Influence of different conjugation methods for activating antibodies on polymeric nanoparticles: Effects for...

Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies.