Evaxion A/S (EVAX) Earnings Call Transcript & Summary

[Audio Gap] during the past 1.5 years. So 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. So we will have 3 of those, and we really encourage people also online. To ask the questions, please write them. We will not do it with audio, but please write in the messaging device in there and we will read them out loud and try and answer the best possible and also for the crowd here, feel free to ask questions, we would like to have a nice discussion. So [ at Evaxion, ] what are we doing? We are doing immunotherapy, and we really aspire to lead the exploration of AI in developing new vaccines, and that goes for both immuno-oncology and also for the infectious disease area. So today, we have a few examples of -- in the oncology arm of the company. And then we also have a few examples in the infectious disease area. So what we will 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. And 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. So 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 antigens and also infectious disease antigens, so both for bacteria and virus. And that will come as we get the speakers on stage. So 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 coating frame is where the action happens. So that part is consisting of 3 different parts. So we have what we call an APC targeting unit or antigen presenting cell targeting unit, that's green part. Then we have a [ multimerization ] unit. And then we also have, like I said, for an antigen of choice. So 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 [indiscernible] 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. And we have seen a lot of examples of how CCL19 can enhance the immune response we get for both cancer antigens and bacterial antigens. So this DNA plasmid is what we give to the patient. So we immunize with the DNA plasmid, inserted into the muscle or injected into the muscle. And this plasmid will encode a protein product. So the muscle cells will generate this product [indiscernible]. This protein product has been depicted up here. Again, it consists of this antigen presenting cell targeting unit depicted in green. Then 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. And we will have 3 cases presented today by some of our top scientists. We will have cancer to begin with, then we'll show some viral examples and 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 ]. Really nice introduction. Yes. So to understand how this APC-targeting molecule can actually enhance the immunogenicity and the efficacy of DNA vaccines harbor in so many different antigens, it's important that we look at what is the mechanism of action of DNA vaccines. So 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 [indiscernible] 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 [ to deliver ] in our vaccine and will release them in a nonspecific manner to the environment. Those antigens then can be engulfed by the [indiscernible] 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 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 antigens that we have [ to deliver ] in the vaccine. Now you may know that DNA cancer vaccines historically have not been really efficient in the clinic, and that is because it's a step of loading the antigen presenter cells with the antigens is really [indiscernible]. So targeting and focusing on this specific step, we can actually enhance the efficacy of any DNA vaccines. And that is what we aim to do with our APC targeting technology. Yes, we can see how that goes. So the first step is the D&A uptake, and that happens exactly the same as it would in any DNA vaccine. The DNA will go into the [indiscernible] APCs or into the muscle cells. But now the muscle cells can actively secrete the antigens. And that increases the amount of vaccine antigen studies available in the immunization side. 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 manner. Furthermore, our APC targeting unit also can recruit additional APCs to the immunization side. So summarizing, the APC targeting unit enhances the amount of antigen that we find in the immunization side. It mediates the interaction via receptor internalization, and it also recruits additional APCs. And all of these 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. So 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 have said so far. So CCL19 is a small globular protein from the family of cytokines, as you can see here in [indiscernible]. It has a secretion signal that carries the protein outside the cell. Yes, and it interacts with the receptor CCR7, which sits in the surface of APCs. The interaction with the receptor mediates the internalization of CCL19. And furthermore and very importantly, it's a very potent [indiscernible] migration of APCs. So when fusing an antigen to this molecule, we can benefit of all these characteristics and use it to deliver our antigens very specifically to this [ cancer ]. Yes. And now I would like to show you a bit with some data how we can investigate this in the lab and how it looks. So here, I would like to illustrate the recruitment of the APCs by our APC-targeting vaccine. So we have here this small plastic [ cassette ] that has 3 different compartments. In the middle one, we can place or see our APCs embedded in a 3D metrics. 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. So here in this little picture, it's -- of the dots that you're seeing is the cell like an APC, and they are sensing our APC targeted protein on the left. Yes. So when you look -- so it has [indiscernible]. 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 directions the cells are moving when you just look in the microscope. But we can actually use the [ software ] to place those cells in a coordinate system and then generate [indiscernible] that allow us to follow the speed, the direction and like where in the space the cells are going. So 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. And you can see that by this very round cloud that is forming -- yes, that is -- that the cells are forming. And now I would really like to play some videos. And you could see how it looks when the cells sense the APC targeting units, CCL19. Otherwise, you will have to believe me. Here we go. Maybe -- well, maybe we can -- we will take it after. And we can play the movies. But you guess more or less how the setup is looking and what is the idea. And you saw how those [indiscernible] looks around the center of the coordinate axis. When we have in the left, in this case, an APC targeting unit, CCL19 and it's very clear that the cells are very directed into the left. And they cannot extend a former 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, its transfer antigens can recruit APCs, while the antigens alone don't have that characteristic. So we can say that APC targeting vaccines contain in CCL19 recruits APCs and is something that is drive by the APC targeting molecule, in this case, CCL19 as the antigens alone cannot drive this process. So now we have almost seen an introduction of what is the mechanism of action of an APC targeting vaccine and some data on this. And I would like to move into the first practical case that is the delivery of cancer-specific antigens using this DNA platform. And I would like to introduce our product, EVX-03, is 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. So 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. So 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. And we have here in this cartoon in yellow and in blue, we have 2 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. So induction of this cancer-specific T cells, together with tumor elimination is how we are evaluating our [indiscernible]. So a really brief introduction of what are neoepitopes and [ ERVs ], the cancer-specific antigens that are contained in our EVX-03 product. So 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 immune system to eliminate the cells. Neoepitopes are the result of mutations acquired in the genome of cancer cells that then 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're looking at the expression of ancient viral DNA that we all carry in our genomes, but 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. Yes. So 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 ], and I will show some data using a mouse models. Here, we have taken a tumor that comes from a mouse and sequence 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. And finally, that has resulted in the generation of a DNA vaccine with clinical applicability that we are happy to take into clinical trials. So let's look into some of the early data where we did our screening for the most optimal APC targeting unit. So here, we are looking at an animal model, where we have a prophylactic vaccination setup. That means that we are first immunizing with the DNA for the animal [indiscernible] and immune response and then after we are challenging them with some tumor cells. Then we will follow the development of these tumors and use tumor volume as a main measure to determine the efficacy of our vaccines. And 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 any other examples of other APC targeting molecules. Yes. So here is how the graph 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, much more smaller and actually, the majority of the animals do not develop a tumor at all. So the efficacy of the vaccine is really improved. It's important to say that there are other APC targeting unit that also has some good effect and that can improve the efficacy of cancer neoepitopes [indiscernible]. So we select CCL19 to move forward. So next, I would like to show some data on how efficient is actually our EVX-03 product containing the neoepitopes. And here, we have done a titration study where we have reduced the dose of DNA that we immunized animals with to see how until which dose will retain some -- both antitumor effect, but also some nice immune responses. So 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 has the ability to recognize the antigens that we have delivered with our vaccine. So 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 mice. The first one on the left is our negative control with all of our animals' developed tumor, and none of them are tumor-free at the end of the study. Moving to the right, we can say with 5 micrograms of EVX-03, the animals do not develop any tumors, and most of them, 12 out of [ 13 remained ] tumor-free at the end. And that effect is maintained when we reduced the DNA dose down to even 0.5 micrograms of DNA, where we can see 5 out of 15 animals still remain tumor-free. The graph on the right then represents the T cells that are -- that have the ability to recognize some of these neoepitopes that we deliver in [ our vaccine ]. And you can see that they are also present even when we reduced the dose of the DNA quite significantly. And 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. And this setup represents much more the reality of a cancer patient in clinic. Here, we will show a readout both the tumor growth in [indiscernible] , but also cytotoxic T-cells. That means cells that are not only able to recognize neoepitopes, but they also secret the cytokines that mediate tumor cell killing. And 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. And that is also able to induce very potent, both CD8 and CD4 T cell responses, the 2 types of immune cells that mediate tumor killing. So as a summary, APC targeting of cancer neoepitopes is a really potent way of improving the efficacy of DNA vaccines. 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 also here some prophylactic experiment when we first vaccinate the animals, then challenged 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. So 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 [indiscernible].

I think while we are waiting because it's a very important video. It is human antigen presenting cells moving towards actually the human backbone we have -- that we will use in our patients in our clinical trial. And we are preparing that to start in the end of this year. So we have tested that compound that will go into people. So I think it's a very important movie to see that the human antigen presenting cells move towards our product.

But 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.

Your CD8 and CD4 response in mice is nicely correlating with your tumor [indiscernible]. Are you also expecting to use this as [indiscernible] in patients [indiscernible] CD8 and CD4 and the response and as prognostic marker or responder marker?

Yes. That is part of the evaluation that the patients in that clinical trial will go through on that. And 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 are raised from the mutations in the specific cancer.

I was just wondering, is there a need also to use an adjuvant? And do you use an adjuvant also in your experimental setting with mice?

We have -- yes, we have explored different adjuvants or delivery methods, we could say in our in vivo models. And I think in the clinic, we have selected a jet injection that suits with pressure [indiscernible] the DNA molecules into the muscle. So it's a way of enhancing the transfection of the muscle cells in the [indiscernible].

So the expression is always in the muscle cells? Or do you also expecting some expression of the DNA construct directly [indiscernible] cells? And what is the percentage of your feeling that everything goes through muscles first? And...

No. Of course, the DNA will go into the cells that are present on injection point. And it is very likely that it will get into the APCs, the antigen presenting cells that are [indiscernible]. But the amount of antigen presenting cells that we find in healthy muscle is very low compared to the muscle cells that we have [indiscernible]. So just statistically, we would expect to have more DNA going into the muscle. However, we have not analyzed what is the relevance of these 2 types of cells in the efficacy of the [indiscernible]. Yes. If any questions around? Yes. Then, thank you very much. And then we will transition into our break. Okay. Great. So if you remember now here, we have our cells [indiscernible] in this little yellow shown in the middle, and [ they are sensing ] on the left, APC targeting molecule, CCL19. See if we get some movement now, yes. So each one of these is an APC that is moving around. This video when you follow over time, this is over 12 to 16 hours, then you can visualize this little movie. It's a bit difficult though to see what is exactly the direction, at least the movement directed towards the specific side [indiscernible]. Okay. Great. And then we came to this little diagram where we could see that when you have only the antigens, then the cells move [ over, ] they are alive, they are having a good time. They create this very round cloud center in the middle. Yes, there we go. And when then the APC targeting unit, CCL19 that I hope is obvious that they are very biased towards where they should go. And they are being recruited to the source of the APC targeting element. Yes. So as you see here now, we are probably the first part of our molecule that contains CCL19 and the [indiscernible] also to make it evident that this would be the case to whatever antigen comes [indiscernible] after. But when adding something -- we have some neoepitopes in this case, they -- yes, this molecule still has the ability to recruit the human APCs. Yes. And as [ Stine ] says, these ones are APCs coming from a human [indiscernible] and they are sensing human construct that resembles what a -- could be, for example, an EVX-03 final product. Yes, some questions?

For how long do the muscle cells expressed this construct?

They should express it for at least 10 days after a single immunization. But our dosing schedule contains several DNA immunizations. So it's a bit difficult to say and check how that accumulates. But one single immunization with the DNA will for sure lead to expression of the protein product for up to 10 days.

And have you experiment with the difference of adding something so that the cells actually terminate so they're actually going to [indiscernible] and create some kind of stress signal versus them just expressing [indiscernible]?

We have not experimented that, but I would say that the delivery process creates some cell death and some stress, no matter what, whether it's just an injection because of the pressure that the fluid will create or if we use the jet injector with its pressure into the muscle. That process per se will create some stress that will also make...

These cells are not expressing the antigen [indiscernible] start during the acute. So I wonder if there's a difference there because...

But you will have the same stress if you deliver just the antigens. So in that case, a comparison. So there's stress on the cell that you would experiment in the immunization side is and specific of the APC targeting unit. So when delivering only the antigens on a regular DNA vaccine, we expect it to be equivalent to the stress on that, that you will experience when delivering the APC targeted DNA. Does that answer your question, really? No...

Do we have more questions? Do we have questions online?

We just heard there's an issue getting [indiscernible] if you can check that.

So far, there's no questions online, it looks like. But if you do, please raise your hand.

Great. We see your hand, Richard?

I was wondering what kind of mouse models have you used in your preclinical tests?

I know we were claiming that please don't do the audio, but I think we have [indiscernible] -- so Richard, if you have a question, just speak up. As I mentioned earlier, this is our first research [indiscernible]. So we are testing all the small things we can improve for next time. We have sound now. So please ask your question.

Just wondering if you could comment on what kind of mouse models you've been using in your preclinical tests?

I can maybe answer that. We have so far used the syngeneic CT26 mouse model, and we have also played around with the [ B16S ] model for clearing tumors using new antigens, if that answers your question. Excellent. Good. I hope -- despite the technical issues that Marina has convinced you that APC targeting can do something to recruit the antigen presenting cells that we really, really want affect. And we will see some more data on how it can improve some of our other vaccines. We have a break now. We're a little bit ahead of time. The intent was that we start again at [indiscernible]. But I think if we can start at half past one, that also leaves a little extra time for our guest speaker from [indiscernible] that will explain about the RNA-LNP technology, and we will follow up with our in-house proof of concept. So I give Marina a hand [indiscernible]. [Break]

So this next session will be about RNA and also touching upon APC targeting. And I am delighted to introduce Sir Ansgar Santel for Pantherna, Head of Translational Research. So you will introduce us to your technology. And I will follow on with some proof of concept in preclinical models.

Yes. Thank you, [indiscernible]. And thank you also to the entire Evaxion team for inviting Pantherna to your [indiscernible]. Really [indiscernible] to work with such an exciting company and teaming up with our technology and your technology in order to make really new advances mainly in vaccination. May I have the first slide? There we go. So yes, my name is Ansgar, I work for Pantherna. We are a company based in the capital region of Germany, Berlin [indiscernible]. And we actually are a small company. We are a team of 12, as you can see, junior and senior scientists. We are a small international team, and just some fun facts at the beginning. We are -- what is Pantherna, as I said, we are a start-up company, also mature startup. We started already late 2017. We are currently a seed-stage company. And our mission is to build new mRNA therapies. And our first endeavor is basically building a [indiscernible], specifically for the endothelium in the lung on an mRNA basis. And this, we want to achieve on these 2 platform pillars shown here on the slide, the PTX mRNA and the PTX_LNPs and this actually gives you already an idea what we are dealing with, and you're probably already familiar with it. COVID-19 and the pandemic and the vaccination, we all experience what it means to get an mRNA-LNP. And this is also what we want to do, not only for vaccination, but also the vaccination space, to build new LNPs. So what is an LNP, a lipid nanoparticle. In this case, it represents a drug. The drug is built of the mRNA or can be also any other nucleic acid, which becomes formulated with the carrier. This carrier built of specific lipids and these specific lipids, if they mix with this state-of-the-art microfluidic mixing system, so you bring different chemical compounds together, you generate particles, which have certain physical, chemical property. And they are basically the final drug product. The mRNA in our case, along with the collection of lipids. And by varying with collection of lipids, you can basically build new mRNA therapies. So I mentioned already, the company is built on 2 pillars, the mRNA platform and the LNP platform. The mRNA platform is actually something what we develop in order to ensure a robust expression of the desired gene product. In our case, as I mentioned, for the lead candidate [indiscernible] agonist. But this is only half of the story. The drug needs a carrier because the carrier is responsible for selectively bringing the drug and mRNA to the desired cell type. And in other case, we employed standard and novel lipid components in order to generate novel LNPs with defined properties depending on what type of administration would you use. You can imagine a medicine, some drugs need to be applied intravenously. This is something what we follow with our lead candidate. And for example, for [ Itraxan ] and the vaccination report, it's also important to do an intramuscular application. And we want to find and develop new solutions for all these type of administration routes. Important for you is to remember, so our LNP platform comes in 3 flavors, shown and depicted here. The so-called cationic LNPs, the neutral LNPs and the anionic LNPs. And I stress this because this neutral LNPs play a specific role which we actually use in collaboration with Evaxion to see what they can really do. And of course, as a start-up company, we need some kind of intellectual property situation. And 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. And then 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. And the combination of even gets room for new IP but as PTX and move back to some scientific data. So what is this PTXmRNA all about? And 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. And since we have the idea of doing this for endothelial cells, so the endothelial cells are the interlining of all our blood vessels. We try to find combinations which are very robust for the cell type. And this was as shown here in the slide, examined just by fewer transactions. We combined different 5' and 3' UTRs, transacted every quarter, construct that is shown in the middle in order to see which combination turns out to be more robust. And there is one where we found, yes, you can see there's one sticking out over time. This is a good combination. And this can also be just applied not only in endothelial cells, but also beyond in endothelial cells. Also these UTRs that come from, in one case, you see at the 5' MCP 1 [indiscernible] and the case of the 3' UTR, it's from an endothelial gene. And also near just by a pure in Vitro transaction, you show the [ bones ] of these mRNA, if you compare it to standard off-the-shelf available mRNA construct. In this case, you see [ CFP ] as a reporter, if you transact these constructs and here are just these examples show over time and also with respect to the intensity of expression you see that the PTXmRNA is superior to the standard. But 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. But 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. So every LNP or mRNA-LNP is composed of, of course, you see on the left-hand side, the mRNA, but then 3 types of lipid. You need these different types of lipids in order to ensure that these particles can form in a specific manner, but also that they have taken up in a functional manner. So you can imagine the particle like bacteria, it's just eaten up, but this doesn't help us. We need that the mRNA becomes released into the endothelial. And in order to ensure a stable particle, which can capture the nucleic acid, deliver it to the right cell type, where it's taken up and the [cargo], namely the mRNA released in a functional manner. This is ensured just by the correct combination of 3 type of lipids. And the most important one, which is also responsible for the complexation of the nucleic acid can be a cationic or so-called ionizable lipid. And I will explain on this why this is so critical later. But important to understand, this is needed in order to bind the mRNA, the negatively charged mRNA. Then you need a second type, the so-called helper lipids. This helper lipids comprise a group of lipid, only use 2. One of them is cholesterol. This is important to do what I mentioned that once these particles are taken up by itself, that the mRNA is released in a way that it can be translated and work functionally. And then there is a third lipid, so-called PEG lipid. And a PEGylated lipid is just to maintain the integrity of the particles. Once we are formulated, the mRNA in these particles, you have to remain -- the integrity of this particle has to be remained and not only outside the body, but also inside the body. And therefore, this is important that you include this type of lipid. And this is what I want you to remember, this are basically the main lipids you need in order to build a functional lipid nanoparticle. And beyond just testing different combinations of these lipids in order to find new lipid nanoparticles with new properties, we also 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. And in order to make a drug, you want to -- you have to make sure that the process and manufacturing is established that we always have a nicely characterized particle. And this is a very complicated slide but it just illustrate something. If you look at the last [indiscernible], you see these curves which are pretty wide. This indicate that you can just buy a simple formulation, general particles of a wide range of size. This is something that you do not want to do. You want to have some in the mono disperse. So all has the same size manner. And our lipid chemists have found some tricks in this particular case, it's employed for our lead candidate, [indiscernible] which I will introduce to you later. In a way that -- and this is shown in the graph on the right-hand side with the blue curve and the orange curve. These are just 2 different methods of measuring the size of the particle, but you see they have this uniform curve. And that 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 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, but it nicely illustrates how these parts will look like. Yes, indeed, this is what we already know from the media when we talk about combination of all these vaccine. These are really this nice spheric structures what we achieved. 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 of how in our case, [ Panora ], looks like, you have some core structures where the mRNA is basically bond to these cationic or ionizable lipid and surrounded by layers of lipid membrane. So this is just a quick run through what are mRNA-LNPs. Now a few words what we really want to do in order to find new mRNA therapies based on mRNA-LNPs. And this little sketch illustrates our approach for 1 to 5, namely we play with this lipid [ loyalties ] like what I said, is cationic, ionizable [indiscernible] lipids, [ places ] around and generate new particles where we know they have different sizes, different charge, different physical chemical parameters. And these are testing the first step just in [indiscernible] in order to see how this behaves. Let me 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 consume, let's say, in a [ nation ] or something like that. But our main focus was at the beginning, just to do intravenously. So a systemic application but we also had an interest in finding routes for intramuscular, so local administration. So this is one way how you can basically move forward. Finding formulations, [indiscernible] and step 2 on the in [ vivo ] level then you might have identified certain type of [organs], but it's [organs] not tell you what are the other cell types. And are these cell types also relevant for certain [ authorities ] which we want to correct with the mRNA therapy. Then we dive deeper with other molecule means to decide the cell types, which are targeted with these LNPs, look then for an interesting target and indication, apply this in disease models and hopefully come up with a new drug development program. But 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. And this is basically how we came up is Evaxion. Evaxion said, hey, we come with the idea of building maybe a new vaccine, want to see how this goes? Can you offer a solution. And from these approaches, namely going from LNP to disease or from disease to LNP, we came up basically with a core of candidate formulations as some are shown here. Like according to our approach, namely from LNP to disease, we developed our lead candidate program, PANO-4. 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, but we also knew, especially since there's a lot of knowledge in this LNP that neutral LNPs are also important, especially for muscle delivery. And this is how we embarked on the journey, so here again -- may be in touch with other companies like with Astellas who had an interest in regenerative medicine. So in this case, also in interest in intramuscular applications and also like to vaccination purposes. And 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. So what is this all about? Again. So we did, as I said, just some screening of LNPs, which are applicable for intramuscular application. And 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. And shown in the diagram is if you apply -- so in orange, our underlying lead candidate formulation, a cationic one, there's no expression in the muscle if you use the neutral one. Then you see robust express and even over time. So we made basically the basis for offering Evaxion,some interesting formulations. So I mentioned several times neutral LNPs right at the beginning, you might remember, I mentioned we use 3 flavors. And I just want to explain what this actually means, which is different flavor of cationic, anionic and neutral LNPs. So we started off with cationic LNPs, right, 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. [indiscernible] an overload of anionic lipids in order to generate an LNP, which has an overall negative charge. You all have to consider at the beginning, you will have the negatively charged nucleic acid, which you need to formulate. But at the end of the day, you have the spheric little structures, and they have a certain charge and this charge is very critical for the delivery properties. And then I mentioned this several times, these neutral LNPs. And neutral LNPs can be generated or at least we do it, like the standard, which is very use so-called ionizable lipids, Ionizable lipids or pH-sensitive lipid means depending on what is the surrounding pH, is it basic or acidic, the lipid is protonated or is positively charged or not. And this is important because at some point, you need some positive charge in order to bind the negatively charged RNA. And this is one manner -- and this is also -- if you follow the scientific literature, the whole field banks on this support, 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. But it also turned out and COVID told us a story, if you apply the intramuscularly, they can also do we took at the vaccination. So we can offer this, but at the same time, we also apply to 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 has a neutral LNP. But in this case, we call this a neutral LNP with an hydrophilic surface, let's put it this way. So hydrophilic, so it can dissolved in water. In contrast to the second one or the original one which is using an ionizable lipid, you generate neutral lipids but with a hydrophobic surface of the [indiscernible]. So it's important to remember, these neutral lipids are in these 2 [styles]. And these 2 [styles], we offered to Evaxion to see how does it work in your [ update ]. And all these complicated slide just to give you an insight, how do the vaccines, which we all experience look like. Again, important message is on the right-hand side, up. Ionizable lipids, pH-sensitive lipids were employed for the product like [indiscernible] or [ Cominati ] then we need these helper lipids, 2 helper lipids and a PEGylated lipid. So this carrier for the vaccine and [indiscernible] fiber is composed of 4 lipids, and they are mixed in a very specific ratio. And to illustrate in an easy way how our formulation compared to those, I [indiscernible] this pie diagram. So on the right-hand side, the well-established validated mRNA-LNPs, and you'll see how they're using the different lipids and the different ratios. In blue is indicating the main important lipid in order to find [indiscernible], which is ionized with pH-sensitive lipid. And we offered Evaxion the very same and you see it also according to the structure of [indiscernible] a very same similar formulation, but the [indiscernible] on the left-hand side is completely different. So this is use of this cationic lipid and we have a way in order to make it neutral. Yes. And 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 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 PANO-4 and PANO-4 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 3 lipids. So it's simpler compared to these neutral LNPs. But again, it's the same story. You need a main lipid, in our case, a cationic lipid. You need a core lipid and a PEGylated lipid and the mRNA we are employing is a fusion mRNA encoding an agonist for the endothelial type 2 receptor. And yes, we have done a lot of pre-clinical work on this. And if you're interested, I invite you to visit bioarchive where you can read the full story. But it is our candidate for that 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. So the cLNP formulation is the underlying formulation for PANO-4 due to this cationic over character, you see it's driving the formulator the expression to the lung. This is shown with in this part of diagram. But 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 the reported mRNA. And with the sequencing approach, you see that we cluster and each dot represents an individual cell that takes you basically and proves that those cells have taken up the mRNA, which we want to be expressed. And to give you an idea how this really behave in [vivo], just simple western blot, if you inject into mice intravenously, these LNP, you can see a nice expression on the left-hand side in a dose-dependent manner. This big black bars or bands reflects the expression of the transient expression of these [ agonists ]. On the right-hand side, you can also follow this over time. And we have employed this in various mouse model 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 [ type 2 ] pathway and that this also leads into some pharmacological activity. But 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 accretive to pathology, especially in the course of acute respiratory distress syndrome. And many people die of these because people develop -- it's basically flooding of the lung, and you need to prevent this flooding. And because of the flooding of the lung, is that inflammatory trigger opens up to send barriers between the endothelial cells of the vessels, right? And this is shown here. There are certain antagonistic proteins, they inactivate this type 2 receptor, which leads them to openings, the barriers between the cell and this leads to vascular leakage. And 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 occurred, 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 these carrier delivers the mRNA is shown here to the endothelial cells of the lung, the protein, in this case, comes [indiscernible] agonist becomes expressed, secreted and reactivated [ type 2 ] receptor, signaling pathway in order to restore vascular barrier function. And 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 cycle. We are now at the process of launching the CMC campaign and do the IND-enabling studies in order to test this concept in the clinic. And 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 because those patients or most of these patients are still dying of this complication on the ICU unit. And these patients required ventilation -- mechanistic ventilation in order to stabilize this patients. And with our concept, we believe just by addressing this very first critical [pattern of Etiological], step, which is the development of lung edema, and neutrophil inflows, by preventing this one, we can also prevent the patient from getting worse, basically. And with this, I will close and thanks for your attention and Evaxion 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. And 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.

Yes, about question for your lead candidate, essentially the fact that it is positively charged, makes it target the lung cells. Is that correct?

That's correct.

And can you explain -- I don't quite see that connection. How -- you know why that works?

I assume also some kind of luck and experience. [indiscernible]. So it's a cationic -- the cationic charge is definitely important in order to get an interaction with the surface of the [indiscernible]. And -- but basically, this is what we found by chance. In addition, what's also important, I haven't pointed that out in detail for the overall physical chemical characterization, the size of the particles, I have mentioned this in detail, what we are dealing here is particles around 80 nanometers. So it's a combination. And last but not least, it's also the systemic growth. You can imagine if you apply a drug intravenously, the first big vascular [indiscernible] which you hit is a lung. So it's probably a combination of all these effects. What's important is that you find the right LNP with the right charge, with the right size. So then these capillaries also at lung are targeted and also, in a way, when it won't interfere with a lung function.

So for our lead candidate, do you expect that you need to do repeated dosing? Or would one infusion be sufficient? [indiscernible] that you induce an ongoing [indiscernible] response, if you have to do this repeated infusions.

This is exactly also a very important field in general for the mRNA-LNPs. Can you apply these over a longer period. And our idea was to find also the right indication in this manner that we do not need to treat patients forever or for a very long time. So we've picked this acute setting, absolutely right, and we know also in this acute setting, we had this 1-week period, where the patient is in a critical phase where we basically need to treat the patient. And now we do [ oncology ] 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. Some of these investigations are running. They are also important in preparation of the IND-enabling toxicology study. But the important point is that we just want to cover this critical 1 week period. So it's the semi acute setting.