OK, well, welcome, everyone very happy to be here to give you a little bit of an update on cancer immunotherapy. And, this is a really exciting opportunity, for a couple of reasons, Obviously, For those of you who know some things about human oncology, immunotherapy is really rapidly becoming what we call the fourth pillar of, of cancer care, along with surgery, radiation therapy and chemotherapy. And really great strides have been made in immunotherapy in the last couple of decades on the human side.
And it's very exciting that some of these advances are starting to trickle down into veterinary medicine as well. So it's a really opportune time for us to get a little bit of a primer on immunotherapy and the ways it has been used in veterinary cancer and, the way it's going to be used in a in a very, very near future. So very exciting.
So just a quick outline. This couldn't be quicker, I think, just in order to to make sure everybody's on the same page, I do have to unfortunately for some of you go back to do a very, very basic review of immunology. Probably some stuff that you learned back in vet school.
And that will actually sort of do a little history lesson about all the different things that have been looked at as, ways to treat, canine cancer either, on purpose, or perhaps even accidentally, that shows some promise, in dogs. And I do think for those of you who might be larger animal or mixed practitioners, I have one or two little tidbits that also apply to horses. That could be interesting as well.
So with that, let's go ahead and jump in. So we'll start with just a quick review of, of basic immunology principles. And again, it's not too bad, only a few slides.
But as I mentioned at the at the very beginning I, I do wanna say that all different kinds of, cancer immunotherapy approaches are actually being investigated in humans. So it's a really, really, exciting time to to sort of be thinking about this. And this includes monoclonal antibody therapy, which is coming in veterinary medicine.
Vaccine approaches, viruses, sort of non-specific immunotherapy. cytokine therapy, cell based therapy, all different kinds. Of things that are really sort of revolutionising the human cancer space.
And again, we're really excited that some of these are starting to trickle down into Vet Med as well. So very quickly, when we sort of take a big picture of the immune system in general, we sort of tend to initially divide it into two big buckets. And one of these buckets is what's called the innate immune system, and the other is what's called the adaptive immune system.
And innate immunity is generally what we consider to be the first line of defence against pathogens. So the adaptive immune system actually takes a little bit of time to sort of rev up and start to do its job. And in the meantime, it's this innate immune system, things like neutrophils, macrophages and K cells that actually sort of bear the brunt of trying to control an infection.
And while they're doing that, they're also able to help to rev up that, that adaptive immune system that we're going to talk about, in a moment and again, some components of the innate immune system, primarily macrophages and and K cells can actually play an important role in in tumour control or, in the case of macrophages, even tumour promotion, which is kind of a scary thing that we'll talk about quite a bit. And then again, the second part of the immune system is the adaptive immune system, and that can likewise be subdivided into two big components, one of which is what's called the humeral immune system. These are antibodies, and the other is cell mediated immunity, which is generally, mediated by T cells.
And here's a little slide that looks at this. So here we've got a little mouse, but this could be a dog or a human just the same way. When the body is reacting to a foreign pathogen again, there's two main ways this can happen.
One is through the Humoral immune response, which is mediated by B cells. When they encounter antigens and certain cytokines, they then differentiate into plasma cells, and those plasma cells make antibodies. And those antibodies can then bind to the pathogen and in some cases, sort of directly kill the pathogen.
Or they can, sort of serve as a little flag to other parts of the immune system to say Hey, this doesn't belong here. And then the immune system, other parts of the immune system can go in and pick off those antibody tagged. pathogens now on the on the T cell side of the equation or the cell mediated side of the equation.
Once again, we can subdivide our T cells into two main groups, and these are generally referred to these days as helper T cells and cytotoxic T cells. And as the name implies, the main job of helper T cells is to help promote the development of a robust immune response, whether that's a humoral immune response or a cell mediated immune response. But in certain circumstances that we'll talk about, a subset of these cells can actually be immunosuppressive, too.
And then we've got our cytotoxic T cells, and these are the ones that can actually recognise, foreigners, usually in the context of a of A of a host cell. So this is primarily something that happens with viruses so it can recognise a funky looking viral protein that's being presented on the surface of a host cell and then say, Hey, that cell doesn't look right. We better kill it.
And obviously you can imagine how this could apply to tumours as well. So just a couple other things. You may hear me use these terms, so I'm gonna introduce them.
Now, these these helper T cells are often characterised by the expression of, a cell surface marker called CD four. So sometimes you'll hear me refer to these as CD four T cells. The cytotoxic T cells tend to express a marker called CD eight, and you may hear me refer to these instead of as cytotoxic T cells as CD eight T cells.
So just a quick review of of antigen presentation. So I mentioned this very briefly. So there's two different kinds of antigen presentation that we think about.
And I'm actually gonna start on the right side of this diagram. With again back to our let's say, virus infected cell, certain kinds of cells, can be called antigen presenting cells, and their job is actually to chew up proteins and show them to the immune system. And in most cases they actually start by showing them to, our helper T cells, our CD four T cells and the cells that are capable of doing this primarily are things like certain kinds of macrophages.
a macrophage derived cell called a dendritic cell. And even some B cells can sometimes do this. And again, they do this so they can gobble up, proteins, all different proteins, whether they're foreign or not.
And then show them to the immune system in the context of what's called Class two Major histocompatibility complex or class two MH C. So little bits of protein get get shown on the surface here, and they get presented to all the different, helper T cells. And then some of these helper T cells that recognise this foreign antigen are gonna get revved up and start, making cytokines and proteins and things like that that can help to rev up an immune system.
But as I mentioned previously, these cytotoxic T cells generally recognise those same antigens or related antigens in the context of MH C Class one and every cell in the body actually has MH C class one, you know primarily so again it helps with the body's surveillance against pathogens. So there are lots of different strategies that have evolved over the millennia for tumours to figure out how to avoid being picked off by the immune system. So anytime there's a mutation in a tumour that codes for a protein, theoretically, that protein could look forward to the immune system and should be actually picked off by the immune system.
So there are lots and lots of ways that tumours have figured out how to stop that from happening. So one of them is through the secretion of certain cytokines that tend to dampen the immune response. And again, here's just a couple of the names I don't I'm not gonna go through an, a long list of them, But you can see there's quite a few there, immune checkpoints, which we'll talk about more later, then, immunosuppressive cell recruitment.
So actually, tumours can, send signals out into the world to actually encourage immunosuppressive cells. Like certain kinds of macrophages that actually tend to dampen an immune response. These, this certain subset of helper cells that actually are immunosuppressive Get those guys actually into the the the micro environment of the tumour to help to, keep those, cytotoxic T cells and antibodies away.
And then you can actually have, down regulation by the tumour cells of the target antigen. So again, if you've got one little antigen that looks forward to the immune system, there's ways that the tumour cells can actually say, Oh, I don't wanna show that too much to the immune system. I'm gonna get picked off, and there's ways that that can actually be reduced.
So the, as sort of a cloaking device for the immune system. And then similarly, you could actually have components of MH C that are down regulated that, means that not only are the proteins not there but the little framework on the surface of the cell that needs to be present so that the immune system can see that protein could also be missing. And again, this is just a, a very, sort of brief list.
There's lots of other ways that this can happen, but it just goes to show you really for any tumour in the body to actually evolve to to be clinically relevant. It has figured out how to defeat the immune system already, So we're we're really up against it. When we're trying to come up with a way to use the immune system to combat these tumours when they have already evolved, lots of different strategies to avoid getting picked off, just something to keep in mind.
So one quick example that about sort of how this is relevant that I think is really neat. And this is actually a paper from, Doctor Anne Avery's lab here at at Colorado State University. So she actually looked at at, canine B cell lymphomas.
And as I mentioned previously, there's a subset of B cells that can actually act as pro pro professional antigen presenting cells so they can actually show antigen to the rest of the immune system. And they do that in the context of MH C class two. So it turns out that different, canine lymphomas can have different amounts of MH C class two on their surface, and those tumour cells that have high levels of MH C class two, actually end up having a conferring a better outcome to their hosts than if you're a canine lymphoma that has low levels of M MH C class two.
And we think The reason for this is that again, these class two high expresses can probably, probably look more foreign to the immune system. And they're more easy to get picked off by the immune system, especially when chemotherapy is given, then those ones that have low levels of class two. So really kind of cool practical example where hey, it looks like this component of the immune system actually has a really important role in dictating how well a patient's actually gonna do with chemotherapy.
And this is something that we can measure, quite easily with flow cytometry using our sort of standard diagnostic panel that, that we use, routinely here for our B cell lymphomas. So another, group of cells I wanna introduce are what are called myeloid derived suppressor cells. And these are one of these immunosuppressive, groups of cells that are generally immature macrophages or immature neutrophils that can actually get into tumours and do bad things actually make tumours worse so they can increase blood vessel growth.
They can cause immunosuppression. And there are actually been several studies that have been performed in dogs with cancer that actually show that the more of these you have either in the blood or in the tumour tissue. The worse you do.
Especially in dogs with mammary cancer. So this is something that again exists. We think of the immune system as being a good thing, but here's a component of the immune system that actually does bad things when it comes to cancer.
And again, even in dogs, it's actually associated with a worse outcome. So ways to get rid of these or turn them off or change them could actually be a very important immunotherapy strategy. And that gets into this, which is also important.
So, macrophages, which are these sort of non-specific immune cells that we mentioned at the very beginning part of the, the innate immune system actually come in two basic flavours and we're not gonna kind of get into the weeds here. But just like with myeloid derived suppressor cells, there's a subset of, of macrophages that are actually bad, and it's these ones that are referred to as M two macrophages. So these are tumour promoting macrophages.
So these actually, make tumours grow better, increase blood vessel growth, potentially hasten metastasis and actually do a whole bunch of things to dampen the immune system to actually make these tumours more hidden from the immune system. Conversely, these M one macrophages are ones that are actually also called inflammatory macrophages. So these are ones that actually sort of rev up the immune system and, maybe make the tumour a little bit more, obvious to the immune system and promote a good immune response so you can get what kind is usually present in most tumours.
It's these M two macrophages, these bad macrophages, and once again, strategies to get these, keep these out of tumours or change them to make them look more like these guys. Is a very interesting, treatment strategy for some kinds of cancer. So once again, not only in dogs or not only in people and mice in dogs.
Actually, there have been studies suggesting that these, M two macrophages are bad in dogs. And, even if you just look at total macrophage or monocyte numbers in the blood. So there's two studies once again coming out of this institution, Colorado State University, that have actually looked at blood monocyte numbers both in lymphoma.
I'm sorry in osteosarcoma and lymphoma and actually shown that the more monocytes are floating around in the blood, the worse the outcome is in patients with osteosarcoma and lymphoma. These are dog patients. And we think that the reason for this is that, the more monocytes you have floating around the blood, the more of them have the potential to get sucked into the tumour and turned into these bad tumour promoting M two macrophages.
So something that you can just read off a off a a plain old, complete blood count that you're doing in your clinic every day. That could actually give you a little bit of information about prognosis that could be important to confer to an owner. So are there strategies that we could use to actually maybe keep these cells out of, the tumour tissue?
Yeah. Here's one right here and again. This comes from, the lab of doctor Steve Dow, who is an immunologist here at CS U.
And he's actually used a repurposed blood pressure drug called losartan. Which it turns out, actually, has the ability to block signalling through what's called a chemokine receptor, which is a and chemokines are this group of, of molecules that have the potential to attract and recruit certain kinds of white blood cells. And this particular chemokine, which is called CCR two, is very important for macrophages monocyte recruitment into tumour tissues.
And if you block this chemo kind, you can actually reduce the number of monocytes in tumour tissue, and that actually results in slower tumour growth. And again, this is a drug that you can actually give to canine patients, which is a really interesting drug repurposing strategy for cancer. So we'll talk a little bit more about this.
So it turns out, that when you combine, losartan with palaia, which is a drug that I think we all know about. It's a kinase inhibitor that actually, interferes with not only so it's approved for canine mast cell tumours, but it has a potential to interfere with blood vessel growth and actually may also have some immunomodulatory effects. When you combine high dose losartan with palaia, about 50% of dogs with metastatic osteosarcoma are going to have either their disease stabilise or, as in this case actually experience meaningful tumour shrinkage, and this is significantly higher than with palaia by itself.
So one important thing to know is the dose of losartan that's actually used when we give it this way is 10 times higher than the typical anti hypertensive dose, and that can be very scary for some people. But we've utilised this in an awful lot of dogs, and surprisingly, we do not see problems with hypotension or other things like that, although we are careful to look for them. So again, a really interesting strategy that relies on locking, or keeping these tumour promoting macrophages out of the tumour tissue as a way to treat canine cancer.
So I mentioned previously that there is a subset of these helper T cells that are actually not helpers at all. They're actually bad T cells, and they actually suppress the immune system. And these are what are called regulatory T cells or T regs.
You'll hear me use that term in the older literature. You may have learned these as what are called suppressor T cells, Same thing, and again, here's, sort of some of the markers that they express CD four, CD 25 which is actually the high affinity interleukin two receptor and a transcription factor called Fox P three. So that's not all that important.
But fox P three is something pretty easy for us to be able to stain for in tissue sections. So we'll often use that, And again, these are bad T cells. These are T cells that you would also like to try and keep out of your tumour if possible.
I'm gonna skip that. So why do they matter in in cancer patients? Because there are many, many tumour types where the more of these regulatory T cells you have either in the tumour tissue or in the blood, the worse you do.
And the same thing is actually true in dogs. And again, what these do is suppress tumour specific T cells like CD eight T cells. And they are also capable of inhibiting innate immune responses, so interfering with the function of things like NK cells and the good and one type macrophages.
And again, if you can deplete these, regulatory T cells, you can actually have improved outcomes. What do we know about in dogs. So again, this is work from Doctor Barbara Biller, who was also in Steve Dow's lab here at CS U.
So, it turns out that dogs with osteosarcoma actually have more regulatory T cells floating around in their blood than normal dogs do. And the more regular toy T cells you have in the blood, the worse your outcome is. So these are dogs that have fewer numbers of regulatory T cells.
These are dogs that have larger numbers of regulatory T cells, and you can see a statistically significant difference in outcome in these patients that are treated with a combination of amputation and chemotherapy. So once again, this isn't something you can just read off a off a complete blood count the way MONOCYTE count is. But this is something that can be measured pretty readily with flow cytometry, final concepts that we'll talk about.
So are there ways that we might be able to modulate tumours to actually wake them up and sort of make them more obvious to the immune system? So one of them is through what's called danger signalling. So, basically, if tissues are damaged, either because of infection or because of trauma or because of something else?
A lot of the components in that tissue, so stuff that spills out of the cells into the surrounding area can actually modulate the immune system. And some of these are fungal or viral or bacterial products that can actually signal to cells in the innate immune system to stimulate the immune system. But, as I mentioned previously, direct damage to tumour cells.
So whether that's physical damage, whether that's lysis by viruses, whether it's something like radiation, for example, can all potentially send signals to the immune system to say, Hey, there's some damaged tissue out there really got to get in there and find out what's going on and potentially help to clean it up and that can actually stimulate a nice immune response. And so this is the way things like oncolytic viral therapy works. So there are ways that you can take attenuated viruses and actually shoot those into tumours on purpose to try and stimulate this kind of danger response.
And this is something that is being looked at in some very early studies in dogs and, just a final idea before we move on to some of the therapy options that we're talking about. So there's this sort of misconception that all chemotherapy is immunosuppressive, and I sort of say that in quotes. So there's a lot of chemotherapy that can be myelosuppressive, right?
So in other words, you can. The primary blood cell target of most chemotherapy drugs is actually neutrophils and neutrophils are certainly part of the the innate immune system. Absolutely.
But interestingly, most chemotherapy drugs do not have a profound effect on B cells or T cells. So, for example, if you take dogs with lymphoma that are getting chemotherapy and give them vaccines, they mounted a fine antibody response to those vaccines. You give a dog chemotherapy and you take their T cells out and ask how they work.
They pretty much work like like any other dogs, T cells so different story with things like glucocorticoids. But as far as sort of conventional chemo drugs, they don't really suppress the innate immune system as much as we used to think. And very interestingly, there are some chemotherapy drugs that can actually be immunos stimulatory so, and it really depends on on the mechanisms by which these tumour cells die, but a chemotherapy can actually, kill tumour cells.
Sometimes in a way that actually makes those tumour cells more obvious to the immune system and can actually stimulate a pro. A pro inflammatory, immune response. So rather than being immunosuppressive, certain chemotherapy drugs can actually be tumour immune promoting.
And again, this is just sort of like a ranked list of the drugs that tend to be the most tumour promoting. And just for funsies down here at the very bottom. What are one of the drugs that we see doxorubicin.
It's the drug. Adriamycin is a drug we use every day in the clinic, to treat dogs with cancer. So it's actually conceivable that some of the anti tumour effects that we see with doxorubicin could be actually related to, helping to stimulate an immune response, which is kind of just a neat, a neat thing to know about.
So, let's do a quick, little review of some of the immunotherapy approaches that have actually been looked at in dogs. So we already talked about losartan. So I'm not gonna go back to that.
That's one that's very interesting, but we will sort of talk about quite a few other things and sort of where they are in the framework of Hey, is this something I can actually do today? Is this coming? how practical is this for me?
Actually, to be able to do so? The way that we can divide up types of immunotherapy is is basically into active or passive and then into specific or non-specific. And there's some examples that you can see up here of all these different approaches.
So, an example of passive non-specific immunotherapy would be something like metronomic chemotherapy that can deplete regulatory T cells or checkpoint inhibitors, which we'll talk about at the very end. Then we've got active non-specific where you could give something that is, rather than depleting a cell or blocking an interaction, you're actually giving something to actively stimulate the immune system. So bacterial products or recombinant cytokines and these are things that have been looked at in in animals, then you can have passive but specific kinds of immunotherapy and the classic example of this would be tumour targeting monoclonal antibodies.
And this is something that sort of has come and gone in, veterinary medicine. But we actually make see, some lymphoma targeting antibodies on the way back that we may be able to use in the near future. And then there are active, specific approaches where you're actively trying to stimulate the immune system, but doing it in a specific rather than a non-specific way and tumour vaccines and cell based therapeutics, which we're not really going to have a lot of time to talk about today, but is really exciting in people are a couple of examples of that.
So one of the very first types of immunotherapy that was ever looked at actually dates to before the turn of the last century, which is pretty amazing to think about. And this was a guy named William Coley who was a surgeon practising in, New York City, who observed. And so if you can, if you can think about it back in the 18 nineties, this was really before the advent of antisepsis.
So an awful lot of patients who had surgery for anything got post-operative infections, and some of these were really horrendous and in fact, fatal. But Coley made this really interesting observation when he was operating on some patients with cancer that certain patients with cancer that actually developed a post operative infection could have really remarkable regression of their tumours as a result of that infection. And he was actually so impressed with some of the responses that he saw that he started purposefully infecting his patients with bacteria in order to try to stimulate this.
At the time, he didn't know it was an immune response but stimulate this response. And yep, some patients died, but these were usually patients with incurable cancer, and some patients again had these remarkable responses. So he then tried to refine this kind of after the turn of the century in the 19 hundreds and 19 tens to use sort of killed products.
He killed bacteria, things like that, and they never really worked quite as well. But he still saw some activity way back when, So this was really the first non surgical cancer therapy that was ever invented, and it happened to be immunotherapy quite interesting. And really, what happened?
Well, around 1915, World War Two, A little bit after that, people actually discovered the X ray and when the X ray was discovered. People started shooting x-rays at tumours and actually saw, you know, really interesting nice tumour control. And this basically fell out of fashion and disappeared off off of the everyone's radar for I don't know, seven decades.
So people sort of stopped looking at this until sort of, ways to modulate the immune system became to be looked at again, maybe in the 19 seventies, But we can actually thank William Coley for starting this whole thing out. So are there any examples of this in the veterinary literature or the human literature that that might be an approximation of what Coley used to see? I think the one that's that's potentially the most interesting is this observation, which is again here at Colorado State University.
For decades, we've been exploring ways to, do limb preserving surgery in dogs with bone cancer. So are there ways that you can actually take out the diseased bone and replace it or stabilise what's left in a way, that sort of gives the dog a functional limb, and this is something that's done in humans as well? And this is something that's primarily, efficacious in dogs who have tumours of the distal radius where the distal radius, with a tumour in it can be removed.
A cadaver bone or a metal spacer can be put in place of that, Then one or two giant plates are put across. This defect, the carcass is fused and the dog wakes up and, goes home, and this can be very effective. The outcome is about the same when combined with chemotherapy as dogs that get a conventional amputation.
But the rate of complication is very, very high in these patients. So, probably 75% plus of dogs are gonna develop some kind of complication. And the most common complication is a post surgical infection.
So a chronic, long term post surgical osteomyelitis. And that can really be a very, very problematic, management issue for these dogs. However, there is a really interesting, sidebar to this.
And that is if you're a dog that has a limb salvage procedure performed and you get a post operative infection, you live twice as long as if you do not get a post operative infection, and this has nothing to do with the likelihood that the tumour is gonna grow back at the infected site. This is actually a prevention of metastasis that occurs, which is really quite remarkable. The same observation has been made in humans who have a similar limb salvage procedure performed.
So, Joe sat Nick here was a graduate student in in in both my lab and Steve Dow's lab who actually tried to work out what was going on in mice. To sort of explain this phenomenon. So he actually developed a model where he could give mice osteomyelitis, and then actually track the osteomyelitis using, a bacteria that are expressing, or making light that you can see with a special camera.
And actually, when you, give mice osteomyelitis either with staph aureus or with pseudomonas so both a gramme negative or gramme positive organism and then put tumours on them. In this case, these are osteosarcoma tumours. You can see that those osteosarcoma tumours actually grow more slowly.
So he was actually able to recapitulate what we observed in dogs in this mouse model. And it turns out that one of the things he observed is that there are fewer blood vessels in the tumours, that are grown in mice that have an infection. And once again it goes back to these innate immune cells.
So mice with infections that have tumours actually have much higher levels of blood monocytes and the type of monocytes that they actually have in their bloods are these activated proinflammatory so-called, M, one monocytes. So this chronic bacterial osteomyelitis actually gives you more M one monocytes, which are the kinds of monocytes that you want in your tumour because they help to suppress tumour growth and stimulate the immune system. So fun.
Little fact again, are we going around purposefully infecting our patients with bacteria in order to get this anti tumour response? Well, that's an interesting question. So not not as such.
But there are some really interesting ways that this has been looked at. So one of them is is this example here, which is actually very recent. So a group from University of Pennsylvania, Doctor Nicola Mason's group has actually, given a, a modulated or an attenuated listeria bacterium intravenously to dogs with osteosarcoma.
And in this very early study, 18 dogs with osteosarcoma got chemotherapy followed by this listeria, and they were compared to historical dogs. So not a not a randomised placebo controlled trial, but historical dogs that just got regular old chemotherapy. And in this early study, it appeared that the dogs who got, listeria actually had an outcome.
That was Super Year, in fact, to those dogs that got just conventional chemotherapy. So a very, very interesting observation that goes in line with with what Coley observed and what we observed in a in dogs and mice with, with bacterial osteomyelitis. So this looked great.
This was very exciting. The the group that was doing this and and some commercial entities were actually very interested in trying to commercialise this as, a therapy for dogs with cancer, but unfortunately, sort of subsequent to that initial work. There have been several reports of actually dogs developing long term chronic, abscesses full of listeria after treatment with, this product.
And, as a result of these observations, really, the the folks who were developing it thought that that was really associated with an unacceptable risk to the patient and even potentially to the to others in the household. And as a result, clinical development of of this really encouraging product needed to be halted, for safety reasons, but a really inter, interesting proof of concept that that, bacterial therapy could actually be a very, very potent way to treat cancer. In dogs.
So stay tuned for more of that. Are there potentially less aggressive ways or or less, infectious ways that we could, sort of mimic the same idea? One of them is something that was looked at actually a very long time ago.
And this is, a drug. That's, as you can see called here Liposome tripeptide phosphatidyl ethanolamine. Say that 10 times fast.
I'm going to abbreviate this from now on and call it MTP. So MTP is actually a synthetic. a product that is made to mimic what a mycobacterial cell wall looks like.
So mycobacteria the things that cause tuberculosis obviously are known for inciting a very, very potent granulomatous response in tissues. That's a hallmark of mycobacterial infection that we probably remember from veterinary pathology. So what?
What makes up a granulomatous response? It's mostly activated macrophages. So long time ago.
Back in the 19 seventies and eighties, people thought that, hey, if we could actually rev up these macrophage use the drug like this, that's safe. That this doesn't involve giving live tuberculosis. Maybe we could stimulate macrophages, to to be better at controlling cancer.
And this particular drug, MTP. Was put into a liposome, which is sort of like a fat droplet that very specifically targets macrophages. Because of some some ways that the cell surface was modulated with the idea that maybe it'll go to the lungs and actually, change.
The way those lung macrophages function in a way to keep tumours, at bay tumour metastasis at bay. And this is actually just a little, electron micrograph down here showing a picture of one of these liposomes. Here's, a transmission electron micrograph that's actually shown pulmonar macrophages that have eaten up these liposomes.
And so their cargo has been delivered into the macrophages, hopefully stimulating them. So a large number of different studies were actually done by one of my mentors, Doctor Greg McEwan at the University of Wisconsin. And this is the summary of a large number of different studies that were done again in dogs with osteosarcoma Looking at the efficacy of this therapy.
So just to summarise here, this first Kaplan Meyer line is dogs that get amputation alone. So their median survival time is about four months. Dogs that get chemotherapy or dogs that get just MTP with no chemotherapy.
Both have the outcome that's been reported median survival time around a year or so. But those dogs that get a combination of, chemotherapy and MTP had a median survival time of around 18 months, which is, you know, really quite remarkable and also noticed the tail of this curve. So about 40% of these dogs were long term survivors.
So very exciting information and similar data was actually generated in heo sarcoma. And, early stage, melanoma in dogs. And these results were actually encouraging enough that it led to, the approval of this drug for the treatment of human, osteosarcoma.
And this drug is actually approved. by the EME A for the treatment of, osteosarcoma, in in humans. And again it's sold under the brain brand name NAC.
So you might ask yourself, Hey, what a great thing. So I could actually buy this drug and give it to my patients. Well, hypothetically, yes, you could, but it's horrendously expensive.
The last time, I, I, priced it out. I think the average cost to treat a 30 kg dog with this drug would be on the order of about $70,000. And again, that's about about 70,000 pounds.
So not not something that's within the reach of most donors, Unfortunately, so we continue to look for drugs that work in a similar way that might actually end up being more affordable for our patients. Another non-specific immunotherapy that you may have heard of is actually this drug. Amy, which is also called Aldara and Aldara, is an, is an agonist, a stimulator of of a toll, like receptor seven, which is one of these no, receptors that's present on cells of the innate immune system like macrophages and NK cells.
And it's used in humans to treat things like bones, disease, superficial basal cell actinic keratosis, which is an an early, sun induced change in the skin and genital warts. But there actually are some studies that have been looked at using this drug, in animals as well. And it seems to be fairly effective for these early preinvasive cutaneous squamous cell carcinoma in bats.
And there actually is also some work looking at it as a topical therapy for sarcoid in horses. And one of the things that's important to know about is that this drug does not penetrate enormously deeply into the tissues. So it tends to be far more effective for the flat sarcoid that we observe, rather than the sort of big nodular very, very thick sarcoid where it's hard to get good tissue penetration.
But this is certainly something that could be considered as a topical therapy for these two diseases and animals. So other bacterial products that are that are currently available, at least in the United States. So there's a product called Regress and V, and there's a product called XY that are both available and they are primarily used in horses.
So regress and V is is ABC G, which is again a mycobacterial product, and XY is a, propion Ofter Acne's product And if I remember correctly, I'm not a horse doctor, But I think this is primarily used for things like shipping fever in horses where a little boost of non-specific immunity might actually help to keep them from getting respiratory infections. And, these are often used again intralesion for the treatment of equine sarcoid. And some, equine practitioners actually have a lot of success using these types of of, products.
It appears to very be very operator dependent. So, in other words, the more familiarity you have with with giving them, the more careful you are with sort of how you inject them. The better they tend to work, for an individual practitioner.
But these are definitely forms of immunotherapy that are used every day to treat animals with cancer in the clinic. So, I mentioned very briefly this concept of metronomic chemotherapy. I could actually do a whole hour on metronomic chemotherapy for you guys.
But one of the ways that metronomic chemotherapy appears to work is actually by depleting regulatory T cells. So it also appears to have some effects against blood vessels, which are very important. But yes, it can actually deplete regulatory T cells.
And there is one particular drug out of all the metronomic chemotherapy drugs that have been looked at in dogs, where we've actually been able to demonstrate this phenomenon. And that's cyclophosphamide. And again, this is work that comes from, Barb Biller's lab here at Colorado State University when she was here, in collaboration with Steve Dow and she was actually able to show much like her data in osteosarcoma, that dogs with soft tissue sarcomas actually have more circulating regulatory T cells in their blood.
And you can give, metronomic cyclophosphamide and actually see a reduction in those regulatory T cells in the blood and the dose of cyclophosphamide that's actually required to do. This is a dose of 15 milligrammes per metre squared per day. So lower doses of cyclophosphamide were not effective or not as effective in depleting regulatory T cells or actually interfering with blood vessel growth in the soft tissue sarcomas.
So this is actually the dose of metronomic cyclophosphamide, which we recommend for the treatment of cancer in dogs based on on this data. So another thing that that we were able to look at was actually a combination of palaia and metronomic cyclophosphamide for its effects on regulatory T cells and what we were able to, to, demonstrate was that actually palaia by itself was capable of depleting regulatory T cells in dog blood. And when you combined it with, cyclophosphamide, you actually saw additive depletion of those regulatory T cells.
So in addition to having an effect against me, cell tumours In addition to having an effect on blood vessels, potentially PALAIA also appears to have an immunomodulatory effect at least partially through depletion of regulatory T cells. So we might be doing immunotherapy in our patients anytime we give, either metronome cyclophosphamide or palaia and not even realising. So, the more you deplete regulatory T cells in this study, the more interferon gamma was actually present in the blood and interfere on gamma is one of these pro inflammatory cytokines that's potentially a surrogate for again, a robust, anti tumour immune response, which is very exciting.
So antibodies I mentioned previously, So tumour targeting antibodies are actually an exciting way that cancer is being treated in humans. Quite a bit. And there's a number of different ways that these antibodies may be working depending on what they're doing, so they can have direct effects by sort of crossed crosslinking their target on the surface of cells and causing, causing cell death.
They can inhibit growth factor signalling those, antibodies that are targeting growth factor receptor interactions, and they can also have immune effects. So again, if we go back to our basic immunology or may may remember a couple of concepts, one of them is called antibody dependent cellular cytotoxicity. So if there are cells in the body that have antibodies bind to them, certain cells of the immune system, like NK cells and macrophages may think that those cells are foreign and pick them off.
That's a DC C, and certain antibodies can also fix complement that can result in cell lysis. So, tumour targeting antibodies can work in a variety of different ways, and perhaps the one that has gotten the most attention over the years for cancer is an antibody targeting a cell surface protein called CD 20 which is present on the surface of all B cells, including B cell lymphoma cells and the antibody that That sort of does this. On the human side that has gotten the most press is an antibody called rituximab, and this has been approved for about 20 years for the treatment of human, B cell lymphoma.
And in multiple multiple studies. It's been shown that when you combine conventional chemotherapy with rituximab, you end up in with significantly improved outcome. And that's demonstrated by this red line here in a human clinical trial.
And again so quite often we receive comments for or questions from sort of Internet savvy owners about Hey, my dog's got B cell lymphoma. Can I give it rituximab? Is there some rituximab like drug that we can give to my dog?
And, the sad thing is that rituximab actually doesn't work in dogs, so rituximab does not bind or does not react with the dog version of CD 20. And even if it did, this is a human protein. So it would be recognised as foreign by the dog immune system and get picked off after just probably one infusion.
So we cannot give rituximab to dogs. However, a few years ago, there were two different monoclonal antibodies that actually were approved by the US Department of Agriculture for the treatment of dog lymphoma. So one was an anti CD 20 antibody, and the other was an antibody targeting CD 52 which was expected to actually potentially, B two T cell lymphomas.
And these were approved again, only conditionally, based on the fact that they were safe and that they were reasonably expected to be efficacious in in quotation marks. And actually the the company aano Therapeutics was actually selling these monoclonal antibodies for the treatment of dog lymphoma. And there was a lot of hype and a lot of excitement about these, But, after a short period of time, all of a sudden this, press release appeared on their website, which actually suggested that, when they did, additional studies didn't seem like either antibody was improving the outcome in dogs with lymphoma.
And when they went back and looked, it actually didn't even appear that these antibodies were binding the way they should to dog lymphoma cells. So there was a lot of hype associated with these antibodies, but at the end of the day, it actually did not appear that they were efficacious at all or even specific for their targets. So that got everybody pretty depressed.
However, there is another antibody out there that is actually now owned by alanko, which is both specific. It is, also capable of depleting, normal B cells in dogs and, in mouse models of canine lymphoma is associated with an anti tumour effect. So this is an antibody that, we don't really know where it is in the development, pathway, but actually appears to be, potentially efficacious.
And we're eagerly awaiting some more information about, when an antibody like this could possibly be, at least looked at in some additional studies in dogs with the intent of of eventually potentially making them available for, for treatment of dogs with B cell lymphoma. So it could be very exciting. Now, let's move on to to vaccine based approaches and and the one that I think probably all of us are the most familiar with, is this which is a DNA vaccine, that actually targets or that is approved for the treatment of canine melanoma, and this is a product that's referred to as concept in, in the veterinary world.
And this is actually a DNA vaccine that is, made to engender an immune response against a protein called tyros inase and tyros. Inase is a differentiation antigen. That is very important.
In actually, a pigment manufacturing. So it's one of the enzymes that actually catalyses the development of of pigmentation in tissues. And as a result, it's, its expression is restricted mostly to melanocytes and actually certain cells.
In, certain neuroendocrine tissues as well has a little bit of this. And one of the things that you might actually notice is that, this, antigen that they use is actually human tyros. So they're using human thy Rosina as a vaccine for dogs.
And the idea behind that is there are some subtle differences between human tyros and canine tyros that make this tyros inase antigen look forward to the immune system. And the idea is all right. If you vaccinate dogs with human tyros inase and generate an antihuman ty Rosina immune response, maybe some of that immune response will actually leak over into, the dog version of the protein and that actually appears to be true.
So in mouse models, if you vaccinate mice with human tyros inase you can actually engender a very nice response against, mouse tyros inase. And that's indicated here by this depigmentation that's occurring. So these are T cells actually picking off the pigment cells in these black mice.
So, this, this human tyros inase is actually capable of inducing antibody responses in dogs, and it's capable of inducing T cell responses in dogs. So that's been demonstrated very nicely. Experimentally and then this has been used in studies in dogs.
So in order to get full approval of this vaccine for the treatment of dogs with melanoma in the United States, a single arm non randomised study was performed looking at this vaccine as an adjuvant to local therapy. So either, surgery or radiation therapy in dogs with stage two or three, oral, malignant melanoma. And in this study, these are the dogs that received the, the, onset vaccine.
Here is the control group, which again was not randomised, not concurrent. This is a historical control group, and there did appear to be a significant advantage in terms of outcome to those dogs that received, the vaccine. So this is obviously very encouraging and led to the full approval of this product.
However, subsequent to this publication, there have actually been a number of publications. These are retrospective studies that have, at least in part, evaluated whether there appeared to be any benefit to the administration of on SE in dogs with malignant melanoma. And these three retrospective studies have not demonstrated a significant improvement in outcome, in these dogs.
So, really, what has been lacking since the very beginning is an appropriately performed, randomised prospective study where a whole bunch of dogs were all treated with the same kind of surgery and then randomised to get vaccine or or placebo. That didn't happen in the in the registration trial for the product and certainly has not happened in these three retrospective studies either. So I, I think there there's always going to be a little bit of a question here about about truly how efficacious this product is.
But I do think it's incredibly important to note that we have never identified anything else that is effective in the Post Surgical treatment of dogs with melanoma. And this is an incredibly safe product. So in the absence of anything that we know works better, I do think this is a very reasonable thing to consider because of its It's really, really, excellent safety profile.
And again, the lack of of anything else that we know is helpful. So, you know, definitely a reasonable consideration in these patients. The last thing that I'd like to mention very quickly, which is really quite exciting is this concept, which is, something that you may have heard about from from human oncology, This concept of utilising what are called immune checkpoint inhibitors.
So one of the things that that, I sort of alluded to at the very beginning of this talk is that tumour cells, and and tumours in general have evolved a large number of different ways to avoid being picked off the by the immune system. And some of these involve, these so called immune checkpoints so tumour cells can secrete a variety of different molecules that can engage these, these immune checkpoint receptors on T cells. Certain cells of the immune system so again, Bad macrophages, M, two macrophages, regulatory T cells.
Myelo drive suppressor cells can also potentially, participate in this process and actually again engage some of these inhibitory receptors on T cells. So why would these receptors be here in the first place? Well, if you think evolutionarily, it's it's actually makes a lot of sense.
So obviously it's very important for, an organism, whether it's a dog or a cat or a person to be able to respond appropriately to a pathogen. Right. So we need to be able to rev up a good immune response and really be able to control an infection.
But there has to be some kind of off switch. There has to be some way to turn that immune response off when the infection has been handled, or you're gonna have runaway tissue destruction. You're gonna have autoimmunity.
You're gonna have a whole bunch of things. So these regulatory these counterregulatory pathways evolve as a way to turn off the immune response when the danger is finished, when the pathogen has been eliminated and tumours have figured out how to subvert these pathways in order to make themselves, less visible to the immune system and the two molecules that have actually, received the most press are these two CTL a four and PD one. And there are actually antibodies that have been developed targeting these two, inhibitory receptors that have really shown some pretty remarkable, anti tumour effects in humans.
So just a few examples here. So antibodies is either targeting the receptor, which is called PD one, or it's ligand, which can be made by tumour cells called PDL one, actually are showing really remarkable activity in some human cancers, such as malignant melanoma, non small cell lung cancer, kidney cancer, bladder cancer, a variety of other cancers as well. And again for for many tumour types that this new form of therapy is really revolutionised, the management of of certain cancers.
It's really quite exciting. So the question is, where are we on the veterinary side with sort of having these kinds of of therapies available. So there are actually several groups groups in the United States groups in Japan, a group in Australia that have been able to demonstrate that, PD one blocking antibodies and actually CTE four blocking antibodies are capable of stimulating an immune response in in dog cells.
So this is a quick little study that actually looked at, proliferation of dog T cells, in a dish and actually demonstrated that in the presence of a PD one blocking antibody, these, T cells proliferated much better. And if you looked at, a secretion of a cytokine called interferon gamma, which is a good, surrogate for how active or how potent are these T cells You can see in the presence of this antibody? These T cells may made a tonne more interference.
Gamma. So again, this is in a dish. You take cells out of the body, stick them in a dish, this is what you see.
So that's great and very exciting. However, we're really fortunate that one of these PD one blocking antibodies, now has conditional approval from the US Department of Agriculture for the treatment of dog cancer. And this is a molecule that's called Gil VTM.
And I have heard that, Gil, that mad may be available at least in the EU. Under certain circumstances for the for the treatment of dogs as well. So this actually may be relevant information.
And again, this is a canine antibody that targets PD one, for the treatment of dog cancer. So gilt map is given at a dose of 10 milligrammes per kilo, as an intravenous infusion over 30 minutes. So it's a It's a short term intravenous infusion, and it's given every other week.
So this again has conditional approval from the US Department of Agriculture. And actually, a randomised placebo controlled trial that will hopefully lead to full approval of this product is ongoing in the United States as we speak. So what do we know about how well this antibody is working currently?
So, there's some, evidence in dogs with mast cell tumours of efficacy. So the overall response rate in dogs with mast cell tumours is about 40%. So this is the percentage of dogs that have, you know, quote unquote meaningful tumour shrinkage, and a total of 73% of dogs had either tumour shrinkage or their tumour stopped growing for a clinically relevant period of time.
So that's one of the label indications, and the other indication is actually for stage two and three melanoma, where the overall response rate was about 20%. So 20% of dogs had meaningful tumour shrinkage, and a total of 60% of dogs either had tumour shrinkage or their tumour stopped growing for a clinically relevant period of time. So these small single arm studies you can see about 25 dogs each were performed, which led to again the conditional approval of this product and, its current, licensure, at least conditionally for the treatment of dogs with these two cancers in the United States.
And so, as I mentioned, the registration trial is ongoing. And they're they've chosen to do this in in dogs with melanoma specifically, and there are a variety of what are called investigator initiated to try that are ongoing, trying to determine sort of the best ways to utilise this this really exciting new tool that we have for the treatment of dog cancer. So should we be combining it with other forms of therapy?
Are there other tumour types where it might be effective? Are there tests that we can do to pick out which dogs are going to respond better. This is all a work in progress, and we're really just scratching the surface here.
But it's actually very exciting for us that we have this new immunotherapy tool in our toolbox that we can start to explore in our canine cancer patients. And with that, I thank you very much for your kind attention. Obviously, it's not possible for us to have a real time question and answer, session associated with this due to the fact that, that this is an on-demand presentation.
But I do strongly encourage you to reach out to me by email any time you might have a question and whether it's related to this particular presentation or anything else having to do with cancer. I am always more than happy to answer any questions from my colleagues. I love, talking about about cancer treatment and helping out, helping out folks all over the world.
Who might have questions about cancer treatment in their patients. So again, thank you very much. Look forward to talking to you again.
Cheers.