No abstract

Material Supplementary 7.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.100002

Dendritic cells (DCs) play key role in eliciting antigen (Ag)-specific immune responses, and crucial to this is the uptake of Ag via surface receptors including the heterodimeric integrin CD11c/CD18. Here we report that CD11c/CD18-interacting peptides can be used as targeting moieties to deliver liposomal Ag to antigen presenting cells (APCs) and elicit Ag-specific and antitumor immunity. Two peptides of sequence related to human ICAM-4 and previously reported to bind CD11c/CD18, and a 12-mer cyclic peptide previously identified by phage display to bind CD11c/CD18, were produced synthetically, and tested for their ability to target liposomal Ag. The three peptides were designed to contain a shorter spacer to reduce steric hindrance, and a His-tag to enable engraftment onto liposomes incorporated with chelator lipid. Our results show that the three peptides, denoted as p17, p18 and p30, promote strong binding of liposomes to CD11c 1 and CD11b 1 cells in vitro and in vivo. Vaccination of mice with Ag-bearing liposomes engrafted with the peptides, particularly p18 and p30, induced Agspecific T cell priming and antibody production. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA-derived plasma membrane vesicles (PMVs) engrafted with p18 and p30 peptide showed dramatic antitumor responses, inhibiting tumor growth/metastasis in both the lung and subcutaneous tumor models, with a high proportion of the mice apparently being ''cured'' of their tumors. The engraftment of p18 and p30 peptides onto liposomes and PMVs, thus provides an effective means to target Ags to DCs in vivo, for the development of effective cancer vaccines and immunotherapies.The use of antigen-pulsed dendritic cells (DCs) has shown promise for developing cell-based cancer vaccines and immunotherapies. 1 DCs play a key role in eliciting Ag-specific immune responses, and crucial to this is the uptake of antigen (Ag) via a number of DC surface receptors such as Fc-receptors, C-type lectins, Toll-like receptors (TLRs) and integrin receptor CD11c/CD18. [2][3][4][5][6] The unique ability of DCs to prime naïve T cells makes them ideal targets for Ag-delivery and vaccine development. 7 To better exploit this potential, techniques have been used to target protein-Ag to DCs in vivo, potentially circumventing the need to manipulate DCs ex vivo for cancer immunotherapy. 8,9 Typical strategies involve conjugation of Ag to mAbs, ScFv or Fab fragments specific for a DC surface marker or Ag-uptake receptor. 4,6,10,11 To facilitate the delivery of multiple Ags simultaneously, we previously attached recombinant co-stimulatory molecules and ScFv to Ag-containing delivery vehicles such as liposomes and membrane vesicles. 8,12 The vaccination of mice with such constructs can elicit potent immune responses and inhibit tumor growth in tumor models. 12 However, mAbs, ScFvs, and recombinant proteins for use as DC-targeted conjugates, or as targeting moieties for liposomes, can be time-consuming and expensive to produce for clinical applications.The use of...

Complexes of cationic lipids and DNA (lipoplexes) are widely used for non-viral gene delivery and DNA vaccine development, but cationic lipids are toxic and promote non-specific interactions with cells, leading to poor efficacy. Near-neutral lipoplexes, on the other hand, can obviate toxicity, but a convenient means to target them to specific cells such as dendritic cells (DCs) has been lacking. Here, we show that a His-tagged flagellin-derived peptide (denoted 9Flg), previously reported to promote binding of liposomal antigen to TLR5-expressing cells, can be used to target near-neutral pDNA-lipoplexes incorporating the chelator lipid NTA(3)-DTDA (3(nitrilotriacetic acid)-ditetradecylamine) to DCs and other antigen-presenting cells (APCs). Thus, we show that pDNA-lipoplexes engrafted with 9Flg target pDNA to APCs in vitro and in vivo. Following i.v. administration, radiolabelled 9Flg-lipoplexes exhibited increased accumulation in spleen, lung and liver. Vaccination of C57BL/6 mice with 9Flg-lipoplexes containing either pcDNA3.1-SIIN (pSIIN) or a Kunjin virus replicon-based vector (pKUN), each encoding the epitope OVA(257-264) (SIINFEKL), induced Ag-specific T cell priming, and elicited strong cellular immunity as reflected by a marked increase in the number of Ag-responsive IFN-γ-producing CD8(+) T cells. Importantly, compared to i.m. injection of these SIINFEKL-encoding pDNAs in naked form, the i.v. administration of pSIIN or pKUN in 9Flg-lipoplexes to C57BL/6 mice induced a significantly more potent anti-tumour response in the B16-OVA melanoma tumour model. The targeting of near-neutral 9Flg-lipoplexes bearing pDNA encoding tumour antigens to TLR5 on APCs, therefore, is a powerful approach for developing more effective DNA vaccines and immunotherapies.