 So, I will try to give you an insight into therapeutic monoclonal antibody CRPs and looking a little bit also into the history and into the future and of course trying to then also finalize with some physical candidate challenges that I'm facing as a formulator in the industry. So, I have to show this very slide not very interesting. So, a little bit what exactly I will be presenting. So, I will be giving you an overview on the other antibodies therapeutic market and then also hopefully this is not what you already know by heart but how do therapeutic antibodies really work. So, what do they induce and what is their mode of action and then I will elude on the evolution of antibody structures because we are seeing increasingly really crazy structures and the antibody designers are very creative. And then I would like to finalize with how the monoclonal antibodies are really applied to human beings and how they are formulated and conclude. So, with that, yeah, actually here, I think go here, yeah. So, currently there are 140 therapeutic monoclonal antibody products on global markets. The first monoclonal antibody was generated it already in 1975 based on the novel winning work of Kohler and Milstein on marine hypodoma technology and they won the Nobel Prize indeed then in 1984. And the first monoclonal antibody was fully licensed in 1986. It was Autoclone OKG3 for acute transparent rejection. And then the first monoclonal antibody approved for oncology patients was then we took them up in 1997. And as of March 2021, 100 therapeutic maps have been approved by the US FDA. And the importance of this class of drugs cannot be overstated. More than 50% of the biologics drugs on the market today are maps and within the biologics or within our product the biologics even play a very significant role. Also in terms of finances. So, the market value of monoclonal antibody CRPs is really substantial. In 2020, 10 out of the 20 best selling drugs worldwide were antibody or antibody like molecules. And if you look at the numbers behind, so they say it's behind, this is quite substantial. Given the relatively high price of map projects, the indications are rather in the severe fields like in oncology. So, products here are Obdivo, Ketruda, Rituxan, Avastin, or severe autoimmune diseases. So, Ocrevos, for instance, for multiple sclerosis, Remicade, Enbrel, Hermira, for rheumatic disease, or Stelarra, for Morros Crohn, or Colitis Ulcerosa, or to avoid blindness like Ilea for red macular degeneration. You may have been wondering about the names of the underlying monoclonal antibodies and I should like you to give you a little bit of a lecture here on a fully marine. So, mouse antibody would have the suffix MoMap. And if more and more protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans, they would be called Xemap or Thumap. And an antibody with fully human structure is a UMap or MoMap. The reason for trying to get the antibodies humanized or even fully human is to reduce immunogenicity. However, since November 2021, a new INN nomenclature scheme has been published. And I must say that I'm really a little bit confused about that. But they differentiate in this new nomenclature by suffix and infix. So, the suffix is determined by the nature of the MoMap. So, Tuk stands for an unmodified MoMap and MIG for a mighty specific MoMap and MENT for a fragment. And the infix is determined by the mode of action. So, for instance, STO is for immunostimulatory and TRU for immunosuppressive MoMaps. I have given here an example for the new nomenclature. So, we have TREXA-V-BART and V would be for a viral, anti-viral molecule. And BART would be for an antibody which has been generated artificially or a designed antibody. But to make it more confusing, since old INN names continue to be used, we will have several nomenclatures in parallel in use which does not ease the understanding. But hopefully next time you read about new market entries, you will remind a little bit these rules. Okay. So, next is how do therapeutic antibody work? And here a quite illustrative picture to understand the complexity of therapeutic monoclonal antibodies, a comparison between the well-known aspirin. So, acetyl salicylic acid is quite helpful. Not only the size is substantially different. We are talking here of a difference of 800 times, but also the efficacy is dependent not only on the primary sequence or if you will, a center of virality is typical for small molecules, but on three-dimensional structure and the folding of the monoclonal antibody. So, secondary structure, tertiary structure, quaternary structure really make or trigger the efficacy and the bioactivity. So, let us now spend some time on the structural details of a monoclonal antibody and here the type IgG. It is composed of two heavy chains and two light chains. Each chain consists of variable and constant regions, sometimes also of highly hyper variable regions, and they are desulfide or cysteine, which is both stabilizing via intra-chain bonds, the structure of a chain, and via inter-chain bonds, the whole monoclonal antibody. The hints region separates the so-called FAP FAB from the FC part and the FAP part typically contains the domains responsible for the target recognition, whereas the FC part comprises carbohydrates, and it is important for factor functions and for recycling of the mops and for tissue distribution. There are various FC gamma receptors that upon binding to FCs of antibodies trigger an immune activation or an immune inhibition. We will have a closer look into this in the subsequent slides. The first example of a therapeutic monoclonal antibody that I would like to present is Adalimumab or Hermione. The indication are various forms of arthritis, spondylitis, Crohn's disease, ulcerative colitis, chronic plaque, so riosis. It's a human antibody which binds with specificity to tumor necrosis factor alpha and inhibits its interaction with the P55 and P75 cell surface TNF receptors. Adalimumab also lies in cells expressing tumor necrosis factor on the surface in the presence of complement and now complement. The complement system is a set of plasma proteins produced in the liver that act together to attack extracellular forms of pathogens because you recall the immune defense is essentially targeting at pathogens and not necessarily at cells that we present in the context of our treatments. The complement activation can occur by antibody binding to the pathogen or other cells marked by the antibody or therapeutic antibody and leads to the killing of the pathogen or the target cell by formation of a pore and the subsequent lysis of this very cell. Another example for an important therapeutic monoclonal antibody is Ocrelizumab or Ocribus. The indication is primary progressive or relapsing forms of multiple sclerosis MS. Ocrelizumab is a cytolitic antibody that selectively targets species that express the CD20 antigen and it acts via antibody dependent cytotoxicity, so ADCC. ADCC here is enhanced by the so-called AFUCO platform which at the same time has reduced complement dependent cytotoxic effects. Natural killer cells and K cells recognize these cell-bound antibodies via CD16 FC receptor and lead to the apoptosis of the marked cell. Yeah Ocrelizumab is further a second generation recombinant humanized monoclonal ITG1 antibody and compared to the non-humanized CD20 antibodies such as frituximab Ocrelizumab is expected to be less immunogenic, unwanted immunogenic with repeated infusion and improving so thus the benefit-to-risk profile for patients with MS who definitely have to take the medication lifelong. So now hopefully you are experts in complement activation and in this ADCC so antibody dependent cytotoxicity and with this slide I should like to summarize the antibody effector functions. The therapeutic antibodies may function simply by blocking a ligand or a receptor like the programmed death ligand one or the programmed death one receptor blockers in the checkpoint inhibitor anti-cancer drugs you may have heard about that already and they may function by this famous complement activation, they may function by antibody dependent cytotoxicity so CDC and ADCC or by a combination of both and they may function by triggering phagocytosis by the cells of the innate immune system so that's basically the functionality and I think you also may recognize that not only the binding but really the FC part of our monoclonal antibody may have a very integral part in the mode of action. So in some instances however we do not want to have antibody effector functionality by the FC part and then by antibody design we may modulate or silence and silence this function. Certain mutations or amino acid exchanges will trigger the silencing one of the most known is the LALA mutation where leucine and alanine are exchanged in the FC part. Also via glycoengineering in the FC effector function this effector cell can be modified to give you an example for antibody drug conugates and I will present these later and if C dependent effector function is not desired because it would enhance off target accumulation of the toxin. So well now the evolution of antibody structures. I would like to familiarize with you with some evolutions that antibody design has enabled and I can tell you that in the Novartis biologics early portfolio we hardly have classical mono specific antibodies or non-designed antibodies anymore. We already heard about the switch from early mouse antibodies to humanized or human antibodies which induce less unwanted imagosonicity so you see that on the left hand side of the slide. The predominant antibody subtype used in therapeutics is IgG1. In fewer cases we have IgG2s and IgG4s and I'm not really aware of the use of IgG3s. Quite important are the species on the right hand side. Antibody fragments for instance the FAB2 fragment also FAB2 antibody is the antigen binding fragment of an antibody that can be obtained by cleavage with the enzyme pepsin. The FAB2 fragment is a protein constructed of two FAB fragments which are held together by disulfide bridges or alternatively by adhesive domains. In contrast to classical full-size antibodies such as immunoglobuli G, FAB2 fragments do not elicit acytotoxic response via activation of the complement system which we now are experts on because they lack large portions of the FC fragment. Another very important species nowadays are the single chain variable fragments or SCFV fragments or SCFV antibodies. They are artificially produced antibody fragments that covalently link the antigen-recognizing variable domain of the light and heavy chains of a classical antibody. Due to their smaller size they may also have a different tissue penetration property so for instance might be more penetrating also into the brain. What is depicted here is that antibody engineering really creates much more crazy formats as you really see with all these schematics. Building up on the resounding therapeutic success of monoclonal antibodies and supported by accelerating progress in engineering methods the field of multi-specific therapeutic antibodies is growing rapidly. Multi-valent by specific IGD modified formats predominant today as I alluded with a clear tendency for more target antigens and further increased valencies in newer constructs. This is augmented by adding additional functionalities by drug conjugation or attaching radio ligands to mumps or fragments and these developments will be alluded in examples in the next slides. A first generation by specific antibody also known as a quadroma consists of one heavy and one light chain of two different monoclonal antibodies. The two arms of the antibodies are each directed against different antigens. The FC part or the foot of the antibody is formed together from the two heavy chains of the antibodies and represents the third binding site of the by specific antibody. This structure makes it possible for example to place the paratope of an antibody directed against a tumor antigen and the paratope of another antibody directed against a lymphocyte antigen. Each one arm of the by specific in the each on one arm of the by specific antibody. In this example it is then possible for the antibody to bind to a tumor cell with the corresponding tumor antigen and to a lymphocyte. Antigen presenting cells such as B cells or microphages can then bind via the FC section of the by specific antibody and form a three cell complex. The three cell complex usually results in improved activation of the body's own immune cells against the tumor cells. However the production of this kind of by specifics is not trivial as homodimers so no longer heterodimers with two binding sites in the FAP region or mispaired antibodies in the FAP region need to be minimized. But there are technologies like here in this case the knob and hole formats which have brought some solutions. The by specific antibodies of newer generations deviate from the previously shown structure and they can be composed of two single chain variable fragments so I see a three fragments. This concept is called bytes by specific teal cell engager. The first example for such a bytes plenatomo map was generated by Micromet and later Amgen bought this company. The variable domains of the VH and VL of a CD19 specific monoclonal antibody with B cell affinity and a CD3 specific map with T cell affinity were more or less converted here into a single chain antibody. Another interesting format are so-called antibody drug coniogates ADCs. The first generation of these ADCs were using lysines to bind via linkers the toxin and the toxin was supposed to accumulate via the so-called magic bullet antibody at the target cell and kill the cell in a directed way. In the case of Godzilla the toxin is called the DM1 and is a microtubule inhibitor. Newer developments comprise cleavable linkers which are cleaved only up on internalization by the target cells and most importantly target integration of cysteine thio mops which enable a more precise DAR. DAR is the drug to antibody ratio by the conjugation so with this targeted integration of cysteines this ratio could be made more precise and instead of DARs here in this case of Godzilla of 0 to 7 or on average 3.5 the controllable DARs of exactly 2 or 4 are feasible. Also the conjugated toxins are in evolution and there are even attempts to exchange the toxin by an antibiotic and thus also enable quite toxic antibiotics to be targeted applied. This would be then no longer ADCs but antibiotic coniogates or AVCs. So with that I would like to make here actually a cut and also talk about challenges that industry and more specific drug product development faces when developing mops. Is there something? So it is not only about designing the right functional molecule but it should also be stable and very importantly can be applied in a patient-friendly manner since really non-compliance is one of our major challenges in health systems. So with that I'm showing you here the application forms of mops unfortunately the oral tablet or capsule is still the holy grail. There are attempts but still antibodies are typically given parenteral either by infusion or injection and meanwhile there are also self-injection systems on the market and feasible. However due to the fact that we want to be more patient-centric and enhance compliance and there is a clear trend towards a subcutaneous application which means that we need to go higher in concentration in order to have in the same or have in a volume that can be applied subcutaneously the same dose that we have been given before via infusion. And yeah this renders these high concentrations bringing further challenges. The solubilized proteins are closer together which promotes protein-protein interaction and the monoclonal antibody clusters can also form mild to severe turbidity measured by light scattering and furthermore these clusters can also end up in quite viscous formulations which are then difficult to apply. Let's depict it here and ultimately depending on the antibody viscousities of more than 20 centipwas can be rising if we concentrate to 100 mL or above and this as I said may hamper the fact the syringeability or the application by syringes. And yeah the following image here is intended to illustrate the mechanism involved in the aggregation of therapeutic proteins. So it's an instability reaction. The proteins can either partially unfold and form clusters or form clusters of refolded proteins through these mentioned protein-protein interactions. And in the end over a cascade process quite large insoluble and soluble agglomerates and aggregates can again can be residing. And the consequences are for sure not only that we may have a two-bit solution but most importantly is the threat of an immune reaction. As you recall the innate immune system typically fights against pathogens and if we have aggregates of proteins they look a little bit like a virus and then the innate immune system of the patient may fight against it and create immune reactions but also reduce the efficacy of our monoclonals. So this is absolutely to be avoided. As stabilizers the formulator typically uses surfactants besides of course trying to use an appropriate pH value and there are more exotic recipients under investigation. I should like to share with you one uncomfortable situation. What is if the stabilizer itself is destabilized and that's here the case unfortunately for our cash cow polysorbates which we add to prevent the aggregation and unfortunately due to lipases that may result as host cell proteins from the drug substance manufacture and are not removed during the polishing process. So these lipases may degrade our polysorbate or via oxidation and then instead of having particles from proteins we may have particles generated from the polysorbate and you can imagine that something like that wouldn't be accepted by the market. So with that I'm at the end of my presentation and I hope that I could give you some insights into industry life and industry research and I'd like to summarize that monoclonal antibodies are key modalities to treat unmet medical needs and the field of antibody engineering constantly results in evolving complex molecules with even more complex physical chemical properties which we need to understand and due to the need for highly concentrated formulations put in formulators face new challenges and these new challenges might be aggregates leading to immunogenicity and therefore it's my mantra that we all should know much more about monoclonal antibodies not only of the design but also the physical chemistry in order to enable effective and patient-friendly monoclonal antibody therapies on the market and with that I should like to thank you for your attention and if there are any questions I have put in here also my contact details. And yeah with that I don't know whether how much time we have still for the Q&A and thank you very much.