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TIL trial: Dr. Klemen

Doc Talks

In this DocTalk, Dr. Nicholas Klemen from NIH discusses the TIL trial with PALTOWN Scientific Director Dr. Manju George.  Recorded in November 2023.

Manju George
Hello, everyone. Welcome to Doc Talks. Today we have Dr. Nicholas Klemen with us. I’m Dr. Manju George, the Scientific Director of Paltown Development Foundation, the nonprofit that supports COLONTOWN. It’s great to have everyone here. If you haven’t watched the first session with Dr. Klemen about trials at the NIH, the video is posted on COLONTOWN University in the Lecture Hall, please make sure to watch it. So Dr. Klemen take it away.

Dr. Nicholas Klemen
All right. Well, thank you so much for having me back. It’s really thank you for the opportunity to speak. I do apologize in advance. I’m still coughing and a little hoarse. I had some daycare-itis last week, two kids in two different daycares. So twice the fun. So please bear with me if I’m coughing every now and again. So yeah, looking forward to telling everybody a little bit about TIL therapy for metastatic colorectal cancer. I have no disclosures for myself, although our group does have a collaborative agreement with Iovance Biotherapeutics, which I’ll talk a little bit about what they’re doing.

Dr. Nicholas Klemen
So, just an outline of what we’re going to discuss today. I’m going to the very basics and I’m gonna try to keep this sort of very straightforward and not get into a lot of jargon and in depth, science. There’s a lot of material out there, available for those who are interested in that. But I’m going to talk a little bit about what TILs are- tumor infiltrating lymphocytes. Talk about how we build a TILL therapy, what it looks like clinically to get it and then I’ll talk about how it all started in melanoma and then how we’re trying to make it happen in colorectal cancer. And then the last thing I’ll talk about are just some clinical challenges in doing this.

Dr. Nicholas Klemen
So what are TILs. So, the first part is the easiest so TILs stands for tumor infiltrating lymphocytes, and the tumor infiltrating part just means that these are lymphocytes that are going into the tumor. So just in this simple cartoon, you can see the pink cells are sort of inside the tumor in various places. And it was first observed decades ago that there were these lymphocytes into tumors and for a long time, people didn’t really understand what they were doing or why were they there. Sorry for the image is being a little bit grainy. These are just histopathologic slides. So if you have one of your tumors removed, a pathologist looks at it under a microscope. So this is sort of like a high level view of what one of colorectal tumors might look like under a microscope. And the pathologist uses a special dye that can stain for a marker that’s on on TILs, and that dye appears to be brown in this. So here you can see the top three are just examples of three specimens where there are very few TILs. And on the bottom, these are examples of specimens where there are lots of TILs. So there’s a lot of variation. And as I’ll show you later, in colorectal cancer, it’s well established that this is an important prognostic marker. Patients who have many TILs in their tumors when they’re taken out initially, do have better outcomes.

Dr. Nicholas Klemen
Now, okay, so we talked about what the tumor infiltrating part of it is. That’s the easy part. Well, what are lymphocytes? Well, lymphocytes are a special type of white blood cell. And the main three lymphocytes that we think of are T cells, which can include CD8 sort of classically called cytotoxic lymphocytes, CD4 T cells, which are also called helpers. And there are lots of other types but the CD8s and the CD4s are the main two. And then there are other important ones that are regulatory lymphocytes. I could talk all day about those. There are also B cells which of course make antibody, those are B lymphocytes, and then there are natural killer cells. The important thing to know is that lymphocytes are the driver of the adaptive immune system.

Dr. Nicholas Klemen
Okay, so what’s the adaptive immune system? Well, you can really broadly think of your immune system as having two big, separate components. There’s the innate immune system and there’s the adaptive immune system. The innate immune system is kind of what you’re born with. It doesn’t require training. It’s like automatic. So it’s a rapid response. But it’s a very nonspecific response, if that makes sense. And the strength of the response is fixed. So it’s not like it gets better over time. It’s always the same. And there’s no memory with the innate immune system. So the innate immune system is more primitive, it’s more primordial. The adaptive immune system, the response can be delayed initially, but it’s highly specific to the specific thing that’s causing the immune system to go active, right, so if it’s like a virus or something. And then it’s adaptive so that it can change over time, and there is a memory function in it. So that’s a lot of text and description verbally, but I guess you could think of an analogy of your immune system as an analogy, think about your body and responding to threats, right? If you’re walking around the woods, and you hear and then turn around and see a giant bear charging at you. It doesn’t matter if you speak English or Spanish or whatever. Doesn’t matter if you’ve been to school or not. You’re going to run away. Everybody knows that this bear is a threat. Now, you know, you see some guy standing there with an axe. That also looks like something that is probably a threat. Although, you know, it takes some sophistication because on October 31, this is a lot less threatening. But then on the other end of the spectrum, maybe this guy here right, this is a scam artist. So a lot of people fall victims to scam artists because they cannot detect that very subtle threat that a scam artist presents. And so the immune system sort of works in the exact same way. The equivalent of the charging bear for your immune system is bacteria in your blood. So if your body sees bacteria in the blood, say E. coli cells. E coli is so different than anything that should be in the human body that the immune system does not need any special tools to go after that. They just blast it away immediately. Now you can think of the way a virus works, virus particles go in and so now this red cell is infected with virus particles. Now the cell itself, right, it’s your cell. So how does your immune system figure out how to kill your cell? Well, it can figure that out because the virus DNA is foreign, right? So this CD8 T cell can taste little pieces of this protein that are presented on the surface of the HLA molecules and can say that’s a completely foreign DNA fragment. So we’re going to kill that cell. Now cancer is really the most difficult thing for the immune system to get rid of because cancer cells really, they are your own cells. And the DNA is really almost normal. The genes usually will change by just like a single spot in the DNA and so it’s very hard for your immune system to detect a cancer cell. Now sometimes cancer cells do things that can trigger the immune system, like if they look like they’re sick or dying and things, but to actually specifically detect the cancer, it’s challenging. And really, in truth, T cells are the only immune cells that can “see”, the specific mutations in cancer now, give you this example here.

Dr. Nicholas Klemen
So a normal cell, right? You’ve got your DNA, the DNA becomes RNA, the RNA becomes protein, and then you make a finished protein product. And now what the body always is doing is taking little pieces of this protein and putting it in the MHC molecules. And that’s where the T cell then can come and taste that protein. If it’s on the MHC and can say, hey, that’s a normal protein. In the case of a cancer cell, you have the DNA it’s all the same, but there’s one spot where there’s a mutation. That one mutation then can go into RNA and for example, a K RAS right, a lot of people have K RAS mutations, and that’s a single change in the gene there. So now the RNA is a little bit different and then this amino acid fragment is all the same, except for one amino acid is different. Well, that becomes this finished protein. And now when those get presented, sometimes a T cell can sense those. T cells are really the only immune cells that are capable of detecting a mutation like this, no other cells can do that and so that’s why T cells are so powerful and that’s the main thing we’re interested in TILs. So when we say TILs what we are mainly interested in are the CD8 T cells and the CD4 T cells. Now there is also natural killer cells in TILs but sort of a separate discussion and we are not convinced they do all that much.

Dr. Nicholas Klemen
Okay, so how is till therapy built? Very simply, and I’m sorry, this is a little bit grainy, but very simply, the way that it works is that we remove a tumor from the patient’s body doesn’t have to be big. I’ll talk a little bit more about that later.

Dr. Nicholas Klemen
This is a cartoon of a cross section of the tumor. So here you can see the blue cells are tumor cells. These green cells maybe are myeloid cells, or some other type of cell. And then the little red ones here. These are, let’s say TILs, they’re maybe T cells and we can put this in a dish and give a special media with special growth factors. And these little red cells will grow in numbers and start leaving the tumors in the dish and they start killing the cancer cells. And they will eventually expand into large numbers and once we get enough of the cells we then reinfused them back into the patient after lympho depletion, which I’ll talk about a second. So that’s very simply how it’s done. So get again, just to sort of verbalize it involves a tumor, surgical excision of about a one centimeter one and a half centimeter tumor. That’s all we need. The tumor is cut into pieces put into dishes, the T cells then migrate out of the tumor, begin to multiply. Step four is I’ll talk a little bit more about later, but we have to identify which of the tills can see the cancer. And then we can either expand the cells for immediate use or we can freeze them actually and you can cryopreserve them for a long time and know and they’re fine and they’re happy.

Dr. Nicholas Klemen
Okay, so what does TIL therapy look like clinically? So it’s one treatment right? So normally when you get chemotherapy, you go in for cycles, and it’s every two weeks or every three weeks, and you go on a break and then you get the next cycle and it’s like over a period of six months or sometimes longer. That’s the way chemotherapy which is systemic therapy is normally done or even like BRAF inhibitors, you can take a pill you take it every day, every two weeks. It’s a constant thing. TIL therapy is a one time thing. It’s one treatment. Because it’s a living drug, right when you take a pill it’s a it’s a it’s a drug that gets metabolized that eventually leaves your body and it no longer does anything. When we give TIL we’re giving live T cells, those cells have the ability to live in the body to grow in the body and expand in the body. So when we talk about TIL therapy we’re talking about one treatment, but that one treatment is a big deal. So first it involves lympho depletion, which is five days of chemotherapy that we give before the cells, before the TIL. We also do Pembrolizomab we do four doses of that the first dose is given the day before the TIL, we have you come back to the NIH, if we do it here, we have people come back every three weeks for four doses total. You get the TIL infusion on day zero and it’s kind of anticlimactic. It’s a huge process but it’s just that you end up getting a IV bag basically and it’s full of white cells and they go in your body and that’s it. Then we give an immune stimulant for a few days after the cells go in. And we give this stimulant called IL2 and that is interleukin 2 and that helps the TIL cells to grow and be happy in your body. And then the last part is just recovery. Waiting for the body, the blood counts to normalize after the lympho depletion, waiting for fevers and cytokines to come back to normal. And so it’s a lot. So this therapy involves a lot of back and forth to the NIH, on our TIL protocol back and forth. And 1) it involves a single inpatients day of about two and a half weeks. So it’s a serious commitment.

Dr. Nicholas Klemen
So why is chemotherapy given before TILs? Well? I guess the simplest way to think about it would be if you were going to move into this house what would be the first thing you would do? And we would say well, let’s clean it out. Right? You got to make space. So that’s the most obvious reason why we give chemotherapy before TILs. It provides space for TILs. The chemotherapy gets rid of most of the existing immune cells in the body. And so that provides space for the T cells that we’re giving. Now a little bit more sophisticated explanation for that is that the chemotherapy promotes homeostatic expansion of TILs. What does that mean? Your body is always trying to achieve homeostasis, which means it’s at a balance, right? So if you’re too hot, your blood vessels dilate and you sweat because your body is now cooling you down. If you’re too cold, your body shivers to make more heat, right. If you have too much fluid in your body, you make more urine. If you don’t have enough fluid, your kidneys hold on to the urine. And so if you have too many T cells in your body, like if we just gave you TIL– billions and billions of T cells, but didn’t do anything else, your body would say hey, we got way too many immune cells. We don’t need this many immune cells we are going to let a whole bunch of them die. But on the other hand, if you give this chemotherapy first, now the body says Whoa, we don’t have any immune cells, right? We have a shortage of immune cells, we need to make more immune cells, we need to make them fast. So then your body is going to release all sorts of hormones that make T cells grow and be happy and we infuse the TILs just as that is happening. And so we think that that helps them grow and be happy. And then it’s also this chemotherapy potentially helps deplete a lot of the immunosuppressive cells that are in your body and in the tumor, right. So the body has regulatory T cells, which can shut down the immune system. The tumor can have macrophages and other cells that suppress the T cells. Because you may be wondering, well, if the T cells were in the tumor in the first place, why weren’t they working? Well, the answer was that they were there but they were suppressed or they were outnumbered and so depleting all the immunosuppressive elements can help release the brakes. So the expected side effects of TIL therapy, a lot of it is due to this chemotherapy. So because we give you the chemotherapy, it suppresses the counts that results typically in some anemia, so patients often require a few transfusions of red blood cells. Sometimes they require a transfusion of platelets. The chemotherapy also knocks down the neutrophil count. And those are white blood cells that fight bacteria. And so that leaves you vulnerable to a bloodstream infection for about a week. And sometimes that happens, then we give antibiotics and it’s fine. And those counts come back, but during that time, that’s part of the reason why it’s an inpatient stay to watch patients when this is the case. Now there are also immune side effects of the cells and the interleukin 2. So the cells, this can result in cytokine storm, which is something that happens with CAR-T a lot where you’re releasing a lot of immune hormones in the body and so essentially it’s like having the worst flu of your life for a few days. But it’s transient and gets better after a while. And so, this whole process also results in some fatigue and some loss of appetite. So it’s a it’s quite a slog. It was two and a half weeks in the hospital sometimes longer. Now, there are also unfortunately unexpected side effects of TIL therapy. And so, one of the things that can happen is organ failure, resulting from an overstimulated immune system. So what we’re trying to accomplish here is we’re trying to rev up the immune system to fight the cancer. But if the immune system gets overstimulated, it can be dangerous. And the two common problems we see is breathing issues, if there’s so much inflammation in the lungs, that can be a problem and it can require intubation in the worst case scenario. Usually, all these toxicities get better with time, but it can be problematic in the short term. And the kidneys also can take a hit from all this and in the worst cases, it can require dialysis for a month, although it’s pretty rare. Also, in some patients an unexpected side effect is a very delayed recovery of blood counts. So some patients who’ve had a really extensive prior chemotherapy when we give a lympho depleting chemotherapy for the protocol, they don’t recover as fast as we would expect. Now we’re getting good at learning how to dose reduce the chemo so that if we think hey, this patient’s had a lot of chemo before, we should maybe give less so that they recover better. We’ve gotten pretty good at doing that, but it’s still a potential risk that can happen. And then finally, there’s a risk of autoimmune side effects because we do get Pembrolizumab as a part of our protocol. Pembrolizumab has autoimmune side effects, and now TIL alone almost never causes autoimmunity. But the combination of them, it’s theoretically there could be even a higher risk of autoimmune side effects, so that’s not totally clear. So I’ve sort of talked to you about what TIL looks like clinically and you’re probably all thinking that doesn’t sound so great. So let’s talk about the data and why we’re excited about this treatment and why we think that this is worth pursuing, scientifically and clinically. And you can’t tell the story of TIL without starting with melanoma because that’s where this all started.

Dr. Nicholas Klemen
So up until recently, the median survival of metastatic melanoma was six to 12 months. And there were really no good long term effective therapies. And there are very few patients that made it out to five years. TIL therapy was first pioneered and described here in the Surgery branch by my boss who is still my boss, Steve Rosenberg. And this is from the seminal paper back in 1988 in the New England Journal. Here you can see this patient with metastatic melanoma back then they didn’t even do the CAT scans. But you can see these two big lung metastases this patient had and here you can see two years later, those lung metastases are completely gone. Here’s a patient with melanoma who had a whole bunch of metastatic tumors in the liver. And here’s 30 days after TIL treatment and you can see they’ve mostly resolved and this patient went on to have a complete response and was cancer free for years after this. And so this figure is why this job was so appealing to me, and why I wanted to dedicate my research efforts to TIL therapy, into cell therapy. A single infusion of TIL and in melanoma, in about 25% of patients will cause a complete response and what’s amazing about these complete responses is that they’re almost always durable. Meaning if you get a complete response, the cancer usually never comes back. So this figure here in the gray bar on the top, these are the complete responders. So we call them a complete responder at some point. And then if you follow these patients here, you can see five years and then it jumps to like nine years or something. But you can see all these little lines here. These little lines mean the patients were censored. So that means you know they were seven years after their treatment, so we can’t say they’re alive 10 years after the treatment, but we can say they’re alive seven years after treatment. So you can see all these patients got TIL and had a complete response. And now over five years later, their cancer has never come back. And so that’s really an amazing result in solid tumors, because you don’t normally see that you almost never see that with chemotherapy, right? Chemotherapy really never does that. And even some of the patients who had partial responses as you can see here, and some of the patients that even didn’t have an objective response by RECIST. Some of these patients actually had pretty good responses. And maybe they had shrinkage in a lot of tumors and maybe one tumor grew. And we took that tumor out surgically and that was the last thing they had, and nothing ever came back. So so these are the results that really excite us in melanoma. And so and then there was also more recently very recently, there was a randomized controlled trial where they took patients with melanoma who were refractory to immune checkpoint blockade, meaning they all got PD-1. And then they said okay, well that didn’t work. Let’s try something else. So then they randomized the patients to TIL or to ipilimumab and again you can see TIL was way better. And you can see that in about 25% of the patients they never progressed, the cancer was under control for years. So that’s the story of melanoma and so now I think finally till is being commercialized again. I don’t get any money from Iovance and no financial interest to me. I’m not like advertising for them here but they’re the company that’s hopefully going to be FDA approved this year. Or soon, I guess to do this for melanoma. The way it’ll work is that the tumors potentially get resected at any hospital right? So you go to your local hospital, you get a tumor resected. That tumor gets shipped to the Iovance facility. They then grow the TIL and get them all ready for you then they put them in a bag and freeze it and they ship that bag of TIL back to your hospital. And then your doctor at the hospital will do what we do here. They give the chemo, they give the IL2 and then give the cells.

Dr. Nicholas Klemen
I think this this really changes things, right, because for melanoma, patients sometimes get metastasectomy for Stage IV melanoma and attempt to cure the disease and in the past, those specimens were just discarded. But in the future, you can especially see now this is an option. You don’t necessarily want to throw those away. Those are potentially an invaluable patient resource. Of course, they’re not discarded. They’re studied by pathology and whatever, but they’re not preserved. And so with TIL therapy an option those specimens are invaluable.

Dr. Nicholas Klemen
Alright, so now, I’ve given all this background and so let’s talk about how are we going to do TIL therapy for colorectal cancer. So one of the main differences between colorectal cancer and melanoma is the number of mutations. So this is a figure that’s sort of commonly shown, but this shows on the x axis on the bottom, this shows the median number of mutations in the cancer and then on the y axis you’re seeing, this is the response rate to PD-1 blockade. And so you can see that metastatic melanoma is way up here. There’s a lot of mutations and they respond very well to PD-1 blockade and they also respond well to TIL And as I’m sure a lot of you know, colorectal cancers that are MMR deficient, which in the metastatic setting is only about 4%. But those 4%, they have a lot of mutations, and those patients do respond beautifully to PD-1 blockade. But if you look at the vast majority of colorectal cancers, 96% of them, the MMR proficient ones, they have a much lower burden of mutations. And they essentially never respond to PD-1 blockade. So that’s the main difference between melanoma and colorectal cancer, at least in terms of the immune system. But what’s interesting is that we’ve known for over 20 years that TILs are prognostic in MMR proficient colon cancer. So this is work first described by a European guy named Jerome Galon, and he published in Science back in 2006 and he basically showed that if you look at a colorectal tumor, like a primary tumor, and you look at the infiltration of CD3+ TILs in the center of the tumor and in the invasive margin, the patients who have more TILs did a lot better and in fact, he shows that looking at TILs, gives you better prognostic information that the TNM staging. So if theTNM staging, if you have no cancer in lymph nodes, you’re stage II, if it’s in your lymph nodes you are stage III, and then there’s like different subcategories. They generated this thing called the Immuno Score, which is actually better prognostic than the TNM staging. So they would show for example, that if you’re stage II with a bad immune score, you’re going to do worse than a stage III with a good immune score. Now, this is used in Europe. We don’t use it much here, but the bottom line is, this is MMR proficient colon cancers. We’ve known for a long time that even though they don’t respond to PD-1, they don’t have a lot of mutations that the immune system is clearly at least prognostic.

Dr. Nicholas Klemen
And now one of the other interesting things is that when we looked in the surgery branch, at the TILs taken from patients with these low mutational burden, MMR proficient (MSS) colon cancers, the run of the mill, not the the rare MMR deficient (MSI-H) ones. If you grow the TILs, and you look, we show in almost 90% of the patients, you could find at least one type of T cell that was recognizing a mutationn, in 90% of patients that were there. And so this sort of led us to an obvious way to change our treatment. So when we were doing melanoma, which we don’t do much anymore, but when we were doing TIL for melanoma, imagine this. This is a simplification but I think the concept is accurate. So we grow from one patient all the TILs, right. The TILs are all different. A lot of them recognize different things in the cancer, or they don’t recognize the cancer at all. So there’s a whole bunch of different types of T cells. And so some of those T cells are going to recognize the cancer and some don’t. And so you can imagine in melanoma, that maybe the red ones are the ones that recognize the cancer and maybe the blue ones don’t. And so, you know, most of these are recognizing the cancer and so what we do in melanoma is we just sort of take them the all, kind of kitchen sink approach, just take all these grow them up and then give them to the patient, it works great. Well, that works if you’ve got tons of mutations. But if you don’t have tons of mutations, as in the case of colorectal cancer, what you’ll find is that if you look at all the TILs, well, we can almost always find at least one clone that does recognize the cancer the vast majority do not. And so maybe a better strategy is to take these red ones, which are the tumor reactive clones, and grow those selectively. And now, we end up with a cell product that is selected for the ones that recognize the cancer, grow those and give those to the patient.

Dr. Nicholas Klemen
And so the first time we tried this, was with a patient with metastatic cholangiocarcinoma, she had lung and liver metastases. And here you can see, this is flow cytometry data. I’m not going to show a lot of science, but I figured I’d show this. Each dot represents a single cell. So these are her TILs. And so what this shows you this is a gene that she has in her body, we all have this gene, it’s called ERBB2IP. And I don’t actually remember exactly what it does, it doesn’t really matter. But basically these are her normal TILs, so here you can see they have this VBeta 22, which is the type of receptor they have. And this is TNF, which is tumor necrosis factor. Necrosis is a fancy way of saying kind of an ugly cell death. TNF is a protein that helps make tumor cells die an ugly death. So it’s a good protein to have. So here you can see nothing happens. If you take the mutated version of this gene that she had in her cancer and you expose her T cells to this mutated gene, you now see all of the T cells turn on and they start releasing TNF. And so we gave this patient her TIL treatment that was selected to be almost entirely these cells. And here you can see one of the liver tumors in 2013. And here 10 years later, they’re all gone and haven’t come back. So that was sort of one of our early attempts doing this.

Dr. Nicholas Klemen
And so now this is one of our big approaches for colorectal cancer. And so here’s a simplified diagram: we remove a tumor from a patient with colorectal cancer. And we plate the tumor in different spots so that you can see here, we’ve got different types of T cells. Now we grow the cells in the lab. And so here you can see there’s, 2 wells grew out green cells and 2 grew out yellow cells and 2 grew out blue cells. And then what we do is we check to see which of those recognize the cancer and if it turns out that maybe only the blue cells recognize the cancer, then we select for those and we treat the patient with those and the rest we just throw away. And so this is just an example of one patient with colorectal cancer. This patient had two liver tumors. Here’s another cut with two other liver tumors and here’s some shots of some lung tumors. And now this is again eight months after a single infusion of TILs. Here you can see there’s a little speck here, but this liver tumors gone. Here you can see this big liver tumor is shrinking a lot. This little one is almost gone, and the lung tumors are mostly gone too. We don’t know what will happen, because it’s still early, but this is a nice response. And so we have seen in colorectal cancer, we have a few patients who are years after a single treatment. So we do know that it’s possible for this to work as well as it does in melanoma, but we just haven’t figured out how to make it work that well. reproducibly yet. So that’s our main focus. And so how likely is TIL to work for a patient with metastatic colorectal cancer. Well, the first point I would want to say is that if we knew this would work, it wouldn’t be experimental. It would just be the treatment that you get. But we’ve spent the better part of 10 years studying this. I will say the vast majority, especially of our early attempts were unsuccessful and there are a lot of reasons for that I could sort of talk about all day. But the bottom line is most of them were unsuccessful. But we did have a few home runs in the sense that patients who’ve had one treatment that has lasted years, and it’s those patients that really have shown us that this can work and that’s what really drives us to keep pushing this forward. So we’re constantly trying to find new and better ways to grow this the TILs and to select them. And we’ve made a lot of progress on that front. And so over the tire entire experience, we actually haven’t even published our experience on this yet. So a lot of this is unpublished data, and so lot of people don’t know about it. And I would say over the entire experience, our average response rate is probably around 15 to 20%. I would say early on it was lower than that. And I can tell you sort of anecdotally more recently it’s been a lot higher than that. But hard to know whether that success is because we’re getting lucky or because our methods are actually better. It’s too early to say.

Dr. Nicholas Klemen
The final thing I wanted to talk about and then I’m planning to leave 20 minutes for discussion questions that you have have some. But I want to talk about the clinical challenges for this because there are many. The reality is we are very selective here about enrolling patients on these TIL protocols. And it’s not because we want to be, but it’s because we have to be. It’s a really complicated process. So we’ve got first the initial workup. So when I first see a patient, we got to know how soon does this patient need treatment? Because it takes us months to put this together. So if the patient needs a treatment in a month or in two weeks, it’s not feasible. We also have to know if they’re eligible for this study and we have eligibility criteria, age has to be younger than 72. And there’s some other ones that I’ll detail a little bit. So then if we say okay, well, patient may be reasonable to proceed, then we got to know is the surgery safe? Because the first thing we have to do is resect the tumor and can we stop the chemotherapy because we have to stop the chemotherapy at least a month before the surgery. Now this is a big sticking point because if if all the tumors are in inconvenient locations, we’re not going to put a patient through a major operation to get a tumor for TIL. And so this is a big issue that can come up. Then we’ve got to know well look, it’s going to take us three months to do this. So it doesn’t do anybody any favors if we take a tumor out and try to go to grow TILs, but if the patient doesn’t have anything that they can do in the meantime, and the cancer is growing, then they’re not going to be eligible once we get the TILs. So, again, this is another reason why we’re trying to predict all these things when we first see patients who say are we gonna be able to do an operation? Okay, what are the patient going to do to bridge while we’re getting this ready? Then let’s say we finally get the cells to grow. They don’t grow in everybody, but they grow in most people. And we say okay, now can we stop the chemotherapy? Is the patients still eligible? Because sometimes patients have progressed and they’re not eligible. We’ve got to make sure they’re going to tolerate the treatment. And so that’s even before they come in for the inpatient admission, so you can see that there’s a lot of considerations and so when we do this initial workup, we do turn a lot of people away and we don’t want to, but it’s just a reality. If we do not think we can get a patient to the end goal, the end goal being cells get in the body and the patient makes it out of the hospital safe, then we won’t start down this road.

Dr. Nicholas Klemen
So what are some of the key considerations for tumor harvest? As I mentioned, the patient needs to be off systemic treatment for at least a month, the longer the better, honestly. The tumor has to be at least one and a half centimeters. Bigger is not necessarily better. So a small tumor works just fine. Unfortunately, we can’t use the primary tumors themselves. Because they potentially are contaminated. So during the TIL culture, the bacteria from the colon or rectal primary tumor would contaminate them. The tumor can’t have previously been irradiated, can’t have like Y90, things like that. And then again, the key is that the purpose of the surgery is to take a tumor out for TIL we only do it if it’s minimal morbidity. So there are a lot of patients that need to get back on chemo ASAP. And we don’t want to put somebody through something that requires, 4-6 weeks recovery or longer, because we know these people often need to get back on their chemo. So we like doing minimally invasive lung surgeries. I do minimally invasive liver surgeries. We’ll do peritoneal tumors, something that patients recover quickly from. And again, it’s important also because not only patients need to get on chemo, but sometimes the TILs don’t grow. And so if you’ve gone through surgery, major surgery and then the TILs don’t grow, that’s not great.

Dr. Nicholas Klemen
Now, there is a reason why I’m particularly excited though about TIL therapy as an option for patients with colorectal cancer. And that’s because in colorectal cancer, as you know, metastasectomy is now a pretty routine part of clinical practice. So you can think about how complicated this is for most of our patients. But if we do metastasectomy and grow TIL from that, makes it much easier, right? Because all this is out the window. If the metastasectomy is clinically indicated, then if we can grow TILs from that fantastic and if we can’t, then no harm, no foul, and we can also cryopreserve the cells and so if we can get TILs this way then the question is just is the patient eligible? Do they want this? When do they want it and are they going to tolerate it? If the answer is yes, we get started. So we have a few patients who we took out their last tumor and they’re you know, NED but if they recur, you know, this will be an option for them if they choose. 

Dr. Nicholas Klemen
And so what are some of the key moments to consider joining a TIL study? You know, I’d say one of them, as I just mentioned, is again metastasectomy. In other words, that’s a elective surgery, non emergent elective surgery to remove a metastatic tumor. Commonly liver, also lung or lymph node, whatever. Another key moment to consider is if a patient has been off chemo for a long time, and now they have a new recurrence. That’s sometimes a good opportunity to join until study, you would want to talk to your oncologist to make sure they would be comfortable delaying resuming chemo for one to two months. And then also patients who are playing stop systemic therapy and let’s say go on a chemo holiday. That’s also a good time to consider joining a TIL study. And then what are some of the common barriers to joining a TIL study. So if tumors are causing major problems, like a bowel obstruction, significant infectious risk is a big no no for us, because again, we’re getting rid of your immune system, so you’re vulnerable to infection. So if you already have an infectious risk, that’s a big problem. So if you have infected fluid in the abdomen or liver infections can be a big problem. If there are problems with the immune system, like let’s say, HIV infection, or if patients are on immune suppressive medications like high dose steroids, that’s a problem. Patients who have more than three brain metastases. And then points 5 and 6, you know, declining performance status, like losing weight, appetite, fatigue, intractable pain or significant disease progression. Those are just signs that it’s unlikely that we’re going to be able to get to that a patient experiencing those symptoms, get them to the finish line. And we don’t want to put you put anybody through unnecessary agony and waste time for something that they ultimately are not going to get to try. The final comment, I just wanted to very briefly make, in the to zoom out 30,000 foot view again and the two big areas of Immunotherapy are cellular therapy which I’ve been talking about today and checkpoint blockade. Cellular therapy, you can think of TIL, TCR-T and CAR-T. So TIL we talked about today came first in 1988. TCR T therapy actually came second back in 2006. And that was done here as well. And in CAR- T has been around since 2011. The reason I mentioned that is because everybody’s sort of heard of CAR-T, but even oncologists are like “oh you guys have car T for this ?” Oh it’s kind of like CAR- T but 20 years younger. I just wanted to highlight that we do have this option of TCR-T which is basically it’s just like TIL but we get the receptor from TILs that recognize shared mutations, and we can engineer blood cells, without the need of a surgery, to recognize those mutations. And so, you know, the pros and cons of TIL is that we have the most experience with TIL but the con is going to require surgery and they don’t always grow well. The pro of TCR-T therapy is that there’s no surgery required so we can potentially get to treatment much faster. But there are two big cons, one is that the patient needs to have the target and an HLA match. And the other problem is that all all of our eggs are in one basket which is more of a theoretical problem, but we’re targeting one mutation, so that if the cancer cell loses that mutation, then the treatment may fail. To contrast these, CAR- T cells basically use a completely synthetic receptor and they can only target cell surface proteins. So CAR- T cannot target mutations in cancer. And so that’s a big problem for solid tumors. And so that’s why there’s some excitement for CAR-T ni solid tumors, but we’re less optimistic. But we do think TCR-T is promising. And so every time we do a TIL harvest we potentially can find receptors that can be used to treat other patients. So these are a library, a bunch of receptors that we have now, so we can target K RAS G12D, KRAS G12R, KRAS G12 V,

TP53 mutations, and we find all these receptors from individual patients. So this, this receptor here came from a patient with colorectal cancer. This patient got TIL and is five years out of their treatment, but we are using the receptor taken from those TILs and using that to treat other patients. So, that’s a really cool way that this therapy can can go forward and how each patient that we treat potentially can be additive to help us treat others. So that’s the end of my talk. As I said the last time feel free to reach out, our referral office email is here. And you can always email me directly. I’m happy to respond to emails, if you’re interested or have questions.

Manju George
And thank you very much, Dr. Klenman, especially since you’re sick and you still made the time to talk to us. Thank you very much. So I have a couple of general questions, and then we can look at the questions in chat. So can you talk a little bit about MHC and HLA, people are a little bit confused and what things to keep in mind and how is all that related to TILs or TCR- T?

Dr. Nicholas Klemen
Yeah, I sort of glossed over it, but the idea is that we’re really trying to differentiate how does the immune system figure out what is a normal cell and what is an abnormal cell. And the way in the case of T cells that recognize cancer, the way that it can do that is by sensing the individual mutations that are driving the cancer. So with these mutations, the problem is that they’re not necessarily on the surface of the cell, right? That’s the way CARs (Chimeric Antigen Receptors) work because they recognize things on the surface of the cell. So it’s something obvious that shouldn’t be there potentially, and you can target it with an antibody. These are mutations that are hidden inside the cell so the way the immune system works is it’s constantly kind of reaching inside of itself right? Every cell in your body is constantly it’s like taking a hand and reaching inside and pulls like little pieces of itself out and holds it up in the air and says Hey, T cell come smell this. Make sure it smells okay. And the T cell comes and checks it and if it smells bad, the T cell will kill that cell, right that your body’s constantly doing that. Every cell does that. And that hand is MHC. Every cell in your body has MHC and they’re constantly presenting little pieces of peptide. And that’s how your body detects viruses, right? Because you get infected with a virus so that MHC reaches in randomly grabs a piece of protein. Maybe that was a piece of viral protein, right? Holds it out and now the T cell says, Hey, here’s a viral protein, because otherwise, how would the T cell know what’s inside the cell? So that’s how MHC works. And that’s why T cells are so powerful because they can detect those subtle mutations, whereas no other immune cell really can can detect those.

Manju George
Okay, thank you very much, so we can look at the questions here. So the first question is, if you have multiple mutations, and TILs are selected against, for example, a KRAS mutation, are other populations of cancer cells affected or do you need a cocktail of TILs?

Dr. Nicholas Klemen
So when we, we sort of try to do some diversity. So when we when we check for mutations, we often have multiple different pieces of the tumor and will say, Oh, these T cells are recognizing this mutation. And these T cells are recognizing this mutation and these ones are recognizing a third mutation, we’ll often kind of put them all together. We do think that diversity may be good. And so we’ll go with that. And then the other thing we’ve been doing more recently, which some of you have heard, follow some of the science in the field, but you can generate organoids from patient tumor products. And an organoid is basically like a tumor that we grow in a dish. It sort of sounds obvious to check the T cells against your your tumor, right? I mean, that’s like the most obvious thing to do is like, why not just take a piece of the tumor and see if the T cells recognize that? It’s really obvious. The reason why we couldn’t always do it before is because you couldn’t get the cells to grow that well. Which is sort of a bad reason, right? It’s like a technical problem. But now we have these organoid platforms. And so from every patient, we’re trying to grow organoids and we check the T cells against that. And we don’t even necessarily know what that T cells are recognizing, but we’re finding that the T cells that recognize those organoids in particular may be very, very good T cells, so we think that’s a really good way to select them. Anyway, the long story short is I think it does give us a good cocktail of TILs and we do try to achieve that.

Manju George
So as against collecting TILs from a patient’s tumor, or like having the engineered T cells, or off the shelf products, you probably have more diversity if you’re collecting TILs from the patient. Is that true?

Dr. Nicholas Klemen
Yeah, there’s more diversity in a TIL product than a TCR product.

Manju George
Okay, the next question is, how severe is the immune depletion in terms of immunosuppression both immediately and long term?

Dr. Nicholas Klemen
So, in the immediate term, in the week after the chemotherapy, the neutrophil count is essentially zero. So that’s why we keep patients as an inpatient. Now, there is a risk of a bacterial infection, but if we do surveillance blood cultures, and if we spot one, we treat them with antibiotics and it squashes it and the blood counts come back seven to 10 days after the chemotherapy is done. And then long term. We do have patients take a prophylactic medication for like six months, when we wait for the CD4 T cells to come back because they can be kind of slow. But clinically the patients I would say it doesn’t really affect them. Now, the caveat there is we have had patients who we’ve treated who’ve had probably too much prior chemotherapy or their cancer has really progressed to the point that it was really causing a lot of problems. In some patients like that, sometimes their recovery can be a lot different, and not well. But if patients are kind of in good shape coming in long term, they fully recover and feel fine.

Dr. Nicholas Klemen
I mean, we just, you know, so yeah, I would say long term not really an issue.

Manju George

Okay, okay. So this is almost like bone marrow transplantation, the the lympho depletion that happens before that, is it?

Dr. Nicholas Klemen
It’s like a mini bone marrow transplant. We don’t have to give like stem cells or anything, but it’s the same idea.

Manju George
Okay. The next question is, Is this likely to be effective for treatment-resistant MSI high, KRAS G12D mutated CRC?

Dr. Nicholas Klemen
Yeah, it’s a good question. I mean, I think probably. I would have to imagine that it would be more likely to work than a run of the mill patient. And the reason we don’t know is we just haven’t gotten those patients. I don’t think we’ve ever had a referral from somebody who had a checkpoint refractory MMR deficient tumor but I think it would be more likely to work, probably.

Manju George
Okay. The next question is, can you please explain the clinical difference between TIL and TCR? I think your slides really very nicely explained it. The next question is, TILs do not live forever. So does the body learn to make more tumor specific cells or does it kill the cancer quickly so it does not recur?

Dr. Nicholas Klemen
I’m not sure they don’t live forever, actually. Most of them don’t survive long, for months. I mean any immune response, there’s like a big curve right? So that you get like a bunch of cells and then they taper off. But there’s always a tail. I actually do think the TILs probably live for years. And they probably control the cancer for years. Yeah, but the body also could potentially make more more cells, that’s also possible.

Manju George
So the next question is, who would be a patient that should be considered for TIL therapy? I think you kind of answered that in your talk, too.

Dr. Nicholas Klemen
Yeah. As I mentioned in my last talk, oncologists, and it’s not a knock against oncology, most people just don’t know what’s happening from a research standpoint. It’s not their job to know but also, like I said, we haven’t even published our experience, and I don’t know what other researchers are doing. I wasn’t a doctor, I would sort of assume that all doctors know like, really exciting trials and would be getting their patients on them. It’s just not the case. And so, the time to think about this is honestly kind of early, when there’s metastatic disease in play. The problem is when patients are early in the metastatic treatment course where there isn’t a lot of disease and they have good options, the oncologists don’t always have a sense of urgency, right? They may say, well, maybe we can treat this with surgery or the cancer is not growing that much and we’re going to control this for a while. And it’s not until all the chemo options are exhausted, and the cancer has really gotten out of control, that sometimes then we get those referrals and at that point, it’s very difficult for us, it’s more difficult than maybe it would have been. So I would just say when should a patient be considered, I highlighted those three points, when to think about jumping on a TIL trial. The earlier the better and especially if there’s an opportunity to get a tumor grown for TIL early on. You don’t have to use it, right?. It’s not a contract. I mean, we grow TIL from a patient and they don’t want to come here again then that’s their right and we’re happy with that.

Manju George
Okay, the next question is, have you had any success with BRAF mutated CRC?

Dr. Nicholas Klemen
Yeah, we pretty much only exclusively treat MMR proficient colorectal cancer and a lot of our patients have BRAF mutations and KRAS mutations.

Manju George
So, the person who asked about the MSI high immune checkpoint inhibitor refractory, that patient is saying that she wants to chat, so maybe you got a patient. This is a question from another patient. She says, so I’m currently NED, negative Signatura and not doing chemo. If my Signatura turns positive, and eventually a met appears on scans, is it best not to do anything and just let it grow to 1.5 centimeters so it can be harvested for a TIL trial?

Dr. Nicholas Klemen
Yeah, that’s a tough question. And it’s something you have to talk with your oncologist about. I can imagine two scenarios. I mean, if if there’s literally only one tumor, and it’s growing, then it certainly would be very reasonable, I think to watch one tumor and then say, okay, good. It’s like 1.5. let’s take it out, or maybe just a couple tumors. But I would say if there’s like a whole bunch of tumors growing and your oncologist is getting nervous. It’s just it’s just a discussion, it would all depend on what systemic options do you have left? What’s the molecular profile of the tumor? Does the oncologist feel like yeah, we can wait, versus no. So you know, short answer, maybe I guess.

Manju George
What’s happening in research to improve the response rates in CRC for TILs and is prior exposure to Immunotherapy and exclusion?

Dr. Nicholas Klemen
So the second question is no, it’s not an exclusion. Unless patients have had a really bad side effect. Like sometimes checkpoint inhibitors can cause permanent adrenal dysfunction, so patients are then on steroids for the rest of their life. If there was something like that we may not be able to treat patients, but otherwise it’s not an exclusion. And then in research to improve the response rates, it’s all about patient selection, right? I mean, think about this, if I said to you, okay, we’re going to take PD-1 inhibitors, and do a randomized trial of just all comers with metastatic colorectal cancer. The response rate would be like 1% Right? And if you did a survival curve, there would be basically over superimposing. And then you would incorrectly conclude that PD-1 blockade is not useful for colorectal cancer, but if you can identify patients, as we all know, that for MMR deficient patients, even though they’re only 4%, it’s a game changer for those people. And so, this is one of the same lessons here is that we are getting better at selecting the TILs, taking the T cells that are healthier, determining tumor reactivity, figuring out better ways to grow them. But then the other thing, in the future is if we can figure out a better way to select the patients who are likely to benefit from this, then it would become much more powerful. We can say, look, it’s not an option for everybody, but let’s say it’s an option for 30%. And if in that 30%, we can get the response rates up to 60, 70% that would be a really powerful thing versus, kind of a blunt tool that’s maybe not that useful for everybody.

Manju George
Part of that question is, if a patient progresses during bridging, they have the tumor removed, they’re getting chemo, and then they’re progressing. If the TILs are ready, can they move forward with the TIL trial?

Dr. Nicholas Klemen
Yeah, it’s not necessarily about progression that’s the problem, right? It’s just if the progression makes us nervous about doing the treatment, or the progression does something that makes the patient ineligible. So we have, like all clinical trials, there’s a lot of rules about lab values have to be a certain amount, kidney function has to be good. And, there can’t be these Infectious Risks. So if all those are still fine, even if it’s progressing, that’s not a problem. But if those things are changing, or the patient’s not doing well, then then it makes us nervous.

Manju George
okay. So, from what you’re saying, it looks like if, if there are if lots of patients when they have a metastatic lesion that can be removed if they come to the NIH and have it removed there, then your chances of finding more tools that are you know, that either you can grow and preserve or, you know, use it for other people or the same patient that will significantly increase right.?

Dr. Nicholas Klemen
We, I’m sorry to say one more time,

Manju George
Like, meaning if you get a large number of metastatic tissues that you can collect and then isolate those TILs and then you can have a library of TILs that are capable of being used in other people or for the same patient, then how applicable all of this is for larger number of patients would greatly increase.

Dr. Nicholas Klemen
So we won’t use the TILs for other patients but we do use the receptors and the TCR can come from different patients. So you can get it from a patient with breast cancer and use it in patients with colon cancer. One we’ve got from a patient with colon cancer, and we use it in pancreas. It doesn’t actually matter what tumor type it is, it’s the mutation they share. But yeah, the more patients we do this with the more receptors we find and we’re building this bank of receptors and if people have a match for one of our receptors and other people are doing this too, but then you don’t even need surgery. We can we can set up the treatment more quickly.

Manju George
Okay, we will go to the last question. So the patient asked if you have stable disease in lungs, with multiple lung mets, but are becoming intolerant to current standard of care chemo, would TILs be an option, or do you have to have some disease progression?

Dr. Nicholas Klemen
Yeah, it would be it would be an option. Yeah. I mean, there are rules about enrolling people on trials, there’s some subtleties. I mean, disease can’t be shrinking, right? We wouldn’t, if we went to enroll you the same day. I mean, we would do a tumor harvest and everything, but if we brought a patient in for treatment, and all their cancer was shrinking, we would say look, come back in six weeks and let’s scan you again. Because if we do this treatment and the cancer is shrinking, nobody’s gonna know whether it was the treatment or whether it was what you had before. But otherwise, yeah, that’s not an issue.

Manju George
Okay, you have stable disease, that’s fine, and you have lesions that you can harvest, that’s still okay. You don’t have to wait for things to grow more, to be eligible for TIL trials.

Dr. Nicholas Klemen
Yeah, great. Okay. There is that there is that month long washout, so it’s okay.

Manju George
So when they’re off chemo, waiting for surgery everything should not grow too much.

Dr. Nicholas Klemen
Well, again, yeah. I mean, it’s really not about the growth. It’s just about the consequences, the growth causes to be an issue.

Manju George
Yeah. Okay. Okay. Thank you so much. We are three minutes past the hour. And thank you very much. This was very informative.

Dr. Nicholas Klemen
Thank you for having me. Yeah. So I’m always available, feel free to reach out.

Manju George
Okay, thank you. And a video recording of this will be posted on Colontown University. Take care and hope you will recover from your infection soon. Thank you. Yeah. Okay. Take care. Bye.