Protein-protein interactions and self-assembly of a T-cell Receptor-like protein

Abstract

Protein engineering enables the creation of tailor-made proteins for an array of applications,
and successful formulation is central to translating new molecules to the point of being suitable
for delivery to patients. Molecules not found in nature present new challenges for formulation;
unlike natural proteins they have not undergone evolutionary optimisation for stability in physiological conditions, which requires a delicate balance of weak interactions. Often designed to
have a high degree of specificity, solution stability is usually not accounted for in the process
of designing new synthetic molecules. ImmTACs stand out as promising therapeutics for cancer and other treatments, while also presenting unique challenges for stability, formulation and
delivery.
We have shown that ImmTACs behave as Janus particles in solution, leading to self-association
at low concentrations, even when the net surface charges suggest that the molecule should be stable. The formation of small but stable oligomers for a molecule referred to as ImmTAC1 has been
confirmed by a range of techniques including static and dynamic light scattering, and analytical ultracentrifugation. Through structural analysis of existing X-ray crystallogrpahy structures
complimented by modelling using AlphaFold, the highly anisotropic nature of the surface charge
distribution is observed, providing a rational explanation for this self-assembly behaviour, consistent with the Janus particle assembly observed for inverse patchy particles.
We shed further light on this phenomenon by comparing two ImmTAC molecules. Despite
sharing a nearly identical structural template, ImmTAC1 and ImmTAC2 exhibit markedly different solution behaviors due to variations in their charge distributions. ImmTAC2, characterized
by pronounced charge patchiness and a near-neutral net charge at physiological pH, is significantly less stable than ImmTAC1, which has a more evenly distributed charge profile and a net
negative charge. Using size and polydispersity analysis through light scattering techniques, we
demonstrate that ImmTAC2 presents more pronounced aggregation behaviour than ImmTAC1.
The addition of Tween 80 effectively mitigated aggregation of ImmTAC2 at low concentrations,
improving homogeneity by reducing surface-induced aggregation. Additionally, ImmTAC2 displayed enhanced stability at pH 8.5, where increased net charge promotes electrostatic repulsion. The findings highlight that aggregation of ImmTACs is driven by a combination of charge
patchiness, proximity to its isoelectric point, and surface effects. These insights have critical im-
ii
plications for the formulation of ImmTAC-based therapeutics: strategies that modulate protein
charge (e.g. pH adjustment) can significantly improve stability and solubility, ensuring better
therapeutic performance and manufacturability.
Finally, Monte Carlo simulations were employed to investigate the aggregation behavior of
ImmTAC particles under varying electrostatic screening conditions and particle concentrations.
A coarse grained model was developed for ImmTAC-like particles to study the effects of charge
distribution and Debye length on clustering, revealing a strong dependence on electrostatic interactions. Simulations highlighted a delicate balance between long-range repulsion and shortrange attraction in determining cluster size and stability. ImmTAC2-like particles, characterized
by lower net charge, and more extreme anisotropy exhibited a higher propensity for aggregation, in agreement with experiments. Two-particle simulations further confirmed stronger selfinteraction behaviour in ImmTAC2 due to reduced electrostatic repulsion. These results provide
critical insights into the role of charge anisotropy and electrostatic interactions in ImmTAC aggregation, emphasizing the importance of tuning charge distributions and screening conditions
to optimize stability for formulation and therapeutic applications.

Date of Award	30 Sept 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jennifer McManus (Supervisor) & Shahid Uddin (Supervisor)