In WP1, novel methods mapped macrophage and monocyte subsets in human carotid plaques. Advanced multispectral and high-plex imaging identified eight distinct populations, including pro- and anti-inflammatory and lipid-associated macrophages. A refined antibody panel was established, integrating new markers to characterize immune cells in relation to plaque features and patient traits. Disease-relevant in vitro macrophage models, including inflammatory lipid-associated macrophages (ILAMs), are under development to capture key aspects of human plaque biology.
In WP2, strategies were developed to identify HuAbs binding macrophage subsets in vulnerable plaques. A next-generation sequencing (NGS) pipeline was implemented for phage display outputs using PacBio long-read sequencing, enabling full-length coverage of scFv antibody sequences, accurate annotation, and clonotype identification. Collaboration with IMGT ensured precise filtering, annotation, and clustering of sequences, complemented by Sanger-sequenced clones to validate recurrent clones and affinity-impacting mutations. This enabled selection of the top 100 enriched scFv sequences, now undergoing validation through flow cytometry, immunohistochemistry, and in vitro assays.
Within WP3, we focused on optimizing production and purification of selected HuAb fragments. Novel vector systems and tag-based purification strategies improved yields and stability, enabling efficient tracer grafting and milligram-scale production for biological validation.
In WP4, proteomic studies of aortic samples identified proteins differentially expressed in diseased versus healthy vessels. Computational analyses confirmed that two of the most promising antibodies recognise Galectin-3, a protein implicated in plaque inflammation. Biochips have been designed to further assess antibody-target interactions and specificity across macrophage subsets.
In WP5, two lead antibodies (P3, C10) targeting Galectin-3 were radiolabelled with zirconium-89 and tested in animals. Both showed high stability and suitable pharmacokinetics. PET imaging revealed uptake in plaque-prone regions, with P3 showing promise for detecting early atherosclerotic lesions. Therapeutic studies in advanced mouse models are planned.
