Scientist Stories: Tobiloba Oni, Unleashing the Antitumor Immune Response

Well, good afternoon everyone. Welcome to today's webinar on unleashing the anti-tumor immune response. My name is Olivia Cordine. I am currently the Scott Cook Insignia Oxbow Fellow at the Whitehead Institute and today I'll serve as host and moderator. So today's webinar is the third in a series of talks that we've organized to share some of Whitehead Institute science with with the outside wider community. Our topic this afternoon is unleashing the anti-tumor immune response. In a few moments, we'll get to hear from our presenter today, Whitehead Fellow Toboloba Ony. He will be highlighting the general principles of the anti-tumor immune responses and discuss his lab strategies to unleash the immune attack on the cancer of the pancreas. So first, I'd like to let you know that this webinar is being recorded for future viewing. Also, I know Toby will be happy to take any and all questions at the end of today's presentation, but please submit those questions in the Q&A box at the bottom of the screen so that we are ready to ask Toby those questions at the end. So with that, I'd just like to introduce our speaker today, Toby. So Toby joined the Whitehead Institute as the Valhalla Fellow in February of 2021. The Oni Lab seeks to develop new methods of understanding and detecting and even potentially treating pancreatic cancer. Toby grew up in Ikere, Nigeria, where he earned his Bachelor of Science in Biology in 2011 from the State University of New York at Plattsburgh, where he received a Senior Leadership Achievement Award. He went on to then earn his PhD in Cellular Molecular Biology in 2020 from Stony Brook University, where he was selected as a scholar in biomedical science. He most recently performed his graduate research under David Tuveson, director of Cold Spring Harbor Laboratory Cancer Center. So Toby, thank you for being here with us today. Excited to hear what you have to share.

Thank you for the introduction, Olivia, and thank you very much, everyone, for being here. I would like to congratulate Olivia for Being one of the latest members of the Whitehead is very well deserved, and it's certainly an achievement I hope to follow up on. Today, what I thought I would do is really to talk to you about unleashing the anti-tumor immune response. My lab just started about 3, 4 months ago. Instead of diving through any data that we don't have at the moment, I really wanted to give you a background on anti-tumor immune response and why this topic is so important. Then I would like to talk very briefly about some of the strategies we're thinking about really pursuing to really unleash the immune response against cancer. Particularly pancreas cancer. As many of you know, the immune system is a collection of tissues and cells and organs that really allow us to fight dangerous pathogens like SARS-CoV-2. But that's not only what it does, it also is able to actually fight against cancer. In fact, there are many immune cells, some of them that I've depicted here, they really actually are able to, to, you know, have effective functions to kill cancer cells. And right now we're only just realizing that the promise of this therapy to defeat such a large enemy. And so speaking about cancer, it's worthy to note that cancer is the number 2 killer of human beings. In the world. And in fact, 1 in 3 Americans will develop cancer in your lifetime. And so this is a huge public health problem that really needs to be addressed. And obviously one way to address that is therapeutics, but there are two other important things that I must mention just because they're important also. One is prevention, the other is early detection. But today I'm going to really focus on the therapeutics, particularly how we can use the immune cells to really drive therapies towards one of, you know, some of the worst cancers that exist. And so when you think about traditional cancer therapies, you think about 3 things. You think about radiation, surgery, chemotherapy. We burn the tumor with radiation, we cut it out or slash it out with surgery, and we poison it with chemotherapy. The benefit of the first two, radiation and surgery, is that they work really well if the tumor is localized, if it's not spread to other organs or invaded other tissues. This particular strategies, are only helpful when the cancer is not metastasized. Well, it turns out that most cancers actually kill people because of metastasis. That's where chemotherapy comes in. Chemotherapy works to a degree, but we all know that there's a lot of collateral damage when there's chemotherapy, as well as ineffectual therapies. For several cancers. And so in the past 20— in the past 20 years, 2 decades, basically, we've been able to really realize that, well, we could go after cancer cells if we understand the pathways that keep them alive. And particularly, this led to development of a lot of targeted therapies targeting pathways within cancer cells that can kill them. What is common between, you know, the traditional cancer therapies and targeted therapies is that it's focused on the tumor. And when the tumor evolves, invariably what ends up happening is that the therapy stops working. And so, you know, this has been a challenge to really update on this. Well, in the last decade, we've realized increasingly that there's a different paradigm, which is not to really focus directly on the tumor cell, but rather to harness the immune system, to treat the immune system, stimulate it to eliminate the cancer cells. And this is the basis, this is the fundamental basis of immunotherapy. And so I bring this image again here, and you'll see this image again and again, which shows that there are multiple immune cells, T cells, B cells, NK T cells, macrophages, neutrophils, that are able to actually eliminate cancer cells. And so fighting cancer with the immune system does not only mean that we're only doing T-cell therapy, but rather that we're able to harness the different parts of the immune system to, to a concerted goal. So I'm going to give you just like a, a brief, um, kind of, um, idea about the 5 types of immunotherapy And I'll tell you about a particular immunotherapy that has really sparked our imagination about the promise of this sort of therapies. And so the first is adoptive cell therapy, and this basically involves taking cells from a patient, either engineering the cells ex vivo or modifying the cells ex vivo in one way or the other, and then putting it back into the patient. This works really great, and in many cases, it's very expensive. Then there's targeted antibodies, and this were actually, you know, quite early forms of immunotherapy that involves using antibodies to targeted proteins on the cancer cells. Then you have vaccines, which include BCG for bladder cancer and Cipollasi T for prostate cancer. Prostate cancer, and this involves vaccinating with some sort of antigen-presenting cell or even the tumor cell itself, and in case of BCG, vaccinating with some bacterial products, which will wake up the immune system and allow it to target the cancer cells. And then we have oncolytic virus therapies, such as TVEC for melanoma, where you have this induction of inflammatory cascade by putting in the virus into the tumor, allowing the tumor to lyse and present and allow the dendritic cells to present antigens to T cells. And it basically starts this wheel that will drive autonomous response to the cancer. And finally, and the most important for this decade, are immunomodulators. And these are cytokines, for example, particularly immune checkpoint therapies, which is— which will be the basis of many of the things I'll talk about today. So when we think about immune checkpoint therapies, these are basically therapies that allow the immune system to work freely without being inactivated or basically to release the brake on the immune system. And this type of therapies really work particularly by targeting T cells. And so if you compare some of the traditional therapies I've told you about, so chemo here in purple in melanoma, basically, you know, was the standard of care for advanced melanoma. And then we had targeted therapies targeting BRAF. And while these therapies extended, you know, the, you know, life of these patients by a few months, ultimately it didn't have a big effect on overall survival. But we can start to see something important about immune checkpoint therapies. Because you see this elongated tail, which shows that even if some patients did not respond, some of the patients that responded, they basically had very durable response. So the response was long, and, and these patients were in remission for a long period of time. And in fact, if you combine some of these checkpoint immunotherapies, you actually have increased effect to this degree. And this really conveys the point that these therapies are the future of oncology because they're conferring durable responses that we've not really seen before, especially in these really aggressive cancers. And the idea of durability makes sense because you, you think about it and you say, well, the immune system, the immune system has memory. Right? So it remembers the, the tumor, and so even if the tumor comes back, the immune system can respond to it. And secondly, the immune system is adaptable. So even if the cancer changes, as cancers usually do, the immune system will change with it. And so this sort of therapies really give us the, the idea of what could be if we could really identify how to make— how to really direct immune therapies to cancer cells. And this is not just lines. This is not just like, you know, graphs where you're comparing different things. It's beyond that. These are lives of people who have been affected by cancer and who in some cases are basically cured. You probably know of an example, which is the former President Jimmy Carter shown here laughing. When he was 91 years old, he was diagnosed with brain metastasis from his melanoma. Amongst other treatments, he had one of these checkpoint blockades. He's about 96 now and he's still laughing and he's still building houses somewhere. And so this really illustrates the fact that beyond just like, you know, looking at the efficacy on a population level, these are real lives of people that could really be transformed if we can figure out how to really expand these therapies to all cancers. And so the good news is that it works. The bad news is that it doesn't work for all cancers, and it also doesn't work for all patients, even in cancers where it works. And so I've shown here some FDA-approved indications for PD-1 blockade antibody called Pembrolizumab. And you see several tumors here where it's been approved, but I just identify here just two of, two of the most lethal cancers that exist: pancreas cancer and glioblastoma. These therapies don't work in those tumors. And so you can kind of just imagine if we can really take what we've learned from melanoma and use that to build new immunotherapies or combination immunotherapies that will work for pancreas cancer and glioblastoma, that will be really transformative. And that's the sort of, you know, that's the sort of questions that is driving a lot of people to work in this field. And so the question everyone is trying to answer is, well, why is it that some people respond and others don't? Why is it that pancreas cancer does not respond but melanoma does? Well, to really answer this question, it's really important to put this in a context of how does anti-tumor immunity work? What are the central principles of anti-tumor immunity. I've really summarized this in 4 steps. 4 principles of anti-tumor immunity. First, the immune system has to recognize the tumor and differentiate it from normal. Second, the immune system has to be activated, and so it needs to switch on. Third, the immune system needs to go to where the cancer is, so it has to traffic or infiltrate into the cancer. To kill the cancer cells by eliminating it. And so I call this RAGE because these are the 4 principles you need for the immune cells to rage against the tumor cells. And again, I show here, because this is very important, that multiple immune cells are capable of enacting anti-tumor immunity, not just T cells. And so I'm going to go through the steps one by one and give you little examples of how these principles really shape how tumors develop, how immune checkpoint inhibitors work, and finally, how we can change this kind of ineffectiveness of ICTs and make it better in many other tumors. And importantly, this will also give us an idea of how to design new immunotherapies, because now if we know what works, if we know the principle of anti-tumor immunity, then we can really go after these tumors. And so the first, you know, the first principle when it comes to raging against cancer is recognition. So the immune system, as I said earlier, has to differentiate between tumor and normal cells. And there are many things that differentiate tumor and normal cells, even though, you know, cancer is very close to, to self because it arises from our tissues. But there are certain things that cancer cells have and make that normal cells tend not to have. And an example of that are peptides that are derived from a lot of mutations that accumulate in cancer cells. And these peptides are processed, shown here, and are loaded onto MHC Class I and displayed on the cell surface. This is what T cells recognize. T cells cannot recognize anything else. Particularly conventional T cells. And so it's very important that cancer cells display this on their surface to really allow T cell recognition of the cancer cells. So we also know that it's not only mutant peptides that are seen by the immune system. In fact, there are other modifications on proteins, even on normal proteins, that are not present on normal cells but are present in tumor cells. But this is the dark horse of immunotherapy. People don't really know much about this, and we need to do a lot more work to understand the immune responses to these aberrant post-translational modifications. But in this case, I'm going to focus more on what is known, on what we know, and then go out from there. One more thing I would say here is that different immune effector cells have different substrates of recognition. So whereas T cells see mutant peptides, B cells can see many other things. B cells can see proteins, they can see sugars, they can see lipids. You know, NK T cells see lipids, and there are all these different modes of recognition that we must take into account if we're trying to really accommodate the whole immune effect of cells in antitumor immunity. So just to give you an example, as I said earlier, CD8 T cells recognize peptide antigens on MHC class I, and these peptide antigens supposedly on tumor cells and not on normal cells. And so this confers specificity to the response, and this is why the CD8 T cells can specifically recognize tumor cells. And you can imagine that if tumor cells get rid of their MHC class I and cannot display anything on their surface, then you have no recognition. And so this is actually, you know, by understanding this principle, we understand very quickly how tumor cells can evade anti-tumor immunity. So the second thing is activation. So activation of immune cells is super important because you don't want your immune cells to be on all the time. You only want to switch them on when you need them and when the occasion calls for it. Otherwise, you cause autoimmunity. The mechanism of switching on T cells is very simple. You need signal 1, you need signal 2. In fact, you also need signal 3, but here I'm going to concentrate on just signal 1 and signal 2. The first signal is the recognition. You bind with the T cell receptor, the TCR, to the MHC class I, and then the binding of CD28 to B7 or CD80/CD86 mediates that second signal, which now allows that T cell to be activated. So the T cell not only needs to be activated, in the secondary lymphoid tissue, it also needs to keep being activated even when it becomes an effector T cell. And so this activation is not something that happens once. It's something that needs to be— the switch needs to be on while the T cell is enacting its function as an effector cell. And so this brings us to immune checkpoint inhibitors again, and it explains exactly why these therapies work, because it turns out that tumor cells, either by themselves or by remodeling their ecosystem, they really inhibit T cell activation. And so this second part is important because this is the major way that tumor cells and their microenvironment turn off T cells. And they do this by the interaction between, for example, PD-1 and PD-L1, which are the checkpoint molecules. And this also happens with CTLA-4 and in competition with, uh, CTLA-4 and CD28. And this basically makes sure that you don't get priming of the T cells. And so what you start to see here is that now when you use anti-PD-1 antibodies, then you can block that interaction between PD-1 and PD-L1, and that allows the T cell to turn back on. And become activated. The same happens with CTLA-4 antibodies. When you use CTLA-4 antibodies, you release that brake on the immune system, on the T cell, and you allow it to activate. And so the next important thing is that it's not just PD-1 and CTLA-4. There are multiple other checkpoints. So you have TIM-3, LAG-3, TIGIT, and many others. And some are stimulatory, some are inhibitory. And so the idea here is that by targeting these checkpoints and either encouraging the stimulatory checkpoints or inhibiting the inhibitory checkpoints, we can really reverse this inactivation of the immune system, particularly T cells. And the second part of this is obviously that the immune microenvironment matters. And it matters because you have immunosuppressive cells and molecules in the tumor microenvironment that prevent the activation or inactivate T cells. And so this is another form of interfering with the antitumor immune response. But interfering with the activation. The next one is to go. The cancer cells are embedded in this tumor microenvironment and the T cells need to find them. The T cells need to leave the secondary lymphoid organ, traffic and infiltrate into the tumors.. And so in this particular process, chemokines, selectins, and integrins are very important players. And what we notice also is the fact that just like the tumor environment blocks activation, the tumor microenvironment also impedes immune infiltration, and there are many examples of this. So for example, we see that the CAFs in the microenvironment produce a lot of extracellular matrix, which might in some cases disrupt the infiltration of the T cells. Another means of blocking this is basically by using chemokines that also block the infiltration of the T cells. A very good example is CXCL12, and this is, you know, very beautiful work done by Doug Fearon from Cold Spring Harbor. Which really shows that, that this interaction between CXCL12 and the CXCL4 on the T cells really inhibits their infiltration into, into the tumor nest. And so this critical aspect really mediates anti-tumor immunity, because if the T cells or the immune cells cannot be close to the tumor, or they cannot enact their effective function by being in the tumor microenvironment, then they're ineffective. It doesn't matter what you do. Even if they're activated, they stay outside. And finally, it's important then for the immune effector cells to enact their effective function. And so exerting the effective function requires multiple things. Number one, it requires all of the above that we've talked about, but also it's important to note that different immune cells have different ways of enacting their effective functions. So for example, T cells will, you know, dock with tumor cells and secrete granzyme and perforin, for example, and induce apoptosis. They can also do this with interferon gamma and TNF alpha here. Other immune cells have different methods. For example, macrophages, they're big eaters. They can eat the tumor cells in the right conditions. And so tumor cells have found out ways to either directly inhibit this effect of functions or to, again, use their microenvironment for this purpose. One of the ways this is possible is really by activating autophagy. This is something that has been recently found that activating autophagy actually blocks the effect of TNF-alpha from the T cells. This reduces or even in some cases, completely blocks the elimination of these cancer cells. Another mechanism is tumor cell resistance to apoptosis. So I previously mentioned that tumor cells, when they're in contact with T cells, one of the things that T cells use to really exact effective function is to induce apoptosis in these cancer cells. When tumor cells become resistant to apoptosis by a variety of means, then they become resistant to this form of killing. And so it's important that even if tumor cells are resistant to a form of killing by a certain immune cell, By taking advantage and leveraging other immune cells, perhaps we can reverse this sort of resistance. Another well-known mechanism is the interaction between Fas ligand and Fas. And it's possible that the tumor cells could express Fas ligand, which engages with Fas in the T cells and induces their cell death. And this basically automatically blocks any effect of function activation and all that because the tumor— the T cell is dead. So knowing what we know now based on these principles of immune activation and immune effect of functions, we can now go back and actually quickly review why a disease so lethal like pancreas cancer is resistant to ICTs. Why is this disease resistant to, to, um, immune checkpoint blockade, um, and actually other forms of immunotherapies too? So we're going to run the tape back and, and go back to, to, to the four principles. One, we know that one of the mechanisms to differentiate between tumors and normal cells is by the expression of these mutant proteins in tumor cells. The more mutant proteins you have, the more recognition you can induce. And that's why, if you see here, you see some correlation It's not perfect, but especially at the extremes, it's very consistently true. You see that the, for example, melanoma in red, red arrow right here, shows high mutational burden, very high mutational burden. Whereas pancreas cancer, black arrow right here, doesn't have that high of a mutational burden.. And, and so this already tells us that recognition is disparate between these two malignancies. So that is one of the, the issues with pancreas cancer. And in fact, if you see right next to pancreas cancer, glioblastoma, which is equally as— equally deadly, um, like pancreas cancer, also has low recognition. So now we can begin to appreciate, um, the reason for this, for the low response of pancreas cancer and this deadly malignancy. And next thing is, we know from different studies now that there are impaired activation. So there are multiple immune checkpoints like I told you about before. PD-1 is one of them. Many groups now are reporting that TIGIT is an important checkpoint in the anti-tumor immune response in pancreas cancer. And so low recognition, you already have a lot of immune checkpoints, immunosuppressive microenvironments, which includes TGF-beta, Tregs, myeloid-derived suppressor cells, and importantly, cancer-associated fibroblasts. All of these cells, at least subtypes of them, are able to promote an immunosuppressive microenvironment and block the activation of T cells. And finally, I'm going to combine the last two principles, which is the trafficking and infiltration and elimination. And so first, you know, we've already established that fibrosis and deposit— deposition of extracellular matrix is actually a mechanism to increase the immune resistance and promote T-cell exclusion from the microenvironment. And the other mechanism we talked about is chemokines such as CXCL12. Well, it turns out that pancreas cancer has both of them. In abundance. And so this is an image I took, and this is a human pancreas cancer patient, and this is a tissue where you actually see really nicely that the stroma, which is where the ECM is deposited, is just abundant. And the cancer cells in purple, and they're denoted here with the arrow, are not as abundant as the surrounding stroma. So you can imagine how hard it is for immune cells, particularly T cells, to access this microenvironment. And finally, we know also in pancreas cancer that there's elevated autophagy, and this autophagy promotes resistance to TNF-alpha-mediated killing, and also it's been shown that Elevated autophagy also promotes the reduction of MHC class I expression on the cell surface, which also impairs the first principle, which is the principle of recognition. And so altogether, we have a clearer view of why this disease is really hard to treat with immunotherapies. But that's not the end, and that's where our lab comes in. So in the next few slides, I'll give you a very short peek into what my lab will be doing and what we're currently doing to really, you know, think differently and really harness, you know, our expertise to understand better how to trigger immune response and conquer all these four hallmarks or these four principles. And so one thing that we noticed right away is that, well, you know, the mutant peptides that are derived from the mutations are probably not abundant in pancreas cancer. They're there, but they're not abundant, and so there's low recognition. But based on lots of work from other people and our current work, We now know that there are tons of aberrant post-translational modifications on these cells. So these are modifications that we find in the tumor cells that we don't find in the normal cells. And we know that these modifications are substrates for immune response. So what are the mechanisms of anti-tumor immune responses to these aberrant post-translational modifications. And so this is a critical question my lab is trying to answer, because if you can increase the threshold and increase the recognition, then, then, you know, you can imagine a combinatorial approach where you can actually use some of the lessons we'll learn from post-translational modifications to really understand how to combine activation and recognition and really elicit immune response in pancreas cancer. And so we know there are many post-translational modifications. This is just some of them, but the ones we're focusing on right now is glycosylation. And glycosylation, we know very well, alters— is altered in pancreas cancer and actually in cancers in general. And so we know that glycosylation, like many post-translational modifications, are not encoded in the gene. They're not directly templated in the genome. Rather, they're determined by the levels of glycosyltransferases, glycosidases, and substrates that allow these marks to be added onto proteins and other macromolecules. And so, just to give you an example, the epigenetic state of cancer cells, the aneuploidy that is found in almost all cancer cells, mutations, those can contribute to skewing the expression and levels of glycosyltransferases and glycosidases. Altered metabolic states in cancer can also alter the substrates and also the mutations in consensus sequences, particularly on proteins, and also changes to Golgi and ER homeostasis can alter the glycosylation of cancer cells. And so the big question then is, how do we understand how immune cells respond to this altered glycosylation, and how can we translate this to effective immunotherapies? And this, this will be the last part that I'll talk about, and this will be very brief. So the main things we're doing, and this is a major aspect of our work, is basically to use similar strategies that immunologists have used to understand the immune responses to protein antigens, and that is to use model antigens, to put in a foreign glycoantigen in the cancer cells, put them in the right microenvironment, and ask the question, what is going on? What's the functional response? What are the cells responsible? And so this functional profiling is a key part of what we're currently doing, and it's starting to yield some very interesting insights. Into how this post-translational modifications can powerfully trigger anti-tumor immunity, and we're starting to now understand the cell types that are responsible for these responses. And one of the things we're really excited about doing is to leverage our expertise in antibody screening to really now develop novel antibodies to activate the immune response to glycoantigen by using various constructs to induce response by the immune cells that are typically responsible for this activation and immune response. And so what we hope to achieve is to really understand the ins and outs of immune response to post-translational modifications, which is really not well understood at the moment, and then hopefully develop new tools to facilitate this immune response in cancers that particularly don't respond to the conventional immunotherapies. And so in summary, we seek to establish a fundamental understanding of the antitumor immune response to glycoantigens, and we will expand this to other post-translational modifications as well. We think people have forgotten about PTMs, and I really hope our data and our work will really inspire renewed focus on post-translational modifications. And finally, With our antibody strategies, both using conventional antibodies and various other types of novel antibodies, we seek to really show an example of how you can leverage this immune response to glycoantigens and use that to trigger immune response, very powerful immune response, in malignancies such as pancreas cancer and potentially glioma. And with this, I want to thank you for listening. I think this is an area that we're very excited about. So I know there are some scientists in the audience. If you're thinking in this area and you're thinking in this kind of field, reach out to me. I would love to talk about our strategies in more details, and we really think we're on to something that could be potentially clinically relevant.

So thank you. Excellent. Thank you, Toby, for that beautiful talk and a clear explanation of what's going on in the anti-tumor immune response. So please continue to put questions in the Q&A, and we'll get to as many of these as we can. Perhaps I'll kick it off with a question of my own. So you paint such an interesting picture of how the cancer is able to evade the immune system. The immune system is trying. Now we are trying to intervene into this path and this battle of immunity and anti-immunity. How do you think these types of therapies can overcome that back and forth battle as the cancer is ever evolving, trying to evade whatever we throw at it?

That's a great question, and that's something that actually drives some of the work we're doing. We know that in some cases in the clinic, there have been very clear demonstrations of adaptive resistance and also acquired resistance to these immunotherapies, especially the checkpoint blockade. What is clear though is that compared to other forms of therapy, you know, it's very clear that this type of therapies do a way better job at, you know, staying with the cancer and evolving with the cancer. When it doesn't, then, you know, it's important for us to understand why. And how to, you know, trigger that immune response again. And that's the beauty of this because, you know, in a situation where the immune response is lost, because let's say, you know, you go back to the 4 principles, you know, let's say you lose MHC class I, and so you don't, you know, the cancer cell does not express the antigen anymore, then you have to go back to the Joint Board and say, well, Maybe if we understand that the immune response to post-translational modifications, can we induce that? And that way there's a double whammy that the cancer cell cannot escape. And the same is true for the other principles: activation, you know, going, trafficking, and also elimination. Every single step of that, if you think about it, you know, The fact that, you know, if the tumor cell evolves something to block that, then you ask the question, well, what about other immune cells? You know, macrophages, what about them? They eat the cancer cells, they can do that. Whereas, you know, the T cell takes a different approach. So it's really the diversity of responses and also the ability to kind of understand what's going on and adapt according to that, I think that will really be the central benefit of this sort of approach.

Excellent. So we're getting some questions in the Q&A about epigenetics and these PTMs related to cancer, including, could there be a correlation between post-translational modifications and epigenetic factors in how cancers respond to the immune system?

Oh, absolutely. And there are several people studying this that I know of at the moment. It's very clear. I mean, so number one, if you look at different tissue types, you see that there are different expressions of glycosyltransferases, which tells you that, you know, just in normal biology, this is something that is important for, you know, tissue type function. And then when you look at tumors, you realize that a lot of these glycosyltransferases are changing dramatically and consistently, and you're actually able to predict the types, you know, if you have like transcriptional subtypes of a tumor, you can predict based on the glycosyltransferases expressed you can predict the subtype. So epigenetic factors for sure have a role to play in, in this. And also importantly, these glycosyltransferases are also triggered by oncogenes. So if you activate KRAS, some of the most important, some of the most highly expressed genes are going to be glycosyltransferases.

Excellent. Sort of related question asks, are these PTMs more unique and distinct, something that only happens in the tumor? Is it more of a shift in the ratio of how much post-translational modifications exist?

That's another great question. Thank you for that. In a lot of the cases that we've been able to see, we do see that you get low expression in some normal tissues and you get super high expression in cancer. And maybe in inflammatory states, you could also get high expression of some of these glycosyltransferases. Recently, what we found is that we were able to find some unique glycoepitopes, some unique glycoattachment on proteins that we've not been able to find in other normal tissues. And so the limitations of PTMs and why people have not really studied them a particular glycosylation is that it's really hard to characterize it. It's because they're not templated, you know, it's really hard to really characterize them. And so because of that, it's been hard to actually ask the question, what are all the structures that are changed in cancers versus normal cells, like in a very comprehensive way? And we hope to be able to do that with newer technologies.

Excellent. This next question is asking for a bit of more clarity, want to hear more about your thinking on the topic of how this foreign glycoantigens will work given that the tumor is already evading the immune system.

Yeah, I think about this similarly to the checkpoint blockade immunotherapy where recognition is intact. You have recognition. What is impaired is activation. So probably initially during the life of the tumor, you get immune response, and that immune response gets lost because the immune cells get inactivated one way or the other. So by understanding the basics of this kind of immune response to PTMs, particular glycans, aberrant glycans, then we can really understand how the tumor cells or the microenvironment turns it off and how we can turn it back on. So I think about this very similarly to ICTs, and, and indeed, um, it's possible that we'll find similar things.

That brings us right to this question of the tumor microenvironment. Is there any concerns that it could mask these unique glycosylation events?

Not particularly that I've thought about. Masking, I don't know in what context, but I anticipate that it's possible that maybe you don't get release of this glycoantigens because it's binding to some structure in the microenvironment so that it doesn't really get out and/or It doesn't get picked up by dendritic cells or other cell types that could present antigens. Or it's possible that it doesn't get to the cell that actually is responsible for the effector response. So it needs to get out of the— it needs to get beyond the tumor to be, you know, to be recognized by the immune cell. And if that doesn't happen, then it's possible that, you know, if it's stuck in a microenvironment, then it's possible that, you know, you don't get immune responses to it. But the evidence so far suggests that a lot of those glycan marks are— you actually see them in circulation, so they get out. So in terms of masking, it's unclear what exactly the questionnaire meant. But from that perspective, I don't anticipate that we will get masking. It's possible that we get inhibition of activation. And that just means that we need to understand the activation and then find ways to activate it, to basically reverse that inactivation. And then pair that with biologics to induce like a very effective immune response.

Excellent. This next question is asking about anti-fibrotic, and would that be necessary to add on to immunotherapies to increase efficacy?

Yeah, you know, this is something that many of my colleagues and friends are studying very heavily, and it seems that this will be very important in terms of, you know, allowing immune cells to come in and really, you know, giving them a platform to actually enact their effective function. Sunil Hingarani has done some work on one of the ECM proteins aileronin, and has shown that actually if you decrease that with PEG-PH20, you can potentially increase immune response in pancreas cancer. You can allow the T cells to come in. So that is certainly something that should be explored, and I'm pretty sure a lot of people are currently looking at this.

Great. Next question asks, would the dependence of glycoantigens on the epigenetic state make it more Is it susceptible to the emergence of resistance, considering how dynamic the epigenome is and its sensitivity to the environment?

Yeah, that's another great question. What we're starting to see just by analyzing, you know, datasets— and several other people are doing this too— what we're starting to see is that these epigenetic states are linked to many other functions important for the tumor cell. So if the therapy forces them to go away from it, then it might render them susceptible to something else. So it will be something that will be important to really pay attention to. But one more thing I would say about that is that some of those glycans are also functional. They are actually selected for for in the tumor evolution. So they're helping the tumor do something. And so even if they're— if the immune system can recognize them, they're still helping the immune system— the cancer cell to survive in some way or the other. And so by reducing that, you know, it also might reduce their fitness. And so it's multifactorial. I'll say it that way.

Interesting. This next question asks about the PTMs as use beyond recognition handle. So could they be used as any diagnostic tool for cancer progression?

Could you look for abundance of a PTM and predict what might happen with certain treatments? Yes, we're— it's part of the work we're doing in the lab, and I focused this talk on anti-tumor immunity, so I didn't get to talk about some of our work on early detection. But this is something really dear to my heart and, and something I'm really passionate about also. And we think we can, you know, use these antibodies to comprehensively determine, you know, kind of early cancer. And if you can determine early cancer, well, maybe you don't need immunotherapy, maybe you just need surgery and radiation, and then, you know, you go home. And so that's certainly something we're looking into.

Just a couple more here. A bunch of interest in your talk, clearly. So can you comment a little bit about glycoRNAs in cancer?

Yeah, it's a fascinating new discovery that Ryan Flynn and Carlin Bertozzi, you know, put out a couple weeks ago. It has potential to be a tool for cancer. It's still very unclear, you know, how exactly, like, what roles glycol RNAs play in cancers, and it will be interesting to see the, you know, kind of the set of glycol epitopes that are present on glycoRNAs, because if it's true that, you know, all this glycan that are specific to tumors are abundant on the cell surface, then, you know, they must be also on this RNA species that are displayed on cell surface, especially since some of them are sialic acids, which are very enriched in tumor cells.

Excellent. So one question here is asking for some clarity. They want to understand how you would go about inducing glycoantigens just in the tumor but not in normal tissues.

Yeah, so, you know, I kind of like, you know, talked about it like a cold shower, like quickly in, quickly out, but what we're doing is actually to use foreign glycoantigens that some human beings express, other human beings don't express it, and rodents don't express it. And so we use that as a model to basically have a defined glycoantigen that we can put into tumor cells specifically by using transplantation models. And so we put them into tumor cells and we put them back into the pancreas orthotopically, and we use this organoid system that I didn't get a chance to talk about today that really recapitulates the microenvironment, all the cellular diversity that you find in the actual tumors, both in mice and in patients. You find it when you transplant this— these organoids. And so we're using this as a system to really ask the question, What are the cell types that are involved? How can we, you know, trigger them in cancers that have been already established? And how can we focus the immune system, you know, away from, you know, ineffective immune responses to maybe neoantigens or peptides, but rather to PTMs? And how can we find collaboration between those those two things. One thing I would quickly mention is something that we often forget, that there is linked recognition. You know, when you think about how some vaccines work, you think that, you know, really inducing responses to glycans requires inducing antibody responses of the class-switching variety to glycans requires T cell help, and to really get that, you get, you know, you attach your glycans to a protein or to some very immunogenic antigen. And the same might be true of cancer because these glycoantigens are present on proteins derived from cancer cells. They're present on lipids derived from cancer cells, and there are different immune cells that could see aberrant lipids, there are different immune cells that can see aberrant peptides, and there is linked recognition conferred by this glycan moiety on that particular conjugate. So there's— the possibility here is huge, and I, you know, I want more people to study this. You know, I want people to get in touch with me and let's, like, study this because I think it's very important.

Excellent. I think that's a great note to end today's webinar on. So thank you so much, Toby, for that great discussion and presentation, and for all of you for joining us. We look forward to seeing you again in the fall when we resume the webinar series. Until then, take care and have a great summer.