| 1 | HMP Education | Immunomodulation of NK cells for the treatment of lymphoid malignancie... | 8102 | 92 | 1 | 28.4 | negative | 21:05 | All right, I'd like to also thank the organizers for inviting me to talk at the scientific session on natural killer cells. My talk's a little bit different from the other talks you've heard about B.C.L. disease pathogenesis and how that might contribute to lymphomas. Rather, it's about how one aspect of the immune system might be used to fight lymphoid diseases. The overriding objectives or take home messages here with my talk are really twofold. One, I hope that everyone has an improved understanding of end-case-albiology. So what an end-case-al is and what it does. And number two, an improved understanding using a few examples of how drugs or agents, either that are being used in clinical practice, being evaluated in clinical trials, or being developed pre-clinically may augment end-case-al responses to lymphoid tumors. So I think just by way of introduction, there's a lot of hematologists in this class and end-case-als are under the microscope large granular lymphocytes, at least the majority of those. We can look by flow cytometry to more specifically identify end-case-als as CD56 positive, CD3 negative, a T cell marker lymphocytes. And end-case-als, similar to T cells, can be divided into functionally distinct and developmental subsets. I'm not going to go into the details of this, but I would like everyone to appreciate that. End-case-als express specialized organelles called cytotoxic granules. And in these granules are the mediators or effector proteins of inducing cell death, enzymes, and perforate. And they also constitutive of the expressed interferon gamma mess and journey. So they're really poised to rapidly respond to a tumor target. So how does an end-case-al recognize a target cell? Well, unlike the B cells we just heard about, or T cells which are in the adaptive branch immune system, they don't chop up their DNA, clonally rearrange it into a receptor that's specific for a single protein. And rather, they use a large family of various inhibitory and activating receptors synthesized to signals and decide whether they're encountering a friend or a foe. Just as an example, Kier, killer like immunoglobulin-like receptors that are inhibitory, recognize MHC class 1 on all of our nucleated cells. That's an off signal to the end-case-al. And in turn, activating receptors that can bind to activating ligands, mediate an on or a positive signal. So in the setting of health, these are integrated and the off signal wins and the end-case-al moves on. There's a couple of ways in the setting of disease, either viral infection or malignant transformation that this process can be altered and really tipped in favor of activating the end-case-al. The first is the loss of the inhibitory receptor ligand. And this is termed missing cell, primarily because MHC class 1 or class 1 like ligands are typically lost on these cells. And the other is a process called abnormal cell for induced cell, where there's an increase in the expression of these activating ligands and these end-case-als can be better triggered. So what happens when an end-case-al is triggered? There's various effector functions that can be activated. These include production of effector cytokines like interferon gamma. And in addition, these cytotoxic granules are released and mediate the apotonic like cell death of that target cell. In this process of degranulation, a protein called C107A or lamp 1 is released onto the cell surface and we can use this to track recently degranulated end-case-als. This is not a simple process, so my cartoon is very simple. This is actually a very complex process. And this is just a few of the inhibitory and activating receptors that have been identified in end-case-als that the end-case-al integrates in order to make that decision. The second concept I wanted everyone to understand is that end-case-als can be function-enabled to better respond by cytokines from accessory cells. And so in this situation, accessory cells like dendritic cells or macrophages when they encounter a virus produce pro-inflammatory cytokines. And those cytokines bind to constitutively expressed receptors on natural killer cells, inducing functional responses such as cytokine production, chemokine production to bring in additional immune cells and also induce the end-case-als to proliferate and expand. Activation markers come up that we can track and effector death receptors like trail and fast ligand can also be induced. And then for this particular audience, in this particular topic, I think introducing the concept of antibody-dependent cellular cytotoxicity is very important. This evolved as a cooperative effort between the B cells in our immune system that produce antibodies in an infected cell expresses protein antigens on its surface. These antibodies combine to that and end-case-als come in. And through their CD-16 or FC-GAM receptor 3A, a low affinity FC receptor, they're able to be triggered by antibody-coded targets and kill those cells by the mechanism play just mentioned. So there's a few different approaches that you could envision as far as how we can manipulate this system to enhance end-case-al responses to lymphoid malignancies. The first is to enhance the activating receptor signals or the triggering. The second is to improve the functional status or the functional competency of these end-case-als, either using cytokines or drugs that might do that. And the third is to block negative regulators of inhibition or activation and attempt to tip that balance again in favor of anti-tumor or anti-lymphoid tumor immune responses. So I did want to start here talking about retuximab. I mean, this is one of the first antibodies developed for cancer therapy. It recognizes CD-20. It's FDA-approved. And I'm sure everyone in the room is familiar with this. It acts through multiple different mechanisms. And one of those mechanisms is ADCC mediated by end-case-als and other effectors. And this is a movie. I don't know if someone can activate it. I can't. I just wanted to show you because a picture really does meet a thousand words is that what we're looking at here are end-case-als, shown with red dots, marking their cytotoxic granules and tumor cells in green, and what you're, if you guys could play that again, I don't know if it'll loop. What you're seeing here is the, can you click that through one more time? You're seeing the end-case-al, come in, meet up with this green tumor cell that's a live die. That end-case-al is going through that decision-making process, then mobilizes its cytotoxic granules as you can see here in red, mediates that lethal blow, and that vital die leaks out of that apototic cell. And so, let's see here. So getting back to retuximab. And I think one of the first clues that ADCC was important in the context of retuximab is looking at FC Gamma receptor polymorphisms. And I won't go into this because this has been well described in the literature over the last ten years in too much detail, but individuals are found that have polymorphisms in this gene that result in higher finity for the antibody. And individuals end-case-als with the amino acid 158VV SNEP or version of this protein have increased binding to monoclonal antibodies. They have increased cytotoxicity at limiting concentration of antibodies. And when they're better activated in vivo, and patients get single agent retuximab, and this has been borne out in improvements in clinical outcomes in several different clinical studies using single agent retuximab. So how can we improve in that? There's a number of antibodies that have been developed to essentially engineer enhanced ADCC. So an improved recognition by FC receptors. And I'm listing off a few of them here. These are in clinical trials. And they'll be interesting to see how they compare to retuximab, and in particular how they compare when coupled with other agents that might augment and case-al or other anti-tumer immunifactors. Moving beyond monospecificity, additional types of antibodies are in development that seek to even better target NK cells to lymphoid and malignant tumor cells. So here there are bi-specific and tri-specific or bikes and triches that have been developed that utilize the single chain FV of an anti-CD16 antibody, and the single chain FV of a CD19 antibodies are put together. And there's also a triple version that includes CD22. And what this allows is a high affinity interaction of this reagent with the FC receptor, and redirects the lysos of these NK cells, again CD19 positives tumor cells. This is data from Jeff Miller's lab at the University of Minnesota. And what they're showing here is that when in vitro using primary human NK cells and primary ALL and CLL cells, that the percentage of NK cells that degranulate or responds to these tumor cells increases dramatically in the presence of these bi- and tri-specific reagents to a greater degree than even retuximab, which is shown here in this third column. So I think these will be exciting and interesting drugs to see in the future in clinical trials and see how they stand up to other triggering approaches. Okay, so moving on to augmenting the NK cell functional response. There, I think everyone in this room is probably also familiar with a class of drugs called immumulomodulatory drugs. Lendolidamide is one example of that, and this particular drug has a wide level of activity against a number of different myeloid and lymphoid diseases, as is shown here. And an equally diverse number of potential mechanisms of action. And I think one of the big challenges here is, Ben, to put together what is the mechanism of action that results in clinical activity in all of these different diseases. Lendolidamide has been shown preclinically to augment NK cell functionality, and I just wanted to review that quickly here. This is one of the first studies published demonstrating that Lendolidamide can enhance the ADCC of NK cells in combination with retuximab. So what this group did was use primary CLL cells as targets, and we're looking at the level of killing of those targets by NK cells with increasing concentrations of Lendolidamide within without retuximab, and you can see that there's a steady increase in the ability of those NK cells to kill those CLL cells in the combination. Similarly, if you, there's a synergistic combination of Lendolidamide and retuximab using CD20 positive mantle cell and phoemacellins as targets. Lendolidamide's modulation of NK cells doesn't appear to end there in a study that was published in JCO by the Children's Oncology Group. This group used single agent Lendolidamide in relatively high doses in a dose escalation format where they gave these patients with solid tumors or MDS 21 days of Lendolidamide in 28 day cycles. And what they observed is that the percentage of natural killer cells from baseline within those patients to day 21 increased dramatically and significantly both in the percentage and the absolute numbers of these cells that were in the blood. In addition, these cytotoxic effector molecules such as granzyme B were also increased, and this translated it increased killing of prototypic NK cell target cells. This may be working through cytokines such as IL-15 because they also observed a correlation in the increase of IL-15 plasma levels going from baseline measurements to day 21 measurements. Lendolidamide and retuximab have been combined already in a number of different clinical studies, and I'm highlighting a few of them here. They have promising preliminary clinical results, and it will really be interesting to see what the NK cell correlative studies show as far as the ability of Lendolidamide to augment the numbers and function has been observed in that pediatric oncology study. I think there's still a lot of questions about this class of drugs and how it works and how it works on NK cells, what's the molecular mechanism of enhanced ADCC? It's not clear. Lendolidamide has also been shown to enhance the ability of T cells from cancer patients and to recognize their tumor cells in the context of CLL. There's been a number of studies from John Gribin's lab to this end. And then I think importantly, we need to seek confirmation of NK cell modulation in vivo in patients treated with lymphoid doses. The COG study I showed you, the highest dose level gave the equivalent of 100 milligrams of Lendolidamide, which is much higher than we typically use in our diseases, and there also may be disease specificity. Okay. Moving on to cytokines, there's been a long history over the last two decades demonstrating that cytokines such as IL-2 and IL-15 can augment NK cell functionality. These two cytokines bind to receptors on the NK cell surface, and these cytokines share a couple of signaling components, the IL-2, 15 receptor beta and the common gamma chain. IL-15 is distinct in that it has a high affinity receptor and is trans-presented to this intermediate affinity receptor on the NK cell. Both of these cytokines used in relatively high doses augment a number of different functional properties of NK cells that would be beneficial for anti-tumor immunity. There's been a number of different clinical trials using recombinant human IL-2, which have shown some degree of NK cell modulation, but overall have been relatively disappointing from a clinical standpoint. And over the last ten years, what we've come to realize is that one of the negative regulators of anti-tumor immunity, T-regs, are exquisitely efficient at utilizing IL-2, and that what's likely going on in this situation is that T-regs are expanding along with NK cells, and these T-regs are able to mediate control over NK cell anti-tumor responses. So there's been a big push to look at IL-15 more closely in the clinic because it doesn't have this fact on T-regs and may be uniquely altering NK cell and CDA positive T-cell responses. This is data from our lab, just showing that IL-15, just a brief exposure to IL-15 in this case hours, can increase the degranulation of both of the major NK cell subsets in the peripheral blood. There's pretty dramatic changes here that are summarized. In addition, there's an increase in the killing after just a few hours. That's pretty dramatic in both of these different NK cell subsets against K-562 leukemia cells, but also primary acute myeloid leukemia blasts and lymphoid cell lines. IL-15 is being explored in the clinic. There's been a couple of phase one studies that have been initiated, both in solid tumors and in AML in the context of an allege-NAK NK cell infusion. These studies are sponsored by the NCI, and there's also additional compounds in development. Altor biosciences has a compound, Alt-803, which I think really combines in a clever way than physiologic workings of IL-15 by fusing IL-15 and its high affinity receptor. And so what this does is trans-percent IL-15 in a very similar way as NK cell C in vivo when IL-15 is expressed on, for example, dendritic cells. And there's other cytokines in development as well. So just in the last couple of minutes, I wanted to introduce you to one new aspect of basic NK cell biology, NK cell memory. Traditionally, memory in the immune system has been limited to T cells and B cells. Adaptive lymphocytes. And more recently, three different groups and three different contexts have shown that NK cells can remember their prior activation. And then what I'll share with you is one of those cytokine-induced NK cell memory. This was first identified by Wayne Yokayama's lab several years ago. And what they showed was that NK cells that are exposed to cytokines in the context of an infection as how this usually occurs in an organism become activated and highly activated. These NK cells contrary to what was stopped before actually can persist. And they differentiate into memory-like NK cells. And they persist in mice for up to six months. And when you then give them a repeat stimulation, either through cytokine receptors, activating receptors, tumor targets, they exhibit an enhanced responsiveness. So we explored this in human NK cells, thinking that this was a potential translatable concept for NK cells. And taking human NK cells, exposing them to IL-12, IL-15 and IL-18 results in a highly activated NK cell pool. And we compared that to controls, which were just kept alive with low doses of IL-15 from the same donor. At 16 hours, at the end of this initial activation, the majority of these NK cells down here are activated, whereas these NK cells remain quiescent. After seven days of resting in vitro, these cells appear the same as far as their interferon gamma production, so they're returned to a quiescent state. These cells can be rested either in vitro or more recently, we have data that I won't show you that they can be transferred to non-skid-gamicy mice, immunodeficient mice, and also survive there. If we restimulate them at various times after their initial activation and give them a recall response, we see enhanced interferon gamma production here in each of these different time points, and that's summarized here in this graph. We also see enhanced responsiveness to tumor targets, as well as primary AML blasts, as well as enhanced cytotoxicity against these same targets. It's not just cytokine production. I think most intriguingly for today's topic, we also see enhanced responsiveness through CD16 after these cells become activated, return to a resting state, and then their CD16 is ligated, suggesting that this might be an approach to enhance ADCC down the road. Just to summarize what I've talked about today, there are approaches to enhance activating receptor signaling or triggering that are promising, both with next-generation antibodies that are optimized for ADCCs, as well as these bike-and-trike reagents. In addition, there's approaches that we can use to combine with enhanced triggering in the form of immunomodulatory drugs, cytokines, as well as more novel approaches with combinations of cytokines. This is something I actually didn't think I had time to talk about, but I will briefly mention because of that question you guys saw at the beginning of the session. There's also clinical different approaches to block these inhibitory receptors on NK cells to then tip the balance in favor of NK cell activation, and inhibitory receptor antibodies that are being explored include Kier, NKG2D, and of course PD1, as well as key-reg depletion strategies. We're also working on Alligenic NK cell therapy, and our group is not, but I'm sure you've heard a lot about CAR-modified T cells, these chimeric antigen receptor modified cells, and that NK cells can also be modified with CARs, and it'll be interesting to see how those compare to CAR-modified T cells in the future. With that, I'd just like to acknowledge my lab. Our collaborators at both Washington University and other institutions, the lymphoma group at Washington University, that's led clinically by Nancy Bartlett and then different funding for our research. So thanks very much. | ↗ |