NorthWest Biotherapeutics Inc (NWBO)

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ASCO 2023 Transcript: Dr. Marnix Bosch Speaks in the Industry Expert Theater

DCVax-L Mechanism of Action, Immunological Effects, and Clinical Trial External Controls Methodology

https://www.youtube.com/watch?v=BWwdnedw_PA

Slides
https://nwbio.com/wp-content/uploads/NWBT_ASCO_slides_06032023_FINAL.pdf

Well good afternoon everybody. I think you're going to need your headsets . . . or maybe you don’t. So thank you all for coming, ladies, gentlemen, friends, colleagues, ex-colleagues. It's really exciting to, again be here at ASCO, and being able to present some of our work to you.

Today we really have three topics that all tie together, I think in a very nice fashion, and I'll walk you through those. And there really are, sort of three separate topics. One is the mechanism of action of DCVax. We've talked about that a little bit in the past, but we haven't really filled you in on all the work that we've been doing to demonstrate that DCVax indeed has all the characteristics that it needs to induce the immunological and clinical effects that we're all seeing, and we're expecting. Then, I'll highlight the immunological effects that we're actually seeing in patients. And I'll talk a little bit in depth about the clinical trial external controls methodology.

I truly believe that we're at the beginning of a revolution here, where hopefully, five or ten years from now, there will be no more need for placebo-controlled clinical trials, because really, in the end, once you know, sort of, what the outcome is of a disease, especially if the patients are given standard of care, at some point, there will be no more need for for placebo controls, and all patients will be able to participate in the new inventive treatments that we’re coming up with. And we think that's an important development, and we're happy to be one of the leaders in that particular field.

This is a the obligatory disclaimer. I don't need to read this whole thing. This is the overview of the presentation. I'll first give an introduction about DCVax-L. A lot of you are familiar with the product, but I still think it's useful to give you a bit of an introduction because that leads into the rest of the presentation. The mechanism of action, I'll go into some detail, and therefore, first I will again give a bit of an introduction on what the mechanism action should be. And then I'll show you that it actually works in the way that we expect. Again, for some of you that will be a bit repetitious because you may be familiar with it, but because of some of the detailed explanations I'm giving later, I think it’s still useful to go through that. The immune monitoring section is entirely new to everybody here in the audience. And then I'll talk, rather extensively, about the use of external controls and clinical trials, and some of the things that we did that we believe are important also, for other people to follow, so that the design can be, and the execution can be, sufficiently rigorous and well controlled. I'll talk about two of our compassionate use cases, and make some observations about that. And then of course, I'll end with some general conclusions and a broader perspective.

So what is DCVax-L? DCVax-L is composed of a group of autologous dendritic cells, that are loaded with autologous tumor cell lysate. So both components are autologous. The tumor cell lysate is the source of the antigens. They come from the patient's own tumor, and we use the tumor tissue that's resected at the time of surgery, to make the lysate. So it comes from the patient, and it goes back to the patient.

The dendritic cells come from the white cells that are in the blood, we obtained that through a leukapheresis. And we need that, because the immune system of the dendritic cells, needs to match with the immune system of the patients, once we get it back. So if you give dendritic cells from somebody else to a patient, the likelihood is that, that match will not be there, and therefore, you don't get the stimulation of the immune response that you need. The tumor lysates, we prefer that very much over using a limited number of antigens, because tumor lysate is a broad spectrum source of antigens. Every single antigen that's present in the tumor, will be present in that lysate. And I'll show to you the evidence why that is actually important.

For one, the reason is that, at least we presume, that it makes it more difficult for the tumor to escape from the immune attack that we're targeting. But of course, therefore we need to show that we actually have a broad array of antigens, but also that the patients are responding to a broad array of antigens. And I'll show both of those factors to you later in this presentation. And of course, the tumor lysate provides the correct antigens, and not the wrong antigens. It's intended as active in treatment following surgery. We need the tumor cell lysates from the from the resected tumor to make the treatment.

And we have used it to date to treat almost 600 patients with GBM, both in clinical trials, and in compassionate use programs, and also tens of patients with other cancers, including: Merkel cell carcinoma, breast cancer, thyroid cancer, and several others. The trial results from the phase 3 trial have now been published in JAMA Oncology, with Dr Linda Liau from UCLA, as the first author, showing both clinically meaningful, and statistically significant, extension of survival, in both newly diagnosed, and recurrent GBM.

And just to tell you a little bit about the manufacturing, of which I gave long presentation last year. And manufacturing takes about eight days, provides several years of doses that are frozen down, and can then be shipped as an off-the-shelf treatment to patients over time. And that's exactly how we did it in the clinical trial.

So how does, how is, DCVax-L supposed to work? How do the dendritic cells actually interact with the T cells, and induce the immune response that were after, leading in the end, to the induction of cytotoxic T cells that can travel to the tumor, and kill tumor cells? One of the hallmarks of this mechanism of action is the so-called multiplier effect. One dendritic cell leads to hundreds and hundreds of T cells that are responsive to the tumors.

How does that actually work? So the dendritic cell takes up, and presents the tumor target proteins from the lysate in the context of its own MHC molecules. I'm not going to talk about MHC, but that MHC word is important because that's how the antigens are being presented. Then the antigens are presented to the anti-cancer T cells that become activated, because the resting anti-cancer T cells attach to the dendritic cells through a number of molecules called co-stimulatory molecules. Those activated anti-cancer T cells then divide rapidly, that's the multiplier effect, and acquire a capability to kill tumor cells. Something we call cytotoxic activity. And then those multiplied T cells then can travel anywhere, including to the brain.

Activated T cells are one of the few agents that can actually travel across the blood-brain barrier. If you've heard about drug development for glioblastoma, you know that the blood-brain barrier is a huge hindrance towards the development of drugs. Because most drugs, well actually you can inject them, you can ingest them, but they won't get to the brain. Whereas, activated T cells travel everywhere, including to the brain, and including to the tumor. And we have evidence. I'll show you a picture that they actually end up there, and start killing the tumor cells.

So let's first talk about antigen uptake and presentation. Because yes, we incubate the dendritic cells with the lysate that contains the peptides and the antigens. But how do we actually know that those peptides and antigens are taken up by the dendritic cells and then presented on their surface? And this is a series of experiments that show you how we actually have gathered that information. How we know that the information I just told you, is actually correct?

So we did proteomics analysis on the following materials to determine protein content, and diversity. Diversity is a very important key word here. We did a proteomics study. I.E., proteomics means that you characterize every single protein that's in that particular preparation. So proteomics on the tumor lysate will tell you every single protein that's in the tumor lysate. We used unpulsed dendritic cells, so dendritic cells that were never incubated with tumor lysate, but also on dendritic cells that were pulsed with tumor lysate.

And then we did another trick. We took the peptides off of those dendritic cells and asked what's in those peptides. Because then we know what's actually being presented. So from a single sample, this is one experiment that we did. We found that there were 25,000 MHC class 1 associated peptides. Those are short peptides that are important for cell killing . We also found almost 13,000 Class 2 associated peptides. Those are more important in inducing T Cell help. Helper T cells are cytotoxic T cells that interact to induce the tumor killing effect. Of those 25,000, a lot of those were up-regulated on the dendritic cells after pulsing, suggesting that they actually came from the tumor lysate. And the same is true for 7,000 or more that were more abundant on dendritic cells after pulsing. That's not everything, because then we found that we had 400, (399) tumor-associated peptides, 400 different tumor-associated peptides.

So these are all presented on the dendritic cells. They're all presented to the immune system. The immune system now has the capability to react to every single one of those antigens. So we truly do get a broad spectrum immune response, just by the function of using the tumor lysate to pulse the dendritic cells, rather than what has been tried many other times, using a single antigen, or a small repertoire of antigens. And some of those peptides, that you can see that are either, two-fold, three-fold, five-fold, enriched on those dendritic cells are listed here.

And these are just amino acid sequences, so I don't expect you to read those. And these are just examples. For MHC Class 2, like I said, are important for the interaction with the helper T cells. There were 220, and these are the ones that are detected. There is a certain limit of detection, level of detection as well here, and anything below that limit we won't be able to detect, but they can still be there. So this is a low estimate of the number of tumor-associated peptides that are presented by these dendritic cells. Remember these are the ones that were taken off the dendritic cells, and these are the ones that are actually presented. And we've got a few examples here, again as well, with the degree of up-regulation on it on a two-log scale.

So where does that take us? These are some antigens that we found. Because once you have those amino acid sequences, you can go to a database and you ask: Where does that amino acid sequence come from? Which protein was that derived from? And we found, not to our surprise, that there were peptides that came from proteins that were previously identified in glioblastoma. Okay, so you take a tumor lysate from glioblastoma, you put it on those dendritic cells, you find antigens that were already known to be associated with GBM. Interesting, exciting, not that novel perhaps. Some of those is an inhibitory protein known for GBM. One, I really love this name; the Disheveled Associate Activator of Morphogenesis number 2. There must be a number 1 out there somewhere as well, which promotes gliomogenesis. So these are known MHC associated, GBM-associated peptides.

So what else have we found? We also found examples of peptides that were known to be associated with other tumors. So if we had taken a repertoire of known GBM proteins, we would have missed these, because we would have never anticipated that these would be presented by dendritic cells. And that these might be as relevant for the activation of the immune system against GBM as the ones that were known to be associated with GBM. My motto is always: Don't pretend to know more than you do. And this was something that we truly did not know before. There's this, this ligate that's expressed in multiple cancers, a protein associated with poor prognosis in ovarian cancer. So an attack against these tumors might attack GBM as well. And probably will attack GBM as well.

So a few of the summary observations are: that the lysate preparations releases multiple cancer-related proteins, from which these peptides can be processed. (I’m sorry, like many of you, I'm still recovering from a cold) These dendritic cells pickup, process, and present hundreds of tumor-specific peptides to T cells. Literally hundreds. Imagine now, that you have, that you're a tumor, and you're attacked from a hundred different sites. It's almost impossible to down-regulate those targets, and escape from that immune pressure, which is why we think the product works as well as it does.

It presents peptides from both known GBM-associated tumor antigens, and tumor antigens which have not been reported in GBM before. And the presentation of these peptides, not only through class 1, remember important for cytotoxic T cells, but also through Class 2, to CD4 T cells, suggests that we also get T-cell memory responses in vivo. Something of course, that we believe is incredibly important in a chronic disease like GBM, where you can't just eradicate it at once. You needed ongoing response that continues to hammer away at the tumor. You need to set up that battle in the brain, where the immune system can continue to eradicate tumor cells, because they will continue to replicate otherwise. So this immune memory is actually critical for the, for the potential success.

So now we have these in the context of MHC Class 2, and MHC class 1, we have these peptides that interact with the T Cell receptor. But that's not sufficient for a dendritic cell to activate a T Cell. We need at least two signals. In fact we need three. We'll only talk about two today. We need the T cell receptor with the multiple peptide antigens. We need to demonstrate that happens, but we also need co-stimulatory signals. We have CD40 here, we have CD80 here ,CD82 here, and they're given different names here, but they're the same proteins. And we need those because the T cell, like I said, needs at least two signals to become activated.

So what do we know about those? What you see here on these little plots? This is an indicator for the size of the cells. It's called forward scatter on the x-axis. And this is the degree to which the cell's label positive with the different markers that we have here expressed on the y-axis. In this case, it's CD40 here, and CD141. So, if they're sitting in the top right corner, you can see that they're large cells. They're moving in this direction, and they're positive for this particular marker. So for CD40, CD141, CD82, CD86, MHC class 2. We have these pictures. We have it for many more of these particular kind of markers that are so critical for those interactions to have a positive outcome. So here you see the function of those molecules. CD40 and tracks with CD40 ligand, CD41 is directly involved in antigen presentation. CD82 stabilize the dendritic cells interactions.

The longer those interactions take place, in the, what's called the immunological synapse, the more, the stronger the T cells will be activated. So all of these molecules are critical for this, for the effect that we're seeing. And then there’s CD83, which is a marker for activation or maturation of dendritic cells. Immature dendritic cells don't activate the immune response very well. Mature or activated dendritic cells do. And we showed it ourselves, do express CD83, as expected.

So then the question is: okay now we've got all this, right? We've got the peptides on the class 1, and Class 2 molecules, we got the co-stimulatory molecules. What has actually happened when you put those cells together, and you stimulate, and re-stimulate them a few times in vitro? Can we now actually generate cytotoxic T cells like we're hoping that we're going to be doing in the patient? The short answer to that is: yes.

These are complicated experiments. This was done by Daphne Franz and Mark Lowdell, and you can, I'll just make this relatively short. But you can see that if you have all the right components here, which is the lysate from the MCF7 cells, the matured dendritic cells, and you have the T cells present, that you get a degree of cell killing that kills 60% of the cells. It doesn't work if you leave out the lysate. It doesn't work if you leave out the T cells. It also works if you use only the CD8 positive T cells. So you don't need CD4 cells at the end of the cell killing process. You do need them to get to that stage, and you can do it, an assay for either 16 hours or 24 hours. At 24 hours, your background becomes a little bit more disturbing, so we tend to do these in relatively short period of time, like 16 hours. So we find that killing of target cells is observed, if the T cells were stimulated by dendritic cells loaded with tumor cell lysate.

So yes, we have all the components, now we show that they actually work the way that they're supposed to work. But that doesn't mean that we’re getting a lot of T cells yet. This is work done by Lekhana and Andrew at Flaskworks, the company that was helping us present last year, and they're both here in the audience. This is what we call the multiplier effect, or at least it's evidence of the multiplier effect. Whether it is the multiplier effect, we really don't know at this point in time. But it’s strongly suggestive.

What this is, is a T-cell stimulation assay where T cells are dividing. And every time they divide, they move a little bit to the left in the spectrum. This is a die that gets diluted so every generation of T cells has its dye diluted by two fold, and therefore you get all these multiple peaks, and you see that there's like first generation, second generation, third generation, fourth, et cetera, et cetera, et cetera. For this particular question, we're mostly interested in the late generation T cells because those are the ones that have undergone the most divisions, so they have the most daughter cells at this point.

So what do we see when we do this assay with T cells loaded with lysate, dendritic cells loaded with lysate or dendritic cells not loaded with lysate? We see that the dendritic cells loaded with lysate, these ones here, generate significantly more of these late generation T cells. So there is an additional T Cell similar stimulatory capacity that we still need to identify, and still need to understand. But it's something that we observed, and we think that is very interesting, and could be, at least in part, responsible or co-responsible for the multiplier effect that I told you is so important for the mechanism of action.

So the conclusion is that, these lysate loaded dendritic cells have acquired this additional T cell stimulator capacity, which result in more late generation T cells, that we need to fight the tumor. More is better in this particular case.

In summary, the dendritic cells in DCVax-L expressed or regular requisites co-stimulatory molecules to perform this productive interaction. They also express CD141, a molecule involved in effective antigen presentation to T cells. If we mix those dendritic cells the load of once with the tumor lysate, then we have this additional T Cell stimulatory capacity, and we can then induce killer T cells the CTL Cytotoxic T Lymphocytes. And the combined, these effects enhance the multiplier effect, or are responsible for the multiplier effect. In conclusion, mechanism action studies demonstrate uptake, presentation for broad range of antigens, which is important to prevent tumor escape.

I will next talk about immune monitoring data that demonstrate activation of a wide repertoire of T cells, which can travel to the brain. This is data that we have not shown to you before. So what is in vivo immune monitoring? What we wanted to know is: when we inject these dendritic cells into the patient, what happens to the T Cell response? Are we inducing a T Cell response? The hypothesis is: yes that we are.

Linda Liau has shown that, in the past, that indeed in her early stage trials this was actually, this actually happened. But that's really proof of concept. That is, okay if I do this, and I do some complicated experiments, then I can demonstrate it actually happens. What we did here, was take all of the T cells from a patient out of the blood, well not all the T cells, but let's say several million T cells, and ask: Are they expanded? Are they not expanded? Has these are expansion occurred like we expect? What is happening in these patients?

Here's the experiment that we did: Broad Spectrum Immunological Stimulatory Approach, with all the tumor antigens, that I just explained. We also use an analysis that allows monitoring of the entire T cell response, as opposed to monitoring response to a single antigen. Single antigen is easy to do. This is hard to do. Okay, now what you need to realize is: each T cell that emerges from the thymus during tautology, during development, has a unique T Cell receptor and DNA sequence. So, before you get hit with any pathogen or any infection as a young kid, you have all these T cells circulating, and whenever you get an infection, or another stimulation, the T cells that respond to that pathogen are the ones that start to expand. And that's how you can fight an infection. That's how you fight the Coronavirus for instance. So, that's a normal process that happens during, as a response to an infection.

We are asking the question: Does it also happen in response to DCVax? The hypothesis was that it did. But of course, when a T Cell divides in response to team stimulus, that specific T-cell DNA sequence becomes more numerous. It's a T cell has specific DNA sequence T Cell receptor, and multiplies a thousand times. You now you have a thousand. So sequencing all the T cell receptors allows you to identify newly expanded T cell clones that weren't there to begin with, or identify T cell clones that have further expanded, like from baseline.

Here's some of the data from three patients. What you see here is the number of expanded T-cell clones between baseline and month 4, or baseline and month 8. Now remember, we give several immunizations. We give day 0, day 10, day 20, month 2, month 4, month 8, and so. And then we measure at discrete time points: what happened to the T Cell repertoire. And here we measure the number of expanded T cell clones. And you can see that, between baseline and month 8, 800 T cell clones have expanded in this particular patient. More interestingly, about 250 of those expanded ones had never been seen before in that patient. So they weren't there at baseline, at least not at a detectable level. They're now very detectable at month eight. This is a newly induced T-cell response. This is enhancement of a T-cell response that was already existing. We think that both are important.

Glioblastoma is not known to be a very immunogenic tumor, but it doesn't mean that it does not induce a bit of a T-cell response, already. It’s sitting in the brain. It's well protected from the immune system, but that protection is not completely 100%, black and white. So it's not surprising that there are already T cell clones present in the patient, that we are expanding once we immunize it with the lysate from the same tumor, that the patient already carried in his brain.

Patient 2 and also about 800, and this patient is really taking the cake, with more than 1200 expanded T cell clones, of which 500 or more, are new, that weren't seen at baseline. So these bars represent a total number of expanded T cell clones, as well as the pre-existing. (and yeah thank you I'm getting my medication here I need to take your drugs that's what they tell me) Now, the background, in patients where we would not be doing this, is roughly 2 to 20 clones. I'll show you a picture to illustrate that. So this is the background stimulation. This is what's happening when you give DCVax. Here's a more, sort of better, illustration of the T cell dynamics. You're all probably old enough to remember the old 1980s commercial, “This is your brain, and this is your brain on drugs.” Well, this is your blood, and this is your blood on DCVax-L. Sorry, I had to throw that in there.

Here you see, so an orange, here you see expanded T cell clones. And here you see the converse. It's contracted to T cell clone, so T cell clones that were detectable at baseline without no detection. In small, and smaller numbers, they don't disappear. They get out-competed by the ones that are expanding. And so you see, in a normal patient, between sort of Time Zero, and month 4, you see basically almost nothing happening. There's one newly expanded T cell clones. Here, there's two that are contracting. Here, you see literally hundreds that are expanding. And the ones, here on the left, are all the new ones. And here, you see the ones that are. that are less numerous following the simulation. So there is a shift in the T Cell response. There's a dynamic going on that results mostly in the uh generation of a new immune response against the tumor antigens presented by DCVax.

Oops sorry. Here's example of three patients, and these two are very strong responders. You can see that all these new clones here. All these expanded clones here, that were already existing. Same for this patient. This patient, like any drug is a slightly less strong responder. You still see new T cell clones. You still see expanded T cell clones. It's still quite different from the patient where there had been no stimulation. It’s not as potent, and not as powerful. And of course, we're very interested why some patients respond stronger than others. This is true for any drug, and every drug, so it's not a surprising finding. But we think it's a very interesting finding.

This, by the way, is a comparison between baseline and month 8. Very exciting. Once you have these T cell receptors sequenced, you can go to a database, and you ask: Oh the T Cell receptor, is that known to already be associated with a T Cell response that has already been characterized to a known antigen? Those algorithms, and those predictions are, I would say, they're, they're limited, and they're only indicative, at this point in time. But within that, we could still make the following observations: we found T cell responses there, for potentially responding to a very broad range of antigens. Not surprising, we give a broad range of antigens, we get a broad T-cell reaction. The predicted epitopes include several known tumor antigens. Surprise, surprise, some known to be associated with GBM, but some not previously identified in GBM. Just like we found, in the proteins present, presented by the dendritic cells, we also found several viral antigens, both CMV and EBV, and both CMV and EBV have been found in the past to be associated with GBM, so that's not a surprise either. And then we found multiple molecules that we really don't know what they do. So we'll be looking into that some further as well.

Then this is a picture that you may have seen before. It is a picture by Linda Liau, that she shows a lot, and I've shown it a couple times. And these are, this is a section of the brain tumor from a patient operated post-vaccination. And you can see that post vaccination, you have a lot of T cells that are getting, actually to the tumor. There's very few prior, there's a lot after, and it includes both CD4 cells, and cd8 cells. So now we have the whole chain complete. We know how dendritic cells work. We know what they present. We know how they interact with T cells. We know to induce T cells responses, expansion in the patient. And we now know that to actually make it to the brain. So that completes the whole sequence of events. That's important for how the dendritic cells, and the specific DCVax works

So just a summary of these data before I go to the clinical trial. T cell receptor sequencing demonstrates extensive expansion of specific T cell clones in response to a broad repertoire of antigens. And I love this quote, so I put it in. This was by the technician of the company that actually did this work. He said this level of clonal expansion, especially of the new detective clones, provide strong evidence for a novel stimulus of the immune system during this interval. I couldn't have said it better myself. So these observations together all support the postulated mechanism of action of DCVax.

So let me move on to the clinical trial methodology. Okay, just mention the data out real quick to you. Most of you are familiar with the data, so I'm not going to spend much time on that. But, one thing that was overlooked by a lot of people that had the chance to read our paper in JAMA Oncology, was that there was a lot of methodology explained in the online only supplement. But I understand that not everybody gets to the online only supplement. And it also introduced some new statistical methodologies that are not typically, that are typically used in health economics analysis, but not always used in analysis of clinical trial data. So therefore we thought it would be useful to spend some time on those here today.

Now, of course you are aware, sorry, of all of this, and primary endpoint, overall survival. Newly diagnosed GBM versus external controls that I will be talking about next. And the secondary endpoint is in recurrent GBM, also compared to external controls. There is good reasons for the use of external controls, I won't go into that. That's all extensively explained in the paper. And these are just the two observations: that the findings, in both newly diagnosed, and recurrent disease are both clinically meaningful, and statistically significant, showing extension of survival. In this case, of about three months. In this case, with more than five months. And a five months increase in survival in recurrent GBM, is not something that you see every day. In this landmark survival data, you see that we have more than a two-fold increase of the relative rate of survival at 5 years. 5-year survival in GBM is a rare finding, in any case. In recurrent disease, 30-month survival of 21, of 11%, ie., doubling of overall survival. It's quite, we would say meaningful and significant.

So how did we actually do this? We have the DCVax-L trial was originally designed as a placebo. That was then deleted because those placebo patients mostly got treated with DCVax, so you can’t do that comparison anymore. But, so we used external controls, and we got those external controls for newly diagnosed GBM, from 5 other randomized clinical trials in newly diagnosed GBM, where the patient's got the treatment, but the other half, they basically got a placebo. We use the data from those placebo patients. Those generate the external controls, that then our newly diagnosed patients treated with DCVax-L, are compared to. Now, then we had a crossover arm, by which the placebo patients could also get DCVax. So we needed external controls for that as well. 10 trials with recurrent GBM, randomized controlled clinical trials. There is no placebo in most of those, so those patients got standard of care, mostly, or physicians choice.

Again, the control patients from those trials, form the external controls, that then ,the recurrent patients from our trial are compared to. This is a concept that, that is, very important. And that, I wanted to explain one more time, because it's important background for the rest of the the small presentation that I still have left.

Now, we showed you how we did the analysis. There was the analysis against external controls, Kaplan Meyer analysis. But when you use external controls, you have to be conscious of bias. By the way, when you do a randomized trial, you also have to be conscious of bias. That is something that tends to be forgotten. Sometimes, where people say, “Oh it's a randomized trial, and therefore it's perfect.” It really isn’t. And, I I feel that the field has sometimes lost sight of that. But okay, we are very conscious of the fact for, consciously aware of the fact, that we need to control for bias. And we used four layers of methods to make sure that we had at least thought about that, and addressed it to the extent possible, to minimize both known, and unknown biases, and to closely match the internal control population, and the DCVax-L patients.

So we match the comparator clinical trials, from which external control population was drawn. And I'll tell you how we did that, in the next slide. Then we validated whether the external control population was a useful, and meaningful control population, by comparing it to the trials from which we drew those individual control populations . . . pulled them, and then compared them, again, to the treatment arm of those trials. I'll show that to you as well. Then we did some sensitivity analysis, because yes, there are known, and unknown biases, when you do it this way. We are aware of that, we address them, and then we adjusted also for individual patient characteristics. We did not not have access to individual patient data. It turns out that there's mechanisms, if you don't have that to address differences in prognostic factors, it's called the MAIC - matching adjusted indirect comparison. And we did that as well.

So here's how we selected those comparator trials: We used an independent expert firm, and the company was not actually not involved in that. We asked the question: What are trials that can provide controls that demonstrate what the outcome is with standard of care for patients that participate in clinical trials? So we set up the criteria . . . at least the experts did. . .14 criteria: seven for newly diagnosed, and seven for recurrent disease. They needed to be contemporaneous. We did not want to use historical controls; standard of care changes, outcome in disease changes, just because surgeons get better, oncology, oncologists get better. They had to be contemporaneous, and they were. We needed the reported outcomes, and Kaplan Meyer curves. We needed the same standard of care. Now in GBM, that's easy, because everybody gets these two protocols. If you're on a trial, you get the same protocol, that's what you get. It needed to be a randomized study design. We need patients that matched on age to patients that we had in our trial. We need a Kaplan Meyer available, for survival, and for subgroup analysis. And they had to be in the English language.

The data that we got got from here are actually very high quality data, because there are very few patients lost to follow up. It's not that this was muddied, because some patients were never tracked for a long time. And these patients, we have all the data for all these patients, for a whole period of time, for all of these trials. Therefore, the comparative studies weren’t actually fit for this particular purpose: to compose an external control arm for our trial, and those compared to our trials. Not just the criteria were selected, but the actual trials were identified prior to unblinding the data. We did not know what the data looked like before the trials were identified by the external experts. This is, this is a critical point. You can't look at the data first, and then say, “okay which trials are are a good fit for me?” You have to do it beforehand, and that's exactly what we did.

So what were these trials? These are trials that everybody knows about, because these are the big trials in GBM, and newly diagnosed GBM. Dose-dense Temozolomide trial. Bevacizumab trial for newly diagnosed, the Celldex trial with rindopepimut, the anti ETF arm peptide trial, tumor treating fields by Stupp et al, for the Optune device, and ICT 107, which is the Patrick Wen trial.

The number of patients in those trial add up to 1366. That is the power of using external controls; you're not stuck with 100 patients that you had in your trial, that you could use as controls. You're adding tremendous statistical power by doing this, but you're also adding a much more realistic comparison, because your numbers are bigger. Look how tight the outcomes are in this trial, median is 16.5 months to 95% confidence interval is 16.0 to 17.5 months. If you're a patient with GBM, youre put in clinical trial, you're given Placebo, we know how long you're going to live, this is the answer. And recurrent disease variation looks to be a little bit broader but actually turns out that when you match all the numbers it really isn't 7.2 months survival for first recurrence, and the 95% concentration, interval 7.2 to 8.2 months. So we feel that this is a very very solid database, against which you can compare the outcome of your trials.

So this is the validation that I that I mentioned where we compare it. Now, the treatment arm, this is validation of the external external, external control population actually work as an external control population? We can test that by comparing the treatment arm of these trials, to the external controls as a whole, through the 1366, instead of to their own internal control population. And it turns out, the outcome is exactly the same, every single time. All of these trials basically, unfortunately, were negative. There was no survival advantage in any of these trials. And as you can see, if you can see these statistics, the hazard ratio shows that too. They're all very close to 1, or even above 1, except for one trial, the Optune trial. And low and behold, that was the only trial that was positive, out of all of these trials. So whether you compare that trial, to its own controls, or to the ECP, the outcome is exactly the same. And that's true for all the other trials as well. So the ECP is hereby validated. This is a good external control population.

We did the same thing, of course, for the recurrent disease, and the outcome there, is exactly the same. Let's talk about biases. Like I said, every trial has biases, and they need to be addressed. And we are aware of that, and we did that . In this case, I'm just giving you an example of a known bias. In our trial, patients who had disease recurrence post chemoradiation, were excluded from the trial. They were often put into compassionate use protocols, but they were excluded from the trial itself. Now, the five trials that we used for external comparisons, also excluded external controls. But two trials also . . . sorry, also excluded patients with Progressive Disease. But two trials did not explicitly state that, so we could not be sure that they excluded those patients, so we said, “okay, let's remove those from the ECP. Let's not use those. Let's only use the other three.” Does that make a difference on the outcome? The answer to that is: absolutely not. The hazard ratio instead of 0.8, is now 0.77, so the effect is actually stronger. The p-value remains exactly the same, and the confidence interval is almost exactly the same as well. One of the known biases hereby addressed, and dismissed.

Unknown biases, remember that all of those five trials, had slightly different inclusion criteria because they're all testing different drugs. So you need to, like, tweak your inclusion criteria a little bit, to make sure that your patient population, is the right patient population, for what you're testing. So they were all slightly different. So therefore, we removed each of these individually, one by one, to make sure that not one trial, because of its inclusion criteria, may be skewed to data, to the extent that it would favor us, or disfavor us. The answer to that is: if you remove each of these, one by one, doesn't make one bit of a difference. Hazard ratio remains around 0.8, a little bit higher, a little bit lower. The p-value still remains 0.001, .002. There's an outlier 0.007, highly significant. And the difference, really is the same, that it was before.

So what we did in these different sensitivity analyses, like I said, this is only one example of one, where we address a known bias, is that we actually did address this very carefully. And we come to the conclusion that, the sensitivity analysis that we did, don't reveal any biases that our data could have been subjected to, and it could have skewed the results in one way or the other.

And then, the last thing we did, because no matter how carefully your comparator trials are chosen, there's always going to be differences in the patient population, in terms of, for instance, prognostic factors. We know that age is a prognostic factor, extent of resection is a prognostic factor, we know that. And you can, using the matching adjusted indirect comparison, you can adjust for that. And you adjust for that by, it's actually a very complicated statistical method, but it works really well.

Like I said, it's being used a lot in health economics analysis in Europe. All countries have payers, the HTA’s, health technology associations. And they determine whether a drug should be paid for, or not. And they use this technology a lot, so it's a well-established technology. But in a slightly different field, related field, and and used in reimbursement decisions. It can adjust for even small difference in patient characteristics. The net effect is that the sample size is reduced, so you lose some statistical power, because when you adjust, your population becomes smaller, and smaller, and smaller, every time, because the differences weren't really that big with the comparator cohort. The loss in sample size wasn't that great, but there was a loss of sample size, irrespective.

But when we did this for these known prognostic factors, which is quite a bit, age, sex, race, MGMT methylation status, KPS score, extent of resection, or residual disease, we did either one, the difference remains statistically significant. It still favors DCVax. The outcome is still these effects is clinically meaningful, and statistically significantly better against the pool controls, but also against each of the comparator studies. So again, we address the fact. what our one study was maybe a strong component in making up this control population? The answer to that is: no.

Conclusions from these external control methodology? Sort of enlightenments, I hope. The survival of GBM patients participating in clinical trials, as a control subject, is remarkably consistent. . . for us, I mean. That's unfortunate for the patient population. But for us, it creates a landscape in which internal control populations can be used as synthetic control arms. Against this background, DCVax-L is associated with statistically significant, and clinically meaningful extended survival, both in newly diagnosed, and recurrent disease. And these results are robust, and they hold up well against multiple analyzes, to address both known, and unknown sources of bias.

So that's what I wanted to talk about, about, the clinical trial. And now I just want to highlight two cases that we've treated in a compassionate-use setting. There are, by the way, several people here in the room, that are long-term survivors, who have been treated with DCVax-L. I think we have a 20-year survivor, here in the room, somewhere. There he is, Brad, so happy to have you. And there’s, there's several more as well. And they will be all in our booth. And if you're interested, they'll be happy to talk to you.

So, this lady is also here in the audience; Sarah. So these are, these are real patients, these are real people. [applause] Sarah came to us in 2012, had only received chemo and radiation, and then Temozolomide. And the first batch of DCVax-L was made in 2013. Let's get, repeated treatments, here, became, uh once every six months, or so. The second batch was made, and we are now 11 years out. And as you can tell, she's alive, and doing well. I don't have that many scans, but I have one scan that looks really clean. For those of you who understand what is what a scan, what GBM looks like, no, no evidence of progression to date.

Here's a 70 year old gentleman, his daughter is here, actually. This patient came to us at third recurrence, at 70 years old. Third recurrence, typically is is not a time, that’s, that’s, the time when the doctor tells you to get your your affairs in order. This patient had two course of DCVax. I believe the second course of DC facts was made after the fourth recurrence, with a new preparation of tumor tissue. And again, this patient is also alive, and doing well. And here's a series of scans that shows that the enhancing matter has reduced over time, and this base here, actually the DCVax was given as a monotherapy.

So let me just summarize this part of the talk, give a broader perspective, and then I'll wrap it up. And then, if there's questions, we'll be happy to address those individually, with people in the booth. And we prefer that, to doing it here, because it gives us much more meaningful interaction with people that are truly interested.

So these are anecdotal observations, but we think they're meaningful. The drug is well tolerated, and can be effective in older patients patients, patient that you'll probably not treat with many other drugs at that stage in life, including patients, with substantial co-morbidities. And we've got a few examples of those as well. When a patient experiences recurrence, we can make a new batch of DCVax-L, and those patients can respond again, to a whole new host of tumor antigens, that are in the recurrent tumor, which may be different from the original tumor, because tumors change over time. They have plasticity. But when they experience recurrence before all doses are used, we can also continue to treat them with the original DCVax. And that can still extend survival, and we think, again, that is because we're presenting hundreds of antigens to the tumor, or to the to the immune system, that the immune system can respond to.

So broader perspective: we get a broad spectrum immune response. I think that ,that, I think I've made that very clear by now. We think it's suitable for combinations, with a wide range of other treatments. I did not have time to go into the safety of DCVax-L, but it's extremely well tolerated . There were very few Serious Adverse Events, that were associated with the treatment. Among thousands of doses that we have administered, both inside, and outside the clinical trial. So good candidate for combination with: checkpoints Inhibitors, oncolytic viruses, cytokine therapy, chemotherapy, etc.

Like I said, when a patient has recurrence, new batches can be made. The treatment targets are not lost, the tumors still have treatment targets. That, that’s not the case when you give a small molecule against a a kinase, like a kinase inhibitor. If the, the tumor develops a Superfluous Pathways to overcome that blockage of kinase inhibitor, then you cannot use that drug again. But with DCVax-L, you can continue to use it.

We can apply it to any solid tumor. You saw that, even from a GBM lysate, we already got antigens that are present in other tumors. So if we use lysate from another tumor, we'll get new new antigens, again that are more probably more appropriate to that particular tumor, that will also, again, contain antigens that we would not have known existed beforehand. It's very easy to administer treatment; it's administered as an intradermal injection. We ship the treatment, every single dose, and it can be, and has been, administered in community settings. We don't need an academic hospital, even though they can do very well, as well, of course.

Let me just acknowledge the patients and their families, both patients and families that are here, and patients and their families that could not be here with us. The clinical trial investigators; we had we had 90 hospitals where this trial was conducted, and we're very proud of that amount of collaboration that we found in the field. At UCLA, of course, Dr. Linda, Liau Robert Prins, have stood really at the beginning of this particular type of therapy. Dr. Ashkan was the principal investigator in Europe. Mike Scott, Alessandra and the team at Advent, some of which are here as well. We've got people from Flaskworks here, including Lekhana and Andrew. Mark and Daphne are not here. We have people from Cognate Bioservices, that have worked on this a lot during the trial. And of course, the team at Northwest Biotherapeutics. And with that, I'd like to thank you for your time, and attention here today, and wrap it up. Thank you very much. [Applause]