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Thank you very much.
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So thanks everybody for coming.
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And today I really want to talk to you about some bioluminescent tools that Promega has developed to help you with your antibody cell and gene therapy development.
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So here's an overview of the talk.
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What I'm really aiming to show you is how our assay platforms can simplify potency testing and mechanism of action studies.
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I'm going to briefly introduce you to why we use bioluminescence, why we like it so much and I'm going to give you some focus areas.
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So we're going to talk about some a peptide application, antibody applications and a cell-based application as well.
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And why should you pay attention for the next 20 minutes, very important.
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It's just after lunch after all.
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And essentially what we want to show is that we can give you more reliable data faster and help you streamline in your workflows for both biotherapeutic R&D and also QC.
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So I don't want to give you a physics lecture here.
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You're probably all quite aware that in bioluminescence you don't need to put any external energy into the system, unlike with fluorescence.
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And really the take home message here is that means that the background is effectively 0 and you're not going to have any problems with fluorescent interference.
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That means that our bioluminescence assays are extremely sensitive.
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They've got very high signal to background ratios.
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You've got a really wide dynamic range, generally around 8 to 10 logs.
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They are amenable to multiplexing with other fluorescent assays and there's no phototoxicity.
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So for those reasons, Promega has really focused on our bioluminescent capabilities.
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And what we've done over the years is we've developed a whole suite of assays that can help you with monoclonal antibody development, looking at things like FC effector activity, cytokine modulation, immune checkpoint blockade and agonism.
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We've also got some tools to look at bispecific antibodies, conjugated mabs, and more recently cell therapy, gene therapy and RNA therapeutics.
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I'm not going to be able to talk you through all of these, so I've just picked out a few examples.
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If I don't mention your favourite therapeutic modality, please come chat to us.
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We're at booth 11 and we can give you more details there.
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OK, so let's start off with the peptide therapeutics.
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GLP-1 has been in the news a lot lately.
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It's obviously a great target for obesity and what R&D have done is they've developed a bioassay to look at GLP 1 potency.
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This assay takes advantage of the fact that GLP-1R signals through the cyclic AMP signalling pathway.
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And what they've done is they've engineered a NanoLuc luciferase reporter such that when GLP-1 binds to its receptor, you get a luminescent output that's proportional to the amount of GLP-1 that's present.
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And we've then got generated some nice data to show that this assay gives you nice dose response curves and you can get rank order potency testing various different GLP 1R agonists including Semaglutide.
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So moving on to some antibody therapeutics, we've also just recently released a similar bioassay for myostatin, which is an important target for muscle wasting diseases.
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And here myostatin or activin will bind to their receptor and these signal through SMAD and same thing again, we've engineered a NanoLuc luciferase reporter to report on activity of that pathway.
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This assay can show both activation.
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On the left, you can see both activin and myostatin give you nice dose response curves using this assay.
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And then you can also look at blockade.
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So if you stimulate your cells with myostatin in order to produce a luminescence signal, you can then titrate in something like landogrozumab, which is a myostatin blocker.
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And then you can actually see inhibition which increases as you increase the dose.
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So sticking with antibody therapeutics, a lot of things a lot of people want to look at whether or not their antibody is internalised or not.
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And this is an assay that's actually still in R&D.
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It's just moving into early phase product development.
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And essentially what we've done is we've created a fab fragment that is bound to NanoLuc Luciferase.
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And what you do is you take your therapeutic antibody, you pre incubate it with that NanoLuc fab fragment and that labels it with the luminescent Lumina floor.
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You then add that antibody to your cells.
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If the antibody binds to the antigen and becomes internalised, it'll also bring that label in with it.
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You wait for the desired time frame, give your cells a wash to remove any unbound labelled antibody and then you add in both the substrate and an extracellular NanoLuc inhibitor.
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And what that does is it dampens down any signal from the bound but non internalised antibody.
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And then you read the luminescence and that tells you what portion of your antibody has been successfully internalised.
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And we've used this to demonstrate that the internalisation of two ADCs, Kadcyla and Enhertu essentially mimic the parental antibody.
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And you can see that they internalise much better than a control IgG, which only internalises at much higher concentrations.
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We've also used our new GloMax bioluminescence imager to actually image that bioluminescence.
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And you can see here that it forms punctate signalling in green and that at least partially colocalises with LysoTracker, which is in red.
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So that shows that this antibody is being internalised.
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OK, so moving on.
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Another thing that lots of people want to measure when they are designing therapeutic antibodies, it's FC binding and also to test whether or not that binding is functional.
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And Promega has developed a whole suite of assays.
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So you've always got a trade off with bioassays between how quantitative they are versus how physiologically relevant they are.
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And as you move down the curve towards more physiologically relevant assays, you lose a little bit in terms of your quantitativeness.
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So starting off, we'll start off on the most quantitative assay, which is a biochemical binding assay using Lumit technology.
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You may have come across this, it's been out for a few years now, but it's still fairly new.
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It essentially uses our NanoLuc luciferase.
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And what we've done is we've split it into two component parts called large bit and small bit.
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Those two bits have extremely low affinity for one another.
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So on their own, they're not going to give you a signal at all.
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And then what we've done for this assay is we've taken a human recombinant FcRn and labelled that with small bit and then we've taken a tracer antibody labelled that with large bit.
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So we then incubate those two together, they'll bind to one another and you get NanoLuc luciferase reconstituted and a light signal.
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You then take your unlabelled test antibody, you put that in, if it binds to FcRn, it'll out compete the tracer antibody and you'll see a loss of signal.
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And these protocols are really fast.
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So when you compare it to an ELISA where you've got lots of washing and blocking steps, the Lumit assay is a true homogeneous no wash assay.
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It's very fast.
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It's very sensitive.
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It's going to take you probably 30 minutes to 60 minutes to complete.
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And you don't need any fancy equipment to read it.
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Just a standard plate reading luminometer is all you need.
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So here's some data to show that the Lumit FcRn assay can pass out things like antibody oxidation and the shift in binding ability.
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So if you treat for increasing amounts of time with hydrogen peroxide to oxidise your antibody, you'll see that the binding shifts, so it doesn't bind so well anymore.
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We've used this same assay technology to develop different biochemical assays for FC Gamma R1, R2A and R3A.
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We are developing more, but if your favourite receptor isn't there, let us know.
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We might be able to develop a custom assay for you.
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Moving on, we also have surrogate reporter assays which will tell you whether or not your binding is functional or not.
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These assays are all designed in the same way.
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So you have target cells which express the antigen of interest and on the right-hand side you've got your effector cells which are engineered to express a luciferase upon binding.
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So what you need is your antibody that binds to both the antigen and to the FC gamma receptor and then you get a signal.
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And we've got lots of different available effector cells.
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We started off of course with ADCC, probably everyone's familiar with that Promega bioassay.
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We've also developed assays for ADCP and we've got a whole host of available target cells or you can supply your own target cell if you want.
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Just to give you an example, this is an ADCC antibody.
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All of the bioassays have been qualified according to the guidelines and they all are specific.
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So we test everything to make sure that you must have the target cells, and you must have the antibody and you must have the correct antigen on those target cells.
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And if not, you see a flat line.
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We've also qualified in terms of specificity, accuracy, precision, linearity and range.
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So you can see the data here.
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You can detect very nicely relative potency of your antibody and that's extremely linear.
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And it's also reproducible day-to-day analyst to analyst.
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So finally, our most biologically relevant assays which are quite new, these actually only launched into catalogue in January this year.
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These have been designed around the need to be able to demonstrate that the mechanism of action is indeed target cell killing.
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And at the moment the current methods tend to be quite laborious.
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You have to do things like radio labelling and they might not be particularly high throughput.
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So the assay principle uses again our NanoLuc luciferase, but in this case we've used a high affinity version of that peptide.
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So Lumit uses the low affinity version, HiBiT is the high affinity version.
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So here we end up with spontaneous complementation between HiBiT and large bit.
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And what Promega has done is we've generated a number of target cells with different antigens and they have been genetically engineered to express the HiBiT protein.
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So if the cells are alive and happy, you've got detection reagent with large bit in the media.
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You don't see any signal.
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If you treat with your antibody and the effector cell comes in and kills those cells, then the HiBiT is released into the media.
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You get a light signal that's proportional to the amount of cell death that's occurred.
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And that cell death is specific only to the target population.
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You won't get anything from effector cell death.
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So here's some ADCC assay data here we've got pre-qualified ADCC competent PBMCs from our catalogue.
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And then we've got two different antibodies cetuximab and trastuzumab and two different target cell lines A549 and SKBR3.
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And you can see that both give you a nice dose response curve.
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We've also been able to adapt this to look at ADCP.
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So this is actually a loss of signal assay.
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So you can imagine that if your antibody triggers phagocytosis, then instead of the cells lysing open, they're actually engulfed and the protein is destroyed.
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So if you've got no phagocytosis and then you lyse your cells, at the end you'll see a very bright light signal.
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If, on the other hand, you have had phagocytosis and the HiBiT has been destroyed, that signal will be lost.
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And on the right here you can nicely see that rituximab is able to induce ADCP.
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You see a loss of signal again, there's also a very new application.
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We've just generated some data here.
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I've not included it, but if you want to come and talk to us at the stand.
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But here what you do, if you're looking at bystander effect, effectively what you want is for your ADC to be up taken into the cells, the payloads to be released and then to be taken up by adjacent cells that perhaps don't express the target antigen.
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And what we do is we have in this case the target cells are not labelled with HiBiT.
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So they're not giving you any signal upon death.
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But the other cells, there's a mixed population of cells in the well, the cells that do not express the target antigen do express the HiBiT.
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So that means that if you get bystander killing, you will see a light signal.
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If there's no bystander killing, no light signal.
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So moving on to some bispecific antibody data.
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So we've also used our Lumit assay to create a number of off the shelf assays to detect cytokine release.
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So in this case, you've got two different antibodies labelled with the two bits and these, remember these are the ones which have got very low affinity from one another and they bind to different epitopes on the same analyte, in this case as cytokine.
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So when the cytokine is present, they will bind simultaneously reconstitute the NanoBiT and you get a luciferase signal that's proportional to the amount of analyte that's present.
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So in the kind of workflow in a bispecific type workflow, what you do is you plate your target cells, you add CD8 positive T cells, you add your bispecific. After the desired treatment time, you would add the Lumit labelled cytokine antibodies, wait for that reaction to equilibriate, add the detection reagent and then read your plates.
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So again, this is a homogeneous rapid assay that doesn't require any washing and it's very fast, very sensitive.
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This is some data to show that you can detect an increase of IL-2 production and interferon gamma production upon treatment with Blincyto.
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And you can see a list of ready to use assays here.
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These are also on our website.
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We have got additional targets constantly in development and again, a custom assay services department.
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So if your favourite target isn't there, do talk to us because we might be able to make that for you.
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You can also measure cell death that's triggered by bispecific activation using our HiBiT target cells.
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So here you can see a CD3, CD19 bispecific antibody.
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And as you titrate in more antibody, of course, you get more increasing amounts of cell death.
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So finally, I'm just going to show you, I think one more data slide to show that this also works for cell therapy.
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And on the left, you can see cytokine release, this is TNF alpha.
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In this case, as you increase the effector to target ratio, you get, you see more cytokine release.
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And on the right-hand side, as you increase the ET ratio, you get more cell death as shown by the HiBiT cell killing assay.
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So this is our current portfolio.
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It is expanding.
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We do continue to create different HiBiT target cells.
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We've also got available you can purchase ADCC qualified PBMCs.
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So what we've found during our assay development is that a number of PBMCs available from commercial suppliers are not functional in these types of assays.
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They don't they aren't competent to induce killing.
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So what we've done is we've screened a lot of banks and then we've we provide those for sale as pre qualified to for use in our assays.
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We've also got some CD8 positive T cells that you can purchase from us and some macrophages.
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And then if you want to use these for CAR-T or TCR-T assays and obviously those will be user supplied.
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So thank you very much.
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Thank you for your attention.
