It's my pleasure to introduce Jim Markert next, he's going to talk about his experiments at the University of Alabama with Oncolytic HSV immunotherapy for adults and children. So here are my disclosures, sadly Amgen had spent their billion dollars and I will be keeping my day job. We're going to talk about some of our initial clinical experience as well as the current clinical trials and where we think the data is pointing us in terms of future trials. So HSV is something, perhaps the most well studied virus in malignant glioma and here on this table here you can see GTO7 which Bob mentioned and then 1716 was a version of this that was used in Great Britain. We've made a virus M032, Nino didn't mention it but he's got a virus in trial RQNestin 34.5 version 2 and there's Dr. Toto in Japan is studying G47 Delta and we have an upcoming trial with C134. So there certainly is a good developing experience with this virus in glioma. And as you've heard, Imligec or TVEC has been approved by the FDA and it's certainly in use for melanoma. So here's the GTO7 construct or genome just to use as kind of a basis and in this virus there are two copies of the viral neurovirulence gene 34.5 that are deleted and here you can see this schematically demonstrated here as well as the LACZ gene as well for ribonucleotide as well as a LACZ insertion in the ribonucleotide reductase gene and HSV1716 which was being studied in Britain about the same time as this had the 34.5 deletion but they did not take out the ribonucleotide reductase gene. Bob and I talked about this at that time and we did not want to be famous for killing a bunch of people back then so we went with a more conservative construct. Different generation viruses have sought to increase the activity of the HSV by somehow compensating for the 34.5 loss and Nino's virus that has nestin expressed under 34.5 does that. G47 delta also has a mechanism of compensating for the 34.5 loss and C134 which I'll talk about at the end of the talk also has this approach. The other approach that's been used is the approach that we've used with our IL-12 virus is to try to somehow increase the activity of the virus against the tumor using another mechanism. So these are the studies that we did with G207 and the first study Bob and I did, here's Bob sculpting away here and Mike Medlock helped us and we did 21 patients and we showed in fact that the virus was safe and we had some really remarkable responses in that early study. Then we went on and studied this in a trial similar to the one that Fred talked about, similar design and we put in a catheter, treated the patients and then came back and resected so that we could get some biologic information and that was very helpful for telling us that really there was an immune response being evoked against the tumor as a result of the viral infection or injection. And then finally we did a study that was in part due to the work of Ralph Wechselbaum, one of my collaborators, who showed that you could increase the replication of the virus and increase the anti-tumor response by using it with radiation right afterwards and we were able to successfully do this as well. So from that work we inspired this gentleman here at my institution, Greg Freedman, who's a hemodoc and then Jim Johnston who's a faculty in my department to look at this in children and so it turns out Greg found out that pediatric xenolines were much more sensitive to G207 than the adult tumors were and when he looked at this in a variety of ways, it looked like the pediatric tumors were more than 20 times more sensitive to OHSV killing than the adult specimens and in fact, gamma stem cells from the pediatric brain tumors were also more sensitive to the gamma 134.5 delayed HSV so it seemed like a good idea to try to test this in children. So we designed a trial that the primary objective was similar as to some of our earlier studies with G207 to determine the safety and tolerability, just like all phase one studies, in patients that had any kind of recurrent or progressive malignant supertentorial brain tumors. So since these were children, glauma malignant glauma is not as common a disease in that group and so we decided to accept any kind of histology as long as it was supertentorial. And these patients would get infused with the virus in up to four different intratumoral catheters and some patients would get followed with a dose of radiation. And so this study has started enrollment. We also had some secondary objectives for this and these were kind of standard to look for any kind of immune response that might develop, to look for shedding of the virus because perhaps, although we didn't see this ever in adults, maybe this would be present in children. And then we wanted to look at the tumor genotype to see if we got any clues about what tumors might be sensitive to the virus and look at the tumors themselves for evidence of different HSV entry molecules. So we're still enrolling patients in the trial, but if you look at some of our early patients, we are seeing some interesting results here. Here's a patient with a highly perfused tumor over here. They received the virus and within three days they're already starting to develop a hole in the perfusion and by day 36 there's this rather large area of non-perfused necrotic looking substance. If we look at the patient's peripheral blood cells, similar to what Fred was alluding to, we do see an increase both in absolute T lymphocytes as well as activated T lymphocytes in the blood. This patient eventually went on and had pseudoprogression that was significant enough that she underwent a resection of the tumor and we saw in the cavity mostly necrotic tissue and some hemorrhage. There was evidence. If you look at the tumor histology before administration of the virus and compared to the after administration, there were definitely focal areas of scattered CD8 cells that were increased after viral treatment, going along with the hypothesis that you've heard about oncolytic viral therapy being a mechanism of turning a cold tumor into a hot tumor and producing some infiltration of T lymphocytes into the tumor. Here's another patient who had, interestingly, a very different kind of response but also a positive response. This patient, here you can see their tumor two days prior to G207. This is another patient with a glioma and what you see is this development. This patient never really had the pseudoprogression response that we usually think of when we think of a positive reaction to the virus, but the patient develops this kind of Swiss cheese-looking appearance within the tumor and, interestingly, I've spoken to other investigators who have seen this. The poliovirus group notices this a lot and you can see that after 12 months after treatment with no other treatment, the amount of flare change is diminished, the amount of enhancement is diminished and you're seeing more and more of these little bubbles. So additionally, this patient had an increased improvement in their KPS with treatment, which is obviously critical as well. Here's a third patient that was treated and you can see this patient had some stabilization and then actually had a decreased enhancement of their tumor over time and this was five months after treatment out here. And if you look at, interestingly, this patient, they had a secondary lesion that was not treated and this lesion increased in size with this sort of cystic-looking center that we've seen not infrequently in our ongoing viral treatment patients. You can see perfusion actually has somewhat decreased over time. Because of this pseudo-progressive type response, the patient received a half a dose of Avastin and then came back in for another MRI scan and, in fact, not only is there a decrease in the size of the tumor, but the necrotic center seems to be also folding and vaginating as well and still not much in the way of perfusion. So this is interesting, again, because this is at a distant site and shows further evidence perhaps that we may be developing an antitumor immune response. Let's leave children and move on to adults right now and M032 is the virus I mentioned before that we made that made IL-12. You've heard a little bit about IL-12 from Nino already and it seems to be a very promising cytokine for producing an antitumor immune response. We've looked at the virus versus other viruses and are stable and it certainly does seem to produce a better response in the preclinical area. These are my collaborators in this effort here. We went on and showed that this virus, compared to earlier generation viruses, actually replicated better and produced better antitumor responses, even when compared to viruses that expressed other cytokines. And nicely, when we injected the primate A. aeotus, we did see a little bit of inflammation, but when we looked at this later, we didn't see any neuronal dropouts, so that combination was really what we were after. So we opened a trial with this after going through a lot of hoops with the NIH and then FDA and we started the trial at 10 of the 6 platforming units. Again, we were using up to 4 different loci for injecting the virus and then we infused it over 6 hours. We didn't want to infuse it over a longer period of time for fear of inducing an antitumor interferon response. We've treated 6 patients to date. The 7th is scheduled for later this month. We're using a CRM method of dose escalation and we're already going to be up to 10 of the 8th platforming units, so we're very excited about that. We haven't had any DLTs to date and we had some clear radiographic evidence of activity in several patients. Here's one. Here's a patient that was treated with radiation in temozolomide and then Avastin, failed Avastin, came to get treated with the M032 and then one month after that, the patient developed what looked like to be pseudoprogression. I don't have that here, but Avastin was restarted and maintained for 9 months and then stopped. And 11 months after treatment, so 2 months after the Avastin was stopped, we can see we still have what looks to be a nice antitumor response going on here and the patient survived for an additional 6 1⁄2 months without additional treatment. So clearly, we wouldn't expect Avastin as a re-treatment option to work on its own and certainly not to have this kind of effect, so we think this is a positive sign. When we looked at this patient here, we looked at their T cell subsets and what we found was in particular, the effector memory subset was upregulated one month after treatment compared to prior to treatment. Here's another patient that was treated. Here you can see their tumor here and again, this kind of cystic-looking change occurs over time. There's a lot of enhancement around here at day 28 consistent with the pseudoprogression, but if you look at the FLIR T2 images, there's a big hole right in the middle of the tumor and so we really believe that the virus is having an impact. And we look at our virus in just a small number of patients here. It seems like we are having increased survival, but obviously, we can't tell until we do a phase 3 study. Here's a study that's being done in canine glioma. I apologize for the pictures, they must have changed from a Mac, but we've just treated the third patient in this study, Renee Chambers, who was a neurosurgeon on my faculty, was originally a veterinarian and then went back to medical school and got interested and she's maintained the interest in animal studies and so she's developed this study. We're looking at this M032 virus in canines. Just treated the third patient, so we're excited to see whether we have any interesting results in here and we might be able to use this to help inform our adult human studies. Finally, I mentioned this earlier, this virus C134. Kevin Cassidy, a collaborator of mine now at Nationwide Children's, developed this chimeric virus that expresses the PKR evasion gene IRS1, which is a CMV gene instead of the native 34.5 gene and here you can see how he originally looked at TRS1 too, but IRS1 had a better safety profile and plugging this in where 34.5 had been resulted in a virus that had much better replication but did not regain the neurovirulence of the native wild type virus. So, we've gone and looked at that and gotten an FDA approval, it looks very promising in preclinical studies and now we're just getting it through our IRB right now, we're looking forward to studying that virus as well. So we're going to go ahead and continue to evaluate the safety of these different approaches as are others in their studies. I think that critical are next-gen sequencing opportunities to look at both the viral immune response to the virus as well as the tumor and to try to educate us about the genomics of the tumors that seem to respond best. And I think you've heard much today already about the use of immunomodulatory agents and oncolytic viruses and certainly will be interested in doing this. I think it's going to be important that as Nino showed in one of their gene therapy studies that we really move up into the up-front patient population. Here you can see a graph that demonstrates how the cell number in a tumor decreases over time and then increases later. So at the time of initial resection, you get a lot of the tumor cells out, you treat with radiation and chemotherapy, and you can really reduce the number of tumor cells down to a more manageable number here. We tend to be treating out here, which is bad both because, one, we have a larger number of tumor cells we have to impact, but two, the immune system has been more devastated by the various treatments that have preceded this time. And so I think it's going to be really critical that we get our OV therapy into this window in the future. So in addition to timing of therapy, we need to learn more about the role of foreign gene expression like IL-12. What other opportunities are there out there? Perhaps some of the checkpoint inhibitors, perhaps other cytokines. As was mentioned previously, what about some of these less debilitated HSV, C134, RQ-Nessen, and so forth? And then we need to think about what other immunomodulatory drugs might we use, checkpoint inhibitors that Bob and Sam Rabkin looked at in their excellent cancer cell paper, as well as vaccines and other agents that might help modulate the immune response to the virus and tumor. And also, what is the best delivery method? I like the ESEA method that Fred talked about during his talk, and how often do we have to give the virus? So even in the area of immunotherapy, GBM remains a formidable opponent. Checkpoint inhibitors haven't really demonstrated promise yet alone, but we think that if we combine these kinds of immunotherapeutic agents with oncolytic viruses, we might be able to achieve this holy grail of turning cold tumors into hot tumors. I want to thank all the people that have been involved with me in these studies over the years. It wouldn't have been possible to do this without their great assistance. Thank you.

Oncolytic HSV immunotherapy

clinical trials

G207

tumor necrosis

immune response