Background:Liposomes and liposome-derived nanovesicles such as archaeosomes and
virosomes have become important carriers systems in vaccine
development and the interest for liposome-based vaccines has markedly
increased. A key advantage of liposomes, archaeosomes and virosomes in
general, and liposome-based vaccine delivery systems in particular, is their
versatility and plasticity. Liposome composition and preparation can be
chosen to achieve desired features such as selection of lipid, charge, size,
size distribution, entrapment and location of antigens or adjuvants.
Depending on the chemical properties, water soluble antigens (proteins,
peptides, nucleic acids, carbohydrates, haptens) are entrapped within the
aqueous inner space of liposomes, whereas lipophilic compounds
(lipopeptides, antigens, adjuvants, linker molecules) are intercalated into
the lipid bilayer and antigens or adjuvants can be attached to the liposome
surface either by adsorption or stable chemical linking. Co-formulations
containing different types of antigens and/or adjuvants can be combined
with the parameters mentioned to tailor liposomal vaccines for individual
applications.
Special emphasis is given in this review to cationic adjuvant liposome
vaccine formulations. Examples of vaccines made with CAF01, an adjuvant
composed of the synthetic immune stimulating mycobacterial cordfactor
glycolipid trehalose-dibehenate (TDB) as immunomodulator and the
cationic membrane forming molecule dimethyl-dioctadecylammonium DDA
are presented. Other vaccines such as cationic liposome-DNA complexes
(CLDC) and other adjuvants like muramyl dipeptide, monophosphoryl lipid
A and listeriolysin O are mentioned as well.
The field of liposomes and liposome-based vaccines is vast. Therefore, this
review concentrates on recent and relevant studies emphasizing current
reports dealing with the most studied antigens and adjuvants and pertinent
examples of vaccines. Studies on liposome-based veterinary vaccines and
experimental therapeutic cancer vaccines are also summarized.
Background: Solid tumors grow within a complex microenvironment composed of diverse
cell types such as fibroblasts, endothelial cells, mast cells, macrophages and immune
cells that are attracted by tumor cell derived factors and embedded in an extracellular
matrix. Molecular and cellular interactions between epithelial cells and cells
surrounding the tumor stroma promote growth, invasion and spread of tumors. To delay
or impede tumor growth, the tumor microenvironment (TME) is increasingly being
explored as a potential therapeutic target for which novel strategies are developed.
This article reviews how key interactions between tumor cells and surrounding
mesenchymal and immune cells in the TME can promote tumor progression and it
highlights cellular and molecular elements that might represent novel therapeutic
targets. Special emphasis is given on therapies targeted towards tumor-associated
macrophages. As main class of drugs the bisphosphonates are covered with their
properties to repolarize a pro-tumorigenic, immunosuppressive environment to a tumor
growth inhibiting and immunocompetent microenvironment. Properties and advantages
of liposome-encapsulated bisphosphonates as macrophage depleting or modulating
agents as well as the latest developments towards clinical applications of compounds
targeting cellular and molecular components of the TME are described and reviewed.
Background: Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process
they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-b signaling and activation of the b-catenin pathway can induce EMT in intratumoral cancer cells.
Methods: We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).
Results: Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating b-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-b was identified as the main inducer of this EMT-associated phenotype.Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-b levels and tumor grade.
Conclusions: Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.
Liposomes are the most extensively studied carrier systems to deliver drugs to macrophages. Upon delivery to macrophages, depending on the encapsulated drug, various functions in the human immune system could be affected and macrophage associated diseases could be treated. Here we report a new, specific and efficient approach of macrophage specific drug delivery by coating liposome surfaces with albumin. Liposomes modified with polyethylene glycol (PEG) as hydrophilic spacer were reacted with activated albumins to create a flexible layer of covalently bound albumin molecules on the liposome surface. Albumin coated liposomes were taken up faster and more efficiently than uncoated control liposomes by murine macrophages, probably because of better recognition and enhanced macrophage avidity. Liposome uptake in vitro was significantly higher in macrophages as compared to three other cell types tested (MS-1 endothelial cells, NIH 3T3 fibroblasts, LLC tumor cells), suggesting specificity for macrophages. In vivo, splenic macrophages phagocytosed BSA coated liposomes (BSA-L) at faster rates compared to conventional liposomes (L) and PEG liposomes (PEG-L). Finally, to prove the effectiveness of this new macrophage specific drug carrier, we encapsulated the bisphosphonates clodronate and zoledronate in BSA-L and compared them with conventional liposomal encapsulation. In cell culture, the treatment of RAW 264.7 macrophages with clodronate or zoledronate encapsulated in BSA-L led to cytotoxic activity within a very short time (1h and 3h, respectively) and the IC50 concentrations were reduced 8- and 50-fold respectively, compared to L. When tested in mice, clodronate encapsulated in BSA-L depleted splenic macrophages at a 5-fold lower concentration of conventional clodronate-liposomes. Altogether our results highlight the pharmaceutical benefits of albumin coated liposomes for macrophage specific drug delivery.
Tumor-associated macrophages (TAMs) play a key role in tumor progression and metastasis as they produce cytokines and chemokines that promote many functions associated with tumor malignancy. These factors include basic fibroblast growth factor (bFGF), VEGF, angiopoietins, interleukins, TNF-a , thymidine phosphorylase (TP), matrix metalloproteinases, nitric oxide (NO) and many others. These molecules, which are expressed by TAMs in a coordinated spatial and temporal fashion, promote proliferation and migration of endothelial cells, matrix remodelling and the eventual formation of stabilized vessels. The liposome encapsulated bisphosphonate clodronate (Clodrolip) is highly cytotoxic to macrophages, a property that is extensively exploited in immunology research. We hypothesized that the elimination of TAMs and thus the abolishment of cytokine production by Clodrolip could inhibit tumor growth.
To this end we treated tumor bearing mice with Clodrolip alone, with anti-VEGF scFv antibodies and with combinations of both. Tumor growth inhibition experiments in the highly vascularized syngeneic F9 teratocarcinoma and the human A673 rhabdomyosarcoma mouse models revealed significant tumor growth inhibition, ranging from 75% to > 92%, depending on therapy and schedule. The strongest effects were observed with the combination therapies. Immunohistologic evaluation of the tumors showed significant depletion of F4/80+ and MOMA-1+ TAMs and, surprisingly also of the CD11c+ subset of tumor associated dendritic cells. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs in the A673 model showed reduction rates of 85% to > 94%, even nine days after completion of therapy.
Based on these results we conclude that Clodrolip mediated depletion of TAMs together with other anti-angiogenic and/or cytotoxic treatment regimens represents a new and highly effective therapeutic modality for the successful treatment of solid tumors and prevention of metastasis.
Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study,
functionalized Antennapedia (Antp, aa 43--58) and HIV Tat (aa 47-57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage.
Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes.
Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per
small unilamellar liposome (100 +/- 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes
was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-
deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate
proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N(4)-octadecyl-1-b-D-arabinofuranosylcytosine-
(5'- 5')-3'-C-ethinylcytidine showed a reduction of the IC(50) by 70% on B16F1 melanoma cells compared with unmodified liposomes.
PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of
a large variety of liposome-entrapped molecules.
7) Simultaneous inhibition of tumor growth and tumor angiogenesis by target specific immunoliposomes.
Cytotoxic Tumor Targeting
with scFv Antibody-modified Liposomes
Cornelia Marty and Reto A. Schwendener
Abstract
Specific targeting of liposome-formulated cytotoxic drugs or antigens to receptors expressed
selectively on target cells represents an effective strategy for increasing the pharmacological
efficacy of the delivered molecules. We have developed a feasible technique to selectively
attach antibodies and fragments thereof, but also small-mol-wt ligands such as peptides, carbohydrates,
or any molecules that recognize and bind target antigens or receptors to the surface of
small unilamellar liposomes. Our concept is based on the site-specific functionalization of the
ligands to be attached to the liposomes by thiol groups. These thiol groups can easily be introduced
to antibodies or peptides by addition of cysteines, preferably at sites that do not interfere
with the receptor binding domains. Optimally, the site-specific modification is introduced at
the C-terminal end of the ligand, separated by an inert spacer sequence located between the
thiols and the specific part of the ligand. The thiol-reactive molecules on the liposome surface
are maleimides that are linked to phospholipids composing the liposome bilayer membrane.
We illustrate the coupling method of a functionalized single-chain antibody fragment with binding
specificity to ED-B fibronectin, an isoform of fibronectin exclusively expressed in tumor
tissues, to long circulating small unilamellar poly(ethylene glycol) liposomes.
In vivo Antigen Loading and Activation of Dendritic Cells via a liposomal Peptide Vaccine mediates protective antiviral and anti-tumour Immunity.
Burkhard Ludewig, Federica Barchiesi, Marcus Pericin, Rolf M. Zinkernagel, Hans Hengartner.
Abstract
Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC)transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore,important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.
A liposomal Peptide Vaccine inducing CD8+ T Cells in HLA-A2.1 transgenic Mice, which recognise human Cells encoding Hepatitis C Virus (HCV) Proteins.
Engler OB, Schwendener RA, Dai WJ, Wolk B, Pichler W, Moradpour D, Brunner T, Cerny A.
Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8(+) T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-g-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-gamma-secreting cells by 2-10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2-23% of the CD8(+) T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8(+) T cell against HCV.
