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Ashish Kamat: Hello, and welcome to UroToday's Bladder Cancer Center of Excellence. I'm Ashish Kamat, Professor of Urologic Oncology at MD Anderson Cancer Center in Houston. And it's a distinct pleasure to welcome today, one of the recipients of the prestigious Bladder Cancer Research Innovation Award, that BCAN awarded this year in 2023, Assistant Professor, Chris Garris, who is going to talk to us today about overcoming immune suppressive myeloid cell barriers in bladder cancer.

So Chris, welcome to the platform, and take it away.

Christopher Garris: Thank you, Dr. Kamat, for the generous introduction. And so, thank BCAN for this great honor of this Research Innovation Award, and I'll here to discuss briefly the background of my research, and the goals of this project, with a lens towards forward-thinking for developing novel therapies for bladder cancer.

So to start, as a brief overview, I'll go over some of the work about immune agonism in cancer therapy. Some of our work describing anti-tumor myeloid phenotypes, our experimental and therapeutic systems, to identify IL-12 inducing drug combinations. And then, this therapeutic platform we call CANDI, that we use for a combinational immune stimulation. And then lastly, what as a core component of this Research Innovation Award, is this systematic discovery of genetic regulators of IL-12 production.

So BCG has been used as a therapy for non-muscle invasive bladder cancer for quite some time, but there are also fundamental limitations. But at the very least, it has sort of shown that immune agonism, through this attenuated bacterium, can deliver cures to a certain number of patients. However, there are fundamental limitations with this. As you see, that about 30 to 50% of patients eventually are refractory to BCG therapy, and there's been a very prolonged shortage of BCG in the clinic. So we like to pivot, eventually, to therapies that are more readily available, and can also, likewise, activate the immune system in similar ways.

So the mechanisms for BCG therapy are not precisely clear, but the immune activation seems to be the leading theory about how it works. Because deficiency of T cells, IL-12 or interferon gamma, which are classically called type one or Th1 flavored immunity, are associated with BCG response. And basically, the connection of BCG and mycobacterial immunity with Th1 response really highlights the idea that these flavors of cytokines are important in anti-cancer immunity. And that it functions, likely, as an immune stimulant.

So stepping back a little bit to some of my historical work, where we've used models of cytokine observation with interferon gamma and IL-12, both key Th1 flavored immune cytokines, that have established this feedforward loop of immunity in cancer elimination. Whereby, lymphocytes, which are one of your major producers of gamma interferon, can signal to myeloid cells, in this case, a dendritic cell, to produce interleukin 12. And IL-12 licenses cytolytic effector functions and CaM interferon production. So this virtuous loop enables a sustained anti-tumor immune response.

So what we have built over the years for investigating this loop, is cytokine reporter mice, that we could observe the induction of gamma interferon, or IL-12, in vivo, and longitudinally over time. So I won't get into details of those particular studies, but I'll share with you some of the background about identifying specific IL-12 producing DC types in the tumor.

This was done years ago from single cell RNA-seq, identifying that there's an activated dendritic cell subset, that is the prime producer of IL-12 within the tumor microenvironment. And if we activate this pathway called NF-kappa-B-inducing kinase, we can, which is also a strong component of the non-canonical NF-kappa-B response, we could activate IL-12 in situ with the tumor microenvironment, and trigger anti-tumor immune responses. I'll get back to why this particular kinase is important, because that is a major therapeutic target in our combinatorial immune stimulation platform for anti-cancer therapy.

And then, the genetic evidence to say why this kinase is important, is if we have a bone marrow chimera where all hematopoietic cells are missing this kinase, and we treat with anti-PD-1 in an otherwise immunotherapy responsive model, we lose all therapeutic effects of anti-PD-1, and we have a deficiency in CD8 T cell infiltration within these tumors. Really suggesting that NIK deficiency has a role to play in therapeutic anti-tumor immune response.

So to kind of get at this idea about what kind of therapeutic combinations we can have to activate myeloid cells in the tumors, in this case also DCs and macrophages, we have adopted these cytokine reporter approaches to derive in vitro dendritic cells or macrophages from IL-12 reporter mice. And we can give them combinations of stimuli, and then gradually see higher degrees of IL-12 induction in vitro within 96 well plates, and at high parameters. We can also do this like with macrophages, where we can give combinations of drugs, and see which combinations lead to IL-12 induction.

So this allows for a very scalable method to select IL-12 activating stimuli and combinations, that is very cheap to do, and also very fast. So we're able to kind of use this as a tool to identify combination immune activating therapies.

So getting into some of the core goals of our project that we have for the B CaM, we want to determine the therapeutic utility of these IL-12 inducing drugs in mouse models of bladder cancer using this CANDI therapeutic platform. And secondarily, we would want to identify the critical regulators of IL-12 within myeloid cells. And this involves finding new combinations of drugs that can induce IL-12, using our image screening platform. And then, also combining this with CRISPR screens, to identify genetic regulators of IL-12 within macrophages, and DCs.

So central to this, is the idea that you need to have combinatorial immune stimulation in order to effectively get IL-12 responses. So we do this by using this CANDI platform, which stands for a cyclodextrin base, that is full of its adjuvant's nanoparticle dual immunotherapy. So that's what CANDI stands for.

This basically, it uses a standard technique for solubilizing drugs, such as [inaudible 00:07:54] and cyclodextrins, and then linking it into a nanoparticle that can bind hydrophobic groups within its structure. And what we can do here is, we can take different small molecule activators of the immune system, or inhibitors, and then put them into one package, so we can get combinatorial delivery to a target cell. Which from what we have observed in vivo for pharmacokinetics studies is that, it primarily travels to macrophages and dendritic cells, cells that have phagocytic activity.

So as you can see here, if we take our image screening approach, and if we have a compound, in this case, R848, which is a TLR agonist. And then, LCL161, which is a CIP inhibitor, which it serves to raise NIK levels, as I mentioned in my earlier slides, as an immune activating strategy, we see synergistic activation of IL-12 within macrophages treated with this therapeutic combination.

Likewise, if we start adding in more compounds, in this case, a JAK inhibitor, which was quite surprising to us, could increase IL-12 expression levels. This is presumably through IL-10 mediated suppression, as we see that this response can be sensitive to IL-10. And furthermore, if we go into some preclinical models in our preliminary data, we see that this CANDI combination can eradicate tumors quite readily from a single dose, and then mice have memory to rechallenge.

So this is kind of exciting for us, because this is our sort of a first pass for understanding this therapeutic combination that can induce IL-12 and treat cancers. And this platform can be used to mine additional combinations that may be able to better induce IL-12 as we kind of increase its scalability.

Likewise, this CANDI nanoparticle, we have done work on its IV administration blood half-life and tissue biodistribution. In this case, this was done initially in this work with a glioblastoma model, we find that it primarily distributes to the tumor and tumor draining lymph nodes within their system. And then if we do cellular analysis, we find that macrophages and DCs are the two prime cells in the tumor microenvironment that uptake this CANDI nanoparticle. What we're expanding upon within this BCAN grant, is the usage of CANDI for intravesical treatments. And then, how it's pharmacokinetics and pharmacodynamics change, or what they are in this route of delivery.

Likewise, we can use our in vivo cytokine reporters to identify within the tumor the timing and induction of IL-12 and interferon gamma response to therapy, which we will likewise follow up with bladder orthotopic models.

And then, the last part that I will cover, is this approach to identify key genetic regulators of IL-12 within macrophages, which revolve around taking a Cas9 expressing immortalized macrophage lines that we've made, then doing a lentivirus library induction, and then stimulating with our CANDI stimuli, and then sorting out and identifying the top and bottom 10% of IL-12 signals from here.

So this approach is robust, as we show with some of these preliminary datas. We can identify and knock down particular genes in our early studies, and we can also have good transduction efficiencies with homemade mRNAs to modify these. And to bring this to a therapeutic aspect at the end, not all genetic perturbations may be targetable by small molecules. So we've also, likewise, developed a therapeutic lipid nanoparticle system, that we can use to induce IL-12 at very high levels. And then also, show strong therapeutic benefits when given in vivo to mice bearing tumors.

So hits that we identify from these CRISPR screens can, if not available to target through small molecules, can be targeted through mRNA or RNA-based approaches, to make it therapeutically useful as well.

And with that, I'd like to thank BCAN and UroToday for this opportunity to present our work, and for the generous support that they have provided to enable these studies. Thank you very much.

Ashish Kamat: Thank you, Chris. And once again, congratulations on receiving this award. I've been involved with BCAN since its inception, going on now almost like two decades. And also BCG, we've been working with BCG for the past three plus decades, and my lab's done a lot of work with IL-12, and the cytokines and the recombinant. So it's great to see you and your team come up with some sophisticated measures to actually help us understand how this works better. So again, hats off to you, and congratulations on this award.

So Chris, if I could ask you just a few general questions, right? With the whole IL-12 story, and your CANDI platform and everything, and you alluded to a little bit from towards the end about the clinical application. But if you had to look forward five, 10 years down the road, or hopefully even sooner, where do you see this being applied in the clinic for our patients?

Christopher Garris: I think that there is an avenue for clinical translation for this. The CANDI platform, which is based off of cyclodextrins, which are biocompatible, and have low toxicity on their own, we have an imaging agent that is currently in Phase I clinical trials. It's used for bioimaging, but it's based on similar molecular scaffolds. So there is already some precedent for these types of cyclodextrin nanoparticles in clinical use and testing.

And what we also really hope for in the future is, using this sort of cytokine guided approach to identify drug combinations for drug discovery. Because we're not limited to IL-12, actually, we're doing CXCL9 as another aspect as well, which is further much important for bringing T-cells into tumors. So we envision that we could translate these therapeutics into patients within the next five to 10 years, based on the prior history with these molecular scaffolds, and their safety within humans. And then also, designing novel stimulation cocktails that could be used to induce these cytokines more effectively.

Ashish Kamat: Well, that's great. So am I correct in understanding that you could actually have more than one cytokine stimulant scaffold in a true cocktail? Or would this be sort of sequential treatments for patients and when we get there?

Christopher Garris: So it's all in one. You could put up to three drugs so far in this nanoparticle and you could try four and five. We're just at the stage of three right now. We started with one, then we move with two, now we're up to three. So maybe the sky's the limit. It depends on how big we want to make the particle.

Ashish Kamat: Great. And again, talking a little bit about the award, and your interest in bladder cancer. And for the young folks that are listening, because our audience is not just experienced bladder cancer experts, but even residents, and trainees, and fellows that are interested in bladder cancer. Would you be kind enough to share with our audience your journey through how you thought about working in this particular area, and how it brought you to where you are today?

Christopher Garris: Yes. So I started in bladder cancer about five years ago, while I was a postdoc with Dr. Jeffrey Ravetch, who as several years ago also won a BCAN Innovation Award. And what we did for our initial studies, was looking at immune agonism through CD40 agonist antibodies, through intravesical delivery into mouse models of bladder cancer. This has since evolved into a Phase I clinical trial at Sloan Kettering. And my core interests, for actually the past several decades, well, decade and a half, I guess. I'm not that old, still. Is immune stimulation as a form of cancer treatment.

And what really opened the door to me to investigate this in bladder cancer, is the intravesical treatment route. How that could potentially increase the therapeutic index of otherwise toxic drugs, given systemically through IV means.

So the nature of non-muscle invasive bladder cancer, and the fact that you can give local treatments, that it can be highly efficacious, and also, our immune stimulatory, really opens the doors for testing these immunostimulatory cocktails in bladder cancers, as a proving ground before potentially expanding other indications. But it sort of gives a higher degree of safety for immune activating therapies, that can potentially then be expanded beyond, into other cancers as well.

Ashish Kamat: Yeah. No, absolutely. I mean, it's taking our ever evolving understanding of the immune system, and the immune responses, and oncology in general, but here, bladder cancer. And again, having techniques, and avenues such as yours, is kind of honing it, and fine-tuning it, and hitting the target right on the head. So I think that's a really novel approach.

For years, we've struggled with the fact that the best immunotherapy that we have for cancer in general is BCG, and in bladder cancer. And it's a dirty drug. But I mean, some of that, people will often look down on it saying, "Well, it's such a dirty drug." But that's the beauty of it. It activates so many different pathways. And then, when we can tease them out, and hone in on a few of them, I think that's where the future is.

If I could pick your brains a little bit, where do you think the real home run, when it comes to augmenting the immune response with BCG will be? Do you think it's IL-12? Do you think it's clearly not PD-1. We know that now clinically. But where do you think it is?

Christopher Garris: I think it is IL-12. I think it's immune stimulation. And the reason why I spent a little bit of time in my presentation, even though it maybe seemed like a distraction on this NF-kappa-B-inducing kinase, is that, it is seemingly involved in mycobacterial immune responses in humans. So I always like to frame. I'm a PhD, so I don't have clinical practice, but I like to keep all my work quite translational. And I frame my work in mechanistic mouse models, based upon studies in human primary immunodeficiency.

So for example, patients that have dysfunctional mutations in NIK actually suffer from BCG-osis, and the inability to control mycobacterial infections. So that kind of begs the hypothesis that maybe this NIK pathway is important for generating a response to mycobacteria, but that could be also in theory be what BCG's inducing. And what we identified in dendritic cells is that, if you activate NIK, you can get them to have better cross presentation or immune activation. So perhaps, that is a core pathway involved in BCG response, or could maybe be engineered independently of BCG, to deliver effective tumor immunity for bladder cancer patients.

Ashish Kamat: Yeah. No, I believe that pathway was sort of interrogated and investigated when it comes to immunization. Not oncologic treatment, but immunization for newborns when it comes to bladder cancer. So again, what's relevant in kids is general immunity against tuberculosis, is also quite possibly relevant in patients, when it comes to their ability to mount the appropriate immune response to BCG. So really fascinating avenue.

Once again, Chris, congratulations on this prestigious award. Looking forward to seeing what great things you do with it. And thank you for taking the time and sharing with us today.

Christopher Garris: Okay. Thank you very much, Dr. Kamat.