No description available.

No description available.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Outcomes for most patients with acute leukemia remain dismal. In-vitro, vesiculovirus members induced rapid apoptosis of acute leukemia cells. Intravenous injection of lymphoblastic leukemia cells infected ex-vivo with attenuated Vesicular Stomatitis Virus or Maraba Virus followed by gamma-irradiation, controlled leukemic progression in murine recipients. Essential properties of this autologous vaccine [immunotherapy by Leukemia-Oncotropic Virus (iLOV)] and the host’s immune system were characterized. iLOV durability was restricted to the leukemia used to manufacture the vaccine. At administration, virion cell-entry was required but vesiculovirus lifecycle completion was not essential. Apoptotic or necrotic leukemia cells, with/without co-injection of virus, were ineffective vaccines. Similarly ineffective were leukemia cells activated by, or injected with, Toll-like receptor agonists. Naïve recipients of adoptive splenocyte transfer from vaccine-treated immunocompetent donors were protected from leukemic challenge. Efficacy was notably diminished following matched allogeneic bone marrow transplantation; this correlated with isolated depletion of cytotoxic T-cells. iLOV was ineffective in athymic mice. Taken together, iLOV therapy relies on immediate spaciotemporal interactions between infected-dead/dying leukemia cells and the immune system; this promotes adaptive anti-tumor responses. Clinical translation could target patients in remission to control relapse. During the above I discovered that under specific conditions, live vesiculovirus exposed to a precise window of UV fluence reproducibly generates unique “non-replicating rhabdovirus-derived particles” (NRRPs) that maintain cell-entry and cytopathic properties. A gamut of leukemia cells, including multidrug-resistant blasts, underwent rapid NRRPs-induced apoptosis. Normal cell lines and healthy bone marrow mononuclear cells were resistant, in part through interferon-mediated signaling responses. Administering NRRPs intravenously was curative in a murine acute leukemia model, versus uniform disease progression using maximal tolerated dose of replicating virus. Serum levels of an array of immunomodulatory cytokines were significantly elevated after injection of NRRPs. iLOV prepared with NRRPs protected recipients from otherwise lethal leukemia. Intracranial administration of NRRPs proved nonlethal as opposed to neurotoxic live vesiculovirus. Following treatment, neutralizing antibodies were diminished with NRRPs compared to replicating virus. Together, NRRPs exhibit enhanced therapeutic index over replication-competent vesiculovirus. Leukemocidal activity of NRRPs is exerted through a plurality of immune-related and direct cytotoxic effects. This novel approach now extends vesiculovirus-based therapeutics into upfront treatment for acute leukemia.

Glioblastoma is the most common primary brain tumour in adults. Despite the aggressive standard of care currently used, median patient survival following treatment is only 14 months. Innovative treatment options are needed for these patients. Recently, oncolytic viruses have emerged as promising immunotherapies for the treatment of solid tumours. Preliminary work in our lab has demonstrated that Farmington virus, a novel brain-safe oncolytic rhabdovirus, can be engineered to encode a tumour-associated antigen (TAA) to prime and boost antigen-specific adaptive immune responses. Since other rhabdoviruses share this boosting capacity, a heterologous rhabdovirus prime/boost regimen can be designed to combine two powerful oncolytics and a robust anti-TAA adaptive immune response. We evaluated Farmington’s ability to vaccinate against a self- glioblastoma antigen and two foreign glioblastoma-associated antigens. Farmington was able to vaccinate against the foreign antigens, leading to efficacy in prophylactic and therapeutic glioblastoma models. Additionally, treatment with heterologous rhabdoviruses demonstrated efficacy in an aggressive murine mammary carcinoma model. Herein, we demonstrate promising preliminary results for a novel glioblastoma therapeutic approach. Le glioblastome est la tumeur primaire la plus fréquente chez l’adulte. La survie moyenne des patients n’excède pas 14 mois malgré une prise en charge thérapeutique agressive. Par conséquent, la mise au point de traitements innovants et efficaces est une nécessité pour ces patients. Des avancées récentes ont mise en évidence l’intérêt des virus oncolytiques dans le traitement des tumeurs solides. Des travaux préliminaires réalisés au sein de notre laboratoire ont, en effet, démontré que le virus Farmington pouvait être modifié afin d’exprimer un antigène associé aux tumeurs (AAT), pour initier et potentialiser une réponse immunitaire adaptative spécifique. D’autres rhabdovirus possèdent des capacités de potentialisation immunitaire similaires et peuvent être utilisés en association avec le virus Farmington modifié pour amorcer et amplifier la réponse immunitaire oncolytique de l’hôte. Le but de ce projet était d’évaluer le potentiel du virus Farmington comme vaccin contre des antigènes tumoraux d’origine endogène ou exogène associés au glioblastome. Nos résultats ont montré que le virus Farmington a la capacité d’induire une réponse immunitaire prophylactique et thérapeutique contre les antigènes tumoraux exogènes dans des modèles de glioblastome. De plus, l’utilisation de rhabdovirus hétérologues s’est aussi révélée efficace pour le traitement de carcinome mammaire agressif chez la souris. Cette étude préliminaire apporte des résultats prometteurs pour le développement de nouvelles approches thérapeutiques efficaces dans le traitement du glioblastome.

Les anticorps bloquant les points de contrôle immunitaires modifient profondément la gestion des patients atteints de cancer. À la pointe de cette nouvelle classe d'agents anticancéreux, les anticorps anti-PD-1 / PD-L1 peuvent ainsi restaurer une réponse efficace des cellules T antitumorales. En conséquence, ces agents sont maintenant approuvés dans divers types de tumeurs, tels que le mélanome, le cancer bronchique non à petites cellules, le cancer du rein, les tumeurs ORL ou le cancer de la vessie. Ces nouvelles immunothérapies entraînent également de nouveaux profils de réponse tumorale tels que des réponses tumorales retardées ou des pseudoprogressions. Au fil de l’expérience acquise avec ces traitements, il a été observé chez certains patients un état de progression rapide de la maladie, ce qui pourrait suggérer que le blocage de points de contrôle immunitaire pourrait avoir un effet délétère en accélérant la maladie chez un sous-groupe de patients. Ce travail de thèse a permis de caractériser sur le plan clinique et biologique ce phénomène d’accélération de la croissance tumorale sous immunothérapie anti-checkpoint que nous avons définit “maladie hyperprogressive” (HPD). L’analyse transcriptomique d’échantillons tumoraux de ces patients a permis d’orienter vers un rôle spécifique de l’environnement myeloide. / Immune checkpoint blocking antibodies are profoundly changing the management of patients with cancer. At the forefront of this novel anticancer agent class, anti-PD-1/PD-L1 antibodies can exhibit a significant activity by restoring an efficient antitumor T-cell response. As a result, these agents are now approved in various tumor types such as melanoma, squamous, and nonsquamous non–small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC) or bladder cancer. Interestingly, these new immunotherapies also result in novel tumor response patterns such as delayed tumor responses or pseudoprogressions. As experience grows with these therapeutics, anecdotal reports are relating rapid disease progressions, which could suggest that immune checkpoint blockade may have a deleterious effect by accelerating the disease in a subset of patients. This thesis work has made it possible to characterize clinically and biologically this phenomenon of accelerated tumor growth under anti-checkpoint immunotherapy, which we have defined as “hyperprogressive disease” (HPD). Transcriptomic analysis of tumour samples from these patients suggested a specific role for the myeloid environment.

No description available.

archives@tulane.edu / Background: Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million and infects 1.8 million new people each year. Antiretroviral therapy (ART), although effective controlling plasma viremia and transmission, does not purge latent or persistent reservoirs necessary to eliminate infection, and must be maintained for life. It is thus imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Methods: To harness the immunosurveillance capacity of highly functional and persistent CMV-specific adaptive response, rhesus PBMCs were stimulated with rhCMV peptide pools (IE1, IE2, and pp65) to expand rhCMV-specific T cells. These cells were then genetically modified with retroviral vectors expressing a CD4 extracellular domain linked to T cell intracellular signaling domains that instruct CTL activity, converting them into HIV-specific effector cells. Vectors combine CD4 targeting with an maC46 fusion inhibitor to protect against viral entry. In a reversal of the critical step in the HIV viral lifecycle whereby virus targets new CD4+ host cells using its Env glycoprotein, these genetic modifications redirect host immune responses to target and kill Env expressing infected cells. We hypothesize that continuous stimulation of CD4-CAR T cells through their rhCMV-specific TCR will maintain activated T effector memory CTL capable of targeting HIV infected cells. Results: We find that autologous rhPBMCs can be expanded ex vivo with rhCMV peptides up to therapeutically relevant numbers for adoptive transfer. This rhCMV-specific T cell expansion enriches cells in a phenotype consistent with T effector memory differentiation. Following genetic modification and adoptive transfer, cells reach peak expansion at seven days post infusion into ART suppressed or unsuppressed SHIV infected Rhesus Macaques. We observe these cells capable of persisting in vivo for at least 2 years following reinfusion. Furthermore, these cells are maintained in vivo in an effector memory phenotype throughout the duration they were analyzed. Despite this, SHIV plasma viral loads remain unchanged. Conclusion: These studies establish use of rhCMV-specific T cells as an effective way to produce persistent genetically modified cells targeting SHIV. Future studies will need to further increase in vivo expansion, protection, and CTL activity as viral loads remain detectable. / 1 / Nathan Michel Johnson

Cancer immunotherapies focused on tumor-specific T cell responses are promising alternatives to chemotherapy/radiotherapy for various cancers as they can be engineered to specifically target tumours and establish long-term surveillance against relapsing tumours. The premise for the application of active immunotherapies is the recognition of tumor-specific and/or tumor-associated antigens by the immune system. Approaches that have been explored to this end include cancer vaccines and gene therapy/autologous cell transfer (T-cell receptor (TCR) or chimeric antigen receptor (CAR)-based). This study evaluated the use of oncolytic rhabdovirus-based vaccines (ORV) for the treatment of sarcoma with a focus on rhabdomyosarcoma (RMS). Sarcomas are amenable to to oncolytic virus (OV) infection and generate robust T-cell responses against tumour antigens. The ORV strategy undergoing clinical evaluation uses a prime-boost vaccine whereby a non-replicating adenovirus serotype 5 vector (Ad5) encoding an antigen is administered as a priming agent and boosted with a rhabdovirus encoding the same antigen (NCT02285816). However, the prevalence of pre-existing immunity to Ad5 in patients serves as an exclusion criterion and limits its effectiveness as a priming agent. To this end, we have shown that an alternative priming agent and antigen delivery vehicle, anti-DEC205 (aDEC205), targets antigens directly to dendritic cells (DCs), inducing robust immune responses. However, a lack of targetable antigens and methods to identify antigens is a limiting step for the application of ORVs for sarcoma. Thus, the identification of immunogenic sarcoma antigens is a critical step for the study ORVs. Current methodologies have important drawbacks in that they can be prohibitively time-consuming, complex or are ineffective in coupling antigen discovery and immunogenicity. Presented herein is the study of a novel methodology for the discovery of immunogenic antigens by probing for T cell activation marker CD107a and isolation by flow cytometry. In parallel, RMS antigen discovery was also performed via peptide elution and mass spectrometry resulting in the identification of 24 novel murine RMS antigens. Ultimately, therapeutic vaccination with a subset of these antigens encoded into DEC205 and ORV did not yield immune responses in a pre-clinical model; however, this research established immunization tools for further study of immunotherapy in RMS.

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy. / 2025-05-22