Co-delivery of antigen and adjuvant by site-specific conjugation to dendritic cell-targeted Fab fragments potentiates T cell responses

This data is collected within the context of the doctoral thesis of Zacharias Wijfjes, Chapter 3. It covers the development and testing of a site-specific targeted antigen-adjuvant conjugate. The work presents a molecularly defined DC-targeted antibody fragment-antigen-adjuvant (AAA) conjugate. The AAA-conjugate is prepared through a combination of CRISPR/HDR hybridoma genome editing, sortase-mediated chemoenzymatic conjugation, and strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. We successfully generate and characterize the AAA-conjugate and demonstrate unaltered binding capacity due to the cargo being conjugated site-specifically, away from the binding domain of the Fab fragment. Ex vivo, an increase in T cell activation is observed for the AAA-conjugate compared to the antibody fragment-antigen conjugate. We attribute this to a combination of DC maturation induced by the adjuvant and the hydrophobic characteristics of the adjuvant, which seem to enhance cross-presentation. Suboptimal pharmacokinetics hinder in vivo efficacy of the AAA-conjugate, but the data is hinting towards the self-adjuvating capacity of the small fraction of the AAA-conjugate that does reach DCs in vivo. As alternative approach to overcome the suboptimal pharmacokinetics, we present the combination of targeted antigen and targeted adjuvant on separate Fab fragments, which did result in enhanced T cell activation in vivo.