The ability of dendritic cells (DC) to be either tolerogenic or immunogenic offers many
opportunities to manipulate DC function and genetically modify DC to promote allograft
acceptance. However, the greatest challenge in DC gene therapy is the difficulty of
achieving successful transfection. At present, DC-targeting gene delivery strategies are
hindered by the lack of DC-specific target molecules. Therefore, the essential aim of this
study is to isolate and identify peptides capable of targeting immature DC (iDC), in
particular those that internalise well as they are likely to be more effective in gene
delivery.
Phage library of short cyclic peptides expressed on pIII protein of M13 filamentous phage
was used first in selection against streptavidin. Phage bearing HPQ and HPM motifs were
successfully isolated indicating the feasibility of the library in selecting streptavidin-binding
peptides through specific biotin interaction. In order to develop a method for in vitro
selection on cell surfaces, the library was used against a cell line expressing αVβ3 integrin.
One phage clone with encoded sequence of CLSSPALLC bound to the cell surface and
was taken up by the cells expressing the αVβ3 integrin.
Using methods established in the previous panning, the library was panned against iDC,
after two subtractive pannings against PBMC and CD14+ cells. The phage isolated were
characterised by using several methods including flow cytometric analysis and confocal
microscopy. Two phage selected, DC4.25 and DC4.44 with encoded peptide sequences
of CFPPTFPAC and CTPLLSPFC, respectively, have demonstrated the ability to bind to,
and be internalised by, iDC. Soluble biotinylated peptides, which contain the encoded
peptide sequences, were synthesised along with sequence-scrambled control peptides.
Flow cytometric analysis detected non-specific binding of the monomeric peptides to iDC
when used at high concentration. In an attempt to increase the binding avidity of the
peptides to iDC, Streptavidin conjugated with Alexa-488® flurophore was used to form
complexes with the peptides. However, flow cytometric analysis revealed that the
complexes also bound non-specifically to iDC, as binding was also seen in control
peptides. Streptavidin conjugated with HRP was then used to form complexes with the
peptides, in order to increase the sensitivity of the binding assay. The results corroborated
the previous findings, which showed that the peptides bound iDC non-specifically.
The failure of the synthetic peptides to bind to iDC might be due to a number of reasons,
including loss of cyclised conformation of the peptides during synthesis and insufficient
incorporation of sequences that is essential and accountable for the binding, in the synthetic peptides. However, this approach does offer the possibility of identifying cell
binding peptides that may allow delivery of genes and other therapeutic or imaging
agents.