Lipid-based cancer vaccines

Each aim of this project ranges from vaccine formulation to whole-body response; starting from the structure-function relationship of the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) to the immunological action in vivo. The three aims of this work examine DOTAP; the structural specificity, development of a melanoma-specific formulation and development of a joint siRNA/peptide delivery vehicle. The first aim in these studies investigates the impact of DOTAP's chiral center on a peptide based therapeutic vaccine in a murine cervical cancer model. The lipid, (R) or (S)-DOTAP was combined with an MHC Class I restricted peptide (E7), from the E7 oncogene in human cervical cancer. In this work, (R)-DOTAP/E7 was shown to act equally well as the racemic mixture in causing tumor regression, while (S)-DOTAP/E7 had a lesser effect. This data was supported by other studies evaluating the cell-mediated anti-tumor response, and in each, (R)-DOTAP/E7 showed greater efficacy. The second aim investigates the development of a peptide/lipid melanoma vaccine with a different antigenic peptide that poses unique formulation challenges. The amphipathic Trp2 peptide antigen was formulated to address poor aqueous solubility, and yielded stable vaccine particles when mixed with (R)-DOTAP. (R)-DOTAP/Trp2 delivered in high Trp2 doses to tumor-bearing mice showed enhanced tumor growth delay compared to lower Trp2 doses, which was supported by additional immunological assays. The third aim seeks to enhance the versatility of the delivery vector with added siRNA therapy. Formulation of tri-modal particle, including (R)-DOTAP adjuvant, melanoma antigen (Trp2 peptide) and siRNA against PD-L1 (RTP particles) expanded the versatility of the previously developed (R)-DOTAP/Trp2 vaccine. PD-L1 siRNA was incorporated to dampen the inhibitory signal that DCs express when activated, that can inhibit T cell function. The examination of the structure and activity of these particles in vitro and in vivo, led to further understandings of the vaccine. In a solid melanoma model, delivery of RTP showed a tumor growth delay statistically significant compared to particles with control siRNA. In closing, (R)-DOTAP is the immunologically active enantiomer, showing efficacy in cervical cancer and melanoma models, with the potential to deliver a variety of peptide antigens and siRNA, showing tumor growth delay

The Suppressive Tumor Microenvironment: A Challenge in Cancer Immunotherapy

In this review, we introduce the changing public perception of vaccines and immunotherapy in cancer treatments. We discuss the roles that different immunosuppressive cells play in the tumor microenvironment. Tumor associated macrophages (TAMs) and M1 and M2 macrophage phenotypes are discussed in depth. Additionally, the role that myeloid derived suppressor cells (MDSC) and T regulatory cells (Tregs) play in the tumor microenvironment is addressed. Highlighted are examples of therapies used against each suppressive cell type, which vary from the hypothetical to the ineffective; the inefficient to the successful. A variety of treatments have been tried to combat this fundamental problem, indeed the cause that allows cancerous mutated cells to survive, multiply and overtake the body. Efficient methods to disable each particular suppressive type of cell have been introduced; this review summarizes the discussion with a table to guide future development. We see gene therapy as the most innovative and flexible method to lead the charge to specifically modifying the tumor microenvironment

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I–restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8+ splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8+ tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8+ TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8+ splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8+ TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8+ anti-tumor response

Trp2 Peptide Vaccine Adjuvanted with ( R )-DOTAP Inhibits Tumor Growth in an Advanced Melanoma Model

Previously we have shown cationic lipid (R)-DOTAP as the immunologically active enantiomer of the DOTAP racemic mixture, initiating complete tumor regression in an exogenous antigen model (murine cervical cancer model). Here, we investigate the use of (R)-DOTAP as an efficacious adjuvant delivering an endogenous antigen in an aggressive murine solid tumor melanoma model. (R)-DOTAP/Trp2 peptide complexes showed decreasing size and charge with increasing peptide concentration, taking a rod-shape at highest concentrations. The particles were stable for at 2 weeks at 4°C. A dose of 75nmol Trp2 (formulated in (R)-DOTAP) was able to show statistically significant tumor growth delay compared to lower doses of 5 and 25nmol which were no different than untreated tumors. (R)-DOTAP/Trp2 (75nmol) treated mice also showed increased T cell IFN-γ secretion after restimulation with Trp2, as well as CTL activity in vivo. This vaccination group also showed the highest population of functionally active tumor-infiltrating lymphocytes, indicated by IFN-γ secretion after restimulation with Trp2. Thus, (R)-DOTAP has shown the ability to break tolerance as an adjuvant. Its activity to enhance immunogenicity of other tumor associated antigens should be studied further

The Function of Mammalian COPII Components Sec23a and Sec23b.

In all eukaryotic cells, cargo proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus in COPII-coated vesicles. Human diseases have recently been described that result from inherited mutations in the paralagous genes SEC23A and SEC23B, which encode a key component of the COPII vesicle coat. In this thesis, I explore the phenotypes of gene targeted mice deficient for the mouse SEC23 orthologs Sec23a and Sec23b. Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disorder characterized by mild to severe anemia, and recently reported to be due to mutations in the SEC23B gene. Mice homozygous for a gene trap insertion in the last intron of the mouse Sec23b gene (Sec23b gt/gt) were demonstrated to exhibit massive pancreatic degeneration and die within a day of birth, with no evidence of anemia on blood analysis. Chimeric mice generated by transplantation of Sec23b gt/gt hematopoietic precursor cells into wild type mice demonstrated full reconstitution of erythropoiesis, as well as other blood cell lineages. Mass spectrometry analysis of Sec23b gt/gt RBC ghosts prepared from chimeric mice using a comparative SILAC approach detected no proteins that were quantitatively decreased compared to control RBC ghosts. These results suggest divergence in tissue-specific SEC23B function between mice and humans. Humans with mutations in SEC23A are affected with Cranio-Lenticulo-Sutural Dysplasia (CLSD), which is characterized by late closure of the cranial fontanelles and mild mental retardation. Mice with a gene trap insertion in the second intron of the Sec23a gene die between day 10.5 and 11.5 of embryogenesis. Mice heterozygous for both the Sec23a gt and Sec23b gt alleles (Sec23a+/gt Sec23b+/gt) are viable and fertile with no apparent pathologic phenotype. However, Sec23a gt/gt Sec23b+/gt mice die at an earlier time point in embryogenesis than Sec23a gt/gt mice, suggesting functional overlap between SEC23A and SEC23B. These studies have shown that humans and mice with mutations in Sec23a and Sec23b have disparate but tissue specific phenotypes. This demonstrates the importance of the COPII pathway in human disease and could suggest functional divergence of these isoforms over evolutionary time between human and mouse.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97814/1/mpv_1.pd

Economic analysis of short-rotation forests for energy in Ireland

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Economic analysis of short-rotation forests for energy in Ireland

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