Avacta Therapeutics is targeting the tumor microenvironment. My name is Christina Coughlin, and I am the CEO here. Let me take you through our story. Avacta is challenging the current drug delivery models to expand the reach of highly potent therapeutics using our innovative technology of peptide drug conjugates. This is a revolutionary approach, and Avacta is leveraging a key protease, fibroblast activation protein, that is specifically expressed in the tumor microenvironment. It's a tumor-specific release mechanism that enables the delivery of potent warheads directly to the tumor microenvironment, with clinical proof of concept achieved and published at the AACR in April of 2024. This FAP-enabled drug release mechanism can be implemented in two different manners in the clinic, either as a small molecule approach with the peptide linked to a potent warhead. This is in the case of AVA-6000, our lead program that's in the clinic.
Or we can implement this peptide drug release mechanism in conjugation with a biologic-targeted approach, either an antibody or an antibody drug conjugate, or an Affimer or an Affimer drug conjugate. Let me introduce you to our research and development team at Therapeutics in the next slide. Myself, CEO and the head of R&D. Simon Bennett is our Chief Business Officer, with multiple years of experience in deal-making in the pharma and therapeutic arena. Karen Harrison is our Chief Operating Officer and specializes in outsourcing, key for Avacta Therapeutics. And then David Jones and Francis Wilson, who lead our research teams, David in the field of biology and cancer biology, and Francis in the field of medicinal chemistry. Avacta is combining our two innovative platform technologies to deliver these potent warheads directly to the tumor microenvironment.
As I mentioned, pre|CISION technology takes a peptide that is specifically cleaved by FAP in the tumor microenvironment, links it to a warhead, which prevents cellular entry, only to be released then in the tumor microenvironment by the tumors themselves. This can be used alone, in the case of AVA-6000, our lead program, or in combination with biologic-targeted agents. The proprietary platform is the Affimer technology. These are next-generation biotherapeutics that are designed to essentially overcome many of the limitations of antibodies that are used in these. And so combining these two together gives us a next-generation pre|CISION ADC or Affimer DC products. A bit more about FAP, the release mechanism. FAP is a protease, it's an enzyme. It's selectively expressed in human cancers, and it's a member of the DASH family of serine proteases.
It's found on the cell surface in a membrane-bound form on cancer-associated fibroblasts in the tumor microenvironment. There's a key spatial organization that we'll look at here. In most solid tumors, we see some degree of FAP in that TME. Higher amounts are found in more stromal-dependent tumors. An overexpression of FAP has been associated with poor prognosis in a number of different solid tumors. These FAP-targeted therapies, because of the broad expression of FAP in multiple solid tumor settings, will reach a very broad patient population. You can see these are immunohistochemistry stains from two different patients that were treated in the AVA-6000 trial. What you can see here is a patient with undifferentiated pleomorphic sarcoma, very dark expression of FAP within that tumor microenvironment. And here is a patient with pancreatic ductal adenocarcinoma.
Again, dark expression in the stromal compartment, but essentially in those small tumor islands, we don't see the expression of FAP. This is exactly what we see in, a couple of patients actually not treated on the trial, where we assessed spatial organization of the FAP-positive cancer-associated fibroblasts. This is a technique that's different from immunohistochemistry. It's called multiplexed immunofluorescence and allows us to visualize in different colors what we're seeing in terms of specific antigens or proteins in the tumor microenvironment. In aqua, we're looking at a marker of tumor cells, pan cytokeratin. In red, alpha smooth muscle actin, a marker of the cancer-associated fibroblasts, and again, FAP expressed in a subset of the cancer-associated fibroblasts.
What we find here, unpublished as of yet, is that the FAP-positive cancer-associated fibroblasts are located exactly where we'd like them to be, surrounding and outlining those tumor nests of tumor cells that exist in the tumor microenvironment. And what you can see is in those more distal areas of stroma, we don't see as much FAP expression, suggesting that FAP plays a role in the tumor biology. Just as importantly, we see another layer of spatial organization, and here we're gonna swap out that red stain for a stain for blood vessels, asking the question: Where are the blood vessels in relation to tumor cells and those FAP-positive CAFs? What you can see is that the blood vessels are right where we hoped they would be in order to optimize the delivery of those warheads.
So here you can see in high power, in red, the blood vessel, in bright green, the FAP-positive CAFs ready to deliver that warhead through the bloodstream directly to the tumor cells. So spatial organization here of the FAP-positive cells, as well as the blood vessels and the tumor cells, would suggest that given this co-localization, this is how we're seeing that direct concentration of the warhead in the tumor microenvironment. Let's talk a bit more about how we achieve precision delivery of the warheads. This is the mechanism of action of a precision-enabled warhead. What you can see here in red is the warhead. The first one, of course, is doxorubicin for AVA-6000. It is linked with the peptide, and because of this link, this now conjugated drug, the peptide drug conjugate, is incapable of entering a cell, whether it be FAP-positive or FAP-negative.
What happens is the FAP, the enzyme on the cell surface, is able to break this drug conjugate apart, release the peptide, release the warhead. The warhead is now located in the extracellular space and is able to now migrate into either a FAP-positive cell or, importantly, even a FAP-negative tumor cell located in that specific localization. We can implement the precision technology essentially in three different platforms. The first, precision alone. This is a nice illustration of AVA-6000. It is a peptide drug conjugate. We can also extend the half-life by binding these precision-enabled warheads to a protein, such as the Fc region of an antibody. Here, you can see this gives us a significant increase in the half-life that we would expect.
And then we can double-target these to the tumor microenvironment using either the standard approach of a monoclonal antibody or, proprietary to Avacta, are the Affimers, where we'll show you a little bit of how we've been able to achieve the ability to create an Affimer drug conjugate. Peptide drug conjugates and Affimer drug conjugates have several key advantages over the traditional ADC approach. The first is the mechanism of action. The release of the warhead here is extracellular, so we're optimizing the bystander effect.
Now, that is going to enable us to be able to treat patients with very high levels of FAP in both the tumor cells as well as the CAFs, but also because the bystander allows the warhead to now migrate into the FAP-positive or the FAP-negative cells, bystander effect, we can now treat patients and see concentration of warhead even in those mid-level FAP expression tumors. This is a tumor-specific release mechanism. We expect, based on the chemistry, that only membrane-bound FAP in the tumor microenvironment is going to be able to release those high concentrations of warhead. The drug-to-antibody or Affimer ratio, again, an important factor in the ADC space. With the Affimer drug conjugates, we're able to achieve a 2:6 ratio, and that's with multiplexing of the Affimers. And with the peptide drug conjugate, recall, this is a small molecule, so 1:1 is just fine.
What you can see in manufacturing, because of that 1:1 ratio, it's small molecule, so short timelines and really controls the cost, being able to manufacture these and not needing a biologic conjugation. When we move to the biologic conjugation with Affimers, recall the Affimers are able to be manufactured in E. coli. They're thermally stable, they're low molecular weight, and so they really lend themselves well to ease of manufacturing and very efficient conjugation processes. That can help to control the cost here, even though it is a biologic conjugate, very different, again, from traditional ADC approaches. The design of our precision FAP-enabled warhead goes through several key steps in order to get to the final drug candidate and moving to the IND.
The first is to locate where that peptide is gonna be conjugated to the warhead, and that's to optimize that specificity of the peptide being released by FAP. The structure-activity relationship then allows us to look at the efficiency of the FAP enzyme. We then use in vivo pharmacokinetic modulation to look at how do we extend the half-life, if we need to extend the half-life, and how can we get optimized tumor delivery based on the science and the biology of that warhead? And then finally, we use both in vitro and in vivo characterization of the efficacy prior to submitting that IND. In order to perfectly position that warhead peptide conjugate right in the enzymatic groove, we use a docking model of the FAP enzyme. And what you can see here are two different approaches to conjugation of the warhead attachment.
Here in green is the ortho, and here in pink is the para. So we've got multiple different options. We can also utilize a capping group sitting out here, and that's to optimize the efficiency of the enzyme. We wanna see precise cleavage, and when it's together, we want to see a lack of cellular entry. Two important factors in the FAP enablement of warheads. We use then multiple in vitro assessments using these four conditions: so the warhead alone, the warhead with an exogenous FAP added, so being able to cleave the warhead in the test tube. The precision compound alone, so no FAP added, we shouldn't see much killing here, and then with exogenous FAP, but also with a FAP inhibitor.
Both of these measures together and their difference with green and black will show us that this warhead has now been FAP-enabled and should be able to be delivered again directly to the tumor microenvironment. We can modulate the efficiency of the enzyme, and it may be important for us to get different release kinetics and different PK, again, depending on the class of warhead and the biology of how that warhead targets the tumor. In the AVA-6000 phase I trial, this is a FAP-enabled doxorubicin and is our first molecule to go to the clinic. We saw three key findings. I'm gonna show you some of the clinical data that were published here by Professor Banerji and his colleagues at the 2024 AACR Annual Meeting.
First is AVA-6000 delivers high concentrations of doxorubicin directly to the tumor microenvironment, which results in anti-tumor activity in those patients who have overexpression of FAP. Our exposure response modeling or our PK/PD modeling shows that the released doxorubicin is generated primarily by cleavage in the tumor microenvironment, exactly as the chemistry was designed, as opposed to the soluble FAP in the bloodstream. This leads to that distinctive safety profile that we've seen and reported on. The pre|CISION-enabled peptide drug conjugate doxorubicin in AVA-6000 leads to a significant widening of the therapeutic index, so a separation of the toxicity that we would expect with the efficacy that we are seeing from the released doxorubicin. Here you can see the dose levels and the phase I design. In green check marks, you can see the different dose levels that were reported at AACR.
Upwards as we get to the 385, which is almost four times the normal dose of doxorubicin. We are now currently enrolling in the every two weekly regimen, and looking forward to completing and moving to expansion cohorts in the second half of 2024. What you can see here are the baseline characteristics of the patient population that was treated in the phase I, Arm 1 or the every three weekly regimen. Many of the patients had soft tissue sarcoma. Again, looking at those FAP-positive diseases, we also saw GI cancers of colorectal cancer and pancreatic cancers of the biliary tract, and then a group of patients with one each. Many of the patients were heavily pretreated with a median of three prior lines of therapy in a range of zero to seven.
We saw a distinct safety profile, which is quite different than what is seen with standard dose doxorubicin of 75 mg per meter squared, that is dosed every three weeks. Here we're comparing to a phase III trial, in a number of patients, and the Grade three, fours, so those, severe to life-threatening toxicities. And we see a significant reduction in a number of the key toxicities that lead to dose reductions. Our first patient with a deepening partial response, who, at the time of the AACR presentation, had been treated for just under one year on study, is a patient with undifferentiated pleomorphic sarcoma. Sarcomas often metastasize directly to the lungs, and that's what happened, with this particular patient. He had been treated with standard therapy in the localized disease setting.
Once his cancer had become metastatic, he had been treated on a phase I trial of immunotherapy, experienced disease progression, and then enrolled into the AVA-6000 trial. What you can see here outlined in orange are the near complete resolution of multiple pleural metastases in this patient. He's joined by multiple other patients, as you can see here on the waterfall plot, who have experienced tumor shrinkage. At the time of the data cutoff, indicated in asterisks, we had six patients on study. Four of the six patients were the patients that had experienced minor or partial responses on trial. The figure that is important to the interpretation of the precision results and important for the entire pipeline is that concentration of the warhead in the tumor microenvironment.
In the trial, patients were offered the option of undergoing, again, an optional biopsy, 24 hours after the first dose was administered, and you can see a subset of the patients underwent this biopsy. At the same time, a blood sample was taken, and we compared the concentration of doxorubicin in the tumor with the concentration in the plasma. Important to note here, this is a log scale, and so what we're seeing is, regardless of FAP high or FAP mid expression in the tumor microenvironment, we see a two-log difference between the blood concentration and the tumor concentration. So again, pulling that warhead directly into the tumor microenvironment, with some leaking out into the peripheral blood.... A two-log difference was quite impressive to us.
What you can see in this figure is a logistic regression analysis asking the question: what is occurring in therapeutic index of release doxorubicin compared with standard-dose doxorubicin? We're looking at two categorical data sets, neutropenia, severe neutropenia, grade three and four, and response or prolonged stable disease of approximately four months. And we're grouping these as a probability of 100%, or one, versus a probability of zero, or zero. And then looking at the logistic regression according to the AUC that is measured at that first cycle. We're also indicating the dose levels in the AUC levels, where we see the five patients who experienced either partial response or minor response. And what you can see here is this is the curve for the probability of patients experiencing a grade three, four, neutropenia.
Here is the exposure of doxorubicin that we would expect with that standard dose, and we know that about 50% of patients with standard dose would experience grade three, four neutropenia. Here we're seeing this widening of the therapeutic index at these lower dose levels, where we see lower exposures, and yet still that nice activity of stable disease, partial response, or minor response, thus leading to the widening of the therapeutic index. Let's talk a little bit about the second platform for this drug delivery and how we're gonna combine the precision platform with our second novel platform of Affimers. Affimers are a class of engineered antigen-binding proteins, and they have a few key advantages over monoclonal antibodies. What you can see here is just the sheer difference in the size.
The Affimer, a monomer Affimer, is about 1/10 the size of a monoclonal antibody, which should lead to much better tumor penetration. The Affimers are engineered proteins. They specifically bind desired targets with affinities that are in the nanomolar to even the picomolar range. Recall, trastuzumab, which is a key antibody that is used in the HER2 space, has an affinity of five nanomolar. So our Affimers are right there with the affinities that we would expect with monoclonal antibodies. The Affimers, again, are engineered using a scaffold of Stefin A, which is a typical protease that is found in human biology. Here we can insert loops that allow us to create an engineered binding domain. They're highly stable. They have no post-translational modifications.
As I alluded to earlier now, these can be produced at very high levels in bacterial cells, and so we don't need to use the more expensive manufacturing techniques of mammalian cells. Monomers, again, are about a tenth the size, a dimer, about the fifth of a size, and so they should—we should expect optimized tissue penetration. They may enhance the clearance, which we can modulate with half-life extension. So lots of aspects of the Affimers that feed well into the drug delivery mode. We've also engineered them to have multiple cysteines within different locations, the C-terminus and the loop three. The Affimers provide distinct optionality. They're a very modular drug delivery system. We have libraries that we screen. There are multiple formats.
We can take them anywhere from a monomer straight up to a tetramer, where we can mix and match, include two antigens, a single antigen, or another binding domain that allows PK extension. Because they are so small, even a tetramer is gonna be significantly smaller than an antibody, and they have key manufacturing advantages over monoclonal antibodies. Again, produced in bacterial cells, no post-translational modification, thermal stability, solvent stability, which really allow us those efficient conjugation reactions to put the warhead directly onto the Affimer. And so there's multiple critical advantages for drug delivery, again, over monoclonal antibodies. It's a small protein. It has very simple structure and fold, no post-translational modifications. We have a rapid and simple discovery process that's based on phage display libraries and unencumbered IP that is all licensed to Avacta. These are engineered with multiple cysteines for efficient warhead conjugation.
Even precision warheads can be conjugated directly to Affimers. They have flexible formatting, allowing a monomer straight through to a tetramer, multispecifics, and modulating the affinities as well as the PK profile, and the optimized GMP manufacturing that we've discussed. So in summary, Avacta's precision platform is a highly tumor-specific drug release mechanism. It's capable of concentrating anticancer drugs or warheads in the tumor microenvironment versus the plasma, and it can be leveraged in different formats, peptide drug conjugates straight through to even precision-enabled ADCs. Our clinical data released at AACR earlier in 2024 provide clinical proof of concept for AVA-6000 and proof of mechanism that the precision platform is working exactly as it was designed.
Our PK/PD modeling in this early presentation supports the ongoing exploration of an every-2-week dosing schedule to assist in defining the recommended phase II dose, and we remain on track to begin the expansion cohorts in the second half of 2024. Our ongoing work in our two divisions, therapeutics, as we've discussed today, and our diagnostics division, to integrate and plan for the future. We have three upcoming key milestones catalysts in the second half of 2024, and we remain on track for all three of these deliveries. The first is to initiate those expansion cohorts, moving from phase I into disease-specific expansion cohorts. The second is to update the AVA-6000 clinical data, including that every-two-week regimen arm. Third, to release our pipeline update.
We will be looking at the updated pipeline of the Avacta Therapeutics assets with both their stage and their timing to the clinic. Thank you for joining us.