Team:Freiburg/Interview Willbold
“After 24 hours we observed that 90% of the D-peptides were completely unmodified”
During our visit at the Ulm peptide pharmaceutical center many experts recommended to talk to Prof. Dr. Dieter Willbold at the Research Centre Jülich, one of the biggest research centres in Europe. The visit turned out to be a key event, which shaped our understanding of peptides and the advantages of D-peptides even more.
Prof. Willbold is one of the leading experts in the field of D-peptides, is the director of the Institute for Complex Systems at the Research Center Jülich and Professor at the Heinrich Heine University Düsseldorf. Further, he is cofounder and Chairman of the Supervisory Board of Priavoid. He developed an all-D-peptide therapeutic for the treatment of Alzheimer’s disease (AD) that already entered clinical trials. He now aims bring it through the remaining stages of clinical development to make it commercially available. Oral treatment of three different transgenic AD animal models with the compound led to deceleration of neurodegeneration and reduction of cognition and behavior deficits to a point where it couldn’t be distinguished from healthy wild-type mice.
During our visit, Prof. Willbold emphasized the advantages of D-peptides compared to L-peptides: “D-peptides combine the advantages of small molecule compounds with the advantages of therapeutic antibodies. D-peptides have all the benefits of small molecule drugs: they can be administered orally and they can be produced synthetically outside of biological systems, but they are significantly more specific than small molecule compounds. So in D-peptides these good qualities of small molecule compounds are combined with the much higher selectivity of antibodies and proteins. If you would now compare L- and D-peptides it is clear that you don’t want an L-peptide as a potential drug candidate”. Additionally L-peptides may lead to immune responses, which are not evoked by D-peptides, as Mr. Willbold experienced himself: “With our drug candidate we had to administer such exorbitantly high doses that it nearly ruined us financially. We had to give doses of 600 to 1000 mg per kg body weight until we saw any side effects.”
Additional downsides of L-peptides are the required high dosages and protease instability, which do not occur with D-peptides: “After 24 hours we observed that 90% of the D-peptides were completely unmodified. In our evaluation the slightest modification was counted as digestion but still we didn't observe any in those 90%. For comparison: The L-versions of those peptides were degraded in seconds in analogous experiments.” If you compare L- and D-peptides, D-peptides do not only lead to a superiority regarding half-life, moreover they can be administered orally and some of them are able to cross the brain-blood barrier. During our visit we got an in-depth view about the advantages, but also the difficulties of working in the emerging, but widely undiscovered, field of D-peptide therapeutics.
iGEM Freiburg: We came across your name in the context of our research on D-peptides, we were very interested in your compound that is able to directly destroy toxic amyloid beta oligomers and especially the methods for discovering it. We also came across your company and what catched our eye was the announcement “clinical trial phase I completed”. This sounded highly interesting, so we wanted to ask you what the current status of this project is.
Prof. Willbold: In several animal models of Alzheimer’s disease, this D-peptide is capable of improving the cognition of mice that are impaired by the disease. Indeed, it cannot only decelerate the progression of the disease but also ameliorate the cognition to a point, where it is not distinguishable from healthy wildtype mice. This means that it reverses the phenotype of the disease completely.
Of course, this is data from an animal model and we have yet to see how good it can be translated to humans. However, we could show in our clinical phase I trial that the compound is completely safe and that there were no drug-related side effects of the D-peptide. (Phase I clinical trials usually are performed in healthy volunteers to test the safety of a drug before they can be administered to patients.)
iGEM Freiburg: That sounds very interesting and promising! Could you tell us more about how you conceived the idea of D-peptides as therapeutics in the first place?
Prof. Willbold: In 1996 when the freshly invented Mirror-Image Phage Display was published, I immediately decided to try this with amyloid beta as a target molecule. It is necessary for mirror-image phage display that the target molecule is chemically synthesized. This is the most important limitation...at least at the moment, but this might change in the future if the research field of mirror life advances further.
Because amyloid beta has only 42 amino acid residues, it was clear to me that it would be manageable to synthesize it chemically.
iGEM Freiburg: So a very important factor is the target size! This will be crucial to consider. In your opinion, what are the advantages of D-peptide therapeutics as opposed to small molecule compounds or therapeutic antibodies?
Prof. Willbold: That’s a very good question. D-peptides combine the advantages of small molecule compounds with the advantages of therapeutic antibodies. D-peptides have all the benefits of small molecule drugs: they can be administered orally and they can be produced synthetically outside of biological systems, but they are significantly more specific than small molecule compounds. So in D-peptides these good qualities of small molecule compounds are combined with the much higher selectivity of antibodies and proteins. If you would now compare L- and D-peptides it is clear that you don’t want an L-peptide as a potential drug candidate. That is because on the one hand the L-peptide is degraded too fast and on the other hand it can cause immunogenic reactions so that already the second administration could lead to severe side effects and fatal consequences.
Another advantage of the D-peptide as compared to the L-peptide is the lack of an anti-drug-antibody (ADA) answer, a phenomenon that we also observed in the development of our drug candidate. It is also known already from literature, that D-peptides are less or not at all immunogenic.
iGEM Freiburg: Can this phenomenon be completely explained by chirality?
Prof. Willbold: Yes, probably. It is not yet finally resolved, why, but it is obvious that all the proteases in our body are optimized for L-peptides and and for cleaving L-L peptide bonds. This is probably also the reason, why D-peptides cannot be processed and presented on MHC molecules, which is an important process for the induction of antibody production.
iGEM Freiburg: What about the oral uptake of D-peptides? The protease resistance has an important role here, but how can one be sure that D-peptides are really absorbed from the gastrointestinal tract and that they really do pass the blood-brain barrier?
Prof. Willbold: Well, you have to have a bit of luck but we were able to show experimentally in our publications, that if we exposed D-peptides to simulated gastric and intestinal fluids (standard conditions used by pharmacologists) containing the relevant enzymes, that there was almost no digestion. After 24 hours, we observed that 90% of the D-peptides were completely unmodified. In our evaluation, the slightest modification was counted as digestion, but still we didn't observe any in those 90%. For comparison: The L-versions of those peptides were degraded in seconds in the same experiments.
iGEM Freiburg: Did you observe this only specifically with your drug candidate or did you do this experiment with several D-peptides?
Prof. Willbold: We tested it for two different ones: the candidate that is in the clinical trials right now and its predecessor compound, D3, which is kind of the lead compound for the clinical drug candidate. We were able to show protease resistance for both of them.
iGEM Freiburg: Do you know if this was shown in other publications as well?
Prof. Willbold: We knew about the protease stability from the literature already, but still we were quite surprised ourselves that there is such a huge difference between L and D. This might not be the case for each and every protein sequence, but in the literature, there are more such examples described.
iGEM Freiburg: Did you test the immunogenicity also with those two candidates?
Prof. Willbold: Yes, we tested the immunogenicity, because you need to show the safety of a compound that is meant for trials in humans in an animal model first, usually up to the point where the dose is high enough to see the first side effects. With our drug candidate we had to administer such exorbitantly high doses that it nearly ruined us. We had to apply doses of 600 to 1000 mg per kg body weight, until we finally saw side effects during the dose exploration studies. Consequently, the pre-clinical safety tests needed high amounts of the drug candidate and we produced 2 to 3 kg of this D-peptide almost exclusively for the safety tests.
iGEM Freiburg: What are other challenges in the application of D-peptides, next to the considerable costs which you just mentioned? Do you think the financial aspect is a big problem? Are there technical limitations for the production of D-peptides as compared to L-peptides, which can be easily synthesized in vivo?
Prof. Willbold: The prices for the compounds of chemical peptide synthesis don’t vary too much between the L- and D-variant, because the demand is growing. I talked to representatives of different synthesis companies and I was told that there is a significant price difference only for some amino acid building blocks. Overall, however, we don’t experience really big price difference in our orders of L- and D-peptides. Sure, for L-peptides, you always have the option to express them in E. coli or some other expression system, but especially for small peptides, it is more efficient and less time consuming to synthesize them chemically. Especially in the case of drug development, it is cheaper at the moment to synthesize them chemically.
iGEM Freiburg: Are there other challenges in the development of D-peptide therapeutics, especially regarding the medical application? For example the longer half-life: are there scenarios, where this could be problematic for the patient?
Prof. Willbold: Sure, this is an important factor. In most of the cases, you prefer a longer half-life, because for patients it means that they will have to take the medication less often. In our animal experiments and also in the clinical trials we used application intervals of one day.
iGEM Freiburg: Oral or intravenous application?
Prof. Willbold: Oral. With the exception of one animal study, all studies have been performed by oral application of the compound. We think, it is an advantage, if you have to give a drug orally and only once a day.
iGEM Freiburg: But isn’t the penetration of the blood-brain barrier a big problem, when it comes to antibodies?
Prof. Willbold: Definitely. For antibodies, it is mostly the case that only 0.1% of the given antibodies can penetrate the blood-brain-barrier. Consequently, one has to give very high doses to reach a sufficient concentration in the brain. This surely is one of the reasons, why so far, therapeutic antibodies (with the central nervous system as its site of action) have not been successful in AD clinical trials.
iGEM: How do you explain that your drug can cross the blood-brain barrier?
Prof. Willbold: We tested it in detail with the lead-compound, by demonstrating the mechanism with an in-vitro blood-brain barrier model. This does not mean that every D-peptide can cross the barrier, and I would actually doubt this. Our D-peptides have a high proportion of arginines and the mechanism is principally the same as for the HIV-Tat protein. Tat is also used to enable L-peptides to cross the blood-brain barrier. You attach a section of HIV-Tat, which has lots of arginines and lysines, in order to make it permeable for membranes. If something is permeable for membranes, it is also able to cross the blood-brain barrier. In our published studies, we observed that after one to two hours a 1:1 distribution between blood and brain. This is a special and important characteristic of our developed D-peptides. We not only develop drug candidates for Alzheimer's disease, but also for other neurodegenerative diseases, which all need to cross the blood-brain barrier. I would expect also the L-versions of these D-peptides to cross the blood-brain-barrier, but it won’t help them, because they are degraded too fast.
iGEM Freiburg: You mentioned the high doses that have to be administered. We heard from experts, that dosing is a problem for peptide therapeutics. Does this also apply for D-peptide therapeutics?
Prof. Willbold: We definitely don’t need high doses, whereby “high” and “low” is of course relative. For example Penicillin: Those are 1 gram pills, which really is a lot to swallow. So, it always depends - Ultimately it depends on the affinity for the target
iGEM Freiburg: Within what dosage range are we with your therapeutic?
Prof. Willbold: The drug candidate binds its target in the low nanomolar affinity range. Based on the pre-clinical treatment experiments, we expect about 40 mg per day per person to be efficient in humans. If you think about treating someone every day for a whole year, then the costs are feasible. This is magnitudes below modern cancer therapies.
iGEM Freiburg: When we searched the literature for D-peptides, we did not find many papers. What do you think is the reason, that this is a relatively unknown field? Do you think most people ascribe D-peptides no potential, or, is the technology not advanced enough?
Prof. Willbold: I ask myself this question every once and a while. I get invited to peptide conferences more frequently, for example BASEL LIFE. So why are D-peptide therapeutics so uncommon? The inventor of the mirror-image phage display, Peter Kim, moved to MERCK, to develop an anti-HIV drug based on all-D-peptides.
iGEM Freiburg: We’d like to talk more about the optimization of your Ligand. What was your strategy? Did you plan other modifications?
Prof. Willbold: When you work with phage display or mirror-image phage display, you have the advantage to have 1 to 10 billion different peptides to select from. But by far, you don't have all sequence options in the phage library available, so there is a lot of room for subsequent improvement. We also looked at further options, for example cyclization: When I cyclize a peptide, I make it less flexible. If the conformation that is relevant for target binding is still contained in the available conformation space of the cyclized peptide, then the amount of the entropic part of the free binding energy is increased reducing the free binding energy ΔG and the binding affinity is increased. That way, one can reach a lower KD-value. Of course, we tested this and then we varied sequences, changed sequence residues and so on.
iGEM Freiburg: When it comes to optimization of peptide drugs, the usual focus is half-life. What was your focus?
Prof. Willbold: We assumed we had a good half-life with D-peptides. During development, the most important characteristic for us was the efficiency to eliminate toxic amyloid-β oligomers. We do not want to inhibit or slow the aggregation, which would be only preventive. It was connected to a lot of effort to develop tests and assays that can measure this. This is not a simple enzyme, so I don’t have a nice enzyme reaction to evaluate. Rather, I have to find a reliable way to produce the target and measure how efficient the compound destroys it. It took one or two PhD dissertations to obtain an assay, which can do that. Not only every hand counts, but also, every brain.
iGEM Freiburg: Do you think that, because of the stated advantages, D-peptides will revolutionize the market of therapeutics one day, or maybe replace small molecular drugs?
Prof. Willbold: I believe that if we are able to bring our compounds on the market, it will lead to a massive boost for D-peptide drugs in general. There are different therapeutics, where one drug on the market established a whole scene.
iGEM Freiburg: You already mentioned the advantages of peptides compared to low molecular weight drugs. We talked about bispecific peptides in Ulm. We learned that peptide therapeutics can be linked easily, which, compared to small molecules, is a major advantage. Do you think this also works for D-peptides? Did you consider that?
Prof. Willbold: Of course! One of the drug derivatives was the molecule times two, we called it a tandem version. This had a higher affinity to the target, since the target amyloid assemblies are multivalent. Also, cyclization could be combined with tandemization. We tried all of that, but bioavailability suffered a bit with some modifications, which is probably due to the larger size.
iGEM Freiburg: Thank you for the interview.
Prof. Willbold: It was a pleasure.
