It's now my honor to present and introduce Dr. Fred Lange from the MD Anderson Cancer Center, who's going to be talking about oncolytic adenoviruses, clinical trials, and the delivery problem. Thank you very much. It's a great honor to be here, and I appreciate the invitation. It's also a special honor to be speaking with Dr. Martuzzi, who, as you can see, is really the father of oncolytic viral therapy in the world, so it's a great honor to be here. And all that he predicted is actually coming true, and I will be discussing this in terms of oncolytic adenovirus. So these are my disclosures. I have a patent holder for one of the agents, Delta 24, but I don't have any affiliation with the company. So I just wanted to also say that in terms of where this fits in the bigger picture, one might consider these oncolytic viruses as sort of biological therapies, naturally occurring agents in which you're trying to exploit the intrinsic properties of the agent to treat the cancer. And our group got interested in this idea because we see that in these biological agents, one frequently finds that nature or evolution has actually solved many of the hard-to-replicate problems of cancer therapy, so getting into the tumor cell, activating an immune system, and even, as I'll tell you, even traversing the blood-brain barrier. So we've been working with adenovirus. As Dr. Martuzza said, there's a variety of other viruses besides the herpesvirus. Adenoviruses were attractive to us because they're relatively low pathogens, they cause a common cold, and they don't cause major disease in the brain. Adenoviruses are DNA viruses. They have a linear double-stranded genome that is completely understood and manipulatable. They also have an icosahedral non-envelope capsid, which helps the virus get into cells and the biology of which is understood. So we at MD Anderson have been working on this virus called Delta-24-RGD. This is the brainchild of Dr. Foyo, who will be speaking after me, and you'll hear the further developments of this virus. I feel fortunate to have met Dr. Foyo and to have been working with him for the last 15 or 20 years. This is really his great idea, building on the work of Dr. Martuzza, and I've always felt blessed to be able to come along for the ride with him. So Delta-24 has several features which make it ideal for treating tumors. The first, as you heard from Dr. Martuzza, this, like herpes, is a replication-competent virus. So that means that if you inject it into the tumor, the virus will get into the tumor cells, it will replicate in those tumor cells, expand its number, lyse those tumor cells, be released into the microenvironment, and then go on to infect more cells, and in that iterative process eventually be able to potentially spread through the tumor. This virus is also tumor-selective. The basis of that tumor selectivity is a 24-base pair deletion in the E1A gene, which allows this virus only to replicate in cells that have lost or inactivated their RB, which is found in tumor cells, and it's not able to replicate in cells that have a normal RB, which is essentially in normal brain cells or normal cells throughout the body. The virus has also been engineered to have an enhanced infectivity. This was achieved by insertion of an RGD motif into the fiber knob, which allows the virus to enter cells independent of the normal Coxsackie adenoreceptor and to enter cells also based on the integrins on the surface of the cells. Since integrins are very high on tumor cells, this enhances its infectivity and also increases its selectivity for tumors. So after doing a large amount of preclinical work in multiple model systems, we eventually brought this virus to a dose escalation phase 1 clinical trial, which we just recently published in the Journal of Clinical Oncology. This trial was unique in that we had two arms. In the first arm, we basically did a stereotactic biopsy to confirm recurrent glioblastoma. We then injected the virus directly into the tumor and then followed those patients for toxicity and response, as you would do in any phase 1 clinical trial. We also, however, had an arm B or a group B, the purpose of which was to obtain post-treatment biological specimens to prove that the virus could do what we intended it to do. So in this arm, we obtained a stereotactic biopsy. We then implanted a catheter into the tumor, injected the virus into that catheter, cut the catheter, closed the patient up, came back 14 days later, did an on-block resection of the tumor with the catheter in place in order to mark the injection site. That gave us a biological specimen that we could then analyze. And then we injected the wall of the resection cavity and followed the patients. This arm B was critical because it proved to us that the virus could do what it was intended to do in a human patient. So here's an example of a resected tumor. You can see the catheter sticking out of the tumor and going into the tumor. We cut per- we formalin fixed these, cut them perpendicular with the catheter marking an injection site. And as we examined those, we stained them for E1A and hexon, and we were able to see large numbers of cells infected with active virus based on this staining. Most importantly, we thought- saw three zones of viral replication, which exactly replicated or reproduced what we had seen in our animal models. There was a central zone of necrosis where the virus had been and had killed the cells. There was then a surrounding zone of active viral infection as evidenced by E1A and hexon staining. And then there was a zone beyond that where the virus had not yet reached. So we proved in our- in these specimens that the virus was actually to- able to do what it was intended to do. Equally interesting, and although this was only a phase one trial, we actually saw signs of efficacy. Indeed, 20% of the patients lived longer than four years. And most importantly, three of those patients had complete responses that were durable for over three and a half years. Here's an example of one of those responses. You see a patient here. This patient has this posterior temporal tumor with surrounding flare change. We injected this back in 2012, and you can see here, 37 months after, there's no evidence of contrast enhancement. And equally important, there's no evidence of flare on this, indicating that the virus is able to get into the infiltrating tumor cells and eliminate those cells. Interesting, and as Dr. Martuza alluded to, as we were watching these patients, we saw that the patients that responded, all of them went through this period where the tumor got worse before it got better, which has now been labeled as pseudoprogression and is indicative of an immune response. So this was the first clue to us that, in fact, this virus was potentially inducing an immune response. So we actually had several patients in which we removed the tumor at the time of this pseudoprogression, and we were able to show in those specimens that these patients had increased T cells, and in particular, they had increased levels of cytotoxic CD8 T cells indicative of a Th1 immune reaction. We then went back and looked at our pre- and post-treatment specimens from RMB, and again, we were able to show, quite nicely, that there was a statistically significant increase in CD4 cells in our post-treatment specimens, and there was also an increase, again, in the critical cytotoxic CD8 T cells. So this has now led to the concept, or affirming Dr. Martuza's prediction, that this virus actually works by two mechanisms. The first mechanism is direct oncolysis, which results in killing of the tumor cells, but equally important, release or presentation of a large number of tumor antigens. In addition, because the virus is highly immunogenic, it calls into the tumor large numbers of immune cells, which is initially an innate immune response, but that then results in the presentation of antigens to T cells, which results in an adaptive cytotoxic immune response, not only to the virus, but also to the tumor, creating an antiglioma effect. Now we've studied this a lot in preclinical models, and so the next step, we thought, was that if we really wanted to go from a 15% response rate, the three patients that we've seen, to 100%, we had to enhance, we think, at least in part, the antiglioma response. So we thought that one potential way to do that would be to combine Delta 24 with immune activating agents, in particular, the checkpoint inhibitors. And as Dr. Martuza referred to, and as you all know, the T cells, the immune cells, are not only stimulated to activate, but they're also turned off by these negative regulators. So the T cells still are the CTL-4 and the PD-1 checkpoint molecules. And over the last several years, many drug companies have developed inhibitors of these, in particular, the anti-CTL-4 ipilimumab and the anti-PD-1 pembrolizumab. So the idea here, again, is that we would combine Delta 24 with these agents, based on the idea that most of the tumors that respond to these agents by themselves are so-called hot tumors. In other words, they have high antigen expression, and they're calling in, at their very baseline, lots of immune cells, particularly cytotoxic CD8 cells. And so when you give these checkpoint inhibitors by themselves, all of those immune cells are there to attack the tumor. It turns out, however, that glioblastoma is a very cold tumor. At its baseline, it has almost no T cells inside that tumor. So the concept here is that you give the virus. This results in the attraction of all of these immune cells into the tumor with release of antigens. But these cells are being held back from actually attacking the tumor because the virus actually itself upregulates the checkpoint inhibitors, the checkpoints, the PD-L1 and the PD-1 in the tumor, in the reactive immune cells. So now if we hit those cells with the PD-1 checkpoint inhibitor, we would be able to induce an antiglioma response. So in fact, we've actually now started this trial. We've combined Delta-24 with Pembrolizumab in a collaboration with Merck. This is a multicenter trial being carried out both in the United States and Canada. We initially had a dose escalation phase where we escalated the dose in three stages. We've actually completed that stage, and now we're in an expansion phase where we're combining – where we're giving patients the highest dose of Delta-24 with the Pembro. So as you can see here, in this trial, patients undergo a single injection of the Delta-24 into the tumor. After 7 to 10 days, they then receive the Pembrolizumab, and then they receive that drug every three weeks. So we've actually had some interesting results so far in our preliminary analysis. We've enrolled 22 patients. 20 of those patients are alive, with many patients reaching up to one year. And interestingly, as in our previous trial, we're also seeing interesting responses. Here's a case from Toronto of a large left-sided tumor, which the patient was becoming weak. They were again treated with a single injection of the DNx2401 or Delta-24, followed by every three weeks of Pembrolizumab. And you can see after initial increase in size of the tumor, there's been a progressive decrease over approximately seven months to essentially almost no tumor left here. And equally important, all of the flare change has also progressively disappeared. So a lot of potential for this combination. So in addition to trying to activate the immune system more, we also think that part of the area of improvement of this virus or these viral therapies is to increase the delivery so that the virus can get to more parts of the tumor. So as I've told you, up to now, we've been injecting the virus only into one location in the tumor. And we actually think that it would be better if we could get this virus throughout all of the tumor so that more of the heterogeneity and the antigens within the tumor would be released. And the best way to do that would be to deliver the virus intravascularly through the bloodstream. Unfortunately, for adenovirus, this is basically prohibited because of severe immunologic reaction to the virus, which will inactivate it, but also cause peripheral organ toxicity. So as an alternative, we turned to another biological agent, essentially stem cells. And of the different stem cells that are available, our group has been focusing on a stem cell from the bone marrow called mesenchymal stem cells. These are basically the cells in the bone marrow that control the homeostasis of the marrow so that the hematopoietic stem cells can proliferate. And we thought that these stem cells would be of advantage because the bone marrow is easy to access. The isolation and expansion techniques of these stem cells are well known, are easy to do, and the patient can act as his or her own donor, and there's no ethical issues associated with their use. The rationale for using these cells is based on the idea that these bone marrow cells actually leave the bone marrow and circulate through the blood, and then they're called out into peripheral organs during times of tissue injury or stress, potentially to repair that injury. So we hypothesized that since a tumor looks like an injured tissue, that these cells would actually home to a brain tumor, and then we could load them with various agents. So we showed, in fact, that these mesenchymal stem cells are able to home to brain tumors in several papers that we published. We showed that they could home to so-called professional glioma stem cells like U87, where you see here high levels of these fluorescent label cells in the tumor. And we also showed that these mesenchymal stem cells can home to glioma stem cell line, the X lines, as you see by these green label cells within these infiltrative tumors. So we wanted to see if we could load the Delta 24 into these tumors and then deliver them, and so we set up this model in which we implanted the tumors. We then labeled them with green fluorescent protein. We loaded them with the Delta 24 virus. We injected them intra-arterial into the animals, and then we took out the tumors to see if the cells would be in the tumor. And indeed, we were able to show that there was large numbers of green MSC-loaded... Delta 24-loaded mesenchymal stem cells essentially only within the tumor, but not in the opposite brain, indicating that the loading of these stem cells with the Delta 24 did not interfere with their ability to home to the brain tumors. And then we went on to show, in a variety of models, that these cells could actually deliver the virus and could produce a cure of the animals, shown here as two independent experiments showing that 30-40% of the animals were cured with this delivery system. And we published those results in Cancer Research. So now we're moving on to actually take this approach to a Phase I clinical trial. We've partnered with our stem cell group at MD Anderson and have created GMP versions of this virus. And most importantly, we've worked with our endovascular neurosurgeons, Peter Kahn, who is an outstanding endovascular neurosurgeon at Baylor College of Medicine, with the idea of using the endovascular approaches that have been applied to aneurysms and AVMs and now applying that to cancer in a new field or evolving field of endovascular neurosurgical oncology. So we've tested this using the state-of-the-art angiography research lab at Baylor College of Medicine. And we've used this approach, which we like to call endovascular selective intra-arterial or EASIA delivery. And we've tested it in canines using clinical-grade catheters. And we've shown that you can inject these up to 10 to the 8 cells without any strokes or based on angiography, MRI, and necropsy. So in conclusion, Delta-24 represents a potentially new treatment of glioblastoma. Combining Delta-24 with checkpoint inhibitors like pembrolizumab is a rational approach and is showing early signs of efficacy in our current multicenter clinical trial. In addition, I think mesenchymal stem cells may allow us to deliver these Delta-24 intravascularly and actually may be the beginning of a new field of endovascular neurosurgical oncology. And I think that this combination of these kind of agents really shows the power of these biological agents in which we take advantage of the intrinsic properties to treat cancer. So with that, I thank you. And I just want to thank all my collaborators, not only all the people in neurosurgery, but particularly Dr. Foyo, who is really the mastermind of all of the work we've been doing. And also for my collaboration with Peter Kan with the endovascular work. Thank you very much. Thank you.

oncolytic adenoviruses

clinical trials

delivery problem

Delta-24 adenovirus

checkpoint inhibitors