α-Galactosylceramide and peptide-based nano-vaccine synergistically induced a strong tumor suppressive effect in melanoma.

α-Galactosylceramide (GalCer) is a glycolipid widely known as an activator of Natural killer T (NKT) cells, constituting a promising adjuvant against cancer, including melanoma. However, limited clinical outcomes have been obtained so far. This study evaluated the synergy between GalCer and major histocompatibility complex (MHC) class I and MHC class II melanoma-associated peptide antigens and the Toll-Like Receptor (TLR) ligands CpG and monophosphoryl lipid A (MPLA), which we intended to maximize following their co-delivery by a nanoparticle (NP). This is expected to improve GalCer capture by dendritic cells (DCs) and subsequent presentation to NKT cells, simultaneously inducing an anti-tumor specific T-cell mediated immunity. The combination of GalCer with melanoma peptides and TLR ligands successfully restrained tumor growth. The tumor volume in these animals was 5-fold lower than the ones presented by mice immunized with NPs not containing GalCer. However, tumor growth was controlled at similar levels by GalCer entrapped or in its soluble form, when mixed with antigens and TLR ligands. Those two groups showed an improved infiltration of T lymphocytes into the tumor, but only GalCer-loaded nano-vaccine induced a prominent and enhanced infiltration of NKT and NK cells. In addition, splenocytes of these animals secreted levels of IFN-γ and IL-4 at least 1.5-fold and 2-fold higher, respectively, than those treated with the mixture of antigens and adjuvants in solution. Overall, the combined delivery of the NKT agonist with TLR ligands and melanoma antigens via this multivalent nano-vaccine displayed a synergistic anti-tumor immune-mediated efficacy in B16F10 melanoma mouse model. STATEMENT OF SIGNIFICANCE: Combination of α-galactosylceramide (GalCer), a Natural Killer T (NKT) cell agonist, with melanoma-associated antigens presented by MHC class I (Melan-A:26) and MHC class II (gp100:44) molecules, and Toll-like Receptor (TLR) ligands (MPLA and CpG), within nanoparticle matrix induced a prominent anti-tumor immune response able to restrict melanoma growth. An enhanced infiltration of NKT and NK cells into tumor site was only achieved when the combination GalCer, antigens and TLR ligands were co-delivered by the nanovaccine.

Liposomal α-galactosylceramide is taken up by gut-associated lymphoid tissue and stimulates local and systemic immune responses.

OBJECTIVES: α-Galactosylceramide (α-GalCer), a synthetic glycosphingolipid that exhibits potent immunostimulatory effects through activation of natural killer T (NKT) cells, can be used to treat conditions such as atopy, cancer, infection and autoimmunity. Administration of therapeutics through the oral route has advantages such as patient convenience, safety and reduced cost; however, there has been little research to investigate whether oral delivery of α-GalCer is possible. The aim of this study was therefore to determine whether α-GalCer formulated in either DMSO/Tween 80 or in liposomes, could access lymphoid tissue and stimulate immune activation following oral administration. METHODS: Fluorescently labelled cationic liposomes incorporating α-GalCer were prepared, characterized and administered by oral gavage to fasted mice. KEY FINDINGS: Liposomes were detected inside the Peyer's patches (PPs), in the subepithelial dome just under the follicle-associated epithelium. CD11b+ cells and CD11c+ were shown to have taken up the formulation in a higher proportion compared to the total cell proportion in the PPs, suggesting that cells with these markers may be the prominent antigen-presenting cells involved in selective uptake. Finally, the liposomal formulation demonstrated a higher degree of immune stimulation compared to the DMSO/Tween 80 solubilized α-GalCer in the PPs, mesenteric lymph nodes and spleen as shown by the increased expression of IL-4 mRNA expression and increased proportion of NKT cells at 6 h and 3 days after administration. CONCLUSIONS: These results show that oral delivery of a liposomal α-GalCer can stimulate local and systemic immune responses to a different degree compared to the non-liposomal form.

In vivo and in vitro analyses of α-galactosylceramide uptake by conventional dendritic cell subsets using its fluorescence-labeled derivative.

Conventional dendritic cells (cDCs) present α-galactosylceramide (αGC) to invariant natural killer T (iNKT) cells through CD1d. Among cDC subsets, CD8(+) DCs efficiently induce IFN-γ production in iNKT cells. Using fluorescence-labeled αGC, we showed that CD8(+) DCs incorporated larger amounts of αGC and kept it intact longer than CD8(-) DCs. Histological analyses revealed that Langerin(+)CD8(+) DCs in the splenic marginal zone, which was the unique equipment to capture blood-borne antigens, preferably incorporated αGC, and the depletion of Langerin(+) cells decreased IFN-γ and IL-12 production in response to αGC. Furthermore, splenic Langerin(+)CD8(+) DCs expressed more membrane-bound CXCL16, which possibly anchored iNKT cells in the marginal zone, than CD8(-) DCs. Collectively, it is suggested that the cellular properties and localization of CD8(+) DCs are important for stimulation of iNKT cells by αGC.

The nanoparticulation by octaarginine-modified liposome improves α-galactosylceramide-mediated antitumor therapy via systemic administration.

Alpha-galactosylceramide (αGC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because αGC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-γ. Here, we incorporated αGC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. Unexpectedly, the systemic administered R8-Lip incorporating αGC (αGC/R8-Lip) failed to improve the immune responses mediated by αGC compared with soluble αGC in vivo, although αGC/R8-Lip drastically enhanced αGC presentation on CD1d in antigen presenting cells in vitro. Thus, we optimized the αGC/R8-Lip in vivo to overcome this inverse correlation. In optimization in vivo, we found that size control of liposome and R8-modification were critical for enhancing the production of IFN-γ. The optimization led to the accumulation of αGC/R8-Lip in the spleen and a positive therapeutic effect against highly malignant B16 melanoma cells. The optimized αGC/R8-Lip also enhanced αGC presentation on CD1d in antigen presenting cells and resulted in an expansion in the population of NKT cells. Herein, we show that R8-Lip is a potent delivery system, and size control and R8-modification in liposomal construction are promising techniques for achieving systemic αGC therapy.

Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses.

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Pharmacokinetics and tissue distribution of 5-fluorouracil encapsulated by galactosylceramide liposomes in mice.

AIM: To study the pharmacokinetics and tissue distribution of 5-fluorouracil encapsulated by galactosylceramide liposomes (5-Fu-GCL) in mice. METHODS: The concentration of 5-fluorouracil (5-Fu) in serum was detected by high performance liquid chromatography after 5-Fu-GCL (80, 40, 20 mg/kg) and free 5-Fu (40 mg/kg) were injected intravenously into mice. The tissue distribution of 5-Fu-GCL (40 mg/kg) and free 5-Fu (40 mg/kg) was investigated, and concentration-time profile of the two preparations in the liver were studied. Data were analyzed by 3p97 program. RESULTS: Serum concentration-time curves of 5-Fu-GCL and free 5-Fu conformed to one compartment model of first order absorption. 5-Fu-GCL at 80, 40, and 20 mg/kg had T(1/2Ke) of 25.8+/-4.2, 27.3+/-4.4, and 28.2+/-5.6 min; C0 of 24.9+/-4.9, 17.7+/-3.6, and 11.5+/-2.7 mg/L; and AUC of 990.0+/-45.2,622.5+/-38.3, and 340.4+/-25.6 mg x min x L(-1), respectively. In contrast free 5-Fu at 40 kg/mg had T(1/2Ke) of 15.8+/-2.2 min, C0 of 35.8+/-6.2 mg/L, AUC of 639.0+/-35.9 mg x min x L(-1). The tissue distribution of 5-Fu-GCL in the liver and immune organs was significantly increased, while in heart and kidney it was remarkably decreased. The AUC of 5-Fu-GCL in the liver was 3 times higher than that of free 5-Fu. CONCLUSION: The pharmacokinetics and tissue distribution of 5-Fu-GCL appears to be linear-related and dose-dependent, and exhibits sustained-release and hepatic target characteristics.

A phase I study of the natural killer T-cell ligand alpha-galactosylceramide (KRN7000) in patients with solid tumors.

PURPOSE: alpha-galactosylceramide (KRN7000) is a glycosphingolipid that has been shown to inhibit tumor growth and to prolong survival in inoculated mice through activation of natural killer (NK) T cells. We performed a dose escalation study of KRN7000 in advanced cancer patients. EXPERIMENTAL DESIGN: Patients with solid tumors received i.v. KRN7000 (50-4,800 micro g/m(2)) on days 1, 8, and 15 of a 4-weekly cycle. Patients were given 1 cycle and, in the absence of dose-limiting toxicity or progression, treatment was continued. Pharmacokinetics (PK) and immunomonitoring were performed in all patients. RESULTS: Twenty-four patients were entered into this study. No dose-limiting toxicity was observed over a wide range of doses (50-4,800 micro g/m(2)). PK was linear in the dose range tested. Immunomonitoring demonstrated that NKT cells (CD3+Valpha24+Vbeta11+) typically disappeared from the blood within 24 h of KRN7000 injection. Additional biological effects included increased serum cytokine levels (tumor necrosis factor alpha and granulocyte macrophage colony-stimulating factor) in 5 of 24 patients and a transient decrease in peripheral blood NK cell numbers and cytotoxicity in 7 of 24 patients. Importantly, the observed biological effects depended on pretreatment NKT-cell numbers rather than on the dose of KRN7000. Pretreatment NKT-cell numbers were significantly lower in patients compared with healthy controls (P = 0.0001). No clinical responses were recorded and seven patients experienced stable disease for a median duration of 123 days. CONCLUSION: i.v. KRN7000 is well tolerated in cancer patients over a wide range of doses. Biological effects were observed in several patients with relatively high pretreatment NKT-cell numbers. Other therapeutic strategies aiming at reconstitution of the deficient NKT-cell population in cancer patients may be warranted.

Population pharmacokinetics of the novel anticancer agent KRN7000.

PURPOSE: KRN7000 is a novel anticancer agent, acting through stimulation of the immune system. The first clinical trial with this agent, which included pharmacokinetic studies, has recently been completed. The aim of the study presented here was to develop a population pharmacokinetic model for KRN7000. METHODS: Plasma concentration-time data were gathered from 24 patients enrolled in a phase I trial in which KRN7000 was administered as a weekly slow injection at doses ranging from 50 to 4800 microg/m(2). These data were used to build a pharmacokinetic model using the nonlinear mixed-effect modeling (NONMEM) program. The model was validated by performance of 200 bootstraps. RESULTS: A three-compartment model with interindividual variability on the central and two peripheral volumes of distribution (V1, V2 and V3) and on clearance (CL) adequately described the data. The final estimates were: V1 2.34 l, V2 2.61 l, V3 2.13 l, and CL 0.130 l/h. Of 24 covariates tested, including both demographic and pathophysiological factors, none showed a significant relationship with the pharmacokinetic parameters obtained. The bootstrap analysis provided parameter estimates within approximately 15% of the original estimates, indicating stability of the model. CONCLUSION: The pharmacokinetic behavior of KRN7000 in the clinical trial could be described by a three-compartment model. Hence, KRN7000 demonstrates linear pharmacokinetics over the investigated dose range. The pharmacokinetics of KRN7000 are not influenced by patient demographic or pathophysiological characteristics.

Activation of natural killer T cells by alpha-galactosylceramide in the presence of CD1d provides protection against colitis in mice.

BACKGROUND & AIMS: CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of natural killer (NK)1.1(+) T cells. These NK T cells exhibit important immunoregulatory functions in several autoimmune disease models. METHODS: To investigate whether CD1d and NK T cells have a similar role in intestinal inflammation, the effects of the glycolipid, alpha-galactosylceramide (alpha-GalCer), on dextran sodium sulfate (DSS)-induced colitis were examined. Wild-type (WT), CD1d(-/-), and RAG(-/-) mice were examined for their response to either alpha-GalCer or the control analogue, alpha-mannosylceramide (alpha-ManCer). RESULTS: WT mice, but not CD1d(-/-) and RAG(-/-) mice, receiving alpha-GalCer had a significant improvement in DSS-induced colitis based on body weight, bleeding, diarrhea, and survival when compared with those receiving alpha-ManCer. Elimination of NK T cells through antibody-mediated depletion resulted in a reduction of the effect of alpha-GalCer. Furthermore, adoptive transfer of NK T cells preactivated by alpha-GalCer, but not alpha-ManCer, resulted in diminished colitis. Using a fluorescent-labeled analogue of alpha-GalCer, confocal microscopy localized alpha-GalCer to the colonic surface epithelium of WT but not CD1d(-/-) mice, indicating alpha-GalCer binds CD1d in the intestinal epithelium and may be functionally active at this site. CONCLUSIONS: These results show an important functional role for NK T cells, activated by alpha-GalCer in a CD1d-restricted manner, in regulating intestinal inflammation.

Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells.

A short-chain analogue of galactosylceramide (6-NBD-amino-hexanoyl-galactosylceramide, C6-NBD-GalCer) was inserted into the apical or the basolateral surface of MDCK cells and transcytosis was monitored by depleting the opposite cell surface of the analogue with serum albumin. In MDCK I cells 32% of the analogue from the apical surface and 9% of the analogue from the basolateral surface transcytosed to the opposite surface per hour. These numbers were very similar to the flow of membrane as calculated from published data on the rate of fluid-phase transcytosis in these cells, demonstrating that C6-NBD-GalCer acted as a marker of bulk membrane flow. It was calculated that in MDCK I cells 155 microns membrane transcytosed per cell per hour in each direction. The fourfold higher percentage transported from the apical surface is explained by the apical to basolateral surface area ratio of 1:4. In MDCK II cells, with an apical to basolateral surface ratio of 1:1, transcytosis of C6-NBD-GalCer was 25% per hour in both directions. Similar numbers were obtained from measuring the fraction of endocytosed C6-NBD-GalCer that subsequently transcytosed. Under these conditions lipid leakage across the tight junction could be excluded, and the vesicular nature of lipid transcytosis was confirmed by the observation that the process was blocked at 17 degrees C. After insertion into one surface of MDCK II cells, the glucosylceramide analogue C6-NBD-GlcCer randomly equilibrated over the two surfaces in 8 h. C6-NBD-GalCer and -GlcCer transcytosed with identical kinetics. Thus no lipid selectivity in transcytosis was observed. Whereas the mechanism by which MDCK cells maintain the different lipid compositions of the two surface domains in the absence of lipid sorting along the transcytotic pathway is unclear, newly synthesized C6-NBD-GlcCer was preferentially delivered to the apical surface of MDCK II cells as compared with C6-NBD-GalCer.

Metabolism of exogenous galactosylceramide in the twitcher mouse brain.

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-14C] stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.