It's my pleasure to introduce Dr. Chioka from the Brigham, who's going to be talking about GBM clinical trials and viral-based gene therapies. Thank you very much. Thank you. Well, great to be here. And I'm so happy that there's actually an audience on Wednesday afternoon. So I'm going to tell you something a little bit different. This has probably the most controversial title, resurrection of an old paradigm. These are my disclosures. So everybody before and after me has discussed with you this approach, which is to use a replication-competent virus that's been engineered to replicate in cells and biodistribute an infection throughout tumor cells. I'm going to go back to an older technology. The older technology is to use viruses that have been disabled completely of their genetic elements, and we're just using the viruses as a vector to deliver an anticancer gene. And the prototype anticancer gene was thymidine kinase. This was shown by Ed Oldfield, Bob Martuzzi, and others back in the late 1980s, early 90s, that this could be inserted into cancer cells and make cancer cells sensitive to drugs against cyclovir, acyclovir, or valacyclovir. The TK would make these into phosphorylated nucleotides that would get intercalated into areas of DNA damage. But there's no viral replication. And there's a history with this. And so back in the mid-1990s, we did a clinical trial. This was a phase zero clinical trial testing this approach. The key is we were testing this approach using retroviral vectors, replication-defective retroviral vectors. And in fact, what we found was this very poor distribution and poor transduction in the tumor with these retroviral vectors. And there was also a big phase three trial that was carried out in multiple places showing that this was a failure. They did not provoke a good response. It did not show any evidence of efficacy. And that is where the collective consciousness of everybody that does not work in the field stopped and said, gene therapy doesn't work. Forget about it. Let's do something else. But some have gone on and tried to figure out how to make this better. And I think what I'm going to try to tell you is that while the concept was not wrong, the execution or the details on how to make this more effective is where we needed to work on. And that's what we've been doing for the last two decades. When I say we, I say the collective we, not just me. And so one approach has been what Bob Martuzzi and others have discussed, using viruses that can actually replicate and distribute the infection throughout the tumor. But there was others. So for example, there was another clinical trial that was done in Finland showing that retroviral vectors were more effective in infecting tumor cells and distributing a transgene. And then there was others showing, like Fred Lang, that you could deliver P53 into these tumors or a cytokine, a potent cytokine to these tumors and actually finally get into what's called a maximum tolerate dose. Up to that point, nobody had ever shown a maximum tolerate dose. And so that was available. And then over the last several years, there have been additional trials of gene therapy using adenoviral vectors primarily. And the most recent one has been TOCA 511, which is this curious combination of gene therapy and viral therapy. This virus does not kill tumor cells per se, but just distributes the infection throughout the tumor. So I'm going to tell you today about two trials that I've been involved with. One has been for newly diagnosed GBM. There have been very few trials using these gene therapies or viral therapies for newly diagnosed GBM. And then a trial for recurrent GBM. So in this trial, we actually resected the tumor. So we're not doing stereotactic injections. We're bringing the patient to surgery with a presumed glioblastoma. We resect the tumor. And then we freehand inject into the tumor cavity this adenoviral vector that delivers thymidine kinase. And then we give the patient for two weeks the prodrug valacyclovir. And then we combine this also with radiation and temozolomide. And so what happens is that thymidine kinase will phosphorylate valacyclovir. This acts as a false nucleotide. And as soon as you give radiation and open up and cause changes in the DNA, the DNA tries to repair this by using these false nucleotides and lets the cell commit apoptosis or death. And this is also an immunogenic cell death that stimulates the immune response. And this particular process is kind of shown here in this cartoon. And so I'm going to give you the punchline for this trial. This is a Phase II multicenter study that was run at Ohio State. It was run at City of Hope. It was run at Methodist Hospital and the University of Chicago. And as a fifth site, we had the Brigham. The Brigham actually had a prospective database that was collecting patients at the same time. And this prospective database was going to be our comparison cohort. So this is not a randomized trial. But everything I'm going to show you is by comparing the data from our trial with this prospective cohort. And the punchline is here. So in solid red, you see the patients that had the resection of their tumor with standard of care that had an injection of this gene therapy. It's called gene-mediated cytotoxic immunotherapy into the tumor cavity. We had 18 patients in this group. The median survival time was 25.1 months. And they had a growth or resection of tumor. And this is this curve right here. If they had just standard of care alone, which is growth of this resection plus standard of care, their survival was 16.3 months. And this seems to be statistically significant. These curves are fairly widespread apart. However, if they had a so-called subtotal resection, in other words, you left a residual tumor there, there seemed to be no effect of this gene therapy compared to standard of care. These are the dashed curves. I think this is an internal control for our database cohort, which somewhat validates it. But clearly, this is still potentially a source of a lot of bias because you're still comparing your patient to the database. But it's the best we could do at this time. And I think that there's some evidence of increased potential, increased survival. And just to remember, the Rindo-Peppermint Celldex report that failed, those patients lived about 20 months, 21 months. In this case, we were at 25.1 months. So I think this is pretty interesting. If we then look at survivor-defined time points, so this is a group that got gross resection plus this gene therapy. At 12 months, we had almost 90% survivors versus 63.6. At two years, we had over half of our patients were surviving with 27.3 in the standard of care resection group. At three years, over a third of the patients were still surviving with less than 5% in the standard of care resection group. Okay. So this is interesting. But we're still having patients that are failing therapy. Why is that so? So we went back to the lab and modeled this. And we published this in Neuro-Oncology. And basically, what we found is like everybody else, what everybody else is finding, is that what you see is when you apply this therapy, you get an upper regulation of PD-L1 in tumor as well as PD-1 in macrophages and microglia within the tumor itself. And so we did a number of experiments to show that if you do this in combination with anti-PD-1 or anti-PD-L1, that you get a better treatment. So based on this, we're now starting a second clinical trial. This is going to be done at multiple institutions throughout the United States. All the institutions belong to the American Brain Tumor Consortium. It's sponsored by NCIC-TEP. And Advantage Gene provides the gene therapy. And Bristol-Myers Square provides the NeuroMED. So these, again, are patients with newly diagnosed glioblastomas. They'll have standard-of-care surgery restructuring, injection of adenoviral TK into the tumor cavity, followed by radiation, temozolomide, and a volumemab. And then there's a drop-off potential of temozolomide if the patients have an MGMT non-methylated result. And this trial is about to open at our site and is about to open at a couple of other sites, I think including Penn and Henry Ford and others. So now I'm going to tell you about a second trial that's been involved in. So similar to the previous one, this is going to be for recurrent GBM. And this is to deliver interleukin-12 using, again, an adenoviral vector. Now, why interleukin-12? Interleukin-12 is thought to be a very potent master regulator. It upregulates a number of different immune cells in tumor as well as in systemically. One of the problems with interleukin-12 is when it was given as a protein into humans, back in the mid-'90s, it provoked a fairly severe cytokine release syndrome and made the immune system go AWIRE, secreting too many cytokines. And therefore, what we wanted to do is to control the expression of interleukin-12. So the expression of interleukin-12 in this adenoviral vector is controlled by this promoter called a real switch promoter. The real switch promoter is actually off. So when you inject this vector, the interleukin-12 is not made. It's off. And actually, you have to take a drug called Velodimox, VDX, by mouth. And this drug supposedly should cross the membrane barrier, turns on this promoter, and turns on expression of interleukin-12 for about 12 to 24 hours. And so the idea here, again, is you take the drug. It turns on IL-12 expression once you inject the vector to this tumor. So this trial was done at five different institutions, Brigham, Cedars-Sinai, University of Chicago, UCSF, and Northwestern. So these are patients that are scheduled for standard of care resection, either gross total or subtotal. We did not know. We knew that it might Velodimox cross the membrane barrier, but we did not know if that was true in humans. And so we scheduled patients to have an oral dose of the drug three hours before resection. So we then went, took out the tumor by gross total resection or subtotal resection, and then set off the tumor to test for Velodimox, but also test for expression of interleukin-12, interferon gamma, et cetera. We had three cohorts. We kept the dose of adenoviral the same, which is 2 times 10 to the 11th. We actually started to initiate a cover of 20 milligrams of the drug. Then we dose escalated to 40. This was a little bit too toxic to patients. We went down to 30. This was also toxic, so we finally expanded it to 20. So this is sort of like the biologic effect slide. This is by the most important slide of this. So again, here we have the pay of this presentation. So here we have the three doses, 20 milligrams in green, 30 in blue, and 40 in orange. So the first question we ask is if we give Velodimox, do you find it in plasma? Yes, you find it in plasma. Here it is at 20. Here it is at 30. Here it is at 40. The second question, do you find it in the tumor? Yes, we find it in the tumor in a dose response fashion. It's about 40% of the peak plasma level. So clearly we can get that drug into tumor. The second question, do you see any evidence that this drug is actually turning on expression of the gene? And so here we're looking at expression of interleukin-12 in blood of patients. So a baseline before they take the drug, there's no interleukin-12 being made. Now, if you take the drug about 12 hours later at peak, you see expression of interleukin-12 at 20 milligrams. At 30 milligrams, a little bit higher. And clearly at 40 milligrams, there's two stratospheric levels. In fact, the main reason for toxicity at this level was the fact that these patients were getting a cytokine release syndrome. One of the downstream effects of interleukin-12 action is expression of interferon gamma. And so we also asked, do you see interferon gamma expression downstream? And the same thing happens. When the drug is off, when you're not giving the drug, there's no interferon gamma in the blood of these patients. But as you give more drug, you can see interferon gamma levels go up. As soon as you stop the drug, the levels go back down. So we then asked, is there any evidence of survival? So here now we're looking just at the survival of the 20-milligram cord. We went back post hoc and also tried to look at whether there was an effect of dexamethasone. What I'm trying to show you here is it's very interesting. We had not actually used this as a variable. The different sites could use dexamethasone the way they liked. But some sites used less dexamethasone than the others. And we saw this curious relationship that if sites used very little dexamethasone, we seem to have this fairly significant 100% survivorship in patients. Now, I must tell you, this data was censored in October. And I think that we're going to be looking at a new baseline, a new evaluation in two weeks. And I'm pretty sure now that this curve is going to start coming down. But clearly, up to about 16 months from dosing, we still had quite a few patients that had very little dexamethasone alive. If you took an intermediate dose of dexamethasone, you were midway. But if you had the high-dose dexamethasone that we usually give patients, these patients all seemed to pass away. We also looked at potential other biomarkers in blood that could be indicative of a response. And this is an interesting one. This is pre-surgery. It seems, again, that if patients have a cytotoxic to Treg ratio at 14 to 28 days after injection, they seem to be doing better with survival. So, in conclusion, what I think I've shown you is that gene-mediated cytotoxic immunotherapies seem to show an encouraging result in this Phase II trial, 25 months, 25 median overall survival, 25 months, one-third of the patients still alive at three years. We think evasion may be due to PD-L1 upregulation. So, we have a new trial starting with GMCI placenta PD-L1. And also, we have this adenovactor-regulated IL-12 gene delivery that seems to be encouraging in terms of biologic and immunologic activity. Thank you for your attention. Thank you.

gene therapy

viral-based gene therapies

glioblastoma

replication-defective viruses

anticancer genes