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Hello.
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Hi, It's a pleasure to be here to talk a little bit about the research that we're doing with Icosagen.
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Today I'll be talking about one of the research projects that our team has been running where we are focused on targeting cancer cell metabolism as a potential avenue for developing additional therapeutics against cancer.
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But before I get into that, I'll just give a brief overview of what Icosagen is.
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I mean maybe some of you have been working with us through our CRDMO arm.
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The company otherwise is a contract research and development manufacturing organisation.
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So we have developed a variety of different technologies in the space of antigen production, antibody discovery engineering.
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We have capabilities in bi and multi specific antibodies.
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We do ADCs and ADC conjugations and all different analytical work around the space of a biologic drugs in the preclinical space.
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More recently we've established also now a GMP manufacturing site that links the preclinical work that we are able to do.
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So we can have a seamless transition when working with your projects in your preclinical stage and transferring the know how into your CMC process as you're moving ahead with the clinical phase.
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And we are annually partnering with more than 100 different pharma and biotech companies and work in a wide variety of different projects in the space of biologic drug development.
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But in addition to being a CDMO, we are very active also on the space of research.
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We operate a little bit on a unique model.
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We also are a private research institution and are eligible for governmental research funding.
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We're fairly active in different Horizon and EU based research projects and grants and we have quite a variety of different industrial and academic research programmes ongoing including also industrial PhD programmes with multiple universities within Europe as well.
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So if we look about targeting antibodies, then the majority of antibodies are therapeutic antibodies have been targeting the let's say low hanging fruit, the single pass transmembrane proteins.
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At the same time there's a wide variety of different multi transmembrane proteins like the GPCRs, pentaspanners, ion channels and transporters that actually reflect the even larger class of therapeutically relevant proteins.
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At the same time they have been fairly difficult to target with antibodies.
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It's the same time due to the complexity of their of these proteins in the surface of the cell, there are not any good recombinant protein expression platforms to use for developing antibodies reliably and against this class of proteins.
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So what we have opted for is establishing our antibody discovery platforms that are focusing on expressing these types of antigens on the surface of virus-like particles, whether they be HIV Gag derived or Ebola VP40 derived.
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We're able to express and capture these membrane proteins in the composition of these virus-like particles and use them as antigens in different in the antibody discovery process, but also further on in the development process as well.
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So in principle, how the VLP expression works, you Co transfect your membrane protein of interest together with the Gag scaffolding protein into transfectable cell, whether it be a HEK293 or CHO cells.
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The Gag protein starts to assemble underneath the cell membrane and forms a virus like particle that then buds off from the transfected cell encapsulating the cell membrane as an envelope.
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So when you overexpress your target protein on the surface of the cells, very likely your target protein will be encapsulated on the surface of this virus-like particle as well.
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So what I did and what I've been working on previously is we have established an engineered HIV Gag based virus like particle platform where we have a peptide binding domain actually fused to the C terminus of the Gag protein.
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Meaning that this peptide binding domain is able to bind the seven amino acid synthetic peptide that you can fuse to the C terminus of your target membrane protein.
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And that peptide protein interaction then facilitates an increased display or encapsulation of your target membrane protein into the composition of this virus like particle, increasing the display level of your target membrane protein in the composition of such VLPs.
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And this is especially has been useful in the cases where the target membrane protein expression is either low or for whatever reason, the pseudotyping level or the capsulation level of your target protein in the composition of a wild type virus like particle is suboptimal.
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So here's just a few 2 graphs indicating, let's say in an ELISA format the way how the platform works.
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Otherwise the target membrane expression levels let's say are the same on both cases on the surface of the cell.
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But in case of the peptide protein interaction Gag proteins, we can see the increase of display or your target protein on the surface of these particles.
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So we're using these VLPs and a variety of different antibody discovery campaigns and where we in our antibody discovery toolbox, we have different naive libraries, for example, VHH domain libraries and also chicken naive libraries.
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We can also do hyper immune phage libraries.
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We have a B cell panning and cell cloning platform established.
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And we also have synthetic phage libraries as well that we can use in different combinations to develop antibodies.
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And moving ahead and moving on into the topic of today's talk, we know that one hallmark of cancer is the deregulated levels of cellular metabolism of cancer.
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There are several opportunities targeting this type of hallmark with antibodies is that there's can be higher specificity towards the targets.
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This let's say many of these transporter proteins are very conserved.
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They are very homologous family members related to them.
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So small molecule compounds have been shown to be effective, but they don't have the desired selectivity.
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Of course we know that antibodies can have a better PK compared to small molecules.
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But another advantage can be also that antibodies are not so available to the CNS as well.
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So the transfer through the blood brain barrier can be an advantage for these type of antibody based therapeutics.
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But again, there's also multiple challenges in antibodies are targeting this type of pathway with antibodies as well as I was mentioning that the availability with relevant antigens can be poor and it is fairly difficult to raise a wide variety of antibodies against these targets as they are very conserved throughout different species than the antibody discovery process itself can be difficult.
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In our case, we have used often mostly to immunise chicken in the case of developing antibodies against GPCR or transporter proteins.
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Just due to the fact that chickens are evolution in more divergent compared to other mammals and the unique properties of the CDRH 3 of chicken antibodies seems to be favourable in obtaining antibodies that can have functional effects within this class of proteins.
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If we go on now into the more into detail.
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So we have opted as one of the targets to be SLC2A1 also known as GLUT1.
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The GLUT1 has a 12 trans membrane glucose transported protein that is known to be overexpressed in many cancers.
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There have been small molecule inhibitors developed that have shown preclinical efficacy, but they have lacked the desired specificity to have a therapeutic window.
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Also the small molecule compounds have the capacity to pass the blood brain barrier.
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And as we're targeting glucose uptake the effects that limiting glucose uptake in the brain cells can have severe toxic effects as we have seen also in some of our in vivo studies that I'll touch base on a little bit later.
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So the design objective that we had was to develop highly specific inhibitors of GLUT1, evaluate the effects as a combination of therapy with some approved or clinically evaluated small molecule drugs in order to switch off multiple potential metabolic pathways and confirm that there is a therapeutic window within these this combination therapy.
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So GLUT 1 is as I mentioned over expressed in many cancers and many cancers rely on aerobic glycolysis.
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This is also known as the Warburg effect.
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So by limiting glucose uptake in the cells, what we're hoping to see is that we can divert the cancer cell a metabolic pathway and in order to limit the and the transport of these type of glucose into the cell, we are able then to limit cancer cell proliferation.
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So as I mentioned, we use the VLP platform to raise antibodies from chicken.
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We have raised now with all of our antibody discovery patent platforms a variety of different antibodies that are targeting GLUT-1 and low nanomolar binding affinities and also they are demonstrating fairly high specificity.
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If we look here in this HepG2 wild type cells that are expressing GLUT 1 compared to a GLUT 1 knockout cell line and HepG2.
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So we are able to develop highly specific antibodies that are also inhibiting the glucose uptake.
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So here in the red bars, we can see a glucose uptake assay where we can see that some of these antibodies can effectively inhibit glucose uptake into the cells.
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So we chose, we have selected some lean antibodies that we took forward in our analysis.
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And then we established based on the HepG2 GLUT-1 knockout cell lines that were overexpressing GLUT1, GLUT2, GLUT3 and GLUT4 that are the closest family members of GLUT-1.
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And we can see that our antibodies are binding very specifically to GLUT1 and not to the other family members that can be expressed on the surface of cancer cells and also healthy cells.
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So as I mentioned, from the get go the design, we were thinking that probably a single therapy will not have the desired effect.
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So this is something that we were able to see in the cancer cell proliferation was that by treating HT29 cells with glucose uptake inhibiting cells, we don't see much of an impact on cell proliferation in an incucyte based assay.
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So cancer cells have high plasticity or high metabolic plasticity, meaning that once you shut one door, another door opens.
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So it's known for many cancer cells that if aerobic glycolysis is inhibited, for example, oxidative phosphoration can be a pathway that is up regulated.
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So we know that there are multiple OXPHOS inhibitors established that are very widely used in the clinical setting as well.
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So we opted to try what would happen if we inhibit at the same time aerobic glycolysis or we inhibit the Warburg effect and also imply also an OXPHOS inhibitor into this combination.
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And what we were able to see was actually pretty staggering.
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So here on the left graph you see a cell proliferation assay as I showed before, the antibodies itself that are inhibiting glucose uptake or the small molecule OXPHOS inhibitor drug independently do not have an effect on the cancer cell proliferation.
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But as we combine these two drugs, we see a drastic effect in the cell proliferation, and this was also evident in the synergy plot.
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Here we can see a clear, let's say synergistic effect by combining these two drug entities and in the limiting the cancer cell proliferation.
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So together with Eurofins discovery, we also looked at a more broad aspect.
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Is this just a phenomenon that we're seeing for a small amount of different cancer cells or can this be translated to a wider variety of different cancer cells as well?
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And we were able to see it here in the blue dots are the proliferation effects of the isotype antibody control and the drug small molecule drug OXPHOS inhibitor itself.
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And here in the coloured dots are the effects of the GLUT-1 inhibitor antibody combined with the small molecule drug.
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And we can see in especially for example in colon cancer, pancreatic cancer, we can see a clear effect of this drug combination in the proliferation of these cancer cells.
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Many cancers are highly hypoxic.
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So in those cases I think or we believe that this type of a therapeutic combination might be especially useful.
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So we went forward in evaluating this drug combination also in pancreatic cancer model.
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So we used my MIAPACA-2 cells for this and we were able to see again the same effect.
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By combining this OXPHOS inhibitor with glucose transporter inhibition, we are able to see a drastic effect in the cell proliferation.
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So we went forward with the MIAPACA-2 xenograft model.
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And in this animal model we are able to see that again the drugs independently we're not having an effect.
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But with the combination drug we could see a decrease and an effect in cancer cell proliferation in these animals.
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So another aspect that I touched based on in the beginning was the toxicity aspect, right?
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So once you're targeting such a central pathway in a human or in a hidden body, there can be potential drastic side effects.
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For this specific ICO-33 antibody that I'm highlighting here, the animals are tolerating this antibody in very well.
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At the same time, other antibodies that we have developed that are inhibiting glucose uptake will have drastic toxicity impact in the animals.
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So what we think or we're trying to right now demonstrate is also that on the blood brain barrier there's a different glycoform expressed of GLUT-1.
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What we think is happening is that this specific antibody is differentiating between the GLUT-1 that is expressed on cancer cells versus the one that is expressed on the blood brain barrier.
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So that we have a selective glucose inhibitor that is not affecting the GLUT-1 that's expressed on the blood brain barrier, the epithelium, but is able to bind and inhibit glucose uptake that is the GLUT-1 that is expressed on the cancer cells.
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And this is at this point that we are in this programme.
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And we are right now evaluating the further or trying to convolute the different toxicity aspects that might be relevant in also non-human primate studies to demonstrate the safety profile of such targeting of these pathways.
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Just to sum up, we have established a very nice VLP based antigen display platform.
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We have used that very effectively in our antibody discovery campaigns to develop antibodies against GPCRs transporter proteins.
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And in this programme today I demonstrated how we have used this approach to develop antibodies that are able to inhibit glucose uptake in cancer cells and this kind of combination therapy with inhibiting the Warburg effect with OXPHOS inhibitors can have the very attractive strategy against hypoxic tumours.
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So I want to thank everybody from our team and also our collaborators from UT and University of Tartu with the in vivo studies.
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And I'm happy to take any questions if you have some.
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Thank you so much.
