Human Natural Killer Cells: From Bench to Clinic

February 18, 2019

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Yale Cancer Center Grand Rounds | February 12, 2019

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00:00Well, good afternoon and thank you all for joining us today for Kansas Center. Grand rounds were really very fortunate to have a special speaker here today because many of you know today. We are recognizing the branch blanched.
00:21Sure, series for those of you are aware the blanched Almond series was endowed by doctor. Marvin Sears Professor Emeritus at Optomology in memory of doctor, Sears mother plans. Tolman and that a lectureship has brought to our center.
00:42Extraordinary talented individuals focused on research, particularly in the realm of Hematology and he even a logical agencies and we're very fortunate to continue that tradition today with our invited Speaker Doctor Michael Calijuri Doctor Calijuri is.
01:03Resident Inn physician in chief of the city of Hope National Cancer Center and who Mike's career as a physician scientist a leader executive mentor is really unparalleled. Mike was for 19 years at the James cancer ha.
01:23State and ultimately the CEO over the James where he took that institution to an altogether new level in terms of clinical care. Research philanthropy recruitment and really bring it to a great impact in cancer care regionally nationally and internationally.
02:26But for a mentor so I'm very pleased to welcome the 2019 blanched home and lecture doctor. Michael Calijuri probably. Thank you very much very kind introduction. I had the pleasure of having Charlie is the senior resident when I was.
02:47And he continues to do amazing things so it's a pleasure to be here. It's so good to see so many friends and so nice. Voluta comments such a snowy day, we were supposed to meet this morning but you got busy good. I realize some of you might have kids to pick up school closing early and then I.
03:07Was already closed yesterday for today, so that's impressive preventative Madison?
03:15I'm from Buffalo so this is like we used to roll in this kind of weather in the canal. You know, but it's a great pleasure and it's an honor to be here and especially because I'm going to talk about natural killer cells. I see that there's a great deal of interest and that's really neat for me because not a lot known when I started.
03:36And what I want to do is really take you through kind of my story of different discoveries and translation that we've made with natural killer cells through the years and finished with some clinical application. I'm not going to talk about its application in acute my Lloyd leukemia, which is perhaps the most exciting, but probably the best known.
03:57Going with natural killer cells as we learn more and more about the biology. My lab is really for focused since beginning is on understanding receptors because if you understand receptors that sit on the surface of a cell you can understand what that sells sees and if you understand what that liggen binding.
04:17Doing function really start to think about how do we manipulate these to help in cancer and infectious disease?
04:26So first just some disclosures here mine and I really won't be addressing any of this today, and so we'll talk about role of cytokines and natural killer cell development secondary lymphoid tissue, which is where we discovered it sells developed will talk about subsets.
04:47Applications of that receptor biology, so back along time ago. This is what was known about natural killer cells cover of Time magazine. You could take these CD 34 stem cells and pop them into Interleukin 2 and after.
05:07Based at 3 weeks 4 weeks, you get natural killer cells and really described by this CD 56 bright cell, which is an image here NK cell's becomes dim on the surface of the cell. It's it's mature in case I won't hear bout. BCD 56 bright and dim but really the question was is this. The real growth factor for Isle to in feet.
05:28Because we knew that I'll 2 while Olympus cited Tropic hormone was very selectively released by T cells on activation. And yet you have billions and billions of NK cells in your body and turnovers fairly rapid so Mother Nature helped us with that with some genetic disruption experiments because the mouse when knocked out with oil 2.
05:49Find K cells in the mouse knocked out of the aisle 2. Alpha chain, which is one of 3 chains of the aisle. 2 Receptor had NK cells. Both the aisle to knockout and the aisle 2. Alpha chain knockout had NK cells. However, if you knockout. The beta chain of the aisle 2 receptor or the car.
06:10You know the aisle 2 receptor there were no NK cells, so that told us that something that isn't I'll too and doesn't require the Alpha chain, but does require the beta chain in the gamma chain is important for NK cell survival.
06:24And so that receptor schematically looks something like this, where you see the Beta Gamma and those of the signal transducing chains. That's where all the action is but for that Beta Gamma to sea. Isle 2 in your body it needs that third tiny Alpha chain that Alpha chain will not signal transducing confers very hief.
06:45Physiologic doses of I'll 2 and with that transduces a signal but by itself. The Beta Gamma chain can transducer powerful signal, but just requires enormously high doses of I'll 2 and that's why I'll 2 when given exogenously in high doses could make NK cells.
07:04And So what was subsequently discovered was another molecule that did precisely what I told you it had its own Alpha chain. I'll 15 Receptor Alpha chain and unlike the Alpha chain of I'll 2 the Alpha take about 15 by itself combined. I'll 15 with very high affinity and it sits on other cells a ninja presenting cells.
07:25Since the molecule I'll 15 to the beta gamma chain, and that sends a signal and then we can see this here. When we take I'll 15 pop it into culture. Those CD 34 positive cells and now we're looking at a histogram here and you could see the CD 56 bright cell, which is the image here NK cell.
07:46What we noticed was that even though there were pure cells from this uncommitted metaphorical Jenna Tercel there were very few in number without 15 alone and so we asked ourselves. What other receptors might there be in the CD 34 cell that augments the growth of the.
08:07To do that, we actually looked at this cells and what sells what receptors are on this cell that might tell us a little bit more about that.
08:15By the way I'll 15 knockout mice at all 15 receptor Alpha chain. Akamai slack NK cells, confirming what we found in humans. All the work. I'm going to talk about is in humans and then I refer back to the mouse for studies. We go to the mouse for but we work primarily in human natural killer cell development. So to look at what else could be involved in the growth of NK.
08:36We went back and we found that this receptor receptor tyrosine kinase seek it was also on the service of just the bright NK cells. Not on the surface of the more abundant dim NK cells that are floating around in your blood. But when collected on the bright eyed kids if you could see fading as the cells become dimmer.
08:57You look to see if Kit Lige end stem cell factor also known or mass cell growth factor and its sister flip 3 lie again could be important in NK cell development and so here you see kit lag. Endorf Litli again. Here's total cell number from those CD 34 cells initially and here's the eh.
09:17Soak it log into flet login along with CD 34 cells gives you growth here. We're looking at FL alone. But as you can see, there is no natural killer cells, though no natural killer cells here. I'll 15 I showed you very high numbers of very low growth that so together.
09:38NK cells, but combine kale or FL when combined with their look at 15 in both of these stromal cell factors shows that not only do you get the purity of NK cells? But you get synergy and growth. Total numbers so you get high numbers of cells here, so the.
09:58Date that FL in Cal work on the NK cell without affecting it by itself is Twofold. One is through the proliferation pathway, which I'm not showing you but the other is it up regulates the beta and gamma chain on more CD 34 positive cells, allowing a greater number of those cells to be responsive to Interleukin 15.
10:19So the picture, then began to unfold is such where an earlier sell a projector has on its surface kit in or flip 3 and sees flip 3 or kit lie again and.
10:34Pops up, I'll 15 Receptor Beta and gamma chain, and then in the presence of Isle, 15 would produce the natural killer cells and exogenous. I'll 2 and high doses could do this same thing.
10:46So this is interesting, so we wanted to go after this cell to see if we could identify the true natural killer cell precursor in humans because if we could we could start to figure out the developmental pathway beyond this we could figure out a couple of things where to human NK cells develop.
11:03And then what are the molecules involved in their development so in looking for this cell we so let's just look for this? I'll 15 Receptor Beta and gamma chain on the surface of the CD 34 cell. We couldn't see that by flow cytometry at all but we knew it had to be in the 34 population because of this pathway.
11:24As we took the 34 positive cells and using a variety of markers. I'm not showing you. On 3 different populations from these 2 markers 3:45 RA in 3412 and 3 and we popped each of those into culture with Kyle 15 or high dose. I'll too and you see only the third population over here.
11:45Number 3 labeled here CD 34 low are a positive was the one that became natural killer cells and indeed. We further took that population and said what other receptors are on the surface of this population to tell us where this cell might live what we found uniquely was that.
12:06Population of CD 34 positive cells expressed high levels of L selection in beta 7, Integrant and these 2 molecules were known at this time to be important for homing to secondary lymphoid tissue such as tonsil and spleen and lymph. Notes so they've never been CD 34 cells found in any of these tissue.
12:26Time, but we decided to take a look and so we first speech our system work. We had a CD 34 column. We put blood through CD 34 column and you see all 3 populations when enhanced through this. Selective column found in your circulating blood when we did the same thing with the collection of human lymph nodes or tonsil you.
12:47We see this population so the receptor biology brought us to the right population and in fact when you take that population out of the fresh lymph nodes or console and you put it in. I'll 15 you. See you get your CD 56 bright natural killer cells right here.
13:05So that was really interesting and then we said well. Why are they going to lymph nodes and we looked at lymph nodes and we found that there were CD 56 bright immature NK cells in the lymph nodes as well, and they lived around the parafollicular T cell. Rich region of the lymph nodes and so we asked ourselves well if you have the early NK cell here and.
13:26Earliest precursor here in secondary lymphoid tissue. Maybe this is where human NK cells actually develop and mouse. They develop in the bone marrow and it didn't seem to be the case in humans, so a lot of work and by the way I'm showing different pictures of people that responsible for the work.
13:45In the lab we narrow this down to 3 different markers acquisition of CD 34, no seek it. No see 94 CD 34 with Scikit. No seating 94 Los of CD 34 CC kitten CD 94 negative and then just CD 94.
14:06When you took these populations. You said let's look at CD 56 and this is from lymph nodes. You see that we see absent some a lot and then CD 56 bright cells. So we name. These stages 123 and 4 of NK cells develop we could find these all fresh and lymph nodes and tonsil.
14:27Interesting journey characterizing these various stages of NK cell development for number reasons. One reason is when we characterize this population here, we found out that actually before it transforms to an end kasell. It's actually what's called an innate lymphoid cell type 3 ILC threes?
14:48Important in gastrointestinal physiology, and pathophysiology secreting abundant. Interleukin, 22, which drives the epithelial cells of the gut to secrete defensins for protection against bacterial invasion when this cell actually loses a transcription factor called HR.
15:08Drives all the way to do natural killer cell so this. I'll see 3 and NK cells actually live in balance, depending on which tissue there in.
15:17But the picture began to sum up something like this, where T cells. T cells developing in the Thymus B cells in the bone marrow and NK cells, having a projector that left the bone marrow's went to secondary lymphoid tissue and then really preceded in this pathway here.
15:37And over the years, just to summarize work from our lab, predominantly in some work from other labs. We've gone on to further dissect how not only NK cells develop but how human innate lymphoid cells of which there are at least 3 types developed within secondary lymphoid tissue with a variety of different types of discovery.
15:57Focus on one of those I'm here you see we've learned that we go from a projector here to about 3 or 4 different stages. Here we get a projector for both I'll see 3 and NK and when you get to this stage 4, eh, it's quite a unique population. I'll show you how this works so.
16:18Acquire CD 117 that's shown here, then you require and acquire NKPAT and then you acquire CD 16 and once you get to CD 16. You gotta mature NK cells that floating around in your blood remember CD 16 is the receptor for human globulin? What's important for a toxin and or so.
16:39Mediate antibody dependent, killing and this all occurs through these inside shoe. These pathology flow. Cytometric pictures of normal lymph nodes and tonsil as well as well as all your lymph nodes and of course, you have kilograms in kilograms of your lymph nodes. So lots and lots of these cells are develop.
17:00What's interesting is that this stage for a As you seen here we actually have found something very interesting to show you where this work goes into the clinic and into the lab and so, if you look it. Turns out here's your C 4, A is only in tonsil or lymph nodes. You don't see it.
17:21Or bone marrow or other places as well, what we found is that there's a malignant car apart to stage for A and that's this EDDTNK cell lymphoma. This God awful tumor that you see here and it turns out that if you look at 4 different patients with this and you look at their tumors whether it's Blood Bowl.
17:42Blood here or central nervous system with CNS involvement. All of them have their malignant counterpart in this stage for a originally found it's part of the NK Cell Development Paradigm. So we're actually doing lots of work on the Genomic, an epigenomic differences between these normal and malignant popul.
18:02Very interesting findings and moving this out publication soon.
18:08So now you're experts on NK cell development. There's no quiz. But I'm sure you all get. It now will talk a little bit about survival and what goes wrong with NK survival so.
18:21Once we understood it, I'll 15 was critical for the development. We asked you know what? How to NK cells sustain just like your hemoglobin. They remain very, very constant your body despite tremendous dynamics. And we ask the file 15 could be the factor responsible for that. So just taking NK cells and placing them and I'll 15.
18:42Media You can see the tiny amount of pile 15.01 nanograms per mill will sustain their survival for over a week, so that gave us a hint but it's only of course in in vitro experiment to do this properly. You'd want to take normal NK cells. Popham into a mouse that has no I'll 15 and see if.
19:03Switch can't develop in that mouse still surviving that mouse and so we did that experiment. Here's your normal NK cells from a wild type mouse coat them with and I put them into a knockout mouse and say do they survive in the picture tells the story because here's normal mice going into normal mice, so you took him out. You label them with die.
19:24And 36 hours later, you see their .12 is what's in there and a week, 5 days later. It's .1 want essentially the same. You do this into an aisle 15 knockout mouse wild type cells into another 15 and within 36 hours. You're drastically down and within 5 days. They're completely gone, so this told us that I'll 5th.
19:44Very very important for not only in case of development, but in case cell homeostasis survival in people as well.
19:55So where is the Isle 15 coming from obviously not the marrow. These aren't developing the marrow something in the left out and we figured this was an antigen presenting cell most likely dendritic cell so we ended up doing experiment again going to the mouse to prove that when you knockout dendritic cells, which I show here this is the dendritic cell.
20:16There knocked out through this mechanism actually NK cells are drastically reduced if not almost absent so the picture is for 3 log end is important not only for that CD 34 cell, but of course, it sustains the survival and growth of dendritic cells. It induces I'll 15 or sustains out.
20:37The brain surface through that I'll 15 receptor Alpha that high affinity chain and then that with precursors leads to NK cell development and then as NK cells develop and trickle through your secondary lymphoid tissue constantly. They get a hit from Isle 15 and continue with their survival.
20:54What's interesting about I'll 15 is that the M RNA is in virtually every tissue in your body is abundant in brain heart, liver kidney, but very, very little protein anywhere. And so it's modification. It's impossible detect most methods.
21:15For example, so one of the things it must be is that it's being translated post transfer posttranslational regulation post transcriptional regulation and so we looked at that.
21:28And you can see here that what we did is we did a little jockeying with this gene and put basically some constructs into the gene to make it so that the transcript. The abundant transcript was transcribed and not only transcribe but abundant. Lisa created and developed this transgenic mouse simply to ask the question when you alter this site a kind.
21:49K cells as you can imagine NK cells are abundantly increased as shown here here, you're looking at a mouse and you could see a wild type mouse has this much NK same mouse transgenic for the Isle 15, where it's getting abundantly transcribed translated. It's created his huge numbers of NK cells so.
22:10People driving development, sustaining survival you're going to get lots of NK cells. What was perhaps more interesting, however, is this is, after about 3 to 6 months. These mice developed TNKSLLGL leukemia. I think was the first time a single sided kind manipulation was shown to be.
22:30In a mouse and these bice developed massive Lucas Cytosis, sometimes 5600 thousand and I from overwhelming accumulation of these blasts in the body and as I say it was NK cells is shown here, but also NKT which is another innate immune effector cell.
22:51When we looked at the NKT cells. We could determine who is clonal by looking at the T cell gene rearrangement and we could show here that it was in fact model here is an example of the lymphoblast you can see prominent nuclei prominent nucleoli and very blast like picture different from the large gradient lymphocyte in normal.
23:12Further very much very easy to adopt complete transfer and lethal in the syngeneic mice.
23:19That was interesting and specially because we also study AML, which I'm not talking about today. But the leukemia. Ologist Here know that about 30% of AML in some LL have constituent of activation of flip 3, the receptor tyrosine that I talked about.
23:41And when it's the ideas present that is can step receptors constituent. Lee active as if there's abundant. Liggen, always activating the Receptor and that's at least a precursor to malignant transformation into AML and sometimes they LL but fully 30% of AML has this particular mutation usually.
24:02Operative Mutation was interesting to us because we didn't have a constituency active receptor like the TD. But we had constituent Lee active lie again in this transgenic mouse. So it was like the aisle. 15 Beta Gamma Chain was always on and sure enough, we saw this with just this.
24:23Phone we saw this transformation, so this became interesting and relatively easy for us to study and I'll make a Long story short about that kind of working backwards. We notice that the blasts in both human LG L&R. Mice with this, LG L leukemia had a Barent and abundant centrosomes.
24:44Look at the pathway that's important for centrosome development and maintenance and it involved mic and so to summarize what we determined was that I'll 15 was driving NF. Kappa beta increase mic and then together, we're driving up Aurora kinases aid be and that led to.
25:04Ability through this centrosome apparency and part of the story for Leukemia Genesis. What we also found which was interesting was that NF cap would be in Mick. We're teaming up with H Dec, one and this trimer repressor complex pop down and drove near 20.
25:25To the depth which mere 29, B regulates DNMT 3 B in the leukemia, Docs now. This is often mutated in AML and BI. Lo mere 29 be an high DNMT 3. P we got tremendous amounts of meth. Elation at the 5 Prime regulatory region of jeans, which led to further chromosone instability gene silencing.
25:46Yeah, they put this pathway together for the Genesis of LG L leukemia through overexpression of Isle 15 an to focus on this therapeutically. We had determined that bart is a map actually broke up this repress are complex and could lead to dry.
26:07Time be up and EMT 3 beat down and so we focused on that and here you see that simplistic schema where you have partism abt hitting on their oppressor complex. This disappearing near 29 be going way up driving DNMT 3 be way down and so we treat the LG L leukemia.
26:28UCL the DNMT 3 B disappears up here in this gel, so seem to be working. We took this then to the clinic and had huge success, kidding, but didn't give up actually turned out the PK was off.
26:48Developing a nano part is a map and ended up showing that in our animal model. We could get 100% cure of this, LG L leukemia with this nanoparticle map in this gentleman still at OSU is further developing this compound for the clinic. The other thing I just thought I'd show here is.
27:09Very interesting actually came in my lab instead so that happens in 30% of the transgenic mice. What happens, the other 70%. Of course I had never been to the mouse room. So I said, I don't know so she went to the mouse room in a Long story short, she found that these mice were getting this progressive alopecia and ultimately this.
27:30Cutaneous problem denuding of the fur etc. An again telling a long story in a single slide. This turned out to be a great model for CTCL cutaneous T cell phone with something studied so, so incredibly well here.
27:51And as you can see here, the pathology is classic foresee TCL and I could replace these with normal and see. TCL patient and quite indistinguishable. The infiltrators a CD 3, Knoll or a CD 3 CD 4 T cell and again lot of work being done comparing.
28:11In this mouse model and got great strategies now on their way to the clinic to both slow down or prevent the disease very exciting epigenetic modifyers.
28:23So that's a bit about NK cells survival deregulation by the way I'll 15 is abundantly overexpressed in CTCL and you may now know there's a peptide being test that blocks I'll 15 binding and they've had some maybe it's open the trials opened here, but there's been some dramatic responses and multiply relapse.
28:44Suggesting that what we're finding in this transgenic mouse is quite quite important in the human pathogenesis of the disease.
28:53So moving on to a little more defense of what are these NK cells normally doing in the body here you see this is a typical flow of your blood your T cells and B cells are down here. Your NK cells. A few of them in mature ones up here that bright cells as they become dim they acquire CD.
29:13That molecule it hooks up with antibody to do the ADC, killing and so one of the things we were wondering is does it bright cells floating in your blood versus largely in the lymph nodes and the dim cells? Which you don't find in the lymph nodes, but are in your blood abundantly do they have different immune regulatory properties and so?
29:33One of the things we knew is that NK cells can make gamma interferon in a heartbeat like within a minute if their tweak the right way, with Mana Kinz. I'll 12 by 18 IA one. I'll 15 they could make lots of Game Interferon and we knew that monocytes need game interference to contain obligate intracellular.
29:54CB and pneumocystis and listeria etc. And so we said well if they don't make gamma they must get it. Early on from NK cells and could there be a difference between the bright selling them cells to what cells providing that monocyte macrophage with that requisite gamma, interferon to contain these.
30:15And we know these infections are contained because people that lack either the Gamma Receptor Organ Interferon die of overwhelming obligate intracellular pathogens such as tuberculosis so we asked are they the same or different. We set up an assay where we put monocytes in a well needed the bright cells are that.
30:36And then we added the pathogen and to see Monica is delivered is the bright so the dim cell that cell that's delivering the game interferon and it turns out you can see from this slide, even when you just give the monic kinds or if you do that ass. I showed you it's really the bright cells that are making all of the gamut.
30:57Cells that are more abundant in the blood art that made more sense to us because of course. The dim cells live in the blood and Bryce largely live in that parafollicular T cell. Rich region of the lymph nodes so one model is within your lymph node pathogens enter and case.
31:17Those are tweaked with Mana kinds, they make lots of Gamma Interferon, there more Mana cards produce and it's a very nice cycle. We also learned I'm not developing the story here at all today is that In addition to that. Those NK cells that sit in the parafollicular T cell. Rich regional lymph node actually get a signal from T cells as well and.
31:37Tiny amount of Interleukin, 2 that the T cells secrete when it's activated because just the bright cell not the dim cell has the high affinity. I'll 2 receptor on its surface. In addition to the beta gamma itself, so it can compete for I'll too. With the T cell so if it gets the energycap presenting cell for example, gives 12.
31:58Who is Shawn this in our culture constructs you actually get abundant game interferon that can drive this to a TH one important adaptive immune response to certain pathogens so the NK cells involved in both of those the bright cell. I should say is involved in both of those in its niche in the secondary lymphoid.
32:19Sets.
32:20So that's here bright cell doesn't have many granules again receptors tell the story produces lots of side of different cytokines to stimulate the innate and adaptive immune system certain receptors that suggest homing etc and very little CD 16.
32:41Very little granules, so not a killer in contrast, the dim cell was floating around in your blood abundant granules. Lots of different receptors. Here's The Cure Cure Globulin receptors, which are inhibitory signal largely tells NK cells don't kill and I'll refer to that a little later very few cited kinds.
33:02To do an abundance CD 16 to bind all that antibiotic and so there's been a beautiful story developed when the cure is Michmash Mismatched in allogeneic transplant. Many of you hematologist know that certain not going to talk about today is fascinating there's increased survival in patients that uh.
33:23Call T cell depleted mismatch transplant that have cure mismatch as well to talk about the CD 16 receptor largely today. But I'm going to refer back to these inhibitory receptors for a moment and talk about some discoveries. We've made and how we're taking them to the clinic about CD 16, which as I say we know is important.
33:44We could try to get inside the mind of the killer. This is an NK cell is the tumor cells puncturing holes from its perforin release and then cytoskeleton of the tumor and the NK cell recovers. After that can regenerate and kill again? So very interesting tumor license. So I'm going to talk about glioblastoma bad tumor.
34:04Now very fail, we've all known friends and family have had this devastating disease. Horrible survival with this been working on this in our lab now for awhile because I teamed up with a neural neurosurgeon.
34:25Call Kyoka is that Ohio State and he kept noticing that there were Gamma Interferon, producing cells when he would try to treat GBM with uncle lytic virus uncle herpes virus. I'm going to tell you how that stories involve for us, so just the Digress. We do have a car T program very act.
34:46Will soon have car MK 4 GBM at city of hope and this has been very, very interesting study that continues so here's what happens with uncle lytic virus is couple videos in here and just to show you So what we do is you create a virus that only becomes lytic.
35:07Context of a tumor specific promoter which is found in the GBM and not in your normal brain tissue. So herpes virus is, you know is a neurotropic virus right kids die of herpes encephalitis adult style represent satellites. It's attracted to the nervous system so it's a perfect virus to consider therapeutics because it goes to neural tissue and with.
35:28It'll only become lytic in in a GPM versus normal tissue. So what happens is the virus comes along it will infect one cell and then what's critical is that those viruses able to spread along it spreads along as its president. It then becomes lytic so it need.
35:48To do this, it needs time to infect and it needs time to spread before it will become lytic problem with this and why my friend in Tokyo called me is that it turns out NK cells protect you against dying of herpes.
36:07Um anybody gets a cold sore it's a limited infection. It's not ebola right. Whatever you have an infection. But you don't have any be seller. T cell immunity to it. Your first exposure, but it's very limited and it turns out that NK cells are critically important to limit that infection as as evidenced again by Mother Nature.
36:27And the problems we have when NK cells aren't around. This has been shown in several different examples, but how and why they work was really unknown.
36:37So what we found Neo Cap collimated gamma producing cells is that the NK cells were flocking into the brain tumor before the herpes virus got a chance to work on the brain tumor. Massive numbers of NK cells going in there and killing the virus and I'll just illustrate that so here's your.
36:58In the context of normal neural tissue, you get your herpes virus comes in. It's going to again go to infect your GBM and start its processes spreading before it even gets a chance to spread your NK cell is there to kill it.
37:14Such that it's ineffective and we showed in rat models that you deplete NK cells or mouse models. You can get great response of the brain tumor. So we knew we had something but understanding what to do really had to understand what's going on here to really start to and no clue despite that other paper. I think being published in the early 90s.
37:35So gentlemen in our lab function when it had any cloned every single gene of the herpes genome into a GBM cell line and then he took and that is about 70 or 80 jeans and then he took NK cells and he did this, killing assay he said. Which of these jeans are the NK cells getting activated?
37:56And, which ones are they being inhibited by the GPN it turned out there about 3 of the ladder but about 5 of the former the front about 5 jeans that you put that single gene in the GBM. All this messy stuff and you tilt. The scale and that GBM goes to history with the NK cell so he found the jeans and.
38:16And I'm going to refer to 2 called glycoprotein E and glycoprotein, I and these 2 were the most potent if you have GE GI GI by itself did nothing but GE with Georgi alone. You get the kill it and the killing would release the grain size, you get the cell death.
38:35So.
38:37Just going to explain how this work and how we're exploiting it so I said normally what happens is the topic of your NK cell and there's it's CD 16 stick it off of the surface. It binds FC and of course, your tumor cell has tumor specific immune globulin protoxin per se.
38:58CS either CD 20 Hertz, dude on the Antigen specific way. The Fab Fragment of fines to that specific antigen and the end. K cell then binds to a different region. It binds to the FC region here as you could see FC.
39:18Non antigen reason, Anet Locks and loads when you get dimerization shoots a signal to the NK cell. It kills the tumor cell and that's at least in part, how things like Herceptin and Rituxan are working in an anti tumor. It's called antibody dependent cellular site successfully been known for 40 years.
39:37What we found was something completely different and irrelevant to herpes and other pathogens that I'm not going to talk about. But so here's your CD 16. It's binding to that. FC portion of the antibody here. It turns out the GENGI of the herpes genome wooden infects the GBM or whatever it sells infect.
39:58Add GENGI go on the surface that forms a protein that also binds FC.
40:07It's called an FC binding protein so just like CD 16. It's binding FC. But it's binding it in a different region.
40:17And it allows the CD 16 to still bind wenig lab in his Brown to the FC binding protein or the dimer up here of CD 16 with any immune globulin on services limited in your body turns out, I'll show you NK cells are covered in a globulin without any antigen specificity.
40:38The system working before you have any edge of specificity. It binds here it finds this receptor on the surface of the HSV infected GBM and then they lock and load and again. The key with this is this without any engine specificity so before you have immunity to herpes with B cells and T.
40:59Your NK cells, which are floating around with limited globulin stuck to their CD 16 use that dimer to CFC binding proteins. And I'm not going to talk about it today, but FC binding proteins are also on staff. A and they're on strep. So it's a broad mechanism of innate immune recognition of pathogens any pathogen that encode.
41:20See binding protein is recognized by this mechanism.
41:25And then you get your license and we did. This crystal graphic structure of this region was known CD 16 binding to human FC and of this FC binding proteins bind inhuman FC and as it shown here in the crystal in the in silico representations. Here's Gigi from her.
41:46Here CD 16 from the NK cell no overlap whatsoever, so this can and does occur structurally and have lots of proof of this. I'm just going to show you a couple things. Here's how this would work in our cartoons. So now you got your GBM here in the midst of your normal tissue and again, you're HSV is coming in and because of that tumors.
42:06Here it combine only infects will become lytic and only those cells and so the first cell goes to lice, but it can't because the NK cell comes in quickly. Anne Lise is that HSV infected GBM before it can really spread throughout the whole tumor.
42:26So that's why is it goes like the next one notice there's no spreading of the virus. Here's an up close? What's going. This is GB a membrane HSV infected so there's GE. HSV infected there's GI they come together and form a high affinity FC binding protein for emitted globular then the NK cell, which is.
42:49Up there with it, you'll see popping up all over the surface of the GBM here and K cell has CD 16 on its surface. It's gotta admit globulin stuck to it, and it has as I said, this binding area called the bridge area.
43:05Binds the CD 16 and this called CH 2. CH 3 region that binds the high affinity. FC binding protein and then you get your dimerization your activation in your kill it.
43:19So.
43:22Is this real so here we're looking now were staining NK cells to say? Do they have a big lobby on the surface? When they're floating around in your blood and the answer is they do here is imminent globulin staining. Here's your NK cell. Here's your T cell. You can see the drastic difference and that's what it would look like. But it turns out, we're all different we.
43:43Infinity so here's 3 different donors In addition to this donor you see look at the numbers here. This is just indicating the amount of immigrants binding relatively high low low so depending on who you are is how much immunoglobulin is on the surface of your NK cells and if that's the case then.
44:04Killing mechanism I showed you is the case. Then there should be variation between all of us. When we actually see a herpes infected cell whether it's GBM or otherwise. There should be a difference because we have so much only so much in many globulin on the surface or abundant in Munich globulin on the surface of the NK through the CD 16.
44:25It took 25 people randomly and just measured their intensity. How much in England, they have on their surface and the disaster cells do. They all killed GBM the same or is it different? Strikingly different highly correlated so the more I'm going to have a new surface more activation or killing you have at the GB.
44:46Perfect correlation you take that same 25 people and you have them kill an NK sensitive target. K 562 that doesn't have Gigi on its surface. No correlation whatsoever. Same people so that told us that in fact. This is a functional mechanism. That's relevant to people and you can understand.
45:06I don't understand why at least in this case why different people respond to different pathogens differently in this case this by the way is called we've we've got a name for this. It's called FC bridging ADC so because the FC Fragmente is bridging between the end case.
45:27Herpes infected cell so how about in vivo does FC bridging Cellular State, Texas. He protected because we know mice have CD 16. They have NK cells. They bind their own immunoglobulin. This should be able to see weed affect the mouse with herpes NK cells should see it and prevent it well, it turns out that this.
45:48When pathogen and it this FC binding protein doesn't see the mouse. CHCH 3 region, so in fact, you don't get recognition and you get 100% fatality.
46:02But we should be able to take that same mouse and inject it with human human globulin. And if there's enough. It will compete for the CD 16, it does bind human globin mouse CD 60, Bynes went above and we know that CH 2 CH 3. This whole complex recognize the SD binding protein so now if I give one.
46:22This antibody and it could be irrelevant antibody. It should provide protection when I give a fatal dose of the HSV one.
46:32And in fact, I should be able to cleave off the Fab Fragment and just give FC because this is a complete innate immune function and indeed that's in fact? What is the case as you can see here whether I give Retaks and whether I give Derek to mmap weather, I just give the FC.
46:53Portion of any imminent globulin, you get nearly complete or complete protection against the HSV infection. Fatal infection in the mice further if you knockout. The NK cells as shown in the bottom line with something called a sale. GM one before you do the exact same experiment you completely lose the protection because now.
47:14FC fragment before you give the herpes. You've taken out the NK cell that CD 16 is not there and so even though the FC fragments binding to the herpes FC binding protein. There's no killer cell to come in and clear it out.
47:30So therapeutically what could we do to delay those NK cells from killing that GBM and it turns out that there's a natural bacterial pathogen that struck progenies that makes an endopeptidase that cleaves globulin and this actually has been given in vote patients.
47:51This endopeptidase to stop hyper acute rejection of kidney transplants and in vivo in the human body. It cleaves this so you're just left. With this piece, which binds to CD 16 or this piece, which binds that FC binding protein and so, if you purify this you should be able now to take your cartoon and what you should do is.
48:12This FC piece and it should bind only to the herpes infected FC binding protein and block the NK cell from seeing that free site. Here's the FC Fragmente coming in blocking GE GI so now when the NK cell comes by that sites occupied.
48:33Get that sell the virus has time to spread replicate and lice and you could prolong your survival and so we've been working on this to do this in vivo and ultimately in patients for this another pathogenic diseases that I'm not going to talk about today that are relevant to this mechanism used surveillance.
48:54They discovered
48:56So one thing is our lab is developing EGFR car for GBM going to talk about it, but it's relevant to what I'm going to tell you about and it turns out you can get a nice little bump in survival and Xenogeneic. Human GBM when you take NK cells and Expresa car on the surface that can find both mute.
49:16In wild type EGFR in this car that we have can.
49:20It turns out that if you give HSV and NK car sequentially you can get this actually start to get a survival curve with these mice. But what we found is that we took our original herpes virus, which we now have in patients to do start clinical trial.
49:41We actually added to that herpes virus.
49:44An inhibitory lie again that finds the NK cell cure like molecules. I was telling about is called Cal. GR one KLRG. One so we added this eat cat here in to the purpose virus when it infects the GBM it secrete salivated expresses a log in.
50:05In case cell from killing it so it actually without needing to do that difficult cleaving experiment talk about if you change your herpetic virus to express an inhibitory lagom NK cell it holds them off for a bit and we've now shown that actually just by itself. This virus can be cured of in these models.
50:26Wow, scaling up to move this virus to the clinic in the mean time. This is this the so in the mean time. We started our first clinical trial for AP 01. We have with our original herpes virus that is specific for GBM and the idea is that we will next do GBM with EGFR car.
50:47Ultimately, we will combine the HSV one for GBM followed by a NK car therapy or if we have it in time. We will move to are treating recurrent GBM with our uncle itic herpes virus that expresses eat coherent and then.
51:07Car NK cells so I'm going to stop there. I want to talk about today or cited kinds in NK development specifically the role of I'll 15 how we use receptive biology to find where the precursor is where it traffics to wear NK cells develop we talk to live.
51:28Malignant counterparts to those we talked about human NK cell subsets and their role in the immune regulation the immune system prior to Antigen specific immune responses and then clinical application. Receptor biology understanding how in case else herpes virus trying to prevent that.
51:49Like herpes virus from working in GBM and then ultimately combining that with an NK cell car in this particularly terrible disease. So I'll stop there. Thank all the people in my lab 15 of whom move with us to city of hope. This is the real mastermind here Jim why you?
52:09There.
52:11We are responsible for a lot of the work in our laboratory so. Thank you very much for your time in such an honor to be here appreciate.