Intermolecular Protein Interactions and Self-assembly of a Synthetic Therapeutic T-cell Receptor-like Molecule

The emergence of protein engineering has enabled the synthesis of tailor-made therapeutic molecules designed to target specific diseases. One such class of biologics is the immune mobilizing monoclonal T-cell receptor against cancer (ImmTAC). ImmTACs target cancerous cells with high specificity and harness the body’s own immune response. These potentially transformative molecules are unique in the biopharmaceutical space.

The ability to create proteins not found in nature has profound implications for the rational control of phase behaviour, protein-protein interactions (PPIs), and self-assembly. Natural proteins have been finely tuned through evolution to be colloidally stable under physiological conditions, maintaining a delicate balance between solubility and aggregation to ensure proper cellular function. In contrast, engineered proteins like the ImmTACs offer a unique opportunity to explore the effects of deliberate design modifications and the consequences for phase behaviour. These modifications, aimed at enhancing target specificity and immune activation, also impact the intermolecular interactions and hence the propensity of the protein for self-assembly or aggregation.

Here we show that the design of the ImmTAC molecule leads to a highly anisotropic surface charge distribution which in turn results in unexpected phase behaviour. Using a range of analytical tools such as light scattering, analytical ultracentrifugation (AUC), and thermal denaturation, we characterise the protein-protein and protein-excipient interactions and observe the formation of small, but stable oligomers.