Viral Nanoparticles: Cancer Vaccines and Immune Modulators.

In the last decades, viruses have gained great interest in the field of immuno-oncology (I-O) for their ability of interacting both with the immune system and the tumour microenvironment. Those pathogens have naturally evolved and been evolutionary to specifically infect hosts, replicate, deliver their genome, and spread. These properties, initially considered a disadvantage, have been investigated and edited to turn viruses into precious allies for molecular biology serving as gene therapy vectors, adjuvants for the immune system, drug cargos, and, lately, anticancer therapeutics. As anticancer drug, one interesting option is viral engineering. Modification of either the viral genome or the outer shell of viruses can change infectivity and tissue targeting and add new functions to the viral particle. Remarkably, in the field of cancer virotherapy, scientists realized that a specific viral genomic depletion would turn the normal tropism of viruses to conditionally replicate in cancer cells only. This category of viruses, named 'Oncolytic viruses', have been investigated and used for cancer treatment in the past decades resulting in the approval of the first oncolytic virus, a herpes simplex virus expressing a stimulating factor, named T-Vec, in 2015. As such, oncolytic viruses achieved positive outcome but still are not able to completely eradicate the disease. This has brought the scientific community to edit those agents, adding to their ability to directly lysate cancer cells, few modifications to mainly boost their interaction with the immune system. Viruses experienced then a renaissance not only as infecting agent but as nanoparticle and cancer vaccines too. These strategies bring new life to the concept of using viruses as viral particles for therapeutic applications.

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses.

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.

Loss of ß2-integrin function results in metabolic reprogramming of dendritic cells, leading to increased dendritic cell functionality and anti-tumor responses.

Dendritic cells (DCs) are the main antigen presenting cells of the immune system and are essential for anti-tumor responses. DC-based immunotherapies are used in cancer treatment, but their functionality is not optimized and their clinical efficacy is currently limited. Approaches to improve DC functionality in anti-tumor immunity are therefore required. We have previously shown that the loss of ß2-integrin-mediated adhesion leads to epigenetic reprogramming of bone marrow-derived DCs (BM-DCs), resulting in an increased expression of costimulatory markers (CD86, CD80, and CD40), cytokines (IL-12) and the chemokine receptor CCR7. We now show that the loss of ß2-integrin-mediated adhesion of BM-DCs also leads to a generally suppressed metabolic profile, with reduced metabolic rate, decreased ROS production, and lowered glucose uptake in cells. The mRNA levels of glycolytic enzymes and glucose transporters were reduced, indicating transcriptional regulation of the metabolic phenotype. Surprisingly, although signaling through a central regulator of immune cell metabolisms, the mechanistic target of rapamycin (mTOR), was increased in BM-DCs with dysfunctional integrins, rapamycin treatment revealed that mTOR signaling was not involved in suppressing DC metabolism. Instead, bioinformatics and functional analyses showed that the Ikaros transcription factor may be involved in regulating the metabolic profile of non-adhesive DCs. Inversely, we found that induction of metabolic stress through treatment of cells with low levels of an inhibitor of glycolysis, 2-deoxyglucose (2DG), led to increased BM-DC activation. Specifically, 2DG treatment led to increased levels of Il-12 and Ccr7 mRNA, increased production of IL-12, increased levels of cell surface CCR7 and increased in vitro migration and T cell activation potential. Furthermore, 2DG treatment led to increased histone methylation in cells (H3K4me3, H3K27me3), indicating metabolic reprogramming. Finally, metabolic stress induced by 2DG treatment led to improved BM-DC-mediated anti-tumor responses in vivo in a melanoma cancer model, B16-OVA. In conclusion, our results indicate a role for ß2-integrin-mediated adhesion in regulating a novel type of metabolic reprogramming of DCs and DC-mediated anti-tumor responses, which may be targeted to enhance DC-mediated anti-tumor responses in cancer immunotherapy.

Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy.

Cancer immunotherapy requires a specific antitumor CD8+ T cell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ T cell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine in vitro and in vivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ T cell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

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.

Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses.

Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

PeptiVAX: A new adaptable peptides-delivery platform for development of CTL-based, SARS-CoV-2 vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a threat to public health and the global economy, necessitating the development of various vaccination strategies. Mutations in the SPIKE protein gene, a crucial component of mRNA and adenovirus-based vaccines, raised concerns about vaccine efficacy, prompting the need for rapid vaccine updates. To address this, we leveraged PeptiCRAd, an oncolytic vaccine based on tumor antigen decorated oncolytic adenoviruses, creating a vaccine platform called PeptiVAX. First, we identified multiple CD8 T-cell epitopes from highly conserved regions across coronaviruses, expanding the range of T-cell responses to non-SPIKE proteins. We designed short segments containing the predicted epitopes presented by common HLA-Is in the global population. Testing the immunogenicity, we characterized T-cell responses to candidate peptides in peripheral blood mononuclear cells (PBMCs) from pre-pandemic healthy donors and ICU patients. As a proof of concept in mice, we selected a peptide with epitopes predicted to bind to murine MHC-I haplotypes. Our technology successfully elicited peptide-specific T-cell responses, unaffected by the use of unarmed adenoviral vectors or adeno-based vaccines encoding SPIKE. In conclusion, PeptiVAX represents a fast and adaptable SARS-CoV-2 vaccine delivery system that broadens T-cell responses beyond the SPIKE protein, offering potential benefits for vaccine effectiveness.

Personalizing Oncolytic Virotherapy.

The use of oncolytic viruses has become an attractive tool in the clinics for the treatment of various tumor types. Such viruses are genetically modified to conditionally replicate in malignant cells while unharming healthy cells. This platform offers a highly specific tumor killing with exceptional safety profiles. However, the use of oncolytic viruses as sole oncolytic platforms has not achieved full tumor clearance in murine models and in the clinics. In fact, the formation of anti-tumor immune responses is attributed to the effectiveness of oncolytic viruses. In this review, we will discuss the various strategies that scientists have employed to enhance the anti-tumor immune responses driven by oncolytic viruses. Moreover, focus will be drawn into personalizing such anti-tumor responses by the addition of tumor-associated peptides.

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.

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

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.

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 infiltration 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.

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-γ 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.

Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine.

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.

GAMER-Ad: a novel and rapid method for generating recombinant adenoviruses.

Oncolytic adenoviruses have become ideal agents in the path toward treating cancer. Such viruses have been engineered to conditionally replicate in malignant cells in which certain signaling pathways have been disrupted. Other than such oncolytic properties, the viruses need to activate the immune system in order to sustain a long-term response. Therefore, oncolytic adenoviruses have been genetically modified to express various immune-stimulatory agents to achieve this. However, genetically modifying adenoviruses is very time consuming and labor intensive with the current available methods. In this paper, we describe a novel method we have called GAMER-Ad to genetically modify adenovirus genomes within 2 days. Our method entails the replacement of the gp19k gene in the E3 region with any given gene of interest (GOI) using Gibson Assembly avoiding the homologous recombination between the shuttle and the parental plasmid. In this manuscript as proof of concept we constructed and characterized three oncolytic adenoviruses expressing CXCL9, CXCL10, and interleukin-15 (IL-15). We demonstrate that our novel method is fast, reliable, and simple compared to other methods. We anticipate that our method will be used in the future to genetically engineer oncolytic but also other adenoviruses used for gene therapy as well.

Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform.

Oncolytic viruses (OVs) have been shown to induce anti-cancer immunity and enhance cancer immunotherapies, such as immune checkpoint inhibitor therapies. OV therapies can be further improved by arming OVs with immunostimulatory molecules, including various cytokines or chemokines. Here, we have developed a novel adenovirus encoding two immunostimulatory molecules: cluster of differentiation 40 ligand (CD40L) and tumor necrosis factor receptor superfamily member 4 ligand (OX40L). This novel virus, designated VALO-D102, is designed to activate both innate and adaptive immune responses against tumors. CD40L affects the innate side by licensing antigen-presenting cells to drive CD8+ T cell responses, and OX40L increases clonal expansion and survival of CD8+ T cells and formation of a larger pool of memory T cells. VALO-D102 and its murine surrogate VALO-mD901, expressing murine OX40L and CD40L, were used in our previously developed PeptiCRAd cancer vaccine platform. Intratumoral administration of PeptiCRAd significantly increased tumor-specific T cell responses, reduced tumor growth, and induced systemic anti-cancer immunity in two mouse models of melanoma. In addition, PeptiCRAd therapy, in combination with anti-PD-1 immune checkpoint inhibitor therapy, significantly improved tumor growth control as compared to either monotherapy alone.

Novel personalized cancer vaccine platform based on Bacillus Calmette-Guèrin.

BACKGROUND: Intratumoral BCG therapy, one of the earliest immunotherapies, can lead to infiltration of immune cells into a treated tumor. However, an increase in the number of BCG-induced tumor-specific T cells in the tumor microenvironment could lead to enhanced therapeutic effects. METHODS: Here, we have developed a novel cancer vaccine platform based on BCG that can broaden BCG-induced immune responses to include tumor antigens. By physically attaching tumor-specific peptides onto the mycobacterial outer membrane, we were able to induce strong systemic and intratumoral T cell-specific immune responses toward the attached tumor antigens. These therapeutic peptides can be efficiently attached to the mycobacterial outer membrane using a poly-lysine sequence N-terminally fused to the tumor-specific peptides. RESULTS: Using two mouse models of melanoma and a mouse model of colorectal cancer, we observed that the antitumor immune responses of BCG could be improved by coating the BCG with tumor-specific peptides. In addition, by combining this novel cancer vaccine platform with anti-programmed death 1 (anti-PD-1) immune checkpoint inhibitor (ICI) therapy, the number of responders to anti-PD-1 immunotherapy was markedly increased. CONCLUSIONS: This study shows that intratumoral BCG immunotherapy can be improved by coating the bacteria with modified tumor-specific peptides. In addition, this improved BCG immunotherapy can be combined with ICI therapy to obtain enhanced tumor growth control. These results warrant clinical testing of this novel cancer vaccine platform.

ß2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional ß2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional ß2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, ß2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting ß2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

Viral Molecular Mimicry Influences the Antitumor Immune Response in Murine and Human Melanoma.

Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8+ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.

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.

PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification.

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.