Patient-Derived Organoids for Precision Cancer Immunotherapy

Cancer immunotherapy has revolutionized the way tumors are treated. Nevertheless, efficient and robust testing platforms are still missing, including clinically relevant human ex vivo tumor assays that allow pretreatment testing of cancer therapies and selection of the most efficient and safe therapy for a specific patient. In the case of immunotherapy, this testing platform would require not only cancer cells, but also the tumor microenvironment, including immune cells. Here, we discuss the applications of patient-derived tumor organoid cultures and the possibilities in using complex immune-organoid cultures to provide preclinical testing platforms for precision cancer immunotherapy.Peer reviewe

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

Controlled release of enhanced cross-hybrid IgGA Fc PD-L1 inhibitors using oncolytic adenoviruses

Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation

Novel peptide-based oncolytic vaccine for enhancement of adaptive antitumor immune response via co-engagement of innate Fcγ and Fcα receptors

BACKGROUND: Cancer immunotherapy relies on using the immune system to recognize and eradicate cancer cells. Adaptive immunity, which consists of mainly antigen-specific cytotoxic T cells, plays a pivotal role in controlling cancer progression. However, innate immunity is a necessary component of the cancer immune response to support an immunomodulatory state, enabling T-cell immunosurveillance. METHODS: Here, we elucidated and exploited innate immune cells to sustain the generation of antigen-specific T cells on the use of our cancer vaccine platform. We explored a previously developed oncolytic adenovirus (AdCab) encoding for a PD-L1 (Programmed-Death Ligand 1) checkpoint inhibitor, which consists of a PD-1 (Programmed Cell Death Protein 1) ectodomain fused to an IgG/A cross-hybrid Fc. We coated AdCab with major histocompatibility complex (MHC-I)-restricted tumor peptides, generating a vaccine platform (named PeptiCab); the latter takes advantage of viral immunogenicity, peptide cancer specificity to prime T-cell responses, and antibody-mediated effector functions. RESULTS: As proof of concept, PeptiCab was used in murine models of melanoma and colon cancer, resulting in tumor growth control and generation of systemic T-cell-mediated antitumor responses. In specific, PeptiCab was able to generate antitumor T effector memory cells able to secrete various inflammatory cytokines. Moreover, PeptiCab was able to polarize neutrophils to attain an antigen-presenting phenotype by upregulating MHC-II, CD80 and CD86 resulting in an enhanced T-cell expansion. CONCLUSION: Our data suggest that exploiting innate immunity activates T-cell antitumor responses, enhancing the efficiency of a vaccine platform based on oncolytic adenovirus coated with MHC-I-restricted tumor peptides

Protein translocation motifs: a common trait between plant and human pathogens

Some oomycetes and fungal effectors carry RXLR-dEER domains. These domains are necessary for the host cell entry and without any pathogen-encoded machinery. Using this information and bioinformatic based analyses, we identified potential cell entry motifs in the N-terminal regions of 13 secreted proteins from plant pathogens (Alternaria brassiciola, Pythium ultimum. Albugo candida), human pathogens (Aspergillus fumigatus, Cryptococcus neoformans, Coccidioides immitus), beneficial (Trichoderma atroviride) and saprophytic (Aspergillus flavus) microorganisms. Filter binding assays was used to test whether the N-terminal domains of the effector proteins bound phosphoinositides. Twelve out of thirteen of the fusions proteins bind PI3P, and more weakly to PI4P and/or PI5P. The capability to enter the host cell was confirmed by a soybean root uptake or an epithelial cell uptake assays. Mutations in the functional RXLR-like motifs of effectors resulted in a loss of binding. Alanine substitutions in the fungal RxLR motifs also abolished the activity of the N-terminal domain-GFP fusion protein, preventing the protein accumulation in human epithelial cells. To test the hypothesis that the fungal effectors bound external PI3P in order to enter human cells, we incubated the effector-GFP fusions with a mammalian cell culture in the presence of a molar excess of each of the PI3P- and PI4P-binding biosensor proteins. We used a highly specific biosensors for PI3P and PI4P by fusing the PH domains of the human proteins PEPP1 to mCherry. The PI3P-binding proteins completely abolished entry by the six effector-GFP fusions into the epithelial cells. Protein accumulation was strongly inhibited in each case by competing PI3P-binding proteins (which entered the cells instead). To determine if the effector-GFP fusions bound to the same sites on the cells as the PI3P-binding proteins, equimolar concentrations of the biosensors and effectors were incubated with A549 cells at 2°C. The effector fusions colocalized with each biosensor in a punctate pattern on the surface of the cells. At 37°C, the biosensor and the effector fusions were colocalized inside the cells within endosome-like structures. The rule of one of these proteins in the virulence was evaluated by generating a knocked-out mutant of A. fumigatus unable to produce the Af2 effector. Conidia from the wild type and from the mutants were compared with respect to their capability to activate phagocytosis in mammalian epithelial cells and for their intracellular survival

Unimodal pattern of soil hydrophobicity along an altitudinal gradient encompassing Mediterranean, temperate, and alpine ecosystems

Background and Aims: Soil water repellency (SWR, i.e. the reduced affinity for water due to the presence of hydrophobic coatings on soil particles) has relevant hydrological implications on the rate of water infiltration, surface runoff, and overland flow. Here, we test how SWR varies along a 2490 m altitudinal gradient encompassing six ecosystems including Mediterranean, Temperate, and Alpine vegetation types. Methods: Water repellency, measured by the Molarity of an Ethanol Droplet (MED) test, was quantified in 80 soil samples collected for 16 different elevations. Soil quality was assessed by measuring soil texture, pH, organic carbon, salinity, and nutrient availability. Results: SWR showed a unimodal pattern along the 2490 m transect, peaking at intermediate elevations. Unexpectedly, SWR was the highest under broad-leaf deciduous forests, and the lowest under evergreen, sclerophyllous Mediterranean vegetation types. The soil organic carbon content, and the pH were the main determinants of water repellency, showing respectively a positive, and a negative correlation with the SWR. In contrast, soil texture and salinity resulted unrelated to the SWR. Conclusions: With this study we demonstrated a linkage between SWR, vegetation type and soil pH and organic carbon content along the elevation gradient. Further studies are needed to explicitly evaluate the impact SRW on erosion risk at catchment scale in the context of climatic change

Antifungal saponins from bulbs of garlic, Allium sativum L. var. Voghiera

A bioassay-guided phytochemical analysis of the polar extract from the bulbs of garlic, Allium sativum L., var. Voghiera, typical of Voghiera, Ferrara (Italy), allowed the isolation of ten furostanol saponins; voghieroside A1/A2 and voghieroside B1/B2, based on the rare agapanthagenin aglycone; voghieroside C1/C2, based on agigenin aglycone; and voghieroside D1/D2 and E1/E2, based on gitogenin aglycone. In addition, we found two known spirostanol saponins, agigenin 3-O-trisaccharide and gitogenin 3-O-tetrasaccharide. The chemical structures of the new compounds were established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses. High concentrations of two eugenol diglycosides were also found for the first time in Allium spp. The isolated compounds were evaluated for their antimicrobial activity towards two fungal species, the air-borne pathogen Botrytis cinerea and the antagonistic fungus Trichoderma harzianum