RESUMEN
Current coronavirus disease 2019 vaccines face limitations including waning immunity, immune escape by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, limited cellular response, and poor mucosal immunity. We engineered a Clec9A-receptor binding domain (RBD) antibody construct that delivers the SARS-CoV-2 RBD to conventional type 1 dendritic cells. Compared with non-targeting approaches, single dose immunization in mice with Clec9A-RBD induced far higher RBD-specific antibody titers that were sustained for up to 21 months after vaccination. Uniquely, increasing neutralizing and antibody-dependent cytotoxicity activities across the sarbecovirus family was observed, suggesting antibody affinity maturation over time. Consistently and remarkably, RBD-specific follicular T helper cells and germinal center B cells persisted up to 12 months after immunization. Furthermore, Clec9A-RBD immunization induced a durable mono- and poly-functional T-helper 1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants of concern, including Omicron subvariants, and with a robust CD8+ T cell signature. Uniquely, Clec9A-RBD single-shot systemic immunization effectively primed RBD-specific cellular and humoral immunity in lung and resulted in significant protection against homologous SARS-CoV-2 challenge as evidenced by limited body weight loss and approximately 2 log10 decrease in lung viral loads compared with non-immunized controls. Therefore, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal protective immunity against rapidly evolving SARS-CoV2 variants.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Células Dendríticas , Inmunidad Mucosa , Lectinas Tipo C , SARS-CoV-2 , Animales , Ratones , Células Dendríticas/inmunología , SARS-CoV-2/inmunología , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/inmunología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Humanos , Femenino , Glicoproteína de la Espiga del Coronavirus/inmunología , Receptores Mitogénicos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Receptores InmunológicosRESUMEN
CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.
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Lectinas Tipo C , Receptores Mitogénicos , Ácido Úrico , Humanos , Gota/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/inmunología , Receptores Mitogénicos/química , Receptores Mitogénicos/inmunología , Ácido Úrico/química , Ácido Úrico/inmunología , Dominios Proteicos , Cristalografía por Rayos X , Imagen Individual de Molécula , Línea CelularRESUMEN
C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.
Asunto(s)
Lectinas Tipo C , Receptores Mitogénicos , Lectinas Tipo C/inmunología , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Humanos , Receptores Mitogénicos/química , Receptores Mitogénicos/inmunología , Receptores Mitogénicos/metabolismo , Cristalografía por Rayos X , Ligandos , Unión Proteica , Sitios de Unión , Modelos Moleculares , Ácido Úrico/química , Ácido Úrico/metabolismo , Ácido Úrico/inmunologíaRESUMEN
Legionella pneumophila is an intracellular pathogen that can cause severe pneumonia after the inhalation of contaminated aerosols and replication in alveolar macrophages. Several pattern recognition receptors (PRRs) have been identified that contribute to the recognition of L. pneumophila by the innate immune system. However, the function of the C-type lectin receptors (CLRs), which are mainly expressed by macrophages and other myeloid cells, remains largely unexplored. Here, we used a library of CLR-Fc fusion proteins to search for CLRs that can bind the bacterium and identified the specific binding of CLEC12A to L. pneumophila. Subsequent infection experiments in human and murine macrophages, however, did not provide evidence for a substantial role of CLEC12A in controlling innate immune responses to the bacterium. Consistently, antibacterial and inflammatory responses to Legionella lung infection were not significantly influenced by CLEC12A deficiency. Collectively, CLEC12A is able to bind to L. pneumophila-derived ligands but does not appear to play a major role in the innate defense against L. pneumophila.
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Interacciones Huésped-Patógeno , Inmunidad Innata , Lectinas Tipo C , Legionella pneumophila , Enfermedad de los Legionarios , Receptores Mitogénicos , Animales , Humanos , Ratones , Lectinas Tipo C/metabolismo , Legionella pneumophila/inmunología , Enfermedad de los Legionarios/inmunología , Enfermedad de los Legionarios/microbiología , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Receptores Mitogénicos/inmunologíaRESUMEN
Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Inmunoterapia/métodos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Mitogénicos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Linfocitos T/inmunología , Animales , Deriva y Cambio Antigénico , Apoptosis , Proliferación Celular , Citotoxicidad Inmunológica/inmunología , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The myeloid inhibitory C-type lectin receptor CLEC12A limits neutrophil activation, pro-inflammatory pathways and disease in mouse models of inflammatory arthritis by a molecular mechanism that remains poorly understood. We addressed how CLEC12A-mediated inhibitory signaling counteracts activating signaling by cross-linking CLEC12A in human neutrophils. CLEC12A cross-linking induced its translocation to flotillin-rich membrane domains where its ITIM was phosphorylated in a Src-dependent manner. Phosphoproteomic analysis identified candidate signaling molecules regulated by CLEC12A that include MAPKs, phosphoinositol kinases and members of the JAK-STAT pathway. Stimulating neutrophils with uric acid crystals, the etiological agent of gout, drove the hyperphosphorylation of p38 and Akt. Ultimately, one of the pathways through which CLEC12A regulates uric acid crystal-stimulated release of IL-8 by neutrophils is through a p38/PI3K-Akt signaling pathway. In summary this work defines early molecular events that underpin CLEC12A signaling in human neutrophils to modulate cytokine synthesis. Targeting this pathway could be useful therapeutically to dampen inflammation.
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Lectinas Tipo C/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Mitogénicos/inmunología , Transducción de Señal/inmunología , Adulto , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Microscopía Confocal , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Mast cells (MCs) are present in various organs including the skin, peritoneal cavity, lung, and intestine and involved in the development of allergic diseases and host defense against infection. However, the regulatory mechanism of mast cell activation remains incompletely understood. We found in a database that Clec12b encoding a C-type lectin receptor Clec12b is preferentially expressed in skin MCs in mice. However, neither MCs in other tissues such as trachea, tongue, esophagus, or peritoneal cavity nor most lymphocytes and myeloid cells express Clec12b. To analyze the protein expression of Clec12b, we newly generated a monoclonal antibody (named TX109), which recognizes both mouse and human Clec12b. Consistent with the gene expression profile, flow cytometry analysis demonstrated that Clec12b is expressed only on MCs in the skin, but not on any other immune cell types in various tissues, in mice. Similarly, Clec12b is also expressed on skin MCs, but not on circulating lymphocytes and myeloid cells, in humans. Our results suggest that Clec12b plays an important role in the regulation of MCs activation in the skin.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Lectinas Tipo C/metabolismo , Mastocitos/metabolismo , Receptores Mitogénicos/metabolismo , Piel/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Citometría de Flujo/métodos , Humanos , Lectinas Tipo C/inmunología , Mastocitos/citología , Mastocitos/inmunología , Ratones , Receptores Mitogénicos/inmunología , Piel/citología , Piel/inmunologíaRESUMEN
OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.
Asunto(s)
Biomarcadores de Tumor/genética , Células de la Médula Ósea/metabolismo , Lectinas Tipo C/genética , Leucemia Mieloide Aguda/genética , Mutación , Células Mieloides/metabolismo , Receptores Mitogénicos/genética , Antígenos CD34/genética , Antígenos CD34/inmunología , Biomarcadores de Tumor/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Análisis Citogenético , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Inmunofenotipificación , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Células Mieloides/inmunología , Células Mieloides/patología , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Cultivo Primario de Células , Receptores Mitogénicos/inmunologíaAsunto(s)
Anticuerpos/uso terapéutico , Antineoplásicos/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Inmunoconjugados/uso terapéutico , Lectinas Tipo C/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Receptores Mitogénicos/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/administración & dosificación , Anticuerpos/efectos adversos , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Relación Dosis-Respuesta a Droga , Terminación Anticipada de los Ensayos Clínicos , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Primarias Secundarias/tratamiento farmacológico , Neoplasias Primarias Secundarias/genética , Receptores Mitogénicos/inmunología , Adulto JovenRESUMEN
The low 5-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using natural killer (NK) cells, we have developed a trispecific killer engager (TriKETM) molecule containing a humanized anti-CD16 heavy chain camelid single-domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK-cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK-cell specific proliferation, enhanced NK-cell activation, and killing of both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in preclinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.
Asunto(s)
Inmunoterapia/métodos , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/terapia , Receptores de IgG/inmunología , Receptores Mitogénicos/inmunología , Anticuerpos de Dominio Único/farmacología , Animales , Apoptosis , Proliferación Celular , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Lectinas Tipo C/antagonistas & inhibidores , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Receptores Mitogénicos/antagonistas & inhibidores , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Emerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting, and several suitable immunotargets (HAVCR2/CD33 and HAVCR2/CLEC12A) have been identified in adult AML. However, clinical and biologic characteristics of AML differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA sequencing of sorted CD45dim and CD34+CD38-CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principal-component analysis. Known immunotargets of adult AML, such as IL3RA, were not overexpressed in pediatric AML compared with healthy precursors by RNA sequencing. CD33 and CLEC12A were the most upregulated immunotargets on the RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A-mutated infant AML clusters separately by RNA sequencing and overexpresses FLT3, and hence, CD33/FLT3 cotargeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38-CD45RA-CD90+ hematopoietic stem cells (HSCs) and nonhematopoietic tissue, while CD33 and FLT3 are expressed on HSCs. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A and CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33 and FLT3 as immunotargets specific for KMT2A-mutated infant AML.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Lectinas Tipo C/genética , Leucemia Mieloide Aguda/genética , Receptores Mitogénicos/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Adolescente , Niño , Preescolar , Femenino , Humanos , Inmunoterapia , Lactante , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Masculino , Receptores Mitogénicos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Transcriptoma , Regulación hacia ArribaRESUMEN
Although most acute myeloid leukemia (AML) patients achieve complete remissions, the majority still eventually relapse and die of their disease. Rare primitive leukemia cells, so-called leukemia stem cells (LSCs), represent one potential type of resistant cell subpopulation responsible for this dissociation between response and cure. Several LSC targets have been described, but there is limited evidence about their relative utility or that targeting any can prevent relapse. LSCs not only appear to be biologically heterogeneous, but the classic immunocompromised mouse transplantation model also has serious shortcomings as an LSC assay. Out data suggest that the most immature cell phenotype that can be identified within a patient's leukemia may be clinically relevant and represent the de facto LSC. Moreover, although phenotypically heterogeneous, these putative LSCs show consistent phenotypes within individual genetically defined groups. Using this LSC definition, we studied several previously described putative LSC targets, CD25, CD26, CD47, CD96, CD123, and CLL-1, and all were expressed across heterogeneous LSC phenotypes. In addition, with the exception of CD47, there was at most low expression of these targets on normal hematopoietic stem cells (HSCs). CD123 and CLL-1 demonstrated the greatest expression differences between putative LSCs and normal HSCs. Importantly, CD123 monoclonal antibodies were cytotoxic in vitro to putative LSCs from all AML subtypes, while showing limited to no toxicity against normal HSCs and hematopoietic progenitors. Since minimal residual disease appears to be a more homogeneous population of cells responsible for relapse, targeting CD123 in this setting may be most effective.
Asunto(s)
Antígenos CD/análisis , Antígenos de Neoplasias/análisis , Antineoplásicos Inmunológicos/farmacología , Células Precursoras de Granulocitos/química , Leucemia Mieloide Aguda/genética , Terapia Molecular Dirigida , Células Madre Neoplásicas/química , Animales , Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Separación Celular , Activación de Complemento , Citometría de Flujo , Células Precursoras de Granulocitos/efectos de los fármacos , Células Precursoras de Granulocitos/patología , Células Madre Hematopoyéticas/química , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Subunidad alfa del Receptor de Interleucina-3/análisis , Subunidad alfa del Receptor de Interleucina-3/inmunología , Lectinas Tipo C/análisis , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/patología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Receptores Mitogénicos/análisis , Receptores Mitogénicos/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T (iNKT) cells, to cross-priming CD8α+ dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141+ (BDCA3+) DCs - the equivalent of murine CD8α+ DCs - and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional iNKT cells and CD8+ T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141+ DCs and iNKT cells and ultimately elicited a potent Melan-A-specific CD8+ T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8+ T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human iNKT cells to license cross-priming DCs in vivo and adds a new dimension to the current strategy of cancer vaccine development.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epítopos de Linfocito T/inmunología , Galactosilceramidas/administración & dosificación , Trombomodulina/metabolismo , Animales , Antígenos de Neoplasias/administración & dosificación , Biomarcadores , Vacunas contra el Cáncer/inmunología , Humanos , Inmunofenotipificación , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteómica/métodos , Receptores Mitogénicos/antagonistas & inhibidores , Receptores Mitogénicos/inmunología , Análisis de la Célula Individual , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
CLEC12B is a C-type lectin-like receptor expressed on myeloid cells. In this study, we have characterized the porcine homologue of CLEC12B (poCLEC12B). To this end, we have generated constructs encoding a c-myc tagged version of the whole receptor, or its ectodomain fused to the Fc portion of human IgG1, from a cDNA clone obtained from an alveolar macrophage library, and raised monoclonal antibodies (mAb) against this molecule. Using these mAbs, poCLEC12B was found to be expressed on alveolar macrophages and, at lower levels, on blood conventional type 1 dendritic cells (cDC1) and plasmacytoid DCs. No binding was detected on monocytes, monocyte-derived macrophages or monocyte-derived DCs. Engagement of CLEC12B on alveolar macrophages with mAbs had no apparent effect on cytokine production (TNF-α, IL-8) induced by LPS. These results provide the basis for future investigations aimed to assess the role of poCLEC12B in different microbial infections and to evaluate its potential in vaccination strategies targeting DCs.
Asunto(s)
Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Macrófagos Alveolares/inmunología , Receptores Mitogénicos/inmunología , Porcinos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Circulación Sanguínea , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Lectinas Tipo C/genética , Lipopolisacáridos/inmunología , Activación de Macrófagos , Receptores Mitogénicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their inâ vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low inâ vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained inâ vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor inâ vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.
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Anticuerpos Monoclonales/química , Proteínas de Ciclo Celular/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Inmunoconjugados/química , Factores de Transcripción/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Femenino , Semivida , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Ratones SCID , Unión Proteica , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Mitogénicos/inmunología , Resonancia por Plasmón de Superficie , Factores de Transcripción/antagonistas & inhibidores , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The detection of microbes and damaged host cells by the innate immune system is essential for host defense against infection and tissue homeostasis. However, how distinct positive and negative regulatory signals from immune receptors are integrated to tailor specific responses in complex scenarios remains largely undefined. Clec12A is a myeloid cell-expressed inhibitory C-type lectin receptor that can sense cell death under sterile conditions. Clec12A detects uric acid crystals and limits proinflammatory pathways by counteracting the cell-activating spleen tyrosine kinase (Syk). Here, we surprisingly find that Clec12A additionally amplifies type I IFN (IFN-I) responses in vivo and in vitro. Using retinoic acid-inducible gene I (RIG-I) signaling as a model, we demonstrate that monosodium urate (MSU) crystal sensing by Clec12A enhances cytosolic RNA-induced IFN-I production and the subsequent induction of IFN-I-stimulated genes. Mechanistically, Clec12A engages Src kinase to positively regulate the TBK1-IRF3 signaling module. Consistently, Clec12A-deficient mice exhibit reduced IFN-I responses upon lymphocytic choriomeningitis virus (LCMV) infection, which affects the outcomes of these animals in acute and chronic virus infection models. Thus, our results uncover a previously unrecognized connection between an MSU crystal-sensing receptor and the IFN-I response, and they illustrate how the sensing of extracellular damage-associated molecular patterns (DAMPs) can shape the immune response.
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Alarminas/inmunología , Interferón Tipo I/inmunología , Lectinas Tipo C/metabolismo , Coriomeningitis Linfocítica/inmunología , Receptores Mitogénicos/metabolismo , Ácido Úrico/inmunología , Animales , Citosol/inmunología , Citosol/metabolismo , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Noqueados , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/inmunología , ARN/metabolismo , Receptores Mitogénicos/genética , Receptores Mitogénicos/inmunología , Transducción de Señal/inmunologíaRESUMEN
A promising strategy for the enhancement of vaccine-mediated immune responses is by directly targeting protein antigens to immune cells. Targeting of antigens to the dendritic cell (DC) molecule Clec9A has been shown to enhance antibody affinity and titers for model antigens, and influenza and enterovirus antigens, and may be advantageous for immunogens that otherwise fail to elicit antibodies with sufficient titers and breadth for broad protection, such as the envelope protein (Env) of HIV. Previously employed targeting strategies often utilize receptor-specific antibodies, however it is impractical to conjugate a bivalent IgG antibody to oligomeric antigens, including HIV Env trimers. Here we designed single chain variable fragment (scFv) and single chain Fab (scFab) constructs of a Clec9A-targeting antibody, expressed as genetically fused conjugates with the soluble ectodomain of Env, gp140. This conjugation did not affect the presentation of Env neutralising antibody epitopes. The scFab moiety was shown to be more stable than scFv, and in the context of gp140 fusions, was able to mediate better binding to recombinant and cell surface-expressed Clec9A, although the level of binding to cell-surface Clec9A was lower than that of the anti-Clec9A IgG. However, binding to Clec9A on the surface of DCs was not detected. Mouse immunization experiments suggested that the Clec9A-binding activity of the scFab-gp140 conjugate was insufficient to enhance Env-specific antibody responses. This is an important first proof of principle study demonstrating the conjugation of a scFab to an oligomeric protein antigen, and that an scFab displays better antigen binding than the corresponding scFv. Future developments of this technique that increase the scFab affinity will provide a valuable means to target oligomeric proteins to cell surface antigens of interest, improving vaccine-generated immune responses.
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Vacunas contra el SIDA/inmunología , Infecciones por VIH/terapia , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Antígenos Virales/genética , Antígenos Virales/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epítopos/inmunología , Femenino , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Inmunogenicidad Vacunal , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ratones , Prueba de Estudio Conceptual , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Receptores Mitogénicos/inmunología , Receptores Mitogénicos/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/genética , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.
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Anticuerpos Biespecíficos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/farmacocinética , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citocinas/análisis , Citocinas/metabolismo , Células HL-60 , Semivida , Humanos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores Mitogénicos/inmunología , Linfocitos T/metabolismoRESUMEN
Malaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8+ T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin.
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Hemoproteínas/inmunología , Lectinas Tipo C/inmunología , Malaria Cerebral/inmunología , Plasmodium berghei/patogenicidad , Receptores Mitogénicos/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Granzimas/metabolismo , Inmunidad Innata , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos C57BL , Receptores Mitogénicos/genética , Linfocitos TRESUMEN
The development of engineered T cells to treat acute myeloid leukemia (AML) is challenging due to difficulty in target selection and the need for robust T-cell expansion and persistence. We designed a T cell stimulated to kill AML cells based on recognition of the AML-associated surface marker CLEC12A, via secretion of a CLEC12AxCD3 bispecific "engager" molecule (CLEC12A-ENG). CLEC12A-ENG T cells are specifically activated by CLEC12A, are not toxic to hematopoietic progenitor cells, and exhibit antigen-dependent AML killing. Next, we coupled stimulation of T-cell survival to triggering of a chimeric IL7 receptor with an ectodomain that binds a second AML-associated surface antigen, CD123. The resulting T cells, identified as CLEC12A-ENG.CD123IL7Rα T cells, demonstrate improved activation upon dual target recognition, kill AML, and exhibit antitumor activity in xenograft models. Enhanced T-cell activation conferred by CD123.IL7Rα was dependent both on recognition of the CD123 target and on IL7Rα-mediated downstream signaling. Expression of a chimeric IL7R targeted to a second tumor-associated antigen (TAA) should improve T-cell activity not only against hematologic malignancies, but perhaps against all cancers.