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Interleukin-2 (IL-2) therapies targeting the high affinity IL-2 receptor expressed on regulatory T cells (Tregs) have shown promising therapeutic benefit in autoimmune diseases through nonselective expansion of pre-existing Treg populations, but are potentially limited by the inability to induce antigen-specific Tregs, as well as by dose-limiting activation of effector immune cells in settings of inflammation. We recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, which induce selective immune tolerance to co-administered antigens but do not increase total Treg numbers. Here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Tregs when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. Importantly, ImmTOR also increases the therapeutic window of engineered IL-2 molecules by mitigating effector immune cell expansion and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, IL-2 mutein shows potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.
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Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.
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Inmunomodulación , Inmunosupresores/administración & dosificación , Inmunotoxinas/administración & dosificación , Leucemia/terapia , Sirolimus/administración & dosificación , Animales , Anticuerpos Neutralizantes , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunotoxinas/inmunología , Mesotelina , Nanopartículas , Factores de TiempoRESUMEN
Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.
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Antígenos/administración & dosificación , Antígenos/química , Tolerancia Inmunológica , Terapia de Inmunosupresión/métodos , Nanopartículas/química , Animales , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Encefalomielitis Autoinmune Experimental/terapia , Factor VIII/inmunología , Femenino , Hemocianinas/administración & dosificación , Hemofilia A/inmunología , Hemofilia A/terapia , Humanos , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/terapia , Inmunidad Humoral , Inmunosupresores/administración & dosificación , Ácido Láctico/química , Ratones , Ratones Endogámicos BALB C , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Nanopartículas/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Péptidos/administración & dosificación , Péptidos/química , Péptidos/inmunología , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Proteínas/administración & dosificación , Proteínas/química , Proteínas/inmunología , Proteínas Recombinantes/inmunología , Sirolimus/administración & dosificaciónRESUMEN
Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of adeno-associated virus (AAV) gene therapy vectors, enhance levels of transgene expression, and enable redosing of AAV at moderate vector doses of 2 to 5E12â vg/kg. However, recent clinical trials have often pushed AAV vector doses 10-fold to 50-fold higher, with serious adverse events observed at the upper range. Here, we assessed combination therapy of ImmTOR with B cell-targeting drugs for the ability to increase the efficiency of redosing at high vector doses. The combination of ImmTOR with a monoclonal antibody against B cell activation factor (aBAFF) exhibited strong synergy leading to more than a 5-fold to 10-fold reduction of splenic mature B cells and plasmablasts while increasing the fraction of pre-/pro-B cells. In addition, this combination dramatically reduced anti-AAV IgM and IgG antibodies, thus enabling four successive AAV administrations at doses up to 5E12â vg/kg and at least two AAV doses at 5E13â vg/kg, with the transgene expression level in the latter case being equal to that observed in control animals receiving a single vector dose of 1E14â vg/kg. Similar synergistic effects were seen with a combination of ImmTOR and a Bruton's tyrosine kinase inhibitor, ibrutinib. These results suggest that ImmTOR could be combined with B cell-targeting agents to enable repeated vector administrations as a potential strategy to avoid toxicities associated with vector doses above 1E14â vg/kg.
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Biologic drugs have transformed the standard of care for many diseases. However, many biologics induce the formation of anti-drug antibodies (ADAs), which can compromise their safety and efficacy. Preclinical studies demonstrate that biodegradable nanoparticles-encapsulating rapamycin (ImmTOR), but not free rapamycin, mitigate the immunogenicity of co-administered biologic drugs. Here we report the outcomes from two clinical trials for ImmTOR. In the first ascending dose, open-label study (NCT02464605), pegadricase, an immunogenic, pegylated uricase enzyme derived from Candida utilis, is assessed for safety and tolerability (primary endpoint) as well as activity and immunogenicity (secondary endpoint); in the second single ascending dose Phase 1b trial (NCT02648269) composed of both a double-blind and open-label parts, we evaluate the safety of ImmTOR (primary endpoint) and its ability to prevent the formation of anti-drug antibodies against pegadricase and enhance its pharmacodynamic activity (secondary endpoint) in patients with hyperuricemia. The combination of ImmTOR and pegadricase is well tolerated. ImmTOR inhibits the development of uricase-specific ADAs in a dose-dependent manner, thus enabling sustained enzyme activity and reduction in serum uric acid levels. ImmTOR may thus represent a feasible approach for preventing the formation of ADAs to a broad range of immunogenic biologic therapies.
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Anticuerpos/uso terapéutico , Hiperuricemia/tratamiento farmacológico , Nanopartículas/química , Nanopartículas/uso terapéutico , Polietilenglicoles/farmacología , Urato Oxidasa/uso terapéutico , Adulto , Anciano , Anticuerpos/química , Terapia Biológica , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Compuestos Orgánicos/química , Urato Oxidasa/farmacología , Ácido Úrico , Adulto JovenRESUMEN
BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.
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Anafilaxia/inducido químicamente , Sulfatos de Condroitina/análisis , Sulfatos de Condroitina/farmacología , Activación de Complemento/efectos de los fármacos , Contaminación de Medicamentos , Heparina/química , Calicreínas/efectos de los fármacos , Animales , China , Sulfatos de Condroitina/efectos adversos , Complemento C3a/biosíntesis , Complemento C3a/efectos de los fármacos , Complemento C5a/biosíntesis , Complemento C5a/efectos de los fármacos , Industria Farmacéutica , Femenino , Alemania , Heparina/efectos adversos , Humanos , Hipotensión/inducido químicamente , Calicreínas/metabolismo , Persona de Mediana Edad , Sus scrofa , Estados Unidos , United States Food and Drug AdministrationRESUMEN
BACKGROUND: In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions. METHODS: Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case-control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants. RESULTS: A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin. CONCLUSIONS: Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause of the outbreak.
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Anticoagulantes/efectos adversos , Sulfatos de Condroitina/efectos adversos , Brotes de Enfermedades , Contaminación de Medicamentos , Heparina/efectos adversos , Anticoagulantes/química , Estudios de Casos y Controles , Edema/inducido químicamente , Edema/epidemiología , Heparina/química , Humanos , Hipotensión/inducido químicamente , Hipotensión/epidemiología , Náusea/inducido químicamente , Náusea/epidemiología , Diálisis Renal , Taquicardia/inducido químicamente , Taquicardia/epidemiología , Estados Unidos/epidemiología , Urticaria/inducido químicamente , Urticaria/epidemiologíaRESUMEN
In recent months as vaccines against the SARS-CoV-2 virus continue to rollout across the globe, there has been a renewed interest in ways to activate or ignite the immune system. For a vaccine to be effective, it must be immunogenic and specific to provoke the body's defenses to mount an effective response that protects the host from disease. However, there are other situations wherein the immune system mounts an unwanted immune response that can be detrimental to health, either directly, by causing an autoimmune disease, or indirectly, by compromising the safety and/or efficacy of biologic drugs. In these scenarios, it would be desirable to have a 'tolerogenic vaccine' that could selectively and effectively mitigate these unwanted immune responses. ImmTORTM, a nanoparticle technology, is being developed to address the issue of immunogenicity for gene therapy vectors and other biologic drugs. By targeting antigen-presenting cells, ImmTORTM has the potential to amplify the efficacy of biologic therapies and unlock the full potential of such treatments to improve the lives of those who suffer from serious and debilitating diseases.
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Enfermedades Autoinmunes , Productos Biológicos , COVID-19 , Vacunas , Productos Biológicos/uso terapéutico , Humanos , SARS-CoV-2RESUMEN
ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4-CD8- (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.
Asunto(s)
Linfocitos B/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Hígado/inmunología , Sirolimus/farmacología , Linfocitos T Reguladores/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/biosíntesis , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Células Estrelladas Hepáticas/metabolismo , Hepatitis/inmunología , Hepatocitos/metabolismo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Tolerancia Inmunológica/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas , Ovalbúmina/inmunología , Poliésteres , Receptor de Muerte Celular Programada 1/biosíntesisRESUMEN
A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.
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Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.
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The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and hypersensitivity reactions for many biologics. The focus of this review is the development of ImmTOR, a platform technology designed to prevent the formation of ADAs that can be applied broadly across a wide variety of biologics by inducing immunological tolerance with ImmTOR nanoparticles encapsulating rapamycin. The induction of tolerance is antigen-specific and dependent on the incorporation of rapamycin in nanoparticles and the presence of the antigen at the time of administration of ImmTOR. Evidence for the induction of specific immune tolerance vs. general immune suppression is supported by the findings that: (1) ImmTOR induces regulatory T cells specific to the co-administered antigen; (2) tolerance can be transferred by adoptive transfer of splenocytes from treated animals to naïve recipients; (3) the tolerance is durable to subsequent challenge with antigen alone; and (4) animals tolerized to a specific antigen are capable of responding to an unrelated antigen. ImmTOR nanoparticles can be added to new or existing biologics without the need to modify or reformulate the biologic drug. The ability of ImmTOR to mitigate the formation of ADAs has been demonstrated for coagulation factor VIII in a mouse model of hemophilia A, an anti-TNFα monoclonal antibody in a mouse model of inflammatory arthritis, pegylated uricase in hyperuricemic mice and in non-human primates, acid alpha-glucosidase in a mouse model of Pompe disease, recombinant immunotoxin in a mouse model of mesothelioma, and adeno-associated vectors in a model of repeat dosing of gene therapy vectors in mice and in non-human primates. Human proof-of concept for the mitigation of ADAs has been demonstrated with SEL-212, a combination product consisting of ImmTOR + pegadricase, a highly immunogenic enzyme therapy for the treatment of gout. ImmTOR represents a promising approach to preventing the formation of ADAs to a broad range of biologic drugs.
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Anticuerpos/inmunología , Productos Biológicos/efectos adversos , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Nanomedicina , Nanopartículas , Sirolimus/farmacología , Animales , Anticuerpos/metabolismo , Formación de Anticuerpos/efectos de los fármacos , Productos Biológicos/inmunología , Composición de Medicamentos , Humanos , Inmunosupresores/química , Lactatos/química , Poliésteres/química , Polietilenglicoles/química , Sirolimus/químicaRESUMEN
Antigen-specific immune tolerance has been a long-standing goal for immunotherapy for the treatment of autoimmune diseases and allergies and for the prevention of allograft rejection and anti-drug antibodies directed against biologic therapies. Nanoparticles have emerged as powerful tools to initiate and modulate immune responses due to their inherent capacity to target antigen-presenting cells (APCs) and deliver coordinated signals that can elicit an antigen-specific immune response. A wide range of strategies have been described to create tolerogenic nanoparticles (tNPs) that fall into three broad categories. One strategy includes tNPs that provide antigen alone to harness natural tolerogenic processes and environments, such as presentation of antigen in the absence of costimulatory signals, oral tolerance, the tolerogenic environment of the liver, and apoptotic cell death. A second strategy includes tNPs that carry antigen and simultaneously target tolerogenic receptors, such as pro-tolerogenic cytokine receptors, aryl hydrocarbon receptor, FAS receptor, and the CD22 inhibitory receptor. A third strategy includes tNPs that carry a payload of tolerogenic pharmacological agents that can "lock" APCs into a developmental or metabolic state that favors tolerogenic presentation of antigens. These diverse strategies have led to the development of tNPs that are capable of inducing antigen-specific immunological tolerance, not just immunosuppression, in animal models. These novel tNP technologies herald a promising approach to specifically prevent and treat unwanted immune reactions in humans. The first tNP, SEL-212, a biodegradable synthetic vaccine particle encapsulating rapamycin, has reached the clinic and is currently in Phase 2 clinical trials.
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Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/administración & dosificación , Inmunoterapia/métodos , Nanocápsulas/administración & dosificación , Animales , Presentación de Antígeno/efectos de los fármacos , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Composición de Medicamentos/métodos , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Humanos , Hipersensibilidad/tratamiento farmacológico , Hipersensibilidad/inmunología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/inmunología , Linfocitos T Reguladores , Vacunas Sintéticas/administración & dosificaciónRESUMEN
We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.
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Vacunas contra el Cáncer/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/administración & dosificación , Animales , Línea Celular Tumoral , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunoterapia , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Activación de Linfocitos/efectos de los fármacos , Ratones , Proteínas E7 de Papillomavirus/inmunología , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
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.
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Dependovirus/inmunología , Terapia Genética , Vectores Genéticos/inmunología , Inmunosupresores/administración & dosificación , Sirolimus/administración & dosificación , Animales , Evaluación Preclínica de Medicamentos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Nanopartículas , Linfocitos T/efectos de los fármacosRESUMEN
CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica , Péptidos/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Toxoide Diftérico/inmunología , Epítopos/inmunología , Femenino , Genes MHC Clase II , Humanos , Macaca fascicularis , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Nanopartículas , Nicotina/inmunología , Toxoide Tetánico/inmunologíaRESUMEN
Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Nanopartículas , Vacunas Sintéticas/inmunología , Animales , Formación de Anticuerpos , Antígenos/administración & dosificación , Antígenos/inmunología , Células Cultivadas , Citocinas/inmunología , Femenino , Imidazoles/administración & dosificación , Inmunidad Celular , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Bazo/citología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 9/agonistasRESUMEN
Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.
Asunto(s)
Progresión de la Enfermedad , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacología , Melanoma Experimental/patología , Imitación Molecular , Metástasis de la Neoplasia , Animales , Línea Celular Tumoral , Citometría de Flujo , Melanoma Experimental/irrigación sanguínea , Ratones , Resonancia por Plasmón de SuperficieRESUMEN
Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.