Joseph G. Altin

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 metal-chelating lipid, 3(nitrilotriacetic acid)-ditetradecylamine, was incorporated into B16-OVA-derived PMV, allowing recombinant hexahistidine-tagged forms of single chain antibody fragments to the DC surface molecules CD11c and DEC-205, to be conveniently “engrafted” onto the vesicle surface by metal-chelating linkage. The modified PMV, or similarly engrafted synthetic stealth liposomes containing OVA or OVA peptide antigen, were found to target DCs in vitro and in vivo, in experiments using flow cytometry and fluorescence confocal microscopy. When used as vaccines in syngeneic mice, the preparations stimulated strong B16-OVA-specific CTL responses in splenic T cells and a marked protection against tumor growth. Protection was dependent on the simultaneous delivery of both antigen and a DC maturation or “danger signal” signal (IFN-γ or lipopolysaccharide). Administration of the DC-targeting vaccine to mice challenged with B16-OVA cells induced a dramatic immunotherapeutic effect and prolonged disease-free survival. The results show that the targeting of antigen to DCs in this way is highly effective at inducing immunity and protection against the tumor, with protection being at least partially dependent on the eosinophil chemokine eotaxin.

Non-parenchymal cells as mediators of physiological responses in liver

Parenchymal cells (hepatocytes) are the sites at which the principal metabolic functions of the liver are located. In the perfused liver, responses (e.g. vasoconstriction and glycogenolysis) to stimulating agents such as zymosan, platelet-activating factor and arachidonic acid, are inhibited by indomethacin and bromophenacyl bromide, inhibitors of cyclo-oxygenase and phospholipase A2, respectively. Since cultured Kupffer and endothelial cells but not hepatocytes, produce eicosanoids, and since eicosanoids and especially prostaglandins induce similar patterns of responses when added directly to the perfused liver, an involvement of these nonparenchymal cells in mediating the above responses is considered likely. We propose that in most situations the responses induced by these stimulating agents are mediated through a combination of pathways that include interaction of the agents directly with hepatocytes or with vasoactive cells (endothelial and/or smooth muscle cells), or interaction of agents initially with non-parenchymal cells to produce and release eicosanoids, which then subsequently interact with hepatocytes or with vasoactive 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. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8+ and CD4+ T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8+ T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.

Possible involvement of prostaglandins in vasoconstriction induced by zymosan and arachidonic acid in the perfused rat liver

Exposure of perfused livers to zymosan, arachidonic acid or phenylephrine but not to latex particles, stimulates hepatic constriction. The effects of arachidonic acid are rapid, reach a maximum after 2–3 min and then decline. They are blocked by the cyclooxygenase inhibitor indomethacin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. This suggests a role for prostaglandins in this action. Zymosan progressively increases hepatic pressure after a lag time of about 1 min. Perfusion of bromophenacyl bromide, indomethacin and nordihydroguaiaretic acid only partially inhibits the zymosan‐induced vasoconstriction. None of these inhibitors affect the phenylephrine‐induced response. Repeated infusion of arachidonic acid leads to homologous desensitization of the response whereas the response of the liver to phenylephrine is unaffected. The present data indicate that prostaglandins, produced and released within the liver, affect vasoconstriction in this organ.

Histidine-rich glycoprotein binding to T-cell lines and its effect on T-cell substratum adhesion is strongly potentiated by zinc.

Histidine‐rich glycoprotein (HRG), a plasma protein that binds heparin and divalent cations, has been implicated in immune regulation through its ability to modulate complement function, macrophage Fc receptor expression and phagocytosis, and its ability to inhibit the proliferation of human peripheral blood T cells in vitro. In the present work we used fluorescence flow cytometry to study the binding of human HRG to the human T‐cell lines Jurkat and MT4, and to the murine antigen‐specific T‐cell clone D10, and to study the effect of divalent cations zinc and copper on this binding. Our results show that HRG binds strongly to these cell lines at 4°, and that the binding is markedly potentiated by physiological concentrations of zinc (20 μM), and to a lesser extent by copper (10 μM). In contrast to previous studies, HRG binding was largely inhibited by 50 μg/ml heparin, both in the absence and in the presence of zinc, suggesting that HRG interacts primarily through glycosaminoglycans on the T‐cell surface. Studies using confocal fluorescence microscopy indicated that following incubation of MT4 cells with HRG in the presence of zinc at 4°, the HRG was localized exclusively at the plasma membrane, but was actively internalized after incubation at 37°. Interestingly, HRG interfered with the ability of D10 cells to adhere to tissue culture plastic, as well as to laminin‐, collagen‐ or fibronectin‐coated culture dishes. This effect was markedly potentiated by 20 μM zinc, and was partially reversed by heparin. The results suggest that zinc markedly potentiates the binding of HRG to T cells, and that HRG and zinc may play an important role in regulating the adhesion of T cells to other cells and the extracellular matrix.

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. Liposomes engrafted with 9Flg or 42Flg induced functional MyD88-dependent maturation of dendritic cells in vivo. The vaccination of mice with 9Flg liposomes containing OVA induced OVA-specific T cell priming, increased the number of Ag-responsive IFN-γ–producing CD8+ T cells, and increased Ag-specific IgG1 and IgG2b in serum. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA–derived plasma membrane vesicles, engrafted with 9Flg or 42Flg, potently inhibited tumor growth/metastasis and induced complete tumor regression in the majority of mice challenged with the syngeneic B16-OVA melanoma, in the lung and s.c. tumor models. Strong antitumor responses were also seen in studies using the s.c. P815 tumor model. Therefore, vaccination with Ag-containing liposomes engrafted with 9Flg or 42Flg is a powerful strategy to exploit the innate and adaptive immune systems for the development of potent vaccines and cancer immunotherapies.

Differential binding of histidine-rich glycoprotein (HRG) to human IgG subclasses and IgG molecules containing kappa and lambda light chains.

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the κ or λ light (L)-chains. Thus, the on-rate for the binding of HRG to the κ L-chain containing IgG1 and IgG2 (IgG1κ and IgG2κ) was ∼4- and ∼10-fold faster than that for the binding of HRG to λ L-chain containing IgG1 and IgG2 (IgG1λ and IgG2λ), respectively, with the dissociation constants (K d ) in the range 3–5 nm and 112–189 nm for the κ and λ isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3κ and IgG4κ was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3λ and IgG4λ, respectively, with the K d in the range 147–268 nm and 96–109 nm for the κ and λ isoforms, respectively. The binding of HRG to immunoglobulins containing the κ L-chain (particularly IgG1κ) was generally potentiated in the presence of a physiological concentration (20 μm) of Zn2+ (K d decreased to 0.60 ± 0.01 for IgG1κ), but Zn2+ had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the λ L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing κ and λ L-chains, with HRG having a 14-fold lower K d for BJκ than for BJλ when 20 μm Zn2+ was present. HRG also bound to IgM (IgMκ), but the affinity of this interaction (K d ∼1.99 ± 0.05 μm) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn2+. The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the κ and λ L-chains of immunoglobulins.

Engrafting Costimulator Molecules onto Tumor Cell Surfaces with Chelator Lipids: A Potentially Convenient Approach in Cancer Vaccine Development

The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200–300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or “engrafted” protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7.1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II.

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor–ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low‐ affinity receptor–ligand interactions has been difficult. Utilizing a novel chelator‐lipid, nitrilotriacetic acid di‐tetradecylamine (NTA‐DTDA), we have developed a convenient liposome system for directly detecting low‐affinity receptor–ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or ‘engrafted’ directly onto fluorescently labelled liposomes via a metal‐chelating linkage with NTA‐DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence‐ activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4–6‐fold above control cells. Such liposomes could detect directly the low‐affinity interaction of murine CD40 and B7.1 with CD154‐ and CD28‐expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.

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). Peptide-engrafted liposomes were examined for their biodistribution and anti-tumor effects after i.v. administration. Our results show that radiolabelled liposomes engrafted with either p24-NRP-1 or p47-LyP-1 and then injected into mice bearing subcutaneous B16-F1 tumors, show increased accumulation in the tumor. For p24-NRP-1-liposomes, tumor targeting was significantly increased when the stabilizing lipid phosphatidylethanolamine polyethylene glycol-750 (PE-PEG(750)) was used instead of PE-PEG(2000) in the liposome lipid mixture. Importantly, compared to the controls, p24-NRP-1 liposomes containing 10 mol% PE-PEG(750) and loaded with doxorubicin significantly inhibited the rate of tumor growth in the tumor-bearing mice. Our findings demonstrate that the use of drug-containing liposomes incorporating NTA(3)-DTDA and engrafted with NRP-1 targeting peptide is a convenient strategy to enhance the therapeutic effect of non-targeted doxorubicin.