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.Peer reviewe

PeptiCHIP : A Microfluidic Platform for Tumor Antigen Landscape Identification

Publisher Copyright: © 2021 The Authors. Published by American Chemical Society.Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.Peer reviewe

Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients’ primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors