Presentation Information
[3SBT-01-KL]Directed Evolution of Dendritic Cell–Targeting Peptides for Antigen Delivery
○Yoshirou Kawaguchi1, Mina Yokoyama1, Rie Wakabayashi1, Noriho Kamiya1, Masahiro Goto1 (1. Kyushu University (Japan))
Keywords:
Dendritic cells,Directed evolution,Peptides,Ribosome display
[Purpose]
Dendritic cells (DCs) orchestrate adaptive immunity through receptor-mediated antigen capture, processing, and presentation. Among these receptors, C-type lectin receptors (CLRs) are attractive targets for antigen delivery because they couple ligand recognition with endocytic uptake and can shape distinct immunological outcomes. While small-molecule ligands and antibodies have been explored as CLR-targeting agents, peptides occupy an intermediate space, potentially combining the tissue penetrability of small molecules with the affinity and selectivity of larger biologics. Here, we generated CLR-targeting peptide ligands through directed evolution and evaluated their potential as antigen delivery vehicles capable of eliciting receptor-targeted immune responses in vivo.
[Method]
Peptide ligands were generated by ribosome display against recombinant extracellular domains of DC-expressed CLRs. To suppress enrichment of nonspecific binders, we implemented two strategies: (1) introduction of a β-hairpin structural constraint to reduce peptide flexibility and (2) enzymatic cleavage-based elution in place of conventional EDTA-mediated mRNA recovery. Selected peptides were fused to EGFP as a model antigen. Binding kinetics and apparent dissociation constants (KD) were determined by bio-layer interferometry (BLI). Binding to endogenous receptors was evaluated using splenic DCs, bone marrow-derived DCs (BMDCs), and RAW264.7 macrophages by flow cytometry and confocal laser scanning microscopy (CLSM). Selected peptide–EGFP constructs were further examined in murine immunization models.
[Results]
Ribosome display yielded multiple peptide sequences with KD values ranging from tens of nanomolar to low micromolar affinities. Flow cytometry of BMDCs and splenic DCs demonstrated significantly enhanced cell-surface association of peptide–EGFP fusion proteins compared with EGFP alone, indicating recognition of endogenously expressed receptors. CLSM revealed membrane-associated and intracellular fluorescence signals consistent with receptor-associated uptake. Notably, peptides targeting CD207 (Langerin), expressed on epidermal Langerhans cells, induced significantly elevated antigen-specific antibody titers following intradermal immunization compared with antigen alone.
[Consideration]
Consistent binding to endogenous CLRs across multiple assays supports the structural integrity of the recombinant bait proteins and the robustness of the selection strategy. Intracellular fluorescence partially co-localizing with receptor-specific antibody signals suggests receptor-associated uptake, although the precise trafficking pathway remains to be clarified. Importantly, a CD207-targeting peptide with a KD of approximately 0.3 μM significantly enhanced antigen-specific antibody responses in vivo, indicating that sub-micromolar affinity may be sufficient for receptor-mediated antigen delivery.
[Conclusion]
We generated peptide binders capable of targeting dendritic cell CLRs through directed evolution and demonstrated their ability to enhance antigen-specific humoral immunity in vivo. These peptides provide a platform for receptor-directed antigen delivery. Their potential tissue penetrability may enable future applications in transcutaneous vaccine strategies and other immunomodulatory therapies.
Dendritic cells (DCs) orchestrate adaptive immunity through receptor-mediated antigen capture, processing, and presentation. Among these receptors, C-type lectin receptors (CLRs) are attractive targets for antigen delivery because they couple ligand recognition with endocytic uptake and can shape distinct immunological outcomes. While small-molecule ligands and antibodies have been explored as CLR-targeting agents, peptides occupy an intermediate space, potentially combining the tissue penetrability of small molecules with the affinity and selectivity of larger biologics. Here, we generated CLR-targeting peptide ligands through directed evolution and evaluated their potential as antigen delivery vehicles capable of eliciting receptor-targeted immune responses in vivo.
[Method]
Peptide ligands were generated by ribosome display against recombinant extracellular domains of DC-expressed CLRs. To suppress enrichment of nonspecific binders, we implemented two strategies: (1) introduction of a β-hairpin structural constraint to reduce peptide flexibility and (2) enzymatic cleavage-based elution in place of conventional EDTA-mediated mRNA recovery. Selected peptides were fused to EGFP as a model antigen. Binding kinetics and apparent dissociation constants (KD) were determined by bio-layer interferometry (BLI). Binding to endogenous receptors was evaluated using splenic DCs, bone marrow-derived DCs (BMDCs), and RAW264.7 macrophages by flow cytometry and confocal laser scanning microscopy (CLSM). Selected peptide–EGFP constructs were further examined in murine immunization models.
[Results]
Ribosome display yielded multiple peptide sequences with KD values ranging from tens of nanomolar to low micromolar affinities. Flow cytometry of BMDCs and splenic DCs demonstrated significantly enhanced cell-surface association of peptide–EGFP fusion proteins compared with EGFP alone, indicating recognition of endogenously expressed receptors. CLSM revealed membrane-associated and intracellular fluorescence signals consistent with receptor-associated uptake. Notably, peptides targeting CD207 (Langerin), expressed on epidermal Langerhans cells, induced significantly elevated antigen-specific antibody titers following intradermal immunization compared with antigen alone.
[Consideration]
Consistent binding to endogenous CLRs across multiple assays supports the structural integrity of the recombinant bait proteins and the robustness of the selection strategy. Intracellular fluorescence partially co-localizing with receptor-specific antibody signals suggests receptor-associated uptake, although the precise trafficking pathway remains to be clarified. Importantly, a CD207-targeting peptide with a KD of approximately 0.3 μM significantly enhanced antigen-specific antibody responses in vivo, indicating that sub-micromolar affinity may be sufficient for receptor-mediated antigen delivery.
[Conclusion]
We generated peptide binders capable of targeting dendritic cell CLRs through directed evolution and demonstrated their ability to enhance antigen-specific humoral immunity in vivo. These peptides provide a platform for receptor-directed antigen delivery. Their potential tissue penetrability may enable future applications in transcutaneous vaccine strategies and other immunomodulatory therapies.
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