[Description] In this webinar, Vincent Pai discussed how the latest automated single B cell functional screening technology helps researchers meet the demands for new monoclonal antibody candidates.
Iris Kulbatski:
Hello everyone and welcome to today's webinar. I'm Iris Kulbatski, associate science editor for the scientist and I will be moderating our discussion. Today, our speaker, Vincent Pai will present the full Beacon antibody discovery suite for screening primary plasma cells and memory B cells.
Let me introduce our speaker Vincent Pai, Associate Director of Product Management at Bruker Cellular Analysis. He has over 10 years of experience in cell biology, nanotechnology and micro fluidics with a background from the University of California, San Diego and Massachusetts General Hospital. He now leads new product development for antibody discovery applications on the beacon optofluidic platform. Okay, let's make sure our slides are up and running. Perfect, Vincent, the floor is yours.
Ensure broader B cell diversity using automated single B cell functional screening for improved monoclonal antibody discovery success
Vincent Pai:
Thank you. Hi, everyone. Thanks for joining into today's webinar. I wanted to thank The Scientist for helping promote some of the work that we're doing here at Bruker Cellular Analysis and wanted to thank you all for taking the time to listen in on our topic today. The talk today that I'll be presenting is titled ensure broader B cell diversity using automated single B cell functional screening for improved monoclonal antibody discovery success. So this is just our forward looking statement. So Bruker analysis is now part of Bruker’s highly innovative and technology forward culture. And as we have done in the past as ISIL, flexes and percolates. Now as Bruker cellular analysis, we will continue to provide scientific leaders and innovative researchers access to two of our single cell functional multielement platforms, on the left the ISO sparkline of systems and products for bulk and single cell proteomics. And on the right, the single cell optofluidic Beacon platform and products for anybody discovery selling development a T cell profiling. These two platforms are at the forefront of Bruker strategic entry into single the single cell biology and multi omics space, and both technologies will continue to provide innovations across a range of life science applications. For today's talk, I'll be focusing on the beacon optofluidic platform and our antibody discovery application, which enable rapid species agnostic function first single B cell screening, which is powering monoclonal antibody discovery for the therapeutic diagnostic and reagent uses. I wanted to start by first laying out some challenges that researchers face in antibody discovery today.
Challenges in monoclonal antibody discovery
So first, on one side of the spectrum, high value antibody targets are becoming more challenging every day and every year. While we have previously thought about GPCRs ion channels and membrane transporters, once is challenging targets. Many new innovations technologies have have allowed the field to go after these targets with higher campaign success rates. In addition, new targets are now being explored, especially those that are highly homologous, toxic or even have low immunogenicity making them challenging to yield target specific antibodies and even more challenging to screen for them. Second, as the therapeutics diagnostics and reagents continue to evolve and become more sophisticated, since you have the requirements and the ideal antibody characteristics for the end applications, as those have become a lot more demanding as well. Things like cross reactivity strain specificity, host species epitope, specificity, affinity, liabilities, and also function. These are often areas where much time, time and effort are put into characterize antibodies to further down select which candidates to carry forward. As that funnel becomes more aggressive, the input needs to be larger screening larger starting sequence diversity becomes important. In addition, methods to improve enrichment strategies bring down bring down selection upfront sooner, and or even investing in better antibody engineering solutions can help reduce the burden and timeline associated with characterizing any of your recovered hits. Taken together, finally, there's a demand continued demand to drive total campaign costs and durations down to get from a target ID to lead optimization. In particular, 2023 was a pretty tough year for the biotech industry at large, with many macroeconomic challenges and tightening budgets that all drive the need for campaign success, particularly at lower costs. And so today, I'll be focusing on one particular area that Bruker cellular analysis has generated data on to enable researchers to stay ahead of these challenges, especially around the access of broad sequence diversity for antibody discovery.
Why does antibody diversity matter?
And so why does sequence diversity really matter? And at the highest level, sequence diversity is really important to consider within anybody's discovery, especially when considering animal derived antibodies. And even with synthetic libraries. In a nutshell, the more diverse the antibody population, you can screen at the start, the more likelihood that you're going to find a unique candidate, or multiple candidates that ultimately fit the needs characteristics or requirements for the end application. And there are a number of methods to actually look at an expansive sequence diversity, starting within a single animal considerations about what B cell repertoire whether you're looking at memory B cells, or plasma cells, as far as what compartments so whether you're looking at the blood PBMs have blood PBMCs, bone marrow, spleen lymph nodes, to harvest and enrich for the the sequences as well, looking beyond single animals, looking at multiple animals in a single cohort can allow you to take advantage of individual repertoire differences as well. And then finally, beyond that, beyond individual animals, different species also offer a number of unique attributes, in addition to highly diverse repertoires between each of these species. And we'll talk a little bit about that later.
Addressing the major challenges in antibody discovery
And so we believe that the up to be discovery suite of products and workflows on the Beacon system that Bruker cellular analysis offers are among the most events and adaptable platforms for antibody discovery, to uniquely address every one of those major challenges in antibody discovery. Our technology really enables three major advantages that helped with the innovation within anybody discovery. And so first, we really do provide access to a broad diversity of sequences for screening, and coupled this with our high screening throughput to enable more shots on goal to find high value antibody. We do this with our world class reagents, and 10 workflows, and optofluidic chips together. Second, we enable the users to easily scale and adapt their assays to assess B cells more deeply. Users can perform binding assays for soluble recombinant targets, and even against membrane bound targets. And additional assays can be layered sequentially, or even in Multiplex, to further increase the number of readouts and quickly scale the profiling depth per B cell. We also provide a foundation for users to customize more complex functional assays that go beyond binding backed by a world class reagents that enable longer basal survivability and secretion so that you get these readouts for these complex assays. And finally, our platform leverages micro fluidics to really empower users to perform this deep B cell profiling all within a single day workflow on the beacon system, or function. First screening really allows you to take forward the only the most promising candidates that will most likely meet your campaign goals while screening out undesirable antibodies sooner, before you start to perform expensive characterizations, saving you time and costs across the entire preclinical antibody development lifecycle. And so taking together we believe that the beacon and the ABI discovery application really enables rapid sample to the candidate selection. And so whereas traditional hybridoma and even B cell sequencing first approaches may take several months to yield functionally characterize sequences, we believe that they have to be discovery application will allow users to reduce their timeline toward functionally characterized sequences in just two to three weeks, and in particular, just a single day dedicated to the beacon screening itself.
How the Beacon system works for antibody discovery
And so how do we do this? We do this by rapidly screening in immunized sample to start and so listed here are most human and rabbit samples. And we isolate and prepare these B cells to be loaded on onto optofluidic chips or up to select chips on the beacon system. Once on the chip, we can load single B cells into individual data at Penn chambers, allowing us to perform antigen binding and function first acids. Once we identify the hits that we'd like, we can automatically recover these BCR sequences for downstream amplification and library preparation for ultimate Sanger sequencing. And so digging into the upstream just a bit more, we start with these immunized or convalescent patients, or convalescent primary samples containing B cells against your antigen or target of interest. We provide a number of products and protocols to really help you isolate and your bridge Endor if needed, differentiate the cells into antibody secreting plasma cells, which could either be derived directly from bone marrow, spleen or circulating blood or can be activated from memory B cells. Once these antibody secreting cells have been prepared, and are ready to be loaded onto our system, we can pull them onto the beacon system and start to do our screening.
So looking at inside our beacon system itself, our flagship model can actually load and operate up to four up to select chips to really maximize and tailor your screening capacity as well as you throughput. The rest of the system this, this large system really helps perform the automation, liquid handling and imaging that occurs between the microfluidic chips and the microfluidic worlds that we traditionally work in. Diving deeper into each of the optive select chips, you can see that it's comprised of 10s of 1000s to 10s of 1000s of Mattapan chambers, which have different sizes and geometries to enable a number of different applications. In our case within antibody discovery, the sub nanoliter pens really maximize screen throughput and antibody concentration for faster and more sensitive acid readouts. Here, what you can see is kind of our secret sauce, where we can use our OAP technology to draw light cages at massive scale. And these light cages really, they move the beat cells and allow us to isolate hundreds of B cells per minute into these individual Mattapan chambers. Once the B cells are isolated, we can start to bring in or import different kinds of reagents, acid reagents that fall into two, two major assay formats to largely look at binding for antigen specificity. On the right, you can see a quick image or video of a multiplex assay that we performed, where the green signal indicates for IgG secreting cells on the chip, and the red signal indicates antigen specific binding, where you see overlapping signals in yellow, we have high confidence that that B cell that's isolated inside that nano pet is actually coding for an antigen specific IgG antibody. These readouts in this particular time, time course can occur in just under 30 minutes. And additional assay interrogation can be easily performed by simply flushing out these reagents after the assays complete from the channel and then importing new reagents and starting your imaging process again, allows you to quickly adapt and scale for the desired B cell profiling that you might need. However, membrane bound targets like GPCRs, and other transmembrane proteins can't always be expressed easily in a soluble or recombinant format. That's not really a problem for us as we can simply import antigen expressing cells into our channel and screened for membrane bound antigen specificity again in just 30 minutes, and still enable you to flush the cells out of the channel for your next assay as you choose. Finally, we provide a suite of complex functional assays that can be custom designed for any specific targets. If you're sufficiently sophisticated assays require quite a bit of optimization and development as they would even in a well plate format. Whether you're measuring cross reactivity, functional blocking, agonism, antagonism, internalization or something else. All together, we provide a platform that allows you to build, optimize and screen for function first, allowing you to filter down to only the most critical hits that you may need. Finally, once we've completed all of our assays, we have a number of methods to recover the hits of interest. Our simplest method leverages the OAP again to move the B cells out of the pen, or the Nano pens, where we can fluently recover the B cell in a well plate of lysis buffer. Once that unload method is complete, and the user recovers that well plate, we can begin single cell RT and cDNA amplification for each of the single B cells in each well. And we can follow that on with PCR amplification and library preparation for eventual Sanger sequencing. All in we really provide a wide range of features and compatibilities that can be used to tailor a bespoke opt up discovery workflow for any particular campaign that you might be thinking about. To date, broker sell and analysis has really been focused on rigorous development and testing for these end to end solutions. And the reagents that go into this have really tried to ensure high performance when performing antibody discovery campaigns from B cells derived from both from mice, rabbits, and humans. And in particular, you can tailor your workflow to meet any of your specific needs, whether it's a particular animal animal species, or B cell repertoire, your different screening throughputs, that you might require by selecting between two different optive select chip types, and the number of chips that you use per workflow, the types of assays and the number of assays that you might be looking for, as well the unload and VCR sequence recovery methods and your sequencing approaches, whether you're doing saying or for NGS. And so a critical piece of all of our optimized optimization really goes into ensuring that our workflow performs as well as they can every time so that you're able to screen as many B cells and yield as many parent heavy chain link chain antibody sequences from the detected hits that you saw on our system.
Tracking performance metrics during antibody discovery
And so, shown here are four major kind of performance metrics that that we track, and that we measure to and optimize toward, for our three our three main flavors of workflows for screening human memory, B cells, rabbit memory, B cells, and mouse plasma cells. So the first metric here is looking at single B cell screening throughput, which really looks at how many nano pens in each of our Optus LED chips are loaded with a single B cell of the two chips that we offer today. So the optive, select 11k 11k chip, which has 11,000 pins, and our octave select 20k chip, which has 20,000 pins, as the name suggests, we're able to, in addition to being able to run four chips in in parallel for single workflow, we're able to screen anywhere between 5000 to up to 60,000 B cells in a single in a single workflow per day. Then, looking at each of those single B cells that are loaded on those chips, what we can start to look at are how many of the cells are actually secreting IgG antibodies. And so, as shown here, depending on the particular the particular species, as well as B cell repertoire, we can see of the of those 55,000 to 60,000 cells, about 20 to 70% of those cells are secreting IgG antibodies. In particular, one of the key kind of new metrics that we are starting to look at is really the longevity of each of these single B cells secreting IgG over a long period of time, especially as the demands for our antibodies being discovered continue to become more challenging. And so we really want to maintain IgG secretion over in our case for hours. And so while we hadn't done this in our first set of products related to mass plasma cells, recently, we've been looking at rabbit memory cells and human memory B cells capable of maintaining IgG secretion over four hours of about 75% of the population still able to secrete antibody in within four hours. And finally, we really want to be able to recover the sequences that we identify our hits from hits that we identify on the beacon system. And so really, we're looking at close sequence recovery from each of these different species in each of these repertoires. And we're happy to say that we're that we are able to recover anywhere between 50 to 90% of the of the hits that we identify on the system that we target for export or an unload that we're able to recover a colonial paired heavy chain like chain sequence for antibodies. And So taken together really trying to increase the the amounts of B cells that you're looking at, trying to increase the the sophistication that you can, that you can perform to down select on those B cells that you've loaded, and then recover of the hits high quality clonal sequences at the end of the workflow to carry on through to reconfirmation.
Results from recent antibody discovery studies
So coming back to the central theme of this talk, I wanted to share some results of two recent studies that we embarked on to further increase our access really Europe in sequence diversity. In our first study, we aim to validate the usage of our optic memory beat discovery rabbit products with non PBMC sources of B cells and assess whether we'll be able to access additional sequence diversity at a small scale. In our second study, we really investigated an off label use of our rabbit and human memory B cell media's on seven different species, to see whether we can immediately access these sources of species diversity without a significant additional optimization.
Using activated memory B cells
So I'll start with the first study, where we performance standard Opto memory V discovery rapid workflow, leveraging our recommended products, which we target for and have validated for PBMCs. But this time, we applied these directly towards splenocytes and lymph node cells from the same animal. And so following our standard protocols, as well as using our our standard recommended products, we loaded the resultant activated memory B cells from each organ into different optic select chips. And we were able to perform this workflow, identify KLH in this particular case was our target specific hits and export about 64 of these from each of the organs to see that to to assess sequence recovery, as well as sequence diversity. And so at a high level, looking at the unique CDR three sequences that we recovered from each group, we had relatively low duplication rates within each individual B cell sources as shown on the top plot. But ticking together when looking at uniqueness across all three sequences, we only notice that three of the 95 CDR sequences that we recovered are unique CDR three sequences that we recovered, were shared across multiple BCL sources as indicated by these arrows here. Another way to look at sequence diversity is really to look at rarefaction curves, which really show how many unique sequences or clonotypes are identified per particular x export that is performed. And so you can see on the x axis as we perform additional exports for any of these given workflows shown on the right in this legend here, for our two different antigens, looking at KLH, it's in three, we're tracking how many unique or incrementally unique heavy chain and luncheon clonotypes that we identify. And you can see roughly, we're with 100, w two exports, we're able in a single in a single workflow, we're able to to maintain about 70 to 80% sequence uniqueness depending on the particular antigen. However, some of these particular workflows are actually coming from the same sample and are the same animal. And so if you look at these, these sequences together, you can actually see that as you screen more deeply into a particular animal, you start to decrease the amount of additional uniqueness that you might get from, from screening or deeply. And so looking at that even further, if you're able to run multiple workflows, like we are able to, then we can start to also assess how the contribution of individual repertoires add into species diversity and sequence diversity. And so that's the slime that that's layered on here. And you can start to see that despite early tailing off of some of the sequences, where we're screening it within a single individual, by running multiple campaigns on multiple individuals, we can actually recover some of that lost that lost incremental uniqueness and sequence diversity by running multiple cohorts against that same same antigen. And so performing this kind of analysis on this small dataset that we had for the PBMCs, splenocytes, and lymph node cells, we can kind of, we can see that we're able to recover about anywhere between 26 to 29. In this in small study, I have unique content types, as shown here. And when stacked together, and compared to other other individuals, because this these, the sequences all came from the same animal, you can see that we're no not really any different. In terms of uniqueness within this particular scale. I think that there's, there's more work to be done here. To really assess whether or not, we're able to further extend the number of unique sequences and clonotypes that we would be able to find from each of these, each of these compartments in the future. And so kind of wrapping that up in terms of case study one, and this is just a quick look at the dendrogram of our recovered sequences. Overall memory B cells derived from spleen and lymph nodes are compatible with our with our products, and also could potentially provide a source of additional sequence diversity to really fill in the gaps if you're starting to notice from a single compartment, a tail off in the number of unique sequences per number of exports that you're performing.
Identifying additional species for memory B cell studies
And so switching gears into our second study, really focused on off label use of the rabbit and human B cell media, memory B cell media for seven different species. And so we really wanted to understand what animal species might be interested in candidates for an initial pass beyond our current offerings of mouse, rabbit and human with the explicit goal of really broadening that diversity and increasing overall campaign probability of success. And so we decided to start with seven species and in particular stuck with memory B cells just given the availability of off the shelf reagents. And so each particular candidate that we chose had unique properties here. And so starting with mouse and rat, mice and rats, we already have a relatively robust plasma cell isolation and screening strategies on the beacon, but accessing the memory B cells population, in addition, could provide additional diversity within the same animal or compartments. Second, looking at camelids with alpacas and llamas, these offer, of course, special single domain heavy chain only nanobodies, which are very interesting in terms of the therapeutic application as well in the multispecific arena. And we also looked into chickens, which make good candidates for immunizations on targets that can't normally elicit a robust immunogenic response or highly homage homologous to mammalian systems. And finally, cow goat and sheep offer a unique opportunity to help the region antibody markets move from a low quality polyclonal antibody discovery toward more controlled and eat more easily quality controlled monoclonal antibodies. And so over the years, some of our customers have really been pushing the bounds with our technology, and really demonstrating successful antibody discovery within alpacas, as well as rats on vegan to date. However, for folks who were initially starting to develop an optimized reagents for food species, we really wanted to ask what would happen if we used our own human and rabbit men Sample Prep kits in an off label way, and whether these would actually already enable the use of some of our some of the other species. And so that's actually what we set out to do. We ordered a bunch of frozen PBMCs, or in the case of, of mice splenocytes for each of the seven species listed in the previous slide, and use off the shelf kits and reagents to run max for a max isolation for memory B cells. And then we use both the human and rabbit memory be some sample prep kits to perform a standard five day activation. And finally screening on the beacon workflow, where we use off the shelf assay reagents, as well as custom primers for the PCR amplification for eventual NGS sequencing. And again, as I had mentioned in the past looking at for for the main kind of metrics that we look at for workflow performance, we really tried to assess single single cell screenings. throughput. And so for reference, I have listed on the left side of the dotted life, the human memory B cell workflow with an optimized human embryo media, as well as the rabbit, the rabbit workflow, you can kind of see that we're able to load to relatively high numbers above 5000 cells per chip, all of these different species. You also noticed that we're missing now, both the bovine and chicken species here, and that was actually because the human memory and the rabbit memory, neither the human nor the rabbit memory workflow, medias, were able to activate the isolated cells from this population. So those they're not listed here.
Next, we looked at IgG as IgG assay results and the hit rates that we can see. And so as previously shown on human and rabbit, we're looking at the IgG hit rates for the other species. And you can really see that mouse looks fairly promising here without any additional any additional optimization. Whereas rat and I'll pack are quite a bit lower. And in particular, for the alpaca case we're looking at IgG today, so only the heavy chain, heavy chain only single chain antibodies, and then for goat and sheep also relatively middling. But what was interesting to look at was in our more sensitive measure of longevity of IgG secretion for hours after the cell load, so look at how many of those initially secreting IgG secrete errs are still secreting for hours later, we found a very high percentage of the rat IgG secrete errors in the rat population, as well as the alpaca that still maintain secretion, which to us signaled that perhaps that our purity or isolation methodology could use some improvement upfront, but that the media and the activation itself may be sufficient for maintaining the the secretion for these two species. And then finally, we looked at the clonal sequence recovery with our with primers that were quickly designed for each of these species. And in particular, for to note for alpaca again, we only exported the heavy chain only antibodies and clonal sequence recoveries measured here by heavy chain only recovery. But you can kind of see that for bass and rat, we're able to recover sequences on par with what we can do for a human and rabbit with a fully optimized workflows, and a little bit more middling for alpaca and sheep. And so really taking together to come back to the essential question, we really do believe that we had a really great first pass answer to the question of our off label use of our kits, in particular for mouse, this mouse and rat, despite really not being experts in house for brat and having never really performed this in the past. And having a known good memory B cell isolation antibody, we believe that, that the products that we offer as well as the protocols that we offer could be used to to expand memory B cell isolation and screening on the beacon system. So long as you have a good read memory B memory B cell isolation antibody. We were quite pleasantly surprised for for alpacas. Given that there was there's very, there was no optimization in the cytokine mix and an isolation this was the first time that we tried this. And we were seeing not not horrible, but not fully optimized results for for the APAC samples that we're looking at. And so there is some minor optimization and reagents, that that is still to be had. And ultimately this should yield good isolation and survivability. Otherwise, for the other four species that are quite a bit more divergent from the from our traditional mammalian systems. We don't currently recommend use of our human and rabbit up them up the bee discovery Sample Prep kits for these different species. And currently, if there is a desire to move into this work, quite a bit of optimization is necessary relative to the isolation of the memory B cells activation, as well as understanding the survivability and sequence recovery for each of these species. In the next week or two, we're actually going to be aiming to release all of our methods, materials and results so that users of our systems today can really use this as a starting point for their own ventures into these species or even other species.
Concluding comments about the Beacon system for antibody discovery
And so kind of taking a sticking step back And summarizing everything that I had kind of covered today, I wanted to really close today by reiterating that our two recent efforts to further unlock access on our Beacon system to an interest kind of grant sequence diversity, really what we're looking at trying to access sequence diversity from additional compartments or organs, as well as looking at sequence diversity through many different species, and really are trying to access that broad sequence diversity to maximize screening and maximizing your screening throughput for more shots on goal for that particular ideal antibody candidate. We believe that sequence diversity and our ability to adapt disparate features, products and capabilities on our platform can really offer researchers and scientists new opportunities to further increase antibody discovery success rates, and generate new antibody products. If you're interested, please don't hesitate to contact us at Bruker Cellular Analysis. And with that, I'm happy to take questions.
Iris Kulbatski:
Thank you so much, Vincent, for that fantastic presentation. We already have several questions submitted by the audience. So let's get to them.
If you were to take on a new host species for antibody discovery, what are the key agents and optimizations you would recommend as a starting place?
Vincent Pai: Yeah, so as I had shown in a previous slide, some of the work the customization or the custom reagents that we adapted to the system to be able to run that seven species workflow, or I guess, study really come around isolation, antibodies or isolation methods. So either fax or max, looking at your sample prep kit, or your isolate, or your activation media that you're going to use to activate the memory cells into antibody secreting cells, as well as what you would use to maintain that secretion through any sort of assay that you're performing. And then finally, looking at the, your primers For ultimately, your sequencing, if that's part of your workflow. And so those would be kind of a good place to start for your optimizations.
What do you use as input: memory B cells or IgM, IgD, memory B cells?
Vincent Pai: Yeah, that's a great question. So typically, many of our customers do recommend or they are looking at IgG antibodies. And so they may target their particular isolation toward IgG. To start with and not and try to deplete out the IgM IgG s IG A's. However, we'd have looked at IgM and IGA activation rates as well, at least in a small quantity, and we are able to activate those as well. And so it really is up to you to determine what types of inputs you're really interested in looking at, and what kinds of antibodies that you're looking to kind of proceed through your your discovery campaign.
Can you comment on whether a particular organ or compartment yields higher affinity antibodies?
Vincent Pai: um, we can't currently do that. I think this is going to be really dependent heavily on both the particular target that you're looking at, as well as your immunization schedule and your images, the musician strategy itself, as well as kind of what timeline that you're harvesting the samples and how you're enriching. So there are a lot of kinds of different. There are a lot of different kind of factors that play into whether or not you start to see a particular population over a particular timeline, start to show up with higher affinity antibodies, if that's the particular characteristic that you're looking for, to, to crop up in a particular organ. And so I think that there's quite a bit of optimization here. And I think our tool is really well positioned to do that optimization to find that for you. But I think it'll be up to your campaign and your strategies for immunization to really optimize for that.
Are the unique target specific antibodies sequences from PBMC significantly different from those derived from the spleen and lymph nodes from the same animal? Is there an argument for running cells from all compartments to get the most diversity?
Vincent Pai: Yeah, great questions. And so I think that the we didn't look at too closely at the sequences are the distinct sequences that we recover from that that small study for spleen lymph node versus PBMCs. I think Maybe the dataset was a little too small to really assess whether we would get these rare populations or clonotypes that, that we might not necessarily see in, in these other compartments, as well, you know, we did this with a particularly immunogenic antigen KLH, which is, which we use at least as a model at house for a lot of our our testing, which may not be representative or of a particular therapeutic target or something more relevant toward a useful antibody discovery campaign. But towards your second question here, whether there's an argument against running this or argument for running this, I think that's that that remains to be seen. There's a lot of literature out there that suggests that you know, plasma cells and memory B cells do have different antibody repertoires, maybe one has higher affinity antibodies, or maybe some that are more broadly neutralizing or broadly binding. And then also relative to the B cell lifecycle, where they go into each of the the compartments, it can be, it really depends on kind of where they're doing their affinity maturation, how exactly the immunization is playing into their other maturation. And so I would certainly say if if resources are not an issue, trying to really push for additional sequence diversity, where possible, and compartments could be a good way to do so. I think looking at individuals, and also even other species, immunizing multiple species against the same target would be helpful for finding that needle in that haystack.
Can you elaborate on the optimization in reagents in case of llamas?
Vincent Pai: so one of the things that we're really looking at right now is looking at the cytokine mix that we have. And so, you know, we we've, we've optimized and use both human and rabbit cytokines for the human memory B cells and rapid memory B cells specifically. And so it may be that some of those, the cytokine receptors that we're trying to target for B cell activation, and stimulation may not be quite homologous or conserved enough to yield the to yield the kind of the performance that we're looking for. And so we're looking into that, but we're also looking into compatibility in our initial study was just with alpaca, but we really would be like to see a compatibility with with llama as well. And in particular, for, for llamas that, that productive IgG, three, heavy chain only antibody sequence. And so really starting to look at those optimizing for that, and really bumping the numbers that I showed in the human rabbit and mouse data, we really want our template workflow to ultimately look like that.
Did you isolate or enrich memory B cells before after activation? And was there any difference between species?
Vincent Pai: Yeah, so in all of our studies, we do recommend enriching for memory B cells ahead of the activation. And we do that in well plate. However, in this particular case, we did a purely an IgG based isolation rather than an antigen specific isolation or enrichment. And this was just largely so that we can broadly assess sequence diversity, as well as IgG secretion rates of what we hadn't already immunized. Some of these, these animals, especially for some of the larger mammals, it can take up to three months to actually get a solid immunogenic response for antibody discovery in those animals. And so we wanted to kind of do a first pass with, with these naive samples IgG specific only. Are there any differences between the species, we did see some differences, and they're not necessarily are not data that that's shown here, mouse and rat were the only two that were really close to seeing, in terms of with a particular isolation antibody that we had chosen, the same kinds of numbers and population that we expect our have regularly seen from rabbits and mouse, or rabbits and human memory B cell populations in PBMCs. And so the others seem to be quite a bit higher in percentage of cells yielded. And so that really led us to think that the particular isolation antibody that we chose to start may not be yielding a really pure sample of cells, in particular memory B cells at start, and so that might be leading to lower IgG secretion rates that we're seeing on the system.
Can you clarify how many complete heavy and light chain pairs you obtain on average? If for example, you are Identify 100 cells of interest.
Vincent Pai: I apologize that that wasn't super clear when I was coming, covering some of the data there. But on the bottom right, in all of the figures that we were looking at Siegler, I was showing the sequence recovery or that clonal sequence recovery, that percentage is how many pairs that I would or you would be able to obtain on average, for its percentage, so per 100 cells of interest. And so this is really looking at if I saw 100 hits on the system, all the way through the any sort of losses in recovering the sample in, in your cDNA amplification losses that might happen there. And through your PCR amplification, anything in that molecule biology, that's all captured in that percentage. So it is really I see a hit and I want it did I get it is anywhere between 50 to 90%.
With additional sequences yielded, what would be the next steps towards selecting the best candidates to move forward for re expression and characterization?
Vincent Pai: Yeah, um, I think that kind of the next steps is really to see whether or not we can start to layer in antigen specific assays, as well as some of those those function for assays upfront, so that we can really start to whittle down the number of sequences that you're carrying. And so, you know, re-expression, reconfirmation itself can take, you know, upwards to a month, and even characterizations of that nature can cost you anywhere between 500 to $700, depending on the service provider and for per antibody. And so that quickly starts to add up. Otherwise, you'll need a lot of automation, or it'll just take a lot of time and equipment in house to characterize. And so one of the things that we really push for is any ability for you to perform some of those down selection assays on upfront so whether on the beacon system or even through enrichment strategies, then the fewer heads the body sequences that you have to carry every characterize the overall your costs will be and your timelines will be shorter accordingly.
