Optimized armed oncolytic adenoviral vaccines (PeptiCRAd) for an enhanced anti-cancer immune response.

In according to the last available data, cancer is the second cause of death in many countries, following only cardiovascular diseases. As the risk of cancer increases in the elderly and also because of other factors such as tobacco, low vegetable and fruit intake, pollution, new treatments are needed to replace or combine the classical treatments such as surgery, radiotherapy and chemotherapy. Among the new therapeutic approaches, one emerging and promising field is the immunotherapy, which aims to elicit de novo anti-tumour response and/or boost the pre-existing anti-tumour immunity. The cancer immunotherapy consists of different approaches such as oncolytic viruses, which are able to replicate only in cancer cells, and the immune checkpoint inhibitors, which revert or prevent T-cell exhaustion. Both approaches showed efficacy in eliciting anti-tumour immune response, but solid tumours, such as triple negative breast cancer, show poor immunogenic and immunosuppressive and could benefit from a combination of these treatments. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer currently resistant to available treatment approaches. Therefore, in the present study, we decided to evaluate the efficacy of a tumour-specific vaccine platform based on peptide-coated oncolytic adenovirus (PeptiCRAd) that we had previously developed for different tumours. PeptiCRAd is a versatile and rapid system to adsorb tumour-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response toward the tumour epitopes. In fact, the combination in a single treatment of the adjuvancy of the virus with the immunological targeting of tumour-derived peptides converts the powerful anti-viral immune response obtained with viral vaccines into a more efficient anti-tumoural response. In the present work, we adapted PeptiCRAd in a poor immunogenic and high immunosuppressive tumour model such as TNBC and for the first time we improved the PeptiCRAd platform adding on the same oncolytic vaccine tumour peptides restricted for both MHC-I and MHC-II in order to harm TCD8+ and TCD4+ lymphocytes and to obtain a more efficient and complete immune response. Tumour cells evade immune recognition and destruction by down regulating MHC-I and up-regulating PDL-1. Thus, we chose and characterized human (MDA-MB-436) and mouse (4T1) triple negative breast cancer cell lines for the expression of MHC-I, MHC-II and PDL-1, demonstrating that they are a solid model for our immunotherapeutic approach. As PeptiCRAd relies on Ad-5-D24-CpG, we demonstrated that has similar cytopathic effect (CPE) to Ad-5/3-D24, already validated in human model of TNBC. In the first set of in vivo experiments, we observed that oncolytic vaccines coated with a combination of MHC-I and MHC-II peptides induced a stronger response compared to those coated with either MHC-I or MHC-II peptides. We also observed that administration of mixture of equal concentrations of oncolytic vaccines coated with MHC-I or MHC-II peptides was less efficient compared to the double coated formulation. Therefore, we conclude that MHC-I and MHC-II peptides have to be loaded on the same surface to maximize the effect. Next, we evaluated the synergistic effect of administration of the PeptiCRAd-D.C. preparation with anti-PDL1 antibody in TNBC. Our results clearly demonstrated a significant improvement of the oncolytic vaccine efficacy when administrated in combination with anti-PDL1. Finally, we translated our treatment in a relevant human model of TNBC. We performed a Cytotoxic T-lymphocytes (CTL) killing assay in a co-culture experiment with human tumours. In vitro we pulsed with our vaccine human peripheral blood mononuclear cell (PBMCs) HLA-matched with the tumours and we added them to the tumour sample and cancer cells viability was then evaluated. The tumour peptides selected in the above experiment for the PeptiCRAd preparation were selected from well-known human triple negative breast cancer antigens. In addition, one tumour peptide was selected by using an improved version of ligandome analysis. In conclusion, we have demonstrated for the first time the efficacy of PeptiCRAd technology based oncolytic vaccine in a challenging model of TNBC. In addition, we observe that vaccine coating with a combination of MHC-I and MHC-II restricted peptides is more effective than the previously used MHC-I restricted peptides coating, leading to a further improvement of the system

Uncovering the Tumor Antigen Landscape : What to Know about the Discovery Process

According to the latest available data, cancer is the second leading cause of death, highlighting the need for novel cancer therapeutic approaches. In this context, immunotherapy is emerging as a reliable first-line treatment for many cancers, particularly metastatic melanoma. Indeed, cancer immunotherapy has attracted great interest following the recent clinical approval of antibodies targeting immune checkpoint molecules, such as PD-1, PD-L1, and CTLA-4, that release the brakes of the immune system, thus reviving a field otherwise poorly explored. Cancer immunotherapy mainly relies on the generation and stimulation of cytotoxic CD8 T lymphocytes (CTLs) within the tumor microenvironment (TME), priming T cells and establishing efficient and durable anti-tumor immunity. Therefore, there is a clear need to define and identify immunogenic T cell epitopes to use in therapeutic cancer vaccines. Naturally presented antigens in the human leucocyte antigen-1 (HLA-I) complex on the tumor surface are the main protagonists in evocating a specific anti-tumor CD8+ T cell response. However, the methodologies for their identification have been a major bottleneck for their reliable characterization. Consequently, the field of antigen discovery has yet to improve. The current review is intended to define what are today known as tumor antigens, with a main focus on CTL antigenic peptides. We also review the techniques developed and employed to date for antigen discovery, exploring both the direct elution of HLA-I peptides and the in silico prediction of epitopes. Finally, the last part of the review analyses the future challenges and direction of the antigen discovery field.Peer reviewe

Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma

Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and –5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients

Harnessing therapeutic viruses as a delivery vehicle for RNA-based therapy

Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo.Peer reviewe

Systems Biology Approaches for the Improvement of Oncolytic Virus-Based Immunotherapies

Oncolytic virus (OV)-based immunotherapy is mainly dependent on establishing an efficient cell-mediated antitumor immunity. OV-mediated antitumor immunity elicits a renewed antitumor reactivity, stimulating a T-cell response against tumor-associated antigens (TAAs) and recruiting natural killer cells within the tumor microenvironment (TME). Despite the fact that OVs are unspecific cancer vaccine platforms, to further enhance antitumor immunity, it is crucial to identify the potentially immunogenic T-cell restricted TAAs, the main key orchestrators in evoking a specific and durable cytotoxic T-cell response. Today, innovative approaches derived from systems biology are exploited to improve target discovery in several types of cancer and to identify the MHC-I and II restricted peptide repertoire recognized by T-cells. Using specific computation pipelines, it is possible to select the best tumor peptide candidates that can be efficiently vectorized and delivered by numerous OV-based platforms, in order to reinforce anticancer immune responses. Beyond the identification of TAAs, system biology can also support the engineering of OVs with improved oncotropism to reduce toxicity and maintain a sufficient portion of the wild-type virus virulence. Finally, these technologies can also pave the way towards a more rational design of armed OVs where a transgene of interest can be delivered to TME to develop an intratumoral gene therapy to enhance specific immune stimuli

Black porous silicon as a photothermal agent and immunoadjuvant for efficient antitumor immunotherapy

Photothermal therapy (PTT) in combination with other treatment modalities has shown great potential to activate immunotherapy against tumor metastasis. However, the nanoparticles (NPs) that generate PTT have served as the photothermal agent only. Moreover, researchers have widely utilized highly immuno-genic tumor models to evaluate the immune response of these NPs thus giving over-optimistic results. In the present study black porous silicon (BPSi) NPs were developed to serve as both the photothermal agent and the adjuvant for PTT-based antitumor immunotherapy. We found that the poorly immunogenic tumor models such as B16 are more valid to evaluate NP-based immunotherapy than the widely used im-munogenic models such as CT26. Based on the B16 cancer model, a cocktail regimen was developed that combined BPSi-based PTT with doxorubicin (DOX) and cytosine-phosphate-guanosine (CpG). BPSi-based PTT was an important trigger to activate the specific immunotherapy to inhibit tumor growth by featuring the selective upregulation of TNF-alpha. Either by adding a low dose DOX or by prolonging the laser heating time, a similar efficacy of immunotherapy was evoked to inhibit tumor growth. Moreover, BPSi acted as a co-adjuvant for CpG to significantly boost the immunotherapy. The present study demonstrates that the BPSi-based regimen is a potent and safe antitumor immunotherapy modality. Moreover, our study high-lighted that tuning the laser heating parameters of PTT is an alternative to the toxic cytostatic to evoke immunotherapy, paving the way to optimize the PTT-based combination therapy for enhanced efficacy and decreased side effects.Peer reviewe

Mannosylated Polycations Target CD206+Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratiosPeer reviewe

A novel cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella

Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. The intratumoral administration of these repurposed vaccines can induce immune cell infiltra-tion into the treated tumor. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV cancer vaccine platform. The intratumoral administration of this novel MMR-containing PeptiENV cancer vaccine significantly increased both intratumoral as well as systemic tumor-specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, improved tumor growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy. Importantly, mice pre-vaccinated with the MMR vaccine responded equally well, if not better, to the PeptiENV therapy, indicating that pre-existing immunity against the MMR vaccine viruses does not compromise the use of this novel cancer vaccine platform.Peer reviewe

Therapeutic Cancer Vaccination with Immunopeptidomics-Discovered Antigens Confers Protective Antitumor Efficacy

Simple Summary Immunotherapy has revolutionized cancer treatment, yet many tumors remain resistant to current immuno-oncology therapies. Here we explore a novel, customized oncolytic adenovirus vaccine platform as immunotherapy in a resistant tumor model. We present a workflow for customizing the oncolytic vaccine for improved tumor targeting. This targeting is based on experimentally discovered tumor antigens, which are incorporated as active components of the vaccine formulation. The pipeline may be further applied for designing personalized therapeutic cancer vaccines. Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.Peer reviewe

Oncolytic adenovirus drives specific immune response generated by a poly-epitope pDNA vaccine encoding melanoma neoantigens into the tumor site

Background: DNA vaccines against cancer held great promises due to the generation of a specific and long lasting immune response. However, when used as a single therapy, they are not able to drive the generated immune response into the tumor, because of the immunosuppressive microenvironment, thus limiting their use in humans. To enhance DNA vaccine efficacy, we combined a new poly-epitope DNA vaccine encoding melanoma tumor associated antigens and B16F1-specific neoantigens with an oncolytic virus administered intratumorally. Methods: Genomic analysis were performed to find specific mutations in B16F1 melanoma cells. The antigen gene sequences were designed according to these mutations prior to the insertion in the plasmid vector. Mice were injected with B16F1 tumor cells (n = 7-9) and therapeutically vaccinated 2, 9 and 16 days after the tumor injection. The virus was administered intratumorally at day 10, 12 and 14. Immune cell infiltration analysis and cytokine production were performed by flow cytometry, PCR and ELISPOT in the tumor site and in the spleen of animals, 17 days after the tumor injection. Results: The combination of DNA vaccine and oncolytic virus significantly increased the immune activity into the tumor. In particular, the local intratumoral viral therapy increased the NK infiltration, thus increasing the production of different cytokines, chemokines and enzymes involved in the adaptive immune system recruitment and cytotoxic activity. On the other side, the DNA vaccine generated antigen-specific T cells in the spleen, which migrated into the tumor when recalled by the local viral therapy. The complementarity between these strategies explains the dramatic tumor regression observed only in the combination group compared to all the other control groups. Conclusions: This study explores the immunological mechanism of the combination between an oncolytic adenovirus and a DNA vaccine against melanoma. It demonstrates that the use of a rational combination therapy involving DNA vaccination could overcome its poor immunogenicity. In this way, it will be possible to exploit the great potential of DNA vaccination, thus allowing a larger use in the clinic.Peer reviewe