Hello everyone. Welcome to Promega's webinar today titled Innovative ADCC and ADCP Technologies and Services for Cellular Therapies and Therapeutic Antibodies. Over the years Promega has been an innovative provider of developer of tools in the areas of monoclonal antibody therapeutics. We have developed Reporter Bioassays that have enabled customers to screen for potency of their therapeutic antibody molecule both in the development phase pre clinical all the way to QC lotteries. Today's webinar will focus on a newer technology that we have developed that enables mechanism of action based therapeutic. Sorry, screening of therapeutic antibodies both for monoclonal antibody therapeutics as well as for cell therapies. But before we get started, I would like to share some housekeeping information about this platform. Many of you might be familiar so you while you watch the webinar, you can also type in questions to the on the box to the right as it says ask a question. That's where we will either answer those questions via chat or if it's a more elaborate answer, the speakers will hold off until we get to the question and answer session right at the very end where we will have a more in depth discussion. There are also useful resources to the left, the left bottom of your screen you can see associated information, either website information that we have or other information that are hosted elsewhere. So you might find it useful, not necessarily right now, but whenever you have time. Also to remind you of this webinar will be recorded and will be available on demand. So in case you want to repeat something, go back and view information again, you're most welcome and we are here to answer your questions as we go along. So welcome again. Thank you for joining us. Next slide. So at this moment before we start the webinar, I would like to pose a question to all of you and this is a live polling question. The question is which drug development modalities do you specialize in and you see the options there. You can check all that apply starting with CAR-T chimeric antigen receptor therapy or TCR therapy. Or again you can check as many boxes as applicable to you CAR NK, monoclonal antibodies, antibody drug conjugate and if you have any others you can also mention those. So you can go ahead and start submitting your questions. Again, this is all confidential information that's only for our internal purposes. Your identity will will be safe with us, you know. We appreciate your feedback. All right. So the polls are in and we see, I see that about 72% of you are working with monoclonal antibody therapeutics and the emerging areas of cell therapy, the chimeric antigen receptor as well as the TCR therapy replacement of T-cell receptor engineered mediated cell therapy comes close. And then there are other modalities like CAR NK and ADC is also pretty high. I missed that. So that's the second. So the majority of you will find this webinar addressing all your needs. We're going to talk about the use of this hybrid target cell killing approach in the in the modalities that are listed here. So let's move on to the webinar. And before we start the webinar, I would like to introduce our presenters for today. Julia Gilden is a Senior Research Scientist at Promega and has been instrumental in developing a lot of the reporter bioassays that I mentioned for monoclonal Antibody therapeutics. Many of the reporter bioassays are addressing immune checkpoints targeting either Co stimulatory or Co inhibitive inhibitory, factors that as the name suggests either up regulate or down regulate T cell activation. But this technology that she's going to present today is about using a complementation approach wherein we have provide, we have different target cells that have what is known as HiBiT, part of the luciferase enzyme that she'll talk a little more about in a few slides from now and highlight its application either with primary cell killing or else with CAR-T. So that's Julia and she has an extensive immunology background coming from the UC University of California, San Francisco as well as University of Wisconsin, Madison that she did a postdoc. So she'll be able to answer many questions related to that, not necessarily right here, but even offline, if you want to e-mail her and ask questions, that's also welcome. The guest speaker for today is from Charles River Laboratories in Leiden in the Netherlands. His group has been extensively using these target cell killing in both ADCC antibody dependent cell cytotoxicity as well as cellular phagocytosis to characterize monoclonal antibodies. So he will be talking about those areas of application sharing some information that are very critical in enabling you to adopt this technology. So without further ado, we will move on to today's presentation starting with Julia. Thank you once again. OK. Thank you so much for that introduction. And thanks everybody for logging on today to hear about some of our new and a little less new ADCC and ADCP technologies that can be really useful for characterizing therapeutic antibodies. So here at Promega in the Bioassay Group, we now have a pretty long history of creating bioassays that can monitor the many different mechanisms of action or MOA of immuno-oncology type drugs. And this really started with our FC effector portfolio, especially our ADCC reporter bioassay that you're going to hear more about later. But this portfolio has expanded to include lots of different mechanisms of action including our immune checkpoint blockade bioassays, our cytokine bioassays as well as a number of bioassays that now are designed to monitor some more complex kinds of drugs including cell and gene therapies. But when you kind of step back and think about all these different kinds of immuno-oncology drugs, the thing that they have in common is that ultimately the mechanism of action is to lead to death of a tumor cell and particularly many kinds of anti cancer biologics induce cell mediated tumor cell killing. So regardless of whether you're thinking about a bispecific antibody, a plain old monoclonal antibody, maybe something like a till or a CAR-T cell, what all of these kinds of therapies are doing is ultimately lysing a target cancer cell. And for this reason, it's very important to have reliable assays that can specifically monitor target cell killing. I mean, it can be really challenging to do these kinds of specific cytotoxicity assays. Some of the challenges that are frequently encountered include reproducibility, especially if your assay relies on primary cells, specificity of the assay for target cells. So if you're looking at something like, you know, LDH release or an MTT assay, that might ultimately give you information about all cell death in a well and not just that of your target cancer cell. Many of these protocols are lengthy or complex. They might involve labeling of cells with a dye or a radioactive material. They can require specialized instrumentation, especially some of the imaging based methods or impedance based methods. And when relying on primary cells, sourcing of effector cells and and getting good quality that's going to work in your assay reliably is a huge challenge. So we thought that we could address many of these challenges using a technology called nano bit which is a split luciferase technology. And what we've done is we've taken the the NanoLuc luciferase type enzyme and we've engineered it into two components. So the little one here is just an 11 amino acid tag called HiBiT and it has a very high affinity like picomolar affinity for the other component of the enzyme which is LgBit. So these have such high affinity that when they're present together in solution, they spontaneously complement and they yield a bright luminescent enzyme in the presence of substrate. So HiBiT is very versatile. It can be fused to the N terminus or C terminus of a wide variety of proteins, and it can be used to monitor protein abundance as a very wide dynamic range. And we've developed a number of different detection formats that enable all kinds of assays. We have lytic assays, extracellular assays, blotting, a lot of different kinds of applications, and if you have ideas or want to hear more about some of these, we can definitely address that with you at another time. So the application of HiBiT for looking at Target cell killing is this. So we have engineered a panel of common tumor cell lines that express commonly targeted tumor cell antigens like CD19, CD 20, HER2 and we've engineered into them a HiBiT fusion protein. So when this is expressed inside of a live target cell, it is sequestered from the LgBit component of the enzyme that's present in the detection reagent along with the substrate. However, when this target cell is lysed by an effector cell, and the effector cell could really be anything that causes cell lysis. This leads to permeabilization of the plasma membrane of that target cell and it makes the HiBiT fusion protein then available to the LgBiT into the in the media, allowing for complementation and the production of light. And this light is in proportion to the number of target cells killed. So this gives us an assay with a number of really useful features. It's target specific. So because we've only expressed this reporter HiBiT fusion protein inside the target cells, we don't get any contribution to the signal from our effector cells. It's label free. You don't have to, you know, add a fluorescent dye or anything like that to your target cells prior to the assay. It's a very convenient add-mix-read format and it uses a standard plate reading luminometer that you probably already have somewhere available to you. So by taking these HiBiT target cells and again we have probably a dozen or more of them now that we've already engineered, we can kind of mix and match them with different primary effector cells, PBMCs, CDAT cells, CAR-T cells or macrophages to arrive at a really wide variety of different kinds of functional target cell killing assays. And so then you have these primary cell target cell killing assays that are very easily correlated with our surrogate reporter bioassays which rely on luminescence to read out activity of of drugs. So today I'm going to kind of focus in on two of these. The ADCC reporter assay are paired with the PBMC-ADCC assay and then our ADCP reporter and primary cell assays. So I'll talk about how these are are set up and and how they can be correlated with one another. OK. So I'm first going to focus in on the PBMC-ADCC bioassay. So this uses that assay concept that we talked about a few slides ago. ADCC is when a monoclonal antibody, usually an IgG 1, binds to a target cell with its variable region and an FC receptor on an NK cell with its constant region and this activates the FC receptor inducing the NK cell to secrete perforin nd granzymes. They then lysed that tumor cell. Our PBMC-ABCC bioassay uses primary PBMCs as the source of NK cells. Each lot of PBMCs is pre-qualified for use with HiBiT target cells. So this removes that step of having to, you know, maybe screen a lot of different PBMC donors or lots to find one that works well with your drug. Because we're using primary PBMCs in this mixed cell population, it's highly biologically relevant and we have a really homogeneous, simple workflow. So here's how that workflow works. You're going to start by just plating your HiBiT target cells. These are available in a thaw and use format, so there's no cell culture required. Then add the primary effector cells, the PBMCs as well as your test antibody. And then after a 4 to 6 hour incubation, you're going to add that HiBiT extracellular detection reagent and read the plates. So really straightforward to set up. And here's a little bit of what the data looks like. So on the left here we have our Ramos HiBiT target cells which express CD20 and we're using rituximab, an anti CD 20 monoclonal antibody. And this is the kind of response that we get using PBMCs in this assay. On ,the right, this is an adherent cell line SKBR 3, which is a breast cancer cell line and expresses HER2. So our test antibody here is trastuzumab. We can also miniaturize these assays and perform them in a 384 well format and we get very similar performance to what we see in the 96 well assay format. So on the left again both of these graphs are using our Ramos HiBit target cells with rituximab and primary PBMCs. So on the left is our 96 well plate and on the right is the 384 well plate and you can see a nice assay window with both and good agreement between the EC50s. So as good as that data looks, it is dependent on primary cells and so for most folks who are maybe looking at taking an assay into a a like a QC lot release kind of environment, they need something that's going to be more reproducible over time and that's where our ADCC reporter bioassay comes in. So this assay, if you look at the the diagram down here is based on an engineered effector cell that has an NFAT response element driving a Firefly luciferase signal and that's going to read out the activity of the FC gamma R3A receptor which is engineered into that effector cell. So when a monoclonal antibody binds to a target antigen on a target cell as well as that FC receptor, it'll cross link the FC receptor and you get light in proportion to the activity of the antibody. This is an example of some data over here on the right side of the screen where we're using WIL2-S cells as our target cell and rituximab is our test antibody. And what you can see from this is that we have to have all three components of the assay present in order to get a signal. So if we take out the target cells or we have a target cell that's not expressing the target antibody, if we take out the FC receptor on the surface of the effector cell or if we don't include effector cells at all and if we use an irrelevant antibody like trastuzumab instead of rituximab, we don't get a signal. But in the presence of all three of those elements, we get a very nice assay response. And this really tells us that the assay is reflecting the MOA of the drug. It's showing that all all the parts of that binding are intact. And so we can see then how this could be could be bridged to the PBMC target cell killing Bioassay to validate the reporter Bioassay for your quality program. So on the left here we have the the ADCC-PBMC Bioassay. So this is, you know, primary cells truly killing. Now it's a Raji HiBiT target cell and you can see the response there. And then in parallel we've run the ADCC reporter bioassay also using Raji target cells. And again you can see that we get good agreement between the EC50s, but you can see also that we are getting, you know, more precision from the ADCC reporter bioassay. So that just kind of gives you a little bit of a a feeling for how these might be differently applied. So now I'm going to talk a little about the primary macrophage ADCP Bioassay. So ADCP is antibody dependent cell cellular phagocytosis. So in this case, as opposed to ADCC, the effector cell is a macrophage and when the FC region of the antibody binds to an FC receptor user, usually FC gamma R2A on a macrophage instead of inducing granule secretion and lysis, this is going to lead to phagocytosis of the tumor cell and degradation of that cell in the lysosome. And traditionally ADCP is a really challenging cellular function to read out and and the assays are really difficult to run. So the the most common primary cell ADCP assays are based on either flow cytometry or confocal microscopy. And what these techniques have in common is that they require high skill, a lot of hands on time, they're low throughput and they rely on a lot of cell culture. Additionally, if you're using primary macrophages, you're going to have those challenges just like with PBMCs of variability and donor selection that can really slow down a research program. So the way that we've addressed this using HiBiT and the target cell killing platform is a little bit different from the PBMC assay. So we're again using our HiBiT target cells that engineered to express this HiBiT fusion protein. And when you co-culture those with a macrophage in the absence of a monoclonal antibody, there's not a lot of spontaneous phagosytosis of these cells. And so if you come in with a lytic detection reagent that is going to release all this HiBiT then into the supernatant, you get enzyme complementation and light. But in the presence of a monoclonal antibody, this is going to induce phagocytosis or ADCP of the target cell by the primary macrophage. That target cell and the HiBiT inside of it is going to be degraded in the lysosome of that macrophage. And then with the addition of the lytic detection reagent, there will be less HiBiT target cell in the in the what pardon me, less HiBiT in the well and therefore less complementation and less light. So this is a loss of signal assay. The workflow is very straightforward. On the first day we plate primary macrophages, the next day come in with target cells and specific antibody. And then after 24 hours of incubation, we add our lytic detection reagent and read luminescence. And this is a little of what this data looks like. So on the left, you're seeing raw RLUs and on the right, you're seeing percent ADCP. So this is kind of the amount of specific phagocytosis that we're seeing in specific degradation of HiBiT. On the top, I'm showing you our Ramos HiBiT target cells with rituximab. So this is a suspension cell. And on the bottom, these are our SKBR 3 HiBiT target cells with trastuzumab so adherent cell lines. So we can we can use either in this assay. And you can see that we get lots of nice specific ADCP in both target cell systems. Now making this easy and and making the workflow really work depends on the source of the macrophages used in the assay and that's why we have created thaw and use primary macrophages. So these are derived from primary human monocytes and we've grown them and prior preserved them in a way that optimizes their ADCP activity with our HiBiT target cells. We are making large batch sizes, so you can expect consistent performance from these primary macrophages and they have very high viability and a flexible phenotype after thaw. So they have sort of an M0 phenotype and we do recommend maturing them with interferon gamma overnight before your assay. But we leave them as M0 to leave maximum flexibility in the assay for the end user. So the availability of these thaw and use primary macrophages really removes the need to do lengthy cell culture or derivation of your own macrophages prior to running a primary cell ADCP bioassay. Because we're making these big batches, we get really nice reproducibility between vials. So here's an experiment with Ramos HiBiT target cells and our thaw and use primary macrophages and we've just run 3 vials in parallel and you can see that both in terms of RLU and the specific ADCP activity, we get very nice reproducibility from vial to vial. Now in spite of this, when you're looking for an assay to use for something like QC or lot release, it's usually preferable to move away from primary cells. And so that's where we're going to recommend our ADCP reporter bioassay in the THP-1 background. So here we have effector cells that are derived from THP-1. It's a monocyte like cell line, so it's a truly phagocytic background and it has endogenous expression of FCgamma, R1 and R2. It expresses a NanoLuc promoter that responds to ligation of the FCgamma R2A and so when a test antibody is combined with target cells expressing an antigen of interest and the THP-1 effector cells, it results in expression of NanoLuc and light in proportion to the activity of that antibody. This assay has very nice performance characteristics. This is a mock potency series from 50% to 150% where we've done a qualification of the assay. So this was performed by two analysts doing 3 independent experiments each, and you can see that we get very nice recovery of those mock potency samples. The repeatability of that 100% reference control is excellent, as well as the linearity of the assay. This assay is also stability indicating. So here we've used heat treatment of Rituximab to create forced degradation samples. And as we extend the time of that heat treatment from one to three days, you can see the rightward shift of the EC50 in this assay. So that indicates that this bioassay is stability indicating. All right. So to summarize, I have told you about assays for ADCC and ADCP. For ADCC we have the PBMC assay which is our most biologically relevant bioassay for ADCC. It uses pre qualified donors with HiBiT target cells and it's got a really simple workflow. It's homogeneous but for more of like a QC lot release type application we're going to recommend the reporter or assay in the jerk app background which reflects the mechanism of action. But it uses engineered cell lines for maximum reproducibility and it is widely used throughout the industry. In for QCM lot release for ADCP, I told you about our macrophage assay. It uses saw and used primary macrophages to remove the need for lengthy cell culture. They're optimized for use with our high bit target cells. It's an easy no label workflow and a homogeneous plate based readout and then the reporter assay correlate of that is our THP 1 ADCP reporter bioassay. It has the benefit of endogenous FC receptor expression. It reflects the mechanism of action of monoclonal antibodies and its accurate, precise, linear and stability indicating. So with that, I want to take a minute to thank the many contributors to this work both in R&D and on the commercial side. And I want to thank you all for your attention. I'm excited to hear your questions. I think we'll take those at the end, but you can put them in the chat anytime and I will hand things over to David. Hello everybody and thank you very much for for the introduction. My name is David and today I would like to present you the F simulated antibody functions assessment for early discovery programs that we perform here, Charles River. Well, first of all, I would like to 1st use a little bit what we do at Charles River as the audience might not be familiar with all of our offerings and we are very well known for late stage development and safety assessment of therapeutic agents. But we also have a very strong unit within the early development and discovery of new drug products. I want to bring forward the value that we add to these, these recovery programs and especially within the antibody field. First of all, here you got some some data of what we've been doing in the last year. So you can see that since 2017, we've completed over 400 antibody discovery programs out of which we've generated 8 clinical and clinical candidates that have been later on further developed into clinical programs. And we come with more than 40 years of antibody discovery experience. And wrapping it all up, the day home message that I want to deliver to you to the audience also in terms of how Charles River can accelerate your antibody discovery programs is that by having us as partners, you have access to a single point of contact and scientific team that will guide you and ease the communications and the workflows within the discovery programs. So within the early discovery of the antibody programs, we've generated an antibody discovery pipeline in which we can take even rough idea of a target of interest that you might want to further investigate and generate a complete library of antibodies that can be pre tested characterize and functional assets also test for off target binders and ultimately performing the efficacy and safety studies in order to get to the best candidate for your for your target. And all of that within about a year and we can we can deliver this because we we have optimized all the communications and processes that are involved in such complex endeavor to to get it to the most optimal point. So when it comes to functionality and mechanism of action assessment, that's why I specialize mostly and that's why we'll be covering in in the presentation. We we want to always focus in in assets that can bring the most value to to our clients in the least amount of time. So several of these studies involve assessing the mechanism of action of these antibodies. Unlike all the therapeutic agents, antibodies can exert the therapeutic function through several different mechanisms of action and these are all important to take into account when considering whether an antibody is safe to use and also assessing the potency of these antibodies. So the monoclonal antibodies has have been a booming market in the last years and the main, the main focus has been treating different cancers. And for those treatments, well it's been described lately is that they don't exert the anti anti tumor effects only by 1 mechanism of action but through several that can work at the same time and also synergistically. So what you can see represented on the left is all the different ways therapeutic antibody cancer cell line can exert its function. So some of these mechanisms of action work directly through the antigen binding domain. For example, if you are blocking a receptor that is supposed to be important when a ligand is bind to it in the cell proliferation or survival, that's going to have a direct effect on the tumor growth. Some of the alternatives are for example the antibody drug conjugates which by which cytotoxic agent is conjugated to A to a monoclonal antibody that is going to bind to a specific target that is expressed in the cancer. However, there are several others mechanisms of action that work through the FC region of the antibodies. So they are target agnostic and they exert the function by interaction with the patient's innate immune system. So several of these are for example the antibody dependence cellular toxicity, the antibody dependence cellular phicocytosis or the complement dependence cytotoxicity. So for for the purpose of today's presentation, I will be focusing on the antibody dependent cellular phicocytosis or ADCP as I will refer to it from now on. And this is a mechanism by which an antibody of opsonized cell gets file size by a macrophage and it will induces degradation through cell filosome acidification. But it's important is because the ADCP has been described as one of the major mechanisms of action of several biologics that are currently in the market. So this interaction of the antibodies with the patients in the immune system work by binding these FC gamma receptors protein family. As you can see represented in this image, there was there are several proteins that are expressed differentially in the different cell populations that composed the immune system. For the ADCP in particular, the FC gamma receptor 2A is the one that's going to have a bigger importance and it does express, as you can see in macrophages, mono size you know profiles. So depending on the location of these receptors, the signaling can induce different activities and as you can see the same receptor can induce ADCC but also ADCP and it will depend mostly on which cell type is being is expressed in this receptor and it's binding the target cells. So as you can imagine to assess this mechanisms, this mechanisms of action in vitro, there are there are several procedures to follow. But traditionally what is been more common is to use monocyte Dr. macrophages to mimic that primary macrophage phagocytosis that we would observe in Bebo. Now these primary monocytes are can be obtained for by isolating them from peripheral blood or blood drive products like particles and then by means of several different cytokine cocktails can be differentiated into macrophages. Those can be then culture with the target cells and the antibodies of interest that we want to assess the ADCP for. And using flow cytometry we can determine whether the internalization of the target cells has happened either through our localization of these two cell types or by using pH sensitive diets that would only be fluorescence once the the lysosom has been added acidified. However, this approach has several limitations. For example, and this is something that you will be very familiar with if you work with primary cells, that's always an intrinsic donor to donor variability that we cannot really control. And this can affect either the shield of your primary monocytes, for example, but also the ability to differentiate into macrophages or the capacity to follow site the target cells. It's also a limited number of microphages that you can obtain from a single blood source and these workflows are quite lengthy and labor intensive and I will focus on that a bit more later on. In contrast, from Mega has developed several bio assays as you as already explained, in order to make these assessment much more straightforward. So today I'm going to talk about one of these products, it's called FC Gamma Receptor 2A, HADCP Bioassay And the idea is to substitute those primary microphages with Jorga cells that stably expressed the human FC Gamma Receptor 2A with the variant that has higher affinity and that is coupled with an end fighting use of luciferase expression. So the idea of the assay is to have a Co cultural establish between your target cells and these Urica cells in which the antibody would then be added and would be able to recognize the target of interest in your cells and would also bind the Urica cells through the FC region. That would trigger the signaling pathway and in the use of luciferase expression that can later on be easily measured by adding a substrate and measuring the. The enzyme activity. So here I have a comparison between the two different methods. On the top you would find the workflow of what an ADCP assessment wouldn't. Title if you would like. If you would do it through the conventional approach, you first will need to perform your C 14. Positive cell isolation will take approximately a day with several steps and then those cells will need to be differentiated into macrophages for about seven days before they are suitable to be used in the ADCP assessment. Of course, first you will also need to characterize those microphages to make sure that the differentiation process went according to plan and that you have functional cells. After that you can form the assays to establish the culture as your antibody and perform your readout as you can see off mark with some stars. Several critical points in this process where you are going to have unpredictable variability. So as I mentioned before, during your CD 14 positive cells isolation, you might have different contaminants from samples to samples, you may have different chills, you might have different number of cells. All those things are going to affect your your end readout. Similarly for your macrophage differentiation, yeah you can have donor to donor viability there you're going to have cells are not going to differentiate that well and might end up not having enough for your for your asking. And lastly, as you can see in the readout, so if you are going to use flow cytometry for your readout, you also need specialized equipment, specialized specialized state analysis and several times to to to get to your final results. And also for with the flow cytometry readouts, you really need to have proper controls in order to control for the viability between runs that you might have. In contrast, if we were going to go with the Promega FC gamma receptor to a kit, you can see that those timelines are massively reduced. So we don't need to perform any sort of isolation and we can start strict with the Co culture establishment and the readout is can be easily done in a luminescence reader. So we tested this this kit from Promethea. We perform a proof of concept study in which we wanted to to demonstrate that two antibodies have been reported in the literature to induce ADCP with the conventional methodology would result in the same effect when using the the FC gamma receptor to a ADCP bioassay. So we use CO3 cells that are two positive and variety cells which are C20 positive and we we cultured them to obtain enough number of cells. And once that was ready, we for the Cryopreserve SC gamma receptor AH131 Jurga cells that were provided by pro mega and established a good culture with a 5050 ratio of the different culture media for the different cell types. And to that we added concentration curve for trastuzumab which is an Antihertu monoclonal antibody currently in the clinic and Rituximax similarly an anti CD 20 antibody also in the clinic. As for controls, we included matching isotype control to see whether having a specific antibody in the system would induce any sort of response by the binding of the FC region to the Jirga cells. Upon establishment of the culture, we went for a time point of 16 hours and down below you can see the acid timeline with the differences between them, whether you need to work with some material cells or you have suspension cells or Stark cells, but you can see that everything was completed in just two days. These are the results for that study. In the two crafts you can see the luminescence that was measured for the different cultures. And what we observed was that both to sum up and reduximab induced an increase in the luminescence measure for the antibody treatments compared to the Izotype control. And we also observed that we could get a clear dose response curve from which we can go later on in FEAR and EC50 and also compare those if you were doing a comparison study between different antibodies. So what's what we draw from this data is that we really need to have an engagement of the target of the antibody in order for it to trigger a signal in the Urca cells to disperse the luciferase. It's not enough to have an antibody in the system might join the FC region of the of the the FC receptor of the yoga cells because that will not induce any increase in luminescence. All right. So now I will talk about how we have implemented this, this product in some of the studies that we we do here at Charles Ribbon and one example for that will be the safety evaluation study for a monoclonal antibody that is are targeted for anti immune diseases. We've talked a lot about how these antibodies are being developed and used for the treatment of cancer. But there are other therapies in which we wouldn't like to have these ADCP effect on. For example, if you are treating arthrosis or arthritis, you don't want to have an activation of your immune system and you don't want to have your macrophages attacking the the, the patient's cells. So we put a clear focus on safety on the studies that we perform and this would be one example for him. So yeah, I drafted what roughly that kind of study would entitle. So there you can see some assumptions for the study. So we will be testing 40 different test articles and having just two controls or one eye step control and one reference antibody that we would know that we would induce ATCP. Of course because of that donor to donor viability that I've talked about a lot, we tried to contract that by having several donors so that we can get an average measurement for that. So the minimum that we would recommend will be these three different PB and CS, but ideally you would go for at least five. And then we also have the caveat that the maximum capacity of our 4 centimeter readout will be of 696 volts per day and also something that is not mentioning there but we assume that people are not working on on weekends. So what you can see down below will be the the timelines for such a project. Following the traditional approach, we need to perform several brands of macrophage differentiation for the three different donors. We would need to stagger those because we will not be able to measure all the plates just at once. So this will be assuming that you you will be able to recall on us and have blood drawn from same donors in consecutive weeks. As you can see that this would result in approximately 17 days until we collect all the data. Of course there will need to be done some data analysis and reporting to be done, but you get the idea. What would happen then if we would use the Promega FC gamma receptor to a kit? Well, those timelines will be massively decrease plus what you can see coming there. We could be able to test all these 40 antibodies plus the controls in just one goal. So we could perform that in two days providing of course that we've got target cells over in culture and in enough quantity. And also as I mentioned before, the data analysis and reporting will be much more straightforward. So another instance in which we can include this, this assay will be in the functional testing of of target binders. So we are charged, we will have great focus and safety and we do a lot of safety testing even in vitro before having a final candidate. So we can assess whether maternal antibody or for that matter other therapies have certain off target binding that might not be identified early on and that could cause potentially side effects or yeah unsuccessful clinical trials. So we can do that by using the Retrogenesis screening platform. This is proprietary technology in which we can assess the binding of test articles to over 6000 human proteins that are expressed in human cells, so making it a very physiological environment to get a real measurement. So in this example for example, we we tested on EGFR targeting IgG antibody and what we observed was that there was no off target bindings. But what would happen if there was some protein that will be bound to your antibody and how to move forward? Well here's what from Mega becomes really useful. We can perform all these assessment using some of the bio acids that Promega has available in a very quick manner. So we can assess whether that's off target binding might elicit A complement dependent cytotoxicity response or an antibody dependence or cytotoxicity or as well an antibody dependent cell or ficocytosis. And as I mentioned all this will be completed very shortly. So to conclude, yes, I would like to draw your attention to these statements. First of all we run a proof of concept of study and validate the ability of the system to measure the FC gamma receptor engagement by two different antibodies when incubated with respective target cell lines and contrast no signal. Was it that when a non targeting antibody was tested in the system confirming that the need of a stable FC mediated cell to cell binding to trigger the report cell line to induce a luciferase expression. So with this in mind, we can conclude that the FC gamma receptor to ADCP bioacid, it can be used as an alternative in vitro method for the assessment of the ADCP. And here I listed the advantages that we found. So it has a much higher throughput compared to the traditional method of ADCP assessment and it's amenable for miniaturization and automation. So that would increase a lot of the efficiency in these antibody discovery programs. By using this kit, there's no need to solve blood products. It eliminates the donor to donor variability in the assay and there's no need for a specialized equipment and data analysis software. And what is maybe more important is that it reduces the timelines and regions cost. I would like and with that I would like to finish my presentation and thank you all for listening to us. And now we will be opening the floor for any questions that you may have. Thank you, David. That was an excellent presentation on using Promega Bioassays in in advancement of and streamlining the discovery and manufacturing of novel drugs. So as you said, we'll open the floor to questions, but there have been many questions that have come from a very engaged audience on the chat in the chat box. I will start reading some of them and you can start both Julia and David can start responding to some of these. So there was a question about Julia. I guess this is for you. The effector cell to target cell ratio in the ADCC assays. Let's start with the ADCC assays if you're available. Julia Sure. Yeah. So for the primary cell assay we recommend a effector to target ratio of 25 to one but we find that that is a pretty robust factor. So so other ratios definitely can work and give good assay windows. With the reporter assay the the recommendation is to start with six to one, but depending on the target cell being used and the antibody being used the the density of the antigen on the surface, anything between 2 1/2 to one and 25 to one could be optimal. Great. And switching over to the hybrid cells, the question is, is hybrid expression always constant in target cells? Can this be regulated by activation, stress or? Anything so. So our best understanding is that the expression is very consistent and we've you know chosen promoters to do our best to ensure that always is a tricky question in biology of course. So I but in our experience we have not encountered conditions where expression of hybrid changes with anything about culture conditions or or cell stress or anything like that. Along the same lines, the question is about the stability of the hybrid protein in serum or culture environment. Could you comment on that? Yeah, we know, we know it's very stable for at least 72 hours in culture and and likely beyond that we don't see any any decline in signal for for at least 72 hours. OK. Again, sticking to hybrid cells, there's a concern on on safety when cultured. The question mainly is about, yeah, safety. Yeah. So there, these are common human cell lines. So you know, of course, we would always recommend that you defer to your institutional biosafety group to make sure that you're handling things the way that they want. But I can tell you they're common human cell lines and they're made without any viral vectors. There's no antivirus involved. All the engineering is done either by plasmid transfection or by CRISPR. So hopefully that can give you some idea of how to handle them. The question is about. Another question is about the hybrid target cells provided in a while if they can be propagated so. Yeah. So we offer them in two formats. There's the thaw and use format where they're for use in a single assay. So you just thaw them out of the freezer and there's no culture or expansion required or allowed for those cells. But they're also offered as a cell propagation model where you know you would you would then own the cells and be able to expand them and and bank them as you like. And and both work very equivalently in the assays. Thank you. And there are a few questions all related to BBMC donors and how there is variability, how they are pre qualified and all that. Could you shed some light on our process? Sure. Yeah. So when we pre. Qualify PBMC donors. What that means is that we have taken those donors and in fact every individual batch from those donors and tested them against all of our high bit target cell lines. And that ensures that when when you order PBMCS along with a high bit target cell line from us, you can be sure that that those PBMCS actually have activity against that cell line when talking to customers. And and in our own testing one of the things that we encounter is that there's huge variability in the ABCC activity of PBMCS. And so we've tried to kind of take that piece of variability out of the equation for folks by by doing this pre qualification. So we're able to maintain, you know, some donor diversity. We we always have a, you know, half dozen or so donors in that a person could test if they wanted to. But but we have a lot of confidence that that each of those donors does have good ABCC activity if it's used with our hybrid cell. Lines. I'll let Dave, David take this other question. There is a question on whether there are special considerations while using the ADCC assay, the Hybrid assay, if there are special culture medias to be used. David, would you like to comment on your protocol? Yeah. So in my experience, it's always good to do a test on which culture media you want to use when you are establishing aquaculture. You may have very sensitive cells are very delicate and might respond not so well to a changing medium to accommodate for the effective cells that you're going to use. So what we typically do is test several ratios of the different mediums or the medium from the target cells and the medium for your effector cells and see whether that has any effect on the functionality of the cells first, but also in the in the viability of both cell types. So yeah, it would be really dependent on the cells that you're using. The most common satellites don't really have such high demands in terms of media compensation and can do pretty well for short terms in in media that is not optimal for them. Thank you, Julia. The next one is for you. The question is about labels to use that you use while developing the hybrid tag. What kind of fusion proteins that you use while developing the hybrid target cells? Yeah. So we've tested a number of different things and we've we've selected tags based on having low spontaneous release and on having good stability in in the culture media. And so all of our cell lines are built with either a Halo tag fusion or an LDH fusion. Thanks. Sticking with the hybrid target cells, there is a question on how the user can develop their own hybrid target cells. The DIY option that. We provide, yeah, so, so we can advise you on that with either using CRISPR to knock hybrid into the LDH. Locus. Or with using lentivirus to generate them yourself in house. We also are happy to make you a hybrid target cell line through our tailored R&D solutions group where you know we do a lot of custom cell line development. So if if you have a target that you're interested in, definitely get in touch and we can talk about whether we may be have something in stock already that that might work for you or whether a custom approach or DIY approach would be best. Julia, another question is about the PBMCS that we offer, the range of choice that the the the user potentially has between the different phenotypes. Yeah. So. Yeah. So we we have donors with all of the CD16 genotypes that F, the V and the FV heterozygote. We we may have fewer donors total than some vendors that you have experience with. But the reason for that is that we have pre qualified these. So you know if you are just you know getting random leukopacks or or something like that in order to to obtain PPBMCS you're going to have to test a lot to make sure that you're actually getting good ABCC activity. Whereas we've pre selected those for you, pre tested those for you. So we always have, you know, a number of donors in stock and and we find that customers often like to try a few and and choose their favorite to go forward with or maybe have a couple that they test or regularly use it in parallel. But yeah, does that answer the question about diversity? Yeah, I think so. The next question is for David. Does Charles River offer similar services for other modalities like cell therapies? And how do these, the products from Promega help in their development? Could you comment on that, David? Yes, of course. So in particular for cell therapies, we have a very similar approach. We intend to be a single point of conduct in which we can develop the whole therapy. So we can take an idea of what you want to develop a cell therapy for perform all the perform all the development on that regards. In addition to that we also have even a GMP qualified manufacturing facility in the United States for the production of EMP batches of these S Rapids. So with that regards, these from mega products help us not only in the early development phase whether it is with the potency testing, but we can also use these hybrid cell cytotoxicity assays in order to to release these GMP manufactured batches. Thank you, David and thank you Julia. I think we'll stop here. We are already above over the time limit, but again let's have the conversation going on. I would encourage all the attendees again. Once again, thank you for the engagement and for tuning in, but we will keep the conversation going. You can contact, we will contact you for any further you know questions or you can you feel free to reach out to your local Promiga reps and they will forward those questions as well. So we'll provide more information on that. You see the, yeah, the tech serve and the Charles River website, UH e-mail IDs can be reached for further clarification, further information. So thank you once again for joining us. Bye, bye. _1732220647968

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