BackgroundResistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs.MethodsLung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated.ResultsAlthough anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered.ConclusionsWe provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.

We evaluated cross-reactivity of immunoglobulin A (IgA) secreted on the nasal mucosa in mice that were nasally inoculated 4 times with a mixture of inactivated H1N1 influenza A viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing d-octaarginine at 7-day intervals. Three viral strains (A/Puerto Rico/8/34, A/New Caledonia/20/99 IVR116, and A/Solomon Islands/03/2006) and D-octaarginine-linked polymers with different molecular weights were used as antigens and their carriers, respectively. Secretion of intranasal IgA was barely observed when the inactivated virus alone was administered. The polymer induced the production of intranasal IgA specific to the inoculated viruses, irrespective of the viral strain and molecular weight of the polymer. The respective antibodies cross-reacted to recombinant hemagglutinin proteins of not only the viral strain used for immunization but also other H1N1 strains, including A/Puerto Rico/8/34 strain whose hemagglutinin proteins are diverse from those of other strains. Mice with high reactivity of IgA to the inoculated viruses tended to acquire clear cross-reactivity to other viral strains. Notably, IgA induced by inactivated H1N1 A/New Caledonia/20/99 IVR116 strain with the strongest immunogenicity between 3 antigens in the presence of the polymer cross-reacted to recombinant hemagglutinin proteins of the A/Brisbane/10/2007 and A/Viet Nam/1194/2004 strains, which are categorized into H3N2 and H5N1, respectively. Our polymer is a potential candidate for an efficient antigen carrier that induces mucosal IgA having cross-reactivity to antigenically drifted variants, irrespective of the subtype of viral strains.

Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and d-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.

Cell-based therapy using dendritic cells (DC) represents a potent cancer immunotherapy. However, activated DC express indoleamine 2,3-dioxygenase 1 (IDO1), a counter-regulatory and tolerogenic molecule, leading to the inhibition of T cell activation and the promotion of T cell differentiation into regulatory T cells. Silencing the IDO1 gene in DC by small interfering RNA (siRNA) represents a potent therapeutic strategy. We report on the successful and efficient introduction of a siRNA targeting IDO1 into mouse DCs by a means of a multifunctional envelope-type nanodevice (MEND) containing a YSK12-C4 (YSK12-MEND). The YSK12-C4 has both fusogenic and cationic properties. The YSK12-MEND induced an effective level of gene silencing of IDO1 at siRNA doses in the range of 1–20 nM, a concentration that commercially available transfection reagents are not able to silence. The YSK12-MEND mediated IDO1 silencing had no effect on the characteristic determinants of DC phenotype such as CD11c, CD80 and MHC class II. The silencing of IDO1 in DC by the YSK12-MEND significantly enhanced the antitumor effect against E.G7-OVA tumor. Moreover, a decrease in the numbers of regulatory T cells in the tumor was observed in mice that were treated with the IDO1-silenced DC. The YSK12-MEND appears to be a potent delivery system for IDO1-silenced DC based cancer immunotherapy.