Polyarginine-Mediated Protein Delivery to Dendritic Cells Presents Antigen More Efficiently onto MHC Class I and Class II and Elicits Superior Antitumor Immunity

Abstract

Protein transduction domains (PTDs) have been used increasingly to deliver reagents to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DCs) stimulated Ag-specific CD8+ and CD4+ T cells. Although the cytotoxic T lymphocytes (CTL) activity generated was sufficient to prevent engraftment of mice with Ag-expressing tumors, treatment of tumor-bearing mice with TAT-PTD Ag-transduced DCs resulted in tumor regression in some animals. Recently, several other PTDs were reported to promote higher transduction efficiencies than TAT-PTD. To evaluate the role of individual PTDs in induction of immune responses in tumor vaccination studies, we engineered recombinant fusion Ovalbumin (OVA) that contained three differrent PTDs, including the most efficacious known PTD (polyarginine (R9)-PTD). Our results demonstrated that R9-PTD-containing OVA transduced DCs most efficiently, and that transduction efficacy was closely correlated with the extent of Ag-specific CD4+ and CD8+ T-cell activation in vitro and in vivo. Repeated vaccination with R9-PTD-OVA-transduced DC in (OVA-expressing) tumor-bearing mice induced enhanced antitumor immunity, and elicited complete rejection of tumors when DC was co-injected with adjuvants. This vaccination strategy may be clinically applicable, and offers theoretical and practical advantages to those that are in current use.

Overexpression of GRIM-19 in Cancer Cells Suppresses STAT3-Mediated Signal Transduction and Cancer Growth

“…His 6 -tagged recombinant nona-arginine-hemagglutininovalbumin (rR9-HA-OVA), rHA-GFP, rR9-HA-GFP, and rR9-HA-murine Fc receptor-like A (mFCRL) constructs were generated as previously described (21)(22)(23). Leishmania homologue of receptors for activated C kinase (LACK), known as highly immunogenic foreign antigen in Leishmania major (28), cDNA was provided by D. Sacks (National Institute of Allergy and Infectious Disease, Bethesda, MD).…”

“…Similarly, R9-HA-GRIM19 was generated by inserting full-length GRIM19 cDNA (derived from the murine heart tissues with GRIM19-specific primers containing enzyme recognition sites) in-frame into the pR9-HA plasmid. rR9-fusion proteins were purified as described previously (22).…”

“…We have shown that protein transduction domain (PTD)-containing fusion proteins, particularly nonaarginine (R9)-PTD, were efficient and safe protein carriers (∼100%) for any type of cell in vitro and in vivo (21)(22)(23)(24). PTDs are incorporated into cells through macropinocytosis/endocytosis after binding to anionic cell membrane components such as heparan sulfate (25)(26)(27).…”

Cell Surface Heparan Sulfate Proteoglycans Influence MHC Class II-Restricted Antigen Presentation

“…Mitsui et al (38), who showed that OVA is not better presented by DCs when it is coupled to the basic region of Tat. It is well known that the basic region of Tat is able to bind heparan sulfate (24) and it is often used as a cargo for transduction of proteins, which thus transit in early endosomes before their transfer to the cytoplasm (39,40).…”

Enhanced antigen presentation and CTL activity by transduction of mature rather than immature dendritic cells with octaarginine-modified liposomes

“…Since then various research groups, including our own, have applied CPP transduction to DCs as a method of stimulating Ag-specific T cells. Many studies reported that mice immunized with DCs transduced with recombinant CPP-Ag protein suppressed the tumor growth significantly [8][9][10][11][12][13][14][15]. Apart from the recombinant fusion protein, synthetic polypeptide containing CPP tandemly linked to peptide epitopes is another striking approach [14].…”