Liver sinusoidal endothelial cells (LSECs) naturally cross-present antigens and induce T cell tolerance. Targeting LSECs with peptide-coupled nanoparticles offers an efficient strategy to induce antigen-specific immune tolerance. Previous preclinical and clinical studies have shown that peptide-coupled nanoparticles can effectively inhibit T cell responses to the selected cognate peptide epitopes. However, clinical situations such as viral-vector-mediated gene therapy would benefit from simultaneous tolerance induction to multiple epitopes/antigens, posing a significant challenge. In this study, we used mouse models of adeno-associated virus (AAV)-mediated gene transfer to assess the in vivo effects of peptide-loaded nanoparticles designed to tolerize immune responses to transgene- or/and capsid-derived epitopes. We report here for the first time that LSEC-targeting nanoparticles coupled to a single peptide epitope promoted extension of tolerance to multiple relevant epitopes/antigens and simultaneously tolerized both CD4+ and CD8+ T cell responses. This tolerance spreading, which we termed "Tspread," remained specific to the transgene- and capsid-derived antigens conveyed by the administered AAV vector and did not impair immune responses to an unrelated vaccine antigen. These findings delineate a novel LSEC-mediated tolerance spreading with important clinical implications for viral-vector gene therapy, where tolerance to multiple epitopes/antigens is essential for long-term expression of therapeutic genes.

De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and effi- cacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for in vivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular applica-tion. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in condi-tions of a late tumor challenge, i.e., 80 days post-vaccination, be-tween 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector par-ticles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.

Antibodies that recognize the ATP-gated P2X7 ion channel are etablished research tools. Nanobodies correspond to the antigen-binding variable immunoglobulin domain (VHH) of heavy chain antibodies that naturally occur in camelids. Nanobodies display better solubility than the variable domains (VH) of conventional antibodies. Therefore, it is much easier to construct bivalent and multivalent fusion proteins with nanobodies than with VH domains or with paired VH-VL domains. Moreover, nanobodies can bind functional crevices that are poorly accessbile to conventional VH-VL domains. This makes nanobodies particulary well suited as functional modulators. Here we provide protocols to raise antibodies and nanobodies against mouse and human P2X7 using cDNA-immunization. This approach evokes antibodies and nanobodies that recognize the P2X7 ion channel in native confirmation, some of which inhibit or potentiate gating of P2X7 by extracellular ATP. Furthermore, we developed protocols for producing P2X7-specific nanobodies and antibodies in vivo using rAAV vectors (AAVnano). This approach can be used either to durably inhibit or potentiate P2X7 function in vivo, or to deplete P2X7-expressing cells.

Co-signaling immunotherapy, via immune checkpoint inhibitors (ICIs), such as ipilimumab (anti-CTLA-4) or nivolumab (anti-PD-1), targeting immune cell co-signaling molecules ICI, has revolutionized cancer treatment. However, reinvigorating tumor-infiltrating T cell cytotoxicity has revealed cardiac toxicity in a subset of patients, uncovering still not fully deciphered cardiac immune tolerance mechanisms. In this context, we aim to study the impact of co-signalling pathways on the cardiac immune response in cardiovascular diseases. Full T cell activation requires three key signals: (a) antigenic stimulation (TCR) (b) interaction with co-signaling receptors, and (c) cytokine-induced signals. Our hypothesis is that immune cell activation in CVDs, mediated by pathogenic cardiac deregulation of co-signaling pathways, affects lymphatic and vascular remodeling in the heart, leading to poor resolution of cardiac inflammation and edema, and subsequently HF progression. Left ventricular pressure-overload model in mice is induced by surgical transversal aortic constriction (TAC), leading to the development of either concentric hypertrophic cardiomyopathy or eccentric hypertrophic cardiomyopathy (DCM-like) depending on murine strain and gender and to heart failure. Tissular and cellular snapshot of co-signaling pathways during cardiac remodeling has been evaluated by transcriptomic tools. We found that CTLA-4 gene is differentially expressed in cardiac tissue of each strain after TAC. In fact, lower expression of CTLA-4 is noticed in eccentric hypertrophic cardiomyopathy model, associated with higher cardiac total IgG amount and less B cell infiltration, compared to concentric hypertrophic cardiomyopathy. Further work is in progress to unravel mechanisms by which CTLA-4 pathways control cardiac remodeling after cardiac pressure-overload, and how it interferes with other co-signaling pathways.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. ATP promotes tumor growth but also anti-tumor immune responses notably via the P2X7 receptor. ATP can also be catabolized by CD39 and CD73 ecto-enzymes into immunosuppressive adenosine. P2X7, CD39 and CD73 have attracted much interest in cancer as targets offering the potential to unleash anti-tumor immune responses. These membrane proteins represent novel purinergic checkpoints that can be targeted by small drugs or biologics. Here, we investigated nanobody-based biologics targeting mainly P2X7, but also CD73, alone or in combination therapies. Blocking P2X7 inhibited tumor growth and improved survival of mice in cancer models that express P2X7. P2X7-potentiation by a nanobody-based biologic was not effective alone to control tumor growth but enhanced tumor control and immune responses when used in combination with oxaliplatin chemotherapy. We also evaluated a bi-specific nanobody-based biologic that targets PD-L1 and CD73. This novel nanobody-based biologic exerted a potent anti-tumor effect, promoting tumor rejection and improving survival of mice in two tumor models. Hence, this study highlights the importance of purinergic checkpoints in tumor control and open new avenues for nanobody-based biologics that may be further exploited in the treatment of cancer.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. Extracellular ATP is known to signal through plasma membrane receptors of the P2Y and P2X families. Among the P2X receptors, P2X7 has attracted increasing interest in the field of inflammation as well as in cancer. P2X7 is expressed by immune cells and by most malignant tumor cells where it plays a crucial yet complex role that remains to be clarified. P2X7 activity has been associated with production and release of pro-inflammatory cytokines, modulation of the activity and survival of immune cells, and the stimulation of proliferation and migratory properties of tumor cells. Hence, P2X7 plays an intricate role in the tumor microenvironment combining beneficial and detrimental effects that need to be further investigated. For this, we developed a novel methodology termed AAVnano based on the use of Adeno-associated viral vectors (AAV) encoding nanobodies targeting P2X7. We discuss here the advantages of this tool to study the different functions of P2X7 in cancer and other pathophysiological contexts.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, a-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adenoassociated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8(+) T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25(+) T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

Recombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-α secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1β and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1β and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity.

When assessing the immunogenicity of gene therapy drug products, two components need to be considered: immunogenicity toward the vector delivery system and immunogenicity specific for the transgene product. BMN 270 is an AAV5-mediated gene therapy indicated for the treatment of Hemophilia A, and encodes for a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver specific promoter. In study BMN 270-201, the first-in-human clinical trial, prospective patients with severe Hemophilia A were screened for both pre-existing antibodies directed against the AAV5 vector and inhibitors of transduction using a cell-based in vitro transduction inhibition (TI) assay. Patients testing positive in either assay were excluded from the trial, as were patients who had a history of FVIII inhibitors. Following infusion of BMN 270, patient plasma was analyzed for total antibody (TAb) responses specific for the AAV5 capsid and TAb responses directed against FVIII, using bridging ECLA immunoassays. Development of FVIII inhibitors was monitored using the Nijmegen-modified Bethesda assay. Additionally, peripheral blood mononuclear cells were collected for analysis by IFN-γ and TNF-α ELISpot assays for detection of capsid-specific and hFVIII-SQ specific cellular immune responses. The available data indicate that, as expected, all patients develop anti-AAV5 TAb by Week 8 post BMN 270 infusion, the first immunogenicity time-point assessed. One patient screened and confirmed positive at a single time point in the anti-FVIII TAb assay. This response was below the minimum required dilution to determine a titer, and was negative at all subsequent time points. No patients have tested positive in the Bethesda assay for FVIII inhibitors. We investigated the possibility that cell mediated immunity may explain the observed elevation in hepatic enzymes. To date, several patients dosed with BMN 270 experienced asymptomatic elevation of alanine aminotransferase (ALT) laboratory values ranging from 44 to 141 U/L (normal, 6-43 U/L) that resolved without sequelae. Analysis of cellular immune response in these patients by IFN-γ ELISpot assay against peptides spanning AAV5 capsid or the hFVIII-SQ protein was negative, as it was for all other subjects in the study across all time points tested to date. Unlike the IFN-γ assay, intermittent responses were detected across several patients in response to hFVIII-SQ peptide pools in the TNF-α ELISpot assay. In general, these responses were self-limiting and were not temporally associated with increases in ALT or a decline in FVIII activity measures, and did not correlate with results from the IFN-γ assay. Similar TNF-α responses to hFVIII-SQ peptides were also observed in a subset of healthy donors. Overall, immune responses to BMN 270 were characterized by the production of anti-AAV5 antibodies, which is an expected finding following viral vector administration. In conclusion, no consistent association could be made with increases in ALT and cellular immune responses. Our data to date indicate that although antibodies to the AAV5 capsid develop in all patients, concomitant cellular immunity is not readily detectable, and has not been associated with liver function abnormalities nor changes in transgene expression levels. Disclosures Long: BioMarin: Employment. Kim: BioMarin: Employment. Wong: BioMarin: Employment. Yang: BioMarin: Employment. Vettermann: BioMarin: Employment. Pryer: BioMarin: Employment. Hardet: Genethon: Employment. Kuranda: Genethon: Employment. Mingozzi: Genethon: Employment. Pierce: Roche: Consultancy; BioMarin: Consultancy. Schweighardt: BioMarin: Employment.