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TACT projects
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There are 11 PhD students working in the TACT project, investigating different questions related to the development of targeted anti-cancer therapies. We spoke to two PhD students at the University of Strasbourg, Lorenzo Turelli and Ilias Koutsopetras, about their research and its importance to the wider goal of developing more targeted anti-cancer therapies.
Multi-component reactions
EU Researcher: What is the main focus in your research?
Ilias Koutsopetras: I am looking at
the use of multi-component reactions for the conjugation of antibodies, where we add several small-molecule reactants to an antibody to produce a final antibody conjugate with precise modification sites.
EUR: Are you able to modify particular sites within the antibody for connection to a linker?
Ilias Koutsopetras, PHD STUDENT
Lorenzo Turelli, PHD STUDENT
Keywords:Multicomponent reactions, bioconjugation
Keywords: Linker synthesis, antibody-drug-conjugates
IK: One of the biggest challenges we face in developing antibody-drug conjugates (ADCs) is to identify which regions of an antibody we modify for connection to a chemical linker. We have made good progress in this.
EUR: How did you identify those regions?
IK: We tried out a lot of different conditions
and conducted many optimisation trials, then we sent our conjugated antibodies to our analytical collaborators – the Cianferani lab, another member of our consortium in this project. They use a technique called native MS (Mass Spectrometry) to determine the conversion and average degree of conjugation of our antibodies – i.e. the number of sites modified by our multicomponent reaction. Then we move on to LCMS techniques to determine precisely the identity of these conjugation sites, using a technique called peptide mapping.
EUR: Did these results from the LCMS help guide your research? IK: The results from the LCMS tell us in which regions of the antibody we had the conjugation. If we find specific sites of conjugation from our experiments, then we try to reproduce it and see if our method provides a repeatable result. The nature of the conjugation site is important as it can have an impact on the
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Faculty of Pharmacy, 74 route du Rhin, 67401 Illkirch cedex
conjugated antibody’s behaviour in vivo: conjugation in the region responsible for antigen recognition – called the paratope – can lead to diminished affinity, making the antibody less able to recognize and bind its target antigen.
Traceless linkers
EUR: What is the role of a linker in an
LT: I aim to develop new types of acidcleavable linkers responsive in a very narrow pH range: stable in plasma, but rapidly cleaved in the more acidic environment of tumoral cells (pH around 5). Once such selectivity is proved, we attach to the linker a cytotoxic drug and eventually we put in place the bioconjugation to the monoclonal antibody.
antibody-drug conjugate?
EUR: What results have you gained so far?
Lorenzo Turelli: A linker is an essential component of an ADC connecting the monoclonal antibody to a cytotoxic drug. Linkers are mainly divided in two categories: cleavable and non-cleavable. We are mostly interested in cleavable linkers: motifs whose cleavage can be operated by enzymes or chemically (acidic pH, reductive conditions, etc.), as this will dictate how and when the drug will be released.
LT: We have recently developed a new type of acid cleavable linker which proved to be specifically cleaved in a tumoral cell, but stable in plasma. We then attached to it an highly potent drug called MMAF, prior to connecting it through a mAb by means of a specific reaction (CuAAC). This work has been recently published.
EUR: What are the main challenges in developing linkers?
LT: For cleavable linkers, the main challenge is to design a system whose cleavage takes place in the tumoral cell to avoid toxicity. EUR: Is this an issue you’re addressing in your research?
EUR: Have you been able to test this? LT: We tested this novel ADC in vitro, on a cancer cell type. After we got some good results in vitro, we then moved to an in vivo test and compared it with a commercially available ADC called Kadcyla. We found that our new ADC was highly effective, highly potent, leading to full tumour regression within 23 days.
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Targeted therapies to combat cancer
Antibody-drug conjugates are a class of drugs that can be targeted specifically at cancer cells, so reducing the side-effects of treatment. We spoke to Dr Guilhem Chaubet, Lorenzo Turelli and Ilias Koutsopetras about the work of the TACT project in training the next generation of scientists and helping to develop effective, targeted cancer treatments. A lot of
attention in research is focused on the development of antibody-drug conjugates, in which a cytotoxic drug is connected to an antibody via a chemical linker, as a means of treating different types of cancer. Based at the University of Strasbourg, Dr Guilhem Chaubet is coordinator of the TACT project, an EU-backed initiative which brings together both academic and commercial partners. “We’re exploring new possibilities in anti-cancer therapies,” he outlines. There are 11 PhD students in TACT, working on research projects addressing different topics around the development of new anti-cancer therapies. “Some of the projects in TACT are about investigating the influence of a linker on the overall potency of an antibody-drug conjugate, others are looking at the use of nanoparticles,” continues Dr Chaubet. “Researchers are also working with mass spectrometry, which is a sophisticated analytical method used to study antibody-drug conjugates (ADCs).”
Antibody-drug conjugates The wider goal in the project is to develop effective, targeted anti-cancer therapies, which involves several different strands of research. As head of the BioFunctional Chemistry group at the University of Strasbourg, Dr Chaubet is deeply interested in a strategy called bioconjugation. “This is a strategy for grafting a cytotoxic drug onto an antibody in a specific manner at a specific site. Antibodies are massive molecules, they’re gigantic compared with the cytotoxic drugs that we’re trying to attach to them,” he says. There are a number of complex challenges to deal with in this research. “The molecules are
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Group photograph taken of the third TACT project meeting in Strasbourg, 21st to 25th March 2022.
Chaubet. When an ADC gets in contact with the antigen, the whole complex is internalised into a cell, sometimes into highly acidic locations rich in enzymes, conditions which Dr Chaubet is using to help target therapies more effectively. “We can put linkers into our ADCs that are highly sensitive to acidic pH. So when this ADC ends up in a cellular compartment with very low pH, then our linker gets cleaved and the drug is released,” he outlines. This occurs inside the cell, which is key to the therapeutic activity and potency of an ADC. The aim is to deliver a cytotoxic drug just to cancer cells and not to healthy cells, which represents a significant improvement on conventional chemotherapies. “In chemotherapy a really cytotoxic drug is
We can put linkers
into our ADCs that are highly sensitive to acidic pH. So when this ADC ends up in a cellular compartment with very low pH, then our linker gets cleaved and the drug is released. used to kill cancer cells, but because it’s not targeted, some of this cytotoxic drug can also reach healthy cells, especially the cells that replicate and multiply a lot,” he explains. These cytotoxic drugs cannot kill cells when they are grafted onto an antibody. “The only way they can kill a cell is if they are released from the antibody,” continues Dr Chaubet. “This is the importance of the linker, to make sure that these drugs get detached – or cleaved – from the antibody only once they have entered a cancer cell. Otherwise you start having the same side-effects as we observe with chemotherapy.”
Training too small to be seen by the naked eye, so it’s really hard to precisely control where we’re going to put our molecule, the specific site on the antibody,” explains Dr Chaubet. “It’s also quite complicated to control the amount of cytotoxic drugs that we’re going to graft onto the antibody.” These are issues that Dr Chaubet and his colleagues are working to address in the laboratory, alongside investigating other aspects of the project’s overall agenda, such as the development of chemical linkers to connect the cytotoxic drug to an antibody in a covalent manner. These linkers have a strong influence on the therapeutic potency of an ADC. “The linker can balance the hydrophobicity of the cytotoxic drug. Essentially proteins are very polar – they really like water as a solvent. However, the cytotoxic drugs are not that polar, so they don’t really
do well in water,” says Dr Chaubet. “If you start putting a lot of cytotoxic drugs onto your antibody, you’re affecting the solubility of your antibody. This causes heavily loaded antibodies to precipitate, so they’re no longer soluble in water. By using specific linkers, you can effectively balance this to make heavilyloaded conjugates more soluble.” The majority of these linkers are cleavable, so they will get cleaved under certain conditions, such as a change in pH. This point holds wider interest because of the way that ADCs work in vivo. “They’re injected in vivo and they circulate in the body, via the bloodstream. Near a tumour, some of the ADC will escape the bloodstream and bind to this tumour, because the antibody we’re using can selectively recognise certain proteins called antigens, which may be present on the surface of certain cancer cells,” explains Dr
EU Research
testing of new treatments. The students themselves are conducting complex indepth research during their PhD studies, yet alongside the scientific training they also learn some softer skills in TACT. “Training is provided on things like legal affairs, entrepreneurship and how to write grant proposals and presentations,” says Dr Chaubet. This is part of the wider goal of training the next generation of scientific leaders and equipping them with the skills they will need for their future careers, both scientific and non-scientific. “Some of the students may want to go into industry while others may want to stay in academia. We sit down with the students every few months and have a chat about what they want to do in the future,” continues Dr Chaubet.
The TACT project overall includes research into a wide variety of topics related to ADCs, including not just the development of the linkers, but also the eventual analysis and
The training can then be adapted to reflect the students’ priorities, for example by helping them build contacts in industry and present their research at meetings. Communication skills are very important in this context, a topic which is being addressed within TACT. “We’re trying to help the students to develop their communication skills,” says Dr Chaubet. A lot of effort is also being devoted to communicating the project’s work to the broadest possible audience, the majority of whom are likely to not have a scientific background; Dr Chaubet says two main activities have been identified. “The first is with an illustrator who is putting a kind of graphic novel together to communicate our work and our results,” he outlines. “With this graphic novel we’re trying to give a fairly general picture of what cancer really is. The second activity is an exhibition, which we hope will be ready by the conclusion of the project.”
TACT Targeted Anti-Cancer Therapies
Project Objectives
TACT is a European Training Networks funded by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska Curie Action Grant Agreement No 859458. TACT brings together six academic and three industrial European institutions in a research consortium dedicated to the training of eleven PhD students on the development of next-generation treatments against cancer.
Project Funding
This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska Curie Action Grant Agreement No 859458.
Project Partners
• University of Strasbourg (coordinator) and CNRS – France •Q ueen’s University Belfast, University College London and Almac Discovery – U. K. • Wageningen University & Research – the Netherlands • Technical University of Munich and Heidelberg Pharma – Germany • SpiroChem AG – Switzerland
Contact Details
Project Coordinator, Dr Guilhem Chaubet BIOFUNCTIONAL CHEMISTRY Faculty of Pharmacy 74 route du Rhin 67401 Illkirch cedex E: chaubet@unistra.fr : @EtnTact : @BFC_UMR7199 W: http://www.biofunctional.eu/ W: https://tact-etn.eu/ Dr Guilhem Chaubet Dr Alain Wagner
Dr Guilhem Chaubet is a researcher in the Pharmacy Faculty at the University of Strasbourg. His research interests include the development of new tools for the bioconjugation of tyrosine residues, and the application of multi-component reactions to the site-specific bioconjugation of native proteins.
Cartoon representation of an antibody and an ADC, made by Lison Bernet for TACT’s graphic novel.
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