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Rewiring the synthesis of protein-linked glycosylation in eukaryotic biopharmaceutical expression hosts

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Rewiring the synthesis of protein-linked glycosylation in eukaryotic biopharmaceutical expression hosts
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Medical Biotechnology Center, VIB and UGhent, Ghent, Belgium. Glycosylation is the enzymatically catalysed modification of biomolecules with carbohydrate structures. For secreted and membrane-integral proteins, it is the most common post- translational modification. Glycan structures characterize the molecular environment immediately outside of all cell types and hence have critical functions in interactions of any cell with its environment (cell-cell, cell-pathogen, cell- molecule). The field of glycobiotechnology is concerned with understanding and re-engineering of these glycosylation-dominated interactions. In particular, the understanding of the synthetic pathways and functions for eukaryotic N-and O-glycosylation, gained over the past few decades, has enabled the rewiring of these pathways for the benefit of pharmaceutical applications. Based on the conservation of the core pathways between eukaryotes, it has been possible to transfer the efficient synthesis of particular human-specific glycan structures to other eukaryotes such as yeasts and plants. This is enabling the cost-effective production of biopharmaceutical proteins with glycosylation patterns customized to particular therapeutic functionality (e.g. targeting to particular glycan receptors, or customized for particular pharmacokinetic behaviour). I will illustrate our work with regard to the production of human IgG-like glycosylation patterns in yeast1, and the production of mannose-6-phosphate modified lysosomal enzymes for the treatment of human inherited lysosomal storage diseases2. Whereas these earlier synthetic biology endeavours were geared towards efficiently synthesising proteins with complex mammalian glycan structures in other eukaryotes, more recently we have generated mammalian cells and plants in which glycosylation complexity has been reduced to the bare minimum, while still being compatible with eukaryotic cell life and protein productivity. This ‘GlycoDelete’ technology3,4 opens up many new structural biology and biopharmaceutical applications that are currently being explored in our laboratory. 1.Jacobs, P. P., Geysens, S., Vervecken, W., Contreras, R. & Callewaert, N. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat. Protoc.4,58–70 (2009). 2.Tiels, P. et al.A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast- produced recombinant human lysosomal enzymes. Nat. Biotechnol.30,1225–1231 (2012). 3.Meuris, L. et al.GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat. Biotechnol.32,485–489 (2014). 4.Piron, R., Santens, F., De Paepe, A., Depicker, A. & Callewaert, N. Using GlycoDelete to produce proteins lacking plant-specific N-glycan modification in seeds.
type sugar residue synthetic biology Asparagine genome fish eukaryotes protein cells amino acids modifications active site glycosylation level side chains processes
homogeneity Index Glucose sugar glycoproteins capacities secret Oligosaccharide cytoplasm Folding Sterics Glycosyltransferasen molecule antigen enzymes Vaccine P sites protein Click modifications membrane surface steps Disulfide Bonds antibodies Disulfide complexes eukaryotes chemische Reaktion firm Thylakoid branches yield blue Ionenbeweglichkeit cells Signal recognition particle tetra glycomics sequence monoclonal antibodies biosynthesis Weinfehler Stereochemistry Calnexin Stearic acid hydroxyl groups Asparagine conservation case Signal peptide DNA Isomerasen rer important cell overexpression amino acids monosaccharide nanoparticle level glycosylation translation mixture side chains areas type progress Glucosamin precursor asset metabolic pathway standards chemical structures substrates basic yeast Peptide Synthesis Crystallographic host
platform homogeneity sugar residue glycoproteins high-throughput screens bind Elektrophorese genetic information DNA enzymes protein hydrolytic lysosomal enzymes modifications clones storage Dipole Moment Mannose Färben probe Trisaccharide steps fluorescence synthetic biology genome complexes firm Receptor Lectin conditions metabolic pathway standards polymer chemical structures capillaries sequence
homogeneity sugar galactose pharmaceutical company Expansion Tetrasaccharide Fusion bind Glycosyltransferasen molecule Chemotherapie protein fermentation biochemistry surface chalk steps antibodies solutions complexes chemische Reaktion firm immunoglobulin G cultivation cells doses scale modules monoclonal antibodies activities pathogens case cell B cell overexpression electron transfer monosaccharide active site glycosylation level progress cytoxicity Tumor serum Erholung genetic code precursor man pressure Eisfläche conditions metabolic pathway chemical structures yeast
sugar residue glycoproteins glycogen transport gene case blocks formulation Alpha hydrolase Zellwand molecule enzymes cell protein conversions injection lysosomal enzymes level glycosylation vesicles Mannose surface rates steps batch genome antibodies man phosphorylation firm Receptor systems young conditions metabolic pathway phosphate yield orders of magnitude cultivation cells doses chemical structures yeast Biopolymere
sugar inductive species carbon abundant chemische Reaktion source eukaryotes pressure Zellwand hydrolase cellulose enzymes cell biotechnology yeast selective
sugar ligands activities glycogen Elektrophorese case Klinisches Experiment Pharmacology enzymes cell protein level Fibroblast Phosphatase Mannose phase organizations precursor man charge firm Receptor toxicity conditions drugs phosphate cells chemical structures yeast genetic condition
Ricin sugar residue galactose bind domain case Glycosyltransferasen Einschnürung molecule enzymes terminal cell protein active site tool toxin glycosylation DNA and RNA type Trisaccharide organizations Harmful cytoxicity steps Katalase complexes solutions pressure end Lectin branches metabolic pathway cells Indigo chemical structures yeast selective
sugar residue ligands Prolin activities polypeptide chain secret glycoproteins mononuclear operation gene cleavage case Folding enzymes rer terminal cell important protein Offener Leserahmen mass spectrometer parents active site Trypsin glycosylation C-terminus Mannose type biofuels Granulocyte macrophage colony-stimulating factor glycol eukaryotes end Lectin emission proteases metabolic pathway cells pharmaceutical chemical structures Concanavalin A resistance sequence selective
homogeneity monoclonal antibodies sugar residue ligands mechanism factors glycoproteins growth case chemical lead protein injection Redoxreaktion active site level glycosylation biochemistry type Schmerzschwelle Trisaccharide Granulocyte macrophage colony-stimulating factor glycol genetic code steps physiological antibodies ligand end emission Wasserstoff conditions coupling cultivation cells pharmaceutical doses Medicine sequence
purification monoclonal antibodies pharmaceutical company Cross plant Rohtabak antibodies case firm agricultural conditions metabolic pathway function protein overexpression doses transformations
it's pleasure to speak here and I've learned a lot already earlier today and yesterday Maya my presentation will be entirely on medically focused applications of what you could call synthetic biology but let's try to relate a little bit to what we heard yesterday as well so in fact I've heard multiple times that we have 20 amino acids I mean that is certainly a protein level not entirely true we have a closer in the neighborhood to 200 different amino acids in proteins because of a process that is called post translational modification so 20 amino acids are encoded in the genome but then afterwards after the protein is synthesized or during its folding a lot of modifications are added to those proteins and my lab is focusing on the synthetic biology fish of those post-translational modifications and so I just give you a chart here of the different types of post-translational modifications just got this from swiss-prot yesterday on experimentally validated post-translational modified sites you can see there is a whole zoo of modifications that can be added covalently or to two proteins and I will talk about this third most abundant on there which is called n-linked glycosylation which is the addition of sugars to the asparagine side chains in eukaryotic proteins in particular I will spend a little bit of time also on Oh link black oscillation which is the addition of sugars to serine and threonine residues again mostly new carry optic cells so eukaryotic cells do perform these modifications by nature and it's our take on this that we might as well use those natural modifications and re-engineer them to introduce particular functionality into therapeutic proteins so glycans are not
just tiny modifications on a protein just to exemplify that this is the crystal structure of influenza virus hemagglutinin and whatever you see on there which is yellow is n linked carbohydrate and as you can see this covers large areas of the protein surface so this is the immune Adama and antigen in the influenza vaccines that we use today and every area that's covered by yellow here is basically not available for recognition by antibodies because of stearic theory hindrance and this is yet a very conserved picture because in crystallography very often the outer branches of carbohydrates are not captured because of large mobility of these structures so what you see here is effectively the part of the carbohydrate that is rigid enough to yield definable electron density in indy crystallography maps so we are playing with these carbohydrates structures in effect about 55 to 60 percent of all therapeutics we have on the market today biopharmaceutical therapeutics are glycoproteins and that includes all monoclonal antibodies or molecules derived from monoclonal antibodies so it's a very important post translational modification for the biopharmaceutical industry so we've been talking yesterday about standardization and glycobiology is a fairly recent field in its current in its current generation and so the standards for nomenclature of carbohydrates have just been agreed upon about less than 10 years ago you know and there's just a few weeks ago there was an update published on this so just like you have the four letters for the bases in DNA and you have the 20 letters for the amino acids in proteins we now have symbology for carbohydrates which is universally accepted and because carbohydrates or branched structures we cannot suffice with a linear denomination of consisting out of letters we need to capture stereochemistry in this symbology and so this can be found it's a defined by the consortium for functional glycomics in here so effectively what this is useful
for if you would have to capture the entire chemical detail of a carbohydrate of moderate complexity like this one and I think we could all agree that it's not very convenient to write that out every single time that you would have to talk about such structures and so the symbology now effectively allows us to reduce that complexity to something more amenable also to automation into database introduction so you'll see a lot of this in my talk effectively every symbol is a monosaccharide squares have glucose topology so this would be an asset of glucosamine blue M sorry green dots or balls circles would be Manos and mannose differs from glucose because of the actual o-h hydroxyl functionality on the c2 position and then this will be galactose which differs from glucose because of an actual hydroxyl functionality on the c-4 position of the ring and so these are the main topologies in the carbohydrates that I will talk about today so as in other fields we do have models about the biosynthesis of these and carbohydrates and models don't always have to be quantitative in fact we have no idea at all about the quantitative nature of the glycosylation pathway and so this is an entirely qualitative model meaning that we know which enzymes are in which sequence after one another this is a very simplified depiction of the eukaryotic and glycosylation pathway I'll just walk you through it a little bit and like ants so those that finally end up on the asparagine side chains of proteins are synthesized as a tetra deca saccharide precursor so 14 monosaccharides and there is an entire sequence which I have not depicted here of enzymes that build up this lipid linked Dolokhov linked precursor oligosaccharide and then as a protein that is in eukaryotic cells the stein for the plasma membrane or other intracellular membranes or for secretion outside of the cell when such proteins all contain signal peptides that's recognized by secret signal recognition particle in the cytoplasm until the ribosome SRP particle docks to the translocon the sex 61 translocon lindy in the plasmic reticulum membrane then the protein translation resumes and the proteins kind of pumped inside the lumen of the endoplasmic reticulum and at that stage so prior to folding these n-glycan precursors are added to the protein and then this entire process in the endoplasmic reticulum of eukaryotic cells is universally conserved in the whole eukaryotic world and the reason for that most likely is that these glycans play a very important role in catalyzing the protein folding in the endoplasmic reticulum so some of these carbohydrates and I will not go in detail today some of these carbohydrates recruit proteins like calnexin and calreticulin to the folding protein and Witkin axon and calreticulin you get protein disulfide isomerase asst which assists in resolving disulfide bonds that have formed inappropriately in the protein and so after that has happened so in other words you can't touch this very much if you remove glycans by simply mutating the sequence to which they tend to be attached many many proteins will simply not fall anymore in eukaryotic cells this is also one of the main reasons why so many proteins do not fault in e.coli because e.coli does not have this machinery and at all right so that's why the biotech industry needs eukaryotic expression systems on top of e.coli for for manufacturing purposes this is the very single most important reason why that is the case then after this folding catalyzing functions have occurred the protein is then processed to these mannose eight click knack to structure and that's recognized for exports from the endoplasmic reticulum to the Golgi apparatus and in the Golgi apparatus depending on which eukaryotic cell you're looking at there will be an entire battery of glycosyltransferases which I've just simplified here by this swarm of arrows that will process that mannose eight structure into a rather complex n-glycan structures that are then secreted from the cell and this is just depicting what a pathway in a very simplified way would look like in a mammalian cell say a liver cell that produces the majority of protein in our serum if you'd be looking at a yeast cell this battery of glycosyltransferases would be entirely different so the protein secreted modified with entirely different set of glycans and that causes a whole set of problems in the biopharmaceutical industry and this is the reason why still today mammalian cells or the main expression host on the eukaryotic site in in that industry so this is the knowledge that has been gathered by many many colleagues and Giants on whose shoulders we stand for then engineering this pathway even in eukaryotic cells in mammalian cells such pathway will not convert in a with a hundred percent efficiency every intermediate to every product and so to build this we need about 16 enzymatic steps starting from this Manos 8 structure and all of that has to happen in the about 10 to 15 minutes that the protein has between the time point when it enters the goji and the time point when it leaves the goji for secretion from cell so in 15 minutes you need 16 enzymes to work on that substrate in a sequential way to convert all of that to the final product I think you can easily understand that this is not going to happen with a hundred percent efficiency at every step and the consequence is that then you get a the synthesis of a large array of these glycan structures some of which will be the similar to the final product but most of which would be biosynthetic intermediates along that path and this makes it very difficult to assign structure-function relations to glycoproteins to glycoforms so as a goal that we've set for ourselves more than 10 years ago is to design a set of eukaryotic expression systems expression hosts that would produce particular glycoforms single structures with a level of homogeneity that would be sufficient to be able to purify it from that mixture and so that's what I will be talking about today so as every
synthetic biologist one needs analytics high-throughput and highly reliable analytics to quantify what we are doing and so what I did back in the days when I was still a graduate student is to use DNA sequencers capillary DNA sequencers as the analytical platform to very quickly profile carbohydrate mixtures using capillary electrophoresis and so that gave us the throughput there has been used to sequence the human genome and to now be able to do clonal analysis of all these different clones that we generate along this engineering path so we label we basically take off the sugars from the protein if that will be the blue line it would be the protein we take it off with an enzyme then we label these sugars with a fluorophore called apts which imparts negative charge and fluorescence to the carbohydrates then there's a bit of cleanup steps and then we go on the sequencer and so the anode the positive pole will be here the negative one will be there and we just separate them out using electrophoresis and just like you would do for DNA or four proteins we include size standards which are depicted there that's just a hydrolysate of a carbohydrate polymer called starch and and so in this way we can very quickly at high throughput analyze these carbohydrates okay sorry so the platform
that we now have built over the last ten years allows us to make every single of the structures that you see here with a homogeneity of higher than eighty-five percent and as taken us many PhD theses and many postdocs work to arrive at this particular stage and what it allows us to do amongst other things is to target glycoproteins so this would be the protein that the sugar of course not in proportion here but what it allows us to do is to target biopharmaceuticals to particular tissues because different tissues in the human body express a different complement of sugar binding proteins called lectins so by injecting proteins modified with particular carbohydrates we can target therapeutics to particular organs and so I will talk about one particular application we've been working on which was published a few years ago which is the modification of carbohydrates with mannose 6-phosphate residues which allows obviously to target mannose 6-phosphate receptors and this is important in treating a category human disease which is called lysosomal storage diseases and so I'll come back to that as we go our most recent addition to this platform is this and that's I think what looks most like synthetic biology we were at a certain point not happy anymore with what nature had to offer and we basically reduced complexity of this pathway to its bare minimum effectively allowing to produce just these very tiny die and trisaccharides and I also explained to you why that is important at least in our mind for therapeutic applications
okay so the first stage of this work involved the building the construction the transfer of the human and black oscillation gogi pathway module if you wish to the east golgi apparatus and the reason why we wanted to do that is that yeast is tremendously more scalable in manufacturing than mammalian cell cultivation is it's also much cheaper to do that and so this is the scale at which yeast fermentation occurs in the industry this is a factory for human serum albumin which is built in the north of Japan where human serum albumin is produced and literally metric tonne quantities and for use as a serum expanded blood expander during surgery or people just if you don't have enough blood as a surgeon you can take just take the blood you have and expand it by diluting it with a solution of serum albumin and so the doses that you have to inject there or in the hundreds of grams so you can make that in yeast and you can make it in yeast only at for the time being and so being able to construct things like monoclonal antibodies in yeast would change the economics of antibody production quite dramatically but you need to get the glycosylation right and so if this would
be fact it is the crystal structure of the FC part of human igg so for those of you know if for the mathematicians antibodies are the molecules in our serum that help us combat infectious disease amongst other things and they have a typically white shape there's some chalk here so and so human igg has this kind of shape sorry for that a little bit like this so there's a linker region here what you see there is the bottom part of this that's called the FC region and this would be the fat region of which you have to these binds for example things on the surface of pathogens this is where then the so-called effector functions are localized this binds to other proteins on the immune cells and that then triggers reactions like killing activity for the pathogen that sits here okay so what you see here is the bottom part and you can see the blue squares there those are sugars and that's conserved in all antibodies that we have in our body there's a universally conserved carbohydrate there and then linked like an original structures are normally like this and what's been found about 10 years ago that if you remove this little triangle there just one monosaccharides which is called few coats the antibodies become approximately 100 fold more potent in their in their triggering of killing activity for tumor cells so this is called antibody-dependent cellular cytotoxicity if you remove that you get much better antitumor antibodies and so this is now being commercialized by companies like Roche so their newest generation of antitumor antibodies are manufactured in mammalian cell lines that do not make such few calls anymore now yeast doesn't make it in the first place so you don't have to engineer it out which makes this somewhat attractive and also sixty percent of all biopharmaceuticals today or antibodies contain this part of the antibody and molecule so what we've been working on is to you could just call it B sell ice the yeast so you try and make a yeast which looks like a b-cell in terms of glycosylation at least and so this is what yeast normally does it takes man 8 and then it converts this man ate into this hugely complicated so called hypromellose related structures whereas this is what you need on the antibody so at first I couldn't be more different but it all comes from the same precursor structure so by knocking out the first steps of this pathway and then like we do here and then building in the human pathway in a very optimized way you could hope of arriving at least at this structure mind that this will also require you to have all the sugar nucleotides to build these monosaccharides on top of one another which is also add certainly when we started was not obvious at all certainly for galactose it was not at all clear that East made UDP galactose if I turned out it does and we still don't know what it makes it for in nature here so to do this to build this pathway into yeast we need to get all of these glycosyltransferases in the appropriate location in the golgi apparatus at appropriate expression levels and activities and for this part way to work with high efficiency and so we do that by targeting the catalytic domains of these glycosyltransferases to the goji by fusing them to transport a transmembrane signals from yeast glycosyltransferases so enzymes that by nature go to the and golgi apparatus of yeast and exactly how to make that fusion that's part of the part of the optimization work that needs to be done when you do this engineering so this is the current results that we have we are getting from this after about ten years of work I must say so this is what the East glycosylation pathway would look like by nature very complicated and not at all the structures you want and then by gradually building in one black acyltransferase after the other we build up a glycosylation profile looks like this which is in fact better than what you would get from choake one cells which are the mammalian cells that are used today in bio manufacturing so brom logans is working on this finishing his PhD as we speak so we introduce also an a new trick creature and fortune can tell much about yet to remove all the yeast and doggedness clients because if you build that pathway there will be remaining yeast endogenous glycans remaining about ten fifteen percent of the total like in pool and that's really complicated for the pharmaceutical industry to get rid of so you have to engineer it out and we'll be publishing very soon how we've done that now effectively allowing us to make pretty homogeneous glycosylation profiles okay we were talking yesterday yesterday about modularity and orthogonality and so I just thought this is not not published yet but when you engineer this these pathways very often and what we observe is that the new intermediates that we synthesized along that path in this case this Manos 5 structure this has never been seen in evolution by the East glycosyltransferases by the east Koji glycosyltransferases so in other words they're they're also has not been selective pressure for the East glycosyltransferases not to recognize these human intermediates and sometimes they do as you can see here and this is a glycosylation site one site on the human protein called interleukin 22 and what we expected in this train was to get Manos 5 here as we doin almost all other proteins that we've tested but all of a sudden there was like 30 percent or more of this new carbohydrates which we've never seen before and so we did all the structural analysis which quite a lot of work to do including an NMR analysis of this to find out that it was this man 5 structure but now with the tetrasaccharide on top of it involving three sorry for different glycosyltransferases to build this all of a sudden now you make an intermediate from the human pathway which yeast has never seen before and the East cogic likes to transferases start building on top of that they're just illustrating the kind of complexity you can get and the kind of known orthogonality of these modules these glycosylation modules fortunately we could solve that problem now because if you get this this is likely to be very very immunogenic it's an entirely new structure and so you certainly wouldn't want to have that in a therapeutic recreation ok so we can
build the human part way into yeast after a lot of work that that that works allowing us to make antibodies in youth add gram per liter quantities in a very short space of time I mean mammalian cells you need about three to four weeks of fed-batch cultivation to get up to the grand per liter levels that people talk about in yeast is takes three days okay so you have a spacetime yield which is at least an order of magnitude better than with mammalian cells okay and then the second target that I was referring to is to try and reengineering like oscillation pathway to deliver therapeutic glycoproteins to the lysosomes of patient cells now well as lysosomes lysosomes are vesicles inside mammalian cells of which the main function is to degrade biopolymers that the cell doesn't need anymore ok this is in a very simplified way with lysosomes do please one of their functions and so if for this degradation of biomacromolecules to the monomyth building blocks you need depolymerizing enzymes and there's about 40 of them in the human genome that so genes that code for such acid ph optimum degradative enzymes and just one disease is illustrated here of what happens when you don't have such degradative enzymes so this is in the case of pompous disease there you don't have the alpha glucosidase in the lysosomes and this leads to the accumulation of glycogen in the muscle cells of these patients and gradually this becomes toxic to the cells and cells lose their functionality so genzyme one of the main bite a complement of the oldest by tech companies in the world contributed tremendously to the outlook for these patients by realizing that the targeting system for these lysosomal hydrolases involved carbohydrates normally when they come from the goji to the lysosome there is a receptor that recognizes mannose 6-phosphate on the carbohydrates now this receptor that normally targets these enzymes to the lysosomes is also expressed at the cell surface of almost all of our cells so if you inject a glycoprotein such a missing enzyme from the lysosomes if you inject and in the human bloodstream if it's modified with mannose 6-phosphate these receptors will capture it and endocytosis and transport that protein into the lysosomes of patient cells so
these molecules are amongst the most expensive therapeutics we have today so the alpha glucosidase for pompous diseased cells at about 300,000 euro per year lifelong for these patients and in fact because these molecules are made by mammalian cells today their levels of mannose 6-phosphate are very very low on this particular protein is less than five percent on the currently used formulation so that means eight more than eighty percent is cleared by delivering the first few minutes after injection and this is not a very efficient and therapeutic although add the high doses that is used today it has changed the life of patients but you know there's clearly room for improvement in the manufacture manufacturing side so what we've done is to take the East glycosylation pathway and yeast by nature builds mannose 6-phosphate like structures into its cell wall the only difference being that there is another mannose residue sitting in the way substituting the phosphate there and that blocks these sugars from recognizing the human mannose 6-phosphate receptor so what we've done is to build the to pump up the levels of manual phosphorylation in yeast cells and by overexpressing the genes that are the rate-limiting catalyze the rate limiting steps of this pathway and then the question became can we now convert these kind of sugars into the kind of sugars you need for lysosomal delivery we had no enzymes to do that you could do that chemically without any problems whatsoever but there is a problem there is a protein here very labile protein that you have to keep intact during that conversion so we figured we needed new enzymes and to do that and then so this
is just I'll skip this so this became the question how are we going to do this right so as always I biotechnologists I
think that our first reflexes to go out in nature and think about where in nature there might have been evolutionary pressure selective pressure for such enzymes to originate and what we I realized is that actually fungal cell walls fungal cell walls are very very abundant carbon carbon sources in the biosphere and so we thought there must be bacteria that have evolved to grow on fungal cell walls as their sole carbon source and in fact just going through the biological literature you can easily find a number of bacteria like that one is this particular species cellulose in microbial cell lines and so when you grow these bacteria on yeast cell walls as their only carbon source you get the induction of a whole array of carbohydrate hydrolytic enzymes including some enzymes as we found that can indeed catalyze these reactions that we were looking for at so this has been done by these two a very very good scientists in in the lab and it was a project that took quite some time as you
can imagine so this is just showing you that we could find such enzymes and so what you just walk you through this so we have here are the carbohydrates that are made by this yeast cell that we've engineered to express very high levels of mannose 6-phosphate manners and those two peaks here would be structures that contain that so when we take one of these enzymes that we found from this bacterium which we have coined with this name here when you three that they all of a sudden in capite electrophoresis move faster which means that either they are smaller or they have more charge and then when we treat those with calif intestinal phosphatase an enzyme that removes and standing phosphates and now they run slower again showing that indeed these phosphates are not terminal and you can remove them and we found a second enzyme that then also can remove these manuals is there and you also need to get rid of those for these ligands to be recognized by the mannose 6-phosphate receptor so this organism effectively makes two of these enzymes we also crystallized one of them this one here to explain why it works and it's all in the paper if you're interested in that so with two equal I produce enzymes you could basically convert the yeast produced mannose 6-phosphate contain carbohydrates into what we really needed for therapeutic delivery and so this was the effect then if we exposed patient cells so we took the cells from pompous disease patience fibroblast in this case because that's the only cells you can easily get from patients and then we exposed these self to increasing concentrations of our recombinant enzymes and we look at how much and then got into the cells the current therapeutic drug had this behavior here and then our yeast made drugs we needed about 15 times less of that enzyme to reach similar levels of activity intracellularly and this is just because we have about 15 times more mannose 6-phosphate on these on these drugs there's also translated in a mouse model and unpublished yet in a non-human primate model in increased clearance of glycogen from the muscles of of these experimental animals and it's now moving into phase 1 / 2 clinical trials because this isn't genetic disease you can actually do phase 1 and phase 2 at the same time because a precursor drug come in the current drug is the protein part is exactly the same so there's less issues with suspected toxicity of this kind of product so this is being
commercialized in this company called oxy rain and so the R&D division is in in Ghent okay how much more time do I
have right now okay perfect so everything I've said so far is basically taking existing pathways or more or less existing pathways from other organisms and putting them into another one which in our case was yeast and the second story involved discovery of new enzymes that we needed which were not present in the human pathway but which we needed to convert the east pathway into the human pathway so the next part and this is something that started about five six years ago in the lab is when talking to biopharmaceutical industry people very often they'll tell you that they have to live with carbohydrates on their molecules because that that happens to be how nature modifies these proteins and then you have to engineer them to such an extent that they don't harm your function but if you actually could get rid of carbohydrates altogether in eukaryotic for a large number of applications that's what they would prefer but they can't because it introduces so much complexity to the manufacturing processes and it introduces heterogeneity in the pharmacodynamic behavior of these molecules but you can't get rid of them so far because you need them for protein folding so that there has been this cat 22 for a very long time and and so we figured perhaps it was useful if we could find a solution for that and so I'll skip that if you have questions on what applications will be useful we can come to that I'll show you a few so this is what wild-type cells mammalian cells do they'll make n glycans of the very complicated type that I've already shown you they also make oak like ants which can also introduce quite a bit of complexity there's about in the range of about 200 different structures known in humans here there's about 500 actually known on the ogle across relation site so there's tremendous complexity can be introduced so what we made is something we call glycol delete and where we re-engineered this pathway shortcut added basically so that we now construct n-glycans of this trisaccharide shape that is identical to the outer branches of what you normally get in mammalian cells and then we've taken it one step further and that's as yet unpublished where we then completely remove heterogeneity only leave Liam trim only keeping one glute neck residue at the end Black oscillation sites and entirely destroying all a constellation because ogloc oscillation is not important for folding with very few exceptions so you can't get rid of it actually and from mammalian cells and keep the cells alive this is rather drastic and it was not known at the moment when we started whether this would be compatible with cellular life so this is one of the I think a small example of how sometimes this kind of engineering can teach you more about what is really necessary for cells and for you inside their life okay so I think the concept was simple you recognize this from the ER we get this minus eight structure and then we wanted to kind of put a pair of scissors in the Indigo G an enzyme that would take off the glycans entirely but for the last residue here and then we were hoping that the endogenous glycosyltransferases galactica transferase and CLL transferase would take that as a substrate and build this trisaccharide which is identical in structure to what you normally get on the outer branches so this was done by Leon during the lab we still with me right now so then we started playing and you need selective pressure like everything if you will do engineering you need some kind of selective pressure to select for what you want and for sugars such tools are less elaborate and for proteins and nucleic acids but what we do have is cytotoxic lectins sugar binding proteins that have a toxin domain like ricin toxin there ricin toxin binds terminal galactose right and that if that binds it will kill you as you know ricin is very toxic now if you re-engineer such pathway and you make a
mutant that does not make galactose heights anymore then the cells become resistant right and in this way you can select for a glycosylation phenotype in yourselves that you want and we wanted to have these Manos 5 because the scissors that are available and only recognize such structures they wouldn't recognize the wild-type carbohydrates that mammalian cells produce so then we
regard the scissors from this beast which is hypocritical rina and that's a fungus that's used for making cellulases nowadays in the biofuels industry and then we studied the secret tone of this fungus we found that it actually secretes its glycoproteins modified with just a single goodnick residue indicating that it must secrete and end up like holidays of the type that we were after and so we cloned that gene and it has this gene structure of jewish open reading frame coding sequence structure it has the in terminal pre-pro sequence for secretion and the catalytic core and it has a c-terminal pro sequence at peptides that as long as this protein is in the endoplasmic reticulum fold back onto the catalytic quorum if the crystal structure in the meantime who folds back on the graph decor and inhibits the enzyme basically protecting the glycosylation pathway of these eukaryotic cells from this degradative activity and the enzyme this propeptide only gets removed in the trans-golgi operators then fully activating the enzyme and at the goji which means after the stage where glycans are important in the cell for folding catalysis which is in the ER so it can activates in the trans-golgi and then remove the carbohydrates when they're not needed anymore for folding català so again we use the lectin trick to select for the cells we want it in this case we use concanavalin a which binds terminal monocytes and there again when we remove these terminal monocytes with this end of t as we call it this enzyme pair of scissors we get the glute net we don't get any terminal mannose anymore and that makes the cells resistant against concanavalin a selection so we analyze that using this
particular protein which is an important pharmaceutical human gene granulocyte-macrophage colony-stimulating factor gm-csf and so it has to end glycosylation sites which you can separate from one another using cleavage with trypsin protease so that allows you to do simple mass spectrometry on these glycopeptides and so on the parental cell line where we got the high- sugars you get these of course then when we looked at the same glycopeptide from glycol delete cells all of that is gone literally everything there's nothing detectable anymore and we get new carbohydrate structures which are the intermediates towards what we want to build here and now maldi mass spectrometry is not very quantitative I'll spare you the details on how we quantitated this but we have about seventy percent of this about twenty percent of that about ten percent of this just without any further engineering so we are now engineering this further to pump it towards this structure and so we have good ideas on how to do that right now so this reduces heterogeneity acquired tremendously on glycoproteins so this is gm-csf you see the mass here from about 14 and a half kilo dalton to about 20 kilo dalton all of that as heterogeneity introduced by the carbohydrates so it took me how from half a day to actually get any spectrum from that because there's so many different forms sure so many different forms here that it all chance to go into the baseline now in glycol delete you have remaining heterogeneity that most of that is because of the old ligands which are also present on the protein the
sequence here is all glycosylated yeah so france is losing the audience in the
back took this one step further well he
first looked at an application of this again here this is the antibodies which have this conserved glycan so if we produce a human monoclonal antibody in these cells we were looking at would that be helpful in any way for pharmaceutical applications what we
found is this if you inject these antibodies in mice so this is in mice time estimate we haven't done it yet in larger animals but at least in mice and when you inject these proteins from the wild-type cells or from the glycol delete cells what you'll see is that immediately after injection normally you lose about and that's true for all monoclonal antibodies you lose half to two-thirds of your antibody in the first half an hour due to so-called bio distribution but if you actually look in literature about what the mechanism might be for that there is absolutely nothing new there's no rigid scientific experimentation on that and so you lose it but we don't know how at the moment and so exactly at that stage we lose our glycol delete antibody much less than the wild-type produced antibody resulting in about doubled circulating levels of the antibody with the same dose and now this is huge extrapolation okay if you'd be able to do this in patients what it would allow you to do if this were your therapeutic threshold you know your trust level that you need to have forgetting therapeutic efficacy it would allow you to reduce dozing with a factor of 2 and like for antibodies with auntie T and where you treat against the rheumatoid arthritis against tnf-alpha these patients have to go to the hospital every four to six weeks for injections now if you could double that to every eight to twelve weeks this means quite a bit for four patients and we're certainly not at the end of what we can achieve there so I think we could get this even much further reduced and as we go forward in human medicine monoclonal antibody therapy will much more driven by patient comfort rather than efficacy and so this might turn monoclonal antibody treatments into something that you can inject maybe three or four times a year alright so I'm almost done so this is the one step further that Francis took it the frat is also got rid of all glycosylation entirely in these cells and that yet to be published but I can show you the result of that what we did is basically tackle sugar nucleotide metabolism for this and so this is a Western blot which is a method to analyze protein heterogeneity molecular weight heterogeneity and you can see here from wild-type cells this gm-csf is hugely spread out as I already showed you in the mass spectrum as well in glycol delete it's quite a bit reduced but still has remaining heterogeneity due to the older like oscillation and some level of the end like ends which are not entirely detritus trisaccharide structure and now in glycol delete cells it's effectively becoming one band and the remaining hydrogen like a double deletes and the remaining heterogeneity we have is due to site occupancy of the another ligand sites so anglich oscillation sites can be can or cannot be occupied with an end like n so if you take off the end lichens entirely what you'll find is not modified and modified with a single black residue so I think for the first time now we can actually make glycoproteins in mammalian cells with the same level of homogeneity as we could make non glycoproteins in e.coli for the longest time and what we still have remaining is a single glute nak rescue and so that's sugar residue which sits on the end ligands is actually chemo orthogonal to the protein backbone so we can do chemistry on that carbohydrate on that non a saccharide residue to couple whatever you wanted to do it there so that hence my question of lost their of yesterday you know what could you do with more than two or three I mean this is another one that we can add and to it and it's made by Nature in case you're wondering this doesn't do much to the physiology of the self glycol delete adapts completely it's just behaved like what type cells like a double the lead suffers a bit more but as you know eukaryotic cells and living cells go you can evolve them to adapt to that new glycosylation phenotype and we are still doing that so we're proud passage in it through normal cultivation conditions and it's improving still almost back to normal levels of cell growth okay so I had one
more story but I'll skip that I guess I just will tell you that we have implemented glyco delete technology now also in plants seeds and this has been done together with and the picker who is in the Department of marks on Montague so mark as you may have known developed Agri bacterium tumefaciens transformation of plants in the very early days of plant biotechnology and we were we were struck I think as all of you by the the dreadful situation of the Ebola epidemic which happened last year where we had antibodies that could prevent the disease but they couldn't be manufactured fast enough because they were manufactured in a transient expression system in tobacco and so the to do that in two back you have to grow the tobacco plants which takes months right until you have the doses so what we figured this out if we could implement antibody production in legumes seeds in this case soybeans if we could do that you could decouple the production stage from the purification stage of pharmaceutical manufacturing so these seeds as you all know I mean legumes seeds you can store them at room temperature for years and they'll still keep their germinating power which means that all the proteins in there are intact so we can actually store monoclonal antibodies at room temperature for many years in huge quantities using legumes seeds and then having them available when such an emergency happens to purify the proteins and done the one problem was that plant
glycans or allergenic to a large section of the human population if you inject them in the bloodstream of course we eat plants all the time and that's safe but that doesn't mean if you inject them in the bloodstream that it's safe and so we engineered out this allergenicity from the plants in a very simple pathway which is men table in crops okay I'm done this is the funding there is a poster from our group that i want to point your attention to by morgan and then we have a similar meeting to this one in january so if in ghent is not far away from here if you want to join feel free all right thank you