Enhancing T Cell and Antibody Response in Mucin-1 Transgenic Mice through Co-Delivery of Tumor-Associated Mucin-1 Antigen and TLR Agonists in C3-Liposomes.

Mucin-1 (MUC1) is a highly relevant antigen for cancer vaccination due to its overexpression and hypo-glycosylation in a high percentage of carcinomas. To enhance the immune response to MUC1, our group has developed C3-liposomes that encapsulate the MUC1 antigen along with immunostimulatory compounds for direct delivery to antigen-presenting cells (APCs). C3-liposomes bind complement C3, which interacts with C3-receptors on APCs, resulting in liposomal uptake and the delivery of tumor antigens to APCs in a manner that mimics pathogenic uptake. In this study, MUC1 and Toll-like receptor (TLR) agonists were encapsulated in C3-liposomes to provoke an immune response in transgenic mice tolerant to MUC1. The immune response to the C3-bound MUC1 liposomal vaccine was assessed by ELISA, ELISpot, and flow cytometry. Co-administering TLR 7/8 agonists with MUC1 encapsulated in C3-liposomes resulted in a significant antibody response compared to non-encapsulated MUC1. This antibody response was significantly higher in females than in males. The co-encapsulation of three TLR agonists with MUC1 in C3-liposomes significantly increased antibody responses and eliminated sex-based differences. Furthermore, this immunization strategy resulted in a significantly increased T cell-response compared to other treatment groups. In conclusion, the co-delivery of MUC1 and TLR agonists via C3-liposomes greatly enhances the immune response to MUC1, highlighting its potential for antigen-specific cancer immunotherapy.

Liposome Delivery of Natural STAT3 Inhibitors for the Treatment of Cancer.

In the tumor microenvironment, cytokines, growth factors, and oncogenes mediate constitutive activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway in both cancer cells and infiltrating immune cells. STAT3 activation in cancer cells drives tumorigenic changes that allow for increased survival, proliferation, and resistance to apoptosis. The modulation of immune cells is more complicated and conflicting. STAT3 signaling drives the myeloid cell phenotype towards an immune suppressive state, which mediates T cell inhibition. On the other hand, STAT3 signaling in T cells leads to proliferation and T cell activity required for an anti-tumor response. Targeted delivery of STAT3 inhibitors to cancer cells and myeloid cells could therefore improve therapeutic outcomes. Many compounds that inhibit the STAT3 pathways for cancer treatment include peptide drugs, small molecule inhibitors, and natural compounds. However, natural compounds that inhibit STAT3 are often hydrophobic, which reduces their bioavailability and leads to unfavorable pharmacokinetics. This review focuses specifically on liposome-encapsulated natural STAT3 inhibitors and their ability to target cancer cells and myeloid cells to reduce tumor growth and decrease STAT3-mediated immune suppression. Many of these liposome formulations have led to profound tumor reduction and examples of combination formulations have been shown to eliminate tumors through immune modulation.

Intratumoral delivery of antigen with complement C3-bound liposomes reduces tumor growth in mice.

Antigen presenting cells (APCs) initiate the immune response against cancer by engulfing and presenting tumor antigens to T cells. Our lab has recently developed a liposomal nanoparticle that binds complement C3 proteins, allowing it to bind to the complement C3 receptors of APCs and directly deliver antigenic peptides. APCs were shown to internalize and process complement C3-bound liposomes containing ovalbumin (OVA), resulting in a significant increase in activated T cells that recognize OVA. Mice bearing A20-OVA lymphoma tumors were treated with OVA-loaded C3-liposomes, which led to reduced tumor growth in both treated and distal tumors in all mice. Peripheral blood from treated mice had a lower percentage of immunosuppressive myeloid derived suppressor cells (MDSCs), a higher percentage of B cells, and increased anti-OVA IgG1 levels compared to control mice. These results indicate that C3-liposome delivery of tumor antigen to APCs initiates a potent and systemic antitumor immune response.

Complement C3-dependent uptake of targeted liposomes into human macrophages, B cells, dendritic cells, neutrophils, and MDSCs.

Antitumor immunity in cancer patients is heavily modulated by cells of the innate immune system. Antigen-presenting cells, including dendritic cells, macrophages, and B cells, initiate immune recognition of tumor antigen by displaying antigen to effector cells. Countering this immune stimulation are immunosuppressive cells which include M2 macrophages, N2 neutrophils, and myeloid-derived suppressor cells (MDSCs). To create effective cancer immunotherapies, it is critical that we can target these important cell types of the immune system with immunostimulatory compounds. A commonality of these cell types is the complement receptor, which recognizes pathogens that are bound to activated complement C3 in human blood. To target the complement receptor, we have created a liposome that has a small molecule, orthopyridyl disulfide (OPSS), conjugated to its surface. OPSS forms a disulfide bond with activated complement C3, which then targets liposomes for uptake by dendritic cells, macrophages, B cells, MDSCs, and neutrophils in human blood. Internalization is efficient and specific to cells that display the complement receptor. Liposomes are a versatile drug delivery device. Possible applications for this system include delivery of toll-receptor agonists or tumor antigen to antigen-presenting cells and delivery of immunostimulatory drugs to M2, N2, and MDSC immunosuppressive cells.

Complement C3 mediated targeting of liposomes to granulocytic myeloid derived suppressor cells.

In cancer patients, granulocytic myeloid derived suppressor cells (G-MDSCs) expand in number, infiltrating tumor and lymphatic tissues where they suppress an anti-tumor immune response. We report here the development of a liposomal drug delivery system that selectively targets G-MDSCs. The liposomes form a disulfide bond with activated complement C3 after intravenous injection and are taken up by G-MDSCs, which express the receptor for activated C3. In vitro experiments utilizing serum from a C3 knockout mouse demonstrate that G-MDSCs take up these liposomes in a C3-dependent manner. After systemic administration to tumor bearing mice, liposomes were incorporated by 22% of G-MDSCs in the blood and were also present in a percentage of G-MDSCs in the tumor (11%), spleen (22%), liver (35%) and lungs (26%). This liposomal system offers a versatile means of targeted drug delivery to G-MDSCs and could be an important tool for restoring anti-tumor immunity in cancer patients. FROM THE CLINICAL EDITOR: It has been shown that the presence of granulocytic myeloid derived suppressor cells (G-MDSCs) in cancer patients suppress the tumor immune response of T cells. Many drugs can be used to reverse this process. In this article, the authors describe the development of a liposomal drug delivery system for targeted drug delivery to G- MDSCs. This system may prove to be useful adjunct in immunotherapy in the fight against cancers.

Targeting Her-2+ breast cancer cells with bleomycin immunoliposomes linked to LLO.

Bleomycin is a membrane impermeable chemotherapeutic agent that is relatively innocuous extracellularly but highly cytotoxic when delivered directly to the cytoplasm. We report on the development of a liposome delivery system that targets Her-2 overexpressing breast cancer cells and breaches the endosomal barrier, delivering bleomycin to the cytoplasm. The liposomes are conjugated to the antibody trastuzumab, which results in specific binding and internalization of liposomes into Her-2 overexpressing cells. In addition, the liposomes are disulfide bonded to a pore-forming protein listeriolysin O (LLO) which forms pores in the endosome and allows the liposomal cargo to pass into the cytoplasm. We demonstrate specific delivery to Her-2 positive MCF-7/Her18 cells relative to Her-2 negative MCF-7 cells using a fluorescent probe calcein within the immunoliposomes. When calcein is replaced by bleomycin, the liposomes effectively reduce viability of five different Her-2 overexpressing cell lines (BT-474, SKBR-3, MCF-7/Her18, HCC-1954 and MDA-453) while harming to a much lesser extent Her-2 negative breast cell lines (MCF-7, MCF-12a and MCF-10a). The liposomes also affect trastuzumab-resistant cells, reducing MDA-453 cell number by 97% compared to untreated cells. Importantly, the concentration of drug needed to reduce tumor cell growth and viability using this liposome therapy is approximately 57,000-fold less than the concentration needed if drug is delivered extracellularly, raising the possibility of increased therapeutic specificity with decreased side effects.

Listeriolysin O enhances cytoplasmic delivery by Her-2 targeting liposomes.

To enhance cytoplasmic delivery of liposomal contents to breast cancer cells, the authors have attached the pore-forming protein, listeriolysin O (LLO), to thermosensitive liposomes. The antibody trastuzumab (Herceptin) was also conjugated with the outer surface of the liposomes, resulting in highly specific binding and internalization into mammary epithelial cells that overexpress the human epidermal growth factor receptor 2 (Her-2). The liposomes were preloaded with a marker fluorescent dye, and the effect of LLO on the distribution of dye within the cells was monitored using fluorescence microscopy. Owing to the thermosensitive nature of the liposomes, hyperthermia at 42 degrees C triggered the release of the encapsulated fluorescent calcein from the endocytosed liposomes into the interior of the endosomes. LLO, when conjugated to these liposomes, subsequently formed pores in the endosomal membrane, allowing calcein to flow out of the endosomal compartment into the cytoplasm. Her-2-targeted liposomes bearing LLO delivered a 22-fold greater concentration of calcein to mammary epithelial cells that overexpress Her-2 compared to cells with normal Her-2 expression. Thus, the addition of LLO to preformed liposomes offers a method for significantly enhancing delivery of liposomal contents to the cytoplasm of targeted cells.

A two-component drug delivery system using Her-2-targeting thermosensitive liposomes.

We report on a new method for enhancing the specificity of drug delivery for tumor cells, using thermosensitive immunoliposomes. The liposomes are conjugated to the antibody trastuzumab (Herceptin), which targets the human epidermal growth factor receptor 2 (Her-2), a cell membrane receptor overexpressed in many human cancers. Being thermosensitive, the liposomes only release their contents when heated slightly above body temperature, allowing for the possibility of tissue targeting through localized hyperthermia. Using self-quenching calcein, we demonstrate the release of liposome contents into cell endosomes after brief heating to 42 degrees C. To further increase targeting specificity, we incorporate the concept of a two-component delivery system that requires the interaction of two different liposomes within the same endosome for cytoplasmic delivery. Experimental evaluation of the technique using fluorescently labeled liposomes shows that a two-component delivery system, combined with intracellular disruption of liposomes by hyperthermia, significantly increases specificity for Her-2-overexpressing tumor cells.

Methods for the study of redox-mediated changes in p53 structure and function.

It is now generally accepted that both the structure and function of a number of specific transcriptional factors, including p53, are subject to redox regulation in cells in which these factors are expressed. The present chapter describes methods for the analysis of redox changes in the structure of p53 and the effect of redox modulation on binding of p53 to a DNA consensus sequence. In addition, methods are described for studying the effect of redox perturbations of cells on the functioning of p53 in the cell cycle and in apoptosis. By studying the effect ofredox agents on p53, we have concluded that p53 is subject to structural redox modulation and that this modulation affects the functional ability of the protein to bind to DNA, to cause cell cycle arrest, and to trigger apoptosis.

Aminothiol WR1065 induces differential gene expression in the presence of wild-type p53.

The aminothiol WR1065 exerts selective cytoprotective effects in normal cells compared to cancer cells and has clinical applications for the protection of normal cells in cancer patients undergoing radio- or chemotherapy. There is evidence that p53 is activated in response to WR1065. To examine the effects of WR1065 on the signalling pathways controlled by p53, isogeneic human colon carcinoma cell lines (HCT116) differing only in the presence or absence of wild-type p53 were used. Treatment with WR1065 resulted in G1 cell cycle arrest in the p53-positive cell line but not in the p53-negative cell line. Long-term exposure resulted in minimal apoptosis of either cell line. Changes in gene expression in p53-positive or -negative cells treated with WR1065 were examined using commercial human stress and cancer gene arrays (Clontech Atlas arrays). Genes found to be specifically upregulated in a p53-dependent manner included coproporphyrinogen oxidase, ICErel-II cysteine protease, macrophage inhibitory cytokine-1 (also known as placental transforming growth factor beta), S100A4, and Waf1/p21. However, most proapoptotic genes typically upregulated by p53 in response to DNA damage were not activated. These studies show that WR1065 specifically modulates a subset of p53 target genes in a colon carcinoma cell line, consistent with the observation that this agent elicits essentially p53-dependent, cell cycle arrest responses.