Welcome and thank you for attending today's webinar Defining Car T Self Potency Advances in Functional Analysis. My name is Kim Haupt and I'm a product manager at Promega. I will be the moderator for today's presentation. Before we get started, I have a few housekeeping items to review. First, on the media player there is ACC option for closed captioning with many different language options. Second, you will see widgets or windows on your screen which can be rearranged as you desire. You can access all the widgets using the yellow buttons along the bottom of the screen. I want to specifically highlight the resource widget where you can download copies of the slides as well as find relevant application notes, brochures, and web links. Next, there's a survey widget labeled Give Feedback. If you could take a few minutes to complete the survey, we would really value your feedback. Next, you can provide live reactions to the speakers using the Give a Reaction widget. Finally, there is AQ and a widget. You are welcome to submit your questions at any time during our broadcast using the Q&A widget. There will be a live question and answer session with our presenters following their talks. If you submitted a question and it is not addressed during the broadcast, we will follow up with you by e-mail afterwards. Before we get started with the webinar, we have 2 short poll questions for the audience. I'll share the answers afterward. So first question, what methods do you currently use to analyze car T potency? If you could select all that apply that would be great. The options are flow cytometry, cytokine release assays, cell viability assays, impedance based assays, reporter bio assays, or none of these or other. So I'll give a few minutes for folks, few seconds for folks to answer. It'll be really interesting to see what people are doing now as we're gathered here to learn about some different options for car T functional analysis. All right. I'm going to go ahead and move to the results in about 10 seconds. So last chance to submit your answer to the question. OK, So it looks like most people are doing flow cytometry and cell viability assays with cytokine release assays being the third most popular. Thank you so much everybody who answered. All right. And then the second question we have is what do you consider the most critical time points for monitoring car T potency? So choice A less than 24 hours, B 24 hours, C 48 hours or D other. So again, I'll give a couple of couple of seconds for folks to answer which time points are the most important for your analysis of CAR T cells. Thanks to everybody who's responding. All right, about 10 seconds and I'll move on to the answers to share what what everyone has said. All right. So those earlier time points are the most important, that 24 hour time point, most people, the most people are looking at that with less than 24 hours being the second most popular. So all right, thank you everybody for participating in the polling. We really appreciate it and it helps us to understand what your current workflows look like and develop solutions that fit into those. All right. Next, I will be introducing our presenters. We have two presenters today. Our first presenter is Doctor Danielle Califano from Axion Biosciences. Dr. Califano received her PhD from Albany Medical College where she studied T cell migration and autoimmunity. With more than 15 years of experience in immunology research, Doctor Califano is an expert in the use of impotence based assays and flow cytometry, including potency analysis for therapeutic discovery and development. Today, Doctor Califano will be discussing Axion's impotence platform and applications in immuno oncology. Our second presenter today is Doctor Nicholas Hess from Permega. Doctor Hess has a PhD in immunology from the University of Illinois at Urbana Champaign and post doctoral experience from the University of Wisconsin, Madison. He has more than a decade of research experience, expertise in TLR biology, T cell biology and cellular therapy development. Now at Promega Doctor Hess is a product manager working in the targeted cell therapy portfolio. And now we will move ahead to the webinar. Thank you, Kim for the introduction. I'm thrilled you could join us for today's webinar. My name is Danielle Califano, and I am the Business Development Manager at Axion Biosystems. In today's presentation, I will discuss the latest methods for functional analysis of CAR T cells and how these techniques can be combined to accurately characterize CAR T responses. Our agenda for today covers some key aspects for evaluating CAR T cell therapies. First, we'll discuss the importance of in vitro potency testing and product characterization during cell therapy development. We will then explore multiple assays that can be used to monitor potency, including the Maestro Z Cytotoxicity assay, which offers real time label free monitoring of CAR T cell mediated killing, and the Promega Lumit cytokine release assay, a sensitive method for quantifying cytokine production as an indicator of immune activation. Finally, we'll cover a Multiplex approach to measuring CAR T potency, highlighting how combining multiple assays provides a comprehensive understanding of the therapy's effectiveness and mechanisms of action. Immunotherapy has shown great promise for treating cancers and in recent years has drastically changed how we intervene in disease. Cancer immunotherapy techniques such as targeted cell therapies, checkpoint inhibitors and therapeutic vaccines have dramatically improved patient outcomes above conventional therapies. The impact of this can be seen in some key examples. Keytruda, an anti PD1 monoclonal antibody or checkpoint inhibitor therapy, was first approved in 2014 and was used to treat former President Jimmy Carter for Melanoma that had metastasized. Chimera is the first FDA approved cell therapy used to treat pediatric ALL. Emily Whitehead was the first patient to receive this therapy back in 2012 and remains cancer free to this day. As these therapies continue to evolve, there's also need for improved analytical tools and more predictive in vitro assays to measure product efficacy and safety. Cell therapy development, particularly for CAR T cell therapy, involves a multi step process that is closely monitored to assess the treatment's efficacy and manage any potential side effects. This includes measuring the viability and potency of patient cells following collection, monitoring CAR expression and cell performance following transduction, testing modified cells following expansion, and performing quality control testing to ensure the release of a safe and potent cell therapy product. Reliable in vitro potency assays are required to ensure product efficacy at different stages of development. Various assays can be used to evaluate the potency of CAR T cells in cancer therapy. Cytotoxicity assays measure the ability of CAR T cells to kill target cancer cells, providing a direct indication of their therapeutic potential. Cytokine release assays are conducted to measure the production of cytokines such as interference gamma and TNF alpha by CAR T cells upon activation. Reporter assays involve the use of a reported gene that produces a measurable signal such as luminescence or fluorescence. Employing a multi assay approach or assay matrix allows for a comprehensive evaluation by a combining complementary assays tailored to specific product attributes. This approach ensures a thorough understanding of the CAR T cells function and strength. Today I will be discussing both the Meister Z cytotoxicity assay and Permega's Lumet cytokine release assays. Later on Nick will provide information on Permega's target cell killing reporter assays. The Meister Z cytotoxicity assay allows for non invasive continuous monitoring of immunotherapy potency. Axion's Meister Z live cell analysis platforms use impedance biosensor technology to capture your entire experiment, allowing you to analyze the magnitude and kinetics cell mediated killing and its persistence over time. The Meister Z range includes 2 bench top instruments, the Meister Z and ZHT with built in environmental control as well as a multi plate Maestro tray system which goes inside the incubator and accommodates up to 8 plates. All of these systems have built in features necessary for 21 CFR Part 11 compliance. The Meister Z products are based on an impedance measurement using specialized Multi Well Cyto View Z plates with sensitive electrodes embedded in the cell culture substrate. To measure impedance, very small electrical signals are passed between electrodes. Before cells are added, the electrical current easily passes and the impedance is low. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance. Disturbances to cell health, such as adding a drug or therapy, will cause cells to die and detach, resulting in a decrease in the impedance measurement. The impedance assay has proven to be a suitable assay to study cell responses in vitro. This includes proliferation by ability, cytotoxicity, strength of cell to cell coupling, cell signaling, and more. They are used in a number of application areas including immuno, oncology among others. The Meister Z cytotoxicity assay offers crucial advantages that significantly enhance the evaluation of cell based therapies. Continuous monitoring of cell killing captures all stages of an experiment before and after treatment to observe both cell growth and death of target cells. The ability to generate minute by minute data also makes the Meister Z well suited to study both acute and chronic treatment conditions. Such comprehensive monitoring ensures that researchers gain a full understanding of cellular responses throughout the entire experiment. One of the standout features of the Meister Z assay is the ability to facilitate hands free data collection. Researchers simply plate the cells, add treatments and the system automatically collects the data, thus enhancing efficiency and minimizing the risk of human error. The user friendly software provides a straightforward approach to set up execution and analysis of the impedance experiments. With the push of a button, the Maestro system will move the plate into the recording chamber, at which point the integrated barcode scanner scans the plate's unique barcode. Data is automatically collected and linked to the right experiment based on this barcode as soon as the plate docks without any complicated instrument setup. Once the experiment begins, impedance data are continually displayed in real time. With just a click, you can plot the percent cytolysis curves, compute kill time 50 values, and explore all figures and data tables to excel. Furthermore, the Meister Z assay is labeled free, which means that measurements do not impact the biology of the cells. There is no need for optimization of labels, dyes, or incubation times, thus eliminating potential variability and background noise from effector cells. This leads to clearer and more accurate results. The assay also supports multiplexing with endpoint analysis, allowing for the evaluation of multiple parameters with a single plate. Here's an example from a recent publication where researchers from the University of Georgia and other institutions study two different CAR T cell lines and their ability to kill glioblastoma using the Meister Z. In the study, the authors compared retrovirally transduced GG2 targeted CAR T cells to CRISPR edited or virus free CAR T cells. In all conditions, the cell products were cold cultured with glio glioma stem cells and impedance was measured continuously for seven days. At the seven day endpoint, the retroviral and CRISPR edited CAR T cells so similar cytotoxicity. However, when you look at the time course of the killing in the continuous plots as well as the kinetic endpoints such as kill time 50 or the time required to reach 50% cytolysis, it becomes clear that the virus free CAR T cells killed the glioma cells much faster than the retroviral CAR T cells did. These results illustrate the importance of kinetic data, which can protect trends and often missed with endpoint analysis. The Meister Z's label free assays are also non destructive to cells or cell culture media, allowing for multiplexing with other complementary assays such as flow cytometry sequencing, or cytokine and metabolite analysis to correlate the impedance data with other endpoint assays and discover underlying mechanisms. In this example, the authors evaluated CAR T cells from the Co culture experiment by flow cytometry on day 7. They found that the virus free cells contain higher frequencies of activated CD 8 positive cells in comparison to the retroviral CAR T cells, suggesting a mechanism for their greater killing ability. Permega also offers a fast, easy method for detecting CAR T activity with the Lumin amino assay. Lumin Immuno assays provide a simple and fast alternative to conventional conventional amino assay methods, including San Michelizas and Western blots. These assays streamline the process of detecting and quantifying specific proteins, making them highly efficient and user friendly. In this assay, primary antibodies to each target are carefully selected for their specific and sensitive detection capabilities and are labeled with the large bit and small bit subunits of nano bit luciferase. In the presence of the target protein, the subunits come together to form an active luciferase enzyme. The addition of an optimized substrate then generates a bright luminescence signal that is proportional to the abundance of the target protein. This luminescence signal provides a clear and quantifiable measure of the target, making lumit immunoassays a powerful tool for rapid and accurate protein detection. Tumor antigen density can significantly impact the CAR T cell performance. Tumor cells may evade immunotherapy by down regulating antigen expression, which makes it crucial to understand how varying levels of antigen density affect CAR T cell function. In this study, we investigated the influence of target cell antigen density on her two CAR T cell activity using 3 cell types, SCOVE 3, which expressed high levels of her 2A low, her two expressing cell A549, and the cell line MD AM B231 with no her tooth expression at all. To comprehensively evaluate CAR T cell potency, we combined in Peanuts with the Lumen amino assays for TNF alpha and interferon gamma. First, target cells receded into the Cyto VZ plate and cell growth was monitored for 24 hours, after which her two CAR T cells were added at either a 1:00 to 5:00 or 1:00 to 1:00 EDT ratio. Self healing was followed in real time for 72 hours while supernatants were collected at various time points during the cold culture for cytokine release analysis. 1st, we wanted to determine how target antigen density influenced potency using the Maestro Z cytotoxicity assay. Here we show a continuous plot for percent cytolysis generated on the Maestro Z. As expected, percent cytolysis directly correlated with antigen density, with Scope 3 cells exhibiting the highest overall levels of cell death and the MDA MB 231 cells the lowest. This trend was observed for both high and low EDT ratios. Further investigation of the 2448 and 72 hour time points showed that differences in cell killing by CAR T cells were most pronounced across all groups at 48 hours post cold culture. MDA MB 231 cells show 20% cytolysis in both 1:00 to 1:00 and 1:00 to 5:00 E to T conditions, most likely due to nonspecific killing by non transduced T cells present as approximately only 78% of the T cell population was carved positive. Next, to better understand the impact target antigen density had on cytokine release, we collected supernatant at 48 hours post antigen exposure from scope 3A549 or MDA MB 231 cells Co cultured with CAR T cells. Interferon gamma and TNF alpha are both pro inflammatory cytokines that play critical roles in the immune system. It has been shown that when simultaneously present they induce a synergistic state that stimulates an anti tumor response at the one to 1 E to T ratio. CAR T cells cold cultured with SCOV 3 had the highest interferon gamma production releasing 41.6% more interferon gamma compared to a 549 cells. The highest levels of TNF alpha were detected from CAR T cells Co cultured with SCOVE 3 cells at the one to 1 E to T ratio releasing approximately 80% more TNF alpha when compared to CAR T cells Co cultured with A549 cells. Similar trends were observed at the one to five ratio for SCOVE 3 and A 549 cells, but with overall lower total cytokine secretion. As expected, CAR T cells cold cultured with MDA MB 231 cells did not release any detectable TNF or interfering gamma at either E to T ratio. To measure how high levels of her two expression on target cells impacts both CAR T cell mediated cytotoxicity and cytokine release in a multiplexed approach, we used her 2 positive scope 3 cells. Cells were seeded into 3 identical 96 well Cytovy Z plates and after 24 hours her two CAR T cells were added at E to T ratios of one to five or one to one supernatant was collected at 2448 and 72 hours post CAR T cell addition. For endpoint analysis with the Lumen Interferon Gamma Amino Assay kit, we observed comparable measurements across the three identical plates indicating reproducible values of percent cytolysis and cytokine release at the one to 1 E to T ratio. 94% cytolysis was reached 24 hours post CAR T cell addition with robust interferon gamma production at approximately 1000 picograms per mil. The highest levels of interfering gamma production were measured at the 72 hours when Scope 3 cells reached 100% cytolysis, during which interfering gamma production increased by 33% from the 24 hour time point. Similar patterns were noticed noted when the 1:00 to 5:00 EDT group reached near complete cell lysis At the 72 hour time point. Interfering gamma production at 24 and 72 hours was roughly 130 and 200 picograms per meal, respectively. The parallel increase in both cytolysis and cytokine release suggests an effective activation of her two CAR T cells, highlighting their ability to induce target cell death while concurrently producing cytokines indicative of a potent immune response. In summary, the Meister Z allows for simple non invasive real time monitoring of immune cell mediated killing of target cancer cells and provides a sensitive quantitative assay for evaluating immune cell potency in vitro. Using the Meister Z hands free non invasive assay allows for assay multiplexing for a comprehensive characterization of cell based therapies. In this study, we used an integrative approach by combining the Meister C cytotoxicity assay with the Promega Lumit cytokine release assay to demonstrate CAR T cell performance in response to varying target antigen expression, with the tumor target cells with the highest antigen expression yielding the greatest response. By increasing E to T ratios, we similarly demonstrated greater overall cell killing and cytokine activity. By applying these same principles demonstrated in this experiment, future work can continue to explore Multiplex potency assays for the evaluation of CAR T cell therapies. And now for the second part of the presentation, I'll hand it over to Nick. Hello and thank you for attending this webinar. My name is Nicholas House. I'm an Associate Product Manager at Permega covering our biologics portfolio. We just heard about the excellent synergy between Permega's lumen cytokine immunoassays and Axion Biosciences impedance assays with Danielle. In this presentation, I will highlight two additional bioassays offered by Permega to highlight the potency of CAR T cell products. So first, I will highlight the programmatic advantages of using bioassays from Permega and then go into detail on the pre qualification of our T cell activation bioassay to measure the functional response of antiviral vectors. I will then highlight an additional cell killing potency assay that utilizes our unique nano bit luminescent technology to precisely measure the killing of target cells without any unwanted background from the effector cells. To begin this presentation though, I wanted to highlight our mission at Permega, which is to provide innovative biologic reagents and integrative systems to enable support and assist you in your biologic development and commercialization. I hope that you will see as I go through these slides that our team at Permega really takes this mission to heart. To help facilitate this goal, we have a broad portfolio that covers RNA therapy, gene therapy, infectious disease, and monoclonal antibody bioassays. In addition to the bioassays I'll be talking about today focus on the cell therapy field. Both our team and our bioassays are designed to take you from the discovery phase all the way to commercialization where I believe the Permega approach really shines. For example, with all of our bio assays, our limited use label license or LOL allows our bio assays to be used for commercial use with no additional fees. There is no time limit on their usage and in many cases, internal sharing or additional modifications to the bio assay can be completed with the free letter agreement with us. Also, as I'll highlight later in the presentation, if there is a specific need that is not met with our current biologics portfolio, our tailored R&D solutions team can help build that assay for you using Permega technology. Our bio assays are sold in two different formats. The first is our cell propagation model where we allow users to perform standard cell culture freeze and bank the cells at their convenience. We also offer a cell bank option where we do this initial step for you and send you A50 vial cell Bank of the cells, an alternative to users performing the labor intensive task of cell culture. We also offer our bioassays in a thaw and use format. This format has been optimized for efficient use directly from thaw and offers users a quick and simple assay set up with consistent results as well suited for a QC environment. OK. Now I'd like to transition to highlighting our T cell activation bioassay as a means to measure antiviral potency. As Danielle excellently summarized, the CAR T manufacturing cycle is very complex with multiple steps occurring for the cell drug product is even created. But to assist with the development and manufacturing of the lentivial drug substance prior to transduction of your cell product, we have generated several app notes highlighted on the bottom of this slide that use existing premier technology to measure the activity and purity of these lentivial preps. Furthermore, the the scrutiny on the alleged viral drug substance has only intensify with the release of the new FDA CAR T guidance document in 2023. I've included an excerpt of that guidance document on the right side of this slide highlighting the text that specifically calls out the need for an assay to measure the antiviral biologic activity to support future Bailey applications. So in response to this, our R&D scientists have adapted our existing catalogue T cell activation bio assay to support this regulatory need. In the bio assay, the strength of the TCR signal is measured via an NFAT reporter in response to a bispecific T cell engaged like linitumab and then a target cell expressing the antigen of interest. For the CAR T cell workflow though workflow though the same design can be used to measure the activity of the CAR product in the presence of target cells expressing the antigen of interest. To highlight some of the robustness of this assay, first the graphs on the left this slide highlight the low variability of our thaw new cells with a with ACV of approximately 3% in our internal pre qualification assay. When this assay is used in the standard format. This assay is also stability indicating with the heat treatment of lunatubimab at 65°C showing it a nice right shifted EC50. Now to transition this to measure lentiviral potency, we allow these T cell activation effector cells to be transduced with the lentiviral vector for 48 hours prior to the addition of target cells and then followed by an additional 6 hour incubation and the luminescent measurement on any standard luminometer. Now working through some of the data from left to right, the blue lines on the leftmost graph show the excellent dose response we see with this assay when the Moy is varied from point O one to 100 and the effector and target cell ratio is left constant. The red line represents AGFP controlled antivirus that shows no response. As highlighted in the FDA guidance document is the request to show antigen specificity of CAR T cell products as well which we show in the middle graph using CD19 negative Raji cells which yield no response in this CAR transduced T cell activation cell. Finally, we compared this assay with the current standard which is the measurement of CAR expression via full cytometry and shown in the rightmost graph. The increase in luminescence in this assay nicely correlates with the percent CAR positive cells in the assay. To expand on this data a little further, we have done an internal pre qualification of this assay set up and shown a 5% variation in the precision of the assay is also is this assay is also stability indicating with antiviral preps incubated at 65°C for the indicated amount of time in the rightmost graph also showing a corresponding drop in the luminescence and a right shift at EC 50. So these data together highlight the utility of this bio assay and measuring the functional response of a car contract which directly addresses a concern during car manufacturing by regulators. Next, I will transition to talking about our targeted cell Killian portfolio to measure the cell death of the target cell as a car T potency assay. I mean this assay, this user utilizes Primeca's patented nano lock technology, which is a small 100 times brighter blue emitting luciferase that is ATP independent unlike Rinella and Firefly luciferase. From this parent luciferase we have developed nano bit technology that was also highlighted in Danielle's presentation with her lumen immunoassays. So to explain this technology a little further, we split and a lock into 158 amino acid component that we coined large bit and then did direct evolution on the 2nd 13 amino acid fragment to generate fragments with over A5 log affinity range. The lowest affinity fragment which which we call small bit is using our lumen immunoassays. But for our TCK bioassays, we utilize the fragment with the highest affinity for large bit, which we call high bit. So this fragment has AKD of 700 picamolar and it rapidly complexes with large bit when both are present in any extracellular matrix. Now for the TCK bio assays, we exogenously express high bit into the cell line of interest through two different approaches. The first is a random integration of our Halo tag high bit fusion protein and the other is a CRISPR based conjugation of high bit to the C terminus of LDHB. We have tested both of these approaches internally and they both perform functioning equivalent and they allow us flexibility in generating these assays. This assay has very limited leakage of hybrid into the extracellular matrix unless the target cells are lysed by the by activated affector cells and when hybrid is released it is incredibly stable with little degradation over a three day. Allowing this assay to accommodate a range of different time points. At the end of the assay, we have our Bio Glow NBTCK substrate which contains a large big component and our firm is in substrate which is added to the wells and luminescence is detected. That is proportional to the amount of hybrid released into the medium or amount of cell death that that occurred in the assay. This is a gain of signal assay as compared to the class of classical loss of signal assay when used using a Firefly luciferase expressing cell line, which can also give you variable results due due to its lack of stability in the extra their space and its dependence on ATP, which nano luck does not have. We've also optimized protocols for for each of these TCK cells to be used with various effector cells, including PBM, CS, CDA, positive T cells and macrophages, those three that we sell and an optimized CAR T cell product as well a CAR T protocol as well. The workflow for these TCK cells is extremely simple. There's no loading, staining or washing steps needed. Once the assay plate is set up the TC, the TCK cells are thawed and washed to remove residual freezing medium for plating with an optional overnight incubation that can also that can also be performed if it suits your workflow. The cells are agnostic to the effector cells and biologic used. As I mentioned earlier, there's optimized protocols for use with PBM, CS T cells, macripages and CAR T cells, and the assay can be read to any standard luminometer in an endpoint assay format. This slide highlights our current TCK portfolio that covers both hematological blood cancers and solid cancers in purple. All the cell lines we have available for each type of cancer indication with the common antigenic targets expressed by these cancer lines bolded. In some cases such as our RAJI and OCAR 3 lines, we have generated knockouts of the common targets C19C20 for Raji and mesothelin for OCAR 3. I'll show you data in a few slides how we use these knockouts to to show antigenic specificity. So if your favorite cell line or cancer indication is not captured on the slide, we would be happy to discuss generating a new TCK line with you. Or if applicable, we can exogenously express the desired energy of interest in our choke K1 or K562 cell background with several examples where we have already done this highlight in the bottom of the slide. So now for a few data slides, there are two controls we always recommend using with our TCK cells, which is our spontaneous release and maximum release controls. The black line on both of these graphs shows the spontaneous release of high bit from these cells over a 72 hour time course comparison. The teal line on top shows the maximum release condition where TCK cells are treated with a 100 micrograms of mil of digitonin to permeabilize the cells. You can see that we maintain an approximate a hundredfold assay window across these 72 hours and in blue we have cells that were treated with digitonin at time zero and then assayed at random intervals to highlight the stability of high bed in the medium with almost no degradation of the signal. Now in this slide, I'm highlighting the importance of our CD19 and CD20 knockout Raji lines in showing antigenic specificity of a target biologic. So in this case, we are showing that bluenotumab only has efficacy in the green and red lines, which to note cell lines expressing CD 19. So showing this antigen dependence. It's in our key metric of the recent FDA CAR T guidance document that RTCK bio assets help support in which case we are happy to generate work with customers to generate new knockout variants in different TCK lines that meet their needs. Finally to get some to get to some CAR T cell data, The top graph is showing ACD 19 CAR T cell product made in house that contains 41BB and ACD 3 Zeta signaling domain. The data are the results of a 24 hour assay using 2500 target cells per well in a 96 well and varying the effector cell concentration. So using both CD 19 positive and CD 19 negative ratchi lines. This assay shows a dose response of the CAR T cells to easily identify the EC50. The small uptick that you see in luminescence at the 100 to one ET ratio in the CD 19 negative condition is an observation we often see at the highest ET ratios and most likely represents non specific killing of the target cells by the by the non targeted release of perforin and granzyme from the CAR T cells which were previously activated as part of the manufacturing protocol. We show a similar dose response curve with our H929 multiple myeloma TCK line using a BCDMA targeted CAR T cell, this time using as little as 1500 target cells per well in a 96 well format. The signal to background noise is approximately 35 in this assay and we are paying close to 100% license of our target cells based on our maximum heart release condition, which is the percent license is variable across different TCK lines and different biologics. Here are a few more examples using our OCAR 3H929 and Ramos TCK cell lines. So for the middle and rightmost graphs, you see the changes in the EC 50 of the CAR T cells when analyzed at various time points, which hides the sensitivity and robustness of this assay to detect changes over time with longer time points up to 72 hours able to measure the cumulative killing of the CAR T cell product. No, I mentioned this earlier, but at Permega, we have our tailored R&D solutions business that is designed to flexibly meet the needs of of any customer. So if there is an antigenic knockout or a new TCK line that a user requires or a qualification of a of any bio assay cell they they would like us to perform, we can discuss these projects with you and help you achieve your goals in a manner that it suits you best. So please reach out to one of us or a Permega sales Rep and we can discuss this further. In summary, all of Primeca's products give customers the freedom to operate and use ourselves for commercial use without any hidden or additional licenses or fees. We also offer our products in reliable and simple fine use format that is well suited for CR, OS or your QC environment. Also showed you data highlighting how our T cell activation bio asset could be an antivirally transduced with its CAR T cell product and measure the functional activation of that drug substance. Lastly, I presented on our TCK portfolio that designed that is designed to measure car T potency in a simple gain of signal assay. So with that, I want to thank all of you for your time and we will transition to the Q&A session. Thank you. Hi, everyone, and thank you so much, Danielle and Nick for sharing your expertise and potency analysis and your perspectives on the field. It looks like we have quite a few questions submitted for the Q&A section and we'll answer as many as we can. If we're not able to answer all the questions live, as I said before, we will follow up with you by e-mail afterwards. So I'd like to start with a question for Danielle about using different cell types with the Maestro Z system. So specifically, we have a few questions about whether or not the impotence assay is adaptable for suspension cells. So I wonder if you could address address that. Danielle yes, of course, that's a great question. So. The impedance measurements as I mentioned do rely on the cells attachment to the electrodes that are in the plate and so therefore using adherent cells can be a much more straightforward assay. However, with the appropriate modifications, such as using an antibody coded plate to actually tether the cells to the plate, it's easily modified for use with suspension cells as well. Great. That makes sense. Thanks so much for clarifying. And next we have a question for Nick about whether or not the Promega TCK cells will also work in A380 four well format for higher throughput analysis. Yes, thank you, Kim. So in all of our 96 well TCK assays, we usually range between 2003 thousand target cells. The nano luciferase is very bright. So that does that does allow us to actually reduce that number considerably in A380 well format and we do have several, several customers already using it in a 384 screen approach. I'd say one thing to consider though is each TCK line does have slightly different brightness. So there is optimization that needs to happen between different TCK lines, but it is fully able to be used in a three duo format. Good. Yeah, I, I know for some of the screening efforts that will be really helpful. OK. So now another question for Danielle. Beyond CAR T cell studies, are there other areas where the Maestro Z assay and impotence technology could be useful, perhaps by specific antibodies or checkpoint inhibitors? Yeah, absolutely. The impedance assay is very versatile, so it can be applied to various modalities beyond car T cells. So this could include things like your bispecific antibodies, checkpoint inhibitors, other immune based therapies as well as small molecules and more. So essentially the Maestro Z is going to track the presence of the cells on the electrode and thereby it allows it to detect effects whenever the mechanism of action involves cell death. So that capability, I think allows the Maestro Z to monitor cytotoxic effects across very different therapeutic approaches. And then kind of building off of that question, could you share some more details on what data analysis tools are available for use with the Maestro Z system? Yeah. So I don't think I went to to know too much detail on the software in the presentation. So this is a great question, but. The software is. Designed to be very easy to use and user friendly to really streamline the acquisition and analysis workflow. I can kind of highlight a few different capabilities that you have with the analysis software. Beyond just being able to build continuous plots for both the raw resistance curves. You can also, with a click of a button, generate percent cytolysis curves directly in the software. You can also have the software also has built in endpoints to enable quick comparison of treatment groups at different time points during the experiment. In addition to this, there's also built in kinetic endpoints such as area under the curve and kill time 50. So if you want to compare the complete time course between your different groups, you have that accessible to in the software as well. And then finally, I think another analysis tool that would be useful to the, you know, attendees here would be our dose response analysis. So this enables an easy way to quantify potency between groups using the built in dose analysis curve. And so this tool can streamline calculations such as like EC 50s and calculate comparison across different treatment conditions. Great. Thanks for sharing those capabilities. I think indeed as you said, this is important in the workflow for for especially the attendees here. All right. So Nicholas, another question for you, could you speak to whether or not the TCK cells contain a reference material and how you can ensure consistency from batch to batch? Yes, that's a, that's a really good question. So I think one of one of the things Permega does really well is have a very rigorous process to actually make sure that each batch that we make and then sell and sell to everybody is very stable and consistent. You know, we have our own internal processes that essentially maintain this. Our QC standards I think are very high and then we can also work with you and you know, make sure that you don't have to qualify as many batches as possible. If you want a custom batch of X number of vials, we can work with you and essentially make as big a lot as we can for you so that you your number of lots you actually qualify are decreased. But when we work, we work with a lot of different customers on on many different of our bio assays in this approach. And essentially our standards are very similar. We've had really positive feedback from many of our customers using our bio assets for lot release that they see very little, very little, you know lot to lot variation in these bio assets. So that's one of the things that we do, we do really well and I'm happy to talk more about more about that with you. Great. Thanks so much for for that. I have a question here as well about how the Maestro Z delivers consistent results and the reliability there are there. Is there anything you can share, Danielle, to that end? Yes, definitely. So I would say like the Maestro Z is is pretty known for it being very reliable and consistent in delivering reproducible results. I think there's a lot of factors that contribute to the fact. That it's a very. Consistent assay with low variability. One of them being is the fact that because the. Cells are. Completely maintained in the appropriate and stable environments of like your temperature in CO2 during the entire recording phase. It means that there's a lot of more stability with the cells during that recording. In addition to this, the measurements across all the wells of the plate occur simultaneously. So it helps maintain uniformity across the the assay. And then finally, you know the assay itself is automated hands free data acquisition and is completely label free. And so this over overall reduces the amount of cell manipulation required during the assay and during the assay setup which? Just as a. Consequence reduces sources of variation that could potentially be introduced during the assay setup. Great. Another quick question about the infrastructure for the Maestro Z. Is the plate reusable? Right. This is a great question. So we do have a lot of customers who may not be using an entire 96 well plate during every experiment. And so yes, the plate itself is reusable. So if you're using like partial plates on different days, you can reuse the same plate and use part of a plate on day one and another part of the plate on your next experiment just due to contamination issues. We would never recommend using the same well, but the plates are reusable. Great. OK. I think we have time for one more question and there's one here for Nick. Wondering, Nick, if you could answer. Do you offer effector cells that are not CAR T cells that can be paired with the TCK cells? Yeah. So one thing we know about these potency assays that they can be quite variable either 33 variable system between your biologic target cells and effector cells. So along with our our TCK target cells, we do pair them with effector cells. We have primary peripheral PBMCS, we have CDA positive T cells and we have macrophages. And the important thing that we do to kind of eliminate some of this variability is that we pre screen all of our donors and all of our lots with our TCK cells. So when we when you buy a primary cell from us and affect your cell from us, we have already pre screened that with our TCK line. So it's, it is it will work essentially with the pair TCK line. There's a lot of variability between different donors and some of that variabilities you can't get rid of, and it's actually very important to actually capture that of the different donors. For example, we do have PPC donors that are VV homozygous or heterozygous. Or or FF. So you can test different FC gamma receptor 3 potency binding with your monoclonal antibody. So some of that variability is captured and we have multiple different donors to choose from. And then yes, that's how we that's how how we manage our primary cells. Great. All right. With that, I do think we are out of time for today. I want to thank everyone for joining us. If you would like to revisit any portions of this webinar, the recording will be available in a few days and you should receive an e-mail allowing you to access it. And again, if we didn't answer your question, we will follow up with you by e-mail afterward. Thank you all again for joining us and we hope to see you at future Promega Webinars. _1726512796799