RESUMEN
Interleukin (IL)-1 family cytokines potently regulate inflammation, with the majority of the IL-1 family proteins being secreted from immune cells via unconventional pathways. In many cases, secretion of IL-1 cytokines appears to be closely coupled to cell death, yet the secretory mechanisms involved remain poorly understood. Here, we studied the secretion of the three best-characterized members of the IL-1 superfamily, IL-1α, IL-1ß, and IL-18, in a range of conditions and cell types, including murine bone marrow-derived and peritoneal macrophages, human monocyte-derived macrophages, HeLa cells, and mouse embryonic fibroblasts. We discovered that IL-1ß and IL-18 share a common secretory pathway that depends upon membrane permeability and can operate in the absence of complete cell lysis and cell death. We also found that the pathway regulating the trafficking of IL-1α is distinct from the pathway regulating IL-1ß and IL-18. Although the release of IL-1α could also be dissociated from cell death, it was independent of the effects of the membrane-stabilizing agent punicalagin, which inhibited both IL-1ß and IL-18 release. These results reveal that in addition to their role as danger signals released from dead cells, IL-1 family cytokines can be secreted in the absence of cell death. We propose that models used in the study of IL-1 release should be considered context-dependently.
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Células de la Médula Ósea/metabolismo , Interleucina-18/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Células de la Médula Ósea/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Taninos Hidrolizables/farmacología , Macrófagos Peritoneales/citología , Ratones , Transporte de Proteínas/efectos de los fármacosRESUMEN
CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T-cell-activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T-cell-derived cytokines license anti-tumour functions in CD40-activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL-10, in a similar fashion to bacterial LPS, but failed to promote anti-tumour functions. The Th1 cytokine IFN-γ optimally licensed CD40L-induced macrophage anti-tumour functions, inducing a switch from IL-10 to IL-12p70 production, promoting macrophage-mediated Th1 T-cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL-4 and IL-13 promoted IL-12p70 production (albeit without inhibiting IL-10 production) and enhanced Th1 T-cell skewing by CD40L-activated macrophages. However, IL-4 and IL-13 did not enhance tumouricidal activity in CD40L-activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L-activated macrophages. The presence of tumour-infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.
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Citocinas/metabolismo , Inmunoterapia Adoptiva/métodos , Macrófagos/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Células TH1/inmunología , Células Th2/inmunología , Animales , Antígenos de Neoplasias/inmunología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Células Cultivadas , Citocinas/inmunología , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Humanos , Inmunidad Celular , Activación de Macrófagos , Balance Th1 - Th2RESUMEN
IL-1ß is a potent pro-inflammatory cytokine produced in response to infection or injury. It is synthesized as an inactive precursor that is activated by the protease caspase-1 within a cytosolic molecular complex called the inflammasome. Assembly of this complex is triggered by a range of structurally diverse damage or pathogen associated stimuli, and the signaling pathways through which these act are poorly understood. Ubiquitination is a post-translational modification essential for maintaining cellular homeostasis. It can be reversed by deubiquitinase enzymes (DUBs) that remove ubiquitin moieties from the protein thus modifying its fate. DUBs present specificity toward different ubiquitin chain topologies and are crucial for recycling ubiquitin molecules before protein degradation as well as regulating key cellular processes such as protein trafficking, gene transcription, and signaling. We report here that small molecule inhibitors of DUB activity inhibit inflammasome activation. Inhibition of DUBs blocked the processing and release of IL-1ß in both mouse and human macrophages. DUB activity was necessary for inflammasome association as DUB inhibition also impaired ASC oligomerization and caspase-1 activation without directly blocking caspase-1 activity. These data reveal the requirement for DUB activity in a key reaction of the innate immune response and highlight the therapeutic potential of DUB inhibitors for chronic auto-inflammatory diseases.
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Caspasa 1/metabolismo , Endopeptidasas/fisiología , Interleucina-1beta/metabolismo , Animales , Carboxipeptidasas/metabolismo , Endopeptidasas/química , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Hidrazonas/farmacología , Hidroxiurea/análogos & derivados , Hidroxiurea/farmacología , Inmunidad Innata , Inflamación , Interleucina-1alfa/metabolismo , Interleucinas/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Ubiquitina TiolesterasaRESUMEN
AZD0171 (INN: Falbikitug) is being developed as a humanized monoclonal antibody (mAb), immunoglobulin G subclass 1 (IgG1), which binds specifically to the immunosuppressive human cytokine leukemia inhibitory factor (LIF) and inhibits downstream signaling by blocking recruitment of glycoprotein 130 (gp130) to the LIF receptor (LIFR) subunit (gp190) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and is intended to treat adult participants with advanced solid tumors. LIF is a pleiotropic cytokine (and a member of the IL-6 family of cytokines) involved in many physiological and pathological processes and is highly expressed in a subset of solid tumors, including non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer. The aim of this work was to develop a mechanistic PK/PD model to investigate the effect of AZD0171 on tumor LIF levels, predict the level of downstream signaling complex (LIF:LIFR:gp130) inhibition, and examine the dose-response relationship to support dose selection for a Phase II clinical study. Modeling results show that tumor LIF is inhibited in a dose-dependent manner with >90% inhibition for 95% of patients at the Phase II clinical dose of 1500 mg Q2W.
RESUMEN
CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.
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Anticuerpos Monoclonales , Sarcoma , Animales , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Inmunosupresores , Adenosina , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Microambiente TumoralRESUMEN
Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that regulates inflammatory responses to injury and infection. IL-1ß secretion requires the protease caspase-1, which is activated following recruitment to inflammasomes. Endogenous danger-associated molecular patterns (DAMPs) released from necrotic cells activate caspase-1 through an NLRP3-inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3-inflammasome-dependent secretion of IL-1ß from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph-induced IL-1ß release. IL-1ß release induced by other well-characterized NLRP3-inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)-dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation.
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Proteínas Portadoras/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Peritonitis/inmunología , Esfingosina/inmunología , Animales , Western Blotting , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Macrófagos Peritoneales/inmunología , Toxinas Marinas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Peritonitis/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/inmunología , Distribución AleatoriaRESUMEN
Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.
Asunto(s)
Neoplasias , Receptor de Muerte Celular Programada 1 , Humanos , Ratones , Animales , Inmunoterapia , Antígenos CD40 , Linfocitos T CD8-positivos , Citocinas/uso terapéutico , Modelos Animales de Enfermedad , Interleucina-12/uso terapéutico , Células Dendríticas , Microambiente TumoralRESUMEN
PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.
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Inmunoconjugados , Neoplasias , Ratas , Humanos , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inhibidores de Topoisomerasa I , Neoplasias/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC. Statement of significance: This study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.
RESUMEN
Pro-inflammatory members of the interleukin-1 (IL-1) family of cytokines (IL-1alpha and beta) are important mediators of host defense responses to infection but can also exacerbate the damaging inflammation that contributes to major human diseases. IL-1alpha and beta are produced by cells of the innate immune system, such as macrophages, and act largely after their secretion by binding to the type I IL-1 receptor on responsive cells. There is evidence that IL-1alpha is also a nuclear protein that can act intracellularly. In this study, we report that both IL-1alpha and IL-1beta produced by microglia (central nervous system macrophages) in response to an inflammatory challenge are distributed between the cytosol and the nucleus. Using IL-1-beta-galactosidase and IL-1-green fluorescent protein chimeras (analyzed by fluorescence recovery after photobleaching), we demonstrate that nuclear import of IL-1alpha is exclusively active, requiring a nuclear localization sequence and Ran, while IL-1beta nuclear import is entirely passive. These data provide valuable insights into the dynamic regulation of intracellular cytokine trafficking.
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Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Interleucina-1alfa/genética , Interleucina-1beta/genética , Ratones , Microglía/metabolismo , Datos de Secuencia Molecular , Señales de Localización Nuclear , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
BACKGROUND: Cerebral ischemia is a devastating condition in which the outcome is heavily influenced by inflammatory processes, which can augment primary injury caused by reduced blood supply. The cytokines interleukin-1α (IL-1α) and IL-1ß are key contributors to ischemic brain injury. However, there is very little evidence that IL-1 expression occurs at the protein level early enough (within hours) to influence brain damage after stroke. In order to determine this we investigated the temporal and spatial profiles of IL-1α and IL-1ß expression after cerebral ischemia. FINDINGS: We report here that in mice, as early as 4 h after reperfusion following ischemia induced by occlusion of the middle cerebral artery, IL-1α, but not IL-1ß, is expressed by microglia-like cells in the ischemic hemisphere, which parallels an upregulation of IL-1α mRNA. 24 h after ischemia IL-1α expression is closely associated with areas of focal blood brain barrier breakdown and neuronal death, mostly near the penumbra surrounding the infarct. The sub-cellular distribution of IL-1α in injured areas is not uniform suggesting that it is regulated. CONCLUSIONS: The early expression of IL-1α in areas of focal neuronal injury suggests that it is the major form of IL-1 contributing to inflammation early after cerebral ischemia. This adds to the growing body of evidence that IL-1α is a key mediator of the sterile inflammatory response.
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Lesiones Encefálicas/inmunología , Lesiones Encefálicas/patología , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Interleucina-1alfa/inmunología , Interleucina-1beta/inmunología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Receptor 1 de Quimiocinas CX3C , Infarto de la Arteria Cerebral Media , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/citología , Microglía/inmunología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunologíaRESUMEN
Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/inmunología , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Femenino , Humanos , Estimación de Kaplan-Meier , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Ratas Sprague-Dawley , Linfocitos T Reguladores/inmunología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine IL-1 alpha is reported to be a damage-associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL-1 alpha is produced by microglia, the resident brain macrophages. We found that IL-1 alpha is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1 alpha to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen-glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL-1 alpha released significantly less IL-1 alpha than microglia with predominantly cytosolic IL-1 alpha. The remaining IL-1 alpha was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL-1 alpha may serve to limit inflammation following cell death.
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Núcleo Celular/metabolismo , Inflamación/metabolismo , Interleucina-1alfa/metabolismo , Necrosis/metabolismo , Animales , Humanos , Microglía/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.
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Neoplasias Colorrectales/prevención & control , Interleucina-12/administración & dosificación , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/prevención & control , ARN Mensajero/administración & dosificación , Células TH1/inmunología , Microambiente Tumoral/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Femenino , Humanos , Interleucina-12/genética , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , ARN Mensajero/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
Asunto(s)
Afinidad de Anticuerpos , Arginasa/química , Anticuerpos de Cadena Única/química , Regulación Alostérica , Cristalografía por Rayos X , HumanosRESUMEN
BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.
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Células Dendríticas/inmunología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Células Asesinas Naturales/inmunología , Melanoma Experimental/tratamiento farmacológico , Ácidos Esteáricos/farmacología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Microambiente Tumoral/inmunología , Inmunidad Adaptativa , Adyuvantes Inmunológicos/farmacología , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Inmunoterapia , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1ß and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1ß to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.
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Inflamación/enzimología , Inflamación/patología , Sistema de Señalización de MAP Quinasas , Melanoma/enzimología , Melanoma/patología , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL1/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Ligandos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-8B/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.
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Boro/química , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Animales , Células de la Médula Ósea/citología , Boro/farmacología , Compuestos de Boro/química , Compuestos de Boro/metabolismo , Compuestos de Boro/farmacología , Calcio/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Conformación Molecular , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Relación Estructura-ActividadRESUMEN
Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.
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Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Antígenos CD40/agonistas , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Microambiente Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Antígenos CD40/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/patología , Distribución AleatoriaRESUMEN
Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.