Chemo-immunotherapy by nanoliposomal epacadostat and docetaxel combination to IDO1 inhibition and tumor microenvironment suppression.

The over-activation of tryptophan (Trp) metabolism to kynurenine (Kyn) catalyzed by Indoleamine 2,3-dioxygenase-1 (IDO1) enzyme, is one of the main metabolic pathways involved in tumor microenvironment (TME) immune escape and cancer treatment failure. The most efficient of IDO1 inhibitors is Epacadostat (EPA). Since monotherapy with single-agent IDO1 inhibitor regimen has led to an insufficient anti-tumor activity, we examined the efficacy of simultaneous treatment by Liposomal epacadostat (Lip-EPA) as a potent IDO inhibitor, in combination with docetaxel (DTX) as a complement immunogenic cell death (ICD) agent against B16F10 model. First, the in vitro combination index (CI) of epacadostat (EPA) and DTX was investigated by using the unified theory. Then, the in vivo efficacy of the combination therapy was assessed. Results indicated the synergestic cytotoxic effect of the combination on B16F10 compared to normal fibroblast cells (NIH). The immune profiling demonstrated a significant increase in the percentage of infiltrated T lymphocytes and IFN-γ release, a significant decrease in the percentage of regulatory T cells (Treg) population and the subsequent low levels of IL-10 generation in mice treated with Lip-EPA + DTX. Further, a significant tumor growth delay (TGD = 69.15 %) and an increased life span (ILS > 47.83 %) was observed with the combination strategy. Histopathology analysis revealed a remarkable increase in the Trp concentration following combination treatment, while Kyn levels significantly decreased. Results showed that the nano-liposomal form of IDO1 inhibitor in combination with chemotherapy could significantly improve the imunity response and dominate the tumor immuno-suppressive micro-environment, which merits further investigations.

Targeting the tumor microenvironment by liposomal Epacadostat in combination with liposomal gp100 vaccine.

Indoleamine-2,3-dioxygenase (IDO1) pathway has vital role in cancer immune escape and its upregulation leads to immunosuppressive environment which is associated with poor prognosis and progression in various cancers like melanoma. Previously, we showed the antitumoral efficacy of nanoliposomal form of Epacadostat (Lip-EPA), as an IDO1 inhibitor. Herein, we used Lip-EPA as a combination approach with liposomal gp100 (Lip-gp100) anti-cancer vaccine in melanoma model. Here, we showed that B16F10 tumor express IDO1 so using Lip-EPA will enhance the efficacy of vaccine therapy. The biodistribution of ICG-labelled liposomal form of EPA showed the remarkable accumulation of drug at tumor site. In an in vivo study, Lip-EPA enhanced the antitumor efficacy of Lip-gp100 in which the IDO mRNA expression was decreased (~ fourfold) in tumor samples. Also, we identified a significant increase in the number of infiltrated T lymphocytes (p < 0.0001) with enhanced in interferon gamma (IFN-γ) production (p < 0.0001). Additionally, Lip-EPA + Lip-gp100 significantly modulated intratumoral regulatory T cells which altogether resulted in the highest delay in tumor growth (TGD = 56.54%) and increased life span (ILS > 47.36%) in treated mice. Our study demonstrated that novel combination of Lip-EPA and Lip-gp100 was effective treatment with capability of being used in further clinical studies.

Liposomal celecoxib combined with dendritic cell therapy enhances antitumor efficacy in melanoma.

Cancer vaccine efficacy is limited by the immunosuppressive nature of the tumor microenvironment created by inflammation, immune inhibitory factors, and regulatory T cells (Tregs). Inspired by the role of cyclooxygenase-2 (COX-2) in inflammation in the tumor site, we proposed that normalization of the tumor microenvironment by celecoxib as a COX-2 inhibitor might improve the efficacy of Dendritic Cell (DC) therapy in a melanoma model. In the present study, liposomal celecoxib (Lip-CLX) was combined with ex vivo generated DC vaccines pulsed with gp100 peptide (in liposomal and non-liposomal forms) for prophylactic and therapeutic evaluation in the B16F10 melanoma model. Tumor site analysis by flow cytometry demonstrated that intravenous administration of Lip-CLX at a dose of 1 mg/kg in four doses effectively normalized the tumor microenvironment by reducing Tregs and IL-10 production. Furthermore, in combination with DC vaccination (DC + Lip-peptide+Lip-CLX), it significantly increased tumor-infiltrating CD4+ and CD8+ T cells and secretion of IFN-γ. This combinatorial strategy produced an effective prophylactic and therapeutic antitumor response, which reduced tumor growth and prolonged the overall survival. In conclusion, our findings suggest that the liposomal celecoxib targets the inhibitory mechanisms of the tumor microenvironment and broadens the impact of DC therapy to improve the outcome of immunotherapy in solid tumors.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy.

Lack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

Preparation of nanoliposomes containing HER2/neu (P5+435) peptide and evaluation of their immune responses and anti-tumoral effects as a prophylactic vaccine against breast cancer.

HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.

Liposomal gp100 vaccine combined with CpG ODN sensitizes established B16F10 melanoma tumors to anti PD-1 therapy.

OBJECTIVES: Program death 1 (PD-1)/ program death-ligand 1 (PD-L1) pathways, as the main inhibitory checkpoints, induce immunosuppression in the tumor microenvironment (TME). Despite the importance of inhibitor checkpoint receptor (ICR) blockers, their outcomes have been limited by the low immune response rate and induced acquired resistance. Pre-existing tumor-specific T cells is related to the improvement of their therapeutic efficacy. In the present study, we show that the combination of liposomal gp100 nanovaccine with anti PD-1 monoclonal antibody (mAb) potentiates the therapeutic effect in the melanoma model. MATERIALS AND METHODS: In this study, we first decorate the cationic liposome with gp10025-33 self-antigen and then characterize it. Mice bearing B16F10 melanoma tumors were vaccinated with different formulations of gp100 peptide (free or liposomal form) with or without CpG ODN adjuvant in combination with anti PD-1 mAb. RESULTS: Therapeutic combination of liposomal nanovaccine and CpG with anti PD-1 mAb, demonstrated the increased number of tumor infiltrated lymphocytes (TILs) in TME with the highest IFN-γ production and cytotoxic activity, which led to remarkable tumor regression. CONCLUSION: Our results demonstrated the synergism between Lip-peptide+CpG nanovaccine and anti PD-1 regime, which improved the therapeutic efficacy of PD-1 checkpoint blocker in melanoma mice models.

Vaccination with dendritic cells pulsed ex vivo with gp100 peptide-decorated liposomes enhances the efficacy of anti PD-1 therapy in a mouse model of melanoma.

BACKGROUND: Targeting antigens to dendritic cells (DCs) via nanoparticles is a powerful strategy which improves the efficacy of ex vivo antigen-pulsed DC vaccines. METHODS: In this study, liposomes were first decorated with gp10025-33 self-antigen and then characterized. Then, DCs were pulsed ex vivo with liposomal gp100 and injected subcutaneously in mice bearing B16F10 established melanoma tumors in combination with anti-PD-1 therapy. RESULTS: Treatment with liposomal pulsed DC vaccine elicited the strongest anticancer immunity and enhanced intratumoral immune responses based on infiltration of gp100-specific CD4+ and CD8+ T cells to the tumor leading to significant tumor growth regression and prolonged survival rate. Treatment with liposomal pulsed DC vaccine also markedly enhanced specific cytotoxic T lymphocytes (CTL) responses with a significant higher titer of IFN-γ in the spleen. Moreover, a significant increase of PD-1 expressing CD8+ tumor infiltrating lymphocytes (TILs) was detected in tumors. CONCLUSION: Our results demonstrate an optimum dose of liposomal gp100 significantly increases the efficacy of anti-PD-1 therapy in mice and might be an effective strategy to overcome resistance to anti-PD-1 therapy.

Ex vivo-generated dendritic cell-based vaccines in melanoma: the role of nanoparticulate delivery systems.

Melanoma is a poor immunogenic cancer and many treatment strategies have been used to enhance specific or nonspecific immunity against it. Dendritic cell (DC)-based cancer vaccine is the most effective therapies that have been used so far. Meanwhile, the efficacy of DC-based immunotherapy relies on critical factors relating to DCs such as the state of maturation and proper delivery of antigens. In this regard, the use of nanoparticulate delivery systems for effective delivery of antigen to ex vivo-generated DC-based vaccines that also poses adjuvanticity would be an ideal approach. In this review article, we attempt to summarize the role of different types of nanoparticulate antigen delivery systems used in the development of ex vivo-generated DC-based vaccines against melanoma and describe their adjuvanticity in mediation of DC maturation, cytoplasmic presentation of antigens to MHC class I molecules, which led to potent antigen-specific immune responses. As were represented, cationic liposomes were the most used approach, which suggest its potential applicability as delivery systems for further experiments in combination with either adjuvants or monoclonal antibodies.

P435 HER2/neu-derived peptide conjugated to liposomes containing DOPE as an effective prophylactic vaccine formulation for breast cancer.

The present study was aimed to develop an effective nanoliposomal vaccine delivery system with P435 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The nanoliposome formulation composed of DSPC/DSPG/Chol/DOPE and monophosphoryl lipid A was used as an adjuvant. Liposomal formulations were prepared and their physical properties were characterized. Anti-tumoral efficacy of formulations was evaluated by immunization of tumor-bearing BALB/c mice and the generated immune response was studied by using ELISpot and flow cytometry analysis. The results of the study demonstrated Lip + DOPE + P535 formulation caused the lowest tumor size and the longest survival time in TUBO mice model and could make it a promising candidate in developing effective vaccines against HER2-positive breast cancers.

Preparation of nanoliposomes linked to HER2/neu-derived (P5) peptide containing MPL adjuvant as vaccine against breast cancer.

The study was aimed at evaluating antitumor and immunomodulatory effects of liposomal vaccine composed of P5 human epidermal growth factor receptor 2 (HER2)/neu-derived peptide coupled to the surface of high-temperature nanoliposomes containing distearoylphosphocholine:distearoylphosphoglycerol:Chol:dioleoylphosphatidylethanolamine (DOPE) comprising monophosphoryl lipid A (MPL) adjuvant in HER2/neu overexpressing the breast cancer model. BALB/c mice bearing TUBO carcinoma were subcutaneously immunized with formulations containing 10 µg P5 peptide and 25 µg MPL three times with 2-week intervals. To determine immuno responses in immunized mice, the amount of released interferon-γ and IL-4 were measured by the enzyme-linked immunospot method and the flow cytometric analysis on the isolated splenocytes. The results demonstrated that tumor-bearing mice immunized with Lip/DOPE/MPL/P5 formulation had the most released interferon-γ and the highest cytotoxic T lymphocyte responses that led to the lowest tumor size and the longest survival time than those of other formulations. The results achieved by Lip/DOPE/MPL/P5 formulation could make it a suitable candidate to induce effective antigen-specific tumor immunity against breast cancer.

Stimulation of Tumor-Specific Immunity by p5 HER-2/neu Generated Peptide Encapsulated in Nano-liposomes with High Phase Transition Temperature Phospholipids.

BACKGROUND: The aim of this study is to evaluate the possible advantages of liposomes with high transition temperature (Tm) in the function of a vaccine for P5 HER2/neu-generated peptide and its adjuvant action to elicit CD8+ T cell response and its efficacy in TUBO in vivo tumor mice model, which over expresses the HER2/neu oncogene. P5, a hydrophobic peptide, was encapsulated in the nanoliposomes consisting of DSPC/DSPG/Chol(Tm 54°C) with a chaotropic loading system via 7M urea and described by size, zeta potential, encapsulation efficiency, and the structural stability of SDS-PAGE. METHODS: We immunized the mice for three times subcutaneously based on a two-week intervals using encapsulated peptide in the nanoliposomes, empty liposome, P5 in PBS, and PBS. Enzyme-linked immunospot assay, cytotoxicity test, and flow cytometric studies followed by the size of tumor and survival time measurements, which were done in TUBO tumor mice version. RESULTS: Findings of ELISpot and flow cytometric analysis showed that immunization with Lip-p5 nanoliposomes has enhanced the antigen-specific IFN-γ response of CD8+ T cells and induced CTL response, which resulted in a smaller tumor and longer survival time. In addition to increase in amounts of IFN-γ-CD8+ T cells in a group, which was immunized with Lip-P5, our findings also revealed a Th1 shift in the group immunized with an empty liposome with reduced frequencies of IL-4-producing cells and increase of IFN-γ-producing cells. CONCLUSION: The results indicated that simple liposomes consisting of phospholipids with high transition temperature could be an effective vaccine vehicle for tumor-associated antigens for inducing cell mediate immunity.

P5 HER2/neu-derived peptide conjugated to liposomes containing MPL adjuvant as an effective prophylactic vaccine formulation for breast cancer.

Vaccines containing synthetic peptides derived from tumor-associated antigens (TAA) can elicit potent cytotoxic T lymphocyte (CTL) response if they are formulated in an optimal vaccine delivery system. The aim of this study was to develop a simple and effective lipid-based vaccine delivery system using P5 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The conjugated lipid was then incorporated into liposomes composed of DMPC:DMPG:Chol:DOPE containing Monophosphoryl lipid A (MPL) (Lip-DOPE-P5-MPL). Different liposome formulations were prepared and characterized for their physicochemical properties. To evaluate anti-tumoral efficacy, BALB/c mice were immunized subcutaneously 3 times in two-week intervals and the generated immune response was studied. The results demonstrated that Lip-DOPE-P5-MPL induced a significantly higher IFN-γ production by CD8+ T cells intracellularly which represents higher CTL response in comparison with other control formulations. CTL response induced by this formulation caused the lowest tumor size and the longest survival time in a mice model of TUBO tumor. The encouraging results achieved by Lip-DOPE-P5-MPL formulation could make it a promising candidate in developing effective vaccines against Her2 positive breast cancers.

Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN.

Cationic liposomes have been used as efficient antigen delivery systems for cancer vaccination. The current study has investigated whether the incorporation of the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) in cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol enhances the cytosolic delivery of p5 HER-2/neu derived peptide (p5) and promotes cytotoxic T lymphocytes (CTL) response. The p5, which is a very hydrophobic peptide, was encapsulated into liposomes by using three different methods and characterized for their colloidal properties. A chaotropic loading method using 7 M urea provided the highest encapsulation yields. Mice were first immunized with encapsulated p5 in liposomes composed of either DOTAP-cholesterol or DOTAP-cholesterol-DOPE, alone or co-administered with CpG-ODN, as an immunoadjuvant, then, inoculated with a subcutaneous injection of TUBO tumor cells. Results obtained from enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays as well as tumor sizes and animal survival analysis demonstrated that p5 encapsulated in DOTAP-cholesterol-DOPE liposomes co-administered with CpG-ODN greatly enhanced the cytotoxic T lymphocytes response and highly inhibited the tumor progression. The outperformance of DOTAP-cholesterol-DOPE liposomes+CpG-ODN was found to be attributed to its capability in induction of both CD8+ and CD4+ responses. This formulation could be a suitable vaccine candidate against Her2 positive cancers and merits further investigations.