The magic bullet

ILLUSTRATION: ANNEMARIE ROOS

  Proteins can be deployed therapeutically against a wide range of diseases, including cancer, arthritis, allergies and asthma. Thanks to their unique, three-dimensional structures, they are highly targeted and often produce fewer side effects than medicines based on smaller molecules. Researchers therefore sometimes call them ‘magic bullets’. With the advent of artificial intelligence software such as AlphaFold – which helps design new proteins – the number of possibilities for therapeutics is only growing. Yet the great challenge remains getting proteins to work not just in the laboratory but also in the body. To hit the target more often, researchers are developing techniques to protect these bullets on their journey and deliver them into cells: the ultimate ‘magic’.  

Delivery systems Pharmaceutical biotechnologist Enrico Mastrobattista of Utrecht University works on delivery systems to transport proteins efficiently through the body. ‘The problem is that the body breaks proteins down, which means they naturally do not survive for very long in the bloodstream’, says Mastrobattista. ‘On top of that, you need to be able to deliver proteins in a highly targeted way to the right location.’  

To achieve this, the researchers use computers to design new peptides, which they then synthesise in the laboratory. The peptides form cages that enclose the therapeutic proteins, providing stability in the bloodstream. Mastrobattista: ‘The body breaks proteins down using specific enzymes, among other mechanisms – proteases. We design the peptide cages in such a way that they are less susceptible to this degradation.’  

Protein cages Alongside peptides, the researchers also work with liposomes – vesicles made of phospholipids that likewise enclose proteins. To investigate which protein cages are best able to reach the desired destination, the researchers test their various particles in multiple models. They test the particles in two- and three-dimensional cell culture and then move on to animal models, to see whether the particles can also pass through biological barriers at the lungs or the brain. ‘Sometimes we discover that something we have developed does not do what we want, but instead accumulates in a different organ’, says Mastrobattista. ‘Then we turn our idea around. If, for example, we see that our particles end up in the liver, we contact physicians working on liver diseases and explore whether our delivery system could be applied there.’  

Masking Chemist Joachim Van Guyse of Leiden University works with his group on delivery systems that can mask the activity of a protein in the bloodstream and reveal it at the desired location. They do this, among other approaches, by coating the proteins with biocompatible polymers, which protects them against adverse environmental factors (see box: Polymer packaging). ‘To be able to release the proteins at the desired location, we attach them to polymers using selectively cleavable bonds – cleavable linkers‘, says Van Guyse. ‘These specialised chemical bonds break only under certain conditions, such as a change in pH or in response to specific enzymes. This allows us to ensure that the protein is only released once it has reached the right environment.’

Endosomal escape This type of polymer–protein therapeutic acts primarily at the extracellular level. But for intracellular delivery, different routes must be taken. When a cell takes up a protein, it ends up inside an endosomal vesicle that transports it to the lysosomes for degradation. The great challenge is to release the protein from the vesicle in time – so-called endosomal escape – and to do so in a way that causes as little harm to the cell as possible (see box: Endosomal escape). ‘To achieve this, we are developing carriers for proteins that break apart inside the endosome and release small-molecule drugs that cause the vesicle to rupture’, says Van Guyse. ‘Here too we make use of selectively cleavable bonds, this time to attach the small-molecule drug to the protein. Only at an acidic pH – which the endosome automatically encounters during its journey – does the bond break, releasing the small-molecule drug, which then opens up the endosome.’  

‘What you want is for release to occur rapidly in the target cells and not elsewhere’  

Van Guyse regards intracellular delivery as the greatest challenge in the field of protein delivery. ‘What you want is for release to occur rapidly in the target cells and not elsewhere. But that is always a matter of weighing things up. If the technique you are using is too unstable, proteins are released where they should not be. If it is too stable, release is too slow. Finding that balance is extremely difficult.’  

Little machines Given this complexity, Mastrobattista foresees that protein delivery could one day take a very different direction. In the scenario he sketches, cells themselves would be used as drug-delivery machines, migrating in a highly targeted manner to specific locations and secreting the necessary therapeutic proteins there. ‘We are learning more and more about how cells migrate in particular directions via signals and molecular gradients. This is called chemotaxis, and we can synthetically mimic it in the cell. In this way, you could direct a genetically modified cell that produces therapeutic proteins to a specific site in tissues or organs. The ultimate goal would be to turn our own cells into medicine cabinets that you switch on and off at the DNA level.’

Costly As an example, he cites antibody therapy for people with chronic diseases, autoimmune diseases, or inflammatory conditions. These patients must visit hospital regularly for an antibody infusion, or administer injections themselves at home. ‘That is expensive and inconvenient’, says Mastrobattista. ‘We want to solve that with a single injection, consisting of nanoparticles that deliver the genetic code for the antibodies into cells. You could then switch those cells on to produce the antibodies by swallowing a tablet. Although, even for this process you still need to get biomolecules into the cells somehow, which means we will first have to crack the challenge of endosomal escape.’  

‘Suppose we see that our particles end up in the liver, for instance – we then contact physicians working on liver diseases’  

To deliver proteins inside cells, Enrico Mastrobattista and colleagues are working on techniques to help proteins escape from endosomal vesicles after endosomal uptake. They draw inspiration from membrane-active proteins found in nature. ‘Think of melittin, for example, a peptide in bee venom’, says Mastrobattista. ‘This substance disrupts cell membranes.’ The researchers engineered a similar synthetic peptide in the laboratory that only permeabilises membranes at low pH, and demonstrated how it could deliver the Cas9 protein into cells. The peptides formed a complex that protected the protein during endocytosis. The peptides then selectively disrupted the endosomal vesicles in the low-pH environment, releasing the protein so that it could carry out gene correction. Breaking open the endosome was not the actual problem, Mastrobattista explains. ‘You could, in a manner of speaking, open an endosome with soap – but then the cell dies. The method we developed works to a certain extent, but can also be highly toxic at high concentrations. It is a very narrow therapeutic window.’ The peptide – or indeed any technique for intracellular delivery – must therefore be potent enough to help sufficient proteins escape, yet subtle enough not to harm the rest of the cell. The higher the working concentration, the higher the toxicity. ‘We have not yet mastered this balance sufficiently. In that respect, current methods for endosomal escape are very crude: we are firing buckshot to perforate the vesicles. But you do not want the toxic contents of the endosome to be released into the cytosol as well. Finding that subtle equilibrium is the great challenge.’  

Joachim Van Guyse and his research group are developing polymers to protect proteins during their circulation in the body. By encasing proteins in a coat of biocompatible polymers, their circulation time is extended and an adverse immune response is prevented. Van Guyse is currently searching for alternatives to the widely used polyethylene glycol (PEG), which, despite its success in various medicines, is still not optimal. ‘PEG was also present in the COVID-19 vaccines, for example’, says Van Guyse. ‘Increasingly, antibodies are emerging that can recognise this polymer in a vaccine. That can lead to faster clearance from the bloodstream or, again, a possible immune response.’ To circumvent this problem, the researchers are working on two potential alternatives: poly(2-oxazoline)s and polysarcosine. Whereas poly(2-oxazoline)s offer high stability and structural variability, polysarcosine is composed of the endogenous amino acid sarcosine, making it biodegradable. The researchers study the behaviour of these polymers at multiple levels: in cell cultures, in zebrafish embryos – which, owing to their transparency, are well suited to visualising the circulation and distribution of the proteins – and in mice. In doing so, Van Guyse and his colleagues continue to encounter several challenges. ‘Unfortunately, in vitro results rarely match what happens in vivo. It remains challenging to get the proteins to the right location in mice and to achieve an effect that is purely local. The body is a remarkably complex entity that we still do not fully understand.’