New tentacle method captures tumour cells with increased efficiency
The development of an improved ‘superglue’ technology by Dr Mark Howarth and his group could help in cell capture approaches aimed at detecting and treating cancer.
Dr Howarth together with DPhil students Gianluca Veggiani and Jacob Fierer have published their work in PNAS (1). They describe the new tool, developed from an earlier approach in which they engineered a stable interaction that could be used to lock molecules together.
The original method, SpyTag/SpyCatcher, was published in 2012 (2) and has already found a number of uses. It was developed from an adhesion protein found in Streptococcus pyogenes which locks itself together. The group took a domain of the protein and split it into two parts to create protein-peptide ligation between a peptide tag (SpyTag) and a protein domain (SpyCatcher).
As a covalent and irreversible interaction, the SpyTag/SpyCatcher ligation has a major advantage over other types of engineered interactions. But it also has a drawback which the new system addresses.
‘The protein domain is quite big and may interfere with protein folding and function,’ explains Gianluca Veggiani. ‘In the new scenario, we split the domain into 3 partners – two peptide tags and a protein domain. The protein domain is only required to catalyse the reaction and form the bond.’
With this bulky domain required only transiently now, the new system, called ‘SpyLigase’, allows assemblies of proteins to be built up.
Cartoon showing the three-part design of the new method, with SpyTag peptide (blue), KTag peptide (pink) and SpyLigase (green). Residues involved in the reaction are coloured red (Click to enlarge)
The group decided to apply their new method to affibodies (small engineered antibody mimics) and antibodies, and were able to generate large polymers.
‘We think we have chains, although we don’t know about the 3D structure,’ says Gianluca Veggiani. ‘There is a broad range of sizes, going up to at least 20 units.’
To test the biomedical potential of the new method, the researchers looked at how effective it was in circulating tumour cell (CTC) capture, an approach used to monitor treatment in clinical cancer trials.
CTCs are cells that have worked free from a tumour and have the potential to form metastases. They can be detected and captured via specific target molecules on their surface, using magnetic beads coated with antibodies against these molecules. But CTCs are very rare and the expression of markers on the surface can be highly variable making capture difficult.
‘We are limited by the number of copies of target molecules on the cell surface – we need a minimum number to detect a cell and isolate it from the bloodstream,’ explains Gianluca Veggiani. ‘So we thought that if we can bring the cells together with multiple binding events, that would increase the chance of pulling out these cells. They will be bound to the beads more strongly than if it’s a single binding event.’
The researchers coated the magnetic beads with either monomeric affibody or affibody built up into a polymeric assembly using SpyLigase. They then compared how well each type pulled out cells alone as well as cells doped into a blood sample.
Polymeric beads were much better than monomeric beads at extracting cells in both cases, when tested with two clinically relevant markers – Epidermal Growth Factor Receptor (EGFR) or HER2. The difference was particularly marked when cells expressed low levels of the receptor, with almost no recovery using monomeric beads.
Alongside this, the researchers checked the specificity of their system by imaging the cells captured by the beads.
Cartoon of SpyLigase covalently joining KTag on one affibody to SPyTag on another, so directing polymerisation (Click to enlarge)
‘With increasing valency some non-specificity could arise,’ says Gianluca Veggiani. ‘When we imaged cells that had been captured, we could detect a few white blood cells (non-specific capture), but comparable numbers between polymeric and monomeric beads. So the polymers are both effective and specific.’
Dr Howarth says that the experiments are promising in terms of applying the technology to a range of applications in the future.
‘In terms of clinical use, CTC capture is very widely used in clinical trials to follow success of treatment and understand what is going on. It is just starting to be used in clinical practice outside trials, for example for prediction and prognosis.’
‘Research is moving towards using microfluidic devices to isolate cells. Our new approach could be used in this scenario. In fact, in many applications where you have an antibody on the surface, you could do better by building up a polymer as we have done.’
SpyLigase has advantages over other tools for building protein complexes - the interaction is covalent and because the peptide tag can be placed anywhere in the protein, the method could be used to assemble branched structures.
In addition, the use of multi-specific polymers could get round the problem of unpredictability of cancer cell marker expression. ‘If the cells are not expressing one receptor, maybe you have a chance of capturing the cells using another,’ says Gianluca Veggiani, who will be carrying out experiments to test bi-specific polymers of EGFR and HER2.
In the longer term, Dr Howarth says, the group would like to explore protein assembly using SpyLigase in more detail. ‘We are going to be improving our ability to assemble complex protein architectures and our understanding of the specific interactions of the chains and what’s happening at the cell membrane when these interactions form.’
‘We are also going to take the work into more clinical applications with collaborators at the John Radcliffe Hospital. We want to ask whether we can find new CTCs that have been missed by the current approaches and from these, see what we can learn about the course of the disease and how to treat it.’
*joint first author