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BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animales , Ratones , Carcinoma Hepatocelular/genética , Claudina-1/genética , Neoplasias Hepáticas/genética , Carcinógenos , Microambiente Tumoral , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Línea Celular TumoralRESUMEN
Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.
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Anticuerpos Monoclonales , Plasticidad de la Célula , Animales , Ratones , Humanos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Claudina-1 , Cirrosis Hepática/tratamiento farmacológicoRESUMEN
Trauma combined with hemorrhagic shock (HS/T) leads to systemic inflammation, which results in organ injury. Toll-like Receptor 4 (TLR4)-signaling activation contributes to the initiation of inflammatory pathways following HS/T but its cell-specific roles in this setting are not known. We assessed the importance of TLR4 on leukocytes of myeloid lineage and dendritic cells (DCs) to the early systemic inflammatory response following HS/T. Mice were subjected to HS/T and 20 inflammatory mediators were measured in plasma followed by Dynamic Bayesian Network (DBN) Analysis. Organ damage was assessed by histology and plasma ALT levels. The role of TLR4 was determined using TLR4-/-, MyD88-/-, and Trif-/- C57BL/6 (B6) mice, and by in vivo administration of a TLR4-specific neutralizing monoclonal antibody (mAb). The contribution of TLR4 expressed by myeloid leukocytes and DC was determined by generating cell-specific TLR4-/- B6 mice, including Lyz-Cre × TLR4loxP/loxP, and CD11c-Cre × TLR4loxP/loxP B6 mice. Adoptive transfer of bone marrow-derived TLR4+/+ or TLR4-/- DC into TLR4-/- mice confirmed the contribution of TLR4 on DC to the systemic inflammatory response after HS/T. Using both global knockout mice and the TLR4-blocking mAb 1A6 we established a central role for TLR4 in driving systemic inflammation. Using cell-selective TLR4-/- B6 mice, we found that TLR4 expression on both myeloid cells and CD11chigh DC is required for increases in systemic cytokine levels and organ damage after HS/T. We confirmed the capacity of TLR4 on CD11chigh DC to promote inflammation and liver damage using adoptive transfer of TLR4+/+ conventional (CD11chigh) DC into TLR4-/- mice. DBN inference identified CXC chemokines as proximal drivers of dynamic changes in the circulating levels of cytokines/chemokines after HS/T. TLR4 on DC was found to contribute selectively to the elevations in these proximal drivers. TLR4 on both myeloid cells and conventional DC is required for the initial systemic inflammation and organ damage in a mouse model of HS/T. This includes a role for TLR4 on DC in promoting increases in the early inflammatory networks identified in HS/T. These data establish DC along with macrophages as essential to the recognition of tissue damage and stress following tissue trauma with HS.
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BACKGROUND: Increased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease. METHODS: The capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4. RESULTS: RASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro. CONCLUSION: TLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism.
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Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Líquido Sinovial/inmunología , Receptor Toll-Like 4/inmunología , Anciano , Anticuerpos Monoclonales Humanizados/farmacología , Autoantígenos/inmunología , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos Cíclicos/inmunología , Receptor Toll-Like 4/antagonistas & inhibidoresRESUMEN
BACKGROUND: Although the role of TLR4 in driving inflammation and organ injury after hemorrhagic shock and resuscitation (H/R) is well established, the role of TLR2-another receptor for damage-associated molecular pattern (DAMP) molecules-is not. In this study, we used a combination of TLR2 and wild type (WT) mice treated with anti-TLR2 and anti-TLR4 neutralizing monoclonal antibodies (mAb) to discern the contribution of TLR2 relative to TLR4 to the systemic inflammatory response in murine H/R. MATERIAL AND METHODS: WT mice, TLR2, and WT mice receiving an anti-TLR2 or an anti-TLR4 mAB (given as a pretreatment) were sacrificed at 6 or 20 h post-H/R. Bone marrow TLR2/WT chimeric mice were created to assess the importance of immune and nonimmune cell-associated TLR2. RESULTS: TLR2 mice subjected to H/R exhibited significantly less liver damage and lower markers of systemic inflammation only at 20 h. Bone marrow chimeric mice using combinations of TLR2 mice and WT mice demonstrated that TLR2 on non-bone marrow derived cells played a dominant role in the differences at 20 h. Interestingly, WT mice treated with anti-TLR2 mAB demonstrated a reduction in organ damage and systemic inflammation at both 6 and 20 h following H/R. A combination of anti-TLR2 mAB and anti-TLR4 mAB showed that both receptors drive IP-10 and KC levels and that there is cooperation for increases in IL-6, MIG, and MCP-1 levels between TLR2 and TLR4. CONCLUSION: These data also support the conclusion that TLR2 and TLR4 act in concert as important receptors in the host immune response to H/R.
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Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Choque Hemorrágico/tratamiento farmacológico , Choque Hemorrágico/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Médula Ósea , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Resucitación , Choque Hemorrágico/inmunología , Choque Hemorrágico/terapia , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 4/antagonistas & inhibidoresRESUMEN
INTRODUCTION: Toll-like receptors (TLRs) play an important role in the recognition of microbial products and in host defense against infection. However, the massive release of inflammatory mediators into the bloodstream following TLR activation following sepsis is thought to contribute to disease pathogenesis. METHODS: Here, we evaluated the effects of preventive or therapeutic administration of monoclonal antibodies (mAbs) targeting either TLR2 or TLR4 in a model of severe polymicrobial sepsis induced by cecal ligation and puncture in mice. RESULTS: Pre-treatment with anti-TLR2 or anti-TLR4 mAb alone showed significant protection from sepsis-associated death. Protective effects were observed even when the administration of either anti-TLR2 or anti-TLR4 alone was delayed (i.e., 3 h after sepsis induction). Delayed administration of either mAb in combination with antibiotics resulted in additive protection. CONCLUSION: Although attempts to translate preclinical findings to clinical sepsis have failed so far, our preclinical experiments strongly suggest that there is a sufficient therapeutic window within which patients with ongoing sepsis could benefit from combined antibiotic plus anti-TLR2 or anti-TLR4 mAb treatment.
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Anticuerpos Monoclonales/farmacología , Sepsis/prevención & control , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Ratones , Sepsis/inmunología , Sepsis/microbiología , Sepsis/patologíaRESUMEN
Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.
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Anticuerpos Biespecíficos/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Pruebas de Neutralización , Biblioteca de Péptidos , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. METHODS: Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. RESULTS: In vitro TLR4 blockade decreased lipopolysaccharide-mediated ß-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. CONCLUSION: Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.
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Anticuerpos Monoclonales/farmacología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Inmunosupresores/farmacología , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Aloinjertos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Rechazo de Injerto/inmunología , Humanos , Memoria Inmunológica/efectos de los fármacos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Trasplante de Piel , Linfocitos T/inmunología , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Receptor Toll-Like 4/inmunologíaRESUMEN
In order to treat Toll like receptor 4 (TLR4)-mediated diseases, we generated a potent antagonistic antibody directed against human TLR4, Hu 15C1. This antibody's potency can be modulated by engaging not only TLR4 but also Fcγ receptors (FcγR), a mechanism that is driven by avidity and not cell signaling. Here, using various formats of the antibody, we further dissect the relative contributions of the Fv and Fc portions of Hu 15C1, discovering that the relationship to potency of the different antibody arms is not linear. First, as could be anticipated, we observed that Hu 15C1 co-engages up to 3 receptors on the same plasma membrane, i.e., 2 TLR4 molecules (via its variable regions) and either FcγRI or FcγRIIA (via the Fc). The Kd of these interactions are in the nM range (3 nM of the Fv for TLR4 and 47 nM of the Fc for FcγRI). However, unexpectedly, neutralization experiments revealed that, due to the low level of cell surface TLR4 expression, the avidity afforded by engagement through 2 Fv arms was significantly limited. In contrast, the antibody's neutralization capacity increases by 3 logs when able to exploit Fc-FcγR interactions. Taken together, these results demonstrate an unforeseen level of contribution by FcγRs to an antibody's effectiveness when targeting a cell surface protein of relatively low abundance. These findings highlight an exploitable mechanism by which FcγR-bearing cells may be more powerfully targeted, envisioned to be broadly applicable to other reagents aimed at neutralizing cell surface targets on cells co-expressing FcγRs.
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Anticuerpos Monoclonales Humanizados/inmunología , Afinidad de Anticuerpos/inmunología , Receptores de IgG/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Células CHO , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Modelos Inmunológicos , Unión Proteica/inmunología , Receptores de IgG/metabolismo , Resonancia por Plasmón de Superficie , Receptor Toll-Like 4/metabolismo , Células U937RESUMEN
Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.
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Polisacáridos/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Animales , Glicosilación , Humanos , Ratones , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.
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Inflamación/inmunología , Macrófagos/inmunología , Receptores de IgG/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Células CHO , Línea Celular , Cricetulus , Dimerización , Femenino , Humanos , Inflamación/metabolismo , Macrófagos/citología , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de IgG/metabolismo , Receptor Toll-Like 4/química , Receptor Toll-Like 4/metabolismo , Células U937RESUMEN
IL-27 is an APC-derived IL-6/IL-12 family composite cytokine with multiple functions such as regulation of Th1, Th17, and regulatory T cell differentiation, B cell proliferation, and Ig class switching. The IL-27 complex is formed by the association of the cytokine p28 with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27 cytokine and soluble receptor subunits p28 and EBI3 can be secreted independently. The p28 subunit has been shown to have IL-27-independent biological activities. We previously demonstrated that p28 can form an alternative composite cytokine with the EBI3 homolog cytokine-like factor 1 (CLF; CRLF1). p28/CLF modulates NK cell activity and CD4 T cell cytokine production in vitro. In this study we used IL-6-dependent plasmacytoma cell line B9 and CD4 T cells from IL-27Rα-deficient mice to demonstrate that p28/CLF activates IL-27-unresponsive cells, indicating that p28/CLF and IL-27 signal through different receptors. The observation that p28/CLF, unlike IL-27, sustains B9 plasmacytoma cell proliferation prompted us to investigate the effects of p28/CLF on mouse B cells. We observed that p28/CLF induces IgM, IgG2c, and IgG1 production and plasma cell differentiation. p28/CLF therefore has the potential to contribute to B and plasma cell function, differentiation, and proliferation in normal and pathological conditions such as Castelman's disease and multiple myeloma.
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Linfocitos B/citología , Interleucinas/inmunología , Linfopoyesis/fisiología , Células Plasmáticas/citología , Animales , Linfocitos B/inmunología , División Celular , Línea Celular , Femenino , Inmunoglobulinas/biosíntesis , Interleucinas/genética , Quinasas Janus/fisiología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Células Plasmáticas/inmunología , Procesamiento Proteico-Postraduccional , Receptores de Citocinas/deficiencia , Receptores de Citocinas/genética , Receptores de Interleucina , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción STAT/fisiología , Transducción de Señal , Células Th2/inmunología , TransfecciónRESUMEN
The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines. This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography. The IL-27 polyglutamic acid domain is located in a flexible inter-α helix loop, and HA-bound IL-27 retained biological activity. Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL-27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications. IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs could be grafted onto other type I cytokine inter-α helix loops to modify their pharmacological properties.
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Médula Ósea/química , Durapatita/química , Interleucinas/metabolismo , Ácido Poliglutámico/química , Secuencias de Aminoácidos/inmunología , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Linfocitos T CD8-positivos/química , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Células Cultivadas , Durapatita/metabolismo , Femenino , Humanos , Interleucinas/genética , Interleucinas/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Osteoclastos/inmunología , Osteoclastos/metabolismo , Ácido Poliglutámico/genética , Ácido Poliglutámico/uso terapéutico , Unión Proteica/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Células del Estroma/inmunología , Células del Estroma/metabolismoRESUMEN
The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.
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División Celular , Interleucina-17/genética , Animales , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Vectores Genéticos , Humanos , Proteínas Recombinantes/biosíntesisRESUMEN
Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor ß (LIFRß) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRß signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRß with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-ß in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.
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Linfocitos T CD4-Positivos/metabolismo , Subunidad alfa del Receptor del Factor Neurotrófico Ciliar/metabolismo , Factor Neurotrófico Ciliar/metabolismo , Citocinas/metabolismo , Receptores de Citocinas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Receptor gp130 de Citocinas/metabolismo , Humanos , Interferón gamma/biosíntesis , Subunidad alfa del Receptor de Interleucina-11/metabolismo , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
BACKGROUND: IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. METHODS: We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). RESULTS: The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. CONCLUSIONS: Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-ß stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.
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Interleucina-6/metabolismo , Activación de Linfocitos/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Linfocitos T/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Interleucina-6/farmacología , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: Chronic intestinal inflammation culminates in cancer and a link to Toll-like receptor-4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. METHODS: Mice transgenic for a constitutively active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)-DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC-associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. RESULTS: Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis-associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. CONCLUSIONS: Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis-associated cancer.
Asunto(s)
Colitis Ulcerosa/complicaciones , Neoplasias del Colon/etiología , Inflamación/complicaciones , Mucosa Intestinal/patología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/fisiología , Animales , Azoximetano/toxicidad , Western Blotting , Carcinógenos/toxicidad , Colitis Ulcerosa/inducido químicamente , Neoplasias del Colon/metabolismo , Sulfato de Dextran/toxicidad , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas para Inmunoenzimas , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/lesiones , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/genéticaRESUMEN
Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.
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Linfocitos B/metabolismo , Células Dendríticas Foliculares/metabolismo , Centro Germinal/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Anticuerpos Bloqueadores , Afinidad de Anticuerpos , Antígenos de Diferenciación/biosíntesis , Linfocitos B/inmunología , Linfocitos B/patología , Trasplante de Médula Ósea , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Dendríticas Foliculares/patología , Centro Germinal/patología , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Memoria Inmunológica , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Mutación/genética , Quimera por Radiación , Transducción de Señal/inmunología , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.
Asunto(s)
Interleucinas/metabolismo , Células Asesinas Naturales/inmunología , Subunidades de Proteína/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interleucina-6/fisiología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Proteínas Portadoras/biosíntesis , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Glutatión Transferasa/biosíntesis , Humanos , Interleucinas/biosíntesis , Interleucinas/fisiología , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Unión Proteica/inmunología , Subunidades de Proteína/fisiología , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/fisiología , Linfocitos T/metabolismoRESUMEN
Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.