Excellent. Good morning, everyone. It's great to see you all today. My name is Neil Klompas, Zymeworks' President and Chief Operating Officer, and I'd like to welcome you to Zymeworks Early Research and Development Day. We appreciate you taking the time out of your busy schedules to join us, both in person here in New York and virtually via webcast. Before we start, just some housekeeping matters. In today's presentation, we will be making forward-looking statements. We rely upon standard safe harbor disclosures common to our industry and companies at our stage of development in our sector. For more information on risks and forward-looking statements, we encourage you to read our SEC filings available on our website.
Additionally, for anyone watching the webcast live today, if you have any questions on today's material, please email ir@zymeworks.com, and we'll try to answer your questions during the Q&A portion of this event at the end of today's prepared presentation. Today, we are very pleased to share this exciting look into our next generation platforms, and more importantly, a first look at some of the data on our preclinical programs. Before I hand it over to Paul and our research and development team leads, I want to say a few words in light of today's or yesterday's announcement. As you may have seen, yesterday we were thrilled to announce a global licensing collaboration agreement for zanidatamab with Jazz Pharmaceuticals.
I encourage everyone to view the webcast and read the press releases and other relevant disclosures, all of which are available on our investor website, as we will not be spending any time today speaking to the agreement. The focus of today's event is on our early research and development pipeline. However, I would like to briefly frame what this opportunity means to Zymeworks as a company, as I think it is pertinent to today's discussion. With our significantly improved financial position and our newly updated cash runway guidance, which enables Zymeworks to fund planned operations through at least 2026 and potentially beyond, we are now able to enact a focused R&D strategy with emphasis on development opportunities in difficult to treat cancers, where we can utilize our in-house multi-specific antibody therapeutics and antibody drug conjugate platforms, which we will speak to today.
Going forward, it's important to highlight that partnering and business development opportunities will continue to be core to our business and to our long-term strategy of developing these and other novel antibody-based therapeutics, and we hope that this partnership provides an indication of our ability to form, execute, and integrate important partnerships and collaborations that will be helpful to every aspect of our business. The agreement we announced yesterday will not be the end of what we intend to be a core piece of our go-forward strategy. Today, Paul and our research and development leadership team look forward to providing context on the pipeline you see before you on this slide. The team we have with us today was pivotal to the development of zanidatamab zovodotin, and the platforms that enable us to develop these and other therapeutics.
We have an exciting first look today at our preclinical pipeline, and we hope you walk away with a better understanding of the rationale and data behind the therapeutic modalities and clinical candidates that will drive the next wave of growth here at Zymeworks. With that said, let me please introduce Dr. Paul Moore, our new Chief Scientific Officer. Paul brings with him significant experience in biologics, drug discovery and development, spanning a career of 25 years where he has led discovery and development efforts across a range of FDA-approved and clinical-stage biologics. I know I speak for our entire leadership team and board when I say we are thrilled to have him as part of our executive committee and broader team. I look forward to the benefits that his experience brings to Zymeworks.
As you will see from the presentation today, under his leadership, our research and development teams have been working diligently and with a renewed sense of excitement to meet our corporate goal of delivering five new Zymeworks-developed candidates to the clinic in the next five years. Now, let me hand the mic over to Paul and his team. Again, thank you all for attending and listening in online.
Okay. Well, thank you, Neil. That was a nice introduction, and I have to say I'm delighted to be at Zymeworks at this stage in the company's development, working both with Neil and the other senior leadership team, but also very importantly with the talented research team that is in place at Zymeworks. What we're really gonna be focused on is the development of next wave of multifunctional antibody therapeutics in oncology. Since coming on board, I've been, as I've mentioned, extremely impressed with the talent and the tools in place at Zymeworks.
What we really have is a team that's capable of supporting both the development of multispecifics and ADCs, which really kinda sets Zymeworks apart from other companies in the field. Importantly, the infrastructure and the experience to also take molecules from preclinical into development, into clinical studies is also in place at Zymeworks, which really provides us the runway to take our discoveries into the clinic. Key Zymeworks are the proprietary platform technologies that have been developed that enable the design and generation of unique and differentiated functional multifunctional proteins. On this slide, I'd like to focus on the Azymetric and the ZymeLink platform technologies because they're really the foundation of the multispecific and the ADC molecules that we're developing. With the Azymetric, we have the ability to generate bispecific molecules through the efficient heterodimerization of the Fc.
We can then incorporate antibody-based binding specificities tailored to the Fc to generate bispecifics and trispecifics. Importantly, these molecules maintain IgG1-like properties and can be purified as such and manufactured as such. Within our drug conjugate platform, we then also have proprietary technology and expertise to generate antibody or bispecific antibody drug conjugates. And we have a suite of payloads that can be matched, can be plugged into that system. This includes our recently developed topoisomerase I inhibitor-based payload technology that we'll be showcasing in the context of product candidates in the presentation today. In addition, we also have our EFECT and ProTECT platforms that we can also wire into our products. There with the EFECT, we can use that to enhance or diminish effector function both in the context of the ADCs or bispecifics.
We also have our ProTECT platform, which we can employ to limit functional activity of our molecules to the tumor microenvironment. The common theme to our technologies are that they're all antibody-based, so they can all integrate with each other. This really allows us to build molecules that are fit for purpose with the features that our technologies can endow on a molecule. We can take an antibody, and then we can have this choice on how we conjugate, which drug we conjugate to that, and how we develop the ADC. We can also, of course, plug in a second specificity, a second antibody into these platforms and make it bispecific. This really provides us the opportunity to make either multispecifics or ADCs with different features depending on the challenge of the target.
What's really important, though, is that Zymeworks technology works clinically and has yielded therapeutics that have been clinically validated. The first program, of course, that you may be aware of is zanidatamab, which is a biparatopic antibody based on our Azymetric platform, which targets two independent epitopes of HER2 and has shown clinical activity across multiple HER2 tumor types, and that's in clinic, the pivotal clinical studies. We've also developed ADCs, in this case, ZW49. We also have had that come from concept to clinic and was recently shown some clinical data on that. We also have the experience of taking ADCs to the clinic. Our platform technologies have also been validated through external partnerships and other internal programs. On the external partnerships, we've...
In the Azymetric, which is the foundation for multispecifics, there we have T-cell engagers that are in the clinic with Johnson & Johnson. Then additional other undisclosed programs that have been developed by our other corporate partners covering different therapeutic areas. On the antibody-drug conjugate side here, we have also partnered those ProTECT technologies with companies. Exelixis are developing a tissue factor ADC, and they will be presenting clinical data next month at the ESMO meeting in Barcelona. Atara are also developing an EpCAM-2 ADC utilizing technology licensed from Zymeworks. In addition, the flexibility, it can be shown in several different examples of recently presented data or to-be-presented data from Zymeworks. On the Azymetric side, we presented last year work showing conditional co-stimulation using a bispecific approach.
Then we can also restrict cytokines to the tumor microenvironment using our Azymetric. Then on the ADC side, we'll be presenting next month at SITC work on our TLR7 conjugates. What I should also indicate is that with our focus now moving forward with cancer therapeutics, we are gonna focus on cancer indications where we consider the greatest unmet medical need, and that we can measure by patients with the poorest outcome, five-year survival. There, these are challenging diseases to treat. That's the reason they have poor survival. But we think that with the multifunctional proteins that we're developing, we now have the tools available to try and take on these challenges. That'll be a theme that you'll see during the presentation. Let me just.
This may then summarize our overview of our path from the tumors that we want to treat to integrating with our technologies, either ADCs or multi-specifics. Here, what we really like are the features, the optionality that we can do with the two ADCs, the two foundational platforms modalities. With an antibody-drug conjugate platform, we can actually really customize the molecule through our experience in antibody engineering, looking at antibody properties with the proper format or the appropriate format for the challenge with the payload, the linker conjugation, and the DAR. They are all variables that we consider when we design a molecule, and we can customize that antibody-drug. I think, say, we likewise have the ability to really functionally fit that molecule for purpose.
If we need multiple mechanisms of action, or we need multiple specificities to get more precision targeting, you'll see that, then we can do that with the design of our model. Where are we? I think it was five years ago here in 2017 and during that time, we've made a lot of progress. We've had a select product pipeline. We've really developed a lot of good platform technologies. What we really wanna do is invest in technology and infrastructure and accelerate in the next five years our in-house pipeline. As Neil mentioned, we've got a goal of developing five new products into the clinic in the next five years, and that will be a balance of ADCs and multi-specifics.
You'll see some of those initial programs that we're gonna be bringing into the clinic in the presentations that the research leadership team are gonna be giving shortly. This is sort of an introduction to the Nina and Thomas. They really spearhead our multispecific team, and then they will be talking in a little bit of time. Stuart and Jamie will take the podium first, and they're gonna cover our ADC technologies. As we will be revealing for the first time data on our two next IND candidates, ZW191 and ZW171. Then behind that, we'll also be giving you a flavor of the other programs that we're working on, having five INDs in the next five years.
With that, I'll hand it over to Stuart.
Candidates. Delivering targeted chemotherapy through antibody-drug conjugates has emerged as an extremely effective therapeutic strategy. In total, there are 11 FDA-approved ADCs. We have seen an explosion of approvals in the last three years. 7 ADCs. There are a number of other promising ADCs on the cusp of approval, and we look forward to the continued success for this clinically impactful modality. The 11 FDA-approved ADCs represent success in both liquid and solid tumors using a variety of payload mechanisms with a range of potency. The more potent DNA-damaging agents like calicheamycin and the PBD dimers have been successful in liquid tumors targeting CD22, CD33, and CD19.
While the slightly less potent microtubule inhibitors have been seeing success in liquid tumors targeting CD30, BCMA, and CD79B, and in solid tumors targeting HER2, tissue factor, and Nectin-4. The relatively lower potency topoisomerase I inhibitors have seen success in solid tumors targeting HER2 and Trop-2. Despite all of this recent success, the discontinuation rate remains high across multiple payload mechanisms, especially for the more established payload classes like the auristatins, the maytansinoids, the calicheamycins, and the PBD dimers. While still relatively early in its development cycle, the more nascent camptothecin class is on an exciting trajectory with very few discontinued programs to date. Now, the reason for each discontinued discontinuation can be categorized into two main themes.
The first one being on-target toxicity due to target expression on normal tissue, and the second being lack of efficacy despite achieving the maximum tolerable dose dictated by the platform. Discontinuations due to on-target toxicity constitute a very small proportion of total discontinuations, and the majority of discontinuations occur because of lack of efficacy at the platform maximum tolerated dose. We believe the key area for improving on past failures is by selecting the right design criteria for each ADC component. The target, the antibody, the linker conjugation, and the payload all need to be considered in order to drive robust efficacy at tolerable doses. As I just mentioned, building a successful ADC requires the right design. Careful selection and combination of target, antibody, linker conjugation, and payload are critical.
For the target, one needs to consider the expression profile, as this will dictate how to select the antibody binding properties and the payload mechanism and potency. The payload potency and metabolism will dictate the features of the linker and the conjugation method employed. Simply taking an old antibody and repurposing it for ADC use may not yield the best results. Similarly, taking a popular payload and just applying it to an antibody without considering the targets and the tumor biology may not yield a successful ADC. To realize the right design, we need the right tools. There's no one-size-fits-all solution to this, and employing a toolbox of technologies allows for careful tuning of each ADC design feature. In order to bring all of this together, the right combination of design features and technologies, we need the right team.
Having a fully integrated multidisciplinary team that eats, sleeps, and breathes ADCs development will increase the likelihood that a program can be taken from design to discovery through to development and in the hands of physicians to treat patients. At Zymeworks, we're uniquely positioned to build fit-for-purpose ADCs due to the breadth of our internally generated ADC technologies. Our ZymeLink auristatin technologies are most mature and used in our anti-HER2 ADCs, ZW49, and in XB002 and ATRC-301. Similar to our ZymeLink auristatin technology, our ZymeLink hemiasterlin technology is also a microtubule inhibitor and uses the same N-acylsulfonamide spacer to link the payload core to a protease-cleavable linker. Our topoisomerase I inhibitor platform utilizes a novel bystander-active camptothecin payload, and we'll be talking a lot about this technology as it is used in the three pipeline assets we'll be discussing today.
We also have a site-specific conjugation technology that allows for homogeneous conjugation at multiple sites, and when combined with our Azymetric technology, we can precisely control the DAR. We also have an immunostimulatory drug conjugate platform that Paul mentioned, and we'll be presenting a poster on this technology at the SITC annual meeting next month. Zymeworks has been investing in ADC technologies for the last eight years. The ZymeLink technologies were the very first ones to be developed, and the ZymeLink auristatin technology has been applied in three programs now, two in clinical development and one in late-stage preclinical development. In the last three years, we've really focused on developing new technologies to fortify our ADC toolbox. This includes the ones I mentioned earlier, like the site-specific technology, the TLR7 immunostimulatory technology, and the topoisomerase I inhibitor technology.
Now what we're doing, as Paul mentioned, is really shifting our focus from developing technologies and tools to building clinically and commercially relevant ADC assets. We've done it successfully once with ZW49, and we're really excited to unveil three new pipeline assets today in ZW191, ZW220, and ZW251. As I mentioned earlier, the combination of each ADC component is critical to designing a successful ADC. At Zymeworks today, our strategy is to focus on targets with evidence of clinical activity in indications with high unmet need. We believe that known targets are still actionable when armed with the right technology and the right design. The antibody is a far too often overlooked component of an ADC. Many ADCs resort to using repurposed antibodies.
Here at Zymeworks, we focus on specifically discovering and developing optimal ADC antibodies, because antibodies specifically selected for ADC use are gonna increase the likelihood of success of that ADC. For the linker conjugation, we've leveraged validated peptide cleavable linkers and stochastic conjugation for right now. The peptide cleavable linkers are used in more than half of the approved ADCs and provide the benefit of releasing the unmodified payload. Stochastic conjugation is still the gold standard and used in all 11 FDA-approved ADCs. For the payload, we're gonna focus on our novel topoisomerase 1 inhibitor ADC platform because there's undeniable evidence right now that topoisomerase 1 ADCs are providing meaningful clinical benefits to patients. We believe that our topoisomerase 1 inhibitor platform is a critical component to building clinically impactful ADC assets at Zymeworks.
With that, I'm gonna pass things over to my colleague, Jamie Rich, to discuss that novel topoisomerase 1 ADC platform.
Okay. Thanks, Stuart. Hi, everyone. I'm Jamie Rich. I'm the Director of Technology in the therapeutic development group here. I'm gonna start off today by orienting you to Zymeworks' topoisomerase I ADC platform. This is part of some of the pipeline that we'll be discussing a little bit later on. You often hear that HER2 is transforming breast cancer. It's a pretty common refrain these days. The impressive patient responses in these waterfall plots from the DESTINY-Breast studies seen in the upper left of the slide really illustrate what's driving this change. Furthermore, HER2 is also showing really meaningful benefit to patients in several other solid tumor indications, as you can see both in this slide and in clinical data that we've presented or have been presented historically.
I think perhaps most importantly for us, though, there are four other DXd-based ADCs, specifically non-HER2 ADCs and solid tumor indications, and these are also starting to show real benefits for patients. From my perspective, the key takeaway is that all of these data really illustrate just how successful antibody-based targeting of topoisomerase I inhibitors can be, to the point where we don't really feel that that can be ignored. With this in mind, we've developed our own topo I ADC platform and corresponding pipeline and indications that we feel will benefit the most from using this mechanism of action. The clinical stage topo ADC landscape can be subdivided into a couple of different strategies. There is the repurposing of known experimental drugs, and then there is the discovery of fit-for-purpose ADC payloads and linkers.
Companies that are repurposing for ADC use experimental drugs that were previously in the clinic as normally as standalone chemotherapies are more common, and they're grouped on the left-hand side of this slide. I think there's a couple of points about these that bear consideration. First off, in many cases, the payloads are more potent compared to those that are in the approved drugs. That would be SN-38 and Trodelvy and DXd and Enhertu. Secondly, the biggest focus, both pre-clinically as well as clinically, involves the repurposing of exatecan. Exatecan is the most potent of the topo payloads in the clinic, and it's among. It's worth remembering that in order to use exatecan for an ADC purpose, it needs to be engineered, or at least we need a chemistry solution to enable its use.
A different approach just is to search for and to develop novel payloads specifically for use in ADCs, and a smaller number of companies are taking this tack. Daiichi Sankyo and DXd are one of them, and this is also where Zymeworks has purposely decided to position ourselves, mainly because the approach allows us to carefully tune the biophysical characteristics of the small molecule, and thus the biophysical characteristics of the ADC. Also because it enables us to tailor not only potency, but other drug-like properties of the small molecule towards the intended use in an ADC. Being able to tailor these properties is an imperative to us, and ultimately, it's what led us to generate our own platform.
In designing a topo ADC platform, the three elements that require consideration are fairly straightforward, the payload, the linker, and the conjugation chemistry. Together, these components make up the drug linker, and they dictate in a large part how an ADC will work, how it will function. We had specific criteria in mind for our drug linker, and I can walk you through these criteria in the context of each component. The payload. Historically, the payload is what's received the most attention in the ADC field. Our payload criteria were pretty simple. We were searching for a camptothecin-based molecule with moderate potency, strong bystander activity, and good drug-like properties. There isn't really time to go into depth about the drug-like properties comment, but you know, think solubility, permeability, metabolic stability, PK, transporter profile, and so on.
The features of the linker, as I mentioned, are also very important. Our design criteria here included a cleavable peptide sequence that would release the payload unmodified, a so-called traceless linker. We were also looking for a sequence that would be sufficiently stable in circulation to avoid release of the payload prematurely. In terms of chemistry, the maleimide reaction was favored so that we could attach the drug linker to cysteine residues on the antibody. Not only is it simple chemistry that enables us to easily tune the DAR for each of our candidate molecules, it's also very well-validated, and we feel it provides a good balance between safety on the one hand and antitumor activity on the other.
I'll shed just a little bit more light on our general thinking around topoisomerase ADC drug linker design. ADCs are mechanistically complex, and I think it's naive to consider them as a means simply to deliver the payload directly to the tumor through a single mechanism. In fact, I think we know that the vast majority of payload is not delivered directly to the tumor, but it's released elsewhere. Of course, that you know speaks to the need for tolerability. Further to this, you know, we think that the circulating payload likely plays a role in the observed antitumor effects of topoisomerase I inhibitor-based ADCs. As such, it's important that the payload have those good small molecule drug-like properties that I referred to previously.
The effort that led to our topo ADC platform and its subsequent application to our pipeline is summarized on this slide. What we're trying to convey here is that our molecules have been put through a rigorous screening process, and the features that we were looking for were evident not just in a single experiment, but really across a number of complementary experimental systems. The confidence that we have in this approach and in this data comes from, in no small part, the ADC team that we've assembled over the years and developed over the years at Zymeworks. It starts with medicinal chemistry. Of course, it includes antibody generation, bioconjugation, and analytics. Of course, it captures in vitro and in vivo pharmacology groups, as well as the translational sciences team and others.
We started on the effort to develop this platform by synthesizing and screening on the order of about 100 novel camptothecin molecules as potential payloads. We compared them to various benchmarks toxins, including things like DXd, SN-38, exatecan, belotecan, and more. The focus at this stage was primarily on potency, as I mentioned previously, but also on weeding out hydrophobicity because of its potential to lead to undesirable aggregation and off-target toxicities. We identified a selection of payloads with appropriate potency, hydrophobicity, and other preferred properties. We converted those payloads into drug linkers, and then we conjugated them to different antibodies.
The ADCs that we prepared were then put through our biophysical characterization workflow to get rid of anything that was poorly behaved in terms of a drug linker or an ADC. Quite frankly, you know, finding molecules in this class that don't aggregate is one of the biggest challenges. The ADCs at this stage that remained were then assessed in a built-for-purpose tumor spheroid-based potency assay, and we gradually narrowed down the pool of ADCs by assessing in vivo antitumor activity in CDX and PDX models, and then finally, by looking at the top ADCs in terms of tolerability in most mice and rats. Ultimately, a dedicated non-human primate toxicology and toxicokinetics study was conducted, and this yielded us with our lead payload and drug linker.
Overall, the platform development effort was rigorous, and it was comprehensive in scope. It employed eight antibodies, eight different tumor-associated antigen targets, dozens of cell line-derived and patient-derived xenograft models, several specific PK assessments and multiple toxicity studies. Ultimately, this enabled us to launch the three distinct pipeline programs that we'll be talking about today, ZW191, ZW251, and ZW220. In addition, the technology has been applied to several early-stage assets. I'm gonna hand it back to Stuart now, who's gonna talk about ZW191.
All right. Thank you, Jamie. The first program I'd like to introduce you all to is ZW191. This is a potential best-in-class ADC targeting folate receptor alpha. As I just mentioned, ZW191 targets the clinically validated target, folate receptor alpha. The expression profile of FR α is compelling in ovarian cancer and other gynecological cancers, and other solid tumors like non-small cell lung and triple-negative breast. We've paired an internally discovered antibody with our novel topoisomerase I inhibitor platform to generate ZW191. The antibody itself was selected from a large panel to find a unique antibody with optimal internalization, payload delivery, and tumor penetration.
We're really excited by the preclinical data that we have on this program, where we're able to dose non-human primates safely up to and including 30 mg per kg, and we see strong antitumor activities in models with a range of folate receptor alpha expression. Folate receptor alpha is a GPI-anchored membrane protein responsible for the transport of folate into cells. In normal tissue, expression is limited to the apical surface of the intestine, lung, fallopian tube, placenta, choroid plexus, and the luminal surface of the kidney. In cancer, overexpression is observed in numerous gynecological cancers, like I mentioned, ovarian cancer, and in other solid tumors like non-small cell lung and triple-negative breast. The strong overexpression profile in cancer and the restricted expression in normal tissues makes FR α an ideal ADC target.
As I mentioned earlier, and I'll reiterate here, antibodies specifically selected for ADC use are very important to a successful ADC. Here we show that the ZW191 antibody is superior to the antibody utilized in ImmunoGen's anti-folate ADC, Mirvetuximab soravtansine, in two important attributes, internalization and payload delivery. In the left-hand panel, we show that ZW191 internalizes to a greater extent compared to MIRV, mirvetuximab, in a 24-hour period in folate receptor alpha-expressing KB HeLa cells. This is a fluorescence-based assay, and it's clear from the images that both the fluorescent intensity and the breadth of fluorescent distribution is greater for ZW191 compared to mirvetuximab. In the right-hand panel, we use a different assay.
This assay utilizes mass spectrometry to quantify the concentration of intracellular payload present in four different folate receptor alpha-expressing tumor cell lines after treatment with either the ZW191 mab or mirvetuximab conjugated to the same payload at the same drug antibody ratio. As we look at the blue bar, ZW191, in relation to the purple bar, mirvetuximab, we see that the concentration of payload found in all four cell lines is greater for ZW191. I'd like to draw your attention to the CAOV-3, HEC-1-A and H2110 cell lines, which express moderate to low levels of folate receptor alpha, and we see that ZW191 retains impressive payload delivery in these lower expressing cell lines. Internalization and payload delivery are important features that can drive antitumor activity, and the ZW191 antibody was selected to maximize both of those features.
Another important attribute we assessed was the ability of ZW191 to penetrate three-dimensional tumor spheroids. In vitro, we're moving more and more of our assays to a three-dimensional format as we believe this assay format better recapitulates the architecture of tumors. This is a fluorescence-based assay where we look at the fluorescent signal over a 96-hour time course in three different layers of a single tumor spheroid. From the images, you can see that the fluorescent signal for ZW191 is more intense in the outer layer, the middle layer, and the inner layer compared to mirvetuximab.
When we quantify this fluorescent signal in the graph on the right-hand side, we see that at every time point and in every layer of the tumor spheroid, the fluorescent intensity for ZW191 is greater compared to mirvetuximab. This means that ZW191 has the potential to penetrate tumors more deeply and to a greater extent compared to mirvetuximab, and this may contribute to a more robust anti-tumor response. When we designed ZW191, we wanted the ADC to have strong bystander activity, as we believe this is an important feature to target heterogeneous tumors and tumors with low antigen expression. Bystander activity is the ability of an ADC to kill antigen-negative cells in the presence of antigen-positive cells. The antigen-positive cells process the ADC and liberate the payload. The payload then permeates into neighboring antigen-negative cells and elicits its killing effect.
The cartoon on the left depicts the bystander assay, where we compare the viability of antigen-negative cells in monoculture to the viability of antigen-negative cells in co-culture with antigen-positive cells. In the figure on the right, we see that ZW191 exhibits a strong bystander effect, as the viability of antigen-negative cells in co-culture with antigen-positive cells, the blue bar, is 30% lower compared to the antigen-negative cells in monoculture, the green bar. To put this in perspective, we compared the bystander activity of ZW191 to the technology used in Enhertu, DXd, but conjugated to our ZW191 antibody. We see that the folate receptor alpha DXd ADC elicits similar levels of bystander killing as ZW191 in this assay.
Now, moving on to in vitro or in vivo experiments, we assess the ability of ZW191 to reduce tumor volume in four ovarian cancer xenograft models with a range of folate receptor alpha expression. You can see that ZW191, the blue curve, is effective at reducing tumor volume in all four of these models. The left-hand panel is looking at two folate receptor alpha high-expressing models, OV90 and CTG-2025, dosed at a single dose of 6 milligrams per kilogram. We see that ZW191 has similar anti-tumor activity to relevant controls like mirvetuximab in purple and MORAb-202 in red. The situation becomes more interesting on the right-hand panel when we look at two folate receptor alpha mid-expressing models, OVCAR-3 and CTG-0958.
In both models, we see that ZW191 is more effective in reducing tumor volume compared to these relevant benchmarks, suggesting that ZW191 has the potential to target lower levels of folate receptor alpha expression. This data is encouraging that across a range of folate receptor alpha-expressing models, we see robust anti-tumor activity with ZW191. Finally, we assess the tolerability of ZW191 in both rodent and non-human primate toxicology studies. Non-human primates represent a relevant antigen-binding species, and in this 2-dose every 3-week study, we found that ZW191 was tolerated up to 30 mg per kg. In this study, we evaluated how we compared to the DXd technology, but conjugated to our ZW191 antibody at the same DAR, and we saw a similar maximum tolerated dose and similar target organs of toxicity with the folate receptor DXd ADC and ZW191.
From this study, we also see that ZW191 displays linear and stable toxicokinetic profile. Not shown on this slide, but worth mentioning, we also dosed ZW191 in mice and rats, and those represent non-antigen-binding species, so that's gonna be the platform talks that I mentioned earlier. We found that ZW191 was extremely well-tolerated up to 200 milligrams per kilogram. This exquisite tolerability in non-human primates and strong anti-tumor activity in models expressing a range of folate receptor alpha suggest a favorable therapeutic window for ZW191. We believe that ZW191 has some significant commercial opportunity due to the distinguishing features of the antibody paired with our novel bystander-active topoisomerase I inhibitor payload and compelling preclinical data and the expression profile of FRα in cancers with unmet need.
We have potential best-in-class opportunity in folate receptor α-high ovarian cancer and potential first and best-in-class opportunities in folate receptor α-high endometrial, non-small cell lung, and triple-negative breast cancer, and in those indications in folate receptor mid and low expression. To summarize, FRα has a favorable expression profile in ovarian cancer and other solid tumors, and we think the mechanism of topoisomerase I inhibition is favorably positioned in ovarian cancer due to the sensitivity of ovarian cancer to topotecan chemotherapy. We're really excited about the ZW191 asset as we've designed it to have optimal internalization, payload delivery, tumor penetration, and anti-tumor activity. We also believe that our topoisomerase I inhibitor payload is likely to have a differentiated safety profile when compared to MIRV and STRO-002.
We're excited about the numerous best-in-class and first-in-class opportunities for ZW-191, and we're moving full steam ahead with this program, positioning it for a 2024 IND. With that, I'm gonna pass things back to Jamie Rich to talk about our next ADC asset, ZW-251.
Okay, thanks, Stuart. Now I'm gonna spend some time, as was just mentioned, to detail our second program, ZW251, which is potentially a first-in-class Topo I ADC targeting glypican-3 or GPC3. We're really excited about this program for a few reasons. First off, we're excited about the target. GPC3, glypican-3, is an oncofetal protein that's very highly expressed in hepatocellular carcinoma or HCC, which is by far the most prevalent form of liver cancer. Liver cancer patients generally face a fairly poor prognosis, and we view an ADC as having the potential to make a significant difference here. Next, we're excited because we've developed a great antibody that we selected specifically to use as an ADC, and we're coupling with our bystander active Topo technology. Finally, we're still excited about our data.
We have strong in vivo pharmacology data supporting broad antitumor response of this ADC in PDX models and CDX models of hepatocellular carcinoma. We've just initiated our pilot toxicology study in non-human primates. Let's start with a bit of background on the target. GPC3 is a GPI-anchored cell surface proteoglycan. It's expressed in the placenta, it's expressed in some fetal tissues, but its expression is downregulated in normal adult tissue. As I mentioned previously, it's highly expressed in some tumors, most notably in hepatocellular carcinoma on the liver. There are a few important points that relate to the ADC mechanism for this target that I wanna get into. First off, GPC3 expression in HCC is very common with GPC3 overexpression evident in about 70% of tumors.
Furthermore, the expression is not only generally quite high, but it's also relatively homogeneous, and this is exemplified in the leftmost immunohistochemistry image below. It means that an ADC can target a high proportion of the tumor cells directly. Finally, the target is not appreciably expressed in normal adult tissues, and there's very little expression in other kinds of liver damage, which is frequently present alongside of hepatocellular carcinoma. The takeaway is that GPC3 is not only a good marker for HCC, it's also a good target for the delivery of ADC payload, and we have an ADC antibody that's tailored for that purpose. Let's take a minute to go through some of the data that support ZW251 on the next couple of slides.
Codrituzumab is an antibody from Chugai that demonstrate to be in the clinic that an antibody can engage the target. It can engage GPC3. The graph at the left-hand side of the slide here illustrates the strong and equivalent binding to an HCC cell line of ZW251, which is in blue, and codrituzumab, which is in red. The second panel in from the left relates to our optimization of another key ADC-specific property, namely internalization. In this experiment, HepG2 hepatocellular carcinoma cells were incubated with labeled antibody. The amount of internalized fluorescence was determined, and it's represented by the height of the bars in the bar graph.
We actually identified the ZW251 antibody, that's the blue bar, by screening a large panel of GPC3 targeting antibodies, some of which are shown here in black, and by selecting the one with the greatest internalization. That internalization of the antibody or more specifically the ADC is also inferred or evident in the next panel over, the third panel from the left, where the potent cytotoxicity of ZW251 ADC towards tumor spheroids generated from the same HepG2 cell line is shown. The blue line illustrates the cell killing and its concentration dependence, and the orange line corresponds to an isotype control and demonstrates that the cytotoxicity that we're seeing for ZW251 is driven by GPC3 targeting. Finally, at right, we measured strong bystander activity in an HCC co-culture model.
Here you can see that when only antigen-negative cells, the light blue or purple colored bar, are there, that the ADC doesn't kill them. When you apply the ADC to a mixture of antigen positive and antigen negative cells, the dark blue data bar, the ADC eliminates about 80% of the cells that don't express the target. We're seeing a bystander effect there. Taken together, these data highlight not just the ADC properties of the antibody, but also the compatibility of the ZW251 GPC3 targeting antibody and our Topo platform. We're also very pleased to note that ZW251 has strong antitumor effects across a number of cell line-derived and patient-derived xenograft models of hepatocellular carcinoma.
Importantly, this strong tumor regression effect, which is illustrated by the blue data points in the left-hand graphs on the left-hand of the slide, have been consistent across models that feature a range of high, medium, and low GPC3 expression. Furthermore, you can see that the antitumor effects are targeted since the isotype control ADC, again in orange, have negligible impacts on tumor volume. The PK or pharmacokinetics of the ADC is another important consideration. The Tg32 humanized FcRN mouse model for PK is considered a pretty good way, an excellent way, in fact, to estimate the PK of antibodies at an early stage. In this case, the PK observed are independent of the target, owing to the absence of a tumor in the animal and the lack of cross-reactivity between ZW251 antibody and murine GPC3.
It is, however, evident there are no PK liabilities for this antibody, the ZW251 antibody in blue, which overlaps more or less bang on with the clinical benchmark codrituzumab. Together these in vivo data here and multiple other studies that we haven't shown you today emphasize the promise of ZW251, and we look forward to its continued development. To summarize for ZW251, hepatocellular carcinoma is an indication with high unmet need. It could benefit from an ADC, and we believe that a GPC3-targeted topo ADC is the best approach owing to the strength of the target and the platform. We've identified a great ADC antibody. It shows good specificity, strong internalization, and when it's paired with our topo platform, it shows excellent antitumor activity in vitro and in vivo. We're really excited about this first-in-class opportunity. We're looking forward to moving it on.
Now I'm gonna turn it back over to Stuart Barnscher, and he's gonna talk about ZW220.
All right. Thanks, Jamie. Okay, the third ADC asset I'd like to introduce you to today, as Jamie mentioned, is ZW 220, a potential best-in-class ADC targeting NaPi2b. As I just mentioned, ZW220 targets NaPi2b or solute carrier family 34 member two. This target is overexpressed in ovarian cancer, non-small cell lung and other solid tumors. ZW220 has been designed with an internally discovered novel IgG1 antibody conjugated to our topoisomerase I inhibitor platform. Again, we've identified the best antibody that is exquisitely suited for use as an ADC with optimal internalization properties. We have strong antitumor data supporting the activity of this molecule in patient-derived xenograft models of ovarian cancer, and we've just initiated a pilot toxicology study in non-human primates. Just a little bit of information about the target.
NaPi2b is a multipass transmembrane protein, responsible for sodium phosphate transport, and contributes to phosphate homeostasis. It consists of four extracellular loops. As you can see in the cartoon on the right-hand side of the slide, the second extracellular loop is the largest and provides the most epitope space for antibody binding. In normal healthy tissue, NaPi2b is expressed in the epithelial cells in lung tissue, the small intestine and the mammary gland. In cancer, there are very high rates of overexpression in ovarian cancer, non-small cell lung and endometrial cancers, as I just mentioned. As I mentioned earlier, the antibody used in ZW220 was specifically selected from a large panel for use as an ADC. Here we show that ZW220 has strong tumor cell binding and optimal internalization.
We compare our antibody to two relevant NaPi2b antibodies, Upifitamab. This is the antibody that's used in Mersana's Upifitamab rilsodotin, and lifastuzumab. This is the antibody that's used in Genentech's Lifastuzumab vedotin. In the left-hand panel, we're looking at the dose-dependent binding of ZW220 in blue in the NaPi2b-expressing ovarian cancer cell line, IGROV-1. Upifitamab in purple binds to similar levels as ZW220, while lifastuzumab in red binds at slightly lower levels. Now on the right-hand side, we see that ZW220 in the blue bars internalizes to a greater extent, compared to competitor antibodies over a 24-hour period. In the NaPi2b-expressing ovarian carcinoma cell line IGROV-1. Again, this is our fluorescence-based assay, and it's clear from the images that both the fluorescent intensity and breadth of fluorescence distribution is greater for ZW220 compared to Upifitamab or lifastuzumab.
Again, we believe that antibody internalization is a critical attribute and feature that contributes to the antitumor activity of ADCs. It's really important. That figure didn't turn out very well. Similar to ZW191 and ZW251, the ZW220 or ADC was designed with bystander activity in mind, to target heterogeneous tumors and tumors with low antigen expression. In this assay, we determine the bystander activity by comparing the viability of antigen negative cells in monoculture to the viability of antigen negative cells in co-culture with antigen positive cells. We'll fix the figure on the right for when the slides get distributed, but let's just imagine that there's a blue bar that's a lot higher than the green bar in both of these. ZW220 exhibits a strong bystander effect.
The viability of the antigen-negative cells in co-culture is much lower. It's about 20% lower compared to the antigen-negative cells in monoculture. Again, we compared to the DXd technology, and we see similar levels of bystander when that DXd technology is conjugated to our NaPi2b antibody at the same DAR. Next we're gonna look at ZW220 mediated killing of three NaPi2b expressing tumor spheroids. IGROV-1, TOV-21G, and NCI-H441. As I mentioned earlier, we employ a three-dimensional tumor spheroid assay, where a single tumor spheroid consisting of thousands of tightly packed cells is grown in a single well. The images on the left show the untreated tumor spheroids characterized by their large volume and visible proliferation.
Conversely, when we treat these spheroids volume and there's visible evidence of apoptosis and necrosis. Now, when we look at the figures on the right, we see that ZW220 in the blue squares exhibits potent dose-dependent killing in all three tumor spheroids. The NaPi2b high-expressing tumor spheroid, IGROV-1, we see that lifastuzumab vedotin kills at similar levels to ZW220. In moderate expressing TOV-21G and NCI-H441 tumor, NCI-H441 spheroids, we see that ZW220 shows superior tumor spheroid killing compared to the lifastuzumab vedotin benchmark. Now, we wanted to see how this was gonna translate into our in vivo studies, and we looked at the anti-tumor activity in PK of ZW220. On the left-hand side, we evaluated ZW220 in two ovarian carcinoma patient-derived xenografts.
CTG-0958 is a NaPi2b high-expressing model, and CTG-2025 is a NaPi2b low-expressing model. In both models, we see that ZW220 shows robust anti-tumor activity when dosed at a single dose of 6 milligrams per kilogram. In both of these models, lifastuzumab vedotin is ineffective at reducing the tumor volume. On the right-hand side, we evaluated the PK of ZW220 in a Tg32 mouse model. As Jamie mentioned, we consider this model, and many consider this model to be predictive of human PK. In the blue circles, we see that ZW220 shows stable and linear PK over 21 days. The PK profile of ZW220 is comparable to that of the 220 naked antibody, which is in green.
ZW220 PK profile compares favorably with the relevant NaPi2b benchmarks I mentioned earlier, like lifastuzumab vedotin in red and the Upifitamab antibody in yellow. To summarize ZW220, NaPi2b is a target with a compelling expression profile in ovarian cancer and other solid tumors. We think the mechanism of topoisomerase I inhibition again is favorably positioned in ovarian cancer due to the sensitivity of ovarian cancer to topotecan chemotherapy. ZW220 was designed to have strong internalization, and we believe this contributes to the compelling anti-tumor activity we saw in those preclinical models. We have numerous best-in-class and first-in-class opportunities, particularly in NaPi2b expressing ovarian cancers and lung carcinomas. We're gonna continue to drive ZW220 forward with a non-human primate toxicology study already initiated.
Just to summarize here, we've presented three advanced preclinical ADC assets in ZW191, ZW251, and ZW220. In all three of these assets, we've carefully selected the antibody to have strong internalization to facilitate payload delivery and drive anti-tumor activity. We've matched the tumor and target biology with a favorable payload mechanism by leveraging our novel bystander active topoisomerase I inhibitor payload technology. ZW191 is our most advanced asset. Strong anti-tumor activity in preclinical models and non-human primate tolerability suggest a favorable therapeutic index, and we're well underway in our IND-enabling studies for this asset. ZW220 and ZW251, for those programs, we've presented evidence of compelling preclinical anti-tumor activity, and we've initiated non-human primate toxicology studies for both of these programs.
We just told you how we're using our technology today to build a pipeline of exciting ADC assets. Now, I'd just like to take a few minutes to tell you about where we might go in the future. Moving forward, we'll continue to reinforce our unique design philosophy around target antibody, linker conjugation, and payload to develop innovative technologies and use our existing technologies in new ways to develop fit for purpose ADC assets. On the target, we want to explore novel targets. As we continue to address difficult to treat cancers, we will likely need to explore targets with less validation.
For the antibody, we wanna continue our pursuit of optimal ADC antibodies, but now start to leverage our bispecific and biparatopic know-how to engage targets in unique ways to drive better internalization, specificity and potentially even lower the target expression threshold required to see anti-tumor activity. For the linker conjugation, we'll be exploring novel linkers as we believe the linker design is really dictated by the payload potency, solubility, and metabolism. On the payload side, we're gonna be developing novel payloads. We're gonna be adapting MOAs with clinical validation to a novel ADC application. This is gonna provide us more opportunities to match disease and target biology with payload mechanism. With that, I wanna thank you all for your attention for this section, and I'll pass things back to Paul to discuss our multispecific antibody strategy.
Okay. Well, thanks, Stuart. Yeah, we're gonna switch now to the other foundational modality that we have, our multispecifics. Let me introduce that by maybe putting into perspective where the field of multispecifics are and the bispecifics. Similar to what we saw with the ADCs, bispecifics are now starting to accelerate the pace of approvals. You know, in this year alone, there has actually been five bispecifics that have been approved, three of which are CD3 engagers. Still the CD3 engager space is really where there's a lot of focus and a lot of opportunity. That really was heralded by the approval of Blincyto in 2014, a CD3 CD19, which was developed by Micromet and then Amgen.
That really ushered in a huge amount of excitement about CD3 engagers and their potential. There's been, as you can see, a bit of a hiatus between that, and then the more recent approvals. I think that's really because it became much more aware that it's actually much more challenging to develop a CD3 a T-cell engager than maybe was initially appreciated. I think what we feel though is that those challenges still remain. There are still limitations with existing strategies for T-cell engagers, and we feel that there is opportunity to apply Zymeworks technologies to actually advance those kind of molecules and overcome limitations that we'll briefly describe in the next few slides.
This table slide summarizes the technologies in the toolbox for Zymeworks on the multispecifics side. Again, the Azymetric technology really is the foundation of our bispecifics and the cornerstone. The most advanced bispecific, of course, is the biparatopic antibody, zanidatamab. But again, as I mentioned earlier, we've also, our corporate partner, J&J, they've actually developed T-cell engagers using Azymetric platform. What we're gonna be talking about today is in the bispecific or the multi...
More specifically, the multispecifics are T-cell engagers, where we've actually taken it away from just a one by one, but we're actually developed a two plus one T-cell engager platform to sort of maximize therapeutic window. That will be around the discussion of our 171 program that Nina will discuss. Then what we'll also do is talk about our tri T-cell engagers, the tri TCEs, where we're actually incorporating a third specificity into the molecule to give advantages over the existing simply bispecifics. That'll be in two flavors. There'll be the co-stimulation addition, and then there'll also be the checkpoint inhibition into our trispecific. Here is a timeline of the advances made at Zymeworks.
We've made a lot of progress internally, not only in the design and development of bispecifics using Azymetric, but also in the manufacturing and clinical development. In parallel, our corporate partners have also been developing programs, and together we can use these learnings to really accelerate the development of future bispecifics. What we wanna do, as I sort of alluded to, moving forward, is that we wanna move forward with this knowledge base that's been developed on a platform focus, and then move that into more of a pipeline focus. Again, more specifically, we see real opportunity in the T-cell engagers space. There, what we wanna do is we actually wanna use a more trispecific strategy, and we'll talk about that in more detail.
That's gonna be the two plus one TCE and the tri TCE platform. Why do we think that the Azymetric platform is well suited for this challenge? Something to bear in mind is that the flexibility of the Azymetric and the power of that heterodimerization really allows you to engineer and modify different components of the molecule. We can really dial in the change in the CD3 paratope, the features of that, the tumor antigen paratope, and the valency and the geometry and the format can also be supported by this platform. What we can also do is not only design different molecules, but importantly because again of the efficiency of the heterodimerization, we can screen in high throughput.
Nina will discuss this in a little bit more detail when we talk about the development of ZW171, but this together really allows us to power through screening different formats and be almost empirical in our screening format to pick the right molecule to push forward. Let's put this a little bit into the context of the T-cell engagers. Here, blinatumomab, of course, was again a breakthrough for CD3 bispecifics. You know, the CD3 bispecifics did show benefit over chemotherapy. There are challenges, of course, with that molecule. We know it's a short acting mole- a short path length molecule, so that also necessitated continuous infusion. Subsequent molecules including Fc, which we would have with our Azymetric.
Of course, there's also, as you see on the clinical data, the durability of response is not optimal. It would be nice to see responses that were more sustained and longer sustained. With T-cell engagers, there are also more global challenges that are associated with them. Probably the one that you may be most familiar with is cytokine release. That's a key problem in that when you have T-cell activation, T-cell killing, it comes with cytokine release, which to some degree can be good, but you don't want too much of it. In some of the first generation molecules, the balance between activity that you wanted with cytokine release was skewed. You want to try and limit cytokine release while maintaining killing.
What you also don't want to do is you don't wanna have too much killing on normal versus your tumor expressing cell. Getting that difference, differential activity also requires some thinking about design, and you'll see that we've factored that into the design of 171. Another challenge is that many tumors don't have enough T-cells or infiltration, or the T-cells are energized. The T-cells are not strong enough to mount a response. You need to help the T-cells, and signal 1 is not enough from the CD3. The third key problem is that in solid tumors, you often have other components that signal an immune suppressive activity or cell populations or pathways, and that can also then dampen the ability of a T-cell engager to work.
What we've done, and we'll touch on these in more detail when we get into these in more detail. For the first biological problem, where there's a narrow therapeutic window and toxicity due to CRS, what we've done is we've engineered our 2+1 T-cell engager to have a CD3 binding, low affinity CD3 binder, which maintains a killing activity, but has reduced cytokine response, and it's a novel CD3 binding domain. What we also have is we've engineered a 2+1 format to sort of skew binding towards cells that have high level of tumor antigen expression. For the second program.
Second challenge with the limited T-cell activity, what we're doing is we're gonna incorporate co-stimulatory signal into the tri-specific to actually activate and give a second co-stimulatory signal to the T-cell to enhance the fitness on those T-cells. Then third, to overcome the tumor microenvironment where you've got suppressive signals, we're gonna incorporate a checkpoint inhibitor specificity into the tri-specific T-cell engager. That's sort of just an introduction. Nina and Thomas are gonna talk about these programs in more detail, so I'll hand over the podium to them.
Thank you, Paul, and good day, everyone. My name is Nina Bhatt. Zymeworks Director of Multispecific Antibody Therapeutics. Today I need to change the slide first. Today I will provide an overview of the data supporting our preclinical candidate, ZW171, which is a bispecific T-cell engaging anti-CD3 and anti-mesothelin antibody for the treatment of mesothelin-expressing cancers. ZW171 is a bispecific T-cell engager that is built using Zymeworks' asymmetric platform. The illustration on the left shows the ZW171 antibody format, illustrating what is commonly referred to as a two plus one format, where there are two paratopes targeting mesothelin and a single paratope targeting CD3, shown in blue. Heterodimeric heavy chain assembly is facilitated by the asymmetric platform mutations in the Fc. ZW171 facilitates its anti-tumor activity by binding CD3 on T-cells and redirecting T-cell cytotoxicity to mesothelin-expressing cancer cells.
The tumor target mesothelin is a glycoprotein that is highly expressed in several cancer indications, including pancreatic, mesothelioma, ovarian, and many others, for which there is a high unmet medical need. ZW171 has a novel anti-CD3 paratope that was designed to widen the therapeutic window by having low T-cell binding and cytokine release and potent tumor cell lysis. We applied our engineering expertise and the flexibility of the asymmetric platform to modify and perform extensive assessment of various aspects, including valency, geometry, and affinity. ZW171 was recently nominated for preclinical development, with anticipated IND in 2020. To speak further to the target, mesothelin is a GPI-anchored membrane glycoprotein that has normal tissue expression that is restricted to the mesothelial cells of the pleural, pericardium, and peritoneum.
Mesothelin is overexpressed, as I mentioned, in many cancer indications, including pancreatic mesothelioma, ovarian, and others. Mesothelin is known to bind MUC16, and preclinical data has shown that mesothelin plays a role in cell adhesion, tumor progression, metastasis, chemoresistance, and the formation of cancer-associated fibroblasts that induce immunosuppressive environment. Mesothelin is a clinically validated target that has been targeted by different immunotherapeutic strategies, including bispecific T-cell engagers, CAR-Ts, and ADC. Recent clinical data has shown responses in cholangiocarcinoma, mesothelioma, and ovarian cancers with a mesothelin-targeted autologous T-cell therapy. The illustration on the right shows the structure of mesothelin. ZW171 binds the epitope at the N-terminal domain, as highlighted in cyan. This region is also the binding site for MUC16. The anti-mesothelin antibody is able to block this binding in interaction with MUC16.
Based on the restricted normal tissue expression and the abundant overexpression in cancer tissue, mesothelin is an ideal profile for bispecific immune cell targeting and the rationale for why we chose this target. Moving to the engineering and screening of ZW171. We have taken learnings from the clinical experience and the field and applied this to the engineering of ZW171. Specifically, we aim to engineer a molecule with a widened therapeutic window, with enhanced antitumor activity and improved safety profile for the treatment of mesothelin-expressing tumors. As Paul mentioned, protein engineering is a core competency at Zymeworks, and it is this deep experience, expertise, combined with the flexibility of the Azymetric platform, that allows us to screen multiple critical parameters in silico and in vitro in high-throughput fashion.
The workflow starts with paratope screening and optimization in silico, affinity engineering, followed by the generation of an extensive panel of engineered antibodies based on valency, geometry, and affinity. Based on the strength and flexibility of the asymmetric platform, we can easily make to high purity and yield a wide variety of antibody formats that is typically problematic to produce with other bispecific platforms. The asymmetric platform can be used for traditional expression and screening workflows, but also in high-throughput screens, where multiple parameters or antibody formats can be screened in parallel. We then screen the panel of antibody geometries, formats, and affinities in a series of in vitro assays, followed by confirmation of antitumor activity in vivo.
Both the in vitro and in vivo screening are performed under high stringency conditions, meaning that we test in conditions that best represent the challenging tumor physiology, including screening in a large panel of cell lines and in established in vivo models which are less sensitive to antitumor inhibition. By screening under these conditions, we are able to identify ideal antibody properties and, if required, refine this in an iterative fashion with more rounds of engineering and screening. One of the first parameters we optimized was the anti-CD3 paratope. The goals around engineering the CD3 paratope were built off the learnings from the field. In particular, were generation one anti-CD3 paratopes based on the OKT3 and SP34 paratopes are associated with high-affinity CD3 binding and dose-limiting toxicity related to cytokine release syndrome, or CRS.
Thus, we engineered the antibody to have low-affinity CD3 binding that results in low cytokine release, yet maintains potent tumor cell lysis in vitro assay to potentially avoid CRS. In the data presented, we have compared the ZWCD3-1 paratope in blue to a generation one high-affinity paratope in purple and generation two low affinity paratope in orange. As illustrated, ZWCD3-1 has reduced binding to T-cells, stimulates reduced cytokine release in the presence of tumor cells, yet elicits equivalent and potent T-cell cytotoxicity. We believe this profile is ideal to maintain antitumor activity and avoid dose-limiting toxicity related to cytokine release syndrome in patients. Moreover, our CD3 paratope binds a unique and structured epitope compared to first-generation paratopes based on SP34. The structure illustrated is a CD3 crystal structure. Highlighted in yellow is the epitope for SP34, and the ZWCD3-1 paratope is highlighted in green.
This distinct binding geometry may provide more optimized engagement in biology for mesothelin and other tumor targets. Additionally, the antibody is cross-reactive with cynomolgus CD3. We also evaluated aspects of the mesothelin paratope, including affinity as well as different antibody formats, and confirmed our lead properties and lead candidates by evaluating antitumor activity in the high stringency established tumor models that have reduced sensitivity compared to co-implantation models. In the established tumor model, the tumor or tumor fragment, in the case of OVCAR-3, is first engrafted subcutaneously and grown to a size of 100-200 cubic millimeters, and then the human effector cells or human PBMCs, which include T-cells, are added, followed shortly by test article administration to evaluate antitumor activity. Established tumor models can better reflect the challenging tumor structure, environment, and sensitivity for solid tumors.
This contrasts with co-implantation models where tumor and effector cells are administered simultaneously in mice. These models can be more sensitive to growth inhibition and may not best represent the challenging environment of solid tumors. Based on our in vitro screening, we have identified the 2 + 1 format indicated at center and as the lead format. We then evaluated the impact of varying the affinity of the anti-mesothelin paratopes and identified that mesothelin paratope with affinity C mediated the greatest antitumor activity in the mesothelin high expressing OVCAR-3 tumor model.
We compared our lead two plus one format to a different two plus one format, where the anti-mesothelin paratope with affinity C were Fab instead of ScFv, and observed greater antitumor activity with the ZW two plus one lead candidate. We also compared the lead two plus one candidate to the Roche two plus one molecule, which has different affinities for both the CD3 and mesothelin, and again, showed greater antitumor activity with the lead two plus one format. This highlights an important point in that not only is a specific antibody format critical to antitumor activity, but even with very similar two plus one formats, there are specific geometry requirements that likely impact the quality of T-cell synapse formation and antitumor activity.
This brings us to the preclinical candidate molecule, ZW171, that was engineered for enhanced antitumor activity and safety. To go a little more in detail on the format, there are two anti-mesothelin single-chain variable fragments, or scFvs, with one on the N-terminal domain of heavy chain A of the CD3 paratope, and a second scFv on heavy chain B. The reduced CD3 affinity anti-CD3 paratope is an Fab format or fragment antigen-binding fragment attached to heavy chain A. A heterodimeric heavy chain assembly is facilitated by the asymmetric platform, as I mentioned, and with mutations in the Fc that also includes the effector IgG1 knockout mutations that abrogate Fc gamma receptor binding. A summary of the design criteria is indicated.
Again, just to quickly review, we optimize the mesothelin paratope affinity for increased antitumor activity, reduced CD3 paratope binding to reduce the risk of CRS in the clinic. The antibody format and geometry were optimized for enhanced antitumor activity and safety, and the 2 + 1 format facilitates avidity-driven tumor-specific binding and cytotoxicity, thus mitigating the risk for on-target, off-tumor toxicity. Additionally, the format was engineered for improved stability. Next, I will review the data that showcases Zymeworks' advantage that includes greater antitumor activity compared to benchmark and potentially improved safety profile. Speaking specifically to the advantage of the 2 + 1 avidity-driven binding, we observe potent cell cytotoxicity with ZW171 in high mesothelin-expressing cells, but do not observe antitumor activity in low mesothelin-expressing cell lines, which act as a surrogate for normal, healthy tissue that express low levels of mesothelin.
As illustrated in the graph at right, you can see that ZW171 mediates potent T-cell cytotoxicity in high mesothelin-expressing cell line, but little to no activity is observed in low mesothelin-expressing cells, thus mitigating the risk of on-target, off-tumor toxicities. Further in vitro evaluations in high and moderate mesothelin-expressing cell lines show that ZW171 exhibits mesothelin-dependent cytotoxicity in a panel of mesothelin-expressing cancer cell lines, including lung, ovarian, colon, and mesothelioma cancer. ZW171 mediates high potency in both mesothelin high and mesothelin moderate cell lines. Additionally, ZW171 mediates cytokine release and T-cell proliferation in the presence of mesothelin tumor cells and not in the absence of the mesothelin target, thus mitigating the risk of peripheral T-cell activation and cytokine release syndrome.
We then compared ZW171 to clinical benchmark, as shown at right, HPN-TriTac or HPN-536, that has a single anti-mesothelin single-domain antibody and a single anti-CD3 ScFv paratope with an anti-albumin domain at the center for half-life extension. In vitro comparisons showed equivalent cytotoxicity with ZW171 and HPN in a high mesothelin-expressing OVCAR-3. However, when we tested in an established OVCAR-3 model, ZW171 showed greater antitumor activity. Due to the different PK properties of the two antibodies, ZW171 was dosed once weekly IV, while the HPN-TriTac was dosed daily IP for 18 days. While the doses for ZW171 and HPN-TriTac were not exactly exposure-matched, the serum exposure of HPN-TriTac is expected to be equivalent or up to 2 x greater than ZW171. Even with this potential differences in serum exposure, ZW171 mediated greater antitumor activity. Similarly, we compared ZW171 in a mid-mesothelin-expressing colon cancer tumor model, HCT116.
As observed with the OVCAR-3 cell line, we observed equivalent in vitro cytotoxicity in HCT116. However, as with OVCAR-3, we observed greater antitumor activity following treatment with ZW171 compared to the HPN-TriTac at exposure matched doses. PK and exposure analysis from this study confirm that the exposure of ZW171 and HPN-TriTac were comparable. These data highlight two important points. One, the importance of testing in vivo due to the lack of translation between in vitro and in vivo testing. Secondly, the importance of testing in more physiologically relevant established tumor models as the co-administration models may overestimate antitumor activity. We are excited about the opportunity of ZW171 and in treating mesothelin-expressing cancers. Mesothelin, as I mentioned, is expressed in many solid tumors as shown at left.
ZW171 has the potential to be first and or best in class for mesothelin pancreatic, non-small cell lung, triple-negative breast, mesothelioma, ovarian, and other mesothelin-expressing cancers. The estimated yearly incidence of mesothelin in patients ranges from 10,000 in mesothelioma to greater than 100,000 in pancreatic cancer. In summary, mesothelin is a clinically validated target with high expression in many solid tumor types that represent a high unmet medical need. Investigational mesothelin-targeted biologics have demonstrated clinical activity in mesothelin-expressing cancers. ZW171 has been engineered for optimal format, paratope affinity, and stability, with an improved safety profile and widened therapeutic index. This represents a first-in-best-in-class opportunity for the treatment of mesothelin-expressing cancers. ZW171 shows improved antitumor activity compared to clinical benchmark in mesothelin-expressing cancers. Our next milestones, as indicated, include a GLP toxicology study and IND submission anticipated in 2024.
Up next, I'd like to introduce Thomas Schalck, who will present our data supporting our engineering strategies to further address the biological challenges with T-cell engagers in solid tumors.
Thank you, Nina. Good morning, ladies and gentlemen. My name is Thomas Spreter von Kreudenstein, Director of Protein Engineering at Zymeworks, and I would like to shift focus to our next-generation multispecifics and present to you two new projects and technologies that we have been developing. As Paul showed you in the slide earlier today, T-cell engagers are a very promising class of therapeutics, but there are also a number of challenges that have limited their applications. We're developing next-generation tri-specific T-cell engagers to target two key challenges in solid tumors, as outlined here as problem number two and number three. The first project I'd like to present is aiming to address what Paul discussed here as problem number two. To better explain the problem and our approach, I'd like to briefly go back to basic T-cell biology.
Activation of T cells usually requires what is termed signal one and signal two, and as shown here in this figure, signal one is mediated by CD3 or the TCR and can also be termed the ignition, and signal two is mediated by CD28, can also be termed the gas pedal. Both signals together are critical for T cell activation, sustained T cell proliferation, and overall T cell fitness. Now, in the case of a T cell activation by bispecific T cell engagers, the T cell engager will only target CD3 or signal one and rely on natural B7 costimulatory ligands expressed on the tumor for optimal T cell activation.
The problem is now that the number of solid tumors, particularly cold tumors with low T-cell infiltration, are not gonna express costimulatory ligands, and particularly in those settings, because of the lack of costimulation, bispecific T-cell engagers are less active and are not able to induce sustained T-cell activation and proliferation. To address those limitations, we're developing costimulatory tri-specific molecules. They're able to target both CD3 and CD28 to induce better T-cell activation. Oops, that's incorrect. As you are aware, CD28 targeting T-cell engagers are currently also being developed by others. I'd like to spend a minute to better explain our approach and why we believe we have a differentiated approach. I'll ask you to focus on the left. There was supposed to be an animation.
I'm gonna be talking only on the left side first. What you can see here in the figure on the left is that optimal T-cell activation is not sufficient. It's not sufficient to just provide signal one and signal two, but what is critical is to balance signal one and signal two. If the T-cell only as shown here on the left side or the left panel, if the T cells only receive one signal, this leads to T-cell anergy, T-cell death, and lack of sustained T-cell proliferation. At the same time, if the T-cell receives two strong signals, as shown here in the middle, it leads to T-cell overactivation, T-cell dysfunction, and excessive cytokine release.
For optimal T-cell activation, the balance of signal one and signal two is critical, as shown here in the panel in the green in the middle. To develop an effective and safe costimulatory T-cell engager, we believe it will not be sufficient to just deliver two strong signals like, for example, in a combination approach, but it will be critical to balance CD3 and CD28 stimulation, or in other words, to balance signal one and two in one molecule. Our aim here is to develop costimulatory trispecific with a balance of signal one and two to be able to provide optimal T-cell activation in one single molecule. Now I'm focusing on the right here. That was supposed to come up as an animation, as I said. I'm gonna talk about the right panel here now.
To achieve this, we went back to what's known about T-cell engager engineering in our own experience, and we know from the field of T-cell engager engineering and from what Nina has shown you for our 171 program, that both geometry and affinity are critical criteria to develop an effective T-cell engager. We believe that this will likely be equivalently important for the design of any tri-specific molecule. With this in mind, we have developed a novel screening approach, which allows us to interrogate and screen multiple different geometries and affinities, and we believe this will provide a differentiated approach to fine-tune T-cell activation by CD3 and CD28 or signal one and signal two in a single molecule. The first step, we've selected and engineered our own CD28 agonist, and we have specifically selected what's termed a conventional agonist.
Conventional CD28 agonist binds to a different epitope than the known CD28 super agonist, which have had significant toxic liability in the clinic. We further engineered our own CD28 agonist with a broad range of affinities to be able to fine-tune CD28 activation in the tri-specific and to prevent potential CD28-mediated toxicities. In addition, the CD28 agonist can be formatted as scFv or Fab, which allows for flexibility in the design of the tri-specific. Oh, the animation works here. After selecting the CD28 agonist, we designed the costimulatory tri-specific, as shown here in the top panel, and this is just a representation of a subset of formats we're actually screening. What you can see in this panel is that the CD3 and the CD28 Fabs or scFvs are attached at very different positions.
What is unique in our approach, we believe, is that by doing this, we're able to interrogate different CD3 and CD28, but also CD28 TAA and CD3 TAA geometries, which ultimately might result in a better balance of signal one and signal two in a more optimal T-cell activation. The figure on the bottom left shows the design space of CD3 and CD28 affinities we're able to screen with the CD3 affinity on the y-axis and CD28 affinity on the x-axis, and each of the dots represent one format. In a second step, we pick the lead format and further fine-tune CD3 and CD28 affinities, and this is illustrated in the insert in the graph where we show varied CD3 and CD28 affinities in one format.
In this illustration, I should say, we're screening one TAA, but we routinely test different paratopes targeting the same TAA in the same screen. Different variants are expressed in a high throughput workflow and screened for activity in the T-cell dependent cytotoxicity assay on different tumor cell lines. The figure on the right shows a representation of an activity screen. As you can see, the different variants show significant differences in potency, a number of them being significantly more potent than the benchmark bispecific. At the same time, our aim was not to just make more potent T-cell engagers, but what we wanted to do is to develop trispecifics that are active under conditions where bispecifics have shown limitations.
As I discussed in the slide earlier, those limitations particularly include cold tumors with low T-cell infiltration or low effector-to-tumor cell ratio, and also conditions that reflect more longer term T-cell proliferation. To do this, we developed a screening assay at low E:T ratios with longer term incubation, which would better reflect conditions in solid tumors where CD3 bispecifics generally show low activity. As you can see in the left graph for day three, under those more stringent conditions the bispecific benchmark, but actually also tri-specifics were not active. When we take this assay out to seven days, we see activity of the tri-specific but no activity of the bispecific control. In addition, what you can see on the right, we also looked at T-cell proliferation in the same assay.
The tri-specific is significantly better at long-term T-cell expansion, which is in overall in line with the mechanism of co-stimulation driving enhanced T-cell proliferation. Overall, this shows the potential of our costimulatory tri-specifics to be active under conditions that are more relevant for low E:T and cold tumors. In the same assay, we also looked at cytokine release, and as you can see in the top panel, the tri-specific induces increased levels of cytokines and in particular IL-2 levels are significantly increased for the tri-specific, which really is a sign for enhanced CTL, CD28 co-stimulation, and is in line with the mechanism of action for a tri-specific. As a further step, a critical criteria in our design of the tri-specific is obviously safety. In particular, we want to avoid non-specific activation of peripheral T-cells.
To test this, we use cultures of T-cells only and test the cytokine release as a sensitive readout for T-cell activation. As shown in the graph on the bottom, the lead trispecific does not induce any non-specific T-cell activation without the presence of tumor cells. To summarize, a key limitation of bispecific T-cell engagers in solid tumors is lack of durable responses and low activity in cold tumors. Costimulatory trispecifics targeting both signal one and signal two have the potential to reinvigorate cold tumors and potentially show prolonged responses. We are developing a novel and differentiated approach of modular geometry and variety screening of trispecifics, which allows us to optimize and balance T-cell activation by signal one and signal two or CD3 and CD28.
Further, we have shown that the tri-specifics have superior activity to bispecific benchmarks at low effector-to-target ratio and induce enhanced T-cell proliferation. In addition, the lead costimulatory tri-specific show no activation of T-cells without presence of tumor cells, as a critical criteria for safety. Currently, we have different programs at lead selection stage. The second program I'd like to share is our tri-specific T-cell engager with checkpoint inhibition. This program and technology is attempting to address problem number one, as highlighted here by Paul earlier. To explain this observation, the observed clinical limitations, I'd like to briefly go back to basic T-cell biology again.
What you can see here, what happens after a T-cell activation, or what happens after T-cells have been activated is rapid upregulation of PD-1 and PD-L1, and that PD-1, PD-L1 interaction reduces and inhibits further T-cell activation. This is a natural mechanism that prevents autoimmune reaction and is illustrated on the left panel. In tumors shown on the right, the same mechanism will inhibit T-cell engager-mediated tumor cell killing and T-cell activation. In the clinic, it has been shown that in inflamed tumors with a high content of dysfunctional exhausted PD-1 positive T-cells, bispecific T-cell engagers have reduced or no activity. In addition, after dosing, T-cell engager activity leads to rapid upregulation of PD-1, PD-L1 on tumors, in T-cells as part of the mechanism of action, and this can lead to secondary resistance mechanisms of T-cell engagers in the clinic.
To address these clinical limitations, we're developing tri-specific T-cell engagers with checkpoint modulation one molecule. We've designed a tri-specific T-cell engager that engages CD3 and the tumor-associated antigen, but can also bind and block PD-L1. The molecule here shown is just one representation of the structure. We have a number of different geometries that I'll show you in a later slide. There's a few things that we believe make this molecule and the mechanism of action unique and differentiated, and I want to point them out in a bit more detail before I walk you through the data. The first mechanism of action of this tri-specific is blocking the PD-1, PD-L1 action in the synapse, which is shown here on the left.
This restores your original T-cell activation, but you could argue this can certainly be achieved by just a combination of the T-cell engager and an anti-PD-1, PD-L1 antibody. The unique mechanism of action of the molecule is that in addition to checkpoint modulation, it also creates further avidity by engagement of PD-L1, which is expressed on the tumor cell, but which is also upregulated on T-cell after activation. PD-L1 engagement functions like an additional adhesion molecule, strengthening and stabilizing the T-cell tumor cell synapse interaction, and this leads to enhanced T-cell activation and tumor cell killing. Overall, we believe that the molecule function by dual MOA, which makes this unique and has the potential this would be to be superior and significantly different to the combination therapy.
For the design of the trispecific, we used a natural PD-1 domain as an anti-PD-L1 binder and PD-1, PD-L1 antagonist. The figure on the left shows the crystal structure of natural PD-1, PD-L1, with PD-L1 on the left and PD-1 on the right. We have used the PD-1 domain shown in the box here and engineered it for higher affinity to PD-L1. It functions as a natural PD-1, PD-L1 inhibitor, equivalently to an anti-PD-L1 blocking antibody. As shown on the bottom graph, we further engineered this PD-1 domain with a range of affinities to be able to fine-tune efficacy and tolerability of the trispecific. Using this engineered higher affinity PD-1 domain as a natural PD-L1 antagonist allows for significant flexibility in format and geometry design of the trispecific.
The domain overall has a more modular nature than scFvs or Fabs that can be easily fused to different terminal of the T-cell engager. This flexibility allows us to design multiple different tri-specific formats as shown here on the right. As I discussed previously, we believe that geometry and affinity will be critical criteria for the dual mechanism of action to achieve both high avidity and functional PD-1, PD-L1 blockade. To select the best tri-specific, we use a screening approach to fine-tune both mechanism of action. In this screening approach, we are interrogating different formats and geometries, but we're also testing different PD-L1 affinities. It's illustrated here on the left, the tri-specifics are screened in a T-cell-directed cytotoxicity assay. Again, as you can see, the different variants exhibited significantly different activities.
For the lead formats shown here on the two graphs on the right, we consistently see a large gain in potency over the bispecific control, with easily over 100-fold improved potency for the two leads selected here. In addition, you can see the lead molecules also show superior potency with benchmark competitor bispecifics. To better understand the reason and the mechanism of actions for this large potency gain and to also ask the question if the same activity could just be achieved by a combination of a T-cell engager and an anti-PD-L1 antibody, we ran a series of more mechanistic assays. The first assay we looked at, shown here on the left, looks at the ability of the tri-specific to just block PD-1, PD-L1 interaction and restore effective T-cell activation.
In this assay, we have a high expression of PD-1, PD-L1, which reduces the T-cell activation, and we measure the extent of T-cell activation under those suppressive conditions. As you can see on the left, the bispecific is able to induce some T-cell activation, but overall has very low activity. As you can see in the middle, adding the anti-PD-L1 antibody restores a lot of the signal and leads to higher T-cell activation. As you can see on the right, the tri-specific has significantly higher activity.
Interestingly, it is able to not only restore T-cell activation by blocking the PD-1, PD-L1 action, but it's also able to further increase the T-cell activation. In the second assay, we looked at the ability of the tri-specific versus the bispecific to bridge T cells and tumor cells, and this can be viewed as a surrogate measure of how strong or stable the T cell tumor cell interaction mediated by the tri-specific is. What you can see here on the right panel is the amount of cross-linked T cells, the tumor cells, and the bispecific, as expected, is able to cross-link T cells and tumor cells, but the tri-specific is significantly more potent in cross-linking, which again suggests that it's able to induce a more effective and more stable T cell tumor cell interaction.
Those two assays taken together support our hypothesis that the lead trispecifics are functioning by a differentiated dual mechanism of action of both blocking the PD-1, PD-L1 action, but also enhanced avidity, which leads to superior T cell activation. We wanted to better understand if the trispecific can be active under more relevant conditions of suppressive solid tumor microenvironments. It is discussed previously, one key limitation of T-cell engagers is lack of activity in settings of exhausted or dysfunctional T cells. To better reflect this condition, we established assays with exhausted T cells and tested in the first step, just binding of bispecific and trispecific to naive T cells versus binding to exhausted T cells. The graph on the left shows binding of the trispecific and a benchmark bispecific to naive T cells.
As expected, it shows equivalent binding of the tri-specific and the bispecific benchmark to T cells. Binding for both is as expected around two-digit nanomolar range. One thing to note here on the graph on the left is that we also tested binding of an anti-PD-L1 antibody, and it shows no binding to those T cells. As expected, naive T cells do not express PD-L1. The graph on the right shows binding to exhausted T cells, and it interestingly shows significant differences between the bispecific and the tri-specific that I'd like to call out in a bit more detail because we believe this is very relevant for the differences for the differentiated activity and mechanism of action of the tri-specific.
You'll first notice that the clinical anti-PD-L1 antibody atezolizumab shows binding to the exhausted T cells and really confirming that exhausted T cells are expressing PD-L1. This has been described previously, might not be as widely appreciated. Second, as you can see, the tri-specific shows significantly higher binding to exhausted T cells than the bispecific control. This really suggests the tri-specific is able to bind both CD3 and PD-L1 on T cells and achieve a higher binding affinity and avidity than the bispecific. Lastly, what I would like to point out is that the bispecific control seems to overall show reduced binding to exhausted T cells when compared to naive T cells.
This is overall in line with previous observations by others that activated T cells actually down-regulate the TCR and CD3 as a mechanism to reduce T cell activation. Overall, the tri-specific seems to have the ability to better bind to exhausted T cells, which could be critical to retain activity in settings of suppressive tumor microenvironment with exhausted T cells. The next step, obviously, we wanted to understand if the differences in binding will translate into cytotoxicity activity. As you can see in this graph, this assay uses exhausted T cells as effector cells as opposed to naive T cells. We observed a significant increase in potency for the tri-specific versus the bispecific control.
In addition, we also observed that the combination of the bispecific control and the clinical anti-PD-L1 antibody atezolizumab did not show improved potency. In summary, the observations support our hypothesis that the trispecific has the potential to be active on conditions of suppressive tumor microenvironment where bispecific T-cell engagers have shown limited activity. In addition, the superior activity of the trispecific supports the unique activity through a dual mechanism of action of both checkpoint blockade and also enhanced T-cell tumor cell avidity. We were very encouraged by this activity and the unique properties of the trispecific, but one obvious question is how broadly applicable the superior activity enhanced potency actually is. To better evaluate the broader application of the Tri-TC, we screened a larger panel of cell lines with different levels of tumor-associated antigen and PD-L1 expression.
As you can see here on the left, the cell lines have varied expression levels of the tumor-associated antigen from medium to high, and similarly for the PD-L1 expression. On the right, you can see the activity in the T-cell-dependent cytotoxicity assay, and we observed a similarly large potency gain across all cell lines tested, fairly independent of expression levels. What we observed in addition to the data that I'm showing here is that after incubation with the T cell engager, PD-L1 is rapidly upregulated on most of the cell lines. This rapid PD-L1 upregulation might contribute to the broad activity across multiple different cell lines. We believe this is a unique mechanism of our tri-specific, and it suggests that the tri-specific mechanism of action might be broadly applicable and potentially be less susceptible to PD-1, PD-L1 mediated secondary resistance mechanisms.
As a next step, we wanted to understand if the superior in vitro activity translates into in vivo, and we tested the activity of the trispecific in humanized mouse model. The model used an established tumor model, established, so very similar to the models that Nina has shown you for our ZW171 development. As you can see here, the bispecific in this established more difficult model shows only very little tumor control. Whereas the trispecific is able to control tumor growth, and we observed six complete responses out of eight mice. Interestingly, in this model, the combination of the bispecific control and the anti-PD-L1 antibody Atezolizumab did not show improved activity. Which again suggests the trispecific has a differentiated activity to the combination therapy and supports our hypothesis of the dual mechanism of action.
To summarize, we know that PD-1, PD-L1 upregulation in the tumor microenvironment is a key clinical limitation of bispecific T-cell engagers. To overcome these limitations, we're developing novel differentiated trispecific T-cell engagers with a dual mechanism of action of both checkpoint inhibition and avidity-driven T-cell activation. We have demonstrated potent and differentiated activity in vitro and in vivo, and high activity with exhausted T-cells. The activity supports our dual of a novel dual mechanism of action of both checkpoint inhibition and avidity of the trispecifics. In addition, the data suggests superior activity of the trispecific to combination therapy of bispecific with checkpoint inhibitor. It's overall belief that the trispecific T-cell engager, due to the differentiated dual MOA, may provide unique opportunities for targeting microenvironments with exhausted T-cells, and potentially also provide more dual responses.
We have a number of lead molecules at lead selection stage. With that, I thank you, and I'll hand it over to Paul.
I'm gonna just wrap up with, like, a 10-minute sort of wrap up, summary. What you've hopefully heard is the significant progress that we've made with our ADC and multispecific platforms with various product candidate opportunities. Before I recap on those, I wanted to take a step back and just again frame in context our overall focus of which is to, you know, which is to address unmet medical need. We're really developing molecules that really make a difference. We're gonna really focus on the indications with the worst patient prognosis. We then intend to apply our, either our multispecific platform or our ADC platform against targets from those indications. We'll apply the technology that best serves that target, the biology associated with that disease and that tumor type and the disease complexity.
From there, we pick molecules that with the most favored profile for development, and with the intention that they will ultimately enter into earlier lines of treatment and have the potential for accelerated approval. That's always at the back of our mind, is thinking about the tumor target and the disease. As the team shared with you, we're really uniquely placed at Zymeworks to leverage either world-class ADC or world-class bispecifics or multispecifics. Our strategy, as you heard on the antibody-drug conjugates, is to think of the molecule totally. Think about the antibody properties and format, payload, linker conjugation, and DAR. Likewise, on the multispecifics, we really can fine-tune and engineer the molecule to get the right features, multispecific features, ingrained into that molecule to address what the challenges is.
Now, some of the diseases, the tumor types that you heard us describe are listed on the left and so are consistent with our thinking of trying to treat patients with the poorest outcome, overall survival. Now, getting back to the programs that we talked about today, how this integrates with our planning and vision in the near term and the longer term. In the context of the ADC programs, our next IND will be ZW191, the folate receptor targeted topoisomerase program, topoisomerase ADC. That targeting folate receptor is a target, as you heard, is validated as responsive to ADCs clinically.
We believe with an antibody optimized for internalization features and the use of our proprietary topo payload, we can develop a best-in-class molecule for folate receptor alpha-expressing tumors that can also expand into the middle range expressing middle range folate receptor expressing tumors, as our in vivo modeling demonstrated to you. In the context of multispecifics, our next IND will be the ZW171 program, a two-by-one or a two-plus-one mesothelin CD3 trivalent T-cell engager. As Nina described, we feel mesothelin is an excellent target for a bispecific T-cell targeting approach. The target is, like, highly expressed in a range of tumor types, including those hard to treat and with poor overall survival. A lot of engineering has gone into the design of that molecule to really optimize all components.
The CD3 affinity and epitope to limit cytokine release, together with bivalent targeting of mesothelin and the optimal geometry to increase therapeutic window of activity, versus normal tissue. Like, you get a good window of activity between tumors and normal tissue. Importantly, we maintain excellent anti-tumor activity with that molecule. That was a real high bar for us to make sure that we maintained activity in established tumor models, and we see that. We look behind that, like in our sort of research engine, what we've got coming behind that. Stuart and Jamie described additional topo ADC-based product opportunities. There are the two programs we decide to describe were the 251 targeting glypican-3 and 220 targeting NaPi2b. These were again engineered through careful selection of antibody payload.
We're really encouraged about the profile of each molecule and the cancer indications they serve. These molecules are now completing preclinical development and could serve as future IND candidates. As Stuart alluded to, we have other programs behind that that we've also identified tumor antigens for in cases of applying the biparatopic also with the TOPO platform. Moving to the multispecific side is an area that we think is of still great opportunity with redirected T-cell killing. Major challenges remain for many tumors that are not expected to respond to just a CD3 bispecific, either due to the intrinsic fitness of T-cells in the tumor microenvironment or the interplay of or immune inhibitory molecules. Where there this has led to.
This in the field has led to combination approaches where people are thinking about adding co-stimulation along with primary CD3 engagement through a combination of antibodies. We think we can do it in the one molecule, and the benefit of the design and the flexibility of the Azymetric allows you, as Thomas explained, to really fine-tune both signals so you can get that right balance. In addition, we also have employed this for checkpoint inhibitor where we were able to again demonstrate proof of concept where we could actually see enhanced activity and synergistic biology by incorporating PD-1 binding. Moving forward from here, what we really are well-placed to sort of utilize the technologies for more targets.
We really are starting to think more about novel targets. We've initially focused on targets somewhat validated, but as we go into other tumor indications, we will select more novel targets and potentially will select them in combination to further potentially advance therapeutic window there. This may include targeting that enables gated activation to increase therapeutic window or strategies that combines simultaneous tumor cell killing and reversal of immune cell population repression, either through a signal blocking or perhaps direct lysis. To this end, we intend to continue evolving our payload strategies as well, and the competitive edge there within our ADC platform.
Likewise, in the context of multispecifics, we can also consider additional effector cell populations or more specific effector cell populations through avidity-driven cell activity, as Thomas showed with the TCE checkpoint, where we could select the PD-L1 population or really drive that targeting of that population. That could also be extended to other immune subsets through a similar approach that he described. In the near term, we have four exciting molecules, two of which have already been targeted for IND filing. Behind that, as we mentioned, we've got a machine or an engine that's working on additional molecules, and we think that these will continue to fuel our clinical pipeline. We will continue to partner opportunities, moving forward.
This is just to sort of reiterate that partnering will remain a part of Zymeworks. I think our focus moving forward will be deriving more value from product candidates than platform, necessarily just purely platform capabilities, but we will continue to also leverage opportunities, partnering opportunities through that as well. To finalize, I'd just like to finish with this slide that really captures our five-by-five vision for Zymeworks, building on the company's proven success in leveraging bispecific and ADC technology to develop therapeutics, and the advances we've made in the platform technologies, and tools based on the team's capability and infrastructure. We envision transitioning from a company focused on a select product pipeline and platform technology advancement to one focused now on accelerating more program products to the clinic. We have one go.
We have a goal of five new programs in the clinic in five years. We shared with you two of those programs that have already been identified as INDs, one in ADC and the other a novel bispecific, each fit for purpose designed to improve on existing treatment options for patients suffering from hard-to-treat cancers. We're uniquely placed to have both world-class ADC and multispecific platforms to leverage and are well-placed to meet our goal with truly differentiated molecules. With that, I'll let Neil close out.
Great. Thanks, Paul. Thank you, Nina, Thomas, Sue, Jamie, for presenting the exciting work that's coming out of our early research and development groups. Additionally, before we start Q&A, I'd like to thank everyone who tuned in to the webcast and participated live and share some closing thoughts. With yesterday's announcement of our partnership with Jazz Pharmaceuticals and a host of potentially first and best-in-class ADCs and multispecific therapeutics based on our cutting-edge platform technologies, these are exciting times for Zymeworks.
It's fair to say that the combination of exciting and dynamic partnerships coupled with world-class science truly brings Zymeworks back to its roots. By leveraging our core competencies in antibody engineering to create the right antibodies, as you've heard today, and coupling these antibodies with our leading and proprietary platforms, we are focused on developing meaningful novel therapeutics that we believe will deliver meaningful clinical benefits when these programs enter the clinic and deliver real shareholder value. We are focused on our two key research areas, ADCs and multi-specific antibodies, to drive pipeline expansion. As I noted at the start of this session, going forward, we are fully committed to the use of partnerships and collaborations as a key strategy to bring these novel therapeutics to patients, setting up success for years to come.
With that, I'll open up the floor to Q&A, where we will have Paul and our research team leads join us up front and available to answer questions on today's presentation. As a reminder for those listening in to the webcast, if you have questions, please email ir@zymeworks.com, and we will do our best to answer your questions. Thank you, everyone. Excellent. We'll open up to questions from the floor here in New York.
Hey, good morning, everyone, and thanks again for taking the time to host this event. Charles Zhu from Guggenheim Securities. Maybe one scientific philosophy type of question. When you're designing some of these next gen molecules, whether it's an ADC or a T-cell engager, how should we think about the relative contribution of potential clinical activity from the various components or specifically from, let's say, the antibody itself? Where I'm coming from with this is when we look at zanidatamab versus ZW49 as an example, zanidatamab without the payload does have activity. So from that perspective, how should we think about that with your emerging ADCs and similarly for ZW171, given where you're binding on mesothelin, is there contribution of activity through the blockade of MUC16 activity as well? Thank you.
Charles Zhu, thanks for that question. Understand what you're getting at is that I think a lot of that depends on the dose that you're gonna be dosing your molecule at, okay? We do believe that there's gonna be certain features that are additive to the sort of primary mechanism of action of a T-cell engager. The potential that the specificity against the target could have in and of itself biology that you could also be getting a benefit from, right? You asked the question specifically about ZW171 and mesothelin. There we do believe that we're binding an epitope there with that design and that particular molecule because it is a block.
That antibody does bind to an epitope that blocks engagement and that it's bivalent, that we may actually, in that case, have the opportunity to see that. That may not always feature in all the programs that we have. It may be that mechanism is not necessary if you can get the power of the other modality that you're trying to exploit in the design of your molecule. Okay. Yeah. I don't know if maybe on the ZW171 if it was something else.
Yeah. You said it well.
Okay. Yeah.
Great. Thank you.
All right. Thanks. Akash from Jefferies. It's kind of interesting. Like ZW49 was a really elegant molecule, right? Like, but you were going for something very different. You're going at a DAR2 higher internalization, non-greasy, and it's you've completely flipped the switch with these next gen ADCs. You know, just conceptually speaking, like, you know, you're trying to avoid the eye toxicity, but you avoided the liver, you avoided the lung toxicity, which is a big deal. Why not take, let's say, ZW49 and then just put a topo toxin and put a DAR2, right? Why go to a DAR8 and making it greasy, given, like you probably are still increasing net exposure either way?
Yeah. No, we think a lot about the design of our ADCs and, with our programs going forward, we do think, you know, totally about the ADC, right? We think about the antibody, we think about the payload and how we attach it and how that total molecule will give us the effect that we want. I think at the time, depending on the timeframe of when we've got our knowledge base, we will design the molecule to the best of our knowledge at that time and what information we have. I think moving forward, we're attracted with the topo platform for these targets. We think ZW49 is a great program itself with the design of that molecule. We kind of consider them, you know, independently when we're looking at different targets. I guess I'll let...
I mean, I don't know if you or Jamie wanna elaborate on that.
Yeah. I think we talked about this maybe before, Akash, too, and there's this dichotomy exists in kind of the hydrophobicity and the greasiness.
On antibody, you probably want something that's a lot less greasy, so you have a lot more antibody-like properties of your ADC. We really do think we have that with ZW49, and that's using the topoisomerase I inhibitor platform. On antibody, we wanted to have as antibody-like properties of our final ADC product. Now it's the payload when it's released that happens to be hydrophobic, right? That can then permeate neighboring cells and have a bystander effect. I would call them kind of like tweaks as opposed to like wholesale changes in philosophy. It might seem like that a little bit, but I think you know, the mechanism of action of topo I versus MTI really will dictate kind of like where we apply this.
We think that, you know, the ZW191, the ZW251, and the ZW220 are really good places to apply topo.
Right. Just to follow up on that. ZW49 and then, you know, the Exelixis tissue factor ADC, both of them showed eye toxicity, right? To me it seems like it's probably the toxin, but it could theoretically be the linker. And it also could be a non-target effect, right? Like Tivdak, even with the antibody itself was showing eye toxicity. You know, if you're using topo as a toxic-
David Martin from Bloom Burton. When you were looking at the efficacy of 191 compared to mirvetuximab, you're comparing head-on to that antibody. When you went to the toxicity comparison, you compare it to your antibody with DXd toxin on it. Why not compare to mirvetuximab on the toxicity front?
Yeah, I think Surena can best place.
Yeah. The Mirvetuximab soravtansine kind of like tox profile is a pretty known entity, right? We know how it's performing in clinical trials and we know how it performed pre-clinically. We know the doses where they see toxicity, and we know the tox profile. That was kind of a known entity. when-
Would you say comparing across, you are less toxic or equal toxic or?
I think it's, you know, we're in preclinical models. What we can say is that we think that in our preclinical models, we compete really well with the DXd technology, which is in the same class of molecules. We thought that was a much more important comparison than saying, you know. The other thing too is that Mirvetuximab soravtansine causes ocular tox, and there's really poor models of ocular tox preclinically. We could have dosed the monkeys with Mirvetuximab soravtansine, saw a tox profile that actually isn't consistent with the clinical tox profile and said, "Well, we're, you know, somewhere in the range," sort of thing. We thought the comparison we made was relevant.
Okay. Second question is more high level platform related. With the ADCs, you talk a lot about bystander effect. Is there any bystander effect you'd get with the T-cell engagers, or is it something that will be missing from that therapeutic class?
Yeah, I can take that question. I think that is one. The feature of T-cell engagement, an attraction of that technology in one principle is that you will only hit the cell that expresses the target, and you will spare the cell that doesn't have it, right? I think on direct engagement that you will have the. You are focused on the target cell that you co-engage the T-cell with that cell, right? I think.
What really attracts me to the platform technologies that Zymeworks is, I think by adding in other features into your molecule, they may lend you some benefit of cells that are in the tumor microenvironment. If you can actually start resurrecting T-cells, PD-1, you know, antigen positive, you know, T-cells that recognize tumor antigen TILs, they may, you know, by expanding that, they may have actually intrinsic ability to take out more of the tumor. That's somewhat hypothesis in principle, that could be an extended obvious primary mechanism of action of T-cell engagement and activation. Billy?
Hey, this is Ken Shields from SVB Securities. Congrats on these early programs. I'd say, relative to these early developments, how committed are you to ZW49, and if you think the clinical profile you've seen with the drug is competitive and compelling?
Paul, why don't I take that one?
Yep. Yes.
Great question. Thank you. No, we are fully committed to ZW49 going forward. We believe we have an exciting compound, and we look forward to advancing it as we move forward. I think the additional capital and resources that we have following yesterday's announcement open up some great new opportunities to advance our emerging pipeline, and that definitely includes ZW49.
Thanks.
Hi, guys. This is James Shin from Wells Fargo. Just wanted to go back to ZW191 real quick. You mentioned a cysteine conjugation. Is that a cathepsin B linker? In your non-human primate studies, can you clarify and state whether or not you saw any ocular neutropenia? I ask because I believe cathepsin B linkers are sort of related to some of the ILD and neutropenia issues. I just wanna make sure that's there or not. What is your assay that you've determined for FRα high? Because right now, I believe the two comparisons you made to STRO-002 and Merck, they're using different IHC-based assays. Just wanna get your thoughts there. Thank you.
Yeah. There's quite a few questions there. I think most of those will fall on the ADC team. Yeah, I think, you know, we think about a lot of those features about the IHC. You know, that's something you mentioned about IHC, I think. Obviously, we're using. I mean, I'll let Stuart and Jamie elaborate on that. But I think that we're, you know, obviously we're just looking side by side, and we're using relatively, you know, assays that give us a sense of the relative expression of those targets and those preclinical models. As we move forward into clinical development, we will be thinking about a select and a specific biomarker strategy to support that will wire into our phase one study as, you know, when we get there.
I think regarding the more specific questions on the technology and the details, I'll hand that to, I think Stuart or Jamie should take that.
Sure. I mean, I can take the protease question. Stuart, maybe you want to take the IHC. The linker, as we discussed is a peptide-based linker. It is cleaved by proteases, and I would include cathepsin B among those. I sort of shy away from just focusing on cathepsin, but that's accurate, I think. In terms of whether or not we saw any ocular tox, we did not see any ocular tox in the NHP study?
Just to be really clear, about the IHC, we're not using the Roche Ventana assay and PS2+ scoring. It's internal kind of, you know, research level IHC scoring. It's, you know, think of it as relatively high, right? We're not using the strict scoring that ImmunoGen uses.
I guess with the T-cell engaging bispecifics, you kinda talked about some of the problems you're trying to overcome in the solid tumor space. I guess it would seem conceptually that kind of problem two and problem three, those concentric circles are kind of overlapping. I guess how do you think about whether or not you wanna go with a costim feature or with a checkpoint inhibitor? Then maybe just on the TCE side again, how comfortable are you guys with some of the pharmacologic modeling for selecting an appropriate starting dose, specifically for solid tumors? I know some of this has been tried to work out in a lot of heme malignancies, but yet we've seen a lot of companies kinda get locked into these multi-year dose escalation phase 1 protocol.
How complicated is some of the modeling with respect to selecting a starting dose for solid tumors? Thanks.
Yeah. Sure. So I think your first question was, you know, would you go with the costim or would you go with the checkpoint inhibitor? I think there, you know, we're encouraged with both. The preclinical profile we're seeing for both. We are gonna be continuing to do studies with those models. We've actually got tumor-associated antigens that we think are attractive for both of those approaches. That data will kind of drive, will be data-driven on which one we take forward. You know, we're very excited with both. I think, you know, in particular, the PD-1 just, you know, it's got some striking in vivo data we've already seen, and that profile across, you know, multiple cell lines is, you know, really eye-catching.
We think the CD28 program as well could be, you know, very beneficial. Of course, we can learn from what others are doing in combination and see if there are certain tumor types. Again, it may really come down to that. There's gonna be certain tumor types that will benefit from one over the other, on that one. Maybe we'll take that question and elaborate on that if Nina and Thomas have anything.
Yeah. One thing to add on the target, I think. You know, obviously, it has to fit with the biology and the safety profile, particularly. You know, particularly if we think of the CD28 costimulatory. That's an important feature. You know, as we've mentioned, we are focused on indications with high unmet medical need and, you know, maybe save for a few indications or cold or hot, you know, most tumors actually have a continuum. You know, I think as long as you pick the target wisely, you know, these approaches can be used really for any indication. Then it comes down to, you know, patient selection and setting up your clinical trial appropriately.
Yeah. On your second question, I mean, that's something you know, I've been working on, you know, T-cell engagers in, you know, my career and, you know, seeing that evolve, how you know, design the starting dose. Again, there's a lot we can learn. I think, not to say too much, but we can think about those MABEL approaches, and it tends to be more of a MABEL approach that you use. There's increasing, I think, models and in vitro and assays that you can deploy to sort of get a logical starting dose. Of course, that has to be. You know, you have to run that through the FDA.
I think we will really be, you know, as safe as possible, but design our study as a way that we can get into the therapeutic doses as soon as possible.
Hi. Yigal Nochomovitz from Citi. I just had a few fairly specific questions on some of the data that you presented. I was intrigued by the experiment where you compared 171 to the HPN536 molecule, where you really didn't see any difference on cytotoxicity, but a substantial difference on tumor reduction in vivo. I was wondering if you expand a little bit on what you think is different about 171 that is providing that interesting signal in vivo, but not cytotoxically. Thanks.
Yeah. I mean, we've been really obviously excited with that data and also kinda put our. You know, think about it, you know, because it's quite startling, the difference that we see there. I think, you know, if you looked as we would develop that design of that molecule earlier in the slide deck, Nina shared that geometry, that format is actually really important. The way that we've got the two binding domains for mesothelin in that is actually also important. Although we didn't see activity with the HPN molecule, we actually didn't see activity with other formats as well.
I think what it might be is what we like to think is that we've actually struck just the right molecule structure for mesothelin, and that's reflected then in the functionality of the really strong antitumor activity we see in those models. I think that's more, we've just hit the right format that we believe, and that whether it's the strength of the synapse and the ability to get into the tumor, because it's an established tumor model, the right balance with the T-cells, it just seems to have, you know, really remarkable activity. As you observe, you know, as we shared, we see it in more than one model, so it's not just an artifact of one model. We see it, you know, repeated.
I think it's just again, you know, We believe that with the Azymetric platform and that ability to screen different formats, we could plug in Fabs, single chains, different positions, and that allowed us to find molecules that had a good activity. We also relied on the in vivo model to select a molecule, and it was only then that we also compared some other molecules and then we saw that this one worked really well. Yeah. Nina or Thomas?
Yeah. I think exactly like, you know, the format has a strong impact on the synapse formation, number one, which is what Paul was highlighting. I think also the avidity driven binding and its ability to, you know, bind mesothelin high, but also maintain binding and maintain, you know, the T-cell synapse formation is, you know, likely a component. There's the potential for less effects, you know, due to mesothelin shedding as kind of reducing antitumor activity also because of that avidity driven binding. There's, you know, a number of kind of design features that are distinctly different between the two molecules.
Okay. The other thing I noticed in the data, which admittedly it's a bit of a subtle observation, but I noticed that for ZW220, you still had some killing of the antigen-negative cells. For ZW251, and I know it didn't show up on the slide, but I saw it on the download. For ZW251, there was actually no killing of the antigen-negative. Just wondering what was going on there, if there's an explanation for that subtle difference.
Yeah, that one. Sure, we'll take that one.
Okay.
Yeah. So, that was in the bystander assays, right?
I believe so, yeah.
Yeah, yeah. Oftentimes what we see, depending on the tumor cell line, is that there is a phenomenon called macropinocytosis. That's where kind of you can think of cell lines as being a little bit thirsty and kind of taking up non-specifically, you know, things in the dish. Sometimes we will see with antigen-negative cell lines, if we were to run 30 antigen-negative cell lines with one of our antibody-drug conjugates, some of those cell lines would be flat, you know, no change, no reduction in viability, and some of them would have this kind of modest reduction in viability.
It's not anything that we read into too much, and it's, like you said, it's a very subtle observation and one that, I'm surprised you made, but it's very interesting to us and, like, we care about it, so it's cool that you noticed it too. But it's nothing to read too much into.
Okay. Just really fast, just one housekeeping question. I think you said on the slides that for 191 it was DAR 8, and then in the cartoons you had DAR. You did have DAR 8 for the other two, but you didn't specify. Can you just clarify if the 251 and 220 are also DAR 8? And for the topo-1, are they all the same molecular structure? Are they just all topo-1 inhibitors, but slightly different structures for the three?
Yeah. That one I'm going to hand over to Stuart as well. Or Jamie.
You take the payload.
All right. The payload that we're using is in fact all the same.
Okay.
With respect to the DAR for those two programs, you know, I think we talked a little bit about how we try to approach these things holistically. Every candidate is different, and we go with whatever is best. We're actually right in the midst of our toxicity studies, and so we'll be selecting that DAR in the very near future.
DAR can be viewed as the final fine-tuning knob that we have to kind of address efficacy and toxicity, and we do that kind of at the very end.
Thank you.
Sure.
Hey, guys. This is Brian Cheng from JPMorgan. First question is on ZW220. It's interesting you're seeing responses in the NaPi2b low model. Compared to UpRi, which really been showing responses for you in the clinical studies, is the benefit you saw driven more by the bystander effect? I don't know if you have any thought on that. And more importantly, are there any specific learnings based on what UpRi had shown in the past? Do you see more of a need to tackle the NaPi2b low population first?
Yeah. I mean, again, I think our design, our thinking on the design on that is that we really sort of build our own molecule.
Okay.
Design that to have the features that we really desire, right? We obviously are gonna compare against competitor molecules and take the learnings from that. We're a little bit more focused on how we design ours. I think our approach, we really believe in all the features of the design of the molecule. You know, I think what the team, the ADC team have done has been really careful picking the antibodies. It's a different. It's a unique antibody. That already adds in a difference between any other molecule 'cause we really feel we picked. You know, we were very careful in our selection of that antibody and that it would support ADC. We think that, you know, we have the choice of payloads.
We are using the topo here because we think for that disease indication that where NaPi2b is expressed that works. We'll also, as Jamie and Stuart alluded to, dial in the DAR. Once we've made our molecule, we will benchmark it against the competitors just to give us, you know, confidence whether we see better activity in certain tumor models or whether we see, you know, comparable tumor activity in models. That is one driver for us. It's really that we're confident in how we've designed our molecule to meet what we think is the need for that design. Yes.
On 191, what kind of toxicity did you see at MTD in non-human primates? Can you compare and contrast against to what Mirv had shown? Is the profile at MTD the same as what Mirv shown at the respective MTD?
Yeah. No, that's good. I mean, I think we shared with you some of those findings. I think Stuart would be closer again to the details of the tox studies or Jamie. I'll let them take this one.
Yeah, sure. I think we're not gonna be disclosing, you know, all of the tox findings from that study. We are, you know, scheduling our GLP tox study. One thing we will say as another gentleman asked, the Mirvetuximab soravtansine has a very distinct toxicity profile in patients. It's mostly driven by ocular AEs, which you don't observe in non-human primate studies. Again, you know, our tox profile just based on the fact that we have a different payload with a different mechanism of action that has really no evidence of ocular tox, you know, that's really where the statement of, you know, a differentiated tox profile to mirvetuximab comes from. We won't really, you know that until we till we move in.
The other statement around, you know, the tox profile of ZW191 is consistent with the findings we had with the DXd payload conjugated to our antibody is true. We didn't see anything in terms of target organs of toxicity, magnitude of toxicity that was really different. That's really encouraging to us 'cause we think that, you know, the DXd platform in HER2 allows for these kind of like higher doses. We've got high DAR. They're seeing good efficacy. We could have that too with ZW.
Just one last one. Just on the overall strategy, your targets are already validated by some. How do you decide which one to prioritize? What is giving you the confidence to move ZW191 and ZW171 into IND first? I think you said 2024. What needs to be done between now and your IND?
Sure. Yeah. I mean, I think there are certain internal measures that we want to reach when we pick a molecule that we're gonna take into the clinic. You know, they're quite stringent. You know, on the ZW171, I can talk about the ZW171 first. I think you saw that we really took quite a bit of time to actually design that molecule because we really wanted to check the boxes of having, you know, a differential profile on normal versus, you know, or low versus high. That question of can you actually, you know. One of the challenges with T-cell engagers has been, you know, they're so potent that they'll actually even take out a cell that has a low level of expression.
That was really the thinking behind the 2+1 format there, is that could you get a day where you needed to have, you know, only when you've got a higher level of expression can you kinda get a gate where you would get to a certain level and that you would now get activity. That took some design. I mean, we are able to screen with the Azymetric platform, so we can do things that we feel others can't do. But that's, you know took time and care and design. That, and then on the mesothelin, that's a target that's a very attractive target.
It's got pan-tumor, it's highly expressed on other tumors, and that really fit the profile of what we wanted for a 2 + 1 engager, and it was in tumors that it could be tractable with that format. I think on the other side, as we're looking, we've got the 220 and the 251, and we've got the NaPi2b molecules that are both being developed in the preclinical setting. So far, the profiles of those molecules look very encouraging, and we're gonna continue developing them.
At some point, if they continue to check the boxes in our internal, you know, barriers of what we want, we see a molecule, we'll make a decision on whether one of those two should also go into the clinic.
Yeah, Akash, just a couple of follow-ups. It's really interesting, you presented this data at World ADC, and you kind of went through bin 1 and bin 2 of your HER2 toxins. You know, it was really interesting 'cause bin 2 was super efficacious across the board. When it actually came to some of the toxicities, that was also the one that was leading to the most changes in body weight. To me, the difference between bin 1 and bin 2 is really just the potency of the toxin. It was interesting. In your presentation, it seemed like the ones that were more potent was bin 1, but they were actually having less efficacy in the studies, which was kind of a little surprising to me.
I just wanted to confirm, was bin one versus bin two, was bin two the more potent toxin in those studies? Then just, as we go to your next-gen ones, can you confirm that at your go-forward dose, you don't have any significant changes in body weight in some of these, female mice and rat studies?
Yeah. Well, thanks for that question. That's great that you're digging into our presentations even not today. That's complementary. I would think Jamie would probably be the best person to answer that one or.
Sure. Thanks for the question, Akash. Although, I think I might need a little bit of clarification. You're right, you're correct in going back to those presentations from the past. We had the bin one and bin two molecules. There's a couple of things to consider. One is that we had a little bit of disconnect between the type of potency assay we did and the actual relative rank of potency, right? Stuart and I both sort of alluded to, but didn't get into too much detail around the development of the three-dimensional spheroid-based assays, which ultimately are what predicted or correlated really nicely with the in vivo outcomes. We had some reversal of potency with depending on the assay, but then ultimately these 3D assays correlated very well with the efficacy models that we ran.
That's one aspect. With respect to tolerability, the bin two molecules were less tolerated, you're correct. I think you asked us with our go-forward molecule whether or not we saw any substantial problems with tolerability. You know, the answer to that is no. We did not. It's not to say that we can feed animals infinite amounts of the molecule, but right.
Okay, thanks. That's very helpful. Maybe on just the bispecific engagers of the two here. You know, everyone kinda has a theory on CRS, and to me, the only thing that I've seen that at least shows signs of possibly being right is, you know, Roche dosing Kymriah and then putting their TCB platform. You're basically reducing the CD3 binding density. I think even IGM's, we can argue on the efficacy, but they're showing some signs of life there.
Mm-hmm.
By like physically preventing this CD3 binding density. And then of course, pulse dosing, right? So you guys going with a lower affinity binder, right? If you look at most of these bispecifics, there's really no dose response, and you seem to have. Right? So if you look at Regeneron CD20 CD3, when they were at 1 mg per kg or 40 mg per kg, they were having a pretty equivalent amount of CRS. So do you kind of ascribe to this like different lytic threshold where you can, you know, one lytic threshold where you can actually kill the cell and the other one where you're actually leading to it? And where do you come down on the CD3 binding density kinda hypothesis?
Okay. There's sort of two components, I think, of that question. One is, and I think this is kind of what I was trying to explain earlier, you know, on cytokine release, I guess, is what you're really getting at. I don't think it's always true that the toxicity is purely cytokine release, okay? I think there are other things you have to think about when you're thinking about T-cell engagers purely than just cytokine release, okay? That's one component we think about, but it's also the component of being able to differentiate. It targets tumor cells versus normal tissue, right?
That's where in the design of ZW171, where we've incorporated in the two bivalent strategy for targeting the tumor cell, so that only when you have a certain threshold, that binding is avidity-driven binding. It's not just necessarily one arm binding or the other. It requires both to bind. That will come when you've got a certain level of target antigen that can then support synapse formation or support binding. Okay? As Nina showed there in models that mimic the level of mesothelin in normal cells, we don't see killing. Then on cells that have higher, we see killing. Models as well have shown that T-cell engagers, unless they engage their target, they're not. You know, if your molecule designed properly, it would cause T-cell activation. This.
The molecule itself doesn't have intrinsic ability to trigger cytokine or T-cells unless it's co-engaged with its target.
Okay.
That tells you, like what you're saying about, you know, Roche's molecule where they had this high level of targets, would be cleared out first so that it didn't trigger the systemic release. That is something that's maybe more unique to that kind of target because it is more systemically expressed.
Mm-hmm.
If you're looking at a target that's more localized and so more in a tissue-restricted manner, there that challenge may not happen. There's certainly, I know from experience, that that can be the case, okay, for molecules that you develop. That then really depends on the profile of that target expression.
I think with mesothelin, we feel in that case, that target has a profile that we feel, you know, if you're careful then with the CD3 and the amount of cytokine you engage when you do have productive synapse formation, that if you can limit that amount of cytokine to the amount of killing, if you can switch the balance so that you can get more killing per cytokine release, that's where you wanna try and work in and see if that can then result in having a product profile that gives you the killing but limits the cytokine release.
Okay. That's really helpful. Just one last one, if I may. I know Regeneron's really excited about the CD28, but I think a lot of investor. It's interesting data in solid tumors, probably not that different from the PSMAxCD 3 data.
Mm-hmm.
that we've seen from Amgen.
Mm-hmm.
You know, what Regeneron's saying is, like, when we get responses and we get these weird side effects, it's a non-target effect, and the patients who are getting the responses are the ones that are also getting the side effects.
Mm-hmm.
It's kind of acceptable.
Mm-hmm.
When you think about your kind of conditional activating CD28 platform.
Mm-hmm.
Like, right, how do you find a therapeutic window on CD28 here? How do you have the confidence that as you're increasing target expression, you're not gonna have that same kind of uncontrolled immune response? You know, how do you-
Yeah.
You know, what's the therapeutic window with that product?
Yeah. I mean, I can take that one to begin with. I mean, I think it's a very good question.
Sure.
Yeah.
No, go ahead.
Yeah.
I can chime in too if you want.
Yeah.
You got it covered.
No, well, go ahead, Thomas, if you could.
Yeah. I think there are fundamental differences in mechanism, right? Regeneron makes a bispecific CD28 T-cell engager, which really relies on T-cells that are already pre-activated and they're there. So you're really engaging a very different type of T-cells. It's a hypothesis, but that's probably also why they're seeing those toxicities. What we're trying to do, we're providing a CD3 and a CD28, so you will really induce a very different response, and I don't think those toxicities will be applicable to what we're trying to do. Sorry, Paul.
Yeah, no, I think that's good. Yeah, no, I think you captured it.
I think that's actually for us exactly a reason for doing this because looking at those toxicities, you're right about kind of those seem to be significant toxicities.
This is Tom Cujino from Barclays. Just quick follow-up on the DAR. So, just wondering how well you can control the DAR, how narrow you can control with the kind of chemistry as compared to, if you may compare to the ZW49.
Yeah. You know, I think that we can do that. I think, Stuart and Jamie can probably answer that better than, you know, in more technical detail than I can.
Sure.
I mean, I can tackle that. Both ZW49 and our 191 molecule employ the cysteine conjugation interchain on the same conjugation. So we can tune that to various extents. I think with our topo platform, our anticipation is that typically we'll use DARs that range sort of between 4 and 8. But again, we tune this on the basis of the product, not some preexisting knowledge. Does that help answer your question?
Yeah. Yeah. That's very helpful.
I'll do a small plug for the site-specific conjugation technology.
Sure.
Cysteine insertions when paired with Azymetric, we can actually get homogeneous DARs of 1, 2, 3, 4, 5, 6, 7. Eight is easy 'cause we just use the interchain those whole ones.
Hi, this is James Shin from Wells Fargo again. I was looking at the T-cell tumor cell bridging assay, and I noticed slightly above 10 EC50 concentration, the trispecific and the bispecifics, the lines start to overlap. Is that sort of indicative of, like, the T-cell engagement being hindered. Is it like a bivalent structure where there's some sort of aberrant binding that just like it starts, like, working better? Like, what's going on there?
Yeah, I mean, I think, those hook effects, you can often see those, you know, with T-cell engagers, and it tends to be as if, you know, and I think in some of those models when you're getting to those artificially high concentrations in vitro, that they start saturating, and they do tend to bind to one, sort of more of an equilibrium of level of molecules that allow you to see the synapse formation. If you go too high, you start occupying too much in one cell and not the other, and it doesn't allow you to form that.
I think, you know, the fact that we've shifted the curve to the left there is really what we're looking to see, the strength of the synapse at a more kind of, what we'd think maybe is a more physiologic concentration.
Hi. On behalf of Josh Schimmer from Evercore. How do you discern whether your ADC is truly targeting the tumor as opposed to acting as a depot effect for the payload or chemo? Along that line, how does the answer to this question impact development strategy, if at all?
Yeah, I think that's an excellent question. I know, Jamie, you just published a paper sort of touching on that subject that maybe you wanna. It's also relevant to that question. I think at least from my perspective, that seems to be a component is how much of it is purely, you know, on target and how much of it is, you know, delivering chemotherapy or a payload to that area and, you know, having it, you know, in that patient who has cancer. I think we, you know, we will use the profile of what's the safety profile overall of that molecule and does it have anti-tumor activity in a range of models that we can best, you know, demonstrate.
We'll look at you know, look at those and really be driven by that. I think those other components are slightly more, you know, conceptual. It's harder to actually validate. But that's pretty much my personal opinion on that one. I don't know if Jamie or Stuart have something to elaborate on.
Yeah, I mean, I can comment on that briefly. I mean, I think I sort of alluded to this at one point, wherein I think any ADC has to have some level of targeting effect and some level of depot effect. Actually, the ability or the interplay of those two things really also depends heavily on the payload. In terms of how do we identify whether or not target engagement is playing a role in the ADC effect, I think that really speaks to whether or not you're seeing expression-dependent efficacy to some extent.
We really have focused a lot on our payload properties like I contributed to the sort of drug-like characteristics of the small molecule, because I think that for topoisomerase-based ADCs, that depot effect is gonna be an important thing. Whereas if we were looking at a much more cytotoxic, much more potent payload, it's unlikely that the tolerable dose might get to a level where we would see the effect of the small molecule to the same extent.
I think I'll just add, in our preclinical models where we assess antitumor activity, there's two ways in which we can start to understand kind of the you know a non-kind of directed effect. The first one is by using an isotype control conjugated to the payload we're using at the same DAR. I think in a number of the ZW251 programs, you can see the isotype control. Sometimes there's a bit of a response when compared to vehicle and sometimes not so much. Some of that is model dependent.
The other way we can look at it is by taking the actual molecule, whether it's ZW191, ZW251, or ZW220, and putting it into a model where you know, it's a cancer model grown in mice, but the target maybe isn't present or is present at extremely low levels. We have done that, and we don't see it. Like, the target needs to be there to see an effect. As Jamie mentioned, it really is difficult to quantify the contribution of a possible depot effect in some of these preclinical models.
Yeah. I can add to that in the sense that, you know, I think some people are actually led astray by focusing too much on those models because we're dealing with entirely different systems of distribution. Also, the interspecies difference between albumin, for example, is a big player in a lot of these molecules.
And perhaps-
I just want to add just to follow up on one of Paul's comments that just last week on the thirteenth Jamie and one of his colleagues in the ADC group had a paper published in Cancer Cell outlining some of the evolving understanding in the field of ADCs. We definitely point you at that peer-reviewed publication for more insight into that area.
Just one more question.
Sure.
With regard to 171, solid tumors such as pancreas tumors can be highly glycosylated, and this can impact antibody binding. Does the two-by-one help with this targeting?
I think, you know, you mentioned pancreatic cancer and, you know, a lot of people are. You know, that is a challenging tumor type, and that is actually in our lens of tumor types that we want to tackle. We hope that the design of the two-by-one will lead to, you know, responses in that patient population. We realize that just as we talked about, there may be certain tumor types or certain patients that will, you know, like you mentioned, glycosylation or challenges with specific tumors.
There, it may be that we have to design our molecule with a certain additional feature, and that's where we think with the tri-specifics, that's another area of opportunity for us that we can actually, you know, counter some of those resistant mechanisms, whether it is checkpoint inhibition or lack of T-cell fitness or some other structural component. You know, that's part of our thinking moving forward as to wire that into our molecules. But we do think that the two-by-one, the mesothelin target, we do have opportunity there. We think that that strong antitumor activity that we see in vivo models, we didn't run a pancreatic model, I don't think. But you know, we're really impressed with that antitumor activity. As well as a model can mimic antitumor activity, we're encouraged there. You know, we are optimistic.
Again, we'll have to wait and see what the data looks like. You know?
Right.
Yeah. I'll add that I mean, we have no data that suggests that there's a difference in binding or a negative consequence of binding to highly glycosylated tumor cell lines. You know, to further add, yes, we showed an ovarian cancer model, and ovarian is another indication that's heavily glycosylated. You know, we see strong antitumor activity in that indication.
I do have a question from the email. On slide 35, where you guys were talking about ZW191 and the folate program. Do you have any hypothesis why you saw better efficacy in mid versus high expressing folate?
Yeah. I mean, I think it again speaks to our care in the design of our molecule, where we really picked an antibody as well as the payload and the design features that really fulfilled what you want in a molecule to deliver a payload or a toxin. I think there, by having that real efficient internalizer along with the other features as in ZW191, really allows us to start hitting tumor types that you know may not be hit with other ADCs targeting folate receptor.
Following up, you know, on ZW191. You guys did present safety data with mirvetuximab. Do you have any comparisons with STRO? Or do you have any thoughts on how it may compare?
Yeah. I'll hand that one to Stuart and Jamie.
Yeah. I'll maybe just mention that the comparison to STRO is very difficult preclinically because it's very difficult and kind of bespoke technology to recreate that molecule. We don't have any direct comparisons. Where we think we do have potential differentiation is in the potential of a differentiated tox profile. The AEs for STRO seem to be driven quite substantially by neutropenia, which they saw preclinically, and they're seeing substantially in the clinic.
Yeah. Stuart and I have a fair amount of experience working with the hemiasterlin class because of the design of the hemiasterlin that part of our sort of portfolio of toxins. There have to be some similarities there in terms of platform. Yeah, it's very clear that the safety profile of STRO is differentiated from even other MTI-based ADCs.
Great. If there's no further questions, again, I'd like to thank everybody for joining us both online and here in person in New York. Again, we'll make ourselves available for a follow-up following the meeting. Please continue to reach out to our investor relations group. Everybody, have a great day. Thank you again.