Oncolytics Biotech Inc. (ONC) Earnings Call Transcript & Summary

Good afternoon, and welcome to the Oncolytics KOL Webinar. [Operator Instructions] As a reminder, this conference is being recorded, and a replay will be made available on the Oncolytics Biotech website following the event. I would now like to turn the call over to your host, Dr. Matt Coffey, President and Chief Executive Officer of Oncolytics Biotech. Please go ahead, doctor.

Thank you, operator. So we're really quite excited about what we're accomplishing as a company. And I think today is the first time, I think, we've been able to express exactly how an agent like ours is actually able to cause a survival benefit. And I think, with our colleagues that will be presenting today, you'll have an opportunity to actually see how significant this actually is. Now just a quick look to our forward-looking statements. What we plan on talking today is just an overview of what it is that we're trying to accomplish and how significant the results that we're presenting today actually are, not only in terms of what we're doing as we move into the Phase III environment for a registration study in metastatic breast cancer, but more globally. I think today's results really allow us to understand how significant this product is and how it allows us to not only have a positive impact with agents like immune checkpoint inhibitors, but more globally. And one of our colleagues, Dr. Vile, will talk about how these findings, how the product causes an inflammatory event within the tumor, can be used in other areas. And he'll be talking specifically to CAR T. But today, some other results came out that this also works with CDK4/6 inhibitors, with PARP inhibitors, with checkpoint inhibitors. Basically, what we're able to do is prime the immune system so that it can more readily engage cancer cells. Basically, what we're trying to do is label the product or label the tumor as an entity that should be removed and something that your immune system should be targeting. And cancer is very good at evading the immune system. And I think what we're doing is we're pulling that cloak of invisibility off and what we're allowing is the immune system to see the tumor for the first time and engage it in a way that causes a benefit in terms of not only progression-free survival, but ultimately, into an overall survival benefit for these patients. And then lastly, Tom Heineman will be joining us to talk really about how we can use the product, where we're going, what this actually means, the big sort of reveal, if you will. Now in terms of an overview, we're an oncolytic virus. So what it does is it will move through the bloodstream following an IV administration, and it will set up shop in permissive cancer cells. Normal tissue doesn't support the replication of the virus, so it's very specific. What we're doing is we're engaging the immune system through the specific replication of the virus. And these infected cells basically signal to the entirety of the immune system that there is a problem. The virus will start replicating through its own genetic cycle which is composed of double-stranded RNA. And this double-stranded RNA looks very foreign to the cell. It's one of the biggest immunological triggers because your cells would never normally see double-stranded RNA unless they were infected by a virus. Now because it is such a threat, there's a lot of pattern recognition receptors that actually recognize double-stranded RNA as a threat. Things like toll-like receptor 3, PKR, MDA5, RIG-I, all of these are surveillance systems for our cells to see this and signal to the immune system that there's a problem. Now the wonderful thing about this is it's the cancer cells that are now signaling. These cancer cells are now basically labeled or colored as being malicious in intent or horrible or something that the immune system should be targeting to eliminate. Because, for the first time, these cancer cells are viewed by the immune system as a threat and something that should be eliminated. So these infected cells are releasing things like interferon. They're releasing things like cytokines and chemokines that alert the immune system to come rushing in. And really, that's what we're doing. We're labeling the tumor as different. So there is viral lysis or there is viral killing. But for the most part, what I want you to consider is that this is actually labeling the tumor as foreign so that the immune system can eliminate it for the first time. Now this picture, I love. And it is from the AWARE study, and this is a person with HR+ disease. And we're using an antibody here that turns brown so you can actually see which cells are infected and which ones aren't. So this is really what's happening to the immune system. We're making something very visible. And you can see this here with the brown, basically, dye that what you're using. What we're getting is selective replication within the tumor cells, but not the normal [ cells ] surrounding normal tissue. So you can look at this picture and say, okay, the brown ones are the cancer cells, the blue ones are the normal tissue. And this is what we're doing for the immune system. We're making these cells very, very visible so that they know they have a target that they can eliminate. Now the important thing for this also is this is systemic delivery. And by that, I mean, we're giving the patients 250 ml sf saline with the virus in it and we're perfusing the entirety of the body. And the virus will only replicate in the tumor tissue, which makes it look this foreign. Now the other thing that's interesting about this is because we're not a modified organism, we're considered almost a vaccine. There's no special handling. This is something the patient can go and get in conjunction with other chemotherapeutic agents, other immunological agents. But at the end of treatment, they can go to home to their family. And this is very important because, at end of life, these types of treatments, we want the patients to be supported by their family, their loved ones, their community. And because it's considered basically just a vaccine, they're able to do so. Now what we demonstrated today, I think, is very significant. We have 3 publications at AACR over the weekend that demonstrated not only does it work with checkpoint inhibitors in humans, but it also has the potential to work with CDK4/6 inhibitors, which kill immunologically; PARP inhibitors, which kill immunologically. So basically, we can use this product alongside all the trending, all of the leading immuno-oncology products because we are priming the immune system. We're setting these patients up to succeed with other interventions. And as again, you can see here, it is very selective to the tumor. Now importantly, we're working with our investigators to determine endpoints that allow us to identify who's likely to respond and who's not. And this is very significant because, again, it shortens the cycle to get this into the Phase III registration program. Now what really led to this was a study conducted by the NCIC called IND 213, and this looked at women with late-stage metastatic breast cancer. And what we found here in these patients was a near doubling of their overall survival. So these women were able to be given the product, and the people who were fortunate to be on the test arm, they had an extra 10 months added to their life, if you will. So that's an extra Christmas or birthday. It's a meaningful amount of time that these people spent with their loved ones. The problem we had with the study and what AWARE really answers is we didn't appreciate how significant the immune system was in getting this to work. And the work that Dr. Prat did, I think, is the first time that we've been able to definitively demonstrate that it's the engagement of the immune system that's providing the survival benefits. So without further ado, I'll actually flip the page over to Dr. Prat, who can go through this, I think, very compelling result. [ Dr. Prat? ]

Thank you. Thank you very much, Matt. It's a pleasure to be here today presenting the results of cohort 1, cohort 2 of the AWARE-1 study. This is a window of opportunity trial run by SOLTI. SOLTI is an international competitive group, nonprofit that runs clinical trials, mostly in Spain and Portugal. And we are known around the world because of our studies that, in general, like these window studies, have a lot of translational work behind. So this is a huge effort from many investigators that have been participating, including patients into this study and collecting the samples. So regarding the design of this trial. As I said, this is a window of opportunity study that evaluates the safety and biological effect of pela plus/minus atezolizumab, an anti-PD-L1 antibody, on the tumor microenvironment. And the aim is to recruit 38 patients, 38 women with early stage breast cancer. Today, we're going to go over the 2 cohorts that have been recruited completely, cohort 1 and cohort 2. Those 2 cohorts have 10 patients each, both are including patients with hormone receptor positive, HER2- disease. Cohort 1 treated patients with pela, days 1, 2, 8 and 9, together with letrozole. Cohort 2, the same exact treatment, but atezolizumab was added on day 3. The treatment duration was 21 days, and we had a biopsy before therapy; a biopsy at day 3; and a biopsy at surgery, around 21 days of initiating therapy. The primary objective of this study was to look at an early read on an early biomarker of response, which is the CelTIL that I will discuss in the next slide. But basically, this is an easy biomarker that can be evaluated under the microscope in an H&E slide looking at tumor cellularity on one hand and tumor-infiltrating lymphocytes on the other hand. So the primary objective was to evaluate the changes in the CelTIL score at 3 weeks compared to baseline in each cohort. And we call success criteria in the protocol as an increase of 30% -- relative increase of 30% in CelTIL score in at least 50% of patients from each cohort. Regarding the CelTIL score, we have now substantial data published and reported. On one hand, our group reported in Annals of Oncology in 2017, that these early changes at day 15 of targeted therapy, in particular, tumor cellularity and tumor-infiltrating lymphocytes, when you combine both variables, you can identify patients that at day 15 of targeted therapy have low tumor cellularity, high immune infiltration. And these are the patients that have more likelihood of achieving a pathological complete response after standard neoadjuvant therapy in this study with chemo. This was our first publication. We wanted to link this early readout of biomarker with survival. And this is something that we have recently published very recently, a few weeks ago in [ NCBI ], where we validated these in tumor samples from a Phase III clinical trial in HER2+ disease, showing that CelTIL score, changes in CelTIL score at here, at week 6, can predict event-free survival, long-term event-free survival and long-term overall survival. The biomarker runs as a continuous fashion, meaning that the more score, the better outcome. So no specific cut points have been defined to date. Coming back to the AWARE-1 study, regarding the primary objective. Cohort 1 and cohort 2 achieved the study's primary endpoint. Again, 30% or more increase in CelTIL score from baseline in at least 50% of the patients. You can see here that the plot to fit individual patient. Cohort 2 achieved this in 60% of the patients, so above what we aimed for, which was 50%. Cohort 1, 40% of the patients showed a relative increase in the CelTIL score of 30% or more. So there is a relative increase as a continuous variable, just that the limit of 50% was not met in cohort 1. Most strikingly in my opinion, and Matt already stated that, is when we look at the tumor biopsies before at day 3 and after 21 days, and in particular, when we looked at pela replication using the immunohistochemistry-based assay. To me, it was very striking when we saw that, it was one of those moments when we say, wow. What we observed is that in 50% of -- around 52% of tumor cells at day 3 had pela replication and 42% at surgery. And of course, not -- this was not observed in the baseline untreated samples. And this was found in both cohorts, cohort 1 with pela only, and cohort 2 with pela and atezolizumab. So very striking results in my opinion. At the same time, we've been working on doing more translational work that we are presenting at AACR. On one hand, we looked at the intertumoral CD8+ T cells, where we observed that, in all patients, this amount of intertumoral CD8 T cells is increased by an average of 11-fold increase with a range of 1.1 up to 24.3. Also, we looked at the ratio of CD8+ T cells to FOXP3+ regulatory T cells. And also this ratio was higher, especially in cohort 2 compared to cohort 1. And finally, we also looked at the numbers of memory T cells, CD45RO and CD8+ T cells was also numerically higher in cohort 2 compared to cohort 1. So again, more immuno-filtration, more CD8 T cells, less regulatory T cells in this day 21 tumor samples. At the same time, it was important to evaluate PD-L1 expression, protein expression by immunohistochemistry using 2 different IHC assays. One is the VENTANA SP142 assay, which is the companion diagnostic of atezolizumab in advanced triple-negative disease and the 28-8 assay. And what we observed in both cohorts is that, at baseline, these tumors are hormone receptor-positive, HER2-negative. So PD-L1 positivity was not observed in any of the screening or based on samples. In general, there was an increase in PD-L1 positive tumor cells. And in 20% up to 40%, especially in cohort 2, 40% of tumor samples at surgery met the criteria to be called PD-L1 positive tumors, meaning 1% or more of PD-L1 positive tumors, immune cells in the microenvironment. So definitely more evidence that more immuno-filtration, but also more immune cells that are PD-L1 positive at surgery. And finally, another piece of evidence, to me, very striking, and when we saw it, is the T cell repertoire turnover. Using here the adaptive assay, what we observed overall, and can go in detail into the data, but we observed more T cell repertoire turnover, as indicated by this index called Morisita in patients in cohort 2, especially, compared to patients in cohort 1, indicating more overall change in the T cell population during therapy in both. Because we evaluated this in blood as well as in tumor, comparing baseline samples compared to 21-day samples. In cohort 2 also, patients also have more expanded T cell clones and more newly created clones relative to cohort 1, also in blood and in tumor tissue. And finally, regardless of cohort, what we observed is that treatment with pela transforms the T cell population in both blood and tumor tissue, where more than 75% of the T cells are considered new. And it can be shown here in the fraction new plot. So again, have pela together, or not, with atezolizumab can really make a change regarding the T cell repertoire. I think this is very striking data in patients, suggesting that we are getting where we are supposed to get, not only more immuno-filtration in the tumor, but a different type of T cell repertoire. So my conclusions about these results is, on one hand, cohort 2 met the study success criteria of 30% or more increase in CelTIL score in at least 50% of the patients, although cohort 1 almost made this cut point. I would not be as worried about the meeting or not the primary endpoint, CelTIL should be evaluated as a continuous variable, as we have shown in the past. In addition, pela and atezolizumab act synergistically to establish a more favorable immunologic response in both the tumor and also in the blood by looking at T cell repertoire, as demonstrated. And here is a summary of regulation of PD-L1 in tumor tissue, increased CD8+ and memory T cells in tumor tissue, a favorable CD8 Treg ratio, indicating a less immunosuppressive tumor microenvironment, and the dramatic changes in the T cell population that we observed in the TCR analysis. Therefore, in my opinion, the combination of pela and atezolizumab warrants for further investigation as a potential breast cancer treatment approach with the current ongoing BRACELET-1 study design to generate the data to support the results of the results that Matt already shared with you, the IND 213 study, and also evaluate the value of pela to act synergistically with Taxol together in this scale with an anti-PD-1 -- anti-PD-L1 therapy, which is avelumab. With that, and here, I think we're going to have questions afterwards. So I'll pass the word to my colleague, Professor Vile, Director of Mayo's Immuno-Oncology and Gene and Virotherapy programs and also Co-Director of the Cancer Immunology and Immunotherapy Program. Richard?

Thank you very much, indeed. It's a pleasure to be here to talk about some of the data that we've been accruing with pela. As you just heard, I'm at the Mayo Clinic in Rochester, Minnesota, Director of the Immuno-Oncology Program there. And I'm going to talk today about essentially the -- a poster that we presented at the recent meeting, the CAR-TCR Summit Europe 2021 back in February, where we've been investigating the ability of pela to interact with CAR T cell therapies. And as Matt said at the top of the meeting, not just CAR T cells, but sort of other immunotherapies in general. So the issue why we were so excited with working with oncolytics and reovirus in particular with CAR T cells is that it's well-known that CAR T have excellent efficacy in liquid tumors, hematological tumors, but they have had real problems in treating solid tumors. And that's for a variety of reasons, some of which are clearly defined and some of which are slightly unclear at this stage. But it is clear that the CAR T cells become very exhausted very early on. They are -- they tend to be short-lived. And the best clinical responses are associated with high levels of longevity and persistence of CAR T. But CAR Ts, at least in some of the clinical studies and also in the preclinical studies, show that when they encounter solid tumors, they tend to become exhausted and die very quickly. In addition, CAR Ts have to deal with a highly immunosuppressive tumor microenvironment. And although they may be able to traffic tumors once they get there, there's clearly a highly attritional environment associated with immunosuppressive cytokines, immunosuppressive cells and so on. So using an oncolytic virus or like a reovirus was very attractive to us and other people because, again, as Matt said, clearly, by infecting tumor cells with an inflammatory viral infection, that's exactly what the immune system needs in order to see that a tumor suddenly is not a self and harmless entity but needs to be rejected. And so we hypothesized that by using pela systemically or even intratumorally to try and stimulate an inflammation within the immunosuppressive tumor environment, we would turn these cold tumors, those are tumors without inflammation, without an immune infiltrate, and we would turn them hot because the virus would replicate. The immune system would see the double-stranded RNA. Those cells would become labeled as foreign and as the site of infection. And once that occurs, our hypothesis was that, that inflammation would then recruit a whole variety of immune -- of pro-immune T cells and other cell types to the tumor. And obviously, if we had adoptively transferred CAR T cells into the animal in the preclinical scenario or into the patient, then that inflammatory environment induced by the viral replication and the viral presence would recruit those CAR T cells or other activated T cells to the tumor site. And as we've just heard, that's really now being supported by the clinical data from the AWARE-1 trial. The 2 sort of wow moments that we heard about was the systemic administration of pela would allow viral replication in tumor cells following systemic administration, and that the T cell populations were clearly being changed and altered in terms of a pro-inflammatory type, tumor rejection-type response in those patients. So we have studied this in preclinical models now. We've combined oncolytic viruses with CAR T cells. And in a recent publication last year, we showed that, indeed, pela can induce intratumoral inflammation. But there's some sort of qualifications for that, and that we want to make sure that we can induce the right sort of inflammation at the right time relative to the CAR T cell at administration. Or -- I'm sorry. So perhaps our sort of wow movement in the preclinical studies is shown here. Here, we took C57 black mice and we gave them a subcutaneous tumor. In this case, it's a melanoma engineered to express a target for CAR T cells, EGFRvIII. And we grew these tumors subcutaneously for 8 days. So they're truly established now and they have this immune-suppressive microenvironment. We then treated these mice either with CAR T by themselves, with virus by itself or we mixed the virus with the CAR Ts in an attempt to try and get the CAR T to carry the virus to the tumor. And then in this experiment, we also boosted at day 15 with either nothing, virus or a different virus that the CAR T cells were loaded with. And I'll just take you through these results here. This is a fully immune-competent mouse with a tumor. There's no total body radiation or preconditioning to allow the CAR Ts to populate the mouse here. And in this model, if you just give the mice PBS, they will be -- their tumors will be too large by about day 30. And in this model, where systemic administration of the virus, we deliberately made a low dose of virus so that the virus itself has relatively little effect, although there is a slight effect over the PBS. So these are the controls. When we give the CAR T cells, these are just CAR T directed against the EGFRvIII target, we do see a small but significant increase in median survival. And in this experiment, one of the mice survived long term. When we loaded the CAR T cells with pela, if you just focus for a moment on the purple and the brown lines, we saw significant increases in both median survival and overall survival now, with about half or over half of these mice were actually cured, indicating that there's something about the loading of the virus under these CAR T cells that actually makes these CAR T significantly more active. And we've got some -- we're working in the lab, and we've got some data to show that we think that the virus is activating these CAR T cells to become better killers and possibly more persistent. But what was really exciting to us was that, having administered these CAR Ts, either loaded or not -- or sorry, having administered these CAR T cells loaded with pela, if at day 15 now we gave no boost, or we boosted with a heterologous virus, in this case, VSV. Then in this case, the boost had no effect if the boost was with a virus different from pela. But in this group here, when we actually loaded the CAR T with the pela and we boosted with a pela, you can see that up to day 50 and now this has gone out to day 80, 100% of these mice were cured of their tumors. So we believe that there is a very important interaction of the virus with the CAR T cells, both at the stage of administration of the CAR T and then boosting with a pela makes a significant impact on the activity of those CAR T. And we did a second experiment where we reduced the number of CAR T that we actually gave. So that instead of curing 100% of these mice, in a few mice, we actually -- we give the CAR T loaded with the pela. And as you saw before, those tumors appear to go away. But then in a few mice, in this case, 3 mice, these tumors start to recur. And as we start to measure them, they start to grow again. If now we gave a second reboost with, in this case, a heterologous virus, VSV, these recurrent tumors just continue to grow, or this recurrent tumor continued to grow. If we gave no second boost, again, the tumor continues to grow. But if now, we give a second boost with pela, this tumor which had been treated with CAR T loaded with pela in the first place, now this tumor regressed completely. Again suggesting that we're setting up some sort of population of CAR T which have already seen the reovirus and which can be boosted with them. And we are now investigating exactly what the mechanism of this effect is. So we're very excited. The use of a virus like pela, which labels the tumor cells as the site of an infection, labels them as foreign and labels them as bad and, as Matt was saying, they need to be rejected. We believe that, that can really be combined with CAR T cell therapy, using T cells which are able to see sites of infection come in, do their own activity. And we believe that this loading of the CAR T with the virus itself probably helped by delivering the virus to the tumor, shielding it from neutralizing agents in the blood. But also clearly affects the differentiation of the CAR T cells such that we can give systemic boosts to treat tumors as we go on. And so we will continue to do this in these preclinical studies, and we would be very keen to try and translate this in the near future into patients. So that's me. I will hand it over to Dr. Heineman.

[indiscernible]

Dr. Heineman, you're on mute. Can you please unmute yourself? We still can't hear you.

We will go to a brief holding slide just for a few minutes while we correct the technical issue. [Technical Difficulty]

Okay. Good afternoon, everyone, or good morning. Very sorry for the technical issues. This is a -- I blame it on the -- as a byproduct of the COVID era, but I'm hoping everyone can hear me okay now. In any case, I'm very happy to have the opportunity to conclude our program today, at least the formal part of our program, by providing you with some perspectives on the data that you've just seen as well as on pelareorep's outlook in both the shorter and longer terms. Okay. I'd like to begin, if I may, by again showing some of the key data that Matt showed at the beginning from the IND 213 study in metastatic breast cancer. I'm showing this again because this figure depicts probably the most important data from the IND 213 study in terms of defining the future development of pelareorep. First and most importantly, these data illustrate pelareorep's potential in breast cancer as they show an almost doubling of survival compared to chemotherapy alone. And secondly, these data strongly suggest the survival benefit conferred by pelareorep is due to an immunologic mechanism of action, as evidenced by the observation that the survival curves begin to separate several months after beginning therapy, which is typical of immunologic killing as opposed to the more immediate effect that you might expect with cytotoxic killing. In any case, as persuasive as these data were, additional work was needed to confirm and define the pelareorep-induced immune responses that may have been responsible for the clinical benefit seen in the IND 213 study. So to that end, and to fill that gap, the AWARE-1 study was conducted, as was described a couple of minutes ago by Professor Prat. And this study tested the hypothesis that pelareorep induces anti-tumor immune responses that could explain the survival benefits seen in the IND 213 study. As you heard in Professor Prat's presentation, the AWARE-1 study clearly demonstrated that IV pelareorep not only infects breast tumors at a high level but also stimulates multiple potentially protective immune responses, including increased T cell infiltration into tumors, which is what you're seeing in the picture on the right-hand side of the slide here. It also increased CelTIL scores, and it activated both the innate and adaptive immune responses. So in essence, these data from the AWARE-1 study confirm that pelareorep can, in fact, induce immune responses with the potential to provide the survival benefit seen in the IND 213 study. In actuality, as Dr. Prat described in some detail, the AWARE-1 data actually go one step further. They demonstrate that by adding a checkpoint inhibitor to pelareorep, it's possible to improve upon the already substantial immune responses induced by pelareorep alone. As you saw from Professor Prat's data, these enhanced immune responses include an even greater impact from the CelTIL score when adding a checkpoint inhibitor to pelareorep; an even more robust generation of new and expanded T cell clones; increased reduction in the immunosuppressive nature of the tumor microenvironment; and up-regulation of PD-L1 expression, which is actually seen with both pela and with pela in combination with the checkpoint inhibitor. So the takeaway from this study is that the AWARE data demonstrates that, first, that pelareorep induces potentially protective immune responses on its own, and it's equally clear from these data that these responses are enhanced by the addition of a checkpoint inhibitor. So together with the 213 data, the AWARE data strongly support the rationale for going forward with the ongoing BRACELET study, which the design of which is shown here on this slide. Okay. So the BRACELET study, which is currently enrolling, is designed to evaluate the impact of adding a checkpoint inhibitor, in this case, avelumab, to pelareorep and chemotherapy in HR+/HER2- breast cancer patients who have advanced or metastatic disease. Okay. And the way we view the BRACELET study is as the last piece of the puzzle needed to set the stage for a licensure-enabling Phase III study. So in actuality, this study is very similar to the IND 213 study that Matt mentioned earlier. However, it addresses a couple of key points that will inform the design of the pivotal Phase III study. First, it evaluates the potential contribution of adding a checkpoint inhibitor to pelareorep and the primary endpoint of its effect on tumor responses. And it is doing this in the HR+/HER2- breast cancer population, which is a target population that we will be evaluating Phase III. So needless to say, it's very important to evaluate the contribution of a checkpoint inhibitor in patients because of the compelling AWARE-1 data that we just saw in which a checkpoint inhibitor clearly enhance the already strong immune responses induced by pelareorep alone. And secondly, this study, and this is very important too, will provide valuable biomarker information and other translational data that will be applied in designing an appropriate Phase III study of pelareorep with or without a checkpoint inhibitor. So once we have these data in hand from this study and in combination with the AWARE data and the IND 213 data, we will be in an excellent position to design the best possible Phase III study. Okay. So I hope it's clear that the path forward in breast cancer that I just described is, in fact, very clear and remains our near-term priority. However, it's also very important to take note of the data that Professor Vile presented. Because the data that he presented, along with the work from other investigators, clearly indicate that pelareorep has the potential to act synergistically with entirely different classes in immunotherapeutics beyond just the checkpoint inhibitors. In particular, Professor Vile's data showed that pelareorep has the potential to enhance the efficacy of CAR-T cell therapy. In fact, as he described, very clearly, I thought, pelareorep not only enhanced the efficacy of CAR-T cell therapy in solid tumor models, but actually led to a high rate of cures in these models. So pelareorep synergy with CAR-T cell therapy appeared to be, in addition to inducing cures in the animals in these models, appear to be specific and was not achieved by combining oncolytic viruses other than the reoviruses with CAR-T cell therapy. So these data clearly emphasize the potential of pelareorep to expand the applicability of CAR-T cells into solid tumors and to enhance the efficacy of CAR-T cells in general. And as I'm sure you're all aware, and as Dr. Vile noted, the applicability of CAR-T cell therapy in solid tumors has been extremely problematic so far. So this provides a potential path forward for addressing this difficult issue. Consequently, Oncolytics is pursuing a partnership strategy to further develop pelareorep as an enabling technology for CAR-T cell therapy. Okay. And as Matt alluded to, even beyond checkpoint inhibitors and even beyond CAR-T cell therapy, data presented at this meeting, that is the ACR 2021 meeting, and elsewhere indicate that pelareorep has the potential to partner with other therapeutic approaches to enhance immune-based anticancer effects. These include bispecific antibodies, which, when combined with pelareorep, show increased infiltration of T cells into tumor and enhanced tumor regression and prolonged survival and solid tumor models; a PARP inhibitor, which showed immune-based synergistic interactions with pelareorep to increase cancer cell apoptosis. And this is presented, I think, as Matt noted in the poster at this AACR. And then another poster at this AACR is CDK4/6 inhibitor showed that when it was combined with pelareorep, it had enhanced immune-mediated increases in cancer cell death. So in summary, these data, and in particular, the CAR-T data that Dr. Vile presented, indicate that pelareorep has potential to serve as a partner therapeutic across multiple different classes of cancer treatments. So finally, while our focus remains on breast cancer, at least in the nearer term, we cannot ignore the fact that pelareorep has the potential to be an infective immunotherapeutic in other cancers as well. Consequently, we have ongoing programs to explore potentially several other cancers, including GI cancers, through the GOBLET study as well as in myeloma. So thank you very much for your participation and for your attention, and I'll hand it back now to our operator.

[Operator Instructions] I'll turn it over to Tim McCarthy of Lifesci Advisors to read those questions.

Okay. Thank you. The first question we have is from John Newman of Canaccord Genuity. Can you discuss why adding a checkpoint inhibitor to pelareorep boosted CelTIL above pelareorep alone?

Thank you. Thank you for the question. I mean we do have prior data suggesting clearly that anti-PD-L1 not only activates the current TILs that might be in the tumor, but also might be able to increase this amount. I think what we're seeing here is a synergy of both therapies. It is true that in the AWARE study, we don't have an anti-PD-L1 alone therapy as a control, but I think the data clearly tells us that there is a synergy between the 2. We should not expect by just administering atezolizumab monotherapy to have these increases in CelTIL, even endocrine therapy. We have performed prior studies, window of opportunity studies, in the same population with letrozole only, and we don't see increases in CelTIL. We have regarded this previously. So although we don't have a control, that's a fair point. I do see -- I do think that what we're seeing is a synergy. The exact underlying mechanisms of the synergy is still under investigation, of course, but I think the data, going in that direction.

Okay. The second question is from Patrick Trucchio from H.C. Wainwright. Regarding the result in Cohort 2, I'm wondering by what proportion would patients treated with checkpoint inhibitor treatment alone be expected to meet the primary endpoint. How do we know the result in Cohort 2 represent synergy between pela and CPI treatment based on CelTIL score?

This is a very good point, very similar to the previous question. It is fair to say that, number one, we don't have that control, so it's a fair point. But I do think that we don't -- we would not expect with an anti-PD-L1 to see, first, certain amount of CelTIL. I think that's something that I would not expect in my experience. And at the same time, what we're seeing in plasma and in tumors regarding the T cell repertoire and their changes also, I think, point out to this potential synergy. So again, I don't think we have that control to really tell us. We have not run at all a prior study with a window opportunity trial with just anti-PD-1 or anti-PD-L1 drug. We've done it with letrozole, as I said. But I do think that anti-PD-1, together with endocrine therapy, has not shown activity in hormone receptor positive HER2 negative disease in the neoadjuvant setting, on the advanced setting. So I think this tells overall that what we're seeing is a synergy.

Thank you. Next question comes from Wangzhi Li from Ladenburg. For AWARE-1 results, overall, high percentages of tumors are positive for reoviral protein. So some patients showed 50% plus positive cells, whereas some patients showed less than 20%. Any insight into what contributed to that variability?

Personally, I do not have an insight of the amount. Definitely, Oncolytics has a lot of experience, not only an expected amount of infection, so I would refer it to Matt or Tom to comment. But if -- to me just to -- it's not just probably the amount of infection, but what biology this induces in the macro environment. But Matt, maybe you can comment more.

No. And I think what's striking about this result, and certainly, the pathologist who does the work for us, we've not seen this level of infection in any other tumor type before. If you look at multiple myeloma, if you look at GBM, if you look at GI malignancy, we're typically seeing 15%, 20% of the cells actively infected. Whereas breast cancer, we're seeing some samples come back at more than 90%. I think breast cancer might just be a tissue that is acutely susceptible to the infection. And I want to point out when we are seeing this active replication of the virus, those cells are basically undergoing an apoptotic pathway. At that point, they're no longer viable. So to say 90% of the cells were infected is really to say 90% of the tumor is no longer viable as tissue that it's going to what you said and will no longer be the viable tissue, if you will. I think what we do know is much of this tissue, whereas we're saying 50%, 60%, 70% are infected with protein, almost all of them are replicating the genome. And it's a double-stranded RNA, which leads to the immunogenic signal, and I think that's what people have to differentiate. The double-stranded RNA, which we're seeing in almost 100% of the tumor tissue, basically is signaling through these pattern recognition receptors like toll-like receptors 3, like MDA5, like RIG-I, and it's this double-stranded RNA that's recruiting. Now in terms of why some tissue is susceptible, some isn't, when we look at standard cell line panels, we do see some tissue that isn't susceptible that does not replicate the virus. I think Richard can speak to this. B16 is a very difficult model for us because we don't get a lot of viral protein synthesis, but we do get a lot of accumulation of inflammatory cells, and I think that's just the natural variability. But I think the fact that more than 70% of these tumors are seeing increases in CelTIL score and because this is a continuous variable, any increase in inflammation is a positive for these patients. Richard, you've probably worked at the virus more than anyone hands on. Is there anything you'd like to add?

Yes. I mean, I think what you say is absolutely true. Our experience is that there's a range of cancers -- there's clearly a range of cancer cell types. And susceptibility to infection can be modulated by a whole variety of things, levels of receptors and so on. But one of the biggest indicators for us is whether those cells retain their sensitivity to type I interferons or whether they've lost them. So in those cells which have big defects in type I interferon alfa or interferon b2 responses, the virus tends to be able to replicate well. But in those cells like, as you mentioned, B16, where there's still an intact in type I interferon response, the virus tends not to replicate well. But that said, even in those cells like B16, where you don't see lots of replication, the reason you don't see lots of replication is because they induce interferon at high levels because the tumor cells retain their sensitivity to interferon. And so the end result is the same in those cells which are -- which have lost interferon sensitivity and which show high levels of viral replication. That replication then stimulates all of those triggers that you were talking about earlier, including double-stranded RNA. So you get the alarm signal. And in those tumors where the virus is shut down by the interferon, it's shut down by the interferon, but the interferon itself is a very positive pro-inflammatory signal. So in a way, it's a win-win situation. We want replication but -- obviously, we want replication. But if we don't see replication, the reason for that is probably a stringent interferon response, which of itself is good news.

Thank you.

Okay. The next question is a follow-up question from Patrick Trucchio from H.C. Wainwright. Was this the first study that's kind to demonstrate potential for an oncolytic virus in combination with CAR-T in solid tumors. What intrinsic properties could make pela more attractive as compared to other oncolytic viruses? What would explain why pela boost appears superior to the VSV boost?

So every oncolytic virus has its own identity, its own -- I hesitate to say positives and negatives because positives and negatives change depending on the context and so on. We think pela is particularly attractive to the CAR-T cell combination in a sense because it is a double-stranded RNA virus, of which, again, sets off these alarm signals in tumor cells and which can then set up a microenvironment within the tumor, which is very conducive to the attraction -- recruitment of activated T cells, and the CAR-T are highly activated. The way that we make them in vitro before we put them into the mouse means that they're highly activated. There are lots of issues in terms of timing and when the CAR-Ts are given to the virus. We do think that pela is particularly attractive also for the CAR-T because we can load the CAR-T with the virus. It doesn't seem to touch the T cells. It doesn't replicate well in those cells, doesn't kill them. So it allows those cells plenty of time to get to the tumor. And again, we see high levels of what we call hand-off on the loaded T cells into the tumor. So the first major advantage is the virus combined to the T cells and they can hitchhike onto the T cells, get into the tumor and release the virus there. So we can set up foci or viral infection within the tumor through the CAR-T carriage. And then the second point is why is the boosting with pela but not VSV, we need to do more work on exactly what is going on. But we think that the virus that is loaded onto the CAR-T leads to recognition of that same virus in vivo by those CAR-T. And those CAR-T then become virus-specific, if you like. And then when we boost with the virus that was loaded, we get a reactivation of the CAR-T. If we boost with a different virus, i.e., in this case, VSV, those CAR-T don't see that boost and are not reactivated in vivo.

Okay. Thank you. We have another question here. Will pela infect T cells? Can you define more about load CAR-T with pela?

So it's just one of these questions, if you have enough virus on enough -- on few enough T cells, you will get infection and so on. But at the MOI that we use, which is sort of between 0.1 and 1, maybe going up to 10, we get very little infection of the T cells with the pela. Again, we're not sure whether that's a lack of a receptor for the virus or exactly what that is. But it's certainly the level of infection is very low and the level of productive infection, i.e., if you look at viral production from those loaded CAR-T cells, over time, that is extremely low. So it's different from other systems, which have been reported, which actually infect the T cells and sort of use the T cell as a sort of a Trojan horse to get into the tumor. Here, we think that the effects we're seeing are very much sort of surface adhesion followed by release in the tumor.

Thank you. We have another follow-up from Wangzhi Li from Ladenburg. For CAR-T combo, would you know if reovirus infected and replicate inside the CAR-T cells or just stay on the surface outside of the CAR-T cells for the synergy?

So that's more or less what I just said for the previous question. I think our experience is that it essentially adheres to the -- to the T cells to carry the virus in vivo with a sort of protective effect as well. I think it shields the virus from a lot of the factors in the blood, which are potentially damaging to the virus. We don't see high levels of viral replication at least at low MOIs of the experiments that we've done.

Okay. We have an investor question. Is the fact that Cohort 1 did not meet the success criteria imply that without a checkpoint inhibitor, you would not be able to replicate the results of the Phase II breast study?

Thank you for the question. I think overall, the effect in Cohort 1 and Cohort 2 are very similar. CelTIL increases in a substantial proportion of patients. It's true that we predefine a particular cut point, not to say if you have success or not. But if you look at the CelTIL as a continuous variable, which should be looked at as a continuous variable, our prior work has shown that the association of CelTIL with patients' outcome, either PCR, pathological complete response and more recently survival, is not about a particular cut point, it's about looking at the variable as a continuous variable. So I think I would not call Cohort 1. I mean, formally, because the protocol was written as is, we need to call it not a successful cohort. But if you look at the data, data clearly shows a clear tendency. And actually, 40% of the patients in that cohort showed an increase in CelTIL. It just did not meet the 50%. So I'm less concerned -- I'm not confident at all about the results in Cohort 1. I think they resemble very well the Cohort 2. Definitely, they meet 60% of the patients achieved the CelTIL score increase of 30% or more.

And to Professor Prat's point, I think as a company now, we're feeling very confident that the 213 randomized study was a success because we did have this inflammation. I think in terms of being able to understand the shape of the curve, the delayed clinical benefit, I think now in the context of our AWARE-1 can be very easily explained. The majority of these patients are seeing an increase in CelTIL. And the fact that it's a continuous variable, really what that's saying is any increase in CelTIL is positive for the patients. So we set preordained endpoints. We did not meet it on the first cohort despite the fact most of the patients did have an increase in CelTIL, whereas we clearly met it with the second cohort. And it does seem that the checkpoint enhances or deepens the CelTIL response, but the fact Cohort 1 did have a demonstrable benefit in terms of CelTIL. And I think now in the terms of the 213 data, I think we can all very clearly say it was an immunological response that resulted in the overall survival benefit. And I think that was really the power of the AWARE-1 study. But more importantly, I think AWARE-1 demonstrates how, really quite awesome, the viruses at promoting inflammatory events. The design that Dr. Prat created for this study allows us to, in almost real time, measure the inflammatory events in this tissue, and we do know that breast cancer is the only area we've seen single agent responses following IV administration. I think the AWARE-1 very clearly demonstrates why we have seen the survival benefit.

Okay. So that's all the questions we have. I'll turn it back to Matt for any closing.

So I wanted to thank everyone for participating. Professors Prat and Vile and Tom, I think it was nice to be able to contextualize what these results mean not only in terms of the Phase III going forward is that we do have, I think, a very clear picture that checkpoint inhibition does benefit the inflammatory response that we're seeing. I think here, what we're clearly seeing is a benefit that supports what we're doing or why we saw that success of 213. And I think it paints a very good picture of not only how we can benefit women with HR-positive disease. But I think more generally, that we can use this across immunological therapies. Basically, any therapy that requires a T cell response, I think, will benefit from a priming with the virus because we do get this tremendous turnover in the T cell repertoire. We do get an education in immune system. And I think now based on the AWARE-1 results and how people like Professor Vile have been able to exploit those results and move us in new areas, I think we can rationalize and expand the work that we're doing and very clearly and very confidently move into that Phase III environment, knowing what the right backbones are. And with that, Tim, I think I'll sum it up.

Well, great. So thank you. That concludes the program. Thank you, and have a great day.

Thanks, everybody.