Presentation Information
[P04-484]Selective antigen delivery to Langerhans cells using CD207-binding peptides
○Mina Yokoyama1, Yoshirou Kawaguchi1, Rie Wakabayashi1, Noriho Kamiya1,2, Masahiro Goto1,2,3 (1. Graduate School of Engeineering, Kyushu University (Japan), 2. Center for Future Chemistry, Kyushu Univercity (Japan), 3. Next Generation Transdermal Absorption Research Center (Japan))
Keywords:
peptide,directed evolution,Ribosomal display,targeting
Introduction
Langerhans cells (LCs) are specialized antigen-presenting cells residing in the epidermis and express the C-type lectin receptor CD207, which mediates antigen uptake and internalization. Because epidermal LCs are positioned to encounter antigens at the skin surface and regulate subsequent immune responses, CD207 provides a rational molecular target for skin-based antigen delivery strategies. Current approaches for targeting LCs have reported carbohydrate ligands or monoclonal antibodies against CD207. While carbohydrate moieties are small molecular size, their limited interaction interface may restrict binding affinity and specificity. In contrast, antibodies provide high affinity but are constrained by large molecular size and limited tunability. On the other hand, peptides offer an alternative modality with moderate size and structural versatility, enabling precise control of binding properties.In this study, we explored CD207-targeting peptides using the ribosome display and evaluated their potential for antigen delivery to LCs.
Method
The extracellular domain of mouse CD207 (mCD207) was expressed in E. coli and refolded. Using this domain, three rounds of ribosome display-based bio-panning were performed. Two peptide libraries were employed: disulfide-cyclized peptides (Library 1) and beta-hairpin-structured peptides (Library 2). Selected peptides were fused to the N-terminus of a model antigen (EGFP), and their binding affinity to the mCD207 extracellular domain was measured by bio-layer interferometry (BLItz). Selective antigen delivery to mCD207-positive cells was evaluated by flow cytometry using transiently transfected Expi293 cells and mouse-derived epidermal cells. Mice were intradermally immunized with WT-EGFP (15 µg) or peptide 4–EGFP (15 µg) at a 1-week interval. Antigen-specific serum IgG levels were quantified by enzyme-linked immunosorbent assay.
Result and Discussion
After three rounds of bio-panning, enriched peptide candidates were obtained. Quantification of the recovered mRNA revealed that the recovery amount increased with increasing amounts of added the mCD207 extracellular domain. These results suggest a concentration-dependent enrichment of peptide binders toward mCD207. Binding affinity analysis using the BLItz system demonstrated that four candidate peptides (peptides 1–4) exhibited affinity for mCD207, with dissociation constants ranging from 300 nM to 3 µM. Peptide 1 was derived from Library 1, whereas peptides 2–4 were obtained from Library 2. In antigen delivery assays using mCD207-expressing mammalian cells, peptides 3 and 4 selectively bound to mCD207-positive cells, with minimal binding to mCD207-negative controls. Furthermore, peptides 3 and 4 also showed preferential binding to primary mouse LCs in ex vivo epidermal cell suspensions.In vivo immunization studies demonstrated that peptide 4–EGFP induced an approximately 100-fold higher EGFP-specific serum IgG response compared to EGFP alone. These findings indicate that peptide-mediated targeting of CD207 improves antigen delivery efficiency and enhances humoral immune responses.
Conclusion
mCD207-binding peptides were identified through ribosome display–based bio-panning. Peptide-fused antigens enabled selective delivery to LCs and enhanced antigen-specific antibody responses in mice.
Langerhans cells (LCs) are specialized antigen-presenting cells residing in the epidermis and express the C-type lectin receptor CD207, which mediates antigen uptake and internalization. Because epidermal LCs are positioned to encounter antigens at the skin surface and regulate subsequent immune responses, CD207 provides a rational molecular target for skin-based antigen delivery strategies. Current approaches for targeting LCs have reported carbohydrate ligands or monoclonal antibodies against CD207. While carbohydrate moieties are small molecular size, their limited interaction interface may restrict binding affinity and specificity. In contrast, antibodies provide high affinity but are constrained by large molecular size and limited tunability. On the other hand, peptides offer an alternative modality with moderate size and structural versatility, enabling precise control of binding properties.In this study, we explored CD207-targeting peptides using the ribosome display and evaluated their potential for antigen delivery to LCs.
Method
The extracellular domain of mouse CD207 (mCD207) was expressed in E. coli and refolded. Using this domain, three rounds of ribosome display-based bio-panning were performed. Two peptide libraries were employed: disulfide-cyclized peptides (Library 1) and beta-hairpin-structured peptides (Library 2). Selected peptides were fused to the N-terminus of a model antigen (EGFP), and their binding affinity to the mCD207 extracellular domain was measured by bio-layer interferometry (BLItz). Selective antigen delivery to mCD207-positive cells was evaluated by flow cytometry using transiently transfected Expi293 cells and mouse-derived epidermal cells. Mice were intradermally immunized with WT-EGFP (15 µg) or peptide 4–EGFP (15 µg) at a 1-week interval. Antigen-specific serum IgG levels were quantified by enzyme-linked immunosorbent assay.
Result and Discussion
After three rounds of bio-panning, enriched peptide candidates were obtained. Quantification of the recovered mRNA revealed that the recovery amount increased with increasing amounts of added the mCD207 extracellular domain. These results suggest a concentration-dependent enrichment of peptide binders toward mCD207. Binding affinity analysis using the BLItz system demonstrated that four candidate peptides (peptides 1–4) exhibited affinity for mCD207, with dissociation constants ranging from 300 nM to 3 µM. Peptide 1 was derived from Library 1, whereas peptides 2–4 were obtained from Library 2. In antigen delivery assays using mCD207-expressing mammalian cells, peptides 3 and 4 selectively bound to mCD207-positive cells, with minimal binding to mCD207-negative controls. Furthermore, peptides 3 and 4 also showed preferential binding to primary mouse LCs in ex vivo epidermal cell suspensions.In vivo immunization studies demonstrated that peptide 4–EGFP induced an approximately 100-fold higher EGFP-specific serum IgG response compared to EGFP alone. These findings indicate that peptide-mediated targeting of CD207 improves antigen delivery efficiency and enhances humoral immune responses.
Conclusion
mCD207-binding peptides were identified through ribosome display–based bio-panning. Peptide-fused antigens enabled selective delivery to LCs and enhanced antigen-specific antibody responses in mice.
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