biostudies-literatureWijfjes ZEuropean Research CouncilDutch Research Council (NWO)948-962https://www.ebi.ac.uk/biostudies/studies/S-EPMC12057635biostudies-literatureUnknown6(6)The aim of therapeutic cancer vaccines is to induce tumor-specific cellular immune responses. This requires tumor antigens to be efficiently processed and presented by antigen-presenting cells, in particular dendritic cells (DCs). In addition, DCs require maturation to upregulate the surface expression and secretion of T cell costimulatory molecules, which is achieved by co-administration of adjuvants in vaccines. Peptide-based antigen vaccination is an attractive strategy due to the established biocompatibility of peptides as well as the dosing control. To enhance the efficacy of peptide-based vaccines, antigens can be targeted to DCs. Antigen-adjuvant conjugates are known to enhance T cell activation by ensuring DC maturation upon antigen delivery. In this study, we aim to combine these two approaches in a single molecule, and present a DC-targeted antibody fragment-antigen-adjuvant (AAA)-conjugate. We generate the AAA-conjugate through a combination of site-specific sortase-mediated chemoenzymatic ligation and click chemistry. <i>Ex vivo</i> T cell activation assays show enhanced efficacy of the AAA-conjugate compared to non-adjuvanted control conjugates. The <i>in vivo</i> performance of the AAA-conjugate was suboptimal, which we hypothesize to be a consequence of the hydrophobic character of the conjugate. <i>In vivo</i> efficacy was rescued by co-administration of antibody fragment-antigen conjugates and antibody fragment-adjuvant conjugates, in which the antigen and adjuvant were separatedly delivered using two different DC-targeting molecules. In conclusion, this study provides a proof-of-concept for effective <i>in vivo</i> antigen-specific T cell activation by targeted delivery of both antigen and adjuvant to DCs in a single or separate molecule using site-specific protein engineering.RSC chemical biologyCo-delivery of antigen and adjuvant by site-specific conjugation to dendritic cell-targeted Fab fragments potentiates T cell responses.PMC1205763518058101069163679921Turlings FWijfjes ZPeters RJRWHagemans IMVerdoes MFennemann FLvan Dalen DEsser-Kahn AManna SScheeren FAFigdor CGRamos Tomillero IClassens Rvan der Schoot JMSvan Dalen FJSchouren Kvan Dinther EAWLe Gall CMfalseCo-delivery of antigen and adjuvant by site-specific conjugation to dendritic cell-targeted Fab fragments potentiates T cell responses.The aim of therapeutic cancer vaccines is to induce tumor-specific cellular immune responses. This requires tumor antigens to be efficiently processed and presented by antigen-presenting cells, in particular dendritic cells (DCs). In addition, DCs require maturation to upregulate the surface expression and secretion of T cell costimulatory molecules, which is achieved by co-administration of adjuvants in vaccines. Peptide-based antigen vaccination is an attractive strategy due to the established biocompatibility of peptides as well as the dosing control. To enhance the efficacy of peptide-based vaccines, antigens can be targeted to DCs. Antigen-adjuvant conjugates are known to enhance T cell activation by ensuring DC maturation upon antigen delivery. In this study, we aim to combine these two approaches in a single molecule, and present a DC-targeted antibody fragment-antigen-adjuvant (AAA)-conjugate. We generate the AAA-conjugate through a combination of site-specific sortase-mediated chemoenzymatic ligation and click chemistry. <i>Ex vivo</i> T cell activation assays show enhanced efficacy of the AAA-conjugate compared to non-adjuvanted control conjugates. The <i>in vivo</i> performance of the AAA-conjugate was suboptimal, which we hypothesize to be a consequence of the hydrophobic character of the conjugate. <i>In vivo</i> efficacy was rescued by co-administration of antibody fragment-antigen conjugates and antibody fragment-adjuvant conjugates, in which the antigen and adjuvant were separatedly delivered using two different DC-targeting molecules. In conclusion, this study provides a proof-of-concept for effective <i>in vivo</i> antigen-specific T cell activation by targeted delivery of both antigen and adjuvant to DCs in a single or separate molecule using site-specific protein engineering.2025-01-01T00:00:00Z2025 Jun2026-05-29T17:03:14.118Z2026-04-08T05:27:08.426ZS-EPMC120576354034317410.1039/d5cb00014a