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Good afternoon.
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Thank you for the kind introduction.
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Before diving into the functional assays, I would like to start with a very brief update on the company.
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So some of you may know us as immune experts.
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We were founded in 2014 and over the years we have been acquired several times and at the end of last year, we underwent an official rebranding and now what was formerly called Immune Experts is part of IQVIA Laboratories.
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So we are now IQVIA Laboratories in vitro immunology.
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So IQVIA provides services throughout drug development from discovery to the clinical phase.
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And our in vitro immunology team is particularly involved in the discovery phase, providing services to aid in lead selection by evaluating the risk of immunogenicity of a compound or evaluating the functionality in order to select the best leads to move forward to preclinical stages.
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We can also be involved in some instances in clinical trials, but being a non regulated environment, we can only be involved exploratory readouts.
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So the way we typically work or operate is that we have a drug in development which is provided by the customer and this can come in all kinds of format.
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It can be a biologic, a small molecule, it can be an mRNA vaccine candidate for example.
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And these drugs in development will then be used in our in vitro assays using cellular material.
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In most cases we work with PBMCs from our bio bank that I will elaborate a bit later during the presentation, but they can also be used in combination with fresh whole blood or in some instances cellular material derived from animals such as non-human primates or other animals.
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And in more rare cases, we also work with cell lines.
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Now the readouts, they are quite classical.
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So they involve for example flow cytometry, but also the analysis of cytokine production in the Multiplex or single Plex format.
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It can be based on live-cell imaging or plate readers for example.
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So this is very typical.
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And at the end what we provide is deliverable is obviously the data, and this can come in different formats.
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It can be the raw data if you prefer to do in house analysis or we can do the full analysis including statistical analysis and provide the data as a PowerPoint overview report or as a full written report in form of or in the format of a scientific paper.
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Now our activities are generally subdivided into different teams.
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So we have the immune-functionality with a particular focus on immune-oncology, but also inflammation and autoimmunity.
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And this will be today's topic.
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But we also have an immunogenicity team which addresses questions of unwanted or wanted immunogenicity as well as immunotoxicity.
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Then we have our consultancy services under the name of Immune Academy and the consultancy service can include assay development but also assay transfer to your facilities as well as trainings, for example for PBMC isolation.
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And last but not least, we can also provide cells cell supply services, either the Cryopreserve PBMC from our bio bank or specifically isolated or fax sorted subsets of cells from isolated from fresh blood.
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Now to dive into the topic of functional immune-assays.
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What you see here is a brief overview of different assays that we can provide.
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So this list is not entirely complete and it's here separated by cell type.
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So for instance, we can have very common assays, I would call them such as the mixed lymphocyte reaction, which can be very simple, but they can also be complexified or customised, for example by including our cell population such as regulatory T cells or M2 like macrophages.
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It really depends on what is the mode of action of the compound and what cell population needs to be present.
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Now most of these assays can be ready to use as off the shelf assays, but typically they need to be customised especially in the context of modern immune-oncology where some of the compounds have very specific mode of actions where we use these assays then as a basis to develop a new assay or complexify an existing assay.
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I will not have the time to address all of them today obviously, but I made a selection of them to illustrate how they can be used as standalone assays or combined into an entire pipeline.
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But if you have questions regarding any of the assays that I'm not going to focus on, feel free to discuss it later.
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One of the pipelines that we can propose is the target peptide identification and functional peptide evaluation.
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So this is a pipeline that can be, for example, applied for the development of therapeutic cancer vaccines.
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For the development of therapeutic cancer vaccines, it all starts with the identification of the target peptide.
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And this can rely on different techniques, for example, in silico predictions for which we team up with experts, namely Martin Nielsen or it can rely on immunopeptidomics, which is particularly important now days also with the technological advancement.
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And here we team up with a Belgian company called Immune Spec who perform immunopeptidomics for Class 1 and Class 2 presented peptides.
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And then the third option here is the in between immunogenicity assessment, which we perform in house and which I will elaborate a little bit further on.
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Once we identified target or received directly a target sequence from a customer, what we can do is expand or enrich the peptide reactive T cells and this is done in DC-T cell assay.
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So this is an assay that I will elaborate a little bit further and show some examples of how this looks.
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And once we identify the immunogenic peptides that could be used as candidates for therapeutic vaccination, we can evaluate the potency in a cytotoxicity assay, namely a killing assay for which I will also be showing you some results.
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So it all starts with the immunogenicity assessment of a candidate peptide.
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So how we proceed here is we select donors for which we have PBMCs in our biobank isolate monocytes, which are differentiated into immature dendritic cells.
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And these immature dendritic cells are then loaded with the peptide of interest or for example, electroplated with an mRNA construct depending on the format that is relevant here.
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At the same time, the dendritic cells will undergo maturation so that the next day we can co-culture them with autologous T cells.
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And this is where the enrichment of the peptide reactive T cells we start.
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This is an assay which takes approximately one week, but we can also perform multiple rounds of stimulation.
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And this is something which may depend on the prevalence of the peptide reactive T cells.
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So this is just illustrated here.
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And we can evaluate after each round of the enrichment whether we have sufficient response to isolate these cells, for example, for a cytotoxicity assay.
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And then after one or two rounds of stimulation of the T cells of the expansion, we can perform a Fluorospot assay or for example, tetramer or dextramer staining combined or not with intracellular cytokine staining to evaluate whether a specific peptide led to the enrichment of a peptide reactive T cells and whether these T cells show a cytotoxic response.
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Once we identified then the candidate peptides and enrich the T cells to generate the peptide reactive T cells, we can continue with the killing assay.
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So in the killing assay it can come in different setups, for example with a cell line.
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But if we need a very high sensitive assay, we prefer to perform this in an autologous setup.
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So we isolate from the same donor for which we enrich the T cells.
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Autologous B cells.
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These cells are fluorescently labelled and co-cultured with the T cells after loading with the peptides in the present of cytotoxicity dye and then imaged over time in a live cell imaging system in order to be able to monitor killing over time And to give you an example of such a pipeline, here you see a case study for a MART-1 short peptide and for the MART-1 short peptide what we did is electroporate dendritic cells.
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We have a construct to express the peptide.
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We use the GFP report to validate the transfection of the electroporation was successful.
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And then in order to show that the peptide was truly presented by the class one HLA at the surface of the cells.
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We performed immunopeptidomic analysis with our collaborator.
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And as you can see in the top middle section, we identified the peptide sequence matching the MART 1 short peptide, indicating that it was indeed presented at the surface of these cells and these peptides loaded dendritic cells, whether it was with an mRNA construct or peptide pool.
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But in Co-cultured with autologous CD8 T cells and subjected after the enrichment to an interferon gamma fluorospot assay.
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And what you can see is that already after one round of enrichment for the MART 1 peptide or after two rounds in the case of some other test peptides, we can detect interferon gamma production by the peptide reactive T cells
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Alternative to the fluorospot read out. We can also perform a tetramer staining to validate whether we have truly enriched peptide reactive T cells.
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And the last part is the killing assay on the right hand side.
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So over time we will monitor the cytotoxicity.
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So we load the B cells, seed them as 10,000 cells per well and then monitor for each well the cytotoxicity over time.
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And we can see that the response of the T cells also depends on the concentration of peptides that was used for the loading.
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And similar results were obtained for different effector to target ratios.
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Now switching gears a bit to our non-human primate assays.
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So non-human primates are particularly relevant in pharmacology and toxicology studies.
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However, there is a high demand and a shortage of non-human primates as well as some ethical concerns.
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So when there was a particular shortage and high prices associated to these studies, we developed several in house immunogenicity and functional assays using Sino material most notably.
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And these assays aimed at contributing to the replacement, the refinement and the reduction of non-human primate use in drug development.
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And as you can see on the lower section, we have some immunogenicity assays, for example, T cell proliferation mixed lymphocyte reaction, but also some functional assays including M2 like macrophages derived from cyno PBMCs, for which you work with a third party that provides us the cryopreserved PBMCs.
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Now, we continuously try to optimise these assays because the material is quite limited.
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So this means that we are also limited in screening size.
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And here I would like to just give you an example of what we recently did is basically generating M2 like macrophages from cynos, which is very similar to human.
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We were, I would say, playing a little bit around with the cell culture conditions in order to see whether we could optimise the generation of cyno macrophages.
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And as you can see here, depending on the condition, we were able to increase to some extent the yield of M2 macrophages that were generated from a specific amount of dendritic cells, still with a very high viability.
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But what was even more striking is when we look at the phenotype of the cells, we typically have, as you see in the reference condition, a mix of less well differentiated cells and nicely differentiated into like macrophages.
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And depending on the cyno that you're looking at, this can be much more pronounced or less pronounced.
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And this is impossible for us to predict since we don't isolate and quality control the cells ourselves.
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But as you can see on the right side is that we've adapted culture conditions.
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You can really minimise the heterogeneity of the cells with more M2 like macrophages or less well differentiated or more differentiated cells compared to the reference condition.
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And the same is confirmed by looking at phenotypic markers where the heterogeneity which can be of different intensities across cynos will reduce if we adapt the culture condition.
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So by improving the culture conditions, we are able to increase the screening capacity, but also to reduce the amount of material that we need from these cynos.
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We attempted last year also to perform a Cyno MAPPS assay in the context of immunogenicity.
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So here we loaded cyno dendritic cells with Infliximab which we have been commonly using in human MAPPS assays and we were actually able to detect predicted sequences that are actually the same sequences that what we see on the human samples.
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Meaning that the MAPPS assay can already be used in a non-human primate assays before starting the cyno samples in order to guide the lead selection before proceeding and after I say that it's relevant particularly context of immuno oncology is the mixed lymphocyte reaction that we have also been trying to perform with cyno material.
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However, when we mixed cyno PBMCs, we had very low degree of reactivity which was a bit surprising, which might be related to an inbred nature of these cynos.
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We don't really know for sure.
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And to overcome this obstacle, what we did is co-culture cyno on PBMCs with human dendritic cells.
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And in this setup we used several immune-oncology drugs such as checkpoint blockade and we were able to see that we were still able to modulate the reactivity as we would see in the human setup.
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And this can be combined either with flow cytometry based readout or the analysis of cytokine production.
13:51
Now the last section addresses neutrophils because neutrophil functional assays are particularly challenging.
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So neutrophils can have a variety of functions and are relevant in not only in inflammation, but also autoimmunity and immune-oncology.
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We have access to very fresh blood, which is really essential for performing these studies.
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And these studies address mainly the activation of neutrophils but also the viability mostly in an inflammation context as well as more complex assays such as neutrophil migration.
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But the neutrophil activation assay or in this case the NET formation assay, what we propose to do is perform live cell imaging based analysis in order to monitor NET formation over time.
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So here we can co culture the neutrophils with a drug of interest to see where we can reduce NET formation or the release of myeloperoxidase and lactoferrin which can be assessed by ELISA.
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We can also look at production of reactive oxygen species by live cell imaging with fluorescent probe.
14:53
Or combine this to an assessment of activation markers which I evaluated by flow cytometry, namely the shedding of CD62 ligand or the overexpression of CD11b.
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And the last more complicated assay is the trogocytosis assay, where we use target cells which are dually labelled, so on the one hand with an eFluor dye with intracellular labelling or fluorescence labelling, and on the other side we use a Vybrant membrane dye.
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These fluorescently labelled cells can then be incubated with the neutrophils in the presence of a test compound and by performing flow cytometry analysis, we are able to monitor how the neutrophils picked up the membrane fragments from the target cells to monitor trogocytosis and in parallel evaluate activation markers such as the CD62 ligand shedding and the viability of the target cells.
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These assays, as mentioned previously, can be used individually in order to address very specific questions, for example, regarding release of the neutrophils, but they can also be combined in parallel to provide a pool of information regarding the mode of action of a compound.
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So what you see here is for example, that when we start monitoring CD62 ligand shedding so from left to right.
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This goes along with increased rate of trogocytosis or increased lactoferrin production by ELISA.
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Now, as I mentioned, we have a variety of assays and I cannot address all of them, but there is one critical ingredient for all of them.
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It's the quality of the starting material in the beginning.
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So it can be the PBMC for the for most of these assays, but it can also be fresh blood for the neutrophil assays because this will really determine the reproducibility of the data and the variation that we observe across donors and across compounds.
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Now for this, I mentioned previously that we have access to our own biobank.
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So we have a Belgian biobank licence with PBMC preparations of over 1,300 donors.
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And the advantage here is that these PBMCs were all isolated according to a very standardised and optimised procedure with optimised cryopreservation procedures as well.
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And for each of these preparations, we have quality control data, meaning that we know the exact viability, the recovery, but we also know how many or what is the percentage of specific immune cell subsets so that we can predefine how many vials we basically need to perform a specific assay.
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We also have functional data of all of these preparations, meaning that for each of these preparation cells were exposed to specific antigens.
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So we can already preselect donors for example to PBMC reactive or reactive to other antigens.
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We also have for all of them 4-digit HLA typing information, which is particularly relevant in the context of immunogenicity or if you think about the testing of vaccine candidates because we need to select specific HLA types in these instances.
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And with that, I would like to thank you for your attention and invite you to drop by if you have any questions about one of the assays that I couldn't address here today.
17:53
Thank you very much.
