Dendritic cell (DC)-based vaccines have shown promise as an immunotherapeutic modality for cancer and infectious diseases in many preclinical studies and clinical trials. Provenge (sipuleucel-T), a DC-based vaccine based on ex vivo-generated autologous DCs loaded with antigens, has recently received FDA approval for prostate cancer treatment, further validating the potential of DC-based vaccine modalities. However, direct antigen delivery to DCs in vivo via DC-specific surface receptors would enable a more direct and less laborious approach to immunization. In this study, the recombinant extracellular domains (ECD) of human and mouse DC-SIGN (hDC-SIGN and mDC-SIGN) were generated as DC-specific targets for mRNA display. Accordingly, an antibody-mimetic library was constructed by randomizing two exposed binding loops of an expression-enhanced 10th human fibronectin type III domain (e10Fn3). After three rounds of selection against mDC-SIGN, followed by four rounds of selection against hDC-SIGN, we were able to evolve several dual-specific ligands, which could bind to both soluble ECD of human and mouse DC-SIGNs. Using a cell-binding assay, one ligand, eFn-DC6, was found to have high affinity to hDC-SIGN and moderate affinity to mDC-SIGN. When fused with an antigenic peptide, eFn-DC6 could direct the antigen delivery and presentation by human peripheral blood mononuclear cell (PBMC)-derived DCs and stimulate antigen-specific CD8+ T cells to secrete inflammatory cytokines. Taken together, these results demonstrate the utility of mRNA display to select protein carriers for DC-based vaccination and offer in vitro evidence that the antibody-mimetic ligand eFn-DC6 represents a promising candidate for the development of an in vivo DC-based vaccine in humans.

Arginine-rich peptides and small-molecule intercalating agents utilize distinct molecular mechanisms for RNA recognition. Here, we combined these distinct binding modules in an effort to create conjugate ligands with enhanced affinity and specificity using the bacteriophage λ N peptide−boxB interaction as a model system. We first designed and synthesized a series of peptide−acridine conjugates using portions of the RNA-binding domain of N protein (11- and 22- residue peptide segments) and then compared the binding affinity, specificity, salt dependence, and structural properties of the RNA−peptide and RNA−peptide−acridine conjugate complexes using steady-state fluorescence, CD spectroscopy, NMR, and native gel mobility shift assays (GMSAs). These analyses revealed that the full-length peptide−acridine conjugate displayed substantially improved RNA binding affinity (∼80-fold; Kd ∼ 15 pM) relative to that of the peptide alone (Kd ∼ 1.2 nM). In accordance, we also observed specificity enhancement (∼25-fold) as determined via comparison of the binding of the best conjugate to a cognate λ boxB RNA with that to a noncognate P22 RNA hairpin (80-fold vs 3.2-fold enhancement). Furthermore, the observed binding enhancement was unique to the full-length conjugate with a flexible linker, implying that the structural context of the acridine presentation was critical. Taken together, our observations support the idea that peptide- and intercalation-based binding can be combined to create a new class of high-affinity, high-specificity RNA-binding ligands.