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

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

Biohybrid Vaccines for Improved Treatment of Aggressive Melanoma with Checkpoint Inhibitor

Recent approaches in the treatment of cancer focus on involving the immune system to control the tumor growth. The administration of immunotherapies, like checkpoint inhibitors, has shown impressive results in the long term survival of patients. Cancer vaccines are being investigated as further tools to prime tumor-specific immunity. Biomaterials show potential as adjuvants in the formulation of vaccines, and biomimetic elements derived from the membrane of tumor cells may widen the range of antigens contained in the vaccine. Here, we show how mice presenting an aggressive melanoma tumor model treated twice with the complete nanovaccine formulation showed control on the tumor progression, while in a less aggressive model, the animals showed remission and control on the tumor progression, with a modification in the immunological profile of the tumor microenvironment. We also prove that co-administration of the nanovaccine together with a checkpoint inhibitor increases the efficacy of the treatment (87.5% of the animals responding, with 2 remissions) compared to the checkpoint inhibitor alone in the B16.OVA model. Our platform thereby shows potential applications as a cancer nanovaccine in combination with the standard clinical care treatment for melanoma cancers.Peer reviewe

Front cover - Cell Membrane Wrapping: Influence of Cell Membrane Wrapping on the Cell−Porous Silicon Nanoparticle Interactions (Adv. Healthcare Mater. 17/2020)

Biohybrid nanosystems represent the cutting‐edge research in biofunctionalization of micro‐ and nano‐systems. Their physicochemical properties bring along advantages in the circulation time, camouflaging from the phagocytes, and novel antigens. This is partially a result of the qualitative differences in the protein corona, and the preferential targeting and uptake in homologous cells. However, the effect of the cell membrane on the cellular endocytosis mechanisms and time has not been fully evaluated yet. Here, the effect is assessed by quantitative flow cytometry analysis on the endocytosis of hydrophilic, negatively charged porous silicon nanoparticles and on their membrane‐coated counterparts, in the presence of chemical inhibitors of different uptake pathways. Principal component analysis is used to analyze all the data and extrapolate patterns to highlight the cell‐specific differences in the endocytosis mechanisms. Furthermore, the differences in the composition of static protein corona between naked and coated particles are investigated together with how these differences affect the interaction with human macrophages. Overall, the presence of the cell membrane only influences the speed and the entity of nanoparticles association with the cells, while there is no direct effect on the endocytosis pathways, composition of protein corona, or any reduction in macrophage‐mediated uptake

Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine

Publisher Copyright: Copyright © 2022 Feola, Russo, Martins, Lopes, Vandermeulen, Fluhler, De Giorgi, Fusciello, Pesonen, Ylösmäki, Antignani, Chiaro, Hamdan, Feodoroff, Grönholm and Cerullo.Oncolytic Viruses (OVs) work through two main mechanisms of action: the direct lysis of the virus-infected cancer cells and the release of tumor antigens as a result of the viral burst. In this sc.enario, the OVs act as in situ cancer vaccines, since the immunogenicity of the virus is combined with tumor antigens, that direct the specificity of the anti-tumor adaptive immune response. However, this mechanism in some cases fails in eliciting a strong specific T cell response. One way to overcome this problem and enhance the priming efficiency is the production of genetically modified oncolytic viruses encoding one or more tumor antigens. To avoid the long and expensive process related to the engineering of the OVs, we have exploited an approach based on coating OVs (adenovirus and vaccinia virus) with tumor antigens. In this work, oncolytic viruses encoding tumor antigens and tumor antigen decorated adenoviral platform (PeptiCRAd) have been used as cancer vaccines and evaluated both for their prophylactic and therapeutic efficacy. We have first tested the oncolytic vaccines by exploiting the OVA model, moving then to TRP2, a more clinically relevant tumor antigen. Finally, both approaches have been investigated in tumor neo-antigens settings. Interestingly, both genetically modified oncolytic adenovirus and PeptiCRAd elicited T cells-specific anti-tumor responses. However, in vitro cross-representation experiments, showed an advantage of PeptiCRAd as regards the fast presentation of the model epitope SIINFEKL from OVA in an immunogenic rather than tolerogenic fashion. Here two approaches used as cancer oncolytic vaccines have been explored and characterized for their efficacy. Although the generation of specific anti-tumor T cells was elicited in both approaches, PeptiCRAd retains the advantage of being rapidly adaptable by coating the adenovirus with a different set of tumor antigens, which is crucial in personalized cancer vaccines clinical setting.Peer reviewe

A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines

Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate > 8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-gamma enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.Peer reviewe

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids

Background Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms. Methods The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids. Results Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations. Conclusion Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.Peer reviewe

Artificially Cloaked Viral Nanovaccine for Cancer Immunotherapy

Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.Peer reviewe

Influence of Cell Membrane Wrapping on the Cell-Porous Silicon Nanoparticle Interactions

Biohybrid nanosystems represent the cutting-edge research in biofunctionalization of micro- and nano-systems. Their physicochemical properties bring along advantages in the circulation time, camouflaging from the phagocytes, and novel antigens. This is partially a result of the qualitative differences in the protein corona, and the preferential targeting and uptake in homologous cells. However, the effect of the cell membrane on the cellular endocytosis mechanisms and time has not been fully evaluated yet. Here, the effect is assessed by quantitative flow cytometry analysis on the endocytosis of hydrophilic, negatively charged porous silicon nanoparticles and on their membrane-coated counterparts, in the presence of chemical inhibitors of different uptake pathways. Principal component analysis is used to analyze all the data and extrapolate patterns to highlight the cell-specific differences in the endocytosis mechanisms. Furthermore, the differences in the composition of static protein corona between naked and coated particles are investigated together with how these differences affect the interaction with human macrophages. Overall, the presence of the cell membrane only influences the speed and the entity of nanoparticles association with the cells, while there is no direct effect on the endocytosis pathways, composition of protein corona, or any reduction in macrophage-mediated uptake