Okay, Hi everyone. It's my pleasure to welcome and introduce Dr. James Spain from Johns Hopkins. Doctors actually started Education at Johns Hopkins in the biomedical engineering department before going on to do a PhD with Dane which rip at MIT. And then working as a post-doctoral fellow with Chris Garcia at Stanford. Dr. Spangler then went back to Johns Hopkins in 2017 to launch or independent research group. And she is now the Brody Faculty Scholar there and her lab. And he's jointly house between the Chemical and Biomolecular Engineering Department and the biomedical engineering department. So her lab works with mechanistic and structural insights to can redesign existing proteins or design new proteins entirely that are able to therapeutically modulate the immune response. And as an independent investigator, Dr. Spangler is already won several awards, including, but not limited to, a Young Investigator Award from the Melanoma Research Alliance and AV faculty or V Foundation be Scholar word. So with that, let's welcome, Dr. Spangler. Thank you so much, Johnny. Well, first of all, I'm amazed that you memorize that whole thing. Like usually I'm like the person like when I'm introducing people like my sheet and like going off of that, but I'm so excited to be in person. This is actually the first talk I've ever given in my life with a mask on. So it'll be a really cool experience on a lot of levels. But this is also the first in-person talk that I've given in two years. So it's really exciting experience I think for all of us and I'm really excited that it's here at Georgia Tech and just meeting with the faculty and meeting with students throughout the day. I just, it's such a wonderful rich environment, not just for engineering, but for interdisciplinary science and research. And just seeing sort of the ways in which engineering rate just as sort of this vehicle to accomplish so many different goals in biology, in particular, is really what has inspired me my whole life and it's just inspirational. Begin a place where you have all of these centers and you know, sort of mechanisms devoted to bioengineering. So really excited to be here today and thanks again John in tavern for this opportunity. So as Johnny alluded to, I'm very interested in a particular area of the bioengineering space and that is of Biomolecular Engineering and protein design. And so I'm going to tell you a few different stories sort of in that area today. In ways that we can actually use engineering to manipulate the immune response. So protein-based drugs, or biologics, as they're often called, are actually the fastest growing segment of the overall pharmaceutical market. And they actually didn't exist until 1982, which is where you see the biologic sort of come onto the scene. So it was really just monopolized by a small molecules before that. And in 1982, the first protein based drug or therapeutic those approved, was actually just recombinant insulin for people who are diabetic to replace the influent that they lack in their body. And since then there's been kind of a steady number of FDA approvals reaching over 300 more recently. And still obviously small molecules over in terms of sheer number, right? Because they had such a head start, there's an overall advantage. But if you actually look at the sales, right, in terms of how utilized these particular molecules are universally. It turns out that the antibodies actually make up is as of 2019, almost three quarters of the overall pharmaceutical market. And if you look at the top 10 best selling drugs on the market, actually seven of them are protein-based money than anybody. But other types of biologics exist as well, including FCC's proteins or other receptor traps or other types of protein therapeutics. But proteins as they exist in nature, they don't care about your drug, right? They're not programmed or evolved or pressured in any way to actually make good drugs. And in fact, it's quite to the contrary, most proteins in nature actually terrible drugs for several different reasons. The first limitation is what's known as pleiotropy, right? So if your protein right in somebody's body, then you're being pressured to actually be jack of all trades, right? To deal a lot of different things in a small genetic package. And that's great if you're trying to accomplish a lot of different goals. But that's actually terrible, right? If you're going to try to have a specific therapeutic mechanism, because then you're going to have low efficacy because the drugs can be diverted in all different directions, right? Acting on all different cells and receptors. And also you're going to have a lot of off-target effects, right? They could actually be very harmful or detrimental to the, to the host. In addition, anybody who's had the pleasure of expressing proteins. As the woes of aggregation and degradation, right? Things stick together and things fall apart. And that can cause problems, of course, downstream when you want to manufacture these things into drugs. Or if you have an aggregated massive garbage that can actually cause immunogenicity or an immune response rejecting the protein itself. And finally, proteins are notorious unless they have some sort of either serum albumin or Fc fusion to prolong their serum half-life. They actually have very poor PK pharmacokinetic properties, which results in them being cleared from the bloodstream very rapidly. Most proteins, the glow like a 50 Katie cutoff, which encompasses most growth factors and cytokines that we'll talk about later. They actually clear from less than five minutes. And again, that's great. If you want to have a look at diagnostic or something very short lived. But if you want to have a long, durable, sustained response to the target organ and efficient delivery, they're not going to cut it. So as Johnny alluded to in his intro, our mission as protein engineers should we choose to accept it, is to redesign what's in nature or inspired by nature, build our own proteins. They can be therapeutically useful. In my lab has sort of this three-pronged approach. And the first prong is what I'm like, generally referring to as molecular engineering, which is a very loaded term and can mean a lot of different things to different people. What my lab does a lot of is a technique known as East surface display, where we take a protein and we display it lollipop style on the surface of a yeast. And then we come in with some sort of selective pressure shown in gray here. And we pull out the things that bind, we wash away the ones that don't. So through these iterative cycles of Darwin and attached to your right directed evolution, we can actually evolve the protein to have particular function that we would want. So cool idea won a Nobel Prize in 2018 of Frances Arnold and company. But really what unique about what my lab and I think a lot of protein engineering labs are sort of moving toward or evolving toward. One might say, is that we couple this with structural insights and those can come from experimental methods. We do a lot of x-ray crystallography, cryo-EM. We do NMR to get dynamics. Or they can come from computational approaches. So computational protein design, Luxor dynamics simulations, and basically modeling what protein-protein interfaces look like. And why is that useful? Because when we understand right, how proteins interact, we can better design smarter libraries to faster and better get at the answer of how we can discover better drugs. Arrow goes both ways because once we discover these new drugs are these new proteins, we can solve their crystal structures, get at mechanism, understand how they're working and how we can make them better. And why is this useful? Because we want it to go somewhere. And my lab, really being at Johns Hopkins has the unique opportunity to really drive these molecules from the discovery through the design and implementation, all the way through the pre-clinical development in animal models are taking our own human whole blood and looking at our immune response to various perturbations or interventions that we develop to ultimately get that translated into a person. And the last theory I'm going to tell you about today is actually a molecule that was developed in our group that we are currently humanizing and actually have in venture capital investment that we based accompany off of that we're hoping to get into human clinical trials by the end of next year. And so really what's exciting is in the lifetime of the PhD, you can really go from the discovery all the way through the pre-clinical development to actually translate that potentially into a person. And then loopback, right? Say, okay, this is a molecule that we've developed. This, how is it doing right in the patient population? And then go back to the lab and say, how can we make this better? Okay, So as I mentioned, I'm going to talk about three stories, time permitting hopefully today. The first being a combination of antibodies and cytokines were actually fusing them together to form what are called amino cytokines for therapeutic modulation. The second is a collaboration That's long-standing collaboration my lab has with David Baker's group at University of Washington looking at designing de novo proteins that the world has never seen before and your body has never seen before. That could be useful as drugs. And finally, we're going to talk about this by sticky Anybody that my lab has developed targeting cancer metastasis and where we're at currently in terms of the development of that as a drug. So starting with the immune cytokines project, cytokines are the immune system's way of bringing information from outside of the cell, inside of the cell. And the way that it does that is by interacting with these membrane proteins, proteins that have an extracellular domain outside of the cell and an intracellular domain. And what happens is the cytokine is responsible for dimer rising those receptors, bringing them crashing into each other. And the extracellular domains, when they crash in, the intracellular domains also crash into each other, setting off this cascade of events that ultimately converges upon nuclear transcription. And allows for the cell to have some sort of phenotypic response. Now, when you're a cell, right, your life's not that interesting. You're going to proliferate, differentiate, migrate, and die, right? But all of that is dictated by these intermolecular interactions between cytokines and their receptors. So particular cytokine that my lab is very interested in and is on a lot of work with is interleukin two. And IL-2 was the first cytokine to be both genetically and structurally characterized. And for good reason, IL-2 plays an instrumental role in the differentiation and activation and survival of a variety of different immune cells, in particular, T lymphocytes and NK cells. And interestingly, IL-2 is unique in that it actually signals through two different types of receptors. One is the hetero dimer ACH receptor, which just consists of the minimal beta and gamma subunits. And then the hetero trimeric receptor, which also has this Alpha subunit. And what's useful about this Alpha subunit is that it can actually differentiate and distinguish different cell types based on its expression. So whereas cells that express high levels of the heterotrimeric receptor with the Alpha have a ten picomolar affinity. The cells with only beta Gamma, right? Even though they can still signal, they have a much weaker affinity of one nanomolar. So generally, right, when IL-2 is endogenously present, there's a 100-fold higher affinity for IL-2 for cells that express heterotrimeric versus heterodimer. And again, this is useful because you can get selectivity whereas effector cells, so things like CD4, CD8, T cells, and K cells, preoccupation express no, virtually no IL-2 or Alpha. So the express primarily the hetero dimeric receptor T regs constitutively express high levels of the L2 or L3 receptor. And they played an instrumental role in suppressing the immune response. So IL-2 is essentially acting on both sides of the coin, and hence it's been used therapeutically on both sides of the immune system. It's been used to activate effector cells for, as a cancer therapeutic actually since the 990s and metastatic melanoma and metastatic renal cell carcinoma, and also for treatment of infectious diseases in clinical studies, unfortunately, IL-2, right needs to be used at very high doses in order to be active on these effector cells and concurrently, right, while it's activating these effector cells, It's also activating the immunosuppressive harm. And in addition, high doses of IL-2 have been shown to be very toxic. They induce in particular a phenomenon known as vascular leak syndrome, which as the name would suggest, involves your vasculature are becoming very busy. Leaking fluid into the interstitial space lead to multiple organ failure and even death. So even though IL-2 is actually effective, incompletely curing five, the 10 percent of patients who are dosed with IL-2, most of those patients that are responding actually refused the treatment because of the unwanted side effects. Conversely, people said, Okay, well why not use low-dose IL-2, write a lot less IL-2 and activate these regulatory T cells which are much more sensitive for things like transplantation medicine or for autoimmune diseases in particular diabetes. And that could, in theory work and has had some success in the clinic. But the problem is, my low dose is going to be different from your low dose and low dose, right? Everyone else's low dose. And obviously, if you go too high, right, with somebody's dose, then you're going to activate those same pathogenic effector cells that are driving the disease, right? So it's sort of like working on both sides, which can be a good thing. But in the case for therapeutic is really the pleiotropy is what's killing you. And so again, our mission with IL-2 is to be able to decouple the amino stimulatory and immunosuppressive effects of IL-2 to make a more effective targeted therapy. Unfortunately, we actually had a little bit of help in this area from none other than a western blotting antibody, right? You think your western blotting antibodies and you never know how important they are going to be. But there's a particular antibody called S4B sticks that wound up having this phenomenon discovered by underemployment, where it would actually bias cells toward an effector cell response. And conversely, there was kind of Yin and Yang this just 61 was actually biasing the immune response toward activating regulatory T cells. And so of course that beg the question of what, why is this happening? What's the molecular basis for this? So what other way right to get to molecular basis then to solve the crystal structure. And so we solved the crystal structure of the S4B six antibody, which biases toward effector cells. And just x one which biases toward T-Rex bound to the IL-2 cytokine, um, and here it's shown IL-2 cytokine overlaid with the Alpha, Beta and Gamma receptors. And what you can see I'm kind of a high level, right? Is that the S4B six and just explain which have opposite effects are binding on opposite faces of the IL-2, which sort of makes sense. And what was also exciting was kind of seeing the mechanisms through which the antibodies were actually modulating the behavior of IL-2. You can see in the case of the S4B six, it's actually blocking the interaction with the alpha receptor, but not seeming to affect beta gamma. And the just 61 seems to basically blocking that stem region with Beta Gamma, but not necessarily impacting the alpha. So focusing in on the S4B 6 side of things which biases toward effector cells. We were able to, through their structural studies and follow-up studies on binding and function to determine the mechanism through which S4B six is biasing IL-2. The way it's doing that is the S4B six binds to the IL-2 cytokine. And as you saw before, does not impede, but actually enhances its interaction allosterically with the Beta and Gamma receptors, causing it to be very active and promoting the activity of IL-2 or alpha low effector cells. Conversely, when the S4B sticks out too complex comes encounters and elsewhere L for high regulatory T-cell IL-2 or Alpha is actually blocked from interacting with the IL-2 by the S4B six, right, which is covering up that epitope. However, it can of course still interact with the beta, gamma receptors. But now the T regs have lost their edge, right? They used to have the advantage of the IL-2 or alpha receptor, but that's out of the picture. And so with the outer alpha out of the picture and the effector cells being overall more abundant, they get the advantage in terms of immune activation at the expense of t, right? Conversely, looking at the genetics one antibody, there was an interesting allosteric exchange mechanism that was, that came up in terms of how that one was working. And essentially that is the IL-2 bound to the just 61 is completely out of luck on an effector cell because it cannot interact with the beta, gamma receptors and results in activation because that epitope is blocked. However, if you look at an IL-2 or alpha high regulatory T cell, the IL-2 or alpha on the surface can actually grab the IL-2 right through an exchange mechanism, displacing the Jessica one, ejecting it essentially from the IL-2, allowing for the IL-2, IL-2 or Alpha to now form a robust complex, right? High affinity complex with the beta gamma. And that activation actually leads to up-regulation of the owl to our alpha subunit, which can then knock off even more of the antibody. The just 61, allowing for exquisite an exclusive signaling on the aisle two or alpha high T-Rex. So this was excited, right? We've figured out how these different antibodies are manipulating the behavior of IL-2. And we said, let's make a drought, right? Let's make this into some sort of therapeutically useful molecule. And so to that end we said, okay, well we have this antibody cytokine complex. And indeed for S4B six, it was shown to have promised pre-clinically in cancer. And just x one was shown to have promise pre-clinically in GVHD graft versus host disease and diabetes. But unfortunately things fall apart, right? So if you have this antibody cytokine complex, their translation of this complex is very limited. And the first reason is of course, that when it falls apart, you have the same toxic cytokine, IL-2 and rapid clearance as well, right? Because the antibody with its Fc domain and FCR in mediated recycling can actually extend the serum half-life of your IL-2, but you lose all those effects if it falls apart. In addition, dual construct dosing, right, can complicate therapeutic translation because how do you know, right? Do I give extra IL-2 but then I have free able to do I give extra anybody but then the IL-2 is not fully occupied on all of the antibodies sites. So right, there's the stoichiometry issue. And then in addition, there's increased regulatory hurdles, right, in terms of how you would have to present this, the FDA if it's actually two separate molecules. And so we said, well, let's take 1 plus 2 and make three and get an immuno cytokine, right? And this is not like your traditional amino side. So normally when people use the term, you know, cytokine, talking about taking the cytokine and just dangling it off the end, right off the C terminus will be that the heavier light chain. But this is actually on the N terminus. And it's what I call mickey Mousing rates that's making an intramolecular cytokine. So it's actually making these sort of Mickey Mouse ears with the IoT. I love giving this in person because I keep giving it on Zoom. And then my hands go like my arms go out of the screen and nobody knows what I'm talking about, you can actually see what I'm doing. So, so anyway, with the cytokine, right, it's really exciting because the idea is that now it's intra molecularly binding and the, anybody's actually guiding where the IoT was going. So for the S4B 60 immuno cytokine, we were like, All right, this is really exciting, right? We can like actually fuse these two things together, link them, and potentially make a drug out of it. But we actually had to go one step further because the S4B six is against nasa L2, but not cross-reactive with the human, which is often a problem in immune engineering and immune engineering translation. So to solve that, we took this 60 to Him, united cytokine, which is a 62 antibody, is against human IL-2 and actually does the same thing as the S4B six to the human molecule. And we were able to show that this was Mickey Mousing, right? It was intra molecularly binding. The way that we showed that is we took free IL-2. Saw whether it would bind to this complex. And we saw that compared to just free IL-2 binding to the antibody, we saw significantly less interaction. And in addition, we saw it completely ablated the interaction with the IL-2, our alpha subunit. So again, was having the same biasing effects like we talked about for S4 be six. And it was actually enhancing right allosterically or left shifting our curve for IL-2, our beta binding, meaning that we had an increased interaction with Beta, allowing for this to have a bias toward effector cells. And there was an additional layer of engineering that we had to undergo when we switch from the S4B six to the six O2. That was that the S4B six, right, again has this sort of left shift or bias toward effector like cells over T reg cells compared to the endogenous IL-2 cytokine. And so that gives the overall advantage to cells that do not express the L2 or alpha subunit. Unfortunately, when we switch to six O2, although we now got the reactivity with the human eye L2, we've lost that edge, right? You see significantly less bias in the case of the 60 to complex compared to the S4B six complex. So to rectify that, we turn to our trusty friend yeast surface display. We engineered the 600 tear to be more competitive with IL-2 or Alpha. So what we did was we put the six are two single chain variable fragments, which is the minimal region of an antibody that binds, consisting of the variable heavy and light chains on the yeast surface. We then took use error-prone the agenesis specifically focus on the CDR 1s and 3s of the variable heavy and light chain. And we took that library of mutated 60 to antibodies and transform that DNA into competent E cells such that each had a different, wildly different flavor lollipop on its surface. And then ultimately, we selected that for not only things that would bind to IL-2, but would actually be competitive with the IL-2 or alpha subunit. So the idea was that this would be a competition curve, say, between six O2 as we add more IL-2 or alpha, we see less and less finding. But we want to actually move that to the right, right, so that our antibody is more effectively competitive with our IL-2 or alpha subunit. So without going into the details, the painstaking details of my students and post-doc doing all of this work we were able to ultimately evolve SEMP is that right shifted and also improved the binding to the IL-2. And here you can see if we normalize, We're still getting about a tenfold shift in the IC 50. And ultimately we wanted something that would have a closer to one to one ratio, right? The best ratio we can imagine because it's always going to be active on T regs. But we want this is close to one to one as possible, right? So that the effector cells can compete right, effectively against the T regs. And so whereas we started with the amino cytokines 60 to being biased more towards the effector cells were able to get a number of different clones are best one being this F ten that had significantly more bias toward the effector cells. And so ultimately, we moved right from having this sort of very similar behavior on effector like versus tearing, like sells to a molecule that was worst until like cells and ultimately bias toward the effectors. And we actually looked in human PBM see, to see different populations in subsets and how they would respond. And indeed we saw that CD8 positive T cells had pretty much the same response for f ten amino cytokine. But then the T regs lost five logs of activity indicating that this molecule was very effective in biasing toward the CD8 positive T cells. And you can see that dramatic bias. This is on a log scale as well. For the F ten amino cytokine in terms of both CD8 positive effector T cells and CD4 positive effector T cells relative at the expense of T regs. We were also curious what would actually happen right in, on a signaling level when we created these amino cytokines, right? Because in theory, they could actually signal in a different way or through different pathways than the normal IL-2 would. The hope would be that it would actually just behave pretty much the same as IL-2, but have this new bias. And indeed we fortunately saw that. So we performed an RNA seek experiment on in collaboration with Warren Leonard's lab at NIH. And what they were looking at was sort of the six hour in 24 hour response of either a control amino cytokine that was just literally IL-2 fused by violently, but not Mickey Mousing. And then our Mickey Mouse f ten amino cytokine where the F 10, anybody would be biasing. And indeed we saw that at four hours and 24 hours there are pretty much know virtually no differences. My post-doc actually discovered, which is very useful. And you guys want the link I can send it. This software online where you can make Venn diagrams where like the overlapping regions are proportional to their size. So you can see the vast majority of genes from either just the free IL-2 from the control amino cytokine or from the F type of cytokine are vastly overlapping and you can see that on the individual heatmaps as well. So ultimately, we wanted to say, how is this going to work in terms of expanding molecules are extending cells in vivo. And so we actually put this into a mouse directly. And we saw a massive expansion in terms of CD8 and k's at the expense of T regs for both of these, for our F ten amino cytokine. And then ultimately, we wanted to see how this could be used in a therapeutic context. And so we have ongoing studies in a few different singed a mouse models. This is looking at be 16 F ten melanoma, very, very recent data where you can see a decrease in tumor growth without a strong, without any evidence of toxicity to these mice, which is a very exciting place to be for this molecule. Also say that, you know, IL-2 as a monotherapy is very limited and its performance, including clinically. But what's exciting is if we're already seeing this pretty strong activity as a monotherapy compared to how IL-2 would normally do. That really bodes well for how it would do in combination approaches. So on the flip side, right, we were talking about biasing the IL-2 cytokine to actually act favorably for transplantation medicine or auto-immune diseases by increasing the number of T-Rex. And to that end, we actually replace the just 61 amino cytokine with this F5 111, which again is against the human eye. Otzi was opposed to the mouse. And again, we did a bunch of binding studies to sort of indicate or try to identify exactly how this molecule is behaving. And you can see again that it's intra molecularly binding because there's significant reduction in its interaction with the immuno cytokine with free IL-2. There's a little bit of enhancement or about the same binding to IL-2 or alpha. And there's a complete ablation of the Beta binding, right? So it's basically the flip of what we were seeing for the F ten amino cytokine. And we then looked at basic behavior on PBM see, and this was really dramatic because I'd never seen a molecule that was this bias. But, you know, there's always a first time for everything. And here you see that on the T regs, right, we have a little bit of activity, kind of marginal activity compared to IL-2 or the complex. But I'm CD8. And on the CD4s versus T-Rex or CD4s, we see significantly less activity for the F ten amino cytokines shown in purple. How is this going to translate into a mouse model? And again, this is looking at the complex versus the F ten amino cytokine in expanding either T regs or to exit the expensive either CD8 or NK cells in mice. And you see a very strong bias toward expansion of the regulatory cells at the expense of the effector cells. And to look at how this would work in a disease model, we use the dextran sodium sulfate model of colitis, where mice are literally induced to develop a politest like disease by putting DSS and their drinking water. And we saw that dramatically, right? There was significant weight loss that resulted when the mice were just given the DSS without any therapy. But for the F ten amino cytokine, there was a significant delay and reduction in disease severity. And you can see that pretty prominently from the Colin's here. Dissected from the nice. When you can see that the healthy colon right, has these nice crypts and this nice very regular pattern. Obviously the mice that are ravaged by the disease have a much more chaotic, let's say like morphology. But you see a return to that more normalized morphology for the F ten amino cytokine. So really, really exciting. And I think starting to have a very like preclinical, hopefully soon clinical impact for the antibody cytokine fusion proteins. I'm going, I want to move on to the second part of my talk which is going to focus on the these IL-2, IL-4 actually cytokine magnetics that we've developed to basically do better than nature. And so as we talked about before, IL-2 normally has a bias towards cells that have high levels of IL-2 or alpha, or specifically a proclivity towards T regs. What if we were to flip that toward effector cells one approaches as we did with the S4B six. And so subsequent antibody is trying to bias the activity of the cytokine. But what about doing it by creating a brand new cytokine that would be biased toward effector cells. And to that end, we paired up with David bakers lab at University of Washington. And this shows the four helix bundle IL-2 cytokine. You can see the fork Ulysses here. And one of them, right? This green one is primarily interacting with the aisles who are Alpha. So we said why don't we focus on the intermolecular interactions between the IL-2 and the beta gamma chains ignore the alpha peak now distant body, right? All of those different amino acids from the actual cytokine itself. And then build piece by piece. A cytokine to accommodate those same interactions with the beta gamma. And now instead of having the fourth helix on top, try to interact with Alpha, we're going to actually pack that very safely under the crystal hydrophobic core to stabilize that protein. And another thing that we did toward the end of protein stabilization was we said all 37 cytokines known in nature, right? Have this same topology, which is up, up, down, down, meaning that from N to C terminus, the first two helices are facing up, the second two are facing down. And that's very inefficient, right? Because to get from the bottom of the top of the first helix, One way to the bottom of the second, and the bottom of the third, all the way to the top of the fourth, you have all this free spaghetti string, right? Sort of hanging out here in pink all over your protein that's destabilised, right? And so we said, and said, Well, we're making our own cytokine. Why don't we make it smarter than nature and do up, down, up, down, right? So now we just have these nice short compact entry vehicle linkers. And we'll have a much more stable molecule. So David bakers lab popped some of these different constructs out of the computer. And then we took an acid-base leucine zippered dimer of the aisle to our Beta, Gamma receptors and perform subsequent rounds of yeast display to evolve right and enrich the binding, or evolved the binding of the neo kind as we call it, or this new cytokine against the elites who are beta, gamma receptor. After several rounds we bounced out something that had pretty much no interaction or not pretty much like That's a flatline. Yeah. With the IL-2 or alpha receptor and had actually potentiated interaction with the beta gamma. And to that end, we saw a bias toward effector like over T reg cells, which ultimately bias the signaling in the direction that we wanted. And finally, we created a rock, right? We basically made this hyper stable thing that you can actually heat to 80 degrees Celsius for two hours and loses no activity. Whereas IL-2 is dead in the water in five minutes. And we also saw in terms of functional stability, we could literally almost boil this thing to 95 degrees Celsius after the first time that we did these experiments. Note to the y's do this in a thermal cycler. All of the liquid evaporates. And we just ended up with a powder. But no, we could boil this to 95 degrees Celsius in the molecule. So functional, so really, really exciting, right from an engineering standpoint that now we can actually create these hyper stable proteins. Whereas as I was talking about before, right? The proteins in nature are very marginally stable. Can't really play around with them. Too much extent. I'm going of course, again, from a therapeutic angle, we want it to do something useful in a disease. And indeed, we saw that when we treated mice with Neo two compared to either IL-2 were untreated mice in a C 226 highly immunogenic colorectal cancer model on your incent an egg, we saw a decrease, significant inhibition of tumor growth, extension of survival. Same thing in a metastatic melanoma model and then also the toxicity, right? That was what's important because yeah, IL-2 could be effective, but it's also going to be extremely toxic when it is effective. And we saw significantly less toxicity as shown here in terms of lung weight and pulmonary wet weight. And whereas half of the mice in the IL-2 treated group had to be sacrifice due to the weight loss or toxicity alone. Only two in the neo to group had to be sacrificed for that, the other five in the IL-2 group died due to tumor burden. We actually saw 50% long-term survival for up to eight weeks in the very aggressive be 1610 melanoma model in mice. And of course, the obvious question, right, that everyone would come up with at this point is immunogenicity, Gothic Washington blast. But everything, and especially you're going to ask about crazy sequence that nature has never seen before. And although this doesn't necessarily indicate that we won't ever see any toxicity with this molecule. It's actually in a Phase 1 clinical trial. First data will be reported in November. David Baker had some students that created an offshoot company called Neil look IN, OUT based on this molecule and other Neo kinds. And yet there are current clinical trials ongoing, but long story short, we have not seen any evidence of either B cell or T cell immunogenicity against our molecule. So it's really exciting, right, that we have this molecule that could be very therapeutically useful. But what else can we do with it, right? That we've created this rock. And one of the most exciting directions that we've gone with this molecule is to say, look, we've created this hyper stable molecule, all right, and we want to see how it's going to behave. And indeed, we've said, let's play protein Legos. Let's see if we can actually split this molecule right into its component Ulysses and then have it reconstitute in a cottage, conditionally activate Able way. This is sort of like mind-blowing for me. It was at least like, again, everyone learning about these marginally stable proteins that you look at them funny and like, you know, they spit out of the negative 80 freezer and they fall apart. But like no, this is a molecule. You can literally rip it apart and let it come back together. And what's exciting is then you confuse it to antibodies or other targeting domains against different markers that would be on your target cell, such that cells that express not just one, but actually both markers at the same time allow for the molecule to reconstitute on the surface of the cell. Then learning in an immune cell to come and kill your target cell, right? This is a really cool concept for a to be able to do this a protein. And really before these neo kind of think the FID, this was something that was basically like outer space, right? But what's really exciting is we've actually again collaborated with David Baker cgroup to look at different ways to split up this molecule and actually achieved this goal. So the first thing that we did was we said, okay, there's four Ulysses, there's three different ways we can essentially split these up into its components, Ulysses. And we looked at each of these and we said, which one is going to be the most selective, right? The cleanest system. So ideally we would want something that looks like this first one, right? Where the individual pieces by themselves, the H1 plus h3 two-prime for or not active, right? Completely inert. But when we actually put them together in solution, they have some level of activity, obviously not as strong as the intact Neo too, but there's a window right, for targeting them. So we chose this h one plus h 3 to frame 4. And we, as a test case, we actually conjugated these two Darwin's designed anchored proteins to small binding domains against either EGFR her to growth factors that are highly upregulated. And a lot of cancers that we looked at, cells that are expressing our targets versus cells that are not expressing our target. And hoped that we could achieve bias toward our target cells. And indeed, right, I'm sort of abstracting a few years of works ensue a single graph. But this was an exciting moment for us and we were able to show pinpoint specificity for our target cells compared to our off target using these targeted split fusion proteins. So building off of that, we again wanted to try how this, or see how this would work in a therapeutic model. And so we took a PD-L1 high or over-expressing be 16 F ten melanoma model and we fused are splits the h, h1 and h3 true for him for two anti PD-L1 or an anti PD-L1 nano body such that it would bind to these target cells. Um, and indeed we found that the targeted slits were the most effective, shown in red at inhibiting tumor growth. The most effective in slowing and ultimately leading to long-term survival of these animals. And also that there was no consequence on weight loss when you use very high doses of the original and the O2 molecule shown in purple, you ultimately will get toxicity to the animal. But you can use crazy high doses of these conditional splits, right? Because you're just localizing them to the tumor micro-environment. And you can achieve the same therapeutic effect without inducing toxicity. So that was all well and good. But we wanted to go beyond IL-2. Right. I'll just talk has been about IL-2. What about the rest of the cytokine space and cytokine world? And what's really exciting is that the common gamma receptor, which is one of the receptors of the IL-2, signaling receptors of the IL-2 receptor subunit is actually shared by six different cytokines, IL-2, 4, 6 or two that 24791521. And what's exciting is that you can actually swap out, potentially write the aisle to our Beta for a different receptor and actually then create different neo crimes against other cytokines that reflect in a minute other cytokines. And so indeed we preserve the interactions with the green and blue helices here against the IL-2 or Beta. And we modified the yellow and the red slightly to type reverse, we kept the yellow and the red the same rate with the common Gamma. We manipulated the interactions with the green and blue and voila, we've got an IO for mimetic that binds to the 0 for Alpha cytokine. And again, we showed that this was a hyper stable interaction. We showed interaction with the IL-4 receptor subunits on cells. Similar levels of interaction for the Neil For shown in green. And when we heated to 95 degrees Celsius for up to two hours, we lost all of our interaction with the human eye 0 4, or start with the original human IO for molecule. And no difference, no change over four hours with the Neil for. So I have four. It has been shown to play a very important role in healing from traumatic muscle injuries. Things like tearing your ACL. And our collaborators, Jennifer Lee, CFS lab at Johns Hopkins, has done just prolific work in terms of regenerative medicine. And they've developed a model for tearing your ACL or for traumatic muscle wound that actually involves removing a large piece of the quadriceps muscle. And what they've shown is with ECM scaffolds and then doing a subsequent follow-up studies running my son treadmills to look for exhaustion. They see that there's a huge role of T-cells, particularly TH2 responses. That are driven by the IL-4 cytokine. And so you see in red here with the RAG knockout mice, you have dramatically less lower levels of the IL-4 and that leads to dramatically lower amounts of running to exhaustion. So basically indicative of slower and less effective wound healing. So we wanted to see if our Neo4j shown in green could actually recapitulate the same PRO regenerative signature, tea, She's signature that resulted from IL-4. And indeed when we treated with the Neo4j, we saw very similar phenotypes both in the muscle and the spleen. We subsequently said, this is really exciting, right? What else could we do with this hyper stable, crazy powerful amino cytokine? And we said, well, let's throw it in 3D printer, right? Because again, it can deal with these really high temperatures, right? Really high pressure situations. And so we actually literally with 3D printing us, the circular scaffolds at a 120 degrees Celsius. And we showed that our molecule had a beautiful release profile and that was completely active coming out of those scaffolds. So really, really exciting, Great What we can do with these new kinds well beyond what we can do with regular cytokines. And even further, right? We said what would be useful in the case of IL-4 is that IL-4 like IL-2 is a pleiotropic cytokines can actually signal through two different types of complexes. There's the Type one that consists of Alpha or Alpha in the common Gamma. And then there's the type 2 that has alpha or alpha with the 13 are alpha 1. And in this case, right, we had actually engineered this thing and designed it to only recognize the type 1 complex and not the type 2. Type 1 is primarily expressed and not a poetic cells. Whereas the Type 2 is on non-modified cells and the type 1 is associated with M2 polarization, a more pro regenerative TH2 response phenotype. Whereas the nonpolar poetic cells that express type 2 are generally associated with hypersensitivity and hypoplasia. So we created, again this molecule that sort of had a very different profile or trajectory. So you can see in black, right? The IL-2 doesn't care what levels of type 1 or type 2 receptors are on the surface. It's going to be equally active on all of these different cell phenotypes. Whereas we can start to see bias right between the different cell phenotypes to being completely inactive. And type 2 for the i 04 for the Neo4j cytokine because it's got fundamentally different properties in terms the binding. And so we basically completed the trifecta this so in nature, IL-4, right, can signal for either type 1 or type 2. I'll 13. Another natural cytokines can actually only signal through type 2. Now we've completed, right, collect all three we have Neil For, which can only act on the Type 1. So very exciting, right? Sort of the new biology that we can create with these set of hands. How many during this time? I think I'm good to have just one wrap up with this third story that I'm really, really excited about, as I said from a translational angles. So the final story I wanna tell you about is these by specific antibodies that we've designed targeting cancer metastasis. So metastasis, as many of you may have no Nino is the number one killer, right from cancer. So solid tumors, 90 percent of them, an incentive, deaths from solid tumors actually result not the primary tumor, but from it's spreading to other organs in the body, which, you know, as you think about it, right? It's probably intuitive. Depending the primary site might not be as dramatic or detrimental. But once it gets to other organs, particularly the brain, are bones that, that can be very problematic. So if you look at these graphs rate just the whitespace on top. This is looking at survival, mean survival of localized disease versus metastatic disease. And these different bars, if you can see the colors there, the lighter ones represent 2015 and then the darker ones are 2005. So over that 10-year period, right, you're seeing very little progress in terms of increasing the survival rates for a variety of different cancers when they're metastatic. And so we looked at sort of what are the different processes, right, in order for cancer cell to get out of the primary tumor rate it has to go detached from the tumor. Intravasate, get into the blood vessel, circulate through the blood vessel, extravasate out of the blood vessel and then ultimately form the metastatic niche, right? It's new site where it's going to grow into a secondary tumor. And all of these different processes, right, involved 3D cell migration, right, in some way, shape or form. And that's really an instrumental part of this whole process. But some videos here, Let's see if this works. So what, where all of this project originated from was really just a kind of discovery that didn't. He works his lab at Johns Hopkins that does a lot of work with cancer cell migration and understanding cancer cell mechanics. It was this observation that they made with when they seeded sells at slightly higher densities. They were able to see. I think I forgot on this slide, I'm not supposed to use the pointer because it solves that. Okay, great. So if you see the cells at higher densities shown on the right, versus lower densities shrivel up the one on the left, his post. That one's not moving it up, but no. There we go. You see it moving significantly faster when it's seated at higher densities versus lower densities. And so we were wondering what's the cause of this phenomenon. And we sought, was sort of this linear increase. Not anything dramatic, right? But there's a cell speed dependency on the cell seeding density. And what was interesting is they saw this on cells that were metastatic cancer cells. They didn't see it on normal healthy cells, and they also didn't even see it on cancer cells that were non metastatic, right? Something about the seeding at higher densities that was leading to this increased cell speed. And two, without going into too much detail, they wanted to identify what was the culprit for this. And you can see this kind of clearly jumped out that there were two cytokines, IL-6 and i 08, that seem to be sort of a culprits here. And that they were showing up at elevated levels as you increase the cell density. And they wondered, was that actually related to this cell density dependent increase in cell speed. So to get to the bottom of this, they did the knockout experiments where they knocked out either the IL-6 or IL-8 ligands or receptors. And they saw in all cases that the knockout of either ligand in and of itself would somewhat decreased this LCD. So both of them played a role. Unfortunately, when you knocked out them, both the cells are not viable. So you couldn't actually do that experiment. But you could do an inhibition experiment where basically you would introduce total isn't that, which is a commercial anti IL-6 receptor antibody and rep Erickson, a small molecule drug against the receptor. And what they saw was there was a synergistic in a decrease in metastasis resulting from the treatment with the IL-6 and I'll eat inhibition combination but no change in the tumor growth. So you might say, Oh, well, that's not really going to be good as the drought, right? You obviously want to also shrink the primary tumor. The good news is all other drugs right, are targeted at shrinking the primary tumor, right? There's no drug currently that specifically and exclusively targets the process of metastasis. So what's exciting is we can combine this with drugs, right, that very effectively control the primary tumor or respect the primary tumor. And now this could be an adjuvant therapy just focused on metastases. So with the angle of your turning this into a drug, we said, well, we don't necessarily want to stick with this whole like anybody small molecule combination for variety of reasons. The first reason being that small molecule drugs can have one non-specific effects and a lot of different ways. And that the duals construct dosing again, like we talked about before with the immuno cytokines, creates challenges for FDA approval. But really even more than all of that. It was actually to be honest, which many things are in IP issue, right? Like there was total is a map is an approved drug for rheumatoid arthritis, not used in any way in cancer. And rep Erickson has been developed in some phase two studies, although it hasn't done very well on its own. And we actually talk to Jeanette attack, who owns a Roche, I guess there now bought up by their own socialism AB. And we talked to accompany don't pay. They're both like literally miles apart. Just a few miles apart in the Silicon Valley area. And they were like, we're not touching them with a 10 foot pole. Roche actually told us they were not interested in oncology for this molecule, which I don't know. Okay, Sure. Sounds like a good plan for your portfolio, but anyway, we said, you know what the heck with you? We're going to take matters into our own hands and make by specific right, megabytes of the antibody have one arm targeting the 0 6 receptor. One I'm trying it out eight receptor. And we made it in two different formats of divalent and a tetravalent because we didn't know how geometry would actually affect how the molecule worked. And we wanted to have a couple of shots on goal. So again, I'm not going to have time to go through all the detail. We actually just submitted the manuscript recently, though this will hopefully via print very soon. But we wanted to show obviously that are by specific antibodies could bind to both IL-6 and i 08. So we made cell lines expressing exclusively those receptors. We also mean salons expressing both. And we obviously had the original cells. This was like one of my grad students favorite experiments because this is exactly what you wanted and that was very nice. We also wanted to see that it was competitive. And so we did the competition experiments on our IL-6 receptor positive cells and showed that the antibody binding would preclude interaction with the IL-6. And that worked for both of our BI specific shown in green and orange. And same thing on the aisle eight receptor. We showed robust competition with the eight receptor for both of our BY specifics. And ultimately, we wanted to look at the functional response and how we impacted that. And so we looked at tumor cell migration in three-dimensions. Looking at a triple-negative breast cancer cell line MBA and B31. And you can see that normally, right? This is just 24 randomly selected. Trajectories of cells and they're kind of moving around. Some of them are more persistent in one direction, summers moving all over the place, like not really going anywhere productive. But if you add the totals and I'll post up orexin, right antibody, small molecule combination, you see a significant reduction in the cell migration and also much less persistence. And if you add the two anybody separately, again, you get some effect, right? Slowing the motion. You add by specific, right, either be S1 or S2. Pretty much nothing's happening, right? It's either not moving at all, like that, that trajectory, or it's just moving around in circles and not really going anywhere productive, which is really exciting from a cancer metastasis perspective. So again, if you quantify that, just to kind of drive things home, we see that we're doing really well with our biceps. Big one, even better with R by two. And then even better than the combination antibody small molecule, insignificantly better in both cases, then adding the antibody separately. So you might wonder, well, why is this doing so much better than adding the two antibodies separately, right? Shouldn't they have sort of the same. The fact like blocking the 68 actually turns out that it's a receptor expression issue, which one would imagine would be the case that the IL-6 receptor and I'll eat receptor actually expressing very different levels on cancer cells. So if you look on MVA is you really can't even see any of the IL-6 receptor. You see a little bit of the i 08 receptor sort of shifting. But it's just virtually undetectable for the IL-6 receptor. But by having these molecules right, that have both the IL-6 and I'll eat receptor dependency. You have that avidity effect. And what's happening is that the more abundant receptor, right, the eight receptors, GPCR is actually learning in the biases big antibody, which now can actually interact with the IL-6 receptor that's expressed in the same cell because once one arm is tethered or the other one has limited mobility. And that allows it to effectively recognize and block both receptors on the same cell. So ultimately, we wanted to see how this would do in various tumor models. We first looked in an MDA on B23 one scene cells we were looking at in-migration, an ortho topic tumor xenograft, we saw a remarkable amount of potency from this molecule. We're actually kind of floored. So initially we were treating with very, very low levels like 0.03 megs per keg down to 0.1 made per keg. Because we actually saw that the most efficacy was usually with very, very low doses. And when we actually used the combination treatment, the anybody's small molecule, we had to go with 30 makes for a more standard, goes 300 fold higher, and we were getting a worst response. So we did the full tumor model and we've looked at lung that's following the MDA and be 2, 3 one tumor and subsequent metastasis model. And again, you can see significant reduction in the lung that abundance for the BS1 and PS2. The two by specifics at a significantly lower dose. In this case, I'll 30-fold lower dose than Tesla's not separates. As we said, this really did matter that we made it by specific argument is always, well, why don't you just use the two combination of antibodies? If we're just use the combination of antibodies, this project would have been done like this just doesn't work. But clearly with by specific variable to get that efficacy. And ultimately, as I said, we wouldn't want to treat as a monotherapy, right? Because then what about the primary tumor? And so we actually combined this, in this case with a chemotherapeutic gemcitabine, which is standard of care in some models of or in some triple-negative breast cancer. And we saw that our molecule paired perfectly well with this tumor growth, resulting in synergistic reduction in tumor growth, and also virtually no metastases. Okay. So I'm probably a few minutes over and I do want to hopefully have a couple of minutes for questions, but just to conclude, wrap things up, I hope you've been convinced that protein engineering is a very powerful thing from a lot of different angles. Particular, we talked about the cytokine fusions and their role and delivering IL-2. To realize the therapeutic potential of this molecule. We talked about de novo cytokines and how they can use to enhance the stability, selectivity and ultimately therapeutic activity of natural proteins. And finally, these biases again, but he's targeting the synergistic IL-6, IL-8 signaling pathway that inhibit metastases very potently. I'll also like to call this my 2, 4, 6, 8 talk because we got IL-2, IL-4, IL-6, IL-8. That was not intentional. But I will ten is nice. So thanks again for your guys attention. A huge thanks to the people that actually did this work, which we're not me. I've been just blessed to have phenomenal students and postdocs in my lab. This is actually a very dated pick, not very dated, but two years dated. Picture the last time? No, almost more than two years now before masks. So yes. It was July of 2019, So yeah. Been more than two years at this point. So the people in the group are, the composition is a little bit different, but huge thanks to Jacob and a list of phenomenal post stops. You did a lot of work on the amino cytokines. Derek who worked on the immuno cytokine for T regs. Lynn, who's done a lot of the work on the by specific as well as for the cytokine, the medics, Edward and went our master's students that helped out with this project and several undergrads that also helped out. Huge thanks to my collaborators. Mark Coval in the Czech Republic. David Baker at University of Washington might do again, did a lot of the mouse work for the set of kind of medics didn't work. I mentioned with the civic project was Jaffe also, um, as well as all of the funding sources that obviously funded and promoted this work. Thanks so much to all of you guys for your attention. Thanks again to Johnny and the Department for the invitation and happy to take questions if there's time. It blew my mind. Yeah, like that happen. Excellent question and guys, I glossed over a lot of things so that I could cover more ground, but not necessarily as much that. So in those experiments, what we were actually doing was sort of these functional selections, where instead of just adding a single target as bait like the ILD thing, we obviously want to find it out too. We also wanted to be competitive with the Alpha. So we actually added in the competitor alpha and we looked for things that could still bind. So those things would be more effectively competing with the IL-2 or Alpha receptors. So we did like kinda these functional Slash. Yeah. Hopefully no more after the town. Yeah, good question. So right. So the question of endogenous proteins, right? Versus ones that you're adding. Endogenous cytokine levels are actually very loud in your blood. Very, very low in general. So you're usually not really a very elevated levels unless there's some sort of injury or insult that would cause that to be majorly secreted from the T cells. With regard to the dosing regimens, we were actually just using equimolar, which turned out to be pretty much equal molecular weights. Because the, the Neil kinds are a little bit smaller because we cut out some of that spaghetti, but not really that much smaller. So we were just using equimolar amounts in both. The thing was, but this Great question that you bring up about the dosing, because we did actually have to really, really aggressively dose these molecules. Because as we've talked about the clear very quickly, because they're under that 50 Katie cutoff. And so they're very quickly peed out. And so we essentially had to, you know, Joe's these things, it up to like a 100 micrograms per day sometimes just to make sure we have the sustain therapeutic effects. So yeah. Great question. So with the last project that I talked about with the IL6 while a bisphosphate, we actually just started doing some of the PK studies and we were actually kind of floored but it's like all going to the tumor we were like Okay, there's going to be like so I'm going to the kidney or the sleeve, like whatever, like when you put it in the mouse, we were using this law like word near-IR dies. And it was just blowing up the tumor like to the point where we're like we were like blasting like the the imager. But then we couldn't see anything else like it was just completely black everywhere else. So we are getting you the good tumor localization and I think, you know, for different projects, right? You depends on different things. So like the IL-2 won't necessarily go to the tumor and less in the case of the slit cytokines like we targeted there. But in the case of the IL-6, IL-8, by specific, it seems that the cells that are most highly expressing, both of those targets are localized the tumor. And so we're getting really good tumor localization in terms of the PK properties, right? This sort of half-life and longevity with antibodies, of course, we benefit from the Fc domain, which will keep it around a lot longer than one would imagine for a molecule of similar size. And in the case of the medics. And that applies as well for the first project for the amino cytokines that we have the antibody in there. So it's going to behave like the antibody in terms of its half-life. For the medics, we've had to extend the serum half-life using MSA or other approaches, which is what the molecule in clinical trials, well, HSA, but the molecule and clinical trials, this is easy.