Laura Walker: "Broadly Neutralizing Antibodies to Speed Pandemic Responses" (12/8/21)

FACUNDO BATISTA: Good morning. I am your host, Facundo Batista. Together with Richard Young and Kalon Overholt, we organized this MIT course on COVID-19, SARS-CoV-2, and the

pandemic

. On behalf of the three of us, thank you for joining today. The purpose of this course is to learn what we have, what we know about the virus and the

pandemic

from the best scientists from around the world. In addition to vaccines, monoclonal

antibodies

are a very powerful strategy to treat and prevent COVID-19. And who better to talk to us about monoclonal

antibodies

and therapies than our speaker today, Dr.

Laura Walker, a leader in this area. Laura is one of the most dynamic young visionary scientists I have ever met. Laura earned her PhD in immunology and microbiology at Scripps Research Institute. And she was one of the pioneers in the discovery of

broadly

neutralizing

antibodies against HIV. I feel this has opened the way to a possible rational vaccine design for this virus. She then completed her postdoctoral research at the University of California, San Francisco. And in 2012, she made a very brave move by joining Adimab, which today is one of the most prestigious antibody companies in the world, where she is now senior director of antibody science.

Laura's work focuses on understanding human B cell

responses

to viral infection and discovering

broadly

neutralizing

antibodies against our wide variety of emerging pathogens, including Ebola, Zika, yellow fever, and more. recently, SARS-CoV-2, where he directed and discovered several potent monoclonal antibodies. Very recently, Laura has become one of the scientific founders and chief scientific officer of Adagio Therapeutics, a company developing commercialized antibody-based solutions to address the current COVID-19 pandemic. At Adagio, Laura oversees a broad portfolio of research in the area of ​​viral immunology, including the identification of potent and broadly neutralizing antibodies to treat and cure diseases. Laura received the James Houston Antibody Science Talent Award and I believe this is one of the many awards she will receive in her career.

And Laura, we are delighted to have you here. Thank you very much for joining and we look forward to your talk. LAURA WALKER: Thank you, Facundo. And thank you very much for the invitation. I am very happy to speak here today. Let me share my presentation. So, as Facundo mentioned, I'm going to talk about some of the work that we have been doing first at Adimab and then at Adagio, identifying, designing and now developing broadly neutralizing antibodies to combat not only SARS-CoV-2. , but also future emerging viruses similar to SARS. Oh my god, here we go. So, given the general nature of the audience, I thought I would start today with a more general overview of neutralizing antibodies.

More or less what these antibodies are and why we are interested in inducing them through vaccines and developing them therapeutically, at least to combat viral infections. And on this slide here on the top left, you see a cartoon version and kind of an image here on the left, of a human IgG antibody. And the only thing I want to highlight here is that the top half of the antibody, which is called the Fab region, is the part of the antibody that binds to the antigen, the pathogen, the virus, for example. And the bottom half of the antibody, called the FC region, is the part of the antibody that mediates the long half-life that antibodies have and then also interacts with other immune cells to mediate what are called Fc effector functions.

And so antibodies really have two main functions. They may have activity against free viruses, this is called antibody-mediated neutralization. And that's essentially when an antibody binds to an envelope protein on a viral particle, which I'll talk about on the next slide. And that essentially prevents viral entry. And then antibodies can also have activity against infected cells. And again, they bind to the spike proteins. And in this case, it is expressed in infected cells and that can prevent cell-to-cell spread. And it may also allow the antibody to mediate Fc-dependent antiviral activities, for example, by recruiting certain innate immune effector cells such as NK cells or macrophages, which essentially kill the infected cell or may simply phagocytose it.

But today we're going to focus on neutralizing antibodies, and that's because these are the types of antibodies, in general, that are most important for antibody-mediated protection in vivo. And the definition of neutralization that I just put below, which is the loss of infectivity that occurs when an antibody binds to a virus, and that occurs without the involvement of any other agency. Thus, for example, in the absence of other immune cells. And this is just a close-up of a viral particle, and these red, orange, and yellow spots here are the spike proteins, also called envelope proteins. And these are the proteins that interact with the receptors, expressed on the target cells.

I mean, these proteins are what mediate viral entry by binding to the receptor. So what neutralizing antibodies do is bind to these envelope proteins. And they prevent this envelope protein from interacting with the receptor. Therefore, they directly block receptor binding, or they bind and prevent conformational changes necessary for viral entry. These are the types of antibodies we will talk about today in the context of SARS-CoV-2. So just a couple of background slides on SARS-CoV-2 and coronaviruses in general for you. So one of the points I wanted to make clear is that SARS-CoV-2 is actually a member of a very large family of coronaviruses.

There are actually four different types: alpha, beta, delta and gamma. It is the alpha and beta coronaviruses that have been shown, at least to date, to be able to infect human cells. There are four seasonal coronaviruses that circulate and are endemic in the human population. They basically cause common colds. Two of them are alpha coronaviruses and two are beta coronaviruses. And just within the beta coronavirus subgenus, you can see that there are four different lineages. And within each lineage here, only a subset of these viruses are shown. The diversity is absolutely enormous, but within the beta coronavirus genus, it can be seen to comprise the three new pathogenic coronaviruses that have spread to the human population from animal reservoirs over the last 20 years.

And that started with SARS-CoV-1, which emerged in 2002, 2003 and caused the SARS epidemic. MERS, which emerged in 2012 in the Middle East and remains endemic in the Middle East. That's a C-lineage beta coronavirus. And then, of course, SARS-CoV-2, which emerged in late 2019. And that's another B-lineage beta coronavirus, so it's very closely related to the SARS virus. original. And so, as I just mentioned, I mean, I would say, and I think many would agree with me, that it is virtually a certainty that new coronaviruses will emerge in the future, in the same way as SARS-CoV-2. It followed the original SARS and MERS.

And we know this for multiple different reasons. And one is... I just mentioned it, and that is that there is enormous genetic diversity among the coronaviruses that circulate in bat reservoirs. And there have been many studies that have shown that a subset of these viruses, particularly SARS-related viruses, can infect human cells through human ACE2. Therefore, these viruses could, in principle, jump directly from bats to humans without any adaptation in an intermediate host. So these are very high risk pandemic pathogens for that reason alone. And here on the left, you're looking at a phylogenetic tree of many of these SARS-related bat viruses, at least the ones that have been identified to date.

And there are thousands and thousands more who have not been identified. But the point here is that if you look at the lines, these lines indicate where these viruses are circulating geographically. And the point here is that many of these SARS-related viruses are circulating in the same regions of southern China, essentially in the same caves and in the same bats. And this offers ample opportunities for recombination between these different coronaviruses. And that is because coronaviruses have a very modular genome. Basically, a part of one can recombine with a part of another and now we have a new coronavirus.

And this is how this enormous genetic diversity is generated. That's why we know that between 6% and 1/2% and about 23% of bats in China harbor these coronaviruses. And possibly most importantly, serological surveillance studies have shown that a relatively large proportion of people living in rural China (i.e., between half a percent and 3 percent of millions of people) have been really exposed to these SARS bats. -related viruses. And that tells us that these bat viruses are actually spreading to the human population all the time. We don't hear about them because they don't normally cause epidemics or pandemics, because they are not well adapted to humans and they do not transmit well.

But the point is that, because these contagion events occur frequently, the probability that we will see the emergence of another virus, the coronavirus, that has achieved this ideal balance between pathogenicity and transmissibility, is actually very high. So the argument would be that we need broadly active solutions, both in the form of vaccines and therapies. And arguably we need those vaccines and therapies, not only against future emerging coronaviruses, but even against SARS-CoV-2 variants that are now rapidly emerging in the human population. And that could emerge in the future in animal reservoirs, like what we saw in 2020 with mink populations in Denmark, where the virus jumped from humans to mink, diversified, and returned to humans again.

Something like this could happen again. And as you all know, we've seen these waves of variants over time, as shown here in this graph at the top. Right now Delta is dominant. It may soon be displaced by this Omicron variant, and there will likely be variants that displace that variant, given what we know about the evolution of this virus. And more importantly, many of these emerging variants of concern have been shown to be resistant, very resistant to neutralizing antibody

responses

. I talked about this type of antibodies at the beginning. Basically, they show reduced susceptibility and two variants of concern in particular that have emerged to date, and now there is new data on the Omicron variant.

I believe it will soon prove to be the most resilient of all the variants that have emerged so far. But what you can see in this graph below, and this has been shown in many studies. I chose one from the Ragon Institute, since that is where I give this talk. But you can see here that the gamma variant, also known as P1, in the beta variant also known as B.1.351, and which first emerged in South Africa, is significantly less susceptible to neutralizing antibodies relative to the original form of the virus. And more importantly, that resistance is associated with reduced vaccine efficacy, and that's been shown across the board now.

So one of the scientifically interesting things about these variants of concern, at least many of them, is that a large number of them encode convergent amino acid substitutions. And so, although the overall mutation rate of SARS-CoV-2, and other coronaviruses, is not very high, because the virus encodes a corrective polymerase. Here is some data from Betty Korber comparing the genetic diversity of HIV Envs. Of course, this is the most variable of all viruses. You can see that every little dot there is a different variant. You can see their enormous diversity compared to the SARS-CoV-2 isolates on the right, many of them were limited, but more importantly you can see that many of these variants encode the same substitutions.

And so the virus has essentially figured out a way to incorporate a very small number of amino acid substitutions, which have very large phenotypic consequences. And essentially what these substitutions do is allow the virus to evade common classes of neutralizing antibodies. And that's what I'll explain here in the next few slides. And so what we know about the neutralizing antibody response to SARS-CoV-2, and there have been many, many studies published on this by us and many others. So what we know is that the vast majority of neutralizing antibodies target a single subdomain. Here on the left, showing you the structure, the cryo-EM structure of the SARS-CoV-2 spike protein.

And the labeling here is the N-terminal domain and the receptor binding domain in green. Therefore, most of these highly potent neutralizing antibodies attack the receptor binding domain. They essentially target this domain, and most of those neutralizing antibodies do so by blocking the interaction of the receptor binding domain with the receptor. And then on the right, it just shows you some of our own data. This was published inScience Immunology earlier this year, maybe it was last year. I just show you the proportion of binders that target these different epitopes. And you can see that almost all of them join the RBD and compete with ACE2.

And many, many other groups have demonstrated this. This is true not only for SARS-CoV-2, but also for other coronaviruses that have been studied. And so they basically target this kind of small antigenic region of the virus. But when you actually look at the sequences of the antibodies, the neutralizing antibodies, one of the surprising things that we and many others discovered early on is that the response to these regions in the receptor binding domain, the response of the antibodies. is surprisingly restricted in terms of the sequence characteristics of the antibodies and the residues with which they interact.

So when you look through many different individuals, you find antibodies that look almost identical, which is relatively unusual. It's what we call a public antibody response. And because they recognize the receptor binding domain in such a similar way, it is perhaps not surprising that many of these antibodies share the same escape mutations. The same mutations will abrogate the binding of many of these so-called class one antibodies, and the same for class two and three. Basically, there are these three main groups of antibodies. And these escape mutations are shown here, at least some of them. So we know that, for example, this K417N or T substitution, those substitutions that are present in the beta and gamma variants, remove a large proportion of these class one antibodies.

The same goes for E44K, which is a substitution that many of you are probably familiar with and that eliminates many class two antibodies. And these are the escape mutations of many of the class three antibodies. And as I mentioned, the beta and gamma variant have incorporated both the K417 and an N or a T, along with an E44K. And what we and many others have shown is that just these two amino acid substitutions are capable of generating resistance to a large proportion of neutralizing antibodies. Here is more data from the Science Immunology article. Each row here represents a neutralizing antibody that we identified in a COVID-19 patient.

We see similar results with infected people and vaccinated people. And the darker the color here, the stronger the knockout effect. So what you can see is that more than half of the antibodies have reduced activity or completely abolished activity against the beta or gamma variant. I mean, that's true even when you look at these two different substitutions. Therefore, it is not so surprising that we are seeing reduced titers of serum neutralizing antibodies against these viruses, given what we now know about monoclonal antibodies and the properties of those antibodies. I just wanted to include a slide on the Omicron variant.

And that's why, just because of the sequence, I and many other scientists are very concerned, and that's because of what we knew about the neutralizing antibody response. And as I mentioned, the Beta and Gamma variant, which were the most resistant variants described to date, have this K417N and T, and the E44K. But with Omicron, you've now superimposed on that another escape mutation at the class I site, Q493R, and then two substitutions at the class III site. So for Beta and Gamma, class III antibodies were probably mediators and carried a lot of weight in terms of neutralization.

Therefore, the expectation was that this variant would potentially be much more resistant than what we have seen to date. And I think the latest data from Pfizer, and there's another preprint now, that's turning out to be the case. So the other problem when you think about therapeutic antibodies is that antibodies that are... the reason we're seeing the emergence of these variants is because essentially there's pressure... immune pressure on the virus, on these different regions. antigens within the receptor binding domain, which essentially drives escape at those positions. So, think about COVID-19 therapeutic antibodies, for example, antibodies from Lilly, Regeneron, AstraZeneca and Celltrion: these antibodies were extracted.

They were isolated from patients with COVID-19. So you won't be surprised to learn that most of them... they all fall into one of these three main classes of neutralizing antibodies. And it is not surprising that many of them are sensitive to those same mutations. And so we know, for example, and probably the best example would be if you look at the two antibodies in the Lilly cocktail, bam and ete, that are highlighted here, neither of those antibodies are active against the Beta or Gamma variant. . And that's because of the reason I described earlier, which is that that variant has a mutation at the class one site as well as the class II site.

And one of those antibodies is class I and the other is class II. And so the same antibodies that exist in nature that are putting pressure on the virus are the same types of antibodies that certain companies are developing as therapies. So it's not particularly surprising that we're seeing the emergence of resistance to some of these therapeutic antibodies. I will therefore argue that, at least in principle, the inherent characteristics of broadly neutralizing antibodies should offer a higher barrier to resistance. And when I talk about broad neutralization, I mean antibodies that can neutralize, for example, many different viruses related to SARS, like these bat viruses that I mentioned earlier.

And there are two reasons for this. One is that these very broad spectrum antibodies (not always, but usually they target epitopes that are what we call immunorecessive or immunoquiescent), epitopes that are not easily attacked by the endogenous neutralizing antibody response, which means that there is a pressure very limited immunity on these. particular antigenic sites. In other words, there is no reason for the virus to start mutating in these particular regions, because there are not many antibodies that look like these circulating in patients or vaccinated individuals. And the second reason, which is possibly the most important, is that these types of antibodies, by definition, recognize residues that are highly conserved in all of these different strains of the virus, or different variants, for example. (Sorry, this is trying to force myself to quit.) And that is what allows them to be largely neutralizing.

And the reason waste is normally kept is not without reason. Usually it's because they are important, in some way, for viral fitness. And so it essentially makes it harder for the virus to start mutating these epitopes without suffering some kind of fitness cost. And that's what we set out to do. This was in March 2020. We set out to identify an antibody that would neutralize not only SARS-CoV-2, but also the original SARS virus and also many of these other more divergent SARS-like viruses in bats. So what we did was we got a blood sample from a survivor of SARS from 2003.

And then we took those B cells and sorted them with a SARS-CoV-2 spike protein. So the idea with this heterologous probe was to preferentially identify cross-linking antibodies. And so, from this classification effort, we identified 200 antibodies that showed cross-binding activity, of which a subset, seven of them, showed cross-neutralization. That was all described in an article that was published last summer and I won't talk about it today. I'll talk about the second part, which is that we took three of these seven broadly neutralizing antibodies and further optimized them through protein engineering to improve their neutralizing potency while maintaining breadth.

So we improved the affinity, as I'll show you, between 500 and 1000 times, which translated into an improvement in the neutralization potency. And so, from this effort, we identified three therapeutic candidates, which we called Adagio-1, 2 and 3. Adagio-2 became the lead antibody. Adagio-1 became a potential cocktail partner. And then we modified Fc Adagio-1 and 2, so we introduced a two amino acid substitution in the Fc region, which is the region I talked about in the first slide. And it has been shown that these two amino acids can extend the half-life of the antibody, which I'll talk about at the end.

This only shows you the data for the original seven antibodies. These are the ones we isolated directly from the SARS patient before doing any affinity optimization. And this refers to IC50 neutralization. The lower the number, the more potent the antibody. So you want to be down here. And we looked at neutralizing activity against SARS-CoV-2, but also against SARS-CoV-1 and a virus related to SARS in bats called WIV1. Therefore, it is essentially a representative type of pre-pandemic coronavirus. And so, as expected, all antibodies were more potent against SARS-CoV-1 relative to SARS-CoV-2, which makes sense, because that is the virus the person was infected with and the antibodies They were optimized against that virus. - and slightly less potent against SARS-CoV-2 and WIV1.

So what we decided to do was take the three lead antibodies, shown here, and affinity mature them using the Adimab platform. Basically, what we do is take these antibodies and introduce diversity into regions of the antibodies that the antibody then prints to bind to, which are called CDR regions. And then once we've done that, we're essentially creating a single antibody library. So now there are a million versions of that antibody with different amino acid substitutions. And then what we do is we take that library of a million clones or 10 million clones and incubate the library with the receptor binding domain.

And we screen by flow cytometry to identify clones that bind with better affinities than the original clones. So you can see that this red population stands out over the gray one. These are improved clones with respect to the father. And then those yeast cells can be sorted and sequenced to determine the substitutions that mediate the affinity enhancement. And because we have a highly engineered yeast strain, we can induce our yeast to start secreting soluble IgG. And then we can characterize those IgGs. And that's what we did in this case. And once you find improved clones, there's nothing stopping you from further diversifying that clone and re-entering more rounds of selection.

So we ended up doing two of these cycles here for elite clones. And this just shows you here the final round of selection. You can see that the libraries look much better than the main clone, which is good. It means we are selecting these enhanced folders. And as expected, the improvement in flow cytometry translated into an improvement when we analyzed Fab binding using BLI. And as you can see, at least for the first two lineages here, the parents are in light blue and the progeny are in purple. We are obtaining clones that have improved significantly, up to 500 times, compared to the original clone.

And then we take the top folders and look at neutralization. And you can see that in the case of the first two lineages here, we're getting pretty significant improvements (I think up to 70-fold) in neutralizing activity. And in this particular assay, which is a pseudovirus assay with SARS-2, we ran some of these clinical antibodies that we had on hand. And we were pleased to see that Adagio-1 and 2 had potencies comparable to the strongest clinical antibody we had tested. And that's why Adagio-1 and 2 were selected, and we moved forward with them in these other studies that I'll show you.

And then, of course, the next question was, well, how well do these antibodies neutralize true viruses or live, replicating viruses? And do they maintain their neutralization amplitude? And on the left is another phylogenetic tree of many of these SARS-related viruses. It's really the clade 1 viruses that are of most interest, and this is just a subset of these viruses, because it's the clade 1 viruses that can use human ACE2 as a receptor. These are arguably the highest risk viruses. And of all the clade 1 viruses, there are four that were available at that time for neutralization testing. And that was SARS-CoV-1 and SARS-CoV-2, WIV1, which is the virus I mentioned before, and then SHCO14, which is another virus related to SARS in bats that is quite divergent from SARS-CoV -2.

And if you look over here on the right, this is the SARS-CoV-2 data, and this is the original version of the virus, the Wuhan-1 strain. Adagio-1 is in gray and Adagio-2 is in black. And what you can see is that both of our antibodies have potencies, as predicted by the pseudovirus assay, that were very similar to the higher clinical stage antibodies... for example, Regeneron 10933... and more potent than some of the others. such as Regeneron 10987, or S309, which is the precursor to sotrovimab, which is an antibody developed by Vir. But most importantly, you can see that Adagio-2 (hence the black dot here) maintains this very high degree of potency across the board, in SARS-1, WIV1, andSHCO14.

You can see that Adagio-1 is also broad. It hits SARS-1 and WIV1, but does not neutralize SHCO14. That's one of the reasons she wasn't cast as the lead. The Vir antibody is also broadly neutralizing. You can see that she recognizes these three bat viruses, but is significantly less potent than Adagio-2. Therefore, it is between 10 and 50 times less potent, depending on the virus in an assay. All other clinical-stage SARS-2 antibodies failed to recognize these more divergent SARS-related viruses. These are antibodies specific to SARS-2. So, as I mentioned, there were only - we only had four - at that time, four SARS-related viruses available for neutralization testing, but what we found was that the binding affinity to the RBD correlated very well with the ability of the antibody to neutralize.

So, if it linked well with the RBD, in those cases it was neutralizing. And so, as a sort of surrogate for neutralization, we looked at the binding activity of Adagio-1, 2, and 3 in a panel of these clade 1, 2, and 3 viruses. And we weren't expecting binding to clade 2 or 3, because these viruses do not actually use ACE2 as a receptor. So when you look at just the clade 1 viruses, you can see that Adagio-2 is affecting all but one. Adagio-1 is a little less broad. Again, that's another reason why we didn't move forward with that as a leader. S309, which is the very broad Vir antibody, affected all clade 1 viruses.

While, as expected, antibodies that were specific for SARS-2 in the neutralization assays only recognized SARS-CoV- 2 and the closest related viruses: this pangolin virus, for example. So this comes back to a point I made earlier, which is that broadly neutralizing antibodies generally target epitopes that are not the target of the endogenous antibody response. , at least not easily. And that's what you can see here with the Adagio-2 structure. So you can see that the Adagio-2 binding site is distinct from these common classes of neutralizing antibodies (classes 1, 2, and 3) and the residues that are critical for binding, shown here at the top in green . , do not overlap with residues that target common classes of antibodies.

And then on the right here is the second point, which is that broadly neutralizing antibodies, by definition, target highly conserved residues. This is a phylogenetic tree of many of these SARS-related bat viruses. And you can see that the residues that are important for Adagio-20 binding, which we determined by mutational scanning mutagenesis, are highly conserved, again, across all of these clade 1 viruses. And again, that's expected, and that is why the antibody is broadly neutralizing. In contrast, when you look at the residues that are important for binding of these more common classes of antibodies, you can see that they are highly variable residues, essentially indicating that these residues are probably not that important for viral fitness.

The virus can afford to mutate these residues without significantly affecting viral fitness and, in some cases, even improving viral fitness. And for that reason, that's one of the reasons why you see a lot more variability at those sites in circulating SARS-CoV-2 isolates relative to the mutations that we know negatively affect Adagio-2 binding, like shown here. And so we know that the epitope recognized by Adagio-2 is conserved in more than 99.99% of circulating isolates, at least according to the sequences that have been deposited in the GISAID database. This looks at the neutralizing activity of Adagio-10 and 20, as well as other clinical-stage antibodies, against the original virus, which they call the Victoria strain.

I think there is an amino acid difference from the original Wuhan-1 strain, and then there are variants of concern that have been identified until very recently: Alpha, Beta, Gamma and Delta. And I'm highlighting the broad neutralizers in green. What I'm trying to point out here is that, as expected, these broadly neutralizing antibodies neutralize these very divergent SARS viruses. Not so surprisingly, they can also broadly neutralize SARS-CoV-2 variants, which are much less divergent than these bat viruses. And you can see that Adagio-20 is the only antibody that maintains very high potency across all variants of concern to date.

And of course, we're working with Omicron data right now. Adagio-10 loses a bit to Delta, as does S309, but they basically maintain that breadth of reactivity. When you look at the clinical stage antibodies, you can see a different pattern, where many of these antibodies are losing some, if not all, of their activity against one or more of these variants of concern, particularly the beta and gamma variants. for the reasons I described in that previous slide. And this is the reason why the distribution of the Lilly cocktail was suspended, at least for a time in the United States.

This was when the Gamma variant was emerging at relatively high frequencies. And when we look at the mutations in the Omicron variant, I think my expectation and that of many other scientists is that we will see significantly reduced or no activity of the two antibodies in the Regeneron cocktail and the two antibodies. on the Lilly cocktail, but I'm sure the data will be coming soon. Most importantly, we wanted to analyze whether the breadth of neutralization actually translated into breadth of protection. And so we looked at this in collaboration with Ralph Baric's lab and using his model: using mouse-adapted SARS-CoV-2.

So we treated the mice prophylactically with 200 micrograms of Adagio-2 IP, and then we challenged them on day zero with this mouse-adapted SARS-CoV-2. And then we monitored the mice every day for weight changes and respiratory changes. And then the mice were fired on the fourth day to observe lung viral loads and histopathology. And what you can see is that the mice treated with Adagio-2 showed minimal weight loss and are completely protected from respiratory burden, viral replication in the lung, and hemorrhage in the lung. We saw very similar results with SARS-CoV-1, which you can see here, essentially complete protection against clinical measures of SARS disease.

Thus, as expected, it appeared that this breadth of neutralization translated into a breadth of protection. This is just a schedule of our activities in Adimab and Adagio. So we got the SARS sample in March 2020. We isolated binders in April and had neutralization data at the end of April. We finished the affinity maturation that I showed in May. We separated Adagio from Adimab in June. We selected the clinical candidate in July and initiated clinical manufacturing in November. We filed the IND in December and then initiated the first-in-human trial in February and then the phase II/III prevention trial in April.

And we have three clinical trials and different types of arms of these trials going on right now in 2021. So we have completed phase I of the study. I'll show you some PK data on the next slide. We have a phase II/III treatment study, and the primary endpoint is COVID-19-related hospitalization or death through day 29. And then we have a prevention study, both pre- and post-exposure, ongoing . And the endpoint is symptomatic COVID confirmed by RT-PCR up to day 28 for the PEP study and for six months for the PrEP study, the pre-exposure study. This shows you some pharmacokinetic data for Adagio-20.

Again, this is the extended half-life version of Adagio-2. So this looks at the mean concentration of Adagio-20 in phase I volunteers over time, for six months after a single intramuscular injection of 300 mg of the antibody. And what you can see is that this antibody has a remarkably long half-life, averaging almost 100 days. To my knowledge, this is the longest half-life of any of the SARS-2 antibodies that have been reported. And this is due to the modification of the half-life extension combined with the biophysical properties of the antibody. That's the concentration, and of course there's some correlation between the antibody and serum concentration and the neutralizing titers, because this is a neutralizing antibody.

So what we did was we looked at serum neutralization in these Phase I volunteers and we compared those serum neutralization titers to the serum neutralization titers of people who had received the Moderna vaccine. That's mRNA-1273 here on the left. And these are peak titers, so we drew this blood from these individuals just a couple of weeks after they received their second injection. So this is the best case scenario, because we know that these securities decline approximately 10 times over a six-month period. So what you can see is that seven days after we gave the Adagio-20 injection, 300 mg, the titers... the neutralizing titers against both the original virus, the D614G, and the Beta variant, are around of 1 percent. 500, compared to the maximum mRNA titers, which are 1 in 80, and then lower for the Beta variant, around 1 in 50.

And you can see that in the sixth month, we are at titers that are a little bit lower than the maximum titles. for the mRNA vaccine. And this is on day 180. And we still have better titles against Beta, and we're doing longitudinal sampling of these Moderna donors to do a true side-by-side here. But based on this data, based on what we know about the protection that vaccines provide and that neutralizing antibodies are a key correlate, and also the passive transfer studies that have been done with neutralizing antibodies, we expect to see fairly high levels of protection. in people treated with Adagio-20 for at least six months and potentially up to a year.

So I just want to close with one last slide on vaccine design, because I think this is an important point: These types of antibodies, these broadly neutralizing antibodies, are not only valuable if you think about prophylaxis and therapy. , which is what I focused on today, but also to essentially provide templates. You can essentially use them as templates for designing vaccines that induce similar types of antibodies. And here on the left is a receptor binding domain. Here I'm just highlighting these two binding sites in pink. This shows you the structures of some of the broadly neutralizing antibodies that have been described.

So you have Adagio-10 and 20, point to this site here. And then you have many others that point to different sites. And so what people are doing now is using these antibodies (the structures of these antibodies in different strategies that have been used in the context of) in the HIV field, for example, to essentially focus the immune response on these epitopes. particulars to essentially design a pan-SARS type vaccine. Shown on the right are some even broader antibodies that target the stem region. These also affect other beta coronaviruses, although they are much less potent. Most importantly, the thanks.

Much of the work I showed here was done by Garrett Rappazzo, Chengzi Kaku, and Laura Deveau at Adimab. I want to thank everyone at Adagio who did a lot of work clinically developing these antibodies. Many of the neutralization assays were performed in Gavin Screaton's laboratory at Oxford, Dennis Burton's laboratory at Scripps, and Carol Weiss's laboratory at the FDA. We did monkey studies with John Dye. I didn't have time to talk about that today. The mouse study I showed was done with Ralph Baric and the structure I showed was done in collaboration with Jason McLellan. And I'll be happy to answer any questions.

FACUNDO BATISTA: Thank you Laura. It was an incredible talk, a tour de force in such a short period of time. And yes, there are several questions we would like to raise. I mean, you touched on it a little bit on your last slide. That is, are there strategies that can promote the development of vaccination and broadly neutralizing antibodies? And how can you get only those and not just antibodies that bind or neutralize individually? LAURA WALKER: I think there are two approaches that are probably the most promising. I mean, one thing to consider is that SARS-CoV-2, these SARS viruses, I think, are less complicated than inducing broadly neutralizing antibodies against HIV, given that many of the antibodies that have been identified that are broadly neutralizing. ..It doesn't seem to be using super unusual features.

They do not have very high levels of somatic mutation. Probably everyone has the precursors. So I think the barrier to inducing these antibodies is probably lower and maybe less complicated. For example, we may not need germline-directed immunogens, as is being done in the HIV field. One way to think about it is that you have these very dominant, highly variable antigenic sites that I talked about: the class I, II, and III antibodies. One could think, for example, of placing glycosylation sites in these regions to mask those epitopes.particulars to favor obtaining antibodies that bind outside of those sites or use other types of similar strategies.

The other way to potentially approach it is with a heterologous primary drive strategy. So, you may remember the 2009 H1N1 flu and that story, when we were faced with a very divergent flu virus where the head region was completely different than any head we'd ever seen before, and what the immune system does... It will produce antibodies against what was seen before, which in that case was the stem, which is highly conserved. And many people identified these very broad antibodies against the flu stem region. And those antibodies, of course, are now being used to try to design these universal flu vaccines.

And that's essentially the heterologous reinforcement approach, where they prime you with one thing and prime you with another. And the only thing that is the same is that your immune system will recognize precisely that conserved region. And this has now been shown with SARS-CoV-1 patients, that is, people who were infected with SARS-1 and then recently received a booster with one of the mRNA vaccines. And sure enough, that's what you're seeing in the serum: you're getting a reactivation boost from these cross-reactive memory B cells that were originally induced by SARS-CoV-1, but also recognize SARS-CoV-2. , and then they activate.

And then when you look at the serum, you can see that the serum antibodies are neutralizing even more divergent SARS viruses, like SHCO14. So you could imagine a kind of approach like that. I mean, one question that I think will need to be answered is the role of... we think about this phenomenon of original antigenic sin. Basically, we have all been infected with SARS-CoV-2. So when you go in and apply these variant boosts, for example, are you really going to mount a de novo response to the variant virus, or are you just going to reactivate all...recycle your old memory cells that were induced? to the original virus.

And I think that's a question that... I think that's an important question that remains to be answered. And with the flu, of course, there's a big bias toward these pre-existing antibodies. And that's something that we and, I'm sure, many other people are looking at now. FACUNDO BATISTA: Laura, you touch... I mean, there are several questions here. Edward is one of our most active students. He's been connecting each one of them... and he asks you: what do you think about nanobodies? I mean, can nanobodies be used to inhibit responses? Or what are your ideas there? LAURA WALKER: Yeah, I mean, nano...

I think nanobodies... I think nanobodies are quite similar to IgG antibodies. I mean, I think there's a chance that maybe they can recognize epitopes that standard IgGs can't access. That's possible. Perhaps there is some possibility of administering them intranasally. I know it's been talked about. But I don't see any great value right now in nanobodies compared to more traditional IgG antibodies. There is also the possibility of linking them. You can make them somewhat modular, which could improve power and range. Maybe there's some value there. I'm not sure if there are nanobodies in later stage clinical trials.

I know there were some companies working on them, but to date I haven't seen any clinical data. So I think the advantages are potential advantages in recognizing more cryptic epitopes and... or a smaller size that allows for aerosolization, and the ability to link them together to potentially improve the breadth. Yes, I think those are my thoughts. FACUNDO BATISTA: You touched on this a little bit in terms of immune evasion and people are speculating how Omicron came about. Some people say, look, it came from people infected with HIV. But do you think there is room to think about immune selection for the appearance of such a variant?

LAURA WALKER: Oh, definitely. FACUNDO BATISTA: In other words... yes. LAURA WALKER: I mean, I think a lot of these variants of concern that have huge numbers... I mean, one argument is that, yes, they are emerging in chronic patients. Another argument would be that they are emerging in the population and we are not doing enough sequencing to see all the intermediates. And that's why I think it's impossible to say it either way. I think they probably arise in chronic patients, but... and they probably do. Yes, you are having chronic viral replication. You're getting a lot of pressure from these very common types of antibodies.

When you look at the antibody response, the neutralizing antibody response... I mean, that variant has all the mutations that you would have guessed it would have. If I were the virus, those are exactly the mutations I would have chosen... FACUNDO BATISTA: Those that you would choose. LAURA WALKER: --just knowing what I know about the neutralizing response. So I think there is now a lot of evidence that those amino acids are being incorporated for that reason, for immune evasion. And maybe that's a good thing. Maybe it attenuates the virus so it can break through and cause infections, but maybe it causes milder disease, because it inherently doesn't have to replicate to such high titers, because it has this immune evasion ability.

I think we'll know probably in a couple of weeks. FACUNDO BATISTA: And then, I mean, a general question from the students... I mean, surely they have an answer. Why can't we just use antibody cocktails, like putting five... I mean, you're putting Adagio-1, Adagio? Why can't we be more ambitious? I mean, why can't we put in 10 different antibodies? Along the same lines, given their long existence, can we think in the future that we will not be vaccinated with a dose of seasonal antibodies? I mean, what do you think about it? LAURA WALKER: I mean, in principle, you could do...

I mean, I think there's an antibody cocktail against C. diff that has a lot of antibodies. I mean, the problem really is just the complexity of manufacturing, the cost and resources associated with that. And that's one of the reasons we moved forward with monoclonal: it's just doubling the cost, doubling the amount of work, and dealing with all that extra manufacturing. I mean, it's essentially this huge burden. Regeneron was able to do it with Ebola with three. I mean, that wouldn't be feasible at a company like Adagio. We simply wouldn't have the resources. FACUNDO BATISTA: Thinking about that and about future therapy.

I mean, do you think there's room to give antibodies as RNA and leave out all the manufacturing? I mean, what are your views on the future in terms of antibody therapy? There are different ways to administer them, or is the result just protein? LAURA WALKER: Yeah, I mean, I think mRNA would be ideal. I think the problem is that it's not there yet, because the problem with... my understanding is that there are two problems with RNA delivery of antibodies. One is that no... the serum concentrations are not very high. They are peaking at 10 or 20 micrograms per ml.

And I don't know how persistent those titles are. I mean, they decrease. And then the other issue is with immunogenicity, where they haven't gotten to that point yet. And they might be at some point in the future in terms of improving the expression and reducing the immunogenicity of the mRNA. But yes, it would definitely be more ideal than the recombinant protein and presumably cheaper. And you could make... maybe you could make cocktails, although I'm not sure how that would work with string pairing, because you might be struggling. Yes. FACUNDO BATISTA: You have these antibodies, many therapies that seem to be working with a large family of different coronaviruses.

Are you thinking about getting better antibodies that are more broadly neutralizing antibodies, or how hard it is to think about the next epidemic? Like we should start over, or is there a way to predict which antibody will be good at neutralizing the entire panel. LAURA WALKER: Yeah, I think the problem is, if you remember the slide I was showing at the beginning, just looking at the entire coronavirus family, there's too much diversity. I mean, most of them don't even use ACE2 as a receptor. MERS uses DPP4. And so, there are some antibodies that recognize the root that broadly neutralize, broadly neutralize many of the Beta coronaviruses.

I think it's too much to ask to start attacking Alpha and Delta coronaviruses, given the antigenic diversity, but perhaps with some antibodies, you could cover at least the main type of subfamilies that would possibly pose the greatest risk. The SARS viruses... I mean, we've already seen two spillovers that use ACE2. And arguably we are likely to see a SARS-3 at some point. The same thing happens with MERS, like these C Beta lineage coronaviruses. So I think those are probably the families that people are looking for, like the B lineage and the C lineage, simply because all the new viruses to date have come from those two Beta coronavirus lineages.

The problem with broad viruses is the balance between breadth and potency, which is also seen with other viruses. This is seen with the flu, for example, where typically antibodies that are very broad are not very potent, and antibodies that are very potent are not very broad. With Adagio-20, we solved this by engineering, but these stem antibodies are not typically, or at least the ones that have been described to date, are not very potent at all. They are quite wide, but not very powerful. And I think it remains to be seen whether they still protect as well or not, maybe they punch above their weight and have very strong Fc effector functions, and things like that.

That's possible. Yes. FACUNDO BATISTA: And my last question... and it's probably the way to close this incredible course, and we're thrilled to have you join us today, Laura. We know you are very busy. Obviously, for vaccines, this pandemic has been transformative in the sense that they have been rolled out in a matter of a year, and that has revolutionized the field of vaccines. Do you think the same has happened with therapeutic antibodies in the future? In other words, this is probably the first time that people can have access to these therapies. Do you think that in the next pandemics things are going to be much faster in terms of availability of these medications?

LAURA WALKER: I think that's the hope. I mean, Lily was able to develop bamlanivimab in eight months. I mean, that's absolutely unprecedented. I mean, it normally takes 5 to 10 years to develop an antibody. And then there were a lot of shortcuts. A lot of things happened in parallel, but I think a lot of lessons were also learned. And hopefully that will be the case, that we can move even faster next time and, more importantly, be able to deploy the antibodies more effectively, not have to give them IVs to everyone, and all the problems. associated with that. I think we learned a lot from this pandemic.

And I think our argument would also be that if antibodies and vaccines could be identified in advance to cover these important families of viruses that could spread and accumulate, then you could do something like a ring vaccination. As you remember, when there was a cluster of cases in China, the pneumonia cases, you could imagine treating all those patients and all the contacts, and the contacts of the contacts, and maybe you could eradicate it. That would be the most ideal scenario because there will be some delay, whether it's eight months, five months or two months. It probably won't be enough to eradicate the virus, depending on how it spreads, especially if it is a respiratory virus.

So I mean that would be really ideal, I think. FACUNDO BATISTA: Laura, thank you very much for a wonderful talk. Thank you very much for your wonderful work that will influence the lives of many people. And with this we close this course on COVID-19, SARS-CoV-2 2021. Thank you all for attending and we hope to see you eventually next year. LAURA WALKER: Thank you.