunization. Our dosing schedule contains several DNA immunizations. It's a bit difficult to say and check how that accumulates. 1 single immunization with the DNA will for sure lead to expression of the protein product for up to 10 days. Have you experimented with the difference of adding something so that the cells actually terminate, so they actually go into apoptosis and create some kind of stress signal versus them just expressing them and living on? We have not experimented that, I would say that the delivery process creates some cell death and some stress, no matter what. Whether it's just the injection because of the pressure that the fluid will create, or if we use the jet injector with its pressured air into the muscle. That process per se, will create some stress that will also make. Yeah. These cells are not expressing the antigen there. Just messed up during the. Yeah. I wonder if there's a difference there, because. But you will have the same stress if you deliver just the antigens. In that case, the comparison... The stress on the cell death that you would experiment in the immunization side is unspecific of the APC targeting unit. When delivering only the antigens on a regular DNA vaccine, we expect it to be equivalent to the stress and death that you will experience when delivering the APC targeted DNA. I don't... Does that answer your question, or really? No, we- No. Yeah. Do we have more questions? Do we have questions online? We just heard there's an issue getting through online. I don't know if you can check that. Yes. So far, there's no questions online, it looks like, but if you do, please raise your hand. Okay. Great. We see your hand, Richard. Richard? I was wondering what kind of mouse models have you used, in the, your preclinical tests? I know we were, claiming that, please don't, do the audio, but I think we have to now. Yes. Yeah. Richard, if you have a question, just speak up. Can you hear me? Sorry, we haven't turned up. Sorry, we are on mute. One second. As I mentioned earlier, this is our first research and development day, so we are testing all the small things we can improve for next time. We have sound now, so please, ask your question. I was just wondering if you could comment on what kind of mouse models you've been using in your preclinical tests? Yes. Yeah, go ahead. I can maybe answer that. We have so far used this syngeneic CT26 mouse model. We have also played around with the B16 as model for clearing tumors and using new antigens, if that answers your question. Yeah, thanks. Excellent. Good. I hope, despite the technical issues, that Marina has convinced you that the APC targeting can do something to recruit the antigen-presenting cells that we really, really want to affect. We will see some more data on how it can improve some of our other vaccines. We have a break now. We are a little bit ahead of time. The intent was that we start again at 22, 2, but I think if we can start at half past 1, that also leaves a little extra time for our guest speaker from Pantherna that will explain about the RNA LNP technology, and we will follow up with our in-house proof of concept. If we give Marina a hand, we should definitely give. Good. See you at half past. Yeah. Thank for our second session. Just go on 1, 2 seconds. Yes, this next session will be about RNA and also touching upon FC targeting. I am delighted to introduce Ansgar Santel from BioNTech, Head of Translational Research. You will introduce us to your technology, and I will follow up with some proof of concept in preclinical models. Yes. Yeah. Thank you, Stine, thank you also to the entire Evaxion team for inviting BioNTech us. To you, it's really a privilege for us to work with such an exciting company and teaming up with our technology and your technology in order to make really new approaches, maybe in vaccination. May I have the first slide? this is... There we go. Yeah, my name is Ansgar. I work for BioNTech. We are a company based in the capital region of Germany, Berlin-Brandenburg, we're actually a small company. We are a team of 12, as you can see, junior and senior scientists. We are a small international team. Just some fun facts at the beginning. We are, what is BioNTech? As I said, we are a startup company, although mature startup. We started already late 2017. We are currently a seed stage company, our mission is to build new mRNA therapies. Our first endeavor is basically building a Tie2 agonist, specifically for the endothelium in the lung on an mRNA basis. This we want to achieve on these two platform pillars shown here in the slide, the PIONEER mRNA and the PIONEER LNPs. This actually gives you already an idea what we are dealing with, you are probably already familiar with this. COVID-19 as a pandemic and the vaccination, we all experienced what it means to get an mRNA LNP. This is also what we want to do, not only for vaccination, but also as the vaccination space to build new LNPs. What is an LNP? A lipid nanoparticle, in this case, represents the drug. The drug is built of the mRNA, or it can be also any other nucleic acid which becomes formulated with the carrier. This carrier is built of specific lipids, and these specific lipids, if they are mixed with this state-of-the-art microfluidic mixing system, so you bring different chemical compounds together, you generate particles which have a certain physical, chemical property, and they are basically the final drug product. The mRNA, in our case, along with a collection of lipids. By varying this collection of lipids, you can basically build new mRNA therapies. I mentioned already, the company is built on two pillars, the mRNA platform and the LNP platform. The mRNA platform is actually something what we developed in order to ensure a robust expression of the desired gene product. In our case, as I mentioned, for the lead candidate, a Tie2 agonist. This is only half of the story. The drug needs a carrier, because the carrier is responsible for selectively bringing the drug, the mRNA, to the desired cell type. In our case, we employed standard and novel lipid components in order to generate novel LNPs with defined properties, depending on what type of administration route you use. You can imagine in medicine, some drugs need to be applied intravenously. This is something what we follow with our lead candidate. For example, for Evaxion and the vaccination approach, it's also important to do an intramuscular application. We want to find and develop new solutions for all these type of administration routes. Important for you to remember, our LNP platform comes in three flavors, shown and depicted here, the so-called cationic CLNPs, the neutral LNPs, and the anionic LNPs. I stress this because these neutral LNPs play a specific role, which we actually use in collaboration with Evaxion to see what they can really do. Of course, as a startup company, we need some kind of intellectual property secure situation. We secured basically the mRNA constructs we use in order to do enhanced expression. Here, we hope that we will get the patent granted very soon. We try also to protect the LNP process, the process of establishing an LNP for a specific purpose or even for a specific administration route. The combinations of even gives room for a new IP. I skip this and move back to some scientific data. What is this PIONEER mRNA all about? The idea was, at the beginning, to find basically regulatory units. Regulatory units at the 5' and 3' UTR, which allows, as I said, a robust and enhanced expression. Since we had the idea of doing this for endothelial cells, so the endothelial cells are the inner linings of all our blood vessels, we try to find combinations which are very robust for the cell type. This was, as shown here in the slide, examined just by pure transfection. We combined different 5' and 3' UTRs, transfected a reporter construct that is shown in the middle, in order to see which combination turns out to be most robust. There's one where we found, yes, you can see there's one sticking out over time. This is a good combination. This can also be just applied, not only in endothelial cells, but also beyond endothelial cells. Although these UTRs, they come from, in 1 case, you see at the 5' mMCP-1, from a same, a chemokine. In the case of the 3' UTR, it's from an endothelial gene. Also shown here, just by a pure in vitro transfection, you show the robustness of these mRNAs, if you compare just to standard off-the-shelf available mRNA constructs. In this case, we use GFP as a reporter. If you transfect these constructs, and here are just these examples shown over time and also in this respect to the intensity of expression, you see that the PIONEER mRNA is superior to the standard. Again, this is actually only half of the story. The most critical part is to find the right LNP. Like finding the right LNP for the COVID vaccines, we strive for finding the right LNP for certain other diseases, especially ARDS, and I will touch on this at the end of my talk. Now I just want to give you a short introduction, what type of LNPs do we employ, and how can these be used for the Evaxion approach in order to make, maybe one day, another exciting mRNA vaccine? Every LNP or mRNA LNP is composed of course, you see on the left-hand side, the mRNA, but then three types of lipids. You need these different types of lipids in order to ensure that these particles can form in a specific manner, but also that they are taken up in a functional manner. You can imagine a particle, like bacteria, is just eaten up, but this doesn't help us. We need that the mRNA becomes released into the endothelium. In order to ensure a stable particle, which can capture the nucleic acid, deliver it to the right cell site, where it's taken up, and the cargo, namely the mRNA, is released in a functional manner, this is ensured just by the correct combination of these three type of lipids. The most important one, which is also responsible for the complexation of the nucleic acid, can be a cationic or so-called ionizable lipid. I will explain on this why this is so critical later on. Important is to understand, this is needed in order to bind the mRNA, the negatively charged mRNA. You need a second type, so-called helper lipids. These helper lipids comprise a group of lipids. You only use two. One of them is cholesterol. This is important to do what I mentioned, that once these particles are taken up by a cell, that the mRNA is released in a way that it can be translated and work functionally. Then there's the third lipid, the so-called PEG lipid. The pegylated lipid is just to maintain the integrity of the particles. Once you have formulated the mRNA in these particles, the integrity of these particles has to be remained, and not only outside the body, but also inside the body. Therefore, this is important that you include this type of lipid. This is what I want you to remember. These are basically the main lipids you need in order to build a functional lipid nanoparticle. Beyond just testing different combinations of these lipids in order to find new lipid nanoparticles with new properties, we also are engaged in making robust particles, or let's say, very well-characterized particles, because at the end of the day, this is supposed to become a drug. In order to make a drug, you know, you have to make sure that the process and manufacturing is established, that you always have a nicely characterized particle. This is a very complicated slide, but it should just illustrate something. If you look at the left panel, upper left panel, you see these curves, which are pretty wide. This indicate that you can, just by simple formulation, generate particles of a wide range of size. This is something what you do not want to do. You want to have some in the monodisperse, so all has the same size manner. Our lipid chemists have found some tricks. In this particular case, it's employed for our lead candidate, EVX-04, which I will introduce to you later, in a way that... This is shown in the graph on the right-hand side, with the blue curve and the orange curve. These are just two different methods of measuring the size of the particle, but you see they have this uniform curve. This is important because this is how you want to make a product. These particles, so at the end of the day, always show uniformity, monodispersity. To make this slide a little bit easier and to illustrate it in a different manner, you tend to also just do EM. This probably not the right way, especially on the regulatory level, to do this, it nicely illustrates how this particle look like. Yes, indeed, this is what we already know from the media when we talk about Comirnaty and all this vaccine. These are really these nice spherical structures, what we achieve. If you look on the left-hand side in small scale, you see that they almost have the same size. If you dive deeper, and look closer at this, on the right-hand side, you see a really close-up EM picture, how, in our case, EVX-04 looks like. You have some core structures, where the mRNA is basically bound to this cationic or ionizable lipid and surrounded by layers of lipid membranes. This was just a quick run through, what are mRNA LNPs? A few words, what we really want to do in order to find new mRNA therapies based on mRNA LNPs. This little steps illustrates our approach from one to five, namely, we play with these lipid moieties, like what I said, this cationic, ionizable, with helper lipids, with PEG lipids, play with these around and generate new particles, where we know they have different size, different charge, different physical, chemical parameters. These are tested in the first step, just in cell culture, in order to see how these behave. We go deeper, and just depending on what type of therapy we envision, we apply these for biodistribution analysis intravenously or intramuscularly, or you can also pursue, let's say, inhalation or something like that. Our main focus was, at the beginning, just to do intravenously, so a systemic application. We also had an interest in finding routes for intramuscular, so local administration. This is one way how you can basically move forward, finding formulations, dissect them in step two on the in vivo level. You might have identified certain target organs, but these organs do not tell you what are the real cell types, and are these cell types also relevant for certain pathologies which you want to correct with the mRNA therapy. We dive deeper with other molecular means to decipher the cell types which are targeted with these LNPs. Look for an interesting target and indication, apply this in disease models, and hopefully come up with a new drug development program. You can also imagine you can go the other way around, namely, coming with the indication, one, coming with the approach, with the medicine and the target, and find the right LNP. This is basically how we came up with Evaxion. Evaxion, of course, I said, "Hey, we come with the idea of building a maybe a new vaccine. Want to see how this goes. Can you offer a solution?" From these approaches, namely going from LNP to disease or from disease to LNP, we came up basically with a core of candidate formulations, and some are shown here. Like, according to our approach, namely from LNP to disease, we developed our lead candidate program, PAN04. This is one shown on the left-hand side. It's a cationic LNP designed to deliver the mRNA to the lung vasculature or in particular, to the endothelium of the lung. We also knew, especially since there's a lot of knowledge in this LNP field, that neutral LNPs are also important, especially for muscle delivery. This is how we embarked on the journey. I show it here again, folks. When we are in touch with other companies like with Astellas, who had an interest in generate regenerative medicine, so in this case, also an interest in intramuscular applications, and also like for vaccination purposes. You see here we employed certain neutral LNPs, and it's important to remember, these are the ones which we also use in the collaboration with Evaxion. What is this all about? We did, as I said, just some screening of LNPs, which are applicable for intramuscular application. It's shown nicely here. This is how we operate. We do the LNPs, we inject it, in this case, just to mice. We use a reporter mRNA to easily follow the expression. Shown in the diagram is if you apply, shown in orange, our underlying lead candidate formulation, a cationic one, there's no expression in the muscle. If you use a neutral one, you see robust expression even over time. We laid basically the basis for offering Evaxion some interesting formulations. I mentioned several time now, neutral LNPs, or right at the beginning, you might remember, I mentioned we use these three flavors. I just want to explain what this actually means with these different flavors of cationic, anionic, and neutral LNPs. We started off with cationic LNPs, like for our lead candidate product. In this case, we use a cationic lipid, which at the end of the day, at the end of the formulation step, leads to an overall positively charged LNP, so it's positively charged. You can also do the other way around, namely, use a cationic lipid, and this is shown on the right-hand side in blue, but adds an overload of anionic lipids in order to generate an LNP, which has an overall negative charge. You always have to consider at the beginning, you have a negatively charged nucleic acid, which you need to formulate. At the end of the day, you have these spherical little structures, and they have a certain charge. This charge is very critical for the delivery properties. Then I mentioned this several times, these neutral LNPs. Neutral LNPs can be generated, or at least we do it, like the standard, which is where you use a so-called ionizable lipids. Ionizable lipids or pH-sensitive lipids means, depending on what is the surrounding pH, is it basic or acidic, the lipid is protonated or is positively charged or not. This is important because at some point, you need some positive charge in order to bind the negatively charged RNA. These is one manner, and this is also if you follow the scientific literature, the whole field banks on this approach, using ionizable lipids, play with their charges in order to generate neutral LNPs. If you administer these neutral LNPs intravenously, they all end up in the liver. It also turned out, and Kobi told us this story, if you apply it intramuscularly, they can also do be able to get the vaccination. We can offer this, but at the same time, we also applied a different trick using, again, our cationic lipid, along with some steps, also anionic lipids, which can mimic this neutralization step so that we, at the end, have a neutral LNP. In this case, we call this a neutral LNP with an hydrophilic surface. Let's put it this way. Hydrophilic, so it can be dissolved in water. In contrast to the second one or the original one, with using an ionizable lipid, you generate neutral lipids, but with a hydrophobic surface of lipid. It's important to remember, these neutral lipids come in these two styles, and these two styles we offered to Evaxion to see how does it work in your case. This complicated slide, just to give you an insight, how do the vaccines which we all experience look like? Again, the important message is on the right-hand side, up. Ionizable lipids, pH-sensitive lipids were employed for the product like Spikevax or Comirnaty. You need these helper lipids, two helper lipids, and a pegylated lipid. This carrier for the vaccine in Comirnaty and Spikevax, is composed of four lipids, and they are mixed in a very specific ratio. To illustrate in an easy way, how now our formulation compared to those, I applied just these pie diagrams. On the right-hand side, just the well-established, validated- mRNA LNPs, and you see how they're using the different lipids in the different ratios. In blue is indicating the main important lipid in order to bind the nucleic acids, which is this ionizable pH-sensitive lipid. We offered Evaxion the very same, and you see it's also according to the structure of the K, a very same similar formulation, but this hydrophilic on the left-hand side is completely different. This is used of this cationic lipid, and we have a way in order to make it neutral. Yeah, this basically brings me just to the last few slides, introducing to our lead program and what you can do actually, with this mRNA LNPs. Looking forward to the data scheme we present on this formulation, which I have explained to you and which were applied in our nice collaboration. As I said, what we actually embarked is on defining a new therapy for preventing lung edema, called PAN004, and PAN004 looks like this. As I said, in this case, we employ a cationic LNP, so it has an overall cationic charge. The nice thing here with this particular drug is you only need three lipids, so it's simpler compared to these neutral LNPs. Again, it's the same story. You need a main lipid, in our case, a cationic lipid. You need a co-lipid and a pegylated lipid, and the mRNA we are employing is a fusion mRNA, encoding an agonist for the endothelial Tie2 receptor. Yeah, we have done a lot of preclinical work on this, and if you're interested, I invite you to visit bioRxiv, where you can read the full story. This is our candidate product, which we want to bring into the clinic. To give you at least some idea how this cLNP now behaves compared to the nLNPs, just this slide here. The cLNP formulation is the underlying formulation for PAN004, and due to this cationic overall character, you see it's driving the formulation, the expression into the lung. This is shown with this bar diagram. When you apply single-cell RNA-seq, and this is shown with this nice cloud on the left panel, you can define the cell type where the mRNA goes. In this case, we applied the underlying formulation with a reporter mRNA. With the sequencing approach, you see that we cluster eight, and each dot represents an individual cell that takes you basically and proves that those cells have taken up the mRNA, which we want there to be expressed. Just to give you an idea how this really behaves in vivo, just simple Western blot. If you inject into mice intravenously, this LNP, you can see a nice expression on the left-hand side in a dose-dependent manner. These big, black bars or bands reflect the expression of the transient expression of this agonist. On the right-hand side, you can also follow this over time. We have employed this in various mouse models in order to show that we not only deliver the mRNA and express the protein in the lung endothelium, we also have data in order to show that we activate the relevant signaling pathway, the Tie2 pathway, and that this also leads into some pharmacological activity. For the sake of time, I do not want to speak about this. I just want to highlight our mode of action, where we believe this could be actually a new way in order to prevent lung edema, which is a critical pathology, especially in the course of acute respiratory distress syndrome, and many people die of this because people develop effectively, flooding of the lung, and you need to prevent this flooding. The cause for this flooding of the lung is that an inflammatory trigger opens up the cell barriers between the endothelial cells of the vessels, right? This is shown here. There are certain antagonistic proteins. They inactivate this Tie2 receptor, which leads then to opening the barriers between the cells, and this leads to vascular leakage. With our drug, we want actually to restore barrier function in a way, and this is also the edge of the mRNA LNP here. With this particular mRNA LNP, we can basically position the agonist at the site where the pathology occurs, namely in the endothelium of the lung. This is where it becomes expressed. It's not like the biologic, which you give intravenously and it migrates around. Here, we can position the agonist in a way that this carrier delivers the mRNA, as shown here, to the endothelial cells of the lung. The protein, in this case, a compound I, agonist, becomes expressed, secreted, and reactivates the Tie2 receptor signaling pathway in order to restore the vascular barrier function. I want to end with this slide because this is the outlook. This is actually also my job right now as a company. We want to translate this into the clinic. We have done a lot of investigations on the pharmacology side, on the mode of action side, and we are now at the process of launching CMC campaign and do the IND-enabling studies in order to test this concept in the clinic. The idea in the clinic is to prevent. progression of these patients who show the first sign of lung edema and early ARDS, to prevent these patients from severe ARDS. Those patients, or most of these patients, are still dying of this complication on the ICU unit. These patients required ventilation, mechanistic ventilation, in order to stabilize this patient. With our concept, we believe just by addressing this very first critical pathophysiological step, which is the development of lung edema and neutrophil influx, by preventing this one, we can also prevent the patient from, yeah, getting worse, basically. With this, I will close. Thanks for your attention, and thanks Tine, again, and the vaccine team for being here, and I'm ready to take questions. Thank you. Thank you so much for an incredible talk. just a note, we have hopefully reestablished the online system so you can ask your questions by chat. If there are any burning questions now for Ansgar, please don't hold back. Yeah, I have a question. Yeah. For your lead candidate, essentially the fact that it is positively charged makes it target the lung cells. Is that correct? That's correct. Can you explain? I don't quite see that connection, how you know why that works. There was also some kind of luck and/or experience. Yeah. Mother Nature helped us also. The cationic charge is definitely important in order to get an interaction with the surface of the endothelium. Basically, this is what we found by chance or by accident. In addition, what's also important, I haven't pointed that out in detail, is of course, the overall physicochemical characterization, the size of the particles. I haven't mentioned this in detail, but we are dealing here with particles around 80 nanometers. It's a combination. Last but not least, it's also the systemic route. You can imagine if you apply a drug intravenously, the first big vascular bed which you hit is the lung. It's probably a combination of all these facts. What's important is that you find the right LNP, with the right charge, with the right size, so that these capillaries of the lung are targeted, and also in a way that it won't interfere with the lung function. I have a question. For your lead candidate, do you expect that you need to do repeated dosing or would one infusion be sufficient? That's a very good question. Yeah. It's just because, could you risk that you induce an unwanted immune response if you have to do these, repeated infusions? This is exactly also a very important field in general for the mRNA LNPs. Mm. Can you apply these over a longer period? Our idea was to find also the right indication in this manner, that we do not need to treat patients forever or for very long time. We picked this acute setting. Mm. absolutely right. We know also in this acute setting, we have this one-week period where the patient is in a critical phase, where we basically need to treat the patient. Now we do pharmacology studies in order to figure out what is the optimal dosing scenario. At the beginning, we wanted to do something like give one infusion, but we will see. These investigations are running. They're also important in preparation of the IND-enabling toxicology study. The important point is that we just want to cover this critical one-week period, so it's mainly acute setting. Mm. Yeah. Yeah, following up on that, do you know how fast it's taken up by the nucleolar cells and the expression? Yeah. Actually, this is what we know very well from mouse experiments. You also have to consider there we use bolus application, right? Not like in the clinic, where we will apply infusion. The uptake is probably immediately. The earliest time point where we detect still overexpression in the lung, so this is not only the secreted, it's 2 hours. Right, this is very quick. You're absolutely right, especially for this setting, it's also important that this mRNA kicks in, and therefore, also the mRNA is superb in order to do this. You have a gain of function effect, which immediately kicks in, and this is what the doctors actually need in that situation. This one here, we'll check. This is what I cannot read, unfortunately. Good. I think, for now, we'll say thank you again. Great. We're a little bit ahead of time, according to the program that we posted online. Despair not, we will collect all this great information and data in some short videos that you can find later online. We're a little bit ahead. What I'm gonna talk about now is our concept of the mRNA-LNP vaccine using a cancer-specific antigens. We also have a twist on that story is, of course, including our all-time favorite antigen-presenting cell targeting unit, CD19. The data I will show today has never been presented before, other than in the short press releases, et cetera. Please enjoy the graphs. We're excited to share this today. This is what we just heard from Ansgar Santel's technology, where they are mixing mRNA and PIONEER lipids to form this mRNA LNP technology. I hope that you listened that we received two different types of LNPs from Pantherna that we were excited to try. We didn't know which one had what properties, so we tested these blindly in a mouse model. This was done with cancer-specific antigens. The model was that we injected the mice or, sorry, we immunized the mice with these LNPs. 1 group got the first LNP, and another group got the second LNP to raise an immune response against these cancer-specific epitopes. Then we challenged the mice with tumors. Then the major readout was an anti-tumor effect, so tumor inhibition or T cells. Again, T cells are the cells that we really want to boost to kill the cancer cells. First we have a tumor graph and the corresponding T cell data for the LNP01 that we tested in 3 different doses. The short story here is that the only graph you can see in the tumor growth is the untreated group. All 3 doses of the LNP01 we received gave a full anti-tumor response. The matching T cell data that we have on the side, on the right-hand side, on top, we have the CD8. Those are the tumor killing cells. We have a very nice high CD8 response that follows the dose. The highest dose gives the highest CD8 T cell response. In the bottom we have a CD4 response as well, that we will look into why that looks maybe a little bit opposite of the CD8. If we look at the second LNP that we're comparing to, that's the graph down here. Same 3 concentrations that we tested for the LNP 1 in the top graph. Here we see that it is actually only the highest concentration of the LNP 2 that works for our cancer-specific new antigens to give a full tumor depletion in this model. If you also look at the T cell response on the side of the graph, then you can see that the CD8 response, which is the top, so the third graph from the top, is actually lower than for the LNP01, and so is the CD4 response, which is the bottom T cell graph. In conclusion here, we can say that for our cancer neoepitopes, the LNP01 formulation worked the best at the lowest dosage. What we learned is that optimizing or changing the LNP composition can actually improve delivery of the cancer antigens, and it can definitely also enhance the immunogenicity and the T cell response. I don't know if I can reveal from the... You showed us some lovely pie charts earlier in your talk, but, there were two pie charts, exemplifying the. Exactly. LNPs. The LNP02 is the one that's more like the types that are used in the coronavirus vaccine right now, and the LNP is slightly differs. That actually stands out from the more common one. Yes. We're excited to see that we can, if we change the LNPs, we can actually get a better immune response. The next question for us now we're back to our antigen-presenting cell targeting unit. As was explained earlier, our DNA platform carries these three elements, which is an APC targeting unit, a modularization domain, and then you can insert your antigen of choice. What we wanted to ask for our cancer vaccine, Marina has nicely shown this earlier in our EVX-03 program, that when you use CCL19 as APC targeting unit, dimerization unit and cancer specific neoepitopes or IRFs, you get an improved effect when you add this APC targeting unit. The question here was: Can we also do this with RNA? Can adding CCL19 in the end of our antigens, can that improve the efficacy of the vaccine? That we set out to test. Together with the Pantherna, we designed a study where the sequence encoded by the PTX mRNA technology, was a fusion protein with the CCL19 APC targeting unit, a linker, and then these cancer specific antigens. Again, we tested this in mice. We delivered, immunized the mice intramuscularly, waited for a T cell response. We did the tumor challenge and afterwards looked for antitumor response in the T cells. The T cells, we want to boost them as high as possible, mainly CD8 cells. Those are the cells that will kill off the tumor. First we looked at the antitumor response, and even at a low dose for both groups, both with APC targeting and without, we have a completely flat tumor curve. Perhaps here the prophylactic setup was not needed. We will continue with other setups later. How do we see the difference? How do we monitor this? What we did during the study was to do some blood analysis, where we can monitor the antigen specific T cell response over time. We did already a blood sample at day 2 and looked at, is there a difference between adding CCL19 as APC targeting or not? What we saw here in the groups that are shown down here, we have an untreated one, that's the low graph. We have a blue graph, which is without the APC targeting unit, we have the green graph with the APC targeting unit. What we can see here is that early on in our immunization schedule, we have a higher T cell response for the vaccine with the CCL19. We did a follow-up sample on day 6, we saw the exact same pattern. We also analyzed the T cells at the end of the study, so at day 23, we saw the same pattern here. We have some extremely high CD8 responses, we see an increased CD8 response when adding the APC targeting unit. I'm happy to say that this specific experiment was done with the LNP01. If you remember from a few slides ago, that was the LNP that was performing the best. We are super excited to see these results. In conclusion, adding this APC targeting unit to an mRNA cancer vaccine can increase the timing, so improve on early onset, which is important in a cancer setting. We want the T cells as fast as possible to kill off the tumor, it also improves the functional antigen specific T cells. To our knowledge, we are the first ones who have ever shown that adding an APC targeting to an mRNA vaccine can improve, the effect in, on our preclinical model. That was my last slide, and I can see there's some questions online and then maybe also in the crowd. I will leave it at that, and then we'll take questions. Yes, we have one from Thomas Flaten. Yes. What do you think explains the difference in performance of LNP01 and LNP02? Will LNP02 be further optimized to improve T cell response? I would like to invite my collaborator, to the stage here, because that's definitely Ansgar that needs to explain some of these things. Yeah, that's a very good question. Actually, the answer is quite easy. We also would love to know that. This is also something what we will figure out. This is something under investigation, and I think that the profile of set which will be targeted with these different LNPs needs to be investigated in more depth, and this is what we want to apply to understand, the targeting properties in more detail. This is the only thing what I can in this environment disclose. That's what we can disclose for now. Yes. Thank you. Yes. I have a somewhat similar questions also to Ansgar. These new results, they will change in patients or into the personalized nature. In your experience, is the efficacy of your LNPs, is that tied up to the type of disease you're treating or the antigen? That's actually more a question for you. To be honest, this is something where we don't have yet any experience. As you might remember, we followed mainly the intravenous route and find different things. Just by teaming up with vaccine, we now come in this area to learn more about vaccination and the immunology behind, and this is definitely a critical question that could be addressed, but in this case, we need the experts like you. Yes. I think, we have discussed this as well, and I think it could be a tissue-specific targeting that then makes it more indication-specific, but we will play around with this, hopefully in the future. Yeah. This definitely triggers many new ideas, in terms of moving this investigation on this type of vaccination forward, clearly. I think, we have a follow-up from Thomas online. Actually, no, that was the question. Thank you. That's it. Yes. Do we have any other questions on the audience? Yes, one second. Yeah. There's a question here from Volker Ferring. That's our colleague, yes. What are the two positives that make CD8 and CD4 T cells double positive in potential? Yes. We can definitely share that. It's the secretion of the cytokines that we're measuring upon reintroducing the vaccine epitopes. When we put that together with our already immunized immune cells, they recognize it, hopefully, that's the goal. They produce cytokines, and the two cytokines are interferon gamma and TNF alpha, that we are measuring. I think that's the standard in the field. Those are the two that makes them double positive. Interferon gamma and TNF alpha. We learn on immunology. Yes. That's why. Online. Yes. Yes. Good team. Yes? I have a question. Yeah, I get that you cannot disclose too much about LNP 1, but maybe the Spikevax LNP. You said it was an ionizable? Exactly. Lipid. Shown in my slide. Yes. This is what you can read. What do you think has been the thought process behind using that type of lipid for immunization, specifically? Or do you think it's just what they have? I think this LNP field is quite old. In the former times, you didn't call them LNPs. There are different phrases in order to use a word of a liposomal formulation. Many years ago, you applied this, of course, with other nucleic acid entities, like this is also where we come, where we have our experience on sRNAs and RNAi. The same is with these companies like Moderna and BioNTech, who also wanted to develop something for the intravenous route. It turned out these ionizable lipids have certain properties in order to make really good particles, which later then were dubbed and called LNPs. I don't know how actually, Rafael or Jörg, maybe you remember this. I think, these were applied, you know, these work, these type of LNPs, are actually as RNA product. I ended up in the clinic, it's basically the very same makeup, a neutral LNP, but it turned out that these always go into the liver, especially into the hepatocyte. In the case of this drug Onpattro, it's also needed. I think this was just by chance, that they tried it intramuscularly, and it turned out, "Hey, wow! They repurposed liver formulation. Yeah ... weak and clinically validated for other purposes, vaccination, and applied some intramuscularly. You know, they were more advanced, and it, the LNP field is originally derived from liver diseases. For liver, it was always ionizable. At some point, it turned out these are really nice lipids in order to make stable particles, which then also made it into the clinic with an sRNA cargo, not with an mRNA. Excellent. Thank you so much for all the questions. I think from here on, we'll have a break. I will encourage that we will get back 10 minutes before time so that the last two excellent speakers coming with some of our hottest stuff from the lab they have a little bit more time. At 14:50 we will reconvene here. Right? See you then. Great! Next on our agenda today is the last session, that will be focusing on our infectious diseases. We have two speakers. We have the person, we have the speaker that will present on viral targets and bacterial targets respectively. Some of what's presented today has never been presented before, so we are super excited about showing you this disease. I'll just leave it to you guys. You have a smooth transition between the two of your talks, please welcome Gry. Thank you. Thank you so much, team. We have now heard about using CCL19 as an APC targeting molecule for our cancer vaccines, and with the beginning of the COVID-19 pandemic, which you're all aware of, we also tried to move this technology within the viral space and test that. In this presentation, I will show you the two vaccines designs that we have been working with. Both of them contain the CCL19 AP as an APC targeting unit, and they also both of them contain the IG as a dimerization unit. The vaccine that is shown here also contains in silico predicted T cell epitopes from SARS-CoV-2, the SARS-CoV-2 genome. This was designed to, again, just like the cancer vaccines, to activate the T cells, to be able to recognize and also kill virus-infected cells. The other vaccines that we have designed, contains what is called RBD, or receptor-binding domain. That is part of the spike protein on the coronavirus, that binds the, what is called ACE2 receptor on our cells and facilitate the entry of the virus into our cells. The, the purpose of using this vaccine is to activate the B cells and generate antibodies that could then utilize any infection by the coronavirus. This is just a basic, some background. With the COVID-19 epidemic, which is caused by the virus called SARS-CoV-2, a lot of vaccines was developed, and many of these vaccines, they focus on the spike protein. This is the protein on the surface of the coronavirus that, as I just mentioned, binds to our cells, and then, the virus can enter into our cells. There's a problem with using this technology only, and that is that the virus mutates and it mutates quite fast. This has also been the case with the existing vaccines, that the variants are not, that the vaccines available are not protecting against the variants that comes. Another arm of the immune system is our T cells, which has already been introduced in the cancer session that was before. These, so the antigen-presenting cells are presenting bits and pieces, you could say, of the virus to the T cells. This enables also mutants of the SARS-CoV-2 virus are less likely to escape immune recognition because there will be many different proteins that are recognized by the T cells. The activated, especially the activated CD8 T cells, are then able to recognize and also kill the infected cells. This leads that to the conclusion that both antibody and also T cells are very important for long-term immunogenicity against a viral infection. This is not only for SARS-CoV-2, it's against multiple viruses. If we look at our APC-targeting technology, this has also been presented. This directs our antigens to the APCs for enhanced activation of the T cells. I'll now show some of the data we have. In this study, we vaccinated mice with our T cell vaccine. We hereafter looked at the antigen-specific T cells in the mice. As mentioned, the vaccine was designed based on the SARS-CoV-2 genome, and here we used 1 of our AI platform called RAVEN to identify regions in the genome of the coronavirus that was able to activate T cells. As you can also see on this figure, the epitopes, which is kind of put as beads on the string, after the CCL19 and the dimerization unit, comes from many different proteins. We then evaluated the T cell response, so that is the ability of these bits and pieces from the virus to activate the T cells. These bars here, they represent... each bar represent the activity of the T cells for each of these epitopes. As you can see here, the our APC targeting technology are able to induce active T cell response against almost all of the included T cell epitopes. We took this vaccine into another mouse model, and that is a transgenic mouse model, because mice are not normally susceptible towards corona infection, they only infect human cells. This mouse carries the human ACE2 receptor, which enables the virus to also infect the mouse. This study was done in collaboration with Pennsylvania State University. Again, we vaccinated the mice, then we challenged the mice with a lethal dose of the SARS-CoV-2 virus. We monitored the health and the survival of the mice for two weeks after the challenge. The survival graph here shows the percentage of survival of the vaccinated mice. In the top line, you can see that this is the mice that has been vaccinated with our T cell vaccine, and the dotted line is the mice that have been vaccinated with our mock vaccine. That is an empty plasmid containing only CD19, but no antigens. It's quite clear from this graph that our APC DNA delivery technology are able to induce T cells that are actually also capable of protecting the mice from lethal disease. Our second vaccine candidate or product, you could say, for mice that we tested, was this RBD vaccine. Containing it's a B cell-directed or B cell vaccine. Here again, we vaccinated the mice, but instead of evaluating the T cells, we looked at the antibody response in the vaccinated mice. These two graph shows both the level of antibodies, but also the ability, the function of the antibodies. If you look at the blue bars for both of the graphs, this is the antibodies evaluated in mice that have survived a corona infection without any vaccination. This is the natural protection, you could say. The red bar then represents the T cell vaccinated or the B cell vaccinated mice. What we can see to the right graph is the total level of antibodies recognizing this RBD domain of the spike protein. This level is almost equal as for naturally infected mice, also mice surviving an infection. To the left side, you have the, what is called pseudovirus neutralization. This is an assay where we measure the ability of the antibodies to block the binding of the spike protein to the ACE2 receptor in a plate, you could say. This is the function of the antibodies. Here we can see that the APC technology enabled us to get quite functional antibodies that are able to neutralize the viral infection. In summary, these studies show that our APC targeting DNA technology can also be used for viral antigens, both for mounting a strong and specific T-cell response, but also for giving a protection by antibodies. With that, I think we'll move to bacterial antigens and Sophie. Yes, thank you. Last but not least, I will talk about bacterial vaccines. In contrast to some of the other studies that you've heard earlier, where the antigens are basically peptides on a string, these bacterial antigens are large toxins and full-length proteins which makes them very complex and also makes it necessary to be expressed as a structurally intact protein in order to be presented to the immune response in the correct form and also induce the correct immune responses. In order to deliver some of our bacterial antigens, we have played around with the DNA design a little bit, which you can see on the right side of the screen. This would give us a different versions of the proteins to be expressed together with the APC targeting unit, which you can see then on the left, that we have a simple version, which is just a monomeric form, fused to the APC targeting unit, which is the middle graph, and then compared to the dimerized version, which is similar to the other approaches that have been presented in the context of the vaccines against virus and cancer before. We have compared this APC-targeted version expressing bacterial antigens against a version that doesn't have the APC targeting function. Another thing that we have always seen with generating vaccines against bacteria is that we require multiple antigens. When we design our vaccines, we usually have multiple antigen units that we fuse together into one complex, which again makes it even more structurally important to express these bacteria in this, in the right format. When we look at the responses that we want to induce to protect against bacterial pathogens, we mainly focus on antibody-mediated responses, which you can see in different functions on the right, again, of the screen, where antibodies bind to bacterial toxins, thereby neutralizing the toxicity and decreasing the disease symptoms. Antibodies also bind directly to bacterial pathogens and also bacterial infected cells, thereby eliminating the bacteria from the host. In order to produce a very efficient antibody response, we also need some help from T cells. We like to call them helper T cells, which you can see on the left side, that they interact directly with the B cells. The B cells produce the antibodies, which is after stimulation, being stimulated by these helper T cells, to produce a high level of highly functional antibodies. Also with the help of the T cells, we generate a memory response, which induces a long-lived immunity against our bacterial pathogens. In order to produce a protective vaccine against bacterial pathogens, we need both antibody as well as T cell responses. We have tested the efficacy of our DNA delivery technology with our in-house developed Neisseria gonorrhoeae vaccine candidate. We have expressed our Neisseria gonorrhoeae antigens in the DNA plasmids, as I've shown you before, tested a version with the APC targeting unit against a version without the APC targeting unit, to further highlight the impact of targeting our DNA towards the APCs. We measure antibodies as well as T cell responses that are induced after this immunization in mice. As you can see here from the graph, both of the groups that received the DNA vaccine, which are shown in blue, without the targeting unit, and in green with the targeting unit. Both of these groups induce very high levels of antibodies, we see an even an increase in antibody production with the addition of the CCL19 targeting unit. Most importantly, for the T cell help, we also measure a high increase in the functional T cell responses when we add this APC targeting unit to our vaccine designs. As I mentioned before, we usually express multiple antigens in these fusion proteins that are complexes of different proteins that are fused together into 1 sequence, and then we need to make sure that they are structurally intact when they are expressed, and that we also can see responses against each of the individual antigenic components to induce a really appropriate immune response. We have measured the antibody responses against. In this case, we call them EDEN 1 and EDEN 2. These are 2 AI designed antigens fused together, and we can see that we induce antibody responses against each of the individual components. Again, you can see that for EDEN one, there is an increase in the antibody response in the design that contains the APC targeting unit. For some antigens, we see an improved immunogenicity when we have this APC targeting unit, and this could, leads to a broader, protective capacity of the vaccine, because we can better target multiple different antigenic units combined in one vaccine. The same is also true for the T cell responses. Again, we see a strong increase in T cell responses, when we add the APC targeting to our vaccine design. This suggests that we can both deliver, these complex bacterial antigens in our DNA technology, and we can see a definite positive impact by adding the APC targeting unit, for the immunogenicity. This suggests that we have the potential to use a better functional immune response that is better at inducing protection against our bacterial pathogens. This is something that we're still evaluating, so the some of the studies are still ongoing to show this better functionality of the antibody responses by further characterizing the antibody responses, but also by testing them in challenge models, where we then, after immunizing the mice, we infect them with bacteria and measure protection. These studies are still to come, and we're looking forward to the results. That concludes the presentation. Great. Are we doing Q&A? We're doing Q&A, yes. Thank you for some, very exciting, presentations. As I hope you, noticed and felt, this is, brand-new data coming out of the lab. First time for some of this shown today, so, I hope you enjoyed it. If you have any questions, please come with them now, also online. There must be some curiosity around this, super cool data. Maybe a question for both of you. How fast is this response? Can you only use it prophylactically, or can you also? Is it maybe for therapeutic use? Somebody has already the bacteria, and then you go on top, or is it only vaccination? What I'm asking is there is a vision to use it therapeutically, also? You know what I mean? Yeah. Yeah. For the antibody response, you need to maturate the B cells first, and that takes some time. The first time point where we can measure antibodies is 7 days after the first immunization. Mm-hmm. It's quite low, so it requires booster also for memory cells to be generated. You have a longer lasting, yeah, effect of the protection against virus. I think that would not be doable for infections like for the coronavirus or for influenza, where it takes approximately 5, 7 days before the virus starts or you get the symptoms, right? Yeah. I don't think it would work for an ongoing- No ... infection, as you said. In terms of the bacteria, you often have the natural immune response is not enough to protect you against subsequent infections, the same for virus, because they change. Can you overcome a chronic bacterial infection? That was a little bit my question. That's probably not possible. I think it depends if you look on antibodies or if you look on T cells, because if it's, if it's T cells, I think the story might be different, that you could probably also do it therapeutically and activate T cells against an existing infection. It depends on the cause of the infection. If it's a chronic infection or if it's a very long infection, then maybe yes. Yeah, you could do that. Yes. High T cells, I could think, is also against infectious diseases. Excellent. I think I will wrap it up with a single slide. Good. You heard a lot about this module today, our DNA plasmid containing APC targeting unit, modularization unit, and antigens of choice. You've heard about cancer, you heard about virus, and you also heard about bacteria. I hope that we convinced you that this platform can be used for multiple things. We even touched upon that this module and this strategy to target antigen-presenting cells can also be used in an RNA format. Our cancer program that Marina presented earlier today, EVX-03, is being prepared to go into the clinic. We are filing very soon, and we hope to have the first patient in at the end of the year or in the beginning of next year. That is very, very soon. It's a very progressed program. The other programs you've heard about today, the viral one, the bacterial one, are proof of concepts that have been developed the past 6 months in our, in our lab, the viral one, 1 year plus. Brand-new things that we have been excited to share with you guys today. These are our new programs in the future, our EVX-04, our EVX-B3, EVX-B2, and the EVX-V2. Imagine that. More to come, and as already announced, we will hope to collect some more data, maybe in the fall, and do a follow-up on some of the things we've seen today. Thank you for listening, and I think the last thing I would like to do today is to invite all the speakers up, so we can give them one last round of applause. Sophie, Gry, Ansgar, Marina, and I hope you will help me give them a really nice. Me. Me, yes. We talk a lot today. I hope we have some nice things for the speakers. Yes? Great. Yes. As the very last thing... Thank you. That is well deserved. As a very last thing, I hope, thank you also for joining online, and as we mentioned earlier, perhaps you didn't catch it, we will make small videos out of, this and post it online. If you feel like you missed something, I know we... the time schedule, slipped a little bit, you can revisit it online in a week or so. We will have that ready for you, so you can look at it again and again. Then I'll just invite you all for some nice snacks and drinks in our lounge area, and just mingle. Please stick around also for the lovely crew we had today t o help us with the setting up. Thank you, everyone. See you in the lounge.