Using peptides to promote delivery and improve anti-tumour efficacy of liposomal drug.

Liposomal drugs exhibit advantages for cancer therapy, but efficacy is often limited by their rapid clearance from the blood by the reticuloendothelial system, and an inability to target and penetrate tumours. Interestingly, a 21-amino acid SIRP-α- (signal regulatory protein-α) interacting 'self' peptide is reported to inhibit uptake by phagocytes. Also, 'iRGD' a 9-amino acid cyclic peptide that binds αvβ3 integrins and neuropilin-1 (NRP-1), promotes targeting and penetration of the drug into tumours.

Repeated fine-needle aspiration of solid tumours in mice allows the identification of multiple infiltrating immune cell types.

This paper describes a novel method for following the changes in mouse tumour-infiltrating immune cell populations by repeated sampling of tumours by fine needle aspiration (FNA), followed by flow cytometry. Using this technique we were able to collect samples from P815 mouse mastocytomas, and identify and enumerate six tumour-infiltrating immune cell types at multiple time points for each mouse. We demonstrate good agreement between cell percentages obtained from FNA samples and matched whole tumour digests (WTDs).

pDNA-lipoplexes engrafted with flagellin-related peptide induce potent immunity and anti-tumour effects.

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).

Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature.

The use of liposomes to target drugs to tumors represents an attractive therapeutic strategy, especially when used with convenient targeting moieties such as peptides. Here we explored several peptides for their ability to target liposomes to tumors. The metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) was incorporated into liposomes to enable the engraftment of His-tagged peptides containing targeting motifs specific for tumor vasculature markers VEGFR-1 (p39-Flt-1) and neuropilin-1 (p24-NRP-1), or a motif known to accumulate in hypoxic areas of tumors (p47-LyP-1).

Increasing the antitumor efficacy of doxorubicin-loaded liposomes with peptides anchored via a chelator lipid.

The therapeutic efficacy of anticancer drugs like doxorubicin can be significantly increased by their incorporation into liposomes, but an ability to actively target the drug-containing liposomes to tumors could well provide an even greater curative effect. In this work, a commercial preparation of doxorubicin-loaded liposomes (Caelyx) was modified by incorporation of the metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to enable engraftment of histidine-tagged targeting molecules. Our results show that when engrafted with p15-RGR, a His-tagged peptide containing a sequence purported to bind platelet-derived growth factor receptor β (PDGFRβ), NTA(3)-DTDA-containing Caelyx (3NTA-Caelyx) can be targeted to NIH-3T3 cells in vitro, leading to increased cytotoxicity compared with non-targeted 3NTA-Caelyx.

Ag-bearing liposomes engrafted with peptides that interact with CD11c/CD18 induce potent Ag-specific and antitumor immunity.

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.

Antigen-containing liposomes engrafted with flagellin-related peptides are effective vaccines that can induce potent antitumor immunity and immunotherapeutic effect.

The bacterial protein flagellin can trigger immune responses to infections by interacting with TLR5 on APCs, and Ag-flagellin fusion proteins can act as effective vaccines. We report that flagellin-related peptides containing a His-tag and sequence related to conserved N-motif (aa 85-111) of FliC flagellin, purportedly involved in the interaction of flagellin with TLR5, can be used to target delivery of liposomal Ag to APCs in vitro and in vivo. When engrafted onto liposomes, two flagellin-related peptides, denoted as 9Flg and 42Flg, promoted strong liposome binding to murine bone marrow-derived dendritic cells and CD11c(+) splenocytes, and cell binding correlated with expression of TLR5.

Targeting of plasmid DNA-lipoplexes to cells with molecules anchored via a metal chelator lipid.

Background: The ability to deliver plasmid DNA (pDNA) to specific cells in vivo is crucial for achieving efficient targeted transfection with nonviral vectors. We previously used stealth liposomes containing the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to target delivery of antigen and cytokines to immune cells in vivo. In the present study, we utilized liposomes containing NTA(3)-DTDA and the ionizable aminolipid 1,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP) to incorporate pDNA into complexes for targeting to cells.

Liposomal Ag engrafted with peptides of sequence derived from HMGB1 induce potent Ag-specific and anti-tumour immunity.

High-mobility group box 1 (HMGB1) protein is a nuclear binding protein which is released by monocytes and macrophages and is a potent maturation signal for dendritic cells (DCs). Synthetic HMGB1-related peptides are reported to be potent DC stimulants. Two HMGB1-related peptides, denoted as pHMGB-89 and pHMGB-106, were explored for their ability to enhance the immunogenicity of Ag-containing liposomes.

Convenient targeting of stealth siRNA-lipoplexes to cells with chelator lipid-anchored molecules.

A major obstacle for the use of siRNAs as novel therapeutics is the requirement for functional delivery to specific cells in vivo. siRNA delivery by cationic agents is generally non-specific and a convenient targeting strategy has been lacking. This work explored the potential for using the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) with neutral stealth liposomes to target siRNA to cells.

Targeted liposomal delivery of TLR9 ligands activates spontaneous antitumor immunity in an autochthonous cancer model.

Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment.

Liposomal vaccines--targeting the delivery of antigen.

Vaccines that can prime the adaptive immune system for a quick and effective response against a pathogen or tumor cells, require the generation of antigen (Ag)-specific memory T and B cells. The unique ability of dendritic cells (DCs) to activate naïve T cells, implies a key role for DCs in this process. The generation of tumor-specific CD8(+) cytotoxic T cells (CTLs) is dependent on both T cell stimulation with Ag (peptide-MHC-complexes) and costimulation.

The novel chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) promotes stable binding of His-tagged proteins to liposomal membranes: potent anti-tumor responses induced by simultaneously targeting antigen, cytokine and costimulatory signals to T cells.

Recent studies indicate that the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) can be used to engraft T cell costimulatory molecules onto tumor cell membranes, potentially circumventing the need for genetic manipulation of the cells for development of cell- or membrane-based tumor vaccines. Here, we show that a related lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA, which has three NTA moieties in its headgroup instead of one) is several-fold more effective than NTA-DTDA at promoting stable His-tagged protein engraftment. IAsys biosensor studies show that binding of His-tagged B7.

Targeting dendritic cells with antigen-containing liposomes: antitumour immunity.

Dendritic cells (DCs) are antigen-presenting cells that play an important role in the body's immune defence against cancer. Strategies using antigen-primed DCs as tumour vaccines show promise in patients, but the approach is cumbersome to use clinically. Soluble tumour antigens can be targeted to DCs in vivo, but this often induces antigenic tolerance rather than immunity.

Plasminogen is tethered with high affinity to the cell surface by the plasma protein, histidine-rich glycoprotein.

Plasminogen has been implicated in extracellular matrix degradation by invading cells, but few high affinity cell surface receptors for the molecule have been identified. Previous studies have reported that the plasma protein, histidine-rich glycoprotein (HRG), interacts with plasminogen and cell surfaces, raising the possibility that HRG may immobilize plasminogen/plasmin to cell surfaces. Here we show, based on optical biosensor analyses, that immobilized HRG interacts with soluble plasminogen with high affinity and with an extremely slow dissociation rate.

Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy.

Dendritic cells (DCs) are potent stimulators of immunity, and DCs pulsed with tumor antigen ex vivo have applications in tumor immunotherapy. However, DCs are a small population of cells, and their isolation and pulsing with antigen can be impractical. Here we show that a crude preparation of plasma membrane vesicles (PMV) from the highly metastatic murine melanoma (B16-OVA) and a surrogate tumor antigen (OVA) can be targeted directly to DCs in vivo to elicit functional effects.

A novel approach for modifying tumor cell-derived plasma membrane vesicles to contain encapsulated IL-2 and engrafted costimulatory molecules for use in tumor immunotherapy.

The genetic modification of tumor cells and delivery of cytokines have been proposed as useful strategies in the development of anti-tumor vaccines; however, a number of factors limit their use in clinical settings. To facilitate vaccine development, we explored the possibility of modifying plasma membrane vesicles (PMV) by using a novel chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). Our analyses by flow cytometry show that NTA-DTDA can be incorporated into PMV prepared from murine P815 mastocytoma and that the incorporated NTA-DTDA permits anchoring or "engraftment" onto the vesicle surface of hexahistidine-tagged proteins such as recombinant forms of the costimulatory molecules B7.