Next is Dr. Foyer from MD Anderson who needs no introduction but as you know he is a creator of this Delta 24 virus and he will be talking about his work with that. Thank you to the organizers for inviting me to speak after Fred Lang. I always appreciate that because our talks normally are in two versions. He gives the English version and I give the Spanish version. No subtitles though. It is great to be able to work with such a brilliant neurosurgeon, really a leader in the field. Of course without Fred all this Delta 24 would be nothing. He is moving not only the projects that we develop in the lab but he is moving ahead the projects that he develops in his lab and also these delivery things and also these ideas. So it has been a humble experience to be with him. After the talk by both Martusa I almost decided to go home because I think you summarized everything very well and nobody can do this thing better. In addition to Fred I am working also with Candelaria who is my wife and also is an associate professor in neuro-oncology. I am also a co-founder of Dianetrix. Keep this in mind if I exaggerated more than the others. The idea is like Dr. Martusa called this the dark ages in the 90s and it was his seminal paper in 1981 in Science. But at that time while he was already thinking in replicating viruses the majority of us were thinking in gene therapy. Gene therapy was meaning cancer is a disease of the genes. We are going to transfer a gene to the cancer cell and we are going to kill this cell. And then this thing failed mainly because we couldn't infect enough number of cells in vivo to have a therapeutic effect. So this was called the delivery gap and was the main problem for gene therapy and actually many people were thinking that gene therapy for cancer was dead but just wait for the next talk because maybe you are going to see new applications for a new concept. How to solve the delivery gap? So many investigators started to study angiogenesis or by standard effect trying to obtain remote effect by other means. Some of us started to think about the ideas of Dr. Martusa making replication-competent viruses that will multiply the effect after the input dose. So we worked with oncolytic adenoviruses. And then the paper that we published, the seminal paper was this one. It was published in OncoGene. We generated a virus that was capable to destroy Rb null cells but when you restore the Rb activity in these cells, the cells become resistant and this was the basis for the tumor selectivity. And this virus is targeting the Rb pathway and the Rb pathway is abnormal. They say here in 78% of glioblastomas or gliomas. However, the current theories in cancer are that probably the Rb inactivation, P53 inactivation and activation of telomerase is a sine quantum condition for every cancer cell. So probably the oncolytic virus is targeting every cancer cell, not only every glioma cell. This is the mechanisms of the effect, the virus, how the virus, how normally adenovirus work. They express an early protein called E1A that will eliminate the complex RbE2F, allowing for the adenoviral replication. We introduced this mutation in E1A that prevents the binding to Rb. So in a cell in G0, like a neuron, the virus will not replicate and will be eliminated. But in a glioma cell in which the Rb pathway is already inactivated, E2F is free and the virus will replicate. We published the paper in Oncogene and after we do in vivo experiments already with FRET and then this paper was published in the Journal National Cancer Institute. And then this is the paper that showed this magnificent effect, I think, unexpected effect in the U87 model that in which the radiotherapy and the chemotherapy models were capable only to double survival but not produce long-term survival. The problem with this is that all these experiments were done in immune-deficient animals. And then these were presented to every, every expert in virotherapy and gene therapy in USA and nobody told us, hey, what's happening with the immunotherapy component? We published also that the virus can destroy cancerous stem cells. And then now we published this year the paper that FRET has discussed in Journal Clinical Oncology. So I have published only these four papers. People are telling me what you have done in these 17 years. I said that I was working. I mean, these papers were fun, but you had to work and have all the other papers in order to produce the metrics for the production. But these papers were fun. The clinical trial has produced a model, a model that has been, a hypothesis that has been formulated by Peter Forsyth here. And then he's saying in editorial in Journal Clinical Oncology that what happened in our trial was that the viral load started high with the dose, the virus replicate, and then suddenly the virus was eliminated. Then the immune response start to take over after the virus is going down. The immune response is going up. And then the tumor size is increasing because the pseudoprogression and eventually the tumor is shrinkage. This is a model that needs to be tested for sure. But there are things that we should not be discussing any longer. The virus induce an immune response against the tumor. There are enough data already to say that. So there are data from the clinical trials and there are also data from experiments in animals in which using cells that are expressing tumor-associated antigens, it's easy to see that a mouse treated with virus is developing immune response against antigens that are not viral, that are cellular antigens. So I think the idea that if the virus oncolysis is a part of immunotherapy or not is not to be discussed. It has to be accepted, I think. And this is, you know, it was easy. The starting point of immunotherapy was a surgeon, in this case not a neurosurgeon, but a surgeon treating patients with sarcoma in New York that developed this vaccine against tumors using bacterias. So the origin of immunotherapy is using pathogens to trigger an immune response against tumors. The working hypothesis here is that the virus will trigger a dangerous pathogen signals and that will be cooperating or at least coexisting with dangerous signals from the dying cells and that together will be capable to trigger an immune response that go first against the virus, probably, and then later against the tumor. So this is my slide for my talk. This is what I wanted to say here today. Oncolytic virus equals a special case of immunotherapy and more. And the immunotherapists already know this thing. And this is a paper, it's a journal from Europe and also from oncology. It's from December of the last year. And in this review, I don't know if Dr. Martusa has ever seen this, but these are all the immunotherapy strategies and suddenly appear oncolytic virus. It's very interesting because if cancer was a disease of the genes in the 90s, cancer is a disease of the genes, we are going to do gene therapy. Right now cancer is an immunological disorder. And we are going to cure cancer doing immunotherapy. If this is true or not, it's something that we need to demonstrate. But doing experiments in the lab, in a mouse, it is a mouse with no tumor, and then you inject a tumor and suddenly you have an upregulation of immune suppression. In the parenthesis, just by injecting a tumor in a mouse, you modify the immune system of the animal. So how can we take advantage of this using the Delta24RGD concept? This is the idea Fred has mentioned, the idea of Dr. Martusa, also the idea of Dr. Allison, that there are two types of tumors. One tumor is hot, meaning it has lymphocytes before the treatment, probably has a hyper-mutanoma, has many antigens, et cetera. The best case would be melanoma. And there is a cold tumor, like the glioma, in which there is the blood brain barrier, there are no lymphatics, there are no professional representation of antigen cells in the brain, there are no lymphocyte populations in the tumor, et cetera. And then this tumor needs to be modified, transformed into this one, in order that it's sensitive to immune checkpoints strategy. And then when you do this, it really works, and this is the paper in Cell. Also, we have never published, well Martusa was publishing in Nature and Science, I know, but normally people that work with virus on colitis doesn't publish in journals like Cell. This paper is in Cell, and it's showing that there is an objective response of 62%, it's a clinical trial, of melanoma patients treated with the herpes virus, application-competent herpes virus, and immune checkpoint, with a 33% complete response. I think this is unbelievable. There are other cellular receptors in the cells, and in Spain we know this very well, because Godi has explained us all the cellular receptors there. Some of them are positive regulators of the membrane, and there are three that I have interest, GITAR, OX40, and 401BB. These are the receptors in electron microscopy picture, and then when these receptors interact with cells, they are capable to activate T-effector cells, but also decrease the function of T-regulatory cells. So the one that I'm going to explain a little bit more now is called OX40, it's a receptor that binds a ligand with OX40L. When this interaction happens, this is the second signal, the first signal is here with the interaction with antigen. When this interaction happens, OX40-OX40 cell, the AKT pathway is activated in the lymphocyte, the lymphocyte proliferates and generates a clone against the antigen. So it's a positive signal for T-cell regulation, the OX40-OX40 ligand. And then there is a recent paper, I think it's January this year, published in Science Translational Medicine, that show in several animal models that targeting a toll-like receptor 9, by the way, is the one that is targeting the adenovirus, in combination with an antibody that activates the OX40 pathway, is the best strategy to induce spontaneous regression in these mice. So we generated a virus in which we cloned a set of expression for the mutant OX40L in the backbone of the Delta24RGD. The virus is capable to express high levels of the receptor, of the ligand, sorry, in the infected cells. And then, interestingly, in a model, for instance, in which Delta24RGD is inducing necrosis in the tumor, you try to do, you do the same experiment in an animal that is immune deficient. Suddenly the virus cannot induce necrosis. Remember that the mouse cells are very resistant to adenovirus infection. And then you produce increasing survival in the animals. These animals develop memory, and when you do the rechallenge, they survive. But when you rechallenge with another type of tumor, they all die. These are experiments also combining the virus with an anti-PD-L1, in which the increase in survival is significant. Actually, in the first experiment was 100% survival. And I always say this, when you have 100% survival, you go and tell the fellow or the post-doc, do not repeat the experiment, but they repeat the experiment normally. And this is why we have this thing. But yeah, like what Martusa showed before, we are seeing things with immunotherapy that are unbiotherapy that goes close to 100% of curing animals in vitro. This is an experiment to observe how the influence of the, in the tumor population of the virus increase the NK cells, can increase also the, all the lymphocytes, the CD4, the CD8. Interestingly, it changed the landscape in a way that is, you know, that is profound. But when you check the expression, for instance, of PD-1, surprisingly, the PD-1 expression goes high after the virus. So it's not so surprising. I mean, the role of immune checkpoints is to prevent the development of autoimmune disease after a viral infection once the virus is destroyed. So it's not surprising that this immune checkpoint goes up. But really offer a target for the anti-PD-1 therapy in the captive study that Fred was discussing. If not, everything is good. The immune suppression also increased with the virus. So it's not something that is all unregulated. And then in order to prove that the RG-DOX, the data with the RG-DOX are good, we generate another virus that is expressing another ligand, the guitar, that is connected with the OX40. It's also for the TNFR family. And then we do experiments in vivo to see the increased survival. And you can see that the results with the RG-DOX are reproducible. So I'm going to finish here saying that there is enough evidence to show that the immune response is part of the oncolytic virus effect. And that I believe that in addition to the immune checkpoints, the combination of immune checkpoints and virus that offer some advantages but has also some problems. Also what Martuz has said and Gene Harrison recognized, the immune checkpoints are targeting normal cells, are targeting normal lymphocytes, are not targeting cancer cells. So several potential toxicities there. So maybe trying to work with immune modulator with a synopsis that are only expressed during the viral infection may decrease the toxicity. And this is the people in the lab and there is a phenomenon that is we are here with the students that I think they are always much taller than I. And I don't know if I'm undergoing apoptosis or it's because maybe the next generations are better. Thank you very much for your attention.

Delta 24 virus

oncolytic viruses

immune response

tumor selectivity

immunotherapy