Trp2 Peptide Vaccine Adjuvanted with (R)-DOTAP Inhibits Tumor Growth in an Advanced Melanoma Model
Elizabeth A. Vasievich, Srinivas Ramishetti, Yuan Zhang, and Leaf Huang
Molecular Pharmaceutics, 2012, 9 (2), 261–268
Abstract: 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 2 weeks at 4 °C. A dose of 75 nmol of Trp2 (formulated in (R)-DOTAP) was able to show statistically significant tumor growth delay compared to lower doses of 5 and 25 nmol, which were no different than untreated tumors. (R)-DOTAP/Trp2 (75 nmol) treated mice also showed increased T cell IFN-γ secretion after re-stimulation 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 re-stimulation 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.
Enantiospecific Adjuvant Activity of Cationic Lipid DOTAP in Cancer Vaccine
Elizabeth A. Vasievich • Weihsu Chen •Leaf Huang
Cancer Immunology Immunotherapy, 2011, Volume 60, Number 5, 629-638
Abstract: 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-c 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-c 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.
Induction of Cytotoxic T-Lymphocytes and Antitumor Activity by a Liposomal Lipopeptide Vaccine
Weihsu Chen • Leaf Huang
Molecular Pharmaceutics 2008
Abstract: A simple yet effective liposome-based therapeuticvaccine, DOTAP/E7, which contains only two molecules, the cationic lipid DOTAP and a peptide antigen derived from the E7 oncoprotein of human papillomavirus (HPV) type 16 was previously described. In the current study an E7-lipopeptide is incorporated as the antigen instead of the water-soluble native E7 peptide. The lipopeptide consists of an N-terminal α- or ε-palmitoyl lysine connected to the E7 peptide via a dipeptide Ser-Ser linker. The DOTAP/E7-lipopeptide vaccine exhibits an enhanced functional antigen-specific CD8+ T lymphocyte response in vivo compared to the previous DOTAP/E7 formulation. The antitumor activity of lipopeptide formulated in DOTAP liposome was more than twice as potent as that of native E7, likely owing to the increased peptide entrapment efficiency in the liposomal complex. Our results also showed that it is essential to have the dipeptide spacer sequence between E7 peptide and the attached fatty acid to achieve a full immune response. Overall, the improved DOTAP/E7-lipopeptide vaccine described herein showed a significantly enhanced therapeutic effect for the treatment of cervical cancer.
A simple but effective cancer vaccine consisting of an antigen and a cationic lipid
Weihsu Chen • Weili Yan • Leaf Huang
Cancer Immunology Immunotherapy. 57:517-530, 2008
Abstract: Developing a cancer vaccine with a potent adjuvant, which is safe for human use, remains to be an unmet need. In this study, we developed a simple, safe, yet efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and a peptide antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16. The anti-cancer activity of DOTAP/E7 against existing HPV positive TC-1 tumor was compared to that of the previous LPD/E7 formulation, which contains bacterial DNA CpG motifs. Tumor-bearing mice showed significant tumor inhibition following a single vaccination of either formulation at the optimal lipid dose, suggesting that DOTAP alone can provide a potent adjuvant activity without plasmid DNA. E7 peptide formulated with DOTAP induced migration of activated dendritic cells (DC) to the draining lymph node (DLN) and efficiently generated functional antigen-speciWc CD8+ T lymphocyte responses. Accumulation of CD8+ tumor infiltrating T cells and apoptosis at tumor sites were observed after treatment with DOTAP/E7 complexes, which was also associated with a decreased amount of CD25+Foxp3+ regulatory T cells in treated animals. Overall, these results indicate that cationic lipid DOTAP alone serves as an efficient vaccine adjuvant for the induction of a therapeutic, antigen-specific anti-cancer activity.
Mechanism of adjuvant activity of cationic liposomes: Phosphorylation of a MAP kinase, ERK and induction of chemokines.
W. Yan, W. Chen and L. Huang.
Molecular Immunology, 2007, 44 (15), 3672-3681.
Abstract: Cationic liposomes have been effectively used as a delivery system for DNA and protein vaccines. Recently, we reported that strong antitumor immunity could be generated when a peptide antigen (E7) was incorporated into DOTAP (1, 2-Dioleoyl-3-Trimethylammonium-Propane (Chloride Salt)) cationic liposomes. DOTAP liposomes exhibit not only efficient delivery capacity but also a potent adjuvant activity. In this report, the molecular mechanism of the adjuvanticity was studied both in vitro and in vivo. Microarray mRNA analysis demonstrated that several chemokine genes are up-regulated by DOTAP liposomes, including CCL2, CCL3 and CCL4. CCL2 induction was mediated through the extracellular-signal-regulated kinase (ERK) pathway, demonstrated by specific inhibitors of the ERK pathway and siRNA approaches. Furthermore, DOTAP induced CCL2 expression is negatively regulated by the p38 pathway. Consistent with this finding, ERK activation by DOTAP is also negatively regulated by p38. Moreover, PI-3 kinase was shown to be involved in both activation of ERK and induction of CCL2 by DOTAP. DOTAP- induced CCL2 release was also confirmed in the draining lymph nodes. More importantly, inhibition of the ERK pathway completely abolishes the CCL2 accumulation in the draining lymph nodes and attenuates anti-tumor activity of DOTAP/E7. In conclusion, DOTAP is an active lipid stimulator for DC resulting in ERK activation and CC chemokine induction. Our data elucidated one important mechanism of the adjuvant activity of cationic liposomes and could facilitate rational design of synthetic lipid based adjuvants.
Surface-modified LPD nanoparticles for tumor targeting.
S.D. Li and L. Huang.
Annals of the N. Y. Academy of Sciences, 2006 Oct; 1082: 1-8.
Abstract: We have developed a tumor-targeted LPD formulation (liposome-polycation-DNA complex) for siRNA. With surface modification the targeted PEGylated LPD increased the delivery efficiency by four-fold and the gene-silencing effect by two- to three-fold. Downregulation of survivin in human lung cancer cells by targeted LPD induced 90% of apoptosis and sensitized the cells to cisplatin by four-fold. The PEGylated LPD formulation also significantly improved the tumor localization of siRNA in the NCI-H460 human lung cancer xenograft model. The tumor appeared to be the major uptake organ for siRNA formulated in surface-modified LPD. Our encouraging results indicate that surface-modified LPD may be a potent carrier for RNAi-based tumor therapy.
Immunostimulation of dendritic cells by cationic liposomes.
D. P. Vangasseri , Z. Cui , W. Chen, D.A. Hokey, L. D. Falo and L. Huang.
Molecular Membrane Biology, 2006, 23 (5): 385-395.
Abstract: A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-kB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-kB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of costimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-a expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.
Immunostimulation mechanism of LPD nanoparticles as a vaccine carrier.
Z. Cui, Su-Ji Han, D. P. Vangasseri, and L. Huang.
Molecular Pharmaceutics, 2005, Vol. 2, No. 1, 22-28.
Abstract: A novel and improved vaccine delivery system and/or adjuvant is actively sought to enhance the potency of vaccines. Previously, we reported that strong antitumor immunity could be generated when a peptide antigen was incorporated into LPD (cationic liposome-polycationpDNA) nanoparticles. In this study, we found that both the cationic liposome and DNA are required for the full immunostimulation activity of LPD. The unique ability of LPD to readily move into local lymphoid tissues and to activate antigen-presenting cells might be responsible for its strong immunostimulatory activity. Moreover, cationic liposomes stimulate the expression of CD80/CD86 on dendritic cells (DCs), but not the release of TNF-α from DCs, suggesting the existence of a NF-кB-independent immunostimulation pathway for cationic lipids such as DOTAP.
Liposome-polycation-DNA (LPD) particle as a carrier and adjuvant for protein-based vaccines: Therapeutic effect against cervical cancer.
Z. Cui and L. Huang.
Cancer Immunology and Immunotherapy, 2005, 54: 1180–1190.
Abstract: With the successful identification of many tumor-specific antigens, tumor-associated antigens, and the potential of using unfractionated tumor cell derivatives as tumor antigens, a system and/or adjuvant that can deliver these antigens and help them to induce strong effective anti-tumor immune responses is greatly needed. Previously, we reported that a MHC class I restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein, when incorporated into a clinically proven safe LPD (liposome-polycation-DNA) particle, was able to effectively eradicate tumors established in mice. Cervical cancer is the second most common cancer among women worldwide. HPV infection is clearly linked to this cancer. Vaccines based on the early (E) gene products of HPV could be effective in controlling it. However, besides the fact that epitope vaccines have many limitations, particularly concerning the diverse HLAs in humans, the use of the epitope as an antigen prevented us from fully characterizing the immune responses induced by the LPD as a vaccine carrier and/or adjuvant in previous studies. In the present study, by using the HPV 16 E7 protein as an antigen, we first showed that LPD, as a vaccine carrier and adjuvant induced strong and robust immune responses, both cellular and antibody. We then showed that immunization with LPD particles incorporated with either the wild type HPV 16 E7 protein or a potentially safer mutant induced strong immune responses that caused complete tumor regression in a murine model of cervical cancer. LPD could be a potent vaccine carrier and/or adjuvant for many antigens.
Coating of mannan on LPD particles containing HPV E7 peptide significantly enhances immunity against HPV-positive tumor.
Cui Z, Han SJ, Huang L.
Pharmaceutical Research. 2004 Jun;21(6):1018-25.
Abstract: Previously, our laboratory reported that liposome-protamine-DNA (LPD) nanoparticle is an effective delivery system for tumor-associated antigens. Mannan, which potentially targets antigen-presenting cells, was coated on LPD to further enhance its antitumor activity. METHODS: Cholesterol-conjugated mannan was coated on LPD. The abilities of mannan-coated LPD to target antigen-presenting cells, to activate dendritic cells, and to induce antitumor immunity were investigated and compared to those of LPD alone. RESULTS: Both in vitro and in vivo uptake of LPD showed that mannan-coated LPD particles were preferably taken up by dendritic cells and macrophages. In addition, the expression of co-stimulatory molecules CD80/CD86 on DC2.4 cells after co-incubation with mannan-coated LPD was significantly higher than that after co-incubation with LPD. A model major histocompatibility complex class I-restricted peptide antigen from HPV 16 E7 protein was incorporated into LPD to immunize mice against the growth of TC-1 tumor cells expressing E7 protein. Coating with mannan significantly enhanced both preventive and therapeutic activities of LPD/E7. Finally, the release of IFN-gamma from isolated splenocytes was significantly enhanced when mice were immunized with mannan-coated LPD/E7 than with LPD/E7 alone. CONCLUSION: Targeting of the LPD/E7 to local draining lymph nodes by mannan is partially responsible for the enhanced anti-tumor activity.
Lipid–Protamine–DNA-mediated antigen delivery to antigen-presenting cells results in enhanced anti-tumor immune responses.
J. Dileo, R. Banerjee, M. Whitmore, J. V. Nayak, L. D. Falo, Jr. and L. Huang.
Molecular Therapy, 2003, Vol. 7, No. 5, 640-648.
Abstract: Vaccination with antigenic peptides encoding tumor antigens has the potential to be an effective treatment for cancer. To induce tumor-specific cellular immune responses, a peptide antigen must be presented by antigen-presenting cells (APCs) to T-cells in the lymphatic tissues. Effective in vivo delivery of peptide antigens to APCs has been problematic. Here we use a model antigen from the HPV16 E7 protein to formulate LPD/E7 particles that upon iv administration are internalized by CD11c+ and CD11b+ cells in the marginal zone of the spleen. Either iv or sc vaccination with LPD/E7 particles induces E7-specific CTL responses stronger than those obtained using previously described liposome/peptide strategies and prevents the establishment of E7-expressing tumors. Furthermore, the administration of LPD/E7 particles to tumor-bearing mice caused complete tumor regression in 100% of the treated animals. Based on these studies, the entrapment of peptide antigens inside LPD particles may be an effective and generally applicable strategy for the enhancement of peptide vaccine potency.